Regulation of attachment, germination, and appressorium formation by zoospores ofLagenidium giganteum and related oomycetes by chitin, chitosan, and catecholamines

Summary Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, infects the larval stage of most species of mosquitoes and a very limited number of alternate hosts. Host infection by this and other members of Oomycetes is initiated by motile, laterally biflagellate zoospores. Chemical bases for the various degrees of host specificity exhibited by these parasites is not known, but presumably involves receptors on the zoospore surface recognizing compounds either secreted by or on the surface of their hosts. Surface topography had no detectable effect onL. giganteum encystment or appressorium formation. Scanning electron microscopy documented the detachment of flagella during zoospore encystment. Bulbous knobs at the basal end of the detached flagellum were interpreted as encysting zoospores dropping the axoneme and/or the basal body and associated structures to which flagella are attached. Multiple signals appear to be involved in the initial steps ofL. giganteum host invasion. Zoospores of this parasite did not encyst on powdered preparations of chitin or chitosan (deacetylated chitin). Upon dissolution of chitosan in dilute acid followed by drying these solutions to form thin, transparent films, zoospores readily encysted. The degree of reacetylation of these films and the spacing of acetylated and deacetylated residues had no significant effect on zoospore encystment. Zoospores of a strain ofLagenidium myophilum isolated from marine shrimp, that also infects mosquito larvae, encysted on chitosan films. No encystment of spores of the plant parasitePhytophthora capsici was observed on chitin or chitosan films. Simulation of cuticle sclerotization by incubating chitosan films with different catecholamines and tyrosinase significantly reduced zoospore encystment. Zoospores that encysted on chitosan films did not germinate in distilled water. Germination could be induced by adding microgram quantities of bovine serum albumin or proteins secreted by motile zoospores into the water, and to a lesser degree by some amino acids, but not by various cations. Zoospores encysted and germinated on the pupal stage of some mosquito species. Appressoria were occasionally formed, but most subsequently sent out another mycelial branch, apparently without attempting to pierce the pupal cuticle. Methylation of pupal exuviae with ethereal diazomethane or methanol/HCl significantly increased zoospore encystment. Modification of chitin by catecholamines, lipids and protein on the epicuticular larval surface all affected host invasion.Abbreviations BSA bovine serum albumin - CID collision-induced dissociation - DOPA 3,4-dihydroxyphenylalanine - ESI-MS electrospray mass spectrometry - ESI-MS/MS tandem electrospray mass spectrometry - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WGA wheat germ agglutinin - ZAP zoospore aggregation pheromone     

Sialidase-like Enzymes Produced by Group A, B, C, G, and L Streptococci and by Streptococcus sanguis

A group of enzymes were prepared from the culture fluids of streptococci belonging to groups A, B, C, G, and L, and from a strain of Streptococcus sanguis. These streptococcal enzymes (designated St-sialidases) released a substance shown to belong to the sialic acid group from the specific substrate BSM-St, a sialomucoid prepared from bovine submaxillary gland. They were inactive on N-acetylneuramin lactose prepared from bovine colustrum and on a sialomucoid prepared from bovine submaxillary mucin, whereas these substances are susceptible to sialidases produced by group K streptococci and by Vibrio cholerae. Some of the St-sialidases were markedly activated by divalent cations, but others showed little response. The heat stability of the enzymes produced by the different strains varied. The optimal pH was between 5.5 and 6.5 with acetate buffer and was about 7 with phosphate buffer. K(m) values were determined for the St-sialidases with BSM-St as substrate.     

Biotransformation of limonene by bacteria,fungi, yeasts,and plants

The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (alpha-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes.     

Fermentation of D-xylose, xylitol, and D-xylulose by yeasts

Fifteen yeasts which can assimilate D-xylose were examined for the ability to convert this pentose to ethanol. In six of the seven genera investigated the conversion was enhanced when air had access to the medium. Therefore, the ability to convert D-xylose to ethanol under these conditions is probably common among yeasts. Growth under the same conditions on xylitol, a putative catabolite of D-xylose, led to only traces of ethanol. The effects of growth on another putative catabolite, D-xylose, were complex, but some of the strains which were among the better producers of ethanol from D-xylose produced less from D-xylulose.     

Phytoextraction of Ni,Pb and,Cd by duckweeds

Abstract

Heavy metals phytoextraction potential of swollen duckweed (Lemna gibba Linn.) and lesser duckweed (Lemna aequinoctialis Welw.) was determined under greenhouse conditions by exposing to untreated industrial/municipal effluent for a period of 21?days. The nickel (Ni), lead (Pb), and cadmium (Cd) concentrations in water samples were measured weekly and in plant biomass at the termination of experiments. Significant differences (p?<?0.05) between initial and final physicochemical parameters and in heavy metal concentrations of plant and water samples were observed. Periodically measured metal concentrations in mediums revealed that removal percentage was dependent on initial Ni (2.15?mg L^?1), Pb (1.51?mg L^?1), and Cd (0.74?mg L^?1) concentrations. The final metal removal percentages were in the sequence of Ni (97%) > Pb (94%) > Cd (90%) when treated with Lemna gibba L. as compared to control (9–12% reduction). High biomass production of Lemna gibba L. resulted in a large metal reduction in the growth medium and the total plant metal contents were in the sequence of Ni (427?µg) > Pb (293?µg) > Cd (105?µg). The lesser duckweed did not survive under experimental conditions. Based on these results, we concluded that Lemna gibba L. is a good candidate for phytoremediation of wastewater.     

Trapping, parasitism, and poisoning of nematodes by fungi

Naturally occurring toxins and nematode-destroying fungi are the two major factors controlling soil nematode populations. Nematode-destroying fungi fall into two groups - hyphal forms, producing traps which capture nematodes, and endoparasitic forms, both common in natural soils. Nematode-destroying fungi have been used in attempts to control levels of pathogenic nematodes in agricultural soils with limited success.     

Modification, by lithium, of catalepsy induced by central cholinergic stimulants and morphine-like drugs

After unique injection LiCl enhances, in albino rat, catalepsy induced by arecoline and oxotremorine perhaps by adenylcyclase inhibition and/or decrease of acetylcholine synthesis. After repetitive injection of LiCl during 5 days, this phenomenon is not observable, probably owing to increase of acetylcholine synthesis. After unique injection of LiCl enhances catalepsy induced by dextromoramide, probably on account of cholinergic properties of this drug. In contrast catalepsy induced by morphine or pethidine is suppressed. This constatation would depend on opposite influence upon cerebral neuromediators : lithium diminishing cerebral serotonin and striatal acetylcholine levels and morphine increasing them. After repetitive injections these phenomenons are not observable.     

Fermentation of glucose, lactose, galactose, mannitol, and xylose by bifidobacteria

For six strains of Bifidobacterium bifidum (Lactobacillus bifidus), fermentation balances of glucose, lactose, galactose, mannitol, and xylose were determined. Products formed were acetate, l(+)-lactate, ethyl alcohol, and formate. l(+)-Lactate dehydrogenase of all strains studied was found to have an absolute requirement for fructose-1,6-diphosphate. The phosphoroclastic enzyme could not be demonstrated in cell-free extracts. Cell suspensions fermented pyruvate to equimolar amounts of acetate and formate. Alcohol dehydrogenase was shown in cell-free extracts. Possible explanations have been suggested for the differences in fermentation balances found for different strains and carbon sources. By enzyme determinations, it was shown that bifidobacteria convert mannitol to fructose-6-phosphate by an inducible polyol dehydrogenase and fructokinase. For one strain of B. bifidum, molar growth yields of glucose, lactose, galactose, and mannitol were determined. The mean value of Y (ATP), calculated from molar growth yields and fermentation balances, was 11.3.     

Chromosome end formation in phage lambda, catalyzed by terminase, is controlled by two DNA elements of cos, cosN and R3, and by ATP.

The terminase enzyme of phage lambda is a site-specific endonuclease that nicks DNA concatemers to regenerate the 12 nucleotide cohesive ends of the mature chromosome. The enzyme's DNA target, cos, consists of a nicking domain, cosN, and a binding domain, cosB. cosB, situated to the right of cosN, comprises three 16 bp repeat sequences, R1, R2 and R3. A similar sequence, R4, is present to the left of cosN. It is shown here that terminase has an intrinsic specificity for cosN which is independent of the R sites. The interaction with cosN is mediated by binding to target sites that include 12 bp on the 5', and 2-7 bp on the 3' side of the nick. Of the four R sites, only R3 is required for the proper formation of ends. When R3 is present, an ATP-charged terminase system correctly catalyzes the production of staggered nicks in cosN, at sites N1 and N2 on the bottom and top strands, respectively. When ATP is omitted, the bottom strand is nicked incorrectly, at the site Nx, 8 bp to the left of N1. If R3 is removed or disabled by a point mutation, nicking in cosN becomes dependent upon ATP but, even in the presence of ATP, bottom strand nicking is divided between sites N1, the correct site, and Nx, the incorrect one. Thus, R3 is an important regulatory element and must reside in cis in respect to cosN. Furthermore, cosN substrates bearing point mutations at N1 and N2 are nicked at sites Nx and Ny, 8 bp to the left of N1 and N2, respectively. When R3 is present and ATP is added, nicking is redirected to the N1 and N2 positions despite the mutations present. Thus, terminase binding to R3, on one side of cosN, regulates the rotationally symmetric nicking reactions on the bottom and top strands within cosN.     

Suppression of Mitochondrial Function by Oxidatively Truncated Phospholipids Is Reversible, Aided by Bid, and Suppressed by Bcl-XL

Oxidatively truncated phospholipids are present in atherosclerotic lesions, apoptotic cells, and oxidized low density lipoproteins. Some of these lipids rapidly enter cells to induce apoptosis by the intrinsic pathway, but how such lipids initiate this process is unknown. We show the truncated phospholipid hexadecyl azelaoyl glycerophosphocholine (Az-LPAF), derived from the fragmentation of abundant sn-2 linoleoyl residues, depolarized mitochondria of intact cells. Az-LPAF also depolarized isolated mitochondria and allowed NADH loss, but did not directly interfere with complex I function. Cyclosporin A blockade of the mitochondrial permeability transition pore partially prevented the loss of electrochemical potential. Depolarization of isolated mitochondria by the truncated phospholipid was readily reversed by the addition of albumin that sequestered this lipid. Ectopic expression of the anti-apoptotic protein Bcl-X_L in HL-60 cells reduced apoptosis by the truncated phospholipid by protecting their mitochondria. Mitochondria isolated from these cells were also protected from Az-LPAF-induced depolarization. Conversely mitochondria isolated from Bid^−/− animals that lack this pro-apoptotic Bcl-2 family member were resistant to Az-LPAF depolarization. Addition of recombinant full-length Bid, which has phospholipid transfer activity, restored this sensitivity. Thus, phospholipid oxidation products physically interact with mitochondria to continually depolarize this organelle without permanent harm, and Bcl-2 family members modulate this interaction with full-length Bid acting as a co-factor for pro-apoptotic, oxidatively truncated phospholipids.Vascular cells are exposed to oxidizing radicals during normal metabolism, but especially so during physiologic and pathologic inflammatory processes. The double bonds of polyunsaturated fatty acyl residues are particularly prone to attack by radicals because the C-H bond situated between two double bonds is relatively weak, allowing a more facile abstraction of hydrogen to produce a radical (1). Because polyunsaturated fatty acyl residues are abundant and are generally esterified in the sn-2 position of the glycerol backbone, common products of oxidative attack on cells and circulating lipoproteins are phospholipids that have been peroxidized at their sn-2 position. These peroxy radicals abstract hydrogen to form hydroperoxy phospholipids, may be reduced to the corresponding alcohol, rearrange (2, 3), or fragment to generate a host of oxidatively truncated phospholipids (4–7).The shortened sn-2 residue of truncated phospholipids, which may also contain a newly introduced polar oxygen function, does not intercalate into the membrane well and is energetically favored to protrude into the aqueous phase, a conformation that disorders phospholipid packing into a bilayer (8–10). Oxidatively truncated phospholipids are more water soluble than their phospholipid precursors and readily associate with plasma albumin (11), plasma membranes (12), and even traffic into cells to lysosomes (12) or mitochondria (13) depending on the structure of the truncated phospholipid.Phospholipid oxidation products can be cytotoxic (14, 15), and at least some of these are toxic because they initiate the apoptotic process of regulated cell death (13). The manner by which oxidatively truncated phospholipids alter cell viability has been ascribed to solubilization of the plasma membrane (14), adduction of mitochondrial proteins (17), temporary physical distortion of the plasma membrane (18), or activation of acid sphingomyelinase activity that alters plasma membrane microdomains by generating ceramide (15, 19). We found that a common oxidatively truncated phospholipid, containing a 9-carbon azelaoyl fragment derived from fragmentation of sn-2 linoleoyl residues, induces apoptosis by the intrinsic caspase cascade with loss of mitochondrial function and not, apparently, from damage of the plasma membrane (13).Members of the Bcl-2 family modulate mitochondria-dependent apoptosis either by promoting apoptosis (Bid, Bad, and Bax) or obstructing this event (Bcl-2 and Bcl-X_L). Aggregation of Bax on the mitochondrial outer membrane forms ion conducting pores and Bcl-X_L associates with mitochondrial outer membranes to suppress this Bax activity (20). In contrast, Bid promotes apoptosis after cleavage to truncated Bid, a regulatory event catalyzed by activated caspase 8 (21). Bid, alone among Bcl-2 family members, displays homology to plant lipid transfer proteins and both truncated and full-length Bid will incorporate fluorescent phospholipids, and not the cognate fluorescent fatty acid, into mitochondrial membranes (22).We determined whether mitochondrial integrity or function were directly affected by oxidatively truncated phospholipids, and then whether Bcl-2 family members alter these effects as they do in other, established apoptotic signaling pathways. We find that truncated phospholipids accumulated from the extracellular environment depolarize intracellular mitochondria, that these bilayer challenged phospholipids reversibly interact with mitochondria to continually reduce their transmembrane potential, and that Bcl-2 family members modulate this interaction.     

Phylogeny of snake trypanosomes inferred by SSU rDNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 2.4% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannamyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.     

Hemolysis of human erythrocytes by hypochlorous acid is modulated by amino acids,antioxidants, oxidants,membrane-perforating agents and by divalent metals

The optimal conditions under which hypochlorous acid (NaOCl) either hemolyzes human RBC or kills monkey kidney epithelial cells (BGM) in culture had been investigated. While in Hank's balanced salt solution (HBSS), micromolar amounts of NaOCl caused full hemolysis and also killed BGM cells, in D-MEM or RPMI media rich in amino acids, 25-40 mM of hypochlorite were needed to induce cell injury. Cells exposed to high amounts of NaOCl became highly refractory to strong detergents. Hemolysis by NaOCl was strongly inhibited by a large variety of antioxidants. RBC treated by subtoxic concentrations either of peroxide, peroxyl radical, NO, cholesterol, PLA2, PLC as well as by N2, argon or by mixture of CO2 (10%) and O2 (90%) became much more susceptible to lysis by NaOCl. On the other hand, while RBC treated by Fe2+, Co2+, and V2+ and to a lesser extent with Cu2+ became highly resistant to NaOCl hemolysis presumably due to NaOCl decomposition, no such effect was found either with Co2+ or by Mn2+. RBC treated by azide to destroy catalase and then incubated with peroxide and with NaOCl failed to undergo hemolysis due to the ability of peroxide to decompose NaOCl. The inhibitory effects of the divalent metals on NaOCl-induced hemolysis were also substantiated by measuring the decrease in pH and by cyclic voltammetry. The findings that like peroxide, NaOCl also synergizes with membrane-perforating agents and with a protease to kill epithelial cells further implicate such "cocktails" in cell injury in inflammatory conditions. Taken together, because of the capacity of many agents to scavenge NaOCl, tissue damage by NaOCl-generated neutrophils can take place primarily if activated neutrophils closely adhere to target cells to avoid the scavenging effects of amino acids and of antioxidants. Therefore, the significance of the data which had tested the cytotoxic effects of NaOCl using cells suspended only in salt solutions, should be reconsidered.     

Hemolysis of Human Erythrocytes by Hypochlorous Acid is Modulated by Amino Acids,Antioxidants, Oxidants,Membrane-perforating Agents and by Divalent Metals

The optimal conditions under which hypochlorous acid (NaOCl) either hemolyzes human RBC or kills monkey kidney epithelial cells (BGM) in culture had been investigated. While in Hank's balanced salt solution (HBSS), micromolar amounts of NaOCl caused full hemolysis and also killed BGM cells, in D-MEM or RPMI media rich in amino acids, 25-40 mM of hypochlorite were needed to induce cell injury. Cells exposed to high amounts of NaOCl became highly refractory to strong detergents. Hemolysis by NaOCl was strongly inhibited by a large variety of antioxidants. RBC treated by subtoxic concentrations either of peroxide, peroxyl radical, NO, cholesterol, PLA?, PLC as well as by N?, argon or by mixture of CO?, (10%) and 0? (90%) became much more susceptible to lysis by NaOCl. On the other hand, while RBC treated by FE²?, Co²?, and V²? and to a lesser extent with Cu²? became highly resistant to NaOCl hemolysis presumably due to NaOCl decomposition, no such effect was found either with Co²? or by Mn²?. RBC treated by azide to destroy catalase and then incubated with peroxide and with NaOCl failed to undergo hemolysis due to the ability of peroxide to decompose NaOCl. The inhibitory effects of the divalent metals on NaOCl -induced hemolysis were also substantiated by measuring the decrease in pH and by cyclic voltammetry. The findings that like peroxide, NaOCl also synergizes with membrane-perforating agents and with a protease to kill epithelial cells further implicate such "cocktails" in cell injury in inflammatory conditions.

Taken together, because of the capacity of many agents to scavenge NaOCl, tissue damage by NaOCl generated neutrophils can take place primarily if activated neutrophils closely adhere to target cells to avoid the scavenging effects of amino acids and of antioxidants. Therefore, the significance of the data which had tested the cytotoxic effects of NaOCl using cells suspended only in salt solutions, should be considered.     

Growth of and fumitremorgin production by Neosartorya fischeri as affected by temperature, light, and water activity

The effects of temperature, light, and water activity (aw) on the growth and fumitremorgin production of a heat-resistant mold, Neosartorya fischeri, cultured on Czapek Yeast Autolysate agar (CYA) were studied for incubation periods of up to 74 days. Colonies were examined visually, and extracts of mycelia and CYA on which the mold was cultured were analyzed for mycotoxin content by high-performance liquid chromatography. Growth always resulted in the production of the tremorgenic mycotoxins verruculogen and fumitremorgins A and C. The optimum temperatures for the production of verruculogen and fumitremorgins A and C on CYA at pH 7.0 were 25, 30, and 37 degrees C, respectively. The production of fumitremorgin C by N. fischeri has not been previously reported. Fumitremorgin production was retarded at 15 degrees C, but an extension of the incubation period resulted in concentrations approaching those observed at 25 degrees C. Light clearly enhanced fumitremorgin production on CYA (pH 7.0, 25 degrees C), but not as dramatically as did the addition of glucose, fructose, or sucrose to CYA growth medium (pH 3.5, 25 degrees C). Growth and fumitremorgin production was greatest at aw of 0.980 on CYA supplemented with glucose or fructose and at aw of 0.990 on CYA supplemented with sucrose. Growth and fumitremorgin production were observed at aw as low as 0.925 on glucose-supplemented CYA but not at aw lower than 0.970 on CYA supplemented with sucrose. Verruculogen was produced in the highest amount on all test media, followed by fumitremorgin A and fumitremorgin C.     

Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin

Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.     

Activation of ROCK by RhoA is regulated by cell adhesion, shape, and cytoskeletal tension

Adhesion to the extracellular matrix regulates numerous changes in the actin cytoskeleton by regulating the activity of the Rho family of small GTPases. Here, we report that adhesion and the associated changes in cell shape and cytoskeletal tension are all required for GTP-bound RhoA to activate its downstream effector, ROCK. Using an in vitro kinase assay for endogenous ROCK, we found that cells in suspension, attached on substrates coated with low density fibronectin, or on spreading-restrictive micropatterned islands all exhibited low ROCK activity and correspondingly low myosin light chain phosphorylation, in the face of high levels of GTP-bound RhoA. In contrast, allowing cells to spread against substrates rescued ROCK and myosin activity. Interestingly, inhibition of tension with cytochalasin D or blebbistatin also inhibited ROCK activity within 20 min. The abrogation of ROCK activity by cell detachment or inhibition of tension could not be rescued by constitutively active RhoA-V14. These results suggest the existence of a feedback loop between cytoskeletal tension, adhesion maturation, and ROCK signaling that likely contributes to numerous mechanochemical processes.