Enzymology of NAD+ homeostasis in man

This review describes the enzymes involved in human pyridine nucleotide metabolism starting with a detailed consideration of their major kinetic, molecular and structural properties. The presentation encompasses all the reactions starting from the de novo pyridine ring formation and leading to nicotinamide adenine dinucleotide (NAD⁺) synthesis and utilization. The regulation of NAD⁺ homeostasis with respect to the physiological role played by the enzymes both utilizing NAD⁺ through the nonredox NAD⁺-dependent reactions and catalyzing the recycling of the common product, nicotinamide, is discussed. The salient features of other enzymes such as NAD⁺ pyrophosphatase, nicotinamide mononucleotide 5-nucleotidase, nicotinamide riboside kinase and nicotinamide riboside phosphorylase, described under miscellaneous, are likewise presented.Received 30 April 2003; received after revision 9 June 2003; accepted 20 June 2003     

Prevention of estrogenic inhibition of adrenal 686-1686-1686-1686-2686-2686-2in rats

Summary The urinary protein _2u stimulates adrenal ⁵-3 dehydrogenase activity and prevents adrenal enlargement in estrogen-treated adult male rats.Acknowledgements. Dehydroepiandrosterone used in this study was donated by Organon(India) Ltd, Calcutta. This work received financial support from University Grants' Commission, New Delhi. The author's thanks are due to Prof. A. K. Maiti and Prof. C. Deb, Department of Physiology, Calcutta University, for their constant encouragement.     

Investigation of the effects of copper ions on protein aggregation using a model system

Protein aggregation is a notable feature of various human disorders, including Parkinsons disease, Alzheimers disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. -synuclein, amyloid (A) peptide, ₂-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu²⁺ produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu²⁺-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu²⁺-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a structure introduced by the point mutation. The effect of Cu²⁺ on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.Received 4 December 2003; received after revision 7 January 2004; accepted 3 February 2004     

Metabolism in Porifera. IX. Studies on the biological conversion of cholesterol into 19-nor-cholestanol by the spongeAxinella polypoides

Summary The conversion of cholesterol into 19-nor-5-cholestan-3-ol by the spongeAxinella polypoides involves a partial loss (40%) of the 3-hydrogen atom; moreover administration to the sponge of [4-¹⁴C]cholesterol tritiated at C-4 and C-7 showed that the 4- and 7-hydrogen atoms are retained in this conversion. A competitive uptake experiment, [4-¹⁴C]cholesterol vs. [7-³H₂]5-cholestanol, showed that the sponge utilized exclusively cholesterol for the production of 19-nor-5-cholestan-3-ol.Part of this work has been presented at the Nato Conference on Marine Natural Products (Jersey, Great Britain, October, 1976).-This contribution is part of the Programma finalizzato Oceanografia e Fondi marini-sottoprogetto Risorse biologiche C.N.R. Italy.     

Differential chemotactic potential of mouse platelet basic protein for thymocyte subsets

Mouse platelet basic protein (CXCL7/mPBP) was cloned from thymic stromal cells and further identification indicated that it was expressed in thymic monocytes/macrophages (Mo/Ms). Recombinant mPBP was chemoattractive for target cells of polymorphonuclear leucocytes, peritoneal Mo/Ms and splenic lymphocytes with distinct potencies. CXCR2 was identified to be a cognate receptor for mPBP. Mouse thymocyte subsets of CD4^-CD8^- double-negative (DN), CD4⁺CD8⁺ double-positive (DP), CD4⁺CD8^- single-positive (CD4SP) and CD4^-CD8⁺ single-positive (CD8SP) expressed cell surface CXCR2 with different positive percentages and expression levels. mPBP was chemoattractive for thymocyte subsets with the potency order DN>DP> CD8SP>CD4SP, consistent with the levels of CXCR2 expressed on the respective cells. Thus, mPBP in thymus is functionally redundant with chemokine CXCL12/ SDF-1. Moreover, our finding that thymic Mo/Ms can produce mPBP implies that they may have other functions apart from acting as scavengers in thymus.Received 25 March 2004; received after revision 10 May 2004; accepted 8 June 2004     

Lack of tolerance development to tumor necrosis factor α inside the central nervous system

Tumor necrosis factor (TNF) was repeatedly microinfused into the lateral ventricle of guinea pig brains at a dose of 200 ng, 4 times within 150 min, at intervals of 3 days. In comparison to guinea pigs infused with solvent according to the same time schedule, the animals responded to TNF with pronounced fevers. The quantity of the fever response was the same after each of the 4 microinfusions of TNF. Three days after the last infusion of TNF or solvent all animals received an intramuscular injection of bacterial lipopolysaccharide (LPS). The fever in response to LPS was the same in both groups. Thus, the reported development of tolerance to repeated systemic administration of TNF^1–3 does not develop inside the blood-brain barrier. Also, the febrile response to LPS is not influenced by repeated central pre-treatment with TNF, whereas repeated peripheral treatment does have an effect.     

α-1,3-Fucosyltransferase-VII stimulates the growth of hepatocarcinoma cells via the cyclin-dependent kinase inhibitor p27Kip1

After the transfection of -1,3-fucosyltransferase (FucT)-VII cDNA into H7721 human hepatocarcinoma cells, the protein expression of some cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDIs) p16^INK4 and p21^waf1/Cip1 were unchanged. However, CDI p27^Kip1 protein, both the total amount and the amount that bound to CDK2, but not its mRNA, was significantly reduced. The de-inhibited CDK2 stimulated the phosphorylation of retinoblastoma (Rb) protein and facilitated the G1/S transition and growth rate of the cells. The decrease of p27^Kip1 protein, the increase of CDK2 activity and Rb phosphorylation, as well as the cell growth and percentage of S phase cells were correlated to the increased amount of cell surface sialyl Lewis X (SLe^x) antigen in cells with different -1,3-FucT-VII expression. The reduction in p27^Kip1 and the difference in its expression among different transfected cells were blocked by the SLe^x antibody KM93 in a dose-dependent manner, indicating that p27^Kip1 expression was influenced by -1,3-FucT-VII and its product SLe^x. The MEK/MAPK signaling pathway was more important than the PI-3K pathway in the regulation of p27^Kip1 expression.Received 5 August 2004; received after revision 25 October 2004; accepted 11 November 2005     

Potentiating action of hexoprenaline on14C-aminopyrine uptake by isolated rat parietal cells

Summary Hexoprenaline potentiated the¹⁴C-aminopyrine uptake (a reliable index of H⁺ generation) of isolated rat gastric cells stimulated by 10^–6–10^–4 mol/l carbachol, and inhibited that in response to 10^–4 mol/l histamine without and in the presence of propranolol. It is concluded that hexoprenaline acts as a partial agonist on parietal cell H₂-receptors and that -adrenoceptor activation may functionally modualte gastric acid secretion.Acknowledgments. S. Maliski, Institute of Rheumatology, Warszawa, held a fellowship of the Alexander v. Humboldt-Foundation. The study was supported by the Deutsche Forschungsgemeinschaft. The skilful technical assistance of Mrs R. Maier and Mr R. Beer is gratefully acknowledged.     

The free radical oxidation of the tetrahydrocannabinols

Summary The free radical oxidation of ¹ and ⁶-tetrahydrocannabinol has been examined by spin trapping techniques and intermediates that would lead to cannabinol have been trapped. The 1st step in the oxidation of ¹-THC involves the removal of 3-H, while for ⁶-THC, either 2-H or 5-H. Intermediates were isolated which could be pyrolysed to the diene and cannabinol.     

Induction of lymphokine-activated killer cells from nude mouse spleen cells by interleukin-4

The induction of lymphokine-activated killer (LAK) cells from natural killer (NK) lineage cells by interleukin-4 (IL-4) was studied in vitro. Activation of nude mouse spleen cells by IL-4 generated cytotoxic cells, capable of killing NK-sensitive as well as NK-resistant tumor cells. The induction of peak lytic activity was demonstrated after 3 days of culture with IL-4. Surface marker analysis indicated that the majority of precursor cells were aGM1⁺, Thy1^–, and the majority of effector cells were aGM1⁺, Thy1⁺, suggesting that IL-4 induced LAK cells from nude mouse spleen cells were similar to those from normal mouse spleen cells. The induction of nude mouse LAK cells by IL-4 was partially inhibited by anti-IL-4 or anti-interferon (IFN)-, antibody, and it was further inhibited by the combination of two antibodies, suggesting that IFN-, production was associated with LAK induction of NK lineage cells by IL-4.     

Effect of acute administration of Δ1-tetrahydrocannabinol on β-endorphin levels in plasma and brain tissue of the rat

Summary Acute treatment with ¹-tetrahydrocannabinol (¹-THC) elevated the concentration of -endorphin-like immunoreactivity (-ELIR) in plasma and in the hypothalamus, but not in the hippocampus of rats habituated to the injection procedure. These effects were not obtained with the psychotropically inert analog of (¹-THC), cannabidiol. In animals that had not been habituated to the injection procedure, placebo treatment induced a decrease in hippocampal -ELIR.The authors acknowledge the skillful technical assistance of Mrs Willeke Logtenberg.     

Androgen dependency of hepatic hydroxysteroid dehydrogenases in the rat: Prepubertal responsiveness and unresponsiveness towards exogenous testosterone

Summary The prepubertal responsiveness of 3 typical androgen-dependent enzyme activities, namely 20-ketoreductase, 3- and ⁴-3-hydroxysteroid dehydrogenase, towards a large dose of testosterone was investigated. The androgen induced the activity of the 3-enzyme prepubertally in both sexes.This investigation was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 87, Endokrinologie).     

The effect of insulin on the electrophoretic mobility of rat hepatocytes

Summary The addition of insulin (10 U) to a suspension of isolated hepatocytes in Krebs-Ringer bicarbonate solution. causes an increase in the negative electrophoretic mobility of the cells from –1.68 m sec^–1 V^–1 cm to 2.26 m sec^–1 V^–1 cm. This observation supports the findings by other workers that the binding of insulin to its receptor leads to a marked change in the membrane.Acknowledgment. We wish to thank the Medical Research Council for the provision of the microelectrophoresis apparatus and initial running costs of the project.     

C-peptide stimulates Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells

Proinsulin-connecting peptide (C-peptide) exerts physiological effects partially via stimulation of Na⁺, K⁺-ATPase. We determined the molecular mechanism by which C-peptide stimulates Na⁺, K⁺-ATPase in primary human renal tubular cells (HRTCs). Incubation of the cells with 5 nM human C-peptide at 37°C for 10 min stimulated ⁸⁶Rb⁺ uptake by 40% (p<0.01). The carboxy-terminal pentapeptide was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive ⁸⁶Rb⁺ uptake, C-peptide increased subunit phosphorylation and basolateral membrane (BLM) abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits. The increase in BLM abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits was accompanied by depletion of ₁ and ₁ subunits from the endosomal compartments. C-peptide action on Na⁺, K⁺-ATPase was ERK1/2-dependent in HRTCs. C-peptide-stimulated Na⁺, K⁺-ATPase activation, phosphorylation of ₁-subunit and translocation of ₁ and ₁ subunits to the BLM were abolished by a MEK1/2 inhibitor (20 M PD98059). C-peptide stimulation of ⁸⁶Rb⁺ uptake was also abolished by preincubation of HRTCs with an inhibitor of PKC (1 M GF109203X). C-peptide stimulated phosphorylation of human Na⁺, K⁺-ATPase subunit on Thr-Pro amino acid motifs, which form specific ERK substrates. In conclusion, C-peptide stimulates sodium pump activity via ERK1/2-induced phosphorylation of Thr residues on the subunit of Na⁺, K⁺-ATPase.Received 15 June 2004; received after revision 14 September 2004; accepted 14 September 2004     

Die Wirkung einer Leberschädigung durch Thioacetamid auf die mikrosomale Aromatisierung von Testosteron bei der Ratte

Summary Rat liver microsomes, NADPH-regenerating system, and 1, 2-³H-testosterone have been incubated in vitro. The loss of tritium from the steroid, associated with aromatization of testosterone, was linear with time for 20 min and required NADPH. Pre-treatment of the rats with thioacetamide raised the liberation of tritium from 1, 2-³H-testosterone. The results suggest that liver damage by thioacetamide in rats may give rise to increased aromatization of testosterone.     

Tumor necrosis factor-α inhibits collagen synthesis in human and rat granulation tissue fibroblasts

The purpose of the study was to examine the effects of tumor necrosis factor- (TNF-) on collagen gene expression in rat and human granulation tissue fibroblast cultures. The cells were exposed to 0.1, 1, 10, or 100 ng/ml of TNF-, and the rate of collagen synthesis was measured as synthesis of protein-bound³H-hydroxyproline. Total cellular RNA was isolated from fibroblasts, and measurements of specific cellular RNAs from fibroblasts were performed by Northern blot hybridizations using³²P-labeled cDNA probes. In cultures of rat granulation tissue fibroblasts TNF- decreased³H-hydroxyproline production to about 75% of that in controls and it also decreased pro1(I) and pro1(III) collagen mRNA levels, maximally to 33% and 23% of the control levels, respectively. In cultures of human granulation tissue fibroblasts a similar inhibiting effect in the production of collagen was seen. TNF- decreased the production of³H-hydroxyproline to 56% of the control value with a dose of 100 ng/ml also having an inhibiting effect on pro1(I) collagen mRNA levels of up to 43% of the control level. However, no effect was seen on pro1(III) collagen mRNA levels.     

Inhibition of human testicular glucose-6-phosphate dehydrogenase by unsaturated C19-steroids

Zusammenfassung Die Bebrütung einer gereinigten G-6-PDH aus menschlichem Testisgewebe mit 3-substituierten ⁴- oder ⁵-C₁₉-Steroiden zeigte, dass 3-Hydroxy-⁴-oder ⁵-androsten-17-on auch wirksame Inhibitoren der testikulären G-6-PDH darstellen. Zur gleichen Zeit konnten die früher gefundenen Zusammenhänge zwischen chemischer Struktur eines Steroids und seiner biologischen Aktivität im G-6-PDH-Hemmtest uneingeschränkt bestätigt werden.     

Effect of 723-1723-1723-1on ethanol withdrawal in mice

Summary ⁹ (10 mg/kg, i.p.) administered to mice immediately after withdrawal from a 3-day exposure to ethanol vapor was found to intensify withdrawal reactions. No effect was seen when ⁹ was administered chronically during the exposure to ethanol.     

A novel pattern of fast calcium oscillations points to calcium and electrical activity cross-talk in rat chromaffin cells

Slow oscillations of cytosolic calcium ion concentration – – typically originate from release by intracellular stores, but in some cell types can be triggered and sustained by Ca²⁺ influx as well. In this study we simultaneously monitored changes in and in the electrical activity of the cell membrane by combining indo-1 and patch-clamp measurements in single rat chromaffin cells. By this approach we observed a novel type of spontaneous oscillations, much faster than those previously described in these cells. These oscillations are triggered and sustained by complex electrical activity (slow action potentials and spike bursts), require Ca²⁺ influx and do not involve release from intracellular stores. The possible physiological implications of this new pathway of intracellular signalling are discussed.Received 30 July 2004; received after revision 14 October 2004; accepted 1 November 2004     

Unusual high volume of sarcoplasmic reticulum in a wasp leg muscle

Summary Ultrastructural and morphometric examination of a wasp leg muscle showed that it contained a high volume of sarcoplasmic reticulum (volume fraction=0.35, surface density=21.4 m²/m³). As well as being arranged in double or triple layers between the myofibrils, the SR was found in large multilayered accumulations around the nuclei and in the subsarcolemmal space. Fibres of adjacent muscles had the normal volume and arrangement of SR.