Further studies on the surface charge of various strains of Trichomonas vaginalis and Tritrichomonas foetus

The surface charge of three strains of Trichomonas vaginalis and five strains of Tritrichomonas foetus was determined by direct measurement of the mean cellular electrophoretic mobility (EPM) of cells suspended in solutions of different ionic strength and pH. No differences were observed in the mean EPM among the two species, although significant differences among the strains exist. Strains that are more pathogenic to mouse, as measured using the subcutaneous assay, had a surface more negative. Treatment of the parasites with trypsin or neuraminidase reduced significantly their mean EPM and increased their isoelectric point. Tritrichomonas foetus was more sensitive to the enzyme treatment than T. vaginalis. Enzyme-treated cells recovered their normal EPM if, after enzyme treatment, they were incubated in fresh culture medium. The recovery process of trypsin-treated cells was inhibited 10-20% by addition of inhibitors of either protein synthesis (puromycin) or N-glycosylation of proteins (tunicamycin) to the incubation medium, suggesting that a cytoplasmic pool of sialoglycoproteins may exist. The recovering of the EPM of T. foetus and T. vaginalis previously treated with neuraminidase was inhibited by puromycin or tunicamycin about 40-50% and 17-30%, respectively. These observations suggest that sialoglycolipids exist on the surface of both parasite species, and that they contribute more to the surface charge of T. vaginalis than to that of T. foetus.     

Effect of a phorbol ester on basic surface properties of trichomonads

The effect of nanomolar concentrations of 12-O-tetradecanoil-phorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoaTrichomonas vaginalis andTritrichomonas foetus was evaluated by means of measurements of the parasites’ surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites’ net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.     

The effect of tunicamycin on Leishmania brasiliensis. Glycosylation and the cell surface components

Culture conditions of Leishmania cells were developed to allow the study of the effect of tunicamycin (TM) on glycosylation and on the cell surface components. Leishmania incorporate [¹⁴C]-mannose and [³⁵S]-methionine in vitro. The incorporation of [¹⁴C]-mannose is linear for 150 min and is inhibited by TM (2 g/ml) in a time dependent effect which reaches a plateau of 45% inhibition at 36 h. Under the same experimental conditions [³⁵S]-methionine incorporation into protein is slightly affected. This is reflected by an almost identical polypeptide pattern for TM treated and non-treated cells when analyzed on SDS-PAGE. On the contrary, strong differences were detected on the labeled compounds analyzed on SDS-PAGE followed by autoradiography when the precursor used was [¹⁴C]-mannose. A shift in the electrophoretic mobility of most of the glycopeptides synthesized in the presence of TM was observed, which is also reflected in the structure of the main Leishmania cell surface components.The findings are discussed in the light of biological implications.     

Effect of host age at parasitization by Aphidius sonchi on the development, survival, and reproduction of the sowthistle aphid, Hyperomyzus lactucae

Nineteen cohorts of Hyperomyzus lactucae (L.) (Homoptera: Aphididae) reared at various temperaturelight regimes were exposed to attack by Aphidius sonchi Marshall (Hymenoptera: Aphidiidae) at different ages of their development. Irrespective of host age at the initial parasitization and rearing conditions, the rates of reproduction, development and survival were unaffected until the parasites within had completed approximately 30, 40 and 55% of their total development respectively. As a result, aphids parasitized in the first or early second instar failed to reach maturity. Thereafter, the numbers of young produced by parasitized aphids increased rapidly as the age of the hosts at initial parasitization increased, and aphids parasitized 3 days after the final moult or later showed similar potential to contribute to future population increase.In comparison with the effects on host moulting, those on wing development of the potentially alatiform nymphs appeared much sooner after the beginning of parasitization, but again with the time lags being more or less independent of host age at the initial parasite attack. Consequently, the extent of wing development depended entirely on the age of the hosts when attacked.These results showed that the age of the host at the initial parasite attack is crucial in determining the consequences of parasitization on the host.

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Résumé Dix-neuf cohortes d'Hyperomyzus lactucae L. (Homoptera: Aphididae), élevées dans différentes conditions de lumière et de température, ont été exposées à différents moments de leur développement aux attaques d'Aphidius sonchi Marsh. (Hym. Aphidiidae). Quels que soient les conditions d'élevage et l'âge des pucerons quand ils ont été parasités, la survie, le développement et la reproduction n'ont été affectés que lorsque les parasites avaient accompli 30, 40 et 55% de leur développement total. Ainsi, les pucerons parasités au premier stade ou au début du second ne parvenaient pas à atteindre la maturité. Par la suite, le nombre de jeunes produits par les pucerons parasités a cru rapidement au fur et à mesure que l'âge de ces derniers au moment où ils avaient été parasités la dernière mue ou plus tard présentaient la même potentialité pour contribuer à l'accroissement ultérieur de la population.Comparés aux effets sur la mue, les effets sur le développement alaire des larves potentiellement ailées apparaissent beaucoup plus tôt après le moment où le puceron a été parasité, mais là aussi avec des délais plus ou moins indépendants de l'âge de l'hôte lors de la première attaque du parasite. Par conséquent, le degré de développement des ailes dépendait totalement de l'âge du puceron lorsqu'ila été parasité.Ces résultats ont montré que l'âge du puceron lors de la première attaque détermine les conséquences du parasitisme de l'hôte.

     

SEM studies on the leaf surface of promising mulberry (Morus spp.) genotypes

Mulberry (Moms spp.) leaf quality has a great role in silkworm rearing which in turn affects the overall silk yield. In the recent past, many varieties of mulberry have been evolved considering the morphological characters, growth, yield, and quality parameters based on bioassay. The present investigation was carried out on ten promising mulberry genotypesviz. Tr-10, K-2, S-36, S-54, S-1, V-1, Mysore local, S-13, S-34, and RFS-135 to characterize stomatal size and frequency, trichomes and idioblasts using SEM. These new parameters will provide useful information for cultivars identification as well as for selecting mulberry genotypes adapted to different eco-climatic conditions and assessing the feeding quality of leaf for silkworm rearing.     

A comparative analysis of the effects of in-vivo and in-vitro abscisic-acid treatment on the surface electrical properties of barley chloroplast membranes

The effects of in-vivo and in-vitro abscisic acid (ABA) treatments on the surface charge density () of barley (Hordeum vulgare L.) thylakoids were compared using 9-aminoacridine fluorescence. The estimated surface charge density of isolated thylakoid membranes from control (non-treated) barley leaves was-0.065 C · m^-2. The net negative surface charge density decreased after application of various concentrations of ABA (10^-6, 10^-5 M) for 7 d in-vivo, the more pronounced effect being observed at 10^-5 M ABA. When ABA was added to the suspension of isolated thylakoids the opposite effect was observed. The average charge density increased in in-vitro-treated thylakoids at 10^-5 M ABA to -0.081 C · m^-2. The results are discussed in terms of a specific ABA-induced influence of the composition and-or stoicheometry of charged protein complexes within the thylakoid membranes.Abbreviations and Symbols ABA abscisic acid - 9AA 9-aminoacridine - C, C K⁺ and Mg²⁺ concentrations giving equal relative fluorescence - F 9AA-fluorescence intensity - F_max maximum 9AA fluorescence - surface charge density The authors are grateful to Professor L.P. Popova (Institute of Plant Physiology, Sofia, Bulgaria) for continuous support. This work was supported in part by the Bulgarian Ministry of Science and High Education under research contract No. 519.     

蔗糖异构酶PalI在解脂耶氏酵母中的表面展示

【背景】蔗糖异构酶(PalI)生物转化蔗糖是目前生产异麦芽酮糖的主要方法,但在生产过程中存在的蔗糖异构酶转化蔗糖副产物比例较高、游离酶需要分离纯化等问题限制了异麦芽酮糖工业生产的应用。【目的】构建蔗糖异构酶PalI在解脂耶氏酵母(Yarrowia lipolytica) Po1g中的表面展示菌株,以降低蔗糖异构酶转化蔗糖的副产物比例及其纯化成本。【方法】为获得具有生产PalI能力的Y.lipolytica Po1g表面展示菌株,通过重叠延伸PCR将克雷伯氏菌(Klebsiella singaporensis)LX3的PalI的编码基因PalI与全基因合成的来自Y.lipolytica细胞壁的锚定蛋白Pir1融合,转入Y.lipolytica Po1g中构建表面展示菌株。利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid,DNS)比色定糖法测定表面展示的PalI酶活力并对其酶学性质进行探究,通过高效液相色谱法分析其转化蔗糖的产物。【结果】构建了蔗糖异构酶表面展示菌株Pir1-PalI/Po1g,经DNS法测得展示在Y. lipolytica Po1g表面的Pal...     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l--hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.     

Furoquinoline alkaloid accumulation in Fagara zanthoxyloides cell cultures is highly dependent on the presence of exogenous benzylaminopurine

Culturing a non-habituated cell line of Fagara zanthoxyloides Lam. (Rutaceae) in either auxin-free medium or cytokinin-free medium led to opposite effects on furoquinoline accumulation. It appeared that production of skimmianine and -fagarine in the cells was strongly correlated with the presence of exogenous BAP: the levels of both alkaloids were 9 times lower when cells were cultured without cytokinin than in the control culture. NAA removal induced a slight stimulation of skimmianine and -fagarine accumulations, 1.2 and 1.9 times respectively. Culturing the cells in a PGR-free medium generated skimmianine and -fagarine levels that were 3.5 and 2.1 times lower, respectively, confirming the opposite effects of BAP and NAA on furoquinoline accumulation. Growth was only slightly inhibited when cells were cultured for one passage in the PGR-modified media.Abbreviations BAP 6-benzylaminopurine - MS Murashige-Skoog - NAA -naphtaleneacetic acid - PGR plant growth regulator     

Effect of surfactant protein A on the physical properties and surface activity of KL4-surfactant

SP-A, the major protein component of pulmonary surfactant, is absent in exogenous surfactants currently used in clinical practice. However, it is thought that therapeutic properties of natural surfactants improve after enrichment with SP-A. The objective of this study was to determine SP-A effects on physical properties and surface activity of a new synthetic lung surfactant based on a cationic and hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK, KL(4). We have analyzed the interaction of SP-A with liposomes consisting of DPPC/POPG/PA (28:9:5.6, w/w/w) with and without 0.57 mol % KL(4) peptide. We found that SP-A had a concentration-dependent effect on the surface activity of KL(4)-DPPC/POPG/PA membranes but not on that of an animal-derived LES. The surface activity of KL(4)-surfactant significantly improved after enrichment with 2.5-5 wt % SP-A. However, it worsened at SP-A concentrations > or =10 wt %. This was due to the fluidizing effect of supraphysiological SP-A concentrations on KL(4)-DPPC/POPG/PA membranes as determined by fluorescence anisotropy measurements, calorimetric studies, and confocal fluorescence microscopy of GUVs. High SP-A concentrations caused disappearance of the solid/fluid phase coexistence of KL(4)-surfactant, suggesting that phase coexistence might be important for the surface adsorption process.     

The influence of lignification on the development of vascular tissue inVigna radiata L.

Summary During the early development of mungbean seedlings, treatment with L--aminooxy--phenylpropionic acid (AOPP), a potent specific inhibitor of phenylalanine ammonia-lyase, results in an inhibition of anthocyanin and lignin synthesis. The xylem vessels of the hypocotyl and root of AOPP treated seedlings collapse, and the cellulose microfibrils of the unlignified secondary wall are separated from one another and lie disorganized in the lumen of the mature xylem cell. The differentiation of the secondary cell wall appears unaffected by AOPP treatment, as does the ultrastructure of the wall of the mature phloem fibers of the root which is also lignified in untreated tissue. The results are discussed in the light of current thinking on the role and development of lignification in the xylem vessel.Abbreviation AOPP L-aminooxy--phenylpropionic acid     

Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function : Membrane/lipid rafts,mediators of cytoskeletal arrangement and cell signaling

The plasma membrane in eukaryotic cells contains microdomains that are enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR). These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including signaling receptors and ion channels that communicate extracellular stimuli to the intracellular milieu. Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms depends upon interactions with and dynamic rearrangement of the cytoskeleton. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help regulate lateral diffusion of membrane proteins and lipids in response to extracellular events (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand). MLR regulate cellular polarity, adherence to the extracellular matrix, signaling events (including ones that affect growth and migration), and are sites of cellular entry of certain pathogens, toxins and nanoparticles. The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including polarity of endothelial and epithelial cells, cell migration, mechanotransduction, lymphocyte activation, neuronal growth and signaling, and a variety of disease settings. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.