Activity and Heat Stability of Trehalase from the Mycelium and Ascospores of Neurospora

Trehalases from the ascospores of Neurospora tetrasperma and the mycelium of N. crassa were compared. Enzymes from both sources have identical electrophoretic mobilities, K(m)'s, responses to pH, immunological reactions, and activities in low-molarity buffers. Because both enzymes are so similar, conclusions about the properties of the ascospore enzyme may, be made by studying mycelial trehalase. Mycelial trehalase is most active and stable in low-molarity buffers. The enzyme exists in at least three species; the smallest has a molecular weight between 105,000 and 125,000 and is predominant in low-molarity buffers at 37 C. The stability of trehalase to heating at 65 C can be increased by increasing enzyme concentration and by the addition of polyols. Ascospores contain large amounts of trehalose, which protects trehalase from heat inactivation at 65 C. The importance of this phenomenon in vivo and its relationship to the localization of trehalase in ascospores is discussed.     

Expression of Meiotic Drive Elements Spore Killer-2 and Spore Killer-3 in Asci of Neurospora Tetrasperma

It was shown previously that when a chromosomal Spore killer factor is heterozygous in Neurospora species with eight-spored asci, the four sensitive ascospores in each ascus die and the four survivors are all killers. Sk-2K and Sk-3K are nonrecombining haplotypes that segregate with the centromere of linkage group III. No killing occurs when either one of these killers is homozygous, but each is sensitive to killing by the other in crosses of Sk-2K x Sk-3K. In the present study, Sk-2K and Sk-3K were transferred by recurrent backcrosses from the eight-spored species Neurospora crassa into Neurospora tetrasperma, a pseudohomothallic species which normally makes asci with four large spores, each heterokaryotic for mating type and for any other centromere-linked genes that are heterozygous in the cross. The action of Sk-2K and Sk-3K in N. tetrasperma is that predicted from their behavior in eight-spored species. A sensitive nucleus is protected from killing if it is enclosed in the same ascospore with a killer nucleus. Crosses of Sk-2K x Sk-2S, Sk-3K x Sk-3S, and Sk-sK x Sk-3K all produce four-spored asci that are wild type in appearance, with the ascospores heterokaryotic and viable. The Eight-spore gene E, which shows variable penetrance, was used to obtain N. tetrasperma asci in which two to eight spores are small and homokaryotic. When killer and sensitive alleles are segregating in the presence of E, only those ascospores that contain a killer allele survive. Half of the small ascospores are killed. In crosses of Sk-2K x Sk-3K (with E heterozygous), effectively all small ascospores are killed. The ability of N. tetrasperma to carry killer elements in cryptic condition suggests a possible role for Spore killers in the origin of pseudohomothallism, with adoption of the four-spored mode restoring ascospore viability of crosses in which killing would otherwise occur.     

Suppressed recombination and a pairing anomaly on the mating-type chromosome of Neurospora tetrasperma

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, Merino et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.     

Isoforms of trehalase and invertase of Fusarium oxysporum

Enzymatic assays and native PAGE were used to study trehalase and invertase activities, depending on culture age and different sugar conditions, in cell-free extracts, culture filtrates and ribosomal wash of Fusarium oxysporum. The activity of invertase preceded that of trehalase; in the exponential phase of growth, mainly invertase activity was produced, whereas trehalase activity was high in the stationary phase. In this last phase of growth, the activity of intracellular trehalase was repressed by monosaccharides, whereas disaccharides, especially lactose and starch, enhanced the activity of intracellular and extracellular trehalase. However, invertase activity was not repressed under these conditions and had the maximal activity in the presence of saccharose. Intracellular trehalase appeared in a single, high-molecular weight (120 kDa) form, whereas the extracellular enzyme appeared in a single, low-molecular weight (60 kDa) form. The activity pattern of invertase isoforms indicated the occurrence of three forms of intracellular enzyme with the main activity band at 120 kDa and two isoforms of extracellular enzyme. In the ribosomal wash, high-molecular weight isoforms of both trehalase and invertase were identified. A possible role of trehalase and invertase in carbohydrate metabolism of fungal pathogens is also discussed.     

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.     

Chromatographic and enzymatic effects on transfer factor-like activity from human leukocytes and porcine spleen dialysate

1. The effect of dialysable transfer factor (TFd), derived from human leukocytes or porcine spleen cells, was measured using Listeria resistance in mice. 2. The molecular weight range of substance(s) containing TF-like activity is in the less than 3500 MW dialysis fraction on the basis of the capacity of peritoneal macrophages to produce superoxide anion (O2-). This biological activity is removed by heating at 56 degrees C. 3. After Sephadex G-10 chromatography of dialysates the significant activities are found in fractions III and IV of human leukocyte dialysate and in fractions of II and III of porcine spleen dialysate. 4. From enzymatic studies, most of the protective activity of both human leukocyte and porcine spleen dialysate is based on the action of small-molecular weight structures containing peptides and/or polynucleotides.     

The relation between growth, conidiation and trehalase activity in Neurospora crassa

The extent to which trehalose is accumulated in the vegetative mycelium of strains of Neurospora crassa is significantly affected by conidiation. In heavily conidiating strains a rapid decrease in mycelial trehalose occurs following the initiation of conidiation. Meanwhile, trehalase activity in the vegetative mycelium of heavily conidiating strains increases rapidly following the initiation of conidiation, although apparently it is not directly caused by the sporulation process. High levels of both trehalase and trehalose appear concomitantly in the newly formed conidia.     

Conversion of furfural to furoic acid and furfuryl alcohol by Neurospora ascospores

Summary The metabolism of furfural was studied with regard to possible mechanisms by which the chemical induces germination in ascospores. Incubation of ascospores in furfural resulted in the uptake of a small percent of the furfural, and the conversion of the bulk of it to furoic acid which was in turn converted to furfuryl alcohol. Conversion also occurred in Neurospora mycelium and conidia with the order being furfural to furfuryl alcohol to furoic acid. Conversion appears to be a noninducible enzymatic process localized on the outer surface of the cell. Conversion was completely inhibited without preventing germination indicating that conversion is not involved in the breaking of dormancy in Neurospora ascospores.     

Soil fungistasis: role of the microbial nutrient sink and of fungistatic substances in two soils.

Sensitivity of conidia of Cochliobolus victoriae to fungistasis decreased markedly following incubation on moist sand for at least 1 h. Germination was greater on Conover loam or on sand being leached with water than on an alkaline clay loam soil known to produce a volatile fungistatic substance. Evolution of 14CO2 began within 3 min after [14C]glucose was applied to the soils; the rate of 14CO2 evolution was faster with Conover loam. Germination of Thielaviopsis basicola conidia per unit of glucose remaining in agar discs initially containing 0-1% glucose, was lower for discs incubated on the clay loam soil than on Conover loam, and was greatest on a bed of sand undergoing aqueous leaching. Germination of ascospores of Neurospora tetrasperma and conidia of C. victoriae was suppressed on discs of washed, Purified Agar or polyacrylamide gel incubated on or over the clay loam soil, but no suppression resulted when discs were incubated on Conover loam. Extensive aeration of either soil did not remove its fungistatic effect. Fungistasis in Conover loam appears to be caused primarily by nutrient deprivation, whereas volatile fungistatic substances may play a major role in the clay loam soil.     

Trehalase in conidia of Aspergillus oryzae

Horikoshi, Koki (The Institute of Physical and Chemical Research, Bunkyo-ku, Tokyo, Japan), and Yonosuke Ikeda. Trehalase in conidia of Aspergillus oryzae. J. Bacteriol. 91:1883-1887. 1966.-Trehalases (soluble trehalase and coat-bound trehalase) were found in the conidia of Aspergillus oryzae, and the total activity of the trehalases increased during the germination process. The soluble trehalase was purified by diethylaminoethyl-cellulose column chromatography; its optimal pH, Michaelis constant, and heat stability were studied. In vitro, the trehalases were competitively inhibited by d-mannitol, which was also contained in the conidia. Since the trehalose content in the conidia decreased at an early stage of germination, it was assumed that trehalase might begin to hydrolyze trehalose after the inhibitory effect of d-mannitol decreased.     

Antimutagenicity of propionic acid bacteria.

The antimutagenic effect of dialysed cell extracts of 4 strains of propionic acid bacteria was examined against the mutagenicity of sodium azide in the TA1535 tester strain of Salmonella typhimurium using the Ames test. It was noted that dialysates of 2 strains of Propionibacterium shermanii, P. pentosaceum and P. acnes, significantly reduced sodium azide-induced revertants. The dialysate of propionic acid cocci did not show an antimutagenic effect. The inhibitory activity was enhanced if the mutagen and extract were coincubated for 20 min prior to performing the mutagenicity assay. Antimutagenicity of dialysates from P. shermanii VKM-103 against MNNG and 9-aminoacridine was shown in S. typhimurium strains TA1535 and TA97. The antimutagenic activity was found in the protein fraction of the cell extract of P. shermanii. The proteins of the dialysate of P. shermanii were separated using a Toyopearl gel column into 3 main peaks according to their molecular weights. The antimutagenic activity towards sodium azide was found in the second and the third peaks. We suggest that dialysates of the cells of propionic acid bacteria contain several kinds of antimutagenic substances with different molecular weights.     

The infection and perennation of the bitter rot fungus, Gloeosporium album, on apple leaves

In the period from late spring to leaf-fall (May-November) Gloeosporium album Osterw. was regularly isolated from leaves of the apple variety Cox's Orange Pippin affected by the disorder called ‘Cox-spot’. The fungus grew epiphytically on healthy apple leaves, producing a network of mycelium which developed sporulating pustules when in contact with damaged or moribund tissues. Both the imperfect and perfect stages of the fungus were found on overwintered leaves; isolates from ascospores and conidia proved pathogenic on wood and fruit.     

Isolation and identification of the conidial germination factor of Neurospora crassa.

The germination-essential substance (germination factor [GF]) that is lost from conidia of Neurospora crassa on exposure to solutions of low water activity has been isolated and identified as a group of iron-transport compounds, or siderochromes. The principal siderochrome of conidia is ferricrocin, a cyclic hexapeptide. A closely related substance, ferrichrome C, is tentatively identified as a minor constituent. The same substances are also present in extracts of mycelium along with small amounts of a third siderochrome, which has not been identified. The GF activity of culture filtrates is due to coprogen, the only siderochrome previously identified with N. crassa.     

Comparison of responses of ascospores and mycelium by ELISA with anti-mycelium and anti-ascospore antisera for the development of a method to detect Sclerotinia sclerotiorum on petals of oilseed rape

The goal of this project was the development of a serological test for the detection of Sclerotinia sclerotiorum on oilseed rape petals. Since the fungus exists in two forms on petals, as ascospores and mycelium, the responses of anti-mycelium and anti-ascospore antisera against these two kinds of antigens were compared. Two anti-mycelium sera, Smy and Smy', were produced against mycelial soluble extracts at different concentrations (0.3 mg and 0.1 mg of protein ml^-1). Smy gave the greatest response level with eight S. sclerotiorum mycelial extracts tested. It had a very superior level of recognition to that of anti-ascospore serum, Ssp, when mycelium was tested as antigen. In contrast, Ssp and Smy were equally reactive when exposed to ascospores but the sensitivity of the assay was low. For each antiserum, the solution from which ascospores had been removed reacted similarly to the original suspension containing ascospores. A collection of fungi likely to be found on oilseed rape petals was examined. Cross-reactions were produced with both antisera, especially with Botrytis cinerea for which greater cross-reactivities were produced with Smy. The cross-absorption of antiserum Smy with a mycelial extract of B. cinerea considerably reduced this cross-reaction. The choice of antiserum for the development of a reliable detection system is discussed.     

Specificity of sugar cane trehalase

An extract containing trehalase and invertase was prepared from apical internodes of sugar cane. The extract hydrolysed three glucosides: maltose, trehalose and sucrose. By reprecipitation with ammonium sulphate, maltase and trehalase activities appear to be due to different enzymes. As was also shown by differential inhibition and activation and by studies on the behaviour of both enzymes during growth, invertase and trehalase activities are attributed to different enzymes whose activities do not overlap. Invertase-free preparations confirm these results. Sucrose is a simple competitive inhibitor of sugar cane trehalase, excluding a regulatory role for this sugar. Sucrose was found at inhibitory levels in the first four apical internodes. A close correlation between sugar cane growth and invertase and trehalase levels was found in the apical internodes. Invertase has the greatest activity during growing, and trehalase reaches a maximum at maturity, prior to the flowering process. The high levels of trehalase in the flower suggest that the enzyme is involved in flowering or in related processes linked to seed formation.     

Heteroallelism at the het-c locus contributes to sexual dysfunction in outcrossed strains of Neurospora tetrasperma.

Neurospora tetrasperma is naturally heterokaryotic, with cells possessing haploid nuclei of both a and A mating types. As a result, isolates are self-fertile (pseudohomothallic). Occasional homokaryotic ascospores and conidia arise, however, and they produce strains that are self-sterile and must outcross to complete sexual reproduction. Invariably, laboratory crosses employing sibling a and A strains from the same parental heterokaryon restore the pseudohomothallic, heterokaryotic state. In contrast, outcrosses employing a and A strains from different wild isolates typically result in sexual dysfunction. Diverse sexual dysfunction types have been observed, ranging from complete sterility to reduced viability. We report that one type of dysfunction, characterized by spontaneous loss of the heterokaryotic state upon ascospore germination, can result from the interaction of incompatible alleles at heterokaryon incompatibility loci. Specifically, we demonstrate that homoallelism at the het-c locus in N. tetrasperma is required for heterokaryon stability. Heterokaryon incompatibility therefore provides an obstacle to outcrossing in this species, an observation with important implications for fungal life-cycle evolution.     

篮状菌属岛篮状菌组的三个中国新记录种

根据形态学和CaM、Rpb2及rDNA ITS1-5.8S-ITS2序列的分子系统学分析,界定篮状菌属岛篮状菌组Talaromycessect.Islandici的3个中国新记录种,即螨生篮状菌T.acaricola、根篮状菌T.radicus和哒叻篮状菌T.tratensis。螨生篮状菌生长局限,形成绒状菌落,在MEA上产生稀疏的灰绿色分生孢子,菌丝淡绿黄色,分生孢子梗双轮生、三轮生和不规则生,帚状枝排列较松散,瓶梗安瓿形,孢子梭形至椭球形,壁光滑。根篮状菌生长较慢,但在37℃可生长,形成致密短絮状菌落,菌丝体绿黄色,孢子稀疏,灰绿色,分生孢子梗双轮生,帚状枝排列紧密,瓶梗圆柱形至披针形,孢子椭球形,壁光滑。哒叻篮状菌生长缓慢,在25℃培养7 d后未产分生孢子,菌丝体呈浅橙黄色,产生适量橙黄色裸囊壳,子囊孢子椭球形,壁光滑至稍粗糙。     

Evidence for three differentially regulated catalase genes in Neurospora crassa: effects of oxidative stress, heat shock, and development.

Genetic and biochemical studies demonstrated that Neurospora crassa possesses three catalases encoded by three separate structural genes. The specific activities of the three enzymes varied in response to superoxide-mediated stress, heat shock, and development. The three loci, which we designated cat-1, cat-2, and cat-3, map to the right arms of chromosomes III, VII, and III, respectively. The cat-1-encoded enzyme (designated Cat-1; estimated molecular weight, 315,000; pI 5.2) was the predominant catalase in rapid-growth mycelium, and its activity was substantially increased in paraquat-treated and heat-shocked mycelium. Cat-2 (Mw, 165,000; pI 5.4) was absent from rapid-growth mycelium but present at low levels in conidia and stationary-phase mycelium. It was the predominant catalase in extracts derived from mycelium that had been heat shocked for 2 h. Cat-3 (Mw, 340,000; pI 5.5) was the predominant catalase in extracts from mature conidia.     

Intraperitoneal insulin in uraemic diabetics undergoing continuous ambulatory peritoneal dialysis.

The kinetics of absorption of intraperitoneally administered insulin were studied in nine uraemic insulin-dependent diabetics undergoing continuous ambulatory peritoneal dialysis (CAPD). In each of three studies 20 U of regular insulin was directly injected as a bolus into the peritoneal cavity through an indwelling Tenckhoff catheter. In two procedures the insulin injection was followed by the instillation of either 2 litres of 1.5% dextrose dialysates or 2 litres of 4.5% dextrose dialysate. In the third 20 ml of saline was used to flush the tubing. Plasma free insulin values rose more rapidly and reached significantly higher concentrations (55.6 +/- 18.8 mU/l) when the insulin had been injected into an empty peritoneal cavity than when it was followed by dialysate. These differences were observed despite the fact that most of the insulin injected was retained by the patients. Since the plasma insulin values did not differ after instillations of dialysate containing 1.5% and 4.5% dextrose, the osmolality of the dialysate seemed not to affect insulin absorption, and the dilution of the insulin probably delayed its transfer through the peritoneum. These findings suggest that insulin given intraperitoneally to patients undergoing CAPD will be most effective if it is given into an empty peritoneal cavity at least 30 minutes before the dialysate is instilled.