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Research evidence suggests that individuals with schizophrenia are prone to self-stigmatization, which reduces their psychosocial treatment adherence. A self-stigma reduction program was developed based on a theoretical framework proposed by our team. The effectiveness of such program to reduce self-stigma, enhance readiness for change, and promote adherent behaviors among individuals with schizophrenia was investigated. This program consisted of 12 group and four individual follow-up sessions. An integrative approach including psychoeductaion, cognitive behavioral therapy, motivational interviewing, social skills training, and goal attainment program was adopted. Sixty-six self-stigmatized individuals with schizophrenia were recruited. They were randomly allocated to the self-stigma reduction program (N=34; experimental protocol) or the newspaper reading group (N=32; comparison protocol). Measures on participants' level of self-stigma, readiness for change, insight, general self-efficacy, and treatment adherence were taken for six assessment intervals. The findings suggested that the self-stigma reduction program has potential to reduce self-esteem decrement, promote readiness for changing own problematic behaviors, and enhance psychosocial treatment adherence among the self-stigmatized individuals with schizophrenia during the active interventional stage. However, there was a lack of therapeutic maintenance effects during the 6-month follow-up period. Recommendations for further improving the effectiveness of self-stigma reduction program are suggested., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: We reviewed studies from 1990 to 2010 on using aromatherapy for people with anxiety or anxiety symptoms and examined their clinical effects., Methods: The review was conducted on available electronic databases to extract journal articles that evaluated the anxiolytic effects of aromatherapy for people with anxiety symptoms., Results: The results were based on 16 randomized controlled trials examining the anxiolytic effects of aromatherapy among people with anxiety symptoms. Most of the studies indicated positive effects to quell anxiety. No adverse events were reported., Conclusions: It is recommended that aromatherapy could be applied as a complementary therapy for people with anxiety symptoms. Further studies with better quality on methodology should be conducted to identify its clinical effects and the underlying biologic mechanisms.

In this study, we sought to explore the variation in reported rates of retained placenta around the world and over time in the UK. A systematic review of observational studies was performed to obtain retained placenta rates from around the world and annual hospital reports from the Royal College of Obstetricians and Gynaecologists archives were examined to obtain historical retained placenta rates. The data show that the median rate of retained placenta at 30 minutes was higher in developed countries (2.67% vs 1.46%, p < 0.02), as was the median manual removal rate (2.24% vs 0.45%, p < 0.001). In addition to this, there appears to have been a rise in rate of manual removal in the UK from a mean of 0.66% in the 1920s to 2.34% in the 1980s (p < 0.0001).

Objective: Predictors of employment outcomes of individuals with schizophrenia have continued to be studied over the past decade with implications for the development of vocational interventions to help the mentally ill get and keep jobs., Methods: A total of 62 relevant studies since 1998 were systematically reviewed by means of meta-analysis and frequency counts. Frequency count allowed all 62 studies to be included, whereas the meta-analysis excluded studies with inadequate information but made it possible to estimate the magnitude of effects., Results: Both methods resulted in similar findings. In contrast to an earlier review, cognitive functioning received overwhelming support as a significant predictor. Other significant predictors included education, negative symptoms, social support and skills, age, work history (previous history of successful employment), and rehabilitation service to restore community functioning and well-being by occupational therapists, psychiatrists, psychologists, social workers and other mental health professionals. Positive symptoms, substance abuse, gender and hospitalization history were found to be non-significant predictors. The frequency count did not support marital status as a significant predictor but the meta-analysis did., Conclusions: This review highlights increasing sophistication in understanding the links between individual characteristics and functional impairments. It also suggests that more research is needed into other potentially important predictors that may be changeable and relate to recovery. These include attitudes and beliefs about disability payments and psychological processes such as self-stigmatization, negative beliefs, and social skills deficits for which intervention may be possible.

Objective: Torcetrapib, a prototype cholesteryl ester transfer protein (CETP) inhibitor with potential for decreasing atherosclerotic disease, increased cardiovascular events in clinical trials. The identified hypertensive and aldosterone-elevating actions of torcetrapib may not fully account for this elevated cardiovascular risk. Therefore, we evaluated the effects of torcetrapib on endothelial mediated vasodilation in vivo., Methods and Results: In vivo endothelial mediated vasodilation was assessed using ultrasound imaging of acetylcholine-induced changes in rabbit central ear artery diameter. Torcetrapib, in addition to producing hypertension and baseline vasoconstriction, markedly inhibited acetylcholine-induced vasodilation. A structurally distinct CETP inhibitor, JNJ-28545595, did not affect endothelial function despite producing similar degrees of CETP inhibition and high-density lipoprotein elevation. Nitroprusside normalized torcetrapib's basal vasoconstriction and elicited dose-dependent vasodilation of norepinephrine preconstricted arteries in torcetrapib-treated animals, indicating torcetrapib did not impair smooth muscle function., Conclusions: Torcetrapib significantly impairs endothelial function in vivo, independent of CETP inhibition and high-density lipoprotein elevation. Given the well-documented association of endothelial dysfunction with cardiovascular disease and risk, this activity of torcetrapib may have contributed to increased cardiovascular risk in clinical trials.

Objectives: The aim of the present study was to examine and compare the long-term effectiveness of the Integrated Supported Employment (ISE) programme, which consists of individual placement and support (IPS) and work-related social skills training, with the IPS programme on the vocational and non-vocational outcomes among individuals with severe mental illness (SMI) over a period of 3 years., Method: One hundred and eighty-nine participants with SMI were recruited from two non-government organizations and three day hospitals in Hong Kong and randomly assigned into the ISE (n = 58), IPS (n = 65) and traditional vocational rehabilitation (TVR) (n = 66) groups. Vocational and non-vocational outcomes of the ISE and IPS participants were collected by a blind and independent assessor at 7 11, 15, 21, 27, 33 and 39 months after their admission, whereas the TVR groups were assessed only up to the 15th month follow up., Results: After 39 months of service provision, ISE participants obtained higher employment rate (82.8% vs 61.5%) and longer job tenure (46.94 weeks vs 36.17 weeks) than the IPS participants. Only 6.1% of TVR participants were able to obtain employment before the 15th month follow up. Fewer interpersonal conflicts at the workplace were reported for the ISE participants. Advantages of the ISE participants over IPS participants on non-vocational outcomes were not conclusive., Conclusion: The long-term effectiveness of the ISE programme in enhancing employment rates and job tenures among individuals with SMI was demonstrated by this randomized controlled trial.

This study proposed a novel methodology for depicting cerebral small vessels including veins, arterioles, and venules, called 3DDeltaR(2)-mMRA (three-dimensional, steady-state DeltaR(2)-based, and flow-independent microscopic magnetic resonance angiography). The DeltaR(2) map calculated by a fast spin-echo imaging technique before and after the injection of an iron-oxide contrast agent was used to delineate the relative cerebral blood volume, primarily to microvasculature. The proposed 3DDeltaR(2)-mMRA method, which employs 3D reconstruction techniques, can simultaneously provide high-resolution 3D information on the cerebral anatomy, in vivo microvascular architecture, and hemodynamic response, which can be used to evaluate pathological microvascular changes over time in cerebromicrovascular disease. Since spin-echo-based DeltaR(2) imaging was applied, the inflow effects, susceptibility artifacts, and the overestimation of vessel size in brain were reduced. A well-defined three-vessel occlusion model in the rat was performed to evaluate the capability of the proposed method in evaluating alterations to the microvasculature.

The dysregulation of arginine vasopressin (AVP) release and activation of vasopressin V(1A) and V(2) receptors may play a role in disease. The in vitro and in vivo pharmacology of RWJ-676070, a potent, balanced antagonist of both the V(1A) and V(2) receptors is described. RWJ-676070 binding and intracellular functional antagonist activity was characterized using cells expressing V(1A), V(1B) or V(2) receptors. Its inhibition of V(1A) receptor-mediated contraction of vascular rings and platelet aggregation was determined. V(2) receptor-medated aquaresis was determined in rats, dogs and monkeys. V(1A) receptor-mediated inhibitory activity was assessed in vivo in a vasopressin-induced hypertension model and in normotensive rats and in two hypertensive rat models. RWJ-676070 inhibited AVP binding to human V(1A) and V(2) receptors (Ki=1 and 14 nM, respectively). RWJ-676070 inhibited V(1A) receptor-induced intracellular calcium mobilization and V(2) receptor-induced cAMP accumulation with Ki values of 14 nM and 13 nM, respectively. The compound was slightly less potent against rat V(1A) receptors. RWJ-676070 inhibited V(1A) receptor-mediated vasoconstriction in rat and dog vascular rings and AVP-induced human platelet aggregation. Dose dependent aquaresis was demonstrated in rats, dogs and monkeys following oral administration. RWJ-676070 inhibited AVP-induced hypertension in rats but had no effect on arterial pressure in normotensive and spontaneously hypertensive rats but did decrease arterial pressure in Dahl, salt-sensitive hypertensive rats. RWJ-676070 is a new, potent antagonist of V(1A) and V(2) receptors that may be useful for treatment of diseases benefiting from balanced inhibition of both V(1A) and V(2) receptors.

Postischemic cerebral blood flow and blood volume changes have been associated with angiogenesis; nevertheless, the spatiotemporal changes in vascular permeability, vascular density, and vessel size have not been investigated. Here we report a prolonged increase in vascular permeability from day 3 to day 21 after ischemia, in particular in the reperfused outer cortical layers and leptomeninges. Increased cerebral blood volume (CBV) was observed from day 3 to day 14, whereas increased blood volume in small vessels, primarily capillaries, was noticed from day 7 to day 14 in the reperfused cortex. An initial decrease in vascular density and a reciprocal increase in vessel size were observed within the reperfused cortex at days 1 and 3 after ischemia. Immunohistological analysis confirmed a similar decrease in microvessel density and an increase in vessel size in vessels with a diameter greater than 30 microm. These large-sized vessels exhibited intense basic fibroblast growth factor and endothelial nitric oxide synthase immunoreactivity, suggesting the growth of collateral vessels. By contrast, a late increase in vascular density was noticed in the reperfused outer cortex at days 14 and 21 after ischemia. Together, these findings suggest that the early phase of CBV increase is likely because of the improvement in collateral circulation, whereas the late phase of CBV increase is attributed to the surge of angiogenesis.

Treatment of Ru(PPh3)3Cl2 with K(tpip) (tpip(-)=[N(Ph2PO)2](-)) afforded Ru(tpip)(PPh3)2Cl (1), which reacted with 4- t-Bu-C6H4CN, SO2(g), and NH 3(g) to give Ru(tpip)(PPh3)2Cl(4- t-BuC6H4CN) (2), Ru(tpip)(PPh3)2Cl(SO2) (3), and fac-[Ru(NH3)3(PPh3)2Cl][tpip] (4), respectively. Reaction of [Ru(CO)2Cl2] x with K(tpip) in refluxing tetrahydrofuran (THF) led to isolation of the K/Ru bimetallic compound K 2Ru2(tpip)4(CO)4Cl2 (5). Photolysis of cis-Ru(tpip) 2(NO)Cl in MeCN and wet CH 2Cl 2 afforded cis-Ru(tpip) 2(MeCN)Cl ( 6) and cis-Ru(tpip)2(H2O)Cl (7), respectively. Refluxing 6 in neat THF yielded Ru(tpip) 2(THF)Cl (8). Treatment of Ru(CHR)Cl2(PCy3)2 (Cy=cyclohexyl) with [Ag(tpip)] 4 afforded cis-Ru(tpip)2(CHR)(PCy3) [R=Ph (9), OEt (10)]. Complex 9 is capable of catalyzing oxidation of alcohols and olefins with N-methylmorpholine N-oxide and iodosylbenzene, respectively. The crystal structures of 2-7 and 9 were determined.

This study translated and validated the Chinese Version of the Self-stigma of Mental Illness Scale (CSSMIS), which may be used to measure self-stigma of mental health consumers in China. We also examined its correlation with self-esteem, self-efficacy and psychosocial treatment compliance. A cross-sectional observational study was implemented. Some 51 males and 57 females who suffered from severe mental illness were recruited from psychiatric settings in Hong Kong. They were required to complete the Chinese Version of the Self-stigma of Mental Illness Scale, the Rosenberg Self-esteem Scale and the Self-efficacy Scale. Their level of compliance during psychosocial treatment and their demographic information were recorded by their case managers. Exploratory factor analysis revealed two homologous factors for the four subscales of the CSSMIS. Factor 1 was related to the negative beliefs and consequences of having mental disorders, whereas Factor 2 was related to positive beliefs. The perceived stigma subscale and the three self-stigma subscales were strongly inter-correlated. Significant correlations were also found between almost all subscales of the CSSMIS and the remaining scales. The psychometric properties of the CSSMIS are statistically acceptable. The results also suggest that stigma played a detrimental role in undermining self-esteem, self-efficacy and psychosocial treatment compliance. Implications for recovery of mental health consumers are discussed.

Reduction of trans-[OsL2(O)2] (1) (L-=[N(i-Pr2PS)2]-) with hydrazine hydrate afforded a dinitrogen complex 2, possibly "[OsL2(N2)(solv)]" (solv=H2O or THF), which reacted with RCN, R'NC, and SO2 to give trans-[OsL2(RCN)2] (R=Ph (3), 4-tolyl (4), 4-t-BuC6H4 (5)), trans-[OsL2(R'NC)2] (R'=2,6-Me2C6H3 (xyl) (6), t-Bu (7)), and [Os(L)2(SO2)(H2O)] (8) complexes, respectively. Protonation of compounds 2, 3, and 6 with HBF4 led to formation of dicationic trans-[Os(LH)2(N2)(H2O)][BF4]2 (9), trans-[Os(LH)2(PhCN)2][BF4]2 (10), and trans-[Os(LH)2(xylNC)2][BF4]2 (11), respectively. Treatment of 1 with phenylhydrazine and SnCl2 afforded trans-[OsL2(N2Ph)2] (12) and trans-[OsL2Cl2] (13), respectively. Air oxidation of compound 2 in hexane/MeOH gave the dimethoxy complex trans-[OsL2(OMe)2] (14), which in CH2Cl2 solution was readily air oxidized to 1. Compound 1 is capable of catalyzing aerobic oxidation of PPh3, possibly via an Os(IV) intermediate. The formal potentials for the Os-L complexes have been determined by cyclic voltammetry. The solid-state structures of compounds 4, 6, cis-8, 13, and 14 have been established by X-ray crystallography.

A human neuroblastoma cell line, IMR-32, was used as an in vitro model system to study the effects of arsenic trioxide (As(2)O(3)) on aggressive human neuroblastoma. From 0.5 micro M, As(2)O(3) exhibited a dose-dependent inhibition of IMR-32 proliferation. At concentrations of 1.5 micro M or higher, As(2)O(3) up-regulated caspase 3, leading to cellular apoptosis. However, neurofilament-200 kDa and tyrosine hydroxylase were not up-regulated, implying minimal neuronal differentiation. Concomitantly, TrkA was down-regulated and TrkB up-regulated. Pre-treatment with the protein kinase C (PKC) inhibitor Ro-31-8220 partially blocked As(2)O(3)-mediated apoptosis, meaning that As(2)O(3) might signal through PKC activation. The results suggest that As(2)O(3) might be potentially useful in neuroblastoma.

Osteoporosis pseudoglioma syndrome (OPPG) is an autosomal recessive disorder due to mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Here, we report two novel missense mutations found in a southern Chinese family of a non-consanguineous marriage. Three out of four children had blindness, low bone mineral density (BMD) and multiple fractures in their childhood. Genotyping by DNA sequencing demonstrated 2 new mutations in exon 7 of the LRP5 gene. Tryptophans at amino acid residue positions 478 and 504 were replaced by arginine (W478R) and cysteine (W504C), respectively. While the parents that possessed either heterozygous W478R or W504C were apparently normal, all affected subjects were compound heterozygotes for the W478R and W504C mutations in the LRP5 gene. W478R is located immediately C-terminal to the third YWTD repeat of the second YWTD/EGF domain in LRP5, while W504C is located between the third and the fourth YWTD repeats of the second YWTD/EGF domain in LRP5. Using LRP5-related proteins, such as the low-density lipoprotein receptor (LDLR) and nidogen as reference models, a homology model of LRP5 suggested that the observed mutations may affect the molecular interactions of LRP5 and so lead to the observed OPPG phenotypes.

Prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet aggregation and leukocyte activation, is crucial in vascular diseases such as stroke. Prostacyclin synthase (PGIS) is the key enzyme for PGI2 synthesis. Although expression of PGIS was noted in the brain, its role in ischemic insult remains unclear. Here we reported the temporal and spatial expression of PGIS mRNA and protein after 60-min transient ischemia. Northern blot and in situ hybridization revealed a delayed increase of PGIS mRNA in the ischemic cortex at 24- to 72-h after ischemia; PGIS was detected mainly in the ipsilateral penumbra area, pyriform cortex, hippocampus, and leptomeninges. Western blot and immunohistochemical analysis revealed that PGIS proteins were expressed temporally and spatially similar to PGIS mRNA. PGIS was heavily colocalized with PECAM-1 to endothelial cells at the leptomeninges, large and small vessels, and localized to neuronal cells, largely at the penumbra area. A substantial amount of PGIS was also detected in the macrophage and glial cells. To evaluate its role against ischemic infarct, we overexpressed PGIS by adenoviral gene transfer. When infused 72 h before ischemia (- 72 h), Adv-PGIS reduced infarct volume by approximately 50%. However, it had no effect on infarct volume when infused immediately after ischemia (0 h). Eicosanoid analysis revealed selective elevation of PGI2 at - 72 h while PGI2 and TXB2 were both elevated at 0 h, altering the PGI2/thromboxane A2 (TXA2) ratio from 10 to 4. These findings indicate that PGIS protects the brain by enhancing PGI2 synthesis and creating a favorable PGI2/TXA2 ratio.

Objective: Brain expresses abundant lipocalin-type prostaglandin (PG) D2 (PGD2) synthase but the role of PGD2 and its metabolite, 15-deoxy-Delta(12,14) PGJ2 (15d-PGJ2) in brain protection is unclear. The aim of this study is to assess the effect of 15d-PGJ2 on neuroprotection., Methods and Results: Adenoviral transfer of cyclooxygenase-1 (Adv-COX-1) was used to amplify the production of 15d-PGJ2 in ischemic cortex in a rat focal infarction model. Cortical 15d-PGJ2 in Adv-COX-1-treated rats was increased by 3-fold over control, which was correlated with reduced infarct volume and activated caspase 3, and increased peroxisome proliferator activated receptor-gamma (PPARgamma) and heme oxygenase-1 (HO-1). Intraventricular infusion of 15d-PGJ2 resulted in reduction of infarct volume, which was abrogated by a PPARgamma inhibitor. Rosiglitazone infusion had a similar effect. 15d-PGJ2 and rosiglitazone at low concentrations suppressed H2O2-induced rat or human neuronal apoptosis and necrosis and induced PPARgamma and HO-1 expression. The anti-apoptotic effect was abrogated by PPARgamma inhibition., Conclusions: 15d-PGJ2 suppressed ischemic brain infarction and neuronal apoptosis and necrosis in a PPARgamma dependent manner. 15d-PGJ2 may play a role in controlling acute brain damage induced by ischemia-reperfusion.

Objective: Thyrotoxic periodic paralysis (TPP) is a complication of hyperthyroidism association with recurrent, reversible episodes of muscle weakness. Increased sodium-potassium ATPase (Na/K-ATPase) pump activity is postulated to contribute to the hypokalaemic paralytic attacks in TPP. The aim of this study was to determine the genetic predisposition to TPP in relation to Na/K-ATPase genes., Design: A case-control association study., Patients: Ninety-nine male Chinese TPP patients were compared to 84 male Graves' disease (GD) patients without TPP and 100 normal male controls., Measurement: A total of 1500 base pairs upstream of the transcriptional start site of the five Na/K-ATPase genes that are expressed in the skeletal muscles, namely ATP1A1, ATP1A2, ATP1B1, ATP1B2 and ATP1B4, were sequenced in all subjects for mutations or polymorphisms. The single nucleotide polymorphisms (SNPs) of the coding regions of the five genes were also studied for association with TPP., Results: No mutations were detected in the 5' regions of the five genes in any of the patients studied. There was no difference in the distribution of SNPs and SNP haplotypes in the upstream and coding region of these genes between the three groups of subjects., Conclusion: No association between the polymorphisms of ATP1A1, ATP1A2, ATP1B1, ATP1B2 and ATP1B4 genes and TPP could be detected.

Osteoblastic differentiation is an essential part of bone formation. Dimethyl sulfoxide (DMSO) is a water miscible solvent that is used extensively for receptor ligands in osteoblast studies. However, little is known about its effects on osteoblastogenic precursor cells. In this study, we have used a murine preosteoblast cell line MC3T3-E1 cells to demonstrate that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and osterix and is dependent upon the protein kinase C (PKC) pathways. We further demonstrated that prolonged activation of PKC pathways is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCmu.

Bone mineral density (BMD), an important risk factor for osteoporosis, is a complex trait likely affected by multiple genes. The linkage and/or association of 13 polymorphic loci of seven candidate genes (estrogen receptor alpha [ERalpha] and beta [ERbeta], calcium-sensing receptor, vitamin D receptor, collagen type 1alpha1, low-density lipoprotein [LDL] receptor-related protein 5 [LRPS], and transforming growth factor beta1) were evaluated in 177 southern Chinese pedigrees of 674 subjects, with each pedigree identified through a proband having a BMD Z score of -1.28 or less at the hip or spine. A suggestive linkage was detected between the IVS1-351A/G polymorphism of ERalpha and spine BMD, and between the 1082G/A, 1730G/A, and D14S1026 polymorphisms of ERbeta and BMD at both spine and hip. The quantitative transmission disequilibrium test (QTDT) detected total family association between 1730G/A of ERbeta and BMD at spine and hip; between D14S1026 of ERbeta and hip BMD; and between the 266A/G and 2220C/T polymorphisms of LRP5 and hip BMD. Similar total family associations were detected when only the females were analyzed. In addition, the IVS1-397T/C polymorphism of ERalpha was associated with spine BMD, and the 266A/G and 2220C/T polymorphisms of LRP5 were associated with femoral neck BMD in the females. A within-family association was detected with the IVS1-397T/C polymorphism of ERalpha, and the 266A/G and 2220C/T polymorphisms of LRP5 in the females. The effect of each polymorphism on BMD variance ranged from 1% to 4%. In conclusion, ERalpha, ERbeta and LRP5 are important candidate genes determining BMD variation, especially in females.

Novel indolylindazolylmaleimides were synthesized and examined for kinase inhibition. We identified low-nanomolar inhibitors of PKC-beta with good to excellent selectivity vs other PKC isozymes and GSK-3beta. In a cell-based functional assay, 8f and 8i effectively blocked IL-8 release induced by PKC-betaII (IC(50) = 20-25 nM). In cardiovascular safety assessment, representative lead compounds bound to the hERG channel with high affinity, potently inhibited ion current in a patch-clamp experiment, and caused a dose-dependent increase of QT(c) in guinea pigs.

Although mRNA expression of group IIA secretory phospholipase A2 (sPLA2-IIA) has been implicated in responses to injury in the CNS, information on protein expression remains unclear. In this study, we investigated temporal and spatial expression of sPLA2-IIA mRNA and immunoreactivity in transient focal cerebral ischemia induced in rats by occlusion of the middle cerebral artery. Northern blot analysis showed a biphasic increase in sPLA2-IIA mRNA expression following 60-min of ischemia-reperfusion: an early phase at 30 min and a second increase at a late phase ranging from 12 h to 14 days. In situ hybridization localized the early-phase increase in sPLA2-IIA mRNA to the affected ischemic cortex and the late-phase increase to the penumbral area. Besides sPLA2-IIA mRNA, glial fibrillary acidic protein (GFAP) and cyclo-oxygenase-2 mRNAs, but not cytosolic PLA2, also showed an increase in the penumbral area at 3 days after ischemia-reperfusion. Immunohistochemistry of sPLA2-IIA indicated positive cells in the penumbral area similar to the GFAP-positive astrocytes but different from the isolectin B4-positive microglial cells. Confocal microscopy further confirmed immunoreactivity of sPLA2-IIA in reactive astrocytes but not in microglial cells. Taken together, these results demonstrate for the first time an up-regulation of the inflammatory sPLA2-IIA in reactive astrocytes in response to cerebral ischemia-reperfusion.

A human neuroblastoma cell line IMR-32 was used as an in vitro model to examine three naturally occurring retinoic acid (RA) isomers, 9-cis (9c), 13-cis (13c) and all-trans (AT) RA, in mediating growth differentiation and neuronal differentiation. All RA isomers inhibited cellular proliferation, with 13c-RA being most effective. Cyclic AMP-responsive-element-binding-protein (CREB) was activated during RA treatment. AT-RA was a better differentiating agent in inducing the highest expression of the neurotrophic factor receptor TrkA. After prolonged RA treatment, the expression of RA receptors (RARs) was comparable for the three isomers, but retinoid X receptors (RXRs) were differentially regulated. These results imply that distinctive molecular pathways might be involved in the in vitro differentiation of neuroblastoma with different RA isomers.

Families of individuals with mental illness face a range of practical and emotional stresses. Studies that have addressed the sources of these burdens are limited. Literature suggests that burdens could come from the stigmatizing attitudes towards individuals with mental illness and inadequate public resources. Nevertheless, how public attitudes and availability of public resources have affected the burden on patients' families remains to be studied. This study set out to explore the relationship between stigma, accessibility of mental health facilities and family burden through individual interviews of patients' relatives in order to understand the burden on mentally ill patients' relatives from their perspectives. Ten interviewees from two out-patient psychiatric clinics were recruited and interviewed. Each interviewee had at least one family member receiving out-patient psychiatric services. Altogether 11 mentally ill patients were involved. Data analyses showed that much of the burden was related to stigma and to lack of mental health and rehabilitation services. Consequences included social isolation of the families, difficulties experienced by the mentally ill patients when trying to obtain competitive employment and financial difficulties. Subjective burden resulting from social stigma included frustration, anxiety, low self-esteem and helplessness. Implications of the findings to social policy and development of mental health services were discussed.

A retrospective analysis of fresh in vitro fertilization treatment cycles and frozen embryo transfer (FET) cycles from mid-1995 to December 31, 1998, was undertaken. Nurses performed embryo transfer (ET) for government-funded cycles, whereas doctors performed ET for self-funded cycles. During the study period, fresh ET was performed in 1,165 treatment cycles. There were no significant differences in demographic data, ovarian responses and the number of embryos replaced between ET cycles performed by nurses and doctors. Pregnancy rates for ETs performed by nurses and doctors were 16.7 and 15.8% per transfer, respectively, whereas the corresponding implantation rates were 8.3 and 6.9%, respectively. Similar pregnancy and implantation rates were encountered in FET cycles whether ET was performed by nurses or doctors., (Copyright 2003 S. Karger AG, Basel)

Background and Purpose: Angiogenesis occurs after cerebral ischemia, and the extent of angiogenesis has been correlated with survival in stroke patients. However, postischemic angiogenesis is short-lived and may be completely terminated within a few weeks after ischemic insult. The molecular mechanism underlying the dissolution of postischemic angiogenic processes is poorly understood. Although the expression of angiogenic genes has been studied in ischemic stroke models, the activation of angiostatic genes after cerebral ischemia has not been investigated. Thrombospondin (TSP)-1 and TSP-2 are naturally occurring angiostatic factors, which inhibit angiogenesis in vivo. The aim of the present study was to explore the expression of TSP-1 and TSP-2 in relation to the evolution of angiogenic process in a focal ischemia model in rats., Methods: Rats underwent cortical ischemia in the middle cerebral artery territory for 60 minutes and reperfusion for up to 2 weeks. Northern and Western blot analysis were used to study the temporal profile of TSP-1 and TSP-2 expression at the mRNA and protein level, respectively. In situ hybridization and immunohistochemical studies were used to examine the spatial expression patterns. Double immunostaining was applied to define the cellular origins of TSP-1 and TSP-2., Results: A biphasic expression of TSP-1 was noted after ischemia, peaking at 1 and 72 hours. Endothelial cells in the leptomeninges were the only source of the first TSP-1 peak, whereas endothelial, glial, neuronal, and macrophage cells contributed to the second peak of TSP-1 expression. TSP-2 expression occurred much later and in a monophasic manner, peaking 2 weeks after ischemia. TSP-2 immunoreactivity was observed in endothelial, neuronal, and macrophage, but not glial, cells. TSP-1 was expressed before the peak of angiogenesis, whereas robust TSP-2 expression occurred at the peak of angiogenesis and continued into the period when angiogenesis had completely resolved., Conclusions: Robust expression of TSP-1 and TSP-2, 2 major angiostatic factors, was noted in the ischemic brain with different temporal expression profiles from different cellular origins. The expression of these angiostatic factors, especially TSP-2, likely contributes to the spontaneous resolution of postischemic angiogenesis. Further studies are needed to explore the molecular mechanisms that regulate the balance of angiogenic and angiostatic factors in the ischemic brain.

Background and Purpose: Angiogenesis occurs after cerebral ischemia, but the relationship between angiogenesis and cerebral hemodynamic change is unknown. The aim of the present study was to investigate the relationship between ischemia-induced angiogenesis and hemodynamics in a well-defined 3-vessel occlusion model of the rat by using diffusion- (DWI), perfusion-, and T2-weighted MRI (T2WI)., Methods: Rats were subjected to 60 minutes of transient middle cerebral artery occlusion or sham operation. DWI and T2WI were used to characterize the extent of the ischemic lesion from 4.5 hours to 14 days after reperfusion. A flow-sensitive alternating inversion recovery method and dynamic susceptibility contrast MRI were used to evaluate the temporal changes in relative cerebral blood flow (CBF) and cerebral blood volume (CBV), respectively. Rats were randomly selected and killed at each time point for investigation of vascular density and for hematoxylin-eosin staining., Results: Ischemic lesions developed in the ipsilateral cortex, as demonstrated by DWI and T2WI. CBF was significantly increased in the ipsilateral cortex, especially in the cortical outer layer from day 1 to day 14, and peaked on day 7 (P<0.05), while CBV was significantly increased on day 7 (P<0.01). The vascular density on the ipsilateral brain surface was gradually increased from day 1 to day 5, peaked on day 7, and then decreased on day 14. Histology study showed pannecrosis in the cortex from day 1 to day 5 and partial liquefaction of the necrotic tissues on days 7 and 14., Conclusions: A delayed increase in both CBF and CBV is documented in the ipsilateral cortex after transient focal brain ischemia, and such an increase may be associated with angiogenesis.

Background: We tested the hypothesis that bicistronic cyclooxygenase-1 (COX-1)/prostacyclin synthase (PGIS) and COX-1 gene transfer reduce cerebral infarct volume by augmenting synthesis of protective prostaglandins., Methods and Results: We infused into lateral ventricle of a rat stroke model recombinant adenoviruses (rAd) containing COX-1 (Adv-COX-1), COX-1 and PGIS (Adv-COX-1/PGIS), or Adv-PGK control vector, and we determined COX-1 and PGIS protein and eicosanoid levels and infarct volume. COX-1 and PGIS proteins were increased in a time-dependent manner. Adv-COX-1/PGIS infusion selectively augmented prostacyclin levels, with reduction of other eicosanoids in ischemic cortex and a significant reduction of infarct volume, even when the rAd was administered 5 hours after ischemia. Infusion of Adv-COX-1 also increased prostacyclin, suppressed leukotriene levels, and achieved a similar degree of cerebral protection. Its neuroprotection was abrogated by treatment with a selective COX-1 inhibitor., Conclusions: COX-1/PGIS and COX-1 gene transfer reduce cerebral infarct volume by augmenting prostacyclin and suppressing leukotriene productions. COX-1-based gene transfer has potential for treating ischemic stroke.

Human platelets possess two distinct thrombin-activated receptors, PAR-1 (protease-activated receptor-1) and PAR-4, whereas human vascular smooth muscle cells possess only PAR-1. Although such thrombin receptors have been studied extensively in vitro, their physiological roles are still rather ill-defined. We have now employed a potent, selective PAR-1 antagonist, RWJ-58259, to probe the in vivo significance of PAR-1 in thrombosis and vascular injury. RWJ-58259 was examined in two thrombosis models in guinea pigs: the arteriovenous (A-V) shunt assay (monitoring thrombus weight) and the Rose Bengal intravascular photoactivation assay (monitoring time to occlusion). Administration of RWJ-58259 (10 mg/kg, total i.v. dose) did not inhibit thrombus formation in either thrombosis model, although local, intrashunt delivery in the A-V shunt model did elicit a modest antithrombotic effect (thrombus weight reduction from 35 +/- 2 to 24 +/- 4 mg). These results are consistent with the presence of more than one thrombin-sensitive receptor on guinea pig platelets, in analogy with human platelets. Indeed, we were able to establish that guinea pig platelets express three thrombin receptors, PAR-1, PAR-3, and PAR-4. We also examined RWJ-58259 in a vascular restenosis model involving balloon angioplasty in rats. Perivascular administration of RWJ-58259 (10 mg) significantly reduced neointimal thickness (77 +/- 5 microm to 45 +/- 5 microm, P < 0.05), clearly demonstrating an important role for PAR-1 in vascular injury. From these results, it is evident that a PAR-1 antagonist is not especially effective for treating platelet-dependent thrombosis; however, it could well be beneficial for treating restenosis attendant to arterial injury.

Tie-1 and Tie-2 are receptor tyrosine kinases (RTKs) that are exclusively expressed in endothelial cells and play important roles in endothelial cell biology. The authors have reported previously the temporal profiles of Tie-1 and Tie-2 mRNA expression after focal cerebral ischemia-reperfusion. In the current study, the localization of Tie-1/Tie-2 mRNA and proteins were further investigated in the same focal ischemia model. In situ hybridization showed that, after 60-minute ischemia and 72-hour reperfusion, both Tie-1 and Tie-2 mRNA appeared as capillary-like structures in the ischemic middle cerebral artery (MCA) cortex. Western blot analysis showed a biphasic expression of Tie-1 protein in the same region. The first peak, spanning the ischemic and early reperfusion period. was of low intensity and short-lived. The second peak was of greater intensity and spanning the period from 72 to 168 hours after reperfusion. Similarly, Tie-2 expression at the protein level also exhibited a biphasic pattern. Immunohistochemical studies, after 72 hours of reperfusion, showed that although Tie-1 and Tie-2 were detected within the ischemic cortex, they actually were expressed in different populations of endothelial cells in different regions. In agreement with the in situ hybridization study, Tie-1 immunoreactivity appeared as capillary-like structures in cortical layers 2 to 4. Similar capillary-like appearance of Tie-2 immunoreactivity was noted in the outer cortical layers. In addition, Tie-2 immunoreactivity also was observed in cortical layer 6b, where de novo large vessel formation was noted. Cellular colocalization experiments revealed that Tie-2 is expressed in proximity to its antagonist, Angpo-2, as well as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in cortical layer 1, where active vessel remodeling was noted. Interestingly, bFGF only partially colocalized with VEGF, suggesting differential roles for these angiogenic factors during vessel remodeling. Tie-1 protein, to a lesser degree, also colocalized with Angpo-2, bFGF, and VEGF in cortical layer 1. Magnetic resonance imaging (MRI) showed increased regional cerebral blood flow (CBF) corresponding to the expression of these angiogenesis gene products. Together, these findings suggest that the evolving expression of angiogenesis genes underlie the robust vascular remodeling after ischemia and reperfusion.

Retinoic acid (RA), a derivative of vitamin A, is essential for the normal patterning and neurogenesis during development. RA treatment induces growth arrest and terminal differentiation of a human embryonal carcinoma cell line (NT2) into postmitotic central nervous system neurons. Using RNA fingerprinting by arbitrarily primed polymerase chain reaction, we identified a novel serine/threonine-rich protein, RA-regulated nuclear matrix-associated protein (Ramp), that was down-regulated during the RA-induced differentiation of NT2 cells. Prominent mRNA expression of ramp could be detected in adult placenta and testis as well as in all human fetal tissues examined. The genomic clone of ramp has been mapped to the telomere of chromosome arm 1q, corresponding to band 1q32.1-32.2. Associated with the nuclear matrix of NT2 cells, Ramp translocates from the interphase nucleus to the metaphase cytoplasm during mitosis. During the late stage of cytokinesis, Ramp concentrates at the midzone of the dividing daughter cells. The transcript expression of ramp is closely correlated with the cell proliferation rate of NT2 cells. Moreover, overexpression of Ramp induces a transient increase in the proliferation rate of NT2 cells. Taken together, our data suggest that Ramp plays a role in the proliferation of the human embryonal carcinoma cells.

The pharmacology and clinical application of traditional Chinese medicine has been extensively documented. We have used an in vitro model system, PC12 cells, to demonstrate the presence of neuroactive compounds in Ganoderma lucidum (lingzhi). Ganoderma extract induced the neuronal differentiation of PC12 cells and prevented nerve growth factor-dependent PC12 neurons from apoptosis. Moreover, these effects of ganoderma might be mediated via the ras/extracellular signal-regulated kinase (Erk) and cAMP-response element binding protein (CREB) signaling pathways, as demonstrated by the phosphorylation of Erk1, Erk2 and CREB. Thus, our data not only present the first evidence of the presence of neuroactive compounds that mediate the neuronal differentiation and neuroprotection of the PC12 cells, but also reveal the potential signaling molecules involved in its action.

The bFGF/FGFR, VEGF/VEGFR and Angiopoietin/Tie receptor system are crucial for angiogenesis and vascular remodeling. With a rat focal cerebral ischemia model, we previously reported dramatic changes in the vascular density and angiogenesis related genes in the ipsilateral cortex after 60 minutes severe ischemia. While only a small increase in the capillary density was noted in the contralateral cortex with very mild ischemia. In the present study we further reported that only Tie-1 and VEGFR-2 mRNA were significantly changed in the contralateral cortex with a p value of 0.0001 and 0.0168, respectively, and the degree of changes were very small. Interestingly, in contrast to a huge increase in the ipsilateral cortex, Tie-1 mRNA was slowly decreased after the onset of ischemia and stayed below the basal level throughout the remaining periods studied. The mechanism and significance for this decrease is not presently clear. In contrast to the ipsilateral cortex, the Angpo-1/Angpo-2 mRNA ratio was also slightly dropped below the basal level in the contralateral side in most of the ischemia-reperfusion periods studied, which is in line with the notion that small decrease in Angpo-1/Angpo-2 mRNA ratio implied small vascular remodeling activity. It is very likely that increase in this Angpo-1/Angpo-2 ratio is crucial for remodeling into large vessels and increase in Tie-1 may be crucial for capillary density increasing. Nevertheless, the detailed mechanisms and significance of differential expression of these genes and relationship to vascular remodeling remain to be characterized.

Denervation results in a series of changes in skeletal muscle. To elucidate the molecular basis underlying these changes, it is important to identify the profile of altered gene expression in skeletal muscle following nerve injury. In the present study, we have examined the differentially expressed genes in denervated gastrocnemius muscle using RNA fingerprinting by arbitrarily primed PCR. Eight differentially expressed mRNA transcripts have been identified. A bilateral regulatory profile can be observed for the up-regulated genes in both denervated and contralateral control muscle following unilateral sciatic nerve injury. The temporal expression profiles of the denervation-regulated genes in muscle during development, together with their dependency on nerve activity, suggest potential functional roles following nerve injury in vivo. In particular, the identification of two apoptosis-related genes in denervated muscle provides molecular evidence that the apoptotic process is likely to be involved in the intricate changes that lead to muscle atrophy. Our findings not only allow the identification of novel genes, but also suggest possible functions for some known genes in muscle following nerve injury. Taken together, these findings provide important insights into our understanding of the molecular events in denervated muscle and suggest that the differentially expressed genes may play potential roles during muscle denervation and regeneration., (Copyright 2000 Academic Press.)

Retinoic acid (RA), a derivative of vitamin A, is essential for normal patterning and neurogenesis during development. Until recently, studies have been focused on the physiological roles of RA receptors (RARs), one of the two types of nuclear receptors, whereas the functions of the other nuclear receptors, retinoid X receptors (RXRs), have not been explored. Accumulating evidence now suggests that RXRalpha is a critical receptor component mediating the effects of RA during embryonic development. In this study, we have examined the expression profiles of RXRalpha and RARs during the RA-induced neuronal differentiation in a human embryonal carcinoma cell line, NT2. Distinct expression profiles of RXRalpha, RARalpha, RARbeta, and RARgamma were observed following treatment with RA. In particular, we found that RA treatment resulted in a biphasic up-regulation of RXRalpha expression in NT2 cells. The induced RXRalpha was found to bind specifically to the retinoid X response element based on gel mobility retardation assays. Furthermore, immunocytochemical analysis revealed that RXRalpha expression could be localized to the somatoaxonal regions of the NT2 neurons, including the tyrosine hydroxylase- and vasoactive intestinal peptide-positive neurons. Taken together, our findings provide the first demonstration of the cellular localization and regulation of RXRalpha expression in NT2 cells and suggest that RXRalpha might play a crucial role in the cellular functions of human CNS neurons.

The angiopoietin/Tie receptor system may contribute to angiogenesis and vascular remodeling by mediating interactions of endothelial cells with smooth muscle cells and pericytes. The temporal expression of angiopoietin-1 (Angpo-1), angiopoietin-2 (Angpo-2), Tie-1, and Tie-2 mRNA was studied in a focal cerebral ischemia model in rats. The cDNA fragments obtained from reverse transcription polymerase chain reaction amplification were cloned and used as a probe to detect individual genes. Northern blot analysis showed a delayed increase of a 4.4-kb Angpo-1 transcript for up to 2 weeks after ischemia, eightfold higher than the values of the sham-operated controls. A biphasic expression of a 2.4-kb Angpo-2 transcript was noted, peaking at 24 hours (6.4-fold) and 2 weeks (4.6-fold) after ischemia. The expression of Tie-2 mRNA (4.3 kb), a receptor for Angpo-1, and Tie-1 mRNA (4.3 kb) also increased starting 24 hours after reperfusion and remained elevated for up to 2 weeks after ischemia. The temporal profiles of the expression of these genes were different from those of other angiogenic genes such as basic fibrobast growth factor/fibroblast growth factor receptor and vascular endothelial growth factor/vascular endothelial growth factor receptor and proteolytic enzymes (tissue-type plasminogen activator and urokinase plasminogen activator) and their inhibitors (plasminogen activator inhibitor-1). The expression patterns of these genes could be related to progressive tissue liquefaction and neovascularization after ischemia in this stroke model. Differential expression of these angiogenesis genes suggests the involvement of complex regulatory mechanisms that remain to be characterized.

In the present study, we examined the temporal and spatial expression profiles of GFAP mRNA and protein in a focal cerebral ischemia model with ischemic injury confined to the cerebral cortex in the right middle cerebral artery (MCA) territory. Northern blot analysis showed a respective 5.5-fold and 7.2-fold increase in the GFAP mRNA in the ischemic right MCA cortex in rats subjected to 30-min (mild) or 60-min (severe) ischemia followed by 72-hr reperfusion. The GFAP mRNA signal remained elevated up to 2-week reperfusion. Interestingly, increased GFAP mRNA signal was clearly demonstrated for the first time in the left MCA cortex. A significant 1.5-fold and 5-fold increase was observed after 72-hr reperfusion following mild and severe ischemia, respectively. However, unlike the ischemic right MCA cortex, this induction was transient in the non-ischemic left MCA counterpart. In situ hybridization studies further revealed characteristic spatial induction profile following mild vs. severe ischemia. In mild ischemia, following 24-hr reperfusion, increase in GFAP mRNA was observed mainly within the ischemic right MCA cortex. Following 72-hr reperfusion, GFAP mRNA signal was observed in virtually the entire ischemic cortex, particularly the amygdala region, then gradually reduced and restricted to right MCA territory and subcortical thalamic nucleus following 2-week reperfusion. On the other hand, in severe ischemia, following 24-hr reperfusion increased GFAP mRNA signal was observed in area surrounding right MCA territory (infarct region) and outer cortical layers within the right MCA territory. Following 72-hr reperfusion, no signal was detected within right MCA cortex; however, increased GFAP signal was detected throughout the remaining ipsilateral cortex and subcortical region, as well as the contralateral cerebral cortex. GFAP mRNA signals then gradually reduced its intensity and was restricted to area surrounding necrosis and ipsilateral thalamic nucleus following 2-week reperfusion. GFAP-like immunoreactivity was also detected in area expressing GFAP mRNA. It is very likely that de novo synthesis was responsible for this increase. In summary, increased GFAP signal was noted in both ipsilateral and contralateral cerebral following mild and severe ischemia. Although the temporal induction profile for mild vs. severe ischemia was similar, the spatial induction profile was different. The mechanism leading to this differential induction and their physiological and functional significance are not clear at present. It is very likely that some local factors may involve, nevertheless, the detailed mechanisms remain to be fully explored.

Expression of protease-activated receptor-1 (PAR-1), a cell-surface receptor for thrombin, is increased in balloon-injured rat carotid artery and human atherosclerotic tissue. To examine the role of PAR-1 in vascular injury, we compared vascular injury responses in wild-type (WT) and PAR-1-deficient (PAR-1(-/-)) mice. Arterial injury was induced by inserting a flexible guidewire into the common carotid artery and withdrawing it 6 times with rotation. Bromodeoxyuridine, delivered subcutaneously by osmotic minipump, was used to measure cellular proliferation. Mice were perfusion-fixed at 1, 2, 5, 10, and 14 days after injury. Extensive endothelial damage, mural thrombosis, platelet adherence, and medial smooth muscle cell loss and necrosis were apparent at day 1 in both WT and PAR-1(-/-) mice. The incidence of thrombosis or platelet deposition in WT and PAR-1(-/-) mice declined from 100% at day 1 to 25% and 21%, respectively, at 14 days. Endothelial disruption, as assessed by Evan's blue uptake, was maximum at day 1 and declined by day 14. This apparent endothelial regrowth was similar in WT and PAR-1(-/-) mice. Significant medial thickening at 14 days after injury was similar in WT (from 22.8+/-1.7 to 30.7+/-1.9 microm) and PAR-1(-/-) (from 23.2+/-2.1 to 30.5+/-2.2 microm) mice. Medial area also increased in response to injury but to a lesser extent in PAR-1(-/-) mice (from 0.0250+/-0.0044 to 0.0312+/-0.0047 mm(2)) than in WT mice (from 0.0266+/-0.0040 to 0.0398+/-0.0050 mm(2)). Neointima was variable and occurred in 6 of 13 WT and 5 of 12 PAR-1(-/-) mice. However, intimal area tended to be less in PAR-1(-/-) mice (0. 0016+/-0.0007 mm(2)) compared with WT mice (0.0082+/-0.0032 mm(2)), although this difference did not achieve statistical significance (P=0.06). Cell density was significantly greater in normal carotids from PAR-1(-/-) (6.4+/-0.5 x 10(3)/mm(2)) compared with WT (4.3+/-0. 8 x 10(3)/mm(2)) mice and remained elevated after injury. Vessel and lumen diameters tended to increase in WT mice after injury, whereas vessel diameter was unchanged and lumen diameter actually decreased in PAR-1(-/-) mice. Cell proliferation in injured carotid arteries was similar in PAR-1(-/-) and WT mice. These data suggest that PAR-1(-/-) may play a role in vascular injury responses in this mouse model via possible effects on extracellular matrix regulation.

The formation of the neuromuscular junction (NMJ) involves a series of inductive interactions between motor neurons and muscle fibers. The neural signals proposed to induce the mRNA expression of acetylcholine receptors in muscle include neuregulin (NRG). In the present study, we have employed RNA fingerprinting by arbitrarily primed PCR analysis to identify the differentially expressed transcripts following NRG treatment in cultured myotubes. Nine partial cDNA fragments were isolated; the mRNA expression of eight of these genes was found to be up-regulated by NRG. The spatial and temporal expression profiles of these NRG-regulated genes in rat tissues during development suggest potential functional roles during the formation of NMJ in vivo. Our findings not only allowed the identification of novel genes, but also suggested possible functions for some known genes that are consistent with their potential roles at the NMJ. Furthermore, the identification of G-protein beta1 subunit and G-protein-coupled receptor as NRG-regulated genes has provided the first demonstration that activation of the NRG signaling pathway can induce the expression of components in the G-protein signaling cascade.

Protease-induced cell signaling is mediated by specific receptors such as the emerging family of protease activated receptors (PARs). Since proteases are involved in various aspects of vascular injury, we assessed expression of PAR-2, a protease-activated receptor closely related to the thrombin receptor (PAR-1) but activated by an unknown protease, in vascular injury. Rat carotids were subjected to balloon-catheter injury and perfusion fixed at 1, 3, 7 or 14 days after injury. Sections of injured and normal carotid arteries were immunohistochemically labeled with a polyclonal antibody raised against the N-terminal residues 37-53 of human PAR-2. Sections were also labeled with antibodies to factor VIII-related antigen, smooth muscle actin and a proliferating cell nuclear antigen (PCNA). In normal vessels, PAR-2 labeling was diffuse and patchy in medial smooth muscle and endothelium. At one and three days after injury, before appearance of neointima, PAR-2 labeling increased in cells adjacent to damaged or necrotic smooth muscle cells. In addition, proliferating adventitial myofibroblasts labeled strongly for PAR-2. At 7 and 14 days after injury, the media and neointima of injured vessels had increased PAR-2 labeling which was most intense at the luminal edge of the neointima. Double immunohistochemical labeling confirmed the greatest expression of PAR-2 in areas with the greatest density of PCNA-positive cells. In addition, PAR-2 mRNA localization using in situ hybridization paralleled PAR-2 expression. The data suggest an upregulation of PAR-2 in response to vascular injury which is associated with medial smooth muscle damage, proliferating adventitial myofibroblasts and smooth muscle cells of the neointima, particularly those at the proliferating luminal edge of the neointima. Possible functional consequences of this receptor upregulation and its role in the response to vascular injury remain to be determined.

When treated with retinoic acid (RA), a human embryonal carcinoma (EC) cell line, NTera2 cl.D/1 (NT2), differentiates into several morphologically distinct cell types, which include terminally differentiated postmitotic central nervous system (CNS) neurons. Accumulating evidence has demonstrated the significant potential of NT2 cells in studies related to cancer therapy and neurodegenerative diseases. However, preparation of enriched NT2 neurons often requires a lengthy period (ca. five weeks) and depends largely on tedious techniques similar to those used for primary neuronal cultures. Here, we report a rapid protocol for the preparation of these human CNS neurons. Using the method of cell aggregation, enriched NT2 neurons can be obtained in approximately two weeks. We also demonstrated that cell aggregation reduced the time normally required for the induction of neuronal differentiation, as revealed by the early expression of neuronal markers. The period of RA treatment could also be reduced if NT2 cells were maintained as aggregates for a sufficient period of time. Taken together, our findings demonstrated that cell aggregation promoted RA-induced neuronal differentiation of NT2 cells and provided a rapid protocol for the efficient production of NT2 neurons. The ability to produce large quantities of human CNS neurons should facilitate future use of these neurons for basic research and applications in cell therapy.

We developed mice deficient in protease-activated receptor-2 (PAR-2) or PAR-1 to explore the pathophysiological functions of these receptors. In this report, we evaluated mean arterial pressure and heart rate (HR) changes in response to PAR-1 or PAR-2 activation in anesthetized wild-type (WT), PAR-1-deficient (PAR-1(-/-)), and PAR-2-deficient (PAR-2(-/-)) mice. In WT mice, TFLLRNPNDK, a PAR-1 selective activating peptide, caused hypotension and HR decreases at 1 micromol/kg. TFLLRNPNDK also caused secondary hypertension following L-NAME pretreatment. These responses were absent in PAR-1(-/-) mice. In WT mice, SLIGRL, a PAR-2 selective activating peptide, caused hypotension without changing HR at 0.3 micromol/kg. SLIGRL did not induce hypertension following Nomega-nitrol-arginine-methyl ester-HCl (L-NAME). The response to SLIGRL was absent in PAR-2(-/-) mice. SFLLRN, a nonselective receptor activating peptide caused hypotension and HR decreases in WT mice at 0.3 micromol/kg, as well as secondary hypertension following L-NAME. SFLLRN still induced hypotension in PAR-1(-/-) mice, but HR decrease and secondary hypertension following L-NAME were absent. The hypotensive and bradycardic responses to SFLLRN and TFLLRNPNDK in PAR-2(-/-) mice were accentuated compared with WT mice. By using mouse strains deficient in either PAR-1 or PAR-2, we confirmed the in vivo specificity of TFLLRNPNDK and SLIGRL as respective activating peptides for PAR-1 and PAR-2, and the distinct hemodynamic responses mediated by activation of PAR-1 or PAR-2. Moreover, the accentuated response to PAR-1 activation in PAR-2-deficient mice suggests a compensatory response and potential receptor cross-talk.

Vascular expression and cellular functions of the thrombin receptor (PAR-1) and protease activated receptor 2 (PAR-2) suggest similar but distinct vascular regulatory roles. The vascular actions of PAR-1 and PAR-2 in vivo were differentiated by monitoring mean arterial pressure (MAP) and heart rate (HR) of anesthetized mice in response to intravenous SFLLRN (0.1, 0.3, and 1 mumol/kg) and SLIGRL (0.1, 0.3, and 1 mumol/kg), the respective receptor-activating sequences for PAR-1 and PAR-2, and TFLLRNPNDK (0.3, 1, and 3 mumol/kg), a synthetic peptide selective for PAR-1. All peptides dose dependently decreased MAP (order of potency: SLIGRL > SFLLRN > TFLLRNPNDK). SLIGRL induced a more prolonged hypotension with a slow return to baseline, whereas SFLLRN- and TFLLRNPNDK-induced hypotension was followed by a rapid return towards baseline and a sustained moderate hypotension. SFLLRN and TFLLRNPNDK, but not SLIGRL, decreased HR. N omega-Nitro-L-arginine methyl ester HCl (L-NAME), an inhibitor of nitric oxide synthesis, attenuated the cumulative hypotensive response to SLIGRL but had no effect on the SFLLRN and TFLLRNPNDK hypotension. However, L-NAME revealed a rebound hypertension in response to SFLLRN and TFLLRNPNDK but not SLIGRL. In conclusion, activation of either PAR-1 or PAR-2 in vivo results in hypotension. In addition, only PAR-1 activation induced hypertension following L-NAME, reflecting concurrent PAR-1-mediated vasoconstriction. Thus, these different hemodynamic responses in vivo suggest distinct physiological or pathophysiological roles for PAR-1 and PAR-2 in local vascular regulation.

Retinoic acid (RA) induced the terminal differentiation of a human embryonal carcinoma cell line (NT2/D1) into several morphologically distinct cell types, including the postmitotic CNS neurons. Although RA has been suggested to play an important role in brain development, little is known about the molecular mechanism by which RA induces neuronal differentiation. In the present study, RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) was used to identify the transcripts in NT2/D1 cells that were differentially regulated by RA. Northern blot analysis of the differentially amplified PCR fragments revealed 11 genes that were regulated by RA. Of these, seven were up-regulated and four were down-regulated along the course of RA treatment. More importantly, four of the RA-regulated genes that were identified in the present study are novel. Our findings suggested that there are a number of RA-regulated genes that have yet to be identified. RAP-PCR provides a useful tool for studying the patterns of transcript expression during the course of RA treatment and allows the cloning of novel genes involved in the process of neuronal differentiation. Furthermore, it provides a basis for the selection of genes that are involved in the RA-induced signaling pathway in the human CNS.

To explore the potential involvement of neurotrophins in the actions of retinoic acid (RA) on leukaemia differentiation, we examined the ability of RA to regulate the expression of neurotrophins and Trk receptors in several leukaemia cell lines. Expression of TrkA was dramatically induced by RA at both the mRNA and protein level in leukaemia cell lines K562 and KG-1. Furthermore, while no expression of trkB and trkC was detected, constitutive expression of nerve growth factor (NGF), neurotrophin-3 and neurotrophin-4/5 could be detected in leukemia cells. Our findings suggested that NGF/trkA may potentially be involved in the RA-induced differentiation of leukemia cells.

Thrombin's cellular actions are mediated by a novel G-protein coupled transmembrane receptor. We infused SFLLRN, a peptide that directly activates thrombin receptors, into the left circumflex coronary artery (CFX) of anesthetized dogs to evaluate the cardiovascular effects of thrombin receptor activation in vivo. Intracoronary SFLLRN, 0.9, 9 and 90 nmol/min, produced transient, dose-related increases in CFX blood flow, followed by sustained decreases in CFX and left anterior descending (LAD) blood flow. SFLLRN also decreased positive and negative dP/dtmax, arterial pressure, cardiac output and heart rate. Peripheral vascular resistance transiently decreased and then increased. SFLLRN decreased systolic wall thickening (WT) and increased ST segment level within the CFX perfusion area. In contrast, WT was increased, and ST segment was unchanged in the LAD perfusion area. CFX flow, but not LAD flow, increased transiently above control after SFLLRN infusion. FSLLRN, a peptide that does not activate thrombin receptors, had no effect at 90 nmol/min. The response to intravenous SFLLRN was greatly attenuated when compared with intracoronary infusion, and regional changes in coronary flow and function were absent. Decreases in arterial pressure, heart rate, coronary blood flow, and positive and negative dP/dtmax, were inhibited after bilateral vagotomy. Moreover, arterial pressure and peripheral resistance increased in response to SFLLRN after vagotomy. Initial CFX flow increase, regional dysfunction, ST level changes and hyperemic response were comparable but attenuated after vagotomy. Ex vivo platelet function was not affected by SFLLRN up to 100 microM. We conclude that regional myocardial ischemia and cardiac dysfunction result from thrombin receptor-mediated local coronary vasoconstriction. Thus, thrombin generation at a site of vascular injury or thrombus may significantly affect vascular tone and myocardial perfusion.

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.

alpha-Thrombin alters vascular tone via a cell surface receptor. We used isolated guinea pig hearts perfused with buffer at constant flow to assess the effects of thrombin-receptor activation on coronary perfusion pressure, left ventricular function, and electrocardiogram. alpha-Thrombin produced concentration-dependent (0.03-1 U/ml), transient decreases in perfusion pressure followed by sustained increases. Concurrently, alpha-thrombin markedly reduced ventricular function. SFLLRN, a peptide that directly activates thrombin receptors, had qualitatively similar effects, except that it was less potent (0.1-30 microM). FSLLRN, a structurally similar peptide that does not activate thrombin receptors, had no effect. alpha-Thrombin and SFLLRN also changed S-T segment level and T-wave morphology. Previous alpha-thrombin exposure markedly inhibited the response to a alpha-thrombin but only moderately attenuated the response to SFLLRN. However, previous SFLLRN exposure did not alter subsequent response to alpha-thrombin or SFLLRN. Pretreatment with hirudin (3 U/ml), an inhibitor of thrombin's proteolytic action, prevented alpha-thrombin but not SFLLRN responses. Cromakalim (0.5 microM), a coronary vasodilator, reversed the effects of alpha-thrombin and SFLLRN on ventricular function, suggesting that depression of ventricular function resulted, in part, from vasoconstriction-induced myocardial perfusion deficit. Our results show that alpha-thrombin at physiologically relevant concentrations, has marked effects on coronary vascular resistance and ventricular function in isolated guinea pig hearts that are mediated by the proteolytically activated thrombin receptor.

Retinoic acid induced the differentiation of human embryonal carcinoma cells (NT2/D1) into several morphologically distinct cell types, including those resembling terminally differentiated postmitotic CNS neurones. The mechanism by which retinoic acid influences the process of neuronal differentiation in the CNS, however, remains unknown. In the present study, we have examined the ability of retinoic acid to induce the expression of the receptors that mediate the actions of the neurotrophins, using reverse transcription-polymerase chain reaction and Northern blot analysis. Our study demonstrated that the expression of mRNAs for three human trk receptors was significantly induced after treatment with retinoic acid. These findings suggest that the actions of retinoic acid on neuronal differentiation in the human CNS may potentially be mediated by the neurotrophins.