Amino acid composition and terminal sequence analysis of the rabies virus glycoprotein: Identification of the reading frame on the cDNA sequence

The amino acid composition and partial sequences of amino acids at the NH₂- and COOH-termini of the rabies virus (strain ERA) glycoprotein were determined using less than 0.5 mg of the protein. The results provide identification of the reading frame on the cDNA sequence for this glycoprotein.     

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.     

Enzymatic modifications of human plasma fibronectin in relation to opsonizing activity

Plasma fibronectin is one of the largest plasma proteins (Mr approximately 440 000), comprising two approximately equal polypeptide chains which are held together by a disulfide linkage near the C-terminal end of the molecule. The binding of gelatinized latex beads to liver slices as well as the internalization of these particles by macrophages, in the presence of heparin, is greatly enhanced by fibronectin. The question as to whether the entire covalent structure of fibronectin was necessary for opsonizing activity was approached by limited proteolytic degradations of the molecule. Patterns of controlled digestion with trypsin, cathepsin D, Staphylococcus aureus protease, and plasmin all indicate that the minimal unit necessary for retention of opsonic activity is some large (Mr 200 000 and 190 000) single-chain entity. Treatment with plasmin proved to be the most reliable procedure for generating the active split product which could be readily separated from the inactive, disulfide-containing C-terminal fragment. Incorporation of dansylcadaverine into plasma fibronectin (3.5 mol/mol of protein) by fibronoligase (coagulation factor XIIIa) did not affect the opsonic activity of the protein.     

Experimental Mycoplasma pulmonis infection of rats suppresses humoral but not cellular immune response

Humoral antibody response to sheep red blood cells and cellular immune response to bovine serum albumin were studied in Mycoplasma pulmonis infected, adult, male Sprague-Dawley rats. The hemagglutinating antibody response to sheep red blood cells was evaluated at 0, 3, 5, 7, 14, 21 and 28 days postinfection. Antibody titers during all days postinfection were depressed significantly (p less than 0.05) in infected rats as compared to noninfected controls. Cellular immune responses were evaluated by a delayed hypersensitivity response. Rats were sensitized at 0, 3, 5, 7, 14, 21 or 28 days postinfection with bovine serum albumin and challenged with heat aggregated bovine serum albumin 7 days later. Cell-mediated immune responses in infected rats were not significantly different at any point from controls. These results indicate that M. pulmonis infection in rats suppresses the humoral antibody response to sheep red blood cells, but not the cellular immune response to bovine serum albumin.     

Effects of mammal submandibular gland glycoderivatives on human hemostatic system

The aim of this study was to investigate the effects of glycoconjugate components extracted from the submandibular glands of four mammal species (rat, mouse, rabbit, hare) on human hemostatic system. The following analyses were performed: thromboelastography and hemocoagulation screening tests (Thrombin Time, Prothrombin Time, Partial Thromboplastin Time). Findings showed that all glycoconjugates induce modifications of fibrin clot formation time, modulus of elasticity and some hemocoagulation tests (TT, PTT). The anticoagulant effects were of inhibitory type.     

An endogalactosaminidase from Streptomyces griseus.

An endogalactosaminidase has been purified 34-fold from the culture filtrate of Streptomyces griseus. This enzyme cleaves GalN-GalN linkages in oligogalactosaminoglycan, a galactosamine-rich oligosaccharide isolated from the culture filtrate of a Neurospora mutant. Since some or all of the GalN-GalN bonds in this molecule link positions 1 and 4, and are in the alpha-configuration, we are probably dealing with an endo-alpha-(1 leads to 4)-galactosaminidase, bu this characterization is only tentative because the few bonds cleaved by the enzyme could have a different structure. The enzyme is inactive towards N-acetyl-oligogalactosaminoglycan and chitosan. The endogalactosaminidase preparations also cleave high molecular weight galactosaminoglycan (obtained from Neurospora) into fragments greater than or equal to 10(4) daltons in molecular weight, and catalyze the release of Neurospora sporelings from the glass surfaces to which they are anchored. Galactosaminoglycan-cleaving and sporeling-releasing activities elute jointly from DEAE-cellulose columns. This observation provides further support for an earlier proposal that the sporelings are anchored to the glass by means of galactosaminoglycan molecules.     

In vitro metabolism or testosterone by breast microcysts

Breast microcysts are considered to be a normal findings in the adult female breast without any increased risk of developing carcinomatous change. Breast cysts fluid contains steroid but not studies have been reported on the ability of breast microcysts to metabolise steroid hormones. It was, therefore, the aim of this study to identify the metabolites formed on incubation of radiolabelled testosterone with microcysts. In all instances dihydrotestosterone and androstenedione were formed. Oestrogens were not identified. Tis study, therefore, provides evidence for th presence of 5-alpha-reductase and 17-oxidoreductase enzyme systems in breast microcysts.     

The embryonic expression patterns of zfh-1 and zfh-2, two Drosophila genes encoding novel zinc-finger homeodomain proteins.

The zfh-1 and zfh-2 genes of D. melanogaster encode novel proteins containing both homeodomain and C2-H2 zinc-finger DNA-binding motifs. Antisera against these proteins were used to investigate their expression patterns during embryonic development. The zfh-1 gene is expressed in the mesoderm of early embryos and in a number of mesodermally-derived structures of late embryos, including the dorsal vessel, support cells of the gonads, and segment-specific arrays of adult muscle precursors. In addition, zfh-1 is expressed in the majority of identified motor neurons of the developing CNS. The mesodermal zfh-1 expression requires the products of the twist and snail genes. The zfh-2 gene displays a more limited expression pattern, largely restricted to the CNS of late embryos. Ubiquitous zfh-1 expression in transgenic flies bearing an hsp70-zfh-1 construct has specific developmental consequences, including embryonic CNS defects as well as adult eye and bristle abnormalities. The expression patterns of zfh-1 and zfh-2 suggest that both genes may be involved in Drosophila neurogenesis and that zfh-1 may have additional functions in mesoderm development.     

IPA-3 Inhibits the Growth of Liver Cancer Cells By Suppressing PAK1 and NF-κB Activation

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to the high incidences of metastasis and tumor recurrence. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is frequently observed in HCC and is associated with a more aggressive tumor behavior, suggesting that PAK1 is a potential therapeutic target in HCC. In the current study, an allosteric small molecule PAK1 inhibitor, IPA-3, was evaluated for the potential in suppressing hepatocarcinogenesis. Consistent with other reports, inhibition of PAK1 activity was observed in several human HCC cell lines treated with various dosages of IPA-3. Using cell proliferation, colony formation and BrdU incorporation assays, we demonstrated that IPA-3 treatment significantly inhibited the growth of HCC cells. The mechanisms through which IPA-3 treatment suppresses HCC cell growth are enhancement of apoptosis and blockage of activation of NF-κB. Furthermore, our data suggested that IPA-3 not only inhibits the HCC cell growth, but also suppresses the metastatic potential of HCC cells. Nude mouse xenograft assay demonstrated that IPA-3 treatment significantly reduced the tumor growth rate and decreased tumor volume, indicating that IPA-3 can suppress the in vivo tumor growth of HCC cells. Taken together, our demonstration of the potential preclinical efficacy of IPA-3 in HCC provides the rationale for cancer therapy.