Biochimie最新文献

L-asparaginase is a critical therapeutic enzyme for treating acute lymphoblastic leukemia (ALL), a common childhood malignancy. In this study, the L-asparaginase coding sequence from halophilic Vibrio sp. (GBPx3) was cloned, expressed in Escherichia coli, and characterized. The enzyme exhibited a molecular weight of 39.2 kDa and demonstrated a K_m of 4.517 mM, k_cat of 2.88 1/s, and V_max of 0.1055 μmol/min, reflecting high specificity for L-asparagine and minimal activity (0.4%) toward L-glutamine. Optimal activity was observed at physiological conditions (37°C, pH 7.5 and 125-150 mM NaCl), consistent with human serum osmolality. The half-life of the enzyme was 2.64 hours in human serum at 37°C that is longer than the half-life reported for E. coli L-asparaginase. Additionally, the enzyme had no toxic impact on human umbilical vein endothelial cells (HUVEC) and human erythrocytes. The recombinant L-asparaginase was predicted to be 29.3% helix, 35.6% turns, and 35.1% random by circular dichroism spectroscopy. AlphaFold predicted a 3D structure with promising validation scores. The molecular docking study showed that Thr14, Ser60, Thr91, and Asp92 are putative active site residues, with a negative binding energy of -4.5 kJ/mol for the substrate-enzyme interaction. The enzyme's low immunogenicity, high serum stability, and reduced glutaminase activity highlight its potential as a safer therapeutic alternative. Future experiments and protein engineering studies are needed to explore enzyme's in vivo efficacy and improve its clinical effectiveness.

Metformin, initially prescribed as an oral hypoglycemic medication for type 2 diabetes, has recently gained attention for its potential anticancer effects. Its history dates to 1918, when guanidine, a component of the traditional European herb Galega officinalis, was found to reduce glycemia. This review precisely examines the mechanisms underlying Metformin's anticancer effects across various neoplastic conditions. This investigation explores the complex interactions between metformin and major signaling pathways associated with carcinogenesis, including AMP-activated protein kinase (AMPK), mTOR, and insulin-like growth factor (IGF) pathways. The review emphasizes Metformin's diverse effects on angiogenesis, inflammation, apoptosis, and cellular metabolism in cancer cells. Additionally, new data on metformin's capacity to alter the tumor microenvironment and enhance immune surveillance systems against cancer are examined. The review underscores Metformin's potential for repurposing in oncology, emphasizing its clinical relevance as an adjuvant therapy for various cancers. The review provides insightful information about the complex anticancer mechanisms of metformin by combining data from preclinical and clinical studies. These findings not only broaden our knowledge of the effects of metformin but also open new avenues for oncology research and treatment developments.

Oleanolic acid (OA) and hederagenin from Aralia elata have important medicinal value due to their anti-cancer and anti-inflammatory activities. Transcription factors (TFs) can affect the production of active substances by regulating the expression of genes in metabolic pathways. In this study, a transcription factor AeWRKY2 was cloned and overexpressed in A. elata. The result of gene expression analysis and high-performance liquid chromatography (HPLC) showed that the key enzyme genes of all transgenic strains analyzed were up-regulated to varying degrees. The contents of oleanolic acid and hederagenin were higher than those of wild type, up to 2.773 mg·g^-1 DW and 0.325 mg·g^-1 DW, respectively. After one-day MeJA treatment, the contents of oleanolic acid and hederagenin were further increased to 3.31 mg·g^-1 and 0.37 mg·g^-1, respectively.

Skeletal stem cells (SSCs) reside in various niche locations within long bones to maintain bone homeostasis and facilitate fracture repair. Bone fragility, associated with ageing, increases the susceptibility of the femoral head to fractures due to an increase in bone adipocytes and concomitant loss of structural integrity. However, the specific contribution of epiphyseal SSCs to fragility is unknown. To explore this, a comparative analysis was performed on the transcriptional profiles and lineage commitment of Wistar rat femoral SSCs derived from the bone marrow (BM-), diaphyseal cortical bone (CB-) and proximal epiphyseal trabecular bone (PF-SSCs) isolated from the same long bones. SSCs were characterized based on morphology, immunophenotype (CD90/CD45), growth rate (population doubling time), gene expression profiles and differentiation capacity (Oil Red O, Alizarin Red S). qRT-PCR micro-arrays were performed on SSCs to evaluate the expression of stemness, SSC and lineage-specific markers in both undifferentiated and differentiated states. Our findings support the hypothesis that SSCs from different bone regions exhibit distinct transcriptional profiles, reflecting their specific niche environments. CB-SSCs displayed superior osteogenic potential as evidenced by the expression of key osteogenic genes and higher levels of mineralization. In contrast, PF-SSCs had a reduced osteogenic capacity with a higher adipogenic potential. Overall, the study revealed the importance of niche-specific stem cell properties for use in regenerative medicine applications and provides insight into the potential role of PF-SSCs in bone fragility and fracture risk.

In humans, the release factor eRF3a exists in several forms that differ in the length of the polyglycine tract (7, 10, 11 or 12 glycines) in its N-terminal domain. For the 12-Gly eRF3a, an association with cancer risk and a decreased affinity for the cytoplasmic poly (A) binding protein have already been established. In this work, HCT116 colon cancer cells were treated with low doses of doxorubicin, which is known to induce senescence in these cells with high efficiency. The expression of p21 and p53 (senescence marker proteins) as well as lysosomal β-galactosidase activity were reduced when 12-Gly-eRF3a was overexpressed or eRF3a was depleted in cells. If low activity of mTORC1 pathway might be responsible for reduced senescence onset after eRF3a depletion, its activity is maintained in cells overexpressing 12-Gly-eRF3a. In both cases, a defect in termination efficiency could be involved.

This study was undertaken to show whether chromatin phenotype(s) related to histone H1 polymorphism(s) exhibit connection with breeding practice (selection for reduction of body mass) and/or physiological trait (withdraw of the food) of the organism. For this purpose, a genetic diversity between quail lines selected for high and low reduction of body mass after transient food withdrawal was examined using variation in the histone H1 allelic expression. Symmetric and asymmetric expression of the isoforms of histone H1.b and histone H1.d was identified in 2-D PAGE due to various molecular weight and different intensities, respectively. Low (locus H1.d) to moderate (locus H1.b) differentiated populations conform to Hardy-Weinberg equilibrium but distribution of the phenotypes significantly differ between them. Whereas frequency of the same phenotype of histone H1.b is different between selected lines, the frequency of the same phenotypes of histone H1.d is similar in both selected lines. Thus, line-specific promotion of the phenotypes of histone H1.b suggest a response to conducted selection. Unlike this, a line-nonspecific arrangement of the phenotypes of histone H1.d appear due to the stress evoked by food withdrawal ongoing during the selection. Such a result indicate that histone H1 allelic variants possess individual impact on chromatin states and/or processes associated with breeding practices and physiological conditions of the organism. According to this, functional individualization is a characteristic feature of histone H1 polymorphic variants.

The microbial conversion of selenite represents an effective detoxification and assimilation process, although the underlying mechanisms remain incompletely understood. In this study, strain BB61 was a probiotic isolated from piglet feces and identified as Bacillus amyloliquefaciens, which could almost completely reduce 0.1 g/L Na₂SeO₃ to SeNPs within 48h. We investigated the potential mechanisms of selenite reduction in this strain through transcriptome sequencing and qPCR. The transcriptome analysis revealed the up-regulation of 829 genes and the down-regulation of 892 genes in response to 1 g/L Se treatment (padj <0.05) in Bacillus amyloliquefaciens BB61. GO (Gene Ontology) enrichment analysis indicated that DEGs (Differentially expressed genes) were predominantly associated with transmembrane transporters, ion transmembrane transport, cytoplasmic and cell membrane composition, cell movement and localization, and carbon metabolism. Additionally, the KEGG (Encyclopedia of Genes and Genomes) pathway annotation analysis revealed that the DEGs were primarily involved in the pentose phosphate pathway, pyruvate metabolism, pyrimidine metabolism, cofactor biosynthesis, and other pathways (P < 0.05). Among the highly expressed reductases, thioredoxin reductase (TrxA/B), nitrite reductase (NfsA), and selenite reductase (NamA) were all found to be up-regulated. Consequently, this study established a reduction pathway model for Se (IV), offering new insights into the molecular mechanisms underlying the bioreduction of selenite to form SeNPs.

Alternative oxidase (AOX) (EC 1.10.3.11) is a terminal oxidase in the mitochondrial inner membrane that branches the canonical electron transport system (ETS). AOX is ubiquitous in plants, frequently found in fungi and protists and presents a more sporadic distribution in metazoans. More recently, AOX has gained attention due to its potential application in gene therapy for treatment of mitochondrial diseases. Here we characterized the AOX in the basal Dipteran, Pseudolycoriella hygida using a combination of genomic analyses, molecular, functional and in vivo survival assays. AOX is a single copy gene that encodes three developmental stage specific protein isoforms. AOX localizes to the mitochondria in adult thoracic muscles, which present cyanide-resistant respiration that is sensitive to the AOX inhibitor salicylhydroxamic acid (SHAM). Both the cyanide-resistant respiration and AOX levels gradually increase during aging, but are not influenced by thermal stress. Thoracic mitochondria respire using substrates derived from several metabolic routes, such as pyruvate, proline, acylcarnitine, NADH and glycerol-3P, and present values of oxidative phosphorylation capacity ((P-L)/E = 0.70) and coupling (P/L = 4.35; L/E = 0.21). Adult flies exhibit a high survival resistance for SHAM-sensitive complex III inhibition. Together, our results demonstrate the presence of a functional AOX in a terrestrial arthropod and provide insights regarding AOX function in animals and evolution of respiratory systems in metazoans. Psl. hygida emerges as a natural and valuable model for comprehensive AOX research at the whole-organism level which complements models expressing the heterologous enzyme.

Helicases are enzymes involved in all aspects of nucleic acid synthesis, regulation and degradation. As a consequence, several methods were developed to monitor their enzymatic activity. In this report, we described an improvement of bulk fluorescent helicase assays to overcome their specific limitations (cost, health and safety regulations, etc.). Using a real time detection thermocycler to monitor the fluorescence in real-time, we managed to precisely control the initiation of the helicase reaction through temperature tuning. Therefore, we were able to demonstrate that this setup could provide a qualitative and a quantitative evaluation of the helicase domain of the UPF1 helicase (UPF1-HD) and that several fluorophores could be used in parallel during the same run.

Gram-negative bacteria rely on pili assembly for pathogenicity, with the chaperone-usher (CU) pathway regulating pilus biogenesis. Nitazoxanide (NTZ) inhibits CU pathway-mediated P pilus biogenesis by specifically interfering with the proper folding of the outer membrane protein (OMP) usher, primarily mediated by the β-barrel assembly machinery (BAM) complex. In this study, we identified the BAM complex components BamB and the BamA POTRA2 domain as key binding targets for NTZ. Molecular dynamics simulations and Bio-Layer Interferometry revealed that BamB residues S61 and R195 are critical for NTZ binding. NTZ activated the Cpx two-component system and induced inner membrane perturbations, which resulted from the accumulation of misfolded P pilus subunits. Upregulation of the ibpAB gene, which protects the bacteria against NTZ-induced oxidative stress, was also observed. Importantly, NTZ combined with polymyxin B enhanced the latter's antibacterial activity against both susceptible and MCR-positive E. coli strains. This enhancement was achieved through NTZ-induced increases in inner membrane permeability, oxidative stress, and inhibition of efflux pump activity and biofilm formation. This study provides new insights into the antimicrobial mechanism of NTZ and highlights its potential as an antibiotic adjuvant by targeting BamB to inhibit the CU pathway, restoring the efficacy of polymyxin B against multidrug-resistant bacteria.