Immunofluorescent localization of an androgen-dependent isoenzyme of prostatic acid phosphatase in rat ventral prostate

Isoenzymes of rat ventral prostate (RVP) acid phosphatase were isolated and partially purified by ultracentrifugation, Sephadex G-100 column chromatography, and isoelectric focusing. Antisera were raised to the isoenzymes of prostatic acid phosphatase by immunization of New Zealand white rabbits. Rabbit antisera reacting specifically to homologous but not heterologous isoenzymes of acid phosphatase were then reacted with a variety of tissues using indirect immunofluorescence. The tissues included prostate, spleen, bone marrow, liver, kidney, salivary gland complex, small intestine, and adrenal glands. An antiserum against a RVP acid phosphatase isoenzyme with a pI of 4.5 (A-PAP) localized acid phosphatase only in the supranuclear region of rat ventral prostate epithelial cells, and did not react with acid phosphatase in any of the other organs tested. A-PAP did not localize acid phosphatase in the ventral prostate from rats 14 days after castration. A-PAP did localize acid phosphatase in the ventral prostate from castrated animals that were treated with testosterone. These results indicate the A-PAP localized an androgen-dependent isoenzyme of acid phosphatase in RVP epithelial cells that may be secretory in nature. This antiserum should prove to be an ideal marker for studies involving hormonal regulation of prostatic epithelial function in vivo and in vitro.     

输精管结扎4月大鼠血清与前列腺组织的前列腺酸性磷酸酶活力变化

成年Ｗｉｓｔａｒ大鼠２０只，随机分为输精管结扎组（ＶＧ）和假手术组（ＳＯＧ）。术后第４个月检测了前列腺酸性磷酸酶（ＰＡＰ）等代表前列腺功能与肿瘤标志的酶学指标。结果表明，（１）血清ＰＡＰ活力，ＶＧ为４．９６±３．４Ｕ／ｍｌ，ＳＯＧ为５．６３±１．６９Ｕ／ｍｌ，两组无显著差异（Ｐ＞０．０５）；ＶＧ前列腺组织ＰＡＰ活力（１８．００±８．８１Ｕ／ｇ）略高于ＳＯＧ的（１５．０４±１１．９０Ｕ／ｇ），但无显著性。Ｐ／Ｔ百分比值ＶＧ为６５．０２±２５．３６％，ＳＯＧ为６８．６５±１１．６５％，Ｐ大于０．０５。（２）血清与前列腺组织碱性磷酸酶（ＡＬＰ）、乳酸脱氢酶（ＬＤＨ）与磷酸肌酸激酶（ＣＫ）活力，两组均无显著差异（Ｐ＞０．０５）。     

Estrogen receptor expression in prostate cancer and premalignant prostatic lesions.

Estrogens have been implicated in prostatic cancerogenesis and tumor progression. The mechanisms underlying estrogen signaling in human prostate tissue, however, remain poorly understood. Using immunohistochemical and in situ hybridization (ISH) techniques, the present study demonstrates the classical estrogen receptor (ERalpha) in premalignant lesions and prostatic adenocarcinoma through the various stages of the disease. Conversely, the novel characterized ERbeta subtype was undetectable in human prostate tissue. High-grade prostatic intraepithelial neoplasia revealed ERalpha mRNA and protein expression in 28% and 11% of cases evaluated. Focal ER immunoreactivity was detected in a minority of low- to intermediate-grade adenocarcinoma. High-grade (primary Gleason grade 4 and 5) tumors revealed ER protein expression in 43% (62% respectively) of cases. The most significant ERalpha gene expression on mRNA and protein levels was observed in hormone refractory tumors and metastatic lesions, including lymph node and bone metastases. Results of the current study suggest that estrogens can affect prostatic cancerogenesis and neoplastic progression through an ER-mediated process in human prostate tissue.     

雌激素对大鼠腺垂体中瘦素长型受体表达的影响

目的:研究雌激素对卵巢摘除后大鼠腺垂体中瘦素长型受体的调节作用.方法:建立卵巢摘除大鼠模型,用半定量RT-PCR 和免疫印迹技术,检测腺垂体中瘦素长型受体 mRNA 和蛋白表达的变化.结果:注射17β-雌激素组和假手术组中瘦素长型受体 mRNA 和蛋白表达显著高于注射花生油组.结论:雌激素可调节腺垂体中瘦素长型受体的表达,提示雌激素对腺垂体的调节,可能与瘦素相关.     

Early developmental expression of leptin receptor gene and [125I]leptin binding in the rat forebrain

Leptin, via leptin receptors (Ob-R), regulates appetite and energy balance. Of the six isoforms of the receptor identified, so far, only the long form (Ob-Rb) can fully activate downstream signal transduction pathways. Although the expression and function of leptin receptors is well described in the adult brain, little is known about the ontogeny of leptin receptor system around the time of birth. In this study, the mRNA expression patterns of total leptin receptor, Ob-R, and the long signalling form of the receptor, Ob-Rb, were investigated in the brain of embryonic and newborn rats using in situ hybridisation and [125I]leptin binding. On embryonic day 18 (E18), Ob-R mRNA was detected in the choroid plexus and the ependymal layer of the third ventricle by in situ hybridisation. At E21, Ob-Rb mRNA was first observed in the arcuate and the ventral premammillary hypothalamic nuclei while at P3, receptor expression was also found in the dorsomedial nucleus. Other leptin target areas identified were the trigeminal ganglion, the thalamus and the hippocampus. Using quantitative receptor autoradiography specific [125I]leptin binding sites on the choroid plexus were found to increase with age in contrast to the ependymal layer of the third ventricle where levels decreased with age. Together these findings demonstrate that the leptin receptor system is differentially regulated during late gestation and early postnatal life in the rat.     

Carcinoembryonic antigen and human chorionic gonadotrophin in breast carcinoma and prostatic specific acid phosphatase in prostate carcinoma

Tumour markers viz carcinoembryonic antigen (CEA), human chorionic gonadotrophin (HCG) in 30 cases of carcinoma breast and prostatic specific acid phosphatase (PSAP) in 30 cases of carcinoma prostate were studied by peroxidase antiperoxidase technique in paraffin blocks of tissue. Twenty three (76.7%) and 20 (66.7%) cases were positive for CEA and HCG respectively. No correlation was observed between CEA and HCG status, and histological differentiation of the tumours. All the 29 cases (100%) of adenocarcinoma prostate were PSAP positive while a single case, negative for PSAP, was of transitional cell carcinoma.     

Prostate specific antigen and prostatic acid phosphatase measurements for the follow-up of patients with prostate cancer.

The clinical application of 84 prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) measurements for the follow-up of 36 patients with treated prostate cancer was retrospectively examined by case study. Clinicians use several risk markers including serum levels of PSA and PAP to monitor prostate cancer progression or stability. These results of PAP and PSA tests were either utilized during the patient's clinical assessment or they were disregarded. In either case, the results would support or counter the physician's clinical decision for patient management. After predictive value analysis it was concluded that measurement of PSA alone is more useful than parallel measurements of PSA and PAP, when utilized together with standard criteria for assessing treated prostate cancer patients.     

前列腺酸性磷酸酶在慢性痛大鼠脊髓背角和背根神经节的表达变化

目的:观察前列腺酸性磷酸酶(prostatic acid phosphatase,PAP)在多种慢性痛大鼠脊髓背角(spinaldorsal horn,SDH)和背根神经节(dorsal root ganglion,DRG)内的表达变化。方法:应用免疫组织化学染色法以及免疫荧光多重染色技术在多种慢性痛模型大鼠观察PAP的表达变化。结果:在正常大鼠,PAP阳性反应产物主要位于DRG的中、小型的非肽能神经元,PAP阳性神经元约占DRG神经元总数的64±4.3%;在脊髓背角,PAP阳性纤维和终末主要位于Ⅱ层。在神经病理性痛模型大鼠,术侧脊髓背角Ⅱ层的PAP阳性初级传入终末较对侧减少甚至消失,DRG内PAP阳性神经元较对侧明显减少。在慢性炎性痛模型大鼠,双侧脊髓背角和DRG内PAP的表达未见明显改变。结论:PAP特异地定位于DRG神经元以及脊髓背角Ⅱ层,可能与神经病理性痛信号的传递和加工密切相关。     

The effect of leptin receptor deficiency and fasting on cannabinoid receptor 1 mRNA expression in the rat hypothalamus, brainstem and nodose ganglion

Despite ample evidence for the involvement of the endocannabinoid system in the control of appetite, food intake and energy balance, relatively little is known about the regulation of cannabinoid receptor 1 (CB₁R) expression in respect to leptin signalling and fasting. In the present study, we examined CB₁R mRNA levels in lean (Fa/?) and obese (fa/fa) male Zucker rats under basal and food-restricted conditions. Using stereological sampling principles coupled with semi-quantitative radioactive in situ hybridization we provide semi-quantitative estimates of CB₁R mRNA expression in key appetite regulatory hypothalamic and brainstem areas, as well as in the nodose ganglia. Whereas no effect of fasting were determined on CB₁R mRNA levels in the paraventricular (PVN) and ventromedial hypothalamic (VMH) nucleus, in the brainstem dorsal vagal complex or nodose ganglion of lean Zucker rats, CB₁R mRNA levels were consistently elevated in obese Zucker rats pointing to a direct influence of disrupted leptin signalling on CB₁R mRNA regulation.     

Deceased expression of prostatic acid phosphatase in primary sensory neurons after peripheral nerve injury

Prostatic acid phosphatase (PAP) is expressed in nociceptive dorsal root ganglion (DRG) neurons and functions as an ectonucleotidase that dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine to suppress pain via activating A1-adenosine receptor (A1R) in dorsal spinal cord. However, the effect of peripheral nerve injury on the expression of PAP has not been reported until now. In the present study we found that PAP expression in DRG neurons is significantly decreased from the 2nd day after peripheral nerve injury and reaches the bottom at the 14th. In addition, intrathecal PAP injection can reduce mechanical allodynia induced by spared nerve injury. Our findings suggest that the decrease of PAP is involved in pathophysiological mechanisms of neuropathic pain.     

Immunohistochemical acid phosphatase level and tumor grade in prostatic carcinoma

An immunoperoxidase technique to detect prostatic-specific acid phosphatase (PSAP) was used on specimens from 98 cases of prostatic carcinoma that were graded by both the Gleason and the Mostofi systems, to see if tumor grade correlated with amount of PSAP seen in tissue. Most tumors showed strong, diffuse cytoplasmic staining; no significant difference was seen among the various grades. Other than focal, weak staining of renal tubular epithelium, the antibody to PSAP gave uniformly negative results with a variety of normal and neoplastic tissues. In light of the great sensitivity and specificity of this technique, it potential applications include diagnosis of poorly differentiated prostatic malignant neoplasms, whether primary or metastatic.     

Effects of high fat diet,ovariectomy, and physical activity on leptin receptor expression in rat brain and white fat tissue

Aim

To evaluate in a rat animal model whether ovariectomy, high fat diet (HFD), and physical activity in the form of running affect leptin receptor (Ob-R) distribution in the brain and white fat tissue compared to sham (Sh) surgery, standard diet (StD), and sedentary conditions.

Methods

The study included 48 female laboratory Wistar rats (4 weeks old). Following eight weeks of feeding with standard or HFD, rats were subjected to either OVX or Sh surgery. After surgery, all animals continued StD or HFD for the next 10 weeks. During these 10 weeks, ovariectomy and Sh groups were subjected to physical activity or sedentary conditions. Free-floating immunohistochemistry and Western blot methods were carried out to detect Ob-R in the brain and adipose tissue.

Results

StD-ovariectomy-sedentary group had a greater number of Ob-R positive neurons in lateral hypothalamic nuclei than StD-Sh-sedentary group. There was no difference in Ob-R positive neurons in arcuatus nuclei between all groups. Ob-R distribution in the barrel cortex was higher in HFD group than in StD group. Ob-R presence in perirenal and subcutaneous fat was decreased in StD-ovariectomy group.

Conclusion

HFD and ovariectomy increased Ob-R distribution in lateral hypothalamic nuclei, but there was no effect on arcuatus nuclei. Our results are first to suggest that HFD, ovariectomy, and physical activity affect Ob-R distribution in the barrel cortex, which might be correlated with the role of Ob-R in election of food in rats.Obesity is one of the leading health issues worldwide, associated with an increased risk of morbidity and mortality (1). In 1997, the World Health Organization (WHO) formally recognized obesity as a global epidemic (2). Increase in body fat stores and obesity is caused by an imbalance between energy intake and energy expenditure (3,4). Since childhood obesity is a predictor of an increased death rate, the “obesity epidemic” may reverse the current declining rate of death from cardiovascular diseases (5). Factors that contribute to obesity can be environmental (6), social (7), behavioral (8), psychological (9), and genetic (10,11).Women generally have more body fat than men (12). Nevertheless similar odds ratios were recorded in women and men for the association of abdominal obesity with acute myocardial infarction (13). Weight gain is common after menopause, indicating an association between hormones and fat stores (14). A large scale observational study found that both the body mass index and the level of physical activity were independent predictors of mortality and that a higher level of physical activity did not eliminate the risk associated with adiposity. At the same time, women who were both lean and physically active had the lowest mortality (15). In animal studies menopause can be induced by ovariectomy (OVX) (16).Obesity can also be called a disorder of appetite and it is controlled by complex homeostatic mechanisms involving the hypothalamus and brainstem (17). Many gut peptides like cholecystokinin, ghrelin, glucagon-like peptide-1 (GLP-1), and -2 and peptide YY (PYY) act on the brain to control eating behavior (18). There are two different system for controlling feeding behavior: short-term and long-term (19). Short-term regulation involves neural signals from the GI tract and its hormones, like insulin, glucagon, and ghrelin (20). A hormone that functions mainly within long-term regulation is leptin (16 kD), a hormonal product of the obesity (ob) gen, primarily secreted by adipocytes (21) and released in the brain. It generates a feeling of satisfaction and acts like an appetite-suppressing agent. Circulating leptin levels are lower in ovariectomized rats (22).Food intake is regulated via neural circuits located in the hypothalamus (23). Leptin acts via its leptin or Ob receptors (Ob-R) and is primarily expressed in hypothalamic neurons (19) especially in arcuate, ventromedial, and dorsomedial nuclei (24). Leptin is transported across the blood-brain barrier (BBB) by a saturable transporter (25). Ob-R is also detected in nonhypothalamic areas in the mice and in human brain neocortex, cerebellum, entorinal cortex, amygdale, and rostral medulla (26). Adipocytes, endothelial cells, and macrophages also have leptin receptor at its surface, which suggests autocrine and paracrine action for leptin in human adipose tissue (27). Association between the expression of Ob-R in target tissues and physiological and hormonal controlled processes is still unclear. Leptin receptors mRNA is found in each of the major components of the CNS “feeding” circuitry – the brainstem, hypothalamus, and is distributed reward centers (Allan brain) (28). Therefore, the aim of the current study was to evaluate whether HFD affects Ob-R distribution compared with StD specifically in the barrel field and piriform cortex compared to standard feeding centers in the hypothalamus. We supposed that the combination of OVX and HFD is interesting for further research on selected brain regions, which might be alleviated by physical activity. We also supposed that changes in Ob-R level in white fat tissue would correlate with the changes in brain regions.     

Immunohistochemical study of the expression of epidermal growth factor receptor in benign prostatic hypertrophy, prostatic intraepithelial neoplasia and prostatic carcinoma

The present study provides an analysis of immunohistochemical expression and localization of epidermal growth factor receptor (EGFR) in formalin fixed paraffin embedded specimens of prostate. Thirty-five cases each of benign prostatic hypertrophy (BPH) and prostatic carcinoma and 30 cases of prostatic intraepithelial neoplasia (PIN) were taken up for study. Streptavidin biotin peroxidase method was employed for immunohistochemical staining. EGFR positivity was observed in all the cases (100%) of BPH and PIN and in only 10 cases (28.5%) of prostatic carcinoma. In both BPH and PIN the basal cells revealed significantly higher intensity and percentage cell positivity than the luminal cells. Intensity and percentage of positively stained basal cells in BPH was higher than PIN basal cells but the difference was not statistically significant. The intensity and percentage cell positivity of BPH basal cells and PIN basal and luminal cells were significantly greater than the epithelial cells of prostatic carcinoma. Presently, the significance of variable expression of EGFR in various types of prostatic lesions is unknown.     

Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements

Analyzing the status of T-cell receptor (TCR) gene rearrangements has been an essential part of deciphering the stages of thymocyte development, understanding the β vs. γδ lineage decision, and characterizing T-cell leukemias. Methods such as PCR and quantitative Southern blotting provide useful information, but also have significant shortcomings such as lack of quantitation in the case of PCR and technical challenges in the case of Southern blotting. Here we describe a real-time PCR method that overcomes many of these shortcomings. This new method shows comparable results for the fraction of unrearranged TCRγ and TCRβ genes in human thymocytes and peripheral blood T cells as Southern blotting, and has the advantages of being simple to perform, highly quantitative, and requiring nanogram quantities of DNA. We also describe a real-time PCR method to quantitate T-cell receptor excision circles formed during TCRβ rearrangements.     

Long-term effects of neonatal seizures on subsequent N-methyl-D-aspartate receptor-1 and gamma-aminobutyric acid receptor A-alpha 1 receptor expression in hippocampus of the Wistar rat

To evaluate the pathophysiological mechanism of subsequent reduced seizure threshold following neonatal seizures, single or recurrent prolonged seizures were induced to neonatal rats by the inhalant flurothyl. The expression of N-methyl-d-aspartate receptor 1 (NR1) and gamma-aminobutyric acid receptor A-alpha 1 (GABA-A-alpha 1) immunoreactivity in hippocampus were examined by Western blotting analysis at the day 7 (P7) and day 75 (P75) after the last seizure. Whereas there were no significant changes in single seizure group and recurrent seizure group of P75, NR1 expression enhanced significantly in P7 rats of recurrent seizure group. Meanwhile, polypeptide levels of GABA-A-alpha 1 receptor subunit decreased significantly in both single and recurrent seizure-treated P7 and P75 rats. Our results suggest that recurrent or single prolonged seizures during the neonatal period may have long-term effects on the balance between excitatory NMDA system and inhibitory GABA system in hippocampus of rats.