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  • Moderately elevated first trimester fasting plasma total homocysteine is associated with increased probability of miscarriage. The Reus-Tarragona Birth Cohort Study
    Biochimie (IF 3.362) Pub Date : 2020-01-18
    Pere Cavallé-Busquets; Montserrat Inglés-Puig; Joan Fernandez-Ballart; Júlia Haro-Barceló; Alejandra Rojas-Gómez; Carla Ramos-Rodriguez; Monica Ballesteros; Klaus Meyer; Per M. Ueland; Michelle M. Murphy

    The association between elevated early pregnancy fasting plasma total homocysteine (tHcy) and miscarriage risk was investigated prospectively in participants (n=544) from the Reus-Tarragona Birth Cohort study. Pregnancy was confirmed before 12 gestational weeks (GW) by ultrasound scan and a fasting blood sample collected. Pregnancies with complications other than miscarriages were excluded. Miscarriages were diagnosed by ultrasound scan and gestational age at the time of miscarriage estimated by embryo size, where possible. Cases in which blood samples were collected more than a week after the miscarriage, or the miscarriage was of known cause, were excluded. Fasting plasma folate, vitamin B12, tHcy, cotinine (biomarker of smoking), red blood cell (RBC) folate, MTHFR 677C>T (rs1801133) and SLC19A1 80G>A (rs1051266) genotypes were determined. The exposed group consisted of participants with first trimester tHcy ≥P90 (7.1 μmol/L) (n=57) and unexposed of those with tHcy

  • Metabolic memory and diabetic nephropathy: beneficial effects of natural epigenetic modifiers
    Biochimie (IF 3.362) Pub Date : 2020-01-16
    Kriti Kushwaha; Sandeep Sharma; Jeena Gupta
  • Degenerate consensus sequences in the 3′-untranslated regions of cellular mRNAs as specific motifs potentially involved in the YB-1-mediated packaging of these mRNAs
    Biochimie (IF 3.362) Pub Date : 2020-01-13
    Alexander V. Gopanenko; Alexey A. Malygin; Olga A. Kossinova; Alexey E. Tupikin; Marsel R. Kabilov; Galina G. Karpova

    The multifunctional protein YB-1 has previously been shown to be the only protein of the cytoplasmic extract of HEK293 cells, which is able to specifically interact with imperfect RNA hairpins containing motifs that are often found in exosomal (e) RNAs. In addition, it has been revealed that similar hairpins formed by degenerate consensus sequences corresponding to three eRNA-specific motifs are responsible for the cooperative binding of YB-1 to RNA in vitro. Here, using the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method applied to HEK293 cells producing FLAG-labeled YB-1, we identified mRNAs cross-linked to YB-1 in vivo and then carried out a search for the aforementioned sequences in the regions of the YB-1 cross-linking sites. It turned out that many of the mRNAs found cross-linked to YB-1 encode proteins associated with various regulatory processes, including responses to stress. More than half of all cross-linked mRNAs contained degenerate consensus sequences, which were preferably located in 3′-untranslated regions (UTRs), where most of the YB-1 cross-linking sites appeared, although not close to these sequences. Furthermore, YB-1 was mainly cross-linked to those mRNAs with degenerate consensus sequences, which could be classified as packaged because their translation levels were low compared to cellular levels. This suggests that the cooperative binding of YB-1 to mRNAs through the above sequences probably triggers the well-known multimerization of YB-l, leading to the packaging of these mRNAs. Thus, our findings indicate a previously unknown link between the degenerate consensus sequences present in the 3′-UTRs of many cytoplasmic mRNAs and YB-1-mediated translational silencing.

  • Naturally occurring cinnamic acid derivatives prevent amyloid transformation of alpha-synuclein
    Biochimie (IF 3.362) Pub Date : 2020-01-13
    Maria Medvedeva; Kseniya Barinova; Aleksandra Melnikova; Pavel Semenyuk; Vasillii Kolmogorov; Petr Gorelkin; Alexander Erofeev; Vladimir Muronetz

    In search of the compounds that interfere with amyloid transformation of alpha-synuclein, 9 natural and synthetic cinnamic acid derivatives were studied. They are structurally similar to a half of curcumin, which has pronounced anti-aggregatory and anti-amyloid effects. We have shown that some of these derivatives prevent ovine prion protein amyloidization. Subsequently, thioflavin T binding assay showed that 3 out of 9 studied compounds effectively prevented amyloid transformation of alpha-synuclein with IC50 of 13, 50 and 251 μM. Molecular modeling approach revealed possible binding sites of the three selected ligands with alpha-synuclein fibrils, while monomeric alpha-synuclein does not bind to the ligands according to experimental results. This led us to believe that compounds may act by changing the structure of primary aggregates, preventing the formation of full-length fibrils. The inhibiting effect of the ligands on aggregation of alpha-synuclein was further confirmed by monitoring aggregation via turbidimetry, susceptibility to proteolytic cleavage, changes in beta-sheet content, and scanning ion-conductance microscopy. Studied derivatives were not cytotoxic, and, moreover, two studied compounds (ferulic and 3,4-dimethoxycinnamic acid) are found in plant sources and are natural metabolites present in human blood, so they can be promising candidate drugs for synucleinopathies, including Parkinson's disease.

  • Detection and in vitro studies of Cucurbita maxima phloem serpin-1 RNA-binding properties
    Biochimie (IF 3.362) Pub Date : 2020-01-11
    Eugeny A. Tolstyko; Alexander A. Lezzhov; Anna V. Pankratenko; Marina V. Serebryakova; Andrey G. Solovyev; Sergey Y. Morozov

    Apart from being a conduit for photoassimilate transport in plants, the phloem serves as a pathway for transport of proteins and RNAs from sites of their synthesis to distant plant parts. As demonstrated for mRNAs and small RNAs such as miRNA and siRNA, their phloem transport is largely involved in responses to environmental cues including stresses and pathogen attacks. RNA molecules are believed to be transported in the phloem in the form of complexes with RNA-binding proteins; however, proteins forming such complexes are generally poorly studied. Here, we demonstrate that the Cucurbita maxima phloem serpin-1 (CmPS1), which has been previously described as a functional protease inhibitor capable of long-distance transport via the phloem, is able to bind RNA in vitro. Among different RNAs tested, CmPS1 exhibits a preference for imperfect RNA duplexes and the highest affinity to tRNA. A characteristic complex formed by CmPS1 with tRNA is not observed upon CmPS1 binding to tRNA-like structures of plant viruses. Mutational analysis demonstrates that the CmPS1 N-terminal region is not involved in RNA binding. Since antithrombin-III, the human protease inhibitor of serpin family most closely sequence-related to CmPS1, is found to be unable to bind RNA, one can suggest that, in its evolution, CmPS1 has gained the RNA binding capability as an additional function likely relevant to its specific activities in the plant phloem.

  • 更新日期:2020-01-08
  • Hepatic glycerol metabolism is early reprogrammed in rat liver cancer development
    Biochimie (IF 3.362) Pub Date : 2020-01-07
    Florencia Lorenzetti; Alejo M. Capiglioni; Raúl A. Marinelli; María Cristina Carrillo; María de Luján Alvarez

    Evidence shows that oral glycerol supplementation during the early stages of rat liver cancer reduces the growth of preneoplastic lesions. Besides, human hepatocellular carcinoma (HCC) cells display decreased expression of glycerol channel aquaporin 9 (AQP9) and also diminished glycerol-3-phosphate (G3P) content. According to this, we analyzed glycerol metabolism during the initial stages of rat liver carcinogenesis. Wistar rats were subjected to a 2-phase model of hepatocarcinogenesis (initiated-promoted, IP group) or left untreated (control, C group). Different features of glycerol metabolism were compared between both groups. IP animals showed increased plasma free glycerol levels and liver AQP9 protein expression. Also, IP rats showed increased glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) hepatic activities. Gluconeogenesis from glycerol both in vivo and in isolated perfused liver was higher in rats having liver preneoplasia. Nevertheless, preneoplastic foci notably reduced AQP9 and GK protein expressions, displaying a reduced ability to import glycerol and to convert it into G3P, as a way to preserve preneoplastic hepatocytes from the deleterious effect of G3P. In conclusion, the metabolic shift that takes place in the initial stages of liver cancer development comprises an increased hepatic utilization of glycerol for gluconeogenesis. Enhanced glucose production from glycerol is mostly carried out by the surrounding non-preneoplastic tissue and can be used as an energy source for the early transformed liver cells.

  • Strand displacement DNA synthesis by DNA polymerase gp90 exo― of Pseudomonas aeruginosa phage 1
    Biochimie (IF 3.362) Pub Date : 2020-01-03
    Chenyang Mi; Shuming Zhang; Wenxin Huang; Mengyuan Dai; Zili Chai; Wang Yang; Shanshan Deng; Lin Ao; Huidong Zhang

    Strand displacement DNA synthesis is essential for DNA replication. Gp90, the sole DNA polymerase of Pseudomonas aeruginosa phage 1, can bypass multiply DNA lesions. However, whether it can perform strand displacement synthesis is still unknown. In this work, we found that gp90 exo― could perform strand displacement synthesis, albeit its activity and processivity were lower than those of primer extension. Gp90 exo― itself could not unwind Y-shaped or fork DNA. Tail and gap at DNA fork were necessary for efficient synthesis. High GC content obviously inhibited strand displacement synthesis. Consecutive GC sequence at the entrance of fork showed more inhibition effect on DNA synthesis than that in the downstream DNA fork. The fraction of productive polymerase and DNA complex (A values) was higher for fork than gap; while their average extension rates (kp values) were similar. However, both A and kp values were lower than those for the primer/template (P/T) substrate. The binding of gp90 exo― to fork was tighter than P/T or gap in the absence of dATP. In the presence of dATP to form ternary complex, the binding affinity of gp90 exo― to P/T or gap was increased compared with that in the binary complex. Abasic site, 8-oxoG, and O6-MeG inhibited and even blocked strand displacement synthesis. This work shows that gp90 exo― could perform strand displacement DNA synthesis at DNA fork, discovering the presence of new functions of PaP1 DNA polymerase in DNA replication and propagation of PaP1.

  • Multiple catalytic activities of human 17β-hydroxysteroid dehydrogenase type 7 respond differently to inhibitors
    Biochimie (IF 3.362) Pub Date : 2019-12-27
    Terenzio Ferrante; Salvatore Adinolfi; Giulia D’Arrigo; Donald Poirier; Martina Daga; Marco Lucio Lolli; Gianni Balliano; Francesca Spyrakis; Simonetta Oliaro-Bosso
  • Antitermination protein P7 of bacteriophage Xp10 distinguishes different types of transcriptional pausing by bacterial RNA polymerase
    Biochimie (IF 3.362) Pub Date : 2019-12-26
    Maria Prostova; Andrey Kulbachinskiy; Daria Esyunina

    Bacteriophage-encoded transcription antiterminators play essential roles in the regulation of gene expression during infection. Here, we characterize the effects of the antiterminator protein P7 of bacteriophage Xp10 on transcriptional pausing by Xanthomonas oryzae RNA polymerase (RNAP) at different types of pause-inducing signals. When acting alone, P7 inhibits only hairpin-stabilized pauses, likely by preventing hairpin formation. In the presence of NusA, P7 also suppresses backtracking-stabilized pauses and the his elemental pause, but not the consensus elemental pause, suggesting that these pause signals may be mechanistically different. Thus, P7 and other bacteriophage proteins that bind near the RNA exit channel of RNAP have evolved to regulate transcription by suppressing RNAP pausing at a subset of regulatory signals, and to co-opt NusA in doing so.

  • Manipulating and studying triglyceride droplets in microfluidic devices
    Biochimie (IF 3.362) Pub Date : 2019-12-24
    Sébastien Marze; Hoang Thanh Nguyen; Mélanie Marquis

    Triglyceride is the main lipid class in nature, found as droplets in both living systems and man-made products (such as manufactured foods and drugs). Characterizing triglyceride droplets in situ in these systems is complex due to many environmental interactions. To answer basic research questions about droplet formation, structuration, stability, or degradation, microfluidic strategies were developed, allowing well-controlled droplets to be formed, manipulated, and studied. In this review, these strategies are described, starting with the presentation of droplet production devices, with applications essentially related to microencapsulation and delivery, then detailing methods to monitor droplet degradation in situ and in real time, finishing with microfluidic platforms allowing the investigation of many aspects of biological lipid droplets simultaneously.

  • Physico-chemical behaviors of human and bovine milk membrane extracts and their influence on gastric lipase adsorption
    Biochimie (IF 3.362) Pub Date : 2019-12-20
    C. Bourlieu; W. Mahdoueni; G. Paboeuf; E. Gicquel; O. Ménard; S. Pezennec; S. Bouhallab; A. Deglaire; D. Dupont; F. Carrière; V. Vié
  • Cerebrospinal fluid: profiling and fragmentation of gangliosides by ion mobility mass spectrometry
    Biochimie (IF 3.362) Pub Date : 2019-12-17
    Mirela Sarbu; Shannon Raab; Lucas Henderson; Dragana Fabris; Željka Vukelić; David E. Clemmer; Alina D. Zamfir

    The proximity of cerebrospinal fluid (CSF) with the brain, its permanent renewal and better availability for research than tissue biopsies, as well as ganglioside (GG) shedding from brain to CSF, impelled lately the development of protocols for the characterization of these glycoconjugates and discovery of central nervous system biomarkers expressed in CSF. Currently, the investigation of CSF gangliosides is focused on concentration measurements of the predominant classes and much less on their profiling and structural analysis. Since we have demonstrated recently the high performance of ion mobility separation mass spectrometry (IMS MS) for compositional and structural determination of human brain GGs, in the present study we have implemented for the first time IMS MS for the exploration of human CSF gangliosidome, in order to generate the first robust mass spectral database of CSF gangliosides. IMS MS separation and screening revealed 113 distinct GG species in CSF, having similar compositions to the species detected in human brain. In comparison with the brain tissue, we have discovered in CSF several components containing fatty acids with odd number of carbon atoms and/or short glycan chains. By tandem MS (MS/MS) we have further analyzed the structure of GD3(d18:1/18:0) and GD2(d18:1/18:0), two glycoforms exhibiting short carbohydrate chains found in CSF, but discovered and characterized previously in brain as well. According to the present results, human CSF and brain show a similar ganglioside pattern, a finding that might be useful in clinical research focused on discovery of ganglioside species associated to neurodegenerative diseases and brain tumors.

  • Revisiting Prochirality
    Biochimie (IF 3.362) Pub Date : 2019-12-17
    Raymond S. Ochs; Tanaji T. Talele

    We propose a new model for prochirality that satisfies all known examples: the prochiral plane. This plane contains the prochiral carbon and defines two separate faces for chemical modification. We extend this to enzyme catalysis, replacing the “three point attachment” hypothesis and its variants. Once a prochiral substrate is fixed on an enzyme surface, the asymmetry of the enzyme provides reactants exclusively on one side of the prochiral plane, producing an enantiomerically pure chiral product. The aconitase reaction is detailed as an example, using molecular modeling and its known enzymatic mechanism. We show that the prochiral substrate for this molecule is not citrate, but rather cis-aconitate. The number of interaction points of cis-aconitate is not relevant to prochirality, but rather to substrate specificity. A second detailed example is the enzyme fumarase; here the substrate fumarate has only two binding sites, but is nonetheless fixed onto the enzyme and has a defined prochiral plane. We also provide a literature survey of more prochiral substrates, all of which have sp2 hybridized carbon and contain a prochiral plane. An example of a prochiral unnatural substrate for sphingosine kinase 2, fingolimod, has an sp3 hybridized prochiral carbon and also contains a prochiral plane. Finally, we provide an intuitive example of a prochiral physical object, a coffee cup, interacting with one hand and lip.

  • 更新日期:2019-12-17
  • Classical homocystinuria: from cystathionine beta-synthase deficiency to novel enzyme therapies
    Biochimie (IF 3.362) Pub Date : 2019-12-16
    Erez M. Bublil; Tomas Majtan

    Genetic defects in cystathionine beta-synthase (CBS), a key enzyme of organic sulfur metabolism, result in deficiency of CBS activity and a rare inborn error of metabolism called classical homocystinuria (HCU). HCU is characterized by massive accumulation of homocysteine, an intermediate of methionine metabolism, and multisystemic clinical symptoms. Current treatment options for HCU are very limited and often inefficient, partially due to a low patient compliance with very strict dietary regimen. Novel therapeutic approaches are needed to cope with the toxic accumulation of homocysteine and restoration of a healthy metabolic balance. Human CBS is a complex intracellular multimeric enzyme that relies on three cofactors (heme, pyridoxal-5’-phosphate and S-adenosylmethionine) for proper function. Engineering and chemical modification of human CBS yielded OT-58, a first-in-class enzyme therapy candidate for HCU. Pre-clinical testing of OT-58 showed its substantial efficacy in lowering plasma and tissue concentrations of homocysteine, improving metabolic balance and correcting clinical symptoms of HCU. In addition, OT-58 showed great safety and toxicity profile when administered to non-human primates. Overwhelmingly positive and extensive pre-clinical package propelled OT-58 into a first-in-human clinical trial, which started on January 2019. In a meantime, other enzyme therapies based on modified human cystathionine gamma-lyase or erythrocyte-encapsulated bacterial methionine gamma-lyase have shown efficacy in decreasing plasma homocysteine in HCU mice. In addition, gene therapy approaches using adenovirus or minicircle DNA have been evaluated in HCU. In this review, we summarize the current efforts developing novel therapies for HCU to address a high unmet medical need among HCU patients.

  • Deletion of CDR1 reveals redox regulation of pleiotropic drug resistance in Candida glabrata
    Biochimie (IF 3.362) Pub Date : 2019-12-14
    Kseniia V. Galkina; Michiyo Okamoto; Hiroji Chibana; Dmitry A. Knorre; Susumu Kajiwara
  • Four paralogous Gfh factors in the extremophilic bacterium Deinococcus peraridilitoris have distinct effects on various steps of transcription
    Biochimie (IF 3.362) Pub Date : 2019-12-13
    Aleksei Agapov; Andrey Kulbachinskiy

    Gre factors are ubiquitous transcription regulators that stimulate co-transcriptional RNA cleavage by bacterial RNA polymerase (RNAP). Here, we show that the stress-resistant bacterium Deinococcus peraridilitoris encodes four Gre factor homologs, Gfh proteins, that have distinct effects on transcription by RNAP. Two of the factors, Gfh1α and Gfh2β inhibit transcription initiation, and one of them, Gfh1α can also regulate transcription elongation. We show that this factor strongly stimulates transcriptional pausing and intrinsic termination in the presence of manganese ions but has no effect on RNA cleavage. Thus, some Gfh factors encoded by Deinococci serve as lineage-specific transcription inhibitors that may play a role in stress resistance, while the functions of others remain to be discovered.

  • The flanking peptides issue from the maturation of the human islet amyloid polypeptide (hIAPP) slightly modulate hIAPP-fibril formation but not hIAPP-induced cell death.
    Biochimie (IF 3.362) Pub Date : 2019-12-12
    Shadai Salazar Vazquez; Bertrand Blondeau; Pierre Cattan; Mathieu Armanet; Ghislaine Guillemain; Lucie Khemtemourian

    Type 2 diabetes mellitus is a disease characterized by the formation of amyloid fibrillar deposits consisting mainly in human islet amyloid polypeptide (hIAPP), a peptide co-produced and co-secreted with insulin. hIAPP and insulin are synthesized by pancreatic β cells initially as prehormones resulting after sequential cleavages in the mature peptides as well as the two flanking peptides (N- and C-terminal) and the C-peptide, respectively. It has been suggested that in the secretory granules, the kinetics of hIAPP fibril formation could be modulated by some internal factors. Indeed, insulin is known to be a potent inhibitor of hIAPP fibril formation and hIAPP-induced cell toxicity. Here we investigate whether the flanking peptides could regulate hIAPP fibril formation and toxicity by combining biophysical and biological approaches. Our data reveal that both flanking peptides are not amyloidogenic. In solution and in the presence of phospholipid membranes, they are not able to totally inhibit hIAPP-fibril formation neither hIAPP-membrane damage. In the presence of INS-1 cells, a rat pancreatic β-cell line, the flanking peptides do not modulate hIAPP fibrillation neither hIAPP-induced cell death while in the presence of human islets, they have a slightly tendency to reduce hIAPP fibril formation but not its toxicity. These data demonstrate that the flanking peptides do not strongly contribute to reduce mature hIAPP amyloidogenesis in solution and in living cells, suggesting that other biochemical factors present in the cells must act on mature hIAPP fibril formation and hIAPP-induced cell death.

  • SIRT1/PGC-1 pathway activation triggers autophagy/mitophagy and attenuates oxidative damage in intestinal epithelial cells
    Biochimie (IF 3.362) Pub Date : 2019-12-09
    Danyang Liang, Yisha Zhuo, Zeheng Guo, Lihua He, Xueyi Wang, Yulong He, Lexing Li, Hanchuan Dai
  • The Role of Heparin/Heparan Sulphate in the IFN-γ-led Arena
    Biochimie (IF 3.362) Pub Date : 2019-11-30
    Kening Xu, Lan Jin
  • Inactivated AMPK-α2 promotes the progression of diabetic brain damage by Cdk5 phosphorylation at Thr485 site
    Biochimie (IF 3.362) Pub Date : 2019-11-29
    Yan Li, Qiong Xiang, Yu-Han Yao, Jing-Jing Li, Yan Wang, Xian-Hui Li

    Changes in brain energy metabolism in diabetes mellitus, including increased insulin resistance and mitochondrial dysfunction, are critically involved in diabetes-related neurodegeneration, and associate with early cognitive impairment as well. The aim of this study is to detect the specific phosphorylated-Thr485- AMP-activated protein kinase (AMPK-α2), regulated by cyclin-dependent kinase 5 (Cdk5) paly the inhibitory functional role of AMPK-α2, Which is maybe the link to the accelelated diabetic brain damage progression. Here, we used GK rats, the type 2 diabetic animal model for in vivo studies and performed In vitro kinase assay, high glucose treatment, -phosphorylated mutation and protein expression in both HEK-293T and HT-22 cell lines. In vitro, the results show that murine wild-type AMPK-α2 was phosphorylated by Cdk5 at a (S/T)PX(K/H/R) phosphorylation consensus sequence, which was associated with decreased AMPK-α2 activity. Surprisingly, mutation of Thr485 to alanine in AMPK-α2 results in the abolished Cdk5 effects, demonstrating that Thr485-phosphorylation is critical to AMPK-α2 inhibition by Cdk5. In addition, these alterations in AMPK-α2-phosphorylation and -activity induced by Cdk5 is specific at Thr485. Furthermore, in GK rats, the increased phosphorylated- Thr 485 of AMPK-α2 results in the decreased AMPK-α2 activity, which is correlated with the apoptosis of neurons in hippocamps. After high glucose treatment, the decreased survival showed in AMPK-α2T485A HT-22 cells compared to AMPK-α2WT. The down-regulated of p-CREB, SNAP25, synaptophysin as well as synapsin-1were shown in both GK rats and HT-22 cell line. Meanwhile, pre-treated with either the specific Cdk5-inhibitor (roscovitine) or the antidiabetic AMPK-α2-inhibitor (metformin) could restore the alterations in neuronal protein expression. Our results suggest that Cdk5-mediated phosphorylated- Thr485 in AMPK-α2 may be involved in the pathogenesis of diabetic brain damage.

  • Polyamine acetylation and substrate-induced oligomeric states in histone acetyltransferase of multiple drug resistant Acinetobacter baumannii
    Biochimie (IF 3.362) Pub Date : 2019-11-29
    Jyoti Singh Tomar, Ramakrishna Vijayacharya Hosur

    Histone acetyltransferase (Hpa2) is an unusual acetyltransferase, with a wide range of substrates; including histones, polyamines and aminoglycosides antibiotic. Hpa2 belongs to GNAT superfamily and GNATs are well known for the formation of homo-oligomers. However, the reason behind their oligomerization remained unexplored. Here, oligomeric states of Hpa2 were explored, to understand the functional significance of oligomerization. Biochemical analysis suggests that Hpa2 exists as dimer in solution and self-assembles into tetramer in the spermine, spermidine and kanamycin bound form. Stability analysis with denaturants concludes that homo-oligomerization of Hpa2 relies on bound substrate and not on experimental conditions. Homo-oligomerization in Hpa2 depicts direct correlation with its polyamine acetylating capacity. This correlation and in silico model structures suggest that oligomerization of Hpa2 is associated with the hastening of acetylation process. Interestingly, polyamine acetylation down regulates biofilms formation in E. coli BL21/Hpa2-transformants cells. Therefore, we propose that Hpa2 manipulates survival strategies of the bacterium via polyamines and antibiotics acetylation.

  • Vegetable lecithins: a review of their compositional diversity, impact on lipid metabolism and potential in cardiometabolic disease prevention
    Biochimie (IF 3.362) Pub Date : 2019-11-28
    Chloé Robert, Leslie Couëdelo, Carole Vaysse, Marie-Caroline Michalski

    Vegetable lecithins, widely used in the food industry as emulsifiers, are a mixture of naturally occurring lipids containing more than 50% of phospholipids (PL). PL exert numerous important physiological effects. Their amphiphilic nature notably enables them to stabilize endogenous lipid droplets, conferring them an important role in lipoprotein transport, functionality and metabolism. In addition, beneficial effects of dietary lecithin on metabolic disorders have been reported since the 1990s. This review attempts to summarize the effects of various vegetable lecithins on lipid and lipoprotein metabolism, as well as their potential application in the treatment of dyslipidemia associated with metabolic disorders. Despite controversial data concerning the impact of vegetable lecithins on lipid digestion and intestinal absorption, the beneficial effect of lecithin supplementation on plasma and hepatic lipoprotein and cholesterol levels is unequivocal. This is especially true in hyperlipidemic patients. Furthermore, the immense compositional diversity of vegetable lecithins endows them with a vast range of biochemical and biological properties, which remain to be explored in detail. Data on the effects of vegetable lecithins alternative to soybean, both as supplements and as ingredients, is undoubtedly lacking. Given the exponential demand for vegetable products alternative to those of animal origin, it is of primordial importance that future research is undertaken in order to elucidate the mechanisms by which individual fatty acids and PL from various vegetable lecithins modulate lipid metabolism. The extent to which they may influence parameters associated with metabolic disorders, such as intestinal integrity, low-grade inflammation and gut microbiota must also be assessed.

  • A Twist of FATe: Lipid Droplets and Inflammatory Lipid Mediators
    Biochimie (IF 3.362) Pub Date : 2019-11-28
    Eva Jarc, Toni Petan

    Lipid droplets are fat storage organelles present in most eukaryotic cells. They consist of a neutral lipid core containing mostly triglycerides and sterol esters and covered by a monolayer of phospholipids, wherein numerous proteins are embedded. In the cell, lipid droplets have a dynamic life cycle, rapidly altering their size, location, lipid and protein composition in response to environmental stimuli and cell state. Lipid droplets are primarily involved in the coordination of lipid metabolism with cellular requirements for energy production, membrane homeostasis and cell growth. However, they are also directly or indirectly engaged in signalling pathways. On the one hand, lipid droplets sequester lipids and proteins thereby limiting their availability for participation in signalling pathways. On the other hand, the lipolytic machinery provides a highly regulated, on-demand source of signalling lipids: lipids derived from their neutral lipid core, or the phospholipid monolayer, directly act as signalling mediators or are converted into ones. In fact, emerging studies suggest that these organelles are essential for various cellular stress response mechanisms, including inflammation and immunity, acting as hubs that integrate metabolic and inflammatory processes. Here, we discuss the ways in which lipid droplets regulate the availability of fatty acids for the activation of signalling pathways and for the production of polyunsaturated fatty acid-derived lipid mediators. We focus in particular on recent discoveries in immune cells and adipose tissue that have revealed an intricate relationship between lipid droplets and inflammatory signalling and may also be relevant for other tissues and various human diseases.

  • Structural characterization and mechanical properties of chimeric Masp1/Flag minispidroins
    Biochimie (IF 3.362) Pub Date : 2019-11-26
    Shouying Xu, Xue Li, Yizhong Zhou, Ying Lin, Qing Meng

    Dragline silk has the highest tensile strength among the seven types of spider silks due to its abundant polyalanine motifs. Whereas the flagelliform spider silk is most extensible as its composed spidroin is rich in GPGGX motifs. Most of the spider silk proteins are composed of an extensive repetitive domain flanked by N- and C-terminal domains. To obtain artificial fibers with considerable strength and extensibility, herein a kind of chimeric minispidroins were constructed whose repetitive domain (R) mainly consisted of polyalanine motifs and GPGGX motifs. In our study, NT and CT from Araneus ventricosus MaSp1 were fused with different numbers (1, 4, 8) of repeat domains (R), resulting in three chimeric minispidroins. All these chimeric proteins could form silk-like fibers via manual pulling. As the chimeric spidroin was pulled from the protein solution into fiber by shear forces, the secondary structure transformed from α-helix to β-sheet. Among the three types of fibers, the average tensile strength of NTR4CT ranked the highest (149 MPa), which could provide outstanding material with better mechanical properties. In addition, NT was fused with CT and repetitive domain R respectively, namely NC and NR proteins. As a result, NC could form fibers that had much lower properties than NTR1CT, indicating that repetitive domain was responsible for the strength and elasticity of the fibers. However, NR did not form silk-like fibers, suggesting that Araneus ventricosus Masp1 CT controlled fiber formation. These results broaden the limited knowledge of chimeric spider silk sequences.

  • Molecular docking and in vitro evaluation of a new hybrid molecule (JM-20) on cholinesterase activity from different sources
    Biochimie (IF 3.362) Pub Date : 2019-11-23
    Fernanda D’Avila da Silva, Pablo Andrei Nogara, Estael Ochoa-Rodríguez, Yanier Nuñez-Figueredo, Maylin Wong-Guerra, Denis Broock Rosemberg, João Batista Teixeira Rocha

    The main function of AChE is the hydrolysis of the neurotransmitter acetylcholine (ACh) at the neuromuscular and in cholinergic brain synapses. In some pathologies, loss of cholinergic neurons may be associated with a deficiency of ACh in specific brain areas. Consequently, the study of new safe drugs that inhibit AChE is important, because they can increase ACh levels in the synaptic cleft without adverse effects. Here, we evaluated the effects of JM-20 (a benzodiazepine–dihydropyridine hybrid molecule) on cholinesterase (ChE) activities from distinct sources (AChE from Electrophorus electricus (EeAChE), human erythrocyte membranes (HsAChE (ghost)), total erythrocyte (HsAChE (erythrocyte)) and BChE from plasma (HsBChE) and purified enzyme from the horse (EcBChE)). Kinetic parameters were determined in the presence of 0.05-1.6 mM of substrate concentration. The interactions ChEs with JM-20 were performed using molecular docking simulations. JM-20 inhibited all tested AChE but not BChE. The IC50 values were 123.0 nM ± 0.18 (EeAChE), 158.1 nM ± 0.14 (ghost HsAChE), and 172.4 nM ± 0.16 (erythrocytic HsAChE). JM-20 caused a mixed type of inhibition (it altered Km and Vmax of AChE). The molecular docking indicated the binding poses and the most plausible active isomer of JM-20. Besides giving important data for future drug design, our results help us understand the mode of action of JM-20 as a specific inhibitor of AChE enzymes.

  • Prevention of DNA multimerization using phosphoryl guanidine primers during isothermal amplification with Bst exo- DNA polymerase
    Biochimie (IF 3.362) Pub Date : 2019-11-22
    Ravil R. Garafutdinov, Assol R. Sakhabutdinova, Maxim S. Kupryushkin, Dmitrii V. Pyshnyi

    Over the last two decades, isothermal amplification of nucleic acids has gained more attention due to a number of advantages over the widely used polymerase chain reaction. For isothermal amplification, DNA polymerases with strand-displacement activity are needed, and Bst exo-polymerase is one of the most commonly used. Unfortunately, Bst exo-causes nonspecific DNA amplification (so-called multimerization) under isothermal conditions that results in undesirable products (multimers) consisting of tandem nucleotide repeats. Multimerization occurs only for short ssDNA or primer dimers, and the efficiency of multimerization depends significantly on the reaction conditions, but slightly depends on the sequence of DNA templates. In this study we report the prevention of DNA multimerization using a new type of modified oligonucleotide primers with internucleosidic phosphates containing 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups). Primers with one, two or three PG groups located at the 3'- or 5'-ends or in the middle of the primers were designed. It turned out, such bulky groups interfere with the moving of Bst exo-polymerase along DNA chains. However, one modified phosphate does not notably affect the efficiency of polymerization, and the elongation is completely inhibited only when three contiguous modifications occur. Multimerization of the linear ssDNA templates is blocked by three modifications in the middle of both primers whereas specific amplification of the circular ssDNA by rolling circle amplification is not inhibited. Thus, incorporation of three PG groups is sufficient to prevent multimerization and allows to create improved primers for reliable isothermal amplification with Bst exo- DNA polymerase.

  • Mechanism of HrcA function in heat shock regulation in Mycobacterium tuberculosis
    Biochimie (IF 3.362) Pub Date : 2019-11-22
    Owais R. Hakiem, Priyanka Parijat, Prajna Tripathi, Janendra K. Batra

    Molecular chaperones are a conserved family of proteins that are over-expressed in response to heat and other stresses. The regulation of expression of chaperone proteins plays a vital role in pathogenesis of various bacterial pathogens. In M. tuberculosis, HrcA and HspR negatively regulate heat shock protein operons by binding to their cognate DNA elements, CIRCE and HAIR respectively. In this study, we show that M. tuberculosis HrcA is able to bind to its cognate CIRCE DNA element present in the upstream regions of groES and groEL2 operons only with the help of other protein(s). It is also demonstrated that M. tuberculosis HrcA binds to a CIRCE like DNA element present in the upstream region of hrcA gene suggesting its auto-regulatory nature. In addition, we report the presence of a putative HAIR element in the upstream region of groES operon and demonstrate the binding of HspR to it. In vitro, HrcA inhibited the DNA binding activity of HspR in a dose-dependent manner. The current study demonstrates that M. tuberculosis HrcA requires other protein(s) to function, and the heat shock protein expression in M. tuberculosis is negatively regulated jointly by HrcA and HspR.

  • Effect of glucocorticoids on glyceroneogenesis in adipose tissue: A systematic review
    Biochimie (IF 3.362) Pub Date : 2019-11-21
    Mariana Lima-de-Freitas Marcondes-de-Mello, Maria Cristina Serafim-Costa, Mariela Mesquita Alves-e-Silva, Natália Rodrigues Oliveira, Nicola Vasconcellos Bertolucci-Caldo, Ruan Krubniki Ferraz, Valéria Ernestânia Chaves

    Glyceroneogenesis is important for the maintenance of fat content in white adipose tissue (WAT). An increase in WAT, and especially the pattern of fat distribution, specifically in visceral depots, potentially contributes to cardiovascular and metabolic diseases, such as type 2 diabetes mellitus, myocardial infarction and hypertension. Recent studies have shown important differences in glyceroneogenesis of different fat sites under the administration of glucocorticoids (GCs). Such differences need to be analysed with criteria evidencing the parameter studied, the type of corticoid, the form of administration and also the tissue studied. PubMed, Scopus and Virtual Health Library were used to search for articles that analysed the effect of GCs on glyceroneogenesis in different sites of adipose tissue in mammals and primary cultures. GCs decrease the glyceroneogenesis in epididymal WAT (EWAT) and also decrease the expression of the mRNA, content and activity of phosphoenolpyruvate carboxykinase (PEPCK-C), key enzyme of glyceroneogenesis. However, in retroperitoneal WAT (RWAT), although there is no consensus about the effect of GCs on PEPCK mRNA, GCs increase PEPCK-C activity and glyceroneogenesis flux. In inguinal white WAT (IWAT) an in vitro study showed an increase in the PEPCK mRNA induced by dexamethasone. However, prednisolone does not change glyceroneogenesis flux. In interscapular brown adipose tissue (IBAT) prednisolone or dexamethasone does not change PEPCK-C activity in control diet-fed rats but led to a decrease in PEPCK-C activity in fasted- or high-fat/low-carbohydrate diet-fed rats, as well as in suckling rats. Despite that fact that GCs have different potencies, the same dose of dexamethasone reduces PEPCK-C activity in EWAT, but not in RWAT and IBAT from control-diet fed rats. In summary, the data presented in this article show that GCs differentially regulate glyceroneogenesis in different sites of adipose tissue. Further experiments are needed to firmly establish our hypothesis and clarify the mechanisms involved.

  • Identification of novel quinoline inhibitor for EHMT2/G9a through virtual screening
    Biochimie (IF 3.362) Pub Date : 2019-11-20
    M.Ramya Chandar Charles, Arun Mahesh, Shu-Yu Lin, Hsing-Pang Hsieh, Arunkumar Dhayalan, Mohane Selvaraj Coumar
  • Inhibition of base excision repair by natamycin suppresses prostate cancer cell proliferation
    Biochimie (IF 3.362) Pub Date : 2019-11-19
    Judy L. Vasquez, Yanhao Lai, Thirunavukkarasu Annamalai, Zhongliang Jiang, Manqi Zhang, Ruipeng Lei, Zunzhen Zhang, Yuan Liu, Yuk-Ching Tse-Dinh, Irina U. Agoulnik

    Prostate cancer (PCa) progression is characterized by increased expression and transcriptional activity of the androgen receptor (AR). In the advanced stages of prostate cancer, AR significantly upregulates the expression of genes involved in DNA repair. Upregulation of expression for base excision repair (BER) related genes is associated with poor patient survival. Thus, inhibition of the BER pathway may prove to be an effective therapy for prostate cancer. Using a high throughput BER capacity screening assay, we sought to identify BER inhibitors that can synergize with castration therapy. An FDA-approved drug library was screened to identify inhibitors of BER using a fluorescence-based assay suitable for HTS. A gel-based secondary assay confirmed the reduction of BER capacity by compounds identified in the primary screen. Five compounds were then selected for further testing in the independently derived, androgen-dependent prostate cancer cell lines, LNCaP and LAPC4, and in the nonmalignant prostate derived cell lines PNT1A and RWPE1. Further analysis led to the identification of a lead compound, natamycin, as an effective inhibitor of key BER enzymes DNA polymerase β (pol β) and DNA Ligase I (LIG I). Natamycin significantly inhibited proliferation of PCa cells in an androgen depleted environment at 1 μM concentration, however, growth inhibition did not occur with nonmalignant prostate cell lines, suggesting that BER inhibition may improve efficacy of the castration therapies.

  • Cloning, purification and study of recombinant GH3 family β-glucosidase from Penicillium verruculosum
    Biochimie (IF 3.362) Pub Date : 2019-11-19
    Pavel V. Volkov, Alexandra M. Rozhkova, Ivan N. Zorov, Arkady P. Sinitsyn

    A novel bgl1 gene, encoding GH3 family β-glucosidase from Penicillium verruculosum (PvBGL), was cloned and heterologously expressed in P. canescens RN3-11-7 (niaD-) strain under the control of the strong xylA gene promoter. The recombinant rPvBGL was purified and their properties were studied in comparison with those of rAnBGL from Aspergillus niger expressed previously in the same fungal host. The rPvBGL had an observed molecular mass of 90 kDa (SDS-PAGE data) and displayed the enzyme maximum activity at pH 4.6 and 65 °C. The enzyme half-life time at 60 oC was found to be 87 min. Unlike the rAnBGL, the rPvBGL was not adsorbed on microcrystalline cellulose, which gives the latter enzyme an advantage in cellulose conversion with a longer time of hydrolysis.

  • Relationship between acyl-lipid and sterol metabolisms in diatoms
    Biochimie (IF 3.362) Pub Date : 2019-07-07
    Eric Maréchal, Josselin Lupette

    Diatoms are a phylum of unicellular photosynthetic eukaryotes living in oceans and fresh waters, characterized by the complexity of their plastid, resulting from a secondary endosymbiosis event. In the model diatom Phaeodactylum tricornutum, fatty acids (FAs) are synthesized from acetyl-CoA in the stroma of the plastid, producing palmitic acid. FAs are elongated and desaturated to form very-long chain polyunsaturated fatty acids (VLC-PUFAs) in domains of the endomembrane system that need to be identified. Synthesis of VLC-PUFAs is coupled with their import to the core of the plastid via the so-called “omega” pathway. The biosynthesis of sterols in diatoms is likely to be localized in the endoplasmic reticulum as well as using precursors deriving from the mevalonate pathway, using acetyl-CoA as initial substrate. These metabolic modules can be characterized functionally by genetic analyzes or chemical treatments with appropriate inhibitors. Some ‘metabolic modules’ are characterized by a very low level of metabolic intermediates. Since some chemical treatments or genetic perturbation of lipid metabolism induce the accumulation of these intermediates, channeling processes are possibly involved, suggesting that protein-protein interactions might occur between enzymes within large size complexes or metabolons. At the junction of these modules, metabolic intermediates might therefore play dramatic roles in directing carbon fluxes from one direction to another. Here, acetyl-CoA seems determinant in the balance between TAGs and sterols. Future lines of research and potential utilization for biotechnological applications are discussed.

  • Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices
    Biochimie (IF 3.362) Pub Date : 2019-07-06
    Amal Salhi, Sawsan Amara, Pascal Mansuelle, Rémy Puppo, Régine Lebrun, Brigitte Gontero, Ahmed Aloulou, Frédéric Carrière

    Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

  • Targeting TOR signaling for enhanced lipid productivity in algae
    Biochimie (IF 3.362) Pub Date : 2019-06-29
    Laura Prioretti, Frédéric Carriere, Ben Field, Luisana Avilan, Marie-Hélène Montané, Benoît Menand, Brigitte Gontero

    Microalgae can produce large quantities of triacylglycerols (TAGs) and other neutral lipids that are suitable for making biofuels and as feedstocks for green chemistry. However, TAGs accumulate under stress conditions that also stop growth, leading to a trade-off between biomass production and TAG yield. Recently, in the model marine diatom Phaeodactylum tricornutum it was shown that inhibition of the target of rapamycin (TOR) kinase boosts lipid productivity by promoting TAG production without stopping growth. We believe that basic knowledge in this emerging field is required to develop innovative strategies to improve neutral lipid accumulation in oleaginous microalgae. In this minireview, we discuss current research on the TOR signaling pathway with a focus on its control on lipid homeostasis. We first provide an overview of the well characterized roles of TOR in mammalian lipogenesis, adipogenesis and lipolysis. We then present evidence of a role for TOR in controlling TAG accumulation in microalgae, and draw parallels between the situation in animals, plants and microalgae to propose a model of TOR signaling for TAG accumulation in microalgae.

  • 更新日期:2019-11-11
  • Sulfoxides of sulfur-containing amino acids are suicide substrates of Citrobacter freundii methionine γ-lyase. Structural bases of the enzyme inactivation
    Biochimie (IF 3.362) Pub Date : 2019-11-09
    Svetlana Revtovich, Elena Morozova, Vitalia Kulikova, Vasiliy Koval, Natalya Anufrieva, Alexei Nikulin, Tatyana Demidkina

    Interactions of Citrobacter freundii methionine γ-lyase (MGL) with sulfoxides of typical substrates were investigated. It was found that sulfoxides are suicide substrates of the enzyme. The products of the β- and γ-elimination reactions of sulfoxides, thiosulfinates, oxidize three cysteine residues of the enzyme. Three-dimensional structures of MGL inactivated by dimethyl thiosulfinate and diethyl thiosulfinate were determined at 1.46 Å and 1.59 Å resolution. Analysis of the structures identified SH groups oxidized by thiosulfinates and revealed the structural bases of MGL inactivation. The extent of inactivation of MGL in the catalysis of the β-elimination reaction depends on the length of the «tail» at oxidized Cys115. Oxidation of Cys115 results in MGL incapable to catalyze the stage of methyl mercaptan elimination of the physiological reaction.

  • Metabolic shift in the production of corrinoid compounds by Lactobacillus coryniformis in the absence of purines
    Biochimie (IF 3.362) Pub Date : 2019-11-09
    Andrea Carolina Torres, Mariano Elean, Elvira María Hébert, Lucila Saavedra, María Pía Taranto
  • Dietary choline is related to increased risk of acute myocardial infarction in patients with stable angina pectoris
    Biochimie (IF 3.362) Pub Date : 2019-11-07
    Anthea Van Parys, Vegard Lysne, Gard Frodahl Tveitevåg Svingen, Per Magne Ueland, Indu Dhar, Jannike Øyen, Jutta Dierkes, Ottar K. Nygård
  • Development of analytical methods GC-MS vs LC-UV for the serum monitoring of an inflammatory glycotoxin (methylglyoxal): a new biomarker of bovine hepatobiliary distomatosis
    Biochimie (IF 3.362) Pub Date : 2019-11-07
    Nadia. Taïbi, Amina. Taïbi, Rachid. Ameraoui, Mohamed. Abou-mustapha, Mohamed. Hadjadj, Zahra-Mouna. Boutaiba, Amel. Kaced, Souhila. Djema, Qosay-Ali. Al-balas, Ghazi-Ahmad. Al jabal, Miriem. Aissi, Khaled. Harhoura, Safia. Zenia, Farida. Khammar

    Two analytical methods; high performance liquid chromatography and gas chromatography were used to determine the content of 2-methylquinoxaline, a methylglyoxal-derived agent in sera from cattle with fascioliasis. Methylglyoxal is a highly mutagenic and cytotoxic reactive dicarbonyl compound formed by non-enzymatic fragmentation of triose phosphate GAP and DHAP during glycolysis which regularly contributes to repositioning the energetic balance between physiological and pathological situations. The aim of this study was to propose the MGO as a new biomarker in the bovine fasciolosis. Strongly infected animals showed a correlation between the relatively high levels of Fasciola hepatica anti-f2 antibody and methylglyoxal compared to unharmed animals. Also, an acute hyperglycemia was recorded and closely related to hepatic parenchyma hyperplasia, inflammation, bile ducts obstruction and scléro-fibrous foci formation.Unlike HPLC, which has shown analytical flaws and irregularities, GC-MS remains an excellent diagnostic tool for detecting and quantifying methylglyoxal in biological fluids. The developed method has been validated under FDA guidelines. A full scan-range was set from m/z 39 to 144/999 and the molecular weight of the 2-methylquinoxaline was identified according to NIST Database and ES. Methylglyoxal was the only analyte successfully quantified in a relatively short run time. It was linear over a concentration range of 0.057 to 5.7μg.ml-1with mean recoveries and RSD of 118% and 3.63% respectively. The intra and inter-day assays were satisfying and not exceed 3.00%. Results reflect the degree of precision of our method and indicate that MGO was an important contributor to understand the hepatic failure independently of other serum markers.

  • Epigenetically modified N6-methyladenine inhibits DNA replication by human DNA Polymerase iota
    Biochimie (IF 3.362) Pub Date : 2019-11-05
    Shuming Zhang, Bianbian Li, Ke Du, Tingting Liang, Mengyuan Dai, Wenxin Huang, Huizhi Zhang, Yihui Ling, Huidong Zhang
  • Meeting report: Seventh International Meeting on Quadruplex Nucleic Acids (Changchun, P.R. China, September 6-9, 2019)
    Biochimie (IF 3.362) Pub Date : 2019-11-05
    Jean-Louis Mergny

    DNA is prone to structural polymorphism: beyond the iconic Watson-Crick double helix, nucleic acids can adopt a number of unusual motifs, at least in vitro. Scientists around the world gather every two years to discuss two of these oddities: G-quadruplexes and i-DNA. The seventh international meeting on G-quadruplex Nucleic Acids was held in Changchun, in Jilin province of the P.R. of China, approx. 1000 km North-east of Beijing. Nearly 320 participants gathered from Asia, Europe, North America and Oceania. More than 80 talks and as many posters summarized our current knowledge of these unusual DNA and RNA structures. During this meeting, the creation of the G4 society was announced, in order to coordinate efforts and share tools and knowledge in our field (https://www.g4-society.org).

  • Biochemical characterization of the cAMP-dependent protein kinase regulatory subunit-like protein from Trypanosoma equiperdum, detection of its inhibitory activity, and identification of potential interacting proteins
    Biochimie (IF 3.362) Pub Date : 2019-11-05
    Nelson A. Araujo, Mónica Rincón, Eva Vonasek, Maritza Calabokis, José Bubis

    An enriched fraction of an inhibitor of both the catalytic subunit of the cAMP-dependent protein kinase (PKA) from pig heart and a Trypanosoma equiperdum PKA catalytic subunit-like protein (TeqC-like) was obtained from the soluble fraction of T. equiperdum parasites after three consecutive purification steps: sedimentation through a linear 5-20% sucrose gradient, diethylaminoethyl-Sepharose anion-exchange chromatography, and Bio-Sil Sec-400-S size-exclusion high-performance liquid chromatography. The inhibitor was identified as the T. equiperdum PKA regulatory subunit-like protein (TeqR-like) on the basis of western blot and mass spectrometry analyses, and behaved as an uncompetitive or anti-competitive inhibitor of the parasite TeqC-like protein, with respect to a fluorescently labeled substrate (kemptide, sequence: LRRASLG), showing a Ki of 1.17 μM. The isolated protein possesses a molecular mass of 57.54 kDa, a Stokes radius of 3.64 nm, and a slightly asymmetric shape with a frictional ratio f/fo = 1.43. As revealed during the purification steps and by immunoprecipitation experiments, the TeqR-like and TeqC-like proteins were not associated forming a heterooligomeric complex, differing from traditional PKA subunits. Co-immunoprecipitation results followed by mass spectrometry sequencing identified two isoforms of the parasite heat-shock protein 70, α-tubulin, and β-tubulin as candidates that interact with the TeqR-like protein in T. equiperdum.

  • Leucine increases mitochondrial metabolism and lipid content without altering insulin signaling in myotubes
    Biochimie (IF 3.362) Pub Date : 2019-11-02
    Madison E. Rivera, Emily S. Lyon, Michele A. Johnson, Roger A. Vaughan

    Elevated circulating branched-chain amino acids (BCAA) such as leucine have been consistently correlated with increasing severity of insulin resistance across numerous populations. BCAA may promote insulin resistance through either mTOR-mediated suppression of insulin receptor substrate-1 or through the accumulation of toxic BCAA catabolites. Although the link between circulating BCAA and insulin resistance has been consistent, it has yet to be concluded if BCAA causally contribute to the development or worsening of insulin resistance. This work investigated the effect of leucine both with and without varying levels of insulin resistance on metabolism, metabolic gene expression, and insulin signaling. C2C12 myotubes were treated with and without varied concentrations of leucine up to 2mM for 24 hours both with and without varied levels of insulin resistance. Gene and protein expression were measured via qRT-PCR and western blot, respectively. Mitochondrial metabolism was measured via O2 consumption. Leucine at 2mM increased oxidative metabolism as well as gene expression of mitochondrial biogenesis, which was associated with increased cellular lipid content. Despite increased lipid content of leucine-treated cells, neither acute nor chronic leucine treatment at 2mM affected insulin signaling in insulin sensitive, mildly insulin resistant, or severely insulin resistant cells. Similarly, leucine at lower concentrations (0.25mM, 0.5mM, and 1mM) did not alter insulin signaling either, regardless of insulin resistance. Leucine appears to improve myotube oxidative metabolism and related metabolic gene expression. And despite increased lipid content of leucine-treated cells, leucine does not appear to alter insulin sensitivity either acutely or chronically, regardless of level of insulin resistance.

  • Therapeutic efficacy of mesenchymal stromal cells and secretome in pulmonary arterial hypertension: a systematic review and meta-analysis
    Biochimie (IF 3.362) Pub Date : 2019-10-31
    Suleiman Alhaji Muhammad, Abdullahi Yahaya Abbas, Yusuf Saidu, Sharida Fakurazi, Lawal Suleiman Bilbis
  • Heat shock response to exercise in pancreatic islets of obese mice
    Biochimie (IF 3.362) Pub Date : 2019-10-31
    Aline Bittencourt, Helena Trevisan Schroeder, Rossana Rosa Porto, Carlos Henrique de Lemos Muller, Mauricio Krause, Paulo Ivo Homem de Bittencourt
  • Fibroblast-secreted trophic factors contribute with ECM remodeling stimulus and upmodulate osteocyte gene markers in osteoblasts
    Biochimie (IF 3.362) Pub Date : 2019-10-30
    Célio Jr. da Costa Fernandes, Willian Fernando Zambuzzi
  • Protective role of adiponectin against testicular impairment in high-fat diet/streptozotocin-induced type 2 diabetic mice
    Biochimie (IF 3.362) Pub Date : 2019-10-30
    Mayank Choubey, Ashutosh Ranjan, Puran S. Bora, Amitabh Krishna

    Type 2 diabetes (T2D) is the most common endocrine and metabolic disorder, leading to reproductive impairments and infertility in male. Our recent study showed crucial role of adiponectin in the regulation of testicular functions, and the circulating level of adiponectin declines in diabetes. The current study thus aimed to examine the efficacy of adiponectin in improving testicular dysfunction in high-fat diet/streptozotocin-induced T2D mice. T2D was induced in pre-pubertal mice fed with high-fat diet for ∼10 weeks followed by single treatment of streptozotocin. T2D mice showed presence of increased body mass, hyperglycemia, hyperinsulinemia, insulin resistance, increased oxidative stress, and declined serum testosterone compared to vehicle-treated control mice. The spermatogenic, steroidogenic, metabolic, and antioxidative parameters were evaluated in T2D mice treated with adiponectin for both two and four weeks. The exogenous administration of adiponectin to T2D mice showed enhanced serum testosterone and expression of testicular steroidogenic markers proteins, insulin receptor and GLUT8 proteins, increase in intra-testicular concentrations of glucose and lactate and activity of LDH and antioxidant enzymes compared to the levels in untreated T2D mice. This suggests that treatment of adiponectin effectively improves testicular functions by increasing expression of insulin receptor-mediated increased transport of energy substrate (glucose and lactate) and a marked reduction in oxidative stress are the possible mechanism by which adiponectin effectively improves testicular function in T2D mice.

  • RNA structure, maturation, interactions and functions.
    Biochimie (IF 3.362) Pub Date : 2019-07-22
    Yuri Motorin,Séverine Massenet

  • Interactions of the Rad51 inhibitor DIDS with human and bovine serum albumins: Optical spectroscopy and isothermal calorimetry approaches.
    Biochimie (IF 3.362) Pub Date : 2019-09-29
    Denis Velic,Cathy Charlier,Milena Popova,Titouan Jaunet-Lahary,Zakaria Bouchouireb,Sébastien Henry,Pierre Weigel,Jean-Yves Masson,Adèle Laurent,Igor Nabiev,Fabrice Fleury

    Rad51 is a key protein in DNA repair by homologous recombination and an important target for development of drugs in cancer therapy. 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) has been used in clinic during the past 30 years as an inhibitor of anion transporters and channels. Recently DIDS has been demonstrated to affect Rad51-mediated homologous pairing and strand exchange, key processes in homologous recombination. Consequently, DIDS has been considered as a potential revertant of radio- and chemo-resistance of cancer cells, the major causes of therapy failure. Here, we have investigated the behavior of DIDS towards serum albumins. The effects of environmental factors, primarily, solvent polarity, on DIDS stability were evaluated, and the mechanisms of interaction of DIDS with human or bovine serum albumin were analyzed using isothermal calorimetry, circular dichroism and fluorescence spectroscopies. DIDS interaction with both serum albumins have been demonstrated, and the interaction characteristics have been determined. By comparing these characteristics for several DIDS derivatives, we have identified the DIDS moiety essential for the interaction. Furthermore, site competition data indicate that human albumin has two DIDS-binding sites: a high-affinity site in the IIIA subdomain and a low-affinity one in the IB subdomain. Molecular docking has revealed the key molecular moieties of DIDS responsible for its interactions in each site and shown that the IB site can bind two ligands. These findings show that binding of DIDS to serum albumin may change the balance between the free and bound DIDS forms, thereby affecting its bioavailability and efficacy against Rad51.

  • Dose-response estrogen promotes osteogenic differentiation via GPR40 (FFAR1) in murine BMMSCs.
    Biochimie (IF 3.362) Pub Date : 2015-01-13
    Bo Gao,Qiang Huang,Qiang Jie,Long Wang,Hong-Yang Zhang,Jian Liu,Liu Yang,Zhuo-Jing Luo

    Estrogen plays an essential role in bone formation, and estrogen modulation dysfunction is tightly associated with postmenopausal osteoporosis (PMOP). The underlying mechanisms of estrogen-mediated osteogenic differentiation have not been well defined. In this study, murine bone marrow mesenchymal stem cells were induced to undergo osteogenic differentiation, and gene expression analysis or GPR40 expression manipulation was performed. Bilateral ovariectomized or sham-operated C57BL/6 mice were administered GPR40 agonist (GW9508) for bone mineral density analysis. We identified GPR40, a long chain unsaturated fatty acid receptor, to be regulated by estrogen and involved in osteogenic differentiation both in vivo and in vitro. Mechanistically, the Wnt/β-catenin signaling pathway is essential for GPR40 to promote osteogenic differentiation. Furthermore, in vivo GW9508 administration rescued estrogen-deficient bone loss, indicating the essential role of the GPR40 receptor. To our knowledge, this is the first study that provides evidence for GPR40 as a positive regulator of osteogenesis and Wnt/β-catenin signaling. These results indicate that GPR40 may function as an endogenous promoter of estrogen-induced osteogenic differentiation through Wnt/β-catenin signaling activation. Therefore, as the global population of ages and the prevalence of metabolic-related disorders, especially PMOP, increases, our findings suggest that GPR40 is a key in understanding the link between bone and fat. It may also be a useful target for the treatment of bone complications in the future.

  • Caveolin and cavin family members: dual roles in cancer.
    Biochimie (IF 3.362) Pub Date : 2014-09-23
    Reshu Gupta,Chirine Toufaily,Borhane Annabi

    Caveolae are specialized plasma membrane subdomains with distinct lipid and protein compositions, which play an essential role in cell physiology through regulation of trafficking and signaling functions. The structure and functions of caveolae have been shown to require the proteins caveolins. Recently, members of the cavin protein family were found to be required, in concert with caveolins, for the formation and function of caveolae. Caveolins have a paradoxical role in the development of cancer formation. They have been involved in both tumor suppression and oncogenesis, depending on tumor type and progress stage. High expression of caveolins and cavins leads to inhibition of cancer-related pathways, such as growth factor signaling pathways. However, certain cancer cells that express caveolins and cavins have been shown to be more aggressive and metastatic because of their increased potential for anchorage-independent growth. Here, we will survey the functional roles of caveolins and of different cavin family members in cancer regulation.

  • Regulation of CIRL-1 proteolysis and trafficking.
    Biochimie (IF 3.362) Pub Date : 2010-01-27
    Igor E Deyev,Alexander G Petrenko

    Calcium-independent receptor of alpha-latrotoxin (CIRL-1) is an adhesion G protein-coupled receptor implicated in the regulation of exocytosis. CIRL-1 biosynthesis involves constitutive proteolytic processing that takes place in the endoplasmic reticulum, requires the receptor's GPS domain, and yields heterologous two-subunit receptor complexes. It was proposed that the GPS-directed cleavage is based on cis-autoproteolysis. In this study, we demonstrate that activators of protein kinase C - PMA and ionomycin, can inhibit the cleavage of CIRL-1 precursor in transfected cells. Both reagents also downregulate trafficking of CIRL-1 to the cell surface that results in accumulation of the uncleaved receptor precursor inside the cells. Experiments with a non-cleavable soluble mutant of CIRL-1 showed that the downregulation of the receptor trafficking is independent of its cleavage. Our data suggest that the GPS proteolysis of CIRL-1 is not a purely autocatalytic process and may involve auxiliary proteins or factors that become available in the course of CIRL-1 trafficking.

  • 更新日期:2019-11-01
  • Internally quenched fluorogenic substrates with unnatural amino acids for cathepsin G investigation.
    Biochimie (IF 3.362) Pub Date : 2019-05-20
    Katarzyna Groborz,Sonia Kołt,Paulina Kasperkiewicz,Marcin Drag

    Cathepsin G is one of four members of the neutrophil serine protease family and constitutes an important biological target in various human inflammatory diseases, such as chronic obstructive pulmonary disease, acute respiratory distress syndrome and cystic fibrosis. Many studies have been focused on determining its biological roles, the latest ones concerning its involvement in acute myeloid leukemia, and as such, multiple chemical and biochemical tools were developed to investigate cathepsin G. Nevertheless, most of them lack selectivity or sensitivity and therefore cannot be used in complex systems. Here we present the development of an optimal cathepsin G Internally Quenched Fluorescence (IQF) substrate that incorporates unnatural amino acids causing the increase of its selectivity toward neutrophil elastase and potency in in vitro studies.

  • Structural basis for the NAD binding cooperativity and catalytic characteristics of sperm-specific glyceraldehyde-3-phosphate dehydrogenase.
    Biochimie (IF 3.362) Pub Date : 2015-05-06
    M L Kuravsky,K V Barinova,R A Asryants,E V Schmalhausen,V I Muronetz

    Catalytic properties of enzymes used in biotechnology can be improved by eliminating those regulatory mechanisms that are not absolutely required for their functioning. We exploited mammalian glyceraldehyde-3-phosphate dehydrogenase as a model protein and examined the structural basis of the NAD(+) cooperative binding exhibited by its homologous isoenzymes: the somatic enzyme (GAPD) and the recombinant sperm-specific enzyme (dN-GAPDS). Moreover, we obtained a mutant dN-GAPDS, which misses the cooperativity, but exhibits a twofold increase in the specific activity instead (92 and 45 μmol NADH/min per mg protein for the mutant and the wild type proteins, respectively). Such an effect was caused by the disruption of the interdomain salt bridge D311-H124, which is located close to the active site of the enzyme. The thermal stability of the mutant protein also increased compared to the wild type form (heat absorption peak values were 70.4 and 68.6 °C, respectively). We expect our findings to be of importance for the purposes of biotechnological applications.

  • Hepcidin, the iron watcher.
    Biochimie (IF 3.362) Pub Date : 2009-06-27
    Lydie Viatte,Sophie Vaulont

    Hepcidin, a peptide hormone produced by the liver, constitutes the master regulator of iron homeostasis in mammals allowing iron adaptation according to the body iron needs. In recent years there has been important breakthrough in our knowledge of hepcidin regulation that has also implications for understanding the physiopathology of human iron disorders. Different aspects of hepcidin regulation will be considered in this review, including regulation by the iron status and the BMP6/HJV/SMAD pathway. Hepcidin dysregulation in iron disorders will be also discussed. Although much can already be accomplished for treating iron disorders using the knowledge that has currently been developed, additional issues will be challenging for the coming years.

  • Novel chondrogenic and chondroprotective effects of the natural compound harmine.
    Biochimie (IF 3.362) Pub Date : 2012-11-03
    Emilio Satoshi Hara,Mitsuaki Ono,Satoshi Kubota,Wataru Sonoyama,Yasutaka Oida,Takako Hattori,Takashi Nishida,Takayuki Furumatsu,Toshifumi Ozaki,Masaharu Takigawa,Takuo Kuboki

    A significant number of natural compounds have been shown to regulate the behavior of the cells, in collaboration with cellular proteins. CCN2/connective tissue growth factor (CTGF) has been reported to have essential roles in cartilage development, chondrocyte proliferation and differentiation as well as regulation of the extracellular matrix metabolism. Previous studies demonstrated the capability of CCN2 to regenerate surgical defects in articular cartilage of rat knee. Also, transgenic mice over-expressing cartilage-specific CCN2 were shown to be more resistant to aging-related cartilage degradation. We hypothesized that small molecules that induce CCN2 in chondrocytes could be novel candidates to increase the resistance to aging-related cartilage degradation, or even to correct cartilage degenerative changes incurred in OA. Therefore, this study screened a compound library and identified the β-carboline alkaloid harmine as a novel inducer of CCN2 in human chondrocytic HCS-2/8 cells and osteoarthritic articular chondrocytes. Harmine increased the expression of the cartilage markers aggrecan and COL2α1, as well as that of the master regulator of chondrogenesis, SOX-9. Moreover, harmine notably induced chondrogenesis of prechondrocytic ATDC5 cells in micromass cultures. The chondroprotective effect of harmine was investigated under inflammatory condition by stimulation with TNFα, and harmine was shown to ameliorate TNFα-induced decrease in expression of CCN2 and cartilage markers. These findings uncover novel chondrogenic effects of harmine and indicate harmine as a potential drug for prevention and/or repair of cartilage degradation.

  • Design and utility of CCN2 anchor peptide aptamers.
    Biochimie (IF 3.362) Pub Date : 2010-05-04
    Harumi Kawaki,Satoshi Kubota,Eriko Aoyama,Naoya Fujita,Hiroshi Hanagata,Akira Miyauchi,Kenta Nakai,Masaharu Takigawa

    CCN family protein 2/connective tissue growth factor (CCN2/CTGF) consists of 4 conserved modules that are highly interactive with a number of biomolecules. With such interaction, CCN2 exerts multiple functions by forming an extracellular information network. In the present study, we screened for dodecapeptide sequences that bound to each module of human CCN2 by using a bacteriophage display library. Thereafter, consensus amino acid sequences for the binding to individual modules were extracted in silico and utilized to design anchor peptide aptamers that would facilitate the interaction between CCN2 and other molecules. Direct binding of a few peptides to CCN2 was confirmed by surface plasmon resonance analysis. Subsequent biological assay indicated that one such peptide was capable of promoting the proliferation of CCN2-producing chondrocytic cells. This cell biological activity was found to be sequence specific and CCN2 dependent. Since CCN2/CTGF was shown to be effective in articular cartilage/bone regeneration in vivo, utility of such peptide aptamers in CCN2-associated regenerative therapeutics is suggested herein.

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上海纽约大学William Glover