Protein kinase C pseudosubstrate prototope: structure-function relationships

A structure-function study of the protein kinase C (PK-C) pseudosubstrate sequence (R19FARK-GALRQKNV31) has been undertaken. The role of specific residues was investigated using an alanine substitution scan. Arg-22 was the most important determinant in the inhibitor sequence, since substitution of this residue by alanine gave a 600-fold increase in the IC50 value to 81 +/- 9 microM. Substitutions of other basic residue also increased the IC50, 5-, 11- and 24-fold for the Ala-19, Ala-23 and Ala-27 substitutions, respectively. The importance of basic residues in determining the potency of the pseudosubstrate peptide reflects the requirements for these residues in peptide substrate phosphorylation. The residues Gly-24, Leu-26 and Gln-28 were also important for pseudosubstrate inhibitor potency. The large difference in the IC50 value for the [A22]PK-C(19-31) peptide makes it a valuable control in studies employing the pseudosubstrate peptide to explore functional roles of PK-C.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

Summary We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding preserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electronmicroscopical level.     

Adoptive cell transfer studies to examine the role of T lymphocytes in immunity to Trypanosoma musculi

Previous studies in this laboratory utilizing monoclonal antibody-induced immunosuppression have demonstrated that the T-helper lymphocyte is primarily responsible for the T lymphocyte dependency of Trypanosoma musculi elimination from the bloodstream of mice, and that T-cytotoxic lymphocytes play a minimal role in this response. In the current study, these findings were extended by examining the effects of adoptive cell transfers on the course of infection with T. musculi using immune splenocytes enriched for T lymphocyte subpopulations. These studies demonstrated that adoptive transfer of immune splenic T lymphocytes resulted in a specific, dose-related enhancement of kinetics of trypanosome elimination. This effect was found to be due to the presence of L3T4+ T-helper cells in the immune splenocyte population. Adoptive transfer of Lyt-2+ T-cytotoxic cells or lymphokine-activated killer (LAK) cells was ineffective in altering the course of infection. In addition, it was found that immune B lymphocytes were equally capable of adoptively transferring immunity to T. musculi, suggesting that the primary role of the T-helper lymphocyte is to provide help in the induction of parasite-specific antibodies.     

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.     

Nomograms for Calculation of Heat Loss

Two nomograms are presented. The first enables the mean surface and body temperatures and the body heat content of a patient of given weight to be determined from measurements of skin temperature at three sites and of the core (rectal) temperature. The second enables the change in heat content of such a patient to be determined from the change in mean body temperature.     

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.