Search PubMed⌕ Search

Biomedical subjects

O Lenz

Publications and source records attributed to O Lenz.

33 records · Page 2Linked to original sources
PubMed2000

The H(2) sensor of Ralstonia eutropha is a member of the subclass of regulatory [NiFe] hydrogenases.

Two energy-generating hydrogenases enable the aerobic hydrogen bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus) to use molecular hydrogen as the sole energy source. The complex synthesis of the nickel-iron-containing enzymes has to be efficiently regulated in response to H(2), which is available in low amounts in aerobic environments. H(2) sensing in R. eutropha is achieved by a hydrogenase-like protein which controls the hydrogenase gene expression in concert with a two-component regulatory system. In this study we show that the H(2) sensor of R. eutropha is a cytoplasmic protein. Although capable of H(2) oxidation with redox dyes as electron acceptors, the protein did not support lithoautotrophic growth in the absence of the energy-generating hydrogenases. A specifically designed overexpression system for R. eutropha provided the basis for identifying the H(2) sensor as a nickel-containing regulatory protein. The data support previous results which showed that the sensor has an active site similar to that of prototypic [NiFe] hydrogenases (A. J. Pierik, M. Schmelz, O. Lenz, B. Friedrich, and S. P. J. Albracht, FEBS Lett. 438:231-235, 1998). It is demonstrated that in addition to the enzymatic activity the regulatory function of the H(2) sensor is nickel dependent. The results suggest that H(2) sensing requires an active [NiFe] hydrogenase, leaving the question open whether only H(2) binding or subsequent H(2) oxidation and electron transfer processes are necessary for signaling. The regulatory role of the H(2)-sensing hydrogenase of R. eutropha, which has also been investigated in other hydrogen-oxidizing bacteria, is intimately correlated with a set of typical structural features. Thus, the family of H(2) sensors represents a novel subclass of [NiFe] hydrogenases denoted as the "regulatory hydrogenases."

Amino Acid Sequence↗
PubMed2000

Identification of a novel consensus sequence at the cleavage site of the Lassa virus glycoprotein.

The Lassa virus glycoprotein consists of an amino-terminal and a carboxy-terminal cleavage fragment designated GP-1 and GP-2, respectively, that are derived by proteolysis from the precursor GP-C. The membrane-anchored GP-2 obtained from purified virions of the Josiah strain revealed the N-terminal tripeptide GTF(262) when analyzed by Edman degradation. Upstream of this site, GP-C contains the tetrapeptide sequence RRLL(259), which is conserved in all Lassa virus isolates published to date. Systematic mutational analysis of vector-expressed GP-C revealed that the motif R-X (L/I/V)-L(259) (where X stands for L, I, or V) is essential for cleavage of the peptide bond between leucine(259) and glycine(260). This cleavage motif is homologous to the consensus sequence recognized by a novel class of cellular endoproteases which have so far not been implicated in the processing of viral glycoproteins.

Amino Acid Sequence↗
PubMed1999

IGF-1 decreases collagen degradation in diabetic NOD mesangial cells: implications for diabetic nephropathy.

Nonobese diabetic (NOD) mice develop glomerulosclerosis shortly after the onset of diabetes. We showed that mesangial cells (MCs) from diabetic mice exhibited a stable phenotypic switch, consisting of both increased IGF-1 synthesis and proliferation (Elliot SJ, Striker LJ, Hattori M, Yang CW, He CJ, Peten EP, Striker GE: Mesangial cells from diabetic NOD mice constitutively secrete increased amounts of insulin-like growth factor-I. Endocrinology 133:1783-1788, 1993). Because the extracellular matrix (ECM) accumulation in diabetic glomerulosclerosis may be partly due to decreased degradation, we examined the effect of excess IGF-1 on collagen turnover and the activity of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in diabetic and nondiabetic NOD-MC. Total collagen degradation was reduced by 58 +/- 18% in diabetic NOD-MCs, which correlated with a constitutive decrease in MMP-2 activity and mRNA levels, and nearly undetectable MMP-9 activity and mRNA. TIMP levels were slightly decreased in diabetic NOD-MC. The addition of recombinant IGF-1 to nondiabetic NOD-MC resulted in a decrease in MMP-2 and TIMP activity. Furthermore, treatment of diabetic NOD-MC with a neutralizing antibody against IGF-1 increased the latent form, and restored the active form, of MMP-2. In conclusion, the excessive production of IGF-1 contributes to the altered ECM turnover in diabetic NOD-MC, largely through a reduction of MMP-2 activity. These data suggest that IGF-1 could be a major contributor to the development of diabetic glomerulosclerosis.

Animals↗
PubMed1998

Characterization of the active site of a hydrogen sensor from Alcaligenes eutrophus.

A third hydrogenase was recently identified in the proteobacterium Alcaligenes eutrophus as a constituent of a novel H2-sensing multicomponent regulatory system. This regulatory hydrogenase (RH) has been overexpressed in cells deficient in both the NAD+-reducing [NiFe]-hydrogenase and the membrane-bound [NiFe]-hydrogenase. EPR, FTIR and activity studies of membrane-free extracts revealed that the RH has an active site much like that of standard [NiFe]-hydrogenases, i.e. a Ni-Fe site with two CN- groups and one CO molecule. Its catalytic power is low, but the RH is always active, insensitive to oxygen, and occurs in only two redox states.

Binding Sites↗
PubMed1998

A novel multicomponent regulatory system mediates H2 sensing in Alcaligenes eutrophus.

Oxidation of molecular hydrogen catalyzed by [NiFe] hydrogenases is a widespread mechanism of energy generation among prokaryotes. Biosynthesis of the H2-oxidizing enzymes is a complex process subject to positive control by H2 and negative control by organic energy sources. In this report we describe a novel signal transduction system regulating hydrogenase gene (hox) expression in the proteobacterium Alcaligenes eutrophus. This multicomponent system consists of the proteins HoxB, HoxC, HoxJ*, and HoxA. HoxB and HoxC share characteristic features of dimeric [NiFe] hydrogenases and form the putative H2 receptor that interacts directly or indirectly with the histidine protein kinase HoxJ*. A single amino acid substitution (HoxJ*G422S) in a conserved C-terminal glycine-rich motif of HoxJ* resulted in a loss of H2-dependent signal transduction and a concomitant block in autophosphorylating activity, suggesting that autokinase activity is essential for the response to H2. Whereas deletions in hoxB or hoxC abolished hydrogenase synthesis almost completely, the autokinase-deficient strain maintained high-level hox gene expression, indicating that the active sensor kinase exerts a negative effect on hox gene expression in the absence of H2. Substitutions of the conserved phosphoryl acceptor residue Asp55 in the response regulator HoxA (HoxAD55E and HoxAD55N) disrupted the H2 signal-transduction chain. Unlike other NtrC-like regulators, the altered HoxA proteins still allowed high-level transcriptional activation. The data presented here suggest a model in which the nonphosphorylated form of HoxA stimulates transcription in concert with a yet unknown global energy-responsive factor.

Alcaligenes↗
PubMed1998

The inheritance of glomerulosclerosis in mice is controlled by multiple quantitative trait loci.

BACKGROUND: Glomerulosclerosis, the common terminal event in chronic glomerular diseases such as diabetic nephropathy or IgA nephropathy, leads to end-stage renal disease. The considerable variation in both the risk of developing glomerulosclerosis and the rate of progression in individual patients suggest a role for genetic factors which have not been identified so far. In this study we sought to examine the mode of inheritance of glomerulosclerosis in mice. METHODS: F1 animals of a mating between glomerulosclerosis-prone ROP-Os/+ male and non-sclerotic C3H female mice were backcrossed to the ROP strain. We took advantage of the radiation-induced mutation oligosyndactylism (Os) to identify glomerulosclerosis at the age of 3 months. Kidneys were perfused in situ with PBS/Formalin 10%. The extent of glomerulosclerotic lesions was evaluated on PAS stained paraffin sections using computer-aided morphometry. RESULTS: F1 mice did not show any glomerulosclerosis. In the backcross offspring, we found a wide distribution of glomerular lesions between individual animals, ranging from normal to very severe. We calculated that at least 8-10 loci determine the severity of glomerulosclerosis in mice. CONCLUSIONS: Our data show that glomerulosclerosis is inherited in a recessive fashion involving at least 8-10 loci.

Animals↗
PubMed1998

Glomerular endothelial cells synthesize collagens but little gelatinase A and B.

Mesangial sclerosis is a major feature of progressive renal disease. The mesangium contains mesangial cells and is bounded by the peripheral glomerular basement membrane and endothelial cells. Mesangial cells synthesize and degrade extracellular matrix. Whereas both mesangial and endothelial cells synthesize extracellular matrix components, the degradative pathway, well studied in the former, has not been investigated in endothelial cells. This study examines lines of all three glomerular cell types derived from female B6SJLF1/J mice, as well as mRNA levels for collagens alpha1(I), alpha1(IV), alpha3 (IV), alpha5 (IV), and alpha1 (VI), laminin, tenascin, matrix metalloproteinase-2 (MMP-2), and MMP-9. Type I and IV collagen synthesis was confirmed by enzyme-linked immunosorbent assay. MMP-2 and MMP-9 enzyme activity was measured by zymography. It was found that glomerular endothelial cells are a significant source of collagens, laminin, and tenascin. However, they express only low levels of MMP-2 and no detectable MMP-9. Stimulation with exogenous transforming growth factor-beta1 leads to a significant increase in collagen I, tissue inhibitors of metalloproteinase-1, and MMP-9 in conditioned media. These data suggest that glomerular endothelial cells may play an active role in extracellular matrix remodeling in glomerular disease.

Animals↗
PubMed1997

Cardiac angiotensin II receptors: studies on functional coupling in Sprague-Dawley rats and TGR(alphaMHC-hAT1) transgenic rats.

The renin-angiotensin system plays an important role in the pathogenesis of cardiac hypertrophy and chronic heart failure as angiotensin II has been shown to induce cardiac hypertrophy and fibrosis. Besides these structural alterations, functional effects on cardiomyocytes have been reported in different mammalian species. Angiotensin II is known to produce a positive inotropic effect in some species, and differences in atrial and ventricular myocardium have been described. So far, the molecular events which govern angiotensin II-mediated changes in cardiac contractility are not completely understood. In order to study the dependency of the angiotensin II-induced positive inotropic effect on receptor density, we examined the effect of angiotensin II on cardiac function in atria, papillary muscles and isolated ventricular cardiomyocytes from adult Sprague-Dawley rats and TGR(alphaMHC-hAT1) transgenic rats, which expressed the human angiotensin AT1 receptor (hAT1) specifically in the heart. In atrial myocardium from adult Sprague-Dawley rats, angiotensin II (30 micromol/l) produced an AT1-mediated positive inotropic effect (38.5% of control), whereas in papillary muscles and isolated ventricular myocytes, no inotropic response was observed. As shown by polymerase chain reaction (PCR) and radioligand binding, the human angiotensin AT1 receptor was exclusively expressed in transgenic animals, which markedly overexpressed the angiotensin AT1 receptor. However, in transgenic rats the positive inotropic effect in atrial preparations was similar to the controls, and neither in papillary muscles nor in isolated cardiomyocytes the increase in receptor density led to an inotropic effect induced by angiotensin II. These data suggest that the existence of functionally uncoupled receptors rather than the low density of receptors at the ventricular site is responsible for the inability of ventricular myocardium to respond to angiotensin II.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1997

A hydrogen-sensing system in transcriptional regulation of hydrogenase gene expression in Alcaligenes species.

Heterologous complementation studies using Alcaligenes eutrophus H16 as a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophilus. Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ. The product of hoxA is homologous to a transcriptional activator of the family of two-component regulatory systems present in a number of H2-oxidizing bacteria. hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, respectively, which resemble the small and the large subunits of [NiFe] hydrogenases and correlate with putative regulatory proteins of Bradyrhizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU). hoxJ encodes a protein with typical consensus motifs of histidine protein kinases. Introduction of the complete set of genes on a broad-host-range plasmid into A. eutrophus H16 caused severe repression of soluble and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H2. This repression was released by truncation of hoxJ. H2-dependent hydrogenase gene transcription is a typical feature of A. hydrogenophilus and differs from the energy and carbon source-responding, H2-independent mode of control characteristic of A. eutrophus H16. Disruption of the A. hydrogenophilus hoxJ gene by an in-frame deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H2 in response to the availability of the carbon and energy source. RNA dot blot analysis showed that HoxJ functions on the transcriptional level. These results suggest that the putative histidine protein kinase HoxJ is involved in sensing molecular hydrogen, possibly in conjunction with the hydrogenase-like polypeptides HoxB and HoxC.

Alcaligenes↗
PubMed1997

Acute effects of nitric oxide and cyclic GMP on human myocardial contractility.

Evidence that the activity of nitric oxide synthase and the generation of nitric oxide (NO) within the myocardium are enhanced in several cardiovascular disorders is increasing. Findings whether NO exerts a direct effect on cardiac contractility are contradictory. Therefore, the direct effect of the NO donor sodium nitroprusside (SNP) on isometric force of contraction of human atrial and ventricular myocardium was investigated, and the question was addressed whether the effects of NO on cardiac contractility are mediated via cGMP. Experiments were performed on isolated electrically driven (1 Hz, 37 degrees C) human right atrial trabecula and left ventricular papillary muscle preparations from nonfailing and terminally failing hearts. SNP led to a concentration-dependent decrease of force of contraction (FOC) with a maximum effect at 100 micromol/l. In atrial trabecula, SNP (100 micromol/l) caused an acute decrease in basal FOC as well as in FOC after application of isoprenaline or IBMX by 12.5 +/- 5% (P < .05), 16.6 +/- 3.7% (P < .05) and 18.3 +/- 4.2% (P < .05), respectively. The negative inotropic effects could be attenuated by the guanylyl cyclase inhibitor methylene blue. In papillary muscle preparations, NO release caused a maximum decrease in basal and in isoprenaline-enhanced FOC of 11.0 +/- 1.9% (P < .05) and 23.6 +/- 1.5% (P < .05), respectively. In the presence of isoprenaline, the reduction of FOC was less pronounced in failing than in nonfailing papillary muscles. 8-bromo-cGMP caused a 38.2 +/- 5.2% decrease in atrial trabecula contractility. Both SNP and 8-bromo-cGMP caused a shortening of the contractile twitch with a premature onset of relaxation. As determined by radioimmunoassay, exposure of atrial trabecula to SNP (100 micromol) led to a 6-fold increase in myocardial cGMP concentrations, which could be attenuated by methylene blue. In conclusion, NO exerts a negative inotropic effect on human atrial and ventricular myocardium which seems to be mediated via generation of cGMP. The release of NO within the myocardium in a variety of cardiovascular disorders might explain decreases in cardiac contractility. The control of NO release could be an important target for future therapeutical interventions in these pathological conditions.

Cyclic GMP↗
PubMed1996

Investigation of the negative inotropic effects of 17 beta-oestradiol in human isolated myocardial tissues.

1. The aim of the present study was to evaluate the effects of 17 beta-oestradiol in human myocardium. The effects of 17 beta-oestradiol, progesterone and testosterone on force of contraction were investigated in electrically driven isolated atrial trabeculae and ventricular papillary muscles from human hearts in the presence and absence of Bay K 8644, a calcium channel agonist. In addition, the effects of 17 beta-oestradiol, progesterone and testosterone on binding of [3H]-PN 200 110 were assessed in membranes prepared from human ventricular myocardium. 2. 17 beta-Oestradiol elicited a negative inotropic effect in atrial (IC50: 7.1 mumol 1(-1), confidence interval 3.8 to 13.4, n = 3) and ventricular preparations (IC50: 4.6 mumol 1(-1)), confidence interval 2.2 to 9.4, n = 3) as compared with solvent controls. There was no significant difference (P > 0.05) of IC50 values in the absence and presence of isoprenaline (0.0 mumol 1(-1)) in atrial (IC50: 10.8 mumol 1(-1), confidence interval 9.1 to 12.9, n = 6) and ventricular preparations (IC50: 9.4 mumol 1(-1), confidence interval 7.3 to 11.9, n = 8). 3. 17 beta-Oestradiol at 30 mumol 1(-1) induced a significant rightward shift of the concentration-response curves for the positive inotropic effect of Bay K 8644 in atrial preparations (EC50: 0.13 mumol 1(-1), confidence interval 0.08 to 0.19, n = 6; EC50 with 17 beta-oestradiol: 0.58 mumol 1(-1), confidence interval 0.33 to 0.83, n = 6, P < 0.05) and ventricular preparations (EC50: 0.07 mumol 1(-1), confidence interval 0.04 to 0.11, n = 8; EC50 with 17 beta-oestradiol: 0.3 mumol 1(-1), confidence interval 0.18 to 0.49, n = 8, P < 0.05). Testosterone, progesterone at 30 mumol 1(-1) and the solvent control had no significant effect on the concentration-response curves to Bay K 8644. 4. In membranes prepared from human ventricular myocardium the effect of 17 beta-oestradiol on binding of [3H]-PN 200 110, an antagonist at the 1,4 dihydropyridine binding site, was not different from that observed with progesterone, testosterone or solvent controls. 5. In myocardial membranes no specific oestrogen receptors were demonstrated by [3H]-oestradiol binding studies. 6. Thus, the calcium antagonistic property of 17 beta-oestradiol cannot be attributed to a direct interaction with 1, 4 dihydropyridine binding sites.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Effects of angiotensin II and angiotensin-converting enzyme inhibitors on human myocardium.

Human myocardial angiotensin II receptors and the angiotensin AT1 and AT2 receptor subtypes were characterised using the partial angiotensin II receptor agonist [125I][Sar1,IIe8]angiotensin II and the selective antagonists losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'((1H-tetrazol-5-yl)biphen yl-4-yl)- methyl]imidazole) and PD 123177 (1-[(4-amino-3-methylphenyl)methyl]-5-(diphenyl-acetyl)- 4,5,6,7-tetrahydro-1H-imidazol[4,5-c]pyridine-6-carboxylic acid). The density of angiotensin II receptors was higher in atrial than in ventricular myocardium. Angiotensin AT2 receptors were predominant in atria and ventricles (80-85% of total angiotensin II receptors). Only in isolated, electrically driven atrial trabeculae but not in ventricular preparations, angiotensin II did produce a concentration-dependent positive inotropic effect, which was antagonized exclusively by the angiotensin AT1 receptor antagonist losartan and which amounted to about 20% of the positive inotropic effect of milrinone and isoprenaline. The application of the angiotensin-converting enzyme inhibitors captopril, enalaprilat and ramiprilat had no inotropic effect in either tissue. It is concluded that angiotensin AT1 receptors exclusively mediate direct positive inotropic effects in atrial myocardium. Since angiotensin-converting enzyme inhibitors do not produce any inotropic effect, tonic regulation of basal force of contraction by angiotensin II does not occur.

Adolescent↗
PubMed1995

Evidence against a role of nitric oxide in the indirect negative inotropic-effect of M-cholinoceptor stimulation in human ventricular myocardium.

Nitric oxide (NO) has been reported to mediate several effects in response to muscarinic cholinergic stimulation in cardiovascular tissues. Recently, an attenuation of guinea pig cardiac myocyte contraction by NO has been described. The aim of the present study was to determine whether the indirect negative inotropic effect of M-cholinoceptor stimulation in human myocardium is in part due to an effect of endogenous NO. Therefore, the effect of carbachol was studied under control conditions and during inhibition of NO-synthase by pretreatment with NG-monomethyl-L-arginine (NMMA). Functional experiments were performed in isolated, electrically driven (1 Hz, 37 degrees C) left ventricular papillary muscle strips of human myocardium. Since cytokines have been reported to be increased in the serum of patients with heart failure and could induce NO-synthase activity in failing myocardium, we compared samples from nonfailing and terminally failing (classified as NYHA IV) hearts. The indirect negative inotropic effect of carbachol (10 mumol/l) was studied in the presence of the beta-adrenoceptor agonist isoprenaline (0.03 mumol/l). After stimulation with isoprenaline, carbachol significantly (P < 0.05) reduced force of contraction. This effect was diminished in failing myocardium compared to nonfailing, probably due to the diminished inotropic response most likely due to the lower cAMP levels in response to beta-adrenoceptor stimulation in the former condition. Pretreatment with NMMA (100 mumol/l) altered the antiadrenergic effect of carbachol neither in nonfailing nor in failing preparations. Furthermore, inhibition of guanylyl cyclase, the target enzyme of NO, by preincubation with methylene blue (10 mumol/l) for 30 min had no effect on the carbachol-induced decrease in force of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗
PubMed1994

The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation.

Nucleotide sequence analysis revealed a 1,791-bp open reading frame in the hox gene cluster of the gram-negative chemolithotroph Alcaligenes eutrophus H16. In order to investigate the biological role of this open reading frame, we generated an in-frame deletion allele via a gene replacement strategy. The resulting mutant grew significantly more slowly than the wild type under lithoautotrophic conditions (6.1 versus 4.2 h doubling time). A reduction in the level of the soluble NAD-reducing hydrogenase (60% of the wild-type activity) was shown to be the cause of the slow lithoautotrophic growth. We used plasmid-borne gene fusions to monitor the expression of the operons encoding the soluble and membrane-bound hydrogenases. The expression of both operons was lower in the mutant than in the wild-type strain. These results suggest that the newly identified gene, designated hoxX, encodes a regulatory component which, in conjunction with the transcriptional activator HoxA, controls hydrogenase synthesis.

Alcaligenes↗