Characterization of a novel 3-hydroxybutyrate dehydrogenase from <Emphasis Type="Italic">Ralstonia pickettii</Emphasis> T1

We previously reported that the activities of two 3-hydroxybutyrate dehydrogenases (BDH1 and BDH2) were greatly influenced by culture conditions when Ralstonia pickettii T1, a strain growing on extracellular poly-3-hydroxybutyrate (PHB), was grown on different carbon sources such as 3HB and succinate. In this study, knockout mutants of bdh1 or bdh2 were constructed and characterized under different culture conditions. In addition, a novel BDH (BDH3) was found in bdh2 mutants, and bdh3 was cloned. Apparent kinetic parameters for the substrates of BDH3 indicated that the enzyme is suitable for the oxidation reaction of 3-hydroxybutyrate (3HB) to acetoacetate. In Western blotting, it was clear that BDH3 is produced only in cells grown on 3HB or PHB as a carbon source, while BDH1 and BDH2 are produced in cells grown on various carbon sources such as sugars, amino acids, organic acids, 3HB, and PHB. Both the bdh1 and bdh2 mutants lagged behind the wild type in growth rates when the cells were cultured with 3HB, citrate, succinate, or nutrient broth. A test of sensitivity to diamide as an oxidative stress revealed that the lack of BDH1 or BDH2 caused a decline in the capacity to neutralize the stress. These results suggested that BDH1 and BDH2 are needed to regulate the cytoplasmic redox state as well as to utilize 3HB, while BDH3 is specialized to utilize 3HB. The expression of bdh3 may be coordinately regulated with a gene encoding putative 3HB permease.     

Comparison of axonemal proteins from two kinds of Tetrahymena. I. Different characteristics of dyneins in heat stability

Tetrahymena thermophila could still swim after incubation of the cell body at 40°C for 30 min, whereas Tetrahymena pyriformis did not show any motility after the treatment. Turbidity measurements revealed that axonemes of T. pyriformis lost ATP-dependent sliding activity by the heat treatment, whereas those of T. thermophilia still had the activity under the same conditions. In connection with this difference in susceptibility to high temperature, the biochemical characteristics of dyneins were compared between the two species of Tetrahymena. Axonemal dyneins from the two species had significant vanadate-sensitive ATPase activity even after the heat treatment. Native gel electrophoresis and the following two-dimensional electrophoresis showed that the outer arm dynein of T. thermophilia is more stable in maintaining native configuration than that of T. pyriformis against the heat treatment, although both treated dyneins keep three (α, β and γ) subunits. Analysis by peptide mapping demonstrated that β- and γ-subunits of the outer arm dynein are considerably different in amino acid sequences between the two species. These results imply that dynein of T. thermophilia changed their amino acid sequences and biochemical characteristics to adapt to high temperature.     

Characterization of Deciliation-Regeneration Process of Tetrahymena Pyriformis for Cellular Robot Fabrication

Artificial magnetotactic Tetrahymena pyriformis GL (T. pyriformis) cells were created by the internalization of iron oxide nano particles and became controllable with a time-varying external magnetic field. Thus, T. pyriformis can be utilized as a cellular robot to conduct micro-scale tasks such as transportation and manipulation. To complete these tasks, loading inorganic or organic materials onto the cell body is essential, but functionalization of the cell membrane is obstructed by their motile organelles, cilia. Dibucaine HCl, a local anesthetic, removes the cilia from the cell body, and the functional group would be absorbed more efficiently during cilia regeneration. In this paper, we characterize the recovery of artificial magnetotactic T. pyriformis after the deciliation process to optimize a cellular robot fabrication process. After sufficient time to recover, the motility rate and the average velocity of the deciliated cells were six and ten percent lower than that of non-deciliated cells, respectively. We showed that the motile cells after recovery can still be controlled using magnetotaxis, making T. pyriformis a good candidate to be used as a cellular robot.     

梨形四膜虫衰老过程中DNA含量的变化

应用一种新型的细胞核内DNA含量测定方法──图像分析法,测定真核细胞梨形四膜虫衰老过程中DNA含量的变化.根据Beer-Lambert定律,以细胞核在不同生长期内的积分光密度的水平表示核内DNA含量的变化.该方法具有测量速度快,重复性好,操作简单,结果可靠等优点.实验结果表明:四膜虫在进入对数生长期时,DNA含量逐渐达到高峰,随着细胞逐渐老化,细胞分裂次数及核内DNA含量逐步减少.     

Seasonal variations in the encapsulation of eggs of the encyrtid parasitoid Metaphycus stanleyi by the pyriform scale, Protopulvinaria pyriformis

Encapsulation of eggs of the introduced parasitoid Metaphycus stanleyi Compere (Hymenoptera: Encyrtidae) by the pyriform scale, Protopulvinaria pyriformis (Cockerell) (Homoptera: Coccidae) under both greenhouse and field conditions, at Bet Dagan, was found to occur almost all year round (1986–1988). However, encapsulation rates varied considerably during the different seasons and were correlated with the ambient temperatures. The rates of efficient encapsulation (i) in scales infesting Hedera helix and Schefflera arboricola under greenhouse conditions, were lowest during December to May (6–17%) and highest during July to September (78–100%); (ii) in scales infesting avocado in the orchard, were lowest during October to May (0–11%) and highest during June to August (54–57%). Under greenhouse conditions, encapsulation rates did not differ in scales grown on H. helix and on S. arboricola, but were significantly lower in scales grown on avocado. Encapsulation by scales infesting S. arboricola was more frequent at temperatures ranging from 20–28 °C, than from 7–23 °C.The increased resistance by encapsulation of P. pyriformis to successful parasitization by M. stanleyi during the summer, may account for the inability of the parasitoid to prevent the autumn and winter outbreaks of the pest.     

Chemical stimulation of phagocytosis in Amoeba proteus and the influence of external calcium

Summary The process of phagocytosis in Amoeba proteus was examined by following the uptake of Tetrahymena pyriformis and agarose beads. The ciliates are taken up in a time dependent and saturable manner. T. pyriformis apparently emits a water-soluble substance that acts as a chemoattractant to the amoebae. Plain agarose beads are not engulfed by A. proteus, but those beads having reducedglutathione with the -SH group exposed are taken up almost to the same extent as T. pyriformis. Phagocytosis of the glutathione beads is calcium-dependent with maximum bead uptake at 10^-4M Ca⁺⁺. Glutathione applied to A. proteus brings about pseudopod formation, increased phagocytosis and displacement of surface-associated calcium.     

Rotifers as predators on small ciliates

Clearance rates of Synchaeta pectinata, Brachionus calyciflorus and Asplanchna girodi on Tetrahymena pyriformis (46 µm in length) at a density of 10 cells ml^–1, in the presence of algal food, were 2.5 to 6.1 ml rot.^–1 day^–1. Clearance rates of these rotifers were, respectively, about 2, 3, and 13 times lower on Strobilidium gyrans (58 µm in length) than on T. pyriformis, indicating that the saltations of S. gyrans are an effective escape response. Clearance rates of S. pectinata were considerably lower on Colpidium striatum (81 µm) than on S. gyrans, suggesting that S. pectinata may not be able to ingest ciliates of this size. S. pectinata had a clearance rate of 19 ml rot.^–1 day^–1 on S. gyrans at a density of 1.2 cells ml^–1, in the absence of edible algal food. Rotifers may prey extensively on ciliates in natural plankton communities, ingesting 25 to 50 individuals in the 45–60 µm size range day^–1.     

Further studies of the encapsulation of eggs ofMetaphycus spp. (Hym.: Encyrtidae) by the pyriform scale,Protopulvinaria pyriformis (Hom.: Coccidae)

The encapsulation of eggs ofMetaphycus swirskii Annecke &; Mynhardt (Hymenoptera: Encyrtidae) by the pyriform scale,Protopulvinaria pyriformis (Cockerell) (Homoptera: Coccidae), collected in the coastal plain of Israel, was determined during April 1986 to May 1987. The rates of encapsulation were low in November (13.0%) and relatively high in April, May, August and September (32.0–89.0%). The seasonal variations in the encapsulation of eggs ofM. stanleyi Compere and/orM. swirskii byP. pyriformis infesting avocado (Persea americana) and jambolan (Syzygium cumini) were studied at Miqwe Yisra'el (coastal plain) during October 1986 to February 1988. Encapsulation rates were similar in scales infesting either of the two host plants. They were highest during July to August (49.0–75.0%) and lowest during December to February (0.9–10.0%). Encapsulation incidence at Miqwe Yisra'el was correlated with the ambient temperatures (r=0.89). The rate of encapsulation of parasitoid eggs (M. stanleyi and/orM. galbus Annecke) recorded inP. pyriformis sent to Israel from Spain in September 1987 was 42.2%. The high rates of encapsulation ofMetaphycus spp. eggs byP. pyriformis during the summer, may interfere with efficient biological control of the pest.     

Chemotaxis of the unicellular green algaDunaliella salina and the ciliatedTetrahymena pyriformis—effects of glycine,lysine, and alanine,and their oligopeptides

Chemotactic properties of amino acids (L-alanine, glycine and L-lysine) and their oligopeptides (10^–6M) and binding sites to these ligands were investigated in two unicellular models, the heterotrophicTetrahymena pyriformis and the auxotrophicDunaliella salina. Chemotaxis ofDunaliella induced by simple amino acids and their derivatives demonstrated that binding sites (receptors) for food molecules are not only present in the membrane but are also able to induce their basic physiological response. InTetrahymena, substances with special molecular structure and properties (polar, hydrophilic character of the signal peptide chain)-5-L-Lys, 5-Glywere required for chemoattraction, other peptides tested, lacking the required structure, were repellent. Divergences in chemotaxis and binding assays of both species suggest that trends of functional and binding parameters do not run parallel at this level of evolution.     

Expression of heat-shock protein 70 kDa in Tetrahymena pyriformis during cell adaptation to salinity changes in the medium

The alteration of the content of heat-shock protein 70 kDa (Hsp70) was studied in cells of the freshwater ciliate Tetrahymena pyriformis after the salinity of the medium had been changed. It was shown that ciliates acclimated to fresh (0%) or salt (2 and 10%) water have similar levels of constitutive Hsp70. Neither pronounced induction nor a decrease in the Hsp70 level were revealed in ciliates after salinity stress. These data differ from the results we obtained previously with more euryhaline ciliates, Paramecium nephridiatum and P. jenningsi. In those species, we observed both the induced synthesis of Hsp70 after salinity stress and changes (decrease or increase) in the constitutive Hsp70 level after the acclimation of ciliates to the altered medium salinity. We presume that the differences in the chaperone system reaction of these ciliates species may be connected with their different salinity resistances, least of all in P. jenningsi, intermediate in T. pyriformis, and most pronounced in P. nephridiatum.     

Evaluation of endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. tritici in wheat

Endophytic actinobacteria isolated from healthy cereal plants were assessed for their ability to control fungal root pathogens of cereal crops both in vitro and in planta. Thirty eight strains belonging to the genera Streptomyces, Microbispora, Micromonospora, and Nocardioidies were assayed for their ability to produce antifungal compounds in vitro against Gaeumannomyces graminis var. tritici (Ggt), the causal agent of take-all disease in wheat, Rhizoctonia solani and Pythium spp. Spores of these strains were applied as coatings to wheat seed, with five replicates (25 plants), and assayed for the control of take-all disease in planta in steamed soil. The biocontrol activity of the 17 most active actinobacterial strains was tested further in a field soil naturally infested with take-all and Rhizoctonia. Sixty-four percent of this group of microorganisms exhibited antifungal activity in vitro, which is not unexpected as actinobacteria are recognized as prolific producers of bioactive secondary metabolites. Seventeen of the actinobacteria displayed statistically significant activity in planta against Ggt in the steamed soil bioassay. The active endophytes included a number of Streptomyces, as well as Microbispora and Nocardioides spp. and were also able to control the development of disease symptoms in treated plants exposed to Ggt and Rhizoctonia in the field soil. The results of this study indicate that endophytic actinobacteria may provide an advantage as biological control agents for use in the field, where others have failed, due to their ability to colonize the internal tissues of the host plant.     

卡拉麦里南部工业区梭梭和琵琶柴重金属空间分布及污染评价

对五彩湾工业区周边21个采样点的梭梭(Hadoxylon)和琵琶柴(Reaummuria soongorica)的Zn、Cu、Cr、Pb、As、Hg 6种重金属元素含量测定,运用统计学方法和地统计插值法分析其茎叶和根部重金属含量变化情况和空间分布特征,并综合评价其污染程度和潜在生态危害性。结果表明:梭梭和琵琶柴植株Hg含量的最大值和均值都超出新疆土壤背景值0.017 mg/kg,除梭梭根部的均值0.060 mg/kg未超出国家土壤背景值0.065 mg/kg,在二者其他部位的最大值和均值都已超出;琵琶柴整株中Cr最大值72.62 mg/kg和Zn最大值97.61 mg/kg均超出新疆土壤背景值49.3 mg/kg和国家土壤背景值61 mg/kg,Pb元素未被检出。插值精度方面,Hg、As的RMSE较小,分别为0.263和0.443,预测模型中Hg的R~2为0.72,Cu的R~2为0.67,能较好地估计预测样点的重金属含量,Zn的R~2为0.31,精度较低;插值结果,琵琶柴中的Zn、Cr、As、Hg含量较高的区域均在工业园区内部及周围,受人为扰动程度较大。梭梭和琵琶柴中Hg元素分别为中度污染和重度污染;Hg元素为中等潜在危害程度高于其他4种元素。     

Tetrahymena DNA forms a single band in alkaline density gradients

Summary DNA from nuclei ofTetrahymena pyriformis was examined by equilibrium density gradient ultracentrifugation at alkaline pH. The results indicate that the DNA has a uniform distribution of guanine plus thymine in the complementary strands and throughout the nucleotide sequence of the DNA.Abbreviations cDNA chloroplast DNA isolated fromEuglena gracilis - nDNA DNA isolated from nuclei ofTetrahymena pyriformis     

Preventive and therapeutic administration of an indigenous <Emphasis Type="Italic">Lactobacillus</Emphasis> sp. strain against <Emphasis Type="Italic">Proteus mirabilis</Emphasis> ascending urinary tract infection in a mouse model

Probiotics are increasingly being considered as non-pharmaceutical and safe potential alternatives for the treatment and prevention of a variety of pathologies including urinary tract infections. These are the most common infections in medical practice and are frequently treated with antibiotics, which have generated an intense selective pressure over bacterial populations. Proteus mirabilis is a common cause of urinary tract infections in catheterised patients and people with abnormalities of the urinary tract. In this work we isolated, identified and characterised an indigenous Lactobacillus murinus strain (LbO2) from the vaginal tract of a female mouse. In vitro characterisation of LbO2 included acid and bile salts tolerance, growth in urine, adherence to uroepithelial cells and in vitro antimicrobial activity. The selected strain showed interesting properties, suitable for its use as a probiotic. The ability of LbO2 to prevent and even treat ascending P. mirabilis urinary tract infection was assessed using an experimental model in the mouse. Kidney and bladder P. mirabilis counts were significantly lower in mice preventively treated with the probiotic than in non-treated mice. When LbO2 was used for therapeutic treatment, bladder counts of treated mice were significantly lower although no significant differences were detected in P. mirabilis kidney colonisation of treated and non-treated animals. These results are encouraging and prompt further research related to probiotic strains and the basis of their effects for their use in human and animal health.     

Additions to the genusXenidiocercus (coelomycetes) from Ghana

Two new species of the coelomycete genusXenidiocercus are described and illustrated,X. macrospora on leaves ofMacaranga rowlandii andX. pyriformis on leaves ofM. huraefolia. They differ from the type species in having wider and ellipsoidal or pyriform conidia. A key to species ofXenidiocercus andIdiocercus is provided.     

Differential effects of AM fungal isolates on Medicago truncatula growth and metal uptake in a multimetallic (Cd, Zn, Pb) contaminated agricultural soil

Toxic metal accumulation in soils of agricultural interest is a serious problem needing more attention, and investigations on soil–plant metal transfer must be pursued to better understand the processes involved in metal uptake. Arbuscular mycorrhizal (AM) fungi are known to influence metal transfer in plants by increasing plant biomass and reducing metal toxicity to plants even if diverging results were reported. The effects of five AM fungi isolated from metal contaminated or non-contaminated soils on metal (Cd, Zn) uptake by plant and transfer to leachates was assessed with Medicago truncatula grown in a multimetallic contaminated agricultural soil. Fungi isolated from metal-contaminated soils were more effective to reduce shoot Cd concentration. Metal uptake capacity differed between AM fungi and depended on the origin of the isolate. Not only fungal tolerance and ability to reduce metal concentrations in plant but also interactions with rhizobacteria affected heavy metal transfer and plant growth. Indeed, thanks to association with nodulating rhizobacteria, one Glomus intraradices inoculum increased particularly plant biomass which allowed exporting twofold more Cd and Zn in shoots as compared to non-mycorrhizal treatment. Cd concentrations in leachates were variable among fungal treatments, but can be significantly influenced by AM inoculation. The differential strategies of AM fungal colonisation in metal stress conditions are also discussed.     

Metal and antibiotic resistance traits in Bradyrhizobium sp. (cajanus) isolated from soil receiving oil refinery wastewater

Forty isolates of Bradyrhizobium sp. (cajanus) were isolated from the nodules of pigeon pea plants grown in fields receiving petrochemical industrial wastewater for the past 12 years and characterized using standard methods. The heavy metal analysis of field soil and treated wastewater showed their presence in varying concentrations. All isolates showed resistance to one or more metals at concentrations >200 g/ml. Multiple metal resistance was a common phenomenon in these isolates. There was no correlation between extractable soil metal concentration and the ability of the isolates to tolerate metal salts in their growth medium as evidenced from their minimum inhibitory concentration (MIC). However, high incidence of metal resistance and the multiple nature of resistance might have been the result of continuous exposure of these strains to heavy metals in the treated wastewater of Mathura Oil Refinery. These strains were also found to be resistant to one or more of the 13 antibacterial drugs tested.     

Induction of tumor clones in D. melanogaster wts/+ heterozygotes with chemical carcinogens

Ten chemicals were assessed for blastomogenic activity in adult wts/+ heterozygotes of D. melanogaster. All of the strong mammalian carcinogens tested (benzo(a)pyrene (B(a)P), pyrene, aflatoxin B₁, 2-acetylaminofluorene (2-AAF) and cis-dichlorodihydroxydiamminoplatinum IV) were also shown to be strong Drosophila blastomogens. They induced several times more tumors than their counterparts that are less carcinogenic for mammals (4-acetylaminofluorene (4-AAF), aflatoxins B₂ and G₂) and 4-(methylnitrosamino)-1-(-3-pyridine)-1-butanone (NNK). Benzo(e)pyrene (B(e)P) and pyrene demonstrated minor effects. Most tumors were localized on the wing and notum, which are the derivatives of the wing disc. Humeri derived from dorsal prothoracic disc and the abdominal tergites and sternites had the lowest number of tumors. The tumor frequency in the cross of the wild type females with wts^P2/TM6B males was different from that in the reciprocal cross. The former type of cross exhibited consistently higher tumor frequency both in the experimental and control series.     

Phospholipase D in Tetrahymena: activity and significance

We detected phospholipase D in three species of ciliates: Tetrahymena: T. thermophila, T. pyriformis and T. setosa in nutrient medium supplemented with ethanol in in vivo systems, by the appearance of phosphatidylethanol. The calcium ionophore A23187 increased the synthesis of phosphatidylethanol, as compared with untreated controls. We suggest that Tetrahymena possess a calcium sensitive phospholipase D. Propranolol caused the cells in dense cultures to increase their average generation times or die, dependent on the drug concentration. This inhibition could be overcome by the addition of phospholipids or ethanol. Pure phosphatidylethanol had no effect on growth rates or generation times in cultures at high cell density, but postponed cell death in cultures at low cell density by a factor of 10. We suggest that an important role of phospholipase D in Tetrahymena is to supply the cell with diacylglycerol without which it can not enter the mode of proliferation from the lag phase of the culture.