Inhibition of testosterone biosynthesis by ethanol. Relation to hepatic and testicular acetaldehyde, ketone bodies and cytosolic redox state in rats.

In experiments in which liver and testis freeze-stops were performed on pentobarbital-anaesthetized rats, ethanol (1.5 g/kg body wt.) reduced plasma testosterone concentration from 13.1 to 3.2 nmol/litre. 4-Methylpyrazole abolished the ethanol-induced hepatic and testicular increase in the lactate/pyruvate ratio, and the testicular acetaldehyde level, but did not diminish the reduction in plasma testosterone concentration. In testes, but not in liver, ethanol decreased the 3-hydroxybutyrate/acetoacetate ratio, and 4-methylpyrazole did not prevent this effect. In experiments in which freeze-stop was performed after cervical dislocation, ethanol decreased the testis testosterone concentration from 590 to 220 pmol per g wet wt. The effects of ethanol and 4-methylpyrazole on testis acetaldehyde, lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios were the same as found during anaesthesia. The NAD+-dependent ethanol oxidation capacity in testis ranged from 0.1 to 0.2 mumol/min per g wet wt. and seemed to be inhibited by 4-methylpyrazole both in vivo and in vitro. In additional experiments, ethanol doses between 0.3 and 0.9 g/kg body wt. did not alter the plasma testosterone concentration in rats treated, or not treated, with cyanamide, which induced elevated acetaldehyde levels in blood and testes. The results suggest that ethanol-induced inhibition of testosterone biosynthesis was not caused by extratesticular redox increases, or by extra- or intra-testicular acetaldehyde per se. The inhibition is accompanied by changes in testicular ketone-body metabolism.     

Purification and properties of Neurospora crassa laccase

Extracellular Neurospora laccase (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) has been purified to apparent homogeneity by classical purification techniques. The enzyme, which consists of mainly one form, has a molecular weight of 64,800 and contains 11% carbohydrate. The ultraviolet, visible, and electron paramagnetic resonance spectra indicate that both type I and type II copper are present, as described for the Polyporus versicolor enzyme. With the exception of phloroglucinol, only para- and ortho-diphenols serve as effective substrates for the enzyme. Like the extracellular form, intracellular laccase is a glycoprotein as shown by its ability to bind to Concanavalin A Sepharose. Other studies, including gel filtration and ion-exchange chromatography, revealed no differences between the intracellular and extracellular enzymes, suggesting that intracellular laccase is destined for excretion by the cell.     

The effects of two disulphides on cholinesterase activity in the spectrophotometric assay

1. 5,5'-Dithiobis-(2-nitrobenzoate) did not influence serum cholinesterase activity, whereas 2,2'-dithiodipyridine had an inhibitory effect. 2. The lowering of the molar extinction coefficients observed in the presence of physostigmine may be a result of a reaction between thiolate ions with carbamate moieties. 3. The use of 5,5'-dithiobis-(2-nitrobenzoate) is still recommended in investigations, especially where the quantitative aspects are significant.     

Genetic transformation of willows (Salix spp.) byAgrobacterium tumefaciens

Anin vitro transformation method has been developed for stem explants of fast-growing willow clones (Salix spp.) usingAgrobacterium tumefaciens as a vector. Transformants obtained with the strains C58 and GV3101 (pGV3851::pLD1) were selected on hormone-free medium and on medium containing kanamycin, respectively. Transformation was confirmed by Southern blot analysis and nopaline assay. Inoculation of green-house grown plants with nopaline and octopine wildtype strains and shoot or root inducing mutant strains caused undifferentiated tumors at a frequency of 0 to 80%, depending on theSalix genotype and the bacterial strain used.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Km kanamycin - NPT neomycin phosphotransferase     

The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942.

The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.     

Dielectric properties of human blood and erythrocytes at radio frequencies (0.2–10 MHz); dependence on cell volume fraction and medium composition

The dielectric properties of human erythrocytes (red blood cells) suspended in whole blood and in isotonic media at various volume fractions (haematocrits) have been studied in the frequency range 0.2–10 MHz, in which the so-called-dispersion due to the Maxwell-Wagner effect is known to occur. The capacitance and conductance at 25 °C were measured by an instrument interfaced to a computer. The rectangular sample cavity (1 ml volume) contained four pure gold electrode pins, and the sample could be circulated by a roller pump. The frequency-dependence of the permittivity and conductivity were fitted by non-linear least squares regression. Corrections were applied for non-linearity in the dielectric increment at high haematocrit, and for electrode polarisation when diluting the blood in saline. Data were interpreted in terms of a simple equivalent resistor-capacitor circuit. From the measured haematological values the specific membrane capacitance (C_m) and the conductivities internal and external to the cells (_i and _o respectively) were estimated. The conductivities behaved in a predictable manner with a mean of 0.458 S · m^–1 (s.d. ± 0.044) for _i, whereas the value of C_m (and indeed the actual capacitance of the suspension) was dependent on the amount of plasma present. Hence, in stationary normal (anticoagulated) whole blood samples, C_m was as high as 2.98 F · cm^–2 (s.d. ± 0.40), in contrast to about 0.9 F · cm^–2 in blood diluted more than two-fold (to less than 20% hct) in isotonic media. The high value remained when the diluent was plasma. The C_m value returned to a high value when washed erythrocytes were reconstituted with plasma, provided that this was present at above a critical or threshold concentration of about 30 vol % in the medium, irrespective of the haematocrit in the range studied (15–44%). The C_m remained low in serum. When added to washed cells in saline, purified fibrinogen had no effect. However, high C_m values were obtained by fibrinogen supplementation to serum and diluted plasma. Applying moderate flow to whole blood approximately halved its high C_m value in an exponential manner with flow rate, whilst the C_m of washed cells (31–67% hct) slightly increased, and converged to the value for whole blood under flow. We interpret the highapparent C_m value in stationary samples to be a result of rapid cell aggregation in the presence of plasma, where rouleaux formation takes place before visible sedimentation sets in.