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Biomedical subjects

G Assmann

Publications and source records attributed to G Assmann.

At least 199 records · Page 11Linked to original sources
PubMed1996

Compound heterozygosity for a known and a novel defect in the lipoprotein lipase gene (Asp250-->Asn; Ser251-->Cys) resulting in lipoprotein lipase (LPL) deficiency.

Two missense mutations in exon 6 of the LPL gene were identified on separate alleles in a Dutch patient with lipoprotein lipase (LPL) deficiency. The first mutation is a G1003-->A transition resulting in a D250N mutation, which has been shown previously to result in a catalytically defective protein in patients of French-Canadian ancestry. The second mutation, a C to G transition at nucleotide 1007, predicts a S251C residue change in the highly conserved region of LPL surrounding the loop structure the covers the catalytic triad. This mutation constitutes a novel defect among LPL gene mutations reported so far. Site-directed mutagenesis experiments provide in-vitro evidence for the complete loss of LPL activity resulting from this latter missense mutation. The G1003-->A nucleotide substitution underlying the Asp250 mutation deletes a TaqI endonuclease recognition site and the C1007-->G change that leads to the S251C alteration abolishes a HinfI recognition site. This will facilitate rapid screening for these mutations in LPL-deficient patients.

Adolescent↗
PubMed1996

Diadenosine polyphosphates induce transplasma membrane calcium influx in cultured glomerular mesangial cells.

The effects of diadenosine tetraphosphate (AP4A) diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) on the cytosolic-free calcium concentration ([Ca2+]i) were evaluated in cultured rat glomerular mesangial cells (MCs) using the fluorescent dye technique. The addition of 10 mumol L-1 AP4A, AP5A or AP6A significantly increased [Ca2+]i in MCs by 57 +/- 9 nmol L-1 n = 17; P < 0.01), 76 +/- 27 nmol L-1 (n = 9; P < 0.01) or 65 +/- 12 nmol L-1 (n = 18; P < 0.01) respectively. In the absence of extracellular calcium, there was no significant change in [Ca2+]i in MCs after administration of diadenosine polyphosphates, indicating that these agents induce transplasma membrane Ca2+ influx. AP6A significantly enhanced the angiotensin II-induced changes in [Ca2+]i in MCs. The AP5A-induced transplasma membrane Ca2+ influx was inhibited by the P2 purinoceptor blockers suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), but was not affected by the adenosine A1 receptor blocker 8-cyclopentyl-1.3-dipro-pylzanthine (CPDPX). Adenosine triphosphate (ATP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma S) increased [Ca2+]i in MCs, whereas alpha, beta-methylene ATP had no effect on [Ca2+]i in MCs. Measurements of diacylglycerol and phosphatidic acid showed that AP5A and AP6A also stimulated phospholipase C, but had no effect on phospholipase D. The inhibition of phosphatidylcholine-specific phospholipase C significantly reduced the AP5A-induced [Ca2+]i increase. In summary, diadenosine polyphosphates induce Ca2+ influx through P2 purinoceptors and may be involved in the local regulation of vascular resistance evoked by the Ca(2+)-dependent contractile response of mesangial cells.

Adenosine Triphosphate↗
PubMed1996

Structure and chromosomal assignment of the murine sterol carrier protein 2 gene (Scp2) and two related pseudogenes by in situ hybridization.

The murine sterol carrier protein 2 gene (Scp2) has been mapped to chromosome 4C5-->D1 by in situ hybridization. This location is syntenic with the locus of SCP2 on human chromosome 1p32. We found the overall structure of murine Scp2 to be identical to that of the human SCP2 gene. In addition, two Scp2 pseudogenes have been isolated and mapped to chromosomes 3H2-->4 and 9F.

Animals↗
PubMed1996

Elevated low-density lipoprotein-cholesterol in women with polycystic ovary syndrome.

Previous studies have indicated that women with the polycystic ovary syndrome (PCOS) are affected by hypertriglyceridemia and low high-density lipoprotein-cholesterol (HDL-C) level. However, most of these studies did not control for confounding factors such as body mass index (BMI) or differences in ethnicity. Therefore, we compared the lipid data for 26 women with PCOS with those for 1428 female control participants of the Prospective Cardiovascular Münster (PRO-CAM) study who did not use hormonal contraceptives and were of similar age. Data were adjusted for age, BMI and ethnicity. Women with PCOS had higher total cholesterol (5.55 +/- 1.24 vs. 4.99 +/- 0.88 mmol/l, p < 0.05) and low-density lipoprotein-cholesterol (LDL-C) levels (3.61 +/- 1.19 vs. 3.08 +/- 0.82 mmol/l, p < 0.05) than the control subjects. Compared with the women in the control population, those with PCOS more frequently had triglyceride levels exceeding 2.3 mmol/l (11.5 vs. 1.6%, p < 0.001), LDL-C levels exceeding 4.2 mmol/l (30.8 vs. 12.1, p < 0.01), and HDL-C levels below 1.2 mmol/l (46.2 vs. 15.3%, p < 0.001). We conclude that dyslipidemia is found more frequently in women with PCOS, independently of the excess weight that is often found in this patient group.

Adult↗
PubMed1996

Hemostatic variables in the prediction of coronary risk: results of the 8 year follow-up of healthy men in the Münster Heart Study (PROCAM). Prospective Cardiovascular Münster Study.

Myocardial infarction is usually due to the acute formation of an occlusive thrombus within the coronary arteries, in most cases against a background of pre-existing coronary atherosclerosis. Both of these factors may be promoted by a procoagulant state of the hemostatic system. Plasma fibrinogen, factor VIIc, blood pressure, and lipid parameters were measured in 2,781 healthy men aged 40-65 in the Münster Heart Study (formerly known as the PROCAM Study). After 8 years of follow-up 130 coronary events (15 sudden cardiac deaths, 22 fatal myocardial infarctions, and 93 nonfatal myocardial infarctions) were observed. Plasma fibrinogen concentration and factor VIIc activity were significantly greater among men who suffered a coronary event. The mean plasma fibrinogen level of the event group exceeded that of the non-event group by 0.32 g/l [2.59 +/- 0.58 vs. 2.91 +/- 0.58 (mean +/- SD) respectively, P < 0.001]. The incidence of coronary events among men within the upper tertile of fibrinogen concentration was three times higher than among men within the lower tertile. Plasma factor VIIc activity was 112.4 +/- 20.1% in the event group and 108.7 +/- 21.4% in the non-event group (P < 0.05). Among men in the upper tertile of factor VIIc activity, the incidence of coronary events was 1.6 times that of men in the lower tertile. When fibrinogen and low density lipoprotein (LDL) concentration were considered together, there was a graded and dramatic eightfold increase in 8 year risk from 17 per 1,000 of population among men with both fibrinogen and LDL cholesterol in the lower tertiles to 130 per 1,000 in men with both of these parameters in the upper tertile. In men with LDL cholesterol in the lowest tertile, increasing fibrinogen levels did not increase the risk of a coronary event. The coagulation variables, fibrinogen concentration, and factor VIIc activity thus markedly improve our ability to predict coronary risk.

Adult↗
PubMed1996

High density lipoproteins enhance the Na+/H+ antiport in human platelets.

In the present study, we investigated the effect of high density lipoproteins 3 (HDL3) on Na+/H+ exchanger activity and cytosolic pH (pHi) in human platelets. HDL3 alone failed to affect pHi, but preincubation with HDL3 significantly enhanced the Na+/H+ antiport activation brought about by acidification with 100 mM sodium propionate or stimulation with 0.05 U/ml thrombin. the stimulatory effect of HDL3 was unaffected by indomethacin excluding a role for cyclooxygenase products. The HDL3 effect was not mediated by Ca2+/calmodulin-dependent protein kinase as HDL3 failed to increase cytosolic free calcium concentration. However, the potentiating effect of HDL3 was completely blocked in the presence of the protein kinase C inhibitor, bisindoylmaleimide and the phosphatidylcholine-specific phospholipase C inhibitor, D609. Furthermore, the effect of HDL3 was abolished after covalent modification of HDL3 with dimethylsuberimidate and was not observed in platelets from Glanzmann thrombasthenia type 1 which do not express GP IIb/IIIa, as well as in platelets preincubated with anti-GP IIb/IIIa polyclonal antibodies. We conclude that HDL3 enhances the sodium propionate- and thrombin-induced Na+/H+ antiport activity in human platelets via binding to GP IIb/IIIa and activation of protein kinase C and phosphatidylcholine-specific phospholipase C.

Blood Platelets↗
PubMed1996

A new molecular defect in the lecithin: cholesterol acyltransferase (LCAT) gene associated with fish eye disease.

We report a new genetic defect in the lecithin:cholesterol acyltransferase (LCAT) gene associated with classical clinical and biochemical features of fish eye disease. The 63-year-old Australian female proband also suffers from non-insulin-dependent (type II) diabetes mellitus. She presented with corneal opacities, markedly reduced HDL-cholesterol (0.1 mmol/L; < 10% of normal controls), and elevated plasma triglycerides. The presence of diabetes did not explain the lipoprotein profile, which differed markedly in comparison to two female hypertriglyceridemic diabetic subjects. Cholesterol esterification in HDL-like particles was minimal but plasma cholesterol esterification was maintained due to LCAT activity in non-HDL-containing lipoprotein fractions. DNA sequence analysis of the proband's LCAT gene showed two C to T transitions resulting in the substitution of Thr123 with Ile and Tyr144 with Cys. Allele-specific PCR amplification procedures were used to confirm the presence of the mutations in this proband and to screen for additional carriers in her family. Three first degree relatives (mother, brother, son) were heterozygous for the Thr123 --> Ile mutation and her daughter had the Tyr144 --> Cys mutation. Apart from a reduction in HDL-cholesterol levels to half the normal concentration and a 20% reduction in apoA-I levels, their plasma lipids were unremarkable. The proband's son and daughter were further investigated. Both had normal cholesterol esterification rates in plasma and VLDL/LDL-depleted plasma, but reduced LCAT activity (50% that of normal). Thus, the biochemical and phenotypic expression for fish eye disease in the heterozygote subjects was similar, irrespective of the underlying LCAT mutation.

Australia↗
PubMed1996

Characterization of glucuronidated phase II metabolites of the immunosuppressant cyclosporine in urine of transplant patients using time-of-flight secondary-ion mass spectrometry.

The immunosuppressant, cyclosporine, is metabolized in the liver and small intestine to > 30 metabolites. Metabolism and immunosuppressive and toxic potentials of the metabolites are still unclarified. Therefore, search and determination of new metabolites remain an important part of cyclosporine research. In this study, cyclosporine metabolites were determined in 42 urine samples of transplant patients using time-of-flight secondary-ion MS. Besides the known metabolites of phase I and phase II, other groups of new phase II metabolites were detected, and most of them were identified as glucuronidated phase I metabolites. All metabolites were found in the urine of heart, kidney, and bone marrow graft patients, with frequencies in the range of 74% and 12%. The most intensive group of these metabolites was also detected in a HPLC fraction, together with the known glucuronidated AM1c. The concentration of this new metabolic group could be estimated to < or = 5/ml. In conclusion, this work demonstrated that time-of-flight secondary-ion MS is a powerful tool in pharmacological investigations. Furthermore this study showed that phase II metabolism is an important metabolic pathway of cyclosporine in transplant patients.

Animals↗
PubMed1995

The relation of interleukin-6, tumor necrosis factor-alpha, IL-2, and IL-2 receptor levels to cellular rejection, allograft dysfunction, and clinical events early after cardiac transplantation.

Immunoregulatory cytokines have been implicated in the pathophysiology of graft dysfunction after heart transplantation (HTx). In 15 consecutive patients undergoing HTx we prospectively determined levels of interleukin-6 (IL-6), tumor-necrosis-factor-alpha (TNF-alpha), interleukin-2 (IL-2), and soluble-interleukin-2-receptor (sIL-2-R) at eight points in time during biopsy and right heart catheterization and within 12 hr of echocardiography during the first three months after HTx. Blood was taken from the pulmonary arterial line. IL-6-levels correlated positively with hemodynamic and echocardiographic parameters of pump dysfunction--namely, pulmonary capillary wedge pressure, pulmonary arterial pressure, right atrial pressure, heart rate--and negatively with isovolumic relaxation time and stroke volume independent of the degree of cellular rejection as classified by the ISHLT criteria. A similar pattern was found for TNF-alpha- and sIL-2-R, while IL-2 correlated negatively with left and right heart filling pressures and positively with fractional shortening. In the three patients who died of sepsis or multiorgan failure within the study period IL-6-, TNF-alpha, and sIL-2-R-levels were elevated and IL-2-levels were suppressed compared with the 12 patients with a stable clinical course. IL-6 and sIL-2-R correlated positively while IL-6 and IL-2 correlated negatively. In this pilot study, a cytokine pattern with elevated levels of IL-6, TNF-alpha, and sIL-2-R as well as suppressed levels of IL-2 in the early period after HTx corresponds to impaired hemodynamics independent of cellular rejection and may indicate an unfavorable prognosis. These cytokines may therefore be useful for monitoring and warrant further study.

Adult↗
PubMed1995

An immunological enrichment method for epithelial cells from peripheral blood.

The ability of primary tumours to metastasize accounts for the majority of cancer deaths. The emergence of circulating carcinoma cells in the peripheral blood is supposed to be an indicator for cancer cell spread. We have focused on this phenomenon in order to develop a sensitive technique for enriching epithelial derived cells on the basis of a two-layer density gradient and subsequent immune magnetic cell sorting. Epithelial cells are possess a cytoskeleton containing an assembly of intermediate filaments. During carcinogenesis these filaments do not undergo modifications of antibody binding epitopes such as occur in the protein domains of surface markers. We have developed a two-layer density gradient in which the epithelial cells form a single density band. This was demonstrated by recovery experiments using [3H]thymidine-labelled epithelial cells which showed epithelial cells were enriched within this first step by a factor of 20. In a second step the MACS system was applied. Cells were stained with a performed FITC-conjugated mouse anti-human cytokeratin antibody bound to a rat anti-mouse antibody coupled to superparamagnetic particles (immune paramagnetic separation complex; IPSC) and subjected to high gradient magnetic fields. The two-step procedure was confirmed by dispersing 50 epithelial cells in 5 x 10(5), 5 x 10(6), 5 x 10(7), 5 x 10(8), 5 x 10(9) peripheral blood leucocytes. Specific binding of the preformed IPSC was demonstrated by flow cytometry, confocal laser, fluorescent and electron microscopy. The specificity of the method was further proved by dual staining with IPSC and anti-human PSA antibody of epithelial prostatic cells separated from peripheral blood in vitro. By means of this double-step separation method it was possible to isolate up to 15-20 cells out of 50 epithelial cells originally suspended into 5 x 10(7) to 5 x 10(9) human peripheral blood leucocytes. This represented an enrichment factor between 20,000 and 200,000, depending on the initial cell number. The immunologically captured epithelial cells can be used for further cytogenetic investigations such as in situ hybridization (ISH) and/or polymerase chain reaction (PCR) to detect cancer cell specific gene aberrations. This sensitive combined buoyant density immune magnetic cell separation technique is capable of detecting free carcinoma cells in the peripheral blood.

Animals↗
PubMed1995

Double-differential PCR for gene dosage estimation of erbB oncogenes in benign and cancer tissues and comparison to cellular DNA content.

Competitive and differential quantitative PCR methods circumvent the limiting factors of PCR which cause poor reproducibility. We describe the development and performance evaluation of another quantitative PCR method, double-differential PCR (ddPCR). The ddPCR method comprises the co-amplification of the single-copy gene HBB, the erbB-1, erbB-2 and erbB-3 oncogenes and the second single-copy reference gene SOD2 under equal reaction conditions. The ratio of band intensities of the PCR products in silver-stained polyacrylamide gels expresses the average gene copy number (AGCN) per cell of the erbB oncogenes. The coefficient of variability (CV) was less than 25% for an AGCN of 1. The PCR data were in correlation to the results from dot blotting. DNA image analysis did not reveal any correlation between DNA content and gene dosage deviation of the erbB oncogenes. The method was applied to normal breast tissue, benign breast diseases, breast cancer tissue and lymph node metastases. We suggest this method as being reproducible, low cost and rapid, and therefore suitable for clinical studies on erbB oncogene dosage estimation.

Base Sequence↗
PubMed1995

Prognostic relevance of aberrations in the erbB oncogenes from breast, ovarian, oral and lung cancers: double-differential polymerase chain reaction (ddPCR) for clinical diagnosis.

We have determined the average gene copy numbers (AGCN) of the erbB-1 gene, encoding the epidermal growth factor receptor (EGF-R), the erbB-2 and the erbB-3 genes in breast, ovarian, oral, and lung cancer tissue by using double-differential PCR (ddPCR). The ddPCR method comprises the co-amplification of the single-copy gene HBB, the erbB-1, erbB-2 and erbB-3 oncogenes and the second single-copy reference gene SOD2 under equal reaction conditions. In a retrospective study the AGCN of the erbB genes and the time up to the appearance of metastases were subjected to life-table analysis in 128 women with primary breast cancer. Patients whose breast cancer tissue showed an AGCN for erbB-1 of less than 0.4 and greater then 1.6, as expected from the literature, for erbB-2 of greater than 2.0 and for erbB-3 of less than 1.75 had decreased disease-free survival (DFS). The quotient of erbB-1 and erbB-2 AGCN was the most significant in multivariate Cox analysis followed by nodal status and progesterone receptor status. In extensive studies a similar association between erbB AGCN and metastasis was seen in ovarian cancer and oral cancer, though erbB oncogene aberrations in those entities were not as frequent as in breast cancer. The AGCN of erbB oncogenes may not be of prognostic value in untreated lung cancer patients.

Base Sequence↗