Isoenzyme analysis of hyperamylasaemia associated with ruptured abdominal aortic aneurysms

Hyperamylasaemia may occur following abdominal aortic aneurysm rupture and its use as a prognostic indicator has been suggested. However, the isoenzyme responsible for the rise in serum amylase has not been investigated. In this study, isoenzyme analysis was performed on the serum of patients noted to have a raised amylase from their routine biochemistry samples. Individual cases were then reviewed regarding clinical course and outcome. The pancreas has been thought to be the predominant source of the observed hyperamylasaemia. However, in this study a mixed picture of pancreatic and salivary isoenzymes was found. Of the four highest recorded amylase levels two were salivary in origin, one pancreatic and one mixed. The highest recorded amylase level was of salivary origin in a patient that survived without any major complication. The four patients that died all showed evidence of gut infarction/ischaemia. Two had hyperamylasaemia of a mixed pattern, one pancreatic and one of salivary origin.     

Isoenzyme profile of Paracoccidioides brasiliensis

Isoenzyme profiles of 10 strains of Paracoccidioides brasiliensis from different origins (nine strains from patients with different clinical forms of paracoccidioidomycosis and one from the faeces of a penguin) were determined by polyacrylamide gel electrophoresis using 37 different enzymes. Differences in carbonate dehydratase, phosphoglucomutase, phosphoglucoseisomerase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase and beta-esterase were detected among the isolates studied allowing the characterization of nine zymodemes. Two isolates showed identical profiles. There was no correlation between the zymodeme patterns and virulence, clinical forms of the disease nor age of the cultures.     

Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus

The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5.     

Genetic analysis of two cellular degenerations in filamentous fungus Podospora anserina

The filamentous fungus Podopsora anserina presents an unavoidable arrest of vegetative growth (Senescence) determined by a cytoplasmic and infectious factor. Senescence is correlated with a disorganization of the mitochondrial DNA. This disorganization is caused by an event which is not the appearance of the first defective DNA molecules. These ones are generated constitutively and their accumulation during Senescence requires the presence of an additional factor. Life span of the strains is under nuclear and cytoplasmic genetic control. At least 600 nuclear genes influence longevity. Our analysis focuses on the role of the genes involved in cytosolic translation, since mutations in these genes seem to display the most drastic effects on longevity but also on the structure of the defective mitochondrial DNA molecules that accumulate during Senescence. We have detected in some Podospora anserina mutant strains (permissive strains) the presence of a novel cytoplasmic and infectious determinant that entails an easily discernible phenotype associated with a severe growth alteration (Crippled Growth). This growth alteration is not associated with mitochondrial DNA modifications. Only the strains that have an increased translational accuracy present Crippled Growth. However, the Crippled Growth Determinant is found in all the strains during the stationary phase; it is eliminated from the non permissive strains during the exit of the stationary phase. The mutants, that have an increased translational accuracy, probably lack a factor which is needed to eliminate the determinant when cells enter the growth phase.     

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.     

Integrative transformation of the mycotoxin-producing fungus, Penicillium paxilli

A high frequency transformation system has been developed for Penicillium paxilli using pAN7-1. Up to 44% of the primary transformants were heterokaryons. Loss of hygromycin resistance was observed in primary transformants that were sub-cultured on non-selective media, but single spores of these primary transformants were mitotically stable on both selective and non-selective media. A molecular analysis of the transformants generated showed that 78% had single-site integrations, with half of these containing a single copy of pAN7-1. CHEF-gel electrophoresis showed that P. paxilli has at least six chromosomes with a total genome size of about 23.4 Mb.     

Isolation of mRNAs induced by a hazardous chemical in white-rot fungus, Coriolus versicolor, by differential display

During January through December 1993, twelve symptomatic infants and children (6 females, 6 males) with human immunodeficiency virus infection were prospectively evaluated for their cardiovascular clinical manifestations and ventricular functions, using two-dimensional, M-mode and Doppler echocardiographic examination. From auscultation, the pulmonic component of the second heart sound was accentuated in 8 cases and the murmur of atrioventricula valve regurgitation and pericardial friction rub were audible in 7 and 6 patients, respectively. Cardiomegaly and venous congestion were present on chest roentgenogram in 6 cases and electrocardiogram was abnormal in 5. The echocardiogram demonstrated elevated pulmonary arterial pressure in 9 patients. There were 5 cases of non-tamponade pericardial effusion. Five patients had mitral and pulmonary insufficiency while six had tricuspid insufficiency. The ejection fraction and shortening fraction were increased in all. The incidence of pulmonary hypertension was more frequent than previously reported.     

碳氮源对少孢节丛孢生长及捕食线虫能力的影响

[目的]探索不同碳氮源对少孢节丛孢生长及捕食线虫能力的影响.[方法]通过对菌株进行分离、接种与培养,观察、测定少孢节丛孢在含有葡萄糖、蔗糖、乳搪、可溶性淀粉4种碳源和尿素、氯化铵、硫酸铵、硝酸铵4种氮源培养基上生长时的菌落形态、大小、稠密度、菌丝粗细及其捕食线虫的能力.[结果]碳源不同,少孢节丛孢的生长及捕食能力不同.4种碳源中,可溶性淀粉的效果最好,最适合少孢节丛孢的生长,且菌丝捕食线虫的能力最强.3种无机氮源对少孢节丛孢生长及捕食能力的影响没有显著差异,但与有机氮源尿素的影响相比,差异较大.在舍尿素的培养基中,少孢节丛孢生长速度最慢,但菌丝捕食线虫能力最强.[结论]碳源和氮源对少孢节丛孢的生长和捕食线虫能力均有明显影响.     

Corollosporine, a new phthalide derivative from the marine fungus Corollospora maritima Werderm. 1069

Extracts of the culture medium from the marine fungus Corollospora maritima exhibited concentration dependent antibacterial activity against Staphylococcus aureus and other microorganisms. Bioactivity-guided fractionation and purification afforded the new isobenzofuranone or phthalid type compound corollosporine.     

兰州百合与亚洲百合及其杂种F1的蛋白质和同工酶分析

[目的]为选配亲本组合及杂种早期鉴定提供理论依据.[方法]采用聚丙烯酰胺凝胶电泳技术,对兰州百合与亚洲百合及杂交后代的可溶性蛋白质和过氧化物酶进行分析.[结果]以兰州百合为亲本的杂交后代的蛋白质谱中不仅出现了与亲本同源但比亲本染色加深的带,且出现了亲本中不存在的新带.杂种F1 过氧化物酶谱主要表现为亲本的不完全互补型和杂种型.[结论]蛋白质谱和过氧化物酶谱可作为百合杂种鉴定的生化指标及进行目标性状植株的检测.     

Genome size, complexity, and ploidy of the pathogenic fungus Histoplasma capsulatum

The genome size, complexity, and ploidy of the dimorphic pathogenic fungus Histoplasma capsulatum was determined by using DNA renaturation kinetics, genomic reconstruction, and flow cytometry. Nuclear DNA was isolated from two strains, G186AS and Downs, and analyzed by renaturation kinetics and genomic reconstruction with three putative single-copy genes (calmodulin, alpha-tubulin, and beta-tubulin). G186AS was found to have a genome of approximately 2.3 x 10(7) bp with less than 0.5% repetitive sequences. The Downs strain, however, was found to have a genome approximately 40% larger with more than 16 times more repetitive DNA. The Downs genome was determined to be 3.2 x 10(7) bp with approximately 8% repetitive DNA. To determine ploidy, the DNA mass per cell measured by flow cytometry was compared with the 1n genome estimate to yield a DNA index (DNA per cell/1n genome size). Strain G186AS was found to have a DNA index of 0.96, and Downs had a DNA index of 0.94, indicating that both strains are haploid. Genomic reconstruction and Southern blot data obtained with alpha- and beta-tubulin probes indicated that some genetic duplication has occurred in the Downs strain, which may be aneuploid or partially diploid.     

Effect of butyrolactone I on the producing fungus, Aspergillus terreus

Butyrolactone I [alpha-oxo-beta-(p-hydroxyphenyl)-gamma-(p-hydroxy-m-3, 3-dimethylallyl-benzyl)-gamma-methoxycarbonyl-gamma-butyrolactone] is produced as a secondary metabolite by Aspergillus terreus. Because small butyrolactone-containing molecules act as self-regulating factors in some bacteria, the effects of butyrolactone I on the producing organism were studied; specifically, changes in morphology, sporulation, and secondary metabolism were studied. Threefold or greater increases in hyphal branching (with concomitant decreases in the average hyphal growth unit), submerged sporulation, and secondary metabolism were observed when butyrolactone I was added to cultures of A. terreus. Among the secondary metabolites whose production was increased by this treatment was the therapeutically important compound lovastatin. These findings indicate that butyrolactone I induces morphological and sporulation changes in A. terreus and enhances secondary metabolite production in a manner similar to that previously reported for filamentous bacteria.     

Cryogenic X-ray crystal structure analysis for the complex of scytalone dehydratase of a rice blast fungus and its tight-binding inhibitor, carpropamid: the structural basis of tight-binding inhibition

Scytalone dehydratase is a member of the group of enzymes involved in fungal melanin biosynthesis in a phytopathogenic fungus, Pyricularia oryzae, which causes rice blast disease. Carpropamid [(1RS,3SR)-2, 2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropa necarboxamide] is a tight-binding inhibitor of the enzyme. To clarify the structural basis for tight-binding inhibition, the crystal structure of the enzyme complexed with carpropamid was analyzed using diffraction data collected at 100 K. The structural model was refined to a crystallographic R-factor of 0.180 against reflections up to a resolution of 2.1 A. Carpropamid was bound in a hydrophobic cavity of the enzyme. Three types of interactions appeared to contribute to the binding. (i) A hydrogen bond was formed between a chloride atom in the dichloromethylethylcyclopropane ring of carpropamid and Asn-131 of the enzyme. (ii) The (chlorophenyl)ethyl group of carpropamid built strong contacts with Val-75, and this group further formed a cluster of aromatic rings together with four aromatic residues in the enzyme (Tyr-50, Phe-53, Phe-158, and Phe-162). (iii) Two hydration water molecules bound to the carboxamide group of carpropamid, and they were further hydrogen-bonded to Tyr-30, Tyr-50, His-85, and His-110. As a result of interactions between carpropamid and the phenylalanine residues (Phe-158 and Phe-162) in the C-terminal region of the enzyme, the C-terminal region completely covered the inhibitor, ensuring its localization in the cavity.     

Characterization of a split respiratory pathway in the wheat "take-all" fungus, Gaeumannomyces graminis var. tritici

This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a Deltapsi across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.     

Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae

The Cercospora nicotianae SOR1 gene is required for resistance to singlet oxygen-generating photosensitizers. SOR1 was characterized in the wild-type and in five photosensitizer-sensitive mutant strains which are complemented to photosensitizer resistance by transformation with SOR1. Sequence analysis determined that three of the mutants contain SOR1 copies with mutations encoding substitutions in the protein-coding sequence; however, two other mutants had wild-type SOR1 protein and promoter sequences. All five mutants accumulate SOR1 mRNA at levels comparable to that of the wild-type strain. In the wild-type strain, SOR1 accumulation is enhanced two-fold by light, but is unaffected by the presence of cercosporin, the photosensitizer synthesized by C. nicotianae. Southern analysis indicates that SOR1 is present in other fungi that synthesize structurally related perylenequinone photosensitizers.