During mitogen-induced transformation of human lymphocytes, phosphoglycerate kinase (PGK) exhibits new electrophoretic forms (pl=8.5-8.9). Electrophoresis and electrofocusing showed that the new forms are not due to expression of the autosomally linked isozyme found in semen (PGK-B; pl=9.7). The multiple electrophoretic forms are the result of protease modification of sex-linked PGK-A isozyme.When peripheral lymphocytes from young persons are stimulated in vitro with phytohemagglutinin, a selective increase in the levels of the glycolytic enzymes occurs concomitantly with blastogenesis. Human lymphocytes from a geriatric population were also subjected to mitogen stimulation. The initial levels of the enzymes were essentially identical in lymphocytes from young and old subjects as were mitogenfree cultured controls. However, during mitogen stimulation the cells from the old subjects failed to increase the glycolytic enzymes. This inability to activate glycolysis may be related to the decline in cell-mediated immunity which occurs with advancing age. Triosephosphate isomerase (TPI) has an increased thermolabile component in skin fibroblasts from patients with progeria (41.4 per cent)and Werner's syndrome (20.1 per cent) when compared with normal fibroblasts (0-3 per cent). The incorporation of various protease inhibitors failed to affect the percentages of heat-labile triosephosphate isomerases. The labile component appears to be identical to the deamidated …

This work presents the development of a highly sensitive and selective chemical assay for mono(ADP-ribose) residues covalently bound to proteins in vivo. An extensive review of the literature is presented in the introduction of this work. The physiological.functions of mono(ADP-ribosyl)transferase activities associated with certain bacterial toxins (e.g., diphtheria, cholera and pertussis toxins) are well established. However, the roles of endogenous vertebrate transferases are unknown. The elucidation of the roles of these cellular transferases will likely require identification of the physiologically relevant target proteins. Toward this end, it will also be important to identify the types of (ADP-ribose)-protein linkages present in vivo. ADP-ribosylation reactions catalyzed by the different bacterial and vertebrate transferases are specific for different amino acid acceptors in vitro. However, the vertebrate transferases that have been characterized thus far are NAD:arginine mono(ADP-ribosyl)transferases. The work presented here describes the development of a chemical assay for the detection of in vivo modified, ADP-ribosylated proteins containing N-glycosylic linkages to arginine. The assay was applied to the analysis of ADP-ribose residues in adult rat liver. The strategy employed for detection of protein-bound ADP-ribose residues eliminated potential artifacts arising from trapped nucleotides (or their degradation products), since the acid-insoluble material was completely dissolved in …

A new column, Dionex AS4A, (polystyrenedivinylbenzene matrix) used for the separation of ribonucleotides and deoxyribonucleotides for the first time, and previously used for ion analysis was found superior to conventional silica columns because it separates ribonucleotides and deoxyribonucleotides. Resolution of dGTP was not possible with the Dionex column and CTP and GDP often co-eluted. Using conventional silica columns, monophosphates separated from diphosphates and diphosphates from triphosphates. Using the new Dionex column resolves all three simultaneously. The Dionex column resolved nucleotides with sharper peaks than silica columns, and the longer its retention time the better was the resolution. This Dionex column is stable, with 80 runs possible without cleaning while resolving ribonucleotides and deoxyribonucleotides to the picomole level.

The focus of this research is to describe the production and characterization of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in insect cells, using Autographa californica buclear polyhedrosis virus (AcNPV) as an expression vector. All three forms of biological activity of hGM-CSF. Following N-glycanase treatment, the two glycosylated hGM-CSF proteins (15.5 and 16.5 KDa) which bound to Concanavalin A affinity column ran as a 14.5-15.5 KDa band on SDS-PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 KDa species. The N-terminal amino acid sequence of the recombinant hGM-CSF was identical to that of natural hGM-CSF deduced from cDNA. These results demonstrate that baculovirus-produced hGM-CSF could be N-glycosylated in Sf9 cells, the signal peptide of recombinant hGM-CSF could be recognized and cleaved by infected insect cells and the resultant molecule secreted into the medium.

The electrophysiological and morphological properties of small networks of mammalian neurons were investigated with mouse spinal cord monolayer cultures of 2 mm diameter grown on multimicroelectrode plates (MMEPs). Such cultures were viewed microscopically and their activity simultaneously recorded from 2 of any 36 fixed recording sites. The specific aims achieved were: development of techniques for production of functional MMEPs and maintenance of mini-cultures, characterization of the spontaneous activity of mini-cultures, application of inhibitory and disinhibitory agents, development of staining methods for cultured neurons and initial light microscopic analysis with correlation of electrophysiological and morphological characteristics.

Physical and restriction mapping of the salicylate catabolic plasmid SAL from Pseudomonas putida strain PpG 2119 was carried out by standard multiple restriction analysis and by cross hybridization studies using radioactively labeled restriction fragment probes. The total numbers of fragments produced, their respective sizes, the arrangement of the restriction fragments on the plasmid and the map locations of the enzyme recognition sites for Hpal, Xhol, Dral and Smal are given.

A new rapid computer assisted micro-titer plate interferon assay system was developed and characterized for use in high capacity clinical and research applications. The biological aspect of the assay was a modification of the assay methods of Finter, Armstrong and McManus. It was an application of spectrophotometric quantification of the reduction of viral cytopathic effect (CPE) as reflected by neutral red dye uptake by viable cells. A computer program was developed for the extrapolation of raw data to reference interferon units.