In vitro detachment of bacteria from ruminal digesta by buffered sodium oleate solutions

The effectiveness of a buffered sodium oleate solution was evaluated for detaching bacteria from ruminal digesta samples. A response surface derived from an octagonal design was used to determine the pH and concentration combination for maximum detachment of total and cellulolytic bacteria. The total number of bacteria detached increased up to 81% over control with treatment of a pH 8.8 and 1.5% sodium oleate solution. The recovery of cellulolytic bacteria was decreased to 35% of control with treatment of a pH 9.0 and 0.1% sodium oleate solution. Attempts to improve the recovery of viable bacteria exposed to sodium oleate solutions were unsuccessful. This response surface design identified an optimal pH and concentration that were consistent with existing information regarding detachment of total bacteria, and suggested that sodium oleate, at the concentrations tested, was toxic to the cellulolytic population of the rumen.     

Tetrahydropapaveroline and salsolinol alter45Ca2+ efflux within perfused hippocampus of unrestrained rats

The kinetics of⁴⁵Ca²⁺ efflux were examined at circumscribed sites in the perfused hippocampus of the freely moving rat with either one of two tetrahydroisoquinoline (TIQ) products, tetrahydropapaveroline (THP) or salsolinol. Guide tubes for unilateral push-pull perfusion were implanted stereotaxically to rest just above sites within the dorsal hippocampus. Upon recovery from surgery, a tissue site in the hippocampus was prelabeled with 1.0 l of⁴⁵Ca²⁺ (2.0 Ci) injected through the indwelling guide tube. After 16–20 hr had elapsed, successive push-pull perfusions of the site were carried out with an artificial cerebrospinal fluid (CSF), at 5.0 min intervals and at a rate of 20 l/min, in order to obtain a control washout curve of declining radioactivity. On the fifth of a series of 5.0 min perfusions, either THP or salsolinol was added to the perfusion medium in a concentration of 10 or 100 ng/l. Then the hippocampal site was perfused again with control CSF for the collection of an additional three samples. Although THP in both of the test concentrations generally augmented the efflux of⁴⁵Ca²⁺, the temporal course and magnitude of the enhancement depended on the anatomical site of the perfusion. In the more rostral hippocampal planes of AP 3.0 and AP 4.0, THP caused a delayed efflux of the cation, after the perfusion of THP had been discontinued, in nearly half of the loci reactive to the TIQ. Similarly, salsolinol enhanced significantly the efflux of⁴⁵Ca²⁺ in a concentration-dependent manner during the interval of its perfusion within the hippocampal plane of AP 3.0. These results suggest that both THP and salsolinol can affect differentially the kinetics of⁴⁵Ca²⁺ efflux and that these differences are contingent on the circumscribed anatomical site of push-pull perfusion. It is envisaged that a part of the neurochemical effects of the two TIQs when acting centrally are mediated by membrane mechanisms involving Ca²⁺ transport, metabolism or other cellular activity of the cation.     

Sequence comparison with concave weighting functions

We consider efficient methods for computing a difference metric between two sequences of symbols, where the cost of an operation to insert or delete a block of symbols is a concave function of the block's length. Alternatively, sequences can be optimally aligned when gap penalties are a concave function of the gap length. Two algorithms based on the ‘candidate list paradigm’ first used by Waterman (1984) are presented. The first computes significantly more parsimonious candidate lists than Waterman's method. The second method refines the first to the point of guaranteeingO(N ² lgN) worst-case time complexity, and under certain conditionsO(N ²). Experimental data show how various properties of the comparison problem affect the methods' relative performance. A number of extensions are discussed, among them a technique for constructing optimal alignments inO(N) space in expectation. This variation gives a practical method for comparing long amino sequences on a small computer. This work was supported in part by NSF Grant DCR-8511455.     

Class II genes of miniature swine

Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLA ^c haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique genes were obtained which showed no evidence of cross-hybridization, while genes showed extensive cross-hybridization and were frequently detected in the library by more than one human gene probe. These data are consistent with early evolutionary divergence of a genes, prior to mammalian speciation, and with continuing evolution of genes, with possible shared usage of these genes by different a loci. The data also imply that genes can readily be assigned to loci homologous to their human counterparts, but that genes will require further mapping and/or sequence analysis to confirm assignments.     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.