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A new qualitative PCR product detection assay called competitive amplified single mutation detection by selective probe hybridization immunoassay (CASSI) was developed for genotyping the most common apolipoprotein E (apoE) polymorphisms. Single target DNA strands immobilized using biotin on streptavidin-coated microplates were hybridized in separate wells with two distinct, 5'-fluorescein isothiocyanate (FITC)-labeled oligonucleotides, complementary to either the 112Arg or 158Arg encoding site. With this assay, only correctly matched hybrids that form between probe and target DNA can be cleaved with the HhaI restriction endonuclease, leading to loss of probe label in corresponding wells. However, allele-specific, probe-target mismatches due to G-->T exchanges in the HhaI recognition sequences are not cleaved. After digestion, the remaining microplate-adsorbed signal is measured colorimetrically by using anti-FITC, Fab-horseradish peroxidase conjugates. Our results show maximum intensity was detected when the respective probe hybridized incompletely to the target (i.e., no cleavage), and minimum signal was obtained when the probe matched the target completely (complete cleavage); whereas, an intermediate signal was recorded at 50% complementarity (i.e., heterozygote alleles). With this assay, we could demonstrate a high prevalence of the apoE2 allele in patients suffering from coronary artery disease even though they displayed normal triglyceride and cholesterol levels. Corresponding results were obtained by CASSI compared with conventional restriction fragment-length polymorphism analysis.

DNA fragments used as standards in competitive PCR were precisely calibrated using HPLC and commercially available DNA molecular mass markers. The accuracy of calibration was reflected by data that differed by only 2% from the mean when two independently purified and calibrated competitor preparations were compared. Highly dilute competitor solutions were stable at -20 degrees C for up to 1 year in the presence of carrier HindIII-digested lambda DNA, but progressive loss of competitor DNA with increasing storage time was observed when carrier DNA was omitted from the solution. Applying 0.2 U uracil-DNA glycosylase (UDG) per assay of remaining temperature-stable activity did not effect the ratios of synthesized products. This study describes quality management in PCR quantitation that is useful for the measurement of multidrug resistance-associated protein (MRP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts.

In male Wistar rats the influences of age and experimental obesity on the activity of malic enzyme (EC 1.1.1.40) in different organs were studied. Obesity was induced in newborn rats by injection of Na(+)-L-glutamate (2 mg/g b.w. daily) subcutaneously in the first 5 days. The enzyme activity was measured at the ages of 2, 6 and 18 months. In control animals the highest enzyme activities were found in the heart muscle, liver, epididymal fat pad and skeletal muscle after 6 months. After 18 months the activities in these organs are considerably reduced. In the kidneys the activity between the 2nd and the 18th months tends to decrease continuously and only the brain shows an opposite trend. In comparison with the control animals, in glutamate treated rats the enzyme activity doubles nearly in the lipogenic organs liver and fat tissue in all age groups. In liver and fat tissue of 6-month-old rats, previously treated with clonidine to stimulate growth hormone secretion, the activities are lower than in glutamate obese rats without clonidine, but still higher than in normal control animals. The qualification of glutamate obese rats as a model for the study of age-associated diseases like obesity or diabetes mellitus type II needs further investigation.

Glutamate-induced obesity of Wistar-rats is known to develop under normophagic and normoinsulinemic conditions, although hyperphagia and hyperinsulinemia are common to obese individuals. Rats of this obesity model show retarded growth, reduced mass of some organs, carcass and whole body as well as an extraordinary high fat content, whereas protein content is reduced. In this study, nitrogen (N) balance, urinary excretion of urea-N, ammonia-N, creatine-N and alpha-amino acid-N and plasma free fatty acid concentration of growing, glutamate-induced obese rats were determined. The main results were independent of frame of reference (mmol N/kg body mass; mmol N/kg0.75 metabolic body mass; N in % of nitrogen intake): Nitrogen intake, urinary excretion of alpha-amino acids and nitrogen excretion in faeces were equal between lean and obese rats. Nitrogen excretion in urine was elevated in obese rats, mainly resulting from increased amounts of urea and ammonia. Nitrogen balance was positive in both groups, but reduced in obese rats. These data point to normal digestion of food proteins, but an unusual high oxidative desamination rate of the absorbed amino acids in obese rats. Taking into account the various hormonal and nerval alterations in glutamate-induced obese rats, resulting e.g. in increased hepatic insulin concentration, the retained amino acid carbon should be channelled into hepatic fatty acid synthesis. Really, unfasted and overnight fasted obese rats showed elevated plasma free fatty acid concentrations. Channeling of amino acids into lipogenesis may explain the low muscle mass and striking fat accumulation--despite normophagia and peripheral normoinsulinemia--of growing, glutamate-induced obese Wistar-rats.

Development of hyperinsulinemia was investigated which appeared late in the obese state in rats postnatally treated with L-glutamate. Insulin concentrations were estimated in the blood plasma of the caval and portal vein, and morphometric and immunohistochemical measurements of cells in the islets of Langerhans were performed, and also glucose tolerance tests. Not earlier than at 3 months hyperinsulinemia is shown in glutamate obese rats (GOR) in the peripheral blood plasma. Also in the portal blood plasma the insulin concentration is higher (167%) in GOR relative to controls. The insulin concentrations in the portal vein rise further in both animal groups whereas insulin concentration in the peripheral blood remains at the different levels in both animal groups. Impaired glucose tolerance was observed for GOR only. Islets of Langerhans in the Pancreas show enlargement and increased proliferation of B-cells in GOR. In contrast the number of D-cells is diminished. The hyperplasia of islets differs remarkably to hypoplasia of other organs in GOR. We conclude that the peripheral hyperinsulinemia is caused by a permanent hypersecretion of insulin.

The localization of (Na+ + K+)-activated ATPase was investigated in isolated brush borders of rat small intestinal mucosa. The purity of the fractions has been checked by morphological and enzymatic criteria. The brush borders were found to contain a significant quantity of (Na+ + K+)-activated ATPase. Separation of isolated brush borders into their substructures suggests that (Na+ + K+)-activated ATPase is localized deeper within the brush border region than invertase. These findings are discussed in relation to active monosaccharide transport in the intestine.

Injections of monosodium glutamate to neonate rats induce chronic growth hormone deficiency by hypothalamic lesions in the regions of the nucleus arcuatus and the eminentia mediana. The glutamate treated rats develop massive obesity. By this type of obesity hyperinsulinemia in the dynamic phase is not evident. The adipose tissue of the GOR (glutamate obese rat) is characterized by hypertrophic adipocytes and diminished number of adipocytes. In the GOR glucose oxidation and glucose lipid conversion are increased, but insulin sensitivity of glucose metabolism is diminished. The enhanced glucose utilization in adipose tissue of the GOR is discussed as being the consequence of the chronic growth hormone deficiency.

alpha-gliadin was prepared from wheat flour by two different methods. The products were compared electrophoretically and by double radial immuno-diffusion. The alpha-gliadin fraction proved to be identical in the immunological test. Only the alpha-gliadin preparation received by ion exchange chromatography is suitable for further purification by multiple gel filtrations.

1. The condidtions were studied under which the Na+-dependently transported monosaccharides influence the K+-influx across the microvilli membrane of rat jejunum. 2. The Na+-dependent, energy requiring transport of monosaccharides causes a decrease of the K+-influx, whereas monosaccharides metabolizable by the intestinal mucosal cells - under suitable conditions - induce an increase in the K+-influx. 3. These results indicate that the K+-influx may be controlled by an energy dependent system in the microvilli membrane, which is influenced by metabolism and monosaccharide transport. It is discussed that this control system may be a (Na+ + K+)-ATPase.

1. In the rat's jejunum, the changes induced by Na+-dependently transported monosaccharides (10 mM) of the unidirectional Na+- and K+-influx across the microvilli membrane were studied under varying metabolic conditions. 2. The monosaccharide induced change of Na+- and K+-influx is a function of the ion gradients active across the microvilli membrane. 3. In the in vitro preparation during the process of energy-dependent accumulation the monosaccharide induced change of permeability in the microvilli membrane decreases the K+ concentration, while the Na+ concentration increases. 4. The monosaccharide concentration in the tissue reaches a final value which is proportional to the change of Na+- and K+-influx and to the change in ion concentrations, and which is retained even upon compensation of the gradient and at low intracellular K+ concentrations. 5. The correlation between the breakdown of the ion gradient and the rise of monosaccharide accumulation is explained by the monosaccharide induced restriction of the active ion transport and the ensuing change of energy dissipation in favour of the monosaccharide transport. The (Na+ + K+)-ATPase in the microvilli membrane is discussed as being transmitter of the energy from the ion gradients.

1. An energy dependence of the Na+ influx and of the "extra-Na+ influx" across the microvilli membrane was demonstrated in an in vitro preparation of the rat jejunum by adjustment of low ATP/ADP quotients. The monosaccharide influx does not show this dependence. 2. The similar relationship of monosaccharide-dependent Na+ influx and Na+ influx without monosaccharide with the energy state in the mucosa cells suggests a common control system. 3. A constant stoichiometry between monosaccharide and "extra-Na+ influx" can be maintained only under constant intracellular conditions. 4. The changes of the Na+ and K+ influxes by so-called Na+ dependently transported monosaccharides correspond to those which can be elicited by lowering the ATP/ADP ratio in the in vitro preparation. 5. A mechanism is discussed in which an ATP-utilizing reaction is stimulated in the microvilli owing to the monosaccharide transport, thus locally discontinuing the condition for uncoupling of an (Na, K)-ATPase and eliciting an "extra-Na+ influx".