Mechanisms of dimethylbenzanthracene-induced immunotoxicity

Traditional methods for toxicological assessment have implicated the immune system as a frequent target organ of toxic insult following chronic exposure to certain environmental chemicals, radiation or therapeutic drugs (xenobiotics). Immunotoxicity is expressed as autoimmunity, chemical hypersensitivity or immunosuppression. A tiered approach for characterizing chemical and drug-induced immunomodulation has been developed and validated in laboratory animals. Polycyclic aromatic hydrocarbons (PAH) have been studied because of their ubiquitous presence in the environment and carcinogenic potential. Since immunosuppression induced by PAH carcinogens has been implicated as an epigenetic mechanism in the outgrowth of initiated cells, this tiered approach was used to characterize the mechanism of PAH immunosuppressive capacity. Previously, studies in this laboratory have demonstrated that subchronic exposure of B6C3F1 mice to PAH carcinogens suppresses both humoral immunity (HI) and cell-mediated immunity (CMI), concurrently with decreased resistance to tumor challenge. The potent carcinogenic PAH, 7,12-dimethylbenz[a]anthracene (DMBA) was subchronically administered subcutaneously at 5, 50, or 100 micrograms/g of body weight. Natural killer (NK) cell tumor cytolysis, generation of cytotoxic T-cells (CTL), and lymphoproliferation to mitogens and allogeneic splenocytes in mixed leukocyte cultures (MLC) were quantitated 3-5 days after exposure to assess CMI. Mitogen and alloantigen-induced proliferation (MLC) of splenocytes was suppressed up to 90%. CTL and NK tumor cytolysis of radiolabelled target cells were similarly depressed up to 88 and 82%, respectively. Impairment of MLC or CTL responses correlated with increased susceptibility to challenge with PYB6 sarcoma cells. HI was measured by quantitating the number of antibody (IgM) plaque-forming cells (PFC) produced in response to T-cell dependent antigen challenge (sheep erythrocytes) and was similarly suppressed up to 95%. To understand the mechanism of PAH-induced immunotoxicity, splenocytes from DMBA-exposed mice were sensitized to alloantigens in the presence of interleukin-2 (IL-2) because there were indications that T-helper cell function was suppressed. In these preliminary studies, CTL suppression could be completely restored by the addition of the T-cell growth supporting lymphokine (IL-2) during the inductive phase of CTL generation, suggesting that DMBA exposure directly or indirectly induced deficits in T-helper cell function.     

The subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

Incubation of subcellular fractions isolated from rat cerebral cortex with [-³²P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes.A preliminary account of part of this work has been published (1).     

Effects of different proportions of nutrients on insects

Digestibility and nutrient composition of foodstuffs, be they plant or not, and the nutritional requirements of insects vary. The qualitative nutritional requirements among insects are quite similar. Therefore, qualities of foodstuff as measured by its ability to promote or support growth, etc. depend on how well the nutrient composition of foodstuff made available by digestion fits the nutritional requirements of the insect. This is better seen using synthetic diets rather than plants. From such investigations it is found that, provided all essential nutrients are present, the proportions of essential nutrients in a foodstuff contribute more to nutritional quality than do the absolute amounts of nutrients. Thus, nutrient balance per se can affect: rate of food consumption and efficient utilization; parasites with respect to host food; rate of growth and development as such, and with respect to temperature; and food selection in an insect.

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Schlussfolgerung Ich bin der Überzeugung, daß die Kenntnis der Ernährung unser Verständnis der Insekt-Nahrungspflanzen-Beziehung vergrößern und möglicherweise Wege weisen kann, um Insekten zu manipulieren und Pflanzen zu schützen. Dabei brauchen keine geheimnisvollen Dinge betrachtet zu werden, denn die Prinzipien der tierischen Ernährung und Nahrungsaufnahme lassen sich auch auf Insekten anwenden. Bei Insekten wie bei Säugern variieren Verdaulichkeit und Nährwert der Nahrungsstoffe und unterscheiden sich so von einem Tier oder Insekt zum anderen. Auch die Nahrungsansprüche der Insekten sind unterschiedlich. So müssen sowohl die Nahrungsansprüche der Insekten wie der Nährwert der betreffenden Nahrungsstoffe in diesen Beziehungen als Veränderliche angesehen werden. Da jedoch alle Insekten ganz ähnliche Nahrungsansprüche zu haben scheinen, und sehr wahrscheinlich alle Pflanzen ungefähr die 25 Nährstoffe enthalten, welche die Insekten benötigen, erscheint es zweifelhaft, daß qualitative Faktoren eine sehr entscheidende Rolle in den Beziehungen zwischen Insekten und Pflanzen zu spielen vermögen. Höchstwahrscheinlich sind quantitative Faktoren am wichtigsten. Diese hängen davon ab, wieviel von jedem Nährstoff die Verdauung eines Nahrungsmittels für die Ernährung des Insekts verfügbar machen kann; und wie gut sich die Beträge und Anteile der verfügbar gemachten Nährstoffe in die quantitativen Ernährungsansprüche des Insekts, einschließlich besonders der proportionalen Beziehungen, einfügen. Offensichtlich hängt die wirksame Ausnutzung des Nahrungsmittels für die Ernährung von dieser Einpassung ab, wie an gewissen Kriterien der Leistung der Insekten abzulesen ist. Deshalb sind unzweifelhaft die Quantitäten und vorzugsweise die Proportionen der betreffenden essentiellen Nährstoffe das wichtige Forschungsgebiet für Nahrungsuntersuchungen in den Insekt-Pflanzen-Beziehungen.

     

Development of chicken embryos in a pulsed magnetic field

Six independent experiments of common design were performed in laboratories in Canada, Spain, Sweden, and the United States of America. Fertilized eggs of domestic chickens were incubated as controls or in a pulsed magnetic field (PMF); embryos were then examined for developmental anomalies. Identical equipment in each laboratory consisted of two incubators, each containing a Helmholtz coil and electronic devices to develop, control, and monitor the pulsed field and to monitor temperature, relative humidity, and vibrations. A unipolar, pulsed, magnetic field (500-microseconds pulse duration, 100 pulses per s, 1-microT peak density, and 2-microseconds rise and fall time) was applied to experimental eggs during 48 h of incubation. In each laboratory, ten eggs were simultaneously sham exposed in a control incubator (pulse generator not activated) while the PMF was applied to ten eggs in the other incubator. The procedure was repeated ten times in each laboratory, and incubators were alternately used as a control device or as an active source of the PMF. After a 48-h exposure, the eggs were evaluated for fertility. All embryos were then assayed in the blind for development, morphology, and stage of maturity. In five of six laboratories, more exposed embryos exhibited structural anomalies than did controls, although putatively significant differences were observed in only two laboratories (two-tailed Ps of .03 and less than .001), and the significance of the difference in a third laboratory was only marginal (two-tailed P = .08). When the data from all six laboratories are pooled, the difference in incidence of abnormalities in PMF-exposed embryos (approximately 25 percent) and that of controls (approximately 19 percent), although small, is highly significant, as is the interaction between incidence of abnormalities and laboratory site (both Ps less than .001). The factor or factors responsible for the marked variability of inter-laboratory differences are unknown.     

IV. A model system for mass spectrometric sequencing of orthophthalaldehyde peptide derivatives

Presented is a pilot project describing a new strategy for mass spectrometric peptide sequencing. Using orthophthalaldehyde (OPA), two peptides were derivatized and their fluorescent adducts isolated using reversed phase high performance liquid chromatography. The OPA-peptides were permethylated and the derivatized molecules subjected to direct probe mass spectral analysis. The spectra obtained from the OPA-peptides was analogous to those observed for standard $\text{N}$-acyl permethyl derivatives enabling the complete sequence of the peptides to be determined.