N-helix and Cysteines Inter-regulate Human Mitochondrial VDAC-2 Function and Biochemistry

Human voltage-dependent anion channel-2 (hVDAC-2) functions primarily as the crucial anti-apoptotic protein in the outer mitochondrial membrane, and additionally as a gated bidirectional metabolite transporter. The N-terminal helix (NTH), involved in voltage sensing, bears an additional 11-residue extension (NTE) only in hVDAC-2. In this study, we assign a unique role for the NTE as influencing the chaperone-independent refolding kinetics and overall thermodynamic stability of hVDAC-2. Our electrophysiology data shows that the N-helix is crucial for channel activity, whereas NTE sensitizes this isoform to voltage gating. Additionally, hVDAC-2 possesses the highest cysteine content, possibly for regulating reactive oxygen species content. We identify interdependent contributions of the N-helix and cysteines to channel function, and the measured stability in micellar environments with differing physicochemical properties. The evolutionary demand for the NTE in the presence of cysteines clearly emerges from our biochemical and functional studies, providing insight into factors that functionally demarcate hVDAC-2 from the other VDACs.     

Spectrum and distribution of CFTR gene mutations in asthma and chronic pancreatitis cases of North Indian population

Background

Cystic fibrosis transmembrane conductance regulator (CFTR) gene accounts for an autosomal recessive condition called cystic fibrosis (CF). In the Indian subcontinent, CF and its related diseases are under-diagnosed by the medical community due to poor knowledge of the disease and its confounding diagnosis, and also due to poor medical facilities available for these patients, thus causing an increased infant mortality rate with a low life expectancy in general. The aim of the study was to document the spectrum and distribution of CFTR mutations in controls, asthma and chronic pancreatitis cases of North India.

Methods

A total of 800 subjects including 400 controls, 250 asthma cases and150 chronic pancreatitis cases were analyzed for 6 mutations (F508del, G542X, G551D, R117H, W1282X, and S549N) and IVS8 Tn polymorphism.

Results

Out of 800 subjects, 18% [asthma — 24% (n = 250), CP — 29.33% (n = 150) cases and controls — 9.3% (n = 400)] were positive for heterozygous mutation, 0.8% of the (n = 250) asthmatic cases (n = 250) were homozygous for IVS8 T5 polymorphism while no subjects were found positive for W1282X mutation. T5 polymorphism was more common in asthmatic cases while F508del mutation in chronic pancreatitis cases. The carrier frequency of F508del, G542X, G551D, R117H, S549N and T5 was 0.015, 0.025, 0.02, 0.005, 0.005, and 0.022 respectively. The cumulative carrier frequency was 0.093.

Conclusion

CFTR mutations were underestimated in Indian population. The present study will serve in establishment of genetic screening and prenatal setup for Indian population.     

Characterization of M. tuberculosis SerB2, an Essential HAD-Family Phosphatase,Reveals Novel Properties

M. tuberculosis harbors an essential phosphoserine phosphatase (MtSerB2, Rv3042c) that contains two small- molecule binding ACT-domains (Pfam 01842) at the N-terminus followed by the phosphoserine phosphatase (PSP) domain. We found that exogenously added MtSerB2 elicits microtubule rearrangements in THP-1 cells. Mutational analysis demonstrates that phosphatase activity is co-related to the elicited rearrangements, while addition of the ACT-domains alone elicits no rearrangements. The enzyme is dimeric, exhibits divalent metal- ion dependency, and is more specific for l- phosphoserine unlike other classical PSPases. Binding of a variety of amino acids to the ACT-domains influences MtSerB2 activity by either acting as activators/inhibitors/have no effects. Additionally, reduced activity of the PSP domain can be enhanced by equimolar addition of the ACT domains. Further, we identified that G18 and G108 of the respective ACT-domains are necessary for ligand-binding and their mutations to G18A and G108A abolish the binding of ligands like l- serine. A specific transition to higher order oligomers is observed upon the addition of l- serine at ∼0.8 molar ratio as supported by Isothermal calorimetry and Size exclusion chromatography experiments. Mutational analysis shows that the transition is dependent on binding of l- serine to the ACT-domains. Furthermore, the higher-order oligomeric form of MtSerB2 is inactive, suggesting that its formation is a mechanism for feedback control of enzyme activity. Inhibition studies involving over eight inhibitors, MtSerB2, and the PSP domain respectively, suggests that targeting the ACT-domains can be an effective strategy for the development of inhibitors.     

Mineral Release Dynamics of Tricalcium Phosphate and Waste Muscovite by Mineral-Solubilizing Rhizobacteria Isolated from Indo-Gangetic Plain of India

The aim of the present investigation was to evaluate the mineral release abilities of ten rhizobacterial strains isolated from rhizosphere of various crops growing in the Indo-Gangetic Plain (IGP) of India. Their abilities to solubilize inorganic phosphorus (P) and potassium (K) minerals from insoluble tricalcium phosphate (TCP) and waste muscovite (WM) revealed that rhizobacteria significantly solubilized different levels of inorganic P and fixed K, respectively. Some of the rhizobacterial stains have the ability to produce ammonia, indole-3-acetic acid (IAA), and hydrogen cyanide (HCN). The identification based on the 16S rDNA gene sequencing of selected mineral-solubilizing rhizobacteria (MSR) having greater potential to serve as bioagents were identified as Bacillus subtilis (BRHU01, BRHU03, and BHU20), Bacillus tequilensis (BRHU02), Bacillus licheniformis (BRHU04), Bacillus pumilus (BRHU05), Bacillus flexus (BHU02), Brevibacillus formosus (BHU16), Bacillus methylotrophicus (BHU29), and Bacillus amyloliquefaciens (BHU30). Interestingly, inorganic P and K solubilization by these strains belonging to genera Bacillus and Brevibacillus showed significant variations from 0.52 to 14.49 and from 1.62 to 8.60 µg mL^?1, respectively. However, generally, pH values of culture media decreased from near neutral (6.43) to acidic (3.83) with increasing incubation period, and this was inversely correlated with quantities of K solubilized by these rhizobacterial strains. Meanwhile, the electrical conductivity (EC) of broth culture increased from 0.09 to 0.23 dS m^?1 with increasing incubation period. Principal component analysis (PCA) of the MSR revealed three clusters, which exhibited high variance with respect to nutrient release. Taken together, these results suggest that Bacillus and Brevibacillus sp. identified in this study solubilized varying levels of inorganic P and fixed K from insoluble TCP and WM by acidolysis mechanisms.     

Mitochondrial VDAC2 and cell homeostasis: highlighting hidden structural features and unique functionalities

Voltage‐dependent anion channels (VDACs) are the gateway to mitochondrial processes, interlinking the cytosolic and mitochondrial compartments. The mitochondrion acts as a storehouse for cytochrome c, the effector of apoptosis, and hence VDACs become intricately involved in the apoptotic pathway. Isoform 1 of VDAC is abundant in the outer mitochondrial membrane of many cell types, while isoform 2 is the preferred channel in specialized cells including brain and some cancer cells. The primary role of VDACs is metabolite flux. The pro‐ and anti‐apoptotic role of VDAC1 and VDAC2, respectively, are secondary, and are influenced by external factors and interacting proteins. Herein, we focus on the less‐studied VDAC2, and shed light on its unique functions and features. VDAC2, along with sharing many of its functions with VDAC1, such as metabolite and Ca²⁺ transport, also has many delineating functions. VDAC2 is closely engaged in the gametogenesis and steroidogenesis pathways and in protection from oxidative stress as well as in neurodegenerative diseases like Alzheimer's and epilepsy. A closer examination of the functional pathways of VDACs indicates that the unique functions of VDAC2 are a result of the different interactome of this isoform. We couple functional differences to the structural and biophysical evidence obtained for the VDACs, and present a testament of why the two VDAC isoforms with >90% sequence similarity, are functionally diverse. Based on these differences, we suggest that the VDAC isoforms now be considered as paralogs. An in‐depth understanding of VDAC2 will help us to design better biomolecule targets for cancer and neurodegenerative diseases.     

Phenolic acid changes in mycelia of Sclerotium rolfsii as influenced by neem (Azadirachta indica) cake and Zephyarenthes citrina bulb

High performance liquid chromatographic (HPLC) analysis of mycelia of Sclerotium rolfsii grown on neem cake, and Zephyarenthes citrina bulb incorporated media was carried out. Several phenoloic acids, e.g., gallic, tannic, caffeic, cinnamic, chlorogenic and O-coumeric acids, were found in considerable amounts in treated mycelial mat as compared to the control. The amount of phenloic acids increased with increased concentration of both the materials in mycelia of 7 and 14 day-old cultures. Due to anti-oxidant and several other properties of phenolic acids, the senescence of the fungus has been prolonged which may be one probable reason of sustaining the virulence of the pathogen.     

Radiation protection of DNA by ferulic acid under in vitro and in vivo conditions

The effect of ferulic acid was studied on γ-radiation-induced relaxation of plasmid pBR322 DNA and induction of DNA strand breaks in peripheral blood leukocytes and bone marrow cells of mice exposed to whole body γ-radiation. Presence of 0.5 mM ferulic acid significantly inhibited the disappearance of supercoiled (ccc) plasmid pBR322 with a dose modifying factor (DMF) of 2.0. Intraperitoneal administration of different amounts (50, 75 and 100 mg/kg body weight) of ferulic acid 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strands breaks in murine peripheral blood leukocytes and bone marrow cells as evidenced from comet assay. The dose-dependent protection was more pronounced in bone marrow cells than in the blood leukocytes. It was observed that there was a time-dependent disappearance of radiation induced strand breaks in blood leukocytes (as evidenced from comet parameters) following whole body radiation exposure commensuration with DNA repair. Administration of 50 mg/kg body weight of ferulic acid after whole body irradiation of mice resulted disappearance of DNA strand breaks at a faster rate compared to irradiated controls, suggesting enhanced DNA repair in ferulic acid treated animals. (Mol Cell Biochem xxx: 209–217, 2005)     

Amritosides A, B, C and D: clerodane furano diterpene glucosides from Tinospora cordifolia

Four new clerodane furano diterpene glucosides (amritosides A, B, C and D) were isolated as their acetates from Tinospora cordifolia stems. The structures of these compounds were established on the basis of spectroscopic studies.