Glutathione contents of tissues in the aging mouse.

1. Previous results from this laboratory demonstrated that the erythrocyte content of reduced glutathione (GSH) decreased as a function of both increasing cell age and mouse age [Abraham, Taylor & Lang (1978) Biochem. J. 174, 819-825]. In the present investigation glutathione concentrations were determined in other tissues of the C57BL/6J mouse of different ages (6--31 months) throughout the life-span. 2. At all ages the total glutathione and the GSH concentrations in liver were 3 times that in kidney and 10 times that in heart. In the old (31 months) mouse the GSH contents were lower by 30% in the liver, 34% in the kidney and 20% in the heart than in the mature (17--23 months) animals. 3. The oxidized glutathione (GSSG) concentrations of the tissues did not vary with age and constituted less than 3% of the total glutathione. 4. The decreased in GSH concentrations were not due to changes in organ weights, which were constant from 10 to 36 months of age. 5. These findings extend our previous results and indicate that a general characteristic of aging tissues may be a decrease in GSH concentrations. Further, this is consistent with our hypothesis that the reducing potential of tissues decreases in senescence.     

Seasonal species composition of barnacle larvae (Cirripedia: Thoracica) in Rhode Island waters, 1977-1978

Cirripede larvae can occur year-round in temperate and tropicalwaters, often in significant numbers, yet the species compositionof the ‘Balanus sp.’ component is rardy studied.Weekly plankton samples were analyzed qualitatively for larvalcirripede species and stage over a year (1977–1978) attwo Rhode Island stations. Six species of larvae were foundin Lower Narragansett Bay (30° salinity). Semibalanus balanoidesand Balanus balanus have a single winter brood. S. balanoidesis the predominant winter breeder with a minor release of naupliiin early December and major release in March followed by cypridsin mid-April.B. balanus populations release all larvae in Marchwith cyprids in mid-April. Balanus crenatus is mainly a winterbreeder, but has multiple broods; it does not breed in July-Septemberwhen the water temperature is above 18°C. Balanus venustusis the predominant summer breeder, and larvae were observedfrom May through December (water >8°C). Larvae of Chthamalusfragilis and Balanus eburneus occur in low numbers from May-October.At the Pettaquamscutt River site (12 salinity), Balanus improvisuslarvae predominate and early stage nauplii (I-II) occur in samplesyear round (0–27°C). Two peaks of later stage naupliiand cyprids occur in late spring (May) and early winter (Nov.-Jan.).Continued temperatures bdow 5°C or above 20°C appearto inhibit larval development. Comparison of results with existing literature reveals severalsignificant findings. The bimodal rdease of S. balanoides larvaeis unusual and may be in response to the phytoplankton dynamicsof the year; however, the existence of distinct races of S.balanoides may also be a factor. Larvae of B. venustus predominatein the lower bay during the summer, yet this species is unreportedin past studies. B. improvisus nauplii are more cold tolerantthan previously reported. Comparison of findings with reportedbreeding patterns in Florida indicate significant differencesin temperature responses between northern and southern populations. ¹Contribution No. 188 from EPA Environmental Research Laboratory,South Ferry Rd., Narragansett, RI 02882     

Pulsation of nuclei in insect oocytes is reversibly inhibited by cytochalasin B

Oocyte nuclei of the dipteran insect Heteropeza pygmaea display swift pulsating movements during in vitro follicle formation in the ovaries. Low doses of cytochalasin B (CB) completely inhibit the nuclear movements within a few minutes and cause the nuclei to assume spherical shapes. If the drug is removed, nuclear pulsation is resumed within 5–10 min. Phalloidin and colchicine do not affect the nuclear movements. Actin is shown by indirect immunofluorescence microscopy to be present in considerable amounts all over the cytoplasm of the oocytes. It is concluded that microfilaments are responsible for pulsation of the oocyte nuclei, whereas microtubules are not involved.     

Bradykinin-induced oscillations of cell membrane potential in cells expressing the Ha-ras oncogene

Products of ras genes are putative elements of growth factor signal transduction. However, the mechanism of action of these proteins in normal and malignant growth is as yet obscure. To test for functional consequences of ras oncogene expression, electrophysiological experiments were performed on NIH-3T3 fibroblasts transfected with a transforming Ha-ras MMTV-LTR construct expressing the oncogene on treatment with dexamethasone (+ras). Transfected cells in the absence of dexamethasone (-ras) and nontransfected cells in the presence of dexamethasone (oras) served as controls. In -ras and oras, bradykinin induces a single, transient hyperpolarization. In +ras, bradykinin elicits oscillations of cell membrane potential throughout the presence of the hormone by activation of calcium-sensitive K+ channels. The oscillations of cell membrane potential are abolished in the absence of extracellular calcium. As evident from fura 2 fluorescence, bradykinin leads to a transient increase of intracellular calcium both in the presence and absence of extracellular calcium. Oscillations of intracellular calcium could be observed in +ras cells, if bradykinin was applied at reduced extracellular sodium concentration possibly to impair calcium extrusion via the sodium/calcium exchange. Bradykinin induces oscillations of cell membrane potential similarly in -ras cells loaded with GTP[S], a nonhydrolyzable analogue of GTP. Thus, the altered response of ras oncogene expressing cells to bradykinin relates to the GTP binding property of the ras protein. It is concluded that in cells expressing ras oncogene but not in other fibroblasts bradykinin mimicks the effect of growth factors on the cell membrane.     

Activation of cell membrane potassium conductance by mercury in cultured renal epithelioid (MDCK) cells

To elucidate mechanisms of mercury toxicity, the cell membrane potential has been determined continuously in cultured kidney (MDCK)-cells during reversible application of mercury ions to extracellular perfusate. Exposure of the cells to 1 microM mercury ions is followed by rapid, sustained, and slowly reversible hyperpolarization of the cell membrane, increase of cell membrane potassium selectivity, and decrease of cell membrane resistance. Thus, mercury ions enhance the potassium conductance of the cell membrane. Half maximal hyperpolarizing effect is elicited by approximately 0.2 microM. Higher concentrations of mercury ions (greater than 10 microM) eventually depolarize the cell membrane. At extracellular calcium activity reduced to less than 0.1 microM, 1 microM mercury ions still leads to a sustained hyperpolarization and increase of potassium selectivity of the cell membrane. As evident from fluorescence measurements, 10 microM, but not 1 microM mercury ions leads to a rapid increase of intracellular calcium activity. Pretreatment of the cells with either pertussis toxin or cholera toxin does not blunt the hyperpolarizing effect of mercury ions. In conclusion, mercury ions activate the potassium conductance by a mechanism independent of increase of intracellular calcium activity and of cholera toxin- or pertussis toxin-sensitive G-proteins. This activation of potassium conductance may account for early effects of mercury intoxication, such as kaliuresis.     

Reconstitution of an active surface CD2 by DNA transfer in CD2-CD3+ Jurkat cells facilitates CD3-T cell receptor-mediated IL-2 production.

To investigate the requirements for CD2 expression in the activation of T lymphocytes via the CD3-TCR complex, we produced and characterized a series of CD2-variants of the IL-2 producing Jurkat leukemia cell line, J32 (surface phenotype, CD2+, CD3+, CD28+). These mutants were derived by radiation and immunoselection, and were cloned under limiting dilution conditions. A total of 3 out of 30 of these mutants selectively lost the expression of both CD2 surface molecules and CD2 mRNA, and retained the expression of the CD3-TCR complex and the CD28 molecule. A mitogenic combination of anti-CD2 antibodies (9.6 + 9-1) failed to stimulate activation of these variants as measured by mobilization of intracellular Ca2+ and by IL-2 production. The CD2- mutants stimulated with anti-CD3 or anti-TCR mAb revealed an 8- to 32-fold decrease in IL-2 production and IL-2 mRNA accumulation as compared with the parental cells. No alteration of CD3-TCR-induced mobilization of intracellular Ca2+ was observed in the CD2- mutants. Reconstitution of CD2 expression by gene transfer in two J32 CD2- mutants restored IL-2 production and IL-2 mRNA accumulation in responses to both anti-CD2 and anti-CD3-TCR mAb. These results are the first direct demonstration of the requirement for CD2 molecules in optimizing IL-2 response in human T cells stimulated via CD3-TCR complex.     

Rat liver dimethylglycine dehydrogenase. Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone.

Dimethylglycine dehydrogenase (Me2GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8 alpha position to N pi of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) J. Biol. Chem. 259, 12475-12480]. Subcellular fractionation of [14C]riboflavin-labelled hepatocytes and immunoprecipitation with Me2GlyDH-specific antiserum identified a [14C]riboflavin-labelled polypeptide of the size of mature Me2GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [35S]Met-labelled hepatocytes revealed a putative precursor protein to the mature Me2GlyDH in the cytoplasmic fraction. These Me2GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me2GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD-X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) Methods Enzymol. 154, 3-28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me2GlyDH precursor immunoprecipitated from the cytoplasm of [35S]Met-labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease-recognition site Arg(-2)-Ala(-1)-Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me2GlyDH [Wittwer, A.J. & Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108], and a 43-amino-acid leader peptide. The N-terminus of Me2GlyDH contains a conserved amino acid sequence which forms the dinucleotide-binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.