GPR35 as a Novel Therapeutic Target.

G protein-coupled receptors (GPCRs) remain the best studied class of cell surface receptors and the most tractable family of proteins for novel small molecule drug discovery. Despite this, a considerable number of GPCRs remain poorly characterized and in a significant number of cases, endogenous ligand(s) that activate them remain undefined or are of questionable physiological relevance. GPR35 was initially discovered over a decade ago but has remained an "orphan" receptor. Recent publications have highlighted novel ligands, both endogenously produced and synthetic, which demonstrate significant potency at this receptor. Furthermore, evidence is accumulating which highlights potential roles for GPR35 in disease and therefore, efforts to characterize GPR35 more fully and develop it as a novel therapeutic target in conditions that range from diabetes and hypertension to asthma are increasing. Recently identified ligands have shown marked species selective properties, indicating major challenges for future drug development. As we begin to understand these issues, the continuing efforts to identify novel agonist and antagonist ligands for GPR35 will help to decipher its true physiological relevance; translating multiple assay systems in vitro, to animal disease systems in vivo and finally to man.

A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy.

OBJECTIVES: Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons during early or postnatal development. Severity is variable and is inversely related to the levels of survival of motor neurons (SMN) protein. The aim of this study was to produce a two-site ELISA capable of measuring both the low, basal levels of SMN protein in cell cultures from patients with severe SMA and small increases in these levels after treatment of cells with drugs. METHODS: A monoclonal antibody against recombinant SMN, MANSMA1, was selected for capture of SMN onto microtiter plates. A selected rabbit antiserum against refolded recombinant SMN was used for detection of the captured SMN. RESULTS: The ratio of SMN levels in control fibroblasts to levels in SMA fibroblasts was greater than 3.0, consistent with Western blot data. The limit of detection was 0.13 ng/mL and SMN could be measured in human NT-2 neuronal precursor cells grown in 96-well culture plates (3 x 10(4) cells per well). Increases in SMN levels of 50% were demonstrable by ELISA after 24 hours treatment of 10(5) SMA fibroblasts with valproate or phenylbutyrate. CONCLUSION: A rapid and specific two-site, 96-well ELISA assay, available in kit format, can now quantify the effects of drugs on survival of motor neurons protein levels in cell cultures.

Predictors of quality of life following stroke.

PURPOSE: To determine the factors predicting quality of life during the course of rehabilitation following stroke. METHOD: Two hundred and fifteen stroke patients aged 41 93 were studied over a period of three months. Measurement of quality of life, functional ability, social support. demographic and treatment data were taken on admission to the rehabilitation hospital, at two weeks and three months. The instruments used were the Sickness Impact Profile (SIP), the Modified Barthel Index (MBI) and The Social Support Questionnaire, short form (SSQ6). RESULTS: Length of stay, previous stroke, functional ability and social support were found to be significantly correlated with quality of life. Stepwise multiple regression analysis indicated that functional ability, psychological and physical SIP dimensions, social support satisfaction at two weeks and previous stroke explained 47% of the variance in sickness impact at three months following stroke. The factors predicting 53% of the variance in sickness impact at two weeks were baseline functional ability, psychological and physical SIP. CONCLUSIONS: The findings indicate that both psychosocial and physical factors are important in predicting quality of life in stroke rehabilitation. Determining such predictors at an early stage will help to guide clinical decisions throughout rehabilitation.

Neuronal apoptosis inhibitory protein expression after traumatic brain injury in the mouse.

Apoptosis of brain cells is triggered by traumatic brain injury (TBI) and is blocked by caspase inhibitors. The neuronal apoptosis inhibitor protein (NAIP), which has been shown to inhibit apoptosis by both caspase-dependant and caspase-independent mechanisms, is neuroprotective in rat models of cerebral ischemia and axotomy. In order to gain a better appreciation of CNS apoptosis following head injury in general and the possible involvement of NAIP specifically, we have configured a mouse model of TBI. In addition to demonstrating apoptosis, the spatiotemporal expression or levels of a number of proteins with apoptosis modulating effects have been determined. Apoptosis of neurons and oligodendrocytes following TBI was observed in brain sections which were triple-stained with in situ end labeling, bisbenzimide and immunofluorescent stain for neuron specific nuclear protein and myelin-associated glycoprotein, respectively. Further evidence for apoptosis following TBI in this model was obtained in brain samples using ligation-mediated PCR amplification of DNA fragments and gel electrophoresis. The temporal profile of apoptosis was similar to the temporal profile of microglial activation determined by CD11b staining and TNFa expression induced by TBI. NAIP staining in sections of cerebral cortex and subcortical white matter increased at 6 h and decreased towards control levels at 24 h post-TBI. Temporal changes in the expression of NAIP were also observed using Western blot analysis of brain samples removed from injured cortex and sub-cortical white matter. At the time that NAIP expression decreased markedly (24 h post-TBI), procaspase-3 levels also decreased, PARP cleavage increased, and the highest levels of apoptosis were observed. These findings have implications in our understanding of traumatically induced programmed cell death and may be useful in the configuration of therapies for this common injury state.

Needs of Chinese families of critically ill patients.

Critical illness and subsequent hospitalization are stressful for patients and their family members. The purpose of this descriptive study was to identify the family members' perceptions of their immediate needs within 48 to 96 hours following admission of a relative to a critical care unit in Hong Kong and to compare their perceptions with the critical care nurses' perceptions of the family needs. A convenience sample of 37 Chinese family members and 45 registered nurses completed a self-report Chinese version of the Critical Care Family Need Inventory. The 10 most important and 10 least important family needs were identified by family members and by nurses and the results were compared. Conclusions were drawn about the implications for nurses in planning and implementing quality family-centered care for critically ill patients.

Needs of families with a relative in a critical care unit in Hong Kong.

The aim of this study is to explore family members' perceptions of their immediate needs following admission of a relative to a critical care unit in Hong Kong. A convenience sample of 30 family members was drawn from those available during the first 96 hours of hospitalization of their relative. Self-reported questionnaires, consisting of a demographic data sheet, a modified Chinese version of the 45-item Critical Care Family Needs Inventory (CCFNI) and semistructured interviews, are the instruments used to examine family members' perceptions of need importance and to ascertain whether or not these needs are met. Doctors and nurses are identified as the most suitable people to meet most immediate family needs. Conclusions are drawn as to the best focus of nursing interventions in order to provide quality care to patients and families.

IAP family proteins delay motoneuron cell death in vivo.

Neuronal apoptosis inhibitory protein (NAIP), and human inhibitors of apoptosis 1 and 2 (HIAP1 and HIAP2) are three members of the mammalian family of antiapoptosis proteins called 'inhibitors of apoptosis' (IAP). These molecules can prevent apoptosis in vitro and the over-expression of NAIP can decrease ischemic damage in the hippocampus. The goal of our experiments was to determine whether administration of NAIP, HIAP1 and HAIP2 could rescue motoneurons following axotomy of a peripheral nerve. In young rats, an adenoviral gene transfer technique was used to deliver and express these proteins in motoneurons; a fluorescent tracer was simultaneously added as a means for quantitatively assessing the rescue of fluorescently labelled motoneurons in serial sections of the lumbar spinal cord. Control experiments using adenoviral vectors (adv) expressing the lacZ gene showed that 14% of the sciatic motoneuron pool could be transfected indicating the existence of a subpopulation of spinal motoneurons susceptible to this class of viral vectors. The administration of an adv-NAIP, adv-HIAP1 and adv-HIAP2 rescued 30-40% of motoneurons at one week after sciatic axotomy. The efficiency of these proteins was similar to that of two neurotrophic factors, ciliary neurotrophic factor and brain-derived neurotrophic factor, administrated by the same viral technique. The effect of the IAP proteins on motoneuron survival decreased with time but was still present after 4 weeks postaxotomy; the duration of the response was dependent upon the viral titre. These experiments demonstrate that IAP family proteins can prevent motoneuron cell death in vivo and may offer a new therapeutic approach for motoneuron diseases.

The hippocampal neurons of neuronal apoptosis inhibitory protein 1 (NAIP1)-deleted mice display increased vulnerability to kainic acid-induced injury.

The neuronal apoptosis inhibitory protein (NAIP) is a member of a novel family of inhibitor of apoptosis (IAP) proteins. The IAP genes are highly conserved from baculovirus to metazoans and suppress apoptosis induced by a variety of triggers both in vitro and in vivo. Here we describe the generation and characterization of mice with the targeted deletion of NAIP1. We demonstrate that the NAIP1-deleted mice develop normally. However, the survival of pyramidal neurons in the hippocampus after kainic acid-induced limbic seizures is greatly reduced in the NAIP1 knock-out animals. Thus, although NAIP1 is not necessary for normal development of murine central nervous system, the endogenous NAIP1 is required for neuronal survival in pathological conditions.

Chinese elderly patients' perceptions of their rehabilitation needs following a stroke.

Stroke is the third leading cause of death and disability among Chinese elderly patients in Hong Kong and yet the rehabilitation needs of these patients are rarely explored. The aim of this study was to identify the rehabilitation needs of Chinese elderly patients following a stroke. The study adopted an ethnographic approach, information being gathered by the researcher through interviews with 15 key informants selected by purposive sampling. The perceptions of patients as to their own needs were sought at three stages of recovery - in the acute and rehabilitation settings and at 1 month following discharge. Ethical approval was gained from the Chinese University Faculty of Medicine ethical committee and access agreed by the hospital authorities. Verbal approval was gained from the patients before each interview, following confirmation of the voluntary nature of participation and assurance of confidentiality and anonymity. The researcher's role was also clearly stated. Analysis of the interview data produced five categories of patient need at the three stages of recovery, namely informational, physical, psychological, social and spiritual. The most frequently stated, but largely unmet, need in all settings was the need for information, particularly information about the reasons for stroke and about the activities that promote recovery. In the acute and rehabilitation settings patients' responses indicated a need to be respected as individuals, to be addressed by name and to be provided with privacy. Although the Barthel Index administered during interviews charted recovery at different rates, nurses did not always make links between the level of functional ability and the help needed with physical tasks. They also failed to recognize the relationship between physical and psychological needs and the equal importance of both in recovery from stroke. As Chinese elderly patients tend to take a passive role in seeking help and information, nurses play a significant role in the identification of individual rehabilitation needs. Implications for nursing practice are discussed.

Spinal muscular atrophy: molecular pathophysiology.

Spinal muscular atrophy is an autosomal recessive disease characterized by motor neurone loss, muscle atrophy and weakness. Deletion or mutation of the SMN1 gene reduces intracellular survival motor neurone protein levels causes spinal muscular atrophy, most likely by interfering with spliceosome assembly. A range of clinical severity and corresponding survival motor neurone levels is seen because of the presence of copies of the transcriptionally inefficient SMN2 gene and possibly other modifying genes. The delineation of SMN1 as the gene that causes spinal muscular atrophy and the identification of genes that modify spinal muscular atrophy raise the prospect of gene therapy or in-vivo gene activation treatment for this frequently fatal disorder.

A small interstitial deletion in the GPC3 gene causes Simpson-Golabi-Behmel syndrome in a Dutch-Canadian family.

Deletions in the heparan sulphate proteoglycan encoding glypican 3 (GPC3) gene have recently been documented in several Simpson-Golabi-Behmel syndrome (SGBS) families. However, no precisely defined SGBS mutation has been published. We report here a 13 base pair deletion which causes a frameshift and premature termination of the GPC3 gene in the Dutch-Canadian SGBS family in whom the trait was originally mapped. Our analysis shows that a discrete GPC3 disabling mutation is sufficient to cause SGBS. Furthermore, our finding of a GPC3 normal daughter of an SGBS carrier with skeletal abnormalities and Wilms tumour raises the possibility of a trans effect from the maternal carrier in SGBS kindreds.

Adenovirus-mediated gene transfer of inhibitors of apoptosis protein delays apoptosis in cerebellar granule neurons.

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.

Neuronal apoptosis inhibitory protein (NAIP)-like immunoreactivity in brains of adult patients with Down syndrome.

In Down syndrome (DS), enhanced apoptosis (programmed cell death) may play a role in the pathogenesis of characteristic early mental retardation and precocious neurodegeneration of Alzheimer-type. The human IAP (inhibitor of apoptosis proteins) genes (NAIP, c-IAP-2/HIAP-1, c-IAP-1/Hiap-2, XIAP, survivin) are an evolutionary conserved family of proteins which prevent cell death across species, implying that they act at a central, highly conserved point in the cell death cascade. Evidence for downregulation of NAIP-mRNA in fetal DS (23rd week of gestation), as found by subtractive hybridization technique challenged studies at the protein level in adult DS brain specimen. NAIP-like immunoreactivity was determined in four different regions of cerebral cortex and cerebellum in 9 adult DS patients with Alzheimer-like neuropathologic lesions, 9 Alzheimer disease (AD) patients as compared to 9 controls. For the first time, NAIP-IR could be demonstrated in different cortical regions of the human brain. Compared to control subjects, western blotting demonstrated significantly decreased levels in parietal and occipital cortex in DS and in frontal and occipital cortex in AD. While the mode of NAIP action is unknown, inhibition of certain caspases has already been demonstrated for other IAP-family members (c-IAP1, c-IAP2 and XIAP). Although decreased NAIP-IR of certain brain regions in DS and AD awaits further confirmation, the results suggest that alterations of apoptosis regulatory (inhibitory) proteins may be another feature of neurodegeneration in DS and AD.

State self-esteem following stroke.

BACKGROUND AND PURPOSE: Physical rehabilitation after stroke is often highlighted in the absence of consideration of psychosocial factors. This study sought to determine the relationship between state self-esteem and functional independence in patients recovering from stroke. METHODS: In a longitudinal study, data were collected from 152 stroke patients within 48 hours of admission to a rehabilitation hospital and at 2 weeks and 3 months after admission. The Modified Barthel Index was used to assess functional ability. Patients' current feelings of self-worth were assessed with use of the State Self-Esteem Scale. Additional variables included perceived social support, trait self-esteem, age, previous stroke, side of stroke, comorbidity, marital status, and gender. RESULTS: State self-esteem was significantly correlated to functional independence. The results of linear stepwise regression analysis indicated that functional ability and state self-esteem at 2 weeks, as well as the presence of heart disease, were significant predictors (55%) of functional ability at 3 months. For those with a functional ability score of >/=81 on admission to the rehabilitation unit, state self-esteem and functional ability at 2 weeks as well as previous stroke explained 53% of the variance in functional ability at 3 months. When functional ability was

Caring for relatives with serious mental illness at home: the experiences of family carers in Hong Kong.

This article describes a study recently carried out in Hong Kong. The aim of the study was to gain an understanding of the experiences of Chinese family carers in giving care to a relative with serious mental illness at home. Eight primary carers were selected, and data were collected through audiotaped, semistructured interviews in Cantonese. Five main categories emerged from the data: emotional impact, coping and adaptation, psychosocial effect, social support needs, and perceptions of mental illness and mental health services. Conclusions highlight the need for family-oriented mental health services to be developed and for further research to identify the specific nursing interventions that are effective in helping to sustain family caregiving in Chinese families.

Community nurses' assessment of the needs of Hong Kong family carers who are looking after stroke patients.

This research aimed to identify the current practice of community nurses in Hong Kong in assessing stroke patients and their carers, and to pilot a scale for inclusion in the routine assessment of Chinese carers and their families. Qualitative and quantitative methods were used. In Phase 1, information was gained about the current practice of Community Nurses in assessing the needs of carers, and appropriate areas for inclusion in a scale for assessing those needs were identified. In Phase 2, the Carer Assessment Scale was completed by 14 Community Nurses and subsequently administered by a research assistant independently to the carers themselves. The perceptions of the two groups of respondents were compared, agreements and differences noted, and conclusions drawn, as to the real needs of carers.