Cell-specific processing of pro-cholecystokinin and pro-gastrin

The present review argues that the gastrin-cholecystokinin family is a suitable model for the study of cell-specific processing of pro-hormones. First, the homologous active site of the hormones is a precisely defined tetrapeptide amide, which is well preserved during evolution. Second, the genes of both hormones are translated in a variety of cells (neurons, endocrine cells, paracrine cells, lymphocytes, etc,), but to a varying degree during ontogenesis and pathogenesis of various diseases. Third, each pro-hormone contains multiple processing sites (mono- and dibasic cleavage sites, amidation sites and consensus sequences for seryl phosphorylation and tyrosyl sulfation) leaving ample room for variations in the post-translational processing. The review discusses examples of cell-specific processing that appears to be functionally expedient.     

Kinetic analysis of an E.coli phenylalanine-tRNA synthetase mutant.

A mutation in the pheS gene, encoding phenylalanyl-tRNA synthetase, in E. coli NP37 confers temperature-sensitivity on the organism. A five-fold increase in tRNA(phe) levels complements the mutation. Analysis of the kinetic properties of the mutant enzyme indicates that the KM is 20-fold higher than the wild-type and the dissociation constant of the tRNA(phe)-synthetase complex for the mutant is at least 10-fold higher. These results indicate that the mutation in E. coli NP37 directly affects the tRNA(phe) binding site on the cognate synthetase.     

Regulation of γ-Aminobutyric Acid/Barbiturate Receptor-Gated Chloride Ion Flux in Brain Vesicles by Phospholipase A2: Possible Role of Oxygen Radicals

Preincubation of brain membranes with phospholipase A2 (PLA2) has been shown previously to affect the binding characteristics of various recognition sites associated with the gamma-aminobutyric acid (GABA) receptor complex. In the present study, we have investigated the effects of PLA2 (from Naja naja siamensis venom) on the functional activity of the GABA receptor/chloride ion channel. PLA2 (0.001-0.02 U/mg protein) preincubation decreased pentobarbital-induced 36Cl- efflux and muscimol-induced 36Cl- uptake in rat cerebral cortical synaptoneurosomes. The effect of PLA2 was prevented by EGTA and two nonselective PLA2 inhibitors, mepacrine and bromophenacyl bromide. The removal of free fatty acids by addition of bovine serum albumin both prevented and reversed the effect of PLA2. Products of the catalytic activity of PLA2, such as the unsaturated free fatty acids, arachidonic and oleic acids, mimicked the effect of PLA2. However, the saturated fatty acid, palmitic acid, and lysophosphatidyl choline had no effect on pentobarbital-induced 36Cl- efflux. Because unsaturated free fatty acids are highly susceptible to peroxidation by oxygen radicals, the role of oxygen radicals was investigated. Xanthine plus xanthine oxidase, a superoxide radical generating system, mimicked the effect of PLA2, whereas the superoxide radical scavenger, superoxide dismutase, diminished the effects of PLA2 and arachidonic acid on pentobarbital-induced 36Cl- efflux. Similarly, the effect of PLA2 was also inhibited by methanol (1 mM), a scavenger of the hydroxyl radical, and by catalase. These data indicate that exogenously added PLA2 induces alterations in membrane phospholipids, possibly promoting the generation of oxygen radicals and fatty acid peroxides which can ultimately modulate GABA/barbiturate receptor function in brain.     

Influence of SLA haplotype on preimplantation embryonic cell number in miniature pigs

Embryonic cell number in miniature pigs inbred for specific SLA haplotypes (a, c, and d) was determined on Day 6 by nuclear staining and, on Days 9 and 11, by DNA analyses (first day of oestrus = Day 0). Pigs exhibiting first behavioural oestrus at 08:00 h were hand-mated to an SLA homozygous boar 12 and 24 h later. Numbers of embryos flushed from uteri at 08:00-10:00 h on Days 6, 9 and 11 were greater (P less than 0.05) for SLAd females than for SLAa or SLAc females, which did not differ (8.2 vs 6.8 and 6.2, respectively). Recovery rates (embryos recovered/CL number) were similar, averaging 75.8% for all three SLA haplotypes. Embryos from SLAd dams contained fewer blastomeres (23 cells) on Day 6 than did embryos from SLAa (89 cells) or SLAc (79 cells) females. The reduced cell numbers of SLAd vs SLAa or SLAc embryos continued to Day 9 (28 vs 107 and 67 ng DNA/embryo) and Day 11 (167 vs 674 and 586 ng DNA/embryo). These results suggest an effect of the SLA complex on preimplantation embryonic development.     

Asialoglycoprotein receptor phosphorylation and receptor-mediated endocytosis in hepatoma cells. Effect of phorbol esters

The asialoglycoprotein (ASGP) receptor on Hep G2 cells undergoes constitutive recycling and ligand endocytosis in the presence of phorbol dibutyrate, at a 50% reduced rate relative to control cells (Fallon, R. J., and Schwartz, A. L. (1986) J. Biol. Chem. 261, 15081-15089). The relevance of receptor phosphorylation to these events was investigated by selective immunoprecipitation of surface receptors with polyclonal anti-human ASGP antiserum and pulse-chase labeling with [32P]orthophosphate to identify subcellular locations of initial receptor phosphorylation events as well as the eventual fate of phosphorylated receptor during recycling. The surface immunoprecipitation method recovers greater than 95% of surface ASGP receptors and only 5% or less of intracellular (brief[35S]methionine pulse-labeled) receptors. With this assay we detected low levels of ASGP receptor phosphorylation at the cell surface in control cells (0.1 mol of P/mol of R) which were rapidly (less than 1 min) stimulated 20-fold by 400 nM phorbol dibutyrate addition (1.7 mol of P/mol of R). Staurosporine, a protein kinase C inhibitor, blocks this stimulation by phorbol. Receptor phosphorylation at early time points in the presence of phorbol esters was restricted to the plasma membrane. Subsequent chase in the presence of excess unlabeled phosphate and phorbol esters lowered [32P] ATPi specific activity by 68% at 1 h. Surface immunoprecipitation during this chase period showed the phosphorylated ASGP receptors were rapidly lost from the cell surface (t1/2 = 20 min). In contrast, examination of intracellular receptor during the pulse-chase experiment in phorbol dibutyrate-treated cells showed the presence of phosphorylated pool(s) of ASGP receptors which were detectable for 6 h of chase. Since no labeled receptor can be detected at the cell surface at this time, the described intracellular phosphorylated receptors are in a non-recycling pool.     

Effect of gamma radiation on resting B lymphocytes. II. Functional characterization of the antigen-presentation defect

The effect of radiation on three discrete Ag-presentation functions in resting B cells was examined: 1) Ag uptake and processing, 2) expression of processed Ag in the context of functional class II molecules, and 3) provision of necessary co-stimulatory, or "second," signals. Analysis of radiation's effect on B cell presentation of intact vs fragmented Ag or its effect on presentation by Ag-pulsed B cells indicated that damage to Ag uptake and processing could not account for the bulk of the radiation-induced Ag-presentation defect. Experiments with phosphatidylinositol hydrolysis as an indirect measure of TCR occupancy suggested that irradiation caused a fairly rapid (within 1 to 2 h) decrease in the ability of the B cell APC to display a stimulatory combination of Ag and class II molecule. Ag dose-response analyses demonstrated that when presenting a fragment of the Ag pigeon cytochrome c to a T cell clone, 3000 rad-treated B cell APC were able to stimulate approximately 50% as much phosphatidylinositol turnover as unirradiated B cells. It was also found that, in contrast to their inability to initiate T cell proliferation, and similarly to chemically cross-linked splenocytes, heavily irradiated resting B cells plus Ag induced a state of Ag hyporesponsiveness in T cell clones. This effect on T cells had the same Ag- and MHC-specificity as did receptor occupancy required for proliferation, indicating that heavily irradiated resting B cells bear functional class II molecules. Co-culture of T cells with allogeneic B cells and syngeneic heavily irradiated B cells or chemically cross-linked splenic APC plus Ag resulted in T cell proliferation and interfered with the induction of the hyporesponsive state. This co-stimulatory function was radiosensitive in resting allogeneic B cells. Together, these data support the hypothesis that the major functional consequences of radiation to resting B cell APC are a reduction in the effective display of Ag plus class II molecules and, probably what is more important, a loss in the ability to provide APC-derived co-stimulatory signals.     

Upper airway pressure-flow relationships in obstructive sleep apnea

We examined the pressure-flow relationships in patients with obstructive sleep apnea utilizing the concepts of a Starling resistor. In six patients with obstructive sleep apnea, we applied incremental levels of positive pressure through a nasal mask during non-rapid-eye-movement sleep. A positive critical opening pressure (Pcrit) of 3.3 +/- 3.3 (SD) cmH2O was demonstrated. As nasal pressure was raised above Pcrit, inspiratory airflow increased in proportion to the level of positive pressure applied until apneas were abolished (P less than 0.01). However, at pressures greater than Pcrit, esophageal pressures either did not correlate or correlated inversely with inspiratory airflow provided that esophageal pressure was less than Pcrit. When pressure was applied to a full face mask, inspiratory airflow did not occur and Pcrit could not be obtained at pressures well above Pcrit demonstrated with the nasal mask. These results are consistent with the view that the upper airway functions as a Starling resistor with a collapsible segment in the oropharynx. These findings offer a unifying construct for the association of sleep apnea, periodic hypopnea, and snoring.     

Mammalian cell lines can be efficiently established in vitro upon expression of the SV40 large T antigen driven by a promoter sequence derived from the human vimentin gene.

The aim of this study was to investigate a new method to enhance the efficiency to create mammalian cell lines. Cell immortalization was achieved by intranuclear microinjection of a recombinant DNA construct composed of a constitutive promoter controlling the genes encoding immortalizing proteins; the sequences coding for the large T and small t antigens were fused downstream of regulatory elements from the vimentin gene, the activation of which characterizes the vast majority of cells growing in vitro. Data show that the efficiency of the immortalizing procedures using the SV40 early genes could be enhanced by the control elements derived from the human vimentin (HuVim) 5' sequences that contained nucleotides -878 to +93 from the CAP site. This HuVim 830-T/t recombinant was used to create cell lines from numerous primary cultures of different origins: rabbit, porcine and human endothelial cells, rabbit and bovine epithelial cells. A set of large T-expressing cells was derived, and these cells retained characteristics of differential cells: binding of Ulex europaeus lectin and synthesis of Factor VIII for human endothelial cells; network of cytokeratin for bovine oviductal cells and rabbit mammary cells.     

Somatic recombination rather than uniparental disomy suggested as another mechanism by which genetic imprinting may play a role in the etiology of Prader-Willi syndrome

Summary Six Prader-Willi syndrome (PWS) patients with normal karyotypes and their parents were analyzed to determine the nature of the molecular aberrations present in the proximal region of 15q and to determine the parental origin of the aberrant chromosome 15. In addition, the likehood that uniparental disomy plays a significant role in the etiology of PWS patients with normal karyotypes was studied. Restriction fragment length polymorphisms (RFLPs) recognized by seven probes [pML34 (D15S9), pTD3-21, pCGS0.9, pCGS1.1 (D15S10), IR4.3 (D15S11), IR10.1 (DS15S12), p189-1 (D15S13), IR39 (D15S18), and CMW-1 (D15S24)] mapping to the Prader-Willi chromosome region (PWCR) and an additional two probes [pMS1-14 (D15S1); the cDNA of neuromedin B] mapping elsewhere on chromosome 15 were analyzed in the six PWS patients and their parents. Copy number of each locus within the PWCR was determined by densitometry. Molecular rearrangements of the proximal region of 15q were observed in all of the six probands and the origin of the aberrant chromosome 15 when determined was consistently paternal in origin. While data obtained from our six patients does not support the mechanism of disomy, results obtained from three of the six patients show more complex rearrangements hypothesized to have resulted from somatic recombination. These rearrangements have resulted in acquired homozygosity and the lack of a paternal allele at various loci within the PWCR. The presence of only a maternal contribution at certain loci as the result of somatic recombination may be another mechanism by which genetic imprinting plays a role in the presentation of the PWS phenotype.     

Suppression of postprandial glucose, insulin, C-peptide, and glucose-dependent insulinotropic peptide (GIP) in man by oral administration of a prostaglandin analogue (enprostil)

Enprostil, a long-acting, orally active dehydroprostaglandin E2 with cytoprotective and gastric antisecretory properties, is a potent inhibitor of meal-stimulated gastrin release. Recent data have suggested suppression of additional other gastrointestinal peptide hormones following single doses of enprostil. The current investigation was conducted to further clarify the effects of enprostil administration on gastrointestinal hormones and glucose metabolism under physiologic conditions and to determine whether these effects were present following multiple doses of the agent. Enprostil 70 mcg/d and its placebo were each administered for 7 1/2 days to eight normal male subjects in a study of crossover design, each treatment period lasting 7 1/2 days and separated by a 7 day washout period. Subjects received a test meal on days 1 and 8 and an oral glucose challenge on day 3 of each treatment period following enprostil or its placebo. Following the test meal, there was a delay and suppression of the maximum measured serum glucose levels. Mean overall peak glucose concentrations were lower during the enprostil phase compared to placebo (112 vs. 121 mg/dd, P = 0.025) with a trend toward delay in the time to achievement of peak glucose concentrations. Mean overall peak levels for insulin, C-peptide, and glucose-dependent insulinotropic peptide (GIP) were significantly suppressed by 36%, 16% and 60%, respectively by enprostil when compared to placebo. The overall integrated postprandial responses for insulin, C-peptide, and GIP were significantly reduced by 42%, 39% and 90%, respectively while that for glucose above baseline was reduced by 44% (P = 0.098). Similar effects were present following the oral glucose challenge.(ABSTRACT TRUNCATED AT 250 WORDS)