Export of β-1,3-glucanase from mutant rice cells rechallenged and stressed with lysine plus threonine

Mutant rice cells (Oryza sativa L.) grown in liquid suspension cultures exported greater quantities of protein and -glucanases than controls. These mutants were isolated from anther calli resistant to 1 mM lysine plus threonine (LT), regenerated and reestablished as cell suspension cultures from seeds. Cellular protein levels are genetically conditioned, and the levels of extracellular proteins and enzyme activities are inversely related to that of the cellular portions. The rechallenge of cells with 1 mM LT inhibited the expression of both -1,3-glucanases and -1,4-glucosidases but had no significant effect upon the levels of chitinase activity. Mutant cells were more sensitive than controls to stress caused by exogenous LT. In general, under exogenous LT stress the mutant/control ratio for extracellular glucanases increased as the assay conditions were changed from a basic to an acidic pH. The specific activity of glucanases was highest in media and lowest in cells. Both the mutant and control cells exported -glucanases into the suspension medium, but the level of activity in media was greater in that in which the mutant was suspended. The export was probably modulated by the internal protein levels which were highest in mutant cells without LT. Seedlings from mutants with enhanced lysine also had enhanced acidic -glucanase activity.     

Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

Extracellular peroxidases of suspension culture cells of spruce (Picea abies): fungal elicitor-induced inactivation

Extracellular peroxidases of suspension cultures of spruce (Picea abies) (L.) (Karst) become inactivated when the cell suspension is elicited with a cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii. In contrast, cellular peroxidases are induced under these conditions. Both changes of activity are reflected in the isoenzyme profiles.Inactivation of the extracellular peroxidases is caused by an effector, arising from the cells after contact with the elicitor. Formation of the effector is limited to the beginning of elicitation, showing maximal activity at this period of time. Subsequently it becomes increasingly ineffective, probably due to inactivation. The effector is able to also inactivate commercial (horseradish) peroxidase. Inactivation was not the result of the action of a protease present in the medium.The elicitor exerts two different effects on the spruce cell suspension culture. It induces synthesis of enzymes correlated with lignin synthesis and an accumulation of lignin-like material. It also induces secretion of the negative effector which inactivates extracellular peroxidases.The elicitor-induced inactivation is not specific for peroxidases. Other extracellular enzymes, -glucosidase and acid phosphatase (secreted by the cells into the medium) and -amylase and pectinase (from Aspergillus strains) are also inactivated.     

Stable co-transformation of maize protoplasts with gusA and neo genes

An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 × BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing -glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 × 10^–4 (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.     

Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene

We have successfully transferred and expressed a reporter gene driven by an -amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10–12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of -glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice -amylase gene (Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice -amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.     

Combination of induced autolysis and sodium hypochlorite oxidation for the production of Saccharomyces cerevisiae (1 → 3)-β-D-glucan

(1 3)--D-Glucans have received much attention with respect to their biological functions. A novel method to extract (1 3)--D-glucan from Saccharomyces cerevisiae cell wall is proposed in present work, which is based on the combination of induced autolysis and subsequent oxidation of the autolysed cell by sodium hypochlorite to remove undesirable substances. Influences of temperature, pH value and organic solvent on S. cerevisiae FL 1 autolysis were investigated. Results indicated that each factor had its significant effect on induced autolysis and the optimal conditions were 52 °C, pH 5.5 and 1.5% (v/v) ethyl acetate. The kinetic behaviour of the yeast autolytic process under the optimized conditions was further studied. After 36 h of autolysis, 42.0% (w/w) cellular substances were released while the cell wall nearly remained intact. Finally, an ideal glucan yield as high as 22.9% (w/w) was obtained when S. cerevisiae FL 1 was treated by the novel method.     

Control of formation of active soluble or inactive insoluble baker''s yeast α-glucosidase PI in Escherichia coli by induction and growth conditions

Summary Using standard growth conditions (LB medium, 37°C, induction with 5 mM IPTG) yeast -glucosidase PI expressed under the control of the regulated tac-hybrid promoter results in the synthesis of insoluble aggregated -glucosidase granules in Escherichia coli. Under these conditions active soluble -glucosidase amounts to less than 1% of the heterologously produced protein. However, the amount of soluble active -glucosidase was dramatically increased when the strong tac-hybrid promoter was to a limited extent induced. This was achieved at concentrations of 0.01 mM IPTG or of 1% lactose or lower in a lactosepermease deficient host strain containing the lacI ^qrepressor gene on an R-plasmid. The formation of active soluble -glucosidase was almost 100% when E. coli cells induced in this manner were cultivated under conditions that reduced growth rate, i.e. at decreased temperature, extreme pH values or in minimal and complete media supplemented with different carbon sources.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - RB refractile body - t-PA tissue type plasminogen activator - IFN interferon - X--glucoside 5-bromo-4-chloro-indolyl--D-glucopyranoside     

Are active oxygen species involved in induction of β-carotene in Dunaliella bardawil?

The purpose of this work was to test whether induction of massive -carotene synthesis in the alga Dunaliella bardawil is triggered by oxygen radicals. The following results were obtained: (i) The induction of -carotene synthesis is preceded by a lag period of about 4 h during which the cells swell and photosynthesis is partially inhibited, (ii) Addition of promoters of oxygen radicals or of azide (an inhibitor of catalase and superoxide dismutase) during the induction period, under conditions which are suboptimal for massive -carotene accumulation, greatly enhances -carotene synthesis, photodegradation of chlorophyll and inhibition of photosynthesis, (iii) High irradiance, which induces massive -carotene accumulation, also induces a high catalase activity. It is suggested that photosynthetically produced oxygen radicals are involved in triggering massive -carotene accumulation in D. bardawil.     

Five coordinated nitrosylhemoprotein in the whole cells of denitrifying bacterium,Achromobacter xylosoxidans NCIB 11015

The denitrifying bacterium, Achromobacter xylosoxidans NCIB 11015, was cultivated in meat extract-peptone medium and in Mn-free synthetic medium under denitrifying or non-denitrifying conditions. Electron paramagnetic resonance (EPR) spectra for the whole cells of the bacteria thus obtained were measured at 77K. The characteristic three-line signal was observed in the whole cells of the bacteria under denitrifying conditions, but not under non-denitrifying conditions. The three-line signal was more distinctly observed in the cells cultured in Mn-free medium. This signal could be assigned to nitrosylcytochrome c containing a five-coordinated nitrosylheme. The elemental composition in these cells is also discussed.     

Lysine inhibition of Saccharomyces cerevisiae: role of repressible L-lysine ε-aminotransferase

Lysine added to grain mashes under nitrogen-limiting conditions (as in most industrial fermentations) inhibited growth of Saccharomyces cerevisiae. This inhibition was relieved by raising the assimilable nitrogen content. Lysine-induced inhibition is not mediated through accumulation of -oxoadipic acid, an intermediate of lysine metabolism which accumulates by a back up of intermediates in de novo synthesis. Lysine degradation is regulated by the synthesis of L-lysine -aminotransferase, an enzyme that catalyses the first step in one of three possible routes of lysine degradation (not previously reported in S. cerevisiae). Synthesis is repressed under nitrogenlimiting conditions, but derepressed when excess assimilable nitrogen is available. Derepression results in degradation of lysine and decreases inhibitory effects on growth. The toxic compound appears to be lysine itself.The authors are with the Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 0W0. Canada     

The role of retinol in the initiation of sporangiophores of Phycomyces blakesleeanus

The initiation of sporangiophores of Phycomyces was analyzed under oxygen-limiting conditions. Mutants lacking -carotene have a higher oxygen threshold than the wild type depending on the residual amount of -carotene. The supersensitivity to low oxygen tension is specific for sporangiophore initiation and can be suppressed by addition of either retinal, retinol or retinol acetate to the medium. It is suggested that retinol is a natural regulator of differentiation in Phycomyces.     

Construction of an industrial polyploid strain of Saccharomyces cerevisiae containing Saprolegnia ferax β-amylase gene and secreting β-amylase

An industrial polyploid strain of Saccharomyces cerevisiae containing Saprolegnia ferax -amylase gene was developed by using two yeast integrating plasmids. One plasmid was constructed that contains the geneticin resistance gene (Gt^r) as the selection marker and the ribosomal DNA (rDNA) portion that comprises the 18S rDNA as the recombination site. The other plasmid contains the aureobasidin A resistance gene (AUR1-C) as the selection marker and the chromosomal Ty sequence as the recombination site. The -amylase activity of one clone of Saccharomyces cerevisiae transformed sequentially with these two plasmids was approx. 9 times higher than that of Saprolegnia ferax. This type of integration was mitotically stable even after 100 generations of cell multiplication under non-selective conditions.     

In vitro Activation of Protein Kinase C by β-N-oxalyl-l-α, β-diaminopropionic acid,the Lathyrus sativus Neurotoxin

-N-oxalyl-l-,-diaminopropionic acid (l-ODAP) toxicity has been associated with lathyrism; a spastic paraparesis caused by excessive dietary intake of the pulse Lathyrus sativus. We investigated the effect of Lathyrus neurotoxin l-ODAP on protein kinase C (PKC) activity under in vitro conditions. l-ODAP activated phosphorylation activity of purified chick brain PKC. Both lysine-rich (histone III-S) and arginine-rich (protamine sulfate) substrate phosphorylation was enhanced in the presence of l-ODAP. The activation is concentration dependent, and maximal activation is observed at 100 M concentration. Protamine sulfate phosphorylation was enhanced by 47%, whereas histone III-S phosphorylation was enhanced by 50% over PS/PDBu/Ca²⁺ dependent activity. The nontoxic d-isomer (d-ODAP) did not affect both histone III-S and protamine sulfate phosphorylation activity. These results indicate that l-ODAP taken up by neuronal cells could also contribute to PKC activation and so be associated with toxicity.     

Genes encoding α-amylase inhibitors are located in the short arms of chromosomes 3B, 3D and 6D of wheat (Triticum aestivum L.)

Summary Three -amylase inhibitors, designated Inh. I, II and III have been purified from the 70% ethanol extract of hexaploid wheat (Triticum aestivum L.) and characterized by amino acid analysis, N-terminal amino acid sequencing and enzyme inhibition tests. Inhibitors I and III have identical N-terminal sequences and inhibitory properties to those of the previously described 0.19/0.53 group of dimeric inhibitors. Inhibitor II has an N-terminal sequence which is identical to that of the previously described 0.28 monomeric inhibitor, but differs from it in that in addition to being active against -amylase from Tenebrio molitor, it is also active against mammalian salivary and pancreatic -amylases. Compensating nulli-tetrasomic and ditelosomic lines of wheat cv. Chinese Spring have been analysed by two-dimensional electrophoresis, under conditions in which there is no overlap of the inhibitors with other proteins, and the chromosomal locations of the genes encoding these inhibitors have been established: genes for Inh. I and Inh. III are in the short arms of chromosomes 3B and 3D, respectively, and that for Inh. II in the short arm of chromosome 6D.     

Behavioral responses of a leaf beetle to injury-related changes in its salicaceous host

Summary Adults of the leaf beetle Plagiodera versicolora Laich, avoided previously injured shoots of Salix alba Tristis in favor of nearby uninjured ones. The response was rapid and persisted for five days of study. During brief periods of observation, the vast majority of beetles engaged in behaviors other than locomotion such as feeding and resting. Furthermore, the proportions of beetles walking were similar on injured and uninjured shoots. It appears that under these experimental conditions previous injury did not greatly increase searching behavior of the beetles. Leaves on previously injured shoots received less subsequent herbivory than uninjured ones. Findings of these experiments are consistent with patterns of herbivory observed in the field and known reproductive responses of beetles to diets of injured leaves. These results provide new information toward understanding relatively low levels of herbivory on individual willow leaves associated with the feeding of P. versicolora.     

Tree diversity change in remaining primary mixed-broadleaved Korean pine forest under climate change and human activities

Studying biodiversity change in existing typical ecosystems of the world under possible global climate change and local human activities is important for diversity conservation. An adapted forest dynamics model is used to simulate tree diversity change of the remaining primary mixed-broadleaved Korean pine forest (RPMKPF) in northeast China under global climate change and local human activities for the next 50 years. Human activities include logging, which removes all big trees (DBH > 50 cm), removing all individuals of each single species and all species of each functional type (shade tolerant, shade intolerant and medium type tree species). As results for RPMKPF, the index of tree diversity decreases under climate change, but it increases significantly under a combination of climate change and logging. Removing all individuals of each single species significantly affects the tree diversity of the ecosystem. After the removal of shade tolerant species, both and _c indices of tree diversity experience a significant change. The index decreases significantly under climate change when shade intolerant or medium type tree species are removed, but the _c index does not change significantly. The results of this study have implications for tree diversity management in RPMKPF under climate change and human activities.     

Tremorgenic mycotoxins produced by strains of Penicillium spp. isolated from toxic Poa huecu parodi

Seventeen strains of Penicillium spp. have been isolated from Poa huecu Parodi from the Zapala zone, exhibiting toxicity to sheet. The following strains have been identified: P. crustosum, cyclopium, notatum, palitans, puberulum, verrucosum, viridicatum and Penicillium spp. The toxigenic capacity of the strains was studied after growing them under suitable conditions. Toxins produced were analysed by thin layer chromatography (TLC). Penitrem A (PA) and Penitrem B (PB) neurotoxins were identified and quantitated in twelve strains; verruculogen (VERR) and fumitremorgen B (FTB) being present in one of them. The effect of these mycotoxins was studied in mice. Neurological symptoms characteristic of the intoxication by tremorgenic toxins and similar to those observed in sheep suffering from huecu's disease were observed. The possible role of these toxins as causative agents of huecu's disease is discussed.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

The J gene of ?X174: Isolation and characterization of a J gene mutant

Summary The J gene protein of bacteriophage X174 is a component of the mature phage. The previous lack of J gene mutants has prevented an in vivo analysis of J protein functions. A X174 mutant was constructed by inserting an 11 nucleotide sequence into the J gene. This mutant, designated insJ, was viable only in the presence of a wild-type J gene carried on a plasmid that could provide J protein. An analysis of DNA synthesis during insJ mutant infection under non-permissive conditions confirmed that the J protein is not required for viral DNA synthesis.