Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells

The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte.     

HELP (high efficiency liquid phase) new oligonucleotide synthesis on soluble polymeric support.

A simple, rapid and high-yielding method for the synthesis of oligonucleotides by the phosphotriesters approach is described. The use of polyethylene glycol (PEG) as soluble polymeric support preserves some convenient features of the solid-phase synthesis with new interesting advantages. Short oligonucleotides in hundred milligrams scale can be obtained from few grams of functionalized PEG.     

Modulation of the cell-mediated immune function by interferon α, β or γ can partially reverse the immunosuppression induced by human T-cell leukemia virus I in human cord blood cultures

Summary Infection with human T-cell leukemia virus type I (HTLV-I) is associated in vitro and in vivo with a remarkable depression of cell-mediated immune functions. In the present report it is shown that early events following virus-induced suppression of the cell-mediated immune response of freshly isolated cord blood mononuclear cells (CBL) infected with HTLV-I can be partially counteracted by treatment with interferons , or (IFN). All three types of IFN exerted a protective effect on CBL cultures exposed to the virus. This resulted in: (a) a reduced number of virus-positive cells until 4 weeks of culture; (b) delay in the clonal expansion of infected cells (IFN and ); (c) increased natural killer cell activity of CBL, 1 week post-infection (p.i.), mediated by IFN; (d) increase of allospecific recognition of infecting and priming HTLV-I donor MT-2 cells by CBL in a cytotoxic-T-lymphocyte-like response, mediated by IFN and particularly by IFN; (e) phenotype distribution of CBL subpopulations, tested 4 days p.i., more similar to that of non-infected CBL cultures.In contrast, the overall CBL proliferation, that is profoundly depressed during the first week p.i., was not restored by IFN treatments, suggesting that boosting of the cell-mediated killing induced by IFN might involve the maturation of undifferentiated precursor cells rather than stimulation of their proliferation. The improvement of the efficiency of the antiviral immune response induced by treatment with IFN is likely to contribute to the clearance of virus-positive cells during the early phase of infection. This would provide experimental evidence to support an immunopharmacological approach contributing to the conversion of HTLV-I carriers from positive to negative.     

Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina Platensis

A 6.5 kb region from the genome of the cyanobacterium Spirulina platensis was cloned using as a probe the Escherichia coli gene for ribosomal protein S2. Sequence analysis revealed, in this region, the presence of the gene for ribosomal protein S2 and part of the gene for the elongation factor Ts (EF-Ts). The arrangement rpsB-spacer-tsf resembles that reported for E. coli. The deduced amino acid sequences of the platensis S2 and EF-Ts show significant homology with the E. coli counterparts.     

The Salmonella wien virulence plasmid pZM3 carries Tn1935, a multiresistance transposon containing a composite IS1936-kanamycin resistance element

Tn1935, a 23.5-kb transposon mediating resistance to ampicillin, kanamycin, mercury, spectinomycin, and sulfonamide was isolated from pZM3, an IncFIme virulence plasmid from Salmonella wien. Tn1935 possesses the entire sequence of Tn21 and contains two additional DNA segments of 0.95 and 2.7 kb carrying the ampicillin and kanamycin resistance genes, respectively. The latter is part of a composite element since it is flanked by two IS15-like insertion sequences (IS1936) in direct orientation. IS1936 is about 800 bp long and is closely related to IS15 delta, IS26, IS46, IS140, and IS176. Functional analysis of IS1936-mediated cointegrates shows that both insertion sequences are active and able to form cointegrates at the same frequency. Resolution of the cointegrates requires the presence of the host Rec system. The presence of the composite IS1936-element within Tn1935 supports the hypothesis that multidrug resistance transposons evolved by insertion of antibiotic determinants which are themselves transposable.     

Identification, cloning, nucleotide sequence and chromosomal map location of hns, the structural gene for Escherichia coli DNA-binding protein H-NS

Summary Beginning with a synthetic oligonucleotide probe derived from its amino acid sequence, we have identified, cloned and sequenced the hns gene encoding H-NS, an abundant Escherichia coli 15 kDa DNA-binding protein with a possible histone-like function. The amino acid sequence of the protein deduced from the nucleotide sequence is in full agreement with that determined for H-NS. By comparison of the restriction map of the cloned gene and of its neighboring regions with the physical map of E. coli K12 as well as by hybridization of the hns gene with restriction fragments derived from the total chromosome, we have located the hns gene oriented counterclockwise at 6.1 min on the E. coli chromosome, just before an IS30 insertion element.     

Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of newly synthetized oocyte protein and (2) cytochemical F-actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP-associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP-associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to α-chymotrypsin, the amount of newly synthesized actin displayed by cumulus-enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP-associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.     

Phorbol 12-myristate 13-acetate induces resistance of human melanoma cells to natural-killer-and lymphokine-activated-killer-mediated cytotoxicity

Summary Human melanoma cells are sensitive to the lytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells in vitro. The events resulting in tumour cell killing by lymphocytic effectors have not been completely clarified, and the same target cell determinants regulating responsiveness to immune cytolysis have not yet been identified. Indeed, changes in the differentiative status of leukemia cells as well as in the expression of major histocompatibility complex (MHC) antigens have been described to modulate sensitivity to cytotoxic effectors; moreover surface expression of adhesion factors or extracellular matrix proteins by the cancer cells can promote the activation of the cytolytic effectors and has been described to correlate with tumour cell sensitivity to cytolytic cells. We reasoned that treatment with differentiation inducers could modulate melanoma cell sensitivity to NK and LAK cells. The present study demonstrates that human melanoma GLL-19 cells, when treated with the phorbol diester phorbol 12-myristate 13-acetate (PMA) in vitro, undergo growth inhibition and neuron-like differentiation. Moreover PMA treatment induces an evident inhibition of GLL-19 cell sensitivity to NK- and LAK-mediated cytotoxicity. GLL-19 cells express constitutively MHC class I antigens. PMA treatment, however, does not modify the expression of MHC class I and class II DR antigens in human melanoma GLL-19 cells. We have finally evaluated the effects of PMA on the expression at the cell surface of adhesion factors such as ICAM-1, and extracellular matrix proteins such as collagen IV, laminin and fibronectin; we have also studied the expression of the integrin vitronectin receptor, a membrane receptor for adhesive proteins. While adhesion factors and extracellular matrix proteins appear to play an important role in the interaction between immune effector and tumour target, it can be supposed that the modulation of such membrane-associated proteins or glycoproteins induces NK and LAK resistance in cancer cells. We indeed found that PMA treatment induced in GLL-19 a marked reduction of membrane expression of collagen IV and ICAM-1; moreover PMA reduced the cell membrane expression of the integrin vitronectin receptor. On the other hand, membrane expression of fibronectin and laminin was not affected by PMA. These data indicate that the acquisition of a NK- and LAK-resistant phenotype by GLL-19 cells occurs together with cell differentiation, down-regulation of membrane expression of collagen IV, ICAM-1 and vitronectin receptor, but in the absence of changes in MHC antigens.This work has been supported by the Italian Association for Cancer Research (A. I. R. C.) and by Istituto Superiore di Sanità, Italy-USA joint program on New Therapies on Neoplasia.     

Hypothalamic-pituitary somatotropic function in prepubertal hypothyroid rats: effect of growth hormone replacement therapy.

The effect of thyroid hormone deficiency and growth hormone (GH) treatment on hypothalamic GH-releasing hormone (GHRH)/somatostatin (SS) concentrations, GHRH/SS mRNA levels, and plasma GH and somatomedin-C (IGF-I) concentrations were studied in 28- and 35-day-old rats made hypothyroid by giving dams propylthiouracil in the drinking water since the day of parturition. Hypothyroid rats, at both 28 and 35 days of life, had decreased hypothalamic GHRH content and increased GHRH mRNA levels, unaltered SS content and SS mRNA levels, and reduced plasma GH and IGF-I concentrations. Treatment of hypothyroid rats with GH for 14 days completely restored hypothalamic GHRH content and reversed the increase in GHRH mRNA, but did not alter plasma IGF-I concentrations. These data indicate that, in hypothyroid rats, the changes in hypothalamic GHRH content and gene expression are due to the GH deficiency ensuing from the hypothyroid state. Failure of the GH treatment to increase plasma IGF-I indicates that the feedback regulation on GHRH neurons is operated by circulating GH and/or perhaps tissue but not plasma IGF-I concentrations. Presence of low plasma IGF-I concentrations would be directly related to thyroid hormone deficiency.