DNA methylation promotes the expression of PPARγ transcript 1 at least in part by preventing NRF1 binding to the promoter P1 of chicken PPARγ gene.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis. Our previous study revealed that chicken PPARγ has 3 alternative promoters named as P1, P2, and P3, and the DNA methylation of promoter P3 was negatively associated with PPARγ mRNA expression in abdominal adipose tissue (AAT). However, the methylation status of promoters P1 and P2 is unclear. Here we assessed promoter P1 methylation status in AAT of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). The results showed that promoter P1 methylation differed in AAT between the lean and fat lines of NEAUHLF at 7 wk of age (p < 0.05), and AAT expression of PPARγ transcript 1 (PPARγ1), which was derived from the promoter P1, was greatly higher in fat line than in lean line at 2 and 7 wk of age. The results of the correlation analysis showed that P1 methylation was positively correlated with PPARγ1 expression at 7 wk of age (Pearson's r = 0.356, p = 0.0242), suggesting P1 methylation promotes PPARγ1 expression. To explore the underlying molecular mechanism of P1 methylation on PPARγ1 expression, bioinformatics analysis, dual-luciferase reporter assay, pyrosequencing, and electrophoresis mobility shift assay (EMSA) were performed. The results showed that transcription factor NRF1 repressed the promoter activity of the unmethylated P1, but not the methylated P1. Of all the 4 CpGs (CpG48, CpG49, CpG50, and CpG51), which reside within or nearby the NRF1 binding sites of the P1, only CpG49 methylation in AAT was remarkably higher in the fat line than in lean line at 7 wk of age (3.18 to 0.57, p < 0.05), and CpG49 methylation was positively correlated with PPARγ1 expression (Pearson's r = 0.3716, p = 0.0432). Furthermore, EMSA showed that CpG49 methylation reduced the binding of NRF1 to the P1. Taken together, our findings illustrate that P1 methylation promotes PPARγ1 expression at least in part by preventing NRF1 from binding to the promoter P1.

[Regulatory mechanism of interferon regulatory factor 1 by α-synuclein in mouse Parkinson's disease model].

OBJECTIVE: To investigate the molecular mechanism by which α-synuclein (α-Syn) regulates interferon regulatory factor 1 (IRF-1) expression. METHODS: SH-SY5Y cells overexpressing α-Syn and transgenic mouse model carrying human α-Syn gene with A53T mutation (3 and 6 months old) were examined for IRF-1 mRNA and protein expressions using real-time PCR and Western blotting, respectively. The subcellular localization of IRF-1 was determined with immunofluorescence staining and cytoplasmic/nuclear protein isolation. The optimal concentrations of the proteasome inhibitor MG132 (0.01-2.0 µmol/L) and lysosomal inhibitor chloroquine (5-200 µmol/L) for treatment of SH-SY5Y cells for 24 h were determined by examining the cell viability. SH-SY5Y cells were treated with 0.2 µmol/L MG132 and 30 µmol/L chloroquine for 24 h (the maximum dose that did not cause cell damage), and the changes of IRF-1 protein expressions was analyzed. The effects of α-Syn on MDM2 protein expression and IRF-1 ubiquitylation were analyzed using Western blotting and ubiquitylation assay. RESULTS: α-Syn overexpression did not affect the mRNA level of IRF-1 but significantly increased its protein level (P < 0.01). In α-Synoverexpressing SH-SY5Y cells, IRF-1 translocation was observed from the cytoplasm to the nucleus (P < 0.001). Treatment of the cells with 0.2 µmol/L MG132 significantly aggravated α-Syn-induced increase of IRF-1 protein expression (P < 0.01) while 30 µmol/L chloroquine produced no significant changes in IRF-1 level. α-Syn overexpression caused an obvious decrease of MDM2 protein level and further inhibited the ubiquitylation of IRF-1 (P < 0.01). CONCLUSION: α-Syn blocks MDM2-mediated ubiquitylation of IRF-1 through ubiquitin proteasome pathway, thereby enhancing IRF-1 protein expression.

[The role of large-scale testing platform in the prevention and control of the COVID-19 pandemic: an empirical study based on a novel numerical model].

Objective: China adopted an unprecedented province-scale quarantine since January 23rd 2020, after the novel coronavirus (COVID-19) broke out in Wuhan in December 2019. Responding to the challenge of limited testing capacity, large-scale (>20 000 tests per day) standardized and fully-automated laboratory (Huo-Yan) was built as an ad-hoc measure. There is so far no empirical data or mathematical model to reveal the impact of the testing capacity improvement since quarantine. Methods: Based on the suspected case data released by the Health Commission of Hubei Province and the daily testing data of Huo-Yan Laboratory, the impact of detection capabilities on the realization of "clearing" and "clearing the day" of supected cases was simulated by establishing a novel non-linear and competitive compartments differential model. Results: Without the establishment of Huo-Yan, the suspected cases would increase by 47% to 33 700, the corresponding cost of quarantine would be doubled, the turning point of the increment of suspected cases and the achievement of "daily settlement" (all newly discovered suspected cases are diagnosed according to the nucleic acid testing result) would be delayed for a whole week and 11 days. If the Huo-Yan Laboratory could ran at its full capacity, the number of suspected cases could start to decrease at least a week earlier, the peak of suspected cases would be reduced by at least 44%, and the quarantine cost could be reduced by more than 72%. Ideally, if a daily testing capacity of 10 500 tests was achieved immediately after the Hubei lockdown, "daily settlement" for all suspected cases could be achieved. Conclusions: Large-scale, standardized clinical testing platform, with nucleic acid testing, high-throughput sequencing, and immunoprotein assessment capabilities, need to be implemented simultaneously in order to maximize the effect of quarantine and minimize the duration and cost of the quarantine. Such infrastructure, for both common times and emergencies, is of great significance for the early prevention and control of infectious diseases.

Predator prey interactions between predatory gastropod Reishia clavigera, barnacle Amphibalanusamphitriteamphitrite and mussel Brachidontesvariabilis under ocean acidification.

Since the response to ocean acidification is species specific, differences in responses between predator and prey will alter their interactions, hence affect the population dynamics of both species. Changes in predator prey interactions between a predatory muricid gastropod Reishia clavigera and its prey, the barnacle Amphibalanus amphitrite amphitrite and mussel Brachidontes variabilis under three pCO2 levels (380, 950, and 1250 µatm) were investigated. The searching time for barnacles increased and the ability to locate them decreased at higher pCO2 levels. The movement speed and the prey consumption rate, however, were independent of pCO2. There was no preference towards either B. variabilis or A. amphitrite amphitrite regardless of pCO2. Exposure experiments involving multiple generations are suggested to assess transgenerational effects of ocean acidification and the potential compensation responses before any realistic predictions on the long term changes of population dynamics of the interacting species can be made.

Impact of hidradenitis suppurativa on work loss, indirect costs and income.

BACKGROUND: Hidradenitis suppurativa (HS), a chronic cutaneous disease, can negatively affect work life. OBJECTIVES: This retrospective cohort study evaluates the indirect burden among employed patients with HS in the U.S.A. METHODS: Newly diagnosed and general patients with HS, who were employees (age 18-64 years) from a large claims database (Q1 1999 to Q1 2015), were matched 1 : 5 to controls. Income growth and risk of leaving the workforce were assessed among the newly diagnosed HS and control cohorts in the 5-year study period. Income, work loss days and indirect costs (absenteeism and disability) were assessed among the general HS and control cohorts in the 1-year study period. RESULTS: Newly diagnosed (n = 1003, mean age 39·5 years, 66·3% female) and general patients with HS (n = 1204, mean age 39·9 years, 69·1% female) were matched to 5015 and 6020 controls, respectively. Newly diagnosed patients with HS had significantly slower income growth ($324 per year) and higher risk of leaving the workforce (adjusted hazard ratio 1·65, 95% confidence interval 1·45-1·88) compared with controls (all P < 0·05). General patients with HS had more total days of work loss (18·4 vs. 7·7), higher annual total indirect costs ($2925 vs. $1483) and lower annual income ($54 925 vs. $62 357) than controls (all P < 0·001). CONCLUSIONS: Patients with newly diagnosed HS and general patients with HS experienced a greater indirect burden than matched controls.

Validation of combinatorial probe-anchor ligation-based sequencing as non-invasive prenatal test for trisomy at a central laboratory.

OBJECTIVE: To evaluate the clinical validity of a new ultrahigh-throughput non-invasive prenatal test (NIPT) based on combinatorial probe-anchor ligation (cPAL) sequencing of cell-free fetal DNA (cffDNA) using centralized testing. METHODS: Maternal plasma samples were obtained from 10 594 singleton pregnancies in high-risk populations at 20 centers in China, including 8155 that were collected retrospectively and 2439 prospectively. Fetal outcome data and karyotyping results were documented as gold standard and were double blinded during NIPT. The clinical performance of the ultrahigh-throughput sequencing method, cPAL, for NIPT was validated by evaluating its sensitivity, specificity and positive predictive value (PPV) in detecting trisomies 21, 18 and 13 as the centralized testing mode in the reference laboratory. To ensure stable and reproducible performance of centralized cPAL-based NIPT in detecting trisomies, a series of quality-control systems, including sequencing of two sets of artificial samples, were employed and evaluated. RESULTS: Ten prospective cases were excluded from the study because of incomplete clinical data. Four prospective samples failed to generate a NIPT result due to assay failure, presenting a failure rate of 0.16% (4/2429). A total of 168 retrospective cases and 47 prospective cases had a positive NIPT result for trisomy, giving respective positive rates of 2.06% and 1.94%. Four false-positive and no false-positive cases were observed in the retrospective and prospective groups, respectively, resulting in PPV of 97.62% (95% CI, 94.02-99.35%) and 100% (95% CI, 92.45-100%), respectively. In the retrospective group, sensitivity and specificity were, respectively, 100% (95% CI, 97.07-100%) and 99.98% (95% CI, 99.94-100%) for trisomy 21, 100% (95% CI, 97.75-100%) and 99.98% (95% CI, 99.94-100%) for trisomy 18, and 100% (95% CI, 15.81-100%) and 100% (95% CI, 99.95-100%) for trisomy 13. In the prospective group, sensitivity and specificity were, respectively, 100% (95% CI, 90.75-100%) and 100% (95% CI, 99.85-100%) for trisomy 21, 100% (95% CI, 63.06-100%) and 100% (95% CI, 99.85-100%) for trisomy 18, and 100% (95% CI, 2.50-100%) and 100% (95% CI, 99.85-100%) for trisomy 13. CONCLUSION: In this multicenter study with a full quality-control system, NIPT by centralized cPAL-based testing showed high stability and performance comparable to those of previous validation studies in high-risk populations. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Real-world costs of ischemic stroke by discharge status.

OBJECTIVE: The objective of this study was to estimate the acute healthcare costs of ischemic stroke during hospitalization and the quarterly all-cause healthcare costs for the first year after discharge by discharge status. METHODS: Adult patients with a hospitalization with a diagnosis of ischemic stroke (ICD-9-CM: 434.xx or 436.xx) between 1 January 2006 and 31 March 2015 were identified from a large US commercial claims database. Patients were classified into three cohorts based on their discharge status from the first stroke hospitalization, i.e. dead at discharge, discharged with disability, or discharged without disability. Third-party (medical and pharmacy) and out-of-pocket costs were adjusted to 2015 USD. RESULTS: A total of 7919 patients dead at discharge, 45,695 patients discharged with disability, and 153,778 patients discharged without disability were included in this analysis. The overall average age was 59.7 years and 52.3% were male. During hospitalization, mean total costs (third-party and out-of-pocket) were $68,370 for patients dead at discharge, $73,903 for patients discharged with disability, and $24,448 for patients discharged without disability (p < .001 for each pairwise comparison); mean third-party costs were $63,605 for patients dead at discharge, $67,861 for patients discharged with disability and $19,267 for patients discharged without disability (p < .001 for each pairwise comparison). During the first year after discharge, mean total costs for patients discharged with disability vs. without disability were $46,850 vs. $30,132 (p < .001). Mean third-party costs for patients discharged with disability vs. without disability were $19,116 vs. $10,976 during the first quarter after discharge, $10,236 vs. $6926 during the second quarter, $8241 vs. $5810 during the third quarter, and $6875 vs. $5292 during the fourth quarter (p < .001 for each quarter). CONCLUSION: The results demonstrated the high economic burden of ischemic stroke, especially among patients discharged with disability with the highest costs incurred during the inpatient stays.

Novel Polymorphisms of Adrenergic, Alpha-1B-, Receptor and Peroxisome Proliferator-activated Receptor Gamma, Coactivator 1 Beta Genes and Their Association with Egg Production Traits in Local Chinese Dagu Hens.

Adrenergic, alpha-1B-, receptor (ADRA1B) and peroxisome proliferator-activated receptor gamma, coactivator 1 beta (PPARGC1B) genes are involved in regulation of hen ovarian development. In this study, these two genes were investigated as possible molecular markers associated with hen-housed egg production, egg weight (EW) and body weight in Chinese Dagu hens. Samples were analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, followed by sequencing analysis. Two novel single nucleotide polymorphisms (SNPs) were identified within the candidate genes. Among them, an A/G transition at base position 1915 in exon 2 of ADRA1B gene and a T/C mutation at base position 6146 in the 3'-untranslated region (UTR) of PPARGC1B gene were found to be polymorphic and named SNP A1915G and T6146C, respectively. The SNP A1915G (ADRA1B) leads to a non-synonymous substitution (aspartic acid 489-to-glycine). The 360 birds from the Dagu population were divided into genotypes AA and AG, allele A was found to be present at a higher frequency. Furthermore, the AG genotype correlated with significantly higher hen-housed egg production (HHEP) at 30, 43, 57, and 66 wks of age and with a higher EW at 30 and 43 wks (p<0.05). For the SNP T6146C (PPARGC1B), the hens were typed into TT and TC genotypes, with the T allele shown to be dominant. The TC genotype was also markedly correlated with higher HHEP at 57 and 66 wks of age and EW at 30 and 43 wks (p<0.05). Moreover, four haplotypes were reconstructed based on these two SNPs, with the AGTC haplotype found to be associated with the highest HHEP at 30 to 66 wks of age and with higher EW at 30 and 43 wks (p<0.05). Collectively, the two SNPs identified in this study might be used as potential genetic molecular markers favorable in the improvement of egg productivity in chicken breeding.

[Protective effect of compound bismuth and magnesium granules on aspirin-induced gastric mucosal injury in rats].

OBJECTIVE: To investigate the protective effect of compound bismuth and magnesium granules on aspirin-induced gastric mucosal injury in rats and its possible mechanism. METHODS: Acute gastric mucosal injury model was developed with intraperitoneal injection of aspirin in Wistar rats. The rats were divided into normal control group, injury group, sucralfate protection group, compound bismuth and magnesium granules protection group and its herbal components protection group(each group 12 rats). In the protection groups, drugs as mentioned above were administered by gavage before treated with intraperitoneal injection of aspirin. To evaluate the extent of gastric mucosal injury and the protective effect of drugs, gastric mucosal lesion index, gastric mucosal blood flow, content of gastric mucosal hexosamine, prostaglandins (PG), nitric oxide(NO), tumor necrosis factor (TNF), and interleukin (IL) -1, 2, 8 were measured in each group, and histological changes were observed by gross as well as under microscope and electron microscope. RESULTS: Contents of hexosamine, NO, and PG in all the protection groups were significantly higher than those in the injury group (all P<0.01), and content of NO in the compound bismuth and magnesium granules group was significantly higher than that in the sucralfate group ((11.29±0.51) vs(10.80±0.36)nmol/ml, P<0.05). The gastric mucosal lesion index, contents of TNF, and IL-1, 2, 8 were significantly lower in all the protection groups than in the injury group (all P<0.01), and contents of IL-2 and IL-8 in the compound bismuth and magnesium granules group were significantly lower than those in the sucralfate group ((328.17±6.56) vs(340.23±8.05)pg/ml, P<0.01; (170.82±7.31) vs(179.31±7.80)pg/ml, P<0.05). Tissue injury and inflammatory reaction in all the protection groups were obviously mitigated compared with the injury group. CONCLUSION: Compound bismuth and magnesium granules and its herbal components may have significant protective effect on aspirin-induced gastric mucosal injury.

New insights into implication of the SLIT/ROBO pathway in the prehierarchical follicle development of hen ovary.

The SLIT/Roundabout (ROBO) pathway is involved in follicle development of mammalian ovary, and 2 secreted hormones activin A and inhibin A have potential roles in modulation of the SLIT/ROBO system, but the related actions remain poorly understood in bird. The aims of the present study were to examine the spatial and temporal expression of the SLIT ligand genes (SLIT1, SLIT2, and SLIT3) and their receptor ROBO1, ROBO2, ROBO3, and ROBO4 genes in various-sized prehierarchical follicles during hen ovary development and the effects of activin A and inhibin A on the expression of these genes in the cultured hen follicles. Our result demonstrated that the transcripts of the 3 SLIT genes were highly expressed in the developing follicles and expression patterns of the SLIT transcripts were different from those of ROBO genes detected by real-time quantitative reverse transcriptase PCR. Both SLIT and ROBO transcripts were predominantly expressed in oocytes and granulosa cells from the prehierarchichal follicles examined by in situ hybridization. The localization for SLIT and ROBO proteins was revealed by immunohistochemistry similar to the spatial distribution of their transcript. In cultured follicles (4 to 8 mm in diameter), the expression levels of SLIT and ROBO members are hormonally regulated by activin A (10 ng/mL) and/or inhibin A (20 ng/mL) after treatment for 24 h. However, the expression of only SLIT2, SLIT3, and ROBO3 mRNA presented a directly opposite response to activin A and inhibin A hormones. These results indicate that SLIT/ROBO pathway is implicated in the prehierarchical follicular development of the hen ovary by an intrafollicular autocrine and/or paracrine action, and is influenced by activin A and inhibin A hormones.

Association of novel polymorphisms of forkhead box L2 and growth differentiation factor-9 genes with egg production traits in local Chinese Dagu hens.

Transcription factor forkhead box L2 (FOXL2) and growth differentiation factor-9 (GDF9) genes have critical roles in the regulation of hen ovarian development. In the present study, these genes were explored as possible molecular markers associated with BW, hen-housed egg production, and egg weight in Chinese Dagu hens. Samples were analyzed using the PCR-single strand conformation polymorphism (PCR-SSCP) technique followed by sequencing analysis, and two novel single nucleotide polymorphisms (SNPs) were identified within these candidate genes. Among them, an A/G transition at base position 238 in the coding region of the FOXL2 gene and a G/T transversion at base position 1609 in exon 2 of the GDF9 gene were found to be polymorphic and named SNPs A238G and G1609T, respectively. The SNP A238G (FOXL2) leads to a nonsynonymous substitution (isoleucine77-to-valine), and when the 360 Dagu hen samples were divided into genotypes AA and AB, allele A was found to be present at a higher frequency. Furthermore, the AA genotype correlated with significantly higher hen-housed egg production at 30, 43, 57, and 66 wk of age and with a higher egg weight at 43 wk (P<0.05). For the SNP G1609T (GDF9), the hens were typed into TT and TC genotypes, with the T allele shown to be dominant. The TC genotype was also markedly correlated with higher hen-housed egg production and a higher egg weight (P<0.05). Moreover, four haplotypes were reconstructed based on these two SNPs, with the AATC haplotype found to be correlated with the highest hen-housed egg production at 30 to 66 wk of age and with higher egg weights at 43 wk (P<0.05). Collectively, the two SNPs identified in this study might be used as possible genetic molecular markers to aid in the improvement of egg production traits in chicken breeding.

Clinical features of macrophage activation syndrome in the adult northern Chinese population.

OBJECTIVE: Macrophage activation syndrome (MAS) is a relatively rare but potentially fatal complication of childhood rheumatic illnesses. We sought to provide insight for the timely recognition and diagnosis of MAS and efficacious disease management in adults with rheumatic diseases. METHODS: Clinical files for eight adult MAS patients treated at the Second Affiliated Hospital of Harbin Medical University were analyzed for clinical manifestations, laboratory investigations, therapeutic measurements and clinical outcomes. RESULTS: The study included male and female patients with ages ranging from 16 to 59 years old. All patients were diagnosed with underlying rheumatic diseases with five patients having adult-onset Still's disease (AOSD), two patients having systemic lupus erythematosus (SLE) and one patient having Sjögren's syndrome (SS). The interval from fever onset to MAS diagnosis varied from seven days to 40 days. The most common clinical presentations were prolonged high fever, respiratory symptoms and jaundice. No patients had symptoms involving the central nervous system (CNS). Laboratory findings showed peripheral cytopenias, elevated liver enzymes, elevated triglycerides, hypofibrinogenemia and bone marrow hemophagocytosis. Potential effective treatments for MAS include glucocorticoid plus immunoglobulin therapy, but delays in diagnosis and treatment may lead to a fatal disease course. CONCLUSION: MAS in adults may not be as rare as was once thought, although the clinical features of MAS in adults often differ from those seen in children. The MAS mortality in adults is far higher than that for children. A diagnosis of MAS should be considered when a patient with rheumatic disease presents with prolonged high fever, peripheral cytopenia and liver failure. Collection of bone marrow aspirates is critical for accurate diagnosis and MAS therapy should begin as early as possible.

Hypocrea lixii, novel endophytic fungi producing anticancer agent cajanol, isolated from pigeon pea (Cajanus cajan [L.] Millsp.).

AIMS: The aim was to isolate, identify and characterize endophytes from pigeon pea (Cajanus cajan [L.] Millsp.), as novel producer of cajanol and its in vitro cytotoxicity assay. METHODS AND RESULTS: Isolation, identification and characterization of novel endophytes producing cajanol from the roots of pigeon pea were investigated. The endophytes were identified as Hypocrea lixii by morphological and molecular methods. Cajanol produced by endophytes were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). R-18 produced the highest levels of cajanol (322·4 ± 10·6 µg l(-1) or 102·8 ± 6·9 µg g(-1) dry weight of mycelium) after incubation for 7 days. The cytotoxicity towards human lung carcinoma cells (A549) of fungal cajanol was investigated in vitro. CONCLUSIONS: First, a novel endophyte Hypocrea lixii, producing anticancer agent cajanol, was isolated from the host pigeon pea (Cajanus cajan [L.] Millsp.). Fungal cajanol possessed stronger cytotoxicity activity towards A549 cells in time- and dose-dependent manners. SIGNIFICANCE AND IMPACT OF THE STUDY: This endophyte is a potential handle for scientific and commercial exploitation, and it could provide a promising alterative approach for large-scale production of cajanol to satisfy new anticancer drug development.

Functional association of phosphoinositide-3-kinase with platelet glycoprotein Ibalpha, the major ligand-binding subunit of the glycoprotein Ib-IX-V complex.

BACKGROUND: The adhesion receptor glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet-mediated tumor metastasis. The cytoplasmic C-terminal domain of the ligand-binding GPIbalpha subunit contains binding sites for filamin (residues 561-572, critically Phe568/Trp570), 14-3-3zeta (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide-3-kinase (PI3-kinase) regulatory subunit, p85. OBJECTIVES: We previously showed that, as compared with wild-type receptor, deleting the contiguous sequence 580-590 or 591-610, but not upstream sequences, of GPIbalpha expressed as a GPIb-IX complex in Chinese hamster ovary cells inhibited VWF-dependent Akt phosphorylation, which is used as a read-out for PI3-kinase activity. Pulldown experiments using glutathione-S-transferase (GST)-p85 or GST-14-3-3zeta constructs, and competitive inhibitors of 14-3-3zeta binding, suggested an independent association of 14-3-3zeta and PI3-kinase with GPIbalpha. The objective of this study was to analyze a further panel of GPIbalpha deletion mutations within residues 580-610. RESULTS: We identified a novel deletion mutant, Delta591-595, that uniquely disrupts 14-3-3zeta binding but retains the functional p85/PI3-kinase association. Deletion of other sequences within the 580-610 region were less discriminatory, and either partially affected p85/PI3-kinase and 14-3-3zeta binding (Delta580-585, Delta586-590, Delta596-600, Delta601-605), or strongly inhibited binding of both proteins (Delta606-610). CONCLUSIONS: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14-3-3zeta in GPIb-dependent signaling or platelet functional studies involving truncation of the C-terminal residues in cell-based assays and mouse models. The Delta591-595 mutation provides another strategy for determining the function of GPIbalpha-associated 14-3-3zeta by selective disruption of 14-3-3zeta but not p85/PI3-kinase binding.

Compromised ITAM-based platelet receptor function in a patient with immune thrombocytopenic purpura.

BACKGROUND: Receptors on platelets that contain immunoreceptor tyrosine-based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcgammaRIIa, a low affinity receptor for immunoglobulin (Ig) G. OBJECTIVES: We examined the function of GPVI and FcgammaRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. METHODS AND RESULTS: Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen-related peptide, or to FcgammaRII-activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti-GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (approximately 60%) blocked by FcgammaRIIa-blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full-length GPVI on the patient platelet surface was reduced to approximately 10% of normal levels, and a approximately 10-kDa GPVI cytoplasmic tail remnant and cleaved FcgammaRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained approximately 150 ng mL(-1) soluble GPVI by ELISA (normal plasma, approximately 15 ng mL(-1)) and IgG purified from patient plasma caused FcgammaRIIa-mediated, EDTA-sensitive cleavage of both GPVI and FcgammaRIIa on normal platelets. CONCLUSIONS: In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.

Design, synthesis, and biological evaluation of a series of lavendustin A analogues that inhibit EGFR and Syk tyrosine kinases, as well as tubulin polymerization.

A series of N-alkylamide analogues of the lavendustin A pharmacophore were synthesized and tested for inhibition of the epidermal growth factor receptor (EGFR) protein tyrosine kinase and the nonreceptor protein tyrosine kinase Syk. Although several compounds in the series were effective inhibitors of both kinases, it seemed questionable whether their inhibitory effects on these kinases were responsible for the cytotoxic properties observed in a variety of human cancer cell cultures. Accordingly, a COMPARE analysis of the cytotoxicity profile of the most cytotoxic member of the series was performed, and the results indicated that its cytotoxicity profile was similar to that of antitubulin agents. This mechanism of action was supported by demonstrating that most compounds in the series were moderately effective as inhibitors of tubulin polymerization. This suggests that the lavendustin A analogues reported here, as well as some of the previously reported lavendustin A analogues, may be acting as cytotoxic agents by a mechanism involving the inhibition of tubulin polymerization.

Electrical stimulation of acupuncture points enhances gastric myoelectrical activity in humans.

OBJECTIVE: Acupuncture is known to enhance gastric motility. Electrical acustimulation has been shown to reduce gastric tachyarrhymia in vection-induced motion sickness. The aim of this study was to investigate the effect of electrical stimulation of acupuncture points on gastric myoelectrical activity in healthy humans. METHODS: Nine healthy native Chinese were studied. Gastric myoelectrical activity was recorded using surface electrogastrography (EGG). The EGG recording was made in the fasting state, in a study period during which acupuncture points were electrically stimulated continuously, and in a recovery period after stimulation. The percentage of regular slow waves was assessed by computing the percentage of 2 to 4 cycles per minute slow waves in the EGG. RESULTS: Electrical stimulation significantly increased the percentage of regular slow waves, which was sustained in the recovery period. The increase of the regular slow wave activity resulted from the normalization of arrhythmia. CONCLUSION: Electrical stimulation of acupuncture points may enhance the regularity of gastric myoelectrical activity and may be an option for treatment of gastric dysrhythmia.

EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif.

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia.

The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.