Experimental cerebral zygomycosis in alloxan-diabetic rabbits: variation in virulence among zygomycetes

We investigated the potential of 33 different zygomycete isolates to cause cerebral disease following the intranasal instillation of their spores into ketotic rabbits with alloxan induced diabetes. The isolates represented six thermotolerant species of Rhizopus (R. arrhizus, R. chinensis, R. microsporus, R. oligosporus, R. oryzae, and R. rhizopodiformis), Absidia corymbifera, Cunninghamella bertholletiae, and Rhizomucor pusillus. All 13 isolates of the thermotolerant Rhizopus species proved to be cerebral pathogens as confirmed by culture and histopathology. One isolate of R. oligosporus and one isolate of R. rhizopodiformis, however, were less pathogenic than isolates of other Rhizopus species tested. Cerebral pathogenicity was noted with 2 of 5 isolates of Rh. pusillus and only 1 of 13 A. corymbifera isolates. Two thermotolerant C. bertholletiae cultures, recovered from human lesions, did not cause either cerebral or pulmonary disease in ketotic rabbits. The incidence of pulmonary zygomycosis caused by the isolates of the species of the four genera under study was as follows: Rhizomucor 24%, Rhizopus 22%, Absidia 9%, and Cunninghamella 0%. This study confirms the pathogenic potential of the thermotolerant species of Rhizopus to cause cerebral zygomycosis in ketotic diabetic rabbits and also revealed the potential of Rh. pusillus and A. corymbifera occasionally to cause the same disease in animals and humans.     

Differences in thermotolerance induced by heat or sodium arsenite: cell killing and inhibition of protein synthesis

Chinese hamster ovary (CHO) cells became thermotolerant after treatment with either heat for 10 min at 45.5 degrees C or incubation in 100 microM sodium arsenite for 1 h at 37 degrees C. Thermotolerance was tested using heat treatment at 45 degrees C or 43 degrees C administered 6-12 h after the inducing agent. At 45 degrees C thermotolerance ratios at 10(-2) isosurvival levels were 4.2 and 3.8 for heat and sodium arsenite, respectively. Recovery from heat damage as measured by resumption of protein synthesis was more rapid in heat-induced thermotolerant cells than in either sodium arsenite-induced thermotolerant cells or nonthermotolerant cells. Differences in inhibition of protein synthesis between heat-induced thermotolerant cells and sodium arsenite-induced thermotolerant cells were also evident after test heating at 43 degrees C for 5 h. At this temperature heat-induced thermotolerant cells were protected immediately from inhibition of protein synthesis, whereas sodium arsenite-induced thermotolerant cells, while initially suppressed, gradually recovered within 24 h. Furthermore, adding cycloheximide during the thermotolerance development period greatly inhibited sodium arsenite-induced thermotolerance (SF less than 10(-6] but not heat-induced thermotolerance (SF = 1.7 X 10(-1] when tested with 43 degrees C for 5 h. Our results suggest that both the development of thermotolerance and the thermotolerant state for the two agents, while similar in terms of survival, differed significantly for several parameters associated with protein synthesis.     

Thermophilic and thermotolerant bacteria that assimilate methane]

Microorganisms assimilating methane at temperatures above 40 degrees C were isolated from various natural sources: ooze, mud, waste water of coal pits. The bacteria are obligate methylotrophs and are represented by two groups: (a) thermotolerant, growing at 37 to 45 degrees C; and (b) thermophilic, growing at 50 to 62 degrees C. The selective factor used to isolate various physiological forms of methylotrophs is corresponding temperatures of growth which allow to isolate from the same substrate meso-, thermotolerant, and thermophilic forms. Morphological and physiological properties of the strains are described. The thermotolerant cultures of methylotrophs are similar to Methylobacter vinelandii, though differ from it by some characteristics. The thermophilic microorganisms should be classed as a separate species Methylococcus thermophilus.     

Antigenic characterization of some potentially pathogenic mucoraceous fungi

The antigenic profiles of 10 mucoraceous fungi--Absidia corymbifera, Mortierella wolfii, Mucor miehei, M. pusillus, M. racemosus, Rhizopus arrhizus, R. microsporus, R. oryzae, R. rhizopodiformis, R. stolonifer,--Candida albicans and Aspergillus fumigatus were compared by crossed immunoelectrophoresis (XIE). Antigen-rich material was obtained from homogenized hyphae (or yeasts in the case of C. albicans), and antisera by multiple subcutaneous innoculation of rabbits with macerated but viable hyphal fragments of Ab. corymbifera, M. pusillus, R. oryzae or Asp. fumigatus. Unique and common antigens were demonstrable amongst the mucoraceous species although Mort. wolfii revealed little antigenic similarity with the others. Considerable sharing of antigens between Ab. corymbifera and M. pusillus was evident. Little or no cross reactivity was seen between extracts of C. albicans and Asp. fumigatus and the mucoraeceous antisera. R. oryzae and R. arrhizus, now regarded as synonymous, revealed close antigenic similarity. On the other hand, the distinction between both M. pusillus and M. miehei--which are regarded by some as belonging to a separate genus Rhizomucor--and less thermotolerant M. racemosus was reflected in their antigenic dissimilarity. Partial separation and characterization of antigens from the crude Absidia extract was achieved by concanavalin A-Sepharose chromatography. Antigens with and without affinity for concanavalin A could be demonstrated. Cross reactivity between Absidia antigens and M. pusillus antiserum appeared to be contained predominantly in material (possibly carbohydrate) which bound to concanavalin A and could be eluted with alpha-methyl-D-mannoside.     

Thermophilic and thermotolerant fungi in the desert soils of Saudi Arabia

Forty-eight thermophilic and thermotolerant species in addition to 5 varieties which belong to 24 genera were collected from desert soils in Saudi Arabia on glucose-(22 genera and 38 species + 5 varieties), cellulose-(15 genera and 27 species + 4 varieties) and 40% sucrose-Czapek's agar plates (13 genera and 26 species + 4 varieties) at 45 °C. The most frequent species were as follows: Aspergillus fumigatus, A. terreus, Humicola grisea var. thermoidae and Chaetomium thermophile var. copropile on glucose-; A. fumigatus, C. thermophile var. copropile, A. terreus, A. nidulans and C. thermophile var. dissitum on cellulose-; and A. fumigatus and A. terreus on 40% sucrose-Czapek's agar plates. Sixteen species and 4 varieties were particularly thermophilic and these were A. fumigatus, H. grisea var. thermoidae, H. insolens, H. lanuginosa, C. thermophile var. copropile, C. thermophile var. dissitum, C. virginicum, M. pusillus, S. thermophila, S.? pulverulentum, T. thermophilus, T.? emersoni, T. aurantiacus, T. thermophila, M. pulchella var. sulfurea, M. albomyces, ?A. terrestris, C. pruinosum, T. thermophila and P. thermophila. The remaining species showed different degrees of thermotolerant (32 species + 1 variety).     

Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae

Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.     

Ecological and physiological studies on fungi associated with human hair

Thirty-seven species attributed to 19 genera of keratinophilic fungi were recovered from 100 human hair samples collected from the Assiut governorate. The genera Aspergillus followed by Penicillium and Chrysosporium were frequently isolated from 65, 43 and 30% of the samples respectively. Fifteen species and 13 genera of thermophilic and thermotolerant fungi (recovered at 45 degrees C) were identified. The thermotolerant Aspergillus fumigatus was frequently encountered and emerged from 82% of the samples. Thirteen isolates of keratinophilic and 20 isolates of thermophilic fungi were tested for lipolytic and proteolytic activities. All the keratinophilic fungi showed lipolytic and proteolytic activities while 100 and 85% of the thermophilic fungi showed lipolytic and proteolytic activities. Using the paper-disc plate method, 12 types of shampoos and oils were tested for their antifungal activities on 42 strains of keratinophilic and thermophilic or thermotolerant fungi. Three out of four types of shampoo proved to be highly effective against all the test fungi.     

The comparative virulence of thermotolerant Mucorales species in mice

The comparative virulence of thermotolerant Mucorales was determined for cortisone-treated and untreated Swiss mice by intravenous administration of spores. The measure of virulence was based on an LD50 value, calculated after the 30-day observation period. Of the known etiological agents of mucormycosis, Mucor meihei, M. pusillus, Rhizopus arrhizus, R. chinensis, R. cohnii, R. microsporus, R. oryzae, R. rhizopodiformis and Cunninghamella elegans were able to produce fatal infections in mice; whereas, Mucor alternans, M. ramosissimus and Syncephalastrum racemosum were avirulent at dosages of up to 10(5) spores. Of those thermotolerant species which have not been reported to cause mucormycosis in human beings, Radiomyces embreei, R. spectabilis, Rhizopus oligosporus and Thermomucor indicae-seudaticae were found to produce fatal infections in mice; whereas, an isolate of Mycotypha africana was avirulent. Cortisone treatment of mice was found to lower their resistance to infection at a given spore dosage as measured by ET50 values.     

Splicing thermotolerance maintains pre-mRNA transcripts in the splicing pathway during severe heat shock

Thermotolerance, the ability of cells and organisms to withstand severe elevated temperatures after brief exposure to mild elevated temperatures, has been studied in numerous laboratories. Survival thermotolerance is defined as the increase in cell or organism survival at severe elevated temperatures after a pretreatment at mild elevated temperatures. This study examines splicing thermotolerance in Drosophila melanogaster, the ability to splice pre-mRNAs made at the severe temperature (38 degrees C) after a brief pretreatment at a milder temperature (35 degrees C). It is probably one of a number of mechanisms by which cells adapt to heat shock. These experiments demonstrate that pre-mRNAs synthesized at the severe temperatures in splicing thermotolerant cells, although protected in splicing-competent complexes, are not actually processed to mature mRNAs until the cells are returned to their normal temperature. We have also studied the kinetics of acquisition and loss of splicing thermotolerance. As little as 10 min of pretreatment at 35 degrees C was sufficient to provide full splicing thermotolerance to a 30-min severe heat shock of 38 degrees C. Pretreatments of less than 10 min provide partial splicing thermotolerance for a 30-min severe heat shock. Full splicing thermotolerance activity begins to decay about 4 h after the cessation of the 35 degrees C incubation and is completely lost by 8 h after the pretreatment. The kinetics experiments of pre-mRNAs synthesized during the 38 degrees C treatment in splicing thermotolerant cells indicate that one or more splicing thermotolerance factors are synthesized during the 35 degrees C pretreatment which interact with pre-mRNA-containing complexes to keep them in a splicing-competent state. These kinetic experiments also indicate that in cells which are partially splicing thermotolerant, the pre-mRNAs synthesized early during the 38 degrees C incubation are protected, whereas those synthesized late are not. In the absence of splicing thermotolerant factors, the pre-mRNA-containing complexes leave the normal splicing pathway and are allowed to exit to the cytoplasm.     

New developments in oxidative fermentation

Oxidative fermentations have been well established for a long time, especially in vinegar and in L-sorbose production. Recently, information on the enzyme systems involved in these oxidative fermentations has accumulated and new developments are possible based on these findings. We have recently isolated several thermotolerant acetic acid bacteria, which also seem to be useful for new developments in oxidative fermentation. Two different types of membrane-bound enzymes, quinoproteins and flavoproteins, are involved in oxidative fermentation, and sometimes work with the same substrate but produce different oxidation products. Recently, there have been new developments in two different oxidative fermentations, D-gluconate and D-sorbitol oxidations. Flavoproteins, D-gluconate dehydrogenase, and D-sorbitol dehydrogenase were isolated almost 2 decades ago, while the enzyme involved in the same oxidation reaction for D-gluconate and D-sorbitol has been recently isolated and shown to be a quinoprotein. Thus, these flavoproteins and a quinoprotein have been re-assessed for the oxidation reaction. Flavoprotein D-gluconate dehydrogenase and D-sorbitol dehydrogenase were shown to produce 2-keto- D-gluconate and D-fructose, respectively, whereas the quinoprotein was shown to produce 5-keto- D-gluconate and L-sorbose from D-gluconate and D-sorbitol, respectively. In addition to the quinoproteins described above, a new quinoprotein for quinate oxidation has been recently isolated from Gluconobacter strains. The quinate dehydrogenase is also a membrane-bound quinoprotein that produces 3-dehydroquinate. This enzyme can be useful for the production of shikimate, which is a convenient salvage synthesis system for many antibiotics, herbicides, and aromatic amino acids synthesis. In order to reduce energy costs of oxidative fermentation in industry, several thermotolerant acetic acid bacteria that can grow up to 40 degrees C have been isolated. Of such isolated strains, some thermotolerant Acetobacter species were found to be useful for vinegar fermentation at a high temperature such 38-40 degrees C, where mesophilic strains showed no growth. They oxidized higher concentrations of ethanol up to 9% without any appreciable lag time, while alcohol oxidation with mesophilic strains was delayed or became almost impossible under such conditions. Several useful Gluconobacter species of thermotolerant acetic acid bacteria are also found, especially L-erythrulose-producing strains and cyclic alcohol-oxidizing strains. Gluconobacter frateurii CHM 43 is able to rapidly oxidize meso-erythritol at 37 degrees C leading to the accumulation of L-erythrulose, which may replace dihydroxyacetone in cosmetics. G. frateuriiCHM 9 is able to oxidize cyclic alcohols to their corresponding cyclic ketones or aliphatic ketones, which are known to be useful for preparing many different physiologically active compounds such as oxidized steroids or oxidized bicyclic ketones. The enzymes involved in these meso-erythritol and cyclic alcohol oxidations have been purified and shown to be a similar type of membrane-bound quinoproteins, consisting of a high molecular weight single peptide. This is completely different from another quinoprotein, alcohol dehydrogenase of acetic acid bacteria, which consists of three subunits including hemoproteins.     

Sequential cold-sensitive mutations in Aspergillus fumigatus.

Mutants of thermotolerant fungus Aspergillus fumigatus I-21 (ATCC 32722) unable to grow at 37 degrees C were sought. Cold-sensitive mutants were enriched from progeny spores of gamma-irradiated conidia by two or more incubations at various nonpermissive temperatures alternating with filtrations through chessecloth. The approximate minimum, optimum, and maximum growth temperatures of the parent were 12, 40, and 50 degrees C, respectively. Mutants unable to grow at 37 degrees C were not successfully isolated directly from the wild type. A mutant unable to grow at 25 degrees C was isolated and mutations further increasing the cold sensitivity by increments of 3-5 degrees C were found to occur. Mutants completely unable to grow at 37 degrees C were obtained by five sequential mutations. All mutants grew as fast as the wild-type parent at 45 degrees C and higher. Each mutant produced revertants able to grow not only at the nonpermissive temperature used for its isolation but also at lower temperatures.     

Bluetongue epidemiology in the Caribbean region: serological and entomological evidence from a pilot study in Barbados

Variation in the percentage of lambs seroconverting to bluetongue viruses was seen between sites and years in Barbados. Transmission at some sites was nearly absent whereas all lambs at one site became seropositive. The agar gel immunodiffusion test for bluetongue gave consistent results in series of serum samples from 112 of 121 sentinel lambs. Collections of biting midges in association with sheep yielded six species: Culicoides insignis Lutz, C. pusillus Lutz, C. phlebotomus (Williston), C. furens (Poey), C. jamaicensis Edwards and C. trilineatus Fox. The first two species comprised 92% of those caught during a sentinel lamb study and were the predominant species trapped for virus isolation. No viruses were recovered from 5517 C. insignis, 614 C. pusillus, three C. trilineatus and two C. furens placed into pools during two brief intensive trapping operations.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.     

Kinetic studies on the action of Mucor pusillus, Mucor miehei acid proteases and chymosins A and B on a synthetic chromophoric hexapeptide

The action of two milk-clotting fungal proteases from Mucos pusillus and Mucor miehei and of chymosins A and B on the hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, and on kappa-casein were studied. The effects of pH and temperature on the initial rates of hydrolysis of the hexapeptide were examined. Crystalline chymosin and M. pusillus protease exhibited optimal activities around 49 and 55 degrees C, respectively, whereas the optimum temperature for M. miehei protease is higher than 63 degrees C. The optimum pH was about 4.7 for both fungal proteases whereas chymosin A and chymosin B exhibited optimal activities around 4.2 and 3.7, respectively. Kinetic parameters were then determined under optimal conditions and/or at pH 4.7. Fungal proteases had kcat/Km ratios that were similar to each other and that were significantly greater than the ratios obtained for the chymosins. Nevertheless, chymosins had much greater clotting activities towards kappa-casein relative to their proteolytic activities towards the synthetic peptide.     

Differences in thermotolerance induced by heat or sodium arsenite: correlation between redistribution of a 26-kDa protein and development of protein synthesis-independent thermotolerance in CHO cells

In previous studies, we have demonstrated the differences in thermotolerance induced by heat and sodium arsenite (Lee et al., Radiat. Res. 121, 295-303, 1990). In this study, we investigated whether a 26-kDa protein might play an important role in evincing these differences. Chinese hamster ovary (CHO) cells treated for either 1 h with 100 microM sodium arsenite (ARS) or 10 min at 45.5 degrees C became thermotolerant to a test heat treatment at 43 degrees C administered 6 or 12 h later, respectively. After the test heating at 43 degrees C for 1.5 h, the level of 26-kDa protein in the nucleus was decreased by 92% in nonthermotolerant cells, 78% in ARS-induced thermotolerant cells, and 3% in heat-induced thermotolerant cells. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) after ARS treatment eliminated thermotolerance to 43 degrees C and delayed restoration of the 26-kDa protein in the nucleus. In contrast, CHM neither prevented the development of thermotolerance nor inhibited the restoration of the 26-kDa protein in heat-induced thermotolerant cells. However, when cells were exposed to cold (4 degrees C), immediately after initial heating, restoration of the 26-kDa protein and development of thermotolerance did not occur. These results demonstrate a good correlation between the restoration and/or the presence of this 26-kDa protein and the development of protein synthesis-independent thermotolerance.     

Thermotolerant guard cell protoplasts of tree tobacco do not require exogenous hormones to survive in culture and are blocked from reentering the cell cycle at the G1-to-S transition

When guard cell protoplasts (GCPs) of tree tobacco [Nicotiana glauca (Graham)] are cultured at 32 degrees C with an auxin (1-napthaleneacetic acid) and a cytokinin (6-benzylaminopurine), they reenter the cell cycle, dedifferentiate, and divide. GCPs cultured similarly but at 38 degrees C and with 0.1 micro M +/- -cis,trans-abscisic acid (ABA) remain differentiated. GCPs cultured at 38 degrees C without ABA dedifferentiate partially but do not divide. Cell survival after 1 week is 70% to 80% under all of these conditions. In this study, we show that GCPs cultured for 12 to 24 h at 38 degrees C accumulate heat shock protein 70 and develop a thermotolerance that, upon transfer of cells to 32 degrees C, enhances cell survival but inhibits cell cycle reentry, dedifferentiation, and division. GCPs dedifferentiating at 32 degrees C require both 1-napthaleneacetic acid and 6-benzylaminopurine to survive, but thermotolerant GCPs cultured at 38 degrees C +/- ABA do not require either hormone for survival. Pulse-labeling experiments using 5-bromo-2-deoxyuridine indicate that culture at 38 degrees C +/- ABA prevents dedifferentiation of GCPs by blocking cell cycle reentry at G1/S. Cell cycle reentry at 32 degrees C is accompanied by loss of a 41-kD polypeptide that cross-reacts with antibodies to rat (Rattus norvegicus) extracellular signal-regulated kinase 1; thermotolerant GCPs retain this polypeptide. A number of polypeptides unique to thermotolerant cells have been uncovered by Boolean analysis of two-dimensional gels and are targets for further analysis. GCPs of tree tobacco can be isolated in sufficient numbers and with the purity required to study plant cell thermotolerance and its relationship to plant cell survival, growth, dedifferentiation, and division in vitro.     

酵母菌属间原生质体融合构建高温酵母菌株

酿酒酵母(Saccharomyces cerevisiae) A001和克鲁维酵母(Kluyveromyces sp.) Y034属间原生质体融合构建高温酵母菌株。对制备高再生活性原生质体及融合子细胞形态、生理化特征、同工酶性质、遗传稳定性和高温发酵等方面进行了研究。结果表明,融合子AY023和AY680遗传性能稳定,表达了双亲优良性状,获得了在45℃培养条件下产酒率7.4%的属间融合菌株,是目前已见文献报道的产酒率最高的高温(45℃)酵母菌株。     

Influence of thermal conditioning on the heat-induced radioresistance in primordial germ cells of the fish Oryzias latipes

Heat treatment (41 degrees C, 30 min) given before gamma-irradiation results in an increase in radiation resistance of primordial germ cells (PGCs) in Oryzias latipes at a mitotically inactive stage. This may be attributed to the appearance of a shoulder region on the dose-response curve, indicating an increased capacity to tolerate radiation damage. The radiation response curve is biphasic and the conversion of a radiosensitive population to a less sensitive one as a result of heat treatment is suggested. When the PGCs were made thermotolerant by a 'priming' heat treatment (41 degrees C, 10 min) a second heat treatment (41 degrees C, 30 min) at 2 h interval did not induce resistance to radiation. A treatment of 41 degrees C for 30 min without 'priming' gave a thermal reduction ratio of 4.6, whereas with 'priming' the ratio was 1.0. Thus heat induces radiation resistance in PGCs but this induction is suppressed under thermotolerant conditions.     

Production of saccharogenic and dextrinogenic amylases by Rhizomucor pusillus A 13.36

A newly-isolated thermophilic strain of the zygomycete fungus Rhizomucor pusillus 13.36 produced highly active dextrinogenic and saccharogenic enzymes. Cassava pulp was a good alternative substrate for amylase production. Dextrinogenic and saccharogenic amylases exhibited optimum activities at a pH of 4.0-4.5 and 5.0 respectively and at a temperature of 75 degrees C. The enzymes were highly thermostable, with no detectable loss of saccharogenic or dextrinogenic activity after 1 h and 6 h at 60 degrees C, respectively. The saccharogenic activity was inhibited by Ca(2+) while the dextrinogenic was indifferent to this ion. Both activities were inhibited by Fe(2+) and Cu(2+) Hydrolysis of soluble starch by the crude enzyme yielded 66% glucose, 19.5% maltose, 7.7% maltotriose and 6.6% oligosaccharides.