Sequence-based novel genomic microsatellite markers for robust genotyping purposes in foxtail millet [<Emphasis Type="Italic">Setaria italica</Emphasis> (L.) P. Beauv.]

The unavailability of microsatellite markers and saturated genetic linkage map has restricted the genetic improvement of foxtail millet [Setaria italica (L.) P. Beauv.], despite the fact that in recent times it has been documented as a new model species for biofuel grasses. With the objective to generate a good number of microsatellite markers in foxtail millet cultivar ‘Prasad’, 690 clones were sequenced which generated 112.95 kb high quality sequences obtained from three genomic libraries each enriched with different microsatellite repeat motifs. Microsatellites were identified in 512 (74.2%) of the 690 positive clones and 172 primer pairs (pp) were successfully designed from 249 (48.6%) unique SSR-containing clones. The efficacies of the microsatellite containing genomic sequences were established by superior primer designing ability (69%), PCR amplification efficiency (85.5%) and polymorphic potential (52%) in the parents of F₂ mapping population. Out of 172 pp, functional 147 markers showed high level of cross-species amplification (~74%) in six grass species. Higher polymorphism rate and broad range of genetic diversity (0.30–0.69 averaging 0.58) obtained in constructed phylogenetic tree using 52 microsatellite markers, demonstrated the utility of markers in germplasm characterizations. In silico comparative mapping of 147 foxtail millet microsatellite containing sequences against the mapping data of sorghum (~18%), maize (~16%) and rice (~5%) indicated the presence of orthologous sequences of the foxtail millet in the respective species. The result thus demonstrates the applicability of microsatellite markers in various genotyping applications, determining phylogenetic relationships and comparative mapping in several important grass species.     

Utilization of an amphipathic leucine zipper sequence to design antibacterial peptides with simultaneous modulation of toxic activity against human red blood cells

The toxicity of naturally occurring or designed antimicrobial peptides is a major barrier for converting them into drugs. To synthesize antimicrobial peptides with reduced toxicity, several amphipathic peptides were designed based on the leucine zipper sequence. The first one was a leucine zipper peptide (LZP); in others, leucine residues at the a- and/or d-position were substituted with single or double alanine residues. The results showed that LZP and its analogs exhibited appreciable and similar antibacterial activity against the tested gram-positive and gram-negative bacteria. However, the substitution of alanine progressively lowered the toxicity of LZP against human red blood cells (hRBCs). The substitution of leucine with alanine impaired the binding and localization of LZP to hRBCs, but had little effect on the peptide-induced damage of Escherichia coli cells. Although LZP and its analogs exhibited similar permeability, secondary structures, and localization in negatively charged membranes, significant differences were observed among these peptides in zwitterionic membranes. The results suggest a novel approach for designing antibacterial peptides with modulation of toxicity against hRBCs by employing the leucine zipper sequence. Also, to the best of our knowledge, the results demonstrate that this sequence could be utilized to design novel cell-selective molecules for the first time.     

The Effect of Inbreeding on Mortality and Morbidity among Telugu-speaking Populations of Kharagpur, West Bengal, India

Increased mortality and morbidity including congenital malformations among the offspring of consanguineous marriages have been widely reported in human populations from different parts of the world. However, there are few studies on the effect of the intensity of inbreeding and different degrees of inbreeding on mortality and morbidity. The present study is an attempt to examine the effects of inbreeding on mortality and morbidity including congenital disorders in different levels of inbreeding among Telugu-speaking populations of Kharagpur, West Bengal, India, based on data collected through extensive pedigrees. The study reveals that the frequency of spontaneous abortions and stillbirths is higher in the offspring of consanguineous marriages than in that of non-consanguineous marriages. A similar effect is also observed in the infant mortality rate, which is known to have a genetic component, but is not seen in the mortality rate of children and juveniles. The rate of morbidity is consistently higher in the offspring of consanguineous marriages with a sex bias in favour of inbred females. The increased morbidity rates in inbred individuals tend to be inversely correlated with the increase in average autosomal inbreeding coefficient. This appears to strengthen Sanghvi’s hypothesis of a decline in the frequency of deleterious genes with intensification of inbreeding through generations. The present study also confirms an increase in genetic disorders with an increase in inbreeding in almost all populations.     

Isolation and partial characterization of ribonuclease inhibitor from goat liver

Ribonuclease inhibitor (RI), a 50 kDa protein, has been found both in mammalian and nonmammalian tissues. We have isolated RI from goat liver and partial characterization has been accomplished. For the isolation of RI, DEAE cellulose column chromatography followed by affinity chromatography using CNBr activated Sepharose 4B was performed. The inhibition of ribonucleolytic activity of Ribonuclease A has been checked by an agarose gel based assay. The antiangiogenic property of the protein was tested by the chorioallantoic membrane (CAM) assay. Results indicate inhibition of angiogenesis.     

Retinal-Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK_a of 5.8 ± 0.1. It was proposed that this pK_a is associated with the pK_a of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (< 3 M) may be due to an altered conformation adopted by the apomembrane, which is not capable of forming the pigment. Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHR opsin produces pigments with 11-cis retinal and 9-cis retinal owing to the thermal isomerization of these retinal isomers to all-trans retinal. The isomerization rate depends on the pH, and it is faster at a higher pH. The pK_a value of the isomerization process is similar to the pK_a of the binding process of these retinals, which suggests that Lys256 is also involved in the isomerization process. The isomerization is independent of the sodium chloride concentration. However, in the absence of sodium chloride, the apoprotein adopts such a conformation, which does not prevent the isomerization of retinal, but it prevents a covalent bond formation with the lysine residue. The rate and the thermodynamic parameter analysis of the retinal isomerization by NpHR apoprotein led to the conclusion that the apomembrane catalyzes the isomerization via a triplet mechanism.     

A Probabilistic Model of RNA Conformational Space

The increasing importance of non-coding RNA in biology and medicine has led to a growing interest in the problem of RNA 3-D structure prediction. As is the case for proteins, RNA 3-D structure prediction methods require two key ingredients: an accurate energy function and a conformational sampling procedure. Both are only partly solved problems. Here, we focus on the problem of conformational sampling. The current state of the art solution is based on fragment assembly methods, which construct plausible conformations by stringing together short fragments obtained from experimental structures. However, the discrete nature of the fragments necessitates the use of carefully tuned, unphysical energy functions, and their non-probabilistic nature impairs unbiased sampling. We offer a solution to the sampling problem that removes these important limitations: a probabilistic model of RNA structure that allows efficient sampling of RNA conformations in continuous space, and with associated probabilities. We show that the model captures several key features of RNA structure, such as its rotameric nature and the distribution of the helix lengths. Furthermore, the model readily generates native-like 3-D conformations for 9 out of 10 test structures, solely using coarse-grained base-pairing information. In conclusion, the method provides a theoretical and practical solution for a major bottleneck on the way to routine prediction and simulation of RNA structure and dynamics in atomic detail.     

Aflatoxin B1-induced toxicity in HepG2 cells inhibited by carotenoids: morphology, apoptosis and DNA damage

Aflatoxin B1 (AFB1) is a fungal toxin that has been associated with primary hepatocellular carcinoma (HCC) in humans. This study was undertaken to determine the cellular and molecular mechanisms by which the antioxidants beta-carotene and lycopene inhibit AFB1-induced toxic changes in human hepatocytes (HepG2 cells). An in vitro system was optimized to test the chemoprotective effects of lycopene and beta-carotene on HepG2 cells exposed to different concentrations of AFB1. Ultrastructurally, HepG2 cells cultured in the presence of AFB1 showed mitochondrial damage, nuclear condensation and a loss of cell-to-cell contact; the latter was reflected in the observation of dysfunctional gap junctions, resulting in a loss of cell-to-cell communication. At the genomic level, AFB1 formed AFB1-N7-guanine adducts, caused apoptotic cell death and suppressed p53 protein expression. In the presence of the carotenoids, survival of cells exposed to AFB1 was increased, and there was also a significant increase in cellular mitochondrial activity. Our results demonstrate that HepG2 cells pretreated with lycopene and beta-carotene are protected from the toxic effects of AFB1 at both the cellular and molecular levels.     

Maternal methyl supplements increase offspring DNA methylation at Axin Fused

Transient environmental exposures during mammalian development can permanently alter gene expression and metabolism by influencing the establishment of epigenetic gene regulatory mechanisms. The genomic characteristics that confer such epigenetic plasticity upon specific loci, however, have not been characterized. Methyl donor supplementation of female mice before and during pregnancy permanently increases DNA methylation at the viable yellow agouti (A(vy)) metastable epiallele in the offspring. The current study tested whether another murine metastable epiallele, axin fused (Axin(Fu)), similarly exhibits epigenetic plasticity to maternal diet. We found that methyl donor supplementation of female mice before and during pregnancy increased DNA methylation at Axin(Fu) and thereby reduced by half the incidence of tail kinking in Axin(Fu)/+ offspring. The hypermethylation was tail-specific, suggesting a mid-gestation effect. Our results indicate that stochastic establishment of epigenotype at metastable epialleles is, in general, labile to methyl donor nutrition, and such influences are not limited to early embryonic development.