Molecular genetic perspectives on the origin of the Lyngngam tribe of Meghalaya, India

Meghalaya, one of the Northeast Indian states, is inhabited by two major tribal clusters, Khasi and Garo. The disputed origin of the Lyngngam tribe of Meghalaya is a result of their geographic distribution, which is sandwiched between that of the above two major tribal clusters. Our earlier analysis of ethnohistoric, linguistic and demographic data suggested the neighbouring Khasi and Garo as the putative parental population(s) of Lyngngam. In this paper, we have investigated the Lyngngam, Garo and all the 7 subtribes of Khasi of Meghalaya using molecular genetic markers-autosomal, Y-chromosome and mtDNA-to explore the possible origin of the Lyngngam tribe. We obtained admixture estimates for Lyngngam versus the putative parental populations. While autosomal STRs and mtDNA results clearly suggest Khasi origin of the Lyngngam, Y-STR distances show greater proximity of Lyngngam to the Garo. Further, the comparative analysis of the Y-Chromosome and mtDNA haplogroup data on relevant Austro-Asiatic and Tibeto-Burman populations from South and Southeast Asia, published by us earlier, clearly exclude the possibility of Lyngngam origin from outside Meghalaya. The molecular genetic evidence in conjunction with the linguistic, demographic and ethno-historic information clearly suggests Khasi origin of the Lyngngam tribe

Effect of thermal treatment and storage on bioactive compounds, organic acids and antioxidant activity of baobab fruit (Adansonia digitata) pulp from Malawi

Bioactive compounds of baobab (Adansonia digitata) pulp from Malawi were investigated. The effect of thermal treatment and storage on selected quality attributes of the juice was also evaluated. Organic compounds were analysed by HPLC; total phenol content (TPC) and total antioxidant activity (FRAP, ABTS and DPPH) were measured by spectrophotometry. Malawi baobab pulp contains high levels of procyanidin B2 (533 ± 22.6 mg/100 g FW), vitamin C (AA + DHA) (466 ± 2.5 mg/100 g FW), gallic acid (68.5 ± 12.4 mg/100 g FW) and (−)-epicatechin (43.0 ± 3.0 mg/100 g FW) and showed a maximum TPC of 1.89 × 103 ± 1.61 mg GAE/100 g FW. The maximum antioxidant activity was 2.81 × 103 ± 92.8 mg TEAC/100 g FW for FRAP, 1.52 × 103 ± 17.1 mg TEAC/100 g FW for ABTS and 50.9 ± 0.43% DPPH for DPPH. Thermal pasteurisation (72 °C, 15 s) retained vitamin C which further showed extended half-life under refrigeration temperature (6 °C). Procyanidin B2, (−)-epicatechin, TPC and antioxidant activity fluctuated during storage. Antioxidant activity was significantly correlated (p ≤ 0.05) with bioactive compounds and TPC

Role of Disulfide Bonds and Topological Frustration in the Kinetic Partitioning of Lysozyme Folding Pathways

Disulfide bonds in proteins can strongly influence the folding pathways by constraining the conformational space. Lysozyme has four disulfide bonds and is widely studied for its antibacterial properties. Experiments on lysozyme infer that the protein folds through a fast and a slow pathway. However, the reasons for the kinetic partitioning in the folding pathways are not completely clear. Using a coarse-grained protein model and simulations, we show that two out of the four disulfide bonds, which are present in the alpha-domain of lysozyme, are responsible for the slow folding pathway. In this pathway, a kinetically trapped intermediate state, which is close to the native state, is populated. In this state, the orientations of a-helices present in the alpha-domain are misaligned relative to each other. The protein in this state has to partially unfold by breaking down the interhelical contacts between the misaligned helices to fold to the native state. However, the topological constraints due to the two disulfide bonds present in the alpha-domain make the protein less flexible, and it is trapped in this conformation for hundreds of milliseconds. On disabling these disulfide bonds, we find that the kinetically trapped intermediate state and the slow folding pathway disappear. Simulations mimicking the folding of protein without disulfide bonds under oxidative conditions show that the native disulfide bonds are formed as the protein folds, indicating that folding guides the formation of disulfide bonds. The sequence of formation of the disulfide bonds is Cys64-Cys80 -> Cys76-Cys94 -> Cys30-Cys115 -> Cys6-Cys127. Any disulfide bond that forms before its precursor in the sequence has to break and follow the sequence for the protein to fold. These results show that lysozyme also serves as a very good model system to probe the role of disulfide bonds and topological frustration in protein folding. The predictions from the simulations can be verified by single-molecule fluorescence resonance energy transfer or single-molecule pulling experiments, which can probe heterogeneity in the folding pathways

pH Regulates Ligand Binding to an Enzyme Active Site by Modulating Intermediate Populations

Understanding the mechanism of ligands binding to their protein targets and the influence of various factors governing the binding thermodynamics is essential for rational drug design. The solution pH is one of the critical factors that can influence ligand binding to a protein cavity, especially in enzymes whose function is sensitive to the pH. Using computer simulations, we studied the pH effect on the binding of a guanidinium ion (Gdm+) to the active site of hen egg-white lysozyme (HEWL). HEWL serves as a model system for enzymes with two acidic residues in the active site and ligands with Gdm+ moieties, which can bind to the active sites of such enzymes and are present in several approved drugs treating various disorders. The computed free energy surface (FES) shows that Gdm+ binds to the HEWL active site using two dominant binding pathways populating multiple intermediates. We show that the residues close to the active site that can anchor the ligand could play a critical role in ligand binding. Using a Markov state model, we quantified the lifetimes and kinetic pathways connecting the different states in the FES. The protonation and deprotonation of the acidic residues in the active site in response to the pH change strongly influence the Gdm+ binding. There is a sharp jump in the ligand-binding rate constant when the pH approaches the largest pKa of the acidic residue present in the active site. The simulations reveal that, at most, three Gdm+ can bind at the active site, with the Gdm+ bound in the cavity of the active site acting as a scaffold for the other two Gdm+ ions binding. These results can aid in providing greater insights into designing novel molecules containing Gdm+ moieties that can have high binding affinities to inhibit the function of enzymes with acidic residues in their active site

Contrasting Effects of Guanidinium Chloride and Urea on the Activity and Unfolding of Lysozyme

Cosolvents play an important role in regulating the stability and function of proteins present in the cell. We studied the role of cosolvents, urea and guanidinium chloride (GdmCl), which act as protein denaturants, in the catalytic activity and structural stability of the protein lysozyme using activity measurements, spectroscopy, and molecular dynamics simulations. We find that the activity of lysozyme increases on the addition of urea, whereas it decreases sharply on the addition of GdmCl. At low GdmCl concentrations (GdmCl] < 4 M), the activity of lysozyme decreases, even though there is no significant perturbation in the structure of the lysozyme folded state. We find that this is due to the strong interaction of the Gdm(+) ion with the residues Asp52 and Glu35, which are present in the lysozyme catalytic site. In contrast, urea interacts with Trp63 present in the loop region present near the active site of lysozyme, inducing minor conformational changes in lysozyme, which can increase the activity of lysozyme. At higher denaturant concentrations, experiments show that GdmCl completely denatures the protein, whereas the folded state is stable in the presence of urea. We further show that GdmCl denatures lysozyme with the disulfide bonds intact in the protein, whereas urea denatures the protein only when the disulfide bonds are broken using reducing agents

The Pseudouridine Synthases Proceed through a Glycal Intermediate

The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the <i>arabino</i> isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2′. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases

Over expression of minichromosome maintenance genes is clinically correlated to cervical carcinogenesis.

Minichromosome Maintenance (MCM) proteins play important roles in cell cycle progression by mediating DNA replication initiation and elongation. Among 10 MCM homologues MCM 2-7 form a hexamer and assemble to the pre-replication complex acting as replication licensing factors. Binding and function of MCM2-7 to pre-replication complex is regulated by MCM10 mediated binding of RECQL4 with MCM2-7. The purpose of this study is to explore the role of MCMs in cervical cancer and their correlation with the clinical parameters of cervical cancer. We have investigated sixty primary cervical cancer tissue samples, eight cervical cancer cell lines and thirty hysterectomised normal cervical tissue. The expression profiling of MCMs was done using semi-quantitative RT-PCR, immunoblotting and immunohistochemistry. MCM2, 4, 5, 6, 7, 10 and RECQL4 are significantly over-expressed in cervical cancer. Among these, MCM4, 6 and 10 show increased frequency of over expression along with advancement of tumor stages. MCM4, 5 and 6 also show differential expression in different types of lesion, while MCM2 and MCM10 are over expressed in cervical cancer irrespective of clinico-pathological parameters. Our data indicates the role of MCM4, MCM5, MCM6, MCM10 and RECQL4 in the progression of cervical cancer

Graphical representation of normalized expression values.

<p>(A–H) Box plots representing the distribution of normalized expression values of normal (n = 30), tumor (n = 60) and CC cell lines (n = 8). A box in a given box plot represents the interquartile range (25^th percentile to 75^th percentile), the middle line denotes median and the extreme ends of the whiskers marks the minimum and maximum values. P-values indicated over each box represent the asymptotic significance (2-tailed) of Mann-Whitney test comparing normal to tumor and normal to cell lines independently.</p