Food uptake and the fine structure of the dinophytePaulsenella sp. an ectoparasite of marine diatoms

Summary Motile dinospores ofPaulsenella attach to a host diatom frustule, form a feeding tube, drive it between epi- and hypocingulum, pierce the host plasmalemma and suck up host cytoplasm gradually. This mode of endocytosis (myzocytosis) implies that the host plasmalemma is not ingested and that the host cytoplasm within the food vacuole is bounded only by the vacuolar membrane. The feeding tube is formed by the emergence of a preformed microtubular basket consisting of plates of microtubules. At its entrance into the cell body the feeding tube channel is surrounded by an electron-dense ring. Similar sphincters enclose the two exits through which the two flagella emerge. These sphincters are composed of microfibrils which reveal a cross striation when the fixative does not contain calcium ions. The flagellar bases as well as the internal part of the feeding tube are surrounded by a common cavity which is in open connection also with the ampullae of the pusule. The light and electron microscopical observations do not support the assumption that food uptake is driven by a flow of the membrane of the feeding tube channel caused by an interaction with the microtubular basket (as postulated for food uptake inSuctoria) but rather by an hydrostatic gradient which might be caused by rhythmical ion pumping and be based on the existence of the common cavity and the sphincters. Myzocytosis is inhibited by cytochalasin B.—The fine structure of dinospores and trophonts, especially with respect to the cell covering, the amphiesma, and the en- and excystment, is described.     

Preliminary studies on the mechanism of infection and characterization of Malassezia pachydermatis in association with canine otitis externa

A study on the mechanism of infection and the characterization of Malassezia pachydermatis in connection with canine otitis externa was conducted.The ability of this yeast to grow uninhibited in intimate contact with the diversity of other microbial isolants of the canine aural canal was demonstrated. Canine cerumen seemed to promote the growth of this yeast.Although the source of infection is still obscure, M. pachydermatis managed to survive in soil and dust at different temperatures for four weeks.Among the commonly used diagnostic media, Sabouraud's dextrose agar was shown to be the most supportive for the growth of this yeast.The study was presented in parts on the 65th and 66th Conference of Research Worker in Animal disease CRWAD in Chicago, Illinois, 1984 and 1985.     

Different Ks values for hydrogen of methanogenic bacteria and sulfate reducing bacteria: An explanation for the apparent inhibition of methanogenesis by sulfate

Desulfovibrio vulgaris (Marburg) and Methanobrevibacter arboriphilus (AZ) are anaerobic sewage sludge bacteria which grow on H₂ plus sulfate and H₂ plus CO₂ as sole energy sources, respectively. Their apparent K_s values for H₂ were determined and found to be approximately 1 M for the sulfate reducing bacterium and 6 M for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of H₂ consumption by D. vulgaris was five times that of M. arboriphilus, when the hydrogen supply was rate limiting. The apparent inhibition of methanogenesis was of the same order as expected from the different K_s values for H₂. Difference in substrate affinities can thus account for the inhibition of methanogenesis from H₂ and CO₂ in sulfate rich environments, where the H₂ concentration is well below 5 M.     

Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum

Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% -helix, 50%-sheets, 28% random coils, and 3%-turns. This compared to 22% -helix, 44%-sheets, 34% random coils, and no-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25%-helix, 45%-sheets, 27% random coils, and 3%-turns, suggesting a significant alteration at least in the-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at lowpH as indicated by an initial binding rate of 8.4 min^–1 atpH 5.7 compared to 4.0 min^–1 atpH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.     

Characteristics of cellulase preparations affecting the simultaneous saccharification and fermentation of cellulose to ethanol

The cellulase, Spezyme CP from Genencor, widely used for the simultaneous saccharification and fermentation (SSF) of cellulose to ethanol, contained substances inhibitory to the growth of Klebsiella oxytoca P2, emphasising the need to check for inhibition effects in SSF experimentation. Also, the preparation contained enough -glucosidase activity to prevent cellobiose accumulation in SSF with a conventional non-cellobiose fermenting yeast: this finding is relevant to attempts to evaluate novel recombinant cellobiose-fermenting microbial strains.     

Agrobacterium rhizogenes mediated induction of apparently untransformed roots and callus in chrysanthemum

The agropine type Agrobacterium rhizogenes strain LBA9402 induced callus and roots on stems of greenhouse grown plants and on leaf disks of in vitro grown plantlets of chrysanthemum (Dendranthema grandiflora Tzvel.). In this callus and roots no opines were detected, nor were any of the other features of the hairy root syndrome observed. Experiments aimed to identify the nature of the tumour-like growth revealed that induction was correlated with the presence of the TR-DNA on the Ri-plasmid. Root induction was probably the result of auxin synthesis following transient expression of iaaM and iaaH genes, present on the TR-DNA. The chrysanthemum cultivar used, cv. Parliament, showed a high auxin sensitivity compared to tobacco. Analysis of early transformation events using the GUSintron reporter gene revealed that low efficiency gene transfer and transient gene expression took place, but most probably without stable integration of the T-DNA in the plant genome. The results presented here stress the fact that callus formation or root induction as measures for transformation efficiency should be used with caution.     

A novel cyclomaltodextrin glucanotransferase from Bacillus firmus that degrades raw starch

Summary An alkalophilic Bacillus firmus secreting the enzyme cyclomaltodextrin glucanotransferase was isolated from soil. The enzyme attacked raw starch to produce cyclodextrins. Maximum cyclodextrins were produced from tapioca starch followed by potato and corn starch. About 49 % of tapioca starch (at 10 and 50 g/l) was converted to cyclodextrins. The main reaction products were and -cyclodextrins with 40 % and 8 % yield respectively. On prolonged incubation small amount of -cyclodextrin was also produced. The ratio of cyclodextrins was dependent on the initial substrate concentration as well as reaction time.NCL communication number 6203     

Interferometeric sensing of β-galactosidase released by recombinant E. coli responding to an endocrine disruptor, tributyltin

A recombinant E. coli ACV1003 releasing -galactosidase by a SOS regulon system when it is exposed to a DNA-damaging compound, has been used to detect endocrine disruptors such as tributyltin (TBT) and triphenyltin (TPT). Maximum response ratio by E. coli ACV 1003 (recA::lacZ) – indicating the maximum ratio of enzyme produced against an environmental toxicant to that produced in the absence of a toxicant – was estimated as 6.3 with 1.0 g TBT ml^–1 at 37 °C, which was considerably higher than those with other strains. Extracellular -galactosidase activity was 51 unit ml^–1, which was 5% of that obtained by the conventional Miller's enzyme assay using solvents. Such a low enzyme activity can be rapidly determined, not by the usual time-consuming and tedious enzyme assay, but by an alternative interferometric biosensor. Heavily-doped porous silicon to apply to an interferometer was fabricated by etching to produce a Fabry–Pérot fringe pattern, which caused the change in the refractive index of the medium including -galactosidase. The change in the effective optical thickness versus -galactosidase activity showed a sigmoid increase up to the concentration of 250 unit -galactosidase ml^–1.     

Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp.

A cell-bound cyclodextrin-degrading enzyme with a relative molecular mass (M_r) of around 62 000 and an isoelectric point (pI) near 8.0 was isolated and purified to 94% homogeneity from Flavobacterium sp. The enzyme hydrolysed maltooligosaccharides and cyclodextrins to glucose, maltose, and maltotriose. Less glucose, but larger amounts of the line of maltooligosaccharides from maltose to (in case of cyclodextrins) the linearized substrates were found in short-term digests. Digestion of maltotriose yielded glucose, maltose, and some maltotetraose to maltohexaose, i.e. the enzyme catalysed both hydrolysis and transglycosylation. Starch was a poorer substrate, and was hydrolysed to mainly glucose and maltose, presumably by a kind of exo-attack. Pullulan was slightly digested, the products being glucose, panose/isopanose, and larger saccharides containing -1,6-glucosidic bonds. Since maltohexaose to maltooctaose were hydrolysed at higher rates than the cyclodextrins of corresponding lengths, the enzyme of Flavobacterium sp. was proposed to be classified as a decycling maltodextrinase. Correspondence to: H. Bender     

Transference of a crystal protein gene from Bacillus thuringiensis and its expression in Bradyrhizobium sp. cells

The plasmid pHT409 that harbours the cryIA(a) gene for the production of a -endotoxin (crystal protein) from Bacillus thuringiensis was transferred into Bradyrhizobium sp. A conjugal transfer system aiming to introduce the plasmid into the Bradyrhizobium sp. host from colonies of an Escherichia coli donor strain (DH5::pHT409) has been developed. As a result exconjugants were obtained in which the transferred plasmid has been detected by both microbiological and electrophoresis techniques. The cryIA(a) gene when inside the new host had a low expression level which was detected by immunoblotting.     

Production of transgenic rats using cryopreserved pronuclear‐stage zygotes

We investigated the application of cryopreserved pronuclearstage zygotes for the production of transgenic rats. Most of the pronuclearstage zygotes cryopreserved by conventional twostep freezing or vitrification appeared morphologically normal, but the proportion of frozen zygotes that developed into fetuses following transfer (59.7–60.2%) was higher than that of vitrified zygotes (5.5–22.1%). When the frozenthawed zygotes were used for DNA microinjection, 97.5% survived after DNA microinjection and 25.1% of the transferred zygotes developed into fetuses. These proportions were comparable to those of the fresh control zygotes (97.0% and 30.0%, respectively). The integration efficiency of the exogenous DNA into fetuses was similar between the frozen group (3.3% per injected zygote) and the control group (3.5%). These results indicate that pronuclearstage rat zygotes can be successfully cryopreserved by conventional twostep freezing for production of transgenic rats.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

Kinetic properties of bovine brain proteinl-isoaspartyl methyltransferase determined using a synthetic isoaspartyl peptide substrate

Proteinl-isoaspartyl methyltransferase, an enzyme enriched in brain, is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl peptide bonds. We have investigated the kinetics of methylation using a synthetic peptide substrate having the structure Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. Double-reciprocal plots of initial velocity versus concentration of S-adenosylmethionine (AdoMet) at different fixed concentrations of peptide gave straight lines converging at a positive 1/v value and a negative 1/AdoMet value. The product S-adenosylhomocysteine (AdoHcy) was a competitive inhibitor towards AdoMet and a linear mixed-type inhibitor towards peptide. These results are consistent with the rapid-equilibrium random sequential bi-bi mechanism previously proposed for the enzyme, but they also reveal the formation of the deadend, enzyme-peptide-AdoHcy, complex. The rate constants were:V _max=32–34 nmol/min/mg,K ^peptide=7.6–9.4 M,K ^AdoMet=1.9–2.2 M, =0.43–0.53,K ^AdoHcy=0.08 M, =2.9. The interaction factors and indicate that binding of enzyme to peptide increases its affinity for AdoMet and decreases its affinity for AdoHcy. Methylation was linear with time throughout the transfer of 2 mol of methyl groups/mol of enzyme. This absence of burst kinetics suggests that slow release of products cannot explain the low turnover number.Special issue dedicated to Dr. Paul Greengard     

Abundance and half-life of the distinct oat phytochrome A3 and A4 mRNAs

Gene-preferential oligonucleotide probes were used to determined the relative abundance and half-lives of distinct oat phytochrome A (PHYA) mRNAs. Oat PHYA mRNAs are highly conserved in the 5-untranslated region and the coding region, but the 3-untranslated region has an overall lower sequence conservation and was the source of gene-preferential probes. PHYA3 mRNA was estimated to be ca. 61% of the oat PHYA mRNA pool present in poly(A)⁺ RNA from dark-grown seedlings. The half-lives for PHYA3 and PHYA4 mRNAs were both estimated to be ca. 30 min, and a similar short half-life was estimated for the average PHYA mRNA. Sequence comparisons of PHYA mRNAs from four grass species identified conserved sequences within the 5- and 3-untranslated regions that might be important for PHYA mRNA degradation.     

Regeneration of herbicide-tolerant black locust transgenic plants by SAAT

A protocol based on SAAT (sonication-assisted Agrobacterium-mediated transformation) has been developed to obtain herbicide-resistant transgenic black locust (Robinia pseudoacacia L.) plants. Cotyledon explants were co-cultivated with Agrobacterium AGL1 strain carrying the pTAB16 plasmid (bar and gusA genes). The effects of bacterial concentration (OD₅₅₀ of 0.3, 0.6, 0.8) and method of infection (sonication vs immersion) on bacterial delivery were determined by assaying cotyledons for transient -glucuronidase expression 3 days after infection. SAAT increases transient expression efficiency especially at an OD₅₅₀ of 0.6. After determining bacterial concentration and infection method, other factors affecting transformation efficiency, such as explant preconditioning and period of time before applying selection, were tested. From these experiments, the preferred protocol for black locust cotyledon transformation should include sonication of preconditioned cotyledons in AGL1 suspension, coculture for 3 days with 100 µM acetosyringone and transfer to selection medium with 4 mg/l phosphinothricin and 150 mg/l timentin. Of the initial explants, 2% produced at least one transgenic shoot. Genetic transformation was confirmed by Southern hybridization, chlorophenol red assay and herbicide tolerance of the regenerated plants.Abbreviations AS Acetosyringone - BA N-(Phenylmethyl)-1H-purin-6-amine (benzyladenine) - CR Chlorophenol red - 2,4-D 2,4-Dichlorophenoxyacetic acid - GUS -Glucuronidase - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - PPT PhosphinothricinCommunicated by P. Ozias-Akins     

Isolation and biochemical analysis of ethyl methanesulfonate-induced alcohol dehydrogenase null mutants ofArabidopsis thaliana (L.) Heynh

Several mutants have been isolated at theArabidopsis thaliana (L.) Heynh. alcohol dehydrogenase (ADH) gene locus using allyl alcohol selection on ethyl methanesulfonate (EMS)-mutagenized seeds. Eleven mutants were isolated in theADH1-A electrophoretic allele, and 21 in theADH1-S allele. These null mutants are characterized by the absence of measurable ADH activity and genetic data showed that the mutations were confined to theADH1 gene locus ofArabidopsis. Eleven mutants in theADH1-A background were further characterized at the protein and mRNA level. These experiments revealed striking differences in the ADH protein and mRNA content. Some of the mutants did not synthesize any mRNA or ADH-like protein, whereas some of them had a nearly normal level of ADH protein and mRNA. Others had a very low level of both protein and mRNA. ADH null mutants differed physiologically from the wild type by their higher sensitivity to anaerobic treatment in plants and significantly reduced resistance to acetaldehyde in suspension cultures.This research was supported by the Geconcerteerde Onderzoeksactie, Grant 86/91–103, and the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL), Grant 4972A.     

Effect of temperature,hydrogen ion concentration and osmotic potential on zoospore production by threePythium species isolated from pond water

Pythium fluminum produced zoospores most abundantly at 15°C, whereas the optima forPythium group F andP. marsipium were 20 and 25°C, respectively. Increasing the incubation temperature above the optimum resulted in the decrease of the duration of zoospore production. InPythium group F the ability to produce zoospores was not lost even after incubation at 40°C for 24 h. On the other hand,P. marsipium andP. fluminum lost the ability under these conditions. Zoospore production was inhibited at pH 4.5 and 10.5 in all the species tested.Pythium fluminum andP. marsipium were found to have two pH optima for zoospore production (7.5 and 9.5 for the former and 5.5 and 8.5 for the latter). The optimum pH for zoospore production byPythium group F was 6.5–7.5. Moderate osmotic potentials (–0.27–0.47 MPa) appeared to favor zoospore production by the pythia tested. The effect of temperature, pH and osmotic potential on zoospore production was discussed in relation to pollution of pond water.     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole     

Biodegradation of the organochlorine insecticide, endosulfan, and the toxic metabolite, endosulfan sulfate, by Klebsiella oxytoca KE-8

Biodegradation of endosulfan, a chlorinated cyclodiene insecticide, is generally accompanied by production of the more toxic and more persistent metabolite, endosulfan sulfate. Since our reported endosulfan degrader, Klebsiella pneumoniae KE-1, failed to degrade endosulfan sulfate, we tried to isolate an endosulfan sulfate degrader from endosulfan-polluted soils. Through repetitive enrichment and successive subculture using mineral salt medium containing endosulfan or endosulfan sulfate as the sole source of carbon and energy, we isolated a bacterium capable of degrading endosulfan sulfate as well as endosulfan. The bacterium KE-8 was identified as Klebsiella oxytoca from the results of 16S rDNA sequence analysis. In biodegradation assays with KE-8 using mineral salt medium containing endosulfan (150 mg l^–1) or endosulfan sulfate (173 mg l^–1), the biomass was rapidly increased to an optical density at 550 nm of 1.9 in 4 days and the degradation constants for - and -endosulfan, and endosulfan sulfate were 0.3084, 0.2983 and 0.2465 day^–1, respectively. Analysis of the metabolites further suggested that K. oxytoca KE-8 has high potential as a biocatalyst for bioremediation of endosulfan and/or endosulfan sulfate.     

The Genome of the Lepidopteran Mamestra brassicae has a Vertebrate-like Content of Methyl-cytosine

Mamestra brassicae genomic DNAs, isolated from larvae and adult tissues and from in vitro cultured CRL-8003 cells, were enzymatically hydrolysed to nucleosides that were separated by HPLC. HPLC analysis showed that 5mC content in cabbage moth larvae, adults and cultured cells was 8.9±0.5, 9.3%±0.2 and 10.2%±0.4 respectively. Cabbage moth 5mC content results the highest reported till now in insects and it is similar to the typical vertebrate one. Analysis of MspI and HpaII restriction pattern on M. brassicae DNA showed that a portion of its genome was methylated at CpG sites. Moreover, the absence of small digestion products after MspI digestion suggested that CpG are not clustered in the cabbage moth genome. Finally, methylation of repeated DNAs has been studied. Comparison of the restriction pattern of MspI and HpaII after hybridisation with the hobo, mariner, 28S and 5S rDNA probes did not evidence any difference indicating the absence of CpG methylation in all the studied repeated DNAs.