New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

Evaluation of clinical efficacy of platelet substitution in acute leukemia using resonance thrombography

For evaluating the clinical effectiveness of thrombocyte substitution, the resonance thrombogram was registered before and after thrombocyte substitution in 50 patients with acute leukemia and the increase of thrombocytes (corrected increment) determined. Thrombocyte substitution led to a significant diminution of the platelet amplitude (p less than 0.01) and platelet value. The highest value of these alterations of parameters was reached 1 hour after transfusion, it was somewhat lower after 24 hours, yet the initial value was not reached again. Correlations could be found to exist between the platelet amplitude and the corrected thrombocyte increase 1 hour and 24 hours after transfusion. The resonance thrombography enables the successful control of thrombocyte substitution to be made and it is suitable for monitoring hemostasis in severe thrombocytopenia due to production.     

Digestibility of Duddingtonia flagrans chlamydospores in ruminants: in vitro and in vivo studies

Background

Rabies is a widespread disease in African domestic dogs and a serious public health problem in developing countries. Canine rabies became established in Africa during the 20th century, coinciding with ecologic changes that favored its emergence in canids. This paper reports the results of a cross-sectional study of dog ecology in the Antananarivo urban community in Madagascar. A questionnaire survey of 1541 households was conducted in Antananarivo from October 2007 to January 2008. The study addressed both owned and unowned dogs. Various aspects of dog ecology were determined, including size of dog population, relationship between dogs and humans, rabies vaccination.

Results

Dog ownership was common, with 79.6 to 94.1% (mean 88.9%) of households in the six arrondissements owning dogs. The mean owned dog to person ratio was 1 dog per 4.5 persons and differed between arrondissements (administrative districts), with ratios of 1:6.0 in the first arrondissement, 1:3.2 persons in the 2^nd, 1:4.8 in the 3^rd, 1:5.2 in the 4^th, 1:5.6 in the 5^th and 1:4.4 in the 6^th arrondissement. Overall, there were more male dogs (61.3%) and the male/female sex ratio was estimated to be 1.52; however, mature females were more likely than males to be unowned (OR: 1.93, CI 95%; 1.39Conclusion Antananarivo has a higher density of dogs than many other urban areas in Africa. The dog population is unrestricted and inadequately vaccinated against rabies. This analysis of the dog population will enable targeted planning of rabies control efforts.     

Seed characteristics and fatty acid composition of castor (<i>Ricinus communis</i> L.) varieties in Northeast China

Oil content and fatty acid composition were investigated on 12 castor varieties and strains by using the soxhlet extraction method and capillary gas chromatography. This was made to provide a reference and theoretical basis for castorbean breeding with high oil content, determine variability of seed compounds for breeding purposes, and broaden chemical material choices. Results revealed that crude fat percentage in seeds ranged from 18.91 to 35.84% with an average of 25.91%; the absolute content of ricinoleic acid varied between 171.65 g/kg and 314.03 g/kg with an average of 222.43 g/kg, and kernel crude fat percentage was between 24.28 and 46.97% with an average of 34.30%. All these study variables were highest in the 2129 strain. The percentage of ricinoleic acid in crude fat was between 83.85 to 87.62%, and the highest value was found in the zhebi4 accession. The other fatty acids appeared in small concentrations, and showed small amplitude: 1.12 to 1.61%, 1.21 to 1.61%, 3.53 to 4.80%, 5.35 to 6.38%, 0.52 to 0.79%, 0.05 to 0.08% and 0.43 to 0.55%, for palmitic, stearic, oleic, linolic, linolenic, arachidic, and arachidonic acids, respectively. One hundred seed weight was determined for each accession. One hundred seed weight ranged from 25.7 g to 34.0 g with an average of 29.9 g. There was a significant correlation between seed weight and oil content, but the correlation value was low (r=0.51). Cluster analysis by SSPS based on the content of fatty acid composition revealed that the accessions were divided into three independent clusters. These findings will clearly provide useful information for further research in breeding and utilization of castor oil.     

An enzymatic atavist revealed in dual pathways for water activation

Inosine monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the biosynthesis of guanine nucleotides. This reaction involves two different chemical transformations, an NAD-linked redox reaction and a hydrolase reaction, that utilize mutually exclusive protein conformations with distinct catalytic residues. How did Nature construct such a complicated catalyst? Here we employ a “Wang-Landau” metadynamics algorithm in hybrid quantum mechanical/molecular mechanical (QM/MM) simulations to investigate the mechanism of the hydrolase reaction. These simulations show that the lowest energy pathway utilizes Arg418 as the base that activates water, in remarkable agreement with previous experiments. Surprisingly, the simulations also reveal a second pathway for water activation involving a proton relay from Thr321 to Glu431. The energy barrier for the Thr321 pathway is similar to the barrier observed experimentally when Arg418 is removed by mutation. The Thr321 pathway dominates at low pH when Arg418 is protonated, which predicts that the substitution of Glu431 with Gln will shift the pH-rate profile to the right. This prediction is confirmed in subsequent experiments. Phylogenetic analysis suggests that the Thr321 pathway was present in the ancestral enzyme, but was lost when the eukaryotic lineage diverged. We propose that the primordial IMPDH utilized the Thr321 pathway exclusively, and that this mechanism became obsolete when the more sophisticated catalytic machinery of the Arg418 pathway was installed. Thus, our simulations provide an unanticipated window into the evolution of a complex enzyme.     

Bayesian analysis of amino acid substitution models

Models of amino acid substitution present challenges beyond those often faced with the analysis of DNA sequences. The alignments of amino acid sequences are often small, whereas the number of parameters to be estimated is potentially large when compared with the number of free parameters for nucleotide substitution models. Most approaches to the analysis of amino acid alignments have focused on the use of fixed amino acid models in which all of the potentially free parameters are fixed to values estimated from a large number of sequences. Often, these fixed amino acid models are specific to a gene or taxonomic group (e.g. the Mtmam model, which has parameters that are specific to mammalian mitochondrial gene sequences). Although the fixed amino acid models succeed in reducing the number of free parameters to be estimated--indeed, they reduce the number of free parameters from approximately 200 to 0--it is possible that none of the currently available fixed amino acid models is appropriate for a specific alignment. Here, we present four approaches to the analysis of amino acid sequences. First, we explore the use of a general time reversible model of amino acid substitution using a Dirichlet prior probability distribution on the 190 exchangeability parameters. Second, we then explore the behaviour of prior probability distributions that are'centred' on the rates specified by the fixed amino acid model. Third, we consider a mixture of fixed amino acid models. Finally, we consider constraints on the exchangeability parameters as partitions,similar to how nucleotide substitution models are specified, and place a Dirichlet process prior model on all the possible partitioning schemes.     

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

Background

CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively.

Results

Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure.

Conclusion

Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.     

Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51

The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the k _cat/K _m with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate. Received: 7 August 1998 / Received revision: 24 November 1998 / Accepted: 29 November 1998     

Diel movement of smallmouth yellowfish Labeobarbus aeneus in the Vaal River,South Africa

Diel movements of Orange–Vaal smallmouth yellowfish Labeobarbus aeneus (Burchell, 1822) in the Vaal River, South Africa, were determined by externally attaching radio transmitters to 11 adult fish and manually tracking them between March and May 2012. Twenty-four radio telemetry monitoring surveys produced 2 304 diel tracks. At night, yellowfish displayed a preference for slow shallow (<0.3?m s?1, <0.5?m) and fast shallow habitats (>0.3?m s?1, <0.3?m), whereas by day they avoided these habitats, preferring fast deep areas (>0.3?m s?1, >0.3?m). The average total distance of 272?m moved per 24-hour period was three times greater than the diel range, and the average maximum displacement per minute was significantly higher in daytime (4?m) than at night (1.5?m). These findings suggest that L. aeneus is active primarily during the day in fast-flowing, deeper waters, and relatively inactive at night, when it occupies shallower habitats. This behaviour should be further explored to identify causal mechanisms underlying the diel habitat shifts in this species such as water temperature, foraging tactics and/or predator avoidance.