Effect of 2-deoxy-D-glucose on mycotoxins from apples inoculated with Penicillium expansum

Patulin concentration was not significantly different in Bramley and Cox's apples inoculated with Penicillium expansum, and treated with the biocontrol enhancer 2-deoxy-D-glucose (DOG) when compared to no DOG treatment, despite average numerical increases. Some additional small HPLC peaks were detected from some extracts, one of which corresponded to citrinin. This revised version was published online in August 2006 with corrections to the Cover Date.     

Patulin is a cultivar‐dependent aggressiveness factor favouring the colonization of apples by Penicillium expansum

The blue mould decay of apples is caused by Penicillium expansum and is associated with contamination by patulin, a worldwide regulated mycotoxin. Recently, a cluster of 15 genes (patA–patO) involved in patulin biosynthesis was identified in P. expansum. blast analysis revealed that patL encodes a Cys₆ zinc finger regulatory factor. The deletion of patL caused a drastic decrease in the expression of all pat genes, leading to an absence of patulin production. Pathogenicity studies performed on 13 apple varieties indicated that the PeΔpatL strain could still infect apples, but the intensity of symptoms was weaker compared with the wild‐type strain. A lower growth rate was observed in the PeΔpatL strain when this strain was grown on nine of the 13 apple varieties tested. In the complemented PeΔpatL:patL strain, the ability to grow normally in apple and the production of patulin were restored. Our results clearly demonstrate that patulin is not indispensable in the initiation of the disease, but acts as a cultivar‐dependent aggressiveness factor for P. expansum. This conclusion was strengthened by the fact that the addition of patulin to apple infected by the PeΔpatL mutant restored the normal fungal colonization in apple.     

Purification and properties of a β-glucosidase from Penicillium oxalicum autolysates

A beta-glucosidase from the medium of an autolyzed culture of Penicillium oxalicum has been purified by tannic acid precipitation, sephacryl S-200, DEAE-Biogel, CM-Biogel and Mono Q successively. The purification process produced a homogeneous band in the SDS-PAGE that correspond to a Mr of 133,500. The enzyme had a pl of 4, and the active optima were found at pH 5.5 and 55 degrees C. The enzyme hydrolyzed different substrates showing maximum affinity against p-nitrophenyl-beta-D-glucoside with a Km value of 0.37 mM. The beta-glucosidase was inhibited by Glucono-D-lactone but not by glucose in the concentration range of 1 to 10 mM. The enzyme was adsorbed by Concanavalin-A-Sepharose.     

草酸青霉中新型线性五肽的发现及对柑橘采后致腐菌拮抗活性研究

【背景】柑橘是我国南方农村地区的一种重要经济作物,每年因采后真菌感染导致大量柑橘腐烂和劣变,造成了巨大的经济损失,严重影响柑农的积极性。【目的】丝状真菌SG-4对柑橘采后致腐菌橘青霉(Penicillium citrinum)和指状青霉(Penicillium digitatum)具有很强的拮抗作用,为明确其抑菌的物质基础,对该菌进行了生物学鉴定和活性成分分析。【方法】将SG-4菌株的核糖体DNA的内转录间隔区进行测序,结合菌株形态进行鉴定;对其菌体相和培养液进行抑菌活性追踪,利用醇沉淀和反相高效液相色谱分离得到主效物质,通过高分辨质谱和核磁共振等波谱分析方法确定结构;采用离体的牛津杯抑菌圈法和柑橘的体内接种试验来评价其抑菌活性;通过液相分析测定了活性物质在发酵液中的含量。【结果】分子和形态鉴定表明SG-4是一株草酸青霉(Penicillium oxalicum),其抑制柑橘采后致腐菌的活性物质位于培养液的水相提取物中,且具有强极性。进一步研究表明其活性成分为一种新型线性醇溶五肽,含3个具羟基的氨基酸,并且富含甲基化修饰,构成寡肽的第一个氨基酸为β-氨基丁酸(beta-aminobut...     

Alkylresorcinols isolated from rye bran by supercritical fluid of carbon dioxide and suspended in a food-grade emulsion show activity against Penicillium expansum on apples

Apple cultivars are attacked by many fungal diseases, both on the tree and during storage. One of the most serious is blue mould, caused by Penicillium expansum. In this study, 5-(n)-alkylresorcinols (AR) were isolated from rye bran by the supercritical fluid of carbon dioxide and were used for the preparation of bioactive emulsions. These emulsions were applied to harvested fruit of five apple varieties to determine the levels of antifungal activity. A significant inhibition of disease symptoms was obtained after spraying some of the prepared AR emulsions on fruits that had been experimentally infected with Penicillium expansum. The most effective emulsions consisted of 0.025% (m/v) ARs, 0.1% (m/v) xanthan gum, 0.5% (m/v) Synperonic 91/6 or PDMs-copolymer, 0.2% (m/v) Tween 20, 1% (m/v) Trioleate, 2% (m/v) oleylalcohol, 2% (m/v) PEG 400, 5% (m/v) CaCl₂ or NaCl suspended in water.     

The occurrence of β-galactosidase and β-phosphogalactosidase in Lactobacillus casei strains

Abstract Several strains of Lactobacillus casei of different origins were compared and it was observed that lactose metabolism varied from one strain to the other. Certain strains contained a β-galactosidase, others a β-phosphogalactosidase and others contain both. It was shown that the activities present in these last strains are catalyzed by two proteins differing in their electrophoretic mobilities and M _r values. Genetic divergence of the studied strains is considered.     

Carbon,nitrogen and pH regulate the production and activity of a polygalacturonase isozyme produced by Penicillium expansum

The influence of carbon, nitrogen and pH on polygalacturonase (PG) activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized lyophilised fruit tissue and the highest PG activity occurred in apple pectin medium. Nitrogen source influenced PG activity and was highest with ammonia while the greatest mycelial mass was supported by glutamate or glutamine. PG activity and mycelial mass peaked 5 five days after inoculation as polyuronide content decreased and the pH and ammonium levels increased in apple pectin medium. A single active PG isozyme with an isoelectric point of ～7.6 was produced in apple pectin medium and a partial cDNA clone was obtained that was most homologous to the pggII gene from Penicillium. griseoroseum. The results from this study indicate that P. expansum can modulate the activity of PG in response to nutrient sources and ambient pH through signalling pathways that modulate nutrient acquisition, uptake and metabolism.     

Transformation of Penicillium expansum with a heterologous gene which confers resistance to benomyl

Transformation of Penicillium expansum with the pBT6 vector yielded 8 to 34 transformants per g of DNA. Hybridization analyses revealed that homologous recombination occurred in most of the transformants. Twenty-one out of 25 transformants analysed showed hybridization patterns which were indistinguishable from that of the wild type.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

β‐1,3‐Glucanases in wheat and resistance to the Russian wheat aphid

The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko) is one of the most destructive insect pests of cereals world-wide. Although resistant cultivars have been bred, the biochemical mechanism of resistance is unknown. The aim of this work was to gain information on the mechanism of resistance which could contribute to more directed breeding of resistant cultivars in the future. The effect of RWA infestation on the inter- and intracellular β-1,3-glucanase activities was studied in different resistant wheat (Triticum aestivum L.) cultivars containing the Dn-1 gene for RWA resistance and corresponding near-isogenic susceptible cultivars. The activity was determined spectrophotometrically by measuring the release of glucose from laminarin. Infestation differentially induced the intra- and intercellular activities to much higher levels in resistant than susceptible cultivars within 48 h. According to immunological studies induced enzyme activities were due to increased protein levels. The intracellular β-1,3-glucanase contained about 8% exo-activity. The exo-activity made an insignificant contribution to the intercellular activity. The genetic background into which the resistance gene was bred did affect the level of activity that corresponded to the resistance performance. Seven apoplastic isoforms of β-1,3-glucanase, varying from acidic to basic, were resolved by isoelectric focusing. All isoenzymes were equally induced and no specific one could be linked to resistance or susceptibility. The RWA induced β-1,3-glucanase activity in resistant cultivars closely resembles defence responses during pathogenesis and seems to be part of a general defence response like the hypersensitive reaction (HR), which confers resistance to the RWA. This knowledge might be helpful in future to identify genes for RWA resistance. The increased β-1,3-glucanase activity after RWA infestation might serve as an additional measure to biochemically trace resistance in crosses during breeding.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

Early Effects of β,β'-Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l -[³⁵S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.     

Cloning and expression in Enterobacteriaceae of the extended-spectrum β-lactamase gene from a Pseudomonas aeruginosa plasmid

Abstract An extended-spectrum β-lactamase, the gene for which is located on plasmid pMS350 in Pseudomonas aeruginosa strains, hydrolyzes carbapenems and other extended-spectrum β-lactam antibiotics. We cloned the pMS350 β-lactamase gene in an Escherichia coli K-12 strain using the vector plasmid pHSG398, and subcloned it into pMS360, a plasmid with a wide host-range. This resulted in the formation of the recombinant plasmid, pMS363, containing a 4.1-kb DNA insert that includes the extended-spectrum β-lactamase gene. Plasmid pMS363 was introduced into the P. aeruginosa PAO strain or into six species of Enterobacteriaceae, and the specific activities of the β-lactamase and MICs of various β-lactam antibiotics were estimated. The cloned gene was capable of expression in these strains and caused resistance to carbapenem, penem and other β-lactam antibiotics, with the exception of aztreonam.     

Effects of Various Elicitors on the Transcription of a β-1,3-endoglucanase Gene in Citrus Fruit

Very little is yet known regarding the molecular mechanisms involved in pathogen defense responses in citrus fruit. Recently, a basic β-1,3-endoglucanase (EC 3.2.2.39) belonging to the pathogenesis-related (PR) group of proteins, has been purified from Citrus sinensis (L) Osbeck cv. `Valencia' orange callus. Specific antibodies raised against the purified protein were used to screen `Valencia' callus and flavedo cDNA expression libraries, and to isolate its corresponding cDNA, designated gns1. The gns1 gene encodes a predicted polypeptide of 336 amino acids with a molecular mass of 37.3 kDa and a basic pI of 9.19, and shares 55–65% identity with several other plant β-1,3-endoglucanase proteins. Hereby, we show that the expression of the gns1 gene is markedly induced by wounding and inoculation with Penicillium digitatum (Pers. Fr.) Sacc., and following treatments with various elicitors that induce fruit resistance against P. digitatum . These treatments include UV irradiation, application of jasmonic acid (JA), β-aminobutyric acid (BABA), Candida oleophila antagonist yeast cells and hot water rinsing and brushing. Overall, based on various RNA gel blot hybridizations, we assume that gns1 is most likely to be part of the molecular mechanisms involved in pathogen defense responses in citrus fruit. *     

Thiamine increases β-glucosidase production in the newly isolated strain of Fomitopsis pinicola

Aims:  To isolate a high β-glucosidase (BGL)-producing strain and to optimize BGL production in the isolated strain.
Methods and Results:  A high BGL-producing strain was isolated and identified as Fomitopsis pinicola KMJ812 based on its morphology and a comparison of sequence of its internal transcribed spacer rDNA gene. To increase BGL production, F. pinicola was supplemented with various vitamins. Supplementation with thiamine (20 mg l⁻¹) improved BGL production in F. pinicola cultures by 3·7-fold to give a specific activity of 114·4 μmol min⁻¹ mg⁻¹ protein, one of the highest among BGL-producing micro-organisms. The increased production of BGL in the thiamine-supplemented culture was confirmed by 2D electrophoresis followed by MS/MS sequencing. The BGL purified from F. pinicola culture showed the highest catalytic efficiency ever reported.
Conclusion:  Supplemental thiamine remarkably increased BGL production by a novel BGL-producing strain, F. pinicola KMJ812.
Significance and Impact of the Study:  Our results provide a high BGL-producing strain and the production media for BGL production, and should contribute to better industrial production of glucose via biological processes.     

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of β-glucosidase activity in Escherichia coli

Abstract Genomic DNA fragments encoding β-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli . Two clones containing a common fragment encoded a polypeptide of 58000 Da. Cloned β-glucosidase, expressed in E. coli , showed activity against natural β-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids ( M _r 53896) which showed significant homology with β-glucosidases from glycosyl hydrolase family 1.