Domains of human U4atac snRNA required for U12-dependent splicing in vivo

U4atac snRNA forms a base-paired complex with U6atac snRNA. Both snRNAs are required for the splicing of the minor U12-dependent class of eukaryotic nuclear introns. We have developed a new genetic suppression assay to investigate the in vivo roles of several regions of U4atac snRNA in U12-dependent splicing. We show that both the stem I and stem II regions, which have been proposed to pair with U6atac snRNA, are required for in vivo splicing. Splicing activity also requires U4atac sequences in the 5' stem-loop element that bind a 15.5 kDa protein that also binds to a similar region of U4 snRNA. In contrast, mutations in the region immediately following the stem I interaction region, as well as a deletion of the distal portion of the 3' stem-loop element, were active for splicing. Complete deletion of the 3' stem-loop element abolished in vivo splicing function as did a mutation of the Sm protein binding site. These results show that the in vivo sequence requirements of U4atac snRNA are similar to those described previously for U4 snRNA using in vitro assays and provide experimental support for models of the U4atac/U6atac snRNA interaction.     

Manipulation of unfolding-induced protein aggregation by peptides selected for aggregate-binding ability through phage display library screening

A phage-displayed library of peptides (12-mer) was screened for the ability to bind to thermally aggregated bovine carbonic anhydrase (BCA), with a view toward examining whether peptides possessing this ability might bind to partially structured intermediates on the protein's unfolding pathway and, therefore, constitute useful tools for manipulation of the kinetic partitioning of molecules between the unfolded and aggregated states. Two peptides [N-HPSTMGLRTMHP-C and N-TPSAWKTALVKA-C] were identified and tested. While neither showed thermal aggregation autonomously, both peptides individually elicited remarkable increases in the levels of thermal aggregation of BCA. A possible explanation is that both peptides bind to surfaces on molten BCA that are not directly involved in aggregation. Such binding could slow down interconversions between folded and unfolded states and stabilize aggregation-prone intermediate(s) to make them more prone to aggregation, while failing to achieve any steric prevention of aggregation. The approach has the potential of yielding useful aggregation-aiding/inhibiting agents, and may provide clues to whether amorphous aggregates are "immobilized" forms of folding intermediates.     

Testicular development and its relationship to semen production in Murrah buffalo bulls

The objectives of this study were to determine the relationship of age and body weight to testicular development and to establish norms for breeding soundness evaluations of Murrah buffalo bulls. Testicular measurements of 133 Murrah buffalo bulls of various ages were recorded with a caliper and a tape. Semen was collected twice a week for 5 weeks from groups of bulls which were 25-36 (n=17), 37-48 (n=16), 49-60 (n=14), of >60 (n=10) months of age. After examining volume, sperm concentration, and progressive motility semen was diluted in Tris-citric acid-egg yolk-fructose extender and frozen in 0.5 ml French straws. Testicular measurements of buffalo bulls were lower than those recorded for European breeds of cattle bulls. Nevertheless, like cattle bulls, scrotal circumference was highly correlated with other testicular measurements. Also, it had a significant positive relationship with semen volume and sperm concentration per ejaculate. Average sperm output per week in order of increasing age group was 15.3, 18.2, 19.8 and 23.6 x 10(9). Corresponding values for sperm output per week per gram of testis were 59.1, 45.8, 41.1, 36.2 x 10(6) indicating a reduction in spermatogenesis per unit of testis with advancing age. Compared to European breeds, daily sperm output in Murrah bulls was nearly 45% lower, presumably due to their nearly 40% lower scrotal circumference than Holstein bulls of the same age. These results indicate that in buffalo, as in cattle, scrotal circumference is a useful indicator of potential sperm output and may serve as an important criterion for selecting young bulls as AI sires.     

Effect of bioaccumulation of cadmium on biomass productivity,essential trace elements,chlorophyll biosynthesis,and macromolecules of wheat seedlings

Soil contamination with heavy metals has become a worldwide problem, leading to losses in agricultural yield and hazardous human health effects as they enter the food chain. The present investigation was undertaken to examine the influence of cadmium (Cd²⁺) on the wheat (Triticum aestivum L.) plant. Cd²⁺ accumulation and distribution in 3-wk-old seedlings grown in nutrient medium containing varying concentrations of Cd²⁺ (control, 0.25, 0.50, 1.0, 2.5, and 5.0 mg/L) was monitored. The effect of varying Cd²⁺ concentrations up to 21 d on biomass productivity, plant growth, photosynthetic pigments, protein, amino acids, starch, soluble sugars, and essential nutrients uptake was studied in detail to explore the level up to which the plant can withstand the stress of heavy metal. Plants treated with 0.5, 1.0, 2.5, and 5.0 mg/L Cd²⁺ showed symptoms of heavy-metal toxicity as observed by various morphological parameters which were recorded with the growth of plants. The root, shoot-leaf length and the root, shoot-leaf biomass progressively decreased with increasing Cd²⁺ concentration in the nutrient medium. Cd²⁺ uptake and accumulation was found to be maximum during the initial growth period. Cd²⁺ also interfered with the nutrients uptake, especially calcium (Ca²⁺), magnesium (Mg²⁺), potassium (K⁺), iron (Fe²⁺), zinc (Zn²⁺), and manganese (Mn²⁺) from the growth medium. Growth reduction and altered levels of major biochemical constituents such as chlorophyll, protein, free amino acids, starch, and soluble sugars that play a major role in plant metabolism were observed in response to varying concentrations of Cd²⁺ in the nutrient medium. In the present study, the effects of Cd²⁺ on growth, biomass productivity, mineral nutrients, chlorophyll biosynthesis, protein, free amino acid, starch, and soluble sugars in wheat plants was estimated to establish an overall picture of the Cd²⁺ toxicity at structural and functional levels.     

Hydrophobic displacement chromatography of proteins

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.     

The effects of hypergravity and substrate vibration on vestibular function in developing chickens

We used linear vestibular evoked potentials (VsEPs) to characterize peripheral and central vestibular function in birds following embryogenesis at 2G centrifugation or at elevated levels of vibration (+20dB re: background levels). Additionally, we characterized peripheral and central vestibular adaptation to 2G centrifugation in early post-hatch birds. Linear VsEP response peak latencies, amplitudes, thresholds and input/output functions were quantified and compared between experimental and control animals. Birds vibrated throughout embryogenesis and up to one-week post-hatch revealed no changes in linear VsEP response components compared to control siblings. Birds centrifuged at 2G throughout embryogenesis also evidenced no changes in the linear VsEP measured at hatch (P0). Significant changes were seen, however, for linear VsEPs of post-hatch birds placed at 2G for 7 days beginning on post-hatch day 5. Linear VsEPs for these animals displayed significant reductions in response amplitudes associated with peaks P2, N2 and P3, response peaks generated by central neural relays of gravity receptors. The earliest response components, generated by the peripheral vestibular nerve (i.e., P1, N1), were not significantly altered with the 7-day exposure to 2G. Thus, there was no evidence of generalized changes in peripheral gravity receptor excitability or in the rate of maturation in developing animals under increased levels of gravity or vibration. If gravity level plays a critical role in shaping peripheral vestibular ontogeny at magnitudes between 1 and 2G, then it may serve to stabilize function under changing G-fields or it may operate on physiological features that can not be resolved by the VsEP. In contrast, exposure to elevated gravity during post-hatch periods does alter central vestibular function thus providing direct evidence for central vestibular adaptation to the gravitational environment. The fact that central functional change was observed in hatchlings and not embryos, raises the possibility that the first 2-weeks post-hatch may be a critical period of "heightened developmental sensitivity" to hypergravity.     

Striking similarity of murine nectin-1alpha to human nectin-1alpha (HveC) in sequence and activity as a glycoprotein D receptor for alphaherpesvirus entry

A cDNA encoding the murine homolog of human nectin-1alpha (also known as poliovirus receptor-related protein 1 [Prr1] and herpesvirus entry protein C [HveC]) was isolated. The protein encoded by this cDNA proved to be 95% identical in sequence to the human protein and to have similar herpesvirus entry activity. Upon expression of the murine cDNA in hamster cells resistant to alphaherpesvirus entry, the cells became susceptible to the entry of herpes simplex virus types 1 and 2 (HSV-1 and -2), pseudorabies virus, and bovine herpesvirus 1. HSV envelope glycoprotein D (gD), a viral ligand for human nectin-1alpha, is also a ligand for the murine homolog based on evidence that (i) a soluble hybrid protein composed in part of the murine nectin-1 ectodomain bound specifically to purified soluble forms of HSV-1 and HSV-2 gD as demonstrated by enzyme-linked immunosorbent assay, (ii) a soluble hybrid of HSV-1 gD bound to hamster cells expressing murine nectin-1alpha but not to control cells, and (iii) cells expressing both murine nectin-1alpha and one of the alphaherpesvirus gDs were resistant to entry of HSV-1, indicative of interference with entry resulting from interactions of cell-associated gD with the entry receptor. Northern blot analysis revealed that nectin-1 is expressed in most of the mouse tissues examined and at high levels in the brain, skin, and kidneys. Immunocytochemical localization demonstrated the presence of nectin-1 in epithelial cells of the mouse vagina and also in neuronal cells of the central nervous system, suggesting an expression pattern relevant to both infection at a portal of entry and spread of infection to the brain.     

A Molecular Electrostatic Potential Mapping Study of Some Fluoroquinolone Anti-Bacterial Agents

Molecular electrostatic potential (MEP) maps of some fluoroquinolones having varying degrees of activity against the bacterium Staphylococcus Aureus have been studied using the optimized hybridization displacement charges (HDC) combined with Löwdin charges obtained by the AM1 method. The roles of different substitutions at the N₁-position in the parent quinolone ring have been studied. The conformation of the carboxylic group attached to the quinolone ring was shown to be such that there is an intramolecular hydrogen bonding between the hydrogen atom of this group and the oxygen atom of the carbonyl group of the quinolone moiety. The carbonyl oxygen atom of the quinolone moiety, hydroxyl oxygen atom of the carboxylic group and the terminal nitrogen atom of the piperazin ring attached to the quinolone ring appear to be involved in the action of the drugs through electrostatic interactions while the N₁-alkyl substituents seem to be involved in the same through hydrophobic interactions.