A functional study of proximal goat ß-casein promoter and intron 1 in immortalized goat mammary epithelial cells.

Goat ß-casein (CSN2) promoter has been extensively used to derive expression of recombinant therapeutic protein in transgenic goats; however, little direct evidence exists for signaling molecules and the cis-elements of goat CSN2 promoter in response to lactogenic hormone stimulation in goat mammary epithelial cells. Here, we use an immortalized caprine mammary epithelial cell line (CMC) to search for evidence of the above. Serial 5'-flanking regions deleted of promoter and intron 1 in goat CSN2 (-4,047 to +2,054) driven by firefly luciferase reporter gene were constructed and applied to measure promoter activity in CMC. The intron 1 region (+393 to +501) significantly decreased basal activity of the promoter. This finding contradicts other studies of the role of intron 1. The signal transducer and activator of transcription (STAT)5a played a significant role in activating promoter activity by prolactin stimulation. Hydrocortisone enhanced and prolonged the activity of STAT5a and promoter in CMC, but was independent of the glucocorticoid receptor response element. The minimum length of the CSN2 promoter segment in response to lactogenic stimulation was confirmed by 5' serial deletions. A cis-element located from -300 to -90 in proximal goat CSN2 promoter that is absent in bovine and human CSN2 promoter was newly identified. We demonstrated the presence of a STAT5a binding site (-102 to -82) and preservation of the guanosine nucleotide at position -90 based on responses to the presence of lactogenic hormone using internal deletions and point mutations of the predicted STAT5a binding site, and chromatin immunoprecipitation assay. Together, these findings demonstrate that the proximal -300 bp of goat CSN2 promoter containing the STAT5a binding site (-102 to -82) is the response element for lactogenic hormone stimulation. Additionally, intron 1 may be required for tissue or developmental stage-specific expression in mammary gland. The role of the far-distal regions of goat CSN2 promoter in high-level lactogenic hormone induction and specific expression require further examination.

Whole exome sequencing identifies a novel mutation in the transglutaminase 6 gene for spinocerebellar ataxia in a Chinese family.

Autosomal dominant spinocerebellar ataxias (SCA) constitute a heterogeneous group of inherited disorders. The transglutaminase 6 (TGM6) gene was recently suggested as a SCA causative gene in Chinese SCA families. In this study, two affected members of a three-generation Chinese family with SCA characterized by progressive cerebellar ataxia and lower limb pyramidal signs were subjected to whole exome sequencing. Through bioinformatics analysis of the sequence variants in these two individuals, we identified a novel mutation in the TGM6 gene (c.1528G>C) which showed perfect co-segregation with disease phenotype in all nine members of this family. This finding confirms that mutations in TGM6 gene represent an important cause of SCA in Chinese. This study also shows that whole exome sequencing of a small number of affected individuals, leveraged on bioinformatics analysis, can be an efficient strategy for identifying causative mutations in rare Mendelian disorders.

Aquaporin-4 water channel expression by thymoma of patients with and without myasthenia gravis.

BACKGROUND: Neuromyelitis optica (NMO) is a serious idiopathic inflammatory demyelinating disorder characterized by acute transverse myelitis and optic neuritis. A significant proportion of NMO patients are seropositive for NMO-IgG, an autoantibody targeting aquaporin-4 (AQP4) water channel. Paraneoplastic NMO associated various tumors were recently reported. AIM: We studied the expression of AQP4 by thymoma from patients with and without myasthenia gravis (MG). METHODS: Thymoma obtained from thymomectomy in patients with and without MG were studied by immunohistochemistry and western blot. RESULTS: Ten thymoma patients (9 with MG) and two control patients without thymoma or MG were studied. Immunohistochemistry revealed AQP4 immunoreactivity in cell membrane of thymoma cells from all ten thymoma specimens whereas thymic tissues from patients without thymoma or MG were negative for AQP4 immunoreactivity. Western blot revealed that lysates of nine of the ten thymoma specimens reacted with anti-human AQP4 antibody with a band of ~30 kDa compatible with the molecular weight of AQP4. Interestingly, immunofluorescence revealed that IgG isolated from 2 NMO patients seropositive for NMO-IgG bound to cell membrane of thymoma cells from all ten thymoma specimens while IgG from healthy control subject did not. CONCLUSION: Thymoma cells of patients with and without MG express AQP4. AQP4 autoantibodies from serum of NMO patients bound to AQP4 expressed on thymoma cell membrane.

Neuromyelitis optica-IgG in idiopathic inflammatory demyelinating disorders amongst Hong Kong Chinese.

BACKGROUND: Idiopathic inflammatory demyelinating disorders (IIDD) affect the central nervous system. In classical multiple sclerosis (CMS), brain, optic nerves [optic neuritis (ON)] and spinal cord [acute transverse myelitis (ATM)] are affected. In neuromyelitis optica (NMO), optic nerves and spinal cord are predominantly affected. NMO-IgG, an autoantibody targeting aquaporin-4, is a marker for NMO. We studied the frequency and clinical relevance of NMO-IgG seropositivity in IIDD patients. METHODS: Neuromyelitis optica-IgG was detected by indirect immunofluorescence using primate cerebellum. RESULTS: Neuromyelitis optica-IgG was detected in six of 10 NMO patients (60%), six of 10 idiopathic relapsing transverse myelitis (IRTM) patients (60%), two of nine idiopathic relapsing ON patients (22%), one of 11 patients (9%) having single ON attack, one of 30 CMS patients (3%), and none of patients having single ATM attack or controls. Comparing NMO-IgG seropositive (n = 12) with NMO-IgG seronegative (n = 8) patients having NMO or IRTM, NMO-IgG seropositivity was associated with a higher relapse rate in first 2 years, 1.5 and 0.6 attacks/year for seropositive and seronegative groups respectively (P = 0.006), and non-significant trend towards more severe ON and myelitis with poorer clinical outcome. CONCLUSION: Neuromyelitis optica -IgG facilitates diagnosis of NMO spectrum disorders. NMO-IgG seropositivity is associated with higher relapse rate in first 2 years.

Effects of plasticisers and related compounds on the expression of the soluble form of catechol-O-methyltransferase in MCF-7 cells.

Previously we have shown that E2 down regulates S-COMT expression. Here the effects of four phthalate esters and 4-(tert-octyl)phenol on the intra-cellular levels of S-COMT and COMT activity were studied in MCF-7 cells as a measure of estrogenic activity of these compounds. The four phthalate esters caused significant reductions in both S-COMT protein and COMT activity levels. These effects were inhibited by the ERalpha receptor antagonist ICI182780. 4-(tert-octyl)phenol also caused reductions in these parameters, but the effects were not abolished by ICI182780. Assay of S-COMT protein levels represents a simple and convenient method of assessing the estrogenic potential of a compound.

Estrogenic phenol and catechol metabolites of PCBs modulate catechol-O-methyltransferase expression via the estrogen receptor: potential contribution to cancer risk.

Commercial PCB mixtures have been shown to induce liver tumors in female rats and this effect has been attributed to the effects of PCBs on estrogen metabolism. Catechol metabolites of PCBs are potent inhibitors of COMT activity and are likely to contribute significantly to reduced clearance of genotoxic catechol metabolites of estrogen. The effect of PCB metabolites on COMT expression in cultured cells was investigated to explore potential mechanisms by which PCB exposure alters catechol estrogen clearance. We hypothesize that estrogenic PCB metabolites may contribute to reduction of COMT expression via interaction with the estrogen receptor. To test this hypothesis, human MCF-7 cells were exposed to PCB analogues and the expression of COMT determined. Western blot analysis demonstrated that COMT protein levels were statistically significantly reduced by both the phenolic and the catechol compounds, an effect which was abolished by the anti-estrogen, ICI182780. The above suggests that COMT levels may be reduced by estrogenic PCB metabolites, via interactions between PCB metabolites and the ER. It supports the hypothesis that both phenolic and catechol metabolites of PCBs may contribute to PCB-mediated carcinogenesis through reduction of COMT levels and activities and subsequent reduction in clearance of endogenous and xenobiotic catechols.

Comparison of the APACHE II and APACHE III scoring systems in patients with respiratory failure in a medical intensive care unit.

BACKGROUND AND PURPOSE: This retrospective study compared the capability of the Acute Physiology and Chronic Health Evaluation (APACHE) II and APACHE III scoring systems to predict outcome and determined the independent predictors of survival in these scoring systems for patients with respiratory failure in a medical intensive care unit (ICU). MATERIALS AND METHODS: Seven hundred and eight patients with respiratory failure admitted to the medical ICU throughout a 9-year period were studied. Patients with an ICU stay of less than 24 hours, patients under 12 years of age, and burn and surgery patients were excluded. APACHE scores were calculated at 24 hours after admission. Student's t-test was used to compare the total APACHE scores of survivor and non-survivor groups. Multivariate logistic regression analysis was used to determine which variables were predictors of mortality. The discriminative power of APACHE scores to predict in-hospital mortality was studied by the area under the receiver operating characteristic curves of the APACHE II and APACHE III systems, respectively. RESULTS: Both systems showed a significant association between higher scores and higher mortality. The APACHE II system under-predicted the actual hospital mortality rate. The APACHE III systems had a higher discriminative power (area 0.7462) than the APACHE II systems (area 0.6856; p < 0.05). The independent predictors of survival as assessed by APACHE II and III systems were respiratory rate, arterial oxygen pressure, oxygen gradient between alveoli and artery, serum creatinine concentration, and the presence of neurologic abnormalities. CONCLUSIONS: The APACHE III systems has greater discriminative power than the APACHE II systems for predicting in-hospital mortality. The variables of oxygenation, mean artery pressure, respiratory rate, serum creatinine concentration, and Glasgow Coma Scale play important roles in predicting survival for patients with respiratory failure.

Prediction of in-hospital mortality in patients with myocardial infarction using APACHE II system.

BACKGROUND: The acute physiology and chronic health evaluation (APACHE) scoring system has been validated in many different patient populations, however, patients with myocardial infarction (MI) were not included in the original data base. To evaluate the ability of APACHE scoring system in predicting in-hospital mortality, 694 patients with MI were studied. METHODS: Data had been collected prospectively in an ICU computer database in the past 3 years. Patients admitted in coronary care unit with acute MI or acute coronary syndrome who had previous history of MI were all included. Patients were divided into survivor and non-survivor data sets. Multiple logistic regression analysis was evaluated on the variables of APACHE II score to determine which variables could predict in-hospital mortality. A logistic regression model was used to study the mortality curves. The differences of APACHE II scores between survivors and non-survivors were compared. Correlation between observed and predicted mortality was also assessed. RESULTS: According to the statistical analysis, the non-survivors tended to have significantly greater APACHE II scores than those of survivors. The APACHE II values of non-survivors and survivors were 23.64 +/- 9.41 versus 13.35 +/- 7.14 (p < 0.001), respectively. Using multiple logistic regression analysis, we found that age, creatinine, coma scale, sodium and APACHE II score were capable of predicting the in-hospital mortality (p < 0.05). With use of the logistic model, a good correlation of predicted mortality rate to observed mortality rate was found (r = 0.992). This study demonstrated that lower APACHE II scores predicted survival while high scores predicted mortality. Mortality rate increased significantly when APACHE II score was > 25. An APACHE II score greater than 28.25 predicted a more than 50% in-hospital mortality. CONCLUSIONS: This study demonstrates that the APACHE II scoring system is capable of predicting mortality in patients with MI, which makes this modality more applicable in the busy intensive care unit.

Cytochrome P4502D6 (debrisoquine 4-hydroxylase) and Parkinson's disease in Chinese and Caucasians.

Four polymorphic sites (C/T188, C/T2938, G/C4268, G/A1934) in the cytochrome P4502D6 (debrisoquine 4-hydroxylase) gene were investigated for their association with sporadic Parkinson's disease (PD). Three mutant alleles (C/T188, C/T2938 and G/C4268) result in amino acid changes which could alter the substrate specificity or alter its ability to metabolize their substrates; the fourth (G/A1934) causes a loss of enzyme activity. The study was carried out in two ethnically homogenous populations: Chinese (123 PD patients, 124 controls); and Caucasian (95 PD patients, 62 controls). Haplotype status, which took into account amino acid changes at three polymorphic sites, was deduced from genotyping results in order to investigate whether substrate specificity was important rather than loss of enzyme activity. There was no gender difference in the distribution of the alleles in either race. There was, however, significant association among the three polymorphic sites (C/T188, C/T2938, G/C4268) in both ethnic groups. T/T188:C/C2938:C/C4268 is the most common genotype in the Chinese population, in contrast to C/C188:C/T2938:C/G4268 (followed by C/C188:C/C2938:G/G4268) in Caucasians. All 69 of the sub-group of Chinese patients tested were homozygous for the wild-type allele at the G/A1934 polymorphic site. Neither the CYP2D6 allele nor haplotype was associated with PD in either ethnic group.