Hatice Gunes

Identification of extracellular enzyme producing thermophilic bacilli from Balcova (Agamemnon) geothermal site by ITS rDNA RFLP

Aims:  Molecular characterization of extracellular enzyme producing thermophilic bacilli from Balcova geothermal site.

Prolactin receptor gene expression in rat splenocytes and thymocytes from birth to adulthood

In vivo and in vitro studies have indicated that the anterior pituitary hormone prolactin (PRL) is an immunoregulator and functions in the development of the neonatal immune system. In this study, prolactin receptor (PRL-R) expression from birth to adulthood as well as the effect of milk ingestion on the PRL-R expression were examined in splenocytes and thymocytes of neonatal rats. Three approaches were taken to measure PRL-R expression: (i) polymerase chain reaction (RT-PCR); (ii) antibody to PRL-R and Western blotting; (iii) antibody to PRL-R and flow cytometry. RT-PCR analysis revealed the short and long form of PRL-R mRNA in both spleen and thymus at every age tested. However, the long form of PRL-R mRNA was always more abundant than that of the short form. In addition, antipeptide antibody against the long form of PRL-R recognized 84 and 42 kD proteins in the spleen, but only the 84 kD protein in the thymus. A monoclonal antibody U6 recognized 38 and 40 kD proteins in both the spleen and thymus. Although the mRNA level of PRL-R was relatively low at birth and increased with age in both the spleen and thymus, the levels of protein bands detected with both antibodies correlated with development in the spleen; whereas the levels remained steady in the thymus. Therefore, we concluded that the expression of PRL-R at the protein level is developmentally regulated in the spleen but not in the thymus. Finally, milk ingestion in the first seven hours decreased the percentage of cells expressing cell surface PRL-R, suggesting that milk-borne PRL may have a direct effect on lymphocytes.

Prolactin receptor gene expression in rat splenocytes and thymocytes during oestrous cycle, pregnancy and lactation

Much evidence suggests that prolactin (PRL) has an immunoregulatory function. Part of this evidence is that the receptors for PRL are present on lymphocytes. Probably the effects of PRL on cells of the immune system depend on the level and specific forms of PRL‐R present on the target cells. Therefore, PRL‐R expression at both protein and mRNA levels was examined during oestrous cycle, pregnancy and lactation using Western blotting and PCR analysis. Antibody to the long form of PRL‐R detected 84 and 42 kDa protein bands in the spleen but only 84 kDa band in the thymus. The expression pattern of these two protein bands was different in the spleen, suggesting that these two isoforms of PRL‐R long form are differentially regulated by the hormones of oestrous cycle. In addition, depending on the tissue, the level of mRNA for the short and long forms of PRL‐R showed a significant change at different stages of oestrous cycle. Moreover, 42 and 84 kDa PRL‐R bands were detected in both spleen and thymus throughout the pregnancy and lactation; however, the expression pattern of 84 kDa protein band was different between tissues. This finding suggests that each tissue exhibits differential response to hormones which affect PRL‐R content.

Identification of extracellular enzyme producing alkalophilic bacilli from Izmir province by 16S‐ITS rDNA RFLP

Aims:  To screen industrially important extracellular enzymes from the newly isolated alkalophilic bacilli and to characterize them by phenotypic and 16S‐internal transcribed spacer (ITS) rDNA restriction pattern analysis.

Prolactin receptor expression by splenocytes from rats in various hormonal states

Prolactin (PRL) is mitogenic for lymphocytes in vitro, but the responsiveness of lymphocytes depends on the in vivo hormonal status of the rats from which the cells were obtained. Lymphocytes from ovariectomized (OVX) rats, but not from rats in oestrus or from male rats, respond to prolactin; administration of oestradiol to OVX rats diminishes the response. In order to determine if a correlation exists between lymphocyte responsiveness to prolactin and levels of cell surface prolactin receptors (PRL‐R) expression, the percentage of splenocytes and each splenocyte subpopulation expressing surface PRL‐R from rats of various hormonal states (OVX, oestradiol‐injected OVX, oestrus and male) was analysed by single‐colour and dual‐colour flow cytometric analysis. We found that approximately 20% of splenocytes expressed surface PRL‐R regardless of hormonal states (n= 16). The majority (85%) of PRL‐R positive splenocytes were B lymphocytes whereas 11.1% and 4.8% of splenocytes expressing the PRL‐R were CD4 positive T‐helper (TH) and CD8 positive T‐cytotoxic (Tc) lymphocytes, respectively. B lymphocytes also stained more brightly than T lymphocytes. This distribution of PRL‐R expression did not show significant alterations on total splenocytes or TH and Tc lymphocytes during various hormonal stages. However, the percentage of PRL‐R‐positive B lymphocytes increased markedly in OVX rats (twofold), compared to rats at oestrus. In summary, no correlation was found between the responsiveness to prolactin as a mitogen and levels of PRL‐R expression by lymphocytes from rats at different hormonal states. This result suggests that sex steroid hormones may control prolactin responsiveness of lymphocytes by affecting the signal transduction pathway through PRL‐R rather than by altering the level of the cell surface receptor expression.

Induction of interleukin-2 receptor α-chain gene expression by cytochalasin B and 12-O-tetradecanoylphorbol-13-acetate in T lymphocytes

Abstract. The high affinity form of interleukin‐2 receptor (IL‐2R) is composed of two subunits; the α (p55) and β (p75). The α chain, unlike the β, is expressed only on activated T lymphocytes. Therefore, high affinity binding of interleukin‐2 (IL‐2) is controlled by the expression of the IL‐2R α‐chain. In this study, we examined the effect of cytochalasin B (CB) plus 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) on expression of IL‐2 and IL‐2R. Northern blot and flow cytometric analysis showed that the IL‐2R α‐chain was expressed both at mRNA and protein levels. However, IL‐2 gene expression was not induced by this treatment. Unlike the cells treated individually with CB or TPA, cells treated with CB plus TPA accumulated IL‐2R mRNA at all the times examined. In order to determine the percentage of cells that incorporated tritiated thymidine ([3H]dT) in the presence of IL‐2 after treatment with CB plus TPA, autoradiography was carried out. We found that about 11% of the cells were labelled. Because the percentage of labelled cells and cells expressing IL‐2R α‐chain was relatively low (11% and 9% respectively), perhaps CB plus TPA caused IL‐2R expression in only a subset of T cells.