Recolonization of estuarine organisms: effects of microcosm size and pesticides

Two six-week laboratory experiments were conducted to evaluate effects of pesticides and microcosm size on benthic estuarine macroinvertebrate recolonization. Sediments fortified with the pesticides (fenvalerate: controls, 5 (low) and 50 μg g⁻¹ wet sediment (high); endosulfan: controls, 1 (low) and 10 μg g⁻¹ wet sediment (high)) were fine-grained, organically rich (approximately 3.5% organic carbon and 22% dry weight) material. Relative dominance of the four most abundant taxa in both experiments was consistent among treatments with few exceptions. The amphipod,Corophium acherusicum, dominated abundance in both experiments. In the fenvalerate experiment, large trays (400 cm²) contained significantly (p<0.05) more total number of taxa (TNT) than small microcosms (144 cm²) but tray size was not significantly related to total number of organisms (TNO). When size was adjusted to a common unit area, small trays contained significantly more TNO than large containers. Adjusted abundance of small trays was 2.5 times that of large containers; a ratio close to that of microcosm sizes (i.e., 2.8). This result suggests that larval supply may have been inadequate to ‘aturate’ the available sediment in large containers. Fenvalerate significantly reduced abundance in the high treatment compared to both controls and low treatment but low treatment was not significantly different from controls. The amphipod,Corophium acherusicum, accounted for most of the decrease in abundance in response to fenvalerate. The holothruroid,Leptosynpta sp. and the polychaete,Mediomastus ambiseta, increased in abundance significantly with increased concentration of fenvalerate. Combined effects of actual microcosm size and concentration of endosulfan were not significant for TNO or TNT. As in the fenvalerate experiment, adjusted abundance of small microcosms was 2.6 times that of large trays which approximated the ratio of unit area between microcosm sizes. Abundance of a few taxa responded significantly to adjusted and unadjusted unit area. Abundance of the tunicate,Molgula manhattensis, increased significantly with increased concentration of endosulfan. Abundance was affected by sample location (e.g., interiorvs exterior cores) within microcosms. Abundance adjusted to unit area resulted in significantly greater TNO in externalvs internal cores. This has importance for sequential sub-sampling of microcosms to determine temporal dynamics. Statistically significant effects were measured in benthic community structure associated with microcosm size; however, the magnitude was relatively small. There appears to be no major biological reason to select one microcosm size over the other for screening for contaminant effects. Where feasible, the small trays provide savings in sample preparation and analysis, allow more replicates where laboratory space is limiting and generate less chemical waste. These benefits may be off-set by less ‘artifacts’ associated with edge effects of larger microcosms and the need for a larger mass of sediment to accommodate additional analytical requirements (e.g., thin vertical surficial samples to refine contaminant exposure at the sediment/water interface).     

Stream-dwelling insects and extremely low frequency electromagnetic fields: a ten-year study

Changes in the structure of benthic insect communities at an experimental site and at a reference site in the Ford River, Michigan were monitored over a 10-year period to determine whether extremely low frequency electromagnetic fields (ELF) affected those communities. Five of 10 biotic parameters monitored are presented: taxon evenness (J), richness (S), numerical dominance of chironomids, and total insect mass. Data were separated into three seasons because coefficient of variation values were lower in the summer than in the spring and fall. Two-way ANOVA tests for the biotic variables were often significantly different between sites and among years, but the interaction terms were less frequently significant. Biotic parameters were regressed against stream discharge, water temperatures, years, and ELF cumulative ground field exposures. At the experimental site, discharge accounted for more variation than did water temperature or years for all biotic parameters except chironomid numerical dominance in the fall. Intervention analyses, using the B.A.C.I parametric or the R.I.A non-parametric showed significant differences in three of 15 cases; namely, for the highly varying chironomid numerical dominance values in the spring and fall and for the low varying total insect mass values in the summer. For those tests, the Before Impact period spanned April 1984 through May 1986. The After Impact period (full ELF power) spanned June 1989 through August 1993. Trend analysis for total insect mass at the experimental site in the summer showed discharge to be more important than water temperatures or ELF ground field exposures. Natural physical factors appear to be more important than the anthropogenic ELF fields in accounting for seasonal and yearly changes in the community.     

Swim Stress Increases the Potency of Glycine at the N-Methyl-d-Aspartate Receptor Complex

Abstract: We have previously demonstrated that chronic administration of antidepressants results in a reduction in the potency of glycine to displace 5,7-[³H]dichlorokynurenic acid (5,7-[³H]-DCKA) from the strychnine-insensitive glycine recognition site of the NMDA receptor complex. We now report that exposure of rats to the forced swim test results in a 56% increase in the potency of glycine to displace 5,7-[³H]DCKA from frontal cortical homogenates. These data are consistent with the hypothesis that the forced swim test, a preclinical screen sensitive to acute administration of antidepressant drugs and NMDA receptor antagonists, also results in adaptation of the NMDA receptor complex. Moreover, these data lend further support to the hypothesis that glutamatergic pathways are involved in the neurobiological response to stress and, potentially, in the pathophysiology of depression.     

Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine

Summary 1. Site directed mutagenesis was used to alter the structure ofTorpedo californica nicotinic acetylcholine receptor (nAChR) and to identify amino acid residues which contribute to noncompetitive inhibition by quinacrine. Mutant receptors were expressed inXenopus laevis oocytes injected within vitro synthesized mRNA and the whole cell currents induced by acetylcholine (ACh) were recorded by two electrode voltage clamp.2. A series of mutations of a highly conserved Arg at position 209 of the subunit ofTorpedo californica nAChR revealed that positively charged amino acids are required for functional receptor expression. Mutation of Arg to Lys (R209K) or His (R209H) at position 209 shifted the EC₅₀ for ACh slightly from 5µM to 12µM and increased the normalized maximal channel activity 8.5-and 3.2-fold, respectively.3. These mutations altered the sensitivity of nAChR to noncompetitive inhibition by quinacrine. The extent of inhibition of ion channel function by quinacrine was decreased as pH increased in both wild type and mutant nAChR suggesting that the doubly charged form of quinacrine was responsible for the inhibition.4. Further mutations at different positions of the subunit suggest the contribution of Pro and Tyr residues at positions 211 and 213 to quinacrine inhibition whereas mutationsI210A andL212A did not have any effects. None of these mutations changed the sensitivity of nAChR to inhibition by a different noncompetitive inhibitor, chlorpromazine.5. These findings support a hypothesis that the quinacrine binding site is located in the lumen of the ion channel. In addition, the quantitative effect of point mutations at alternate positions on the sensitivity of quinacrine inhibition suggests that the secondary structure at the beginning of M1 region might be sheet structure.     

Glycan modification of a thermostable recombinant (1-3,1-4)-β-glucanase secreted fromSaccharomyces cerevisiae is determined by strain and culture conditions

High level biosynthesis and secretion of the thermostable hybrid (1-3,1-4)--glucanase H(A16-M) has been achieved inSaccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1_p) and theBacillus macerans (1-3,1-4)--glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man_8-13GlcNAc₂, although only traces of Man₉GlcNAc₂ were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3-phosphoglycerate kinase promoter, each in two differentSaccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y. Abbreviations: AMY,B. amyloliquefaciens (1-3,1-4)--glucanse; MAC,B. macerans (1-3,1-4)--glucanase H(A16-M), H(A36-M), H(A78-M),H(A107-M) and H(A152-M), hybrid (1-3,1-4)--glucanases containing 16, 36, 78, 107 and 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC; similar enzyme abbreviations followed by Y, e.g. H(A16-M)-Y, denote the enzymes secreted from yeast cells; PCR, polymerase chain reaction; PGK_p, yeast 3-phosphoglycerate kinase promoter; PRB1_p, yeast protease B promoter; LB, Luria-Bertani medium; SC, minimal medium; CNBr, cyanogen bromide; Endo H_f, endoglycosidase H fusion protein; PNGase F, peptide:N-glycosidase F; HPAEC; high pH anion exchange chromatography; HVE, high voltage paper electrophoresis; CPY, yeast carboxypeptidase Y.     

Measurement of cytokine antibodies. Test development

Several assays have been used for detection of antibodies against cytokines. The choice of assay is greatly dependent on the intended goal, e.g. detection of naturally occurring antibodies or therapy induced antibodies. The different assays can be grouped in 2 categories. The interference or indirect assays are based on the detection of the test sample interference with the biological activity, with detection of the cytokine in EIA or with binding to cellular receptors. In direct assays cytokine antibodies are detected by binding to solid phase fixed cytokines, followed by incubation with a secondary enzyme-labelled anti-human Ig antibody or by binding to¹²⁵I-labelled cytokines in RIA.     

Modulation of cisPlatin cytotoxicity by interleukin-1α and resident tumor macrophages

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1α) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1α in SCC-7 solid tumors. Neither IL-1α nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1α had no direct effect on tumor cell growthin vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC₉₀=6.0 μM), but, the addition of IL-1α (500–2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC₉₀=3.1 μM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1α. The modulation of cisPlatin cytotoxicity by IL-1α exhibited a biphasic dose response that paralleled the IL-1α dose dependent release of H₂O₂by resident tumor macrophages. Further, IL-1α modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H₂O₂ (50 μM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1α treatment for 24 hrs, before cisPlatin, produced drug resistance (IC₉₀=11.1 μM). Our study shows that IL-1α can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explanation for the synergistic antitumor activity of cisPlatin and IL-1αin vivo.     

Cell cultures derived from early zebrafish embryos differentiate in vitro into neurons and astrocytes

The zebrafish is a polular nonmammalian model for studies of neural development. We have derived cell cultures, initiated from blastula-stage zebrafish embryos, that differentiate in vitro into neurons and astrocytes. Cultures were initiated in basal nutrient medium supplemented with bovine insulin, trout serum, trout embryo extract and fetal bovine serum. After two weeks in culture the cells exhibited extensive neurite outgrowth and possessed elevated levels of acetylcholinesterase enzyme activity. Ultrastructural analysis revealed that the neurites possessed microtubules, synaptic vessicles and areas exhibiting growth cone morphology. The cultures expressed proteins recognized by antibodies to the neuronal and astrocyte-specific markers, neurofilament and glial fibrillary acidic protein (GFAP). Poly-D-lysine substrate stimulated neurite outgrowth in the cultures and inhibited the growth of nonneuronal cells. Medium conditioned by the buffalo rat liver line, BRL, promoted the growth and survival of the cells in culture. Mitotically active cells were identified in cultures that had undergone extensive differentiation. The embryo cell cultures provide an in vitro system for investigations of biochemical parameters influencing zebrafish neuronal cell growth and differentiation.