Fungal Proteolytic Enzymes

Two kinds of proteolytic enzyme, tentatively named acid protease A and B which showed a single peak on electrophoresis individually, were isolated from the crude enzyme powder obtained from the broth filtrate cultured with Asper gillus niger var. macrosporus. Acid protease B is similar too the fungal acid protease previously reported, bccause the enzyme exhibits optimum activity on milk casein at about pH 2.6 and 55°C when the incubation was done at pH 2.6. Acid protease A is a new proteolytic enzyme, because the enzyme exhibits optimum activity on milk casein at about 2.0 and 70°C or 60°C when the incubation was done at pH 2.6 or 1.5 respectively.     

Oxidation of Polyamines and Brain Injury

Several amine oxidases are involved in the metabolism of the natural polyamines putrescine, spermidine, and spermine, and play a role in the regulation of intracellular concentrations, and the elimination of these amines. Since the products of the amine oxidase-catalyzed reactions - hydrogen peroxide and aminoaldehydes - are cytotoxic, oxidative degradations of the polyamines have been considered as a cause of apoptotic cell death, among other things in brain injury. Since a generally accepted, unambiguous nomenclature for amine oxidases is missing, considerable confusion exists with regard to the polyamine oxidizing enzymes. Consequently the role of the different amine oxidases in physiological and pathological processes is frequently misunderstood. In the present overview the reactions, which are catalyzed by the different polyamine-oxidizing enzymes are summarized, and their potential role in brain damage is discussed.     

Specificity of DNA Methylation Changes During Fungal Dimorphism and Its Relationship to Polyamines

We utilized our modification of the amplified fragment length polymorphism technique for the determination of changes occurring in the DNA methylation patterns during the dimorphic transition of the fungi Mucor rouxii, Yarrowia lipolytica, and Ustilago maydis. To determine the specificity of differential methylation in regards to dimorphism, we obtained the yeast-like form of the three fungi under conditions that induced mycelial growth, by addition of 1,4-diaminobutanone (DAB), an inhibitor of ornithine decarboxylase in the case of M. rouxii and Y. lipolytica. In an odc null mutant of U. maydis, repression of the dimorphic transition was brought about by limitation in the amounts of exogenous putrescine. Yeasts from the three fungi thus obtained conserved a significant number of the differential DNA fragments with the methylation pattern displayed by normal yeasts, indicating their true correlation with dimorphism. Our results also confirm a role of polyamines in differential DNA methylation and fungal dimorphic transition.     

Microbial Enzymes for Oxidation of Organic Molecules

Abstract

Enzymatic systems employed by microorganisms for oxidative transformation of various organic molecules include laccases, ligninases, tyrosinases, monooxygenases, and dioxygenases. Reactions performed by these enzymes play a significant role in maintaining the global carbon cycle through either transformation or complete mineralization of organic molecules. Additionally, oxidative enzymes are instrumental in modification or degradation of the ever-increasing man-made chemicals constantly released into our environment. Due to their inherent stereo- and regioselectivity and high efficiency, oxidative enzymes have attracted attention as potential biocatalysts for various biotechnological processes. Successful commercial application of these enzymes will be possible through employing new methodologies, such as use of organic solvents in the reaction mixtures, immobilization of either the intact microorganisms or isolated enzyme preparations on various supports, and genetic engineering technology.     

Effects of Polyamines on the Oxidation of Exogenous NADH by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria

The effect of polyamines (putrescine, spermine, and spermidine) on the oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L. cv. OB1) mitochondria, have been studied. Addition of spermine and/or spermidine to a suspension of mitochondria in a low-cation medium (2 millimolar-K⁺) caused a decrease in the apparent K_m and an increase in the apparent V_max for the oxidation of exogenous NADH. These polycations released by screening effect the mitochondrially induced quenching of 9-aminoacridine fluorescence, their efficiency being dependent on the valency of the cation (C⁴⁺ > C³⁺). Conversely, putrescine only slightly affected both kinetic parameters of exogenous NADH oxidation and the number of fixed charges on the membranes. Spermine and spermidine, but not putrescine, decreased the apparent K_m for Ca²⁺ from about 1 to about 0.2 micromolar, required to activate external NADH oxidation in a high-cation medium, containing physiological concentrations of Pi, Mg²⁺ and K⁺. The results are interpreted as evidence for a role of spermine and spermidine in the modulation of exogenous NADH oxidation by plant mitochondria in vivo.     

姜黄素灌胃对糖尿病大鼠心肌酶和氧化指标的影响

目的:探讨姜黄素灌胃对糖尿病心脏病变大鼠心肌酶和氧化指标的影响。方法:将成年雄性SD大鼠随机分为对照组、糖尿病组、姜黄素治疗2周组与姜黄素治疗4周组。取心室肌组织匀浆分别检测心肌酶、超氧化物歧化酶(Superoxide dismutase,SOD)和丙二醛(Malondialdehyde,MDA)的含量。结果:糖尿病组与对照组比较,心肌酶天冬氨酸氨基转移酶(Aspartate transaminase,AST)、乳酸脱氢酶(Lactic dehydrogenase,LDH)、肌酸激酶(Creatinekinase,CK)、肌酸激酶同工酶(Creatine kinase isoenzyme-MB,CK-MB)均增高(P0.01)。心肌氧化代谢产物MDA含量升高(P0.01),抗氧化损伤的SOD含量降低(P0.01);姜黄素治疗组与糖尿病组相比,各心肌酶含量有所下降,治疗4周组有明显统计学差异(P0.01)。同时心肌SOD和MDA含量在治疗组均有显著改善(P0.05或P0.01),治疗4周组心肌组织SOD含量较2周组明显升高(P0.05)。结论:姜黄素灌胃可以减轻糖尿病大鼠心肌损伤,提高抗氧化能力。     

Changes in Ulrastructure following Fungal Invasion and the Possible Relevance of Extracellular Enzymes

Changes in fine structure of fruit tissues were examined byelectron microscopy for the following host-parasite relationships:Sclerotinia fructigena on apple and pear, S. sclerotiorum oncucumber, and Phytophthora palmivora on egg plant. While S.fructigena caused localized degradation of the wall region,S. sclerotiorum caused extensive wall degradation, and infectionby P. palmivora was characterized by disintegration of plasmalemmaand tonoplast. These effects appeared to be correlated respectivelywith pectolytic enzyme activity in vivo with S. fructigenaycellulolytic activity with S. sclerotiorum, and the in vitrolysis of membranes by culture filtrates of P. palmivora.     

Effects of Hydroxybenzoic Acids on Oxidation of Reduced Nicotinamide Adenine Dinucleotide by Enzymes from Tobacco Leaves

Hydroxyhenzoic acids were tested for their effects on oxidation of the reduced nicotinamide adenine dinucleotide (NADH) in the absence of added H₂O₂ and Mn²* by an enzyme preparation from tobacco leaves (Nicotiana tabacum, var. White Gold). For comparison, a commercial horseradish peroxidase was also used. The rate of NADH oxidation was followed spectruphotometrically at 340 nm. Mono- and dihydroxybenzoic acids exerted significant effect on the rate of NADU oxidation, yet their effectiveness was determined by the number and position of the hydroxyl group on the ring. 4-Hydroxybenzoic acid was very effective in stimulating the reaction. Shifting the hydroxyl from the 4- to the 3-position and from the 3- to the 2-position decreased activity. 2,4- And 2,5-dihydroxybenzoic aeids were more active than the other dihydroxy-iscuners in stinulating oxidation of NADH. the dihydroxybenzoic acids with the hydroxyls in adjacent positions were less effective, and their activity was affected by other phenolic activators. In the presence of 4-hydroxybenzoic acid which enhanced oxidation of NADH, 2,4- and 2,5-dihydroxybenzoic acids further stimulated the reaction, but 3,4-, 2,3- and 2,6-dibydoxybenzoic acids were inhibitory. The inhibition by 3,4- and 2,3-dihydroxybenzoic aciils was non-competitive. The enzymes extracted by a L-cysteine-containing buffer showed lower NADH-oxidase activity. The enzyme preparation possessed peroxidase activity. The activity of NADH-oxidase inereased when H₂O₂ and Mi²* were present in addition to 4-hydroxy-benzoic acid. The effect of the position and number of hydroxyl substitution on the rate of NADH oxidation by borseradish peroxidase was also significant. This suggests the involvement of peroxidase in the NADH-oxidase system of tobacco leaves. However, a combination of the inactivated enzyme solution and active horseradish peroxidase with peroxidase activity equivalent to that of the enzyme preparation from tobacco leaves did not reconstitute the NADH-oxidase activity of tobacco leaves. This and other evidence suggests that the soluble NADH-oxidizing zyme system of tobacco leaves is more complicated than peroxidase.     

Inhibition of Vacuolar Ion Channels by Polyamines

In this work, direct effects of cytosolic polyamines on the two principle vacuolar ion channels were studied by means of patch-clamp technique. Fast and slow activating vacuolar channels were analyzed on membrane patches isolated from vacuoles of the red beet taproot. The potency of the fast and of the slow vacuolar channel blockage by polyamines decreased with a decrease of the polycation charge, spermine⁴⁺ > spermidine³⁺ > putrescine²⁺. In contrast to the inhibition of the fast vacuolar channel, the blockage of the slow vacuolar channel by polyamines displayed a pronounced voltage-dependence. Hence, in the presence of high concentration of polyamines the slow vacuolar channel was converted into a strong inward rectifier as evidenced by its unitary current-voltage characteristic. The blockage of the slow vacuolar channel by polyamines was relieved at a large depolarization, in line with the permeation of polyamines through this channel. The voltage-dependence of blockage was analyzed in terms of the conventional model, assuming a single binding site for polyamines within the channel pore. Taking advantage of a simple linear structure of naturally occurring polyamines, conclusions on a possible architecture of the slow vacuolar channel pore were drawn. The role of common polyamines in regulation of vacuolar ion transport was discussed. Received: 1 May 1998/Revised: 25 September 1998     

Transmissible Plasmid Coding Early Enzymes of Naphthalene Oxidation in Pseudomonas putida

The capacity of Pseudomonas putida PpG7 (ATCC 17,485) to grow on naphthalene, phenotype Nah(+), is lost spontaneously, and the frequency is increased by treatment with mitomycin C. The Nah(+) growth character can be transferred to cured or heterologous fluorescent pseudomonads lacking this capacity by conjugation, or between phage pf16-sensitive strains by transduction. After mutagenesis, strains can be selected with increased donor capacity in conjugation. Clones which use naphthalene grow on salicylate and carry catechol 2,3-oxygenase, the initial enzyme of the aromatic alpha-keto acid pathway, whereas cured strains grow neither on salicylate nor naphthalene and lack catechol 2,3-oxygenase, but retain catechol 1,2-oxygenase and the aromatic beta-keto adipate pathway enzymes.     

Polyamines and pancreatic growth induced by caerulein

Activation of polyamine metabolism may be important to initiation of pancreatic cell growth. We are reporting that such activation did occur during pancreatic growth initiation by caerulein, a cholecystokinin analog. Maximal increases in total putrescine (319%), spermidine (63%) and spermine (50%) were observed 12, 96 and 96 hr respectively after the beginning of the caerulein treatment. This time period coincides with pancreatic hypertrophy and hyperplasia as characterized by increased cell mass and DNA content. Rates of pancreatic weight and DNA content increases were significantly correlated with total spermidine and spermine contents. These data suggest that polyamine biosynthesis is closely associated with pancreatic growth.     

Polyamines contained by two densonucleosis viruses.

Densonucleosis viruses 1 and 2 both contain the three polyamines putrescine, spermidine, and spermine in complete virus particles but not in their respective top components. The polyamines, with spermidine predominant, comprise 1.41% of the virus particle by weight, which is sufficient to neutralize 26% of the single-stranded DNA contained within the particles.     

Permeabilization of Fungal Membranes by Plant Defensins Inhibits Fungal Growth

We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 μM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 μM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.     

Blockade of a Retinal cGMP-gated Channel by Polyamines

The cyclic nucleotide–gated (CNG) channel in retinal rods converts the light-regulated intracellular cGMP concentration to various levels of membrane potential. Blockade of the channel by cations such as Ca²⁺ and Mg²⁺ lowers its effective conductance. Consequently, the membrane potential has very low noise, which enables rods to detect light with extremely high sensitivity. Here, we report that three polyamines (putrescine, spermidine, and spermine), which exist in both the intracellular and extracellular media, also effectively block the CNG channel from both sides of the membrane. Among them, spermine has the greatest potency. Extracellular spermine blocks the channel as a permeant blocker, whereas intracellular spermine appears to block the channel in two conformations—one permeant, and the other non- (or much less) permeant. The membrane potential in rods is typically depolarized to approximately −40 mV in the dark. At this voltage, K _1/2 of the CNG channel for extracellular spermine is 3 μM, which is 100–1,000-fold higher affinity than that of the NMDA receptor-channel for extracellular spermine. Blockade of the CNG channel by polyamines may play an important role in suppressing noise in the signal transduction system in rods.     

Selection of Enzymes for Terminal Restriction Fragment Length Polymorphism Analysis of Fungal Internally Transcribed Spacer Sequences

Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzyme-choosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs.Terminal restriction fragment length polymorphism (TRFLP) profiling was originally developed as a means of genotyping mixed DNA samples (30) and is currently being employed in fungal community ecology studies (3, 5, 6, 7, 10, 13, 19, 22, 26, 27, 29, 33, 38), despite a number of technical and conceptual difficulties (11). Briefly, TRFLP profiling involves amplifying the DNA in pools of mixed genetic material with fluorescently labeled primers, digesting the products with restriction endonucleases, and sizing the labeled terminal fragments in a sequencer. The difference in the positions at which the different restriction enzymes cleave DNA is thought to provide enough variability for such DNA mixtures to be characterized and the contributing organisms to be identified.However, the technique is not without its problems. DNA extraction and PCR amplification biases burden most modern molecular techniques, including TRFLPs (18, 25). Additionally, concerns exist regarding the ability of the differences between primer-enzyme pairs (PEPs) to generate sufficiently different fragment sizes (2), the success of enzymatic cleavage (2), the dependency on the detection threshold of the sequencer (4), and the accuracy of DNA sizing (1). The choice of the primer pairs and restriction enzymes to be used has also been a matter of concern since the appearance of TRFLP profiling. Liu et al. (30) performed virtual digestion of all the bacterial RNA sequences in the Ribosomal Database Project database (release V) with 10 different enzymes and four primer pairs. This pioneering work showed the importance of avoiding enzymes with highly conserved target motifs, something that later became recognized as a major source of TRFLP bias (2, 14, 16, 32). Similar studies have been performed by Osborn et al. (36), Dunbar et al. (12), Engebretson and Moyer (15), and Cardinale et al. (8).The first virtual TRFLP analysis involving a database of fungal DNA sequences was performed by Edwards and Turco (14). This consisted of virtual digestion, by use of six restriction endonucleases, of 316 internally transcribed spacer (ITS) sequences belonging to a number of ectomycorrhizal genera. Avis et al. (2) found only small differences in the diversity of the TRFLPs produced in silico by three PEPs when using their own fungal ITS database, although these differences increased with sample number in iterative analysis. Recent advances using automated resources, such as REPK software (9), have allowed optimal enzyme selection for TRFLP profiling of previously defined communities of organisms. This software selects up to four restriction endonucleases capable of discriminating a desired number of sequence groups. However, this system relies on a priori information, which in real biological communities may not available.The aim of the present work was to improve selection of restriction enzymes for use in the TRFLP profiling of the ITS sequences of unknown fungal communities.