Mutagenicity of the racemic mixtures of butadiene monoepoxide and butadiene diepoxide at the Hprt locus of T-lymphocytes following inhalation exposures of female mice and rats.

The purpose of this study was to determine if Hprt mutant frequency (Mf) data from rodents exposed directly to individual epoxy metabolites of 1,3-butadiene (BD) can be used to identify the relative significance of each intermediate in the mutagenicity of BD in mice vs. rats. To this end, the relative contributions of the racemic mixtures of BD monoepoxide (BDO) and BD diepoxide (BDO(2)) to BD-induced mutagenicity was investigated by exposing mice and rats to selected concentrations of BDO and BDO(2) (i.e., 2.5 and 4.0 ppm, respectively) and comparing the mutagenic potency of each intermediate to that of BD (at 62.5 ppm) when comparable blood levels of metabolites are achieved (in the mouse). Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed to rac-BDO (0, 2.5, or 25 ppm) or (+/-)-BDO(2) (0, 2, 4 ppm) by inhalation for 4 weeks (6 h/day, 5 days/week), and then groups of control and exposed animals (n=3-12/group) were necropsied at multiple time points post-exposure for measuring Hprt Mfs in splenic lymphocytes (via the T-cell cloning assay) and estimating mutagenic potencies (represented by the difference in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals). The resulting Mf data, along with the extant metabolism data, suggest that at lower BD exposures (相似文献   

Maximum likelihood estimation of subsequence conservation

A statistical method is presented for comparing protein sequences by partitioning the polymers and estimating each subsegment's degree of conservation. Conservation is measured as a function of the number of transitions occurring in the underlying time homogeneous Markov process assumed to govern amino acid mutations. The Markovian assumption also permits estimation of the ancestral sequence. Partitioning and estimation are carried out via maximum likelihood. The method is contrasted with the commonly utilized percent homology measure. A moving likelihood ratio plot to aid in identifying regions of high conservation is suggested as an analogue to moving hydrophobicity plots. An application is presented which identifies highly conserved regions in thymidylate synthase from L. casei and E. coli.     

Substance P distribution and effects in the canine epicardial coronary arteries

Substance P (SP), a vasoactive neuropeptide detected in animal and human hearts has been reported to increase coronary blood flow in animals. However, no data are available on SP effects on epicardial coronary arteries, the site of coronary disease. To determine the amount and distribution of SP and its action in the large coronary vessels, we studied two groups of dogs. One group was anesthetized for collecting three 1 cm segments of the circumflex coronary artery (CX) and left anterior descending artery (LAD) through a left thoracotomy. These segments represented proximal (I), middle (II), and distal (III) portions of the two arteries. Concentrations (ng/g) of SP-like immunoreactivity (SP-LI) were determined by radioimmunoassay. SP-LI was present in LAD (I: 1.17 +/- 0.20, II: 1.08 +/- 0.36, III: 1.14 +/- 0.25) and CX (I: 1.44 +/- 0.38, II: 1.51 +/- 0.47, III: 0.70 +/- 0.20). SP differences among segments of LAD and segments I and II of CX were not significant, but there was a significant difference between segment III of CX and the others. In the second group of closed chest anesthetized dogs, we examined the effects of intracoronary SP infusion before and during administration of serotonin (5HT). LAD and CX artery responses (% area change) to SP and to SP plus 5HT were examined using quantitative coronary angiography. Intracoronary 133Xe in saline provided coronary flow data. SP infusion produced significant vasodilation in segment II (15% area increase) and III (17%) during the highest dose (1 microgram/min). The three SP doses infused with 5HT (0.05 mg/min) did not produce vasodilation, although LAD segment III constriction from 5HT was abolished during the highest dose of SP infusion. The presence of SP, and its dilatory effect on the coronary arteries, suggests a role in maintaining vasodilator tone in the coronary arteries.     

Application of cross-linked carboxymethyl cellulose degradation by beta-glucosidase and vaginal microbes to toxic shock syndrome.

Eleven bacterial and two yeast strains, four of which were previously identified as having activity on a lightly cross-linked carboxymethyl cellulose (CLD-2) found in one type of superabsorbent tampon, were grown on a variety of substrates, most containing cellulosics. None produced detectable amounts of cellulases, but all elaborated beta-glucosidase. None of these 13 strains nor 3 commercially obtained beta-glucosidase preparations could hydrolyze CLD-2, although a commercial cellulase and two other bacterial preparations known to produce cellulases could. Based on these results, it appears that previous work suggesting that the degradation of CLD-2 by vaginal microbes and beta-glucosidase is implicated in the production by Staphylococcus aureus of toxin causing toxic shock syndrome must be reevaluated.     

Tightly linked flanking markers for the Lowe oculocerebrorenal syndrome, with application to carrier assessment.

The Lowe oculocerebrorenal syndrome (OCRL) is characterized by congenital cataract, mental retardation, and defective renal tubular function. A map assignment of OCRL to Xq24-q26 has been made previously by linkage analysis with DXS42 at Xq24-q26 (theta = 0, z = 5.09) and with DXS10 at Xq26 (theta = 0, z = 6.45). Two additional families were studied and three additional polymorphisms were identified at DXS42 by using a 35-kb sequence isolated with the probe detecting the original polymorphism at DXS42. With additional OCRL families made informative for DXS42, theta remained 0 with z = 6.63; and for DXS10 theta = 0.03 and z = 7.07. Evidence for placing OCRL at Xq25 also comes from a female with Lowe syndrome and an X;3 translocation. We have used the Xq25 breakpoint in this patient to determine the position of OCRL relative to the two linked markers. Each derivative chromosome was isolated away from its normal counterpart in somatic cell hybrids. DXS42 was mapped to the derivative chromosome X containing Xpterq25, and DXS10 was mapped to the derivative chromosome 3 containing Xq25-qter. The markers DXS10 and DXS42 therefore show tight linkage with OCRL in six families and flank the Xq25 breakpoint in a female patient with an X;3 translocation. Linkage analysis with flanking markers was used to assess OCRL carrier status in women at risk. Results, when compared with carrier determination by ophthalmologic examination, indicated that the slit-lamp exam can be a sensitive and specific method of carrier determination in many cases.     

Lactic-acid fermentation as a low-cost means of food preservation in tropical countries

Abstract The range of traditional lactic-acid-fermented foods in tropical countries is briefly reviewed. Recent studies on the lactic acid fermentation of fish and cassava products are described. Lactic-acid-fermented fish products may offer considerable scope for the development of new food products and for the use of under-utilised fish species. Lactic-acid-fermented fish products are common in parts of Asia; methods to improve the product and shelf-life quality, to reduce microbial risks and to accelerate the process are described. This work is based on fish/salt/carbohydrate model systems. The nutritional aspects of cassava fermentation are discussed with respect to factors involved in determining residual cyanide levels; the possible anti-nutritional rôle of condensed tannins is mentioned. The increasing consumption of meat products in tropical countries emphasises the need for a preservation method that does not depend on refrigeration. The possible production of sausage ingredients preserved by lactic acid fermentation, and the associated research needs are described.     

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.     

Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle

The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p -nitroanilide (LPNA). The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N -succinyl alanine alanine phenylalanine proline p -nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio -like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella -like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N -succinyl phenylalanine p -nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.     

Characterization of recombinant polioviruses expressing regions of rotavirus VP4, hepatitis B surface antigen, and herpes simplex virus type 2 glycoprotein D.

Recombinant polioviruses expressing antigens from rotavirus, herpes simplex virus type 2, and hepatitis B virus were generated. Fusion of the heterologous polypeptides to the amino terminus of the poliovirus polyprotein did not prevent myristylation of VP0, suggesting a novel mechanism of myristylation for these recombinant viruses. The effects of the parental genetic background, different foreign sequences, and different insert sizes on growth characteristics were compared. Both the size and the nature of the heterologous sequence appeared to be factors influencing the growth and stability of recombinant polioviruses. All of the recombinants showed a temperature-sensitive phenotype, regardless of the genetic background (attenuated or wild type) from which they were derived. Preliminary studies with transgenic mice carrying the poliovirus receptor gene are discussed.