Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris.
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We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups. (+info)
Examination of low-incidence brain tumor responses in F344 rats following chemical exposures in National Toxicology Program carcinogenicity studies.
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Neoplasms in the brain are uncommon in control Fischer 344 (F344) rats; they occur at a rate of less than 1% in 2-yr toxicity/carcinogenicity studies. Furthermore, only 10 of nearly 500 studies conducted by the National Toxicology Program (NTP) showed any evidence of chemically related neoplastic effects in the brain. Generally, the brain tumor responses were considered equivocal, because the characteristics of potential neurocarcinogenic agents (such as statistically significant increased incidences, decreased latency and/or survival, and demonstration of dose-response relationships) were not observed. A thorough examination, including comparisons with a well-established historical database, is often critical in evaluating rare brain tumors. Chemicals that gave equivocal evidence of brain tumor responses were generally associated with carcinogenicity at other sites, and many chemicals were mutagenic when incubated with metabolic activating enzymes. Other factors that were supportive of the theory that marginal increases in brain tumor incidence were related to chemical exposure were that (a) some of the tumors were malignant, (b) no brain neoplasms were observed in concurrent controls from some studies, and/or (c) brain tumors were also seen following exposure to structurally related chemicals. In 2-yr studies in F344 rats (studies conducted by the NTP), equivocal evidence of carcinogenicity was observed for the following 9 chemicals: isoprene, bromoethane, chloroethane, 3,3'-dimethylbenzidine dihydrochloride, 3,3'-dimethoxybenzidine dihydrochloride, furosemide, C.I. direct blue 15, diphenhydramine hydrochloride, and 1-H-benzotriazole. Glycidol was the only chemical evaluated by the NTP with which there was clear evidence of brain tumor induction in F344 rats. Clarification of the potential neurocarcinogenic risks of chemicals that produce equivocal evidence of a brain tumor response in conventional 2-yr rodent studies may be aided by the use of transgenic mouse models that exhibit genetic alterations that reflect those present in human brain tumors as well as by the use of in utero exposures. (+info)
The evaluation of hypersensitivity tests in cattle after foot-and-mouth disease vaccination.
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The response to passive cutaneous anaphylaxis, dermal hypersensitivity and intravenous provocation tests has been compared in 30, 40, 31 and 24 cattle injected with foot-and-mouth disease vaccine 0, 1, 2 and 3 times respectively, using vaccine components and other substances as test materials. Reaginic antibodies demonstrated by passive cutaneous anaphylaxis in goats, were directed against BHK 21 cell extracts (20), hydroxypropylmethylcellulose (3) and an unidentified vaccine component (3), and distributed in 0, 5, 19 and 75 per cent of the cattle vaccinated 0, 1, 2 and 3 times. None of the animals showed clinical signs of allergy after vaccination. When BHK 21 cell extract was injected intradermally a significant correlation was noted between the development of large weals and the presence of reagins although the size of the weals was not correlated with the reagin titres. In the case of hydroxypropylmethylcellulose a similar trend was evident. The majority of cattle with large dermal weals possessed reagins but the number of reactions was too small for statistical evaluation. Dermal reactions to sodium penicillin, sodium carboxymethylcellulose, saponin and whole vaccine occurred in both unvaccinated and vaccinated cattle but BHK 21 cell lysate and normal bovine serum provoked weals which increased in frequency according to the number of vaccinations experienced. Intravenous hydroxypropylmethylcellulose elicited a response in all the animals previously injected with certain batches of vaccine but cell extract intravenously produced a clinical response in half the tested animals which was uncorrelated with the results of the passive cutaneous anaphylaxis or dermal hypersensitivity tests. (+info)
Preparation of synthetic human islet amyloid polypeptide (IAPP) in a stable conformation to enable study of conversion to amyloid-like fibrils.
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Human synthetic islet amyloid polypeptide (hIAPP) is rapidly converted to beta-sheet conformation and fibrils in aqueous media. Optimal solubility conditions for hIAPP were determined by circular dichroism spectroscopy and transmission electron microscopy. hIAPP in trifluoroethanol or hexafluoro-2-isopropanol (HFIP) diluted in water or phosphate buffer (PB) exhibited random structure which was converted to beta-sheet and fibrils with time. hIAPP, solubilised in HFIP, filtered and lyophilised remained in stable random structure for up to 7 days in water; in PB, insoluble aggregates precipitated from which protofilaments and fibrils formed with time. This suggests that amorphous aggregates of hIAPP could initiate islet amyloidosis in vivo. (+info)
Fluorinated alcohol, the third group of cosolvents that stabilize the molten-globule state relative to a highly denatured state of cytochrome c.
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The effects of 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP) on the conformation of cytochrome c (cyt c) at pH 1.9 were studied using a combination of spectroscopic and physical methods. Analysis varying the HFIP concentration showed that a compact denatured conformation (M(HF)) accumulates in a low concentration range of HFIP in the middle of structural transition from the highly unstructured acid-denatured state to the highly helical alcohol-denatured state of cyt c. This contrasts clearly with the effect of isopropanol (IP), in which no compact conformation accompanied with the transition. Analysis varying concentrations of HFIP and NaCl concurrently showed that the M(HF) state of cyt c is essentially identical to the salt-induced molten-globule (M(G)) state, and the M(G) state in the presence of salt was also stabilized by a low concentration of HFIP. Furthermore, 2,2,2-trifluoroethanol stabilized M(HF) similarly to HFIP, supporting the proposition that the specific effect observed for HFIP is caused by fluorination of alcohol. The mechanism stabilizing compact conformation by HFIP remains unclear, but is probably distinct from that of salts and polyols, which are also known to stabilize the M(G)-like state. (+info)
Pentoxifylline attenuates bacterial lipopolysaccharide-induced enhancement of allyl alcohol hepatotoxicity.
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Small amounts of exogenous lipopolysaccharide (LPS) (10 ng/kg-100 microg/kg) enhance the hepatotoxicity of allyl alcohol in male Sprague-Dawley rats. This augmentation of allyl alcohol hepatotoxicity appears to be linked to Kupffer cell function, but the mechanism of Kupffer cell involvement is unknown. Since Kupffer cells produce tumor necrosis factor-alpha (TNF alpha) upon exposure to LPS, and this cytokine has been implicated in liver injury from large doses of LPS, we tested the hypothesis that TNF alpha contributes to LPS enhancement of allyl alcohol hepatotoxicity. Rats were treated with LPS (10-100 microg/kg iv) 2 h before allyl alcohol (30 mg/kg ip). Co-treatment with LPS and allyl alcohol caused liver injury as assessed by an increase in activity of alanine aminotransferase in plasma. Treatment with LPS caused an increase in plasma TNF alpha concentration, which was prevented by administration of either pentoxifylline (PTX) (100 mg/kg iv) or anti-TNF alpha serum (1 ml/rat iv) one h prior to LPS. Only PTX protected rats from LPS-induced enhancement of allyl alcohol hepatotoxicity; anti-TNF alpha serum had no effect. Exposure of cultured hepatocytes to LPS (1-10 microg/ml) or to TNF alpha (15-150 ng/ml) for 2 h did not increase the cytotoxicity of allyl alcohol (0.01-200 microM). These data suggest that neither LPS nor TNF alpha alone was sufficient to increase the sensitivity of isolated hepatocytes to allyl alcohol. Furthermore, hepatocytes isolated from rats treated 2 h earlier with LPS (i.e., hepatocytes which were exposed in vivo to TNF alpha and other inflammatory mediators) were no more sensitive to allyl alcohol-induced cytotoxicity than hepatocytes from naive rats. These data suggest that circulating TNF alpha is not involved in the mechanism by which LPS enhances hepatotoxicity of allyl alcohol and that the protective effect of PTX may be due to another of its biological effects. (+info)
Neonatal induction of tolerance to T(h)2-mediated autoimmunity in rats.
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Brown-Norway (BN) rats are highly susceptible to drug-induced immune dysregulations and when injected with mercuric chloride (HgCl(2)) or sodium aurothiopropanolsulfonate (ATPS), they develop a syndrome characterized by a polyclonal B cell activation depending upon CD4(+) T(h)2 cells that recognize self-MHC class II molecules. Since peripheral tolerance of T(h)2 cells might be crucial in the prevention of immunological manifestations such as allergy, establishing conditions for inducing tolerance to HgCl(2)- or ATPS-mediated immune manifestations appeared to be of large interest. We report here that BN rats neonatally injected with HgCl(2): (i) do not develop the mercury disease, (ii) remain resistant to HgCl(2)-induced autoimmunity at 8 weeks of age and later, provided they are regularly exposed to HgCl(2), (iii) are still susceptible to ATPS-induced immune manifestations, and (iv) exhibit spleen cells that adoptively transfer tolerance to HgCl(2)-induced autoimmunity in naive, slightly irradiated, syngeneic recipients. These findings demonstrate that dominant specific tolerance can be neonatally induced using a chemical otherwise responsible for T(h)2-mediated autoimmunity. (+info)
The microaerophilic flagellate Giardia intestinalis: Allium sativum (garlic) is an effective antigiardial.
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Whole garlic (Allium sativum L.) extract and some of its components were assayed for antigiardial activity. Whole garlic extract gave an IC(50) at 24 h of 0.3 mg ml(-1). Most of the components assayed were inhibitory to the organism, especially allyl alcohol and allyl mercaptan, with IC(50) values of 7 microg ml(-1) and 37 microg ml(-1) respectively. Studies with calcofluor white indicated that whole garlic and allyl alcohol collapse the transmembrane electrochemical membrane potential (Deltapsi) of the organism, as indicated by uptake of the fluorochrome. Electron microscopy allowed the morphological changes that occur with garlic inhibition to be recorded. Both the surface topography and internal architecture of the organism changed during incubation with the biocides. Both whole garlic and allyl alcohol resulted in fragmentation of the disc and an overexpression of disc microribbons, internalization of flagella, vacuole formation and an increase in distended vesicles. Allyl mercaptan, however, only gave an increase in distended vesicles, suggesting that this biocide has a different mode of action. (+info)