Structure modeling and antidiabetic activity of a seed protein of Momordica charantia in non-obese diabetic (NOD) mice

Momordica charantia is a well known medicinal plant used in the traditional medicinal system for the treatment of various diseases including diabetes mellitus. Recently, a novel protein termed as ADMc1 from the seed extract of M. charantia has been identified and isolated showing significant antihyperglycemic activity in type 1 diabetic rats in which diabetes was induced. However, the structure of this protein has not yet been analyzed. Homology modeling approach was used to generate a high quality protein 3D structure for the amino acid sequence of the ADMc1 protein in this study. The comparative assessment of secondary structures revealed ADMc1 as an all-alpha helix protein with random coils. Tertiary structure predicted on the template structure of Napin of B. Napus (PDB ID: 1SM7) with which the ADMc1 showed significant sequence similarity, was validated using protein structure validation tools like PROCHECK, WHAT_CHECK, VERIFY3D and ProSA. Arrangement of disulfide bridges formed by cysteine residues were predicted by the Dianna 1.1 server. The presence of multiple disulfide bond confers the stable nature of the ADMc1 protein. Further, the biological activity of the ADMc1 was assessed in non-obese diabetic (NOD) mice which are spontaneous model of type 1 diabetes. Significant reduction in the blood glucose levels of NOD mice was observed up to 8 h post administration of the rADMc1 protein. Overall, the structural characterizations with antihyperglycemic activity of this seed protein of Momordica charantia demonstrate its potential as an antidiabetic agent.     

Minocycline,levodopa and MnTMPyP induced changes in the mitochondrial proteome profile of MPTP and maneb and paraquat mice models of Parkinson's disease

Mitochondrial dysfunction is the foremost perpetrator of the nigrostriatal dopaminergic neurodegeneration leading to Parkinson's disease (PD). However, the roles played by majority of the mitochondrial proteins in PD pathogenesis have not yet been deciphered. The present study investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and combined maneb and paraquat on the mitochondrial proteome of the nigrostriatal tissues in the presence or absence of minocycline, levodopa and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP). The differentially expressed proteins were identified and proteome profiles were correlated with the pathological and biochemical anomalies induced by MPTP and maneb and paraquat. MPTP altered the expression of twelve while combined maneb and paraquat altered the expression of fourteen proteins. Minocycline, levodopa and MnTMPyP, respectively, restored the expression of three, seven and eight proteins in MPTP and seven, eight and eight proteins in maneb- and paraquat-treated groups. Although levodopa and MnTMPyP rescued from MPTP- and maneb- and paraquat-mediated increase in the microglial activation and decrease in manganese-superoxide dismutase expression and complex I activity, dopamine content and number of dopaminergic neurons, minocycline defended mainly against maneb- and paraquat-mediated alterations. The results demonstrate that MPTP and combined maneb and paraquat induce mitochondrial dysfunction and microglial activation and alter the expression of a bunch of mitochondrial proteins leading to the nigrostriatal dopaminergic neurodegeneration and minocycline, levodopa or MnTMPyP variably offset scores of such changes.     

Targeting metabolic pathways proteins of Orientia tsutsugamushi using combined hierarchical approach to combat scrub typhus

Orientia tsutsugamushi (Ott) is a causative agent of chigger‐borne zoonosis, scrub typhus which is life threatening and highly pervasive illness in humans. In this report, we have mined and classified the proteins involved in pathways unique to Ott by using high‐throughput computational techniques. The 12 metabolic pathways were found to be unique to the pathogen. Forty‐six proteins were reported to be essential for the pathogen's survival and non‐homologous to the humans. The proteins were categorized into different classes, ie, enzymes, transporters, DNA‐binding, secretory, and outer membrane proteins. Further, in silico analysis of 46 proteins showed that 25 proteins were suitable therapeutic targets with known druggable properties. The structural modeling of B3CSG3 (MurA) protein was carried out and catalytic site essential for its functioning was analyzed. Virtual screening of chemical compounds was performed against modeled structure. The docking study by AutodockVina reported compound from PubChem with CID: 16036947 as best and potential inhibitor by means of docking score and binding affinity. The reliability and stability of the MurA‐16036947 complex were confirmed with molecular dynamics simulation. The report will provide insight to understand the mechanism of pathogenesis of Ott and instigate the development of effective treatment strategies against this disease.     

Molecular docking analysis of hyperphosphorylated tau protein with compounds derived from Bacopa monnieri and Withania somnifera

Tau protein, the major player in Alzheimer’s disease forms neurofibrillary tangles in elderly people. Bramhi (Baccopa Monniera) is often used as an ayurvedic treatment for Alzheimer''s disease. Therefore it is of interest to study the interaction of compounds derived from Baccopa with the Tau protein involved in tangle formation. We show that compounds such as bacopaside II, bacopaside XII, and nicotine showed optimal binding features with the R2 repeat domain of hyperphosphorylated tau protein for further consideration in the context of Alzheimer''s disease (AD).     

Transmitted HIV-1 is more virulent in heterosexual individuals than men-who-have-sex-with-men

Transmission bottlenecks introduce selection pressures on HIV-1 that vary with the mode of transmission. Recent studies on small cohorts have suggested that stronger selection pressures lead to fitter transmitted/founder (T/F) strains. Manifestations of this selection bias at the population level have remained elusive. Here, we analysed early CD4 cell count measurements reported from ∼340,000 infected heterosexual individuals (HET) and men-who-have-sex-with-men (MSM), across geographies, ethnicities and calendar years. The reduction in CD4 counts early in infection is reflective of the virulence of T/F strains. MSM and HET use predominant modes of transmission, namely, anal and penile-vaginal, with among the largest differences in the selection pressures at transmission across modes. Further, in most geographies, the groups show little inter-mixing, allowing for the differential selection bias to be sustained and amplified. We found that the early reduction in CD4 counts was consistently greater in HET than MSM (P<0.05). To account for inherent variations in baseline CD4 counts, we constructed a metric to quantify the extent of progression to AIDS as the ratio of the reduction in measured CD4 counts from baseline and the reduction associated with AIDS. We found that this progression corresponding to the early CD4 measurements was ∼68% for MSM and ∼87% for HET on average (P<10⁻⁴; Cohen’s d, d_s = 0.36), reflecting the more severe disease caused by T/F strains in HET than MSM at the population level. Interestingly, the set-point viral load was not different between the groups (d_s<0.12), suggesting that MSM were more tolerant and not more resistant to their T/F strains than HET. This difference remained when we controlled for confounding factors using multivariable regression. We concluded that the different selection pressures at transmission have resulted in more virulent T/F strains in HET than MSM. These findings have implications for our understanding of HIV-1 pathogenesis, evolution, and epidemiology.     

Boo, a novel negative regulator of cell death, interacts with Apaf-1.

In this report, we describe the cloning and characterization of Boo, a novel anti-apoptotic member of the Bcl-2 family. The expression of Boo was highly restricted to the ovary and epididymis implicating it in the control of ovarian atresia and sperm maturation. Boo contains the conserved BH1 and BH2 domains, but lacks the BH3 motif. Like Bcl-2, Boo possesses a hydrophobic C-terminus and localizes to intracellular membranes. Boo also has an N-terminal region with strong homology to the BH4 domain found to be important for the function of some anti-apoptotic Bcl-2 homologues. Chromosomal localization analysis assigned Boo to murine chromosome 9 at band d9. Boo inhibits apoptosis, homodimerizes or heterodimerizes with some death-promoting and -suppressing Bcl-2 family members. More importantly, Boo interacts with Apaf-1 and forms a multimeric protein complex with Apaf-1 and caspase-9. Bak and Bik, two pro-apoptotic homologues disrupt the association of Boo and Apaf-1. Furthermore, Boo binds to three distinct regions of Apaf-1. These results demonstrate the evolutionarily conserved nature of the mechanisms of apoptosis. Like Ced-9, the mammalian homologues Boo and Bcl-xL interact with the human counterpart of Ced-4, Apaf-1, and thereby regulate apoptosis.     

Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay

We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu²⁺ in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time–response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.     

Dynamics of the T4 bacteriophage DNA packasome motor: endonuclease VII resolvase release of arrested Y-DNA substrates

Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y- or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N- and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism.