The role of vanadium in green plants

In a series of experiments, it is demonstrated that the trace element vanadium (4·10^-7 g-at/l as NH₄VO₃) has a considerable positive influence on the synthesis of -aminolevulinic acid (-ALA) in the autotrophically growing green algaChlorella pyrenoidosa, the effect being visible by an enhanced output of the amino acid into the culture medium in presence of levulinic acid (LA). The level of intracellularly accumulated -ALA, however, is not changed in presence of the metal. The V-effect on exogenous found -ALA is suppressed, when LA is added to the nutrient medium at low pH (pH 5), although V-uptake into the algal cells is not disturbed by LA. As demonstrated in culture media with various nitrogen sources (urea, partially hydrolized urea, ammonium salts), the development of the pH during the cultivation time is important for the presentation of the V-effect on -ALA. It is suggested that vanadium acts as a catalyst in the conversion of 4,5-dioxovaleric acid to -ALA by transamination.Abbreviations -ALA -aminolevulinic acid - LA levulinic acid - DOVA 4,5-dioxovaleric acid     

δ-Toxin,unlike melittin,has only hemolytic activity and no antimicrobial activity: Rationalization of this specific biological activity

The antimicrobial activity of a synthetic peptide corresponding to -hemolysin had been examined. The peptide didnot exhibit antimicrobial activity against gram negative and gram positive micro-organisms unlike other hemolytic peptides like melittin. This lack of antibacterial activity arises due to the inability of -hemolysin to perturb the negatively charged bacterial cell surface and permeabilize the bacterial plasma membrane. However, the red blood cell surface has a structure considerably different from bacteria, and does not act as a barrier to molecules reaching the lipid membrane. Hence -toxin can lyse erythrocytes. Thus, the specificity in biological activity has been rationalized in terms of differences, in the interaction of the toxin with the bacterial and red blood cell surfaces.     

Transgenic sugarcane plants resistant to stem borer attack

A truncated cryIA(b) gene encoding the active region of the Bacillus thuringiensis -endotoxin was expressed in transgenic sugarcane plants (Saccharum officinarum L.) under the control of the CaMV 35S promoter. Genetic transformation was accomplished by electroporation of intact cells. The levels of recombinant toxin were established and biological activity tests were performed against neonate sugarcane borer (Diatraea saccharalis F.) larvae. Transgenic sugarcane plants showed significant larvicidal activity despite the low expression of CryIA(b).     

Synaptosomal non-mitochondrial ATPase activities and drug treatment

Energy-using non-mitochondrial ATPases were assayed in rat cerebral cortex synaptosomes and synaptosomal subfractions, namely synaptosomal plasma membranes and synaptic vesicles. The following enzyme activities were evaluated: Na⁺, K⁺-ATPase; high- and low-affinity Ca²⁺-ATPase; basal Mg²⁺-ATPase; Ca²⁺, Mg²⁺-ATPase. The evaluations were performed after four week-treatment with saline [controls] or -adrenergic agents (-yohimbine, clonidine), energymetabolism interfering compound (theniloxazine), and oxygen-partial pressure increasing agent (almitrine), in order to define the plasticity and the selective changes in individual ATPases. In rat cerebral cortex, the enzyme adaptation to four-week-treatment with -yohimbine or clonidine was characterized by increase in both high- and low-affinity Ca²⁺-ATPase activities. The action involves the enzyme form located in the synaptic plasma membranes. The enzyme adaptation to the subchronic treatments with theniloxazine or almitrine was characterized by increase in Na⁺, K⁺-ATPase or Mg²⁺-ATPase activities, respectively. The action involves the enzymatic forms located in the synaptic plasma membranes. Thus, the pharmacodynamic effects of the agents tested should also be related to the changes induced in the activity of some specific synaptosomal nonmitochondrial ATPases.     

Sequential damage in mitochondrial complexes by peroxidative stress

The biochemical characteristics of the electron transfer chain are evaluated in purified non-synaptic (free) mitochondria from the forebrain of 60-week-old rats weekly subjected to peroxidative stress (once, twice, or three times) by the electrophilic prooxidant 2-cyclohexene-1-one. The following parameters are evaluated: (a) content of respiratory components, namely ubiquinone, cytochrome b, cytochrome c₁, cytochrome c; (b) specific activity of enzymes, namely citrate synthase, succinate dehydrogenase, rotenone-sensitive NADH: cytochrome c reductase, cytochrome oxidase; (c) concentration of reduced glutathione (GSH). Before the first peroxidative stress induction, the rats are administered for 8 weeks by intraperitoneal injection of vehicle, papaverine, -yohimbine, almitrine or hopanthenate. The rats are treated also during the week(s) before the second or third peroxidative stress. The cerebral peroxidative stress induces: (a) initially, a decrease in brain GSH concentration concomitant with a decrease in the mitochondrial activity of cytochrome oxidase of aa₃-type (complex IV), without changes in ubiquinone and cytochrome b populations; (b) subsequently, an alteration in the transfer molecule cytochrome c and, finally, in rotenone-sensitive NADH-cytochrome c reductase (complex I) and succinate dehydrogenase (complex II). The selective sensitivity of the chain components to peroxidative stress is supported by the effects of the concomitant subchronic treatment with agents acting at different biochemical steps. In fact, almitrine sets limits to its effects at cytochrome c content and aa₃-type cytochrome oxidase activity, while -yohimbine sets limits to its effects at the level of tricarboxylic acid cycle (citrate synthase) and/or of intermediary between tricarboxylic acid cycle and complex II (succinate dehydrogenase). The effects induced by sequential peroxidative stress and drug treatment are supportive of the hypothesis that leakage of electrons (as a mandatory side-effect of the normal flux of electrons from both NADH and succinate to molecular oxygen) would be due to alteration in both availability of GSH and the content of components in the respiratory chain associated to energy-transducing system. In this field there is a cascade of derangements involving, at the beginning, the complex IV and, subsequently, other chain components, including cytochrome c and, finally, complexes II and I.     

Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3--glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and pathogenesis-related proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.Abbreviations BMT S-adenosyl-L-methionine: bergaptol O-methyltransferase (EC 2.1.1.-) - 4CL 4-coumarate: CoA ligase (EC 6.1.1.12) - CMT S-adenosyl-L-methionine: caffeate O-methyltransferase (EC 2.1.1.-) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - PR pathogenesis-related - XMT S-adenosyl-L-methionine: xanthotoxin O-methyltransferase (EC 2.1.1.-)     

Up-regulation of glutathione metabolism and changes in redox status involved in adaptation of reed (Phragmites communis) ecotypes to drought-prone and saline habitats

The glutathione (GSH) metabolic characteristics and redox balance in three ecotypes of reed (Phragmites communis), swamp reed (SR), dune reed (DR), and heavy salt meadow reed (HSMR), from different habitats in desert regions of northwest China were investigated. The DR possessed the highest rate of GSH biosynthesis and metabolism with the lowest levels of total and reduced GSH and its biosynthetic precursors, gamma-glutamylcysteine (gamma-EC) and cysteine (Cys), of the three reed ecotypes. This suggests that a higher rate of GSH biosynthesis and metabolism, but not GSH accumulation, might be involved in the adaptation of this terrestrial reed ecotype to its dry habitat. The HSMR shared this profile although it exhibited the highest reduced thiol levels of the three ecotypes. Two key enzymes in the Calvin-cycle possessing exposed sulfhydryl groups, NADP(+)-dependent glyceraldehydes-3-phosphate dehydrogenase (G3PD) and fructose-1,6-bisphosphatase (FBPase), and other two key enzymes in the pentose-phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGD), had very similar activities in the three reed ecotypes. Compared to the SR, the DR and HSMR had higher ratios of NADPH/NADP+ and NADH/NAD+, indicating that a more reduced redox status in the plant cells might be involved in the survival and adaptation of the two terrestrial reed ecotypes to long-term drought and salinity, respectively. These results suggest that changes of GSH metabolism and redox balance were important components of the adaptation of reed, a hydrophilic plant, to more extreme dune and saline habitats. The coordinated up-regulations of the rate of GSH biosynthesis and metabolism and reduction state of redox status of plant cells, conferred on the plant high resistance or tolerance to long-term drought and salinity.     

Unexpected Transformations of Natural Chlorins under Treatment with Dichlorodicyanobenzoquinone

The treatment of natural chlorins with 2,3-dichloro-5,6-dicyanobenzoquinone resulted not only in the intramolecular cyclization of the propionic acid residue in position 17 with the formation of an additional -lactone cycle at the pyrrole ring D, but also in the oxygen-assisted oxidation of 8-ethyl group in ring B to an -methoxyethyl substituent.     

Induction of crassulacean acid metabolism in Mesembryanthemum crystallinum increases reproductive success under conditions of drought and salinity stress

Summary Mesembryanthemum crystallinum L., an inducible crassulacean acid metabolism (CAM) plant, was grown for approximately 5 weeks following germination in well-watered, non-saline soil in a controlled-environment chamber. During this time, plants were characterized by C₃ photosynthetic carbon metabolism. After the initial 5 weeks, CAM was induced by a combination of high soil salinity and reduced soil water content. One group of plants was allowed to engage in CAM by being continuously exposed to normal CO₂-containing air (about 350–400 ppm). A second group of plants was deprived of ambient CO₂ each night (12 h dark period) until completion of their life cycle, thereby minimizing potential carbon gain via dark CO₂ fixation. The capacity to express CAM under conditions of drought and salinity stress markedly improved reproductive success: plants kept in normal CO₂-containing air produced about 10 times more seeds than plants kept in CO₂-free air during dark periods. Seeds from plants deprived of ambient CO₂ overnight had more negative ¹³C values than seeds from plants kept in normal air.     

Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance

Biolistic transformation was used to introduce genes encoding the insecticidal proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA) and cry1Ac Bt toxin (-endotoxin from Bacillus thuringiensis) into elite rice (Oryza sativa) cultivars. Plant transformation was carried out in parallel experiments simultaneously by using either whole plasmids containing suitable gene constructs, or the corresponding minimal gene cassettes, which were linear DNA fragments lacking vector sequences excised from the plasmids. Both transformation methods generated similar numbers of independent transformation events. Selected R₀ clonal plant lines were further characterised for presence and expression of transgenes. Co-transformation of the unselected genes (cry1Ac and gna) with the selectable marker (hpt) was at least as efficient for transformation with minimal gene cassettes as with whole plasmid DNA, and higher levels of accumulation of the insecticidal gene products GNA and cry1Ac were observed in plants resulting from minimal gene cassette transformation. Insect bioassays with major pests of rice showed that transgenic plants expressing gna showed enhanced resistance to brown planthopper (Nilaparvata lugens), and plants expressing cry1Ac were protected against attack by striped stem borer (Chilo suppressalis). Expression of both transgenes gave protection against both pests, but did not increase protection against either pest significantly over the levels observed in plants containing a single insecticidal transgene.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis α-amylase gene

The gene encoding Schwanniomyces occidentalis -amylase (AMY) was introduced into the chromosomal sequences of an industrial strain of Saccharomyces cerevisiae. To obtain a strain suitable for commercial use, an -integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker. The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p). The -amylase activity of Sacc. cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch. occidentalis. The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.     

Calcium-Dependent Regulation of Interactions of Caldesmon with Calcium-Binding Proteins Found in Growth Cones of Chick Forebrain Neurons

1. This study was undertaken to determine if caldesmon, calmodulin, S100, and neurocalcin were present in chick forebrain neurons, and if so, to investigate the interactions of these proteins in the presence of different concentrations of calcium.2. Immunocytochemistry was used to determine the presence and localization of these proteins in cultured forebrain neurons. Western blotting, gel electrophoresis in the presence of different concentrations of calcium, chemical cross-linking, and affinity chromatography were used to investigate the interactions of these proteins with each other.3. Our data show that caldesmon and three calcium-binding proteins (S100, calmodulin, and neurocalcin ) are localized in growth cones and neurites of chick forebrain neurons in culture. In the presence of different concentration of calcium, these calcium-binding proteins have different affinities to caldesmon and to each other. S100 binds with greater affinity than calmodulin to caldesmon, and its ability to bind to caldesmon is regulated by neurocalcin .4. These findings suggest a specific calcium-dependent regulatory pathway for modulating actomyosin during growth cone motility.     

Evidence suggesting energy-dependent formaldehyde transport in an RuMP-type methylotroph (T15)

Formaldehyde accumulation ratios ([¹⁴CH₂O]_i/[¹⁴CH₂O]_o) as high as 12-fold were measured in anaerobic, CH₃OH-energized, whole cell suspensions of the ribulose monophosphate (RuMP)-type methylotrophic strain T15. Uptake kinetics were extremely rapid, enabling the attainment of equilibrium in only 10–30 s. Transport appears to be energy-dependent and associated with the protonmotive force (pmf). Anaerobic incubation with 5 M carbonyl p-(trifluoromethoxy)-phenylhydrazone (FCCP) led to 70%–90% reduction of the accumulation ratio. Though not as pronounced, diminished uptake was also observed in the presence of 140 M nigericin, 161 M valinomycin and 90 mM KSCN, commensurate with their effects on pmf. Accumulation of CH₂O as a function of external pH followed a trend more similar to that of pmf than either pH or . Preventing energization by incubation with 100 M N,N-dicyclohexylcarbodiimide (DCCD) led to nearly 80% inhibition of CH₂O transport. Over short time periods it was possible to chase accumulated ¹⁴CH₂O from previously loaded cells by collapsing pmf; however, this technique also indicated that significant ¹⁴CH₂O incorporation began to occur within 3 min.Abbreviations FCCP Carbonyl cyanide p-(trifluoromethyoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - RuMP ribulose monophosphate - TPP⁺ tetra[U-¹⁴C]phenylphosphonium - pmf protonmotive force     

The Effect of Polyethylene Glycol on Proline Accumulation in Rice Leaves

The regulation of proline accumulation in polyethylene glycol (PEG, –1.5 MPa) treated rice leaves was investigated. PEG treatment resulted in a decrease in relative water content, indicating that PEG treatment caused water stress in rice leaves. Proline accumulation caused by PEG was related to protein hydrolysis, an increase in ornithine--amino- transferase activity, an increase in the content of ammonia, and an increase in the contents of the precursors of proline biosynthesis, glutamic acid, ornithine, and arginine. Results also show that abscisic acid accumulation is not required for proline accumulation in PEG-treated rice leaves.     

Electrophoretic variation at flight-related enzyme loci and its possible association with flight capacity inEpiphyas postvittana

Variations at three flight-related enzyme loci, -glycerophosphate dehydrogenase (-Gpdh), glucose-6-phosphate dehydrogenase (G6pd), and phosphoglucomutase (Pgm), ofEpiphyas postvittana (Walker) moths were investigated using starch gel electrophoresis. Among the three enzyme loci, -Gpdh andG6pd were found to be monomorphic, butPgm was polymorphic, with a total of seven different genotypes and five alleles identified in this study. Comparisons of allozyme variability at thePgm locus showed significant differentiation among five natural populations sampled from geographically distinct localities in New Zealand and Australia and between laboratory populations differentiated by artificial selection on flight capacity. ThePgm polymorphism was shown to be associated with the variation of flight capacity, but the role of the enzyme locus in the evolution of flight behavior is to be demonstrated in this species.This work was supported by the research scholarship of the University of New England.     

Rotala cookii: A new species ofLythraceae from India showingHippuris syndrome

A new aquatic species ofRotala (Rotala cookii) is described from Kerala, India. Growing in the flooded lowlands, along the coastal belt, the plant is a Hippuris mimic.     

Expression of insecticidal activity in Rhizobium containing the δ-endotoxin gene cloned from Bacillus thuringiensis subsp. tenebrionis

Bacillus thuringiensis subsp. tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the -endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.Abbreviations B.t.t. Bacillus thuringiensis subsp. tenebrionis - IPTG isopropyl--D-thiogalactoside     

Stimulation of crystallin synthesis in embryonic brain cells in culture

Summary Expression of -crystallin, a lens-specific protein, in 6-day-old chick embryonic brain cells was examined in situ and in vitro. The presence of minute amounts of -crystallin and its mRNA (-mRNA) in brain cells in situ was demonstrated by immunoblot and Northern blot analysis. In spreading cultures of the brain cells, -crystallin and -mRNA showed a significant increase from their in situ level. Immunohistological staining (peroxidase antiperoxidase) with monospecific anti-serum against -crystallin revealed that -producers were both epithelial cells and dendritic cells. Neither lentoidogenesis nor -crystallin expression was observed. Stimulation of -crystallin synthesis in cultured brain cells differed when compared with transdifferentiating cultures of neural retina cells. In the latter, -crystallin synthesis occurred concomitantly with differentiation of morphologically distinct lens cells containing -crystallin.