Some pathogenic effects of a species ofPleistophora [Protozoa,Microsporida] forAgrotis exclamationis and other noctuids

Larvae of noctuids were inoculatedper os with spores of a species ofPleistophora isolated fromAgrotis exclamationis (L.). The mean median lethal dose for mainly 3rd instar larvae ofA. exclamationis was 1.38×10⁵ spores per larva accumulating 34 or 35 days after inoculation and the mean slope for the regression of mortality on dose was 0.82. Third instar larvae ofA. exclamationis andSpodoptera frugiperda (J.E. Smith) inoculated with 2.5×10⁴ spores gained weight quicker than uninfected ones until between 8 days (A. exclamationis) and 13 days (S. frugiperda) post-inoculation. Thereafter they grew slower than uninfected individuals. Correspondingly, the feeding rate of inoculated larvae ofA. exclamationis was greater than that of untreated ones until 14 days post-inoculation but thereafter was less. Larvae ofNoctua pronuba (L.)Phlogophora meticulosa L. andSpodoptera littoralis (Boisduval) but notAgrotis segetum (Denis & Schiffermüller) were also susceptible to infection. The species ofPleistophora considered here corresponds toP. schubergi noctuidae (Veremtchuk & Issi) in spore morphology, tissue specificity and host range, except that is was non-infective for the typehostA. segetum. It is probably insufficiently pathogenic for use in the biological control of noctuids.     

Structural analysis of cerebrosides from Aspergillus fungi: the existence of galactosylceramide in A. oryzae

Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and ¹H-NMR. We identified them as β-glucosylceramide and β-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of β-galactosylceramide in A. oryzae.     

Cytotaxonomic study of theVicia cracca complex 1. Czechoslovak taxa

Three races ofVicia cracca L. have been found on the territory of Czechoslovakia, two with the chromosome number 2n=14 and one with 2n=28. Diploid races seem to be more primitive and are less widely distributed. They occur mostly in the primary communities while the tetraploid race is very plastic in the ecological respect and is common both in primary and secondary communities. These races are characterized by some morphological and ecological features. As far as the vertical distribution is concerned, one of the diploid races and the tetraploid occur mostly in the lowland and in the colline zone, the second diploid race has a mountain character. The related tetraploid speciesVicia oreophila ?ertová, with the chromosome number 2n=28 is distributed at similar altitudes.     

The effect of ferrous ions,tungstate and selenite on the level of formate dehydrogenase inClostridium formicoaceticum and formate synthesis from CO2 during pyruvate fermentation

In a medium containing a trace element solution and 10^-4 M ferrous ions the growth yield ofClostridium formicoaceticum on fructose was 5.5 g of weight per l; in the absence of metal ion solution it was 1 g per l. The specific activity of methyl viologen dependent formate dehydrogenase under both conditions was 0.28 and 0.03 units per mg of protein, respectively. It could be increased to 9.75 units when the growth medium contained 10^-4 M tungstate and 10^-5 M selenite in addition. Molybdate was only about 40% as effective as tungstate. Tungstate or molybdate could not be replaced by vanadate, selenite not by sulfide. The formate dehydrogenase catalyzed also the reduction of CO₂ to formate. The highest rate of formate synthesis was observed when pyruvate served as the reductant. No pyruvate: formate exchange but rapid pyruvate: CO₂ exchange could be observed with cell-free extracts ofC. formicoaceticum. Pyruvate is fermented byC. formicoaceticum to yield up to 1.16 mole acetate per mole of pyruvate. Resting cells accumulated some formate in addition to acetate.     

Microbial control of the fruit tree leafroller,Archips argyrospila [Lep.: Tortricidae] in California

Field populations of larvae of the fruit tree leafrollerArchips argyrospila (Wlk). were practically eliminated following spray application ofBacillus thuringiensis Berliner serotype III at 192 and 80×10⁶ I.U. per litre on the host trees,Cercis occidentalis. Spray applications of lower rates ofB. thurienginsis serotype III at 18.9 and 32.1×10⁶ I.U. per litre and mist application ofB. thuringiensis serotype 1 at 8.0 and 16.0×10⁶ I.U. per litre gave partial control of populations ofA. argyrospila larvae. A granulosis typeBaculovirus, applied by hand sprayer at 1.4×10⁹ granules/ml produced approximately 50% reduction of 5th instarA. argyrospila larvae onC. occidentalis trees. It was concluded thatB. thuringiensis and the granulosis typeBaculovirus are promising control agents forA. argyrospila larvae.     

Phosphoenolpyruvate synthetase inMethanobacterium thermoautotrophicum

The thermophilic autotrophMethanobacterium thermoautotrophicum assimilates CO₂ via a novel pathway rather than via the Calvin cycle. The central intermediate of this pathway is acetyl CoA which is reductively carboxylated to pyruvate. Cell extracts of the organism contained phosphoenolpyruvate synthetase with a specific activity of 100 nmol min^-1 mg^-1 protein (65°C). Pyruvate kinase and pyruvate, phosphate dikinase were not detected. Phosphoenolpyruvate synthetase was partially purified (50-fold) and the following reaction stoichiometry was established: $${\text{Pyruvate + ATP + H}}_{\text{2}} {\text{O }} \to {\text{ Phosphoenolpyruvate + AMP + P}}_{\text{i}} $$ The enzyme activity was depedent on free Mg²⁺ ions, NH ₄ ⁺ or K⁺ ions, and SH-groups. Mn²⁺, but not Ca²⁺, could partially substitute for Mg²⁺; Na⁺ could not substitute for K⁺ or NH ₄ ⁺ . The pH-optima,V _max-values and the apparentK _M-values for the substrates of the enzyme in both directions were determined. Thermodynamic, kinetic and regulatory features indicate that, in vivo, the enzyme functions in the direction of phosphoenolpyruvate synthesis from pyruvate. Not only is the synthesis of phosphoenolpyruvate via the PEP synthetase reaction energetically favorable; the enzyme also catalyzed this synthesis 100 times faster than the reverse reaction, the apparentK _M value for pyruvate (40 μM) being low and the apparentK _M value for phosphate (100 mM) being high. Furthermore, AMP, ADP, PP and α-ketoglutarate were inhibitors of PEP synthesis, indicating that the enzyme activity may be controlled in vivo. The role of phosphoenolpyruvate synthetase in autotrophic CO₂ assimilation pathway ofMethanobacterium, as expected from previous labelling studies, is confirmed.     

Halimeda biomass,growth rates and sediment generation on reefs in the central great barrier reef province

The average biomass ofHalimeda per m² of solid substratum increased progressively on a series of reefs situated at increasing distances from the shore in the central Great Barrier Reef. There was none on a reef close inshore, increasing to nearly 500 g m^?2 total biomass (?90% calcium carbonate) on an oceanic atoll system in the Coral Sea. The biomass measured contained 13 species ofHalimeda but was dominated by only two species,H. copiosa andH. opuntia, except on the atoll whereH. minima was dominant. Three sand-dwelling species were also present but did not occur anywhere in substantial quantities. Growth rates of the dominant species were measured bv tagging individual branch tips. A mean value of 0.16 segments d^?1 was recorded but 41% of the branch tips did not grow any new segments whilst only 1% grew more than one per day. The number of branch tips per unit biomass was very constant and has been used in conjunction with growth rates and biomass to calculate productivity rates, and thence sedimentation, in the lagoon of one of the reefs. Biomass doubling time of 15 d and production of 6.9 g dry wt m^?2 d^?1 are considerably higher than previously reported values forHalimeda vegetation and there was little seasonal change detected over a whole year. Those values indicate annual accretion of 184.9 g m^?2 year^?1 ofHalimeda segment debris over the entire lagoon floor (5.9 km²) of Davies Reef, equivalent to 0.13 mm year^?1 due toHalimeda alone, or 1 m every 1,892 years when other contributions to that sediment are taken into account.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

Enzyme(s) responsible for tellurite reducing activity in a moderately halophilic bacterium,Salinicoccus iranensis strain QW6

Oxyanions of tellurium, like tellurate (TeO₄ ^2?) and tellurite (TeO₃ ^2?), are highly toxic for most microorganisms. There are a few reports on the bacterial tellurite resistance mechanism(s). Salinicoccus iranensis, a Gram-positive halophilic bacterium, shows high tellurite resistance and NADH-dependent tellurite reduction activity in vitro. Since little is known regarding TeO₃ ^2? resistance mechanisms in halophilic microorganisms, here one of the enzymatic reduction activities presented in this microorganism is investigated. To enhance the enzymatic activity during purification, the effect of different parameters including time, inoculation, different pHs, different tellurite concentrations and different salts were optimized. We also examined the tellurite removal rates by diethyldithiocarbamate (DDTC) during optimization. In the culture medium the optimum conditions obtained showed that at 30 h, 2 % inoculum, pH 7.5, without tellurite and with 5 % NaCl (w/v) the highest enzyme activity and tellurite removal were observed. Results of the purification procedure done by hydroxyapatite batch-mode, ammonium sulfate precipitation, followed by phenyl-Sepharose and Sephadex G-100 column chromatography, showed that the enzyme consisted of three subunits with molecular masses of 135, 63 and 57 kDa. In addition to tellurite reduction activity, the enzyme was able to reduce nitrate too. Our study extends the knowledge regarding this process in halophilic microorganisms. Besides, this approach may suggest an application for the organism or the enzyme itself to be used for bioremediation of polluted areas with different contaminants due to its nitrate reductase activity.     

Antarctobacter jejuensis sp. nov., isolated from seawater in Jeju,Korea

A novel bacterium, designated strain 13-2-B6^T, was isolated from seawater adjacent to Songak Mountain on Jeju Island, South Korea. The novel strain was observed to be Gram-negative, aerobic, rod-shaped and motile with a single polar flagellum. On the basis of 16S rRNA gene sequence similarity, strain 13-2-B6^T was determined to be phylogenetically closely related to the type strain of Antarctobacter heliothermus, currently the sole species of the genus Antarctobacter (family Rhodobacteraceae). Sequence similarity between the 16S rRNA genes of strain 13-2-B6^T and A. heliothermus EL-219^T is 96.9 %. Strain 13-2-B6^T was found to grow optimally at 25–30 °C, pH 7.0–8.0 and 3 % (w/v) NaCl. The predominant isoprenoid quinone in strain 13-2-B6^T was identified as ubiquinone Q-10 and the major fatty acids were identified as C_18:1 ω7c and/or C_18:1 ω6c. Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, two unknown aminolipids, two unknown phospholipids, an unknown glycolipid and an unknown lipid were found to be components of the polar lipid profile. The G + C content of strain 13-2-B6^T was determined to be 62 mol %. On the basis of its phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain 13-2-B6^T is considered to represent a novel species of the genus Antarctobacter, for which the name Antarctobacter jejuensis sp. nov. is proposed. The type strain is 13-2-B6^T (=KCTC 42009^T =JCM 19898^T).     

Naphthalene oxygenase fromCorynebacterium renale: Characterisation and mechanism of oxygenation

The formation ofcis-l,2,-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified fromCorynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption of one mol oxygen. The enzyme contains one g-atom Fe²⁺ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H₂O₂ was present; the generation of superoxide anion on addition of NADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H₂O₂ was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction     

Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1 ^b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1^a/Igh-4^a heavy chain molecules. The Igh-1^a determinants were maintained, but the Igh-4^a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.     

The systematics of autopolyploidy in Epilobium latifolium (Onagraceae)

Diploid (2n = 36) plants ofEpilobium latifolium L. have been found in Alaska and the western Cordillera of North America, and tetraploids in Iceland, western Greenland, and southeastern Quebec. Study of the morphology of these chromosome races revealed a character, the number of pores in the pollen grains, by which they could be distinguished with an average probability of 75%. Tetraploids ofE. latifolium tend to have high percentages of 4-pored pollen, while the diploids usually have only 3-pored pollen. On this basis, the ranges of the races were extrapolated from herbarium material. Detailed comparisons of eight other features of the presumptive diploids and tetraploids showed no significant difference between them. The races appear to form worldwide, allopatric, ecogeographic phases. A high level (57.4%) of quadrivalent formation observed during tetraploid meiosis, and other considerations, indicate that the tetraploids arose by autopolyploidy. The recommendation is made that the races not be given formal taxonomic recognition.     

Field evaluation of a granulosis virus for control ofPieris rapae [Lep.: Pieridae] in the United Kingdom

A purified granulosis virus isolated fromPieris brassicae (L.) was tested in the field against an introduced population ofPieris rapae (L.) larvae on cabbage (cv January King) in small experimental plots at Littlehampton, Sussex. Experiments were designed to compare the relative efficacy of single and multiple applications of virus (2.1×10¹² and 3.7×10¹² or 2.1×10¹⁴ and 3.7×10¹⁴ virus capsules/ha) in reducing numbers ofP. rapae larvae. An experiment was carried out in June 1978 and repeated in August to coincide approximately with the 2 natural generations ofP. rapae in southern England. Larval populations were monitored by regularin situ assessment of plants and by destructive sampling. Within 10 days of spraying virus there was a significant reduction in the mean larval population on all virus-treated plots compared with untreated controls. Sprays of 2.1×10¹⁴ and 3.7×10¹⁴ capsules/ha reduced larval numbers more quickly than 2.1×10¹² and 3.7×10¹²/ha treatments. In the 1st experiment, three sprays of virus at either 2.1×10¹² or 2.1×10¹⁴ capsules/ha gave no increase over the final level of control achieved by a single spray. However, in the 2nd experiment, a single spray of 3.7×10¹² capsules/ha did not significantly reduce the numbers of larvae. It is likely that this failure could be accounted for by a combination of the larger “natural” population ofP. rapae recorded midway through the 2nd experiment and the rapid inactivation of virus deposits which left little infectious virus to infect these larvae. Virus inactivation was so rapid that only 7–33 % of the initial virus deposits remained 1 day after application. These results suggest that further understanding of virus formulation, persistence and dosage rates are needed before such a virus can be used in a rational manner.     

Factors affecting the determination of total mercury in biological samples by continuous-flow cold vapor atomic absorption spectrophotometry

The acidic reduction of Hg using a continuous-flow analytical system was evaluated. With 25% SnCl₂ as the reductant, characteristic concentrations (sensitivities) of 0.44 μg/L (open cell) and 0.29 μg/L (flow-through cell) were obtained using inorganic Hg²⁺ standards in 1.5% HCl. When CH₃Hg⁺ standards were used, absorption signals were an order of magnitude lower, indicating that Sn(II) is incapable of producing Hg° from organic Hg in this acidic reduction system. Addition of CdCl₂ to the SnCl₂ reductant, as suggested by Magos (1) for the reduction of organomercurials under alkaline conditions, was without beneficial effect. Similarly, combining Sn, with another reducing agent (hydroxylamine hydrochloride), or a strong alkaline solution (40% NaOH), in the reaction coil of the flow-through system did not significantly enhance the Hg absorption signal for either inorganic or organic Hg. Recovery of Hg from spiked liver homogenates digested at 70–80°C using a HNO₃/H₂SO₄/HCl procedure and stabilized with 0.5 mM K₂Cr₂O₇ was >85%, using either inorganic Hg²⁺ or CH₃Hg⁺, indicating that this digestion procedure successfully breaks the C-Hg bond to form readily reducible Hg species. Usingl-cysteine to stabilize standards of inorganic Hg²⁺ in HCl caused significant depressions of the Hg absorption signal atl-cysteine concentrations >0.001% (≈0.5 mM); 0.1%l-cysteine caused total suppression of the Hg signal. These results indicate that: (1) acidic reduction of Hg by Sn in this continuous-flow system requires breakdown of organomercurials prior to analysis; (2) tissue digestion using HNO₃/H₂SO₄/HCl followed by the addition of K₂Cr₂O₇ to stabilize Hg²⁺ achieves this breakdown and allows good recovery of total Hg; and (3) use ofl-cysteine to complex and prevent losses of Hg should be avoided in systems using acidic reduction of Hg. Concentrations of endogenous tissue sulfhydryls are generally lower than those associated with depressed absorbance signals during the acidic reduction of Hg.     

DNA sequence analysis of bacterial toxic heavy metal resistances

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be “linked” together on multiple resistance plasmids. For Cd²⁺, AsO₂ ^?, AsO₄ ^3?, Hg²⁺, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. ThecadA ATPase ofS.aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd²⁺ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K⁺ ATPases ofEscherichia coli andStreptococcus faecalis, the H⁺ ATPases of yeast andNeurospora, the Na⁺/K⁺ ATPases of vertebrate animals, and the Ca²⁺ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in themer system include a DNA-binding regulatory protein from themerR gene and a Hg²⁺ transport system consisting of a periplasmic Hg²⁺-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.     

Formation of GA20 glucosyl conjugates in seedlings ofVicia faba

[³H]GA₂₀ (1)¹, fed toVicia faba seedlings, was converted to [³H]GA₂₀ glucosyl ester (5) and [³H]GA₂₀-13-0-glucoside (6). The GA₂₀ glucosyl ester (5) was identified by HPLC-RC and by GC-MS of GA₂₀-Me formed by transesterification of (5). The [³H]GA₂₀-Me was crystallized to constant specific radioactivity with authentic GA₂₀-Me. On HPLC-RC the GA₂₀-13-0-glucoside (6) was shown to have the same retention time as an authentic sample. Subsequent enzymic hydrolysis gave a product with an HPLC retention time identical to that of authentic GA₂₀ (1).