Ryanodine receptor genes of the rice stem borer, Chilo suppressalis: Molecular cloning, alternative splicing and expression profiling.

The ryanodine receptor (RyR) of the calcium release channel is the main target of anthranilic and phthalic diamide insecticides which have high selective insecticidal activity relative to mammalian toxicity. In this study, the full-length cDNA of Chilo suppressalis RyR (CsRyR) was isolated and characterized. The CsRyR mRNA has an open reading frame (ORF) of 15,387bp nucleotides, which encodes 5128 amino acids with GenBank ID: KR088972. Comparison of protein sequences showed that CsRyR shared high identities with other insects of 77-96% and lower identity to mammals and nematodes with only 42-45%. One alternative splicing site (KENLG) unique to Lepidoptera was found and two exclusive exons of CsRyR (I /II) were revealed. Spatial and temporal expression of CsRyR mRNA was at the highest relative level in 3rd instar larvae and head (including brain and muscle), and at the lowest expression level in egg and fat body. The expression levels of whole body CsRyR mRNA were increased remarkably after injection of 4th instar larvae with chlorantraniliprole at 0.004 to 0.4µg/g. This structural and functional information on CsRyR provides the basis for further understanding the selective action of chlorantraniliprole and possibly other diamide insecticides.

[Role of PI3K/AKT signaling pathway in clonogenicity and tumorigenicity of CD90+ stem-like cells of the hepatocellular carcinoma cell line MHCC-97H].

OBJECTIVE: To investigate the influence of the PI3K/AKT signaling pathway on the proportion and characteristics of the stem-like CD90(+) subpopulation of the human hepatocellular carcinoma (HCC) cell line MHCC-97. METHODS: MHCC-97H cultures were treated with the PI3K/AKT pathway inhibitor LY294002. The proportion of the CD90(+) subpopulation was determined by flow cytometry, and the expression of related proteins was measured by Western blot. The clonogenicity of CD90(+) and CD90(-) cells was measured by plate colony formation assay. The tumorigenicity was compared between CD90(+) and CD90(-) subpopulations (with different concentrations) in xenograft experiments in nude mice, and the changes in tumorigenicity after the addition of LY294002 were evaluated. The changes in the expression of CD90, SHP2, P-AKT, and AKT in CD90(+) and CD90(-) cell xenografts after the addition of LY294002 were examined. Data were analyzed using t test. RESULTS: LY294002 was capable of reducing the proportion of CD90(+) HCC stem cells from 2.98%±0.08% to 0.78%±0.08% (t = 32.400, P < 0.01) and reducing the clonogenicity of CD90(+) subpopulation from 95.13%±3.78% to 61.82%±7.23% (t = 7.617, P < 0.01). However, it showed no significant effect on the clonogenicity of CD90(-) subpopulation. LY294002 also reduced the tumorigenicity of CD90(+) subpopulation and the expression of CD90, SHP2, and P-AKT in related HCC stem cells, but it did not significantly affect the expression of AKT. LY294002 had no significant inhibitory effect on the tumorigenicity of CD90(-) cells. CONCLUSION: The CD90(+) subpopulation of MHCC-97H cells has the characteristics of stem cells and is dependent on the PI3K/AKT signaling pathway.

Minor physical anomalies and craniofacial measures in patients with treatment-resistant schizophrenia.

BACKGROUND: Schizophrenia patients have higher rates of minor physical anomalies (MPAs) than controls, particularly in the craniofacial region; this difference lends support to the neurodevelopmental model of schizophrenia. Whether MPAs are associated with treatment response in schizophrenia remains unknown. The aim of this case-control study was to investigate whether more MPAs and specific quantitative craniofacial features in patients with schizophrenia are associated with operationally defined treatment resistance. METHOD: A comprehensive scale, consisting of both qualitatively measured MPAs and quantitative measurements of the head and face, was applied in 108 patients with treatment-resistant schizophrenia (TRS) and in 104 non-TRS patients. Treatment resistance was determined according to the criteria proposed by Conley & Kelly (2001; Biological Psychiatry 50, 898-911). RESULTS: Our results revealed that patients with TRS had higher MPA scores in the mouth region than non-TRS patients, and the two groups also differed in four quantitative measurements (facial width, lower facial height, facial height, and length of the philtrum), after controlling for multiple comparisons using the false discovery rate. Among these dysmorphological measurements, three MPA item types (mouth MPA score, facial width, and lower facial height) and earlier disease onset were further demonstrated to have good discriminant validity in distinguishing TRS from non-TRS patients in a multivariable logistic regression analysis, with an area under the curve of 0.84 and a generalized R 2 of 0.32. CONCLUSIONS: These findings suggest that certain MPAs and craniofacial features may serve as useful markers for identifying TRS at early stages of the illness.

[Long-term prognosis of primary focal segmental glomerulosclerosis in children].

Objective: To investigate risk factors for the long-term prognosis of primary focal segmental glomerulosclerosis (FSGS) and associated with renal prognosis in children. Methods: A retrospective study was conducted by collecting clinical data including general information, clinical features and renal pathological findings of 124 children with primary FSGS in Department of Pediatrics of Jinling Hospital from January 2003 to December 2019. The cumulative renal survival rate was calculated by Kaplan-Meier survival analysis. The risk factors related to renal prognosis were identified by Cox regression risk model analysis and receiver operating characteristic (ROC) curve. Results: Among 124 children, 94 were males (75.8%) and 30 were females (24.2%). The children were 16 (14, 17) years of age at the time of kidney biopsies. There were 102 cases (82.3%) aged from 13 to 18 years. The period of follow-up was 64.8 (32.1, 86.0) months. There were 49 cases (39.5%) with nonspecific variant, 33 cases (26.6%) with tip variant, 22 cases (17.7%) with collapsing variant, 14 cases (11.3%) with cellular variant and 6 cases (4.8%) with periportal variant. The data of Kaplan-Meier survival analysis showed that cumulative renal survival rates of end-stage kidney disease (ESKD) or ≥50% decline in estimated glomerular filtration rate (eGFR) from baseline at the year of 5, 10 and 15 after renal biopsies were 66.9%, 51.4% and 21.0% respectively. Multivariate Cox regression analysis showed that hypertension, glomerular segmental sclerosis ratio, moderate to severe chronic tubulointerstitial lesions were independent risk factors for progressing to ESKD or ≥50% reduction in eGFR from baseline in pediatric FSGS (HR=5.28, 1.03, 7.81, 95%CI 2.77-10.05, 1.01-1.04, 4.08-14.98, all P<0.01). ROC curve analysis showed glomerular segmental sclerosis ratio (AUC=0.734, P<0.05, optimal cut-off value=25.4%, sensitivity=50.0%, specificity=88.6%), moderate and severe chronic renal tubulointerstitial lesions (AUC=0.724, P<0.05, sensitivity=46.3%, specificity=98.6%) had good efficacy in evaluating renal outcomes of FSGS. Conclusions: The long-term prognosis of FSGS in children is poor. The risk factors of poor prognosis in children with FSGS are hypertension, moderate to severe chronic renal tubulointerstitial lesions and glomerular segmental sclerosis (≥25.4%).

AMF1 (GPS2) modulates p53 transactivation.

We have reported that the papillomavirus E2 protein binds the nuclear factor AMF1 (also called G-protein pathway suppressor 2 or GPS2) and that their interaction is necessary for transcriptional activation by E2. It has also been shown that AMF1 can influence the activity of cellular transcription factors. These observations led us to test whether AMF1 regulates the functions of p53, a critical transcriptional activator that integrates stress signals and regulates cell cycle and programmed cell death. We report that AMF1 associates with p53 in vivo and in vitro and facilitates the p53 response by augmenting p53-dependent transcription. Overexpression of AMF1 in U2OS cells increases basal level p21(WAF1/CIP1) expression and causes a G(1) arrest. U2OS cells stably overexpressing AMF1 show increased apoptosis upon exposure to UV irradiation. These data demonstrate that AMF1 modulates p53 activities.

The psychological effect of severe acute respiratory syndrome on emergency department staff.

BACKGROUND: The severe acute respiratory syndrome (SARS) outbreak in 2003 affected 29 countries. The SARS outbreak was unique in its rapid transmission and it resulted in heavy stress in first-line healthcare workers, particularly in the emergency department. AIM: : To determine the influence of SARS on the psychological status, including post-traumatic stress disorder (PTSD) symptoms, of the staff in the emergency department. METHODS: To investigate whether different working conditions in the hospital led to different psychological effects on healthcare workers, the psychological effect on emergency department staff in the high-risk ward was compared with that on psychiatric ward staff in the medium-risk ward. Davidson Trauma Scale-Chinese version (DTS-C) and Chinese Health Questionnaire-12 (CHQ-12) items were designed to check the psychological status of the staff in the month after the end of the SARS outbreak. RESULTS: 86 of 92 (93.5%) medical staff considered the SARS outbreak to be a traumatic experience. The DTS-C scores of staff in the emergency department and in the psychiatric ward were significantly different (p = 0.04). No significant difference in CHQ score was observed between the two groups. Emergency department staff had more severe PTSD symptoms than staff in the psychiatric ward. CONCLUSION: SARS was a traumatic experience for healthcare providers in Taiwan. Most staff in the emergency department and in the psychiatric ward had PTSD. Emergency department staff had more severe PTSD symptoms than staff in the psychiatric ward.

[Application of dysphonia severity index in laryngeal reflux related voice diseases].

Objective:To discuss the validity and reliability of dysphonia severity index in evaluating the effect of diagnosis and treatment of laryngeal reflux related voice diseases. Method:54 cases of voice disease patients accompanied by laryngopharyngeal reflux from January 2016 to June 2017 in department of otorhinolaryngology of our hospital were divided into two groups according to treatment type. 32 cases in the operation group received laser surgery and standard acid suppression therapy for 6 weeks, and 22 patients in the non-operation group received standard acid suppression therapy for 6 weeks. 24 h multichannel impedance pH (MCII-pH) monitoring was carried out. The indexes of reflux symptom, reflux finding score, subjective auditory perception and objective acoustic parameters of voice were measured before and after treatment, and the dysphonia severity index was calculated and analyzed. Result:There was no significant difference in age, sex and course of disease between the two groups (P> 0.05).Compared with pre-treatment, RSI, RFS, Jitter, Shimmer, G and R in two groups decreased significantly after treatment, and MPT, DSI increased significantly (P<0.05). Before treatment, RSI, RFS, Jitter, Shimmer, G and R in the operation group were significantly higher than those in the non-operation group, and MPT, DSI were lower (P<0.05). There were no significant differences in the parameters between the two groups after treatment (P> 0.05). DSI was negatively correlated with GRBAS scoring parameters, Jitter and Shimmer, and positively correlated with RSI, RFS, and MPT (P<0.01). DSI is related to the location of the lesion (P<0.05) The incidence of anxiety was 27.27% in patients with moderate and severe sudden sensorineural hearing loss, and the incidence of depression was 25.25%. The scores of anxiety and depression were statistically significant (P<0.05). The multivariate logistic regression analysis showed that the status of anxiety and depression was accompanied by symptoms and other diseases (P<0.05). There was a significant difference between the effective group, the significant efficacy group and the cured group (P<0.05). The difference between the scores before and after treatment was compared. Differences in the ineffective group compared with the other three groups, and the cured group compared with the other three groups of anxiety, depression were statistically significant. Conclusion:DSI can be used as an objective evaluation index for the diagnosis and treatment of laryngeal reflux related voice diseases, and it is accurate and reliable.

Curvature measurement of human bilateral cochleae.

OBJECTIVE: This study aimed to characterise the geometry of the human bilateral spiral cochlea by measuring curvature and length. METHOD: Eight subjects were recruited in this study. Magnetic resonance imaging was used to visualise the right and left cochlea. Visualisation of the cochlear spiral was enhanced by T2 weighting and further processing of the raw images. The spirals were divided into three segments: the basal turn, the middle turn and the apex turn. The length and curvature of each segment were non-invasively measured. RESULTS: The mean left and right cochlear lengths were 3.11 cm and 3.95 cm, respectively. The measured lengths of the cochlear spiral are consistent with data in the literature derived from anatomical dissections. Overall, the apex turn segment of the cochlea had the greatest degree of curvature (p < 0.05). The mean apex turn segment curvatures for left and right cochleae were 9.65 cm(-1) and 10.09 cm(-1), respectively. CONCLUSION: A detailed description of the cochlear spiral is provided, using measurements of curvature and length. These data will provide a valuable reference in the development of cochlear implantation procedures for minimising the potential damage during implantation.

The risk of colorectal cancer is related to frequent hospitalization of IBD in an Asian population: results from a nationwide study.

BACKGROUND: The occurrence of inflammatory bowel disease (IBD) is higher in Western countries and is increasing worldwide. The incidence of IBDs is about nearly 20-fold in Western countries than Asia and has risen in Taiwan over the past few decades. Epidemiological studies have demonstrated an increased risk of colorectal cancer (CRC) in patients with IBD. The prevalence of IBD as well as IBD-associated CRC is changing and the risk of CRC in patients with IBD appears to be greater in Western countries, but CRC risk in IBD patients is less well understood in low endemic areas, such as Asia. METHODS: This population-based cohort study collected data from the Taiwan Health Insurance Research Database (from January 1998 to December 2011). In total, 10 650 patients with confirmed diagnosis of IBD served as the IBD cohort and 42 600 non-IBD subjects were enrolled. Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) were used to assess the risk of CRC. RESULTS: The incidence of CRC was slightly lower in the IBD cohort compared with that in the non-IBD cohort (0.94 vs. 1.13 per 1000 person-years), with an adjusted HR of 0.99 (95% CI: 0.71-1.37). More than four hospitalizations were associated with a significantly higher risk of CRC in IBD patients in the Cox model (adjusted HR = 3.48, 95% CI: 1.59-7.63). CONCLUSIONS: The risk for CRC was not increased among IBD patients overall, but appeared to be increased with cumulative frequency of hospitalizations for IBD.

Production of active polyomavirus large T antigen in yeast Pichia pastoris.

The coding region of polyomavirus large T antigen was engineered into the genome of the methylotrophic yeast Pichia pastoris by use of the vector pHIL-D2. Expression of large T antigen was induced by methanol under the control of the strong alcohol oxidase (AOX1) promoter. Large T antigen was purified by immunoaffinity chromatography. We showed that yeast-derived large T antigen bound specifically to a DNA fragment that contains the polyomavirus replication origin, protected the four known major binding sites in the origin against DNase I digestion, and could unwind the strands of an origin-containing DNA fragment in an ATP-dependent manner. This system therefore provides a convenient and inexpensive source of biologically active polyomavirus large T antigen for in vitro studies.

Enhanced binding to origin DNA at low pH enables easy detection of polyomavirus large T antigen by gel mobility shift assay of unfixed complexes.

Enhanced, stable binding by polyomavirus large T antigen to the viral DNA replication origin at pH 6 allowed the development of a gel mobility shift assay for the detection of large T antigen. Such assays were not possible at pH 7.6 without previous fixation, due to instability of the complexes. We demonstrated that the gel mobility shift assay at pH 6 is very sensitive, allowing the detection of as little as 5 ng large T antigen, and is highly specific for DNA containing G(A/G)GGC target sequences. This method was used to detect large T antigen in crude cell lysates from transformed yeast cell lines or nuclear extracts from infected insect cells. Large T antigen-DNA complexes remained at or near the loading well in 5% acrylamide or 1.5% agarose gels, indicating that these complexes are very large. Glycerol gradient analysis showed that protein-DNA complexes formed at pH 6 were massive, and that large T antigen also formed large complexes when incubated at low pH in the absence of DNA. These results show that pH has a major effect on binding of large T antigen to its multiple target sites in the viral origin of DNA replication, presumably by affecting protein-protein interactions that are important for the stability of large T antigen-DNA complexes.

Serological and Molecular Characterization of Peanut chlorotic fan-spot virus, a New Species of the Genus Tospovirus.

ABSTRACT To clarify the serological relationship of Peanut chlorotic fan-spot virus (PCFV) with other tospoviruses, antisera were produced against the nucleocapsid (N) proteins of this virus and tospoviruses from four serogroups including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Groundnut ringspot virus (GRSV), and Watermelon silver mottle virus (WSMoV). In immunodiffusion tests, the antisera only reacted with their homologous antigens. Similar results were noticed in indirect enzyme-linked immunosorbent assay and immunoblot tests, with the exception that strong cross-reactions were observed in heterologous combinations between TSWV and GRSV. The results indicated that the N protein of PCFV is not serologically related to those of the tospoviruses from the four serogroups. To further characterize the virus, viral S double-stranded RNA was extracted from PCFV-infected Chenopodium quinoa and used for cDNA cloning and sequencing. The full-length viral strand of the S RNA was determined to be 2,833 nucleotides, with an inverted repeat at the 5' and 3' ends and two open reading frames in an ambisense arrangement. The 3'-terminal sequence (5'-AUUGCUCU-3') of the viral S RNA is identical to those of other tospoviruses, indicating that PCFV belongs to the genus Tospovirus. The N and the NSs proteins of PCFV share low amino acid identities (22.3 to 67.5% and 19.3 to 54.2%) with those of reported tospoviruses, respectively. The phylogenetic dendrogram of the N gene of PCFV compared with those of other tospoviruses indicates that PCFV is distinct from other tospoviruses. In hybridization analyses, an N gene cDNA probe of PCFV did not react with viral RNAs of TSWV, GRSV, INSV, and WSMoV, and vice versa. Thus, based on these results, we conclude that PCFV is a new tospovirus species.

The effectiveness of serum alpha-fetoprotein level in anti-HCV positive patients for screening hepatocellular carcinoma.

BACKGROUND/AIMS: In Taiwan, most cases of hepatocellular carcinoma (HCC) are hepatitis B virus (HBV) or hepatitis C virus (HCV) related. The serum alpha-fetoprotein (AFP) level is an important factor in the diagnosis of HCC. There have been many studies discussing the role of AFP in diagnosing HBV-related HCC, but only few concerning HCV-related HCC. In this study, we aimed at analyzing the distribution of AFP levels in anti-HCV positive patients with and without HCC and evaluating the effectiveness of serum AFP levels in screening HCV-related HCC. METHODOLOGY: From 1993-1996, we collected the AFP data of 205 HCC patients retrospectively, who were anti-HCV positive. For comparison, 131 randomized anti-HCV positive patients without evidence of HCC served as the control group. We analyzed the AFP distribution in both groups over the following ranges: < or = 5 ng/ml, > 5-20 ng/ml, > 20-50 ng/ml, > 50-100 ng/ml, > 100-200 ng/ml and > 200-400 ng/ml, and > 400 ng/ml. RESULTS: The distributions of AFP levels in anti-HCV positive patients with HCC were 13.2%, 21.5%, 11.2%, 4.9%, 4.4%, 7.3%, and 37.6%. The distributions in anti-HCV positive patients without evidence of HCC were 34.3%, 55.0%, 8.4%, 1.5%, 0.8%, 0%, 0%. CONCLUSIONS: We found the differences in AFP to be statistically significant between anti-HCV positive patients with and without HCC. A serum AFP level of more than 200 ng/ml highly indicates HCC. However, there is a large overlap between these 2 groups. Thus, in anti-HCV positive patients, AFP level is not a good single reference for diagnosis of HCC. Anti-HCV positive patients should be routinely screened for HCC by image studies along with serum AFP level.

Clinical impact of upper gastrointestinal tract injuries in glyphosate-surfactant oral intoxication.

Fifty patients with glyphosate-surfactant oral ingestion were studied with upper gastrointestinal (UGI) endoscopic grading using Zargar's modified grading system for mucosal corrosive injury. Esophageal injury was seen in 68% of the patients, gastric injury in 72%, and duodenal injury in 16%. There were no grade 3 injuries. The upper gastrointestinal tract injuries caused by glyphosate-surfactant were minor in comparison with those by other strong acids. The WBC count, amount of glyphosate-surfactant ingested, length of hospital stay and the occurrence of serious complications increased markedly in the group which had grade 2 esophageal injuries. Thus, the severity of the esophageal injuries may be a prognostic factor for the patient with glyphosate-surfactant ingestion. The UGI endoscopy may be indicated for grading esophageal injury in patients who have ingested glyphosate-surfactant in amounts greater than 100 ml. Physicians should pay more attention to the patients with grade 2 or 3 esophageal injuries to prevent serious complications and to provide aggressive supportive care.

Role of Helicobacter pylori in cirrhotic patients with dyspepsia: a 13C-urea breath test study.

The role of Helicobacter pylori in dyspeptic, cirrhotic patients remains unclear. This prospective outpatient study, conducted to assess the relationship of gastroduodenal disease and H. pylori as determined by the (13C) urea breath test, enrolled 109 consecutive cirrhotic patients with dyspepsia. All patients underwent upper-gastrointestinal endoscopy, which revealed respective prevalences of peptic ulcer, gastric ulcer, and duodenal ulcer of 41.3%, 23.9%, and 22.9%; H. pylori infection was found in 52.3%. The rate of peptic ulcer disease in the H. pylori-positive (45.6%) and -negative (36.5%) groups was not significantly different; neither was the prevalence of H. pylori in patients with or without portal hypertensive gastropathy and with or without esophageal varices. The relationship between peptic ulcer disease and H. pylori in dyspeptic patients with cirrhosis appears to be weak. Likewise, no significant relationship was evident between H. pylori and portal hypertensive gastropathy or esophageal varices. This organism may not be a major pathogenetic factor in gastroduodenal diseases in dyspeptic patients with cirrhosis.

Polyomavirus large T antigen binds cooperatively to its multiple binding sites in the viral origin of DNA replication.

Polyomavirus large T antigen binds to multiple 5'-G(A/G)GGC-3' pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a "handover" mechanism mediated by these protein-protein contacts.

Endoscopic stenting in the treatment of bile leakage after laparoscopic cholecystectomy.

BACKGROUND: Bile leakage is one of the most common complications after laparoscopic cholecystectomy surgery, and biliary decompression is a key factor in treatment. We retrospectively investigated 6 patients with bile leakage after laparoscopic cholecystectomy who were treated with endoscopic stent. METHODS: From March 1995 to May 1999, six patients (4 men and 2 women) aged 30-64 years (mean, 51 years) with bile leakage after laparoscopic cholecystectomy were enrolled. Biliary stent (10 French, 6-10 cm) placement with (n = 4) or without (n = 2) sphincterotomy was attempted. The symptoms, results and outcomes were reviewed. RESULTS: The interval from operation to presentation of bile leakage ranged from 1 to 10 days. Bile leakage was detected from cystic duct stump in 5 patients (83%) and from right IHD in 1 patient (17%). Plastic stent placement was successfully in all patients. Endoscopic stenting healed bile leakage successfully in 5 cases (83%). One patient required surgical correction due to persistent bile leakage. The mean duration between stent placement and cessation of bile leakage was 6.8 days (range 1 to 24 days). CONCLUSIONS: Endoscopic stenting is a safe, rapid and effective treatment for bile leakage after laparoscopic cholecystectomy.

AMF-1/Gps2 binds p300 and enhances its interaction with papillomavirus E2 proteins.

The cellular protein AMF-1 (Gps2) positively modulates gene expression by the papillomavirus E2 protein (D. E. Breiding et al., Mol. Cell. Biol. 17:7208-7219, 1997). We show here that AMF-1 also binds the transcriptional coactivator p300 in vitro and in vivo. E2 interacted weakly with p300. These observations led to a model in which AMF-1 recruits p300 into a complex with E2. Cotransfection of AMF-1 or p300 stimulated levels of E2-dependent transcription, while cotransfection of both AMF-1 and p300 showed an additive effect. The functional significance of p300 recruitment for E2 transactivation was evidenced by repression of E2-activated transcription by adenovirus E1A, which inhibits both coactivator and acetylase activities of p300. Antibodies to AMF-1 or E2 immunoprecipitated histone acetylase activity from cell lysates. Western blotting using antibody against acetyl-lysine failed to detect acetylation of AMF-1 or E2 in complex with p300. These results suggest that AMF-1 facilitates the recruitment of p300 and its histone acetylase activity into complexes with E2 and represents a novel mechanism of transcriptional activation.

Mechanical strain increases endothelin-1 gene expression via protein kinase C pathway in human endothelial cells.

Vascular endothelial cells (ECs) are constantly subjected to mechanical strain due to relaxation and contraction of vessel walls. The effects of cyclical strain on endothelin-1 (Et-1) secretion and Et-1 mRNA levels in human umbilical vein ECs were examined. Cultured ECs grown on a flexible membrane base were deformed by negative pressure (16 kPa at 60 cycles/min). Cells subjected to strain showed increased Et-1 secretion (0.54 ng/hr/10(6) cells) compared with unstrained control cells (0.22 ng/hr/10(6) cells). Northern blot analysis of cells strained for 2 hours or longer demonstrated a sustained elevated Et-1 mRNA level at more than double the level in unstrained controls. This strain-induced ET-1 mRNA level returned to its basal level 2 hours after the release of strain. Cells treated with actinomycin D before or during strain treatment showed no strain-induced gene expression. Pretreatment of ECs with a protein kinase C (PKC) inhibitor, Calphostin C, strongly inhibited the strain-induced Et-1 gene expression. Pretreatment of ECs with cAMP- or cGMP-dependent protein kinase inhibitors (KT5720 or KT5823) only partially inhibited the increased Et-1 mRNA levels in strain-treated cells. EGTA strongly inhibited the Et-1 gene expression. The intracellular calcium chelator BAPTA/AM also showed an inhibitory effect on Et-1 mRNA levels. We conclude that mechanical strain can stimulate the secretion of Et-1 from ECs by increasing Et-1 mRNA levels via transcription, and that this gene induction is mediated predominantly via the PKC pathway and requires extracellular Ca2+. This strain-induced Et-1 gene expression in ECs may contribute to the regulation of vascular tone and structure in normal and pathological states of the cardiovascular system.