Biomedical subjects
Publications and source records attributed to U Keller.
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A circular linkage map was developed for Streptomyces chrysomallus, a producer of actinomycin C. The map order of various marker loci was deduced from matings and to a minor extent from protoplast fusions. The map strongly resembles that of Streptomyces coelicolor A3(2). The recombination frequencies were low and highly variable (from 10(-9) to 5 X 10(-6]. Plasmid pIJ303 expressed its thiostrepton resistance gene in S. chrysomallus but did not promote chromosomal transfer or induce the Ltz+ phenotype. The data provide a background of genetics for investigations of antibiotic synthesis in this strain.
Energy metabolism of calf muscle was assessed non-invasively by phosphorus (31P) NMR spectroscopy in eleven patients with symptomatic arterial occlusion and in seven matched controls. Phosphocreatine (PCr) content and pH values decreased during non-ischaemic foot exercise to lower values in severely afflicted patients but in all patients, as a group, they were not significantly decreased compared to controls. In contrast, recovery from ischaemic exercise (arterial occlusion by a tourniquet) demonstrated significant differences between patients and controls. Intracellular pH and PCr recovered more slowly in patients than in controls; PCr recovery proceeded exponentially with a recovery half-time of 203 +/- 74 s in patients compared to 36.7 +/- 5.5 s in controls (P less than 0.02). Phosphocreatine (PCr) recovery after ischaemic exercise correlated significantly with the degree of arterial stenoses as assessed by Doppler ultrasound (r = 0.739, P = 0.019) and by angiography (r = 0.885, P = 0.005), suggesting that the degree of large vessel stenoses limits the postischaemic increase in mitochondrial oxidative phosphorylation. Reactive blood flow after ischaemia failed to correlate with PCr recovery or with the degree of arterial stenoses. Phosphorus (31P) NMR spectroscopy provides, therefore, quantitative parameters of muscle energy metabolism in patients with peripheral arterial occlusions.
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Recent studies in man demonstrated a marked ketogenic effect of increased plasma norepinephrine concentrations as observed in diabetic ketoacidosis. Since this effect may have been due either to increased substrate supply for ketogenesis (lipolysis) or to direct hepatic activation of ketogenesis, the latter mechanism was examined in isolated rat hepatocytes. Incubation of hepatocytes with norepinephrine (10(-7) to 10(-4) M) resulted in a dose-dependent increase in conversion of the long-chain fatty acid [1-14C]palmitate into ketone bodies and CO2. Norepinephrine decreased [1-14C]palmitate conversion into triglycerides without affecting fatty acid uptake. Norepinephrine enhanced ketogenesis from [1-14C]palmitate in a physiologic range of fatty acid concentrations (0.5-2.5 mM), but failed to affect fatty acid esterification to phospholipids or mono- and diglycerides. In contrast to long-chain fatty acids, oxidation of the medium-chain fatty acid [1-14C]octanoate to ketone bodies was not enhanced by norepinephrine, whereas CO2 production increased. The effect of norepinephrine on [1-14C]fatty acid oxidation was blocked by the alpha 1 receptor blocker prazosin. The results demonstrate that norepinephrine diverts long-chain fatty acids into the pathways of oxidation and ketogenesis away from esterification, suggesting enhanced carnitine-dependent mitochondrial fatty acid uptake. The studies using octanoate indicated that norepinephrine also enhanced fatty acid oxidation by increasing the flux of acetyl-CoA through the Krebs cycle. The data suggest that stress-associated sympathetic activation and norepinephrine discharge, as observed in diabetic ketoacidosis, result in direct activation of ketogenesis in the liver.
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It is an open question whether information about hypertension and obesity increases compliance with therapy. Nevertheless, patients increasingly demand precise but simple and comprehensive information. A simple slide programme is described which can be demonstrated in any waiting room. The learning effect was assessed in 1083 subjects, of whom 485 had seen the programme completely; 256 subjects served as controls. The percentage of subjects with good or excellent knowledge about hypertension and obesity rose from 22.8% in the controls to 64.2% in the experimental group. Age was the only factor influencing learning, but this was of no great importance in subjects under 70. In particular, social status did not have any significant effect on learning. This programme may be an ideal tool to inform patients about hypertension and obesity and to study the influence of information on compliance with therapy.
The metabolic fate of 13C1-labeled butyrate in the liver of living rats has been studied by 13C NMR. The formation of the ketone bodies acetoacetate and beta-hydroxybutyrate was observed in vivo as well as resonances from glutamate, glutamine, and carbonate. The observed time course of these metabolites demonstrates the potential of the technique to measure enzyme kinetics in vivo and also to measure the enzyme capacity of a given organ to metabolize a substrate. The in vivo spectra were compared to in vitro spectra of the excised liver and perchloric acid extracts of the liver. Observation of the metabolites and monitoring of their time course in vivo would not have been possible without distinct improvements in the spectral resolution and the spatial localization of the radio-frequency field within the liver. As a novel approach, we have selected the carbonyl region of the 13C NMR spectra for identifying the ketone bodies and other oxidation products in the liver.
Several structural analogs of 4-methyl-3-hydroxyanthranilic acid (4-MHA) that had been established as substrates of the 4-MHA-activating enzyme from Streptomyces chrysomallus (Keller, U., Kleinkauf, H., and Zocher, R. (1984) Biochemistry 23, 1479-1484) were fed in short term labeling experiments to cultures of two actinomycin-producing streptomycetes. Besides inhibition of actinomycin synthesis, the addition of 4-methyl-3-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-aminobenzoic acid, or 4-methyl-3-methoxy-benzoic acid induced the formation of novel compounds. The data indicate that these compounds are structural analogs of 4-MHA pentapeptide lactones, which are the most probable precursors of actinomycins (Katz, E. (1967) in Antibiotics II (Gottlieb, D., and Shaw, P. D., eds) pp. 276-341, Springer-Verlag, New York). Since the structural analogs of 4-MHA are missing the o-aminophenol configuration, the corresponding acyl pentapeptide lactones cannot react with each other to give phenoxazines. Therefore, they accumulate and thus become detectable. Feeding cultures with 4-MHA resulted in an inhibition of actinomycin synthesis apparently at the level of acyl pentapeptide lactone synthesis. In such experiments, the formation of pentapeptide lactones, which are most likely derived from 4-MHA pentapeptide lactones, could be detected. The results provide experimental evidence that the biosynthesis of actinomycins proceeds via the 4-MHA-pentapeptide lactones.
A 4-methyl-3-hydroxyanthranilic acid (4-MHA) activating enzyme was purified 24-fold from a crude protein extract of Streptomyces chrysomallus . The enzyme catalyzes both 4-MHA-dependent ATP/PPi exchange and the formation of the corresponding adenylate. No AMP was formed during the reaction, indicating that no covalent binding of 4-MHA takes place. Besides 4-MHA, the enzyme also catalyzes the formation of adenylates from 3-hydroxyanthranilic acid (3-HA), anthranilic acid (AA), benzoic acid (BA), 3-hydroxybenzoic acid (3-HB), 4-methyl-3-hydroxybenzoic acid (4-MHB), 4-methyl-3-methoxybenzoic acid (4- MMB ), and 4-aminobenzoic acid (4-AB). No such adenylates were formed from 2-aminophenol (2-AP), 2-hydroxybenzoic acid (2-HB), 3-hydroxykynurenine (3-HK), and tryptophan (Trp). 3-HA, 4-MHB, and 4-AB were among the structural analogues of 4-MHA that were the most effective for adenylate synthesis. In the case of 3-HA, considerable AMP release was observed, most probably due to nonenzymatic hydrolysis of the corresponding adenylate. A molecular weight between 53 000 and 57 000 was estimated. The specific activity of the enzyme was correlated with the titer of antibiotic in the cultures, and feeding experiments with whole mycelium of S. chrysomallus showed that 4-MHB was a strong inhibitor of actinomycin synthesis in vivo. The data strongly suggest that the enzyme is involved in the biosynthesis of actinomycin.
The diagnostic value of a direct assay of free thyroxine has been evaluated by comparing the concentration of free T4 with the results of an oral TRH test. 147 patients were referred to an endocrine outpatient department because of suspected thyroid dysfunction. Based on clinical investigation and on the results of an oral TRH test, 75 patients were euthyroid, 12 hyperthyroid and 12 hypothyroid. Preclinical hypothyroidism (3 h- TSH value above 35 mE/l and euthyroid peripheral thyroid hormone levels) was found in 25 patients. In 23, preclinical hyperthyroidism (3 h- TSH value after TRH below the sensitivity of the assay, i.e. less than 1 mE/l) together with euthyroid peripheral thyroid hormone values was found. Free thyroxine was assayed using the solid phase technique (Clinical Assay); intraassay coefficients of variation varied between 4.5 and 7.6% and interassay coefficients of variation between 8 and 13%. A normal range for free thyroxine was found between 8.4 and 26.4 pmol/l, the log mean being 14.9 pmol/l. In 12 hyperthyroid patients the free T4 concentration was above 49.4 pmol/l, while the mean of 12 hypothyroid patients was below 4.4 pmol/l. The mean free T4 concentrations in the syndromes of preclinical hypo- and hyperthyroidism, which by definition are only recognizable by the TSH increase in response to TRH, were within the normal range. Using the sensitive TRH test as a comparison, the present investigation thus confirms the reliability of direct assay of free T4 for the diagnosis of thyroid function in clinical practise.(ABSTRACT TRUNCATED AT 250 WORDS)
A D-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea was purified about 145-fold. The enzyme was able to catalyse both the D-lysergic acid-dependent ATP-pyrophosphate exchange and the formation of ATP from D-lysergic acid adenylate and pyrophosphate. Both reactions were also catalysed to a decreased but significant extent with respect to dihydrolysergic acid. The molecular mass of the enzyme was estimated to lie between 135 and 140 kDa. The involvement of the enzyme in the biosynthesis of ergot peptide alkaloids is discussed.
The authors report marked hypertriglyceridaemia with peak values of up to 14 mmol/l in patients who have undergone bone marrow transplantation (BMT) and who are receiving 925 g/d glucose and 70 g/d crystalline amino acids for parenteral nutrition. Larger doses of insulin lead to only slightly better control of mean blood glucose level and to a clearly less marked increase in triglycerides (TG) in a series of patients otherwise treated identically. Only a slight increase in serum TG was seen in BMT patients receiving 325 g glucose per day. There was no increase in serum TG to values above normal in a small group of surgical patients receiving 700-900 g glucose/d and high doses of insulin. Peripheral insulin resistance is presumed to be the pathogenetic factor in this hypertriglyceridaemia, mia, which can be avoided by higher doses of insulin or decreased glucose load.
The effect of physiological elevation of growth hormone levels on ketone body kinetics was determined using a 14C-ketone body tracer technique in normal and acutely insulin-deficient man. Changes of ketone body production and metabolic clearance rates during growth hormone infusion (plasma levels of approximately 25 micrograms/1) were measured during basal conditions and during heparin-induced elevation of non-esterified fatty acid levels. Growth hormone administration to six subjects fasted overnight resulted in an increase in ketone body production which exceeded that observed in nine control subjects (5.5 +/- 0.5 versus 3.1 +/- 0.1 mumol X kg-1 X min-1, p less than 0.025) after elevation of plasma non-esterified fatty acids. Growth hormone infusion increased glycerol and non-esterified fatty acid concentrations indicating enhanced lipolysis. During somatostatin-induced acute insulin deficiency (n = 7), growth hormone enhanced the increase in total ketone body production observed in six subjects receiving somatostatin alone (8.4 +/- 0.8 versus 4.1 +/- 0.7 mumol X kg-1 X min-1, p less than 0.01). Total ketone body metabolic clearance decreased by 50% during somatostatin and remained uninfluenced by growth hormone. Non-esterified fatty acids and glycerol levels increased during somatostatin, and growth hormone failed to alter non-esterified fatty acid levels significantly. The results demonstrate a stimulatory effect of high physiological growth hormone levels on ketogenesis which is largely explained by an enhancement of lipolysis and thus increase in substrate supply for ketogenesis. Growth hormone administration during acute insulin deficiency enhanced ketogenesis in the absence of alterations in plasma non-esterified fatty acid levels, suggesting a direct hepatic ketogenic effect.
Plasma concentrations of pancreatic glucagon, C-peptide, and pancreatic polypeptide were measured during arginine stimulation in 16 patients with chronic pancreatitis, in eight subjects with idiopathic diabetes mellitus, and in seven healthy controls. The hormone responses were compared with exocrine pancreatic function as assessed using the urinary excretion rate of p-aminobenzoic acid after oral ingestion of n-benzoyl-l-tyrosyl-p-aminobenzoic acid (BT-PABA). The increase in pancreatic glucagon levels during arginine stimulation was significantly reduced in patients with chronic pancreatitis compared to healthy controls, most markedly in those with secondary diabetes. In contrast, the glucagon response was unimpaired in patients with idiopathic diabetes. The arginine-induced increase in plasma glucagon and C-peptide concentrations correlated significantly with urinary PABA excretion in chronic pancreatitis (P less than 0.001, P less than 0.01, respectively). The responses of plasma C-peptide and pancreatic polypeptide separated pancreatitic and idiopathic diabetes less well. Thus, the glucagon response to arginine distinguished secondary diabetes due to chronic pancreatitis and idiopathic diabetes mellitus. The correlation between urinary PABA excretion and glucagon levels suggests that in chronic pancreatitis there is a parallel impairment of exocrine and endocrine function.
Splanchnic extraction rates of adrenaline and noradrenaline were determined in seven normal subjects and in nine patients with cirrhosis of the liver using arterial-hepatic venous catherization . Both catecholamines were effectively removed when the blood passed through the splanchnic area: splanchnic fractional uptake of adrenaline in normal subjects was 90 +/- 3%, and lower for noradrenaline, 68 +/- 4% (P less than 0.001). Net splanchnic extraction rates were higher for noradrenaline (126 +/- 16 ng/min) than for adrenaline (40 +/- 10 ng/min, P less than 0.001), probably due to the higher arterial plasma levels of noradrenaline. Resting arterial adrenaline and noradrenaline levels were significantly higher in cirrhotic patients than in normal subjects (adrenaline: 121 +/- 27 vs 54 +/- 8 pg/ml, P less than 0.05; noradrenaline: 678 +/- 89 vs 251 +/- 26 pg/ml, P less than 0.005). Net splanchnic catecholamine uptake was increased in cirrhotic patients. The results demonstrate that the splanchnic bed in normal and cirrhotic subjects extracts plasma catecholamines efficiently; they suggest that elevated plasma catecholamines in cirrhosis are not the result of impaired splanchnic catecholamine removal.
The effect of semisynthetic human insulin on hepatic glucose output, peripheral glucose clearance, plasma levels of C-Peptide, free fatty acids and amino acids was compared with purified pork insulin using the glucose clamp technique. 8 normal overnight-fasted subjects received intravenous infusions of either human or porcine insulin at 20 mU/m2.min(-1) during 120 min achieving plasma insulin levels of approximately equal to 50 mU/l. Plasma glucose levels were maintained at euglycaemia by variable rates of glucose infusion. Hepatic glucose production measured by continuous infusion of 3-(3) H-glucose was similarly suppressed by both insulins to rates near zero. The metabolic clearance rate of glucose increased during infusion of human insulin by 120%, C-peptide levels decreased by 41% and plasma FFA concentrations fell by 74%. The respective changes during infusion of pork insulin were similar, 118%, 48% and 72%. Both insulins decreased the plasma levels of branched-chain amino acids, tyrosine, phenylalanine, methionine, serine and histidine similarly. Thus, the results demonstrate that semisynthetic human and porcine insulin are aequipotent with respect to suppression of hepatic glucose output, stimulation of glucose clearance, inhibition of insulin secretion, lipolysis and proteolysis.
Protoplasts of Claviceps purpurea were prepared by treatment of mycelium with a lytic mixture of snail gut enzyme and cellulase from Trichoderma viride. Such protoplasts could be efficiently lysed by Triton X-100 treatment at high osmotic pressure without Ca or Mg, allowing the release of intact vacuoles in high yields. Vacuoles obtained from cells grown in modified Vogel medium (vegetative-type cells not producing alkaloids) were isolated and purified by centrifugation from a 5% Ficoll 400 (wt/vol) phase into the interphase between two layers, one containing 0.25 M each of mannitol and sucrose, and one containing 0.5 M mannitol. Vacuoles derived from cells grown in a medium favoring ergot alkaloid synthesis (sclerotia-like cells) were isolated by gentle centrifugation of filtered protoplast lysates without addition of Ficoll 400. Biochemical analyses of the vacuole fraction isolated from either kind of cell revealed their function as compartments harboring several hydrolytic enzymes. However, the enrichment of free amino acids in vacuoles of sclerotia-like cells was less pronounced than that in vacuoles of vegetative-type cells, indicating a difference in metabolic compartmentation in the two types of cells.