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Biomedical subjects

E Schleicher

Publications and source records attributed to E Schleicher.

At least 55 records · Page 3Linked to original sources
PubMed1991

Insulin activates GTP binding to a 40 kDa protein in fat cells.

The first steps in insulin action are binding of insulin to its receptor and activation of the insulin receptor kinase. As there is indirect evidence that further signal transduction might involve a guanine-nucleotide-binding protein (G-protein), we studied whether insulin modulates GTP binding to plasma membrane proteins of fat cells and skeletal muscle. We found that insulin rapidly increased (30 s) binding of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in a dose dependent manner (0.03-2.0 nM). This effect was not altered by pertussis toxin, but it was abolished by cholera toxin treatment of fat cells. Scatchard analysis of the binding data showed that the increased GTP[S] binding is due to a decrease in the Kd for GTP from 100 nM to 50 nM. Furthermore, binding of GTP to these plasma membranes inhibited both the binding of 125I-insulin to the insulin receptor and the stimulation of the insulin receptor kinase, suggesting a feedback interaction between the insulin-stimulated GTP-binding site and the insulin receptor. In order to identify this insulin-stimulated GTP-binding site, plasma membranes were labelled with the photoreactive GTP analogue [alpha-32P]GTP gamma-azidoanilide. We found that insulin selectively stimulated GTP binding to a 40 kDa protein. In conclusion, in plasma membranes of fat cells and skeletal muscle, the insulin receptor interacts with a 40 kDa GTP-binding site. We speculate that this 40 kDa GTP-binding site might be a G-protein which is involved in insulin signal transmission.

Adenosine Diphosphate Ribose↗
PubMed1991

Identification of lymph and blood capillaries by immunohistochemical staining for various basement membrane components.

In the present report we analyzed the presence and distribution of various basement membrane (BM) proteins in normal blood and lymph vessels with special emphasis on BM-associated heparan sulfate proteoglycan (HSPG) when compared to the BM-components collagen IV, laminin and fibronectin. We found that normal lymph capillaries have a BM that contains only collagen IV and small amounts of laminin, but almost no BM-associated HSPG and fibronectin, while blood capillaries showed a BM comprising of all components tested for. Larger lymphatics, however, were indistinguishable from blood vessels on the basis of BM staining. Lymphangiomas showed a BM pattern similar to that of lymph capillaries. Our findings provide evidence that the differential staining of BM-components may represent a reliable method for morphological distinction between blood and lymph capillaries. A comparison of these results with the BM-pattern in other functionally specialized blood vessels (glomerulus, sinusoids) provides evidence that the BM-composition may have some major impact on the functional properties. Thus, it is conceivable that the lack of HSPG in lymph capillaries may be essential for a free influx of fluid and proteins into these capillaries, which may have been extravasated into the interstitium.

Basement Membrane↗
PubMed1991

[The significance of the Maillard reaction in human physiology].

More than 50 years after Maillard's original paper describing the reaction of amino acids with glucose it was found that this reaction also occurs under physiological conditions in the human body. Initially, it was discovered that human hemoglobin contains protein-bound Amadori-products that are increased in diabetic patients with elevated blood glucose levels. Measurements of fructosylated hemoglobin are now widely used as an index of glycemia in diabetes. It was soon recognized that this postribosomal modification is common to other proteins in vivo like albumin, lens crystallins, proteins of the clotting cascade, collagens, lipoproteins, proteins of the cell membrane, and others. This may lead to alterations in structure and function of the respective protein. Later, the realization that long-lived proteins become browned, fluorescent, and insoluble with age, and at an accelerated rate in diabetes, suggested that later stages of the Maillard reaction might proceed in vivo and contribute to some of the pathophysiology associated with both aging and diabetes. Although the contribution of the Maillard products to the development of diabetic late complications is not fully understood, attempts are being made to prevent formation of late Maillard product with aminoguanidine, a drug currently under clinical testing.

Aging↗
PubMed1991

Distribution of basement membrane-associated heparan sulfate proteoglycan in idiopathic and AIDS-associated Kaposi's sarcoma.

In the present study we analyzed the immunohistochemical distribution of different major basement membrane (BM) components with special emphasis on the BM-associated heparan sulfate proteoglycan (HSPG) in early and late stages of Kaposi's sarcoma (KS), both of idiopathic and AIDS-associated origin. In early KS all BM components tested were found surrounding the small clefts of tumour vessels. Heparan sulfate proteoglycan showed the weakest and often fragmented pattern of staining. In the late, nodular sarcomatous form of KS individual tumour cells were surrounded by a BM composed of collagen IV, laminin and fibronectin, while heparan sulfate proteoglycan was not detectable in most cases. Neither between idiopathic and AIDS-associated KS nor between cutaneous and visceral lesions were significant differences in the staining pattern. Our findings of a rather selective expression of various BM-components and the known distribution in normal blood and lymphatic capillaries raises the hypothesis that KS-cells may be derived from cells of lymphaticovenous differentiation.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Differential basement membrane composition in multiple epithelioid haemangioendotheliomas of liver and lung.

We report a case of epithelioid haemangioendothelioma involving both lung and liver. The tumour cells were positive for factor-VIII-related antigen. Immunohistochemical analysis of various basement membrane components in tumour tissue of lung and liver showed striking differences. In the liver tumour there was selective expression of collagen IV, with minimal and focal amounts of laminin and basement membrane-associated heparan sulphate proteoglycan. In the lung tumour nodules, in contrast, all these basement membrane components were present. These patterns of basement membrane expression closely resemble those of normal liver and lung basement membrane respectively. We suggest that this provides evidence that epithelioid haemangioendothelioma arises from local endothelial cell proliferation and that it supports the assumption of a multicentric rather than metastatic origin when multiple tumour deposits are found.

Aged↗
PubMed1991

Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesions.

The immunohistochemical localization of the extracellular matrix was examined in 31 cases with different degrees of human diabetic nephropathy using antisera to human collagen types I, III, IV, V, fibronectin, laminin, and basement-membrane-associated heparan sulfate proteoglycan (HSPG). In normal glomeruli, HSPG was predominantly localized in the glomerular basement membrane and in the mesangium, and to minor extent in the basement membranes of tubules and Bowman's capsule. Collagen IV and laminin were distributed in glomerular basement membrane and mesangium in minor amounts. Interstitial collagens usually do not occur within glomeruli except for collagen V which has a light microscopic glomerular distribution similar to collagen IV. In diabetic diffuse glomerulosclerosis, the enlarged mesangial matrix showed an increased staining reaction for collagen IV, V, laminin, and fibronectin whereas the staining pattern of HSPG was markedly reduced. Early, small nodular lesions in diabetic glomeruli were similarly positive for most of the basement membrane components, whereas HSPG remained absent. With an increase in the diameter of the noduli, however, the staining reaction for all basement membrane components diminished, whereas interstitial collagens V and III, but not collagen I, were present in these noduli in substantial amounts. These initial studies provide evidence that the changes in the glomerular matrix in diabetic nephropathy may be divided into distinct and progressing stages of lesions. The reduced amount of HSPG even in slight, early lesions may represent the morphologic correlate to the impaired filter function of the glomerular basement membrane.

Adult↗
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

[Immunohistochemical localization of various components of the basal membrane and interstitial collagen in diabetic nephropathy].

We analyzed the composition of the extracellular matrix in the glomeruli from 31 cases with different degrees of diabetic nephropathy by immunohistochemical means. While the enlarged mesangial matrix in diffuse glomerulosclerosis showed an increased staining reaction for the basement membrane components collagen IV and V, laminin and fibronectin, the staining pattern of the basement membrane associated heparan sulfate proteoglycan (HSPG) was markedly reduced. Early, small nodular lesions in diabetic glomeruli were similarly positive for most of the basement membrane (BM)-components, while HSPG remained absent. With an increase in the diameter of the noduli, however, the staining reaction for the BM-components diminished, while interstitial collagens V and III were present in these noduli in substantial amounts. Our findings provide evidence that the changes in the glomerular matrix in diabetic nephropathy may be divided into distinct and progressing stages of lesions. The reduced amount of HSPG even in slight, early lesions may represent the morphological correlate to the impaired filter function of the glomerular BM, while the occurrence of interstitial collagens within the glomeruli in late nodular stages may be regarded as the result of an altered pattern of expression of the mesangial cells.

Basement Membrane↗
PubMed1989

Protein glycation: measurement and clinical relevance.

This review of protein glycation deals with the biochemical background of glycated blood proteins, their methods of determination and their clinical significance. General reaction principles for determination of glycated proteins are discussed with special emphasis on the determination of glycated serum proteins in the clinical laboratory. Binding methods like boronate affinity, immunoassay, phenylhydrazine binding and ion exchange chromatography leave the analyte intact, whereas chemical methods like strong or mild hydrolysis or reduction in alkaline medium (fructosamine assay) results in destruction of the glycated protein. As most reactions are nonstoichiometric (except ion exchange chromatography and periodate oxidation), varying results are obtained from laboratory to laboratory. Up to now boronate affinity chromatography, mild hydrolysis yielding hydroxymethylfurfural and the fructosamine assay have been mostly used for determination of glycated serum proteins. The fructosamine assay appears to be most practical, because it is quick, economic and precise, but it suffers from unspecific side reactions. Although other methods like immunoassays or boronate ester formation in solution appear promising, there is currently no commercially available assay for the economic, precise and accurate determination of glycated serum protein. The clinical relevance of glycated serum protein determination is difficult to evaluate because the assays are based on different reaction principles and hence yield variable results. Nevertheless, the following conclusion may be drawn from the reports now available. i) The possibility that glycated serum proteins may discriminate better than glycated haemoglobin between "normal" and "diabetic" is still controversial. ii) Glycated serum proteins are formed faster than glycated haemoglobin, reflecting the changes in glycaemia for shorter periods of time (medium-term control). iii) It has not been yet established, using large cohorts, whether the glycated serum proteins allow the detection or exclusion of diabetes. iv) Determination of glycated serum proteins should not be considered as a substitute for the determination of glycated haemoglobin, but rather as a complementary determination, leading to the improved laboratory control of the diabetic patient.

Biomarkers↗
PubMed1988

Separation of the protein-tyrosine kinase and phosphatidylinositol kinase activities of the human placental insulin receptor.

On immunoprecipitation using a specific antiphosphotyrosine antibody, phosphatidylinositol kinase (EC 2.7.1.67) activity was separated from the protein-tyrosine kinase (EC 2.7.1.112) activity of the wheat germ agglutinin (WGA) -purified insulin receptor from human placenta. This clearly indicates that protein-tyrosine kinase and phosphatidylinositol kinase activity do not reside on the same polypeptide chain as previously has been suggested. Quantitatively, the fraction of phosphatidylinositol kinase that was bound to WGA sepharose and eluted together with the insulin receptor amounted to 2% of the Triton X-100 soluble phosphatidylinositol kinase. The apparent Km values of the bound and unbound phosphatidylinositol kinase with respect to PI and ATP were very similar (0.4 and 0.3 mmol/l and 8 and 7 mumol/l, respectively) suggesting that the WGA-bound phosphatidylinositol kinase is not a different enzyme, but rather represents a small portion of the bulk Triton X-100-soluble phosphatidylinositol kinase that is bound to the lectin tightly associated with the insulin receptor. The synthetic polymer (Glu80Tyr20)n, a model substrate of the insulin receptor tyrosine kinase, at 0.5 mmol/l, inhibited phosphatidylinositol kinase of WGA-purified insulin receptor by 70-90%. This inhibition was not overcome by increasing the concentrations of ATP or PI as one would expect if a functional interrelationship of the protein-tyrosine kinase and the phosphatidylinositol kinase would exist.

1-Phosphatidylinositol 4-Kinase↗
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↗
PubMed1987

Sustained normoglycemia and remission phase in newly diagnosed type I diabetic subjects. Comparison between continuous subcutaneous insulin infusion and conventional therapy during a one year follow-up.

After onset of type I diabetes 7 diabetics were randomized to subcutaneous insulin pump treatment (CSII) (age 12 to 29 years, mean: 21 years) and 7 diabetics to conventional insulin treatment (CI) (age 14 to 28 years, mean: 21 years). HbA1, glycosylated serum proteins and mean blood glucose (MBG) as parameters of metabolic control were determined monthly. After 2 months both groups showed HbA1 values in the normal range. Mean MBG values were (mean +/- SD) 116 +/- 7 mg/dl for CSII and 118 +/- 14 mg/dl for CI. Residual insulin secretion was determined monthly by fasting C-peptide. After 14 days, 5, 7, 8 months fasting C-peptide values were significantly (P less than 0.05) higher in CI. After one year fasting C-peptide was comparable in both groups (CSII and CI mean: 0.06 nmol/l). The administered insulin dose was comparable in both groups with a 55% reduction of insulin dose after 5 months in CSII (0.35 +/- 0.15 U/kg/24 h) and in CI after 7 months (0.31 +/- 0.28 U/kg/24 h). After 12 months of insulin therapy about 60% of the initial insulin dose was injected in both groups. 1 patient on CSII (12 years) and 2 patients on CI (15, 28 years) showed a complete remission (for 3-9 months) with no exogenous insulin and normal HbA1 values. 50% of the patients had episodes where they did need less than 0.2 U/kg/24 h insulin to maintain optimal diabetic control (3 CSII, 4 CI). During the first year of insulin treatment in type I diabetes with CSII as well as with CI a comparable near normalisation of diabetic control could be achieved.

Adolescent↗
PubMed1987

Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells.

It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, we labeled intact rat fat cells with [32P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, we found, in addition to the 95-kDa beta-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor beta-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. The insulin effect starts after 30 sec, is maximal at 150 sec, and declines to almost basal values by 5 min. Furthermore, the antiphosphotyrosine antibody precipitated at least five proteins in the soluble fraction of the fat cell. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32P incorporation into a 116-kDa band, a 62-kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. We suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission.

Adipose Tissue↗
PubMed1987

Stereochemistry and mechanism of reactions catalyzed by tyrosine phenol-lyase from Escherichia intermedia.

Stereochemical studies on tyrosine phenol-lyase from Escherichia intermedia have shown that the alpha, beta-elimination reactions of L-serine and D- and L-tyrosine proceed with retention of configuration at C-beta. Stereospecifically beta-tritiated L-serine is slowly racemized at C-beta. Deuterium from the alpha-position of L-tyrosine is partially transferred to C-4 of the phenol formed when the alpha, beta-elimination reaction is carried out in H2O, although no transfer of alpha-1H in 2H2O was seen. The result favors tautomerization of the p-hydroxyphenyl to a cyclohexadienonyl moiety prior to carbon-carbon bond cleavage. In the conversion of L- to D-alanine catalyzed by tyrosine phenol-lyase, some alpha-hydrogen recycling is observed, pointing to a single-base racemization mechanism. Attempts to demonstrate cofactor motion during racemization by NaBH4 reduction of [3H]PLP-enzyme: D- and L-alanine complexes failed, but showed that, as in other PLP enzymes, the holoenzyme is reduced preferentially from the Re face with respect to C-4' of PLP and enzyme-substrate complexes preferentially from the Si face.

Carbon Radioisotopes↗
PubMed1986

Kinetic analysis of glycation as a tool for assessing the half-life of proteins.

Glycation of proteins, a common postribosomal modification, proceeds via Amadori rearrangement to yield a stable ketoamine linkage of glucose with the protein. Kinetic analysis of the reaction shows that the amount of glycation at steady state is proportional to the glucose concentration, to protein half-life and to the rate of glycation. Thus, when the rate of glycation is determined in vitro and the extent of glycation of a given protein isolated from euglycemic subjects is measured, the half-life may be calculated. As the in vivo situation may not be simulated accurately in vitro, the calculated values may be considered as approximation. When the calculated values were compared with values reported in the literature fairly good agreement was found except for hemoglobin. Studies on stability of glycated albumin show that ketoamine decreases by about 20% when incubated under physiological conditions for 20 days. The method described by us is especially valuable when turnover of proteins in normal and pathophysiological states are compared. The half-life of plasma low-density lipoprotein is longer in patients with hypothyroidism or a high plasma low-density lipoprotein level than in normal subjects. Extending our studies to tissue proteins we did not find a significant increase in half-life of tendon collagen with age. Basement membrane collagen turnover is faster in diabetic patients in bad metabolic control. Thus, the procedure using fructosylamine as endogenous label of protein offers a method of great potential to study the turnover of human body proteins.

Aging↗
PubMed1986

[Myocardial infarct promoting type A behavior--an attempt to modify stress-inducing behavior].

To change some crucial elements of coronary-prone behaviour in cardiac patients is considered an important task of behaviour medicine, to reduce physico-chemical and, in particular, psychosocial "risk factors". The experimental study presented had examined whether the type A behaviour pattern, defined as the essential element of coronary-prone behaviour for its latent provocation of stress reactions, can be positively modified through behaviour therapy-focussed treatment within a clinical setting. A total of 91 persons, i.e. 49 recent-MI patients from an inpatient rehabilitation programme and 42 clients of a smoker counselling centre, were examined to identify type A or type B behaviour patterns (Rosenman u. Friedman, 1964). Of 39 type A post-MI patients, 13 (randomly selected) subjects were included in the "trial group" (VG), and participated, within a 4-8 week inpatient rehabilitation programme, in psychological training of stress management, relaxation, and self-control techniques; 3 patients denied participation; the remaining 23 type A post-MI patients, and the 20 type A smoker counselling clients, served as control group KG (A) and KG (B), who did not receive psychological training. Psychological and physiological parameters were assessed at the beginning (t1), the end (t2), as well as 3 and 6 months (t3 and t4) following completion of the psychological training. The results obtained confirm that the MI group includes significantly more type A persons (80 percent) than the cardiologically inconspicious control group of smokers (48 percent). Analysis of the data obtained at the various measurement points show that, as opposed to KG (A) and KG (B), significant improvements have in fact occurred in the trial group, such as reduced mental and psychovegetative stress reactions, which persisted even 3 months after programme completion.(ABSTRACT TRUNCATED AT 250 WORDS)

Arousal↗
PubMed1985

Limited nonenzymatic glucosylation of low-density lipoprotein does not alter its catabolism in tissue culture.

This study examines the effects of various degrees of chemical modification of low-density lipoprotein (LDL) on its catabolism by various cell types. Moderate glucosylation of LDL does not alter its interaction with the high-affinity receptor present on human fibroblasts at concentration of 5-2000 micrograms LDL-cholesterol/ml. Only heavily glucosylated LDL (more than 12 lysine residues glucosylated per apolipoprotein B) or LDL glucosylated in the presence of Na(CN)BH3, i.e., conditions not expected to occur in diabetes, inhibit receptor-mediated internalisation and degradation. Moderately glucosylated LDL is also readily recognized by cultured rat hepatocytes and porcine endothelial cells. Human monocyte-derived macrophages accumulate cholesteryl ester when incubated with acetylated LDL for 12 days but no enhanced cholesteryl ester formation was found when native or glucosylated LDL (3.3 lysines glucosylated per apolipoprotein B) were used.

Acetylation↗