Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR NMR

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 Å decrease in the separation between phosphorothioate and 2′F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR–tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, ³¹P–¹⁹F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA–peptide complex.     

Effect of glycerol level in Tris-basek extender and equilibration period on quality of frozen goat semen

Twenty ejaculates, 4 from each of 5 native goats, were collected using an artificial vagina, and the effects of glycerol level (4, 6.4 and 9 %) and the equilibration period (1, 3 and 5 h) were studied by split-sample technique. The extender used was Tris egg yolk citric acid fructose glycerol extender. The semen was frozen in 0.5-ml French straws by exposure for 10 min to liquid nitrogen vapor, 5 cm above the liquid nitrogen level. After 14 h of storage in liquid nitrogen, the straws were thawed in water at 37 degrees C for 12 - 15 sec. The percentage of progressively motile sperm (PPM) and the percentage of damaged acrosomes (PDA) were studied after equilibration and after thawing. The mean PPM after thawing was found to be 64.0 +/- 0.90, 66.92 +/- 0.54 and 63.65 +/- 1.07 when semen was frozen with 4, 6.4 and 9 % glycerol and 61.48 +/- 0.81, 65.05 +/- 0.78 and 68.03 +/- 0.87 in 1-, 3- and 5-h equilibrated semen, respectively. The mean PDA after thawing was 7.12 +/- 0.88, 8.23 +/- 0.76 and 10.58 +/- 0.84 when semen was frozen with 4, 6.4 and 9 % glycerol and 7.0 +/- 0.74, 9.0 +/- 0.95 and 9.93 +/- 0.81 in 1-, 3- and 5-h equilibrated semen, respectively. Both PPM and PDA differed significantly (P<0.01) between glycerol levels, between equilibration periods and between stages (after equilibration and after thawing). The PPM also differed significantly due to equilibration period x stage interaction (P<0.01) and glycerol level x stage interaction (P<0.05). The PDA did not differ significantly due to interactions. When the differences between pairs of means were tested by least significant difference, it was found that after equilibration PPM was not significantly affected by either glycerol level or equilibration period, while after thawing, it was significantly higher (P<0.05) for 6.4 % glycerol and 5-h equilibrated semen than for 4 or 9 % glycerol and 1- or 3-h equilibrated semen, respectively. The PDA was lower with 4 % glycerol and 1-h equilibrated semen.     

Characteristics of polymorphism at a VNTR locus 3' to the apolipoprotein B gene in five human populations.

We have analyzed the allele frequency distribution at the hypervariable locus 3' to the apolipoprotein B gene (ApoB 3' VNTR) in five well-defined human populations (Kacharis of northeast India, New Guinea Highlanders of Papua New Guinea, Dogrib Indians of Canada, Pehuenche Indians of Chile, and a relatively homogeneous Caucasian population of northern German extraction) by using the PCR technique. A total of 12 segregating alleles were detected in the pooled sample of 319 individuals. A fairly consistent bimodal pattern of allele frequency distribution, apparent in most of these geographically and genetically diverse populations, suggests that the ApoB 3' VNTR polymorphism predates the geographic dispersal of ancestral human populations. In spite of the observed high degree of polymorphism at this locus (expected heterozygosity levels 55%-78%), the genotype distributions in all populations (irrespective of their tribal or cosmopolitan nature) conform to their respective Hardy-Weinberg predictions. Furthermore, analysis of the congruence between expected heterozygosity and the observed number of alleles reveals that, in general, the allele frequency distributions at this locus are in agreement with the predictions of the classical mutation-drift models. The data also show that alleles that are shared by all populations have the highest average frequency within populations. These findings demonstrate the potential utility of highly informative hypervariable loci such as the ApoB 3' VNTR locus in population genetic research, as well as in forensic medicine and determination of biological relatedness of individuals.     

Human transposon-like elements insert at a preferred target site: evidence for a retrovirally mediated process.

Members of the human transposon-like family of repetitive sequences (called THE 1 repeats) like many other repetitive DNA sequences are flanked by short direct repeats. Comparison of the base sequences of twelve examples of these flanking direct repeats indicates that THE 1 repeats insert into a preferred genomic target site. In one case, we have identified the sequence of an empty site into which a THE 1 element inserted. The sequence of this empty site and sequences of truncated THE 1 LTRs are consistent with a retroviral mechanism for the insertion of THE 1 elements. Truncated transposon structures illustrate for the first time that intermediate structures of retrotransposition may also be integrated into the genome.     

Repetitive nucleotide sequence insertions into a novel calmodulin-related gene and its processed pseudogene

A gene containing a transposon-like human repeat element, called THE 1, has been isolated and characterized. The gene, termed T+, encodes a polypeptide resembling known calcium-binding proteins. The THE 1 element is present in the 3'-untranslated region of its message. The cDNA clone corresponding to the gene's mRNA product led to the identification of this gene. A processed RNA pseudogene related to the authentic gene has also been isolated. In addition to intron processing, this pseudogene differs from the gene in that it contains an interspersed Alu repeat instead of a THE 1 element in the 3'-untranslated region. Thus, we compare a site containing a THE 1 element to an ancestrally related transposon-less target site. The comparison suggests a retroviral-related mechanism of THE 1 insertion. This system is unusual in that the parent gene is associated with three distinct retrotransposition events: the parent gene was converted to a processed RNA pseudogene, an Alu repeat inserted into the pseudogene, and a THE 1 element inserted into the parent gene.     

Chronic obstructive pulmonary disease: A crosstalk on nucleotide compositional dynamics and codon usage patterns of the genes involved in disease

Chronic obstructive pulmonary disease (COPD), a lung disease, affects a large number of people worldwide, leading to death. Here, we analyzed the compositional features and trends of codon usage of the genes influencing COPD to understand molecular biology, genetics, and evolutionary relationships of these genes as no work was reported yet. Coding sequences of COPD genes were found to be rich in guanine-cytosine (GC) content. A high value (34-60) of the effective number of codons of the genes indicated low codon usage bias (CUB). Correspondence analysis suggested that the COPD genes were distinct in their codon usage patterns. Relative synonymous codon usage values of codons differed between the more preferred codons and the less-preferred ones. Correlation analysis between overall nucleotides and those at third codon position revealed that mutation pressure might influence the CUB of the genes. The high correlation between GC12 and GC3 signified that directional mutation pressure might have operated at all the three codon positions in COPD genes.     

Sustained Anti-inflammatory Effect of Resveratrol-Phospholipid Complex Embedded Polymeric Patch

Resveratrol-phospholipid complex (Phytosome®) (RSVP) was found better aqueous soluble and permeable than free resveratrol (RSV). RSVPs were incorporated in polymeric patch prepared by solvent casting method using Eudragit RL 100, PVP K30, and PEG 400 for application on dermal sites for sustained treating of inflammation. Prepared patches were evaluated for various physicochemical properties, surface morphology by SEM, TEM, and compatibility of patch components by FT-IR and DSC studies. Optimized formulation (F9) gave 95.79?±?3.02% drug release and 51.36% (4.28?±?0.48 mg/cm²) skin permeation after 24 h. Skin extract when examined for drug accumulation showed 38.31?±?2.42% drug content. FE-SEM images of the patch taken after drug release and skin permeation studies showed that RSVPs in polymeric patch are stable and retain their structure after 24 h long exposure to physiologic environment. Sustained anti-inflammatory effect was established in carrageenan-induced paw edema model in which test formulation gave 84.10% inhibition of inflammation at 24 h as compared to 39.58% for standard diclofenac sodium gel. The CLSM study confirmed the localization of RSVPs for a longer period, thus enabling drug targeting to the dermis for sustained effect. Skin irritation test on rabbit revealed that the patches are safe for skin application. Histological observations suggested that after exposure to the permeants, the SC integrity had not altered and no evidence of presence of inflammatory cells found. RSVP (Phytosome®) containing patches abled to give sustained therapeutic effect that may be useful in treating acute and chronic inflammation.     

Treponema pallidum TroA is a periplasmic zinc-binding protein with a helical backbone.

The crystal structure of recombinant TroA, a zinc-binding protein component of an ATP-binding cassette transport system in Treponema pallidum, was determined at a resolution of 1.8 A. The organization of the protein is largely similar to other periplasmic ligand-binding proteins (PLBP), in that two independent globular domains interact with each other to create a zinc-binding cleft between them. The structure has one bound zinc pentavalently coordinated to residues from both domains. Unlike previous PLBP structures that have an interdomain hinge composed of beta-strands, the N- and C-domains of TroA are linked by a single long backbone helix. This unique backbone helical conformation was possibly adopted to limit the hinge motion associated with ligand exchange.     

Role of Visual Cues in Host Searching Behaviour of Exorista sorbillans Widemann, a Parasitoid of Muga Silk Worm, Antheraea assama Westwood

Visual cues are known to be used by numerous animal taxa to gather information on quality and location of resources. The present work was carried out to understand if visual cues are involved in host localization by E. sorbillans and if so, does an associative learning influence host localization behaviour. The Uzi fly, Exorista sorbillans Widemann is an endoparasitoid of the silkworm, Antheraea assama Westwood and a serious threat to the silk production industry in general. Associative learning in the fly was studied by using green and orange coloured paper disk in presence or absence of a reward, sugar or silk worm larvae. Training for the rewards in the learning experiment was given for 2 days for 30 min at regular intervals. Flies positively associated colored paper disks with the presence of hosts which indicated that they might employ visual learning of microhabitats associated with host habitats, or the hosts themselves. Our results showed that in context of host foraging, training with colours as host associated cues was more reliable than training with colours in the absence of hosts. Thus associative learning may be considered to be an essential component for survival of Exorista sorbillans.