Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells.

In a previous report, herpes simplex virus type 2 (HSV-2) was shown to increase the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus of nonpermissive rat XC cells (L. Pilon, A. Royal, and Y. Langelier, J. Gen. Virol. 66:259-265, 1985). A series of 17 independent mutants were isolated after viral infection together with 12 spontaneous noninfected mutants to characterize the nature of the mutations induced by the virus at the molecular level. The DNA of the mutants isolated after viral infection was probed with cloned HSV-2 fragments representing the entire genome. In these mutants, no authentic HSV-2 hybridization could be detected. This was indicative of a mechanism of mutagenesis which did not require the permanent integration of viral sequences in the host genome. The structure of the hprt gene was determined by the method of Southern (J. Mol. Biol. 98:503-517, 1975), and the level of hprt mRNA was analyzed by Northern blots. Except for the identification of one deletion mutant in each of the two groups, the HPRT- clones showed no evidence of alteration in their hprt gene. A total of 7 of 12 spontaneous mutants and 11 of 15 mutants isolated from the infected population transcribed an hprt mRNA of the same size and abundance as did the wild-type cells. Thus, the majority of the mutants seemed to have a point mutation in their hprt structural gene. Interestingly, the proportion of the different types of mutations was similar in the two groups of mutants. This analysis revealed that HSV-2 infection did not increase the frequency of rearrangements but rather that it probably induced a general increase of the level of mutations in the cells. This type of response is thought to be compatible with the biology of the virus, and the possible mechanisms by which HSV-2 induces somatic mutations in mammalian cells are discussed.     

The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

Effect of snow removal on leaf water potential,soil moisture,leaf and soil nutrient status and leaf peroxidase activity of sugar maple

Effect of removal of snow cover in winter was investigated in an 80-year-old sugar maple (Acer saccharum Marsh.) stand in southern Quebec. We hypothesized that winter soil frost would induce some of the decline symptoms observed in sugar maple stands in southern Quebec in the early 1980's. Snow was continuously removed from around trees for a one week (partial removal) or for a four-month period (complete removal) during the 1990–1991 winter. Foliage and soils were sampled periodically during the summer of 1991. The complete snow removal treated trees showed decreased leaf water potential and increased peroxidase activity over most of the growing season. Foliar Ca was reduced in both snow removal treatments early in the growing season while foliar N was reduced in the complete snow removal trees late in the growing season. Soil NO ₃ ^– and K⁺ were elevated in both snow removal treatments at various times throughout the growing season. Prolonged soil frost in a sugar maple stand can induce lower leaf water potential, higher leaf peroxidase activity and early leaf senescence during the following growing season. Soil frost may have reduced nutrient uptake without affecting significantly the leaf nutrient status.     

Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme

We enzymatically deglycosylated pig lung angiotensin I-convertingenzyme (ACE) to study the involvement of its glycanic chainsin its physicochemical and catalytic properties. The effectsof endoglycosidases F₂ and H, and of N-glycanase were assessedby ACE mobility in SDS-PAGE. N-Glycanase only was completelyeffective with or without previous denaturation, leading toa shift in ACE M_r from 172 to 135 kDa; endoglycosidase F₂ producedthe same shift but only without previous denaturation. DeglycosylatedACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine,and an identical Stokes radius as measured by size-exclusionhigh performance liquid chromatography. Neuraminidase had noeffect on ACE Stokes radius but slightly decreased its kcatwhich could be related to variations in ionization of the activesite. The isoelectric point of ACE, as, determined by isoelectricfocusing, increased from 4.5–4.8 to 5.0–5.3 aftereither endoglycosidase F₂ or neuraminidase digestion, but stillwith microheterogeneities which thus did not seem to be relatedto ACE glycans. Deglycosylated ACE did not bind onto agaroselectinsin contrast to native ACE which bound strongly to concanavalinA showing interactions involving oligomannosidic or biantennaryand sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin,an inhibitor of N-glycosylation, did not modify ACE secretionby endothelial cells. Thus, ACE glycans have no drastic effectson structural and biological properties of the protein, butthey may have a functional role on intracellular targeting ofboth secreted and membrane-bound ACE isoforms, also for theprotection of the soluble plasma form against hepatic lectinsand the maintenance of its hydrosolubility. converting enzyme (peptidyldipeptidase EC.3.4.15.1) endothelium glycosidases lectins     

Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP

Abstract: As cerebral neurons express the dopamine D₁ receptor positively coupled with adenylyl cyclase, together with the D₃ receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D₃ receptor signaling pathways. In NG108-15 cells transfected with the human D₃ receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [³H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole-induced [³H]thymidine incorporation. This effect was maximal with 100 n M forskolin, occurred after 6–16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D₃ receptor expression up to 135%, but only after 16 h and at concentrations of >1 µ M . Thus, in this cell line, the D₃ receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D₃ receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D₃ receptor can involve both opposite and synergistic interactions with cyclic AMP.     

Development of crystalline peroxisomes in methanol-grown cells of the yeast Hansenula polymorpha and its relation to environmental conditions

The development of peroxisomes has been studied in cells of the yeast Hansenula polymorpha during growth on methanol in batch and chemostat cultures. During bud formation, new peroxisomes were generated by the separation of small peroxisomes from mature organelles in the mother cells. The number of peroxisomes migrating to the buds was dependent upon environmental conditions. Aging of cells was accompanied by an increase in size of the peroxisomes and a subsequent increase in their numbers per cell. Their ultimate shape and substructure as well as their number per cell was dependent upon the physiological state of the culture. The change in number and volume density of peroxisomes was related to the level of alcohol oxidase in the cells. Development of peroxisomes in cells of batch cultures was accompanied by an increase in size of the crystalline inclusions in the organelles; they had become completely crystalline when the cells were in the stationary phase. Peroxisomes in cells from methanol-limited chemostat cultures were completely crystalline, irrespective of growth rate. Results of biochemical and cytochemical experiments suggested that alcohol oxidase is a major component of the crystalline inclusions in the peroxisomes of methanol-grown Hansenula polymorpha. Possible mechanisms involved in the ultrastructural changes in peroxisomes during their development have been discussed.Abbreviations DAB 3,3-diaminobenzidine - OD optical density (663 nm)     

Transient expression of the CD2 cell surface antigen as a sortable marker to monitor high frequency transfection of human primary B cells.

We have used the T cell surface molecule CD2 gene, expressed from the human cytomegalovirus promoter as a reporter to optimize a transfection system for human primary B cells. The CD2-encoding DNA was transfected into cells by electroporation and transient expression was monitored by flow cytometric analysis. By using our optimal electroporation conditions on activated primary B cells, more than 30% of the resulting viable cells expressed CD2 on the cell surface. Moreover, unactivated primary B cells could also be transfected using this system but subsequent expression of CD2 required cellular activation. Magnetic beads or plastic culture bottles coated with anti-CD2 antibodies have been used to selectively purify transfected cells. The high transfection efficiency combined with the ability to specifically purify transfected cells may allow future studies on specific genes transiently expressed in human primary B cells.     

Expression in Escherichia coli and purification of a translocation-competent precursor of the chloroplast protein ferredoxin

The precursor of the chloroplast protein ferredoxin from Silene pratensis was expressed in Escherichia coli. When a low copy number plasmid was used, the preferredoxin level was low, and the protein was soluble. The expression level was increased by using a high copy number plasmid. In protease-deficient cells transformed with the latter plasmid, the preferredoxin accumulated up to 1% of total protein, and it was found in insoluble aggregates. These aggregates were dissolved in 4 M urea, and the protein was purified to homogeneity. Amino-terminal sequencing confirmed the amino acid sequence as deduced from the copy DNA. However, the first methionine residue of the expected sequence was absent in E. coli. The purified precursor was readily imported by isolated chloroplasts and processed to the mature size.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.