Bursal anti-steroidogenic peptide (BASP): modulation of mitogen-stimulated bursal-lymphocyte DNA synthesis

The present study examined the effects of bursal anti-steroidogenic peptide (BASP) on mitogen-induced DNA synthesis in bursa-derived B-lymphocytes in short-term culture. Partially purified extracts of chicken bursa of Fabricius tissue, containing BASP, significantly (P < 0.05) reduced DNA synthesis in bursal-lymphocytes exposed to increasing concentrations of phorbol 12,13-dibutyrate (PDB). Following these initial observations, BASP, further purified from bursal extracts using sequential rpHPLC fractionation, was observed to reduce (P < 0.05) both B-lymphocyte PDB-stimulated DNA synthesis and ovarian granulosa cell progesterone biosynthesis with bioactivity observed at similar retention times in each assay, suggesting that each bioactivity may be due to the same or similar molecules. A similar BASP-enriched fraction was not effective in altering basal levels of DNA synthesis in chick embryonic kidney cells. Subsequently, BASP was further purified by several sequential chromatographic methods including: C-18 rpHPLC (preparative rpHPLC followed by a semi-preparative rpHPLC column), cation exchange chromatography, molecular sieve HPLC chromatography, and SDS-PAGE. Biologically active material was observed at approximately 29 or 34 kDa. Protein concentration was determined and bioactivity was evaluated. Anti-proliferative effects of this partially purified BASP on bursal-lymphocytes was observed at concentrations as low as 1.6 micrograms ml-1, with complete suppression of mitogen-stimulated DNA synthesis observed at approximately 25 micrograms ml-1. This partially purified BASP was also efficacious for attenuation of ovarian granulosa cell progesterone biosynthesis at concentrations as low as 0.4 microgram ml-1, with complete suppression of gonadotrophin-stimulated progesterone biosynthesis observed at approximately 0.8 microgram ml-1. While BASP is efficacious for attenuation of both granulosa cell steroidogenesis and bursal-lymphocyte proliferation, these data suggest that BASP is much more potent with regard to anti-steroidogenic activity.     

Ultraviolet-Induced Photodegradation of Cucumber (Cucumis sativus L.) Microsomal and Soluble Protein Tryptophanyl Residues in Vitro

The in vitro effects of ultraviolet B (280-320 nm) radiation on microsomal membrane proteins and partially purified ribulose bisphosphate carboxylase (Rubisco) from cucumber (Cucumis sativus L.) was investigated by measuring the direct photolytic reduction of tryptophan fluorescence and the formation of fluorescent photooxidation products. Exposure of microsomes and Rubisco to monochromatic 300-nm radiation resulted in the loss of intrinsic tryptophan fluorescence and the production of blue-emitting fluorophores. The major product of tryptophan photolysis was tentatively identified as N-formylkynurenine (N-FK). Even though the rates of tryptophan photodegradation and N-FK formation were similar, the amount of blue fluorescence produced was significantly higher in the microsomes relative to Rubisco. Studies with various free radical scavengers and other modifiers indicated that tryptophan photodegradation requires oxygen and that the subsequent formation of N-FK may involve reactive oxygen species. The optimum wavelengths for loss of typtophan fluorescence were 290 nm for the microsomes and 280 nm for Rubisco. The temperature dependence of tryptophan fluorescence and rate of tryptophan photodegradation indicated an alteration in the cucumber microsomal membranes at about 24[deg]C, which influenced protein structure and tryptophan photosensitivity.     

Acetylene reduction in conifer logs during early stages of decomposition

Acetylene reduction was examined periodically for as long as 68 months in the outer and inner bark, sapwood, and heartwood of decaying logs of western hemlock [Tsuga heterophylla (Raf.) Sarg.] western redcedar (Thuja plicata D. Don), Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco], and Pacific silver fir (Abies amabilis Dougl. ex Forbes) in the western Oregon Cascade Mountains. Tissues from freshly cut logs from sound trees were unable to reduce acetylene. However, after 18 months of decomposition, acetylene reduction was found in all log tissues except heartwood. Over the 68-month study period, no significant relationship between reduction rate and tissue moisture was found. Acetylene reduction rates differed significantly among tissues, log species, and time of exposure to decomposers. Although acetylene reduction generally showed a steady increase with time, tissues of some species showed a more complex, nonlinear pattern of change. Although the amount of nitrogen fixed is low compared to the total present in decaying logs, it might be an important source of readily available nitrogen for the microbiota responsible for decomposition.Paper 2587, Forest Research Laboratory, Oregon State University, Corvallis.Paper 2587, Forest Research Laboratory, Oregon State University, Corvallis.     

The influence of environmental factors on seasonal changes in bacterial cell volume in two prairie saline lakes

Bacterial biovolumes of hypertrophic Humboldt Lake (total dissolved solids = 3.3 g liter^-1; 6 m deep) and oligotrophic Redberry Lake (total dissolved solids = 20.9 g liter^-1; 17 m deep), Saskatchewan, were measured concurrently with a variety of environmental variables to identify the major factors correlated with volume changes. There was no difference (P > 0.05) in mean bacterial volume between Redberry Lake (0.084 ± 0.034 m³ SD) and Humboldt Lake (0.083 ± 0.021 m³ SD). Statistical analyses suggested there were marked differences in the factors associated with the pronounced seasonality of bacterial cell volumes in these two lakes. Variance in bacterial volume in the epilimnion of Redberry Lake was best explained by a multivariate regression model which included ciliate abundance and chlorophyll concentration (r ² = 0.96). The model accounting for changes in hypolimnetic bacterial volume included ciliate numbers and primary production (r ² = 0.94), of the measured variables. Bacterial volume in Humboldt Lake was most highly correlated with primary production (r ² = 0.59). Bacterial production (estimated as the rate of thymidine incorporation into DNA) and growth (thymidine incorporation rate normalized to cell numbers) were not correlated to cell volume, with the exception of cocci volume in Humboldt Lake. Offprint requests to: R.D. Robarts.     

Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity

A cell line RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. RTL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either -naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants.Abbreviations AHH aryl hydrocarbon hydroxylase - B[a]P benzo[a]pyrene - BNF -naphthoflavone - CHSE-214 Chinook salmon embryo cells - CYP cytochrome P450 - DMSO dimethyl sulfoxide - ECOD 7-ethoxycoumarinO-deethylase - EDTA ethylenediaminetetraacetic acid - EROD 7-ethoxyresorufinO-deethylase - FBS fetal bovine serum - HEPES N-[-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] - H4IIE rathepatoma cells - PCB polychlorinated biphenyls - PHH planar halogenated hydrocarbons - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin - TEM transmission electron microscopy - TEFs toxic equivalent factors - TS 0.05 mM Tris 0.2M sucrose - RLE rat liver epithelial cells - RTL-W1 rainbow trout liver-Waterloo 1 cells     

Cultured myometrial cells establish communicating gap junctions

Myometrial cells were isolated and cultured from term rat uterus. The myometrial origin of the cultures was verified by antibody staining of cellular desmin and alpha-smooth muscle actin. The presence of functional gap junctions was indicated by transfer of radiolabeled nucleotide and microinjected Lucifer yellow dye. The cultured cells expressed mRNA recognized by a connexin43 gap junction cDNA probe. To our knowledge, this is the first report that isolated myometrial cells form gap junctions in culture.     

Influence of medium exchange schedules on metabolic, growth, and GM-CSF secretion rates of genetically engineered NIH-3T3 cells.

The metabolic and secretory characteristics of NIH-3T3 fibroblasts transfected with a cDNA encoding human granulocyte-macrophage colony stimulating factor (GM-CSF) were examined as a function of the culture medium exchange schedule. The rates of glucose and glutamine consumption and of lactate and ammonia production were measured over exchange schedules ranging from complete medium replacement weekly (1/week) to complete medium replacement daily (7/week). All measured metabolic rates increased with increased medium exchange rates and accelerated sharply between exchange rates of 3.5/week and 7/week. The lactate/glucose and ammonia/glutamine yield coefficients, however, remained invariant at about 1.9 and 1.0 mol/mol, respectively, under all medium perfusion conditions. A shift-up in medium perfusion rates from 3.5/week to 7/week resulted in increased metabolic rates that resembled those observed in the cultures that were exchanged at the 7/week rate throughout, showing that the metabolic rates could be directly controlled by the perfusion rate. Differential regulation of medium versus serum perfusion demonstrated that increased NIH-3T3 cell metabolism was directly proportional to the serum flux to which the cells were exposed. Thus a limiting serum component is responsible for the altered metabolic and growth rates. The GM-CSF production by the transfected 3T3 cells was stable but exhibited substantial transient increases during periods of cell proliferation, demonstrating that the secretion of transfected gene products can be highly modulated even when the cDNA is driven from a constitutive promoter. These studies show that the metabolic and secretory behavior of genetically engineered cells is influenced by the medium exchange schedule.     

Patch size effects are more important than genetic diversity for plant–herbivore interactions in Brassica crops

1. Considerable evidence suggests that the diversity within plant communities may strongly affect the strength of species interactions, but the majority of studies only considered interspecific diversity. 2. This paper examines the effect of intraspecific genetic diversity within Brassica fields on two Brassica specialists, cabbage root fly, and diamondback moth, and on a parasitoid attacking diamondback moths. Genetic diversity was manipulated both in a replacement and an additive design. 3. Both herbivore densities and parasitism rates were higher in smaller plots, with limited responses to increased within‐plot diversity. All species showed variable densities across genotypes, and preference hierarchies were species specific. 4. Responses to plot size in root flies scaled with the diameter‐to‐area ratio, suggesting that patch detectability affected local density, whereas responses by diamondback moths and parasitoids deviated from this ratio. These species differences could be traced to differences in the residence time within patches, where diamondback moths typically spend longer and more variable time periods in patches than root flies. 5. The lack of response to genetic diversity by both herbivores suggests that egg‐laying rates are affected by decisions on the plant and not by attraction from a distance, neither to the plant itself nor the patch. Patterns of differential attack may then be due to different acceptability for studied genotypes. 6. Future theories on insect responses to spatial heterogeneity should focus on species traits and how traits interact with information landscapes in the field.     

Structure and activity relationships of tartrate-based TACE inhibitors

The syntheses and structure–activity relationships of the tartrate-based TACE inhibitors are discussed. The optimization of both the prime and non-prime sites led to compounds with picomolar activity. Several analogs demonstrated good rat pharmacokinetics.