Control of coenzyme binding to horse liver alcohol dehydrogenase.

The rate of association of NAD(+) with wild-type horse liver alcohol dehydrogenase (ADH) is maximal at pH values between pK values of about 7 and 9, and the rate of NADH association is maximal at a pH below a pK of 9. The catalytic zinc-bound water, His-51 (which interacts with the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of the coenzyme in the proton relay system), and Lys-228 (which interacts with the adenosine 3'-hydroxyl group and the pyrophosphate of the coenzyme) may be responsible for the observed pK values. In this study, the Lys228Arg, His51Gln, and Lys228Arg/His51Gln (to isolate the effect of the catalytic zinc-bound water) mutations were used to test the roles of the residues in coenzyme binding. The steady state kinetic constants at pH 8 for the His51Gln enzyme are similar to those for wild-type ADH. The Lys228Arg and Lys228Arg/His51Gln substitutions decrease the affinity for the coenzymes up to 16-fold, probably due to altered interactions with the arginine at position 228. As determined by transient kinetics, the rate constant for association of NAD(+) with the mutated enzymes no longer decreases at high pH. The pH profile for the Lys228Arg enzyme retains the pK value near 7. The His51Gln and Lys228Arg/His51Gln substitutions significantly decrease the rate constants for NAD(+) association, and the pH dependencies show that these enzymes bind NAD(+) most rapidly at a pH above pK values of 8. 0 and 9.0, respectively. It appears that the pK of 7 in the wild-type enzyme is shifted up by the H51Q substitutions, and the resulting pH dependence is due to the deprotonation of the catalytic zinc-bound water. Kinetic simulations suggest that isomerization of the enzyme-NAD(+) complex is substantially altered by the mutations. In contrast, the pH dependencies for NADH association with His51Gln, Lys228Arg, and Lys228Arg/His51Gln enzymes were the same as for wild-type ADH, suggesting that the binding of NAD(+) and the binding of NADH are controlled differently.     

Song learning with audiovisual compound stimuli in zebra finches

We investigated the effects of audiovisual compound training on song learning in zebra finches, Taeniopygia guttata. In the first experiment, presentation of a stuffed adult zebra finch male was found to be reinforcing to zebra finch males in an operant task. In a separate experiment, zebra finch males were reared without their father from day 7 after hatching onwards. Between 35 and 76 days, they were placed in isolation and exposed to taped songs of a zebra finch male, according to a random schedule (20 presentations/h). For half of the birds, presentation of the song coincided with presentation of a stuffed zebra finch male. For the remaining birds, each presentation of the song was followed by presentation of a stuffed male. The birds were subsequently isolated until day 142, when their own songs were recorded and analysed. Birds in both groups shared significantly more song elements with their tutor songs than with an unfamiliar song. There was no significant difference in song learning between the groups. These results confirm that zebra finches can learn part of their songs from taped tutor songs. Furthermore, simultaneous presentation of the tutor song and a relevant, salient visual stimulus is not superior to sequential presentation. Copyright 1999 The Association for the Study of Animal Behaviour.     

Comparison of trichloroacetic acid with other protein-precipitating agents in enriching abnormal prion protein for Western blot analysis

Detection of the abnormal or the pathogenic form of prion protein (PrP(Sc)) by Western blot (WB) is challenging, especially, for samples derived from cell cultures that contain low levels of PrP(Sc). A variety of PrP(Sc) concentration methods have been reported with various PrP(Sc) recovery efficiencies. Ultracentrifugation is one of the methods used frequently to enrich the pathogenic form of PrP(Sc) prior to WB analyses. The resulting PrP(Sc) pellet is extremely insoluble and often requires sonication to be dissolved, potentially generating aerosols. We modified the common protein-precipitating protocol using trichloroacetic acid to concentrate PrP(Sc) by slow-speed centrifugation, followed by solubilization of the pellets with 6 mol/L urea prior to sodium dodecyl sulphate -- polyacrylamide gel electrophoresis and WB analyses. Comparative studies suggest this simple trichloroacetic acid protocol was more effective in enriching PrP(Sc) presented in cell cultures and brain homogenates than other reported protein-precipitating methods. Furthermore, incorporation of the urea treatment step to dissolve the precipitated PrP(Sc) pellets helped to reduce the infectivity of PrP(Sc).     

Traditional zootherapeutic studies in India: a review

Background

The objective of the present study was to reveal patterns in the treatment of health conditions in a Quechua-speaking community in the Bolivian Andes based on plant use data from traditional healers and patient data from a primary health care (PHC) service, and to demonstrate similarities and differences between the type of illnesses treated with traditional and biomedical health care, respectively.

Methods

A secondary analysis of plant use data from semi-structured interviews with eight healers was conducted and diagnostic data was collected from 324 patients in the community PHC service. Health conditions were ranked according to: (A) the percentage of patients in the PHC service diagnosed with these conditions; and (B) the citation frequency of plant use reports to treat these conditions by healers. Healers were also queried about the payment modalities they offer to their patients.

Results

Plant use reports from healers yielded 1166 responses about 181 medicinal plant species, which are used to treat 67 different health conditions, ranging from general symptoms (e.g. fever and body pain), to more specific ailments, such as arthritis, biliary colic and pneumonia. The results show that treatment offered by traditional medicine overlaps with biomedical health care in the case of respiratory infections, wounds and bruises, fever and biliary colic/cholecystitis. Furthermore, traditional health care appears to be complementary to biomedical health care for chronic illnesses, especially arthritis, and for folk illnesses that are particularly relevant within the local cultural context. Payment from patients to healers included flexible, outcome contingent and non-monetary options.

Conclusion

Traditional medicine in the study area is adaptive because it corresponds well with local patterns of morbidity, health care needs in relation to chronic illnesses, cultural perceptions of health conditions and socio-economic aspects of health care. The quantitative analysis of plant use reports and patient data represents a novel approach to compare the contribution of traditional and biomedical health care to treatment of particular health conditions.     

Structure-based design, synthesis, and biological evaluation of 1,1-dioxoisothiazole and benzo[b]thiophene-1,1-dioxide derivatives as novel inhibitors of hepatitis C virus NS5B polymerase

A novel series of HCV NS5B polymerase inhibitors comprising 1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-dioxides were designed, synthesized, and evaluated. SAR studies guided by structure-based design led to the identification of a number of potent NS5B inhibitors with nanomolar IC₅₀ values. The most potent compound exhibited IC₅₀ less than 10 nM against the genotype 1b HCV polymerase and EC₅₀ of 70 nM against a genotype 1b replicon in cell culture. The DMPK properties of selected compounds were also evaluated.     

Autocatalytic mechanism and consequences of covalent heme attachment in the cytochrome P4504A family

The prosthetic heme group in the CYP4A family of cytochrome P450 enzymes is covalently attached to an I-helix glutamic acid residue. This glutamic acid is conserved in the CYP4 family but is absent in other P450 families. As shown here, the glutamic acid is linked, presumably via an ester bond, to a hydroxyl group on the heme 5-methyl group. Mutation of the glutamic acid to an alanine in CYP4A1, CYP4A3, and CYP4A11 suppresses covalent heme binding. In wild-type CYP4A3 68% of the heme is covalently bound to the heterologously expressed protein, but in the CYP4A3/E318D mutant, 47% of the heme is unchanged, 47% is present as noncovalently bound 5-hydroxymethylheme, and only 6% is covalently bound to the protein. In the CYP4A3/E318Q mutant, the majority of the heme is unaltered, and <2% is covalently linked. The proportion of covalently bound heme in the recombinant CYP4A proteins increases with time under turnover conditions. The catalytic activity is sensitive in some, but not all, CYP4A enzymes to the extent of covalent heme binding. Mutations of Glu(318) in CYP4A3 decrease the apparent k(cat) values for lauric acid hydroxylation. The key conclusions are that (a) covalent heme binding occurs via an ester bond to the heme 5-methyl group, (b) covalent binding of the heme is mediated by an autocatalytic process, and (c) fatty acid oxidation is sensitive in some CYP4A enzymes to the presence or absence of the heme covalent link.     

Congenital deformity of the paw in a captive tiger: case report

Background

Although Morbillivirus and Toxoplasma gondii have emerged as important pathogens for several cetaceans populations over the last 20 years, they have never been identified together in a Mysticete. In particular, morbilliviral infection has been never described in the Mediterranean fin whale population.

Case presentation

On January 2011 an adult male of fin whale (Balaenoptera physalus) stranded along the Tyrrhenian coastline of Italy. During necropsy, tissue samples from heart, skeletal muscle, mesenteric lymph nodes, liver, spleen, lung, and kidney were collected and subsequently analyzed for Morbillivirus and Toxoplasma gondii by microscopic and molecular methods. Following the detailed necropsy carried out on this whale, molecular analysis revealed, for the first time, the simultaneous presence of a Dolphin Morbillivirus (DMV) and T. gondii infection coexisting with each other, along with high organochlorine pollutant concentrations, with special reference to DDT.

Conclusion

This report, besides confirming the possibility for Mysticetes to be infected with DMV, highlights the risk of toxoplasmosis in sea water for mammals, already immunodepressed by concurrent factors as infections and environmental contaminants.