Effect of the specific cholecystokinin-receptor antagonist loxiglumide on bombesin stimulated pancreatic enzyme secretion in man.

We have investigated the effects of the specific cholecystokinin (CCK) receptor antagonist loxiglumide on basal and bombesin stimulated pancreatic enzyme secretion, bilirubin output and plasma CCK release in six healthy subjects. The data were compared with those obtained in control experiments where saline was infused instead of loxiglumide. Basal amylase output (4.7 +/- 0.8 kU/45 min), trypsin output (2.9 +/- 0.8 kU/45 min) and bilirubin output (7.7 +/- 2.8 mmol/45 min) gradually declined during infusion of loxiglumide to values of 1.3 +/- 0.3 kU/45 min, 0.5 +/- 0.1 kU/45 min and 0.4 +/- 0.0 mmol/45 min, respectively, reaching statistical significance (P less than 0.05) in the 30 to 45-min period after the start of the loxiglumide infusion. In the control experiments saline infusion failed to influence basal amylase, trypsin and bilirubin output, while bombesin stimulated amylase output from 4.7 +/- 0.8 kU/45 min to 25.1 +/- 5.1 kU/45 min (P less than 0.05), trypsin output from 2.9 +/- 0.8 kU/45 min to 11.6 +/- 2.0 kU/45 min (P less than 0.05) and bilirubin output from 7.7 +/- 2.8 mmol/45 min to 68.0 +/- 16.0 mmol/45 min (P less than 0.05). Loxiglumide failed to significantly influence bombesin stimulated amylase output (36.7 +/- 9.0 kU/45 min) and trypsin output (8.3 +/- 2.9 kU/45 min), but almost abolished bilirubin output (9.7 +/- 3.6 mmol/45 min) (P less than 0.05). Basal plasma CCK (2.4 +/- 0.1 pM) was not significantly influenced by loxiglumide (2.4 +/- 0.2 pM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH-sensitive dye carboxyseminaphthorhodafluor-1 (SNARF-1) as a possible candidate for flow cytometric pH measurements and compared it with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and 2,3-dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF-1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratiometric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7-8. With SNARF-1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.     

Calcitonin gene-related peptide, neurokinin A and substance P: effects on nociception and neurogenic inflammation in human skin and temporal muscle.

Calcitonin gene-related peptide (CGRP) was injected alone and in combination with substance P (SP) or neurokinin A (NKA) into the forearm skin and temporal muscle of human volunteers. In the skin, 50 pmol of CGRP induced a wheal response and a delayed erythema. No pain was recorded. No interaction between CGRP and SP or NKA was observed. In the temporal muscle, 200 pmol of CGRP alone did not induce pain or tenderness but, in combination with SP or NKA, CGRP elicited a significant pain sensation. It is concluded that CGRP may be involved in neurogenic inflammation and that only SP, of the three peptides present in nociceptive C fibers, seems to be of major importance in relation to cutaneous nociception. Simultaneous neurogenic release of CGRP and other neuropeptides in skeletal muscle may induce myofascial pain.     

Production of optically active 2,3-butanediol by Bacillus polymyxa

Bacillus polymyxa produces (R, R)-2,3-butanediol from a variety of carbohydrates. Other metabolites are also produced including acetoin, acetate, lactate, and ethanol. The excretion of each metabolite was found to depend on the relative availability of oxygen to the culture. When the relative oxygen uptake rate was high, enhanced yields of acetate and acetoin were noted. At an intermediate oxygen availability, the butanediol yield was maximal. When the availability of oxygen was more restricted, higher yields of lactate and ethanol occurred. The cells appeared to regulate themselves such that energy generation is optimal subject to the constraint that the cells do not produce more reducing equivalents than can be oxidized by the electron transport system. The dependence of each product yield on the relative oxygen availability was determined, and this knowledge was used to carry out a fed-batch fermentation that attained a final butanediol concentration of over 40 g/L in 50 h.     

Deconjugation of glucuronides catalysed by UDPglucuronyltransferase

Evidence was found for UDPglucuronyltransferase-catalysed deconjugation of p-nitrophenol-, 4-methylumbelliferone- and phenolphthalein-glucuronides. The evidence is based on the following observations: 1, deconjugation is UDP-dependent and the reactions show Michaels-Menten kinetics with respect to UDP and glucuronide saturability; 2, UDP-glucuronic acid was identified as reaction product; 3, all studies were done in the presence of a beta-glucuronidase inhibitor; 4, induction profiles, using 3-methylcholanthrene and phenobarbital as inducing agents, were identical for conjugation and deconjugation reactions. Optimal deconjugation rates for p-nitrophenol- and 4-methylumbelliferone-glucuronides were at pH 5.1 and for phenolphthalein-glucuronide at pH 6.5. Only conjugation reactions showed latency; the corresponding deconjugation reactions were not latent. UDPglucuronyltransferase is a group of oligomeric isoenzymes with different molecular masses. The molecular masses of the isoenzyme species catalysing the forward and reverse reactions were determined by radiation-inactivation analysis. The molecular masses of the isoenzyme species mediating the catalyses of deconjugation reactions were significantly smaller than those mediating catalyses of conjugation reactions: 66 +/- 4 kDa vs. 109 +/- 7 kDa for p-nitrophenol; 82 +/- 8 kDa vs. 105 +/- 6 kDa for 4-methylumbelliferone; and 74 +/- 8 kDa vs. 159 +/- 14 kDa for phenolphthalein. This suggests that for catalyses of deconjugation reactions only part of a UDPglucuronyltransferase isoenzyme is needed, whereas for forward reactions the complete isoenzymes are required.     

Effect of glucose on ATP dephosphorylation in rat spermatids

Round spermatids were isolated from rat testes and the effects of different energy-yielding substrates on the cellular ATP content were estimated. The ATP content was constant and high (6-8 nmol/10(6) cells) during metabolism of exogenous lactate. During incubation for 30 min in the absence of exogenous lactate, there was a remarkably slow decline of the ATP content, indicating ATP production from other substrates. It was shown that this could reflect beta-oxidation of fatty acids, but not the mobilization of an endogenous pool of acetylcarnitine. Glucose metabolism in the absence of exogenous lactate resulted in a rapid decline of the ATP content. This effect of glucose was correlated with a high fructose 1,6-biphosphate content (6-7 nmol/10(6) cells) and could be prevented by the addition of lactate. It is suggested that metabolism of glucose (and also mannose and fructose, but not galactose) in the absence of exogenous lactate can result in ATP dephosphorylation.     

Human hepatoma (Hep G2) cultures contain salt-resistant triglyceridase ("liver lipase")

The culture fluid of Hep G2 human hepatoma cells contains triglyceridase activity resistant to high-salt concentrations. The lipase binds to Sepharose-heparin columns from which it can be eluted by 0.8 to 0.9 M NaCl. The nature of this lipase was studied using antibodies raised against "liver" lipases from human and rat origin. The anti-rat liver lipase inhibits both the postheparin human and rat plasma enzyme while the anti-human liver lipase has no effect on the rat enzyme. The lipase of the Hep G2 cultures showed affinity to the antibodies raised against rat as well as human "liver" lipase as shown by inhibition experiments. These results show that Hep G2 cells secrete "liver" lipase and that there seems to exist a structural homology between the lipases from rat and human origin.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

A comparison between dopamine-stimulated adenylate cyclase and 3H-SCH 23390 binding in rat striatum

Methods for measuring 3H-SCH 23390 binding and dopamine (DA) stimulated adenylate cyclase (AC) were established in identical tissue preparations and under similar experimental conditions. Pharmacological characterization revealed that both assays involved interaction with the D1 receptor or closely associated sites. In order to investigate whether the binding sites for 3H-SCH 23390 and DA in fact are identical, the antagonistic effects of a variety of pharmacologically active compounds were examined. Surprisingly, the Ki-values obtained from Schild-plot analysis of the antagonism of DA-stimulated AC, were 80-240 times higher than the Ki-values obtained from competition curves of 3H-SCH 23390 binding. Since both assays were performed under identical conditions, the differences in Ki-values indicate the possibility of different binding sites for DA and 3H-SCH 23390 or, that DA and 3H-SCH 23390 label different states of the same receptor.     

The effect of air-borne ammonium sulphate onPinus nigra var.maritima in the Netherlands

Summary As a result of air pollution, considerable deposition of ammonium sulphate occurs on vegetation and soil in the vicinity of chicken farms and fields dressed with animal slurry. A clear relation exists between this ammonium sulphate deposition and the distance to certain agricultural activities. Field investigations and ecophysiological experiments both show that the needles ofPinus nigra var.maritima (Ait.) Melville take up ammonium and excrete potassium, magnesium and calcium. This often results in potassium and/or magnesium deficiencies and may lead to premature shedding of needles. The high levels of nitrogen in the needles are strongly correlated to fungal diseases.Whether the observed cation leaching will result in disturbed nutrient budgets depends mainly on soil conditions. Leaching of K, Mg and Ca from the soil, caused by ammonium sulphate, may further inhibit nutrient uptake.Field investigations show a clear correlation between increased ratios of NH₄ to K, Mg and Ca in the soil solution and the damage to pine forests.