Transformation of T lymphocytes by the v-fos oncogene

Activation of T lymphocytes through the T cell antigen receptor has been shown to stimulate a rapid and transient accumulation of c-fos mRNA and protein. Transfection of a normal murine T lymphocyte clone with the FBJ-v-fos oncogene resulted in generation of a cell line that was morphologically transformed, had lost the requirement for IL-2 for proliferation, and was tumorigenic in adult syngeneic mice; however, the transformed cells retained the ability to proliferate in response to IL-2. The transformed cells did not show constitutive expression of IL-2 or c-fos mRNA, although the promoter regions of both IL-2 and c-fos genes contain AP-1 sites that are expected to be targets for binding of Fos/Jun complexes. In contrast, the transformed T cells showed increased constitutive expression of IL-2R alpha and c-myc mRNA; these genes may represent cellular targets for transformation by v-fos and physiologic activation by c-fos. We discuss the possibility that these transformed cells behave as cells partially activated through the TCR, and that transformation occurs through a mechanism independent of IL-2.     

Extreme differences in charge changes during protein evolution

Summary The maintenance of a proper distribution of charged amino acid residues might be expected to be an important factor in protein evolution. We therefore compared the inferred changes in charge during the evolution of 43 protein families with the changes expected on the basis of random base substitutions. It was found that certain proteins, like the eye lens crystallins and most histones, display an extreme avoidance of changes in charge. Other proteins, like phospholipase A2 and ferredoxin, apparently have sustained more charged replacements than expected, suggesting a positive selection for changes in charge. Depending on function and structure of a protein, charged residues apparently can be important targets for selective forces in protein evolution. It appears that actual biased codon usage tends to decrease the proportion of charged amino acid replacements. The influence of nonrandomness of mutations is more equivocal. Genes that use the mitochondrial instead of the universal code lower the probability that charge changes will occur in the encoded proteins.     

Extraretinal photoreceptors in the brain of the crayfish Cherax destructor.

Two clusters of red-brown pigmented cell somata lie among other cell somata along the anterior margin of the cerebral ganglion in the crayfish Cherax destructor. Electron micrographs show these cells to contain round electron dense pigment granules and that the cell membranes of two or more adjacent cells fold together to form rhabdom-like structures. The pigmented cells specifically bind a monoclonal antibody against the major species of opsin in R1-7 retinula cells of the compound eye of Cherax. When stimulated with light, the pigmented cells respond with a receptor potential-like depolarization. The axons of the pigmented cells terminate in the neuropil of the protocerebral bridge, together with neuronal elements that label with antibodies against serotonin and substance P. We suggest that the brain photoreceptors of the crayfish are important in the entrainment of circadian rhythms.     

Analysis of Adenosine Immunoreactivity, Uptake, and Release in Purified Cultures of Developing Chick Embryo Retinal Neurons and Photoreceptors

We have investigated the presence of endogenous adenosine and of mechanisms for adenosine uptake and release in chick embryo retinal neurons and photoreceptors grown in purified cultures in the absence of glial cells. Simultaneous autoradiographic and immunocytochemical analysis showed that endogenous adenosine and the uptake mechanism for this nucleoside colocalize in practically all the photoreceptors, but only in approximately 20% of the neurons. Approximately 25% of the neurons showed either immunocytochemical labeling or autoradiographic labeling, while greater than 50% of the neurons were unlabeled with both techniques. [3H]Adenosine uptake was saturable and could be inhibited by nitrobenzylthioinosine and dipyridamole and by pretreatment of the [3H]adenosine with adenosine deaminase. Although these observations indicate that the uptake is specific for adenosine, only 35% of accumulated radioactivity was associated with adenosine, with the remaining 65% representing inosine, hypoxanthine, and nucleotides plus uric acid. Adenosine as well as several of its metabolites were released by the cells under basal as well as K(+)-stimulated conditions. Potassium-enhanced release was blocked by 10 mM CoCl2 or in Ca2(+)-free, Mg2(+)-rich solutions. The results indicate that retinal cells that synthesize, store, and release adenosine differentiate early during embryogenesis and are therefore consistent with a hypothetical role for adenosine in retinal development.     

Short- and Long-Term Alterations of Gene Expression in Limbic Structures by Repeated Electroconvulsive-Induced Seizures

Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.     

Monoclonal Antibodies Allow Precipitation of Esterasic but Not Peptidasic Activities Associated with Butyrylcholinesterase

Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.     

Marked Amine and Amine Metabolite Changes in Norrie Disease Patients with an X-Chromosomal Deletion Affecting Monoamine Oxidase

Urinary and plasma amines and amine metabolites were quantified in two individuals with Norrie disease resulting from a deletion in chromosomal region Xp11.3, recently reported to be associated with absence of the gene encoding monoamine oxidase (MAO)-A and nondetectable MAO-A activity in fibroblasts and MAO-B activity in platelets. Marked (four-to 100-fold) elevations in levels of urinary phenylethylamine, o-tyramine, and m-tyramine (which are preferential substrates for MAO-B) and marked reductions (90%) in levels of 3-methoxy-4-hydroxyphenylglycol (a deaminated metabolite of norepinephrine, a preferential substrate for MAO-A) in urine and plasma confirmed the presence of a systemic, functionally significant reduction in the activities of both MAO isozymes. The magnitude of these changes, which are equivalent to those found in subjects taking MAO-inhibiting antidepressants, suggests that early initiation of dietary and drug restrictions may be clinically important in these and other patients with X-chromosomal mutations involving MAO. These findings further support the proposition that the MAOA and MAOB genes are located in close proximity on the X chromosome. Negligible changes in the metabolites of dopamine and serotonin raise the possibility that other metabolic pathways are of importance for their production, that dietary or intestinal bacterial sources contribute substantially to the presence of these amine metabolites in urine, or both.     

Short-term stimulatory effect of Sertoli cell conditioned medium on Leydig cell steroidogenesis is not mediated by inhibin

Addition of concentrated rat Sertoli cell conditioned medium (rSCCM) to isolated Leydig cells from immature rats stimulated steroid production more than 13-fold within 4 h. LH-stimulated steroidogenesis was not enhanced by addition of rSCCM. The biological activity of the concentrated rSCCM was higher after incubation of Sertoli cells with FSH, whereas FSH alone did not stimulate steroid production. This effect of rSCCM was not due to inhibin, since highly purified 32 kDa rat inhibin, in doses equivalent to those present in rSCCM, had no effect on steroidogenesis during the 4 h incubation period. Furthermore, inhibin could be separated from the Leydig cell stimulating factor by anion-exchange chromatography. These results indicate a short-term paracrine control of Leydig cell steroidogenesis by Sertoli cell derived factors, which differ from inhibin.