Inhibitors of Urokinase and Thrombin in Cultured Neural Cells

Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum-free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate-stable complexes with 125I-urokinase or 125I-thrombin. Rinsed glioblastoma possessed two components that complexed 125I-urokinase. One was type 1 plasminogen activator inhibitor (PAI-1), because the 125I-urokinase-containing complexes were immunoprecipitated with anti-PAI-1 antibodies. The other component formed complexes with 125I-urokinase that were not recognized by antibodies to PAI-1 or protease nexin-1 (PN-1). Its identity is unknown. In addition to these cell-bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with 125I-urokinase; one was PAI-1, and the other was PN-1. The secreted PN-1 also formed complexes with 125I-thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes with either 125I-urokinase or 125I-thrombin. However, human neuroblastoma cells did contain very low levels of PN-1 mRNA and PN-1 protein. Added PN-1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN-1 and blocked the ability of PN-1 to form complexes with 125I-urokinase. Thus, cell-bound PN-1 was a specific thrombin inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replication-defective mutants of herpes simplex virus (HSV) induce cellular immunity and protect against lethal HSV infection.

Live viruses and live virus vaccines induce cellular immunity more readily than do inactivated viruses or purified proteins, but the mechanism by which this process occurs is unknown. A trivial explanation would relate to the ability of live viruses to spread and infect more cells than can inactivated virus. We have used live but replication-defective mutants to investigate this question. Our studies indicate that the immune responses of mice to live virus differ greatly from the responses to inactivated virus even when the virus does not complete a replicative cycle. Further, these studies indicate that herpes simplex virus-specific T-cell responses can be generated by infection with replication-defective mutant viruses. These data indicate that the magnitude of the cellular immunity to herpes simplex virus may be proportional to the number or quantity of different viral gene products expressed by an immunizing virus.     

Purification and characterization of cytosolic 6-phosphogluconate dehydrogenase isozymes from maize

Cytosolic isozymes of 6-phosphogluconate dehydrogenase were purified from roots of maize (Zea mays L.). The final preparation contained two 55-kD proteins. Affinity-purified dehydrogenases from a maize line that is null for both cytosolic 6-phosphogluconate dehydrogenase isozymes (Pgd1-null, Pgd2-null) lacked the 55-kD proteins. The substrate kinetics of the purified enzyme were determined.     

A rapid method for counting cell nuclei using a particle sizer/counter

Summary We report here an improved method for nuclei counting utilizing Triton-X 100 to reduce the size of cell debris, thereby allowing the use of a particle sizer/counter. Furthermore, nuclei are completely released within 30 seconds, as compared to 1 hour using hypotonic solution. The method is accurate above 0.3 × 10⁶ cells/mL.     

Studies on the primary sequence requirements for PKC-alpha, -beta 1 and -gamma peptide substrates.

The substrate specificity of purified PKC-alpha, -beta and -gamma has been investigated. A series of synthetic peptides based upon the sequence surrounding serine-7 in glycogen synthase were generated and used to determine the basic residue requirements of these PKC isotypes. While PKC-alpha and -beta are indistinguishable in their phosphorylation of these peptides, PKC-gamma shows a distinct specificity profile for these synthetic substrates.     

Interaction between corticosteroid binding globulin and activated leukocytes in vitro

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.     

Solitary angiomyolipoma of the liver. Report of a case initially examined by fine needle aspiration biopsy

Transabdominal fine needle aspiration biopsy of a solitary space-occupying lesion in the liver produced smears containing irregular bundles of smooth muscle cells with granular or fibrillary cytoplasm and slightly pleomorphic nuclei. In a few bundles, aggregates of mature fat cells were present, which is characteristic for an angiomyolipoma. Histologic examination of the resected mass showed it to be a solitary angiomyolipoma of the liver. The diagnosis was further confirmed by immunohistochemical and electron microscopic studies.     

Regional localization of the genes for human HEXB. PGK, GALA. HPRT, G6PD by somatic cell hybridization

Twenty independent man-mouse (Cl1D,LA/TK-, HPRT-) and man-hamster (CH,HPRT-) hybrids using female human cells with balanced reciprocal translocation XX,t(X;5)(q21;q11) were analyzed for human genes localized on chromosome 5 (HEXB), on chromosome X (PGK, GALA, HPRT, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.5, chr.5q-, chr.Xq+, chr.X). The different results obtained indicate that the genes for human markers HEXB, PGK are on Xq+, and that the genes for human markers GALA, G6PD are on 5q-. These data implicate finally the following localizations: HEXB on 5q11 leads to 5qter; PGK on Xq21 leads to Xpter; GALA, HPRT, G6PD on Xq21 leads to Xqter.