哺乳动物P1精蛋白基因表达的研究

目的:研究哺乳动物P1精蛋白基因在大鼠和小鼠中的表达。方法:用RT-PCR法制备大鼠P1精蛋白(rP)cDNA,用小鼠P1精蛋白(mP1)DNA重组质粒(pUC8)制备mP1cDNA,rPcDNA和mP1cDNA经迪高辛标记后作为探针,用Northern杂交等技术分析基因表达情况。结果:rPcDNA与大鼠睾丸RNA有杂交反应,而与肝、脑RNA无交叉反应;rPcDNA和小鼠睾丸RNA有交叉反应;mP1cDNA探针与性成熟不同阶段小鼠睾丸RNA杂交结果表明:小鼠性成熟过程中,在睾丸出现圆形精子细胞后mP1基因开始转录,而翻译成蛋白质是在性成熟小鼠睾丸内出现长形精子细胞后。结论:哺乳动物P1精蛋白的表达有器官特异性,是睾丸特异表达的基因,P1精蛋白基因在进化上保守,因而在大、小鼠睾丸中均能检测到杂交讯号;精蛋白基因的表达表现为翻译延迟,该基因在圆形精子细胞阶段开始转录,而在长形精子细胞阶段翻译成蛋白质。     

Expression of cyclooxygenase-2 (COX-2), receptors for estrogen (ER), and progesterone (PR), p53, ki67, and neu protein in endometrial cancer

OBJECTIVE: We aimed at investigating by immunohistochemistry the relationship between cyclooxygenase-2 (COX-2) and estrogen (ER), and progesterone (PR) receptors in a single institution series of 90 primary untreated endometrial cancer patients. The simultaneous assessment of p53 protein, ki67, and neu protein has been carried out. METHODS: Immunohistochemistry was performed on paraffin-embedded sections by using rabbit polyclonal antiserum against human COX-2, anti-ER (clone 1D5), and anti-PR (clone 1A6) monoclonal antibodies, anti ki67 (clone MIB-1) and p53 (clone DO-7), and polyclonal antibody anti human c-erbB2/neu. RESULTS: There was no difference in the distribution of COX-2, p53, and neu positive cases according to ER or PR positivity, while the percentage of ki67 positive endometrial tumors was significantly higher in ER negative versus ER positive tumors (54.5% versus 31.6%, P value = 0.044). ER and PR positive tumors showed a statistically significant association with clinicopathological parameters of better clinical outcome. There was no clear association between COX-2 positivity and any of the clinicopathological features. The percentage of ki67, p53, and neu positive tumors was found to be strictly related to more aggressive features. Only advanced stage of disease was found to be a predictor of poor prognosis (P value = 0.034). None of the biological parameters examined was shown to be associated with patient outcome. CONCLUSIONS: We showed that COX-2 expression is not correlated with ER, PR, p53, and neu, thus suggesting that COX-2-mediated activities may follow independent pathways. Our findings provide the rationale to design trials based on the combination of antihormones with inhibitors of COX-2 and neu in recurrent/metastatic endometrial cancer.     

Cytochrome P2A13 and P1A1 gene polymorphisms are associated with the occurrence of uterine leiomyoma

Problem To investigate the association between the occurrence of uterine leiomyoma and two SNPs of the CYP 2A13 and CYP 1A1 genes.Method of study Prospective case control study with 132 women with clinically and surgically diagnosed uterine leiomyoma and 260 controls. Genotyping was performed by polymerase chain reaction (PCR) based amplification of CYP 2A13 and CYP 1A1 genes, and restriction fragment length polymorphism (RFLP) analysis.Results Comparing women with uterine leiomyoma and controls, we demonstrate statistical significant differences of allele frequency and genotype distribution for the CYP 1A1 polymorphism (P = 0.025 and P = 0.046, respectively). Furthermore, for the CYP 2A13 polymorphism we found a significant difference concerning allele frequency (P = 0.033). However, for the genotype distribution, only borderline significance was observed (P = 0.064).Conclusions The CYP 2A13 and CYP 1A1 SNPs are associated with uterine leiomyoma in a Caucasian population and may contribute to the understanding of the pathogenic mechanisms of uterine leiomyoma.     

Monocyte chemoattractant protein-1 (MCP-1/CCL2) in experimental autoimmune orchitis

Experimental autoimmune orchitis (EAO) is characterized by an interstitial mononuclear cell infiltrate and a severe lesion of seminiferous tubules with germ cells that undergo apoptosis and sloughing. The mechanism by which immune cells migrate and extravasate in the testicular interstitium is poorly understood. The aim of this study was to detect the variations in the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor in the testis of rats undergoing autoimmune orchitis. EAO was induced in Sprague–Dawley adult rats by active immunization with an emulsion of testicular homogenate and complete Freund adjuvant using Bordetella pertussis as co-adjuvant. Control rats injected with saline and adjuvants and normal untreated rats were also studied. By ELISA we observed a significant increase of MCP-1 in the testicular fluid (TF) and in the conditioned medium obtained from cultures of testicular macrophages of rats with EAO compared with control groups. By immunohistochemistry, an increase in MCP-1 expression was observed in mononuclear, endothelial, Leydig and peritubular cells. MCP-1 immunoreactivity was also detected in Sertoli cell cytoplasm of rats with severe orchitis. A 2-fold increase in the number of mononuclear cells that express CCR2 was also found in rats with orchitis compared with controls. In conclusion, we demonstrated in vivo that MCP-1 is highly expressed in testicular interstitial cells suggesting that this chemokine has an important role in recruiting immune cells to the testis in rats undergoing autoimmune orchitis.     

The correlation between birth weight and insulin-like growth factor-binding protein-1 (IGFBP-1), kisspeptin-1 (KISS-1), and three-dimensional fetal volume

Purpose: This study aimed to determine the relationship between birth weight, and maternal serum insulin-like growth factor-binding protein-1 (IGFBP-1) and kisspeptin-1 (KISS-1) levels, and first-trimester fetal volume (FV) based on three-dimensional ultrasonography.

Materials and methods: The study included 142 pregnant women at gestational week 11°–13⁶. All fetuses were imaged ultrasonographically by the same physician. Maternal blood samples were collected at the time of ultrasonographic evaluation and analyzed for IGFBP-1 and KISS-1 levels via enzyme-linked immunosorbent assay (ELISA). Maternal and neonatal weights were recorded at birth. Birth weight ≤10th and the >90th percentiles was defined as small and large for gestational age (SGA and LGA), respectively.

Results: Median crown-rump length (CRL), FV, and maternal serum IGFBP-1 and KISS-1 levels were 58.2?mm (35.3–79.2?mm), 16.3?cm³ (3.8–34.4?cm³), 68.1?ng?mL^?1 (3.8–377.9?mL^?1), and 99.7?ng?L^?1 (42.1–965.3?ng?L^?1), respectively. First-trimester IGFBP-1 levels were significantly lower in the mothers with LGA neonates (p?p?>?.05). The maternal IGFBP-1 level during the first trimester was a significant independent factor for SGA and LGA neonates (Odds ratio (OR): 0.011, 95%CI: 1.005–1.018, p?p?=?.007, respectively). There was no significant relationship between SGA or LGA, and CRL, FV, or the KISS-1 level.

Conclusions: As compared to the maternal KISS-1 level, the maternal IGFBP-1 level during the first trimester might be a better biomarker of fetal growth. Additional larger scale studies are needed to further delineate the utility of IGFBP-1 as a marker of abnormal birth weight.     

Cyclooxygenase-2 (COX-2), epidermal growth factor receptor (EGFR), and Her-2/neu expression in ovarian cancer

OBJECTIVES: Cyclooxygenase-2 (COX-2) seems to be involved in critical steps of cancer onset and progression. Abnormalities of epidermal growth factor receptor (EGFR) and Her-2/neu have been actively investigated in ovarian cancer and associated with unfavorable clinical outcome. The involvement of COX-2 in ErbB family pathways has been proposed. We investigated by immunohistochemistry the expression of COX-2, EGFR, and Her-2/neu in a series of advanced primary ovarian cancers. METHODS: The study included 76 consecutive stage IIIC-IV ovarian cancer patients with measurable disease after first surgery. Immunohistochemistry was performed on paraffin-embedded sections with rabbit antiserum against COX-2, murine monoclonal antibody (MoAb) 300G9 against Her-2/neu, and monoclonal antibody 108 against EGFR. RESULTS: No association among COX-2, EGFR, and HER-2/neu was found. COX-2 positivity was found in a statistically significant higher percentage of unresponsive cases (80.0%) than in patients responding to chemotherapy (35.7%) (P = 0.0008). The association between COX-2 positivity and poor chance of response to treatment was retained in multivariate analysis. In the subgroup of patients who underwent explorative laparotomy COX-2-positive cases showed a shorter overall survival (P = 0.049). CONCLUSIONS: COX-2 could represent a possible new marker of sensitivity to platin-based chemotherapy in ovarian cancer. The lack of association of COX-2 with EGFR or Her-2/neu suggests that the ability of COX-2 to predict tumor sensitivity to chemotherapy is not dependent on EGFR or Her-2/neu status and could be independently associated with prognosis. In this context, the availability of agents able to specifically interfere with COX-2, Her-2/neu, or EGFR tyrosine kinase is of potential interest.     

Norepinephrine transporter (NET), serotonin transporter (SERT), vesicular monoamine transporter (VMAT2) and organic cation transporters (OCT1, 2 and EMT) in human placenta from pre-eclamptic and normotensive pregnancies

Pre-eclampsia is one of the most common causes of perinatal and maternal morbidity and mortality. High blood pressure and proteinuria are important clinical signs of pre-eclampsia. Sympathetic overactivity and elevated level of circulating vaso active substances, such as monoamines has been shown. Extracellular concentrations of monoamines are normally kept low by specific transporter proteins of which many are expressed in the placenta. In this study we used in situ hybridization and real-time PCR to study the gene expression of monoamine transporters, such as NET, SERT, VMAT2, EMT and OCT1/2, in normal as well as in pre-eclamptic placentae. We demonstrated high expression of NET mRNA in the trophoblast cells of the anchoring villi and a lower expression intensity in the chorionic villi. SERT mRNA was mainly detected in chorionic villi. VMAT2 mRNA was not detected in the central part of the placenta but was present in the spiral arteries of placenta bed biopsies, in cytokeratin positive cells. EMT mRNA was mainly detected in the intra lobular septa and together with OCT1 and OCT2 mRNAs also expressed in scattered cells of placental vessel adventitias. Moreover, quantitative analysis showed a significant lower expression of NET and EMT mRNAs in pre-eclamptic placentae as compared to the control group. A defective gene expression or function of these monoamines transporters might explain the elevated concentrations of monoamines in pre-eclamptic patients. Monoamine transporters may serve as a protective mechanism preventing vasoconstriction in the placental vascular bed and thereby securing a stable blood flow to the fetus.     

p57~（K1P2）和PHLDA2蛋白表达对葡萄胎的诊断意义

目的研究母源表达的印记基因p57K1P2和PHLDA2（IPL/TSSC3）蛋白表达对葡萄胎的辅助诊断意义。方法收集北京协和医院刮宫组织存档的石蜡包埋标本,其中病理组织学诊断完全性葡萄胎（completehydatidiformmole,CHM）13例,部分性葡萄胎（partialhydatidiformmole,PHM）16例。全部病例行流式细胞术DNA倍体分析,采用免疫组化二步法检测p57K1P2及PHLDA2在病理组织中表达。结果29例病例DNA倍体分析：二倍体15例,三倍体13例,四倍体1例,诊断为CHM15例,PHM14例,病理诊断的符合率为86.2%。部分性葡萄胎p57K1P2和PHLDA2绒毛滋养细胞层免疫组织化学全部阳性（100%,14/14）。PHLDA2染色阳性位于胞质和胞膜,表达为强阳性。p57K1P2染色阳性位于胞核,表达为强阳性。所有完全型葡萄胎绒毛滋养细胞层p57K1P2和PHLDA2免疫组织化学均阴性（100%,15/15）。结论p57K1P2和PHLDA2可能是鉴别CHM和非CHM的标志物,两者免疫组化学阴性可能是CHM的可靠标志物,但有待扩大样本验证。     

Selective P2Y2 receptor agonists stimulate vaginal moisture in ovariectomized rabbits

OBJECTIVE: To determine the expression of P2Y(2) receptors in vaginal and cervical tissues and the effects of P2Y(2) receptor agonists INS45973 and INS365 on vaginal moisture. DESIGN: Pilot in vivo and histological study using animal subjects. SETTING: Experimental laboratory research. ANIMAL(S): Female New Zealand White rabbits were used for in vivo studies and female cynomolgus monkey (Macaca fascicularis) was used for in situ hybridization. INTERVENTION(S): Rabbits were kept intact or ovariectomized. Two weeks after ovariectomy, animals received daily vaginal instillation of vehicle or drugs for 16 days. MAIN OUTCOME MEASURE(S): Vaginal moisture was assessed in rabbits on 4 separate days during the treatment period. The P2Y(2) receptor mRNA distribution was assessed by in situ hybridization of monkey vagina and cervix. RESULT(S): Compared to control, vaginal moisture was significantly diminished in ovariectomized animals treated with vehicle. INS365 (8.1%) and INS45973 (0.9%) increased vaginal moisture in ovariectomized animals to levels that were comparable to or significantly higher than control animals, respectively. In situ hybridization studies indicated that P2Y(2) receptor mRNA was localized to endocervical and cervical gland, epithelium, and stratified squamous epithelium of the vagina. CONCLUSION(S): INS45973 and INS365 may interact with P2Y(2) receptors in the cervix and vagina to stimulate vaginal moisture in the estrogen (E)-deprived state. The P2Y(2) receptor agonists provide a potential nonhormonal alternative for treating vaginal dryness in postmenopausal women.     

Susceptibility to Toxoplasma gondii proliferation in BeWo human trophoblast cells is dose-dependent of macrophage migration inhibitory factor (MIF), via ERK1/2 phosphorylation and prostaglandin E2 production

Introduction

Macrophage migration inhibitory factor (MIF) participates in the immune response to Toxoplasma gondii, triggers ERK1/2 and prostaglandin E₂ (PGE₂) activation, but there is limited information on these mechanisms in human trophoblast. The present study aimed to verify the role of MIF in the ERK1/2 phosphorylation and PGE₂ production, as well as its effect on the susceptibility to T. gondii in BeWo cells.

Methods

BeWo cells were treated with increasing concentrations of recombinant MIF (rMIF) and/or T. gondii-soluble tachyzoite antigen (STAg) and analyzed for ERK1/2 phosphorylation and PGE₂ production by Western blotting and ELISA, respectively. Cells were also treated with increasing concentrations of rMIF, rPGE₂, or ERK1/2 inhibitor and tested for T. gondii proliferation. The supernatants of cells treated with rPGE₂ were assayed for cytokine production by ELISA or CBA.

Results

ERK1/2 phosphorylation and PGE₂ production increased when the cells were treated with low MIF concentrations while the parasitism control occurred only at high MIF concentrations. STAg was unable to change ERK1/2 phosphorylation or PGE₂ release. BeWo cells demonstrated increased T. gondii proliferation and reduced production of pro-inflammatory cytokines when treated with PGE₂, while PD98059 diminished the parasite proliferation.

Discussion

The intracellular mechanisms triggered by MIF are dose-dependent in BeWo cells, and PGE₂ is an important factor for the persistence of T. gondii at the maternal fetal interface.

Conclusion

MIF was unable to control T. gondii infection in BeWo cells at low concentrations since ERK1/2 and PGE₂ expression were activated, demonstrating a critical effect of these mediators favoring parasite proliferation.     

Serum chorionic gonadotrophin (hCG), schwangerschaftsprotein 1(SP1), progesterone and oestradiol levels in patients with nausea and vomiting in early pregnancy

Summary. Serum concentrations of human chorioni gonadotrophin (hCG). schwangerschaftsprotein l(SPl), progesterone and oestradiol were measured in 116 pregnant women experiencing varying degrees of nausea and vomiting or no nausea at all at between 9 and 16 weeks gestation. The patients were categorized into four groups, namely asymptomatic, nausea alone, nausea and vomiting and hyperemesis gravidarum. The distribution of levels for each group were examined in relation to the calculated normal ranges. Statistically higher hCG levels were found in out-patients with nausea alone or nausea and vomiting than in the asymptomatic women. No significant differences were found between the groups for any of the other measured variables, including the progesterone/oestradiol concentration ratio.     

Circulating levels of placental proteins, placental protein 5 (PP5), placental lactogen and Schwangerschafts-protein 1 (SP1), in antepartum eclampsia

Summary. Placental protein 5 (PP5), Schwangerschaftsprotein 1 (SP1) and human placental lactogen (hPL) were measured in 36 serum samples from seven women with antepartum eclampsia. PP5 levels were generally elevated, hPL levels were reduced and SP1 levels were within the usual ranges.     

Gelatinase A (MM-2), gelatinase B (MMP-9) and their inhibitors (TIMP 1, TIMP-2) in serum of women with endometriosis: Significant correlation between MMP-2, MMP-9 and their inhibitors without difference in levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in relation to the severity of endometriosis

Endometriosis is a highly prevalent gynecological condition, where the formation of endometriotic foci is linked with locally increased activity of matrix metalloproteinases (MMPs). In the present study, we tested the hypothesis that raised serum levels of MMPs might reflect the severity of endometriosis. We compared serum levels of MMP-2 and MMP-9, and of their tissue inhibitors TIMP-1 and TIMP-2, in infertile women, matched for age and body mass index, with either mild (stage I, END-I; n = 15) or severe endometriosis (stage IV, END-IV; n = 22). There was no difference in the concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 between the analyzed groups. There was, however, a correlation between MMP-9 and TIMP-1 for the combined group (n = 37) (r = 0.48; p = 0.0032) and in women with END-IV (r = 0.51; p = 0.0163), as well as a highly significant correlation between MMP-2 and TIMP-2 for the combined group (r = 0.69; p = 0.0001), END-I (r = 0.51; p = 0.0406) and END-IV groups (r = 0.77; p = 0.0001). There was also a significant correlation between TIMP-1 and TIMP-2 in the combined and END-IV groups (r = 0.39; p = 0.0182 and r = 0.5450; p = 0.0099, respectively). The balance between MMPs and their inhibitors is preserved in the serum of women with endometriosis, but serum concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 cannot be considered to represent a valid measure of the severity of endometriosis.     

人与大小鼠精子中精蛋白mRNA的分析

目的 :研究人与大小鼠精子中精蛋白 m RAN存在情况。方法 :应用 RT-PCR法分别从人、大鼠和小鼠精子总 RNA中扩增得到精蛋白 (protamine) c DNA片段。结果 :人、大鼠和小鼠精子中均存在精蛋白基因相应的 m RNA,并发现头部畸形率高的精子中精蛋白 m RNA明显减少。结论 :精子的 m RNA有可能代表精子发生过程中某些基因的表达情况     

Serum chorionic gonadotrophin (hCG), schwangerschaftsprotein 1 (SP1), progesterone and oestradiol levels in patients with nausea and vomiting in early pregnancy

Serum concentrations of human chorionic gonadotrophin (hCG), schwangerschaftsprotein 1(SP1), progesterone and oestradiol were measured in 116 pregnant women experiencing varying degrees of nausea and vomiting or no nausea at all at between 9 and 16 weeks gestation. The patients were categorized into four groups, namely asymptomatic, nausea alone, nausea and vomiting and hyperemesis gravidarum. The distribution of levels for each group were examined in relation to the calculated normal ranges. Statistically higher hCG levels were found in out-patients with nausea alone or nausea and vomiting than in the asymptomatic women. No significant differences were found between the groups for any of the other measured variables, including the progesterone/oestradiol concentration ratio.