70-kDa Protein Research Articles

Homology Modelling and Molecular Docking of Some Natural Compounds as Inhibitors of Anopheles gambiae Heat Shock Protein 70KDa and Bifunctional Glutamyl/prolyl-tRNA Synthetase

Homology Modelling and Molecular Docking of Some Natural Compounds as Inhibitors of Anopheles gambiae Heat Shock Protein 70KDa and Bifunctional Glutamyl/prolyl-tRNA Synthetase

Prolactin: structure, receptors, and functions.

Prolactin (PRL) is a 23-kDa protein synthesized and secreted by lactotroph cells of the anterior pituitary gland but also by other peripheral tissues. PRL binds directly to a unique transmembrane receptor (PRLR), and the JAK2/signal transducer and activator of transcription 5 (Stat5) pathway is considered the major downstream pathway for PRLR signaling. To a lesser extent, PRL may be cleaved into the shorter 16-kDa PRL, also called vasoinhibin, whose signaling is not fully known. According to rodent models of PRL signaling inactivation and the identification of human genetic alterations in PRL signaling, a growing number of biological processes are partly mediated by PRL or its downstream effectors. In this review, we focused on PRL structure and signaling and its canonical function in reproduction. In addition to regulating reproductive functions, PRL also plays a role in behavior, notably in initiating nurturing and maternal behavior. We also included recent insights into PRL function in several fields, including migraines, metabolic homeostasis, inflammatory and autoimmune disease, and cancer. Despite the complexity of understanding the many functions of PRL, new research in this field offers interesting perspectives on physiological and pathophysiological processes.

Recombinant expression, downstream optimization, and therapeutic evaluation of recombinant human interleukin-37 for cancer therapy.

Interleukin-37 is a cytokine with potent immunosuppressive properties that has been shown to have potential to treat autoimmune and chronic inflammatory diseases, as well as certain types of cancer. IL-37 is a 19kDa protein which interacts with proteins in receptor-dependent and receptor-independent pathways. The expression of the IL-37 protein cloned into the pET-28a vector was optimized in Rosetta 2(DE3) after comparing its expression with Rosetta-gami 2(DE3) and Rosetta 2(DE3) pLysS, which was then used for the large-scale production of IL-37. IMAC purification of IL-37 yielded > 97% pure 0.9mg/mL protein from auto-induced fermentation. The IC50 value of IL-37 was < 1µM, which was similar to that of doxorubicin, and proliferation of > 80% of all cancer cells was inhibited by 100µg/mL of IL-37 protein. IL-37 may be a promising theragnostic target for cancer due to its comparable IC50 value with that of doxorubicin.

One-step nanoscale expansion microscopy reveals individual protein shapes.

The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.

Allelic Diversity and Development of Breeder-Friendly Marker Specific to floury2 Gene Regulating the Accumulation of α-Zeins and Essential Amino Acids in Maize Kernel.

Maize zeins lack essential amino acids, such as methionine, lysine, and tryptophan. The floury2 (fl2) mutation reduces zein synthesis and increases methionine and lysine content in kernels. In this study, fl2 gene (1612bp) was sequenced in eight wild-type and two mutant inbreds and detected 218 SNPs and 18 InDels. Transversion of C to T at 343bp position caused the substitution of alanine by valine in the fl2 mutant. A PCR-based marker (FL-SNP-CT) was developed, which distinguished the favorable mutant fl2 allele (T) from the wild-type (C) Fl2 allele. Gene-based diversity analysis using seven gene-based InDel markers grouped 48 inbred lines into three major clusters, with an average genetic dissimilarity coefficient of 0.534. The average major allele frequency, gene diversity, heterozygosity, and polymorphism information content of the InDel markers were 0.701, 0.392, 0.039, and 0.318, respectively. Haplotype analysis revealed 29 haplotypes of fl2 gene among these 48 inbreds. Amino acid substitution (Ala-Val) at the signal peptide cleavage site produced unprocessed 24-kDa mutant protein instead of 22-kDa zein found in normal genotype. Eight paralogues of fl2 detected in the study showed variation in exon lengths (616-1170bp) and translation lengths (135-267 amino acids). Orthologue analysis among 15 accessions of Sorghum bicolor and two accessions of Saccharum spontaneum revealed a single exon in fl2 gene, ranging from 267 to 810bp. The study elucidated the molecular basis of fl2 mutation and reported a breeder-friendly marker for molecular breeding programs. This is the first study to characterize fl2 gene in a set of subtropically adapted inbreds.

Endoplasmic reticulum stress and expression of nitric oxide synthases in heart failure with preserved and with reduced ejection fraction - pilot study.

Unfolded Protein Response (UPR), endoplasmic reticulum (ER) stress, and inducible nitric oxide synthase (iNOS) overexpression have been found to influence heart failure with preserved ejection fraction (HFpEF) pathogenesis. Their importance in heart failure with reduced ejection fraction (HFrEF) is not entirely established; there is little data involving a detailed comparison between HFpEF and HFrEF from this perspective. This pilot study aimed to compare circulating levels of Glucose-regulated protein 78kDa (GRP78) (ER - stress marker) and all NOS isoforms between both HFpEF and HFrEF and to analyze the correlation between these markers and the clinical characteristics of the patients. Forty-two patients with HFpEF and thirty-eight with HFrEF were involved in this study. Clinical characteristics and echocardiographic data were obtained. Basic laboratory tests were performed and ELISA tests for iNOS, endothelial NOS (eNOS), neuronal NOS (nNOS), and GRP78. Patients with HFpEF had lower circulating levels of GRP78 and higher iNOS concentrations when compared to HFrEF patients (P = 0.023, P < 0.0001, accordingly). The subgroup of the HFpEF population with eGFR < 60 mL/min/1.73m2 had higher nNOS and eNOS levels than HFpEF patients with normal GFR (P = 0.049, P = 0.035, respectively). In the HFrEF subgroup, patients with coexistent diabetes mellitus had elevated concentrations of nNOS compared to the subpopulation without diabetes mellitus (P = 0.041). There was a positive correlation between eNOS and nNOS concentrations (ρ = 0.86, P < 0.0001). In HFpEF, there is a more intensified iNOS overexpression, while in HFrEF, ER stress is more prominent.

A-106 Determination of Urinary NGAL Reference Intervals from Specimens of Healthy Adult and Pediatric Individuals

Abstract Background Neutrophil gelatinase-associated lipocalin (NGAL) is a 25kDa protein implicated in multiple biological processes, including attenuation of apoptosis and differentiation of renal tubule epithelial cells and nephrons. NGAL is also produced and secreted by injured kidney tubule epithelial cells. As such, NGAL can serve as an early urinary biomarker for acute kidney injury (AKI). This multi-site, cross-sectional study aimed to establish reference intervals for urinary NGAL (uNGAL) in healthy pediatric and adult populations using a particle-enhanced turbidimetric assay. Methods Apparently healthy individuals, aged ≥ 3 months, were eligible for this study. Subjects with an active urinary tract infection (UTI) on the day of sample collection, acute kidney injury (AKI) or a history of AKI, stage 4 or 5 chronic kidney disease (CKD), known congenital anomalies of the kidney and urinary tract, known urothelial, urological, or kidney malignancies were excluded from the study. Subjects with uncorrected congenital heart disease, having undergone solid organ or bone marrow transplantation, receiving renal replacement therapy, and having undergone surgery (urologic, nephrectomy, or correction of congenital heart disease) were also excluded. A total of 688 subjects were screened, 677 were eligible, and 629 (91.4%) were considered evaluable per the protocol. The 59 excluded subjects were ineligible due to missing/incorrect information in the informed consent form, or not evaluable due to meeting exclusion criteria, mostly a positive UTI test. Urine samples were tested for NGAL on a Roche cobas® c501 analyzer in single determinations. Results Tables 1 and 2 describe the NGAL summary statistics and central 95% reference intervals by age and gender. Conclusions These data demonstrate the normal urine NGAL values in an apparently healthy pediatric and adult populations.

Serosurveillance for Plasmodium falciparum Malaria in Peruvian Army Peacekeeping Personnel, Central African Republic, 2021-2022.

Plasmodium falciparum infection threatens military populations deployed to highly malaria-endemic regions, such as Peruvian Army peacekeepers deployed to Central African Republic. During deployment, malaria cases were identified by microscopy and rapid diagnostic tests. After deployment, we performed malaria diagnosis by malachite green loop-mediated isothermal amplification and photo-induced electron transfer PCR assays. We used ELISA to test for P. falciparum C-terminal 19-kDa region merozoite surface protein 1-specific IgG from 97 peacekeepers. Malaria prevalence during deployment was 33.33% and we detected 4 cases after deployment: P. falciparum (n = 2), P. ovale (n = 1), and Plasmodium spp. (n = 1). IgG surveillance showed a seroprevalence of 31.96% in peacekeepers, who had a high P. falciparum exposure during deployment. Our findings reinforce the necessity of active surveillance in military populations to reduce the risk for introduction of new Plasmodium species and strains into the Americas from malaria-endemic areas.

Microglia-mediated neuroimmune suppression in PTSD is associated with anhedonia

Dynamic brain immune function in individuals with posttraumatic stress disorder is rarely studied, despite evidence of peripheral immune dysfunction. Positron emission tomography brain imaging using the radiotracer [11C]PBR28 was used to measure the 18-kDa translocator protein (TSPO), a microglial marker, at baseline and 3 h after administration of lipopolysaccharide (LPS), a potent immune activator. Data were acquired in 15 individuals with PTSD and 15 age-matched controls. The PTSD group exhibited a significantly lower magnitude LPS-induced increase in TSPO availability in an a priori prefrontal-limbic circuit compared to controls. Greater anhedonic symptoms in the PTSD group were associated with a more suppressed neuroimmune response. In addition, while a reduced granulocyte-macrophage colony-stimulating factor response to LPS was observed in the PTSD group, other measured cytokine responses and self-reported sickness symptoms did not differ between groups; these findings highlight group differences in central-peripheral immune system relationships. The results of this study provide evidence of a suppressed microglia-mediated neuroimmune response to a direct immune system insult in individuals with PTSD that is associated with the severity of symptoms. They also provide further support to an emerging literature challenging traditional concepts of microglial and immune function in psychiatric disease.

Multi-omics integrated analysis reveals the physiological response of large yellow croaker (Larimichthys crocea) to starvation and refeeding during overwintering

This study aimed to investigate the physiological response to starvation and refeeding during the overwintering period of large yellow croaker (Larimichthys crocea) using physiological and biochemical assays, microbiomics and transcriptomics. Fish were sampled at the beginning of starvation (Fa0 group), after 8 weeks (Fa8 group), after 16 weeks (Fa16 group), and after 8 weeks of refeeding (Fe group). Results indicated that compared to the Fa0 group, the viscerosomatic index (VSI) and intestinal digestive enzyme activities (trypsin, lipase, and amylase) significantly decreased in the Fa8 and Fa16 groups (P < 0.05), but these indicators recovered in the Fe group. Prolonged starvation significantly reduced triglyceride (TG) levels in both serum and liver, but these levels rebounded after 8 weeks of refeeding. Chronic starvation also led to a notable increase in serum low-density lipoprotein (LDL) levels, accompanied by a significant decrease in liver LDL content. As the duration of starvation increasing, the antioxidant and immune capabilities in the serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD), lysozyme (LZM), alkaline phosphatase (AKP), acid phosphatase (ACP) significantly enhanced (P < 0.05), and these indicators significantly declined in the Fe group (P < 0.05). Intestinal flora analysis showed that the Alpha diversity gradually decreased with the prolongation of starvation but recovered after 8 weeks of refeeding, with the abundance of Akkermansia muciniphila significantly increasing (P < 0.05) in the Fa16 group compared to the other groups. Liver transcriptomic analysis revealed that upregulated genes enriched in pathways such as "Ribosome biogenesis in eukaryotes", "Proteasome", "RNA transport", "Biosynthesis of unsaturated fatty acids", "Fatty acid metabolism", "Fatty acid degradation", and "PPAR signaling pathway" (P < 0.05), while downregulated genes significantly enriched in "metabolic pathways" (P < 0.05) in the Fa16 group. Genes related to immune responses were significantly downregulated (P < 0.05) in the Fe group. The correlation analysis of intestinal flora and transcriptome revealed LOC104925398 (complement C3-like) and LOC113745439 (heat shock 70 kDa protein) exhibited significant positive correlations with Levilactobacillus brevis, unclassified Lachnoclostridium, Escherichia coli, and Akkermansia muciniphila (P < 0.05). In conclusion, prolonged starvation was more advantageous to the safe overwintering of large yellow croaker. Our study revealed the physiological response of large yellow croaker to starvation and refeeding during the overwintering period, providing a theoretical foundation for the formulation of safe overwintering strategies.

Unveiling the role of protein kinase A (PKA) activity in bovine oviductal epithelial cells: implications on apoptotic signaling pathways during the estrous cycle.

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.

Excreted/Secreted 15-kDa Proteins and Serine Protease Peptides from Haemonchus contortus Act as Immune-Response Enhancers in Lambs.

This study assessed the immunoprotective effect in lambs of a native excreted/secreted 15-kDa protein and two synthesised S28 peptides derived from the infective transitory larvae (xL3) and adult stages (AS) of Haemonchus contortus. Twenty-two Pelibuey lambs were divided into negative and positive control groups, as well as immunised lamb groups, with 100 µg of the 15-kDa native protein (15kDaNP) and S28 peptides (S28P). The eggs per gram (EPG) and haematocrit were measured, and AS were counted and morphologically measured. To assess the immunoprotection in lambs, indirect enzyme-linked immunosorbent assays and relative expression analyses of immune cytokines were performed using serum and abomasal samples. Our results showed a 72.28% reduction in adult worms (AW) in the 15kDaNP-immunised group, achieving a high clinical response with 41% haematocrit and low EPG values (436 ± 661). Conversely, the S28P group achieved the highest IgG levels (2.125 ± 0.880 OD), with AW exhibiting the greatest body length (p > 0.05) and upregulation of the IL5 and FCεR1A genes associated with nematode control. The 15kDaNP group showed increased expression of genes related to nematode control and anti-inflammatory responses, including IL4, IL5, IL6, and IL13 (p < 0.05). The S28P and 15kDaNP should be explored as potential vaccines against sheep haemonchosis.

The cytoplasmic tail of myelin protein zero induces morphological changes in lipid membranes

The major myelin protein expressed by the peripheral nervous system Schwann cells is protein zero (P0), which represents 50% of the total protein content in myelin. This 30-kDa integral membrane protein consists of an immunoglobulin (Ig)-like domain, a transmembrane helix, and a 69-residue C-terminal cytoplasmic tail (P0ct). The basic residues in P0ct contribute to the tight packing of myelin lipid bilayers, and alterations in the tail affect how P0 functions as an adhesion molecule necessary for the stability of compact myelin. Several neurodegenerative neuropathies are related to P0, including the more common Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS) as well as rare cases of motor and sensory polyneuropathy. We found that high P0ct concentrations affected the membrane properties of bicelles and induced a lamellar-to-inverted hexagonal phase transition, which caused bicelles to fuse into long, protein-containing filament-like structures. These structures likely reflect the formation of semicrystalline lipid domains with potential relevance for myelination. Not only is P0ct important for stacking lipid membranes, but time-lapse fluorescence microscopy also shows that it might affect membrane properties during myelination. We further describe recombinant production and low-resolution structural characterization of full-length human P0. Our findings shed light on P0ct effects on membrane properties, and with the successful purification of full-length P0, we have new tools to study the role of P0 in myelin formation and maintenance in vitro.

Role of Lipocalin-2 Levels in Non-Alcoholic Fatty Pancreatic Disease and its Association with Non-Alcoholic Fatty Liver Disease

Abstract Background Lipocalin-2 (LCN2) is a 25-kDa secretory protein with great importance as a sensitive and specific screening and diagnostic biomarker for hepatic inflammation, acute hepatic injury, hepatic cancer, and radiation-induced early phase hepatic damage and lipid abnormalities. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis as well as its actual role in the event of inflammation. Aim and Objectives To establish a correlation between the levels of lipocalin as a useful biomarker with increasing severity of steatosis in NAFLD and NAFPD patients. Subjects and Methods This was observational prospective case–control study, was conducted at Hepatology Unit of Ain Shams University Hospital on 50 patients diagnosed with NAFPD and 50 healthy age and sex-matched volunteers without NAFPD during a period of 6 months. All patients were subjected to; Full history taking and thorough clinical examination, Anthropometric measurements, Laboratory investigations, and abdominal ultrasonography. Result There was a significant Positive correlation between Lipocalin 2, BMI, LDL (mg/dL), Cholesterol (mg/dL), Insulin (μU/mL) and HOMA-IR in patient group(P-value ≤ 0.001), Conclusion; lipocalin-2 is a good blood biomarker for detection of hepatic and Pancreatic steatosis. The lipocalin levels increase with increasing severity of steatosis. In addition to this, lipocalin will be useful for early diagnosis of NAFLD, NAFPD and convenient to do with other blood tests. The early intervention may prevent high morbidity and mortality and to manage the disease properly.

Engineering psychrophilic polymerase for nanopore long-read sequencing.

Unveiling the potential application of psychrophilic polymerases as candidates for polymerase-nanopore long-read sequencing presents a departure from conventional choices such as thermophilic Bacillus stearothermophilus (Bst) renowned for its limitation in temperature and mesophilic Bacillus subtilis phage (phi29) polymerases for limitations in strong exonuclease activity and weak salt tolerance. Exploiting the PB-Bst fusion DNA polymerases from Psychrobacillus (PB) and Bacillus stearothermophilus (Bst), our structural and biochemical analysis reveal a remarkable enhancement in salt tolerance and a concurrent reduction in exonuclease activity, achieved through targeted substitution of a pivotal functional domain. The sulfolobus 7-kDa protein (Sso7d) emerges as a standout fusion domain, imparting significant improvements in PB-Bst processivity. Notably, this study elucidates additional functional sites regulating exonuclease activity (Asp43 and Glu45) and processivity using artificial nucleotides (Glu266, Gln283, Leu334, Glu335, Ser426, and Asp430). By disclosing the intricate dynamics in exonuclease activity, strand displacement, and artificial nucleotide-based processivity at specific functional sites, our findings not only advance the fundamental understanding of psychrophilic polymerases but also provide novel insights into polymerase engineering.

Utility of next-generation sequencing for the etiological diagnosis of Orientia tsutsugamushi infection

BackgroundScrub typhus, an acute febrile disease caused by Orientia tsutsugamushi, is transmitted to humans through infected chigger mites. We present a case of scrub typhus in a previously healthy man from Shandong Province diagnosed using next-generation sequencing (NGS) and PCR and review recent literature on NGS for scrub typhus diagnosis. MethodsNGS was utilized for testing whole blood collected on admission. Confirmatory testing was done by detecting IgM and IgG antibodies to Orientia in acute and convalescent sera by ELISA. Orientia 47-kDa protein gene TaqMan and standard PCR of the 56-kDa protein gene and Sanger sequencing were performed on eschar scab DNA. ResultsThe NGS diagnosis was confirmed by 47-kDa protein gene TaqMan and sequencing of a fragment of the O. tsutsugamushi 56-kDa protein gene from the eschar scab. Analysis of this sequence and the NGS data indicated O. tsutsugamushi strain Cheeloo2020 is a novel genotype. Mapping of the NGS data against the O. tsutsugamushi Gilliam strain genome sequence identified 304 reads with high similarity. ConclusionsNGS is not only useful for multiplex diagnosis of scrub typhus, but also provides insight into the genetic diversity of O. tsutsugamushi. The common failure to submit sequences to databases makes it difficult to determine the minimal quantity and quality of NGS data being used for the positive identification of Orientia DNA in clinical specimens.

Pierisin, Cytotoxic and Apoptosis-Inducing DNA ADP-Ribosylating Protein in Cabbage Butterfly.

Pierisin-1 was serendipitously discovered as a strong cytotoxic and apoptosis-inducing protein from pupae of the cabbage butterfly Pieris rapae against cancer cell lines. This 98-kDa protein consists of the N-terminal region (27 kDa) and C-terminal region (71 kDa), and analysis of their biological function revealed that pierisin-1 binds to cell surface glycosphingolipids on the C-terminal side, is taken up into the cell, and is cleaved to N- and C-terminal portions, where the N-terminal portion mono-ADP-ribosylates the guanine base of DNA in the presence of NAD to induce cellular genetic mutation and apoptosis. Unlike other ADP-ribosyltransferases, pieisin-1 was first found to exhibit DNA mono-ADP-ribosylating activity and show anti-cancer activity in vitro and in vivo against various cancer cell lines. Pierisin-1 was most abundantly produced during the transition from the final larval stage to the pupal stage of the cabbage butterfly, and this production was regulated by ecdysteroid hormones. This suggests that pierisn-1 might play a pivotal role in the process of metamorphosis. Moreover, pierisin-1 could contribute as a defense factor against parasitization and microbial infections in the cabbage butterfly. Pierisin-like proteins in butterflies were shown to be present not only among the subtribe Pierina but also among the subtribes Aporiina and Appiadina, and pierisin-2, -3, and -4 were identified in these butterflies. Furthermore, DNA ADP-ribosylating activities were found in six different edible clams. Understanding of the biological nature of pierisin-1 with DNA mono-ADP-ribosylating activity could open up exciting avenues for research and potential therapeutic applications, making it a subject of great interest in the field of molecular biology and biotechnology.

Molecular Cloning, Expression and Enzymatic Characterization of Tetrahymena thermophila Glutathione-S-Transferase Mu 34.

Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68mM with GSH and 0.40mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25°C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.

The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research.

Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin-glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin α7β1, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein-protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.

Comparisons of procyanidins with different low polymerization degrees on prevention of lipid metabolism in high-fat diet/streptozotocin-induced diabetic mice

Procyanidins, which are oligomerized flavan-3-ols with a polyphenolic structure, are bioactive substances that exhibit various biological effects. However, the relationship between the degree of polymerization (DP) of procyanidins and their bioactivities remains largely unknown. In this study, the preventive effects of procyanidins with different DP (EC, PB2 and PC1) on glucose improvement and liver lipid deposition were investigated using a high-fat diet/streptozotocin-induced diabetes mouse model. The results demonstrated that all the procyanidins with different DP effectively reduced fasting blood glucose and glucose/insulin tolerance, decreased the lipid profile (total cholesterol, triglyceride, and low-density lipoprotein cholesterol content) in serum and liver tissue as well as the liver oil red staining, indicating the improvement of glucose metabolism, insulin sensitivity and hepatic lipid deposition in diabetic mice. Furthermore, the procyanidins down-regulated expression of glucose regulated 78-kDa protein (GRP78) and C/EBP homologous protein (CHOP), indicating a regulation role of endoplasmic reticulum (ER) stress. The inhibition of ER stress by tauroursodeoxycholic acid (TUDCA) treatment abolished the effects of procyanidins with different DP in PA-induced HepG2 cells, confirming that procyanidins alleviate liver hyperlipidemia through the modulation of ER stress. Molecular docking results showed that EC and PB2 could better bind GRP78 and CHOP. Collectively, our study reveals that the structure of procyanidins, particularly DP, is not directly correlated with the improvement of blood glucose and lipid deposition, while highlighting the important role of ER stress in the bioactivities of procyanidins.