Production of laminin B2 chain protein and messenger RNA by a murine neutrophil precursor cell line, 32Dc13.

Laminin is a heterotrimeric glycoprotein that plays a central role in promoting neutrophil chemotaxis, motility, and attachment to basement membrane. Rabbit peritoneal exudate neutrophils stain positively for laminin, which is presumed to be of exogenous origin and bound to laminin receptors on the cell surface. We examined 32Dc13 cells, a murine neutrophil precursor cell line, by immunoprecipitation. Northern blot analysis, flow cytometry, and electron microscopy for the endogenous production of laminin. Our results demonstrate that 32Dc13 cells endogenously produce a laminin B2 chain protein and messenger RNA (mRNA) without producing any detectable A or B1 chain protein or mRNA. The B2 chain protein was not secreted by the cells; rather it could be detected on the cell surface after treatment of cells with neuraminidase. These findings suggest the possibility of a novel role for the laminin B2 chain in myeloid development and function.

Cellular mechanisms in the superinfection exclusion of vesicular stomatitis virus.

The superinfection exclusion of VSV has been studied and found to be caused by a combination of three distinct effects on endocytosis by VSV-infected cells: first, a decreased rate of formation of endocytic vesicles as judged by an inhibition of fluid-phase uptake at 2 hr postinfection; second, a decreased rate of internalization of receptor-bound ligands, which was detected at 4 hr postinfection; and third, a competition with newly synthesized virus for occupancy of coated pits, as indicated by electron microscopy of infected cells. At the same time that fluid-phase uptake decreased, numerous uncoated invaginations were observed at the cell surface.

Biological activity and electron microscopy of poliovirus 14S particles obtained from alkali-dissociated procapsids.

Highly purified 14S subunit particles were obtained from alkali-dissociated poliovirus type 1 procapsids (naturally occurring empty capsids in poliovirus-infected cells) to compare their morphological and biophysical properties with those of naturally occurring 14S particles. Procapsid-derived 14S particles (PC-14S), like naturally occurring 14S particles, were capable of self-assembly into an empty shell in buffer or extracts from uninfected cells. These empty capsids always exhibited pIs more acidic than those of procapsids but were themselves distinguishable by their respective pIs. Nevertheless, if PC-14S or naturally occurring 14S particles were incubated with extracts made from poliovirus-infected cells, procapsidlike empty shells were formed. This clearly showed that the 14S particle, however obtained, possesses the information to form an empty shell of correct dimensions but of improper conformation, unless a factor present in poliovirus-infected cells is present. With the electron microscope, the PC-14S subunit frequently was seen as a pentagonal structure with a diameter of 20.4 +/- 1.4 nm, a size somewhat larger than expected for a subunit composing 1/12th of the poliovirus surface. Upon self-assembly in vitro, the empty shell formed exhibited a diameter of 29 +/- 1 nm and a wall thickness of ca. 6 to 7 nm. It was necessary to avoid CsCl banding of procapsids in their preparation as this treatment altered both their pI and their sensitivity to alkali dissociation into 14S subunits. The relevance of these findings to the nature and role of procapsids and the requirement for a morphopoietic factor in poliovirus morphogenesis is discussed.

Induced versus pre-existing asymmetry models for the half-of-the-sites reactivity effect in bovine liver uridine diphosphoglucose dehydrogenase.

Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior is due to induction effects, the magnitude of the induction could be expected to depend on the nature of the covalent modification. On the other hand, if the half-sites reactivity behavior is due to pre-existing asymmetry and there is no communication between catalytic centers, the properties of unmodified sub-units should be independent of the nature of the covalent derivative introduced on the modified subunits. According to the induced asymmetry hypothesis, the catalytic activity of half-sites modified enzyme might be different for different covalent modifications, whereas for the rigid pre-existing asymmetry hypothesis the catalytic activity of half-sites modified enzyme should be the same regardless of the modifying group. During the course of catalytic site thiol group modification by a number of thiol specific reagents, the loss of enzyme activity was equivalent to the degree of modification for most of the reagents employed. However, with iodoacetate and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, half-sites modification of UDPglucose dehydrogenase reduced catalytic activity by 58 and 78%, respectively, of the initial activity. These observations are consistent with a model in which there is communication between catalytic sites. Electron microscopy shows that the six subunits of UDPglucose dehydrogenase are arranged as a hexagonal planar ensemble.