P0 phosphorylation in nerves from normal and diabetic rats: Role of protein kinase C and turnover of phosphate groups

The effects of phorbol ester and forskolin on the net phosphorylation and turnover of P₀ phosphate groups was studied in normal and exprimentally diabetic rats. In sciatic nerve segments isolated from normal rats and incubated with [³²P]-inorganic phosphate, phosphorylation of the major peripheral myelin protein, P₀, was increased 2–5 fold in a time and dose-dependent manner by phorbol 12,13 dibutyrate (PDB). This increase was blocked by the protein kinase inhibitors, H-7 and staurosporine. Both the basal and PDB-stimulated phosphorylation of P₀ were significantly greater in segments of sciatic nerve from streptozotocin-induced diabetic rats. Prolonged exposure of nerve segments to PDB abolished the stimulated phosphorylation of P₀ and immunoblots of nerve proteins revealed a decrease in the content of the protein kinase C -isoform. The adenylate cyclase activator, forskolin, had no affect on the PDB-stimulated phosphorylation of P₀ in normal nerve but decreased phosphorylation in diabetic nerve. To measure turnover of P₀ phosphate groups, nerves were incubated with³²P and incorporated label was then chased in radioactivity-free medium for up to 4 hours. P₀ from normal nerve prelabeled under basal conditions lost 25% of its radioactivity during this time. In contrast, nearly all of the additional phosphate groups prelabeled in the presence of PDB disappeared after 2 hours of chase. P₀ phosphate groups from diabetic nerve displayed similar turnover kinetics. When forskolin was added to the chase medium, the turnover of P₀ phosphate moieties was accelerated in normal, but not in diabetic nerve. These findings clearly establish a prominent role for protein kinase C in P₀ phosphorylation, provide evidence for heterogeneous turnover of P₀ phosphate groups and suggest that cyclic AMP-mediated processes may modulate P₀ phosphorylation. Further, these results indicate that the metabolism of P₀ phosphate moieties is perturbed in nerve from diabetic animals.Special issue dedicated to Dr. Marjoris B. Lees.     

Sampling dust from human dwellings to estimate the prevalence ofDermatophagoides mite and cat allergens

Summary Quantification of specific allergens in household dust samples may provide important information for selecting appropriate allergen control methods, and monitoring efficacy and compliance. The purpose of this study was to investigate the source of variation in mite and cat allergen measurements associated with dust sample collection. Discrete and composite dust samples were collected on a filter using a special vacuum sampling device. Aqueous extracts of the dust samples were prepared thenDer p I,Der f I, andFel d I were quantitated by enzyme immunoassays (EIA). Mite and cat allergens were frequently detected in dust samples from human dwellings, and the amounts of these allergens varied significantly (p<0.01) among dwellings. The differences of allergen measurements among duplicate samples taken immediately and up to three weeks later appear to be much smaller than differences among houses and between rooms. Variation among dust samples taken from living rooms and bedrooms of the same dwelling suggest differences in allergen reservoirs. Composite samples formed by sampling specific objects within a room may provide a reliable estimate of allergen exposure in that room. Dust samples from discrete objects are useful to find and monitor specific reservoirs of mite and cat allergens.     

DNA Fragmentation Induced by Cytotoxic T Lymphocytes Can Result in Target Cell Death

Cytotoxic T lymphocyte (CTL)-mediated lysis is accompanied by fragmentation of target cell DNA into an oligonucleosome ladder, a hallmark of apoptosis. Is this a fortuitous coincidence, or could CTL be inducing lysis by activation of the suicide signal? In this report we demonstrate that CTL-mediated target cell death can be blocked with the drug aurintricarboxylic acid (ATA). The abrogation of death correlates with the inhibition of DNA fragmentation. While ATA prevented DNA fragmentation, it failed to significantly alter protein, RNA, or DNA synthesis in the cell lines over the dose range used. In addition, there was no inhibition of cell-cell interaction or granule exocytosis during CTL-mediated killing. ATA also significantly inhibited the cytolysis and DNA fragmentation mediated by isolated cytolytic granules, as well as the granular protein fragmentin. We developed an assay in which target cells could be separated from CTL after binding and programming for lysis. Once they had received the "kiss of death," target cells could be rescued from lysis (as indicated by inhibition of DNA fragmentation and increased target cell viability) by treatment with ATA. These results suggest that ATA blocks target cell death by inhibition of DNA fragmentation, and further, that chromatin degradation is a cause rather than a result of cell death in CTL-mediated lysis.     

Somatic embryo maturation from long-term suspension cultures of white spruce (Picea glauca)

Summary The production of cotyledonary somatic embryos of white spruce from cultures grown long-term as suspensions was investigated. We report the effects of removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from the maintenance medium (ordinarily containing both 2,4-D and benzyl adenine) before (&#x00B1;)-ABA-stimulated maturation. In particular the use of a 1-wk culture period without 2,4-D was found to improve the production of normal-looking cotyledonary somatic embryos. Using high performance liquid chromatography analyses of culture supernatants, it was determined that this affect was not related to altered ABA metabolism. Germination of cotyledonary somatic embryos from cultures pretreated by the 1-wk culture period without 2,4-D was improved compared with similar embryos from cultures that had not been pretreated.     

LIGHT-HARVESTING PIGMENT-PROTEIN COMPLEXES OF THE ULVOPHYCEAE (CHLOROPHYTA):CHARACTERIZATION AND PHYLOGENETIC SIGNIFICANCE1

Photosystem II light-harvesting complexes were isolated from a number of ulvophycean algae. Some of these light-harvesting complexes displayed unusual features, most notably a high apparent molecular weight (ca. 58,000) when isolated by lithium doderyl sulfate polyarrylamide gel electrophoresis. Other ulvophycean light-harvesting complexes had a low-molecular weight (ca. 30,000). The distribution of the high-molecular weight complex was limited to certain members of the Caulerpales and Blastophysa rhizopus (Siphanocladales). Within the Caulerpales, there were also spectral differences between the high-molecular weight and low-molecular weight light-harvesting complex types. The differences in light-harvesting complexes in the Ulvophyceae suggest that there are two lines of evolution in the Caulerpales and that Blastophysa may be an intermediate between the Siphon-ocladales and the Caulerpales.     

Utility of field-based artificial streams for assessing effluent effects on riverine ecosystems

Experimentation using field-based artificial streams provides a promising, complimentary approach to biomonitoring assessments because artificial streams provide control over relevant environmental variables and true replication of treatments. We have used large and small artificial stream systems, based in the field, to examine the effect of treated bleached kraft pulp mill effluent (BKME) on the benthos of three large rivers in western Canada. Under natural regimes of temperature, water chemistry, and insolation, these artificial streams provide current velocities and substrata to food chains or food webs that are representative of those in the study river. With these tools we have shown that BKME stimulated mayfly growth in the Thompson River above that which could be accounted for by fertilization of their algal food supply. In contrast, moulting frequency was inhibited at high BKME concentrations. Results from artificial streams also indicate that increased algal biomass and abundances of benthic communities downstream of BKME outfalls were induced by nutrient enrichment from the effluent. BKME treatments did not change diatom species richness in the Fraser River, or diatom species diversity in either the Athabasca or Fraser Rivers. Artificial streams provide a means of understanding the mechanisms of stressor effects over a continuum ranging from single stressor effects on specific taxa to the effects of multiple stressors on communities and ecosystems. Because riverside deployment provides environmental realism within a replicated experimental design, this approach can (i) address questions that cannot be examined using laboratory tests or field observations, (ii) improve our mechanistic understanding of stressor effects on riverine ecosystems, and (iii) can contribute directly to the development, parameterization, and testing of models for predicting ecosystem-level responses.     

Modulation of the adhesion of hemopoietic progenitor cells to the rgd site of fibronectin by interleukin 3

The integrins are a class of adhesion molecules which have been implicated in the homing of hemopoietic stem cells and in their restriction within the bone marrow. Integrins function as mediators of cell-extracellular matrix (ECM) interactions and also of cell-cell interactions. They are unique membrane receptors which are capable of activation, change in affinity, and change in expression. Because of their broad potential for modulation we examined the effect of a cytokine growth factor which is present constitutively in the marrow, interleukin 3 (IL3), on integrin-mediated adherence of hemopoietic progenitor cells to the matrix component fibronectin (FN). The multipotential murine cell line B6Sut and the committed granulocyte progenitor cell line FDCP-1 were used. Both of these cell lines have been shown to bind to FN-coated dishes and to dishes coated with the 120 kDa and 40 kDa chymotryptic fragments of FN. It was found that after a brief withdrawal of IL3 the cells lost 80% adherence to the 120 kDa FN fragment containing the RGD cell binding site. This loss of binding was not related to a loss of viability, appeared unrelated to the growth/survival activity of IL3, and was quickly reversible by readdition of the growth factor. Adhesion of these cells to the RGD site was likely mediated by α5b?1 integrin which was identified in the cell membrane of both cell lines, but present in low copy number in B6Sut cells. Two antibodies against the external and internal domains of α5 and one antibody against b?1 were used to study expression of the integrin. By flow cytometry the expression of α5 was found to decrease in both cell lines by 4 h in the absence of IL3. The relative mean fluorescence intensity for B6Sut cells decreased from 1.0 (control cells always in the presence of IL3) to 0.6 over 4 h, and for FDCP-1 cells the decrement was from 1.0 to 0.8. The loss of RGD-mediated adhesion in the absence of IL3 appeared to proceed through this decrement in expression of the integrin; a loss of affinity of the receptor for its substrate was not detected. The general modulation of integrin activity by growth factors is of great interest because of its potential negative impact on the endothelium in cytokine-treated patients, and also because of its potential positive impact on engraftment during clinical bone marrow transplantation. © 1995 Wiley-Liss, Inc.     

Effectiveness of acoustic “prey”: Environmental enrichment for a captive African leopard (Panthera pardus)

In the course of developing active naturalistic exercise opportunities for zoo felines at moderate cost, a computer-controlled acoustic prey device was established. Changes in the behavior of a 16-year-old melanistic leopard (Sabrina) were studied as she learned to actively pursue bird sounds and obtained food treats as a function of the activity. By the twenty-ninth day she began to capture all 24 bird parts supplied on the feeder belt and continues to actively use the opportunity on a daily basis. General activity and apparent well-being have been enhanced, while stereotypic behaviors have decreased. © 1995 Wiley-Liss, Inc.     

Creatine phosphokinase activity in dysgenic (mdg/mdg) mouse muscle

The specific activity of creatine phosphokinase (CPK) was measured in the muscle of mdg/mdg and control embryos of 14-18 days' gestation. CPK specific activity values were similar in mutant and normal embryos at the earliest stages examined (14-15 days). However, after 15 1/2 days, the specific activity of the enzyme in the mdg/mdg embryos was approximately 50% lower than in the controls. The dysgenic and normal muscle extracts exhibited comparable stability after storage at -85 C. CPK activity levels in the muscle of adult heterozygotes (+/mdg) and wild-type (+/+) controls were found to be statistically identical. The findings suggest that the mdg mutation does not have a primary or direct effect on CPK activity.