Comparison of the efficacy and level of adherence for morning versus evening versus before bedtime administration of simvastatin in hypercholesterolemic patients.

BACKGROUND: Simvastatin is usually taken in the evening due to the circadian rhythm of hepatic cholesterol biosynthesis. The degree of reduction of low-density lipoprotein cholesterol (LDL-C) and the level of adherence to different administration time remained unknown in the Malaysian population. This study aims to investigate the effect of simvastatin on the percentage changes of lipid profile and the level of adherence to when simvastatin was instructed to be taken at different timing. METHODS: Nine primary care health clinics across Malaysia participated in this study. 147 statin-naive subjects were selected through convenient sampling and randomised into one of the three arms (after breakfast, after dinner or before bedtime). Differences on percentage reduction of LDL-C from baseline and level of adherence among the three groups at week-16 were compared. The main outcomes measured in this study were the percentage change of lipid parameters and the percentage of high-adherence (MMAS=8) at week-16. RESULTS: 59.2% of the patients were male. The mean age of the study population was 53.93± 10.85 years. Most of the patients were Malays (69.4%); followed by Indians (22.4%) and Chinese (8.2%). LDL-C decreased from 4.26 (Standard Deviation, SD1.01) to 2.36 (SD0.69)mmol/L at week-16 for patients taking simvastatin before bedtime; an absolute reduction of 44.95%.The differences of LDL-C percentage reduction between three arms were significantly different (p<0.001). The greatest LDL-C reduction was observed when simvastatin was taken before bedtime and revealed 56.2% patients with high-adherence at week-16. CONCLUSION: Simvastatin showed superior LDL-reduction and higher level of adherence when being instructed to be taken before bedtime.

Avian influenza A H7N9 virus infects human astrocytes and neuronal cells and induces inflammatory immune responses.

Seasonal, pandemic, and avian influenza virus infections may be associated with central nervous system pathology, albeit with varying frequency and different mechanisms. Here, we demonstrate that differentiated human astrocytic (T98G) and neuronal (SH-SY5Y) cells can be infected by avian H7N9 and pandemic H1N1 viruses. However, infectious progeny viruses can only be detected in H7N9 virus infected human neuronal cells. Neither of these viral strains can generate infectious progeny virus in human astrocytes despite replication of viral genome was observed. Furthermore, H7N9 virus triggered high pro-inflammatory cytokine expression, while pandemic H1N1 virus induced only low cytokine expression in either brain cell type. The experimental finding here is the first data to demonstrate that avian H7N9 virus can infect, transcribe, and replicate its viral genome; induce cytokine upregulation; and cause cytopathic effects in human brain cells, which may potentially lead to profound central nervous system injury. Observation for neurological problems due to H7N9 virus infection deserves further attention when managing these patients.

Molecular characterization of glycation-associated skin ageing: an alternative skin model to study in vitro antiglycation activity of topical cosmeceutical and pharmaceutical formulations.

BACKGROUND: Glycation is a nonenzymatic reaction that cross-links a sugar molecule and protein macromolecule to form advanced glycation products (AGEs) that are associated with various age-related disorders; thus glycation plays an important role in skin chronological ageing. OBJECTIVES: To develop a novel in vitro skin glycation model as a screening tool for topical formulations with antiglycation properties and to further characterize, at the molecular level, the glycation stress-driven skin ageing mechanism. METHODS: The glycation model was developed using human reconstituted full-thickness skin; the presence of Nε -(carboxymethyl) lysine (CML) was used as evidence of the degree of glycation. Topical application of emulsion containing a well-known antiglycation compound (aminoguanidine) was used to verify the sensitivity and robustness of the model. Cytokine immunoassay, quantitative real-time polymerase chain reaction and histological analysis were further implemented to characterize the molecular mechanisms of skin ageing in the skin glycation model. RESULTS: Transcriptomic and cytokine profiling analyses in the skin glycation model demonstrated multiple biological changes, including extracellular matrix catabolism, skin barrier function impairment, oxidative stress and subsequently the inflammatory response. Darkness and yellowness of skin tone observed in the in vitro skin glycation model correlated well with the degree of glycation stress. CONCLUSIONS: The newly developed skin glycation model in this study has provided a new technological dimension in screening antiglycation properties of topical pharmaceutical or cosmeceutical formulations. This study concomitantly provides insights into skin ageing mechanisms driven by glycation stress, which could be useful in formulating skin antiageing therapy in future studies.

A comparison of dabigatran and warfarin for stroke prevention in elderly Asian population with nonvalvular atrial fibrillation: An audit of current practice in Malaysia.

INTRODUCTION: Atrial fibrillation (AF) is the most common cardiac arrhythmia with significant morbidity and mortality in relation to thromboembolic stroke. Our study aimed to evaluate the safety and efficacy of dabigatran in stroke prevention in elderly patient with nonvalvular AF with regard to the risk of ischemic stroke and intracranial haemorrhage (ICH) in real-world setting. METHODS: A retrospective cohort study of 200 patients on dabigatran and warfarin from January 2009 till September 2016 was carried out. Data were collected for 100 patients on dabigatran and 100 patients on warfarin. RESULTS: The mean follow-up period was 340.7±322.3 days for dabigatran group and 410.5±321.2 days for warfarin group. The mean time in therapeutic range (TTR) was 52±18.7%. The mean CHA2DS2 -VASc score for dabigatran group was 4.4±1.1 while 5.0±1.5 for warfarin group. None in dabigatran group experienced ischemic stroke compared to one patient in warfarin group (p=0.316). There was one patient in dabigatran group suffered from ICH compared to none in warfarin group (p=0.316). Four patients in warfarin group experienced minor bleeding, while none from dabigatran group (p=0.043). CONCLUSION: Overall bleeding events were significantly lower in dabigatran group compared to warfarin group. In the presence of suboptimal TTR rates and inconveniences with warfarin therapy, non-vitamin-K antagonist oral anticoagulants (NOAC) are the preferred agents for stroke prevention in elderly Asian patients for nonvalvular AF.

Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

BACKGROUND: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis. METHODS: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro. RESULTS: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar. CONCLUSION: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

Probiotic supplementation in the first 6 months of life in at risk Asian infants--effects on eczema and atopic sensitization at the age of 1 year.

BACKGROUND: The role of probiotics in allergy prevention remains uncertain but has been shown in some studies to have a possible protective effect on eczema. OBJECTIVE: We aimed to assess the effect of probiotic supplementation in the first 6 months of life on eczema and allergic sensitization at 1 year of age in Asian infants at risk of allergic disease. METHODS: A double-blind, placebo-controlled randomized clinical trial involving 253 infants with a family history of allergic disease was carried out. Infants received at least 60 mL of commercially available cow's milk formula with or without probiotic supplementation [Bifidobacterium longum (BL999) 1 x 10(7) colony forming unit (CFU)/g and Lactobacillus rhamnosus (LPR) 2 x 10(7) CFU/g] daily for the first 6 months. Clinical evaluation was performed at 1, 3, 6 and 12 months of age, with serum total IgE measurement and skin prick tests conducted at the 12-month visit. The primary and secondary end-points were eczema and allergen sensitization, respectively. RESULTS: The incidence of eczema in the probiotic (22%) group was similar to that in the placebo group (25%) (P=0.53). The median Scoring Atopic Dermatitis score at 12 months was 17.10 (9.74) in the probiotic group and 11.60 (8.40) in the placebo group (P=0.17). The prevalence of allergen sensitization showed no difference (probiotic=24% vs. placebo=19%, P=0.26). The total IgE geometric mean (95% confidence interval) was 18.76 (12.54-24.98) kU/L in the probiotic group and 23.13 (16.01-30.24) kU/L in the placebo group (P=0.15). Atopic eczema (with sensitization) in the probiotic (7.3%) group was comparable to the placebo group (5.8%) (P=0.86). CONCLUSION: Early life administration of a cow's milk formula supplemented with probiotics showed no effect on prevention of eczema or allergen sensitization in the first year of life in Asian infants at risk of allergic disease. Further work is needed to determine whether timing of supplementation, dose and probiotic strain are important considerations.

Homozygous alpha-thalassemia in a growth retarded, non-hydropic premature newborn.

A newborn with homozygous alpha-thalassemia presented with intrauterine growth retardation and presumed persistent pulmonary hypertension. He also had moderate anemia, hepatomegaly and hypospadias. Correlating the newborn's clinical presentation with an underlying cause of anemia was helpful for early diagnosis. Prenatal blood tests must include red cell indices and a mean corpuscular volume value below 80 fL should prompt thalassemia screening in an at-risk population.

Laparoscopic-assisted colon and rectal surgery - lessons learnt from early experience.

INTRODUCTION: Current evidence shows that laparoscopic bowel surgery is associated with a lower incidence of postoperative ileus, lower postoperative pulmonary and wound complication rates, shorter hospital stays and a quicker return to activity than open surgery. This paper aims to report our early experience with laparoscopic-assisted colorectal procedures in our Centre for Advanced Laparoscopic Surgery and the important lessons we have learnt from this. MATERIALS AND METHODS: All laparoscopic-assisted colon and rectal surgical (LAC) procedures performed between January 2000 and December 2003 were reviewed. Clinical and operative records of these patients were reviewed. Data retrieved included patient demographics, selected intraoperative parameters, and postoperative outcomes. In order to provide a comparable reference, an equal number of matched open procedures over the same period were accrued and similarly analysed. All patients were managed on a standard carepath. All data were entered into a database and analysed using a statistical software package. RESULTS: Forty-two laparoscopicassisted colorectal procedures were performed from June 2000 to December 2003. A similar number of diagnosis-matched patients with open colorectal procedures were used as comparison. The diagnoses included cancer (68.5% versus 73.8%), diverticulosis (5.7% versus 9.5%) and polyps (14.3% versus 9.5%). Seven were converted to open surgery because of bleeding, adhesions and locally advanced disease. Laparoscopic-assisted procedures performed included 1 right hemicolectomy, 5 left hemicolectomies, 9 anterior resections, 1 abdominal-perineal resection, 3 sigmoid colectomies, 11 colostomies and 1 Hartmann's procedure. Mean perioperative time (146 min versus 125 min, P = 0.173) was comparatively longer. Mean duration for analgesic requirement (2.25 days versus 2.64 days, P = 0.05), mean length of stay (5.31 days versus 9.07 days, P < 0.05), mean time to commencement of diet (2.91 days versus 4.05 days, P < 0.001) and mean time to first bowel movement (2.57 days versus 4.10 days, P < 0.001) were all comparatively shorter. General morbidity rates (17.1% versus 21.4%, P = 0.35) were lower. No local wound complications were found in our laparoscopic-assisted group. Patients who had undergone open surgery instead of the planned laparoscopic-assisted procedures fared more poorly. CONCLUSIONS: Laparoscopic-assisted colorectal procedures performed in well-selected patients are associated with shorter hospital stays, quicker return of bowel function and lower morbidity when compared to the matched open procedures. Early experience should be acquired from performing technically simple procedures in patients with benign conditions before progressing to definitive resections in those with cancer.

NG108-15 cells express neuregulin that induces AChR alpha-subunit synthesis in cultured myotubes.

A cholinergic neuroblastoma x glioma hybrid cell line NG108-15 is able to form functional synapses, and contains both AChR-aggregating and AChR-inducing activities when cocultured with myotubes. Several lines of evidence indicate that the AChR-inducing activity of NG108-15 cells is derived from neuregulin. The conditioned medium of cultured NG108-15 cells induced the expression of AChR alpha-subunit as well as the tyrosine phosphorylation of erbB-3 receptor. NG108-15 cells expressed neuregulin with a protein of approximately 100 kDa in size and transcripts of approximately 6.8 kbp, approximately 2.6 kbp and approximately 1.8 kbp; mRNAs encoding beta1 and alpha2 isoforms of neuregulin were revealed. NG108-15 cells were induced to differentiate by chemicals, and the chemical-induced differentiation of NG108-15 cells increased the level of neuregulin mRNA expression approximately 3-fold while the expression of a housekeeping gene remained relatively unchanged. The activity of neuregulin in the conditioned medium of NG108-15 cells was reduced by treating the medium with heparin and anti-neuregulin antibody. In addition, NG108-15 cells were transfected with antisense neuregulin cDNA and its expression of neuregulin was reduced, while its neuregulin-induced tyrosine phosphorylation activity was markedly decreased. This is the first direct demonstration that the NG108-15 cell-induced AChR upregulation on cultured myotubes is mediated by neuron-derived neuregulin.

The EGF-like domain of chick acetylcholine receptor-inducing activity (ARIA) contains its full biological activity.

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR) on cultured myotubes. cDNA encoding ARIA contains different domains and the functions of each domain in ARIA activity are not known. We used molecular genetic methods to construct a chimeric fusion protein, designated ARIA(S136-K205)-Fc, that contained the leader sequence, the EGF-like domain of chick ARIA (S136 to K205) and the Fc region of human immunoglobulin. The ARIA(S136-K205)-Fc cDNA was transfected into HEK 293 cells and stable cell lines secreting soluble ARIA(S136-K205)-Fc were obtained. The secreted ARIA(S136-K205)-Fc has a molecular mass of approximately 60 kDa and can be purified by protein G chromatography. The purified ARIA(S136-K205)-Fc retained its full biological activity of chick ARIA that included: (i) induction of tyrosine phosphorylation of erbB 3 receptor in C2C12 myotubes; and (ii) approximately 12-fold stimulation of AChR alpha-subunit mRNA synthesis when applied onto cultured chick myotubes. This Fc-tagged ARIA could be rapidly purified and provides a very useful ligand for identifying its true receptor(s) on muscle cell surface.

Expression and cellular localization of COX-1 and -2 in Helicobacter pylori gastritis.

BACKGROUND: There are conflicting reports on the expression of cyclooxygenase in Helicobacter pylori infection. AIM: To evaluate the expression of COX-1 and COX-2 in H. pylori gastritis at messenger RNA (mRNA) and protein levels. METHODS: Endoscopic gastric biopsies were obtained from patients with non-ulcer dyspepsia. The levels of COX-1 and COX-2 mRNA were compared between H. pylori-infected and uninfected specimens using reverse transcription-polymerase chain reaction. The immunohistochemical findings were correlated with the cellular localization of cyclooxygenase mRNA using in situ hybridization. RESULTS: A total of 40 H. pylori-infected and 40 uninfected specimens were studied. mRNA of COX-2 but not COX-1 was elevated in H. pylori-infected mucosa. COX-1 was localized to the mononuclear inflammatory, endothelial and smooth muscle cells in the lamina propria. COX-2 was barely detectable in uninfected mucosa but was strongly expressed in the foveolar and glandular epithelia in H. pylori gastritis. CONCLUSION: Cyclooxygenase-1 is expressed in the mononuclear inflammatory, endothelial and smooth muscle cells in the lamina propria irrespective of the H. pylori status. By contrast, H. pylori induces COX-2 expression in the foveolar and glandular epithelia.

Up-regulation of cyclooxygenase-1 and -2 in human gastric ulcer.

BACKGROUND: The expression of cyclooxygenase (COX) in human gastric ulcers is unknown. AIM: To study the expression and cellular localization of cyclooxygenase in human gastric ulcers. METHODS: A total of 38 surgical gastric ulcer specimens were studied; 20 were Helicobacter pylori-positive and 18 were associated with NSAID use. Twenty non-ulcerated, histologically normal gastric specimens were used as controls. The cellular localization of COX-1 and COX-2 were determined by immunohistochemistry and double immunofluorescence. Cyclooxygenase messenger RNA (mRNA) was measured by reverse transcription-polymerase chain reaction and localized by in situ hybridization. RESULTS: In control specimens, COX-1 was detected in stromal cells in the lamina propria. There was focal and weak immunostaining for COX-2 in the foveolar epithelium. At the ulcer edge, COX-1 was significantly increased in lamina propria cells whereas COX-2 was strongly expressed in the hyperplastic foveolar epithelium in H. pylori- and non-steroidal anti-inflammatory drugs (NSAID)-associated ulcers. At the ulcer base, there was strong expression of COX-1 and COX-2 in myofibroblasts, macrophages and endothelial cells in the granulation tissue, irrespective of H. pylori status or NSAID use. Messenger RNA of COX-1 and COX-2 were demonstrated by reverse transcription-polymerase chain reaction. Double immunofluorescence and in situ hybridization confirmed the cellular localization of cyclooxygenase at protein and mRNA levels, respectively. CONCLUSION: Both COX-1 and COX-2 are up-regulated in human gastric ulcers.

Denervation decreases the ipsilateral expression of AChE in chick lumbaric motor neurons.

In vertebrate neuromuscular junctions, acetylcholinesterase (AChE; EC 3.1.1.7) is highly concentrated at the synaptic basal lamina and the postsynaptic muscle fiber. The postsynaptic muscle cell is the primary source of AChE. However, several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE monoclonal antibody in immunohistochemical staining, we found that the AChE-positive cells were labeled only at the motor neurons of the chick spinal cords. When the protein extract of chick spinal cords was analyzed by a Western blot analysis, a protein band of approximately 105 kDa was recognized. In denervated chicks, the expression of motor neuron AChE, as recognized on a Western blot, decreased by approximately 50% 4 days after denervation. The AChE expression in denervated chick spinal cords, however, was restored to control level 10 days after denervation. The decreased AChE expression was restricted to the ipsilateral side of the denervated chick spinal cord while the contralateral side was relatively unchanged. In comparison with the contralateral side, the level of AChE protein and enzymatic activity expressed in the ipsilateral spinal cord was approximately 50% lower. This is the first demonstration to show that the ipsilateral and contralateral sides of chick spinal cords respond differently after nerve denervation.

Cross-sectional study of near-work and myopia in kindergarten children in Singapore.

INTRODUCTION: In view of the high and increasing myopia rates amongst young Singaporean children, we aimed to assess the relationship between near-work and myopia in 414 pre-school children aged 4 to 6 years. MATERIALS AND METHODS: We measured near-work indices such as tuition classes outside school and other possible risk factors via a questionnaire. We then measured myopia with a hand-held autorefractor. RESULTS: Children who had 3 or more hours per week of near-work classes outside school had a higher rate [odds ratio 1.39 (95% confidence interval 1.02, 2.53)] of myopia. CONCLUSIONS: This suggests that there may be an association between near-work and myopia, even at such a young age. Given the increasing emphasis on near-work in Singapore, it may be important to call for increased visual health awareness, although further studies will be needed to establish if near-work causes myopia.

A microbiological study of vaginal discharge in women attending a Malaysian gynaecological clinic.

Vaginal discharge is a common complaint of women attending gynaecological clinics. The purpose of this study was to compare the occurrence of commonly implicated microorganisms in vaginal discharge amongst women with or without the complaint, attending a gynaecological and family planning clinic. The association of Gardnerella vaginalis with bacterial vaginosis was also studied. It was found that there were no significant differences between the cases and controls in the isolation rate of Gardnerella vaginalis, Torulopsis glabrata, Ureaplasma urealyticum, Mycoplasma ssp and Group B streptococcus (p greater than 0.05). Only the isolation rate of Candida albicans was significantly higher in the cases than controls (p less than 0.01). However, there was a significant association of G. vaginalis with bacterial vaginosis.

Detection of Helicobacter pylori from endoscopic biopsies and the biochemical characteristics of these isolates.

Helicobacter pylori (formerly Campylobacter pylori) has been recently described as a gastritis-associated bacterium. We examined endoscopic biopsies of 100 patients with dyspepsia and found H. pylori in the gastric antrum of 34 (34%) by either culture, urease tests and/or histology. Thirty-one out of 41 patients (75.6%) confirmed to have chronic active gastritis histologically had H. pylori in their gastric antrum compared to 3 out of 59 patients (5.1%) with dyspepsia but normal histology (p less than 0.01). Histological examination, using gram stain and the Warthin-Starry Silver stain, detected 29 of the 34 positive cases (85.3%); urease test, 26 cases (76.5%) and culture, 22 cases (64.7%). A combination of histological examination and urease test increased the detection rate to 97.1%. Therefore we felt that for the detection of H. pylori in endoscopic biopsies, culture, which is time consuming and expensive, is not necessary in routine diagnosis as it did not improve the diagnostic rate over a combination of histology and urease test. A comparative study on three media (blood agar, chocolate agar and Skirrow's agar) used in the isolation of the organism showed that non-selective blood agar and chocolate agar were superior to Skirrow's agar. The strains isolated appeared to be homogeneous in their morphological and biochemical characteristics.

Avian influenza H5N1 virus induces cytopathy and proinflammatory cytokine responses in human astrocytic and neuronal cell lines.

It has previously been reported that the avian H5N1 type of influenza A virus can be detected in neurons and astrocytes of human brains in autopsy cases. However, the underlying neuropathogenicity remains unexplored. In this study, we used differentiated human astrocytic and neuronal cell lines as models to examine the effect of H5N1 influenza A viral infection on the viral growth kinetics and immune responses of the infected cells. We found that the influenza virus receptors, sialic acid-alpha2,3-galactose and sialic acid-alpha2,6-galactose, were expressed on differentiated human astrocytic and neuronal cells. Both types of cells could be infected with H5N1 influenza A viruses, but progeny viruses were only produced from infected astrocytic cells but not neuronal cells. Moreover, increased expression of interleukin (IL)-6 and/or tumor necrosis factor alpha (TNF-alpha) mRNA was detected in both astrocytic and neuronal cells at 6 and 24 h post-infection. To examine the biological consequences of such enhanced cytokine expression, differentiated astrocytic and neuronal cells were directly treated with these two cytokines. TNF-alpha treatment induced apoptosis, as well as proinflammatory cytokine, chemokine and inflammatory responses in differentiated astrocytic and neuronal cells. Taken together, our findings reveal that avian influenza H5N1 viruses can infect human astrocytic and neuronal cells, resulting in the induction of direct cellular damage and proinflammatory cytokine cascades. Our observations suggest that avian influenza H5N1 infection can trigger profound CNS injury, which may play an important role in the influenza viral pathogenesis.

Chick muscle expresses various ARIA isoforms: regulation during development, denervation, and regeneration.

Acetylcholine receptor inducing activity (ARIA) is a glycoprotein released from the motor neuron to stimulate the synthesis of acetylcholine receptors (AChRs) on the postsynaptic muscle fiber. Transcripts encoding ARIA were detected not only in brain but also in muscle, and immunohistochemical staining showed that muscle-derived ARIA was restricted to the neuromuscular junctions. RT-PCR analysis revealed three biological active isoforms of ARIA in chick muscle, namely ARIA beta 1, ARIA alpha 2, and ARIA beta 2 that were classified based on their variation in the carboxylterminus of the EGF-like domain. The expression of these ARIA isoforms in muscle change during development denervation, and nerve regeneration. ARIA beta 1, ARIA alpha 2, and ARIA beta 2 were expressed in embryonic and young chick muscles, while ARIA beta 1 was the major isoform expressed in adult chicken. The embryonic-like expression of ARIA alpha 2 and ARIA beta 2 was induced after nerve injury in adult chicken. However, the prominent expression of ARIA beta 1 in adult-like profile was restored after nerve regeneration. A splicing variation in the region between Ig-like and EGF-like domains of ARIA was also revealed; a zero-amino acid insertion (ARIASP0), a 17-amino acid insertion (ARIASP17), or a 34-amino acid insertion (ARIASP34) were identified. Unlike ARIASP0, the expression of ARIASP17 and ARIASP34 was found in muscle and sciatic nerve only. The expression of ARIASP0, ARIASP17, and ARIASP34 in chick muscle remained unchanged during development and after nerve injury. Moreover, the specific expression of these ARIA isoforms in cultured myotubes was not affected by drug treatments or by coculturing with neurons. Our findings provide strong evidence that muscle ARIA may play an important role in the formation of neuromuscular junctions.

Agrin-deficient myotube retains its acetylcholine receptor aggregation ability when challenged with agrin.

Agrin is a synapse-organizing molecule that mediates the nerve-induced aggregation of acetylcholine receptors (AChRs) and other postsynaptic components at the developing and regenerating vertebrate neuromuscular junctions. At the neuromuscular junction, three different cell types can express agrin, i.e., neuron, muscle, and Schwann cell. Several lines of evidence suggested that neuron-derived agrin is the AChR-aggregating factor, but the possible roles of muscle-derived agrin in the formation of AChR aggregate are not known. By using the recombinant DNA method, a clonal stable C2C12 cell line transfected with antisense agrin cDNA was created. RNA dot blot and western blot analysis indicated that the expression of agrin in the transfected cell was abolished by DNA transfection. When the agrin-deficient C2C12 cells were induced to form myotubes and subsequently cocultured with agrin cDNA transfected fibroblasts, AChR aggregates were formed in the cocultures. In addition, acetylcholinesterase (AChE) aggregates in agrin-deficient myotubes were also induced by exogenous agrin and the AChE aggregates were colocalized with the AChR aggregates. The agrin-deficient myotubes could also respond to neuron-induced AChR aggregation after coculturing with neuroblastoma cells. Thus, the agrin-deficient myotubes retain their ability to exhibit the agrin- or neuron-induced AChR aggregation. This result suggests that the formation of postsynaptic specializations during development and regeneration is mediated by neuron-derived agrin but not the agrin from muscle.

Association between cyclo-oxygenase-2 overexpression and missense p53 mutations in gastric cancer.

Wild-type p53 competitively binds to the promoter region of COX-2 in vitro and inhibits its transcription. We examined the association between p53 mutation and COX-2 expression in gastric cancer. COX-2 over-expression was seen in 19 (48.7%) cases. These tumours had more lymph-node metastasis (P = 0.048) and tended to have a poorer survival (P = 0.07). Missense mutations of p53 were detected in 20 (51.3%) patients and had a significantly stronger COX-2 expression than tumours without p53 mutation (P = 0.016). Our results suggest a link between p53 mutation and COX-2 overexpression in gastric cancer.