Natural purified porcine gastric inhibitory polypeptide (GIP) stimulates exocrine pancreas secretion due to contamination with a cholecystokinin-like substance

The effects of purified natural gastric inhibitory polypeptide-enterogastrone III (GIP-EG III) and a fraction which is further purified by high pressure liquid chromatography (GIP-HPLC) were investigated on the endocrine and exocrine isolated perfused pancreas of rats. At the dose of 5 ng/ml used for both GIP preparations, only GIP-EG III significantly stimulated volume and amylase secretion of the exocrine pancreas. The response of insulin release to stimulation by GIP-EG III or GIP-HPLC was not significantly different. In the presence of cholecystokinin-octapeptide (CCK-8) at a concentration which gave half-maximal stimulation of amylase secretion, GIP-EG III almost doubled the response of the exocrine pancreas, whereas GIP-HPLC had no additional effect. CCK-8 alone significantly increased total insulin output under hyperglycemic conditions. We conclude that porcine GIP purified by gel chromatography contains a CCK-like substance which can be removed by further purification on high pressure liquid chromatography without affecting the insulinotropic activity. Some of the reported effects of GIP could be due to contamination.     

Phorbol esters activate proteoglycan metabolism in human colon cancer cells en route to terminal differentiation

Tumor-producing phorbol esters [e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA)] induce changes in a human colon cancer cell line, VACO 10MS, that mimic terminal differentiation: a rapid blockade of DNA replication and cell division, a marked increase in cell adhesion properties with striking changes in morphology, and the acquisition of ion-transporting activities. The present report shows that the triggering of this terminal differentiation sequence by TPA is associated with a rapid release of heparan sulfate proteoglycans from the cell surface that is soon followed by an acceleration of proteoglycan synthesis. The activation of the release mechanism is independent of ongoing protein synthesis, whereas the resynthesis of the proteoglycans requires the production of new proteins. A persistent high rate of proteoglycan synthesis and release appears correlated with the progression of the colon cell into the terminal differentiation state. Bryostatin 1, an agent which has been shown previously to block the TPA-induced terminal differentiation of this cell line, also largely prevents the TPA effects on proteoglycan metabolism. Since both TPA and bryostatin 1 produce their effects through the activation of members of the protein kinase C class of enzymes, it is proposed that the differentiation state of these colon cancer cells may be regulated by a differential activation of isozymes or a ligand-directed phosphorylation of proteins that are involved in proteoglycan metabolism.     

Desmoplakins of Epithelial and Myocardial Desmosomes are Immunologically and Biochemically Related

Abstract. Guinea pig antibodies against desmoplakins from bovine muzzle epidermis showed specific reaction in several epithelial tissues with desmoplakin I (M_r 250,000) and desmoplakin II (M_r 215,000). By immunofluorescence microscopy, prominent punctate staining was observed in various lines of cultured epithelial cells, revealing desmosomal junctions at sites of established cell-to-cell contacts as well as hemidesmosomes and internalized desmosome-derived membrane domains. On frozen tissue sections punctate staining was observed along plasma membranes of epithelial cells, and electron microscopy using the immunoperoxidase technique revealed that the antibodies were specifically localized at the plaques associated with desmosomes and hemidesmosomes. Of a large number of non-epithelial cells examined positive staining was only observed on desmosome-like junctions of myocardial cells and Purkinje fiber cells. In both epithelial and myocardial tissues the antibodies showed a broad range of cross-reactivity between diverse vertebrate species such as man, cow, rodent, and chicken, indicating that desmoplakins contain determinants strongly conserved during evolution. When binding of these antibodies to cytoskeletal polypeptides separated by gel electrophoresis and blotted on nitrocellulose paper sheets was examined, specific reaction was noted with desmoplakin I and, to a variable degree, also desmoplakin II from various epithelial cells. Reaction was also observed with a myocardial polypeptide from bovine and human hearts which had a similar M_r value (250,000) and isoelectric pH range as desmoplakin I. We conclude that desmoplakins are the major proteins present in the desmosomal plaques of both epithelial and myocardial cells and that the desmoplakin polypeptides present in these two different cell types are very similar, if not identical.     

Human β-galactosidase and α-neuraminidase deficient mucolipidosis: Genetic complementation analysis of the neuraminidase deficiency

Summary Human -galactosidase and -neuraminidase deficient mucolipidosis [ML(gal^-neur^-)] is an inherited lysosomal enzymopathy which recently was designated as a sialidosis. We analyzed the neuraminidase deficiency of this disorder with genetic complementation analyses using a heterokaryon enrichment procedure. The genetic defects of two apparent variants of this disorder complemented the defects of the neuraminidase deficiency diseases, sialidosis I and mucolipidosis I, resulting in the restoration of neuraminidase activity in heterokaryons. The neuraminidase deficiency, therefore, may not be the primary defect in ML(gal^-neur^-) and is not an appropriate test for determining carrier status. The clinical and biochemical characteristics of this disorder suggest that a post-translational or processing event for these enzymes may be defective. The defect, however, is different from I-cell disease and pseudo-Hurler polydystrophy, two disorders of post-translational lysosomal enzyme biosynthesis, since complementation studies demonstrated recovery of intracellular -galactosidase and -neuraminidase levels in heterokaryons. The lack of human -galactosidase expression in man-mouse somatic cell hybrids formed from fibroblasts of the infantile onset type disorder suggests that the defect is not corrected by the mouse genome. The ML(gal^-neur^-) disorder therefore appears to be a distinct subtype of the inherited neuraminidase deficiencies in which the defect may occur in a post-translational or regulatory step which coordinately affects the expression of lysosomal -galactosidase and -neuraminidase.     

Proliferative characteristics of clonal endothelial cell strains

We have utilized clonal strains of bovine fetal aortic endothelial cells to study cellular senescence in a differentiated cell type of physiological significance. Serial subcultivation of nine endothelial clones derived from three fetal calf aortas revealed proliferative life-spans in vitro of 53–125 population doublings (PDs), compared with 60 and 143 PDs for two lines of bovine fetal lung cells and 85 and 147 PDs for two lines of bovine vascular smooth muscle cells. Serial growth curves showed marked reductions associated with endothelial cellular senescence both in cellular growth rate and culture plateau density. Studies of the 24-hour [³H]-thymidine labeling index versus percentage of proliferative life-span completed indicated that clonal endothelial cultures contained a large proportion (greater than 90%) of rapidly cycling cells until about 75% of the life-spans were completed. Senescent endothelial cells showed evidence of large increases in cell area, cell volume, and protein content. In those clones examined, one specialized endothelial function, Factor VIII antigen expression, was retained qualitatively throughout the life-spans.     

Coupling between H+ transport and anaerobic glycolysis in turtle urinary bladder: Effects of inhibitors of H+ ATPase

Summary The coupling between H⁺ transport (J _H) and anaerobic glycolysis was examinedin vitro in an anaerobic preparation of turtle urinary bladder.J _H was measured as the short-circuit current after Na⁺ transport was abolished with ouabain and by pH stat titration. The media were gassed with N₂ and 1% CO₂ (PO₂<0.5 mm Hg) and contained 10mm glucose. Under these conditions,J _H was not inhibited by 3mm serosal (S) cyanide or by 0.1mm mucosal (M) dinitrophenol. Control anerobic lactate production (J _lac) of 47 bladders was plotted as a function of simultaneously measuredJ _H. The slope ofJ _lac onJ _H was 0.58±0.12 with an intercept forJ _lac atJ _H=0 of 0.55 mol/hr. Values for J _lac/J _H were determined in groups of individual bladders whenJ _H was inhibited by an opposing pH gradient (0.55±0.16), by acetazolamide (0.58±0.19) and by dicyclohexylcarbodiimide, DCCD (0.58±0.14). The constancy of J _lac/J _H indicates a high degree of coupling betweenJ _H andJ _lac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the J _lac/J _H values can be used to estimate the stoichiometry of H⁺ translocation. The movement of slightly less than 2 H⁺ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited byM addition of oligomycin but was inhibited byM addition of DCCD and Dio-9, inhibitors of H⁺ flow in the proteolipid portion of H⁺-translocating ATPases. DCCD inhibited anaerobicJ _H without change in J _lac/J _H or basalJ _lac and, therefore, acted primarily on the H⁺ pump.S addition of vanadate also inhibitedJ _H, but the inhibition was associated with an increase inJ _lac. The site of this apparent uncoupling remains to be defined. The acidification pump of the luminal cell membrane of the turtle bladder has H⁺-ATPase characteristics that differ from mitochondrial ATPase in that H⁺ transport is oligomycin-resistant and vanadate-sensitive. As judged from the flows of H⁺ and lactate, the H⁺/ATP stoichiometry of the pump is about 2.     

Assignment of the gene coding for human catalase to the short arm of chromosome 11

Human and murine catalases can be separated electrophoretically as single bands of different mobility. In man-mouse somatic cell hybrids, however, detection of human catalase is precluded by the complexity of banding patterns resulting from interference of a catalase-modifying enzyme activity. We have identified human catalase in hybrid clones by Laurel electrophoresis employing a specific anti-human catalase antibody, and by exploiting heat stability differences. Catalase co-segregates with LDH A and is probably located on the short arm of chromosome 11.     

Tyrosine metabolism for cuticle tanning in the tobacco hornworm, Manduca sexta (L.) and other Lepidoptera: identification of beta-D-glucopyranosyl-O-L-tyrosine and other metabolites

When [${}^{14}\text{C}$]tyrosine and [${}^{14}\text{C}$]glucose were fed or injected into feeding fifth-instar larvae of the tobacco hornworm, Manduca sexta (L.), they were incorporated into a conjugate identified in hemolymph and carcass extracts as β-d-glucopyranosyl-O-l-tyrosine. In wandering larvae and pupae, the conjugate was hydrolyzed, and tyrosine was hydroxylated and decarboxylated to dihydroxyphenylalanine and 2-(dihydroxyphenyl)ethylamine. None of these metabolites were formed in fourth-instar larvae or in adults. [${}^{14}\text{C}$]Phenylalanine was hydroxylated to tyrosine in all stages of insect development. β-d-Glucopyranosyl-O-l-tyrosine was also detected in 18 other species of Lepidoptera but not in species from other insect orders. This conjugate appears to be the major tyrosine storage metabolite for production of tanning diphenol substrates in Lepidoptera.     

Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii

A membrane-bound cytochrome oxidase from Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using an ascorbate-TMPD oxidation assay. The oxidase was ‘solubilized’ from a sonic-type electron-transport particle (R₃ fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27–70% (NH₄)₂SO₄. The highly purified cytochrome oxidase has a V of 60–78 μgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN₃ and NH₂OH; NaNO₂ (but not NaNO₃) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c₄ and cytochrome o; cytochromes a₁ and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c₄?o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a₃ oxidase of mammalian mitochondria.