Requirement of hydD, hydE, hypC and hypE genes for hydrogenase activity in Helicobacter pylori

Helicobacter pylori possesses a membrane-bound, nickel containing, hydrogen uptake hydrogenase enzyme; its synthesis requires structural as well as accessory proteins, the latter needed for the complete maturation of the enzyme. Our lab previously characterized mutants in the accessory hyp genes, hypA, hypB, hypD and hypF that were all severely affected for hydrogenase activity, and in some cases (hypA and hypB mutants) also affected for urease activity. This finding prompted us to disrupt the two remaining unstudied hyp genes of H. pylori, hypC and hypE, in order to see if the same pleiotropic effect would be observed. In both mutants hydrogenase activity was abolished but urease activity remained unaffected. Addition of 5 microM nickel into the growth medium partially restored the hydrogenase activity in the hypE mutant and to a lesser extent in the hypC mutant. In addition, we also disrupted the genes HP0634 (referred as hydD in the H. pylori 26695 genome database) and HP0635 (whose function was unknown, referred to here as hydE) to address their possible roles in the hydrogenase synthesis/maturation process. In both cases, hydrogenase activities were abolished and addition of nickel could not restore the activity, suggesting that these proteins are involved in the hydrogenase synthesis process rather than in nickel mobilization/insertion steps.     

AgNORs in cardiomyocytes from surgical patients with coronary heart disease.

AIM: To evaluate the interphase ribosomal RNA cistron activity of cardiomyocytes in surgical patients with chronic ischaemic heart disease by means of the nucleolar organiser region silver staining (AgNOR) technique. METHODS: Nucleoli were investigated in myocardial samples obtained from 46 patients with chronic ischaemic heart disease before, during, and soon after cardioplegia ischaemia. Cryostat sections of 10 microns thickness were air dried, fixed in methanol/glacial acetic acid (3:1) for 15 minutes, rinsed carefully with distilled water, incubated in 2 N formic acid for 10 minutes, and impregnated with silver colloid solution for 2.5-3 minutes at 68-70 degrees C. The lightly counterstained sections were examined under oil immersion at x1000 magnification. For the estimation of AgNOR numbers at least 100 silver stained cardiomyocyte and fibroblast nuclei were counted in each section. On the basis of these data, the mean number of AgNORs in each nucleus was determined. The Student's t test was used to compare the groups tested. RESULTS: The initial mean numbers of AgNORs varied greatly, demonstrating a difference between groups of patients with or without antecedent myocardial infarction (9.5 v 11.0; p < 0.05). During myocardial arrest, the numbers of AgNORs in cardiomyocytes were decreased in all but seven patients, while those in fibroblasts tended to increase. At the stage of reperfusion and myocardial warming, in all but three patients the numbers of AgNORs in cardiomyocytes either normalised or were even higher than the initial value. CONCLUSIONS: The AgNOR count in cardiomyocytes is a very sensitive test for the measurement of cardiac function in surgical patients with chronic ischaemic heart disease and could be useful for monitoring myocardial status during the course of surgery, including cardioplegia. The high risk group for surgery included patients with antecedent myocardial infarction and severe heart failure. It is thought that a reversible nucleolin/fibrillarin/pre-rRNA/small nucleolar RNA modification might account for this fast decline then rise in the AgNOR count in cardiomyocytes at the stages of cardioplegia and reperfusion, respectively.     

Morphological detection and quantification of lipoprotein(a) deposition in atheromatous lesions of human aorta and coronary arteries

Summary Lipoprotein(a), as an atherogenic particle, represents an independent risk factor for coronary heart disease. In the present study the morphological distribution of apoprotein (a) and apoprotein B within the arterial wall is described. Apoprotein B, a constituent of very low-density lipoprotein, low-density lipoprotein and lipoprotein(a) has previously been demonstrated in atheromatous lesions. Lipoprotein(a) possesses an additional protein, designated apoprotein (a). Autopsy material (n=74) from the left coronary artery and from the thoracic aorta has been examined by means of immunohistochemistry and both apoprotein (a) and apoprotein B were detected, primarily associated with the extracellular matrix and accumulating in lesions in the arterial wall. The staining pattern for both antigens was almost always found to be congruent, suggesting that the detection of (a)-antigen has to be attributed at least in part to the presence of lipoprotein(a). It is concluded that both low-density lipoprotein and lipoprotein(a) have an important role in the pathogenesis of atherosclerosis.     

Thermogenic effect of adrenaline: interaction with insulin

Summary The contribution of insulin (3.6 pmol sd kg body mass^–1·min^–1 to adrenaline-induced (0.164 nmol · kg fat free mass^–1·min^–1) thermogenesis was studied in ten postabsorptive healthy volunteers using two sequential protocols. Variables considered were oxygen consumption as well as carbon dioxide production, heart rate, blood pressure, plasma concentrations of glucose, insulin, glycerol, free fatty acids,-HO-butyrate and lactate. Adrenaline increased plasma concentrations of glucose, glycerol, free fatty acids, and-HO-butyrate, and heart rate and metabolic rate during normo-insulinaemia [61.3 (SEM 6.6) pmol·^–1]. Similar effects were observed during hyperinsulinaemia [167.9 (SEM 18.7) pmol·^–1], but the effect of adrenaline on oxygen consumption was reduced. On average, metabolic rate increased by 12.9% during normo-insulinaemia and by 8.9% during hyperinsulinaemia. We concluded that relative hyperinsulinaemia resulted in decreased adrenaline-induced thermogenesis and therefore increased whole body anabolism.     

Structural analysis of alkene/maleic anhydride copolymers

Structural investigations on maleic anhydride (MAn) copolymers with ethene, propene and styrene, their products of hydrolysis, and their methyl half-esters by means of ¹³C NMR spectroscopy are presented. The spectra of 2,3-diethylsuccinic acid and its anhydrides in the erythro- and threo-configuration and of butylsuccinic acid and its anhydride were obtained and compared with the spectra of the copolymers. In each case the results show the formation of both threo(trans)- and erythro(cis)-structures. At a polymerization temperature of 60°C the proportions were 88% threo to 12% erythro for ethene/MAn copolymers and 80% threo to 20% erythro for propene/MAn copolymers, a ratio which was confirmed also by the hydrolyzed forms. Copolymerization at 150°C leads, in the case of propene/MAn, to 67% threo and 33% erythro. The production of ethene/MA copolymer via the copolymerization of ethene and fumaric acid half-ester and its saponification to ethene/fumaric acid leads to a ratio of 38% threo to 62% erythro. These results are in accordance with the thermal stabilities of the configurations. In the case of styrene/MAn copolymer it is not possible to obtain a unique interpretation of the configurations from the spectra. For the conversion of propene/MAn and styrene/MAn copolymers by means of methanol to half-esters it can be derived from the ¹³C NMR spectra how many of the half-ester moieties are obtained in the neighbourhood of the methyl and phenyl groups, respectively, of the olefin.     

The time course of glycogen depletion in single fibers of chronically stimulated rabbit fast-twitch muscle

A time course study was conducted to investigate the possibility of a relationship between fiber degeneration and glycogen depletion in chronically nerve-stimulated extensor digitorum longus muscle of the rabbit. Muscles were stimulated 12 h daily at 10 Hz using alternating one-hour periods of stimulation and rest. When measured for the first time after 3 h (1 h stimulation, 1 h rest, 1 h stimulation), microphotometry revealed complete glycogen depletion of all fiber types (fast glycolytic, FG; fast oxidative glycolytic, FOG; slow oxidative, SO). Different responses were noted beginning at day 4. At this time point, all FOG and SO fibers recovered their glycogen stores with some of the FOG population attaining levels higher than the FOG fibers in the unstimulated, contralateral muscle. Approximately 28% of the FG fibers recovered to normal glycogen values, whereas 58% remained depleted and 14% displayed overshoting glycogen levels. Fifteen percent of all fibers were glycogen-depleted after 12 days of stimulation. At this time, classic fiber types could no longer be distinguished. Fiber degeneration, which was recognized by the invasion of nonmuscle cells, began after 6 days and was restricted to the glycogen-depleted fibers. By this time, there was also a significant increase in DNA content. Exhaustions of glycogen, the main fuel of the FG fibers, is believed to cause a collapse of energy-supply and ATP-driven ionic pumps. The latter could be the initial step of fiber deterioration.     

Metabolism of phenylalanine in liver diseases

Summary 1. Amethod for the determination of phenylalaninehydroxylase-activity in needle biopsy material of human liver was developed and tested. The kinetic data of the enzyme were determined. TheK _m for the substrate phenylalanine is 1,32 mM, for the cofactor 0,08 mM.2. The activity of phenylalaninehydroxylase was determined in biopsies from normal liver, liver cirrhosis, alcoholic hepatitis and other liver diseases. In all liver diseases the enzyme activity related to wet weight or DNA is reduced. The capacity for the hydroxylation of phenylalanine (of a cirrhotic liver) amounts to about 20% of normal liver.3. After an oral load with L-phenylalanine (100 mg/kg) the phenylalanine- and tyrosine-concentration in blood plasma was followed for 5 hours. Patients with liver cirrhosis or acute hepatitis show significant higher concentrations of phenylalanine, and significant lower concentrations of tyrosine than normal persons.4. From the decline of phenylalanine-concentration after the maximum the phenylalanine-elimination rate and from the increase of tyrosine concentration the tyrosine-production rate were calculated. Between phenylalanine-elimination rate and tyrosine-production rate a strong correlation exists. In patients with cirrhosis or acute hepatitis significantly reduced phenylalanine-elimination rate and tyrosine production rate were found compared with persons without liver disease. Patients with alcoholi hepatitis show no significant difference compared with normal persons.5. There is no correlation between the activity of phenylalaninehydroxylase in liver tissue and phenylalanine-elimination rate and tyrosine-production rate, respectively, calculated from the concentration of this amino acids in serum after a phenylalanine load.6. We conclude from these findings that the increased serum concentration of phenylalanine in some liver diseases, especially liver cirrhosis, can partly be explained by the diminished metabolism of phenylalanine in the liver. Portocaval shunts probably contribute to the elevated level of phenylalanine in serum.Mit Unterstützung der Deutschen Forschungsgemeinschaft und der Sandoz-Stiftung für medizinische Forschung     

The effect of erroneous responses on response monitoring in patients with major depressive disorder: a study with event-related potentials

Perceived failure is reported to have detrimental effects on subsequent performance in patients with major depressive disorder. We investigated the error-related negativity (ERN)/error negativity (Ne), an electrophysiological correlate of response monitoring, using a 64-channel EEG. Sixteen patients with DSM-IV major depressive disorder and 16 matched controls participated in an Eriksen flanker task with continuous performance feedback that signaled monetary reward. Compared to controls, patients with major depressive disorder showed a less negative ERN/Ne in error trials following error trials. This result might reflect impaired response monitoring processes in major depressive disorder resulting from an underactivity in a central reward pathway and/or a deficit in strategic reasoning.     

Comparative genomic hybridization analysis of hepatoblastoma reveals high frequency of X-chromosome gains and similarities between epithelial and stromal components

Hepatoblastoma (HB) is the most common liver tumor in childhood and differs in its environmental risk factors and genetic background from hepatocellular carcinoma. HB is associated with inherited conditions such as familial adenomatous polyposis and Beckwith-Wiedemann syndrome, suggesting the importance of genetic abnormalities in the pathogenesis and progression of this disease. It has a very polymorphous morphology. A diverse range of cytogenetic alterations has been reported to date, the most frequent being trisomy 2 and trisomy 20. Thirty-five HB specimens from 31 patients (22 purely epithelial, 4 purely mesenchymal, 9 mixed) were examined by comparative genomic hybridization (CGH), a technique that enables us to screen the entire tumor genome for genetic losses and gains. Our aims were as follows: (1) to characterize chromosome abnormalities that appear in this tumor and (2) to identify possible differences between different histologic subtypes of HB. We found significant gains of genetic material, with very little difference in the number and type of alterations between the different histologic components of HB. The most frequent alterations were gains of Xp (15 cases, 43%) and Xq (21 cases, 60%). This finding was also confirmed by fluorescent in situ hybridization performed on nuclei extracted from 6 specimens. Other common alterations were 1p-, 2q+, 2q-, 4q-, and 4q+. We found no difference between different histologic subtypes, a finding that may be in agreement with the hypothesis of a common clonal origin for the different components. An hitherto-unreported high frequency of X chromosome gains may support the assumption that X-linked genes are involved in the development of this neoplasm.