4-Nitrophenol isolated from diesel exhaust particles disrupts regulation of reproductive hormones in immature male rats

In previous studies, we found that 4-nitrophenol (PNP) isolated from diesel exhaust particles exhibited both estrogenic and anti-androgenic activities. This compound is also a degradation product of the insecticide parathion. Here, we investigated the in vivo effect of PNP on reproductive function in immature male rats. Twenty-eight-day-old rats were injected subcutaneously with PNP (0.01, 0.1, 1, or 10 mg/kg) daily for 14 days. Plasma concentrations of luteinizing hormone (LH) were significantly lower in all PNP dosage groups than in the control group, and follicle-stimulating hormone (FSH) was significantly decreased in rats treated with 0.1, 1, or 10 mg/kg PNP. However, plasma concentrations of testosterone were significantly increased by 10 mg/kg PNP, and plasma concentrations of immunoreactive (ir)-inhibin were also significantly increased in the 0.1, 1, and 10 mg/kg PNP groups. Plasma concentrations of prolactin were significantly increased by 10 mg/kg PNP, and plasma concentrations of corticosterone were significantly increased in all treatment groups. These findings clearly show that PNP influences the hypothalamic–pituitary–gonadal axis in immature male rats, with decreased secretion of LH and FSH and increased secretion of testosterone and inhibin. PNP, therefore, appears to disrupt endocrine activity in the immature male reproductive system.     

加味左金丸对大鼠胃癌前病变增殖细胞核抗原及B淋巴细胞瘤/白血病-2蛋白表达的影响

[目的] 观察加味左金丸对大鼠萎缩性胃炎(CAG)癌前病变增殖细胞核抗原(PCNA)及B淋巴细胞瘤/白血病-2(Bcl- 2)蛋白表达的影响,并探讨其治疗CAG癌前病变的可能机制。[方法] 采用SP法检测大鼠胃癌前病变自然恢复组,加味左金丸高、中、低剂量组,维甲酸组和正常组大鼠胃黏膜组织中PCNA、Bcl -2 蛋白表达的情况,并计算细胞增殖指数(PI)。[结果] 各治疗组PI及Bcl- 2 蛋白阳性率与自然恢复组比较差异有统计学意义(P<0 .01或<0 .05);其中加味左金丸各治疗组PI值与维甲酸组比较差异有统计学意义(P<0 .05)。[结论] 加味左金丸可能通过对抗PCNA、Bcl 2的激活而抑制PCNA、Bcl -2蛋白的表达发挥治疗作用。     

胆囊癌组织中增殖细胞核抗原和癌基因蛋白的表达及相关性研究

为探索胆囊癌中PCNA和c -erbB - 2表达及其分化、浸润及转移的关系和临床意义。应用HE和免疫组化 (S -P)方法对 98例胆囊癌进行组织学分型、分级、浸润和转移的观察 ,对其中 85例进行PCNA和c -erbB - 2免疫标记。女性 5 7例 ,男性 4 1例。组织学以腺癌为主 6 3例 (占 6 4 3% ) ,免疫组化标记 85例 ,高分化 38例 ,中分化2 7例 ,低分化 2 0例。PCNA阳性表达率为 88 2 % ,其中高表达率在三种分化中分别为 2 9 9%、5 1 9%和 5 5 % ,各组织学分级之间的差异有显著性 (P <0 0 5 ) ;c-erbB - 2阳性率为 36 5 % ,与组织学分级有关 (P <0 0 5 ) ;PCNA高表达的胆囊癌 ,其c-erbB - 2阳性率显著高于低表达者 (P <0 0 5 ) ,PCNA的表达与胆囊癌的组织学分级和转移有关 ;c-erbB - 2的阳性表达与胆囊癌增殖活性、组织学分级和浸润深度有关 ,提示c -erbB - 2阳性表达预后不良     

Association between shear stress, angiogenesis, and VEGF in skeletal muscles in vivo

OBJECTIVE: To investigate the hypothesis that capillary proliferation in skeletal muscles, induced by a long-term increase in blood flow which elevates capillary shear stress, is associated with capillary expression of vascular endothelial growth factor (VEGF). METHODS: Adult rats received prazosin in drinking water ( approximately 2 mg per day) or had extensor digitorum longus (EDL) muscles stimulated by implanted electrodes for up to 14 days. At intervals, serial frozen sections of EDL were stained for alkaline phosphatase to identify capillaries, proliferating cell nuclear antigen (PCNA), and VEGF-A protein. Shear stress was estimated from capillary red blood cell velocities and diameters, measured by direct observation of epi-illuminated EDL. RESULTS: Chronic stimulation and prazosin treatment both increased capillary: fiber ratio by approximately 40% after 14 days. In stimulated muscles, the percentage of capillaries positively stained for VEGF increased within 3 to 4 days, while the density of PCNA-positive capillaries had increased 20-fold after 2 days. With prazosin, VEGF-positive capillaries increased after 2 and 4 days, accompanied by a threefold increase in PCNA. By 14 days, PCNA labeling and VEGF were still high in stimulated muscles, but no longer different from controls with prazosin. After 3 to 4 days of treatment, capillary shear stress in resting muscle was 57% higher than in controls as a result of stimulation, but 4 times higher with prazosin. CONCLUSIONS: Higher capillary shear stress with prazosin than with stimulation may upregulate VEGF expression in the early stages of treatment. Greater proliferation of capillaries preceding a higher proportion of VEGF-positive capillaries in stimulated muscles, in the presence of a modest increase in shear stress, suggests that angiogenesis was initiated by other factors in addition to shear stress.     

凋亡抑制基因survivin bcl-2 bax在肺癌中表达的研究

目的检测肺癌患者癌组织和癌旁组织中survivin、bcl-2及bax的基因表达,探讨它们的相关性及与肺癌发生、发展的关系。方法应用TUNEL原位细胞凋亡检测方法及免疫组化方法,对1998—2004年华中科技大学同济医学院附属协和医院收治的163例肺癌患者手术常规石蜡包埋组织中癌基因survivin、bcl-2及bax的表达进行检测,并与其中106例患者的癌旁组织对比,分析免疫组化结果及其与肺癌的病理特征和预后关系。结果在癌旁肺组织中,survivin、bcl-2、bax蛋白阳性表达率分别为1·9%(2例)、29·2%(31例)、93·47%(99例);肺癌组织内,三者阳性表达率分别为69·9%(114例)、62·0%(101例)、52·8%(86例)。异常增高的survivin、bcl-2表达呈明显相关。结论细胞凋亡和增殖失控,相关基因survivin、bcl-2和bax蛋白异常表达在肺癌发生发展中起重要作用。survivin、bcl-2可能在肺癌癌变及浸润过程中起着重要作用,可作为判断肺癌生物学行为和预后的参考指标。     

Activated peripheral blood mononuclear cells detected in lupus patients using cDNA coding for proliferating cell nuclear antigen

 The expression of proliferating cell nuclear antigen (PCNA) mRNA in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) was measured by dot blot hybridization using a PCNA cDNA, and correlated with the percentage of PCNA-positive cells detected immunohistochemically using a monoclonal anti-PCNA antibody. PCNA-positive PBMCs were detected in 72.2% of SLE patients (n = 36), which is significantly more than among healthy controls. In addition, among those in whom PCNA expression was detected, the percentage of PBMCs expressing PCNA was significantly higher in SLE patients (mean 2.5% vs. 0.15%). The level of PCNA mRNA was increased in PBMCs from 83.3% of SLE patients, and was significantly correlated with the percentage of PCNA-positive cells (r = 0.54, P < 0.01) and with the disease activity score (r = 0.56, P < 0.01). A longitudinal study of two SLE patients confirmed that PCNA mRNA expression and the percentages of PCNA-positive cells varied in parallel with disease activity. Thus, an analysis of activated PBMCs from SLE patients using PCNA cDNA may be a useful method by which to estimate SLE disease activity. Received: January 25, 2002 / Accepted: March 30, 2002 Acknowledgments This work was supported in part by a 1999 research grant from the Mixed Connective Tissue Disease Research Committee of Japan, Ministry of Health and Welfare, and a 1999 Grant-in-Aid for Scientific research (C) (2), Ministry of Education. Correspondence to: Y. Takasaki     

p27kip1 Regulates cdk2 activity in the proliferating zone of the mouse intestinal epithelium: potential role in neoplasia

BACKGROUND & AIMS: Reduced p27(kip1) expression is a marker of poor prognosis in colorectal neoplasia, and inactivation of p27 in mice (p27(Delta51/Delta51)) causes increased intestinal epithelial cell proliferation and small and large intestinal neoplasia in a diet-dependent manner. Here, we addressed the role of p27 in untransformed intestinal epithelial cells in vivo and the consequence of its targeted inactivation. METHODS: A sequential fractionation procedure was used to isolate murine intestinal epithelial cells relative to their position along the crypt-villus axis, and the levels of cyclins, cyclin-dependent kinases (cdks), and cdk inhibitors and of the complexes formed among them was determined by immunoprecipitation-immunoblotting and kinase assays. RESULTS: As cells exited the proliferative crypt compartment, expression and activity of both cdk2 and cdk4 decreased, in parallel with reduced expression of cyclin A and proliferating cell nuclear antigen (PCNA); expression of cyclin D1, D2, and cyclin E showed little change. As expected, expression of the cdk inhibitors p21, p57, and p16 was highest in differentiated villus cells. Unexpectedly, p27 protein expression was highest in cells of the proliferative crypt compartment where it bound both cdk2 and cdk4. Cdk2 activity was increased in crypt cells from p27(Delta51/Delta51) mice, although cyclin D-associated kinase activity was unchanged (indeed, cyclin D1/2-cdk4 complex levels were reduced). Importantly, cdk2 activity was unchanged in crypt cells from p21(-/-) mice, which do not develop intestinal tumors. CONCLUSIONS: We propose that p27 contributes to intestinal epithelial homeostasis by regulating cdk2 activity in proliferating cells, thus gating cell cycle progression and suppressing intestinal neoplasia.     

A Case of Massive Hepatic Necrosis (Scarred Liver) with Elevated α-Fetoprotein and Seroconversion from HBe Ag to HBe Ab

A 39-year-old male with general malaise visited our clinic with high levels of aminotransferases (AST 776 IU/1, ALT 1,069 IU/I) and a prolonged prothrombin time. Serum-fetoprotein (AFP) level was markedly elevated (2,214 ng/ml) and human hepatocyte growth factor (hHGF)was also high (0.81 ng/ml). Hepatocellular carcinoma was found to be negative from imaging techniques. AFP bands separated by lectin affinity electrophoresis had a benign pattern. Laparoscopic diagnosis was scarred liver and histologically massive hepatic necrosis with a strongly positive AFP stain. Levels of hHGF and AFP fell into a normal range after treatments with glucagon-insulin and prostaglandin Ei. At second laparoscopy, regenerated nodules were confirmed by improved staining with Indocyanine Green. Hepatocyte regeneration was also histologically proven with a weakly positive AFP stain. The proliferating cell nuclear antigen (PCNA) was stained, and hepatocytes with PCNA positive nuclei were not seen at the time of the first laparoscopy, whereas hepatocytes with PCNA positive nuclei were found to be many at the time of the second laparoscopy. Seroconversion from HBe Ag to HBe Ab was observed 4 months after the maximum rise of AFP (6,341 ng/ml), when levels of ALT and DNA polymerase were within normal ranges. This is a patient with a marked rise in his serum level of AFP in association with seroconversion from HBe Ag to HBe Ab, and the elevation of AFP in the present case was thought to reflect severe liver injury with concomitant regenerative activities of injured hepatocytes rather than simple hepatocyte regeneration after hepatic necrosis was resolved.     

Effect of millimeter wave radiation on the expression of proliferating cell nuclear antigen,CDK4 and P16 in rat hepatocellular carcinoma

OBJECTIVE: To study the effects of millimeter wave (MMW) radiation on rat hepatocellular carcinoma. METHODS: Forty male Wistar rats were randomly divided into four groups, and the liver region of the rats was directly radiated by MMW (35.8 GHz, 100 mW/cm²) for 20 min twice per week. Rats in groups 1 to 3 were fed with diethlnitrosamine (DEN). Group 1 was a tumor control group with sham radiation. Group 2 was given radiation for 10 weeks starting from week 5 and group 3 was radiated for 5 weeks starting from week 10. Group 4 was a normal control group radiated for 5 weeks and given distilled water. At week 14, all rats were sacrificed. A serological test for γ‐glutamyltransferase (γ‐GT) and immunohistochemical staining of proliferating cell nuclear antigen (PCNA), CDK4 and P16 in liver tissue were performed. RESULTS: The serum level of γ‐GT in group 1 (18.44 ± 4.88 U/L) was higher than that in group 2 (13.75 ± 2.41 U/L, P < 0.05), and the level of γ‐GT in group 3 (16.43 ± 2.12 U/L) was lower than that in group 1, but the difference was not significant. Based on histological examination of the livers, adenocarcinoma only developed in group 1. In the other DEN‐induced tumor groups, only eosinophilic and basophilic nodules were formed in the liver; no carcinoma cells were found. Expression of proliferating cell nuclear antigen (PCNA) and CDK4 staining in liver tissue in groups 2 and 3 were significantly lower than those in group 1, but the expression of P16 in the former was higher than that in the latter. CONCLUSIONS: Millimeter wave radiation partially inhibits cell proliferation and suppresses DEN‐induced hepatocellular carcinoma in rats.     

Enhancement of methyl-aminolevulinate photodynamic therapy by iron chelation with CP94: an in vitro investigation and clinical dose-escalating safety study for the treatment of nodular basal cell carcinoma

PURPOSE: Methyl-aminolevulinate (MAL) photodynamic therapy (PDT) is a cancer therapy that combines the selective accumulation of a photosensitizer in tumor tissue with visible light (and tissue oxygen) to produce reactive oxygen species. This results in cellular damage and ablation of tumor tissue. Combining iron chelators with MAL has the potential to increase the accumulation of the photosensitizer protoporphyrin IX (PpIX) by reducing its bioconversion to heme. This paper investigates this method of enhancement both in vitro and for the first time clinically for the treatment of nodular basal cell carcinoma (BCC). METHODS: Enhancement of MAL-induced PpIX accumulation by the iron chelator CP94 was quantified fluorometrically in human cultured cells (including three dermatological cell types). An open, dose-escalating, pilot study was then conducted in patients with nodular BCC, to determine the safety of this pharmacological modification. RESULTS: Large enhancements in PpIX accumulation were observed in the cultured cells when co-incubated with the iron chelator CP94. Clinically the addition of CP94 was found to be feasible and safe. In addition greater reductions in tumor depth were observed in the CP94 co-incubated tumors. CONCLUSION: Iron chelation by CP94 is an effective enhancer of MAL-induced PpIX accumulation in vitro. This method of enhancement was safely applied to a clinical PDT protocol with no unexpected adverse effects reported. Although the clinical investigation was only intended to be a small pilot to assess safety, enhancements in tumor clearance were observed both clinically and histologically when CP94 was included in the photosensitizing cream.     

Conditional deletion of beta-catenin reveals its role in liver growth and regeneration

BACKGROUND & AIMS: The Wnt/beta-catenin pathway plays a role in liver growth and development. To address this conclusively, we used a conditional knockout approach to delete beta-catenin in the liver. METHODS: Floxed beta-catenin (exons 2-6) mice were intercrossed with Albumin-Cre recombinase transgenic mice; considerable beta-catenin deletion was evident 15 days after birth by Western blot and immunohistochemistry analyses. RESULTS: Although these mice were viable, there was a significant decrease in their liver weight/body weight ratio by 14% at 1 month and 28%-35% by 2-6 months of age, which was sustained throughout their normal life span. There was an accompanying decrease in basal hepatocyte proliferation showed by Ki-67 staining. Additional analysis revealed several known and novel genes to be down-regulated in these mice that play a role in normal liver homeostasis. When subjected to two-thirds partial hepatectomy, the Ctnnb1(loxp/loxp); Alb-Cre(+/-) mice were sick and lethargic, especially during the first 2-3 days only. These mice display a 2-fold decrease in the number of Ki-67- or PCNA-positive cells at the time of peak hepatocyte proliferation at 40 hours, which coincided with decreased cyclin A, D, and E expression. However, a rebound increase in hepatocyte proliferation was evident in the knockout mice at 3 days. Also, increased apoptosis was observed in the knockout livers during regeneration at all stages. CONCLUSIONS: Thus, beta-catenin is essential for normal liver growth and development. Also, although regeneration is delayed in the absence of beta-catenin, it does occur suboptimally, showing its redundancy in the liver.     

Stimulatory effect of insulin on theca-interstitial cell proliferation and cell cycle regulatory proteins through MTORC1 dependent pathway

The present study examined the effect of insulin-mediated activation of the mammalian target of rapamycin complex 1 (MTORC1) signaling network on the proliferation of primary culture of theca-interstitial (T-I) cells. Our results show that insulin treatment increased proliferation of the T-I cells through the MTORC1-dependent signaling pathway by increasing cell cycle regulatory proteins. Inhibition of ERK1/2 signaling caused partial reduction of insulin-induced phosphorylation of RPS6KB1 and RPS6 whereas inhibition of PI3-kinase signaling completely blocked the insulin response. Pharmacological inhibition of MTORC1 with rapamycin abrogated the insulin-induced phosphorylation of EIF4EBP1, RPS6KB1 and its downstream effector, RPS6. These results were further confirmed by demonstrating that knockdown of Mtor using siRNA reduced the insulin-stimulated MTORC1 signaling. Furthermore, insulin-stimulated T-I cell proliferation and the expression of cell cycle regulatory proteins CDK4, CCND3 and PCNA were also blocked by rapamycin. Taken together, the present studies show that insulin stimulates cell proliferation and cell cycle regulatory proteins in T-I cells via activation of the MTORC1 signaling pathway.     

Histone Demethylase JMJD2D Interacts With β-Catenin to Induce Transcription and Activate Colorectal Cancer Cell Proliferation and Tumor Growth in Mice

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Taurocholic acid prevents biliary damage induced by hepatic artery ligation in cholestatic rats

Background

Ischemic injury by hepatic artery ligation (HAL) during obstructive cholestasis induced by bile duct ligation (BDL) results in bile duct damage, which can be prevented by administration of VEGF-A. The potential regulation of VEGF and VEGF receptor expression and secretion by bile acids in BDL with HAL is unknown.

Aims

We evaluated whether taurocholic acid (TC) can prevent HAL-induced cholangiocyte damage via the alteration of VEGFR-2 and/or VEGF-A expression.

Methods

Utilizing BDL, BDL + TC, BDL + HAL, BDL + HAL + TC, and BDL + HAL + wortmannin + TC treated rats, we evaluated cholangiocyte apoptosis, proliferation, and secretion as well VEGF-A and VEGFR-2 expression by immunohistochemistry. In vitro, we evaluated the effects of TC on cholangiocyte secretion of VEGF-A and the dependence of TC-induced proliferation on the activity of VEGFR-2.

Results

In BDL rats with HAL, chronic feeding of TC prevented HAL-induced loss of bile ducts and HAL-induced decreased cholangiocyte secretion. TC also prevented HAL-inhibited VEGF-A and VEGFR-2 expression in liver sections and HAL-induced circulating VEGF-A levels, which were blocked by wortmannin administration. In vitro, TC stimulated increased VEGF-A secretion by cholangiocytes, which was blocked by wortmannin and stimulated cholangiocyte proliferation that was blocked by VEGFR-2 kinase inhibitor.

Conclusion

TC prevented HAL-induced biliary damage by upregulation of VEGF-A expression.