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The 6-kDa early secretory antigenic target (ESAT-6) and culture filtrate protein-10 (CFP-10), expressed from the region of deletion-1 (RD1) of Mycobacterium tuberculosis H37Rv, are known to play a key role in virulence. In this study, we explored the thermodynamic and biochemical changes associated with the formation of the 1 : 1 heterodimeric complex between ESAT-6 and CFP-10. Using isothermal titration calorimetry (ITC), we precisely determined the association constant and free energy change for formation of the complex to be 2 x 10(7) M(-1) and -9.95 kcal.mol(-1), respectively. Strikingly, the thermal unfolding of the ESAT-6-CFP-10 heterodimeric complex was completely reversible, with a T(m) of 53.4 degrees C and DeltaH of 69 kcal.mol(-1). Mixing of ESAT-6 and CFP-10 at any temperature below the T(m) of the complex led to induction of helical conformation, suggesting molecular recognition between specific segments of unfolded ESAT-6 and CFP-10. Enhanced biochemical stability of the complex was indicated by protection of ESAT-6 and an N-terminal fragment of CFP-10 from proteolysis with trypsin. However, the flexible C-terminal of CFP-10 in the complex, which has been shown to be responsible for binding to macrophages and monocytes, was cleaved by trypsin. In the presence of phospholipid membranes, ESAT-6, but not CFP-10 and the complex, showed an increase in alpha-helical content and enhanced thermal stability. Overall, complex formation resulted in structural changes, enhanced thermodynamic and biochemical stability, and loss of binding to phospholipid membranes. These features of complex formation probably determine the physiological role of ESAT-6, CFP-10 and/or the complex in vivo. The ITC and thermal unfolding approach described in this study can readily be applied to characterization of the 11 other pairs of ESAT-6 family proteins and for screening ESAT-6 and CFP-10 mutants. 相似文献
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Nicole J. Sylvain Mootaz M. Salman M. Jake Pushie Huishu Hou Vedashree Meher Rasmus Herlo Lissa Peeling Michael E. Kelly 《生物化学与生物物理学报:生物膜》2021,1863(5):183573
Stroke is the second leading cause of death and the third leading cause of disability globally. Edema is a hallmark of stroke resulting from dysregulation of water homeostasis in the central nervous system (CNS) and plays the major role in stroke-associated morbidity and mortality. The overlap between cellular and vasogenic edema makes treating this condition complicated, and to date, there is no pathogenically oriented drug treatment for edema. Water balance in the brain is tightly regulated, primarily by aquaporin 4 (AQP4) channels, which are mainly expressed in perivascular astrocytic end-feet. Targeting AQP4 could be a useful therapeutic approach for treating brain edema; however, there is no approved drug for stroke treatment that can directly block AQP4. In this study, we demonstrate that the FDA-approved drug trifluoperazine (TFP) effectively reduces cerebral edema during the early acute phase in post-stroke mice using a photothrombotic stroke model. This effect was combined with an inhibition of AQP4 expression at gene and protein levels. Importantly, TFP does not appear to induce any deleterious changes on brain electrolytes or metabolic markers, including total protein or lipid levels. Our results support a possible role for TFP in providing a beneficial extra-osmotic effect on brain energy metabolism, as indicated by the increase of glycogen levels. We propose that targeting AQP4-mediated brain edema using TFP is a viable therapeutic strategy during the early and acute phase of stroke that can be further investigated during later stages to help in developing novel CNS edema therapies. 相似文献
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S. K. Yasin Baksh Ravindra Donde Jitendra Kumar Mitadru Mukherjee Jitendriya Meher Lambodar Behera Sushanta Kumar Dash 《Physiology and Molecular Biology of Plants》2021,27(3):543
Rice is an important source of calorie for the growing world population. Its productivity, however is affected by climatic adversities, pest attacks, diseases of bacterial, viral and fungal origin and many other threats. Developing cultivars that are high yielding and stress resilient seems a better solution to tackle global food security issues. This study investigates the potential resistance of 24 rice cultivars against Xanthomonas oryzae pv. Oryzae (Xoo) infection that causes bacterial leaf blight disease and submergence stress. Bacterial leaf blight (BLB) resistance genes (Xa4, xa5, xa13, Xa21, Xa38) and submergence tolerance (Sub1) gene specific markers were used to determine the allelic status of genotypes. The results displayed presence of Xa4 resistance allele (78.95%), xa5 (15.79%) but xa13 and Sub1 tolerance allele were not found in any genotype. However, a new allele for Xa21 (84.21%) and Xa38 (10.52%) were identified in several genotypes. Phenotypic screening for both stress conditions was done to record the cultivars response. None of the genotypes showed resistance against Xoo, although varieties viz., Tapaswini and Konark showed moderate susceptibility. Likewise, survival percentage of genotypes under submergence stress varied from 0 to 100%. Tolerant checks FR13A (100%) and Swarna Sub1 (97.78%) exhibited high survival rate, whereas among genotypes, Gayatri (57.78%) recorded high survivability even though it lacked Sub1 tolerant its genetic background. A total of six trait specific STS and two SSR markers generated an average of 2.38 allele per locus. Polymorphism information content (PIC) value ranged from 0.08 to 0.42 with an average of 0.20. Structure analysis categorized 24 genotypes into two sub-populations, which was in correspondence with Nei’s genetic distance-based NJ tree and principal co-ordinate analysis (PCoA). Swarna Sub1 could be differentiated clearly from BLB resistant check, IRBB60 and other 22 genotypes without having Sub1 gene. Analysis of molecular variance (AMOVA) revealed more genetic variation within population than among population. Principal component analysis (PCA) showed that 9 morphological traits collectively explained 76.126% of total variation among all the genotypes studied. The information from this study would be useful in future breeding programs for pyramiding trait specific genes into high yielding cultivars that fall behind with respect to stress resilience. Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00951-1. 相似文献
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Prashanta Kumar Panda Srimanta Patra Prajna Paramita Naik Prakash Priyadarshi Praharaj Subhadip Mukhopadhyay Biswa Ranjan Meher Piyush Kumar Gupta Rama S. Verma Tapas K. Maiti Sujit K. Bhutia 《Journal of cellular physiology》2020,235(3):2776-2791
Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells. 相似文献
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Prawit Kongjan Sompong O‐Thong Meher Kotay Booki Min Irini Angelidaki 《Biotechnology and bioengineering》2010,105(5):899-908
Hydrolysate was tested as substrate for hydrogen production by extreme thermophilic mixed culture (70°C) in both batch and continuously fed reactors. Hydrogen was produced at hydrolysate concentrations up to 25% (v/v), while no hydrogen was produced at hydrolysate concentration of 30% (v/v), indicating that hydrolysate at high concentrations was inhibiting the hydrogen fermentation process. In addition, the lag phase for hydrogen production was strongly influenced by the hydrolysate concentration, and was prolonged from approximately 11 h at the hydrolysate concentrations below 20% (v/v) to 38 h at the hydrolysate concentration of 25% (v/v). The maximum hydrogen yield as determined in batch assays was 318.4 ± 5.2 mL‐H2/g‐sugars (14.2 ± 0.2 mmol‐H2/g‐sugars) at the hydrolysate concentration of 5% (v/v). Continuously fed, and the continuously stirred tank reactor (CSTR), operating at 3 day hydraulic retention time (HRT) and fed with 20% (v/v) hydrolysate could successfully produce hydrogen. The hydrogen yield and production rate were 178.0 ± 10.1 mL‐H2/g‐sugars (7.9 ± 0.4 mmol H2/g‐sugars) and 184.0 ± 10.7 mL‐H2/day Lreactor (8.2 ± 0.5 mmol‐H2/day Lreactor), respectively, corresponding to 12% of the chemical oxygen demand (COD) from sugars. Additionally, it was found that toxic compounds, furfural and hydroxymethylfurfural (HMF), contained in the hydrolysate were effectively degraded in the CSTR, and their concentrations were reduced from 50 and 28 mg/L, respectively, to undetectable concentrations in the effluent. Phylogenetic analysis of the mixed culture revealed that members involved hydrogen producers in both batch and CSTR reactors were phylogenetically related to the Caldanaerobacter subteraneus, Thermoanaerobacter subteraneus, and Thermoanaerobacterium thermosaccharolyticum. Biotechnol. Bioeng. 2010;105: 899–908. © 2009 Wiley Periodicals, Inc. 相似文献
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A co-culture of bacteria responsible for the conversion of butyrate to methane and CO2 was isolated from a cattle-waste treatment plant. The non-methanogenic partner of the co-culture was Syntrophomonas wolfei and the methanogenic partner was Methanobacterium formicicum. Although butyrate degradation occurred at pH<6.0 and below 45°C, methanogenesis was observed at pH>6.5 and above 40°C. 相似文献
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