Crystallization and preliminary X-ray crystallographic studies of UDP-N-acetylenolpyruvylglucosamine reductase.

The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP-N-acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X-ray crystallographic studies of the enzyme. MurB crystallizes in 14-20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP-N-acetylglucosamine enolpyruvate.

The glutamyl binding site of trypanothione reductase from Crithidia fasciculata: enzyme kinetic properties of gamma-glutamyl-modified substrate analogues.

Trypanothione reductase, central to the redox defense systems of parasitic trypanosomes and leishmanias, is sufficiently different in its substrate-specificity from mammalian glutathione reductase to represent an attractive target for chemotherapeutic intervention. Previous studies of the physiological substrates trypanothione (N1,N8-bis(glutathionyl)spermidine) and N1-glutathionylspermidine disulphide established that the spermidine moiety of these substrates can be replaced by the 3-dimethyl-propylamide group (N1-glutathionyl-N3-dimethyl-propylamide). With this modification, the specificity for the gamma-glutamyl moiety of the substrate was examined.

Enhanced antiopiate activity and enzyme resistance in peptidomimetics of FMRFamide containing (E)-2,3-methanomethionine.

FMRFamide is a molluscan peptide that has shown antiopiate activity in a number of mammalian test systems. The current study determined the antiopiate potency of FMRFamide and two conformationally constrained peptidomimetics of FMRFamide containing stereoisomers of (E)-2,3-methanomethionine. Morphine abstinence signs were observed after varying doses (0.

Tyr-MIF-1 precipitates abstinence syndrome in morphine-dependent rats.

The endogenous neuropeptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-amide) has been shown to counteract various opiate actions. In the current study, third ventricular injection of 35 micrograms Tyr-MIF-1 protected by the enzyme inhibitor bestatin precipitated an abstinence syndrome in morphine-dependent rats, but not in opiate-naive rats. The results raise the possibility that Tyr-MIF-1 might participate in opiate dependence and opiate abstinence syndrome.

Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase.

The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.

Synthesis of substrate analogues for trypanothione reductase.

The synthesis and chemical characterisation of a range of substrate analogues for trypanothione reductase are described, with the spermidine portion of trypanothione replaced by the 3-dimethylaminopropylamide moiety. Using 1-hydroxybenzotriazole/N-hydroxysuccinimide coupling, products were obtained which had a range of replacements of the gamma-glutamyl groups of the enzyme substrate. The materials were characterised by FPLC, 1H/13C NMR spectroscopy and FAB mass spectroscopy.

Enhanced antiopiate activity in peptidomimetics of FMRFamide containing Z-2,3-methanomethionine.

FMRFa is a molluscan peptide that has shown antiopiate activity in a number of mammalian test systems. The current study determined the antiopiate potency of FMRFa and two conformationally constrained peptidomimetics of FMRFa containing stereoisomers of Z-2,3-methanomethionine. Morphine abstinence signs were observed after varying doses (0.

Development of standard healthcare EDI messages.

The development of standard healthcare messages requires a full understanding of user needs, an appropriate organisation structure for developing consensus and a suitable methodology. This paper presents the current state of development in the European Standardisation Organisation's Working Group on Healthcare Messages and Communications (CEN TC251 WG3).

Investigation of syntaxes for existing interchange formats to be used in healthcare.

This paper gives a summary of the work done by CEN TC 251 PT 004. The project team objective has been to investigate syntaxes of existing interchange formats (IFs) to be used in healthcare. A set of evaluation criteria have been developed based on functional requirements from two healthcare domains.

Heavy metal and selenium levels in young cattle egrets from nesting colonies in the northeastern United States, Puerto Rico, and Egypt.

Colonially-nesting species of herons and egrets breed mainly in coastal areas, along rivers or near other large bodies of water. Such areas are also preferred for human development, exposing nesting birds to various pollutants. From 1989-1991, the concentrations of heavy metals and selenium were studied in the feathers of fledgling cattle egrets Bubulcus ibis, a terrestrially-feeding insectivore, from New York and Delaware in the northeastern United States, from Puerto Rico, and from Egypt.

Rationally designed selective inhibitors of trypanothione reductase. Phenothiazines and related tricyclics as lead structures.

Trypanothione reductase, an essential component of the anti-oxidant defences of parasitic trypanosomes and Leishmania, differs markedly from the equivalent host enzyme, glutathione reductase, in the binding site for the disulphide substrate. Molecular modelling of this region suggested that certain tricyclic compounds might bind selectively to trypanothione reductase without inhibiting host glutathione reductase. This was confirmed by testing 30 phenothiazine and tricyclic antidepressants, of which clomipramine was found to be the most potent, with a K(i) of 6 microM, competitive with respect to trypanothione.

Transneuronal labeling of cochlear nucleus neurons by HRP-labeled auditory nerve fibers and olivocochlear branches in mice.

Auditory nerve fibers were labeled by extracellular injections of horseradish peroxidase into the spiral ganglion in mice. The labeled fibers were traced in an anterograde direction through the auditory nerve into the cochlear nucleus. In almost half of the injections, the labeled endings of auditory nerve fibers contacted cochlear nucleus neurons that were also labeled with horseradish peroxidase and were presumably transneuronally labeled.

Unmyelinated axons of the auditory nerve in cats.

This paper describes some central terminations of type II spiral ganglion neurons as labeled by extracellular injections of horseradish peroxidase (HRP) into the auditory nerve of cats. After histological processing with diaminobenzidine, both thick (2-4 microns) and thin (0.5 microns) fibers of the auditory nerve were stained.

Synapses formed by olivocochlear axon branches in the mouse cochlear nucleus.

Cochlear nucleus branches of thick olivocochlear axons were labeled by injections of horseradish peroxidase into the spiral ganglion of the cochlear basal turn in mice. Six labeled axons were traced by light microscopy, and selected portions of seven branches were sectioned serially for electron microscopic examination. Axonal branches most frequently terminated near certain granule cell regions of the ventral cochlear nucleus.

Brainstem branches from olivocochlear axons in cats and rodents.

Horseradish peroxidase was used to label axons of olivocochlear (OC) neurons by intracellular injections in cats and extracellular injections in rodents. These axons arise from cell bodies in the superior olivary complex and project to the cochlea. En route to the cochlea, the thick axons (greater than 0.

Projection of septal organ receptor neurons to the main olfactory bulb in rats.

The septal organ of Masera is a distinct patch of olfactory epithelium on the septal wall of the nasal cavity. It lies ventral to the main olfactory receptor sheet. Central projections of the septal organ to the main and/or accessory olfactory bulb were studied with punctate HRP injections into the posterior dorsomedial olfactory bulb of rat pups.

Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis.

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars.

High-resolution 2-deoxyglucose autoradiography in quick-frozen slabs of neonatal rat olfactory bulb.

We have used rapid freezing and freeze-substitution fixation to permit electron microscopic study of [3H]2-deoxyglucose autoradiographs. The techniques minimize diffusion of label into processing fluids and, by inference, migration of label within tissue. Slabs of olfactory bulbs from 12-day-old rats were quick-frozen after one hour of exposure to physiological olfactory stimuli.

Specific olfactory receptor populations projecting to identified glomeruli in the rat olfactory bulb.

A critical gap exists in our knowledge of the topographical relationship between the olfactory epithelium and olfactory bulb. The present report describes the application to this problem of a method involving horseradish peroxidase conjugated to wheat germ agglutinin. This material was iontophoretically delivered to circumscribed glomeruli in the olfactory bulb and the characteristics and distribution of retrogradely labeled receptor cells were assessed.

Effects of sensory deprivation on the developing mouse olfactory system: a light and electron microscopic, morphometric analysis.

Closure of the nostril by electrocauterization on postnatal day (PN) 1 or 2 was used to study effects of olfactory deprivation on developing olfactory epithelium (OE) and bulb (OB) in CD-1 mice. No damage was observed in OE sections 1 or 3 days after closure, and at PN 30 no difference was found in the number of OE receptors between closed and open sides. Odor deprivation and a decrease in functional activity in experimental bulbs was evident from deoxyglucose autoradiographs at PN 21 and PN 30.

Critical re-appraisal of fluorometric method for determination of selenium in biological materials.

The reaction of 2,3-diaminonaphthalene (DAN) with Se(IV) to form a fluorescent Se-DAN complex is the basis of a fluorometric method for determination of Se. With the aid of metabolically incorporated 75Se the method was critically re-examined. The study showed that loss of 75Se was negligible when liver or blood was microwave-dried or thermally dried at temperatures up to 120 degrees C; during HCl reduction of Se(VI) to Se(IV), a temperature up to 210 degrees C could be used with no loss of 75Se; it was unnecessary to perform pH adjustment of solution for formation of Se-DAN complex before solvent extraction; it was unnecessary to carry out the chelation/extraction step in diffuse light; solvent phase containing Se-DAN complex could be left in contact with aqueous phase up to one week under fluorescent light with no effect on analytical results.