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  • vH + -ATPase-induced intracellular acidification is critical to glucose-stimulated insulin secretion in beta cells
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2020-01-04
    Akshata R. Naik, Brent J. Formosa, Rishika G. Pulvender, Asiri G. Liyanaarachchi, Bhanu P. Jena

    Swelling of secretory vesicles is critical for the regulated release of intra-vesicular contents from cells during secretion. At the secretory vesicle membrane of the exocrine pancreas and neurons, GTP-binding G proteins, vH+-ATPase, potassium channels and AQP water channels, are among the players implicated in vesicle volume regulation. Here we report in the endocrine insulin-secreting MIN6 cells, the similar requirement of vH+-ATPase-mediated intracellular acidification on glucose-stimulated insulin release. MIN6 cells exposed to the vH+-ATPase inhibitor Bafilomycin A show decreased acidification of the cytosolic compartment that include insulin-carrying granules. Additionally, a loss of insulin granules near the cell plasma membrane following Bafilomycin A treatment, suggests impaired transport of insulin granules and consequent decrease in glucose-stimulated insulin secretion and accumulation of intracellular insulin. These results suggest that vH+-ATPase-mediated intracellular acidification is required for insulin secretion in beta cells.

  • Effect of castration on pelvic neurons in the male pig
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2020-01-01
    Jerzy Kaleczyc, Natalia Kasica-Jarosz, Zenon Pidsudko, Agnieszka Dudek, Magdalena Klimczuk, Waldemar Sienkiewicz

    Abstract The present study investigated the influence of castration performed at neonatal age on neuronal elements in the anterior pelvic ganglion of the male pig with immunohistochemistry and quantitative real-time PCR (qPCR). The ganglia were examined 3 and 6 months after surgery. In 3-month-old castrated pigs (3MCP) 74% of adrenergic and 31% of cholinergic neurons stained for caspase-3 (CASP-3), and much greater numbers of perikarya than in the control animals expressed CGRP, galanin (GAL) and VIP (peptides known to have neuroprotective properties). In 6-months-old castrated pigs (6MCP), an excessive loss (90%) of neurons and intraganglionic nerve fibres was found. The survived adrenergic and cholinergic neurons also expressed CASP-3, CGRP, GAL or VIP. The qPCR results corresponded with immunofluorescence findings. In 3MCP, genes for CASP-3 and CGRP were up-regulated, while the expression of those for DβH, VAChT, GAL, VIP and SP displayed statistically insignificant variations. In 6MCP, distinctly up-regulated were genes for CGRP, GAL, VIP, SP, DβH and VAChT, while the expression of casp3 gene was down-regulated. The study revealed for the first time the excessive loss of pelvic neurons following castration, and a realistic assumption is proposed, that the neurons died due to apoptosis triggered by androgen deprivation.

  • Zonisamide enhances neurite outgrowth from adult rat dorsal root ganglion neurons, but not proliferation or migration of Schwann cells
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-12-26
    Shizuka Takaku, Kazunori Sango

    Zonisamide, an anti-epileptic and anti-Parkinson’s disease drug, displays neurotrophic activity on cultured motor neurons and facilitates axonal regeneration after peripheral nerve injury in mice, but its underlying mechanisms remain unclear. In this study, zonisamide enhanced neurite outgrowth from cultured adult rat dorsal root ganglion (DRG) neurons in a concentration-dependent manner (1 μM < 10 μM < 100 μM), and its activity was significantly attenuated by co-treatment with a phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002 or a mitogen-activated protein kinase (MAPK) inhibitor U0126. In agreement with these findings, 100 μM zonisamide for 1 h induced phosphorylation of AKT and ERK1/2, key molecules of PI3K and MAPK signaling pathways, respectively in mouse neuroblastoma × rat DRG neuron hybrid cells ND7/23. In contrast, zonisamide failed to promote proliferation or migration of immortalized Fischer rat Schwann cells 1 (IFRS1). These findings suggest that the beneficial effects of zonisamide on peripheral nerve regeneration may be attributable to its direct actions on neurons through PI3K and MAPK pathways, rather than the stimulation of Schwann cells.

  • PUFAs supplementation affects the renal expression of pannexin 1 and connexins in diabetic kidney of rats
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-12-20
    Martina Luetić, Marija Vitlov Uljević, Tomislav Mašek, Benjamin Benzon, Katarina Vukojević, Natalija Filipović

    Abstract In diabetic nephropathy (DN), intercellular communication is disrupted. Connexins (Cx) have a crucial role in that process. Dietary ratios and supplementation with polyunsaturated fatty acids (PUFAs) can alleviate diabetic complications and cause alterations in Cx levels. Although pannexins (Panx) share similarities with members of the Cx family, their function in diabetic nephropathy has still not been fully determined. We studied the influence of PUFA supplementation on the immunoexpression of Px1 and Cx family members in diabetic kidneys of rats. Four groups of rats in experimental DM1 model were supplemented with different dietary n-6/n-3 ratios; ≈7 in control (C) and diabetic groups (STZ), ≈ 60 in the STZ + N6 group and ≈ 1 (containing 16% EPA and 19% DHA) in the STZ + N3 group. Immunoexpression of Cx40, Cx43, Cx45 and Panx1 was evaluated in the renal tissue of diabetic rats using immunohistochemistry. Diabetes significantly decreased the protein expression of Cx40 and Cx43 and increased Panx1 protein expression in the renal cortex (p < 0.05–p < 0.01). There was a significant impact of diet on Cx and Panx1 immunoexpression. Dietary supplementation with a high n-6/n-3 ratio downregulated the protein expression of Cx45 and Panx1 in diabetic rats (p < 0.05–p < 0.01), while Cx43 immunoexpression was increased in diabetic rats fed with high and low n-6/n-3 ratios (p < 0.01–p < 0.001). Hyperglycaemic conditions in DN interfere with cell-to-cell communication and disturb the connection between cells and their immediate environment due to variations in connexin and pannexin immunoexpression. These variations can be regulated by PUFA dietary intake, suggesting their beneficial effect and possible therapeutic option.

  • An adaptation of Twort’s method for polychromatic staining of epoxy-embedded semithin sections
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-12-17
    Vasiliy N. Manskikh, Eugene V. Sheval

    Epoxy-embedded semithin sections are useful for the analysis of cell and tissue organization, as well as for the processing of samples for transmission electron microscopy. Because only a very limited number of staining protocols have been developed for epoxy-embedded sections; semithin sections are used infrequently compared to conventional paraffin sections. Here, we describe a simple and reproducible polychromatic protocol for the routine staining of epoxy-embedded semithin sections by adapting Twort’s staining method (mixture of neutral red and fast green FCF). The method can be used for the visualization of cellular organization as well as for the detection of elastic and collagen fibers. The proposed protocol demonstrated the best results for samples fixed for transmission electron microscopy, which suggests, as we demonstrated here, that this staining protocol can also be used for correlative light and electron microscopy.

  • Abnormal expression of chondroitin sulfate sulfotransferases in the articular cartilage of pediatric patients with Kashin–Beck disease
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-12-16
    Jian Lei, Siqi Yan, Yuan Zhou, Liyun Wang, Jinghua Zhang, Xiong Guo, Mikko J. Lammi, Jing Han, Chengjuan Qu

    The objective of this study is to investigate the expression of enzymes involved in the sulfation of articular cartilage from proximal metacarpophalangeal (PMC) joint cartilage and distal metacarpophalangeal (DMC) joint cartilage in children with Kashin–Beck disease (KBD). The finger cartilage samples of PMC and DMC were collected from KBD and normal children aged 5–14 years old. Hematoxylin and eosin staining as well as immunohistochemical staining were used to observe the morphology and quantitate the expression of carbohydrate sulfotransferase 3 (CHST-3), carbohydrate sulfotransferase 12 (CHST-12), carbohydrate sulfotransferase 13 (CHST-13), uronyl 2-O-sulfotransferase (UST), and aggrecan. In the results, the numbers of chondrocyte decreased in all three zones of PMC and DMC in the KBD group. Less positive staining cells for CHST-3, CHST-12, CHST-13, UST, and aggrecan were observed in almost all three zones of PMC and DMC in KBD. The positive staining cell rates of CHST-12 were higher in superficial and middle zones of PMC and DMC in KBD, and a significantly higher rate of CHST-13 was observed only in superficial zone of PMC in KBD. In conclusion, the abnormal expression of chondroitin sulfate sulfotransferases in chondrocytes of KBD children may provide an explanation for the cartilage damage, and provide therapeutic targets for the treatment.

  • RNAscope dual ISH–IHC technology to study angiogenesis in diffuse large B-cell lymphomas
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-12-13
    Tiziana Annese, Roberto Tamma, Michelina De Giorgis, Simona Ruggieri, Eugenio Maiorano, Giorgina Specchia, Domenico Ribatti

    Diffuse large B-cell lymphomas (DLBCLs) are the most common types of Non-Hodgkin’s lymphomas and are highly heterogeneous in terms of phenotype and treatment response. The natural course of DLBCLs tumor progression is featured by a flow of events leading to the enhancement of proliferative and invasive capabilities and, therefore, towards the establishment of a more aggressive phenotype. Angiogenesis is a constant hallmark of DLBCLs progression, has prognostic potential and promote DLBCLs dissemination. The study of DLBCLs angiogenesis mechanisms, and the tumor endothelium characterization, will allow us to identify new prognostic/predictive biomarkers to proper patient selection to antiangiogenic treatment. In our previous work, by RNAscope technology, we have demonstrated that Janus kinase (Jak) and signal transducer activator of transcription pathway (STAT) is one of the proangiogenic pathways activated in DLBCLs and it drives neoangiogenesis occurred by vasculogenesis mechanism. Here, we describe a detailed protocol to perform RNAscope technology alone and in combination with immunohistochemistry (called dual RNAscope ISH–IHC) in DLBCLs formalin-fixed, paraffin-embedded sections. We propose dual ISH–IHC as an extremely powerful method to study angiogenesis in DLBCLs, because it allows one to answer important biological questions that are difficult to address using other single methods.

  • Evaluating nuclear translocation of surface receptors: recommendations arising from analysis of CD44
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-12-13
    Rick F. Thorne, Yan Wang, Yan Zhang, Xueli Jing, Xu Dong Zhang, Charles E. de Bock, Camila S. Oliveira

    CD44 is a transmembrane receptor that acts as adhesion protein, fundamentally recognizing hyaluronan, an essential component of the extracellular matrix. It has a well-established functional association with cancer metastasis, particularly the CD44 variant forms which are considered essential markers of cancer stem cells. CD44 itself lacks intrinsic kinase activity but rather engages in signalling through specific interactions with kinases and other signalling components. Proteolysis within its transmembrane region also leads to release of the CD44 cytoplasmic domain, which can translocate to the nucleus and regulate transcription. A third signalling modality has been reported where the intact CD44 receptor translocates to the nucleus. Here, we investigated the latter using imaging techniques together with biochemical analyses. Our findings support observations where CD44 is cleaved prior to nuclear translocation and challenges the evidence for the presence of intact CD44 receptors in the cell nucleus. Conclusions regarding the presence of intact CD44 in the cell nucleus as a signalling modality, therefore, require re-evaluation. We highlight artefacts and common technical issues associated with these experiments that can lead to misinterpretation.

  • Characterization of NCC-RbC-51, an RB cell line isolated from a metastatic site
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-11-28
    Hemanth Ravishankar, Abubakar Siddiq Mangani, M. Bhavani Shankar, Mayur Joshi, T. Devasena, Sowmya Parameswaran, Krishnakumar Subramaniam

    Retinoblastoma (RB) is a childhood eye tumor, caused by the RB1 gene mutation. Since RB is a rapidly proliferating tumor, the patient presents with a Group-D/E tumor at the time of diagnosis. Enucleation is preferred in most unilateral cases to prevent metastasis. Various cell lines have been established to study the tumor’s growth pattern and target the cancer cells. The commonly used cell lines are WERI-Rb-1 and Y79, both isolated from the primary tumor of RB. Cell lines established from the metastatic site of RB have not been characterized before. In this study, we have characterized NCC-RbC-51, derived from RB tumor to cervical lymph node site and investigated its potential to represent a highly aggressive and metastatic tumor. We compared the proliferative and invasive properties of NCC-RbC-51 with a cell line isolated from the primary site, WERI-Rb-1. NCC-RbC-51 had higher rates of proliferation and apoptosis and had better invasive ability. Copy number variation analysis and the pathways predicted from these show that the pathways altered in NCC-RbC-51 could contribute to its metastatic nature. In all, the results suggest that NCC-RbC-51, a cell line isolated from metastatic site, could be a potential model to study aggressive/invasive RB.

  • Unequivocal imaging of aluminium in human cells and tissues by an improved method using morin
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-08-28
    Matthew J. Mold, Manpreet Kumar, William Chu, Christopher Exley

    Aluminium is biologically reactive and its ability to potentiate the immune response has driven its inclusion in both veterinary and human vaccines. Consequently, the need for unequivocal visualisation of aluminium in vivo has created a focused research effort to establish fluorescent molecular probes for this purpose. The most commonly used direct fluorescent labels for the detection of aluminium are morin (2′,3,4′,5,7-pentahydroxyflavone) and lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzene-1-sulphonic acid]. While the former has gained popularity in the detection of aluminium in plants and predominantly within root tips, the latter boasts greater sensitivity and selectivity for the detection of aluminium in human cells and tissues. Herein, we have developed a simplified morin staining protocol using the autofluorescence quenching agent, Sudan Black B. This modified protocol improves tissue morphology and increases analytical sensitivity, which allows intracellular aluminium to be detected in monocytes and when co-localised with senile plaques in human brain tissue of donors diagnosed with familial Alzheimer’s disease. Overall, our results demonstrate a simple approach to minimise false positives in the use of morin to unequivocally detect aluminium in vivo.

  • Correction to: Unequivocal imaging of aluminium in human cells and tissues by an improved method using morin
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-11-20
    Matthew J. Mold, Manpreet Kumar, William Chu, Christopher Exley

    After publication of our article, it has come to our attention that our Conflict of Interest statement should read.

  • Glycolaldehyde induces sensory neuron death through activation of the c-Jun N-terminal kinase and p-38 MAP kinase pathways
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-11-16
    Tomoyo Akamine, Shizuka Takaku, Mari Suzuki, Naoko Niimi, Hideji Yako, Keiichiro Matoba, Daiji Kawanami, Kazunori Utsunomiya, Rimei Nishimura, Kazunori Sango

    Glycolaldehyde (GA) is a highly reactive hydroxyaldehyde and one of the glycolytic metabolites producing advanced glycation endproducts (AGEs), but its toxicity toward neurons and Schwann cells remains unclear. In the present study, we found that GA exhibited more potent toxicity than other AGE precursors (glyceraldehyde, glyoxal, methylglyoxal and 3-deoxyglucosone) against immortalized IFRS1 adult rat Schwann cells and ND7/23 neuroblastoma × neonatal rat dorsal root ganglion (DRG) neuron hybrid cells. GA affected adult rat DRG neurons and ND7/23 cells more severely than GA-derived AGEs, and exhibited concentration- and time-dependent toxicity toward ND7/23 cells (10 < 100 < 250 < 500 µM; 6 h < 24 h). Treatment with 500 µM GA significantly up-regulated the phosphorylation of c-jun N-terminal kinase (JNK) and p-38 mitogen-activated kinase (p-38 MAPK) in ND7/23 cells. Furthermore, GA-induced ND7/23 cell death was significantly inhibited due to co-treatment with 10 µM of the JNK inhibitor SP600125 or the p-38 MAPK inhibitor SB239063. These findings suggest the involvement of JNK and p-38 MAPK-signaling pathways in GA-induced neuronal cell death and that enhanced GA production under diabetic conditions might be involved in the pathogenesis of diabetic neuropathy.

  • Selective autophagy of cytosolic protein aggregates involves ribosome-free rough endoplasmic reticulum
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-11-12
    Sujin Park, Christian Zuber, Jürgen Roth

    Autophagy is a degradative cellular process that can be both non-selective and selective and begins with the formation of a unique smooth double-membrane phagophore which wraps around a portion of the cytoplasm. Excess and damaged organelles and cytoplasmic protein aggregates are degraded by selective autophagy. Previously, we reported that in fed HepG2 cells, cytoplasmic aggregates of EDEM1 and surplus fibrinogen Aα–γ assembly intermediates are targets of selective autophagy receptors and become degraded by a selective autophagy called aggrephagy. Here, we show by multiple confocal immunofluorescence and colocalization panels the codistribution of cytoplasmic protein aggregates with the selective autophagy receptors p62/SQSTM1 and NBR1 and with the phagophore marker LC3, and that phagophores induced by vinblastine treatment contain complexes of protein aggregates and selective autophagy receptors. By combined serial ultrathin section analysis and immunoelectron microscopy, we found that in fed HepG2 cells, a basically ribosome-free subdomain of rough endoplasmic reticulum (RER) cisternae forms a cradle that engulfs the cytoplasmic protein aggregates. This RER subdomain appears structurally different from omegasomes formed by the RER, which were suggested to provide a membrane platform from which the phagophore is derived in starvation-induced autophagy. Taken together, our observations provide further evidence for the importance of RER subdomains as a site and membrane source for phagophore formation and show their involvement in selective autophagy.

  • 3D analysis of capillary network in skeletal muscle of obese insulin-resistant mice
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-08-31
    Nejc Umek, Simon Horvat, Erika Cvetko, Marko Kreft, Jiří Janáček, Lucie Kubínová, Tatjana Stopar Pintarič, Ida Eržen

    In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.

  • Metalloproteinase 14 (MMP-14) and hsa-miR-410-3p expression in human inflamed dental pulp and odontoblasts
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-09-05
    Aniela Brodzikowska, Agata Gondek, Beata Rak, Wiktor Paskal, Kacper Pełka, Agnieszka Cudnoch-Jędrzejewska, Paweł Włodarski

    The objective of this study is to evaluate MMP-14 expression in odontoblasts and in the bulk of dental pulp of teeth with pulpitis; to determine the expression of microRNA-410 (miR-410) in pulp tissue, since sequence analysis suggests that miR-410 has potential binding site on MMP-14’s 3′UTR, and hence, can regulate expression of the latter one. Tissue samples of dental pulp from teeth with pulpitis and healthy (control) were formalin fixed and paraffin embedded (FFPE). Samples were examined using immunohistochemical staining for MMP-14 and the expression of miR-410 was evaluated using qRT-PCR. In both, healthy and inflamed pulp odontoblasts stained more intensively than remaining pulp tissue, but this difference was not statistically significant. More positive staining was observed in inflamed pulps compared to healthy pulps. Expression of miR-410 was found significantly lower in inflamed pulps than in healthy ones. In the two examined zones, odontoblasts and remaining pulp, miR-410 was expressed on a similar level. No statistically significant correlation of miR-410 and MMP-14 expression was found. We showed that inflammation changes the MMP-14 expression in pulp tissue and odontoblasts. This study demonstrates for the first time miR-410 expression in human dental pulp and that expression of this microRNA was downregulated in inflamed dental pulp and odontoblasts.

  • Scaffold protein Lin7 family in membrane skeletal protein complex in mouse seminiferous tubules
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-08-13
    Akio Kamijo, Yurika Saitoh, Takeharu Sakamoto, Hiroshi Kubota, Junji Yamauchi, Nobuo Terada

    The membrane skeletal complex, protein 4.1G–membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell–cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.

  • Aging results in accumulation of M1 and M2 hepatic macrophages and a differential response to gadolinium chloride
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-11-06
    Steven A. Bloomer, Eric D. Moyer, Kyle E. Brown, Kevin C. Kregel

    Macrophages have vital roles in innate immunity by modulating the inflammatory response via their ability to alter their phenotype from pro-inflammatory (M1) to anti-inflammatory (M2). Aging increases activation of the innate immune system, and macrophage numbers increase in the aged liver. Since macrophages also produce free radical molecules, they are a potential source of age-related oxidative injury in the liver. This study evaluated macrophage phenotype in the aged liver and whether the increase in the number of macrophages with aging is associated with enhanced hepatic oxidative stress. Hepatic macrophage phenotype and oxidative stress were evaluated 2 days after a single intraperitoneal injection of saline or gadolinium chloride (GdCl3, 10 mg/kg) in young (6 months) and aged (24 months) Fischer 344 rats. GdCl3 has been shown to decrease the expression of macrophage-specific markers and impair macrophage phagocytosis in the liver. Saline-treated aged rats demonstrated greater numbers of both M1 (HO-1+/iNOS+) and M2 (HO-1+/CD163+) macrophages, without evidence of a phenotypic shift. GdCl3 did not alter levels of dihydroethidium fluorescence or malondialdehyde, suggesting that macrophages are not a major contributor to steady-state levels of oxidative stress. However, GdCl3 decreased M1 and M2 macrophage markers in both age groups, an effect that was attenuated in aged rats. In old animals, GdCl3 decreased iNOS expression to a greater extent than HO-1 or CD163. These results suggest a novel effect of aging on macrophage biology and that GdCl3 shifts hepatic macrophage polarization to the M2 phenotype in aged animals.

  • GASMoC method: a phenol-free technique to detect acid-fast bacilli
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-10-25
    A. Gomes, P. Amaral, R. Santos, S. Santos, F. Tortosa, P. Mendonça, A. Marques-Ramos

    The genre Mycobacterium includes a series of pathological species, such as M. tuberculosis, which is important for pathology laboratories to detect for a correct diagnosis. The Ziehl–Neelsen technique (ZNT) is the most commonly histochemical method used to detect these bacilli and uses a heated mixture of carbol-fuchsine, which contains basic fuchsine and phenol. Whereas the former component is responsible for the pinkish staining of acid-fast mycobacteria, the role of phenol is not completely understood and it has been suggested that its exclusion does not impact the detection ability of the ZNT. Since phenol is highly toxic and induces several injuries, the goal of this study is to determine the detection capacity of mycobacteria through a method that excludes the use of phenol. Accordingly, the GASMoC method, a modified ZNT that employs a solution of aqueous basic fuchsine heated at 37 °C, was tested on histological samples positive for mycobacteria and the results were compared to that of the ZNT. Data demonstrated that the GASMoC method was able to detect acid-fast bacilli (AFB) in all analyzed cases. Remarkably, microscopic inspection of mycobacteria was easier when the GASMoC method was applied. In conclusion, our study demonstrates that AFB detection does not require phenol and that the GASMoC method, a phenol-free technique, may substitute the ZNT in pathology laboratories.

  • Tricellular tight junction protein LSR/angulin-1 contributes to the epithelial barrier and malignancy in human pancreatic cancer cell line
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-10-24
    Takuro Kyuno, Daisuke Kyuno, Takayuki Kohno, Takumi Konno, Shin Kikuchi, Chihiro Arimoto, Hiroshi Yamaguchi, Masafumi Imamura, Yasutoshi Kimura, Masuo Kondoh, Ichiro Takemasa, Takashi Kojima

    Lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 is a crucial molecule of tricellular contacts in the epithelial barrier of normal cells and the malignancy of cancer cells. To investigate whether LSR/angulin-1 affects the epithelial barrier and malignancy in human pancreatic cancer, human pancreatic cancer cell line HPAC was used. Treatment with EGF or TGF-β increased the expression of LSR, but not tricellulin (TRIC), and induced the localization of LSR and TRIC to bicellular tight junctions from tricellular tight junctions. TGF-β receptor type-1 inhibitor EW-7197 prevented changes of the distribution and the barrier function of LSR by TGF-β. Knockdown of LSR increased cell migration, invasion, proliferation and EGF ligand amphiregulin expression and decreased the epithelial barrier. Treatment with amphiregulin induced cell migration and invasion and knockdown of amphiregulin prevented the increases of cell migration, invasion and proliferation caused by knockdown of LSR. Treatment with LSR ligand peptide angubindin-1 decreased the epithelial barrier and the expression of LSR, but not TRIC, and increased cell invasion. Knockdown of TRIC decreased cell migration and the epithelial barrier. In immunohistochemical analysis of human pancreatic cancer tissues, LSR and TRIC were found to be localized at the cell membranes of normal pancreatic ducts and well-differentiated pancreatic ductal adenocarcinomas (PDAC), whereas in poorly differentiated PDAC, LSR was weakly detected in the cytoplasm. Amphiregulin was highly expressed in the cytoplasm of well- and poorly differentiated PDAC. In pancreatic cancer, LSR contributes to the epithelial barrier and malignancy via growth factors and may be a potential targeting molecule in the therapy.

  • Molecular and ultrastructure study of endoplasmic reticulum stress in hepatic steatosis: role of hepatocyte nuclear factor 4α and inflammatory mediators
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-10-21
    Salwa M. Abo El-khair, Fatma M. Ghoneim, Dalia A. Shabaan, Ayman Z. Elsamanoudy

    Endoplasmic reticulum (ER) stress could participate in high-fat diet (HFD)-induced hepatic steatosis. The current study aims to investigate the role of ER stress as well as inflammation as possible pathophysiologic mechanisms of HFD-induced hepatic steatosis at ultrastructure and molecular levels. Fifteen control rats on ordinary diet and 30 HFD-fed rats were enrolled in the study. Histological and EM examinations of rats’ liver were carried out. Molecular study of TNF-α, CRP, and HNF4α by RT qPCR as well as biochemical investigation of liver function and lipids profile were done. Hepatic steatosis was induced with lipid droplets accumulation at histological level and mega-mitochondria with reduced ER-mitochondrial distance at EM level. Increased gene expression of TNF-α and CRP was significantly correlated with the reduced HNF4α expression and with other ER stress markers. In conclusion, endoplasmic reticulum stress, confirmed at ultrastructure level, plays an important role in pathogenesis of HFD-induced hepatic steatosis. HNF4α downregulation as well as increased expression of hs-CRP and TNF-α enforce the concept of interplay between ER stress, hepatic subclinical inflammation, and disturbed gene expression regulation in the pathogenesis of HFD-induced hepatic steatosis.

  • The effect of GnRH antagonist cetrorelix on Wnt signaling members in pubertal and adult mouse ovaries
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-10-19
    Filiz Tepekoy, Fatma Uysal, Nuray Acar, Ismail Ustunel, Gokhan Akkoyunlu

    Wide application of gonadotropin-releasing hormone (GnRH) agonists and antagonists for clinical purposes determines their effects on ovarian signaling pathways. Our study aimed to determine the localization, expression levels of Wnt signaling members in the pubertal and adult mouse ovary and the impact of GnRH antagonist cetrorelix on these signaling members. 0.5 mg/kg of cetrorelix was injected to 3-and 6-week-old mice for 2 weeks. At the end of injection, ovaries from 5 (5Ce)- to 8-week (8Ce)-old mice were embedded in paraffin for immunohistochemistry and homogenized for western blot to compare with control (5C–8C) and sham groups (5S–8S). WNT2 and WNT4 showed higher expression in thecal and stromal cells in adult mouse ovaries and only WNT4 expression was affected by cetrorelix. FZD1 was localized mainly in oocytes of pubertal ovaries and granulosa cells and oocytes of adult ovaries. FZD1 was reduced by cetrorelix in pubertal ovaries. FZD4 was abundantly localized in thecal and stromal cells of all groups and protein level was not affected by cetrorelix. LRP-6 was expressed mainly in oocytes and stromal cells of pubertal, oocytes of adult ovaries and its expression was reduced by cetrorelix in adult ovaries. CTNNB1 intensity in granulosa cells was the lowest in pubertal and the highest in adult ovaries and its expression was decreased by cetrorelix in adult ovaries. Cetrorelix affected the expression of specific members of the Wnt signaling depending on the developmental stage of mice, pointing out its possible interaction with gonadotropins during pubertal and adult stages.

  • Age-related glomerular lesions with albuminuria in male cotton rats
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-10-18
    Osamu Ichii, Teppei Nakamura, Takao Irie, Yuki Otani, Marina Hosotani, Md. Abdul Masum, Rashedul Md. Islam, Taro Horino, Yuji Sunden, Yaser Hosny Ali Elewa, Yasuhiro Kon

    The increased prevalence of aging-related chronic kidney disease (CKD) among humans is a problem worldwide. Aged cotton rats (Sigmodon hispidus) are considered novel model animals for studying CKD, especially as the females develop severe tubulointerstitial lesions with anemia. To investigate the renal pathologic features in aged male cotton rats and their characteristic glomerular injuries, the animals were divided into young, adult, old-aged, and advanced-aged groups (1–4, 5–8, 9–12, and 13–17 months, respectively) and pathologically analyzed. Anemia and renal dysfunction, as indicated by hematologic and serologic parameters, were significantly milder in the advanced-aged males than in the old-aged females. The males had increased urinary albumin-to-creatinine ratios from the old-age period, with the advanced-aged males having significantly higher levels than those in the old-aged females and young males. The old-aged females did not show clear glomerular injuries, whereas the advanced-aged males showed membranous lesions characterized by irregular and thickened glomerular basement membranes (GBMs). Characteristically, several large-sized projections from the GBM toward the podocytes were observed by microscopy, and podocytes covering these projections effaced their foot processes. The advanced-aged males showed aging-related IgG immune-complex depositions in the paramesangial regions and along the GBM. Furthermore, the positive reaction for podocin (a podocyte molecule) was granulated along the GBM. Thus, we clarified the albuminuria associated with altered glomerular structures in advanced-aged cotton rats, and that these phenotypes were closely associated with aging. These data help to clarify the aging-related pathogenesis of glomerular injury.

  • LacdiNAcylation of N -glycans in MDA-MB-231 human breast cancer cells results in changes in morphological appearance and adhesive properties of the cells
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-10-12
    Kiyoko Hirano, Yoshio Takada, Kiyoshi Furukawa

    We demonstrated previously that the expression of the disaccharide, GalNAcβ1 → 4GlcNAc (LacdiNAc), on N-glycans of cell surface glycoproteins in MDA-MB-231 human breast cancer cells suppresses their malignant properties such as tumor formation in nude mice. Here, we report changes in the morphological appearance and adhesive properties of two kinds of clonal cells of MDA-MB-231 cells overexpressing β4-N-acetyl-galactosaminyltransferase 4. The clonal cells exhibited a cobble stone-like shape as compared to a spindle-like shape of the mock-transfected cells and the original MDA-MB-231 cells. This was associated with an increased expression of cell surface E-cadherin, a marker of epithelial cells, and a decreased expression of N-cadherin, vimentin, α-smooth muscle actin and ZEB1, markers of mesenchymal cells. In addition, the clonal cells showed a lower migratory activity compared to the mock-transfected cells by wound-healing assay. These results suggest that mesenchymal–epithelial transition may be occurring in these clonal cells. Furthermore, increased adhesion to extracellular matrix proteins such as fibronectin, collagen type I, collagen type IV, and laminin was observed. The clonal cells spread and enlarged, whereas the mock-transfected cells demonstrated poor spreading on laminin-coated plates in the absence of fetal calf serum, indicating that expression of LacdiNAc on cell surface glycoproteins results in changes in cell adhesive and spreading properties particularly to laminin.

  • Potential role of IL-37 signaling pathway in feedback regulation of autoimmune Hashimoto thyroiditis
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-10-04
    Cui-Ping Ren, Li Sun, Feng-Chun Liu, Chun-Lin Zuo, Miao Liu, Wenda Gao, Ji-Jia Shen

    IL-37, the anti-inflammatory cytokine of the IL-1 family, plays several key roles in the regulation of autoimmune diseases. Yet, its role in Hashimoto’s thyroiditis (HT) is not clear. In the present study, we found that, in tissues from HT patients, most of the follicular epithelial cells were positive for both IL-37 and single Ig IL-1-related receptor (SIGIRR) by immunohistochemical staining, while the infiltrating lymphocytes and other inflammatory cells hardly expressed any. Meanwhile, mRNA expression levels of IL-37 in peripheral blood mononuclear cells (PBMC) of HT patients were significantly higher than those in normal controls measured by quantitative real-time PCR. Finally, we studied the possible role of IL-37 in IFN-γ-stimulated rat FRTL-5 cells. The results showed that IL-1β, TNF-α, and MCP-1 mRNA levels were significantly decreased, while the expression of IL-4 mRNA was dramatically up-regulated in IFN-γ-stimulated rat thyroid cell line FRTL-5 pre-treated with IL-37. The current study, for the first time, demonstrated that the IL-37 network is involved in Hashimoto’s thyroiditis, and IL-37 signaling pathway may ameliorate the excessive autoimmune responses in this chronic lymphocytic thyroiditis.

  • Mapping of the cystine–glutamate exchanger in the mouse eye: a role for xCT in controlling extracellular redox balance
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-08-08
    Renita M. Martis, Paul J. Donaldson, Bo Li, Martin Middleditch, Prasanna K. Kallingappa, Julie C. Lim

    The cystine–glutamate exchanger (system xc−) is responsible for the exchange of extracellular cystine for intracellular glutamate. In this study, we mapped the expression of xCT, the light chain subunit of system xc− in the different tissues of 3–6-week-old mouse (C57BL/6J) eye and have used an xCT knockout mouse to verify labelling specificity. Moreover, using the xCT knockout mouse, we investigated whether xCT was involved in maintaining extracellular redox balance in the eye. xCT transcript and protein were present in the cornea, lens and retina of wild-type mice, but not knockout mice. xCT was localised to the corneal epithelium, and the lens epithelium and cortical fibre cells but was absent in the iris. xCT localisation could not be determined in the ciliary body or retina, since xCT labelling was also detected in the knockout indicating a lack of specificity of the xCT antibody in tissues of a neural origin. Intracellular cysteine and cystine concentrations were similar in the wild-type and xCT knockout mouse for the cornea, lens, and retina. While extracellular cysteine levels were similar between the plasma, aqueous humour, and vitreous humour of the wild-type and xCT knockout mouse, extracellular cystine levels in the plasma and aqueous were significantly elevated in the xCT knockout mouse relative to the wild type. This suggests that loss of xCT results in an increased oxidative environment, particularly within the anterior chamber of the eye in which the aqueous humour resides. How this oxidative shift impacts ocular tissues that interface with the aqueous humour over time will be the focus of future work.

  • Mast cell chymase: morphofunctional characteristics
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-08-08
    Dmitri Atiakshin, Igor Buchwalow, Markus Tiemann

    During degranulation, mast cells secrete a specific set of mediators defined as “secretome” including the preformed mediators that have already been synthesized by a cell and contained in the cytoplasmic granules. This group includes serine proteases, in particular, chymase and tryptase. Biological significance of chymase depends on the mechanisms of degranulation and is characterized by selective effects on the cellular and non-cellular components of the specific tissue microenvironment. Chymase is known to be closely involved in the mechanisms of inflammation and allergy, angiogenesis, and oncogenesis, remodeling of the extracellular matrix of the connective tissue and changes in organ histoarchitectonics. Number of chymase-positive mast cells in the intra-organ population, and the mechanisms of biogenesis and secretome degranulation appear to be the informative criteria for interpreting the state of the internal organs, characterizing not only the diagnostic efficacy but also the properties of targets of pharmacotherapy. In this review, we discussed the current state of knowledge about mast cell chymase as one of the mast cell secretome proteases. Main issues of the reviewed publications are highlighted with our microscopic images of mast cell chymase visualized using immunohistochemical staining.

  • Long noncoding RNA CASC2 promotes paclitaxel resistance in breast cancer through regulation of miR-18a-5p/CDK19
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-07-27
    Pengfei Zheng, Liangpeng Dong, Bin Zhang, Jinfang Dai, Yifu Zhang, Yanan Wang, Shuang Qin

    Breast cancer is one of the most prevalent cancers in women. Chemoresistance is a major obstacle for the treatment of breast cancer. We investigated the role of long noncoding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) in paclitaxel (PTX) resistance in breast cancer. CASC2 expression was increased in PTX-resistant clinical samples and cell lines. PTX induced CASC2 expression in a concentration-dependent manner. Downregulation of CASC2 increased PTX toxicity and decreased IC50 value, while upregulation of CASC2 decreased PTX toxicity and increased IC50 value in MCF-7/PTX and MDA-MB-231/PTX cells. Moreover, downregulation of CASC2 decreased tumor growth in xenograft mice implanted with MCF-7/PTX cells. miR-18a-5p possessed a putative binding site in 3′-UTR of CASC2 and cyclin-dependent kinase 19 (CDK19). In PTX-resistant breast cancer cells, miR-18a-5p expression was decreased. CASC2 and miR-18a-5p could negatively regulate the expression of each other. CDK19 expression could be negatively regulated by miR-18a-5p, but positively regulated by CASC2. miR-18a-5p mimics or downregulation of CDK19 decreased tumor growth in xenograft mice implanted with MCF-7/PTX cells. In summary, we identified that CASC2 activated PTX resistance in breast cancer through regulation of miR-18a-5p/CDK19. We highlight the importance of CASC2/miR-18a-5p/CDK19 axis in the chemoresistance of breast cancer and provide potential targets for the improving chemotherapy of breast cancer.

  • Triple labelling of actin filaments, intermediate filaments and microtubules for broad application in cell biology: uncovering the cytoskeletal composition in tunneling nanotubes
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-08-08
    Nataša Resnik, Andreja Erman, Peter Veranič, Mateja Erdani Kreft

    We report a protocol for simultaneous triple labelling of intermediate filaments, microtubules and actin filaments. The described procedure offers an optimal preservation of the structure and antigenicity of individual representatives of cytoskeletal elements and is applicable for labelling of tissue samples and cultured cells. Namely, we demonstrate that using this protocol the cytoskeletal elements are well-preserved and detectable in the whole mount urinary bladder tissue pieces, cryosections of the urinary bladder, and in cultured normal and cancer urothelial cells including their delicate intercellular connections such as tunneling nanotubes (TnTs). The protocol uncovers for the first time the co-distribution of actin filaments, intermediate filaments and microtubules in TnTs, which were up to now known as mono- or bi-cytoskeletal structures. Presented triple labelling protocol provides an efficient tool for studying co-distribution of actin filaments, intermediate filaments, and microtubules and therefore offers new insights into their cellular and tissue distribution.

  • Life time of some RNA products of rDNA intergenic spacer in HeLa cells
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-07-25
    T. Vacík, S. Kereïche, I. Raška, D. Cmarko, E. Smirnov

    In human cells, the intergenic spacers (IGS), which separate ribosomal genes, are complex approximately 30 kb-long loci. Recent studies indicate that all, or almost all, parts of IGS may be transcribed, and that at least some of them are involved in the regulation of the ribosomal DNA (rDNA) transcription, maintenance of the nucleolar architecture, and response of the cell nucleus to stress. However, since each cell contains hundreds not quite identical copies of IGS, the structure and functions of this locus remain poorly understood, and the dynamics of its products has not been specially studied. In this work, we used quantitative PCR to measure the expression levels of various rDNA regions at different times after inhibition of the transcription by Actinomycin D applied in high doses. This approach allowed us to measure real or extrapolated half-life times of some IGS loci. Our study reveals characteristic dynamic patterns suggestive of various pathways of RNA utilization and decay.

  • FoxN1 mediates thymic cortex–medulla differentiation through modifying a developmental pattern based on epithelial tubulogenesis
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-09-24
    Juan J. Muñoz, Esther Tobajas, Sonia Juara, Sara Montero, Agustín G. Zapata

    The mechanisms that determine the commitment of thymic epithelial precursors to the two major thymic epithelial cell lineages, cTECs and mTECs, remain unknown. Here we show that FoxN1 nu mutation, which abolishes thymic epithelium differentiation, results in the formation of a tubular branched structure according to a typical branching morphogenesis and tubulogenesis developmental pattern. In the presence of FoxN1, in alymphoid NSG and fetal Ikaros−/− thymi, there is no lumen formation and only partial apical differentiation. This initiates cortex–medulla differentiation inducing expression of medullary genes in the apically differentiating cells and of cortical genes in the non-apically differentiating cells, which will definitely differentiate in wt and postnatal Ikaros−/− mice. Therefore, the thymus development is based on a branching morphogenesis and tubulogenesis developmental pattern: FoxN1 expression in the thymic primordium inhibits tubulogenesis and induces the expression of genes involved in TEC differentiation, which culminates with the expression of functional cell markers, i.e., MHCII, CD80, Aire in both postnatal Ikaros−/− and WT thymi after arrival of lymphoid progenitor cells.

  • Nitric oxide synthase and VEGF expression in full-term placentas of obese women
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-09-24
    Eleonora Salvolini, Arianna Vignini, Jacopo Sabbatinelli, Guendalina Lucarini, Veronica Pompei, Davide Sartini, Anna Maria Cester, Andrea Ciavattini, Laura Mazzanti, Monica Emanuelli

    An adequate placental vascularization allows the proper development of the fetus and it is crucial for the gestational success. A number of factors regulate angiogenesis, including vascular endothelial growth factor (VEGF), which induces the synthesis of nitric oxide (NO), a potent vasodilator produced by three different nitric oxide synthase (NOS) isoforms. NO is essential to maintain a low vascular resistance in the fetoplacental circulation, although at high concentrations, it may combine with excess superoxide to produce peroxynitrite, which reacts with proteins giving rise to nitrotyrosine. Since obesity, whose incidence is increasing worldwide, is characterized by a low-grade inflammatory state and increased levels of oxidative and nitrative stress, both affecting placental function, our aim was to evaluate the expression of VEGF, eNOS, and iNOS in full-term placentas obtained from normal weight and pre-pregnancy obese women by means of immunohistochemistry and real-time PCR. Moreover, we assessed the NO levels and the nitrotyrosine immunoexpression in the same sample groups. Our results show a significantly higher immunohistochemical expression of VEGF and eNOS in the endothelium of placentas from obese women than in controls, whereas the immunoexpression of iNOS was comparable in the two groups. These data agree with those of the gene expression analysis, thus suggesting the possible existence of a compensatory mechanism for changes in placental blood flow associated with obesity. As concerns nitrotyrosine and NO levels, we observed a significant increase in placental tissue from obese women which may contribute to the development of metabolic and cardiovascular diseases both in the mother and the offspring.

  • Spatial distribution and correlation of adipocytes and mast cells in superficial fascia in rats
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-09-23
    Dandan Zhang, Yingyue Dong, Yanfei Zhang, Xueying Su, Tongsheng Chen, Yuanyuan Zhang, Bihan Wu, Guoheng Xu

    Superficial fascia has abundant preadipocytes capable of spontaneous and induced differentiation and is thought to be a novel origin of adipocytes. This study aimed to quantitatively evaluate the spatial distribution and correlation of adipocytes and mast cells in rat superficial fascia. Panoramic images were obtained from whole-mounted fascia stained by toluidine blue. Adipocytes increased gradually in superficial fascia of growing rats. Abundant mast cells, with the degranulation and exocytosis of abundant secretory granules, appeared in fascia where partially differentiating adipocytes and mature adipocytes occurred. Quantitative histological analysis by variance–mean ratio and Morisita index of dispersion indicated that both mast cells and adipocytes in fascia were distributed individually in cluster, but not random or uniform. Spearman’s correlation coefficient revealed that the spatial cluster distributions of mast cells and adipocytes positively correlated with each other and correlated with increased number and size of adipocytes and adipogenic areas in fascia. Morphometry analysis indicated the strong correlation between fascial adipocytes and mast cells during the periods of rat growth. The correlation coefficient increased significantly at 8 weeks compared to 4 weeks, consistent with the increasing trend of the number and size of fascia adipocytes in growing rats. This finding provided the first quantitative histological analysis for the spatial distribution and correlation of fascia adipocytes and mast cells, which could be the histocytological basis for further exploring spatial and functional interactions between fascial mast cells and adipocytes. Also, the present data were informative for the research on physiologies and pathologies of fascia and fascia-related connective tissues.

  • Outgrowth, proliferation, viability, angiogenesis and phenotype of primary human endothelial cells in different purchasable endothelial culture media: feed wisely
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-09-21
    Barbara Leopold, Jasmin Strutz, Elisa Weiß, Juergen Gindlhuber, Ruth Birner-Gruenberger, Hubert Hackl, Hannah M. Appel, Silvija Cvitic, Ursula Hiden

    Function and dysfunction of endothelial cells are regulated by a multitude of factors. Endothelial cell research often requires in vitro cell culture experiments. Hence, various culture media specifically designed to promote endothelial cell growth are available. These strikingly differ in their composition: complex media contain endothelial cell growth supplement (ECGS), an extract produced of bovine brain with undefined amounts of biologically active compounds, whilst defined media contain selected growth factors in defined concentrations. We here compared the effect of seven purchasable endothelial cell culture media on colony outgrowth, proliferation, viability, in vitro angiogenesis and phenotype of mature primary human endothelial cells using feto-placental endothelial cells isolated from chorionic arteries (fpEC). The effect of media on colony outgrowth was additionally tested on umbilical cord blood-derived endothelial progenitor cells (ECFCs). Outgrowth, purity, proliferation and viability differed between media. Outgrowth of fpEC and ECFCs was best in a defined medium containing EGF, FGF2 and VEGF. By contrast, established fpEC isolations proliferated best in complex media containing ECGS, heparin and ascorbic acid. Also viability of cells was higher in complex media. In vitro angiogenesis was most intense in a defined medium containing the highest number of individual growth factors. FACS analysis of surface markers for endothelial cell subtypes revealed that endothelial phenotype of fpEC was unaffected by media composition. Our data demonstrate the fundamental effect of endothelial cell culture media on primary cell isolation success and behaviour. Whether the composition of supplements is suitable also for individual experiments needs to be tested specifically.

  • Nanoscale analysis reveals no domain formation of glycosylphosphatidylinositol-anchored protein SAG1 in the plasma membrane of living Toxoplasma gondii
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-09-21
    Yuna Kurokawa, Tatsunori Masatani, Rikako Konishi, Kanna Tomioku, Xuenan Xuan, Akikazu Fujita

    Glycosylphosphatidylinositol (GPI)-anchored proteins typically localise to lipid rafts. GPI-anchored protein microdomains may be present in the plasma membrane; however, they have been studied using heterogeneously expressed GPI-anchored proteins, and the two-dimensional distributions of endogenous molecules in the plasma membrane are difficult to determine at the nanometre scale. Here, we used immunoelectron microscopy using a quick-freezing and freeze-fracture labelling (QF-FRL) method to examine the distribution of the endogenous GPI-anchored protein SAG1 in Toxoplasma gondii at the nanoscale. QF-FRL physically immobilised molecules in situ, minimising the possibility of artefactual perturbation. SAG1 labelling was observed in the exoplasmic, but not cytoplasmic, leaflets of T. gondii plasma membrane, whereas none was detected in any leaflet of the inner membrane complex. Point pattern analysis of SAG1 immunogold labelling revealed mostly random distribution in T. gondii plasma membrane. The present method obtains information on the molecular distribution of natively expressed GPI-anchored proteins and demonstrates that SAG1 in T. gondii does not form significant microdomains in the plasma membrane.

  • Tuftelin and HIFs expression in osteogenesis
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-09-13
    Jan Bobek, Veronika Oralova, Adela Kratochvilova, Ivana Zvackova, Herve Lesot, Eva Matalova

    Tuftelin was originally discovered and mostly studied in the tooth, but later found also in other organs. Despite its wide distribution among tissues, tuftelin’s function has so far been specified only in the formation of enamel crystals. Nevertheless, in many cases, tuftelin was suggested to be associated with cellular adaptation to hypoxia and recently even with cell differentiation. Therefore, we aimed to investigate tuftelin expression along with hypoxia-inducible factors (HIFs) during the early development of the mandibular/alveolar (m/a) bone, when osteoblasts started to differentiate in vivo and to compare their expression levels in undifferentiated versus differentiated osteoblastic cells in vitro. Immunohistochemistry demonstrated the presence of tuftelin already in osteoblastic precursors which were also HIF1-positive, but HIF2-negative. Nevertheless, HIF2 protein appeared when osteoblasts differentiated, one day later. This is in agreement with observations made with MC3T3-E1 cells, where there was no significant difference in tuftelin and Hif1 expression in undifferentiated vs. differentiated cells, although Hif2 increased upon differentiation induction. In differentiated osteoblasts of the m/a bone, all three proteins accumulated, first, prenatally, in the cytoplasm and later, particularly at postnatal stages, they displayed also peri/nuclear localization. Such a dynamic time–space pattern of tuftelin expression has recently been reported in neurons, which, as the m/a bone, differentiate under less hypoxic conditions as indicated also by a prevalent cytoplasmic expression of HIF1 in osteoblasts. However, unlike what was shown in cultured neurons, tuftelin does not seem to participate in final osteoblastic differentiation and its functions, thus, appears to be tissue specific.

  • Cellular localization and regulation of receptors and enzymes of the endocannabinoid system in intestinal and systemic inflammation.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2018-09-10
    Magdalena Grill,Carina Hasenoehrl,Melanie Kienzl,Julia Kargl,Rudolf Schicho

    Surveys suggest that Cannabis provides benefit for people with inflammatory bowel disease. However, mechanisms underlying beneficial effects are not clear. We performed in situ hybridization RNAscope® combined with immunohistochemistry to show cell-specific distribution and regulation of cannabinoid receptor 1 and 2 (CB1, CB2), G protein-coupled receptor 55 (GPR55), and monoacylglycerol lipase (MGL) mRNA in immune cells using murine models of intestinal and systemic inflammation. In healthy animals, the presence in enteric ganglia is high for CB1 mRNA, but low for CB2 and GPR55 mRNAs. MGL mRNA is predominant throughout the intestinal wall including myenteric neurons, epithelium, circular and longitudinal muscular layers, and the lamina propria. Within the immune system, B220+ cells exhibit high gene expression for CB2 while the expression of CB2 in F4/80+ and CD3+ cells is less prominent. In contrast, GPR55 mRNA is highly present in F4/80+ and CD3+ cells. qRT-PCR of total colonic segments shows that the expression of GPR55 and MGL genes drops during intestinal inflammation. Also at cellular levels, GPR55 and MGL gene expression is reduced in F4/80+, but not CD3+ cells. As to systemic inflammation, reduced gene expression of MGL is observed in ileum by qRT-PCR, while at cellular levels, altered gene expression is also seen for CB1 and GPR55 in CD3+ but not F4/80+ cells. In summary, our study reveals changes in gene expression of members of the endocannabinoid system in situ attesting particularly GPR55 and MGL a distinct cellular role in the regulation of the immune response to intestinal and systemic inflammation.

  • In focus in HCB.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-11-25
    Douglas J Taatjes,Jürgen Roth

  • Diversity of enteroendocrine cells investigated at cellular and subcellular levels: the need for a new classification scheme.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2018-10-26
    Linda J Fothergill,John B Furness

    Enteroendocrine cells were historically classified by a letter code, each linked to a single hormone, deduced to be the only hormone produced by the cell. One type, the L cell, was recognised to store and secrete two products, peptide YY (PYY) and glucagon-related peptides. Many other exceptions to the one-cell one-hormone classifications have been reported over the last 40 years or so, and yet the one-hormone dogma has persisted. In the last 6 years, a plethora of data has appeared that makes the concept unviable. Here, we describe the evidence that multiple hormone transcripts and their products reside in single cells and evidence that the hormones are often, but not always, processed into separate storage vesicles. It has become clear that most enteroendocrine cells contain multiple hormones. For example, most secretin cells contain 5-hydroxytryptamine (5-HT), and in mouse many of these also contain cholecystokinin (CCK). Furthermore, CCK cells also commonly store ghrelin, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), neurotensin, and PYY. Several hormones, for example, secretin and 5-HT, are in separate storage vesicles at a subcellular level. Hormone patterns can differ considerably between species. Another complication is that relative levels of expression vary substantially. This means that data are significantly influenced by the sensitivities of detection techniques. For example, a hormone that can be detected in storage vesicles by super-resolution microscopy may not be above threshold for detection by conventional fluorescence microscopy. New nomenclature for cell clusters with common attributes will need to be devised and old classifications abandoned.

  • Blank spots on the map: some current questions on nuclear organization and genome architecture.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2018-09-22
    Carmen Adriaens,Leonid A Serebryannyy,Marina Feric,Andria Schibler,Karen J Meaburn,Nard Kubben,Pawel Trzaskoma,Sigal Shachar,Sandra Vidak,Elizabeth H Finn,Varun Sood,Gianluca Pegoraro,Tom Misteli

    The past decades have provided remarkable insights into how the eukaryotic cell nucleus and the genome within it are organized. The combined use of imaging, biochemistry and molecular biology approaches has revealed several basic principles of nuclear architecture and function, including the existence of chromatin domains of various sizes, the presence of a large number of non-membranous intranuclear bodies, non-random positioning of genes and chromosomes in 3D space, and a prominent role of the nuclear lamina in organizing genomes. Despite this tremendous progress in elucidating the biological properties of the cell nucleus, many questions remain. Here, we highlight some of the key open areas of investigation in the field of nuclear organization and genome architecture with a particular focus on the mechanisms and principles of higher-order genome organization, the emerging role of liquid phase separation in cellular organization, and the functional role of the nuclear lamina in physiological processes.

  • Murine CLCA5 is uniquely expressed in distinct niches of airway epithelial cells.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2014-09-13
    Kristina Dietert,Lars Mundhenk,Nancy A Erickson,Katrin Reppe,Andreas C Hocke,Wolfgang Kummer,Martin Witzenrath,Achim D Gruber

    The murine mCLCA5 protein is a member of the chloride channel regulators, calcium-activated (CLCA) family and is suspected to play a role in airway mucus cell differentiation. Although mCLCA5 mRNA was previously found in total lung extracts, the expressing cells and functions in the naive murine respiratory tract are unknown. Therefore, mCLCA5 protein expression was identified by immunohistochemistry and confocal laser scanning microscopy using entire lung sections of naive mice. Moreover, we determined mRNA levels of functionally related genes (mClca3, mClca5, Muc5ac and Muc5b) and quantified mCLCA5-, mCLCA3- and CC10-positive cells and periodic acid-Schiff-positive mucus cells in naive, PBS-treated or Staphylococcus aureus-infected mice. We also investigated mCLCA5 protein expression in Streptococcus pneumoniae and influenza virus lung infection models. Finally, we determined species-specific differences in the expression patterns of the murine mCLCA5 and its human and porcine orthologs, hCLCA2 and pCLCA2. The mCLCA5 protein is uniquely expressed in highly select bronchial epithelial cells and submucosal glands in naive mice, consistent with anatomical locations of progenitor cell niches. Under conditions of challenge (PBS, S. aureus, S. pneumoniae, influenza virus), mRNA and protein expression strongly declined with protein recovery only in models retaining intact epithelial cells. In contrast to mice, human and porcine bronchial epithelial cells do not express their respective mCLCA5 orthologs and submucosal glands had fewer expressing cells, indicative of fundamental differences in mice versus humans and pigs.

  • Murine mCLCA5 is expressed in granular layer keratinocytes of stratified epithelia.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2009-12-17
    Josephine Braun,Melanie K Bothe,Lars Mundhenk,Carol L Beck,Achim D Gruber

    CLCA proteins represent a large family of proteins widely expressed in mammalian tissues with a unique expression pattern for each family member analyzed so far. However, their functions in normal and diseased tissues are poorly understood. Here, we present the cellular expression pattern of mCLCA5 in murine tissues using immunohistochemistry, confocal laser scanning microscopy and immune electron microscopy with specific antibodies and RT-qPCR following laser-capture microdissection. The mCLCA5 protein was localized to granular layer keratinocytes of virtually all stratified squamous epithelia of the body. Biochemical protein characterizations revealed that the amino-terminal cleavage product is fully secreted by the cell, while the carboxy-terminal cleavage product remains associated with the cell. The results imply that mCLCA5 may play a role in maturation and keratinization of squamous epithelial cells.

  • Correction to: Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2018-07-11
    Manja Luckner,Gerhard Wanner

    Unfortunately, part of the legend to Fig. 6 has been incorrectly published.

  • Correction to: Expression of matrix metalloproteinase 12 is highly specific for non-proliferating invasive trophoblasts in the first trimester and temporally regulated by oxygen-dependent mechanisms including HIF-1A.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2017-12-14
    Ursula Hiden,Christian P Eyth,Alejandro Majali-Martinez,Gernot Desoye,Carmen Tam-Amersdorfer,Berthold Huppertz,Nassim Ghaffari Tabrizi-Wizsy

    In the original publication, the contribution of Dr. Christian Eyth as equal first author was not indicated. This has been corrected confirming that U. Hidden and C. Eyth contributed equally to this work.

  • In focus in HCB.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2019-11-05
    Douglas J Taatjes,Jürgen Roth

  • Intracellular localization of protein C inhibitor (PCI) and urinary plasminogen activator in renal tubular epithelial cells from humans and human PCI gene transgenic mice.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2008-01-15
    Zhenhu Song,Ning Ma,Tatsuya Hayashi,Esteban C Gabazza,Yoshiki Sugimura,Koji Suzuki

    Urinary plasminogen activator (uPA) is a serine protease that plays important roles in various extracellular proteolytic processes. In humans, protein C inhibitor (PCI) is known to regulate the activity of the serine proteases involved in blood coagulation, wound healing, and tumor metastasis, whereas PCI is not present in murine plasma or tissues other than the reproductive tissues. The large amount of uPA-PCI complexes found in human urine suggests that these complexes are formed in the kidneys. In the present study, we performed immunofluorescence double labeling and electron microscopic immunocytochemistry using renal tissues from humans and human PCI gene transgenic (PCI-TG) mice. In human renal tissues, PCI and uPA colocalized in the cytoplasm of renal proximal tubular epithelial cells (RPTECs), and juxtaposition of PCI and uPA immunoreactive particles was detected in the microvilli and lysosomes in the RPTECs. The intracellular distributions of PCI and uPA in the RPTECs from PCI-TG mice were similar to those observed in human RPTECs. These findings hint at the physiological roles of uPA and PCI in human kidneys, and also suggest that the PCI-TG mice will be useful for evaluating the roles of PCI in human physiological and pathological conditions.

  • Soluble mucus component CLCA1 modulates expression of leukotactic cytokines and BPIFA1 in murine alveolar macrophages but not in bone marrow-derived macrophages.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2018-04-04
    Nancy A Erickson,Kristina Dietert,Jana Enders,Rainer Glauben,Geraldine Nouailles,Achim D Gruber,Lars Mundhenk

    The secreted airway mucus cell protein chloride channel regulator, calcium-activated 1, CLCA1, plays a role in inflammatory respiratory diseases via as yet unidentified pathways. For example, deficiency of CLCA1 in a mouse model of acute pneumonia resulted in reduced cytokine expression with less leukocyte recruitment and the human CLCA1 was shown to be capable of activating macrophages in vitro. Translation of experimental data between human and mouse models has proven problematic due to several CLCA species-specific differences. We therefore characterized activation of macrophages by CLCA1 in detail in solely murine ex vivo and in vitro models. Only alveolar but not bone marrow-derived macrophages freshly isolated from C57BL6/J mice increased their expression levels of several pro-inflammatory and leukotactic cytokines upon CLCA1 stimulation. Among the most strongly regulated genes, we identified the host-protective and immunomodulatory airway mucus component BPIFA1, previously unknown to be expressed by airway macrophages. Furthermore, evidence from an in vivo Staphylococcus aureus pneumonia mouse model suggests that CLCA1 may also modify BPIFA1 expression in airway epithelial cells. Our data underscore and specify the role of mouse CLCA1 in inflammatory airway disease to activate airway macrophages. In addition to its ability to upregulate cytokine expression which explains previous observations in the Clca1-deficient S. aureus pneumonia mouse model, modulation of BPIFA1 expression expands the role of CLCA1 in airway disease to involvement in more complex downstream pathways, possibly including liquid homeostasis, airway protection, and antimicrobial defense.

  • Real-time RT-PCR quantitation of mCLCA1 and mCLCA2 reveals differentially regulated expression in pre- and postnatal murine tissues.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2002-07-18
    Ina Leverkoehne,Bettina A Horstmeier,Georg von Samson-Himmelstjerna,Bob J Scholte,Achim D Gruber

    Members of the recently discovered chloride channels, calcium-activated (CLCA) gene family are thought to contribute to transmembrane trafficking of anions and other cellular functions. Previous northern blot and in situ hybridization studies revealed expression of the murine putative chloride channel mCLCA1 (alias mCaCC) in numerous epithelia and few other cell types. However, the subsequent cloning of mCLCA2 which shares 96% cDNA sequence identity with mCLCA1 suggested that the distribution pattern proposed for mCLCA1 in fact represented the sum of both mRNA species. In this study, a real-time RT-quantitative PCR assay was established to specifically quantify mCLCA1 and mCLCA2 expression in 19 pre- and 44 postnatal murine tissues. Different expression levels of mCLCA1 and mCLCA2 were found in most tissues analyzed. Particularly strong and virtually exclusive expression was found for mCLCA1 in spleen and bone marrow and for mCLCA2 in lactating and involuting mammary glands. In contrast, other tissues including intestine and trachea were found to express equally moderate levels of both homologues. Moreover, mCLCA2, but not mCLCA1, seems to be involved in stage-specific organogenesis in fetal tissues. These results indicate that, in spite of their extremely close sequence homology, mCLCA1 and mCLCA2 are involved in different, yet unidentified pathways.

  • GFP associates with microfilaments in fixed cells.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    H D Schmitz,J Bereiter-Hahn

    The discovery of the green fluorescent protein (GFP) and its use as a marker for proteins in cells revolutionised cell biology. Among its applications are the intracellular localisation of proteins and the investigation of the organisation, regulation and dynamics of the cytoskeleton. GFP itself is considered to be an inert protein, homogeneously distributed within the cytoplasm. Here we investigated the intracellular distribution of GFP in an amphibian and in various mammalian cell lines (XTH2, CHO-K1, HaCaT, MDCK, NIH-3T3) by confocal laser scanning microscopy. After paraformaldehyde fixation GFP became associated with microfilaments in all the cell lines investigated. This interaction was not impaired by detergent treatment (1% Brij 58 for 10 min). In contrast to the F-actin binding of GFP in fixed cells, association of GFP with stress fibres was not detectable in living cells. The actin-binding property of GFP might contribute also to the interaction of fusion proteins with microfilaments. Thus, careful controls are unavoidable in investigating (weak) actin-binding proteins in fixed cells. Because no association of GFP with microfilaments was detectable in living cells, it is recommended to monitor the intracellular distribution of GFP-tagged proteins in vivo.

  • Differentiation of the smooth muscle cell phenotypes during embryonic development of coronary vessels in the rat.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    A Ratajska,M Zarska,C Quensel,J Krämer

    Smooth muscle cell (SMC) maturation during embryonic development of coronary arteries and veins was studied in rats using different markers of the contractile phenotypes. The spatio-temporal pattern of distribution of these markers compared with the developing tunica media was examined. Alpha-smooth muscle actin (alpha-SMA) was the first marker of the SMC in the tunica media of coronary arteries found in ED16 hearts, followed by smooth muscle myosin heavy chain isoform which occurred on ED17. Subsequently 1E12 antigen was expressed in coronary artery wall in ED18 hearts, and finally smoothelin. The markers occur within the proximal part of the coronary arteries and deploy toward the apex. They are also found within the great vessels. None of the markers except for the alpha-SMA were found in coronary veins during embryonic life. We conclude that the SMC population of the developing tunica media of coronary vessels differentiates by the acquisition of particular markers and this process lasts till the end of the prenatal and early postnatal life.

  • Comparison of long-term transgene expression after non-viral and adenoviral gene transfer into primary articular chondrocytes.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    R Dinser,F Kreppel,F Zaucke,C Blank,M Paulsson,S Kochanek,P Maurer

    Different gene transfer approaches to achieve long-term transgene expression in cultured primary bovine chondrocytes were compared using enhanced green fluorescent protein (EGFP) as a reporter. Transduction with a high-capacity adenoviral vector was 82% efficient when analysed by fluorescence microscopy, while up to 42% of plasmid-transfected cells were EGFP positive with FuGene as a transfection reagent. Rapid dominant marker selection of plasmid-transfected cells was achieved in monolayer culture. With either method of gene transfer, a high proportion of the chondrocytes remained transgene positive during prolonged alginate culture. Transgene transcription in single cells was quantified with a confocal laser scanning microscope. Detection of EGFP expression was more sensitive with this method, identifying more transgene-expressing cells than conventional fluorescence microscopy. Long-term EGFP expression was higher in adenovirally transduced chondrocytes embedded in alginate as compared to plasmid-transfected cells cultured in monolayer or in alginate. Both the adenoviral and the plasmid-based approach appear suited for studies of the molecular and cellular mechanisms by which mutations in cartilage matrix proteins cause disease.

  • Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    R Koopman,G Schaart,M K Hesselink

    The objective of the present study was to develop a stain permitting automated quantification of myocellular lipid depositions in skeletal muscle sections together with immunolocalisation of other myocellular constituents by fluorescence microscopy. Lipid droplets were detected in skeletal muscle by oil red O (ORO). Conventional ORO was modified to diminish background staining, prevent crystallisation of ORO and to optimise lipid retention in cryosections. These modifications resulted in a punctate staining of lipid droplets, rather than the somewhat diffuse staining by conventional ORO. Small cavities in muscle sections (like the lumen of small blood vessels) lack ORO when using the protocol presented here. In addition a staining protocol is presented combining ORO with immunofluorescence. This combination permits multiple staining studies in the same section. Thus, lipid droplets can be studied together with immunolabelling of proteins involved in lipid handling and metabolism. This will extend our knowledge on the subcellular localisation of lipid handling proteins (i.e. enzymes and fatty acid transporting proteins) in relation to the localisation of lipid depositions. In conclusion, the protocol presented here permits examination of ORO-stained lipid droplets in skeletal muscle sections together with multiple staining of other immunodetectable proteins present in skeletal muscle by quantitative fluorescence microscopy.

  • Cytokine expression in rat molar gingival periodontal tissues after topical application of lipopolysaccharide.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    M Miyauchi,S Sato,S Kitagawa,M Hiraoka,Y Kudo,I Ogawa,M Zhao,T Takata

    It is well known that proinflammatory cytokines produced by host cells play an important role in periodontal tissue destruction. However, the localization of the cytokines in in vivo periodontal tissues during development of periodontal disease has not been determined. Immunohistochemical expression of proinflammatory cytokines including IL-1alpha, IL-1beta, and TNF-alpha was examined at 1 and 3 h, and 1, 2, 3, and 7 days after topical application of lipopolysaccharide (LPS; 5 mg/ml in physiological saline) from E. coli into the rat molar gingival sulcus. In the normal periodontal tissues, a small number of cytokine-positive epithelial cells were seen in the junctional epithelium (JE), oral sulcular and oral gingival epithelium, in addition to macrophages infiltrating in the subjunctional epithelial area and osteoblasts lining the alveolar bone surface. Epithelial remnants of Malassez existing throughout periodontal ligament were intensely positive for IL-1beta but negative for the other two cytokines. At 3 h after the LPS treatment, almost all cells in the JE were strongly positive for the cytokines examined. In addition, several cytokine-positive cells, including neutrophils, macrophages, and fibroblasts, were seen in the subjunctional epithelial connective tissue. At day 2, expression of the cytokines in the JE gradually decreased, while cytokine-positive cells in the connective tissue increased in number. Positive staining of the cytokines was seen in osteoclasts and preosteoclasts which appeared along the alveolar bone margin in this period. The number of cytokine-positive cells decreased by day 7. These findings indicate that, in addition to macrophages, neutrophils, and fibroblasts, the JE cells are a potent source of TNF-alpha, IL-1alpha, and IL-1beta reacting to LPS application, and suggest that JE cells may play an important role in the first line of defense against LPS challenge, and the proinflammatory cytokines transiently produced by various host cells may be involved in the initiation of inflammation and subsequent periodontal tissue destruction.

  • Developmental expression of CLC-K1 in the postnatal rat kidney.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    K Kobayashi,S Uchida,S Mizutani,S Sasaki,F Marumo

    CLC-K1, a kidney-specific chloride channel, has been demonstrated to be involved in the urine concentration mechanism. Here, we investigated the developmental expression of CLC-K1 in the rat kidney. Using immunohistochemistry, we showed that CLC-K1 was not present in the thin ascending limb of Henle's loop during the early prenatal stages but was significantly expressed during the adult stage. CLC-K1 started to appear at day 5 and its expression increased during further development. In developing rats this increase coincided with the increase in the urine-concentrating capacity as the animals matured. We also investigated the expressions of other channels and transporters, including NKCC2, AQP-1, and AQP-2. NKCC2 was strongly expressed throughout the inner medulla in neonatal rat kidneys but was entirely undetectable at the adult stage. The decline in its expression took the form of a gradual recession from the inner medulla together with reciprocal increases in the expression of CLC-K1. AQP-1 was weakly expressed in the inner medulla during early development and showed a rapid increase in expression at a later stage. The collecting duct cells significantly expressed AQP-2 even at birth and maintained its expression throughout the development. These results suggest that CLC-K1 expression is one of the major determinants of the urine-concentrating capacity of the developing rat kidney.

  • Recycling cultured cells for immunofluorescent labeling.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    J Espada,A Juarranz,A Villanueva,M Cañete,I Andrés,J C Stockert

    A method to use sequential rounds of immunofluorescent labeling in cell cultures is presented. The method is based on the utilization of a non-liquid reducing agent, sodium dithionite, in conjunction with ionic or non-ionic detergents (SDS or TX100, respectively) at room temperature. This method preserves cell morphology and substrate antigenicity, and operates through the complete extraction of most primary and secondary antibodies. Using this protocol, the sequential immunolocalization of different proteins is possible, without signal interference with previous immunolabeling rounds. In addition, the method is also useful to recycle blotted membranes in immunoblots.

  • Protein phosphatase 2Calpha expression in normal human tissues: an immunohistochemical study.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    B Lifschitz-Mercer,Y Sheinin,D Ben-Meir,L Bramante-Schreiber,L Leider-Trejo,S Karby,N I Smorodinsky,S Lavi

    Protein phosphatase (PP2Calpha) is a member of the mammalian serine threonine-specific protein phosphatases family. We produced monoclonal antibodies against the recombinant PP2Calpha and evaluated the immunoreactivity of normal human tissues. The reactivity was strong in normal skin, the digestive tract, lung, kidney, breast, prostate, endocrine glands, and brain, while it was moderate in the ovary, testis, and liver. Epithelial cells revealed high levels of PP2Calpha expression, but stromal cells, including fibroblasts and endothelial cells, showed no or little PP2Calpha expression. Given the broad reactivity in endocrine and secreting epithelial cells, we propose that PP2Calpha expression might contribute to secretory cell function.

  • Immunolocalization of tubulin isoforms and post-translational modifications in the protists Tritrichomonas foetus and Trichomonas vaginalis.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    L C Lopes,K C Ribeiro,M Benchimol

    In the present report we show the distribution of multiple tubulin isoforms in Trichomonas vaginalis and Tritrichomonas foetus, flagellated parasitic protists of the urogenital tracts of human and cattle, respectively, using immunofluorescence and immunoelectron microscopy. We used several monoclonal and polyclonal anti-tubulin antibodies from different sources and recognizing variant tubulin isoforms. Our results demonstrate that: (1) there is a heterogeneous distribution of the different tubulin isoforms in the main microtubular cell structures, such as axostyle, flagella, basal bodies, and mitotic spindle, (2) the axostyle-pelta junction is a structure with high affinity for glutamylated tubulin antibodies in T. foetus, (3) the spindle labeling is positive to anti-glutamylated tubulin and anti-alpha-tubulin (TAT1 and purchased from Amersham) antibodies in T. vaginalis but it is negative in T. foetus, (4) the nuclear matrix and the cytosol presented positive reaction using glutamylated and TAT1 (anti-alpha-tubulin) antibodies only in T. vaginalis, and (5) the Golgi complex exhibited staining using the glutamylated tubulin antibody. The present data corroborate with the idea of the existence of a heterogeneous population of microtubules in these protists and of a subset of intracytoplasmic microtubules. Microtubule diversity may reflect distinct tubulins, diverse microtubule-associated proteins, or a combination of both.

  • Morphology of the mitochondria and endoplasmic reticula of chorion laeve cytotrophoblasts: their resemblance to villous syncytiotrophoblasts rather than villous cytotrophoblasts.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    S Matsubara,T Takayama,R Iwasaki,H Minakami,T Takizawa,I Sato

    We examined the morphological features of the mitochondria and endoplasmic reticula of chorion laeve cytotrophoblasts from term human fetal membranes, and compared them with those of syncytiotrophoblasts and cytotrophoblasts from human placental villi. Ultrastructural enzyme histochemistry of cytochrome c oxidase and glucose-6-phosphatase were used as cytochemical markers for these intracellular organelles. Chorion laeve cytotrophoblasts possessed abundant endoplasmic reticula, and small mitochondria with a few cristae, which were characteristic of villous syncytiotrophoblasts rather than villous cytotrophoblasts. As for these organellar structures, statistical analysis confirmed similarities between chorion laeve cytotrophoblasts and villous syncytiotrophoblasts, but significant differences between laeve cytotrophoblasts and villous cytotrophoblasts. Though these two cytotrophoblasts originated from one common cell in early placental development, they exhibited quite different organellar morphology during placental/chorioamniotic differentiation. Considering previous data, we concluded that chorion laeve cytotrophoblasts were metabolically active cells, similar to villous syncytiotrophoblasts, performing many functions in fetal membrane physiology.

  • Enzyme histochemically detectable NAD(P)H oxidase in human placental trophoblasts: normal, preeclamptic, and fetal growth restriction-complicated pregnancy.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-08-02
    S Matsubara,I Sato

    The purpose of the present study was to determine the subcellular localization of NAD(P)H oxidase, a reactive oxygen species (ROS)-producing enzyme, in the human placenta at various gestational ages. Ultrastructural enzyme histochemistry for NAD(P)H oxidase, using cerium as a capturing agent, was carried out. Placentas from patients with severe preeclampsia and patients who delivered infants with fetal growth restriction (FGR) were also studied. Electron-dense precipitates indicating NAD(P)H oxidase activity were visible in the microvillous membranes of the placentas, especially on the surface plasma membrane of the syncytiotrophoblast microvilli, after 25 weeks of gestation. The distribution pattern and enzyme intensities were apparently the same among normal, preeclamptic, and FGR placentas. Cytochemical control experiments ensured the specific detection of NAD(P)H oxidase activity. These observations indicated that syncytiotrophoblasts possessed NAD(P)H oxidase activity, and thus ROS-generating activity. Placental NAD(P)H oxidase may play a role in placental lipid peroxidation and the placental defense mechanism.

  • Guanylin and uroguanylin in the parotid and submandibular glands: potential intrinsic regulators of electrolyte secretion in salivary glands.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-07-17
    H Kulaksiz,U Rausch,R Vaccaro,T G Renda,Y Cetin

    The intestinal peptides guanylin and uroguanylin regulate the electrolyte/water transport in the gastrointestinal epithelium via activation of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis gene product. Because a major but incompletely understood function of the salivary glands is the CFTR-mediated secretion of an electrolyte-rich fluid, we investigated the rat and guinea pig parotid and submandibular glands for expression, cellular distribution, and subcellular localization of guanylin and uroguanylin. RT-PCR analyses with guanylin and uroguanylin-specific primers revealed that both peptides are highly expressed in the parotid and submandibular glands. At the translational level, western blotting analyses with peptide-specific guanylin and uroguanylin antibodies identified the expected 12.5-kDa immunoreactive peptides in these organs. At the cellular level, guanylin and uroguanylin were exclusively confined to epithelial cells of the intralobular and interlobular ducts. At the subcellular level, the immunoreactivities were localized by preembedding immunoelectron microscopy to small vesicles which were concentrated at the apical part of the secretory epithelial cells. The expression and cell-specific localization of guanylin and uroguanylin in the salivary glands indicate that these peptides may be specifically involved in the regulation of CFTR-mediated electrolyte/water secretion in the salivary gland ductal system.

  • Immunohistochemical localization of estrogen receptor-alpha in sex ducts and gonads of newborn piglets.
    Histochem. Cell Biol. (IF 2.64) Pub Date : 2001-07-17
    M Nielsen,I B Bøgh,M Schmidt,T Greve

    Estrogen receptor-alpha (ER-alpha) expression in piglet uteri has previously been reported from day 15 after birth. Nevertheless, uterine tissue has been reported to be estrogen sensitive from the day of birth. Since estrogen action in the uterine tissue is suggested to be mediated principally by ER-alpha, the present study aimed to evaluate the presence of ER-alpha in uteri of 1- to 2-day-old piglets by means of immunohistochemistry. In addition, sex ducts and gonads of both sexes were examined. The results clearly demonstrate the presence of ER-alpha immunopositive cells in uterine tissue, which explains its estrogen responsiveness. Immunostaining was most intense in the glandular epithelial cells and is suggested to indicate participation of ER-alpha in adenogenesis. In oviducts, almost all epithelial cells were immunostained moderately positive, while the stroma cells were stained comparably more positive. The functional significance of this intensity difference is uncertain but could indicate that part of the estrogen action on the epithelium is mediated through the stroma cells, as is known for the uterus. In ovaries, the surface epithelium and stroma cells were immunostained, whereas germ and granulosa cells were immunonegative. It is speculated that ER-alpha might be involved in yet unknown intraovarian mechanisms. In male sex ducts, immunostaining was virtually confined to the epithelium of efferent ducts. All cells in the epididymis as well as in vas deferens were immunonegative. The unique presence of ER-alpha in efferent ducts corresponds with localization in other species, where it has been shown to be involved in fluid reabsorption. The obtained data on localization of ER-alpha correspond with the present knowledge, obtained in ER-alpha knockout mice, of the biological function of ER-alpha within male and female gonads and sex ducts.

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上海纽约大学William Glover