Wild-type Saccharomyces cerevisiae organisms contain three kinases which catalyze the phosphorylation of glucose: two hexokinase isozymes (PI and PII) and one glucokinase. Glucose transport measurements for triple-kinaseless mutants, which lack all three of these kinases, confirm that the kinases are involved in the low apparent Km transport process observed in metabolizing cells. Thus kinase-positive cells containing one or more of the three kinases exhibit biphasic transport kinetics with a low apparent Km (1 to 2 mM) and high apparent Km (40 to 50 mM) component. Triple-kinaseless cells, however, exhibit only the high apparent Km component of kinase-positive cells (60 mM). Kinetic analysis of glucose transport in the triple-kinaseless cells shows that glucose is transported by a facilitated diffusion process which exhibits trans-stimulated equilibrium exchange and influx counterflow.
Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Secmediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knockout mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.
While the composition and function of the major thylakoid membrane complexes are well understood, comparatively little is known about their biogenesis. The goal of this work was to shed more light on the role of auxiliary factors in the biogenesis of photosystem II (PSII). Here we have identified the homolog of LOW PSII ACCUMULATION 2 (LPA2) in Chlamydomonas. A Chlamydomonas reinhardtii lpa2 mutant grew slower in low light, was hypersensitive to high light, and exhibited aberrant structures in thylakoid membrane stacks. Chlorophyll fluorescence (Fv/Fm) was reduced by 38%. Synthesis and stability of newly made PSII core subunits D1, D2, CP43, and CP47 were not impaired. However, complexome profiling revealed that in the mutant CP43 was reduced to ~23% and D1, D2, and CP47 to ~30% of wild type levels. Levels of PSI and the cytochrome b6f complex were unchanged, while levels of the ATP synthase were increased by ~29%. PSII supercomplexes, dimers, and monomers were reduced to ~7%, ~26%, and ~60% of wild type levels, while RC47 was increased ~6-fold and LHCII by ~27%. We propose that LPA2 catalyses a step during PSII assembly without which PSII monomers and further assemblies become unstable and prone to degradation. The LHCI antenna was more disconnected from PSI in the lpa2 mutant, presumably as an adaptive response to reduce excitation of PSI. From the co-migration profiles of 1734 membrane-associated proteins, we identified three novel putative PSII associated proteins with potential roles in regulating PSII complex dynamics, assembly, and chlorophyll breakdown.
Background/Aims: Nasopharyngeal cancer (NPC) is one of the common human malignant diseases all over the world, and chemotherapy remains the main therapy for NPC. However, the survival and life quality of NPC patients are still very poor. Thus, novel and selective anti-tumor agents are pressingly needed. Our previous study identified pectolinarigenin as a novel effective anti-tumor drug candidate for NPC. In this study, we further investigated its anti-tumor activities and explored the potential molecular mechanism. Methods: NPC C666-1 cells were cultured and treated by pectolinarigenin. Cell proliferation assay, colony formation assay, Transwell assay and wound healing assay were conducted and cell apoptosis was detected by flow cytometry. Mitochondrial transmembrane potential and ROS were also observed. NPC subcutaneous xenograft mice model was established to evaluate the anti-tumor effect of pectolinarigenin in vivo. Results: We observed that treatment of pectolinarigenin inhibited cell viability and cell migration of NPC C666-1 cells in concentration- and time-dependent manner. Pectolinarigenin induced cell apoptosis in C666-1 cells detected by flow cytometry analysis, which was associated with the activation of mitochondrial-related apoptosis and the accumulation of reactive oxygen species (ROS). Pectolinarigenin also activated caspase signaling pathway. The in vivo experiment of subcutaneous xenograft mice model also indicated that the administration of pectolinarigenin could decrease the tumor growth of NPC and no severe toxicity was observed. Conclusions: Based on our findings, we conclude that pectolinarigenin could suppress the tumor growth of NPC, which verifies it as a new therapeutic agent for treating this devastating disease.
Periodontal ligament stem cells (PDLSCs) possess extensive regeneration potential. However, their therapeutic application demands a scaffold with appropriate properties. HydroMatrix (HydM) is a novel injectable peptide nanofiber hydrogel developed recently for cell culture. Our aim was to test whether HydM would be a suitable scaffold for proliferation and osteogenic differentiation of PDLSCs. PDLSCs were seeded on non-coated or HydM-coated surfaces. Both real-time impedance analysis and cell viability assay documented cell growth on HydM. PDLSCs showed healthy, fibroblast-like morphology on the hydrogel. After a 3-week-long culture in osteogenic medium, mineralization was much more intense in HydM cultures compared to control. Alkaline phosphatase activity of the cells grown on the gels reached the non-coated control levels. Our data provided evidence that PDLSCs can adhere, survive, migrate, and proliferate on HydM and this gel also supports their osteogenic differentiation. We first applied impedimetry for dental stem cells cultured on a scaffold. HydM is ideal for in vitro studies of PDLSCs. It may also serve not only as a reference material but also in the future as a promising biocompatible scaffold for preclinical studies.
Background and AimsThe Glutathione S-transferase P1 (GSTP1) polymorphism have been considered a risk modifier for developing head and neck cancer (HNC) in many studies; however, the results of such studies are inconsistent. The aim of this study was to evaluate the possible association between the GSTP1 Ile105Val polymorphism and risk of HNC.MethodWe performed a search in the relevant electronic database and a meta-analysis based on 28 published case–control studies that included 6,404 cases and 6,523 controls. To take into account the possibility of heterogeneity across the studies, a Chi-square based I2-statistic test was performed. Crude pooled odds ratios (ORs) with 95% confidence intervals (CIs) were assessed using both fixed-effects and random-effects models.ResultsThe results of this meta-analysis showed that the GSTP1 Ile105Val polymorphism was not significantly associated with risk of HNC in the overall study population (pooled OR 1.00, 95% CI 0.92–1.09) or in subgroup analyses stratified by ethnicity, sample size, tumor site or publication year. Moreover, substantial evidence of heterogeneity among the studies was observed. Publication year was identified as the main cause of heterogeneity.ConclusionThis meta-analysis does not support a significant association between the GSTP1 Ile105Val polymorphism and risk of HNC.
Purpose The long non-coding RNA MALAT1 is a predictive marker in several solid tumors with highly conserved sequences. However, the role of non-coding RNA in development of laryngeal or hypopharyngeal cancer remains unclear. Methods Tumor tissues and adjacent non-cancer tissues of 24 patients were collected. We detected the expression of MALAT1 in laryngeal cancer tissues and hypopharyngeal cancer tissues. Moreover, we developed a MALAT1 silencing model in human laryngeal tumor cells by transfecting MALAT1 small interfering RNA into human laryngeal carcinoma cell line Hep-2 and pharyngeal carcinoma cell line FaDu with Lipofectamine 2000 system. Cell cycle analysis, Cell Counting Kit-8 assay, Transwell assay, quantitative reverse transcription PCR, and wound-healing assays were performed to evaluate the impact of MALAT1 depletion on laryngeal or hypopharyngeal cancer cell's growth, proliferation, apoptosis, invasion and migration. Results MALAT1 was significantly up-regulated in laryngeal and hypopharyngeal carcinoma cells. MALAT1 down-regulation induced the increased apoptosis of both cell lines and suppressed cells' proliferation. Cells were arrested in G1/G2 phase and cells of S phase were significantly decreased. Down-regulation of MALAT1 expression can also inhibit the migration and invasion of laryngeal squamous cell carcinoma cell (Hep-2) and hypopharyngeal cancer cell (FaDu). Conclusion In summary, our deactivation model of MALAT1 disentangled the active function of it as a regulator of gene expression governing the hallmarks of laryngeal and hypopharyngeal cancer. Blocking this long non-coding RNA may restrain the development of laryngeal cancer.
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