Suppression of excitatory synaptic transmission in the centrolateral amygdala via presynaptic histamine H3 heteroreceptors.

The central histaminergic system has a pivotal role in emotional regulation and psychiatric disorders, including anxiety, depression and schizophrenia. However, the effect of histamine on neuronal activity of the centrolateral amygdala (CeL), an essential node for fear and anxiety processing, remains unknown. Here, using immunostaining and whole-cell patch clamp recording combined with optogenetic manipulation of histaminergic terminals in CeL slices prepared from histidine decarboxylase (HDC)-Cre rats, we show that histamine selectively suppresses excitatory synaptic transmissions, including glutamatergic transmission from the basolateral amygdala, on both PKC-δ- and SOM-positive CeL neurons. The histamine-induced effect is mediated by H3 receptors expressed on VGLUT1-/VGLUT2-positive presynaptic terminals in CeL. Furthermore, optoactivation of histaminergic afferent terminals from the hypothalamic tuberomammillary nucleus (TMN) also significantly suppresses glutamatergic transmissions in CeL via H3 receptors. Histamine neither modulates inhibitory synaptic transmission by presynaptic H3 receptors nor directly excites CeL neurons by postsynaptic H1, H2 or H4 receptors. These results suggest that histaminergic afferent inputs and presynaptic H3 heteroreceptors may hold a critical position in balancing excitatory and inhibitory synaptic transmissions in CeL by selective modulation of glutamatergic drive, which may not only account for the pathophysiology of psychiatric disorders but also provide potential psychotherapeutic targets. KEY POINTS: Histamine selectively suppresses the excitatory, rather than inhibitory, synaptic transmissions on both PKC-δ- and SOM-positive neurons in the centrolateral amygdala (CeL). H3 receptors expressed on VGLUT1- or VGLUT2-positive afferent terminals mediate the suppression of histamine on glutamatergic synaptic transmission in CeL. Optogenetic activation of hypothalamic tuberomammillary nucleus (TMN)-CeL histaminergic projections inhibits glutamatergic transmission in CeL via H3 receptors.

Oxytocin Receptor in Cerebellar Purkinje Cells Does Not Engage in Autism-Related Behaviors.

The classical motor center cerebellum is one of the most consistent structures of abnormality in autism spectrum disorders (ASD), and neuropeptide oxytocin is increasingly explored as a potential pharmacotherapy for ASD. However, whether oxytocin targets the cerebellum for therapeutic effects remains unclear. Here, we report a localization of oxytocin receptor (OXTR) in Purkinje cells (PCs) of cerebellar lobule Crus I, which is functionally connected with ASD-implicated circuits. OXTR activation neither affects firing activities, intrinsic excitability, and synaptic transmission of normal PCs nor improves abnormal intrinsic excitability and synaptic transmission of PCs in maternal immune activation (MIA) mouse model of autism. Furthermore, blockage of OXTR in Crus I in wild-type mice does not induce autistic-like social, stereotypic, cognitive, and anxiety-like behaviors. These results suggest that oxytocin signaling in Crus I PCs seems to be uninvolved in ASD pathophysiology, and contribute to understanding of targets and mechanisms of oxytocin in ASD treatment.

Proinflammatory activation of microglia in the cerebellum hyperexcites Purkinje cells to trigger ataxia.

Specific medications to combat cerebellar ataxias, a group of debilitating movement disorders characterized by difficulty with walking, balance and coordination, are still lacking. Notably, cerebellar microglial activation appears to be a common feature in different types of ataxic patients and rodent models. However, direct evidence that cerebellar microglial activation in vivo is sufficient to induce ataxia is still lacking. Here, by employing chemogenetic approaches to manipulate cerebellar microglia selectively and directly, we found that specific chemogenetic activation of microglia in the cerebellar vermis directly leads to ataxia symptoms in wild-type mice and aggravated ataxic motor deficits in 3-acetylpyridine (3-AP) mice, a classic mouse model of cerebellar ataxia. Mechanistically, cerebellar microglial proinflammatory activation induced by either chemogenetic M3D(Gq) stimulation or 3-AP modeling hyperexcites Purkinje cells (PCs), which consequently triggers ataxia. Blockade of microglia-derived TNF-α, one of the most important proinflammatory cytokines, attenuates the hyperactivity of PCs driven by microglia. Moreover, chemogenetic inhibition of cerebellar microglial activation or suppression of cerebellar microglial activation by PLX3397 and minocycline reduces the production of proinflammatory cytokines, including TNF-α, to effectively restore the overactivation of PCs and alleviate motor deficits in 3-AP mice. These results suggest that cerebellar microglial activation may aggravate the neuroinflammatory response and subsequently induce dysfunction of PCs, which in turn triggers ataxic motor deficits. Our findings thus reveal a causal relationship between proinflammatory activation of cerebellar microglia and ataxic motor symptoms, which may offer novel evidence for therapeutic intervention for cerebellar ataxias by targeting microglia and microglia-derived inflammatory mediators.

MicroRNA-363 inhibits angiogenesis, proliferation, invasion, and migration of renal cell carcinoma via inactivation of the Janus tyrosine kinases 2-signal transducers and activators of transcription 3 axis by suppressing growth hormone receptor gene.

Renal cell carcinoma (RCC) is the most common malignancy involving the kidneys and a major cause of cancer mortality. The involvement of microRNA (miRNA) expression in the tumorigenesis and progression of RCC has been previously highlighted. Therefore, we conducted this study to investigate whether microRNA-363 (miR-363) affects the development of RCC via the Janus tyrosine kinases (JAK2)-signal transducers and activators of transcription (STAT) axis by targeting the growth hormone receptor (GHR), by observing the changes that occurred in the RCC and the normal adjacent tissues of patients with RCC. RCC cells were transfected with a series of miR-363 mimic, miR-363 inhibitor, or small interfering RNA against GHR to determine the influence of miR-363 on the expression of GHR and JAK2-STAT3 axis-related genes with the use of reverse transcription quantitative polymerase chain reaction and Western blot analysis. The angiogenesis, viability, invasion, and migration of cells were evaluated by means of in vitro angiogenesis, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT), wound-healing, and Transwell assays. The results revealed reduced miR-363 expression and elevated GHR expression in RCC. It was also found that miR-363 altered the activation of the JAK2-STAT3 axis through the inhibition of GHR. Cells treated with the miR-363 inhibitor presented with increased capillary vessels, cell viability, invasion, and migration, whereas it was on the contrary in the RCC cells with overexpressed miR-363. These results implicated that the overexpression of miR-363 could specifically bind to GHR to downregulate the expression of GHR, which, in turn, inactivates the JAK2-STAT3 axis, thereby influencing the angiogenesis, cell invasion, and migration abilities in RCC.

Facilitation of spinal α-motoneuron excitability by histamine and the underlying ionic mechanisms.

Spinal α-motoneurons directly innervate skeletal muscles and function as the final common path for movement and behavior. The processes that determine the excitability of motoneurons are critical for the execution of motor behavior. In fact, it has been noted that spinal motoneurons receive various neuromodulatory inputs, especially monoaminergic one. However, the roles of histamine and hypothalamic histaminergic innervation on spinal motoneurons and the underlying ionic mechanisms are still largely unknown. In the present study, by using the method of intracellular recording on rat spinal slices, we found that activation of either H1 or H2 receptor potentiated repetitive firing behavior and increased the excitability of spinal α-motoneurons. Both of blockage of K+ channels and activation of Na+-Ca2+ exchangers were involved in the H1 receptor-mediated excitation on spinal motoneurons, whereas the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were responsible for the H2 receptor-mediated excitation. The results suggest that, through switching functional status of ion channels and exchangers coupled to histamine receptors, histamine effectively biases the excitability of the spinal α-motoneurons. In this way, the hypothalamospinal histaminergic innervation may directly modulate final motor outputs and actively regulate spinal motor reflexes and motor execution.

Aberrant Promoter Methylation of PCDH17 (Protocadherin 17) in Serum and its Clinical Significance in Renal Cell Carcinoma.

BACKGROUND Current studies indicated that PCDH17 functions as a tumor suppressor, which is frequently inactivated by aberrant promoter methylation in urologic tumors. The main purpose of this study was to investigate the methylation status of PCDH17 in serum and its clinical significance in renal cell carcinoma (RCC). MATERIAL AND METHODS The methylation status of PCDH17 in serum samples of 142 RCC patients and 34 controls was evaluated by methylation-specific PCR (MSP). Then we correlated PCDH17 methylation status with the clinicopathologic features of RCC patients and patient outcomes. RESULTS We found that PCDH17 was more frequently methylated in RCC patients than in controls. Moreover, PCDH17 methylation in serum was significantly correlated with advanced stage (p=0.044), higher grade (p=0.019), lymph node metastasis (p=0.008) and tumor progression (p<0.001). In addition, patients with methylated PCDH17 had shorter progression-free survival (p<0.001) and overall survival (p=0.017) than patients without, and PCDH17 methylation in serum was an independent prognostic factor for worse progression-free survival (HR: 4.215, 95% CI: 1.376-9.032, p<0.001) and overall survival (HR: 5.092, 95% CI: 1.149-12.357, p=0.046) of patients with RCC. CONCLUSIONS The present study indicates that PCDH17 methylation in serum is a frequent event in RCC and associated with risk factors of poor outcomes. Moreover, PCDH17 methylation in serum is a potential prognostic biomarker for patients with RCC after surgery.

Evolving renorrhaphy technique for retroperitoneal laparoscopic partial nephrectomy: single-surgeon series.

OBJECTIVES: To evaluate renorrhaphy techniques and to analyze surgical outcomes in retroperitoneal laparoscopic partial nephrectomy. METHODS: A retrospective study from January 2008 to December 2011 analyzed 526 patients with renal tumors in whom renorrhaphy was changed from one layer, interrupted, figure-of-eight (n = 228) suture to two layers, continuous, unknotted (n = 298) suture. All procedures were carried out by the same laparoscopic surgeon (XZ). Patient demographics, tumor characteristics, operative outcomes and perioperative renal function were compared. RESULTS: Median follow up for one layer, interrupted, figure-of-eight suture and two layers, continuous, unknotted suture was 31 and 28 months, respectively. The two layers, continuous, unknotted suture group had shorter warm ischemia time (P = 0.021), faster removal of Jackson-Pratt drains (P = 0.029) and shorter hospital stay (P = 0.037) than the one layer, interrupted, figure-of-eight suture group. There was a trend towards a better preservation of glomerular filtration rates in the two layers, continuous, unknotted suture group (P = 0.045). In a multivariable model, the two layers, continuous, unknotted suture technique was a statistically significant independent predictor of warm ischemia time (P = 0.01), hospital stay (P = 0.001) and estimated glomerular filtration rates (P = 0.043). CONCLUSIONS: Two layers, continuous, unknotted suture renorrhaphy allows better outcomes than one layer, interrupted, figure-of-eight suture renorrhaphy in retroperitoneal laparoscopic partial nephrectomy. A longer clinical follow-up evaluation is warranted.

A role for the cerebellum in motor-triggered alleviation of anxiety.

Physical exercise is known to reduce anxiety, but the underlying brain mechanisms remain unclear. Here, we explore a hypothalamo-cerebello-amygdalar circuit that may mediate motor-dependent alleviation of anxiety. This three-neuron loop, in which the cerebellar dentate nucleus takes center stage, bridges the motor system with the emotional system. Subjecting animals to a constant rotarod engages glutamatergic cerebellar dentate neurons that drive PKCδ+ amygdalar neurons to elicit an anxiolytic effect. Moreover, challenging animals on an accelerated rather than a constant rotarod engages hypothalamic neurons that provide a superimposed anxiolytic effect via an orexinergic projection to the dentate neurons that activate the amygdala. Our findings reveal a cerebello-limbic pathway that may contribute to motor-triggered alleviation of anxiety and that may be optimally exploited during challenging physical exercise.

A comparative study of vestibular improvement and gastrointestinal effect of betahistine and gastrodin in mice.

Betahistine and gastrodin are the first-line medications for vestibular disorders in clinical practice, nevertheless, their amelioration effects on vestibular dysfunctions still lack direct comparison and their unexpected extra-vestibular effects remain elusive. Recent clinical studies have indicated that both of them may have effects on the gastrointestinal (GI) tract. Therefore, we purposed to systematically compare both vestibular and GI effects induced by betahistine and gastrodin and tried to elucidate the mechanisms underlying their GI effects. Our results showed that betahistine and gastrodin indeed had similar therapeutic effects on vestibular-associated motor dysfunction induced by unilateral labyrinthectomy. However, betahistine reduced total GI motility with gastric hypomotility and colonic hypermotility, whereas gastrodin did not influence total GI motility with only slight colonic hypermotility. In addition, betahistine, at normal dosages, induced a slight injury of gastric mucosa. These GI effects may be due to the different effects of betahistine and gastrodin on substance P and vasoactive intestinal peptide secretion in stomach and/or colon, and agonistic/anatgonistic effects of betahistine on histamine H1 and H3 receptors expressed in GI mucosal cells and H3 receptors distributed on nerves within the myenteric and submucosal plexuses. Furthermore, treatment of betahistine and gastrodin had potential effects on gut microbiota composition, which could lead to changes in host-microbiota homeostasis in turn. These results demonstrate that gastrodin has a consistent improvement effect on vestibular functions compared with betahistine but less effect on GI functions and gut microbiota, suggesting that gastrodin may be more suitable for vestibular disease patients with GI dysfunction.

Laparo-endoscopic single site anatomical retroperitoneoscopic adrenalectomy using conventional instruments: initial experience and short-term outcome.

PURPOSE: To our knowledge we present the initial experience with and the short-term outcome of laparo-endoscopic single site anatomical retroperitoneoscopic adrenalectomy using conventional instruments. MATERIALS AND METHODS: Between June 2009 and April 2010, 25 patients underwent laparo-endoscopic single site anatomical retroperitoneoscopic adrenalectomy. A TriPort™ Access System was inserted through a 2.5 to 3.0 cm transverse skin incision below the tip of the 12th rib. Adrenalectomy was done using a 5 mm 30-degree laparoscopic camera and 2 conventional laparoscopic instruments. After Gerota's fascia was incised we explored the first dissection plane between the perirenal fat and the anterior renal fascia at the superomedial side of the kidney. The adrenal gland was identified at the initial stage of the operation. RESULTS: Laparo-endoscopic single site anatomical retroperitoneoscopic adrenalectomy was successfully accomplished in 23 patients. An additional 5 mm port was required in 1 of the 2 unsuccessful cases and in the other it was necessary to convert to standard anatomical retroperitoneoscopic adrenalectomy. Median incision length was 3.0 cm, median operative time was 55 minutes and median estimated blood loss was 15 ml. No major intraoperative complications occurred. In the initial 10 cases median operative time was significantly longer (62 vs 50 minutes) and median blood loss was significantly higher (75 vs 10, each p <0.001) than in the subsequent 15. In 3 cases pheochromocytoma was successfully excised without undesirable hemodynamic oscillation. Postoperative complications developed in 2 patients, including angina and contralateral atelectasis in 1 each. CONCLUSIONS: In properly selected patients laparo-endoscopic single site anatomical retroperitoneoscopic adrenalectomy with conventional instruments is feasible, safe and effective, causes minimal morbidity and results in excellent cosmesis.

Long-term results of a prospective, randomized trial comparing retroperitoneoscopic partial versus total adrenalectomy for aldosterone producing adenoma.

PURPOSE: The indication for laparoscopic total or partial adrenalectomy in patients with aldosterone producing adrenal adenoma remains controversial. We compared retroperitoneoscopic partial and total adrenalectomy for aldosterone producing adrenal adenoma in a prospective, randomized, multicenter trial. MATERIALS AND METHODS: Patients with aldosterone producing adrenal adenoma were randomized to retroperitoneoscopic partial or total adrenalectomy. Patient characteristics, surgical data, complications and postoperative clinical results were analyzed statistically. RESULTS: From July 2000 to March 2004, 212 patients were enrolled in this study, including 108 and 104 who underwent total and partial adrenalectomy, respectively. The 2 groups were comparable in patient age, gender, body mass index and tumor site. Mean followup was 96 months in each group. No conversion to open surgery was needed and no major complications developed. Partial adrenalectomy required a shorter operative time than total adrenalectomy but this did not attain statistical significance. Intraoperative blood loss in the partial adrenalectomy group was significant higher than in the total adrenalectomy group (p <0.05) but no patient needed blood transfusion. All patients in each group showed improvement in hypertension, and in all plasma renin activity and aldosterone returned to normal after surgery. No patient required potassium supplements postoperatively. In the total and partial adrenalectomy groups 32 (29.6%) and 29 patients (27.9%), respectively, were prescribed a decreased dose of or fewer antihypertensive medicines at final followup. CONCLUSIONS: Retroperitoneoscopic partial adrenalectomy is technically safe. It has therapeutic results similar to those of total adrenalectomy in patients with primary aldosteronism due to aldosteronoma.

Laparoendoscopic single-site retroperitoneoscopic adrenalectomy: a matched-pair comparison with the gold standard.

BACKGROUND: Laparoscopic adrenalectomy has become the gold-standard for the surgical treatment of most adrenal lesions. This study evaluated the operative outcome of laparoendoscopic single-site (LESS) retroperitoneoscopic adrenalectomy (LESS-ARA) in comparison with the current standard operation procedure. METHODS: Between June and December 2009, 19 patients underwent LESS-ARA, and their outcomes were compared with a contemporary 1:2 matched-pair cohort of 38 patients who underwent standard ARA by the same surgeon. In LESS-ARA, a multichannel port was inserted through a 2.5- to 3.0-cm transverse skin incision below the tip of the 12th rib. The LESS-ARA procedure was performed using a 5-mm 30º laparoscopic camera and two standard laparoscopic instruments. The following parameters were compared between the two groups: demographics, details of the surgery, perioperative complications, postoperative visual analog pain scale score, analgesic requirement, and short-term measures of convalescence. RESULTS: The finding showed that LESS-ARA and standard ARA were comparable in terms of the estimated blood loss (30 vs 17.5 ml; p=0.64), postoperative hospital stay (6 vs 6 days; p=0.67), and postoperative complications (2 vs 3 patients; p=1.00) for patients with similar baseline demographics and median tumor size (2.1 vs 3.0; p=0.18) cm. The intraoperative hemodynamic values were similar in the two groups. The LESS-ARA group had a longer median operative time (55 vs 41.5 min; p=0.0004), whereas the in-hospital use of analgesics was significantly less (5 vs 12 morphine equivalents; p=0.03). CONCLUSIONS: The LESS retroperitoneoscopic adrenalectomy approach is feasible and offers a superior cosmetic outcome and better pain control, with perioperative outcomes and short-term measures of convalescence similar to those of the standard approach, albeit with a longer operative time.

Anatomical variation of the posterior lumbar tributaries of the left renal vein in retroperitoneoscopic left living donor nephrectomy.

OBJECTIVES: To increase awareness of the anatomical variation of the posterior lumbar tributaries of the left renal vein in retroperitoneoscopic left living donor nephrectomy. METHODS: A total of 61 cases of retroperitoneoscopic left living donor nephrectomy were carried out from March 2008 to June 2010. The anatomical variations of the posterior lumbar tributaries of the left renal vein in these patients were noted. RESULTS: According to the variation of posterior lumbar tributaries, there were seven types in total, including five main types (accounts for 95.1%, 58/61 cases) and the type of reno-hemi-azygo-lumbar trunk (AZV; accounts for 16.4%, 10/61 cases). According to the number of posterior lumbar tributaries, no lumbar vein covers accounted for 16.4% (10/61 cases), one lumbar vein accounted for 47.5% (29/61 cases), two lumbar veins accounted for 32.8% (20/61 cases) and three lumbar veins accounted 3.3% (2/61 cases). According to the operation time during the process of managing posterior lumbar veins, it was type 4 (AZV) on which the surgeon spent the most time (P<0.05), and type 5 (no lumbar vein) on which the surgeon spent the least time (P<0.05). CONCLUSIONS: This is the first report of the anatomical variation of the posterior lumbar tributaries of the left renal vein in retroperitoneal laparoscopic left living donor nephrectomy. Detailed knowledge of these anatomical variations will undoubtedly help surgeons to avoid the potential risk of vein damage during nephrectomy and to obtain a longer renal artery for the following renal transplantation.

CD146 as a Prognostic-Related Biomarker in ccRCC Correlating With Immune Infiltrates.

BACKGROUNDS: CD146 is highly expressed in various malignant tumors and associated with the poor prognosis. However, the role of CD146 in clear cell renal cell carcinoma (ccRCC) is still unknown. This study aimed to identify the role of CD146 in ccRCC by integrated bioinformatics analysis. METHODS: CD146 mRNA expression and methylation data in ccRCC was examined using the TIMER, UALCAN, and MethSurv databases. CD146 expression in paraffin-embedded tissues (140 cancer samples and 140 paracancer tissues) from our cohort were examined by immunohistochemistry assay. The LinkedOmics database was used to study the signaling pathways related to CD146 expression. TIMER and TISIDB were used to analyze the correlations among CD146, CD146-coexpressed genes, tumor-infiltrating immune cells, and immunomodulators. The relationship between CD146 and drug response in renal cancer cell lines was analyzed by the CTRP and CCLE databases. RESULTS: The mRNA and protein levels of CD146 were elevated in ccRCC tissues than that in paracancer tissues. The DNA methylation of CD146 in ccRCC tissues were lower than that in normal tissues. Importantly, high CD146 expression was associated with poor prognosis in patients with ccRCC. Furthermore, multivariate Cox regression analysis showed that CD146 was an independent prognostic factor in ccRCC. GO and KEGG pathway analyses indicated the co-expressed genes of CD146 were mainly related to a variety of immune-related pathways, including Th1 and Th2 cell differentiation, Th17 cell differentiation, and leukocyte transendothelial migration. Our data demonstrated that the expression and methylation status of CD146 were strongly correlated with immune infiltration levels, immunomodulators, and chemokines. Further, the sensitivity and resistance of renal cancer cell lines to some drugs were related to CD146 expression. CONCLUSIONS: Our study highlights the clinical significance of CD146 in ccRCC and provides novel insights into the immune function of CD146 in the tumor microenvironment.

FGL1 as a Novel Mediator and Biomarker of Malignant Progression in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC), which is the most prevalent renal cell carcinoma subtype, has a poor prognosis. Emerging strategies for enhancing the immune response in ccRCC therapy are currently being investigated. Fibrinogen-like Protein 1(FGL1) is a novel mechanism that tumors may use to evade the immune system by binding LAG-3 and negatively regulating T cells. In this study, we aimed at investigating the underlying mechanism of FGL1 in ccRCC, and its expression and prognostic value. We found that FGL1 was upregulated in tumor tissues and plasma specimens of ccRCC patients. High FGL1 expression predicted a poor prognosis for ccRCC patients. We also discovered that overexpression of FGL1 enhances RCC cell migration, invasion, and metastasis by activating the epithelial-to-mesenchymal transition (EMT). Consistent with these results, we identified a significant positive correlation between expression of FGL1 and EMT-related genes through tissue microarray analysis. Gene-expression analysis revealed that FGL1-deficient ccRCC cell lines had altered transcriptional output in inflammatory response, cell-cell signaling, negative regulation of T cell activation, and intracellular signal transduction. Depletion of FGL1 significantly inhibited tumor growth and lung metastasis in orthotopic xenograft mouse model. Infiltration of myeloid-derived CD11b+ and Ly6G+ immune cells in tumor microenvironment (TME) was strikingly decreased when FGL1 expression reduced. Therefore, increased FGL1 expression in ccRCC is positively correlated with poor prognosis. Mechanistically, FGL1 facilitates the EMT process and modulates TME, which promotes ccRCC progression and metastasis. Consequently, targeting FGL1 can potentially improve clinical outcome of ccRCC patients.

Effects of raf kinase inhibitor protein expression on metastasis and progression of human epithelial ovarian cancer.

Loss of function of metastasis suppressor genes is an important step in the progression to a malignant tumor type. Studies in cell culture and animal models have suggested a role of Raf kinase inhibitor protein (RKIP) in suppressing the metastatic spread of prostate cancer, breast cancer, and melanoma cells. However, the function of RKIP in ovarian cancer (OVCA) has not been reported. To explore the potential role of RKIP in epithelial OVCA metastasis, we detected the expression levels of RKIP protein in tissue samples from patients with epithelial OVCA. Consequently, the expression of RKIP is reduced in the poorly differentiated OVCA than in the well-differentiated and moderately differentiated OVCA. In addition, in vitro cell invasion assay indicated that the RKIP expression was inversely associated with the invasiveness of five OVCA cell lines. Consistent with this result, the cell proliferation, anchorage-independent growth, cell adhesion, and invasion were decreased in RKIP overexpressed cells but increased in RKIP down-regulated cells. Further investigation indicated that RKIP inhibited OVCA cell proliferation by altering cell cycle progression rather than promoting apoptosis. Furthermore, the overexpression of RKIP suppressed the ability of human OVCA cells to metastasize when the tumor cells were transplanted into nude mice. Our data show the effect of RKIP on the proliferation, migration, or adhesion of OVCA cells. These results indicate that RKIP is also a metastasis suppressor gene of human epithelial OVCA.

[Relationship between raf kinase inhibitor protein and metastasis of ovarian carcinoma].

OBJECTIVE: To investigate the relationship between raf kinase inhibitor protein (RKIP), a novel metastasis suppressor gene, and metastasis of ovarian carcinoma. METHODS: Immunohistochemistry, RT-PCR, and western blot analysis were performed to examine the expression of RKIP in clinical samples of ovarian tumors and five human ovarian carcinoma cell lines. Stable cell lines over-expressed or deleted of RKIP were cloned to investigate the function of RKIP in ovarian cancer cells. The recombinant plasmids expressing sense (ss) or antisense (as) RKIP cDNA or empty vector was transfected into ovarian cancer cell line SKOV3 by lipofectamine. The expression level of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in ovarian cancer cells were detected by western blot analysis. Assays of cell proliferation, soft-agar colony formation, cell adhesion, and cell invasion in vitro were used to examine the malignant phenotypes of the transfected cells. Flow cytometric analysis was performed to observe the effect of RKIP on cell cycle distribution before and after transfection. RESULTS: (1) The expression levels of RKIP protein in ovarian carcinoma tissues from patients were found to be reduced than those in ovarian benign tumor and borderline tumor. SKOV3 clones stably expressing full-length recombinant ssRKIP, asRKIP, and their respective empty vector were obtained. (2) RKIP was able to block basal levels of MEK and ERK in ovarian cancer cells. The expression level of phosphorylation MEK in ssRKIP#1 and ssRKIP#4 cells were 0.35, 0.34; while the expression level of phosphorylation ERK in ssRKIP #1 and ssRKIP #4 cells were 0. 48 and 0.46. (3) Abilities of cell proliferation in the ssRKIP vector-transfected cells were decreased compared with that in the non-transfected cells (P < 0.01). (4)Anchorage-independent growth in the ssRKIP#1 and ssRKIP#4 cells (83.7 +/- 5.7, 106.0 +/- 9.2) were decreased compared with that in the empty vector-transfected cells (158.3 +/- 14.6, P < 0.01). (5)Cell adhesion in the ssRKIP#1 and ssRKIP#4 cells [(68.3 +/- 0.8)%, (64.1 +/- 0.9)%] were decreased compared with that in the non-transfected cells [(100.0 +/- 1.1)%, P < 0.01]. (6) Cell invasion in the ssRKIP#1 and ssRKIP#4 cells (24 +/- 5, 25 +/- 4) were decreased compared with that in the non-transfected cells (68 +/- 5, P < 0.01). (7) ssRKIP cells had a significant increase in the G1 phase and decrease in the G2 + S phase. CONCLUSION: RKIP could inhibits the metastasis, but also the growth of ovarian cancer cells. patients were found to be reduced than those in ovarian benign tumor and borderline tumor. SKOV3 clones stably expressing full-length recombinant ssRKIP, asRKIP, and their respective empty vector were obtained. (2) RKIP was able to block basal levels of MEK and ERK in ovarian cancer cells. The expression level of phosphorylation MEK in ssRKIP#1 and ssRKIP#4 cells were 0.35, 0.34; while the expression level of phosphorylation ERK in ssRKIP #1 and ssRKIP #4 cells were 0.48 and 0.46. (3) Abilities of cell proliferation in the ssRKIP vector-transfected cells were decreased compared with that in the non-transfected cells (P < 0.01). (4) Anchorage-independent growth in the ssRKIP#1 and ssRKIP#4 cells (83.7 +/- 5.7, 106.0 +/- 9.2) were decreased compared with that in the empty vector-transfected cells (158.3 +/- 14.6, P < 0.01). (5) Cell adhesion in the ssRKIP#1 and ssRKIP#4 cells [(68.3 +/- 0.8)%, (64.1 +/- 0.9)%] were decreased compared with that in the non-transfected cells [(100.0 +/- 1.1)%, P < 0.01]. (6) Cell invasion in the ssRKIP#1 and ssRKIP#4 cells (24 +/- 5, 25 +/- 4) were decreased compared with that in the nontransfected cells (68 +/- 5, P < 0.01). (7) ssRKIP cells had a significant increase in the G1 phase and decrease in the G2 + S phase. CONCLUSION: RKIP could inhibits the metastasis, but also the growth of ovarian cancer cells.

DNA polymorphisms in exon 1 and promoter of the CDH1 gene and relevant risk of transitional cell carcinoma of the urinary bladder.

OBJECTIVE: To investigate the association between DNA polymorphisms, including single-nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms, in exon 1 and promoter of the CDH1 gene, and the risk of transitional cell carcinoma (TCC) of the urinary bladder (TCCB). PATIENTS, SUBJECTS AND METHODS: This was a hospital-based case-control study of 180 patients with TCCB and 110 normal controls. Genomic DNA was extracted from blood samples of all participants and genotypes determined using polymerase chain reaction and DNA sequencing techniques. Haplotypes were analysed using appropriate software. RESULTS: SNPs were detected at -160A/C, -73A/C and 178C/T; an inserted oligonucleotide of 5'-CCGTGCCCCAGCC-3' was identified at 234 bp. The -160A allele frequency in the case group was 0.67, statistically higher than in the control group (0.42; P < 0.001), and higher in invasive carcinoma (0.77) than in superficial carcinoma (0.60). For -73C/A and 178C/T SNPs there was no difference among genotypes. The 234 repeat oligonucleotide insertion (2I) frequencies in cases was 0.27, statistically higher than in the control group (0.17; P = 0.01). The most common haplotype in controls was C-A-T-I (28%), the frequency of which was higher than in the TCCB group (6%). The A-A-T-2I was the only variation distribution carrying the -160A allele and was at a statistically higher frequency in the TCCB group (37%, the most common haplotype in cases) than in the control group. CONCLUSION: The -160A, 234 2I allele and haplotype A-A-T-2I were risk factors of TCCB. Haplotype C-A-T-I might act as a protective factor for TCCB.

Histamine excites rat lateral vestibular nuclear neurons through activation of post-synaptic H2 receptors.

Through whole-cell patch recordings in brainstem slices, the effects of histamine on neuronal activity of the lateral vestibular nucleus (LVN) were investigated. Bath application of histamine elicited a concentration-dependent excitation of both spontaneous firing (n=19) and silent (n=7) LVN neurons. Moreover, histamine induced a stable inward current in the LVN neurons (n=5) and the histamine-induced depolarization of membrane potential persisted in the presence of tetrodotoxin (n=4), indicating a direct post-synaptic effect of the histamine on the LVN neurons. Selective histamine H2 receptor antagonist ranitidine effectively blocked the histamine-evoked excitatory responses on the LVN neurons (n=4), but selective histamine H1 receptor antagonist triprolidine did not (n=4). In addition, selective histamine H2 receptor agonist dimaprit (n=3) rather than 2-pyridylethylamine (n=4), a selective histamine H1 receptor agonist, mimicked the excitatory action of histamine on LVN neurons. The results demonstrate that histamine excites the LVN neurons via post-synaptic histamine H2 receptors and suggest that the central histaminergic projection arising from the hypothalamus may modulate LVN neurons activity and actively influence the vestibular reflexes and functions.

Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits.

The subthalamic nucleus (STN) is an effective therapeutic target for deep brain stimulation (DBS) for Parkinson's disease (PD), and histamine levels are elevated in the basal ganglia in PD patients. However, the effect of endogenous histaminergic modulation on STN neuronal activities and the neuronal mechanism underlying STN-DBS are unknown. Here, we report that STN neuronal firing patterns are more crucial than firing rates for motor control. Histamine excited STN neurons, but paradoxically ameliorated parkinsonian motor deficits, which we attributed to regularizing firing patterns of STN neurons via the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) channel coupled to the H2 receptor. Intriguingly, DBS increased histamine release in the STN and regularized STN neuronal firing patterns under parkinsonian conditions. HCN2 contributed to the DBS-induced regularization of neuronal firing patterns, suppression of excessive ß oscillations, and alleviation of motor deficits in PD. The results reveal an indispensable role for regularizing STN neuronal firing patterns in amelioration of parkinsonian motor dysfunction and a functional compensation for histamine in parkinsonian basal ganglia circuitry. The findings provide insights into mechanisms of STN-DBS as well as potential therapeutic targets and STN-DBS strategies for PD.