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Biomedical subjects

K D Gerbitz

Publications and source records attributed to K D Gerbitz.

At least 73 records · Page 4Linked to original sources
PubMed1990

Competitive inhibition by glucose of myo-inositol incorporation into cultured porcine aortic endothelial cells.

To explore the significance of hyperglycaemia as a causal factor for the appearance of diabetic angiopathies we investigated aspects of myo-inositol metabolism in porcine aortic endothelial cells. myo-Inositol was shown to be a long-living metabolite. Its uptake into the cells was mediated by a high-affinity, Na(+)-dependent uptake system inhibitable by ouabain with an apparent KM of 18.6 mumols/l, which was responsible for more than 80% of total uptake at physiological myo-inositol concentrations. Inhibition of inositol uptake by D-glucose was exclusively competitive with an apparent Ki of 24 mmol/l as shown by Lineweaver-Burk- and Dixon-plot analysis. The specificity of competitive inhibition was studied. L-Glucose which is stereochemically related to myo-inositol in the same way as the D-isomer proved to be an equally potent inhibitor. The hexoses D-galactose, D-mannose and D-fructose inhibited myo-inositol uptake to a minor extent. D-allose and 3-O-methyl-D-glucose had no inhibitory effect indicating that the OH-group of the carbon atom in 3 position is essential for the interaction with the carrier. The acyclic hexitol sorbitol also did not compete. As expected, the aldose reductase blocker sorbinil did not influence the carrier since there is no polyol pathway operating in porcine aortic endothelial cells. In accordance with the results of the uptake experiments, the incorporation of exogenous myo-inositol into membrane phosphatidylinositol was reduced at elevated extracellular glucose levels. The results raise the possibility that hyperglycaemia impairs endothelial inositol supply.

Aldehyde Reductase↗
PubMed1990

Mitochondrial myopathies: divergences of genetic deletions, biochemical defects and the clinical syndromes.

Genomic Southern analysis of muscle mitochondrial (mt) DNA from 16 patients with mitochondrial myopathies was performed; 14 of 16 patients had chronic progressive external ophthalmoplegia (CPEO), while 2 patients had mitochondrial myopathies without CPEO. Eleven patients with CPEO, including 5 who exhibited the complete triad of symptoms characteristic of the Kearns-Sayre syndrome (i.e. CPEO, retinal degeneration and heart block) had heteroplasmic mtDNA with deletions ranging from 2.0 to 8.0 kb in length. There was no clear-cut correlation between the size and location of the deletions, on the one hand, and the histochemical and biochemical data or the severity of the disease, on the other.

Adolescent↗
PubMed1990

Effect of nonenzymatic glycation on the structure of immunoglobulin G.

Incubation of human immunoglobulin G with glucose in vitro leads to covalent incorporation of the sugar concomitant with marked changes in molecular structure. After six to ten days of glucose incubation, absorption at 350 nm and fluorescence at 440 nm upon excitation at 370 nm markedly increased, indicating formation of nonenzymatic browning products. Furthermore, immunoglobulin G derivatives of a molecular mass of 500,000 Da appeared during glucose incubation as revealed by gel filtration. Electrophoretic examination of the 500-kDa protein revealed the formation of covalently bound immunoglobulin G polymers. Compared with nonglycated monomeric immunoglobulin G, functional properties of the 500-kDa protein, such as binding of protein A and fixation of complement are markedly reduced.

Binding Sites↗
PubMed1990

Mutations of the mitochondrial DNA: the contribution of DNA techniques to the diagnosis of mitochondrial encephalomyopathies.

We performed restriction analysis and Southern blotting of the muscle mitochondrial DNA from 34 patients suffering from different myopathies. In 13/21 patients with chronic progressive external ophthalmoplegia the muscle mitochondrial DNA was shown to be heteroplasmic. Further mapping by use of several restriction enzymes yielded large deletions in muscles from 10/13 chronic progressive external ophthalmoplegia patients. Most of the deletions spanned large parts of the mitochondrial genome, leading to loss of mitochondrial genes encoding several subunits of the respiratory chain complexes I (NADH-dehydrogenase), IV (cytochrome c oxidase) and V (ATP-synthetase), as well as of several tRNAs. Comparison of the mapping data with the histochemical and biochemical results did not provide a clear correlation between the location of the mitochondrial genetic defects and the functional deficiencies of the affected respiratory chain complexes. In the majority of patients with chronic progressive external ophthalmoplegia, but without a family history of the disease, restriction analysis reveals large mutations of the mitochondrial genome, while other methods are necessary for the localization of defects in all cases with maternal transmission of the disease. The same holds true for all other kinds of mitochondrial myopathies based on defects within the nuclear DNA or on derangements of the "cross-talk" between the nuclear and the mitochondrial genomes.

Adult↗
PubMed1990

Impairment by glycation of immunoglobulin G Fc fragment function.

Incubation of human immunoglobulin G (IgG) with glucose in vitro leads to the formation of glycated IgG concomitant with marked changes in functional properties of the Fc fragment. After 22 days of incubation in the absence and presence of 13.9, 27.7 and 55.5 mmol/l glucose, respectively, protein A binding was reduced by 42, 66 and 83%, depending on the glucose concentration employed. Binding of complement by IgG was abolished after incubation of the immunoglobulins for 13 days at 13.9 mmol/l glucose. In contrast, functional properties of the Fab region were unaffected upon glycation, as revealed by determination of antigen-binding capacity, antibody affinity and antibody concentration. The functional changes of the Fc fragment were observed at glycation levels comparable to those found in diabetics.

Antigen-Antibody Complex↗
PubMed1990

Differential kinetics of glucose metabolism in porcine retinal and aortic endothelial cells.

Rates of glucose utilization, measured by steady state lactate production, and the rates of initial uptake of [14C]glucose were determined in cultured porcine retinal endothelial cells. The apparent Km for the steady state process proved to be lower than that determined for the uptake step (0.20 vs. 0.86 mmol/l glucose), suggesting that the carrier-mediated uptake of glucose into retinal endothelial cells cannot be the rate limiting step of overall glucose consumption. Thus, porcine retinal endothelial cells differ from porcine aortic endothelial cells, in that the aortic endothelial cell membrane acts as a barrier for the uptake of glucose into the cell. Accordingly, dexamethasone, which is known to reduce glucose uptake and degradation in aortic endothelial cells, does not influence glucose utilization in the retinal cells.

Animals↗
PubMed1989

Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan.

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.

Amino Acids↗
PubMed1989

Characterization and localization of basement membrane-associated heparan sulfate proteoglycan in human tissues.

A polyclonal antiserum was raised in rabbits against basement membrane heparan sulfate proteoglycan (HSPG) purified from extracts of isolated porcine glomeruli. The antiserum was characterized by enzyme immunoassay, immunoprecipitation, and immunohistological methods. It was shown to recognize specifically the core protein of HSPG from porcine, rat, bovine, and human glomerular basement membrane, but it did not bind to HSPG from guinea pig or rabbit kidney. The affinity-purified antiserum did not cross-react with other basement membrane proteins like laminin, fibronectin, or collagen type IV. Immunohistochemical studies on tissue sections from several human organs revealed specific basement membrane staining, although the intensity of the reaction differed among the organs tested. Whereas glomerular and other capillary basement membranes showed prominent staining with antibody to the core protein of the proteoglycan, those from nerve, skeletal, cardiac, and smooth muscle reacted only weakly. Adipocytes and liver sinusoid walls were not stained. Independent of the extent of HSPG staining the basement membranes of all different tissues tested reacted strongly with an antiserum against type IV collagen.

Antibody Specificity↗
PubMed1988

Pathobiochemical aspects of diabetic nephropathy.

Diabetic nephropathy develops in many diabetic patients as consequence of glomerulosclerosis. On the basis of a series of recent observations it is suggested that a combination of metabolic and hemodynamic changes is responsible for the pathogenesis of diabetic nephropathy. Since the glomerular filtration unit has been characterized to consist of collagen type IV and minor components like laminin, fibronectin and heparan sulfate proteoglycan, influence of diabetes on basement membrane (BM) components has been studied. Biochemical alterations of glomerular BM consist of an increased nonenzymatic glucosylation of type IV collagen leading to unphysiological crosslinking. This, in turn, may result in alteration of the size selective properties of the glomerular filtration unit. Changes in composition of glomerular BM have recently been described. An increased synthesis of type IV collagen with concomitant decrease of heparan sulfate proteoglycan may lead to alteration of the charge selective barrier and consequently to increased permeability of the glomerular BM. Permanently unbalanced synthesis of BM components finally results in obliteration of the capillary lumen. In late state nephropathy intrinsic basement membrane components are no longer produced. Instead, massive accumulation of PAS positive material occurs.

Animals↗
PubMed1988

Human nesidioblastosis tissue as an immunogen for generation of islet cell specific monoclonal antibodies.

Nesidioblastosis pancreas was used as an immunogen in BALB/c-mice to generate monoclonal antibodies against structures of human islet cells. Seven clones were selected by screening 311 growing hybridomas for reactivity with the rat insulinoma cell line, RIN m5F, and with cryostat sections from human pancreas. None of the selected clones reacted with pancreatic hormones or endocrine-specific peptides. Monoclonal antibodies cross-reacting with the RIN-cell line also bound to different endocrine organs or cell lines, while some RIN-cell-negative clones bound only to Langerhans islets from human pancreas.

Animals↗
PubMed1988

Is serum fructosamine assay specific for determination of glycated serum protein?

We compared the fructosamine activity in sera from healthy and diabetic subjects with the degree of protein glycation detected by a liquid-chromatographic method. The latter technique measures furosine as a specific product after hydrolysis of epsilon-amino-fructose-lysine. Our results indicate that the fructosamine assay measures the extent of glycation of purified human serum albumin correctly. On the other hand, we found no correlation between the two methods for sera from healthy subjects, although for diabetics' sera the values obtained with both methods were related. However, only about half of the reducing activity (fructosamine) was due to specific nonenzymatic glycation of proteins in healthy subjects and well-controlled diabetics. The remaining unspecific activity varied from serum to serum. It was not reducible with NaBH4 and was independent of the glycation of albumin, which normally accounts for about 80% of glycated serum proteins. The fructosamine assay is therefore of limited specificity for the exact measurement of glycated proteins in serum.

Blood Proteins↗
PubMed1986

Enolase isoenzymes as tumour markers.

alpha- and gamma-enolase isoenzyme substance concentrations were measured in serum and plasma from healthy subjects and from 174 patients with different solid tumours. While alpha-enolase was found to be increased in the plasma of patients with tumours of quite different origin, gamma-enolase apparently reflected malignancies of the neuroendocrine system. Before the beginning of the cytotoxic therapy gamma-enolase was increased above the upper limit of the reference range (10 micrograms/l) in 27/27 patients (100%) suffering from small cell lung cancer. Most patients with squamous cell carcinoma of the lung or with prostatic cancer exhibited normal gamma-enolase, while both tumour types produced high plasma substance concentrations of the alpha-isoenzymes of enolase.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1985

Pancreatic B-cell peptides as parameters for diagnosis and localisation of hormone secreting tumours.

Insulin, C-peptide and proinsulin were measured in peripheral blood of 11 patients suffering from different types of hormone-producing pancreatic B-cell tumours. While proinsulin was elevated in 10/11 patients during prolonged fasting, C-peptide and insulin levels were found within the reference range in 5/11 and 3/5 cases respectively. A rapid insulin assay performed during surgery was helpful for localisation and identification of the respective tumours.

Adenoma, Islet Cell↗
PubMed1984

Brain-specific proteins: solid-phase immunobioluminescence assay for neuron-specific enolase in human plasma.

We raised specific antibodies in rabbits against pure alpha alpha- or gamma gamma-isoenzymes of enolase (EC 4.2.1.11) purified from human brain. After being specifically immunoabsorbed by the respective Sepharose-coupled enolase isoenzymes, the purified antibodies are desorbed by acidification and coated onto polystyrene tubes. Each sample containing the isoenzyme activity to be determined is incubated in the respective coated tube. The coating is then washed, and the reaction of the antibody-bound enzyme is initiated by adding 2-phosphoglycerate, ADP, and pyruvate kinase. The accumulation of ATP is measured by following the increase in light emission in the firefly luciferase bioluminescence system. The assay is as specific as the antibody used for coating. Its detection limit is about 5 X 10(-9) U, corresponding to about 0.1 pg or 10(-18) mol of enzyme protein per assay. Activities of enolase isoenzymes in human plasma can be evaluated separately, rapidly, and precisely. We used the assay to measure enolase isoenzyme activities in plasma of patients suffering from different types of malignant tumors.

Adenosine Triphosphate↗
PubMed1981

Enzymic determination of branched-chain amino acids.

Transamination of branched-chain amino acids to (alpha-oxoglutarate, catalyzed by leucine aminotransferase coupled to the glutamate dehydrogenase reaction, is used in an enzymic assay for determination of branched-chain amino acids in serum and tissue homogenates. The coefficients of variation of the method within-day and day-to-day are 2.4 and 6.5%, respectively. Analytical recovery of physiological concentrations of branched-chain amino acids added to serum is near 100%. Measurements in serum of healthy subjects revealed normal values similar to those found by use of other methods. During prolonged fasting the concentration of these amino acids in serum first increases, reaching a maximum by three days, followed by a successive decline.

Adult↗
PubMed1980

[Pancreatic B cell peptides: kinetic behaviour and concentrations of proinsulin, insulin and C-peptide in plasma and urine, problems of assay methods, clinical significance and literature review (author's transl)].

The kinetics and the concentrations of B-cell peptides in plasma and urine are reviewed. Methodical problems in the determination of proinsulin, free and antibody-bound insulin and C-peptide are discussed, together with the possible application of these methods in the control of insulin-dependent diabetics.

Amino Acid Sequence↗