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Journal Article 2012; 33(4): 406-411 PubMed PMID: 22936257 Citation  Keywords:  Cells, Cultured, Female, Humans, Metalloproteases:classification, Placenta:enzymology, Pre-Eclampsia:enzymology, Pregnancy, Pregnancy Trimester, Third:metabolism, RNA, Messenger:analysis, Trophoblasts:enzymology,.   

OBJECTIVES: Preeclampsia is a specific pregnancy disorder which could be due, at least in part, to impaired invasion of trophoblastic cells. Since matrix metalloproteinases (MMPs) are the predominant proteases involved in trophoblastic invasion, we investigated and compared expression of MMP-1, 2, 7, 9 and 12 of cytotrophoblastic cells (CTB) purified from preeclamptic (PE) placentas to control CTB.

MATERIAL AND METHODS: In order to evaluate invasive properties of cells, purified CTB were seeded on collagen-coated insert following boyden chamber principle and matrix metalloproteinases (MMPs) expression was evaluated by qPCR.

RESULTS: Our results showed that PE CTB are less invasive than control CTB in vitro. In parallel, expression of MMPs, except for MMP-2, tends to be decreased in PE CTB compared to control CTB.

CONCLUSION: At the exception of MMP-2, this study confirms the importance of MMPs in development of PE....

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Journal Article 2008; 29(6): 884-888 PubMed PMID: 19112410 Citation  Keywords:  Analysis of Variance, Animals, Dopamine:genetics, Gene Knockdown Techniques, Inverted Repeat Sequences:genetics, Myelin Proteins:metabolism, Nogo Proteins, PC12 Cells, RNA Interference:physiology, RNA, Messenger:analysis, Rats,.   

OBJECTIVES: Reticulon proteins, which are localized in the endoplasmic reticulum, have recently been shown to be involved in hormone secretion, in particular RTN1 and RTN3. The aim of the present study was to examine the effects of Nogo-A gene expression knockdown by RNA interference (RNAi) on dopamine release in PC12 cells.

METHODS: A small hairpin RNA (shRNA) eukaryotic expression vector, targeting the Nogo-A gene, was constructed and transfected into cultured PC12 cells by lipofecamine2000. Inhibition of Nogo-A gene expression was detected by semi-quantitative reverse transcription PCR and Western blot analysis. Following transfection, dopamine release was detected by high performance liquid chromatography.

RESULTS: The pGenesil-1-Nogo-A-2 plasmid was identified by gene sequencing. After transfection of the recombinant vector in PC12 cells, Nogo-A gene expression was significantly inhibited (p<0.01). Compared with the empty vector control group, dopamine release significantly decreased within 48 hours after transfection.

CONCLUSION: Results from this study suggest that Nogo-A might be involved in the mechanism of DA release in PC12 cells....

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Journal Article 2008; 29(6): 911-916 PubMed PMID: 19112407 Citation  Keywords:  Analysis of Variance, Animals, Capsaicin:pharmacology, Cells, Cultured, Dose-Response Relationship, Drug, Drug Interactions, Galanin:administration & dosage, Ganglia, Spinal:cytology, Irritants:pharmacology, RNA, Messenger:analysis, Rats, Rats, Wistar, Se.   

OBJECTIVES: To determine the effect of exogenous galanin on capsaicin-evoked substance P (SP) release from primary cultured embryonic rat dorsal root ganglion (DRG) neurons.

METHODS: DRG was dissected out from embryonic 15-day-old Wistar rat and cultured as dissociated cells for 2 days then exposed to galanin (1 nmol/L, 10 nmol/L, 100 nmol/L). After 4 days incubation with exogenous galanin, the levels of mRNAs for SP and vanilloid receptor 1 (VR1) and protein for VR1 were estimated by RT-PCR and Western blot, respectively. Basal SP release and capsaicin-evoked SP release levels were measured by radioimmunoassay (RIA).

RESULTS: The amount of VR1 mRNA and VR1 protein expression and capsaicin-evoked SP release in cultured DRG neurons increased significantly after incubation with exogenous galanin compared with control DRG neurons at the same time point, whereas the amount of SP mRNA and basal SP release were not affected after incubation with exogenous galanin.

DISCUSSION: Exogenous galanin may promote capsaicin-evoked SP release from primary cultured DRG neurons. The elevation of the levels of VR1 mRNA and VR1 protein induced by exogenous galanin implicated that VR1 may be involved in the mechanisms of SP release evoked by capsaicin....

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Comparative Study 2008; 29(6): 929-938 PubMed PMID: 19112387 Citation  Keywords:  Adenoma:classification, Gene Expression Regulation, Neoplastic:physiology, Ghrelin:genetics, Humans, Immunohistochemistry, Pituitary Neoplasms:classification, RNA, Messenger:analysis, Somatotrophs:metabolism,.   

OBJECTIVES: It has been suggested that ghrelin synthesized locally in pituitary regulates the function and growth of pituitary cells in autocrine/paracrine way and might be an important factor of pituitary tumorogenesis. The expression of ghrelin receptor in neoplastic cells of pituitary adenomas has also been demonstrated. In vitro studies confirmed that ghrelin stimulates the proliferation of somatotroph cells in GH3 cell line. The presence of both ghrelin mRNA and protein was detected in a number of benign and malignant neoplasms as well as in neoplastic cells of the tissues which do not express ghrelin in physiological conditions. This study showed the presence of ghrelin mRNA and its protein in different types of pituitary adenomas.

DESIGN: The samples of 37 pituitary adenomas were obtained during standard neurosurgical tumor removal. The study tissues included 20 somatotroph tumors (15 patients treated and 5 patients untreated with octreotide LAR before the surgery), 12 nonfunctioning adenomas, 4 prolactinomas and 1 ACTH-secreting tumor. The control included samples of normal mucous membrane of the stomach and normal pituitaries. Expression of ghrelin mRNA was studied in 28 pituitary adenomas by RT-PCR. Immunohistochemical evaluation of ghrelin presence was performed in 34 tumors.

RESULTS: The presence of ghrelin gene transcripts was demonstrated in 10 out of 15 examined somatotroph tumors (obtained from patients treated with octreotide LAR before the surgery) and also in 2 out of 4 samples of prolactinomas, 7 out of 8 of nonfunctioning tumors and in 2 samples of normal pituitary. Immunohistochemical analyses revealed the presence of the protein in all 5 examined somatotroph tumors obtained from patients not treated prior to the surgery and in 10 out of 15 tumors obtained from patients treated with octreotide LAR. The peptide was detected also in 10 out of 12 examined nonfunctioning tumors and in 2 examined PRL-secreting tumors. The immunostaining for ghrelin was not shown in normal pituitaries.

CONCLUSIONS: The study demonstrated that ghrelin gene is expressed in somatotroph adenomas, both treated and untreated with octreotide LAR before the surgery, and also in other types of pituitary adenomas (prolactinomas and nonfunctioning adenomas)....

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Journal Article 2006; 27(3): 345-349 PubMed PMID: 16816835 Citation  Keywords:  Aged, Aged, 80 and over, Female, Humans, Immunohistochemistry, Male, Middle Aged, RNA, Messenger:analysis, Receptor, Endothelin A:genetics, Receptor, Endothelin B:genetics, Reference Values, Reverse Transcriptase Polymerase Chain Reaction, Tissue Distribu.   

OBJECTIVES: Endothelin is a potent peptide mediator that is synthesized by a number of cells. Previous studies have revealed the occurrence of endothelin in nerve cell bodies of some peripheral ganglia. Endothelin mediates its effects via two distinct receptor subtypes ETA and ETB. The present study was designed to investigate the presence of these two receptors in the human trigeminal ganglion.

METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to show the presence of mRNA encoding ETA and ETB receptors in the human trigeminal ganglion. To localize the protein immunocytochemistry with antibodies against the endothelin receptors was used.

RESULTS: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed mRNA for both receptor subtypes in the human trigeminal ganglion. Immunocytochemistry revealed numerous cell bodies containing the ETA and the ETB receptor proteins.

CONCLUSIONS: The expression of ETA and ETB receptors in the human trigeminal ganglion suggests a role for endothelin in autonomic and sensory neural transmission....

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Journal Article 2006; 27(3): 381-386 PubMed PMID: 16816827 Citation  Keywords:  Carcinoma:genetics, CpG Islands:physiology, DNA Methylation, Exons:physiology, Female, Gene Expression Regulation, Neoplastic:physiology, Humans, Neoplasm Proteins:genetics, Ovarian Neoplasms:genetics, RNA, Messenger:analysis, gamma-Synuclein:genetics,.   

BACKGROUND: The synucleins are a family of small cytoplasmic proteins that are expressed predominantly in neurons. Recently, their expression has been found also in many human cancers.

AIM: To understand the molecular mechanisms underlying the abnormal expression of SNCG in malignant ovarian tumors, in this study, we examined the methylation status of a CpG island located in exon 1 of SNCG gene in a panel of ovarian malignant tumors to determine if DNA methylation is related to clinical and histological tumor characteristics.

METHODS: SNCG mRNA expression in tumor samples was assessed by RT-PCR. Methylation status of SNCG gene was studied using methylation specific PCR (MSP).

RESULTS: Study group included 43 ovarian carcinoma samples (40 primary and 3 metastatic). The expression of SNCG mRNA was detectable in 33 of 43 ovarian cancer cases (76.7%). There were no significant differences in the mRNA expression between serous or nonserous tumors. The presence of SNCG mRNA in tumor samples was not correlated with age and menopausal status of patients, also no correlation was found with clinical stage or histological grading of malignant tumors. In 29 of 43 (67.4%) cases of tumors unmethylated product of MSP amplification was present. In a group of SNCG mRNA-positive tumors there were 75.7% (25 of 33) cases with demethylated or hypomethylated exon 1 of SNCG. The differences between the groups were statistically significant (chi2 =4.46; p=0.034). Demethylation of SNCG was not related to clinical tumor stage (p>0.05), but it was strongly associated with tumor grading (chi2=6.66; p=0.035). Aberrant methylation of SNCG was more often seen in tumors of women after menopause (78.2% vs 55%). In postmenopausal women 18 of 33 (62.1%) tumor samples synuclein-gamma mRNA expression was found, however the differences were not statistically significant. No correlations between SNCG hypomethylation and patient age, clinical stage and tumor grading were found. In 9 of 43 samples (21%) both products of amplification with methylated or unmethylated primer sets were found. In all of these cases SNCG mRNA was present.

CONCLUSIONS: SNCG mRNA is expressed in a substantial proportion of malignant ovarian tumors and demethylation is an important event in abnormal synuclein-gamma expression in most of these cases....

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Journal Article 2005; 26(5): 541-547 PubMed PMID: 16264403 Citation  Keywords:  Aging:metabolism, Animals, Brain Chemistry:physiology, Digoxigenin, In Situ Hybridization, Liver:metabolism, Male, Mice, Mice, Inbred C57BL, RNA Probes, RNA, Complementary:biosynthesis, RNA, Messenger:analysis, Receptors, Cytoplasmic and Nuclear:biosynthe.   

OBJECTIVES: The distribution of Liver receptor homolog 1 (LRH-1) mRNA was studied in mice brain with the aim to establish whether this nuclear hormone receptor is expressed also in the brain in addition to liver and classical steroidogenic tissues.

METHODS: Expression of LRH-1 mRNA in juvenile (30 days old) and adult (60 days old) mouse brain was examined using non-radioactive in situ hybridization with digoxigenin labeled cRNA probes and with RT PCR using specific primers.

RESULTS: LRH-1 was strongly expressed throughout the brain. Semiquantitative RT PCR revealed very strong expression of LRH-1 mRNA in cerebrum in comparison to liver and testis, and in situ hybridization revealed that LRH-1 mRNA is uniformly expressed in most brain areas.

CONCLUSIONS: LRH-1 is strongly expressed throughout the mouse brain suggesting important roles for this transcription factor, although its precise roles in the CNS remain to be elucidated....

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Journal Article 2004; 25(5): 361-367 PubMed PMID: 15580171 Citation  Keywords:  Altitude, Animals, Body Weight:physiology, Cysteamine:pharmacology, Growth Hormone:genetics, Hypothalamus:drug effects, Hypoxia:metabolism, Male, Pituitary Gland:metabolism, RNA, Messenger:analysis, Rats, Rats, Sprague-Dawley, Somatostatin:drug effects,.   

OBJECTIVES: The aim of this study was to investigate the response of the growth hormone (GH) in rat anterior pituitary to intermittent hypoxia (IH) and its modulation by hypothalamic somatostatin (SS).

SETTING AND DESIGN: To observe the hypoxic response, rats were exposed to simulated altitude hypoxia (2 km or 5 km) in a hypobaric chamber for various days (4 h/d); to clarify SS-involvement, rats were pretreated with SS antagonist (cysteamine, CSH, 200 mg/kg/d, s.c.) then exposed to IH (5 km) for 2d. The GH mRNA and immunostaining GH in pituitary as well as immunostaining SS in median eminence (ME) of hypothalamus were assayed by RT-PCR and immuno-histochemistry, respectively.

RESULTS: IH of 5 km altitude (IH5) markedly suppressed the body weight gain (BWG) of rats from 1d to 10d, and it was returned to control level henceforth, while no significant influence was showed in the group of IH of 2 km altitude (IH2). IH5 for 2 and 5d significantly decreased GH mRNA expression in the pituitary. The pituitary immunostaining GH was remarkably increased in groups of IH2 for 5, 10, and 15 d, and in groups of IH5 for 2, 5, and 10d. Immunostaining SS in ME was significantly reduced in group of IH2 for 5d, and in groups of IH5 for 2d and 5d. Pretreatments (s.c.) with SS antagonist (CSH) significantly reversed IH5-caused increase of immunostaining GH and reduction of mRNA levels in pituitary.

CONCLUSIONS: IH may cause a short-term and recoverable suppression of GH release, and reduce GH mRNA expression in anterior pituitary, which may depend on the intensity and duration of the hypoxia. This suppression may be due to a modulation of hypoxia-activated SS....

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