A T-to-A substitution in the E1 alpha subunit gene of the branched-chain alpha-ketoacid dehydrogenase complex in two cell lines derived from Menonite maple syrup urine disease patients

We cloned and sequenced cDNAs of the E1 alpha and E1 beta subunits of the branched chain alpha-ketoacid dehydrogenase complex (BCKDH) in two cell lines derived from two different Menonite MSUD patients (GM 1655, GM 1099). A T-to-A substitution which generates an asparagine in place of a tyrosine at amino acid 394 of the mature E1 alpha subunit was present in both alleles in these two cell lines, whereas cDNAs of the E1 beta subunit in these cell lines were identical to that of normal human lymphoid cell line and that of the clone from a human placenta cDNA library. It is suggested that the Menonite MSUD is caused by the missense mutation of the E1 alpha subunit of the BCKDH complex.     

Relation between cytochrome P-450 increase and endoplasmic reticulum proliferation in hepatocytes of mice treated with phenobarbital: a microphotometric and morphometric study.

To obtain detailed information on phenobarbital (PB)-induced cytochrome P-450 (P-450) increase and endoplasmic reticulum (ER) proliferation in hepatocytes, we estimated microphotometrically the amount of P-450 per unit cytoplasmic volume and morphometrically the area of ER per unit cytoplasmic volume in hepatocytes adjacent to the portal area or central venule (1 periportal or 1 perivenular cells) and in the second and third layers from the portal area or central venule (2, 3 periportal or 2, 3 perivenular cells) from mice injected with 35, 50, 100, or 150 mg/kg PB once a day for 3 days. By dividing the P-450 amount by the ER area, the number of P-450 molecules per unit ER area was also calculated. In 1 and 2, 3 perivenular cells, except for 2, 3 perivenular cells after injection of 150 mg/kg PB, the amount of P-450 increased with ER proliferation and the number of P-450 molecules in ER remained unchanged after injection of 50, 100, or 150 mg/kg PB. In 2, 3 periportal cells, however, the P-450 amount and the number of P-450 molecules in ER increased markedly without or with some ER proliferation after injection of 50, 100, or 150 mg/kg PB; the P-450 increase appears to be generally independent of ER proliferation. The 1 periportal cells are probably exceptional hepatocytes that usually did not respond to PB stimulation.     

Mutants with reduced Ca activation inParamecium aurelia

Summary Two heat-sensitive pawn mutants ofParamecium aurelia are capable of avoiding reactions when grown at 23°C but not at 35°C. Electrophysiological analyses show that Ca activation is reduced in the mutants even when they are grown at 23°C. The maximal rate of rise and the peak of the evoked action potential (Ca-spike) in the mutants are smaller than those of wild type in a K-solution. After suppression of K conductance by either TEA⁺ or Ba⁺⁺, the action potentials of the mutants peak at the same level as that of wild type. However, the maximal rate of rise of the mutants remains only about half that of wild type. Thus, the mutations affect Ca activation but not K activation.Incubation at a high temperature (35°C) further reduces Ca activation to almost zero in the mutants but has little or no effect on wild type. This almost complete loss of Ca activation explains the lack of avoiding reactions when the mutants are grown at high temperatures. A double mutant containing two heat-sensitive mutations shows extremely reduced Ca activation even when grown at 23°C.     

A mutant of Paramecium with increased relative resting potassium permeability

Fast-2, a membrane mutant of Paramecium aurelia, is due to a single-gene mutation and has behavioral abnormalities. Intracellular recordings through changes of external solutions were made. The mutant membrane hyperpolarized when it encountered solutions with low K⁺ concentration. This hyperpolarization and other associated activities were best observed in Ca- or Na-solutions devoid of K⁺. Membrane potential was plotted against the concentration of K⁺ (0.5 to 16 mM) in solutions of fixed Na⁺ or Ca⁺⁺ concentration. The slopes of the curves for the mutant membrane were steeper than those for the wild type at the lower concentrations of K⁺. Inclusion of 2 mM tetraethylammonium chloride (TEA-Cl) counteracted the mutational effects. Spontaneous action potentials in Ba-solution and the electrically evoked action potentials in various solutions are normal in this mutant. We conclude that the resting permeability to K⁺ relative to the permeabilities to Na⁺ and Ca⁺⁺ has been increased by the mutation.     

Effect of NADH dehydrogenase-disruption and over-expression on respiration-related metabolism in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> KY9714

The function of type II NADH dehydrogenase (NDH-2) in Gram-positive Corynebacterium glutamicum was investigated by preparing strains with ndh, the NDH-2 gene, disrupted and over-expressed. Although disruption showed no growth defects on glucose minimum medium, the growth rate of the over-expressed strain was lower compared with its parent, C. glutamicum KY9714. Ndh-disruption and over-expression did not lead to a large change in the respiratory chain and energetics, including the cytochrome components and the H⁺/O ratio. However, in the strain that lacked NDH-2, membrane l-lactate oxidase activity increased, while NDH-2 over-expression led to decreased l-lactate and malate oxidase activities. In addition, relatively high cytoplasmic lactate dehydrogenase (LDH) activity was always present as was malate dehydrogenase, irrespective of NDH-2 level. Furthermore, l-lactate or malate-dependent NADH oxidase activity could be reproduced by reconstitution with the membranes and the cytoplasmic fraction isolated from the disruptant. These results suggest that coupling of LDH and the membrane l-lactate oxidase system, together with the malate-dependent NADH oxidase system, operates to oxidize NADH when the NDH-2 function is defective in C. glutamicum.     

Estrogen replacement suppresses stress-induced cardiovascular responses in ovariectomized rats

We assessed the hypothesis that chronic estrogen replacement in ovariectomized rats has the beneficial effect of suppressing stress-induced cardiovascular responses through endothelial nitric oxide synthase (eNOS). We employed a radiotelemetry system to measure blood pressure and heart rate (HR). Female Wistar rats aged 11 wk were ovariectomized and implanted with radiotelemetry devices. After 4 wk, the rats were assigned either to a placebo-treated group (Placebo; n=6) or a group treated with 17beta-estradiol (Estrogen; n=8) subcutaneously implanted with either placebo- or 17beta-estradiol (1.5 mg/60-day release) pellets under anesthesia. These rats underwent either of the two types of stress after 4 wk of estrogen or placebo treatment. Cage-switch stress and restraint stress rapidly and continuously elevated the mean arterial pressure (MAP) and HR both in the Placebo and Estrogen groups. However, the MAP and HR responses to cage-switch stress and the MAP but not HR response to restraint stress were attenuated significantly in the Estrogen group compared with the Placebo group. A NOS inhibitor, NG-nitro-L-arginine methyl ester, given in drinking water, reduced the difference in the pressor response to cage-switch between the Estrogen and Placebo groups. In addition, Western blot analysis showed that eNOS expression in the mesentery was increased in the Estrogen group compared with the Placebo group. Thus for the first time we showed that mesenteric eNOS overexpression could explain at least partly why chronic estrogen treatment suppressed the enhanced cardiovascular responses to psychological stress in the ovariectomized rat.     

A versatile data acquisition system for physiological modelling of laboratory fermentation processes

Summary A versatile data acquisition system for physiological modelling of laboratory fermentation processes is given. This system meets the stringent qualifications of modern modelling techniques in terms of signal to noise ratios and sampling rates using readily available equipment. The data are portable to any data processing system for modelling and analysing the culture characteristics. Two completely equipped fermentation systems are sampled with a sampling period of six seconds. The data acquisition system is used to acquire data of a set of continuous fermenters showing sustained oscillations. Oscillation data was used to show the performance of the data acquisition system. With the high sampling rate signal to noise ratios of 30 dB or higher are achieved. It was possible to determine periods of oscillatory behaviour and delay times. The system has shown to be versatile in alaboratory set up. Addition or subtraction of sensors or complete fermentation systems can be done without major alterations to the system.     

Detection of Staphylococci in Mouse Phagocytic Cells by in Situ Hybridization Using Biotinylated Dna Probes

In situ hybridization was used to detect intracellular Staphylococcus aureus and S. epidermidis in mouse phagocytic cells after experimental infection of C3H mice with Staphylococci via abdominal or intravenous injection. Isolated ascites or whole blood were tested by the phagocyte smear technique, using bacteriolytic enzymes to preserve phagocytic cell morphology. The exposed bacterial DNA was visualized as intracellular hybridized signals by use of biotinylated DNA probes and by immunocytochemistry using streptavidin-alkaline phosphatase conjugates as detector molecules. These DNA probes, prepared from randomly cloned genomic DNA fragments of S. aureus and S. epidermidis, were strain-specific and did not cross-hybridize either in situ or on dot-blot hybridization. This technique of in situ hybridization with phagocyte smears is useful for detection and diagnosis of intracellular bacteria regardless of viability.     

The Short‐Term Effect of Diacylglycerol Oil Consumption on Total and Dietary Fat Utilization in Overweight Women

Diacylglycerol (DAG) is a natural component of edible oils with metabolic characteristics distinct from those of triacylglycerol (TAG). Consumption of DAG oil (containing >80% DAG) induces greater fat oxidation than consumption of TAG oil. We compared the effects of 4 days of DAG oil consumption with those of TAG oil consumption on total and dietary fat oxidation over 24 h in overweight women using a whole‐room respiratory chamber. Overweight (BMI (kg/m²) ≥25) females participated in this double‐blind, crossover‐controlled trial. The subjects consumed test diets containing either TAG or DAG oil as 15% of their total caloric intake (mean test oil intake was 33.0 ± 3.1 g/day) during each 4‐day treatment. Fat oxidation and energy expenditure were measured in a respiratory chamber on the 4th day of each treatment. Compared with TAG oil, DAG oil consumption significantly increased total fat oxidation and dietary fat oxidation in overweight subjects. Total energy expenditure (TEE) and carbohydrate (CHO) oxidation did not significantly differ between DAG oil and TAG oil consumption in overweight subjects. Compared with TAG oil, DAG oil consumption enhanced total fat oxidation and dietary fat oxidation in overweight subjects. The enhanced fat metabolism in overweight subjects that consumed DAG oil partly explains the greater loss of body weight and body fat related to DAG oil consumption in weight‐loss studies.     

Microwave dielectric study on bound water of globule proteins in aqueous solution

A dielectric relaxation peak due to bound water of globule proteins in aqueous solution was observed at first by the use of a time domain reflectometry. This peak locates around 100 MHz as well as that of the aqueous DNA solution and the moist collagen, and has a relaxation strength in proportion to surface of the globule protein except for trypsin and pepsin of hydrolase. It is suggested that this peak is caused by orientation of bound water molecules on the protein surface. The number of bound water molecules estimated is in good agreement with that obtained by other method such as x-ray analysis. The solution exhibits another peak below 100 MHz, which is caused by the rotation of globule protein supplemented by migration of the counterion. Its relaxation time is completely proportional to the molecular weight of the protein. © 1994 John Wiley & Sons, Inc.