RV-Typer: A Web Server for Typing of Rhinoviruses Using Alignment-Free Approach

Rhinoviruses (RV) are increasingly being reported to cause mild to severe infections of respiratory tract in humans. RV are antigenically the most diverse species of the genus Enterovirus and family Picornaviridae. There are three species of RV (RV-A, -B and -C), with 80, 32 and 55 serotypes/types, respectively. Antigenic variation is the main limiting factor for development of a cross-protective vaccine against RV.Serotyping of Rhinoviruses is carried out using cross-neutralization assays in cell culture. However, these assays become laborious and time-consuming for the large number of strains. Alternatively, serotyping of RV is carried out by alignment-based phylogeny of both protein and nucleotide sequences of VP1. However, serotyping of RV based on alignment-based phylogeny is a multi-step process, which needs to be repeated every time a new isolate is sequenced. In view of the growing need for serotyping of RV, an alignment-free method based on “return time distribution” (RTD) of amino acid residues in VP1 protein has been developed and implemented in the form of a web server titled RV-Typer. RV-Typer accepts nucleotide or protein sequences as an input and computes return times of di-peptides (k = 2) to assign serotypes. The RV-Typer performs with 100% sensitivity and specificity. It is significantly faster than alignment-based methods. The web server is available at http://bioinfo.net.in/RV-Typer/home.html.     

Requirement for a macromolecular factor for sodium azide activation of guanulate cyclase.

Sodium azide, a highly nucleophilic agent and a potent metabolic inhibitor, markedly increased guanylate cyclase activity from supernatant fractions of rat liver homogenates. The effect of sodium azide was not observed with partially purified guanulate cyclase from liver or crude soluble guanylate cyclase from cerebral cortex. However, the effect of sodium azide could be restored by the readdition of a fraction isolated from rat liver homogenates. The macromolecular factor required for the sodium azide effect was separated from soluble guanylate cyclase of rat liver with DEAE-cellulose column chromatography, and some of its properties were examined. The factor was nondialyzable and heat labile.     

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.     

Rearrangements and insertions in the Moloney murine leukemia virus long terminal repeat alter biological properties in vivo and in vitro.

The effects of rearrangement and insertion of sequences in the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. The alterations were made by recombinant DNA manipulations on a plasmid subclone containing an M-MuLV LTR. Promoter activity of altered LTRs was measured by fusion to the bacterial chloramphenicol acetyltransferase gene, followed by transient expression assay in NIH 3T3 cells. M-MuLV proviral organizations containing the altered LTRs were also generated, and infectious virus was recovered by transfection. Infectivity of the resulting virus was quantified by XC plaque assay, and pathogenicity was determined by inoculating neonatal NIH Swiss mice. Inversion of sequences in the U3 region containing the tandemly repeated enhancer sequences (-150 to -353 base pairs [bp]) reduced promoter activity approximately fivefold in the transient-expression assays. Infectious virus containing the inverted sequences (Mo- M-MuLV) showed a 20-fold reduction in relative infectivity compared with wild-type M-MuLV, but the virus still induced thymus-derived lymphoblastic lymphoma or leukemia in mice, with essentially the same kinetics as for wild-type M-MuLV. We previously derived an M-MuLV which carried inserted enhancer sequences from the F101 strain of polyomavirus (Mo + PyF101 M-MuLV) and showed that this virus is nonleukemogenic. In Mo + PyF101 M-MuLV, the PyF101 sequences were inserted between the M-MuLV promoter and the M-MuLV enhancers (at -150 bp). A new LTR was generated in which the PyF101 sequences were inserted to the 5' side of the M-MuLV enhancers (at -353 bp, PyF101 + Mo M-MuLV). The PyF101 + Mo LTR exhibited promoter activity similar (40 to 50%) to that of wild-type M-MuLV, and infectious PyF101 + Mo M-MuLV had high infectivity on NIH 3T3 cells (50% of wild type). In contrast to the nonleukemogenic Mo + PyF101 M-MuLV, PyF101 + Mo M-MuLV induced leukemia with kinetics similar to that of wild-type M-MuLV. Thus, the position of the PyF101 sequences relative to the M-MuLV LTR affected the biological behavior of the molecular construct. Furthermore, PyF101 + Mo M-MuLV induced a different spectrum of neoplastic disease. In comparison with wild-type M-MuLV, which induces a characteristic thymus-derived lymphoblastic lymphoma with extremely high frequency, PyF101 + Mo M-MuLV was capable of inducing both acute myeloid leukemia or thymus-derived lymphoblastic lymphoma, or both. Tumor DNA from both the PyF101 + Mo- and Mo- M-MuLV-inoculated animals contained recombinant proviruses with LTRs that differed from the initially inoculated virus.     

Fine structure and histochemistry of the epidermis of the fish Monopterus cuchia

An attempt is made to correlate fine structure with the histochemical reactions of the epidermis in the synbranchiform fish Monopterus cuchia. Three sources of mucus are identified. Superficial epithelial cells produce weakly acidic glycoprotein which is secreted at the surface as the external mucous layer or cuticle. Numerous large unicellular mucous glands have a secretion which is strongly acidic and sulphated, although the basal and peripheral parts of these cells, which contain most of the rough endoplasmic reticulum, react strongly for neutral glycoprotein; Golgi cisternae appear to be involved in a change of histochemical reaction from neutral to strongly acidic as the secretion is formed. A second, slender, type of mucous gland cell, not previously reported, gives a weaker reaction for sulphated acidic glycoprotein and has cytoplasm with numerous Golgi cisternae and free ribosomes, producing electron–dense secreted drops. Sacciform cells, with a protein–aceous secretion, have a characteristic fine structure with membranous "bubbles" at the surface of the cytoplasm. Ionocytes, sensor) cells and intrusive leucocytes have been identified in the epidermis.     

Surface secretions of the skin of Blennius (Lipophrys) pholis L.

Mucus is secreted to the surface of the body and fin webs of Blennius pholis by superficial epithelial cells and by goblet cells. Some goblet cells secrete sulphated acid glycoproteins, others produce a mucus which is neutral or mixed in its reactions. The superficial epithelial cells of these areas secrete sulphated acid glycoproteins, seen by electron microscopy as electron-lucent or moderately lucent vesicles; this secretion is not normally visible external to the skin in transmission electron microscope (TEM) sections. These cells do not react to the bromphenol blue test for proteins. Over part of the surface of the pelvic fins and the distal parts of the rays of the pectoral fins, the skin contains no goblet cells and bears a thick external secretion, or cuticle, containing protein and glycoprotein which is mainly neutral in reaction, although some cells at the edges of the region secrete weakly sulphated or non-sulphated acidic glycoprotein. The protein content of the columnar superficial epithelial cells of these regions correlates with the fibrous nature of the secreted cuticular layer as seen by TEM; the columnar cells are characterized by extensive ribosomal endoplasmic reticulum and vesicles which stain darkly with phosphotungstic acid, less so with uranyl acetate. The distal part of the cell, containing these vesicles, reacts positively to the PAS stain. In some places the borders of the zones with fibrous cuticle are characterized by cuboidal superficial epithelial cells which give a strong positive reaction to alcian blue at pH 1.0, indicating the presence of sulphated acid glycoproteins, but also react positively to the bromphenol blue test for proteins.     

Fungal infections of ear with special reference to chronic suppurative otitis media

Fungus were found to take important role in ear infections of the 344 patients (CSOM 286, Otomycosis 44, Otitis externa 14), significant fungal infections (with positive smear and culture) were detected on 49%, 79.5%, 66.6% patients respectively. 84.8% patients were detected both by smear and culture, 14.1% patients by culture and 0.1% patients in smear preparation only. In CSOM patients, age predominated in 20–27 yrs group, sex in male below 30 yrs, and Aspergillus flavus, A. niger, Penicillium, A. fumigatus in mycelial fungus, Candida albicans, C. parapsillosis in yeast. But in 18 post antibiotic fungus infected patients Penicillium and A. niger were the important isolates. In otomycosis and otitis externa patients A. niger took the main role.     

Cross-protection against Salmonella enteritidis infection in mice.

Mice were immunized subcutaneously with live vaccines and live vaccines with complete adjuvant incorporating Salmonella enteritidis Se 795, Salmonella typhi-murium M206, Salmonella gallinarum 9R or Salmonella pullorum Sp223. They were challenged along with unvaccinated controls with 100 LD50 of virulent S. enteritidis 5694 SMR subcutaneously on the 21st day post-vaccination. The humoral immune response was studied by assessing the sequential level of agglutinins, complete and incomplete somatic antibodies, opsonophagocytic antibodies, cytophilic antibodies and bactericidal antibodies before and after challenge. The level of these antibodies and the protection afforded by the particular vaccine is correlated and the possible involvement of a humoral mechanism is discussed.