Myeloblastosis associated virus (MAV) proteinase site-mutated to be HIV-like has a higher activity and allows production of infectious but morphologically altered virus.

We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associated virus (MAV), containing a genetically altered proteinase (PR). A site-directed mutant of MAV-PR that shows an increased proteolytic activity in vitro (about 20 times higher kcat/Km) as a consequence of substituting five amino acids from the substrate-binding pocket with those corresponding to the HIV-1 PR was cloned into a full-sized MAV plasmid. In particular, the wild-type MAV-PR gene was replaced with the mutant one. Despite encoding for an enzyme with increased PR activity, mutant plasmid-transfected turkey fibroblasts displayed an unimpaired virus production in cell cultures. Further, the mutant progeny virus was infectious and its pattern of gag processing products appeared identical to that of wild-type virus. However, by electron microscopy we found that the predominant morphology of mutant viral particles was altered. Instead of a centrally collapsed avian retroviral core, a more diffuse core was visualized for wild-type mutant virions, similar to that observed in mammalian C-type retroviruses.

The 96-kilodalton antigen as an integral membrane protein in pathogenic Entamoeba histolytica: potential differences in pathogenic and nonpathogenic isolates.

A surface antigen (EH-96) of Entamoeba histolytica was demonstrated to be a plasma membrane antigen by immunoprecipitation of metabolically 35S-labeled antigen from live trophozoites, Triton X-114 detergent extracts, and plasma membrane-enriched fractions prepared by concanavalin A membrane stabilization and differential centrifugation. In addition, the antigen was localized to the plasma membrane by electron microscopy with colloidal gold. Antigen from E. histolytica strains immunoprecipitated with specific immunoglobulin M (IgM) or IgG2b monoclonal antibody was identical by one-dimensional peptide mapping with N-chlorosuccinimide. Additionally, antigen from different axenically cultivated amebae was demonstrated to be identical by N-chlorosuccinimide peptide mapping, as were peptide maps of IgG and IgM monoclonal antibody-purified antigen. The 96-kilodalton (kDa) surface antigen was identified on four axenically cultivated pathogenic isolates and on three polyxenically cultivated pathogenic isolates (zymodeme II) of E. histolytica but was absent or present in lesser quantity on six nonpathogenic polyxenically cultivated isolates. The 96-kDa antigen was detected in liver abscess fluid from four patients with amebic abscesses by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Two-dimensional gel electrophoresis profiles of the 96-kDa antigen purified from abscess material or from polyxenically cultivated trophozoites demonstrated that the antigens were related to the 96-kDa antigen found in axenically cultivated organisms.

Adhesion of type A Pasteurella mulocida to rabbit pharyngeal cells and its possible role in rabbit respiratory tract infections.

Pasteurella multocida serotype A was found in association with the mucosal epithelium of the nasopharynges of rabbits with respiratory tract infections. The bacteria specifically attached to squamous epithelial cells of the pharyngeal mucosa both in vivo and in vitro and to some tissue culture cell lines such as HeLa. All strains with serotype A capsules were adhesive. With the exception of one serotype D strain, strains with capsular serotypes B, D, and E were at least 10-fold less adhesive. Bacterial adhesiveness was much reduced after pronase digestion, heat treatment, and homogenization, but removal of the hyaluronic acid capsule increased adhesion. Electron microscopy revealed that fimbriae were produced by an adhesive pasteurella strain, but not by two nonadherent strains. The attachment of the former strain to pharyngeal and HeLa cells was inhibited by N-acetyl-D-glucosamine. Together, these findings suggest that this amino sugar may be a component of the receptor on both animal cell surfaces and that the fimbriae may be the adhesions. It is proposed that bacterial attachment has a role in colonization and infection of rabbit upper respiratory mucosae.

Persistent infection of BHK-21 cells with pneumonia virus of mice.

Trypsinization of BHK-21 cells 72 h after primary infection with pneumonia virus of mice yielded clones of persistently infected cells which specifically adsorbed murine erythrocytes. We describe one clone of cells, the progeny of which, after more than 100 passages, still bore viral antigen demonstrable by immunofluorescence and immune electron microscopy, but produced little or no detectable infectious virus.

Electron microscopic studies on assembly of herpes simplex virus upon removal of hydroxyurea block.

The release of hydroxyurea-treated, herpes simplex virus-infected cells from the drug-induced block resulted in the prompt assembly of infectious virus. Electron microscope observations at sequential intervals following removal of the drug revealed considerable synchrony of replication. This synchrony permitted stages in the complex process of core assembly to be examined in detail. The data suggest that after partial or complete assembly the nucleoprotein enters the differentiated capsid to become enfolded.

Electron microscopic appearance of silver sulfadiazine-treated Enterobacter cloacae.

Upon exposure of Enterobacter cloacae silver sulfadiazine, a number of ultrastructural changes involving the cell envelope take place. Foremost among these is a modification of the cell wall from an undulating structure to one which is smooth and has become enlarged. Strains of E. cloacae resistant to silver sulfadiazine do not exhibit these changes.

Properties of silver sulfadiazine-resistant Enterobacter cloacae.

Two silver sulfadiazine-resistant isolates of Enterobacter cloacae obtained in a burns unit where the drug was in use were studied. These strains were resistant to elevated levels of the drug, and they were cross-resistant to silver benzoate, but not to silver nitrate. Growth of the strains in nutritionally poor defined media sensitized them to the inhibitory action of the drug. Exposure of the bacteria to penicillins rendered them susceptible to silver sulfadiazine. The resistant bacteria harbored episomes for resistance to carbenicillin and kanamycin; however, resistance to silver sulfadiazine could not be transferred by these episomes. Twenty-three strains of E. cloacae isolated in a general hospital were sensitive to much lower levels of the drug (

Silver sulfadiazine: effect on the ultrastructure of Pseudomonas aeruginosa.

Pseudomonas aeruginosa exposed to silver sulfadiazine (AgSu) were examined in an electron microscope. The treated cells were distorted in shape, and structures (blebs) protruded from the cell surface. These "blebs" appeared to arise from the cell wall. A strain of P. aeruginosa resistant to AgSu did not display these changes. Upon exposure of P. aeruginosa to silver nitrate, none of these changes was seen; rather, such cells are characterized by large, central aggregations of nuclear material. The results are consistent with previous findings which suggested that AgSu acted at the cell surface.

Effect of hydroxyurea on Staphylococcus epidermidis and Micrococcus lysodeikticus: thickening of the cell wall.

Hydroxyurea-sensitive strains of Staphylococcus epidermidis and Micrococcus lysodeikticus showed marked thickening of cell walls and reduction in deoxyribonucleic acid synthesis when grown in the presence of hydroxyurea.

An effect of hydroxyurea on Staphylococcus epidermidis: temporary loss of viability.

Although hydroxyurea (HU) is recognized as a specific inhibitor of deoxyribonucleic acid (DNA) synthesis, it did not have this effect in a strain of Staphylococcus epidermidis. In this case, HU caused a loss of colony-forming ability but did not prevent cell division in liquid medium. DNA synthesis apparently was affected only secondarily. About 10% of the population recovered colony-forming ability during the latter part of the growth cycle or when growth was slowed by chloramphenicol. Recovery also occurred when HU was removed from the medium. HU prevented cellular autolysis.