DNA-enzyme-mediated cleavage of human immunodeficiency virus type 1 Gag RNA is significantly augmented by antisense-DNA molecules targeted to hybridize close to the cleavage site

DNA-enzymes (Dzs) usually cleave short synthetic target RNAs very efficiently, but this activity diminishes significantly when tested on full-length RNAs, primarily because of the rigid secondary structures near the target sequence. We identified two Dzs, one each for 81-17 and 10-23 Dz, which cleaved the human immunodeficiency virus type 1 (HIV-1) Gag RNA poorly. We sought to use short oligodeoxynucleotides (ODNs) with the hope that it will facilitate Dz-mediated cleavage. The efficiencies of several ODNs were analyzed for their ability to augment the 8-17 Dz-mediated cleavage. We observed that ODNs that hybridized close to 5' and 3' ends of the target sequence were able to enhance significantly 8-17 Dz-mediated cleavage activity in a dose-dependent manner. The same was true for 10-23 Dz with ODNs that hybridized close to the target site. Thus, it was possible to enhance significantly the cleavage activity of poorly cleaving HIV-1 Gag-specific Dzs by using sequence-specific ODNs. This combination of antisense and catalytic Dz will, in principle, result in more effective gene suppression that could be exploited for therapeutic purposes.     

Primary cell culture from fish gills and kidney using fish serum

A novel, cost effective and time saving technique for primary cell culture from gills and kidney using fish serum has been developed. Single cell suspension of gills and kidney of Clarias gariepinus was obtained by trypsinization. Minimum essential medium supplemented with 10% fish serum in place of 10% foetal calf serum and 20% fish muscle extract, yielded confluent monolayer on 6th and 8th day in gill and kidney tissue respectively at 28 degrees C. Fish serum can be successfully used as media supplement for cultivation and maintenance of primary cell culture from fishes.     

Naturally occurring anti-alpha-galactosyl antibodies: relationship to xenoreactive anti-alpha-galactosyl antibodies.

Antibodies produced by an individual without a known history of sensitization to the relevant antigen are called "natural" antibodies. Some natural antibodies, called xenoreactive antibodies, react with the cells of foreign species. Most xenoreactive antibodies in humans and higher primates bind to a nonreducing terminal galactose expressed by pigs and other lower mammals. Although human natural antibodies which bind to one or more of a variety of terminal alpha-galactosyl structures have been identified previously, the antigen recognized by anti-alpha-galactosyl antibodies on the cells of foreign species is thought to be exclusively Galalpha1-3Gal. Thus, anti-alpha-galactosyl antibodies which do not react with Galalpha1-3Gal are thought to be nonxenoreactive. Here, we identify natural antibodies in human serum which bind to Galalpha1-6Hexosepyrranosides but not Galalpha1-3Gal, indicating that these antibodies are not xenoreactive. Various lower mammals were found to have natural anti-Galalpha1-2Gal antibodies in their sera, suggesting that at least some anti-Galalpha1-2Gal antibodies might not be xenoreactive and indicating, surprisingly, that anti-alpha-galactosyl antibodies are much more phylogenetically disperse than previously known. Also surprising was the finding that some natural antibodies which bind to Galalpha1-3Gal in vitro do not bind to porcine xenografts. These studies show that naturally occurring anti-alpha-galactosyl antibodies in mammalian serum include antibodies with a greater variety of reactivities than previously thought, only some of which would bind to a porcine xenograft. Further, these studies show that the methods used to detect anti-alpha-galactosyl antibodies of relevance in xenotransplantation must be carefully evaluated to avoid detection of anti-alpha-galactosyl antibodies which would not bind to a porcine organ and which therefore are not involved in xenograft rejection.     

The thrombomodulin-protein C system is essential for the maintenance of pregnancy

Disruption of the mouse gene encoding the blood coagulation inhibitor thrombomodulin (Thbd) leads to embryonic lethality caused by an unknown defect in the placenta. We show that the abortion of thrombomodulin-deficient embryos is caused by tissue factor-initiated activation of the blood coagulation cascade at the feto-maternal interface. Activated coagulation factors induce cell death and growth inhibition of placental trophoblast cells by two distinct mechanisms. The death of giant trophoblast cells is caused by conversion of the thrombin substrate fibrinogen to fibrin and subsequent formation of fibrin degradation products. In contrast, the growth arrest of trophoblast cells is not mediated by fibrin, but is a likely result of engagement of protease-activated receptors (PAR)-2 and PAR-4 by coagulation factors. These findings show a new function for the thrombomodulin-protein C system in controlling the growth and survival of trophoblast cells in the placenta. This function is essential for the maintenance of pregnancy.     

Thermal cycling aids folding of a recombinant human beta-casein with four extra N-terminal amino acid residues

Due to the limited secondary structure, it is believed that the caseins of milk, particularly the beta-caseins (beta-CN), may be in a mostly random-coil conformation or in various structures that result from random association of hydrophobic residues. However, the self-association of the human proteins with increasing temperature (T) and in the presence of Ca2+ is reproducible, implying that they normally fold into fixed tertiary structures. A nonphosphorylated recombinant human beta-CN with four extra amino acids at the N-terminus (GSHM-) was prepared and studied by laser light scattering, analytical ultracentrifugation, fluorescence spectroscopy, turbidity, and circular dichroism. In 3.3 M urea or at 4 degrees C, the protein was monomeric, as expected. Increasing T both without and with the addition of Ca2+ ions caused self-association as it does for the nonphosphorylated native beta-CN but with a somewhat different interaction pattern. However, returning the protein to its monomeric state by reequilibration at 4 degrees C followed again by increasing T caused a shift in the pattern. Such thermal cycling eventually caused the protein to equilibrate to a particular conformation where no more change could be observed. The resulting interaction pattern was similar to that of the native protein but differed particularly in that there was more extensive self-association for the recombinant mutant. The equilibration to a stable conformation was more rapid in the presence of Ca2+ ions. This suggests that the native protein normally folds into a particular conformation which may be aided by Ca2+ in the mammary gland. Further study of a recombinant form with the native amino acid sequence is needed.     

Sequential pathological studies in Asian water buffaloes infected intratracheally with Absidia corymbifera

Zygomycosis was produced experimentally in Asian water buffalo calves (Bubalus bubalis) by intratracheal inoculation of sporangiospores of Absidia corymbifera. Infected animals exhibited dullness, depression, partial anorexia, initial pyrexia, mucopurulent nasal and ocular discharge and coughing during the first week. There was no mortality at any stage of the experiment, which continued for 30 days. The gross and microscopic lesions were restricted to the lungs and there was no dissemination of the fungus to other organs. Gross and microscopic changes in the lungs were observed up to the 20th day post-infection. Gross lesions consisted of pneumonic consolidation of the antero-ventral lobes of the lungs. Microscopic changes consisted of granulomatous reactions with well developed pulmonary granulomas. Distorted hyphae of A. corymbifera were demonstrated in tissue sections up to 15 days post inoculation. Re-isolation of the fungus was achieved consistently for up to 15 days. It is concluded that intratracheal inoculation of A. corymbifera in buffalo calves leads to significant pathological changes in the lungs, but the disease appears to be self limiting 20 days following inoculation.