Genetic structure of natural populations of the red-spotted newt,Notophthalmus viridescens

Genetic variation is described at 15 loci in 2 neotenic and 12 nonneotenic populations of red-spotted newts. Though high levels of genetic similarity (I=0.990) were found among all populations, allele frequencies at six of the eight most polymorphic loci show significant heterogeneity across populations. Change in allele frequencies at two of these loci (Pep-2 and Ldh-1) is significantly correlated with latitude. Interspecific homologies are established for newt peptidases based on substrate specificities and lactate dehydrogenases based on tissue distribution, thermal stability, and kinetic properties. Nonneotenic populations are highly variable (H=0.157) and neotenic populations are only slightly, but significantly, less variable (H=0.120). The high levels of heterozygosity detected in nonneotenic populations may result from large effective population size and/or environmental heterogeneity. The unexpectedly high heterozygosity values obtained for the neotenic populations may indicate adult dispersal or the presence of some previously undetected red efts at these localities. In any case, a major change in life history has apparently had little effect on the genetic structure of these populations.This research was supported by grants from the Blakeslee Fund of Smith College.     

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differentiation of a histiocytic lymphoma cell line by lipomodulin,a phospholipase inhibitory protein

When U 937 cells, a human histiocytic lymphoma cell line, were cultured with purified lipomodulin for 3 days, morphological and functional differentiation was induced as detected by microscopical examination of Giemsa stained smears, expression of mature monocyte antigen, and antibody dependent cellular cytotoxicity tests. Essentially similar differentiation was observed by the treatment with dexamethasone for 6 days and this differentiation by dexamethasone was blocked by monoclonal anti-lipomodulin antibody. Furthermore, the synthesis of immunoprecipitable lipomodulin in these cells was induced by dexamethasone treatment. These results, taken together, suggest that the induction of lipomodulin synthesis might be the primary event in dexamethasone-induced cellular differentiation of U 937 cells.     

Coordination environment for the type 3 copper center of tree laccase and CuB of cytochrome c oxidase as determined by electron nuclear double resonance

A new rhombic EPR signal was recently discovered in the partially reduced type 2 copper-depleted Rhus vernicifera laccase (Reinhammar, B. (1983) J. Inorg. Biochem., in press). The signal originates from one of the type 3 Cu(II) ions that becomes EPR-detectable as a result of the selective reduction of the other copper ion in the exchange-coupled Cu(II)-Cu(II) pair. The 14N and 1H and 63,65Cu electron nuclear double resonance (ENDOR) of this uncoupled Cu(II) now have been collected and represent the first ENDOR measurements of a type 3 copper site. The data indicate that the copper is coordinated by at least three nitrogenous ligands, at least one of which is an imidazole. H/D exchange suggests a nearby H2O or OH-, perhaps as a fourth ligand. A similar EPR signal is seen for CuB of reduced cytochrome c oxidase under turnover conditions. The 14N ENDOR, and, therefore, the structure, of this site corresponds extremely closely to that of the laccase type 3 (Cu(II).     

Identification of a spleen focus-forming virus in erythroleukemic mice infected with a wild-mouse ecotropic murine leukemia virus

An NFS/N mouse inoculated at birth with an ecotropic murine leukemia virus (MuLV) obtained from wild mice (Cas-Br-M MuLV) developed a lymphoma after 18 weeks. An extract prepared from the lymphomatous spleen was inoculated into newborn NFS/N mice, and these mice developed erythroleukemia within 9 weeks. Spleens from the erythroleukemic mice contained ecotropic and mink cell focus-inducing (MCF) MuLVs; however, when these viruses were biologically cloned and reinoculated into newborn NFS/N mice, no erythroleukemia was induced. In contrast, cell-free extracts prepared from the erythroleukemic spleens induced erythroleukemia within 5 weeks. Analysis of cell-free extracts prepared from the erythroleukemic spleens showed that they contained a viral species that induced splenomegaly and spleen focus formation in adult mice, with susceptibility controlled by alleles at the Fv-2 locus. The spleen focus-forming virus coded for a 50,000-dalton protein precipitated by antibodies specific to MCF virus gp70. RNA blot hybridization studies showed the genomic viral RNA to be 7.5 kilobases and to hybridize strongly to a xenotropic or MCF envelope-specific probe but not to hybridize with an ecotropic virus envelope-specific probe. The virus described here appears to be the fourth independent isolate of a MuLV with spleen focus-forming activity.     

Multiple Molecular Forms of Enkephalins in the Guinea Pig Hippocampus

Abstract: The combined techniques of HPLC and radioimmunoassay were used to identify and quantitate enkephalin-related peptides in the guinea pig hippocampus. Both met- and leu-enkephalin were identified, in approximately a 2:1 ratio, as well as a third enkephalin-like molecule that is neither met- nor leu-enkephalin. The third enkephalin elutes earlier than met- or leu-enkephalin from a reversed-phase column, has a molecular weight similar to the other enkephalins, and is as active as these enkephalins are in inhibiting binding of labeled opiates to rat brain membranes. All regions of the hippocampus (dentate gyrus, CA1–2, CA3–4, and subiculum) contain all three immunoreactive peptides. Immunocytochemical techniques, using antisera raised against met-enkephalin, show with one antiserum immunoreactivity in the granule cell-mossy fiber system, and with the other scattered immunoreactive cells mostly in the CA2 region. Enkephalins are not confined to the mossy fiber system, as previously suggested, but may be a component of another hippocampal innervation.     

Synthesis and spectroscopic analysis of modified bile salts

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.     

Activation of human monocyte cytotoxicity by natural and recombinant immune interferon

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.