Endosymbiotic bacteria and their relationship with chlorella in the ciliate Climacostomum virens

Structure of cytoplasmic bacterial symbionts of chlorella-free ciliate Climacostomum virens has been investigated. It is shown that ciliates are not able to support simultaneously growth and duplication of two different symbionts--bacteria and chlorella. Cells of C. virens lost bacterial symbionts after an artificial infection with chlorella by microinjection. Competitive relationships between two endopionts are discussed.     

Simple synthesis of climacostol, a defensive secretion by the ciliate Climacostomum virens.

Climacostol [1,3-dihydroxy-5-[(Z)-2'-nonenyl]benzene, 1], a defensive secretion by the protozoan ciliate Climacostomum virens against predators, was synthesized in a 43% overall yield in three steps by starting from methyl 1,3-bis(tert-butyldimethylsilyloxy) phenylacetate (3).     

Defense function of pigment granules in the ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus (Rhizopodea) and Climacostomum virens (Ciliata)

The defense function of pigment granules in the red ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus and Climacostomum virens, was investigated by (1) comparing normally-pigmented and albino mutant cells of B. japonicum as the prey of these predators and (2) comparing resistance of the predators to blepharismin, the toxic pigment contained in the pigment granules of B. japonicum. Normally pigmented cells which contained more blepharismin than albino cells were less vulnerable to A. proteus than albino cells, but not to C. virens. C. virens was more resistant than A. proteus to the lethal effect of blepharismin. The results indicate that pigment granules of B. japonicum function as defense organelles against A. proteus but not against C. virens and suggest that successful defense against a predator depends on the susceptibility of the predator to blepharismin.     

Infection of algae-free Climacostomum virens with symbiotic Chlorella sp. isolated from algae-containing C. virens

The ciliate Climacostomum virens normally contains algae as symbionts in its cytoplasm and retains them over many generations. An aposymbiotic strain of C. virens which cannot re-establish a new symbiotic association by ingestion of algae derived from green Climacostomum was recently isolated in our laboratory. Results of infection experiments showed that all newly ingested, potentially symbiotic algae were digested in food vacuoles. To clarify whether these ciliates have completely lost their ability to sustain symbiosis with algae or whether this ability can eventually be re-established, infection experiments were performed using a microinjection technique. We have achieved successful infection of algae-free Climacostomum using this method. The endosymbiont population was established in ciliates from as few as 3-5 injected algae, which have retained an intact perialgal vacuole membrane around them. To our knowledge, this is the first evidence of successful infection of aposymbiotic ciliates with algae by microinjection.     

Simple Synthesis of Climacostol,a Defensive Secretion by the Ciliate Climacostomum virens

Climacostol {1,3-dihydroxy-5-[(Z)-2'-nonenyl]benzene,1}, a defensive secretion by the protozoan ciliate Climacostomum virens against predators, was synthesized in a 43% overall yield in three steps by starting from methyl 1,3-bis(tert-butyldimethylsilyloxy) phenylacetate (3).     

Photo-oxidative stress in symbiotic and aposymbiotic strains of the ciliate Paramecium bursaria.

We tested the hypothesis that photo-oxidative stress is greater in symbiotic representatives of the freshwater ciliate Paramecium bursaria than in aposymbiotic (i.e., without Chlorella) ones. The level of oxidative stress was determined by assessing reactive oxygen species (ROS) with two fluorescent probes (hydroethidine and dihydrorhodamine123) by flow cytometry in exponential and stationary growth phases of both strains. Photo-oxidative stress was assessed in the laboratory after exposure of the ciliates to photosynthetically active radiation (PAR: 400-700 nm) and PAR+ultraviolet radiation (UVR: 280-400 nm). Additionally, both strains were screened for their antioxidant defenses by measuring the activity of the enzymes catalase, superoxide dismutase (SOD), and glutathione reductase. The results showed that aposymbiotic ciliates had higher levels of PAR-induced oxidative stress than symbiotic ones. Significant differences in PAR-induced oxidative stress were also found in both strains when comparing exponential and stationary growth phases with generally higher values in the former. After exposure to UVR, aposymbiotic ciliates in the stationary phase had the highest levels of ROS despite an increase in SOD activity. By contrast, exposure to UVR decreased catalase activity in both strains. Overall, our results suggest that in this ciliate symbiosis, the presence of symbionts minimizes photo-oxidative stress. This work represents the first assessment of photo-oxidative stress in an algal-ciliate mutualistic symbiosis.     

Actin in the ciliated protozoan Climacostomum virens: purification by DNAse I affinity chromatography, electrophoretic characterization, and immunological analysis.

The anti-actin monoclonal antibody (mab) JLA20 (Lin: Proc. Natl. Acad. Sci. U.S.A. 78:2335-2339, 1981) labels a 43 kD protein on Western blots of Climacostomum cell extracts; this protein does not react with an anti-alpha-smooth muscle actin mab (Skalli et al.: J. Cell Biol. 103:2787-2796, 1986) nor with an anti-alpha-sarcomeric actin mab (Skalli et al.: Am. J. Pathol. 130:515-531, 1988). This protein binds to DNAse I and can be purified by DNAse I affinity chromatography. The affinity-purified actin also reacts with mab JLA20. Two-dimensional gel analysis reveals that Climacostomum actin focuses as three spots which are more basic than the mammalian actin isoforms. After addition of KCl, the affinity-purified actin polymerizes into filaments as shown by electron microscopy after negative staining.     

Comparison of virulence of clones of two Trichomonas vaginalis strains by the subcutaneous mouse assay

No statistical differences in virulence were found among five clones isolated from each of two Trichomonas vaginalis strains JH31A and Balt 42. The former strain, isolated from a patient showing no cervical epithelial abnormalities, caused relatively small subcutaneous lesions in mice [mean volume for the noncloned strain, 75.45 +/- 4.43 mm3 (n = 70); mean of means for cloned populations, 77.28 +/- 3.14 mm3 (n = 230)]. The latter, Balt 42, isolated from a woman with an in situ carcinoma of the cervix uteri, produced large subcutaneous abscesses in mice [mean volume for the noncloned strain, 202.28 +/- 12.53 mm3 (n = 55); mean of means for cloned populations, 200.48 +/- 13.72 mm3 (n = 264)]. The apparent homogeneity of T. vaginalis strains with regard to virulence reinforces the dependability of the subcutaneous mouse assay.     

Microtubules, Microfilaments, and Membranes in Phagocytosis: Structure and Function of the Oral Apparatus of the Ciliate Climacostomum virens

Climacostomum virens uses oral membranelles to drive suspended food particles into its buccal cavity. The cavity leads to a buccal tube which extends into the cell by as much as half a cell length. The inner end of this tube is delimited by a haplokinety (two rows of basal bodies). Internal to this zone is the cytostome and cytopharynx where food vacuoles form. The buccal tube is encircled by a ring of fibrous material, the cytostomal cord, in the region of the cytostome immediately below the haplokinety. Ribbons of postciliary microtubules extend from the kinetosomes of the haplokinety, attach to the cytopharyngeal membrane, and pass under the cytostomal cord. They become broader and expand into the cytoplasm. Cytopharyngeal vesicles pass between the microtubular ribbons and fuse with the cytopharyngeal membrane to generate membrane for forming food vacuoles. The cytopharyngeal vesicles contain a material which is secreted into the forming food vacuoles. Ciliates continue to feed after incubation in a medium containing cycloheximide, indicating that they draw on a pre-existing pool of membrane when forming the food vacuole.     

Comparison of two independent cDNA clones reported to encode beta 1,4 galactosyltransferase

The identity of cDNA encoding beta 1,4 galactosyltransferase (EC 2.4.1.38) has been controversial, since two independent and unrelated cDNAs have been cloned (GTcDNA-1 and -2), both of which are thought to encode beta 1,4 galactosyltransferase. We have resolved this issue by examining the expression of the corresponding mRNAs in tissues possessing varying levels of galactosyltransferase activity. The expression of GTcDNA-1 parallels the level of galactosyltransferase activity assayed enzymatically, while the expression of GTcDNA-2 is unrelated to the level of enzyme activity, being virtually undetectable in tissues with abundant galactosyltransferase activity. GTcDNA-2, therefore, does not likely encode beta 1,4 galactosyltransferase, but rather, encodes a product that indirectly influences enzyme activity following cellular transfection.     

3个中山杉杂种无性系及其亲本的光合特性比较

对‘中山杉405’(Taxodium‘Zhongshanshan 405’)、‘中山杉406’(T.‘Zhongshanshan 406’)和‘中山杉407’(T.‘Zhongshanshan 407’)及其母本墨西哥落羽杉(T.mucronatum Tenore)和父本落羽杉〔T.distichum(L.)Rich.〕在7月份、9月份和10月份的光合参数进行了测定,并比较了3个无性系净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)和水分利用效率(WUE)的杂种优势。结果显示:不同月份3个无性系及其亲本的Pn值有明显差异,导致光响应曲线明显不同,但3个无性系的光响应曲线基本一致;3个无性系的Pn值在不同月份都高于落羽杉,且‘中山杉405’的Pn值高于另2个无性系。3个无性系及其亲本的光补偿点总体上表现为7月份最高、10月份最低,其中7月份墨西哥落羽杉的光补偿点显著高于3个无性系及落羽杉(P<0.05);3个无性系和墨西哥落羽杉的光饱和点则为9月份最高、7月份最低,而落羽杉的光饱和点为9月份最高、10月份最低。7月份3个无性系与其亲本的Pn日变化曲线均呈不规则型,低谷出现在中午12:00左右;9月份和10月份3个无性系和墨西哥落羽杉的Pn日变化曲线均呈单峰型,峰值出现在上午10:00;落羽杉的Pn值从上午8:00开始呈持续降低的趋势,且全天都明显低于其他树种。不同月份3个无性系的Pn、Gs、Tr和WUE的杂种优势有明显差异,总体上看,‘中山杉405’的Pn、Gs和Tr在3个月份中均保持较高的杂种优势率,而‘中山杉406’9月份的杂种优势率最高,‘中山杉407’则在7月份杂种优势率最高;3个无性系的WUE仅在10月份有明显的杂种优势,且‘中山杉405’和‘中山杉406’的杂种优势率远高于‘中山杉407’;‘中山杉405’各项指标的杂种优势率总体上高于另2个无性系。研究结果表明:3个中山杉无性系的光合能力接近其母本墨西哥落羽杉、高于父本落羽杉,其中‘中山杉405’杂种优势明显。     

Labile anion binding by roots of two plum clones

The anion-exchange capacities (AEC) of roots of intact plants of two plum clones [Marianna 2624 (Prunus cerasifera × P. munsoniana) and Myrobalan 3-J (P. cerasifera)] were assessed with an anionic dye, eosin Y. The positively charged eosin-specific adsorption sites were metabolically dependent and also affected by nutrient status. Myrobalan 3-J roots adsorbed twice as much exchangeable NO₃ as did Marianna 2624. Since we previously showed that net nitrate uptake by Myrobalan 3-J persisted at half the ambient nitrate concentration as that characteristic of Marianna 2624, the data provide circumstantial support for a functional role of labile anion binding in active uptake at dilute concentrations of ambient nitrate.     

A Redescription of Climacostomum virens (Ehrenberg) Stein and Proposal of a New Heterotrich Ciliate Family, Climacostomidae fam. n.

SYNOPSIS. Structure of the vegetative and asexually dividing forms of the large ciliate Climacostomum virens is redescribed with emphasis on stomatogenesis. These findings are discussed in relation to the taxonomic and possible evolutionary position of Climacostomum among heterotrichous ciliates. Comparative considerations of the buccal and somatic structures as well as of the stomatogenic patterns in this and other closely related ciliate genera indicate the need for placing Climacostomum and Fabrea in a new family, CLIMACOSTOMIDAE . The morphologic evidence suggests that Climacostomum may represent a line linking Fabrea and Stentor.     

Fitness comparison between clones differing in their ability to produce sexuals in the aphid Thopalosiphum padi

This study aimed at evaluating the intrinsic rate of increase (r_m) of clones of the cereal aphid Rhopalosiphum padi (L.) that differ in their ability to produce sexuals. The value of r_m was measured for wingless parthenogenetic females in experiments conducted at two temperatures (15 °C and 20 °C). We studied six holocyclic clones, five androcyclic clones and five anholocyclic clones and showed that life-cycle has no significant effect on the age at first reproduction, on fecundity nor on longevity. As a result there is no influence on (summer) fitness: on the one hand, holocyclic clones do not compensate their lower fitness in mild winters by a higher fitness during the summer. On the other hand, anholocyclic and androcyclic clones do not seem to obtain any advantage from living on a more restricted range of hosts: there appears to be no cost of polyphenism for holocyclic clones.     

Kinetics of zinc absorption by excised roots of two sugarcane clones

Summary Sugarcane clones differ in regard to susceptibility to zinc (Zn²⁺) deficiency in the field. Excised roots of clone H57-5174 actively absorbed Zn²⁺ at a rate twice that of H53-263 roots. The maximum rates of Zn²⁺ uptake (Vmax) and the Km values also differed markedly between these two clones. H53-263 roots have a 6-fold greater affinity for Zn²⁺ than do those of H57-5174. H57-5174 readily develops Zn²⁺ deficiency symptoms in the field but H53-263 rarely does so. A partial explanation for these varying responses appears to lie in these data.Journal Series No. 1536 of the Hawaii Agricultural Experiment Station.     

Characterisation of two novel CYP4 genes from the marine polychaete Nereis virens and their involvement in pyrene hydroxylase activity

Cytochrome P450 enzymes (CYP enzymes) catalyse the initial step in biotransformation of xenobiotics like polycyclic aromatic hydrocarbons (PAHs). The marine polychaete Nereis virens has a high capacity for biotransformation of PAHs. In the present study, the complete cDNA sequences of two novel CYP genes isolated from N. virens gut tissue are reported. One named CYP342A1, the first member of a new family and the other named CYP4BB1, the first member of a new subfamily. This is the first investigation of specific CYP enzymes from marine polychaetes in which catalytic activity has been determined. Both CYP enzymes had monooxygenase activity and catalysed hydroxylation of pyrene to 1-hydroxypyrene. Based on the present results it is likely that both CYP4BB1 and CYP342A1 are involved in xenobiotic biotransformation. Furthermore, site-directed mutagenesis of the conserved cysteine residue of the heme binding domain resulted in complete loss of monooxygenase activity of both CYP enzymes, indicating that this cysteine residue is indispensable for monooxygenase activity of invertebrate CYP enzymes, as has been well documented in vertebrates. Considering the important role of CYP enzymes in biotransformation of xenobiotics and the presence of N. virens in estuarine environments that accumulates organic xenobiotics, our results are important in understanding the molecular mechanism of biotransformation in marine polychaetes.     

Comparison of two kinds of Biolog microplates (GN and ECO) in their ability to distinguish among aquatic microbial communities.

We compared the abilities of Biolog's GN and ECO plates to distinguish among aerobic and heterotrophic bacterial communities in samples from six aquatic environments. The Biolog system is based on interpreting patterns of sole-carbon substrate utilization indicated by color development in a 96-well microtiter plate. Whether of fresh or saltwater origin, bacterial communities utilized > 95% of substrates in both types of plates. Samples from any one environment exhibited similar time courses of average well color development (AWCD) in both GN and ECO plates. Principal component analysis was performed on data sets resulting from combinations of algorithms (AWCD and curve-integration methods) and levels of color development (end-point and set-point approaches). In all cases, the two types of plates demonstrated an equal capacity to discriminate among the heterotrophic expressions of the six microbial communities. Substantial deviation from an anticipated 1:1 correspondence occurred when color development of 25 substrates common to both types of plates was compared. The discrepancies likely are related to the different formulations of low-nutrient media in GN and ECO plates.     

Coordination ability of pentapeptides with two dehydro-amino acid residues inserted into their sequences

The study on the binding ability of tested ligands have shown that insertion of two dehydro-amino acid residues into peptide sequences makes them more effective in metal ion binding than ligands with one dehydro-amino acid residue. The ligand with two Z(Delta)Phe residue form more stable complexes than his analogues with one Z(Delta)Phe residue. Interesting is this that position of Z(Delta)Phe residue in peptide chain have impact on Cu(II)-complexes formation.     

Preincubation of human resting T cell clones with interleukin 10 strongly enhances their ability to produce cytokines after stimulation

Interleukin 10 (IL-10) has been described as a cytokine inhibitory factor downregulating IL-2 secretion and inducing T cell anergy. The data reported in this study show that preincubation of resting T cells from the human CD4+ clone SP-B21 (and clone TA-23.6) with IL-10 strongly enhances their capacity to further produce IL-2, interferon gamma (IFN-gamma), IL-4 and tumour necrosis factor alpha (TNF-alpha) after subsequent activation. In contrast, when IL-10 was added during the activation step, the previously reported specific inhibition of IL-2 synthesis was observed. Flow cytometric analysis of intracellular IL-2- and IL-4-producing cells revealed that preincubation with IL-10 increased the number of cytokine-producing cells, but did not affect their individual ability to produce these cytokines. We further show that IL-10 plays a dose-dependent role of viability maintenance factor. This effect relates to a direct anti-apoptotic effect of IL-10, which is likely independent of the expression of bcl-2, bcl-x and fas. Such paradoxal properties of IL-10 on T cells should be considered when aiming at using IL-10 as an immunosuppressive molecule in the treatment of diseases.