In-Vivo Functional Study on the Involvement of CFTR,SLC26A6, NHE-1 and CA Isoenzymes II and XII in Uterine Fluid pH,Volume and Electrolyte Regulation in Rats under Different Sex-Steroid Influence

Precise control of uterine fluid pH, volume and electrolytes is important for the reproductive processes. In this study, we examined the functional involvement of multiple proteins including Cystic Fibrosis Transmembrane Regulator (CFTR), Cl^-/HCO₃^- exchanger (SLC26A6), sodium-hydrogen exchanger-1 (NHE-1) and carbonic anhydrase (CA) in the regulation of these uterine fluid parameters. Methods: Adult female WKY rats were divided into intact, non-ovariectomised at different oestrous cycle phases and ovariectomised treated with sex-steroids. Following oestrous phase identification or sex-steroid treatment, in-vivo uterine perfusion was performed with and without the presence of these inhibitors: glibenclamide, DIDS, ACTZ and EIPA. The pH, volume, Cl^-, HCO₃^- and Na⁺ concentrations of the perfusate from different groups were then analyzed. Meanwhile, the expression of CFTR, SLC26A6, NHE-1, CAII and CAXII was visualized by immunohistochemistry (IHC). Results: Parallel increase in the pH, volume, Cl^-, HCO₃^- and Na⁺ concentrations was observed at estrus (Es), proestrus (Ps) and following 17β-oestradiol (E) treatment, which was inhibited by glibenclamide, DIDS and ACTZ while parallel reduction in these parameters was observed at diestrus (Ds) and following progesterone (P) treatment which was inhibited by ACTZ and EIPA. CFTR and SLC26A6 expression were up-regulated under E dominance, while NHE-1 expression was up-regulated under P dominance. Meanwhile, CA isoenzymes were expressed under both E and P influence. Conclusion: CFTR, SLC26A6 and CA were involved in mediating parallel increase in the uterine fluid volume, pH and electrolyte concentration under E while NHE and CA were involved in mediating the reduction of these parameters under P.     

Genistein-induced fluid accumulation in ovariectomised rats' uteri is associated with increased cystic fibrosis transmembrane regulator expression

OBJECTIVE:

High genistein doses have been reported to induce fluid accumulation in the uteri of ovariectomised rats, although the mechanism underlying this effect remains unknown. Because genistein binds to the oestrogen receptor and the cystic fibrosis transmembrane regulator mediates uterine fluid secretion, we hypothesised that this genistein effect involves both the oestrogen receptor and cystic fibrosis transmembrane regulator.

METHODS:

Ovariectomised adult female Sprague-Dawley rats were treated with 25, 50, or 100 mg/kg/day genistein for three consecutive days with and without the ER antagonist ICI 182780. One day after the final drug injection, the animals were humanely sacrificed, and the uteri were removed for histology and cystic fibrosis transmembrane regulator mRNA and protein expression analysis using real-time polymerase chain reaction and Western blotting, respectively. The cystic fibrosis transmembrane regulator protein distribution was analysed visually by immunohistochemistry.

RESULTS:

The histological analysis revealed an increase in the circumference of the uterine lumen with increasing doses of genistein, which was suggestive of fluid accumulation. Moreover, genistein stimulated a dose-dependent increase in the expression of cystic fibrosis transmembrane regulator protein and mRNA, and high-intensity cystic fibrosis transmembrane regulator immunostaining was observed at the apical membrane of the luminal epithelium following 50 and 100 mg/kg/day genistein treatment. The genistein-induced increase in uterine luminal circumference and cystic fibrosis transmembrane regulator expression was antagonised by treatment with ICI 182780.

CONCLUSION:

Genistein-induced luminal fluid accumulation in ovariectomised rats'' uteri involves the oestrogen receptor and up-regulation of cystic fibrosis transmembrane regulator expression, and these findings reveal the mechanism underlying the effect of this compound on changes in fluid volume in the uterus after menopause.     

Viral messenger RNAs in six lines of adenovirus-transformed cells

We have compared the proteins translated in vitro using virus-specific mRNA from six lines of adenovirus-transformed cells (F4, F17, F19, REM, T2C4, and 293) with those made using RNA from productively infected cells. The mRNAs for proteins E1A-58K, 54K, 48K, and 42K, and for E1B-15K, were detected in all cell lines and for E1B-57K in at least five of the lines. Neither of the minor E1A mRNAs (for proteins 25K and 28K) found under specific conditions in infected cells were found in any transformed cell line. Two cell lines, F4 and T2C4, contained small amounts of other mRNAs encoded within region E1B including in the latter case one not detected in infected cells. The lines F4, REM, and T2C4 contain at least some E4 mRNAs, again including in the latter case one not detected in infected cells. T2C4 cells also contain E3 mRNAs, E2A-72K protein mRNA, and two E2B mRNAs, although the E2B proteins are not identical to any found in infected cells. The F19 and T2C4 lines express mRNAs encoded between 11 and 17 map units, the “IVa₂ region,” but do not include the mRNA for polypeptide IVa₂ itself. Some early mRNAs, including the “immediate early” mRNAs, have not been detected in any of these lines of transformed cells: nor did the cell lines contain any mRNAs found exclusively at late times during productive infection. Comparison of these results with the properties of the cells tentatively suggest a correlation (although not necessarily causal) between the expression of additional viral genes and increased tumorigenicity.     

Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p

We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A)⁺ RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A)⁺ RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A)⁺ RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways.     

The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins

To help account for the variable quality and quantity of RNA in human brain, we have studied the effect of premortem (agonal state) and postmortem factors on the detection of poly(A)⁺mRNA and eight mRNAs. For comparison, the influence of the same factors upon gene products encoded by the mRNAs was studied immunocytochemically or by receptor autoradiography. Brain pH declined with increasing age at death and was related to agonal state severity, but was independent of postmortem interval and the histological presence of hypoxic changes. By linear regression, pH was significantly associated with the abundance of several of the RNAs, but not with poly(A)⁺mRNA, immunoreactivities, or binding site densities. Postmortem interval had a limited influence upon mRNA and protein products. Freezer storage time showed no effect. Parallel rat brain studies showed no relationship between postmortem interval (0–48 h) and amounts of total RNA, poly(A)⁺RNA, or two individual mRNAs; however, RNA content was reduced by 40% at 96 h after death. pH is superior to clinical assessments of agonal state or mode of death in predicting mRNA preservation. It provides a simple means to improve human brain gene expression studies. pH is stable after death and during freezer storage and can be measured either in cerebrospinal fluid or in homogenised tissue.     

Influenza virus polymerase confers independence of the cellular cap-binding factor eIF4E for viral mRNA translation

The influenza virus mRNAs are structurally similar to cellular mRNAs nevertheless; the virus promotes selective translation of viral mRNAs despite the inhibition of host cell protein synthesis. The infection proceeds normally upon functional impairment of eIF4E cap-binding protein, but requires functional eIF4A helicase and eIF4G factor. Here, we have studied whether the presence of cis elements in viral mRNAs or the action of viral proteins is responsible for this eIF4E-independence. The eIF4E protein is required for viral mRNA translation in vitro, indicating that cis-acting RNA sequences are not involved in this process. We also show that PB2 viral polymerase subunit interacts with the eIF4G protein. In addition, a chimeric mRNA containing viral UTR sequences transcribed by the viral polymerase out of the infection is successfully translated independently of an impaired eIF4E factor. These data support that the viral polymerase is responsible for the eIF4E independence of influenza virus mRNA translation.     

Isoflavone Genistein Induces Fluid Secretion and Morphological Changes in the Uteri of Post-Pubertal Rats

A reported increase in the incidence of infertility following high genistein intake could be related to alteration in the normal fluid volume and morphology of the uterus in adult female. In view of this, we investigated the effect of this compound on fluid secretion, fluid volume and morphology of the uterus in post-pubertal rats. Methods: Ovariectomised SD rats were treated with 17-β oestradiol (E) (0.8 X 10^-4 mg/kg/day) and genistein (0.5, 5, 10, 25, 50 and 100 mg/kg/day) for three days. Following drug treatment, in-vivo uterine perfusion was performed and the rate of fluid secretion and the volume of fluid in the uterus were determined via changes in weight (μl/min) and F-dextran concentration of the perfusate respectively. The animals were then sacrificed and the uteri were removed for weight determination, morphological analyses and proliferative cell nuclear antigen (PCNA) expression analyses by Western blotting. Results: Subcutaneous genistein treatment resulted in a dose-dependent increase in fluid secretion rate, fluid volume and uterine wet weight. Treatment with 100 mg/kg/day genistein resulted in a remarkable increase in the rate of uterine fluid secretion, the volume of the uterine luminal fluid as well as the circumference of the uterine and uterine glandular lumen suggesting an excessive fluid accumulation. Meanwhile, there were evidence of glandular hyperplasia and an increase in the expression of PCNA following treatment with 50 and 100 mg/kg/day genistein. Conclusion: High genistein intake could potentially cause adverse effects on the uterus by inducing excessive fluid secretion and accumulation as well as hyperplasia.     

Gender and Androgen Treatment Influence the Expression of Proto-oncogenes and Apoptotic Factors in Lacrimal and Salivary Tissues of MRL/lprMice

The objectives of this study were to (1) determine whether Fas antigen, Fas ligand, p53, and proto-oncogene mRNAs may be detected in lacrimal and submandibular glands of the MRL/lprmouse model of Sjögren's syndrome, and (2) examine whether gender and androgen or cyclophosphamide therapy influence the mRNA expression of these apoptotic factors. Tissues were obtained from treated or untreated MRL/lprmice after the onset of disease and processed for the analysis of mRNAs by RT-PCR and Southern blot hybridization. Our results demonstrated that (1) Fas antigen (exons 1 → 2 or 3 → 7+), Fas ligand, c-myb, c-myc, bcl-2, Bax, p53, and androgen receptor (AR) mRNAs are present in exocrine tissues of MRL/lprmice; (2) the amounts of c-myb, c-myc, bcl-2, p53, and AR mRNA are higher (P< 0.05) and the level of Fas antigen (exons 1 → 2) mRNA is lower (P< 0.05) in lacrimal glands of female compared to male mice. In contrast, the content of c-myb and p53 mRNA is greater (P< 0.05) in submandibular tissues of female relative to those of male mice; and (3) testosterone or cyclophosphamide treatment led to a significant (P< 0.05) decline in the mRNA levels of c-myb, bcl-2, and/or AR, but an increase (P< 0.05) in the mRNA amount of Bax, in lacrimal, but not in salivary, glands of female mice. These findings demonstrate that gender-associated differences exist in the expression of apoptotic factor mRNAs in exocrine tissues of autoimmune mice and that some of these differences appear to be due to the influence of androgens.     

Molecular cloning and expression of toll-like receptor 4 (tlr4) in the blunt snout bream (Megalobrama amblycephala)

Toll-like receptors (TLRs) play a pivotal role in teleost innate immune system. In this study, Megalobrama amblycephala (ma) tlr4 gene was cloned, its putative polypeptide product characterized, and expression analysed. Matlr4 cDNA is 2862 bp long, with an open reading frame of 2364 bp encoding 787 amino acids. MaTlr4 is a typical TLR protein, including the extracellular part with nine leucine-rich repeat motifs, a transmembrane region and a cytoplasmic Toll/interleukin-1 receptor domain. MaTlr4 has the highest level of identity (94%) and similarity (97%) with the grass carp Tlr4.2 homolog. This was also corroborated by the phylogenetic analysis, which placed MaTlr4 in a cluster with other cyprinid homologs. Matlr4 mRNA was ubiquitously expressed in all examined tissues and during all sampled developmental stages. The observed peak in matlr4 mRNA expression during gastrula and somite stages is in good agreement with its proposed role in the development of the neural system. Temporal expression patterns of matlr4 and maMyD88 mRNAs and proteins were analyzed in liver, spleen, head kidney, trunk kidney and intestine after Aeromonas hydrophila infection. And mRNA expression varied between different time-points. Both MaTlr4 and MaMyD88 protein expressions at 12 hpi were significantly enhanced in head kidney and intestine. These results indicate that matlr4 is involved in the immune response in M. amblycephala, and that it is indeed a functional homologue of tlr4s described in other animal species.     

Expression of pulmonary aquaporin 1 is dramatically upregulated in mice with pulmonary fibrosis induced by bleomycin

Introduction

Pulmonary fibrosis occurs due to fibroblast proliferation and collagen production in the lung and begins with alveolar inflammatory edema. Aquaporins (AQPs) play pivotal roles in lung fluid transport. In this study we establish the experimental model for pulmonary fibrosis in C57BL/6 mice to investigate expressions of AQP1 and AQP5 in lung tissue.

Material and methods

Mice in model groups were treated intratracheally with bleomycin with the dose of 5 mg/kg body weight. The mice were sacrificed at 1 week, 2 weeks and 3 weeks respectively. The left upper lungs were harvested for histopathologic H-E and Masson''s staining. The mRNAs of AQP1 and AQP5 were analyzed by real-time polymerase chain reaction (real-time PCR) and the proteins of AQP1 and AQP5 were analyzed by western blotting.

Results

Real-time PCR showed that AQP1 mRNA in bleomycin 1 w, 2 w, and 3 w groups increased by 377%, 880% and 823% respectively compared to that in the control group (p < 0.01). Western blotting showed that the expression of AQP1 protein in bleomycin 1 w, 2 w, and 3 w groups increased by 53%, 144%, and 141%, respectively (p < 0.05). AQP5 mRNA in bleomycin 1 w and 2 w group decreased by 78% and 66%, respectively (p < 0.05). In bleomycin 2 w and 3 w groups it decreased by 69% and 80% (p < 0.05).

Conclusions

The expression of AQP1 dramatically increased in pulmonary fibrosis. AQP1 plays an important role in the progress of pulmonary fibrosis.     

Na+-H+ exchange activity in taste receptor cells

mRNA for two Na(+)-H(+)-exchanger isoforms 1 and 3 (NHE-1 and NHE-3) was detected by RT-PCR in fungiform and circumvallate taste receptor cells (TRCs). Anti-NHE-1 antibody binding was localized to the basolateral membranes, and the anti-NHE-3 antibody was localized in the apical membranes of fungiform and circumvallate TRCs. In a subset of TRCs, NHE-3 immunoreactivity was also detected in the intracellular compartment. For functional studies, an isolated lingual epithelium containing a single fungiform papilla was mounted with apical and basolateral sides isolated and perfused with nominally CO(2)/HCO(3)(-)-free physiological media (pH 7.4). The TRCs were monitored for changes in intracellular pH (pH(i)) and Na(+) ([Na(+)](i)) using fluorescence ratio imaging. At constant external pH, 1) removal of basolateral Na(+) reversibly decreased pH(i) and [Na(+)](i); 2) HOE642, a specific blocker, and amiloride, a nonspecific blocker of basolateral NHE-1, attenuated the decrease in pH(i) and [Na(+)](i); 3) exposure of TRCs to basolateral NH(4)Cl or sodium acetate pulses induced transient decreases in pH(i) that recovered spontaneously to baseline; 4) pH(i) recovery was inhibited by basolateral amiloride, 5-(N-methyl-N-isobutyl)-amiloride (MIA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), HOE642, and by Na(+) removal; 5) HOE642, MIA, EIPA, and amiloride inhibited pH(i) recovery with K(i) values of 0.23, 0.46, 0.84, and 29 microM, respectively; and 6) a decrease in apical or basolateral pH acidified TRC pH(i) and inhibited spontaneous pH(i) recovery. The results indicate the presence of a functional NHE-1 in the basolateral membranes of TRCs. We hypothesize that NHE-1 is involved in sour taste transduction since its activity is modulated during acid stimulation.     

Bcl-2、Bax在NHE-1抑制导致的大鼠肺动脉平滑肌细胞凋亡中的作用

目的:探讨Bcl-2、Bax蛋白表达在Na⁺/H⁺交换器-1(NHE-1)抑制而诱导大鼠肺动脉平滑肌细胞(PASMC)凋亡中的作用。方法: 荧光指示剂(Fura-2/AM)测定法检测转染NHE-1特异性核酶基因的大鼠PASMC内Ca²⁺(i)变化;RT-PCR方法检测细胞内bcl-2和baxmRNA表达变化, 免疫组化法检测细胞内Bcl-2和Bax蛋白表达变化。 结果:转染NHE-1特异性核酶基因后, 大鼠PASMC内i显著升高, bcl-2mRNA及蛋白表达显著降低, baxmRNA和蛋白表达显著增加。结论: NHE-1抑制诱导的PASMC凋亡与i增加、bcl-2表达降低及bax表达增加有关。     

NHE-1与大鼠肺动脉平滑肌细胞增殖和凋亡

目的:探讨Na⁺/H⁺交换器-1(NHE-1)、细胞内pH对肺动脉平滑肌细胞增殖和凋亡的作用。方法:实验分对照组和低氧3周组,大鼠常压低氧3周后,分离肺动脉平滑肌层,测定细胞内pH,并应用RT-PCR技术,检测NHE-1mRNA表达的变化。体外培养肺动脉平滑肌细胞,诱导细胞酸化后,原位细胞凋亡检测不同浓度及时间NHE-1特异性抑制剂作用后,细胞凋亡率的改变。结果:低氧组肺动脉平滑肌细胞内pH及NHE-1mRNA表达均明显高于正常对照组。随药物浓度增加和作用时间延长,细胞凋亡率明显增高。结论:NHE-1参与调节细胞内pH,在肺动脉平滑肌细胞增殖和凋亡中起重要的作用。     

Molecular characterization of the newly identified human parvovirus 4 in the family Parvoviridae

Human parvovirus 4 (PARV4) is an emerging human virus, and little is known about the molecular aspects of PARV4 apart from its incomplete genome sequence, which lacks information of the termini. We analyzed the gene expression profile of PARV4 using a nearly full-length HPV4 genome in a replication competent system in 293 cells. We found that PARV4 utilizes two promoters to transcribe non-structural protein- and structural protein-encoding mRNAs, respectively, which were polyadenylated at the right end of the genome. Three major proteins, including the large non-structural protein NS1a, whose mRNA is spliced, and capsid proteins VP1 and VP2, were detected. Additional functional analysis of the NS1a revealed its capability to induce cell cycle arrest at G2/M phase in ex vivo-generated human hematopoietic stem cells. Taken together, our characterization of the molecular features of PARV4 suggests that PARV4 represents a new genus in the family Parvoviridae.     

Temporal regulation of the rate of vesicular stomatitis virus mRNA translation during infection of Chinese hamster ovary cells

Vesicular stomatitis virus (VSV) mRNAs on polysomes 3 hr after infection are not translated in vivo as efficiently as mRNAs 2 hr after infection. There are 14?, 12?, 11?, and 8-fold increases in the amounts of L, G, N, and M messengers, respectively, on polysomes between 2 and 3 hr after infection. However, there are only 3?, 5?, 3?, and 4-fold increases in the rates of synthesis of the respective proteins. The in vivo translational efficiences of these messengers, which is a measure of their capacity to act as template for protein synthesis, are therefore lower at 3 hr after infection than at 2 hr. Since the mRNAs isolated 3 hr after infection are as active in a wheat germ cell-free protein synthesizing system as the mRNAs isolated at 2 hr, the decreased efficiency of VSV translation in vivo at 3 hr is not due to changes in mRNA primary structure. The observed difference is more likely due to a reduced efficiency of the host cell translational system to translate VSV mRNA at 3 hr relative to 2 hr. It was found that at 2 hr after infection, the majority of VSV mRNA is not associated with polysomes, but at 3 hr, the majority of viral messages is polysome bound.