The occurrence of fungi along the Red Sea coast and variability among isolates of Fusarium as revealed by isozyme analysis

Nineteen identified species which belong to nine fungal genera were recovered from 14 samples collected from different sites of the Red Sea governorate. The aquatic fungal genera were Allomyces, Dictyuchus, Saprolegnia and Pythium while, the terrestrial fungal genera were Aspergillus, Penicillium, Fusarium, Neurospora and Rhizopus. Aspergillus was the most frequent genus, represented by seven species, of which A. niger, A. flavus and A. ustus were the most common. Penicillium was of occurred less frequently and was represented by two species, while Fusarium was isolated unfrequently and contributed four species. The remaining genera were unfrequent or rare and were each represented by one species. In addition, two electrophoretic isozyme patterns, esterase and glutamate oxalate transaminase (GOT), were determined to measure variability among 10 isolates of Fusarium. The results revealed that the tested fungi differed from each other in one or more esterase bands, except that F. moniliforme isolated from Safaga and from 40 Kilometers south of El-Kaussier yielded similar banding pattern. The activity of GOT was observed in the samples of F. solani and F. oxysporum and not detected in other isolates of Fusarium. The results indicated that F. solani differed from F. oxysporum in the isozymes of GOT, while no differences were observed between the isolated of the same species.     

Lactate and malate dehydrogenase and alpha-esterases in oligospermia

Lactate dehydrogenase (LDH), malate dehydrogenase, and alpha-esterase were studied electrophoretically in a total of 99 semen samples obtained from normal, vasectomized, oligospermic, and infertile males. The enzymatic patterns were compared with total sperm count and percentage sperm motility. Lactate dehydrogenase X was absent in semen samples from oligospermic as well as vasectomized males. An extra LDH band (between the second and third LDH bands) was detectable in samples which had a low sperm count but higher motility (80 to 90%). This extra band was absent in samples with higher sperm count regardless of the level of percentage motility, suggesting that the extra band possibly may be related to motility factors of low sperm count but not normal sperm count. The fastest moving alpha-esterase bands were absent in samples from oligospermic or vasectomized males. These bands were also absent in samples from infertile patients having a very low sperm count. It is not clear whether the fast-moving esterase bands are related to sperm count or possible differences in hormonal levels of individuals with low or zero sperm count samples. The patterns of malate dehydrogenase did not differ in individuals with zero, low, or normal sperm count.     

Interaction between citrate synthase and malate dehydrogenase. Substrate channeling of oxaloacetate

The interactions between pig heart citrate synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between citrate synthase and mitochondrial malate dehydrogenase occurred but no interaction between citrate synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and citrate synthase-coupled systems was tested using polyethylene glycol precipitates of citrate synthase and mitochondrial malate dehydrogenase, and citrate synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated citrate synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of citrate synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with citrate synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with citrate synthase.     

Structural studies of malate dehydrogenases (MDHs): MDHs in Brevundimonas species are the first reported MDHs in Proteobacteria which resemble lactate dehydrogenases in primary structure

The N-terminal sequences of malate dehydrogenases from 10 bacterial strains, representing seven genera of Proteobacteria, were determined. Of these, the enzyme sequences of species classified in the genus Brevundimonas clearly resembled those malate dehydrogenases with greatest similarity to lactate dehydrogenases. Additional evidence from subunit molecular weights, peptide mapping, and enzyme mobilities suggested that malate dehydrogenases from species of the genus Brevundimonas were structurally distinct from others in the study.     

Physical properties and chemical compositions of cytoplasmic and mitochondrial malate dehydrogenase from Physarum polycephalum

The malate dehydrogenase isoenzymes from Physarum polycephalum have been purified to homogeneity as confirmed by gel filtration chromatography, polyacrylamide gel disc electrophoresis and analytical ultracentrifugation. Certain physical and chemical parameters of the malate dehydrogenase isoenzymes reported here include sedimentation, molecular weight and subunit molecular weight. Most unique of the differences between the isoenzymes were the widely separate isoelectric points of 9.83 for mitochondrial malate dehydrogenase and 6.14 for the supernatant malate dehydrogenase. The amino acid analyses of each form were done revealing the isoenzymes were unquestionably unique proteins differing in the content of ten amino acids.     

Recognition of partially-folded mitochondrial malate dehydrogenase by GroEL. Steady and time-dependent emission anisotropy measurements

The binding of partially-folded mitochondrial malate dehydrogenase (mMDH) to GroEL was assessed by steady and nanosecond emission spectroscopy. Partially-folded intermediates of mMDH show significant residual secondary structure when examined by CD spectroscopy in the far UV. They bind the extrinsic fluorescent probe ANS and the protein-ANS complexes display a rotational correlation time of 19 ns. Similar rotational correlation time (phi = 18.6 ns) was determined for partially-folded species tagged with anthraniloyl. GroEL recognizes partially-folded species with a K(D) approximately 60 nM. The rotational correlation time of the complex, i.e., GroEL-mMDH-ANT, approaches a value of 280 ns in the absence of ATP. Reactivation of mMDH-ANT by addition of GroEL and ATP brings about a significant decrease in the observed rotational correlation time. The results indicate that partially-folded malate dehydrogenase is rigidly trapped by GroEL in the absence of ATP, whereas addition of ATP facilitates reactivation and release of folded conformations endowed with catalytic activity.     

Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase and citrate synthase are sequential enzymes in the Krebs tricarboxylic acid cycle. We have shown [Lindbladh, C., Rault, M., Hagglund, C., Small, W. C., Mosbach, K., Bülow, L., Evans, C., and Srere, P.A (1994) Biochemistry 33, 11692-11698] that a fusion protein of yeast mitochondrial citrate synthase and yeast mitochondrial malate dehydrogenase channels oxaloacetate between the active sites. A Brownian dynamics simulation model of porcine mitochondrial enzymes of citrate synthase and malate dehydrogenase was used [Elcock, A. H., and McCammon, A. M. (1996) Biochemistry 35, 12652-12658], showing that a positive electrostatic surface potential between the active sites of the fusion protein could account for the channeling of oxaloacetate we observed with the yeast fusion protein. Since the data were established with a yeast fusion protein and the model was with porcine fusion protein, we have now prepared and studied the porcine fusion protein. The channeling of the oxaloacetate intermediate was the same for the porcine fusion protein as it was for the yeast fusion protein. This channeling behavior is eliminated at high ionic strength. A fusion protein of porcine citrate synthase and porcine cytosolic malate dehydrogenase does not exhibit any channeling of oxaloacetate. A model of the fusion protein with the cytosolic malate dehydrogenase shows no clear positive electrostatic potential surface between the two active sites, thus distinguishing it from the fusion protein with the mitochondrial malate dehydrogenase. These results establish the electrostatic nature of channeling in mitochondrial fusion proteins.     

Internal quality assurance activities of a surgical pathology department in an Australian teaching hospital

Parotid and mandibular saliva was obtained from red kangaroos by concurrent acetylcholine isoprenaline stimulation. Salivary proteins were separated by horizontal electrophoresis on either cellulose acetate or starch gels and assessed by specific staining techniques for 23 enzymes commonly found in mammalian tissues and body fluids. Parotid saliva was positive for acid phosphatase, alpha-amylase, carbonic anhydrase, glucose-6-phosphate dehydrogenase, sorbitol dehydrogenase and superoxide dismutase activities. Mandibular saliva was positive for alcohol dehydrogenase in addition to the above six enzymes. The kangaroo salivas lacked activity for alkaline phosphatase, beta-galactosidase and non-specific esterase which occur in saliva from some mammalian species.     

Isoenzyme analysis of Arthrobotrys, a nematode-trapping fungus

Extraction and isoenzyme analysis of four isolates of Arthrobotrys including A. musiformis, A. robusta and A. conoides were conducted. Among the 14 enzymes studied by starch gel electrophoresis, using morpholine-citrate as gel/electrode buffer, the following nine enzymes showed interpretable banding patterns: alpha-esterase, fumarase, hexokinase, isocitrate dehydrogenase, leucine aminopeptidase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase and phosphoglucoisomerase. All isolates studied displayed typical isoenzyme phenotypes for each species. Two isolates of A. conoides differed in their alpha-isoesterase banding patterns, but no differences were observed for the other enzymes. The assay was satisfactory for enzyme extraction and resolution of Arthrobotrys and could be used in future taxonomic and genetic studies of this organism.     

Homotropic activation via the subunit interaction and allosteric symmetry revealed on analysis of hybrid enzymes of L-lactate dehydrogenase

L-Lactate dehydrogenase from Bifidobacterium longum shows homotropic activation by pyruvate as well as heterotropic activation by fructose 1,6-bisphosphate. Hybrid enzymes were produced from the wild-type subunit and a mutant subunit, whose substrate specificity was altered to that of malate dehydrogenase, and separated to analyze the substrate-induced homotropic activation mechanism. Oxamate, a competitive inhibitor of L-lactate dehydrogenase, was used to mimic the substrate-induced activation of the wild-type subunit as "a regulatory subunit." The malate dehydrogenase activity of the mutant subunit as "the catalytic subunit" of the hybrid enzymes was measured, and the activity of the mutant subunit was activated on the addition of oxamate. Thus, we directly observed the inter-subunit homotropic activation transmitted from the wild-type to the mutant subunit. Moreover, "isomeric" hybrid enzymes that have different structural subunit arrangements but identical subunit compositions showed identical kinetic natures. This indicates that the enzyme maintains its subunit symmetry during the allosteric transition.     

Action of L-acetylcarnitine on different cerebral mitochondrial populations from cerebral cortex

The maximum rate (Vmax) of some mitochondrial enzymatic activities related to the energy transduction (citrate synthase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase) was evaluated in non-synaptic (free) and intra-synaptic mitochondria from rat brain cerebral cortex. Three types of mitochondria were isolated from rats subjected to i.p. treatment with L-acetylcarnitine at two different doses (30 and 60 mg.kg-1, 28 days, 5 days/week). In control (vehicle-treated) animals, enzyme activities are differently expressed in non-synaptic mitochondria respect to intra-synaptic "light" and "heavy" ones. In fact, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glutamate-pyruvate-transaminase and glutamate-oxaloacetate-transaminase are lower, while citrate synthase, cytochrome oxidase and glutamate dehydrogenase are higher in intra-synaptic mitochondria than in non-synaptic ones. This confirms that in various types of brain mitochondria a different metabolic machinery exists, due to their location in vivo. Treatment with L-acetylcarnitine decreased citrate synthase and glutamate dehydrogenase activities, while increased cytochrome oxidase and alpha-ketoglutarate dehydrogenase activities only in intra-synaptic mitochondria. Therefore in vivo administration of L-acetylcarnitine mainly affects some specific enzyme activities, suggesting a specific molecular trigger mode of action and only of the intra-synaptic mitochondria, suggesting a specific subcellular trigger site of action.     

Regulation of progesterone biosynthesis in human placental mitochondria by Krebs cycle metabolites

1. 2-Oxoglutarate, succinate, fumarate, malate and citrate, cis-aconitate and isocitrate stimulate conversion of cholesterol to progesterone in human placental mitochondria. 2. The stimulatory effect of dicarboxylic and tricarboxylic acids depends on the activity of malate dehydrogenase (decarboxylating) (NADP+) (EC 1.1.1.40) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42), respectively.     

Identification of a specific effector of the small GTP-binding protein Rap2

The coupled processes of the chloroplast trans-envelope transport of malate and oxaloacetate and their interconversion as catalyzed by the stromal NADP-linked malate dehydrogenase are quantitatively analyzed by means of a steady-state model. The equation for the NADP-malate dehydrogenase reaction is developed. The empirical dependence of enzyme activity on NADPH and NADP+ is used to determine its actual activity. The trans-envelope counter exchange of malate and oxaloacetate is described by a kinetic model of the translocator. Kinetic parameters are derived from known data, except for the Km value and the maximum rate for oxaloacetate transport, which are estimated from oxaloacetate-dependent malate formation in isolated intact chloroplasts. Using the kinetic properties of the system and the known metabolite concentrations, the model demonstrates that photosynthetically generated NADPH can be exported efficiently from the chloroplasts to the cytosol by the malate-valve system. The transfer capacity of the malate valve is estimated not to exceed 20 mumol (mg Chl)-1 h-1 (or 5% of the electron transport) under normal physiological conditions. The possible role of the malate valve in leaf cells under normal conditions and during stress is discussed.     

Identification of metabolic pathways of brain angiotensin II and angiotensin III: predominant role of angiotensin III in the control of vasopressin secretion

A complex seven species model community, including bacteria and fungi, was selected from organisms isolated from the walls of an industrial flowing water system. Growth rates of the species were determined in single and mixed batch culture growth. The rates were found to be significantly higher in mixed culture for Pseudomonas alcaligenes and Flavobacterium indologenes and higher in single culture for Xanthomonas maltophilia, Rhodotorula glutinis and Fusarium solani, whereas no significant difference was recorded for Alcaligenes denitrificans and Fusarium oxysporum. All species attached to PVC in single and mixed culture to form biofilms. Xanthomonas maltophilia, Alc. denitrificans, Ps. alcaligenes and F. solani biofilm cell densities cm-2 were significantly higher than attachment of the component species in mixed culture. Statistical analyses showed a significant difference in rate of colonization between single and mixed cultures for some species. No significant difference was noted between mixed culture cell densities cm-2 at laminar flows of Reynolds number 2.7 and 5.4.     

Preparation and properties of mitochondria derived from synaptosomes

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

Instrumental and metabolic evaluation of patients affected by peripheral arterial occlusive disease (PAOD) following surgical revascularization surgery

Experiments were performed on eight subjects affected by peripheral arterial occlusive disease (PAOD) of the lower limbs. Each patient was submitted to Ecodoppler, angiography and the "Treadmill test". Two bioptic muscle of these patients. A sample was used for the spectrophotometric and spectrophotofluorimetric determinations of: glycogen, pyruvate, lactate, citrate, alpha-ketoglutarate, malate, aspartate, glutamate, AMP, ADP, ATP and creatine phosphate (CP). The other bioptic sample was used to determine the following enzyme activities: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, malate dehydrogenase, total NADH cytochrome c reductase, cytochrome oxidase, aspartate aminotransferase and alanine aminotransferase. Patients showed an increase in lactate dehydrogenase, total NADH cytochrome c reductase and succinate dehydrogenase activities, a decrease in glycogen, ATP and CP concentrations. Telethermographic data showed patient muscle thermic emission quantitatively different from control group. The telethermographic test can be used as an additional diagnostic tool to determine and monitor the efficiency of a muscle undergoing metabolic failure.     

Esterase patterns of species in the Drosophila buzzatii cluster

A comparative analysis was made of the esterase isoenzyme patterns of eight iso-female lines, four of Drosophila serido (B31 D1, A55, B59, Q1, B50Q3), two of D. koepferae (B20D2 and B25D7), one of D. seriema (A95) and one of D. buzzatii (Buz). In all, 43 bands in the spectrum of esterase isoenzymes were detected by electrophoresis in polyacrylamide gels. They showed variations in specific reactions with alpha and beta-naphthyl acetate, number of patterns yielded in their intra-isofemale line combinations, frequencies of such combinations and the thickness and staining degree of some bands, in different individuals, lines and species. Among bands detected exclusively in males, seven may be considered sex-specific (5 alpha-esterases and 2 beta-esterases). These male-specific alpha-esterases have in common the inability to cleave beta-naphthyl acetate in the absence of alpha-naphthyl, denoting a possible common function. The similarity index (SI) and analysis of dependence were calculated in an attempt to quantify the differentiation of the iso-female lines studied, on the basis of esterase bands. SI mean value allowed the separation of the isofemale lines into five classes. Each species had its own pattern of esterase bands, but some bands were shared. A divergence hypothesis for the isofemale lines and the species is discussed.     

Oxidoreductases and hydrolases as marker enzymes for ultracentrifugation of islets of Langerhans of rats

Using quantitative fluorometric micro methods the presence of glutamate dehydrogenase, acid galactosidase, and acid glucuronidase was detected in pancreatic islets of the rat. Some properties of these enzymes and of malate dehydrogenase, 6-phosphogluconate dehydrogenase, and acid phosphatase were investigated. It has been shown that subcellular fractions of homogenates of islets of Langerhans can be characterized by using glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, and acid hydrolases as marker enzymes for mitochondria, cytosol, and lysosomes, respectively. The degree of contamination from acinar tissue in the islet preparations was calculated from the amylase activity of the homogenates.     

Tricarboxylic acid cycle in rat brain synaptosomes. Fluxes and interactions with aspartate aminotransferase and malate/aspartate shuttle

The flux through different segments of the tricarboxylic acid cycle was measured in rat brain synaptosomes with gas chromatography-mass spectrometry using either deuterated glutamine or [13C]aspartate. The flux between 2-oxoglutarate and oxaloacetate was estimated to be 3.14 and 4.97 nmol/min/mg protein with and without glucose, respectively. These values were 3-5-fold faster than the flux between oxaloacetate and 2-oxoglutarate (0.92 nmol/min per mg protein) measured in the presence of glucose. The pattern of intermediates labeling suggests that the overall rate-controlling reaction involves either citrate synthase or pyruvate dehydrogenase but not 2-oxoglutarate or isocitrate dehydrogenase. The enrichment in [3,3,4,4-2H4]glutamate from [2,3,3,4,4-2H5]glutamine was as rapid as in [2,3,3,4,4-2H5]glutamate, which indicates that the aspartate aminotransferase reaction is severalfold faster than the flux through the tricarboxylic acid cycle. [13C]Aspartate was rapidly converted to [13C]malate, suggesting that in intact synaptosomes aspartate entry into the mitochondrion is very slow. The finding that aspartate is taken up by mitochondria as malate, along with the observed high enrichment in [3-2H]malate (from [2,3,3,4,4-2H5]glutamine), is consistent with the substantial synaptosomal activity of the malate/aspartate shuttle.     

Tissue-specific esterases in the Xiphophorine fish Platypoecilus maculatus, Xiphophorus helleri, and their hybrid

Tissue-specific esterases of the xiphophorine fishes Platypoecilus maculatus (platyfish), Xiphophorus helleri (swordtail), and their F1 hybrid have been analyzed using disc electrophoresis. Seven esterase zones (resolved into a maximum of nine bands) exist in these fishes, and these have been classified by employing specific inhibitors. Five of the seven zones, EST-1, EST-2, EST-5, EST-6, and EST-7, appeared to be carboxylesterases; while the two remaining zones, EST-3 and EST-4, were classified as cholinesterases. In the liver of the platfish, all seven esterase zones were detected, while the liver of the swordtail exhibited only five esterase zones. EST-1 and EST-3 were lacking in the liver tissue of the swordtail. All seven esterase loci were expressed in the liver tissue of the F1 hybrid. The reciprocal crosses gave the same results. In the fin, skin, skeletal muscle, and eye tissues from all three genotypes, three major esterase zones, EST-2, EST-5, and EST-7, were detected. In addition, EST-1 was frequently detected in all these tissues of the platfish and the F1, but was lacking in the swordtail. Serum from three genotypes showed one prominent esterase zone, EST-5; however, trace activity of EST-2 and EST-7 zones could also be detected. It seems that in all tissues of the F1 hybrid there is expression of all the esterase genes from the platfish. The results of the present study are discussed in the comparison to those from other studies on teleost esterases.