Lipopolysaccharide-Enhanced Expression of Interleukin-6 in Dibutyryl Cyclic AMP-Differentiated Rat C6 Glioma

Abstract: Rat C6 glioma synthesizes a low basal level of interleukin-6 (IL-6). Stimulation with 10 µg/ml of lipopolysaccharide (LPS) and induction of differentiation with 1 m M N ⁶, O ^2'-dibutyryl cyclic AMP (dbcAMP) for 48 h increased the secreted activity to 400 and 800 U/ml, respectively. An LPS stimulation of dbcAMP-differentiated cells strongly enhanced the secreted activity. Depending on the dbcAMP concentration, the cell number, and the stimulation time, the secreted IL-6 level increased up to 120,000 U/ml. After 48 h of costimulation with 10 µg/ml of LPS and 1 m M dbcAMP, northern blotting and immunoassay demonstrated an eightfold increase in IL-6 mRNA concentration and IL-6 immunoreactivity, whereas titration of the biological activity indicated a 100-fold increase in the secreted IL-6 activity. The enhanced secretion of IL-6 is correlated with the induction of differentiation. Chromatography on heparin-Sepharose and on DEAE-5PW separated the secreted activity into several fractions, indicating that they differ in heparin affinity and charge either by posttranslational modifications or by binding to a carrier protein. Each of the partially purified IL-6-like activities could be neutralized by an anti-murine IL-6 antibody. Our observations demonstrate that in vivo inflammatory signals can trigger astrocytes and their precursors to secrete substantially different levels of immunoregulatory cytokines depending on their degree of differentiation.     

DNA immunization confers protection against murine cytomegalovirus infection.

The murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) encodes an 89-kDa phosphoprotein (pp89) which plays a key role in protecting BALB/c mice against the lethal effects of the MCMV infection. In this report, we have addressed the question of whether "naked DNA" vaccination with a eukaryotic expression vector (pcDNA-89) that contains the MCMV IE1 gene driven by a strong enhancer/promoter can confer protection. BALB/c mice were immunized intradermally with pcDNA-89 or with the plasmid backbone pcDNAI/Amp (pcDNA) and then challenged 2 weeks later with either a lethal or a sublethal intraperitoneal dose of the K181 strain of MCMV. Variable results were obtained for the individual experiments in which mice received a lethal challenge. In four separate trials, an average of 63% of the mice immunized with pcDNA-89 survived, compared with 18% of the mice immunized with pcDNA. However, in two other trials there was no specific protection. The results of experiments in which mice were injected with a sublethal dose of MCMV were more consistent, and significant decreases in viral titer in the spleen and salivary glands of pcDNA-89-immunized mice were observed, relative to controls. At the time of peak viral replication, titers in the spleens of immunized mice were reduced 18- to >63-fold, while those in the salivary gland were reduced approximately 24- to 48-fold. Although DNA immunization elicited only a low level of seroconversion in these mice, by 7 weeks postimmunization the mice had generated a cytotoxic T-lymphocyte response against pp89. These results suggest that DNA vaccination with selected CMV genes may provide a safe and efficient means of immunizing against CMV disease.     

EFFECTS ON FITNESS COMPONENTS OF P-ELEMENT INSERTS IN DROSOPHILA MELANOGASTER: ANALYSIS OF TRADE-OFFS

We analyzed the trade-offs between fitness components detected in four experiments in which traits were manipulated by inserting small (control) and large (treatment) P-elements into the Drosophila melanogaster genome. Treatment effects and the interactions of treatment with temperature, experiment, and line were caused by the greater length and different positions of the treatment insert. In inbred flies, the treatment decreased early and total fecundity. Whether it increased the lifespan of mated females depended upon adult density. Analysis of line-by-treatment-by-temperature interactions revealed hidden trade-offs that would have been missed by other methods. They included a significant trade-off between lifespan and early fecundity. At 25°C high early fecundity was associated with decreased reproductive rates and increased mortality rates 10–15 days later and persisting throughout life, but not at 29.5°C. Correlations with Gompertz coefficients suggested that flies that were heavier at eclosion also aged more slowly and that flies that aged more slowly had higher fecundity late in life at 25°C. The results support the view that lifespan trades off with fecundity and that late fecundity trades off with rate of aging in fruitflies. Genetic engineering is an independent method for the analysis of trade-offs that complements selection experiments.     

Evolutionary plasticity in prokaryotes: A panglossian view

Enzyme directed genetic mechanisms causing random DNA sequence alterations are ubiquitous in both eukaryotes and prokaryotes. A number of molecular geneticist have invoked adaptation through natural selection to account for this fact, however, alternative explanations have also flourished. The population geneticist G.C. Williams has dismissed the possibility of selection for mutator activity on a priori grounds. In this paper, I attempt a refutation of Williams' argument. In addition, I discuss some conceptual problems related to recent claims made by microbiologists on the adaptiveness of molecular variety generators in the evolution of prokaryotes. A distinction is proposed between selection for mutations caused by a mutator activity and selection for the mutator activity proper. The latter requires a concept of fitness different from the one commonly used in microbiology.     

The differential genetic and environmental canalization of fitness components in Drosophila melanogaster

Canalization describes the process by which phenotypic variation is reduced by developmental mechanisms. A trait can be canalized against environmental or genetic perturbations. Stabilizing selelction should favor improved canalization, and the degree of a trait's canalization should be positively correlated with its impact on fitness. Here we report, for Drosophila melanogaster, measurements of environmental canalization for five fitness components. We compare them with measurements of genetic canalization, and we discuss the impact of inbreeding on both. In three experiments we measured the variation of fitness components within lines nested within temperature, treatment, and experiment. Lines differed in the position of a P element insert or in genetic background. Within lines flies were genetically nearly identical. We designated trait variation within lines as environmental canalization. The canalization of the traits increased with their impact on fitness, and the pattern was similar to that found for the canalization of fitness components against genetic differences, measured as the variation among lines nested within temperature, treatment, and experiment. This suggests that developmental mechanisms buffer the phenotype against both genetic and environmental disturbance. The results also suggest, less strongly, that inbreeding weakens canalization.     

Advantages of the scanning ion microscopy for mapping halogen corticoids in normal and transformed cells in culture

Summary— The intra-cellular distribution of eight halogen glucocorticoids was investigated by ion microscopy in two cellular varieties of cultured non-cancer cells (fibroblast 3T3) and cancer cells (human breast tumor cells MCF-7). Two types of ion microscopy helped to determine this distribution, a direct imaging ion microscope (SMI 300) with low spatial resolution, and a scanning ion microscope (IMS4F), featuring high resolution, serving to obtain maps representing the intra-cellular distribution of the fluorine elements and drugs present in these monolayer cultured cells. The fluorine images representative of the drugs containing fluorine showed that these drugs are essentially concentrated in the cell nuclei. In these nuclei, the distribution of these drugs is different from that of heterochromatin and of the nucleolus.     

Dietary subacute zinc deficiency and potassium metabolism

In a controlled animal experiment the effects of dietary subacute Zn deficiency on growth, Zn concentration, and tissue 42-K distribution were studied. Growth retardation caused lower body weight because both skeletal and heart muscle showed a reduction in cell mass. Zn concentrations were reduced in most tissues, however, they remained unaltered in heart muscle. 42-K activity increased in skeletal muscle and pancreas. We hypothesize the latter reflects the organs rate of metabolism, inducing the exocrine pancreas to increase Zn absorption; in skeletal muscle it may induce also alterations in cell potentiation, causing restless behavior. As suggested by the calculated specific K activity (Bq/mol), the K uptake was highest in liver and bone, high in pancreas and skeletal muscle and low in heart muscle. The latter suggests K retention in heart muscle. Specific activity in plasma and jejunum remained unaltered: K status and absorption seem unaffected. Zn deficiency causes different 42-K activities in the various tissues, that respond by alterations in K metabolism without the induction of K deficiency.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.