Hepatic mitochondrial coumarin 7-hydroxylase: comparison with the microsomal enzyme

The specific activity of cytochrome P450-linked coumarin 7-hydroxylase (COH) of hepatic mitoplasts from DBA/2N mice is up to 55% as great as the microsomal activity. According to Western blot and immunodiffusion analysis and inhibition studies with anti-P450Coh and metyrapone, the mitoplastic P450Coh had the same molecular weight and immunochemical and catalytic properties as the corresponding microsomal enzyme. The inducibility of the two proteins by pyrazole and their genetic regulation, as studied with DBA/2N and AKR/J mice, appears to be similar. However, the mitochondrial electron transfer system was not able to support the COH activity of reconstituted microsomal P450Coh although the enzyme was fully active with the microsomal NADPH-cytochrome P450 reductase. This indicates some differences between the two proteins with respect to their interaction with the electron transfer system. This was confirmed by the ability of anti-adrenodoxin reductase antibody to effectively inhibit the mitoplastic COH but not the COH reconstituted with purified microsomal P450Coh and NADPH-P450 reductase. We have previously found that P450Coh does not react with anti-P450b or anti-P450c antibodies, which recognize respective isoforms in rat liver mitoplasts. While P450Coh from microsomes and mitoplasts possess a number of properties in common, the mitoplast P450Coh represents a new subspecies of mitochondrial P450. Some characteristics of mitoplast P450Coh may be the result of post-translational modifications necessary for processing and translocation into the mitochondria.     

Inward rectifier K channels in renal epithelioid cells (MDCK) activated by serotonin

Summary The present study has been performed to test for the effect of intracellular calcium and of serotonin on the channel activity in patches from subconfluent MDCK-cells. In inside-out patches, inwardly rectifying potassium-selective channels are observed with open probabilities of 0.01±0.01, 0.24±0.03 and 0.39±0.07, at 100 nmol/liter, 1 mol/liter or 10 mol/liter calcium activity, respectively. The single-channel slope conductance is 34±2 pS, if the potential difference across the patch (V ) is zero, and approaches 59±1 pS, ifV is –50 mV, cell negative. In the cell-attached mode, little channel activity is observed prior to application of serotonin (open probability=0.03±0.03). If 1 mol/liter serotonin is added to the bath perfusate, the open probability increases rapidly to a peak value of 0.34±0.04 within 8 sec. In continued presence of the hormone, the open probability declines to approach 0.06±0.02 within 30 sec. At zero potential difference between pipette and reference in the bath (i.e., the potential difference across the patch is equal to the potential difference across the cell membrane), the single-channel conductance is 59±4 pS. In conclusion, inwardly rectifying potassium channels have been identified in the cell membrane of subconfluent MDCK-cells, which are activated to a similar extent by increase of intracellular calcium activity to 1 mol/liter and by extracellular application of 1 mol/liter serotonin.     

M?nnchen-überschu? bei Schwarzspechtnestlingen (Dryocopus martius)?

Zusammenfassung Von 1979 bis 1991 wurde auf einer etwa 200 km² großen Mischwaldfläche in Süddeutschland bei 445 Schwarzspechtnestlingen (140 Bruten; Alter der Jungvögel: 14 bis 20 Tage) eine Geschlechtsbestimmung vorgenommen. Das Gesamtgeschlechterverhältnis (0,92 :1 ; alle Daten von 1979 bis 1991 zusammengefaßt) sowie die Geschlechterverhältnisse der Einzeljahre wichen nicht signifikant von einem ausgeglichenen Geschlechterverhältnis ab. Eine statistische Überprüfung aller zum sekundären Geschlechterverhältnis beim Schwarzspecht vorliegenden Literaturangaben ergab ebenfalls in keinem Fall einen statistisch signifikanten Unterschied zwischen den ermittelten Geschlechterverhältnissen und einem Verhältnis von 1:1. Die mehrmals in der Literatur vertretene Meinung, bei Schwarzspechtnestlingen sei ein -Überschuß festzustellen, ist somit nicht länger haltbar.

---

Is there a surplus of males in Black Woodpecker nestlings (Dryocopus martius)?

Summary A population of Black Woodpeckers was studied in an area of about 200 km² in southern Germany from 1978 up to 1991. Once a year the nesting trees were visited to record the age and number of young and to ring the nestlings. In 1979 the sex of seven birds (two broods) was determined additionally (crown feathers showing red from tenth day in , bluish-black in — only nape is red). From 1980 onwards the young of all broods censused were sexed (445 birds; 140 broods; age of the birds: 14 to 20 days). In contrast to several studies which had pointed to a -biased secondary sex ratio in the Black Woodpecker, but corresponding to findings in various bird species sexually monomorphic in size, neither the overall sex ratio (0,92 :1 ; pooled data from 1979 to 1991) nor the ratios of single years showed any significant deviations from 1:1. Likewise, we did not find any differences between the sex ratios of Black Woodpecker nestlings reported in the literature and a balanced ratio. For these reasons it is not tenable any longer to speak about a surplus of in Black Woodpecker nestlings.

     

Restriction fragment length polymorphism in the mitochondrial DNA of cloned cattle

The clonal origin of 4 Holstein bulls was determined by hybridization experiments with 2 different minisatellite probes, and all 4 animals showed identical genomic DNA fingerprints, hence confirming monozygosity. Extra-chromosomal differences were observed among these 4 Holstein bulls. Mitochondrial DNA restriction fragment length polymorphisms with restriction endonucleases Avall and Hhal sites were found, and these polymorphisms can be explained by the loss of a single site for each of these 2 enzymes. Since mitochondrial DNA are maternally transmitted, all 4 bulls would produce genetically equivalent spermatozoa and offspring. The combination of embryo cloning and specific cytoplasmic markers would provide an ideal system for the study of maternal cytoplasmic effects on quantitative traits.     

Foetal circulatory responses to arrest of uterine blood flow in sheep: effects of chemical sympathectomy.

Acute foetal asphyxia, caused by arrest of uterine blood flow, increases both sympathetic activity and peripheral vascular resistance and decreases blood flow to peripheral organs (Jensen et al., J. Dev. Physiol., 9, 543-559). The rapidity and uniformity of this peripheral vasoconstriction suggest that the sympatho-neuronal system may reflexly cause these initial blood flow changes during acute asphyxia. To test this hypothesis, we studied 5 intact and 6 chemically sympathectomized (6-hydroxy-dopamine, 46.1 +/- 6 mg/kg foetal weight) chronically prepared normoxaemic foetal sheep in utero at 0.9 of gestation. Organ blood flows (microsphere method), plasma concentrations of catecholamines, vasopressin, and angiotensin II, acid-base balance and blood gases were measured before, during and after arrest of uterine blood flow for 2 min, i.e., at 0, 1, 2, 3, 4 & 30 min. In intact foetuses there was a progressive increase in arterial blood pressure and a rapid circulatory centralization in favour of the brain stem and heart and at the expense of most of the peripheral organs. The changes in peripheral blood flow during and after asphyxia were well reflected by those in the skin and scalp. In chemically sympathectomized foetuses, arterial blood pressure fell transiently at 1 min of asphyxia and cardiac output was redistributed towards the carcass and intestinal organs at the expense of the heart, spinal medulla, and placenta. We conclude that in foetal sheep at 0.9 of gestation, the short-term adaptation to arrest of uterine blood flow is a rapid and profound peripheral vasoconstriction to effect an increase in arterial blood pressure. This initial response during circulatory centralization, which is necessary to increase or maintain blood flow to the heart, brain stem, and placenta, is blunted by sympathectomy. Thus, the foetal sympatho-neuronal system is important for short-term adaptation to and intact survival of asphyxia.     

Scale-up and optimization of culture conditions of a human heterohybridoma producing serotype-specific antibodies to Pseudomonas aeruginosa

Summary Three different stirred bioreactors of 0.5 to 12 l volume were used to scale up the production of a human monoclonal antibody. Inoculation density and stirrer speed were evaluated in batch cultures, whereas dilution rate and pH were optimized in chemostat cultures with respect to high specific antibody production rate and high antibody yield per time and reactor volume. The cell line used for the experiments was a heterohybridoma, producing immunoglobulin M (IgM) against lipopolysaccharide of Pseudomonas aeruginosa. Cells were cultured in spinner flasks of 500 ml liquid volume for adaptation to stirred culture conditions. Subsequently cells were transferred to the 1.5-1 KLF 2000 bioreactor and to the 12-1 NLF 22 bioreactor for pilot-scale cultures. Chemostat experiments were done in the 1.5-1 KLF bioreactor. Cell density, viability, glucose and lactate and antibody concentration were measured during culture experiments. In batch cultures in all three stirred bioreactors, comparable maximal cell densities and specific growth rates were achieved. Chemostat experiments showed that at a pH of 6.9 and a dilution rate of 0.57 per day the specific antibody production rate was threefold higher than similar experiments done at pH 7.2 with a dilution rate of 0.36 per day. By optimizing pH and dilution rate in chemostat cultures the daily yield of human IgM increased nearly threefold from 6 to 16 mg/day and per litre of reactor volume. The yield per litre of medium increased twofold. Correspondence to: U. Schürch     

Effect of BHT 920 on calcium-activated K+ channels in renal epithelioid MDCK cells.

In Madin Darby canine kidney (MDCK) cells, epinephrine has been shown to increase intracellular calcium, activate calcium-dependent K+ channels and hyperpolarize the cell membrane. The present study has been performed to test for the possible involvement of alpha 2-adrenergic receptors. To this end, the effects of alpha 2-adrenoceptor agonist BHT 920 have been studied on cell membrane potential, ion channel activity and intracellular calcium: Similar to epinephrine, BHT 920 hyperpolarizes the cell membrane, increases intracellular calcium and activates inwardly rectifying K+ channels (single channel slope conductances 30-80 pS). Half-maximal hyperpolarization is achieved at concentrations between 10 and 100 nmol/l. The hyperpolarizing effect of BHT 920 is abolished in the presence of alpha 2-adrenoceptor antagonist yohimbine (100 nmol/l) but not in the presence of alpha 1-adrenoceptor antagonist prazosin (100 nmol/l). At extracellular calcium activity below 100 nmol/l BHT 920 still leads to a transient hyperpolarization of the cell membrane but, in contrast to epinephrine, is unable to significantly increase intracellular calcium or significantly activate the calcium-sensitive K+ channels. The observations indicate that stimulation of alpha 2-receptors participates in the epinephrine-induced increase of intracellular calcium, channel activation and hyperpolarization.     

Structure and solution properties of tamarind-seed polysaccharide.

The major polysaccharide in tamarind seed is a galactoxyloglucan for which the ratios galactose:xylose:glucose are 1:2:25:2.8. A minor polysaccharide (2-3%) contains branched (1----5)-alpha-L-arabinofuranan and unbranched (1----4)-beta-D-galactopyranan features. Small-angle X-ray scattering experiments gave values for the cross-sectional radius of the polymer in aqueous solution that were typical of single-stranded molecules. Marked stiffness of the chain (C infinity 110) was deduced from static light-scattering studies and is ascribed partially to the restriction of the motion of the (1----4)-beta-D-glucan backbone by its extensive (approximately 80%) glycosylation. The rigidity of the polymer caused significant draining effects which heavily influenced the hydrodynamic behaviour. The dependence of "zero-shear" viscosity on concentration was used to characterise "dilute" and "semi-dilute" concentration regimes. The marked dependence on concentration in the "semi-dilute" region was similar to that for other stiff neutral polysaccharide systems, ascribed to "hyper-entanglements", and it is suggested that these may have arisen through a tenuous alignment of stiffened chains.