Anaerobic biotransformations of pollutant chemicals in aquifers

Summary Anaerobic microbial communities sampled from either a methanogenic or sulfate-reducing aquifer site have been tested for their ability to degrade a variety of groundwater pollutants, including halogenated aromatic compounds, simple alkyl phenols and tetrachloroethylene. The haloaromatic chemicals were biodegraded in methanogenic incubations but not under sulfate-reducing conditions. The primary degradative event was typically the reductive removal of the aryl halides. Complete dehalogenation of the aromatic moiety was required before substrate mineralization was observed. The lack of dehalogenation activity in sulfatereducing incubations was due, at least in part, to the high levels of sulfate rather than a lack of metabolic potential. In contrast, the degradation of cresol isomers occurred in both types of incubations but proved faster under sulfate-reducing conditions. The requisite microorganisms were enriched and the degradation pathway forp-cresol under the latter conditions involved the anaerobic oxidation of the aryl methyl group. Tetrachloroethylene was also degraded by reductive dehalogenation but under both incubation conditions. The initial conversion of this substrate to trichloroethylene was generally faster under methanogenic conditions. However, the transformation pathway slowed when dichloroethylene was produced and only trace concentrations of vinyl chloride were detected. These results illustrate that pollutant compounds can be biodegraded under anoxic conditions and a knowledge of the predominant ecological conditions is essential for accurate predictions of the transport and fate of such materials in aquifers.     

The regulation of xylanolytic enzyme formation by Butyrivibrio fibrisolvens NCFB 2249

Butyrivibrio fibrisolvens NCFB 2249 formed xylan-degrading enzymes on a wide range of carbohydrate growth substrates. The specific activities of α-L-arabinofuranosidase and β-D-xylosidase were increased (up 20-fold) after growth on xylan or xylose-containing saccharides. Xylose was not an effective substrate for xylanase production although its formation was induced on xylobiose and higher DP xylose-containing saccharides. Acetyl esterase activity was also highest after growth on xylan. The synthesis of xylanase and β-xylosidase was repressed by glucose and hemicellulosic pentoses and although α-L-arabinofuranosidase formation was also subject to catabolite regulation, xylose did not repress its synthesis.     

Studies on the immediate and delayed leucocytosis elicited by brief (30-min) strenuous exercise

Eight healthy male volunteers exercised for two 30-min sessions starting 3 h apart on an electronically braked cycle ergometer at a work load (mean 155.9 W, SD 33.4 W) which required an oxygen consumption that was 70% of their maximal rate of oxygen uptake. Venous blood samples were taken through an indwelling cannula over a period of 6 h beginning shortly before the first bout of exercise and were analysed for routine haematological parameters and for lactate, noradrenaline, adrenaline and cortisol. Both bouts of exercise induced an immediate leucocytosis due to rises in lymphocytes and neutrophils but only the first exercise bout induced a substantial delayed neutrophilia. In at least five subjects, changes in lymphocyte and platelet numbers were correlated (Spearman's rank procedure, P less than 0.05) with simultaneous changes in the plasma concentrations of lactate, noradrenaline and adrenaline over the 6-h period studied. Increases in the plasma concentration of cortisol due to exercise correlated positively with the percentage changes in neutrophil numbers at 3 h and 6 h. These results are consistent with the suggestion that the immediate and delayed leucocytosis induced by exercise are mediated respectively by catecholamine and by cortisol.     

Peptidergic neurons of the crab,Cardisoma carnifex,in defined culture maintain characteristic morphologies under a variety of conditions

Summary Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, superlarge, found occasionally, has a soma diameter greater than 40 m and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with l-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26° C instead of 22° C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K⁺]_o medium ([K⁺]_o=15–110 mM; standard=11 mM) has no long-term effect, but growth is arrested by [K⁺]_o greater than 30 mM. Cultures were also grown in media in which [Ca²⁺]_o ranged from 0.1 M to 26 mM (standard=13 mM). Outgrowth occured from all neuronal types in all [Ca²⁺]_o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.     

Passive immunization protects guinea pigs from lethal toxoplasma infection

Abstract The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti- Toxoplasma immunoglobulins or immune cells that had been enriched for B cells prior to transfer. Cells residing in the spleen, lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 × 10⁷ sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cell-mediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.     

Somatic embryogenesis and plant regeneration from zygotic embryo explants in mexican weeping bamboo,Otatea acuminata aztecorum

An efficient protocol has been developed for the in vitro propagation of Mexican Weeping Bamboo through somatic embryogenesis from zygotic embryo explants. Mature seeds and excised embryos were cultured in the light or in the dark on both Murashige and Skoog's and Gamborg's B5 basal media with various supplements. Optimal somatic embryogenesis and plant regeneration were obtained by culture in the dark on Murashige and Skoog's basal medium supplemented with 3 mg/1 2,4-dichlorophenoxyacetic acid, 0.5 mg/1 6-benzylaminopurine and 2.0% sucrose. More than 95% of the germinating somatic embryos developed shoots and roots, and were transferred to soil with 85% success.     

Limitations of the scrape-loading/dye transfer technique to quantify inhibition of gap junctional intercellular communication

Gap junctional intercellular communication (GJIC) is recognized as playing an important role in normal cell proliferation and development. Chemically induced alteration of GJIC has been proposed to be associated with abnormal cellular growth and/or tumor promotion. Several in vitro assays are currently used to determine the effects of chemicals on GJIC between cultured mammalian cells. One of these assays, the scrape-loading dye transfer (SLIDT) technique, is based on monitoring the transfer of the fluorescent dye Lucifer yellow from one cell into adjacent cells via functional gap junctions. The objective of our study was to evaluate and compare various approaches for quantifying results obtained with the SL/DT technique. Confluent cultures of either WB rat liver epithelial cells or LC-540 rat leydig cells were exposed to the animal tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), solvent (0.1% ethanol), or culture medium for one hour at 37° C prior to analysis of GJIC. Inhibition of dye transfer was clearly evident following TPA exposure. Quantification of this dye transfer was assessed via four approaches: manually counting the number of labeled cells; measuring the distance of dye travel from the scrape line; quantifying the amount of cellular dye uptake; and determining the distribution of dye away from the scrape line. Our results suggest that while the SL/DT technique can be effectively used as a tool to determine the qualitative presence or absence of GJIC, its use in quantifying changes in GJIC following chemical exposure is limited. Since concentration-dependent responses are critical in chemical testing, application of the SLIDT method should be restricted to a screening assay for qualitatively assessing the presence or absence of GJIC. Another assay (e.g., electrical coupling, microinjection, metabolic cooperation, radioactive metabolite transfer, or fluorescence redistribution after photobleaching) should be considered to quantify changes in GJIC and construct chemical concentration-response curves.Abbreviations FBS, fetal bovine serum - GJIC, gap junctional intercellular communication - HBSS, Hank's balanced saline solution - SL/DT, scrape-loading/dye transfer - TPA, 12-O-tetradecanoylphorbol-13-acetate.     

Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

Human adenovirus type 9-induced rat mammary tumors.

Following subcutaneous inoculation of newborn Wistar-Furth rats with human adenovirus type 9 (Ad9), 16 of 16 female and 0 of 11 male rats developed mammary tumors. Tumor-positive animals usually developed tumors in multiple glands. Histopathological analyses indicated that three general categories of tumor could be identified. Mammary fibroadenomas were the most common tumor type encountered, but phyllodeslike tumors and solid sarcomas were also frequently found. In situ hybridization and immunohistochemical techniques established that benign fibroadenomas were derived from mammary fibroblasts (collagen type I- and vimentin-positive cells) and that malignant tumors were derived from myoepithelial cells (collagen type IV-, vimentin-, and muscle-specific actin-positive cells). The fact that mammary tumors were limited to female rats suggested that female hormones are essential for tumor growth and development. In this regard, ovariectomy of Ad9-infected female rats prevented tumor development, while subsequent diethylstilbestrol (DES) treatment elicited tumor formation. In addition, Ad9-infected and castrated male rats which received DES also developed mammary tumors. Established male mammary tumors regressed when DES treatment was stopped and reappeared after DES treatment was resumed. Together, these results indicate that estrogen is required for both initiation and maintenance of Ad9-induced mammary tumors. Southern blot analysis of high-molecular-weight tumor DNA showed that mammary tumor cells contained single or multiple integrated copies of the entire Ad9 genome. RNase protection experiments established that estrogen receptor as well as Ad9 E1a and E4 mRNAs were expressed in mammary tumors, but Ad9 E3 and, surprisingly, E1b mRNAs were not expressed at detectable levels.