Biochemical and biophysical research communications最新文献

SLC17A3 localized to the apical membrane of the renal proximal tubules has been implicated in the urinary excretion of drugs and endogenous/exogenous metabolites transported into the tubules by OAT1 and OAT3. Because SLC17A3 mediates the facilitated diffusion of organic anions, which requires a sensitive and rapid assay, no system has been established to evaluate its transport activity in mammalian cells. In this study, we demonstrated that the exposure of cells expressing click beetle luciferase (bLuc) and SLC17A3 to D-luciferin produces marked bioluminescence, which enables the evaluation of SLC17A3 function. The bioluminescence intensity increased under depolarized membrane potential conditions, consistent with the unique feature of SLC17A3 as a voltage-dependent organic anion transporter. SLC17A3-mediated bioluminescence was saturable with an apparent Michaelis-Menten constant (K_m) of 8.1 μM. The inhibitory effects of various compounds including OAT1/OAT3 substrates and inhibitors on bioluminescence were in good agreement with those reported in SLC17A3-expressing Xenopus oocytes using radio-labeled substrates. Interestingly, we found that sulfinpyrazone and lesinurad, uricosuric drugs that inhibit SLC22A12/URAT1, are potent SLC17A3 inhibitors, suggesting the possibility that they alter the pharmacokinetics of OAT1/OAT3-substrate drugs and urate. Taken together, the bioluminescence-based SLC17A3 functional assay is robust and reliable. This strategy enables the study of its transport activity and the identification of potential SLC17A3-mediated drug-drug interactions. This approach also provides an opportunity to elucidate the molecular mechanisms involved in the urinary excretion of organic anions.

Gentisic acid (GA), a cytochrome P450 metabolite of the antiplatelet drug aspirin, exhibits smooth muscle relaxant, antiatherogenic, and antioxidant activities. It also has a protective role in hypertrophic heart failure, suggesting its role in the management of myocardial infarction (MI). This study aimed to explore the protective activity of GA in isoproterenol (ISO)-induced MI in Sprague-Dawley (SD) rats in-vivo, followed by mechanistic investigation ex-vivo. SD rats were pretreated with different doses (5, 10, 15, and 20 mg/kg, i.p.) of GA for 21 days, followed by subcutaneous administration of ISO (85 mg/kg) on the 20th and 21st days. At the end of the experiment, electrocardiograph (ECG), blood pressure, myocardial injury marker enzymes, infarct size, lipid profile, and histological changes in myocardium were carried out. The possible underlying mechanisms were explored ex-vivo. GA prevented the ISO-induced changes in ECG parameters in rats in a dose-dependent manner. GA also reversed the fall in blood pressure associated with ISO treatment. GA diminished the elevated cardiac biomarkers and limited the infarcted area size (8 %) indicated by decrease in heart weight to body weight ratio. GA ameliorated the inflammation, edema, and necrosis and reduced collagen fiber deposition associated with ISO-induced MI. The results suggest that GA is an effective cardioprotective agent in rats by reversing ischemic changes in ECG and correcting histopathological and biochemical changes.

The death of retinal ganglion cells (RGCs) is a key factor in the pathophysiology of all forms of glaucoma. RGC culture serves as a simple system for establishing and testing candidate therapies. This study aimed to explore the differentiation of primary retinal progenitor cells (RPCs) into RGC-like cells induced by low-dose cytarabine (Ara-C). RPCs were isolated from the retina of newborn rats and cultured in vitro. Different concentrations of Ara-C were added to the culture medium to induce the differentiation of RPCs into RGC-like cells. Differentiation efficiency was assessed through immunofluorescence staining and cell counting. The addition of Ara-C significantly increased the number of Brn3a/RBPMS double-positive cells. The RPC-RGCs induced displayed characteristic features of RGCs, with roughly 80.9 % ± 6.2 % of the cells positive for both TuJ1/NeuN and 77.5 % ± 4.9 % for Brn3a/RBPMS. The study demonstrates that the addition of Ara-C to primary cultures of rat RPCs can enhance their differentiation into RGC-like cells, providing a simple and rapid method for obtaining RGC-like cells with a relatively high purity. This method shows considerable promise for advancing glaucoma research and potential therapeutic strategies to restore vision after RGC loss.

Plant Toll/interleukin-1 receptor (TIR) domains function as NADases and ribosyl-transferases generating second messengers that trigger hypersensitive responses. TIR-X (TX) proteins contain a TIR domain with or without various C-terminal domains and lack the canonical nucleotide-binding site and leucine-rich repeat domain. In a previous study, we identified an Arabidopsis thaliana activation-tagging line with severe growth defects caused by the overexpression of the AtTX12 gene. Here, we investigated the domains and specific amino acid residues required for the growth inhibition activity of AtTX12 and its homolog AtTX11. C-terminal truncation analysis revealed that the AtTX12C173Δ mutant, lacking 30 C-terminal amino acids, retained partial activity, whereas the C163Δ, lacking 40 amino acids, lost activity entirely indicating that the fifth α-helix within the TIR domain is critical for activity, while the sixth α-helix in the extra domain is dispensable. The substitution mutagenesis revealed that residues essential for enzymatic activities (E79 for NADase, C76 for 2',3'-cAMP/cGMP synthetase), self-association (H25, E43, K142/G144, K150), and undefined roles (I97) were crucial for growth inhibition activity with varying effects. Temperature sensitivity tests revealed that the AtTX12 N36D mutant, which exhibited moderately strong growth inhibition activity at normal temperatures, became inactive under high-temperature conditions in which Enhanced Disease Susceptibility 1 (EDS1) is almost non-functional. In contrast, wild-type AtTX12 retained activity under elevated temperatures, implicating N36 in maintaining temperature-insensitive functionality. Furthermore, a slightly reduced growth inhibition phenotype induced by AtTX12 overexpression in the eds1 mutant was consistently observed under both normal and high temperatures. These results suggest that AtTX12-mediated growth inhibition integrates EDS1-dependent (temperature-sensitive) and EDS1-independent (temperature-insensitive) pathways. Our findings suggest that attenuated AtTX11/12 mutants could be used to optimize the growth-defense trade-off, enhancing plant defense with minimal growth penalties.

Angiogenesis, the process of new blood vessel formation, involves endothelial cell proliferation and migration, accompanied by the remodeling of the extracellular matrix (ECM). Type IV collagen, a major ECM component, plays a critical role in vascular basement membrane regeneration, influencing cell polarity, migration, and survival. This study examines the regulatory role of Notch signaling, mediated by Notch3, in type IV collagen expression using TIG-1 fibroblasts and a co-culture angiogenesis model with human umbilical vein endothelial cells (HUVECs). Using small interfering RNA (siRNA) to suppress Notch3 expression, we observed a significant reduction in COL4A1 gene expression, which encodes the α1 chain of type IV collagen. Conversely, transient expression of the Notch3 intracellular domain (NICD3) activated Notch signaling, resulting in increased COL4A1 expression. In the co-culture model, pre-treatment of TIG-1 cells with Notch signaling inhibitors, including siNotch3 and DAPT, decreased the number of α1(IV)-positive TIG-1 fibroblasts adjacent to HUVECs. This reduction highlights the essential role of Notch3-mediated signaling in promoting type IV collagen expression during angiogenesis. Our findings suggest that Notch signaling regulates type IV collagen expression levels, supporting basement membrane formation and vascular maturation. These results provide insight into the molecular mechanisms of angiogenesis, potentially contributing to therapeutic strategies targeting vascular-related pathologies.

Stimulator of interferon response cGAMP interactor 1 (STING1), as an innate immune adaptor protein that mediates DNA sensing, has attracted tremendous biomedical interest. However, several recent researches have revealed the key role of STING1 in regulating the metabolic pathway. Here, we investigated its role in adipocyte differentiation. Preadipocytes with lentivirus-mediated Sting1 knockdown or overexpression were constructed to examine the effect of STING1 on adipocyte differentiation in vitro. Proteomics was performed in adipocytes to explore the mechanisms by which STING1 exerts pro-adipogenesis effects. Coimmunoprecipitation (CoIP)/mass spectrometry (MS) assay were used to identify the interacting partners of STING1. Our results showed that STING1 was upregulated during adipogenic differentiation of 3T3-L1 and white adipose tissue-derived stromal vascular precursor cells (WAT-SVF), accompanied by upregulation of adipocyte marker genes, peroxisome proliferator-activated receptor gamma (Pparg) and CCAAT/enhancer-binding protein beta (Cebpβ). Knockdown or overexpression of Sting1 altered adipogenesis in adipocytes. Mechanistically, proteomics and CoIP/MS assay revealed that STING1 targets non-muscle myosin protein (MYH9) to block its expression, which enhances AKT/GSK3β signaling and mediates β-catenin accumulation, affecting adipogenesis-related genes in adipocytes. These findings suggest that STING1 targeting combined with MYH9 regulates adipocyte differentiation through the AKT/GSK3β/β-catenin pathway. This is a new potential target for the treatment of hypertrophic adipose tissue, or obesity.

Gastrointestinal T cells (GI-T) play a critical role in mucosal immunity, balancing tolerance and pathogen defence. T cells recognize antigens via T cell receptors (TCRs). Next-generation sequencing (NGS) is utilized to analyse TCR repertoires in contexts such as health, haematological diseases, autoimmunity, and inflammation. While some studies have explored T cell involvement in GI conditions, the integration of different techniques and examination of diverse tissues remain underdeveloped. In our "proof of concept" study, for the first time, we combined flow cytometry, TCR sequencing and transcriptomics to analyse T cell repertoires from bulk sorted T cells and from single cells. This combination provides information about both, specificity and functionality of particular T cells. We focused on biopsy samples from the stomach, colon, and compared these to blood samples from patients with and without inflammation associated with erosive gastritis. This combined approach allows unique insights into T cell biology. Through TCR clonotype analysis, we identified oligoclonal expansion in inflamed biopsies, with minimal TCR clonotype overlap between individuals, highlighting personalized immune responses. Gene expression analysis revealed upregulation of T cell activation and signalling and chemokines in inflamed biopsies. Single-cell sequencing provided deeper insights into specific T cell populations, identifying dominant clonotypes with cytotoxic function. Our findings underscore the importance of studying affected sites to fully understand T cell responses and localized immune reactions. Our approach opens unique possibilities for studying TCR and gene expression from limited biopsy material, potentially leading to personalized therapies and biomarkers for gastrointestinal diseases.

Small RNAs (sRNAs) have emerged as key regulators of transcriptional factors and components within regulatory networks that govern bacterial biofilm formation. This study aimed to explore the regulatory role of the PA3299.1 sRNA in controlling biofilm formation in P. aeruginosa. Results showed that PA3299.1 expression was significantly elevated in both substratum-attached and colony biofilms compared to planktonic growth. Further investigation revealed that strains overexpressing PA3299.1 exhibited enhanced biofilm formation, while its deletion resulted in a substantial reduction in biofilm development. PA3299.1 was found to regulate the expression of AlgU and MucA, the sigma and anti-sigma factors, integral to the biofilm developmental network. In summary, this research identifies PA3299.1 as a critical regulator of biofilm formation and potentially a contributor to the pathogenicity of P. aeruginosa, that could help to develop new therapeutic strategies to manage biofilm-associated infections.

Agonistic monoclonal antibodies targeting 4-1BB have shown much preclinical promise, but their clinical development has been limited by obvious toxicity or unremarkable efficacy. Here, we generated two humanized anti-B7H3 × 4-1BB bsAbs (HK056-001/002) by fusing an anti-4-1BB scFv to the C-terminus of an anti-B7H3 with an intact Fc fragment from human IgG1 or IgG4. The two bsAbs were able to stimulate the 4-1BB signaling pathway, which was strictly dependent on B7H3 expression. In particular, HK056-001 retained Fc function and induced an ADCC effect in tumor cells, whereas HK056-002 did not. Strikingly, HK056-001 showed superior antitumour activity to HK056-002 both in vitro and in vivo. HK056-001 enhanced antitumour immunity and induced lasting antigen-specific immune memory to prevent tumor regrowth upon rechallenge, even at a dose as low as 2 mg/kg. Furthermore, HK056-001 did not induce nonspecific production of proinflammatory cytokines and had no apparent ability to induce ADA production. In addition, HK056-001 has no significant liver toxicity in human 4-1BB-KI BALB/c mice bearing CT26-B7H3 tumors. The optimal anti-B7H3 × 4-1BB bsAb HK056-001 exhibited synergistic antitumour effects by inducing an ADCC effect (innate immunity) and activating the 4-1BB signaling pathway (adaptive immunity) upon cross-bridging with B7H3 with no obvious toxicity, which could potentially provide a better therapeutic window compared to what is seen with 4-1BB agonists.

Regulatory subunits of protein phosphatase 2A (PP2A) define the substrate and functional specificity of the PP2A holoenzyme within specific organelles. While PP2A regulates osteoblast differentiation, the roles and localization of its regulatory subunits in osteoblasts remain unclear. Here, we identified PTPA, a PP2A regulatory protein, predominantly localized to the Golgi apparatus, closely overlapping with the Golgi marker Giantin. Disruption of the Golgi structure by Brefeldin A caused PTPA to disperse into the cytoplasm. PTPA overexpression inhibited osteoblast differentiation by downregulating key transcriptional regulators. The Golgi-specific localization of PTPA suggests it may influence bone-related protein secretion and maintain Golgi integrity. These findings highlight the critical role of PTPA in osteoblast differentiation and its association with the Golgi apparatus.