Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen

17beta-Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17beta-estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17beta-estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17beta-estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17beta-estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17beta-estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17beta-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.     

A study on the localization and distribution of GnRH and its receptor in rat submaxillary glands by immunohistochemical,in situ hybridization and RT-PCR

We investigated the rat submaxillary gland for the presence of GnRH and GnRH receptors, the localization and colocalization of GnRH, GnRH receptor and their mRNA, and studied the sequence of GnRH receptor complementary DNA (cDNA) by immunohistochemistry, in situ hybridization and RT-PCR. The results showed that GnRH and GnRH receptor immunoreactive materials were colocalized in the epithelial cells of the serous acinus and glandular duct. The GnRH and GnRH receptor mRNA hybridization signals were detected in the above cells. The sequence obtained from the RT-PCR product was identical to the published cDNA sequence of GnRH receptor in the rat pituitary. The results suggested that the rat submaxillary gland was capable of synthesizing GnRH and GnRH receptors. GnRH may be involved in the functional regulation of the submaxillary gland through autocrine or paracrine activity.     

Association of hepatitis virus infection,alcohol consumption and plasma vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical workers

Urinary 8-hydroxydeoxyguanosine (8-OHdG) DNA adduct has been used as a biomarker in epidemiological studies. However, the determinants for urinary 8-OHdG have not been clearly identified. We tested urinary 8-OHdG levels in 205 male workers who had been exposed to vinyl chloride monomer (VCM). Epidemiological information was obtained by an interviewer-administered questionnaire. Hepatitis B surface antigen (HBsAg) and anti-hepatitis C antibody (anti-HCV) were also determined by immunoassay. Plasma antioxidants including Vitamins A and E, alpha- and beta-carotenes were assayed by high performance liquid chromatography. Median of urinary 8-OHdG level was 9.8 ng/mg creatinine (range, 1.4-60.1). Multiple linear regression analysis showed that alcohol drinkers had higher urinary 8-OHdG than those who did not, but there was no dose-response between the amount of alcohol consumption and urinary 8-OHdG. Workers with positive HBsAg, anti-HCV and elevated plasma Vitamin A level were independently associated with higher levels of urinary 8-OHdG, whereas age, smoking, body mass index, plasma alpha- and beta-carotenes, Vitamin E levels, or VCM exposure did not show such an association. The results suggest that active inflammation of hepatitis B and C, alcohol consumption and higher Vitamin A level can induce oxidative stress. Thus, we conclude that potential determinants need to be considered in epidemiological studies when urinary 8-OHdG is used as a biomarker.     

Activation of protein kinase A and clustering of cell surface receptors by N-methyl-N'-nitro-N-nitrosoguanidine are independent of genomic DNA damage

Alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cellular stress leading to chromosomal aberrations, mutations and cell death. Previous reports from our laboratory have shown that low concentration of MNNG induces untargeted mutation (UTM), which occurs on intact DNA in mammalian cells through changes in gene expression profile. It also causes the activation of cAMP-protein kinase A (PKA) and up-regulation of POL-beta, which is demonstrated to play a role in DNA repair system. In order to find out the possible initial signal involved in UTM, we try to investigate whether the activation of PKA-CREB signal pathway is closely related to DNA damage. Our data shows that the treatment of low concentration MNNG (0.2 microM) activates PKA-CREB pathway in a comparable level both in a nuclear and enucleated cell system. And similar to the cell response caused by UV, the clustering of cell surface receptors of epidermal growth factor (EGF) and tumor necrosis factor alpha (TNFalpha) was also observed in cells exposed to MNNG. It was further demonstrated that the clustering of the surface receptors is independent of the genomic DNA damage, as this phenomenon was also observed in enucleated cells. These observations indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damaging property.     

Lipid bilayer vesicle fusion: intermediates captured by high-speed microfluorescence spectroscopy

The fusion of lipid bilayers can be visualized under the fluorescence microscope, but the process is very fast and requires special techniques for its study. It is reported here that vesicle fusion is susceptible to analysis by microspectrofluorometry and that for the first time, the entire fusion process has been captured. In the case of giant (>10- micro m diameter) bilayer vesicles having a high density of opposite charge, fusion proceeds through stages of adhesion, flattening, hemifusion, elimination of the intervening septum, and uptake of excess membrane to generate a spherical product very rapidly. These investigations became possible with a fluorescence microscope that was modified for recording of images simultaneously with the collection of fluorescence emission spectra from many (>100) positions along the fusion axis. Positively-charged vesicles, composed of O-ethylphosphatidylcholine and dioleoylphosphatidylcholine, were labeled with a carbocyanine fluorophore. Negatively-charged vesicles, composed of dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine, were labeled with a rhodamine fluorophore that is a resonance energy transfer acceptor from the carbocyanine fluorophore. An electrophoretic chamber allowed selection of pairs of vesicles to be brought into contact and examined. Spectral changes along the axis of fusion were captured at high speed (a few ms/frame) by operating a sensitive digital camera in the virtual-chip mode, a software/hardware procedure that permits rapid readout of selected regions of interest and by pixel binning along the spectral direction. Simultaneously, color images were collected at video rates (30 frame/s). Comparison of the spectra and images revealed that vesicle fusion typically passes through a hemifusion stage and that the time from vesicle contact to fusion is <10 ms. Fluorescence spectra are well suited to rapid collection in the virtual-chip mode because spectra (in contrast to images) are accurately characterized with a relatively small number of points and interfering signals can be removed by judicious choice of barrier filters. The system should be especially well-suited to phenomena exhibiting rapid fluorescence change along an axis; under optimal conditions, it is possible to obtain sets of spectra (wavelength range of approximately 150 nm) at >100 positions along a line at rates >1000 frames/s with a spectral resolution of approximately 10 nm and spatial resolution at the limit of the light microscope ( approximately 0.2 micro m).     

Experimental investigations of multiple steady states in aerobic continuous cultivations of Saccharomyces cerevisiae

The steady-state behavior of a glucose-limited, aerobic, continuous cultivation of Saccharomyces cerevisiae CEN.PK113-7D was investigated around the critical dilution rate. Oxido-reductive steady states were obtained at dilution rates up to 0.09 h(-1) lower than the critical dilution rate by operating the bioreactor as a productostat, where the dilution rate was controlled on the basis of an ethanol measurement. Thus, the experimental investigations revealed that multiple steady states exist in a region of dilution rates below the critical dilution rate. The existence of multiple steady states was attributed to two distinct physiological effects occurring when growth changed from oxidative to oxido-reductive: (i) a decrease in the efficiency of ATP production and utilization (at ethanol concentrations below 3 g/L) and (ii) repression of the oxidative metabolism (at higher ethanol concentrations). The first effect was best observed at low ethanol concentrations, where multiple steady states were observed even when no repression of the oxidative metabolism was evident, i.e., the oxidative capacity was constant. However, at higher ethanol concentrations repression of the oxidative metabolism was observed (the oxidative capacity decreased), and this resulted in a broader range of dilution rates where multiple steady states could be found.     

pPIC9-Fc: a vector system for the production of single-chain Fv-Fc fusions in Pichia pastoris as detection reagents in vitro

Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates. For ScFv fragments, there is no such universal system available up to now. A vector system was constructed based on pPIC9- Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of mouse IgG1 and His-tag were cloned into the Pichia expression vector pPIC9. A model ScFv was introduced into pPIC9-Fc, which can bind Glutathione-S-transferase (GST) from Schistosoma japonicum, to yield the expression cassette pPIC9-ScFv-Fc. Following fermentation in a 5-liter reactor, the fusion was expressed at high levels in the methylotrophic yeast Pichia Pastoris, secreted as a dimeric form in the culture, and purified by Ni2+-NTA column chromatography. The expression yield can reach 10-30 mg/liter of culture medium. The ScFv-Fc fusion retains the biological binding ability of the parent ScFv, and can be applied as anti-GST antibodies for the detection of GST and GST-fusion proteins. Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-mouse antibodies.     

Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.