Hypoxia exposure and B-type natriuretic peptide release from Langendorff heart of rats.

AIM: We studied whether available oxygen without induced mechanical stretch regulates the release of the biologically active B-type natriuretic peptide (BNP) from Langendorff heart. METHODS: Rat hearts were isolated and perfused with a physiological Krebs-Henseleit solution at a constant hydrostatic pressure in Langendorff set-up. The basal O2 level of perfusate (24.4 ± 0.04 mg L-1 ) was gradually lowered to 3.0 ± 0.01 mg L-1 over 20 min using N2 gas (n = 7). BNP and O2 level were measured from coronary flow. During control perfusions (n = 5), the O2 concentration was kept at 26.6 ± 0.3 mg L-1 . RESULTS: A low oxygen concentration in the perfusate was associated with a significant increase in BNP release (F = 40.4, P < 0.001). Heart rate decreased when the oxygen concentration in the perfusate reached 9.1 ± 0.02 mg L-1 and continued to fall in lower oxygen concentrations (F = 14.8, P < 0.001). There was also a significant but inverse correlation between BNP and oxygen in the coronary flow (R2  = 0.27, P < 0.001). CONCLUSION: In the spontaneously beating Langendorff rat heart, a decreasing concentration of oxygen in the ingoing perfusion increased the secretion of BNP. The effect of oxygen was independent of mechanical stretch of the heart as it occurred even when the heart rate decreased but the pressure conditions remained constant. The difference in the oxygen capacitance of blood and Krebs-Henseleit solution appears to be a major factor affecting secretion of BNP, which is correlated with the oxygen tension of myocardial cells and affected both by the oxygen concentration and capacitance of solution perfusing the heart and by the coronary flow.

Regulation of hepatic tyrosine aminotransferase in the frog Rana temporaria.

The regulation of hepatic tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) in the rat has been extensively studied but little is known about the enzyme from other sources. We have studied the regulation of this enzyme in the frog Rana temporaria and in this paper we report that: 1. Cortisone acetate, adrenocorticotropic hormone and alpha-methyl-p-tyrosine, an agent known to induce hepatic tyrosine aminotransferase in the rat via activation of the pituitary-adrenal axis, have no effect on the activity of the enzyme in the frog. 2. Dibutyryl-3',5'-cyclic AMP induces the enzyme to about 2-fold. 3. Injection of tyrosine methyl ester and a protein-rich diet result in an increase in the enzyme activity. This increase is of the same order of magnitude as that caused by dibutyryl cyclic AMP. 4. Glucose significantly reduces tyrosine aminotransferase activity in frog liver. These results suggest that cyclic AMP induces the enzyme via a mechanism independent of glucocorticoids. The frog offers a model for studies on the regulation of hepatic tyrosine aminotransferase in vivo without interference from secondary effects mediated by the adrenals.

Analysis of a Drosophila cyclin E hypomorphic mutation suggests a novel role for cyclin E in cell proliferation control during eye imaginal disc development.

We have generated and characterized a Drosophila cyclin E hypomorphic mutation, DmcycEJP, that is homozygous viable and fertile, but results in adults with rough eyes. The mutation arose from an internal deletion of an existing P[w+lacZ] element inserted 14 kb upstream of the transcription start site of the DmcycE zygotic mRNA. The presence of this deleted P element, but not the P[w+lacZ] element from which it was derived, leads to a decreased level of DmcycE expression during eye imaginal disc development. Eye imaginal discs from DmcycEJP larvae contain fewer S phase cells, both anterior and posterior to the morphogenetic furrow. This results in adults with small rough eyes, largely due to insufficient numbers of pigment cells. Altering the dosage of the Drosophila cdk2 homolog, cdc2c, retinoblastoma, or p21(CIP1) homolog dacapo, which encode proteins known to physically interact with Cyclin E, modified the DmcycEJP rough eye phenotype as expected. Decreasing the dosage of the S phase transcription factor gene, dE2F, enhanced the DmcycEJP rough eye phenotype. Surprisingly, mutations in G2/M phase regulators cyclin A and string (cdc25), but not cyclin B1, B3, or cdc2, enhanced the DmcycEJP phenotype without affecting the number of cells entering S phase, but by decreasing the number of cells entering mitosis. Our analysis establishes the DmcycEJP allele as an excellent resource for searching for novel cyclin E genetic interactors. In addition, this analysis has identified cyclin A and string as DmcycEJP interactors, suggesting a novel role for cyclin E in the regulation of Cyclin A and String function during eye development.

Ectodysplasin, a protein required for epithelial morphogenesis, is a novel TNF homologue and promotes cell-matrix adhesion.

In the mouse Tabby (Ta) mutant and human X-linked anhidrotic ectodermal dysplasia (EDA) syndrome development of several ectodermal organs such as hair, teeth, and sweat glands is impaired. The gene behind Tabby and EDA has been cloned, and several alternative transcripts have been isolated. The protein product named ectodysplasin had no obvious function or prominent homology to other known gene products apart from a short collagen-like sequence. We have isolated two novel Ta transcripts which are variants of the longest isoform of Tabby, named Ta-A. In situ hybridizations revealed Ta-A to be the major transcript in the developing embryo. It was detected in the endoderm of early embryos and subsequently in specific locations in the neuroepithelium and ectoderm. Unexpectedly, sequence analysis of the most C-terminal domain of Ta revealed that ectodysplasin is a novel member of the tumor necrosis factor (TNF) ligand superfamily. Mouse ectodysplasin was biochemically and functionally characterized, and shown to be a glycosylated, oligomeric type II membrane protein (N-terminus inside), all characteristics typical to TNF-like proteins. Members of the TNF family are critically involved in host defence and immune response often mediating either apoptosis or cell survival. Expression of Ta in several epithelial cell lines did not result in prominent changes in cell morphology and did not promote apoptosis. Instead, it was shown to promote cell adhesion to extracellular matrix, a function consistent with its postulated role in epithelial-mesenchymal interactions regulating the development of ectodermal appendages. Ectodysplasin is the first TNF-like signaling molecule described known to be required for epithelial morphogenesis.

Lack of correlation between serum dopamine-beta-hydroxylase activity and blood pressure in middle-aged men.

The activity of serum dopamine-beta-hydroxylase (DBH) was measured in 1194 asymptomatic middle-aged men with diastolic blood pressure ranging from 75 to 125 mm Hg during the baseline examination of a multifactorial intervention program for primary prevention of coronary heart disease. No correlation was present between serum DBH activity and systolic (r = -0.01, NS) or diastolic (r = +0.02, NS) blood pressure. No significant differences in serum DBH activity was observed between individuals with blood pressure in the lower, middle or upper deciles. Serum DBH activity was similar in subjects with normal blood pressure, in individuals with widely fluctuating blood pressure and in patients with fixed hypertension. The results suggest that serum DBH activity cannot be used as an aid in the diagnosis of essential hypertension of middle-aged men.

Are there isoenzymes of cytochrome c oxidase in Paracoccus denitrificans?

We have used a gene replacement strategy to delete the previously isolated gene [(1987) EMBO J. 6, 2825-2833] for the cytochrome c oxidase subunit I from Paracoccus denitrificans. The resulting mutant was still able to synthesize active cytochrome c oxidase. This led us to look for another locus which could completely suppress the mutation. In this study we report the isolation of a second gene encoding subunit I. An open reading frame coding for cytochrome c 550 was found upstream from this gene. We suggest that there are isoenzymes of cytochrome c oxidase (cytochrome aa3) in this bacterium.

Purification and properties of human liver tyrosine aminotransferase.

Tyrosine aminotransferase (EC 2.6.1.5) of human liver was purified 2200-fold by successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, CM-Sephadex C-50 and hydroxyapatite to a specific activity of 64 units/mg of protein. The purified enzyme had a molecular mass of 95 500. The Km-values were 1.04 X 10(-3) mol/l, 0.17 X 10(-3) mol/l and 0.69 X 10(-6) mol/l for tyrosine, 2-oxoglutarate and pyridoxal 5'-phosphate, respectively. In the final purification step the enzyme activity was divided into two major fractions and a minor third one. On isoelectric focusing, three distinct fractions of specific tyrosine aminotransferase activity were obtained. The isoelectric points of these fractions were 4.9, 5.1 and 5.3, respectively. These findings imply that the human tyrosine aminotransferase consists of three subforms. No differences in properties studied could be found between the subforms. The coenzyme, pyridoxal 5'-phosphate, could be removed by dialysis.

Biotin concentrations in maternal plasma and milk during prolonged lactation.

We followed a group of exclusively breast feeding mothers for 12 months after delivery. They numbered 200 at delivery, 140 at 4 months of lactation, 116 at 6, 36 at 9 and 7 at 12 months. The milk concentration of biotin was very low at delivery and subsequently the (geometric) mean value remained at around 4.5 micrograms/l. The mean plasma value was 250 ng/l at delivery and stable (around 330 ng/l) throughout the lactation. There were large intra- and interindividual variations in the concentrations in both milk (from 0 to 27 micrograms/l) and plasma (from 142 to 1090 ng/l). The variations in the milk concentrations result in a similar variation in the biotin intake of the exclusively breast-fed infants. The current recommendations for biotin intake were not met by any of those infants but no manifestations of biotin deficiency were recorded.

Fetal and maternal catecholamines in cesarean section.

The maternal and fetal sympathoadrenal system was studied by radioenzymatic measurement of the release of NA and A in 20 women with full-term uncomplicated pregnancies at cesarean section. Atropinization was performed 30 min or immediately before the induction of anesthesia. Before induction the mean level of maternal plasma NA in the i.v. atropinization group was 1.9 +/- 0.3 pmol/ml and in the i.m. group, 1.2 +/- 0.2 pmol/ml (mean +/- SE) (p greater than 0.05), and the levels of plasma A were 0.5 +/- 0.2 and 0.2 +/- 0.1 pmol/ml, respectively (p greater than 0.05). No significant rise was observed to be connected with the induction of anesthesia. Fetal umbilical arterial NA and A concentrations were higher than maternal values. CA concentration measured in the umbilical artery was 3-6 times higher than the corresponding values measured in the umbilical vein, indicating metabolization of CA in the placenta. Individual variation of fetal NA and A was very high, suggesting great individual alterations in the activity of the fetal sympathoadrenal system.

Enhancement of biotinidase activity in mouse serum by inhibitors of methylation.

DL-ethionine increases the activity of liver biotinidase, an enzyme which hydrolyzes biotinylesters and biotinylpeptides. Chronic DL-ethionine feeding increases transiently the activity of biotinidase in mouse and rat liver, after which it remains elevated in the serum. In the present work we show that both isomers of DL-ethionine are equally good enhancers of the liver biotinidase, while, 3-ethylthiopropionate, the toxic metabolite of DL-ethionine, has no effect on the biotinidase activity of either liver or serum. We have also employed two different combinations of inhibitors of the hydrolytic pathway of SAH, a transmethylation product and potent inhibitor of methylation. It was found that these inhibitors (EHNA and Ara-A, 2-deoxycoformycin and adenosine) increase the activity of serum biotinidase as was the case with ethionine. Because SAH does not ethylate biomolecules, these changes in biotinidase activity, which can not be prevented by adenine, biotin or lecithin are most probably related to the inhibition of methylation.

Heterogeneity of hepatic tyrosine aminotransferase. Separation of the multiple forms from rat and frog liver by isoelectric focussing and hydroxylapatite column chromatography and their partial characterization.

L-Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5; TAT) and other enzymes that transaminate tyrosine in rat liver cytosol have been separated into four fractions by isoelectric focussing. One of the forms is probably identical to mitochondrial L-aspartate:2-oxoglutarate aminotransferase (EC 2.6.1.1.; mASAT). The other three forms have pI's of 4.72, 4.98 and 5.30 and Km values of 1.3 and 0.3 mM for tyrosine and alpha-ketoglutarate. These heat stable forms have little or no ASAT activity. Rat liver TAT is also separated into three peaks by hydroxylapatite. Each fraction gives only one peak of activity when electrofocussed separately. In the frog, three groups of peaks of TAT activity have been separated by hydroxylapatite column chromatography. The first group is connected with ASAT activity. These peaks (pI's 6.35, 6.50 and 6.90) are heat stable and have a Km value for tyrosine of 4 mM. These fractions probably represent cytoplasmic ASAT (sASAT). The second group of peaks has at least two subforms (pI's 9.0 and 9.4, Km for tyrosine 15 mM). These forms probably represent mASAT. The third group consists of three forms that resemble the major forms of rat liver TAT. These results indicate that heterogeneity is common to many aminotransferases and independent of regulation by glucocorticoids.

Evidence for inhibition of dopamine-beta-hydroxylase in vivo after sub-acute pyrazole treatment in rats.

Pyrazole, a widely used inhibitor of alcohol dehydrogenase, has been shown to cause a decrease of brain and heart noradrenaline (NA). An attempt to explain the mechanism of this effect is now described. L-DOPA (50-200 mg/kg, s.c.) was unable to restore brain or heart noradrenaline levels in pyrazole pre-treated rats. After monoamine oxidase inhibition with tranylcypromine or pargyline there was a slight increase in brain NA in these rats but no further increase was observed in response to L-DOPA (30 mg/kg). Brain dopamine levels were relatively higher in pyrazole pre-treated rats. This difference was particularly clear in the hypothalamus but not present at all in striatum. It was impossible to duplicate the above results using nialamide as the monoamine oxidase inhibitor. After depletion of monoamine stores by reserpine (2 x 2 mg/kg) or oxypertine (75 mg/kg) and treatment with tranylcypromine and L-DOPA it is possible to get an indication of the maximal rate of synthesis of NA. In pyrazole treated rats synthesis of NA in brain was 70% reduced and about 50% reduced in heart. Synthesis of dopamine from L-DOPA was unimpaired. Dopamine-beta-hydroxylase activity in the hypothalamus of rats treated for four days with pyrazole (100 mg/kg i.p.) was more than 40% reduced. This inhibition could not be obtained by addition of pyrazole to samples of purified dopamine-beta-hydroxylase. The results strongly suggest that the reason for the decrease in brain and peripheral NA seen after pyrazole administration in rats is due to inhibition of dopamine-beta-hydroxylase.

Effect of methylglyoxal bis(guanylhydrazone) on polyamine metabolism in normal and regenerating rat liver and rat thymus.

1. Injections of sublethal doses of methylglyoxal bis(guanylhydrazone), a potent inhibitor of putrescine-activated S-adenosylmethionine decarboxylase in vitro, resulted after a few days in an immense increase in the activity of S-adenosylmethionine decarboxylase in normal and regenerating rat liver and in rat thymus. The increase in the activity of S-adenosylmethionine decarboxylase was at least partly due to a marked lengthening of the half-life of the enzyme. 2. In regenerating liver and thymus there was also a moderate stimulation of the activity of ornithine decarboxylase (EC 4.1.1.17) and a marked accumulation of tissue putrescine. 3. Injection of methylglyoxal bis(guanylhydrazone) into the rat also markedly decreased the activity of diamine oxidase (EC 1.4.3.6) in thymus. 4. In no cases where doses of methylglyoxal bis(guanylhydrazone) close to the LD(50) dose for the rat were used was it possible to lower tissue spermidine content to any significant extent. 5. Methylglyoxal bis(guanylhydrazone) seemed to act as a competitive inhibitor for the substrate S-adenosylmethionine and as an uncompetitive inhibitor for the activator putrescine in the decarboxylation of S-adenosylmethionine in vitro. 6. In the diamine oxidase reaction, with putrescine as the substrate, methylglyoxal bis(guanylhydrazone) was a non-competitive inhibitor for putrescine.

Transient activation of RNA polymerase in Escherichia coli B after infection with bacteriophage T4.

Sköld and Buchanan(1) have reported that there is a rapid loss of RNA polymerase activity in Escherichia coli B after infection with T4 bacteriophage. More recent studies on the mechanism of this inactivation have been made in this(2) and other laboratories.(3, 4) In this communication, we report the observation of a transient stimulation of RNA polymerase activity when measurement is made immediately after infection and when cells are ruptured by a gentle lysis procedure. The increase in activity is independent of the synthesis of protein. The activity in the extracts of infected cells is lost by treatment of the extract with antibody to E. coli RNA polymerase and is refractive to the inhibitory action of the antibiotic rifamycin. Hybridization experiments indicate that an RNA transcribed almost exclusively from a T4 DNA template is the product of incubation of extracts of infected cells with a reaction mixture containing an exogenous primer (salmon sperm DNA). These findings are consistent with the hypothesis that one of the first steps in phage infection is the formation of a transcription complex containing T4 DNA and E. coli RNA polymerase.

Pharmacological and toxicological properties of 4-hydroxypyrazole, a metabolite of pyrazole.

The effects of 4-hydroxypyrazole (4-HP), a principal metabolite of pyrazole, were studied in mice. The compound was toxic, much more so than pyrazole with an LD50 of 1.1 mmol/kg (92 mg/Kg) and doses greater than 1.5 mmol/kg (126 mg/kg) were almost invariably fatal. Toxicity seemed to be centered on the liver with microscopic evidence of centrolobular necrosis apparent. Mouse liver catalase was almost totally inhibited 1 hour after administration of 1 mmol/kg of 4-HP. Tryptophan pyrrolase was also inhibited 4-HP seemed to penetrate into the brain as judged by inhibition of brain catalase activity. A slight increase in brain serotonin concentration was found but 4-HP had no effect in the doses used (1.5 mmol/kg or 4 x 1.0 mmol/kg) in brain or heart noradrenaline. We conclude that the pyrazole-induced decrease in brain noradrenaline is not mediated via 4-HP. Furthermore, simultaneous treatment with methanol and pyrazole, which prevents the formation of 4-HP, did not prevent the decrease in brain noradrenaline levels. Since methanol prevented the pyrazole-induced decrease in brain catalase activity, we can also rule out the possibility that the decrease in brain noradrenaline is secondary to pyrazole-induced inhibition of brain catalase. It is concluded that though 4-HP is an active metabolite of pyrazole, causing, in particular, the hepatotoxicity of the parent molecule, it is not responsible for all the varied biological of pyrazole.

Partial purification and properties of frog liver tyrosine aminotransferase.

Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.