Microbiological evaluation of the intraruminal in sacculus digestion technique.

The influence of nature of the feed sample, feeding frequency and pore size on the influx of bacteria and protozoa into synthetic fiber bags suspended in the rumens of sheep fed different diets was studied. Counts of total culturable bacteria in bags with a pore size of 10 microns were less than 30% of the ruminal counts for animals that were fed the lucerne hay and high-roughage diets. The maximum count (62 and 82% of the ruminal count) for these specific diets was obtained by using bags with a pore size of 53 microns. Protozoal counts in bags with pore sizes of 30 and 53 microns were equal to or higher than the ruminal counts for the lucerne hay and high-roughage diets but less than half of the ruminal count for the low-roughage diet. An interaction between incubation time, feeding frequency of the host animals, and the microbial populations developing inside the bags was also demonstrated. The results clearly show that the microbial population inside the bag differed from that of the surrounding ruminal ingesta and that caution must be taken in interpreting results on feed evaluation and especially on rates of degradation when using the in sacculus technique. Factors influencing the influx of bacteria and protozoa into bags with different pore sizes and containing a variety of substrates are discussed together with suggestions for the use of this technique.     

Dihydropyridine calcium antagonists depress the amplitude of the plasma melatonin cycle in baboons

An investigation into the effects of verapamil and some dihydropyridine derivatives on plasma melatonin levels was undertaken in baboons. In a number of separate experiments, groups of young male chacma baboons (mean body weight 13 kg) received intraperitoneal injections of the drugs, under ketamine anaesthesia, roughly 30 minutes prior to the following time points: 1200, 1800, 0000, 0200, 0600 and 1200 h. Lights went off at 1800 h and came on at 0600 h. The drugs used, and their respective dosages (expressed per kg body mass), were verapamil up to 4 mg/kg, nifedipine at 0.2 mg/kg, nitrendipine at 0.5 mg/kg and nisoldipine at 0.1 mg/kg. Blood samples, taken at the said time points, were assayed for melatonin. The nighttime peak of the plasma melatonin cycle was significantly depressed by all three dihydropyridine calcium antagonists (up to 40%), while verapamil, even at the relatively high total dose of 24 mg/kg per day, had no significant effect on the circulating plasma melatonin levels.     

Serological properties and processing in Escherichia coli K12 of OmpV fusion proteins of Vibrio cholerae

Summary Fusion proteins comprising the amino-terminal 99 amino acids of the bacteriophage MS2 replicase and various portions of OmpV a major outer membrane protein of Vibrio cholerae were expressed in Escherichia coli K12. These fusions were expressed under the control of the P_L promoter of bacteriophage , and expression was controlled using a cI_ts repressor. Fusions occurring within the secretory signal sequence of OmpV gave rise to the production of mature OmpV. The efficiency, however, decreased with progressive deletion of the signal sequence within the fusions. The reactivity of various OmpV fusions with antisera raised against purified OmpV and whole bacteria demonstrated the existence of two antigenic domains: one present in the denatured form and another in the membrane-associated form of OmpV. These domains correspond to markedly hydrophilic regions of the protein as would be predicted for surface-exposed epitopes.     

Proton nuclear magnetic resonance spectroscopy of horseradish peroxidase isoenzymes: correlation of distinctive spectra with isoenzyme specific activities

High-resolution proton NMR spectra are reported for the paramagnetic ferric native and cyano complexes of the five major horseradish root peroxidase (HRP) isoenzymes (A1, A2, A3, B, and C). Axial imidazole resonances are observed in the native and cyano-complex spectra of all the isoenzymes, thus indicating the presence of a common axial histidine ligand. Proton NMR spectra outside the usual diamagnetic region are identical for sets of A1 and A2 isoenzymes and for the B and C isoenzyme set. Variation in heme residue chemical shift positions may be controlled in part by porphyrin vinyl side chain-protein interactions. Diverse upfield spectra among the isoenzymes reflect amino acid substitutions and/or conformational differences near the prosthetic group, as signals in this region must result from amino acid residues in proximity to the heme center. Acid-base dependence studies reveal an "alkaline" transition that converts the native high-spin iron (III) porphyrin to the low-spin state. The transition occurs at pH 9.3, 9.4, 9.8, and 10.9 for respective HRP A1, A2, A3, and C isoenzymes, respectively. Significantly, this ordering also reflects specific activities for the isoenzymes in the order A1 = A2 greater than A3 greater than B = C. Identical proton NMR spectra for A1/A2 and B/C isoenzyme sets parallel equivalent specific activities for members of a particular set. Proton NMR spectra thus appear to be highly sensitive to protein modifications that affect catalytic activity.     

Flavocytochromes c: transient kinetics of photoreduction by flavin analogues

Kinetics of reduction of phototrophic bacterial flavocytochromes c by exogenous flavin semiquinones and fully reduced flavins generated by laser flash photolysis have been studied. The mechanisms of reduction of Chromatium and Chlorobium flavocytochromes c are more similar to one another than previously thought. Neither protein is very reactive with neutral flavin semiquinones (k less than 10(7) M-1 s-1), and the reactions with fully reduced flavins are slower than expected on the basis of comparison with other electron-transfer proteins of similar redox potentials. Deazaflavin radical is reactive with the flavocytochromes c by virtue of its low redox potential, but this reaction is also slower than expected on the basis of comparison with other electron-transfer proteins. These experiments indicate that the active site for reduction of flavocytochrome c is relatively buried and probably inaccessible to solvent. Fully reduced FMN does not show an ionic strength effect in its reaction with flavocytochrome c, which demonstrates that the active site is uncharged. Sulfite, which forms an adduct with protein-bound FAD, partially blocks heme reduction. This shows that heme is reduced via the FAD. The rate constant for intramolecular electron transfer between FAD and heme must be on the order of 10(4) s-1 or larger.     

Isolation and characterization of soluble cytochromes, ferredoxins and other chromophoric proteins from the halophilic phototrophic bacterium Ectothiorhodospira halophila

A cytochrome c-551 and a pair of 'high redox-potential' ferredoxins (iso-high-potential iron-sulfur proteins) were found to be the major soluble electron-transport proteins in Ectothiorhodospira halophila. Smaller amounts of 'bacterial' ferredoxin and cytochrome c' were also observed. With the exception of cytochrome c-551, these proteins are commonly encountered in the purple sulfur bacteria, family Chromatiaceae and less frequently in the purple bacteria, family Rhodospirillaceae. In addition to the cytochromes and ferredoxins, E. halophila synthesizes substantial amounts of a small yellow-colored protein, which has a chromophore spectrally similar to flavins having oxygen, nitrogen or sulfur substituents in place of the 8-methyl group such as roseoflavin and the methanogen cofactor F-420. A purple-colored protein was only partially purified, but it is spectrally similar to iron proteins having a tyrosine ligand, such as transferrin, catechuate dioxygenase, and especially the purple acid phosphatases. Neither the yellow protein nor the purple one has previously been observed in phototrophic bacteria, but may in some way be required for survival in extremely halophilic habitats. The only feature common to halophiles including E. halophila is the very acidic nature of their proteins.     

Soluble cytochrome composition of the purple phototrophic bacterium, Rhodopseudomonas sphaeroides ATCC 17023

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c2 and c' have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c' found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c' and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c1 and chloroplast cytochrome f.     

Quantitative ultrastructural characteristics relating to transport between luteal cell cytoplasm and blood in the corpus luteum of the pregnant rat

The synthesis and secretion of progesterone by the corpus luteum (CL) may be limited or controlled by transport mechanisms operating between circulating blood and luteal cell cytoplasm. To examine this possibility, the structural features involved in transport, including membrane surface areas and diffusion distances, were quantitated in the CL of 16-day pregnant rats. One ovary from each of eight rats was fixed by perfusion via a cannula inserted into the parametrial artery, and two CL from each ovary were processed for electron microscopy and examined with standard morphometric techniques. For comparison, one CL from each of a further eight ovaries was diced into small cubes, fixed by immersion, and analyzed similarly. In perfusion-fixed CL, there was a substantial volume of vascular space (20% of the total) and interstitial space (5%) and an extensive surface area of capillaries (441 mm2 per CL). The luteal-cell membrane had numerous projections which increased its surface area by a factor of 3.08. Almost 60% of the luteal-cell surface directly faced a capillary, and a further 37% faced interstitial space which probably extended to a capillary surface. Only 3% was in direct contact with a neighboring luteal cell. Despite the extensive interstitial space the harmonic mean thickness, an estimate of likely effective diffusion distance between luteal cell cytoplasm and blood, was only 0.42 micron. This was less than half of the calculated arithmetic mean thickness owing to the presence of surface projections and an uneven capillary endothelium. Results from immersion-fixed CL were qualitatively similar; but the proportion of interstitial space was only 59% of that in perfusion-fixed CL, and the contribution of surface projections to the total area of luteal-cell membranes was significantly reduced. Collectively, these results suggest that membranes and spaces between blood and luteal-cell cytoplasm are structured so as to minimize transport distances.     

Associations between marker genotypes, halothane reaction, creatine kinase activity and meat quality characters in a sample of German Landrace pigs

Routine blood typing of German Landrace pedigree populations and an earlier study revealed very low frequencies of the favourable alleles at the marker loci Phi, Pgd and H. The hypothesis was that in this population the whole linkage group of favourable alleles at the halothane and neighbouring marker loci may have been lost as a consequence of intense selection for leanness and type. The present study of 1050 German Landrace pigs at the Relliehausen experimental station, where some effort has been made to maintain a higher frequency of the favourable alleles PhiA (0.48), H- (0.43) and PgdA (0.70) gave quite different results. The frequency of halothane-positive pigs found by using a severe test was only 30%. Only 5.4%, 8.8%, 13.4% and 13.9% of animals with PhiA/A, H-/-, PgdA/A and PhiA/B genotypes respecitively were halothane-positive. Forty to sixty per cent of pigs with these marker genotypes could therefore be expected to be homozygous halothane-negative (N/N) animals. Creatine kinase activity and three selected meat quality characters showed highly significant differences between the A/A and the B/B genotypes for the marker loci Phi and Pgd, with the heterozygotes being intermediate. These differences are greater than those observed between halothane-negative and halothane-positive phenotypes. The only other consistently superior marker genotype in this population was the H blood group genotype H-/-. In contrast to findings from Sweden and Switzerland, the postalbumin locus Po2 and the suppressor locus S for the A-O blood groups did not exhibit useful marker qualities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assay of mephenytoin metabolism in human liver microsomes by high-performance liquid chromatography

The metabolism of mephenytoin to its two major metabolites, 4-OH-mephenytoin (4-OH-M) and 5-phenyl-5-ethylhydantoin (nirvanol) was studied in human liver microsomes by a reversed phase HPLC assay. Because of preferential hydroxylation of S-mephenytoin in vivo, microsomes (5-300 micrograms protein) were incubated separately with S- and R-mephenytoin. After addition of phenobarbital as internal standard, the incubation mixture was extracted with dichloromethane. The residue remaining after evaporation was dissolved in water and injected on a 60 X 4.6-mm reversed-phase column (5 mu-C-18). Elution with acetonitrile/methanol/sodium perchlorate (20 mM, pH 2.5) led to almost baseline separation of mephenytoin, metabolites, and phenobarbital. Quantitation was performed by uv-absorption at 204 nm by the internal standard method. Propylene glycol was found to be the best solvent for mephenytoin, but inhibited the reaction noncompetitively. 4-OH-M and nirvanol could be detected at concentrations in the incubation mixture as low as 40 and 80 nM, respectively. The rates of metabolite formation were linear with time and protein concentration. The reaction was found to be substrate stereoselective. At substrate concentrations below 0.5 mM S-mephenytoin was preferentially hydroxylated to 4-OH-M, while R-mephenytoin was preferentially demethylated to nirvanol at all substrate concentrations tested (25-1600 microM). These data provide a mechanistic explanation for the stereospecific pharmacokinetics in vivo. The dependence of both metabolic relations on NADPH and the inhibition by CO suggest that they are mediated by cytochrome P-450-type monooxygenases.(ABSTRACT TRUNCATED AT 250 WORDS)