Synthesis of shorter active analogues of Bactenecin7: The effect of change of N-terminal configuration on antimicrobial activity

Summary Bactenecin7, a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa=hydrophobic residues). A series of peptides, Xaa-Pro-Arg-Pro (Xaa=D-Ala, D-Leu, D-Val, D-Phe and D-Lys) were synthesized to investigate the effect of change ofN-terminal configuration on antimicrobial activity. The conformational preferences of these peptides in water and TFE were examined by circular dichroism. All the synthetic peptides with D-amino acid substitution atN-terminal showed potent antifungal activity againstAspergillus niger, Aspergillus flavus andFusarium moniliforme at the concentration level of 8–10 μg ml⁻¹. But the same tetrapeptides were unable to kill or suppress the growth of gram-negative and gram-positive bacteria such asEscherichia coli HB101,Pseudomonas aeruginosa, Klebsiella pneumoniae andStaphylococcus aureus even at the concentration level of 400 μg ml⁻¹. The present study reveals that the change of configuration at theN-terminal of tetrapeptide has negative impact on antibacterial activity but enhanced antifungal activity.     

Screening Antimicrobial Activities of Basic Protein Fractions from Dry and Germinated Wheat Seeds

Two small cationic peptide fractions (5 kDa) were isolated from dry and germinated seeds of wheat, named WAP and GWAP, respectively. The antifungal and antibacterial activities of the peptides were analyzed using disk diffusion and turbidity measurement assays. The peptides in vitro exhibited effective antifungal activity against four plant pathogenic fungi at minimum concentration of 15 g(protein) cm^–3. Their antimicrobial activity was negatively affected by the presence of 5 mM CaCl₂. The peptides were less effective against Gram-negative bacterium Erwinia carotovora subsp. carotovora, but they demonstrated inhibitory activity against Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus. The antimicrobial activity of GWAP was more effective than WAP.     

Stimulation of growth and phospholipase A2 by the peptides mastoparan and melittin and by the auxin 2,4-dichlorophenoxyacetic acid

The peptide mastoparan from wasp venom and the peptide melittin from bee venom stimulated growth in etiolated zucchini (Cucurbita pepo L.) hypocotyls. Both peptides were only effective in hypocotyls with abraded cuticles. At concentrations of 2 g ml^–1 peptide growth was stimulated 72% by mastoparan and 50% by melittin after 2 h as compared to the controls. Mastoparan (5 g ml^–1), melittin (10 g l^–1) and 2,4-dichlorophenoxyacetic acid (5×10^–4 M) stimulated accumulation of ¹⁴C-choline-labeled lysophosphatidylcholine in less than 10 min in cultured soybean cells (Glycine max L.), all to about the same extent. The effects of these peptides are among the first to be reported on plant cells and may be related to important events coupled to growth stimulation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

Increasing glutaminase activity in shoyu koji using a mixed culture of two koji moulds

For improved fermentation of shoyu (soy sauce), a useful koji-making system has been developed using a mixed tane-koji of two shoyu koji moulds, namely Aspergillus oryzae K2 (length of conidiophores about 350 m) and the late-conidiation strain, A. oryzae HG (length of conidiophores about 2500 m). The mixed culture of strains K2 and HG had about twice the glutaminase activity of the single-strain cultures. In addition, the number of conidia in the mixed culture was about 10% of that in a culture of strain K2 alone.     

Antialgal activity of a hepatotoxin-producing cyanobacterium,Microcystis aeruginosa

Antimicrobial activity of toxin produced by a freshwater bloom-forming cyanobacterium Microcystis aeruginosa has been studied. When tested against certain green algae, cyanobacteria, heterotrophic bacteria and fungi, the toxin inhibited growth of only green algae and cyanobacteria. The toxin has been partially purified employing Thin layer chromatography (TLC) and High-performance liquid chromatography (HPLC) techniques and appears to be microcystin-LR (leucine–arginine). Both crude and purified toxins showed toxicity to mice, the clinical symptoms in test mice being similar to those produced by hepatotoxin. Purified toxin at a concentration of 50 g ml^–1 caused complete inhibition of growth followed by cell lysis in Nostoc muscorum and Anabaena BT1 after 6 days of toxin addition. Addition of toxin (25 g ml^–1) to the culture suspensions of the Nostoc and Anabaena strains caused instant and drastic loss of O₂ evolution. Furthermore a marked reduction (about 87%) in the ¹⁴CO₂ uptake was also observed at a concentration of 50 g ml^–1. Besides its inhibitory effects on photosynthetic processes, M. aeruginosa toxin (50 g ml^–1) also caused 90% loss of nitrogenase activity after 8 h of its addition. Experiments performed with ¹⁴C-labelled toxin indicate that the toxin uptake by cyanobacterial cells occurs both in light and dark. These results demonstrate that the toxin is strongly algicidal and point to the possibility that it may have an important role in establishment and maintenance of toxic blooms of M. aeruginosa in freshwater ecosystems. The relative significance of the hepatotoxic effect and the algicidal effect of the toxin is discussed with reference both to survival and dominance of M. aeruginosa in nature.     

Bacterial feeding by the rotifer Brachionus calyciflorus: Clearance and ingestion rates,behavior and population dynamics

Summary The rotifer Brachionus calyciflorus is capable of collecting and ingesting cells or short chains of a laboratory-grown bacterium Aerobacter aerogenes. Clearance rate, the volume of water effectively processed animal ^-1h^-1, does not vary systematically with bacterial density between 0.01 and 100 g dry weight ml^-1. Consequently, ingestion rates are strongly density-dependent, reaching maximal values at the highest food densities tested. Bacterial feeding rates are consistently lower than those determined with larger food types, except in very dense cell suspensions. A. aerogenes in high concentration (100 g ml^-1) induces Brachionus to orient their pseudotrochal cirri to form screens over the buccal funnel; this behavior is at least four times less frequently observed at low (10 g ml^-1) food density. Despite its occurrence, pseudotrochal screening appears ineffective in regulating bacterial ingestion rate. B. calyciflorus can be cultured xenically for greater than 40 generations fed A. aerogenes alone, with no diminution in net reproductive rate or intrinsic rate of natural increase, and no lengthening in cohort generation time.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Ability to use ampicillin as a nitrogen source by the marine cyanobacteriumPhormidium valderianum BDU 30501

The marine non-heterocystous, filamentous cyanobacteriumPhormidium valderianum BDU 30501, which failed to grow in the absence of combined nitrogen in the medium, grew when supplemented with ampicillin. Growth in terms of both protein as well as chlorophyll was proportional to the concentration of ampicillin up to 2000 g ml^–1. With the hydroxylamine assay, the organism was found to produce -lactamase extracellularly with activity reaching a maximum within 6 h of incubation. The maximum production and activity of the enzyme in the culture filtrate was at pH 7 at a concentration of 750 g ml^–1 ampicillin and at 25°C. The crude enzyme was thermolabile, because temperatures above 0°C on storage resulted in the loss of activity within hours. The enzyme could be induced both by ampicillin as well as benzyl penicillin, but ampicillin proved to be a more suitable substrate both in terms of induction as well as activity. This is the first report of an antibiotic serving as a nitrogen source for an organism.     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Effect of zearalenone (F-2) on pea stem,maize root,and rat liver mitochondria

At 5 and 10 g ml^-1 concentration, zearalenone (F-2), a mycotoxin produced by a number of species of the genus Fusarium, causes an inhibition of the oxidative phosphorylation of isolated plant mitochondria, while at 20 and 40 g ml^-1 it causes uncoupling. However, when the mitochondria are pre-incubated for 20 min with F-2, the uncoupling appears to be the prevailing effect. F-2 is also able to inhibit the mitochondrial ATPase activity (Mg²⁺-dependent). Conversely, F-2 (40 g ml^-1) does not alter the ATP level of maize roots and only slightly affects the ATPase activity of pea stem and maize root microsomal fractions. In addition, F-2 (10–40 g ml^-1) inhibits ATP synthesis catalyzed by rat liver mitochondria. It is suggested that the phytotoxicity of F-2, also known for its ability to collapse the transmembrane electric potential of maize roots, may be mainly linked to its ability to increase the proton permeability of the cell, similar to the common uncouplers.Abbreviations F-2 zearalenone - DCCD N,N-dicyclohexylcarbodiimide - PCCP carbonyl cyanide, p-trifluoromethoxiphenylhydrazone - CBT Cerospora beticola toxin     

Inhibition of soil urease activity by aminocresols

Summary The effect on soil urease activity of five aminocresols, at concentrations of 5–100 g/g soil, was examined in the laboratory. Two compounds, 4-amino-o-cresol and 4-amino-m-cresol, significantly inhibited urease activity. The efficacy of 4-amino-o-cresol was compared with that of phenylphosphorodiamidate (PPDA), a known inhibitor, in three U.K. soils. At 50g/g soil 4-amino-o-cresol was as inhibitory as an equivalent concentration of PPDA in a soil with low urease activity, but was less inhibitory in two soils with high urease activity.     

Antifungal activity of allylamines on Epidermophyton floccosum: scanning electron microscopy study

The action of allylamine antifungal agents on Epidermophyton floccosum was studied using scanning electron microscopy. After 7 days of culture on Sabouraud dextrose agar, Epidermophyton floccosum samples were brought in contact with concentrations of 0.2 and 2 g ml^–1 and 0.01 and 0.1 g ml^–1 of naftifine and terbinafine, respectively. Lesions observed after 24 h, 3 and 7 days of contact were mainly on the structure and rigidity of the mycelial and macroconidial wall. They were characterized by hyphal ballooning and twisting and by apical bulbous bulges. Deterioration of macroconidia was characterized by wall exfoliation. The intensity of the deterioration depended on the dose and only slightly on the length of time that the sample and the antifungal drug were in contact.     

Diurnal changes in proline content of desert plants

Summary The diurnal changes in proline and water contents of Zygophyllum quatarense and Francoeuria crispa were investigated under desert conditions. In both plants, the proline content was low before sunrise amounting to 10.42 moles/g fresh weight in Zygophyllum and 6.3 moles/g fresh weight in Francoeuria. By the progress of the day and the intensification of the evaporative power of the atmosphere, the proline content increased considerably in the two plants. The proline content reached maximal values at 5 p.m., reaching 23.36 moles/g in Zygophyllum and 11.16 moles/g in Francoeuria. The proline content of the artificially watered plants was kept at a low level throughout the day. The conditions and role of proline accumulation were discussed.     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

In vitro organogenesis of elephant apple (Feronia limonia)

Elephant apple (Feronia limonia L.). was micropropagated on MS medium containing 4.4 M benzyladenine and 4.6 M kinetin using cotyledon explants taken from in vitro-grown seedlings. Adventitious buds formed on the cotyledon developed into shoots that were rooted in half-strength MS medium containing 0.57 M indoleacetic acid and 0.49 M indolebutyric acid. Plants were successfully established in soil.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - MS Murashige & Skoog     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Endophytic fungi from Musa acuminata leaves and roots in South China

One hundred and sixty-three endophytic fungal cultures were isolated from 200 leaf samples of Musa acuminata trees, which were soaked in 36% formaldehyde solution for surface sterilization. They belonged to the genera of Gloeosporium musae (45%), Myxosporium spp. (11%), Deightoniella torulosa (8.5%), Alternaria tenuis (7.9%), Sphaceloma spp. (7.4%), Aureobasidium spp. (4.3%), Melida spp. (1.8%), Uncinula spp. (1.8%), Penicillium spp. (1.8%), Aspergillus spp. (1.2%), Sarcinella spp. (1.2%), Cladosporium sp. (0.6%), Cephalosporium sp. (0.6%) and sterile mycelium (6.7%). Sixty-eight endophytic fungal cultures were isolated from 100 root samples. They respectively belonged to the genera of Aspergillus spp. (31%), Paecilomyces spp. (16%), Penicillium spp. (15%), Fusarium spp. (10%), Gloeosporium musae (6%), yeast (3%), Deightoniella torulosa (3%), Spicaria sp. (1.4%), Cephalosporrium sp. (1.4%), Meliola sp. (1.4%) and sterile mycelium (10%). Water agar (containing 50 g chloramphenicol ml^–1 and 50 g streptomycin ml^–1) seemed to be a better medium for isolation of endophytic fungi than potato-dextrose agar (PDA, containing 50 g chloramphenicol ml^–1 and 50 g streptomycin ml^–1).