Spatially periodic discrete contact regions in polylysine-induced erythrocyte-yeast adhesion

Cell-cell adhesion occurs when human erythrocytes and yeast cells are suspended together in suprathreshold concentrations of polylysine in saline. The threshold polycation concentration for adhesion depends on cell concentration and decreases with increasing polycation molecular weight. The threshold concentration was similar for erythrocyte-erythrocyte adhesion and for yeast-erythrocyte adhesion. Transmission electron micrographs show that the erythrocytes adhere to yeast as if to engulf the cell. The regions of close contact between the erythrocyte membrane and the yeast cell walls are spatially discrete. The contact separation distance for the asymmetric erythrocyte-yeast adhesion is very similar to that (0.83 micron) observed when polylysine-induced adhesion occurs in the symmetrical erythrocyte-erythrocyte system. The spacing is attributed to the growth of a squeezing wave as an interfacial instability, on the intercellular aqueous layer. Freeze-fracture electron microscopy of cells that were not fixed during preparation for microscopy confirms the discrete nature of contacts between polylysine treated erythrocytes.     

Reduced yield of infectious pseudorabies virus and herpes simplex virus from cell lines producing viral glycoprotein gp50.

Pseudorabies virus (PRV) glycoprotein gp50 is the homolog of herpes simplex virus (HSV) glycoprotein D. Several cell lines that constitutively synthesize gp50 were constructed. Vero cells, HeLa cells, and pig kidney (MVPK) cells that produce gp50 all gave reduced yields of PRV and HSV progeny viruses when compared with the parent cell line or the same cell line transfected to produce a different protein. The reduction in virus yield was greatest at low multiplicities of infection. The Vero and HeLa cells that produce gp50 showed an even greater reduction in HSV yield than in PRV yield. This phenomenon may be an example in a herpesvirus of the interference observed in retroviruses or cross-protection in plant virus systems.     

Characterization and possible opioid modulation of N-methyl-D-aspartic acid induced increases in serum luteinizing hormone levels in the developing male rat

It has been previously reported that the excitatory amino acid, N-methyl-D-aspartic acid (NMDA), elicits prompt increases in serum luteinizing hormone (LH) levels in young male rats. The present studies were carried out to determine whether the effects of NMDA on LH were mediated by the release of LHRH from the hypothalamus. We also examined whether NMDA-sensitive neuronal pathways interacted with the endogenous opioid system regulating LHRH release and the ontogeny of NMDA-evoked increases in serum LH. We found that the age-response curve for NMDA-induced increases in LH was an inverted U; at early ages (10 and 15 days) the amino acid was marginally effective in increasing LH levels, it became maximally effective from post-natal days 20-40 and thereafter rapidly lost its efficacy such that it was virtually inactive in adult animals. Dose-response curves revealed that adult animals were more than 10-fold less sensitive to NMDA than their younger counterparts. Our studies also demonstrated that NMDA increased LH via a direct effect on the hypothalamic release of LHRH since a potent LHRH antagonist competitively inhibited the effects of NMDA. Finally, we observed that morphine competitively inhibited the effects of NMDA on LH release, suggesting a relationship between NMDA-sensitive neuronal pathways and those endogenous opioid-containing systems which are known to regulate LH release.     

Correlation of physical maps and some genetic functions in the genomes of the kappa-theta phage family of Bacillus licheniformis

Summary Natural recombinant genomes between several, phenotypically distinct forms of phages and were isolated and characterized by DNA restriction fragment mapping and electron microscopic heteroduplex analysis. The phenotypes of the recombinants were correlated with the physical maps of the genomes, and several genetic functions were therfore defined and mapped. All genes necessary for the assembly of infectious virus particles map in a contiguous tract of DNA comprising about 20 kb, or nearly one third of the genome length. No DNA homology occurs within these domains of the two genomes, so that homologous recombination does not take place here and phenotypic mixing of the phages is eo ipso excluded. Other regions of heterology contain regulatory genes responsible for the lytic or temperate character of the phages, and for exclusion of phage by .     

A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae

Summary We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.     

Human debrisoquine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22

The enzyme P450db1 (db1) is responsible for the common human defect in drug oxidation known as the "debrisoquine/sparteine polymorphism." Polyclonal antibody against the rat db1 protein was used to screen a human liver lambda gt11 library for the db1 cDNA clone. A cDNA containing the full protein coding sequence was isolated; the deduced NH2-terminal sequence of this cDNA was identical to that derived from direct sequencing of the purified human db1 protein. Comparison of the human db1 with rat db1 revealed 71 and 73% similarities of nucleotides and amino acids, respectively. By use of human-rodent somatic cell hybrids the db1 gene was localized to human chromosome 22 (CYP2D locus).     

Amphiphysin, a novel protein associated with synaptic vesicles.

To obtain access to novel proteins of the neuronal synapse, we have raised antisera against proteins of synaptic plasma membranes and used them for immunoscreening brain cDNA expression libraries. One of the newly isolated cDNAs encodes an acidic protein of 75 kDa with a distinct architecture of structural domains and multiple potential phosphorylation sites. Light and electron microscopy employing monospecific antisera raised against the expression product indicate a synapse-specific, presynaptic localization of this protein in many synapses of the chicken and rat nervous system. Its overall distribution in brain is very similar to that of synaptophysin, a ubiquitous protein of synaptic vesicles. In addition to brain, the protein or its mRNA is expressed in adrenal gland and anterior and posterior pituitary, but was not detected in a variety of other tissues. In controlled pore glass chromatography the native protein copurifies with synaptic vesicles and largely remains associated with them under various washing conditions. However, its amino acid sequence is very hydrophilic and it segregates into the aqueous phase in detergent phase partition. An earlier step of synaptic vesicle purification, sucrose cushion centrifugation, separates a vesicle-bound fraction of this protein from an unbound fraction. This seems to be a new, perhaps peripheral, protein of synaptic vesicles for which we propose the name, amphiphysin.     

Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.

The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.     

Detection of a cytotoxic substance produced by the cyanobacteriumMicrocystis aeruginosa strain PCC 7806: Isolation and differentiation from the peptide toxin microcystin-LR by cytotoxicity assays

Exposure of four permanent cell lines to crude aqueous extracts of the cyanobacteriumMicrocystis aeruginosa (strain PCC 7806) resulted in rounding and lysis of the cells within a few minutes. Cell damage was quantified by determination of lactate dehydrogenase (LDH) activity in the cell culture supernatants. By gel filtration, cation exchange chromatography and SDS-PAGE, the cytotoxic effects could be related to a defined substance with an apparent molecular weight of about 35 kDa, which was not heat resistant. The physicochemical properties very clearly mark off this compound from the peptide toxin microcystin-LR, which did not show any detectable cytotoxic effects.     

Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays.

The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(lambda). Exposure takes place to serial dilutions of the test compound in microtitre wells (250 microliters) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.