Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider

Venom of Loxosceles reclusa free from impurities was expressed from venom glands collected by microdissection. Polyacrylamide gel electrophoresis of the venom at pH 8.3 demonstrated 7 or 8 major plus 3 or 4 minor components. Upon electrophoresis at pH 4.9 two major components plus 3 or 4 minor components were noted. Monophoretic hyaluronidase prepared by Sephadex gel filtration and electrophoresis at pH 8.3 exhibited optimum activity from pH 5.0 to 6.6. Sodium dodecyl sulfate gel electrophoresis of purified hyaluronidase revealed two components with estimated molecular weights of 33,000 and 63,000. The purified hyaluronidase exhibited activity against chondroitin sulfate, types A, B, and C at approximately 20–30% of that upon hyaluronic acid. The enzyme was inhibited 10–20% by the heavy metal ions, Fe⁺³ and Cu⁺². Rabbit antivenom inhibited the spreading effect of whole venom in vivo and completely inhibited hyaluronidase in vitro.Incorporation of [¹⁴C]leucine into the spider venom led to the separation of hyaluronidase from the dermonecrotic activity of the venom.The venom demonstrated activity against carbobenzoxy-l-tyrosine-p-nitrophenyl ester and β-naphthylacetate which was inhibited approximately 65% by 2.5 × 10^?3m levels of EDTA and EGTA but not by 2.5 × 10^?4mo-phenanthroline. The esterase activity resisted concentrations of p-chloromercuribenzoate which totally inactivated papain. The venom appeared devoid of collagenase, dipeptidase, acetylcholinesterase, phosphodiesterase, ribonuclease A, and deoxyribonuclease.     

The mechanism of DNA strand breakage by vitamin C and superoxide and the protective roles of catalase and superoxide dismutase.

Vitamin C breaks DNA only in the presence of oxygen. Superoxide dismutase has no effect on the reaction but catalase suppresses it. Superoxide also gives rise to breaks in DNA suppressible by both superoxide dismutase and catalase. The hydroxyl radical seems to be the agent responsible for strand cleavage itself.     

In vivo assessment of tumor-induced nonspecific suppression of contact sensitivity: I. Correlation with in vitro studies

Fibrosarcoma-bearing BALB/c mice were assessed for 2,4-dinitroflurobenzene (DNFB)-induced contact sensitivity by a quantitative radioisotopic ear assay. Measurement of contact sensitivity was based on the localization of intraperitoneally injected iodinated-human serum albumin ([¹²⁵I]HSA) in the challenged ear. Normal and tumor-bearing mice (TBM) had optimal localization 4 days after sensitization, as determined by challenging with DNFB ear application and measuring increased vascular permeability of the challenged ear over the unchallenged ear. However, at all times TBM responsiveness to challenge was significantly lower than that of the normal population. Kinetic experiments indicated that the most dramatic decrease in TBM primary and secondary cell-mediated immune response to the contact sensitizing agent occurred 15 to 19 days post tumor transplant, flattening out to a consistently low level during the fourth and fifth week of tumor growth. Results from these in vivo experiments strongly corroborate our previous in vitro inhibition data from tumor-induced nonspecific suppressor cells.     

Systematics of the Platyrrhinus helleri species complex (Chiroptera: Phyllostomidae), with descriptions of two new species

Platyrrhinus is a diverse genus of small to large phyllostomid bats characterized by a comparatively narrow uropatagium thickly fringed with hair, a white dorsal stripe, comparatively large inner upper incisors that are convergent at the tips, and three upper and three lower molars. Eighteen species are currently recognized, the majority occurring in the Andes. Molecular, morphological, and morphometric analyses of specimens formerly identified as Platyrrhinus helleri support recognition of Platyrrhinus incarum as a separate species and reveal the presence of two species from western and northern South America that we describe herein as new ( Platyrrhinus angustirostris sp. nov. from eastern Colombia and Ecuador, north‐eastern Peru, and Venezuela and Platyrrhinus fusciventris sp. nov. from Guyana, Suriname, French Guiana, Trinidad and Tobago, northern Brazil, eastern Ecuador, and southern Venezuela). These two new species are sister taxa and, in turn, sister to Platyrrhinus incarum. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 159 , 785–812.     

Effect of phytase supplementation on phosphorus digestibility in low-phytate barley fed to finishing pigs

Forty crossbred barrows (Camborough 15 Line female×Canabred sire) weighing an average of 79.6±8.0?kg were used in a factorial design experiment (5 barleys×2 enzyme levels) conducted to determine the effects of phytase supplementation on nutrient digestibility in low-phytate barleys fed to finishing pigs. The pigs were assigned to one of 10 dietary treatments comprised of a normal 2-rowed, hulled variety of barley (CDC Fleet, 0.26% phytate) or 2 low-phytate hulled genotypes designated as LP422 (0.14% phytate) and LP635 (0.09% phytate). A normal, hulless barley (CDC Dawn, 0.26% phytate) and a hulless genotype designated as LP422H (0.14% phytate) were also included. All barleys were fed with and without phytase (Natuphos 5000 FTU/kg). The diets fed contained 98% barley, 0.5% vitamin premix, 0.5% trace mineral premix, 0.5% NaCl and 0.5% chromic oxide but no supplemental phosphorus. The marked feed was provided for a 7-day acclimatization period, followed by a 3-day faecal collection. In the absence of phytase, phosphorus digestibility increased substantially (P<0.05) as the level of phytate in the barley declined. For the hulled varieties, phosphorus digestibility increased from 12.9% for the normal barley (0.26% phytate) to 35.3 and 39.8% for the two low-phytate genotypes (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 9.2% for the normal barley (0.26% phytate) to 34.7% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In contrast, when phytase was added to the diet, there was little difference in phosphorus digestibility between pigs fed normal barley and those fed the low-phytate genotypes (significant barley×enzyme interaction, P=0.01). For the hulled varieties, phosphorus digestibility was 50.1% for the barley with the normal level of phytate (0.26% phytate) compared with 51.1 and 52.4% for the varieties with 54 and 35% of the normal level of phytate (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 47.1% for the normal barley (0.26% phytate) to 54.4% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In conclusion, both supplementation with phytase and selection for low-phytate genotypes of barley were successful in increasing the digestibility of phosphorus for pigs. Unfortunately, the effects did not appear to be additive. Whether or not swine producers will choose low-phytate barley or supplementation with phytase as a means to improve phosphorus utilization, will likely depend on the yield potential of low-phytate barley and the additional costs associated with supplementation with phytase.     

Membrane lipids and ethanol tolerance in the mouse. The influence of dietary fatty acid composition

Mice of the TO Swiss strain received diets containing different amounts of saturated or unsaturated fat throughout life. These diets produced characteristic changes in cardiac phospholipid fatty acid composition, but produced no significant differences in fatty acid composition of phospholipids from a crude membrane fraction of brain. When littermates of these animals were exposed to ethanol vapour in an inhalation chamber it was observed that mice which had received a diet high in saturated fat lost the righting reflex at an estimated concentration of ethanol in blood higher than that required for mice receiving a control diet, or a diet rich in polyunsaturated fat. Analysis of the brain membrane fraction from those animals which had received ethanol revealed that mice receiving the highly saturated fat diet now had a significantly greater proportion of saturated fatty acids in brain membrane phospholipids. These results are discussed in relation to the hypothesis that brain membrane lipid composition may influence the behavioural response to ethanol.     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.