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Biomedical subjects

H Holzer

Publications and source records attributed to H Holzer.

At least 73 records · Page 4Linked to original sources
PubMed1993

Assay of trehalose with acid trehalase purified from Saccharomyces cerevisiae.

An enzymatic end-point assay of trehalose using acid trehalase from yeast is described. After quantitative hydrolysis of trehalose by acid trehalase, the resulting glucose is assayed with the commercially available glucose oxidase/peroxidase dye system. Pre-existing glucose is determined in a control reaction from which acid trehalase is omitted. When intact cells are analysed for trehalose, pre-existing glucose can be washed out with ice-cold water without reducing the trehalose content of the cells. A convenient method for extraction of trehalose from intact yeast cells is heating for 20 min at 95 degrees C followed by centrifugation. The specificity of the assay is determined by the specificity of the acid trehalase preparation used. As described previously (Mittenbühler, K. and Holzer, H., 1988, J. Biol. Chem. 263, 8537-8543; Mittenbühler, K., 1988, Thesis, University of Freiburg), the following sugars and sugar derivatives do not form glucose when incubated with purified acid trehalase: sucrose, cellobiose, mellobiose, raffinose, maltose, lactose, glucose-6-phosphate, glucose-1-phosphate, galactose. The application of the new trehalose assay to yeast cells grown to different growth stages and at various temperatures is presented.

Biological Assay↗
PubMed1993

A rapid immunocytochemical assay for CMV detection in peripheral blood of organ-transplanted patients in clinical practice.

This study describes clinical experience with a rapid method for diagnosis of cytomegalovirus infection in organ-transplanted patients, based on the detection of CMV-specific antigens in peripheral polymorphonuclear cells with a mixture of monoclonal antibodies. This CMV-pp65 assay was formerly called the "CMV immediate early antigen assay." A group of 180 organ-transplanted patients were examined with this assay; 75 of them could be observed from the date of transplantation. These 75 patients consisted of two groups: 59 kidney transplant patients receiving no CMV hyperimmunoglobulin prophylaxis (group I), 13 heart-transplanted patients, and 3 liver transplanted patients receiving prophylaxis (group II). Group III consisted of 105 patients who had been transplanted ca. 2 years before starting this study. In group I, 26 (44%) were CMV-pp65-positive (13 primary and 13 secondary infections). Fifteen of these 26 (58%) positive patients showed clinical symptoms of CMV infection. Eleven of these 15 (73%) were primary infections. Symptomatic patients had significantly more CMV-pp65-positive cells than asymptomatic patients; 12 patients showed a high number of positive cells and 11 of them developed severe CMV illness. Thirty-three patients were CMV-pp-65-negative (22 CMV IgG-sero-positive, 11 CMV IgG-seronegative). None of them had symptoms of CMV infection. In all patients of group I there were 36 periods of graft dysfunction in which CMV infection had to be differentiated from transplant rejection. In 10 out of 36 there was a CMV-pp65-positive test result and subsequent seroconversion. Treatment of viral infection resulted in improvement of clinical problems. In the remaining 26 episodes no CMV-pp65-positive cells were detected: in 17 cases graft dysfunction was caused by rejection, in 9 cases by other complications. In group II, 13 of 16 patients (81%) were positive in the CMV-pp65 assay (6 primary infections, 7 secondary infections). However, none of them showed clinical signs of CMV infection, regardless of the number of positive cells. No CMV-related graft dysfunction was observed. In group III, CMV infections did not play an important role. The experiences described suggest that this test is a valuable tool in early CMV diagnosis and in differentiating CMV-dependent graft dysfunction from other graft dysfunctions. It allows prompt therapeutic intervention.

Adult↗
PubMed1993

Increased susceptibility for CsA-induced hepatotoxicity in kidney graft recipients with chronic viral hepatitis C.

CsA-induced hepatotoxicity is a rare disorder in renal transplant recipients when low doses are administered and whole blood trough levels of CsA are regularly monitored. However, there is controversy about the clinical value of measuring CsA-metabolites, whose contribution to immunosuppression and toxicity is not fully understood. To assess the relation between low-dose CsA therapy and hepatotoxicity, we studied 128 renal transplant recipients attending our nephrology clinic. Eight of these patients had markedly elevated liver function tests. Three patients while receiving very low doses of oral CsA (< 3.8 mg/kg of body weight) presented marked derangements of CsA metabolism with abnormally increased CsA-metabolite levels. Parent drug levels were in the normal range. All 3 patients had chronic infection with hepatitis C virus and revealed histomorphologic evidence of hepatotoxicity. Hepatic dysfunction normalized when CsA was withdrawn or reduced by 50%. It is likely that hepatitis C virus infection interferes with CsA metabolism and/or biliary CsA-excretion and thus is responsible for CsA and/or metabolite-induced hepatotoxicity despite very low doses of CsA.

Chemical and Drug Induced Liver Injury↗
PubMed1993

Spontaneous bacterial peritonitis in a hemodialysis patient with systemic lupus erythematosus.

Spontaneous bacterial peritonitis (SBP) is defined as infection of preexisting ascites without evidence for any intraabdominal source for secondary infection. SBP is now recognized with rising frequency and has mainly been reported in patients with alcohol-induced cirrhosis of the liver. We report SBP in a female dialysis patient whose ascites was not due to liver disease, but was possibly due to lupus erythematosus or represented 'nephrogenic ascites'. The patient had severe abdominal pain and a positive rebound phenomenon, fever and an elevated peripheral white cell count of 21,000 cells/microliters. Ascitic fluid analysis revealed an exudate with a protein concentration of 5.2 g/dl, 13,000 white cells/microliters with 94% neutrophils and positive cultures for Streptococcus morbillorum. Because of the dramatic clinical features the patient underwent laparotomy which did not reveal a source for secondary infection and in retrospect was unnecessary. The patient responded well to antibiotic therapy. This case report draws attention to SBP as a cause of acute abdomen in patients on chronic hemodialysis.

Aged↗
PubMed1993

Increased red cell 2,3-diphosphoglycerate levels in haemodialysis patients treated with erythropoietin.

The efficacy of recombinant human erythropoietin (rHuEpo) for the treatment of renal anaemia is well established. To assess the effect of rHuEpo treatment on physical performance we evaluated physical working capacity, oxygen uptake and red cell 2,3-diphosphoglycerate (DPG) values at rest and during and after exercise on a bicycle spiroergometer in eight chronically haemodialysed patients. Follow-up examination was carried out after a mean of 14 weeks (range 9-19 weeks), when mean haemoglobin had increased from 7.8 to a stable value of 13.0 g/dl in response to rHuEpo treatment (P < 0.001). Physical working capacity and oxygen uptake at the anaerobic threshold (4 mmol/l blood lactate concentration) increased from 68 +/- 12 to 80 +/- 16 watts and 0.95 +/- 0.14 to 1.10 +/- 0.20 l/min, respectively (P < 0.01). DPG, which determines oxygen affinity to haemoglobin in red cells, increased by 13% from 13.7 +/- 1.5 to 15.5 +/- 2.2 mumol/g Hb (P < 0.05). With maximal exercise mean DPG values significantly decreased to a much lower level without rHuEpo treatment than after correction of anaemia. Therefore rHuEpo treatment results both in better oxygen transport capacity and reduced intraerythrocytic oxygen affinity, which is followed by improved oxygen delivery to tissues per unit of haemoglobin. These effects may explain the improvement of exercise capacity observed in dialysis patients after rHuEpo treatment.

2,3-Diphosphoglycerate↗
PubMed1992

The correlation of interleukin 1 and tumour necrosis factor to oestradiol, progesterone and testosterone levels in periovulatory follicular fluid of in-vitro fertilization patients.

Data has accumulated suggesting reciprocity between cytokines and the reproductive system. The present study was performed in order to evaluate the correlation between interleukin 1 (IL-1) and tumour necrosis factor (TNF) concentrations in follicular fluid and its oestradiol, progesterone and testosterone levels. A total of 39 follicular fluid samples, from eight patients undergoing in-vitro fertilization and embryo transfer were evaluated. All of the patients were treated by a midluteal (long) protocol involving a gonadotrophin releasing hormone agonist (GnRHa) coupled with follicular phase human menopausal gonadotrophin. Mean levels in follicular fluid of IL-1, TNF, oestradiol, progesterone and testosterone were 1.58 +/- 0.42 fmol/0.1 ml, 4.69 +/- 4.18 pg/ml, 28.5 +/- 58.1 ng/ml, 2360.5 +/- 2846.3 ng/ml and 7.22 +/- 7.08 ng/ml respectively. There was a significant (P less than 0.01) positive correlation between IL-1 and progesterone levels. There was no significant correlation between the different lymphokines and oestradiol secretion, oocyte fertilization, embryo quality and pregnancy rates. It is concluded that IL-1 and TNF exist in follicular fluid. It may be hypothesized that IL-1 has a local regulatory action, possibly promoting luteinization.

Adult↗
PubMed1991

Metabolic regulation of the trehalose content of vegetative yeast.

We have investigated the mechanism by which heat shock conditions lead to a reversible accumulation of trehalose in growing yeast. When cells of S. cerevisiae M1 growing exponentially at 30 degrees C were shifted to 45 degrees C for 20 min, or to 39 degrees C for 40 min, the concentration of trehalose increased by about 25-fold; an effect reversed upon lowering the temperature to 30 degrees C. This was compared to the more than 50-fold rise in trehalose levels obtained upon transition from the exponential to the stationary growth phase. Whereas the latter was paralleled by a 12-fold increase in the activity of trehalose-6-phosphate synthase, no significant change in the activities of trehalose-synthesizing and -degrading enzymes was measured under heat shock conditions. Accordingly, cycloheximide did not prevent the heat-induced accumulation of trehalose. However, the concentrations of the substrates for trehalose-6-phosphate synthase, i.e. glucose-6-phosphate and UDP-glucose, were found to rise during heat shock by about 5-10-fold. Since the elevated levels of both sugars are still well below the Km-values determined for trehalose-6-phosphate synthase in vitro, they are likely to contribute to the increase in trehalose under heat shock conditions. A similar increase in the steady-state levels was obtained for other intermediates of the glycolytic pathway between glucose and triosephosphate, including ATP. This suggests that temperature-dependent changes in the kinetic parameters of glycolytic enzymes vary in steady-state levels of intermediates of sugar metabolism, including an increase of those that are required for trehalose synthesis. Trehalose, glucose-6-phosphate, UDP-glucose, and ATP, were all found to increase during the 40 min heat treatment at 39 degrees C. Since this also occurs in a mutant lacking the heat shock-induced protein HSP104 (delta hsp104), this protein cannot be involved in the accumulation of trehalose under these heat shock conditions. However, mutant delta hsp104, in contrast to the parental wild-type, was sensitive towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not the accumulation of trehalose, protects S. cerevisiae from the damage caused by a 50 degrees C treatment.

Glucose-6-Phosphate↗
PubMed1991

Inactivation of enzymes and an enzyme inhibitor by oxidative modification with chlorinated amines and metal-catalyzed oxidation systems.

Oxidative inactivation of various key enzymes and alpha-1-proteinase inhibitor (alpha-1-PI) was studied by treatment with N-chloramines and the metal-catalyzed oxidation (MCO)-systems ascorbate/Fe(III) and ascorbate/Cu(II). Chlorinated amines completely inhibited alpha-1-PI, fructose-1,6-bis phosphatase (Fru-P2ase) and glyceraldehyde phosphate dehydrogenase (GAPD) at a low molar excess, and glucose-6-phosphate dehydrogenase (G6PD) at a high molar excess, but did not impair beta-N-acetylglucosaminidase (beta-NAG), alkaline phosphatase (AP) or lactate dehydrogenase (LDH). MCO-systems affected the activities of Fru-P2ase, GAPD, AP, LDH and G6PD, but not those of beta-NAG or alpha-1-PI. EDTA prevented inactivation of Fru-P2ase, G6PD and LDH by ascorbate/Cu(II) and of Fru-P2ase by ascorbate/Fe(III) suggesting a site-specific oxidation catalyzed by a protein-bound metal ion. In conclusion, N-chloramines and MCO-systems exhibited different properties with regard to oxidative inactivation, sulfhydryl-enzymes were susceptible to both systems, but other enzymes were only susceptible to one or neither system.

Alkaline Phosphatase↗
PubMed1991

Yeast fructose-2,6-bisphosphate 6-phosphatase is encoded by PHO8, the gene for nonspecific repressible alkaline phosphatase.

Yeast fructose-2,6-bisphosphate 6-phosphatase has been purified 7000-fold by heat treatment, poly(ethylene glycol) precipitation, ion-exchange chromatography with Q-Sepharose Fast Flow and Mono Q followed by affinity chromatography with concanavalin-A-Sepharose and gel filtration with Superose 12. The purified dimeric enzyme contains 1.5 mol zinc and 1.3 mol copper/mol subunit. It reacts with fructose 2,6-bisphosphate [Fru(2,6)P2] as well as with p-nitrophenyl phosphate (NpP) showing a pH optimum at pH 6-6.5 with Fru(2,6)P2 [Plankert, U., Purwin, C. & Holzer, H. (1988) FEBS Lett. 239, 69-72] and above pH 9.0 with NpP. The following observations suggest that activity with both substrates depends on the same protein. (a) During 7000-fold purification, the ratio of activity with NpP to that with Fru(2,6)P2 remained constant. (b) The time course of inactivation of enzyme activity in dilute solution at 30 degrees C is similar for both substrates. (c) At increasing temperatures, inactivation of enzyme activity measured with both substrates proceeds at nearly identical rates. (d) Activity with both substrates is found preferentially in the vacuoles. (e) Mutants defective in the nonspecific alkaline phosphatase coded by the PHO8 gene are also defective in Fru(2,6)P2 6-phosphatase activity. (f) A proteinase A mutant, defective in processing and activation of nonspecific alkaline phosphatase coded by the PHO8 gene, also fails to activate Fru(2,6)P2 6-phosphatase.

Alkaline Phosphatase↗
PubMed1991

Characterization of different forms of yeast acid trehalase in the secretory pathway.

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.

Autoradiography↗
PubMed1991

[Special problems of nutrition therapy in patients with terminal kidney insufficiency with chronic ambulatory hemodialysis].

The treatment of end-stage chronic renal failure with intermittent ambulatory hemodialysis induces additional catabolic stimulus. Thus, the protein requirements of maintainance hemodialysis patients with 1.2 to 1.4 g/kg/day are higher than in the predialysis state. An energy intake of 35 to 38 kcal/kg/day contributes to an optimal anabolic utilization of incorporated protein. The catabolic response in case of complications or stress situations demands supplementation of nutrients.

Dietary Proteins↗
PubMed1991

[Clinical experiences with Wegener's granulomatosis from the nephrologic viewpoint].

Immunosuppressive treatment with cyclophosphamide and prednisolone has markedly improved the prognosis of Wegener's granulomatosis (WG). In patients with WG the detection of anti-neutrophil-cytoplasmic autoantibodies (ANCA or ACPA) has become an important parameter to confirm or even routinely establish the diagnosis of WG over the past few years. From 1985 to 1990 we observed and treated 12 patients (5 males and 7 females) aged 14-64 years (mean 41.1 years) with WG. In the same time span an analysis of 35 patients with rapidly progressive glomerulonephritis showed that the octiology was idiopathic in the majority of cases (54.3%), but nevertheless 8 cases (22.8%) were caused by WG. In 9 out of 10 cases the ANCA test was positive; whereas in 8 out of 10 cases we found a close correlation between the serum level of ANCA and disease activity. However, extraordinarily high serum levels (titres up to 1:2560) were recorded in one patient with WG without any clinical symptoms, whilst another patient with severe symptoms of WG showed a titre of only 1:5. 10 out of 12 patients were successfully brought to remission under cyclophosphamide-cortisone treatment. 4 out of 10 patients with renal insufficiency have been retained on the chronic haemodialysis regimen. 2 patients, aged 31 and 51 years, died within 2-5 months after the onset of clinical symptoms of WG.

Adolescent↗
PubMed1989

Purification and characterization of neutral trehalase from the yeast ABYS1 mutant.

Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis. Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0. The apparent Km for trehalose was 34.5 mM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phosphorylated amino acid residue was identified as phosphoserine.

Alkaline Phosphatase↗