Coordination geometry for cadmium in the catalytic zinc site of horse liver alcohol dehydrogenase: studies by PAC spectroscopy

Active site substituted Cd(II) horse liver alcohol dehydrogenase has been studied by Perturbed Angular Correlation of Gamma rays Spectroscopy during turnover conditions for benzaldehyde and 4-trans-(N,N-dimethylamino)cinnamaldehyde. The ternary complex between alcohol dehydrogenase NAD⁺ and Cl^–, and the binary complex between alcohol dehydrogenase and orthophenanthroline have also been studied. The Nuclear Quadrupole Interaction parameters have been interpreted in terms of different coordination geometries for Cd(II) in the catalytic zinc site of the enzyme. Calculation of the nuclear quadrupole interaction for cadmium in the catalytic site of the enzyme with and without coenzyme, based upon the four coordinated geometries determined from X-ray diffraction, agrees with the experimentally determined values. The ternary complexes between enzyme, NAD⁺ and either Cl^– or trifluoroethanol and the binary complex between enzyme and orthophenanthroline have almost identical spectral parameters which are not consistent with a four coordinated geometry, but are consistent with a five coordinated geometry. The nonprotein ligands for the ternary complex with trifluoroethanol are suggested to be an alkoxide group and a water molecule. The Nuclear Quadrupole Interaction parameters for the productive ternary complex between enzyme, NADH and an aldehyde is consistent with the four coordinated geometry predicted from X-ray diffraction data having the carbonyl group of the aldehyde substituting the water molecule as ligand to the metal.Abbreviations LADH Horse liver alcohol dehydrogenase - H₄Zn₂LADH derivative of LADH free of zinc in the catalytic site - ¹¹¹CdZn₂LADH derivative of LADH with ¹¹¹Cd (carrier free) in the catalytic site - Cd₂Zn₂LADH derivative of LDH with 2 mole of Cd(II) per mole LADH in the catalytic site - PAC pertubed angular correlation of gamma rays - NQI Nuclear quadrupole interaction - AOM Angular overlap model - trifluoroethanol 2,2,2-trifluoroethanol - DACA trans-4-(N,N-dimethylamino)cinnamaldehyde - NAD⁺ and NADH oxidized and reduced nicotinamide adenine dinucleotide - NADH₂ reduced 1,4,5,6-tetrahydronicotinamide adenine dinucleotide The experimental work was carried out at the Niels Bohr Institute Risø, 4000 Roskilde and Blegdamsvej 19, 2100 Copenhagen, Denmark Offprint requests to: R. Bauer     

Effect of Dietary and Injectable Vitamin E on the Antioxidant Status of Pigs

An experiment concerning 6 different vitamin E treatments was conducted with 30 young pigs. From 4 to 15 weeks of age the pigs were kept in individual pens and fed a selenium supplemented basal diet consisting mostly of propionic acid treated barley and soybean meal, and containing 4.4 mg vitamin E per kg. The treatments were periods with or without vitamin E supplement (20 mg/kg) or a vitamin E injection (200 mg). Blood samples collected during the period of investigation were examined for vitamin E and for resistance against erythrocyte lipid peroxidation (ELP) in order to evaluate the antioxidant status. Analysis of variance showed a litter effect on ELP values at all the weekly investigations and a treatment effect from two weeks after the experiment had started. Also the blood vitamin E level was litter dependent and influenced by treatment. Paired comparisons by Student’s t-test showed a delay of 1 to 2 weeks in the effect on ELP of a dietary vitamin E supplement. In contrast, both ELP and vitamin E changed very rapidly (hours) after vitamin E injections. Independently of the vitamin E treatments there was a rise in ELP within the first 2 or 3 weeks after weaning; this was taken as an index of a reduced antioxidant status during that period.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

蓖麻蚕两种凝集素在蚕体内的出现与分布

蓖麻蚕Philosamia cynthia ricni血淋巴含两种凝集素,一种凝集兔新鲜红血球,凝血活力被L-鼠李糖和D-半乳糖抑制;另一种凝集戊二醛固定的人和鸡的红血球,凝血活力被岩藻糖抑制.它们在蚕的不同生长阶段及在蚕体各组织中的分布和凝血活力显著不同.血淋巴中这两种凝集素的凝血活力明显比其他组织中高.卵中测不到这两种凝集素活力.本文对这两种凝集素在蚕体中可能的生理功能进行了讨论.     

Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex

Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolateral) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.11.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of Mr 166 000 from the latter fraction was cleaved by trypsin, proteinase K and papain, consistent with an extracellular location of the enzyme at its site of function. In contrast, both the mature form and the transient form of Mr 140 000 from the Mg2+-precipitated fraction were equally well protected from proteolytic cleavage (in the absence of Triton X-100). This indicates that the basolateral plasma membrane is unlikely to be involved in the post-Golgi transport of newly synthesized aminopeptidase N and suggests instead a direct delivery of the enzyme to the apical plasma membrane. A crude membrane preparation from labelled explants was used in immunoelectrophoretic purification of membranes to determine at what stage during intracellular transport newly synthesized microvillar enzymes are sorted, i.e., accumulated in areas of the membrane from where other proteins are excluded. The transient form of aminopeptidase N was only moderately enriched by immunopurification, using antibodies against different microvillar enzymes, but the mature form was enriched approximately 30-fold from explants, labelled for 30 min. This suggests that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.     

Selenium and Vitamin E Deficiency in Pigs

The effect of selenium (Se) and vitamin E (Vit. E) on reproductive performance, growth and health was studied in pigs. Two levels of Se were used, 0.03 and 0.06 nag per kg feed. The major component of the experimental diets was barley originating from soil which had formerly produced crops with a very low content of Se. Prior to seeding, the area was divided into 2 plots, 1 of which was treated with Se in the form of sodium selenite, 100 g Se per ha. The use of Se enriched fertilizer was an effective way of increasing the Se concentration of the grain. Thus the concentration of Se in the barley produced on the treated area was 5 times higher than in barley from the untreated one. Vit. E was added at a level of 30 i.u. per kg feed, and the concentrations were approx. 15 and 45 i.u. in the basal and experimental diets, respectively. The higher level of Se or Vit. E was not significantly associated with milk yield of the sow, litter size, birth weight or haemoglobin levels. However, there was a tendency to an increase in milk yield of the sows following additions of Se plus Vit. E, and litter size was slightly higher from sows which had received an addition of Vit. E. The concentration of Se and Vit. E was much higher in colostrum than in sow milk, and additions of dietary Se and Vit. E were associated with marked increases in the concentrations of these compounds in both colostrum and sow milk. There was a moderately improving effect of a high Se concentration in feed on growth rate and feed utilization. Low dietary levels of Se and Vit. E were followed by increased mortality rate in piglets; iron toxicity in connection with iron treatment was observed in piglets on low dietary Vit. E. Symptoms characteristic of PSE were not observed in the Se and Vit. E deficient pigs.     

A decoy set for the thermostable subdomain from chicken villin headpiece,comparison of different free energy estimators

Background  

Estimators of free energies are routinely used to judge the quality of protein structural models. As these estimators still present inaccuracies, they are frequently evaluated by discriminating native or native-like conformations from large ensembles of so-called decoy structures.     

The influence of temperature and relative humidity on the development of Lepidoglyphus destructor (Acari: Glycyphagidae) and its production of allergens: a laboratory experiment

Laboratory experiments with Lepidoglyphus destructor on a diet of mainly whole wheat were conducted to study the mite's development and production of a specific allergen, Lep d 2, at four different temperatures (5, 10, 15 and 20 degrees C) and three levels of relative humidity (ca. 70-88%). Statistical models were used to analyse the role played by temperature, relative humidity and time in explaining the observed number of L. destructor and the amount of allergen produced. Moreover, the life stage distributions of the mites were determined and related to the population growth. Based on a statistical model the intrinsic rate of natural increase, rm, was computed for a range of different temperatures and relative humidities. High relative humidity in combination with temperatures at about 25 degrees C will lead to the highest rm (ca. 0.15 day-1). The highest concentration of Lep d 2 was 3 micrograms g-1 grain, found at 20 degrees C and high relative humidity at a mite density of 254 mites g-1 grain. The concentration of allergens in the grain was best explained by a model that incorporated both the current and the cumulative numbers of mites.