[Analysis of DMD gene variants in a single center].

Objective: To investigate the DMD genetic variants of the Chinese population with Duchenne (DMD) and Becker muscular dystrophies (BMD). Methods: A cross-sectional study was conducted on 2 690 unrelated patients with DMD and BMD aged 0-18 who visited the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from January 2005 to February 2022. The clinical data, such as gender, age, clinical manifestations, and address, were collected. Multiplex ligation-dependent probe amplification, next generation sequencing panel, Sanger sequencing, and PCR amplification were used to detect the variants of the DMD gene in the patients, whose clinical information and gene detection results were descriptively analyzed. Results: The 2 690 patients included 2 648 males and 42 females, with an age of 6.0 (4.0, 9.0) years. The serum creatine kinase increased in all patients. Pathogenic DMD gene variants were detected in the 2 618 patients, including 1 875 cases (71.6%) large deletions, 231 cases (8.8%) duplications, and 512 cases (19.6%) small variants. Among the deletion variants, the deletion of 3 exons was the most common, accounting for 15.4% (288/1 875); and hotspot deletion involved exons 45 to 50, accounting for 6.3% (119/1 875). Exon 2 was the most common type duplication region, accounting for 13.0% (30/231). Small variants were distributed in all 79 exons of the DMD gene, with no hotspots. In addition, the 46 small variants were previously unreported. Conclusion: Exon deletion is the most common type of DMD gene variant, followed by small variants and exon duplication.

Significance of the plasma membrane H+-ATPase and V-ATPase for growth and pathogenicity in pathogenic fungi.

Pathogenic fungi are widespread and cause a variety of diseases in human beings and other organisms. At present, limited classes of antifungal agents are available to treat invasive fungal diseases. With the wide use of the commercial antifungal agents, drug resistance of pathogenic fungi are continuously increasing. Therefore, exploring effective antifungal agents with novel drug targets is urgently needed to cope with the challenges that the antifungal area faces. pH homeostasis is vital for multiple cellular processes, revealing the potential for defining novel drug targets. Fungi have evolved a number of strategies to maintain a stable pH internal environment in response to rapid metabolism and a dramatically changing extracellular environment. Among them, plasma membrane H+-ATPase (PMA) and vacuolar H+-ATPase (V-ATPase) play a central role in the regulation of pH homeostasis system. In this chapter, we will summarize the current knowledge about pH homeostasis and its regulation mechanisms in pathogenic fungi, especially for the recent advances in PMA and V-ATPase, which would help in revealing the regulating mechanism of pH on cell growth and pathogenicity, and further designing effective drugs and identify new targets for combating fungal diseases.

[Preliminary experience of surgical treatment for torus tubarius hypertrophy in children].

Objective: To assess the incidence of symptomatic torus tubarius hypertrophy (TTH) in recurred OSA in children, and to explore the preliminary experience of partial resection of TTH assisted with radiofrequency ablation. Methods: From January 2004 to February 2020, 4 922 children, who diagnosed as OSA and received adenotonsillectomy at the Department of Otolaryngology, The 4th Medical Center of the PLA General Hospital, were retrospectively reviewed. There were 3 266 males and 1 656 females, the age ranged from 1 to 14 years old(median age of 5.0 years). Twenty-two cases were identified with recurrence of OSA syndrome, and the clinical data, including sex, age of primary operation, age of recurrence and presentation, and opertation methods were analyzed. Follow-up was carried out by outpatient visit or telephone. Graphpad prism 5.0 software was used for statistical analysis. Results: Twenty-two cases were identified as recurred OSA and received revised surgery in 4 922 cases. Among these 22 cases, 11 cases were diagnosed as TTH resulting in an incidence of 2.23‰(11/4 922), 1 case was cicatricial adhesion on tubal torus (0.20‰, 1/4 922), 10 cases were residual adenoid combined with tubal tonsil hypertrophy (2.03‰, 10/4 922). Median age of primary operation was 3.0 years (range:2.4 to 6.0 years) in 11 TTH cases. Recurrent interval varied from 2 months to 5.5 years (2.4±1.9 years) after first operation. Age of revised partial resection of TTH was 7.0±2.7 years (range: 4.0 to 12.0 years). Average time interval between primary operation and revised operation was 3.5±2.1 years (range: 0.5 to 6.0 years). Individualized treatments were carried out based on partial resection of TTH assisted with radiofrequency ablation. All of 11 cases received satisfied therapeutic results without nasopharyngeal stenosis occured. Twenty-two cases were followed up for 1.6 to 13 years (median follow-up time was 6.2 years). Conclusions: TTH contributed to recurred OSA in child. TTH might be misdiagnosed as tubal tonsil hypertrophy. Partial resection of TTH assisted with radiofrequency ablation was a safty and effective treatment.

Correlations of hsa_circ_0046264 expression with onset, pathological stage and chemotherapy resistance of lung cancer.

OBJECTIVE: The purpose of this study was to investigate the correlations of hsa_circ_0046264 expression with the onset, pathological stage, and chemotherapy resistance of lung cancer. PATIENTS AND METHODS: Firstly, gene expression profiling microarrays were applied to screen the differentially expressed circular ribonucleic acids (circRNAs) in the tumor tissues of patients with non-small cell lung cancer (NSCLC). Secondly, quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) assay was adopted to further verify the circRNAs with significant differences. Thirdly, the correlations of hsa_circ_0046264 expression level with the clinical features of NSCLC patients were explored via statistical analysis. Fourthly, Kaplan-Meier survival analysis and receiver operating characteristic (ROC) curve were utilized to investigate the influence of hsa_circ_0046264 expression level on the survival of the patients. Finally, the role of hsa_circ_0046264 in the process of lung cancer was probed using in vitro experimental methods. RESULTS: It was shown in the results of gene microarray assay that hsa_circ_0046264 was the most prominently upregulated gene, and RT-qPCR assay further proved that hsa_circ_0046264 expression was upregulated remarkably in the tissues of tumor patients. Clinical analysis indicated that the expression level of hsa_circ_0046264 was notably associated with the patient's age, tumor size, tumor-node-metastasis (TNM) stage, and lymph node metastasis (p<0.01). In addition, Kaplan-Meier statistical analysis manifested that the patients in hsa_circ_0046264 low-expression group had a markedly longer survival than those in hsa_circ_0046264 high-expression group. In the tumor tissues and serum of the patients, the area under ROC curve of hsa_circ_0046264 was 0.971 and 0.915, the specificity was 0.973 and 0.957, and the sensitivity was 0.951 and 0.927, while the Youden Index was 0.924 and 0.884 respectively. The results of Cell Counting Kit-8 (CCK-8) assay revealed that the proliferative ability of lung cancer A549 cells was significantly enhanced at 36, 48, and 72 h in hsa_circ_0046264 overexpression group. According to the results of wound-healing assay, the migratory ability of A549 cells was distinctly strengthened in hsa_circ_0046264 overexpression group compared with that in the control group (p<0.05). Moreover, the transwell assay results pointed out that the invasive ability of A549 cell lines at 48 h after overexpression of hsa_circ_0046264 was evidently stronger than that in control group (p<0.05). Under the stimulation of different doses of cisplatin, hsa_circ_0046264 overexpression group had a clearly raised survival rate of A549 cells in comparison with control group, and the differences in data were statistically significant (p<0.01). CONCLUSIONS: Hsa_circ_0046264 may serve as a potential biomarker for the diagnosis and prognosis and a possible therapeutic target of lung cancer.

Minimal-access retroperitoneal pancreatic necrosectomy for infected necrotizing pancreatitis: a multicentre study of a step-up approach.

BACKGROUND: Various minimally invasive approaches have been described for infected necrotizing pancreatitis. This article describes a modified minimal-access retroperitoneal pancreatic necrosectomy (MARPN) procedure assisted by gas insufflation. METHODS: This retrospective, observational study documented patients who had undergone a step-up MARPN between 1 January 2010 and 31 December 2016. A minimum follow-up of 1 year was required for inclusion. The step-up approach involved percutaneous catheter drainage followed by the modified MARPN and necrosectomy. If more than one access site was needed it was categorized as complex MARPN. RESULTS: Of 212 patients with infected necrotizing pancreatitis, 164 (77·4 per cent) underwent a step-up approach. The median number of percutaneous catheter drains and MARPN procedures was 3 (range 1-7) and 1 (1-6) respectively. Ninety patients (54·9 per cent) underwent complex MARPN. For residual necrosis after MARPN, three patients (1·8 per cent) underwent sinus tract gastroscopy, and 11 (6·7 per cent) had sinography combined with a tube change. However, operations in 13 patients (7·9 per cent) required conversion to open surgery. Postoperative complications developed in 103 patients (62·8 per cent). The mortality rate was 6·1 per cent (10 deaths). CONCLUSION: A step-up approach using a modified MARPN for infected necrotizing pancreatitis is a reasonable option.

ANTECEDENTES: Los procedimientos mínimamente invasivos se han convertido en los más frecuentes para el tratamiento de necrosis pancreáticas infectadas. El objetivo de este estudio fue presentar un procedimiento de necrosectomía pancreática retroperitoneal de acceso mínimo (minimal-access retroperitoneal pancreatic necrosectomy, MARPN) modificado y asistido mediante insuflación de gases, así como evaluar su seguridad y eficacia. MÉTODOS: Se realizó un análisis retrospectivo y observacional de los datos de un hospital desde el 1 de enero de 2010 hasta el 31 de diciembre de 2016. Se incluyeron en el análisis todos los pacientes en los que realizó un abordaje por etapas, que consistía en el drenaje percutáneo mediante la colocación de un catéter seguido de un procedimiento MARPN modificado, en los que se dispusiese de un seguimiento postoperatorio mínimo de 1 año. El MARPN en el lado derecho y la necrosectomía realizada a través de más de un acceso se clasificaron como MARPN complejo. Se evaluaron los resultados radiológicos y quirúrgicos. RESULTADOS: De 212 pacientes con necrosis pancreática infectada, en 164 (77,4%) se realizó un abordaje por etapas. La mediana del número de drenajes percutáneos y procedimientos MARPN fue 3 (rango, 1-7) y 1 (rango, 1-6), respectivamente. En 90 pacientes (54,9%) se realizó un MARPN complejo. Para la exéresis de necrosis residual después de un MARPN, en 3 pacientes (1,8%) se realizó mediante gastroscopia y en 11 pacientes (6,7%) con un recambio de drenaje bajo control radiológico. En 13 pacientes (7,9%) fue necesaria la reconversión a cirugía abierta. Hubo complicaciones postoperatorias en 103 pacientes (62,8%). La tasa de mortalidad fue del 6,1% (n = 10). CONCLUSIÓN: El abordaje por etapas con un MARPN modificado es seguro y efectivo en el tratamiento de la necrosis pancreática infectada.

[Analysis of curative effect of transoral radiofrequency ablation microsurgery on glottic carcinoma with anterior commissure involvement at the early stage].

Objective: To explore the key technique and outcome of transoral radiofrequency ablation microsurgery for early stage of glottic carcinoma with anterior commissure involvement (ACI). Methods: A retrospective analysis was conducted on 31 patients, who were diagnosed as early stage glottic carcinoma during January 2010 to March 2016 in ENT Department. According to whether the anterior commissure was involved or not, two groups were divided. There were eleven cases with ACI (stages T1a, T1b, and T2). Twenty cases without ACI (stages Tis, T1a, and T2). All the patients received transoral radiofrequency ablation microsurgery and followed up closely.Only one case received radiotheraphy after surgery. SPSS19.0 software was used to analyze data. Results: The follow-up time was 12-67 months, and the median follow-up time was 30 months. Nine among 11 cases with ACI obtained good oncologic outcomes, initial local recurrence was identified in 2/11 cases, including 2 cases of T2. Two cases ultimately required salvage total laryngectomy. Meanwhile, initial local recurrence was identified in 2/20 cases without ACI, including 1 case of T1a and 1 case of T2. One case underwent elective neck dissection, and another one received salvage total laryngectomy.Compared to the patients without ACI, it seemed that the cases with ACI always accomponied with a little higher initial local recurrence and lower overall laryngealpreservation, but the difference had no significance (P>0.05). Conclusions: Transoral radiofrequency ablation microsurgery is an effective treatment for glottic carcinoma with ACI. Its advantages, such as more flexibility and deformability, make it more feasible to operate at the narrow space of anterior commissure assisted with laryngeal endoscopy.Good oncologic outcomes can be obtained by this technique with lower initial local recurrence as well as higher overall laryngeal preservation rate.

[Complications analysis of adenoidectomy and tonsillectomy assisted with ablation on children].

Objective:To analyze the complications of adenotonsilectomy assisted with coblation in children. Method:Complications of 2 089 cases of children with adenoid and tonsil surgery assisted with coblation, in our hospital nearly 10 years, were analyzed by epidemiological methods through the method of retrospective analysis. Result:①the sex ratio of male to female was 2.08：1, average age (5.87±3.12) years old, and most of 2 089 cases 76.35% (1 595/2 089) were 3-7 years old; ②all cases underwent adenoidectomy. Different surgery methods of tonsil consisted of three groups as partial resection associated with ablation was 69.17% (1 445/2 089), ablation (channeling) alone was 22.26% (465/2 089) and total resection was 8.57% (179/208). The amount of bleeding in operation was (8.52±3.18)ml, average operation time was (30.15±8.26) minutes, the postoperative pain score was (3.77±1.61); ③The incidence of postoperative complications: postoperative bleeding (all were secondary bleeding cases) rate was 0.24% (5/2 089), recurrence rate was 0.14% (3/2 089), prevertebral lymphadenitis was 0.96% (20/2 089), the other was 0.29% (torus hyperplasia in 2 cases, dyspnea in 2 cases, 1 cases of angle of mouth burned, nasopharyngeal adhesion in 1 cases). Postoperative fever rate was 9.81% (205/2 089). Conclusion:coblation technique is a good method for the treatment of children's adenoids and tonsil diseases with high efficiency and low complications. But improving the operation procedure proficiency level and skills of operation is an important link to reduce complications.

Impact of complete and incomplete revascularization on short- and long-term quality of life in patients with multivessel coronary artery disease.

OBJECTIVE: In this study, we compared the impact of two main strategies of intervention against multivessel coronary disease (MVD), complete revascularization (CR) and incomplete revascularization (IR), on patients' quality of life. PATIENTS AND METHODS: 210 patients with multivessel disease were selected for this study. The diagnosis was confirmed by coronary angiography, and patients were divided in two different groups according to the type of revascularization method they received: (1) the complete revascularization (CR) group and (2) the incomplete revascularization (IR) group. Patients were asked to fill out SF-36 life quality questionnaire in three-time points: (1) before the operation; (2) one month after the operation; and (3) one year after the operation. RESULTS: Baseline conditions showed no significant intergroup differences. Scores obtained from different sections in the questionnaire were compared between two groups and results revealed that scores in every section were significantly different (p < 0.05) except for the general health condition section. Patients in the CR group scored higher in physical functioning, physical role, body pain, vitality, social role, emotional role and mental health. Scores obtained from different time points also varied significantly (p < 0.05), with those of one month and one year after operation being noticeably higher than the preoperative ones. CONCLUSIONS: Both complete and incomplete revascularization methods can effectively improve the short- and long-term quality of life in patients with multivessel coronary artery disease. However, complete revascularization can produce better results.

[Clinical effect analysis of adenoidectomy and tonsillectomy assisted with ablation on children].

Objective:This study aims to explore the clinical effect of adenoidectomy and tonsillectomy assisted with ablation on children.Method:The investigation took the form of retrospective review of 2 089 cases of children applied with adenoidectomy and tonsillectomy assisted with ablation in our hospital in recent 10 years. We obtained data of these children with epidemiological methods based on analyzing the status of general information and operation selections, and then analyzing the scores of snoring and breath preoperation and postoperation.Result:①General information of 2 089 cases followed with: the ratio of male and female was 2.08∶1，the average onset age was（5.87±3.12）years old, mostly ranged from 3 to 7 years old, which consists of 76.35%（1595/2089）of the group.②Different surgery methods of tonsil consisted of three groups as: partial resection associate with ablation was 69.17%(1445/2089), ablation alone was 22.26%(465/2089) and partial resection alone was 8.57%(179/2089) of the group.③A high level scores of snoring and breath more frequently found in preoperative cases than in postoperative cases(P <0.01).There are no differentiation among the scores of above three groups(P >0.05).The postoperative effect evaluation were related to allergic rhinitis, recurrent of tonsillitis, obesity, circular occipital hyperplasia and nasopharyngeal adhesion.Conclusion:The results suggested that surgery assisted with ablation has its advantage in adenoidectomy and tonsillectomy. Individual therapy for different children will improve the curative effect and relieve the pain of operation, thus is worth a wide application.

[Photodynamic therapy by topical drup for the treatment of juvenile onset laryngeal papillomatosis].

Objective:To observe the therapeutic effect of photodynamic therapy(PDT) on the treatment of juvenile onset laryngeal papillomatosis. Method:Twenty-eight cases of children with laryngeal papilloma were treated,only 2 cases for the first time, and the rest were repeatedly treated outside our hospital, the average hospital surgery were more than 4 times. Under self retaining laryngoscope and microscope and endoscope assisted by semiconductor laser and plasma and cold instrument method，visible tumor resection and local affixed deposited 20% 5-aminolevulinic acid(photosensitizer) 3 hours later, with 635 nm semiconductor laser photodynamic,200-280 mW and can volume density of 80 to 120 J/cm², 20 min irradiation. PDT should be repeated after 25 days until no visible tumor.Then,2 times PDT must be done. Result:In 28 cases, 24 cases were followed up for more than 1 years(12 cases were followed up for 3 years),19 had no recurrence, the cure rate was 79.2%(19/24)；5 cases recurrence, and the recurrence rate was 20.8%(5/24)，among them,2 cases were abandoned because of the relapse，the other 3 cases were cure after 3 times of PDT.The main complications were adhesion of larynx. Conclusion:The preliminary effect of PDT by topical drug for the treatment of juvenile onset laryngeal papillomatosis is encouraging. The principle of PDT and the principle of the recurrence of laryngeal papilloma in children were also introduced in this paper.

Comparison of high-intensity focused ultrasound therapy under nasal endoscopy guidance versus first-line drug treatment in patients with persistent allergic rhinitis.

The aim of this study was to determine whether high-intensity focused ultrasound (HIFU) therapy under nasal endoscopy guidance could provide better efficacy and safety in patients with persistent allergic rhinitis (PAR) than the first-line drugs recommended by the World Health Organization. A total of 120 adult patients with PAR were randomly divided into 2 groups (N = 60 each). One group underwent HIFU therapy under nasal endoscopy guidance using an ultrasound rhinitis therapeutic machine. The other group served as the control group and was treated with corticosteroid nasal spray and oral cetirizine hydrochloride. All patients underwent follow-up treatment for 1 year, after which the efficacy and safety were evaluated. There was no significant difference between the two groups (P > 0.05) in the total effective rate. Moreover, no complications such as nasal adhesion, septal perforation, mucosal atrophy, and hyposmia were observed, indicating that HIFU was as effective as the first-line drug treatments recommended by the World Health Organization for symptom relief in PAR patients. The treatment efficacy, repeatability, safety, economical aspects, ease of performance, and few complications of HIFU therapy strongly suggest that HIFU should be routinely incorporated into clinical practice.

EFFECT OF RELATIVE GENE EXPRESSION ON PLAQUE VULNERABILITY IN PATIENTS WITH STABLE ANGINA AND PATIENTS WITH ACUTE CORONARY SYNDROME.

This study was conducted to investigate the effect of relative gene expression on plaque vulnerability in patients with either stable angina or acute coronary syndrome (ACS). A total of 30 patients with ACS, 28 patients with stable angina and 17 healthy volunteers were selected. High resolution ultrasound was used to detect carotid arterial intima-media thickness (IMT) and plaque score, Sandwich enzyme linked immunoassay to determine the change of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1. The three groups had no statistically significant difference in age, gender, total cholesterol, triglyceride, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol. MMP-9, TIMP-1, MMP-9/TIMP-1 and IMT, total plaque score, soft plaque score and hard plaque score of patients’ acute coronary syndrome were obviously higher than those with stable angina and normal people. It was also found that MMP-9 was in a positive correlation with IMT, total and soft plaques score, TIMP-1 was positively correlated with IMT as was MMP-9/TIMP-1. Regardless of age, IMT was in a positive correlation with MMP-9, TIMP-1 and MMP-9/TIMP-1 in partial correlation analysis. All these findings suggest that ACS patients have remarkably higher MMP-9, 1TIMP-1, MMP- 9/TIMP-1, IMT, total plaque score, soft plaque score and hard plaque score compared to patients with stable angina pectoris and healthy subjects (P<0.05) and there are positive correlations between MMP- 9, TIMP-1, 1MMP-8/TIMP-1, total plaque and soft plaque score.

Excision repair cross-complementing 1 expression protects against ischemic injury following middle cerebral artery occlusion in the rat brain.

To study the effects of excision repair cross-complementing 1 (ERCC1) on the pathophysiological process of brain ischemia, we examined the changes in ERCC1 expression, as well as the functional significance of ERCC1 in the rat brain following middle cerebral artery occlusion (MCAO). The results were as follows: (1) ERCC1 immunopositive cells were widely distributed in various brain regions. ERCC1 expression was localized to the nuclei of neurons and astrocytes. (2) ERCC1 expression, as determined by western blot, increased at 3 days, remaining until 14 days, in the ipsilateral cortex and striatum following MCAO. Immunohistochemical analysis demonstrated that ischemia induced increased ERCC1 expression within the periinfarct core, with increasingly less expression toward the core. (3) Knockdown of ERCC1 expression by intraventricular injection of antisense plasmids increased DNA damage and infarct volume in the ischemic brain. (4) ERCC1 overproduction, by injection of expression plasmids, significantly reduced infarct volume and the accumulation of DNA-damaged neurons. Taken together, these results indicate that both endogenous ERCC1 and exogenous ERCC1 have an important neuroprotective function in the brain. In addition, administration of ERCC1 to the brain could prove to be a successful strategy for neuronal protection against ischemic injury.

A novel DFNA5 mutation, IVS8+4 A>G, in the splice donor site of intron 8 causes late-onset non-syndromic hearing loss in a Chinese family.

We report here the clinical, genetic, and molecular characteristics of a large Chinese family exhibiting non-syndromic, late-onset autosomal dominant sensorineural hearing loss. Clinical evaluation revealed variable phenotypes of hearing loss in terms of severity and age-at-onset of disease in these subjects. Genome-wide linkage analysis mapped the disease gene to the DFNA5 locus with a maximum two-point log odds score of 5.39 at [theta] = 0 for marker D7S2457. DNA sequencing of DFNA5 revealed a novel heterozygous IVS8+4 A>G substitution in the splice donor site of intron 8. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed skipping of exon 8 in the mutant transcript. This mutation faithfully cosegregated with hearing loss in the family. In addition, the mutation was absent in 100 unrelated control DNA samples of Chinese origin. The IVS8+4 A>G mutation is predicted to create a shift in the reading frame and introduce a stop codon at position 372, thereby resulting in a prematurely truncated DFNA5 protein. Up to date, a total of four mutations in DFNA5 have been reported to lead to hearing impairment, all of them result in skipping of exon 8 at the mRNA level. Our findings provide further support for the hypothesis that DFNA5-associated hearing loss is caused by a very specific gain-of-function mutation.

Evaluation of cytotoxic activities of some medicinal polypore fungi from China.

The petrol ether, ethyl acetate and methanol extracts of eight medicinal polypore fungi from China were evaluated for cytotoxic activities using MTT-dye assay. All the petrol ether and ethyl acetate extracts exhibited cytotoxicity against human cervix epitheloid carcinoma cell lines (Hela) and human hepatoma cell lines (SMMC-7721). Cytotoxicity activity was also observed in the methanol extracts of Phellinus conchatus and Pycnoporus sanquineus, but the methanol extracts from Cryptoporus volvatus, Fomitopsis pinicola, Fomes hornodermus, Lenzites betulina, Trametes gibbosa and Trametes orientalis showed weak activity when compared with quercetin.

Vascular endothelial growth factor inhibits outward delayed-rectifier potassium currents in acutely isolated hippocampal neurons.

In the present study, whole-cell patch-clamp recording was used to study whether vascular endothelial growth factor (VEGF) had a regulatory effect on the potassium-channel currents. The outward delayed-rectifier potassium currents (I(K)) were recorded in acutely isolated hippocampal neurons from 14-day-old rat brains. A local application of VEGF at the concentrations from 50 ng/ml to 200 ng/ml dose-dependently inhibited I(K). Administration of VEGF (100 ng/ml) to the neurons only for seconds could significantly reduce I(K) in 26 of 39 recorded cells. The currents could recover to 82.8+/-3.7% of the control level at 60 s after removing VEGF in the buffer. In the I-V curve analysis, VEGF negatively shifted the I-V curve of I(K); the inhibition was gradually enhanced as the membrane potential increased from -40 mV to 50 mV in 13 cells. Thus, the results reveal that VEGF inhibits I(K) in acute, reversible and voltage-dependent manners. Double staining combined with confocal laser scanning microscopy was used to simultaneously detect the distribution of VEGF receptors (flt-1 and flk-1) in the hippocampal section and isolated neuron. Results showed that flt-1-positive staining, but not flk-1, could be observed on the membrane of the hippocampal neuron in both preparations, suggesting the presence of neuronal membrane VEGF flt-1 receptors in the hippocampus. To investigate if the inhibition by VEGF on I(K) is related to the presence of flt-1 receptors, we further did flt-1-receptor immunostaining for the recorded neurons, which was labeled with Lucifer Yellow during the recording. Among nine recorded cells, five showing the inhibition by VEGF had detectable signals for flt-1 receptors on their membrane, whereas the other four showing no inhibition had no flt-1 receptors either. The results suggest that VEGF can acutely inhibit I(K) in the hippocampal neurons probably related to the presence of membrane flt-1 receptors in the neurons.

The effects of simulated microgravity on cultured chicken embryonic chondrocytes.

Using the cultured chicken embryonic chondrocytes as a model, the effects of simulated microgravity on the microtubular system of the cellular skeleton, extracellular matrix, alkaline phosphatase activity, intracellular free calcium concentration and mitochondrial ATP synthase activity with its oligomycin inhibition rate were studied with a clinostat. The microtubular content was measured by a flow cytometer. The decrease of microtubular content showed the impairment of the cellular skeleton system. Observation on the extracellular matrix by the scanning electron microscopy showed that it decreased significantly after rotating, and the fibers in the extracellular matrix were more tiny and disorderly than that of the control group. It can be concluded that the simulated microgravity can affect the secreting and assembly of the extracellular matrix. In contrast to the control, there was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization. Meanwhile a significant drop in the intracellular calcium concentration happened at the beginning of rotation. These results indicate that simulated microgravity can suppress matrix calcification of cultured chondrocytes, and intracellular free calcium may be involved in the regulation of matrix calcification as the second signal transmitter. No significant changes happened in the mitochondrial ATP synthase activity and its oligomycin inhibition rate. Perhaps the energy metabolism wasn't affected by the simulated microgravity. The possible mechanisms about them were discussed.

Decreased E-cadherin augments beta-catenin nuclear localization: studies in breast cancer cell lines.

We showed that the YMB-1-derived breast cancer cell line YMB-S, which proliferates in suspension without aggregation, exhibits complete loss of cell-cell adhesion despite the presence of E-cadherin-catenin complex and expression of free beta-catenin in the cytoplasm. Here, we describe beta-catenin gene regulation, interaction with E-cadherin, immunocytochemical localization, and their relation to growth rate in the YMB-1-derived cell line YMB-A, which forms tight junctions and displays anchorage-dependent growth. YMB-A cells proliferated more slowly than YMB-S cells. E-cadherin and APC gene product expression in YMB-A cells was significantly higher than that in YMB-S cells, whereas expression of beta-catenin, MUC1, and c-myc was lower in YMB-A cells than in YMB-S cells. According to immunocytochemical analysis, beta-catenin in YMB-A cells displayed membranous or submembranous localization, indicating that beta-catenin is mostly tethered to E-cadherin. Inhibition of E-cadherin expression in YMB-A cells by an antisense oligonucleotide did not change expression of whole cell beta-catenin protein, but increased nuclear beta-catenin protein level, c-myc expression, and cell growth rate. These results suggest that decreased expression of E-cadherin and APC and increased amount of beta-catenin in YMB-S cells lead to accumulation of beta-catenin in the nucleus, activate beta-catenin-LEF/TCF signaling pathway, and trigger c-myc proto-oncogene expression. c-Myc overexpression in breast cancer may be related to activated Wnt independent beta-catenin-LEF/TCF signaling.

A novel monoclonal antibody, H9, directed against the core protein of MUC1 mucin.

MUC1 mucin is a target protein for many monoclonal antibodies. Human MUC1 detected by a murine anti-KL-6 monoclonal antibody that recognizes a sialylated carbohydrate chain has been designated KL-6/MUC1. Given the heterogeneous antigenicity of KL-6/MUC1, we established a new murine monoclonal antibody, H9, that reacts with epitope DTRP (Asp-Thr-Arg-Pro) peptides within the immunodominant region of the tandem repeat of MUC1 mucin. The reactivity of the H9 antibody differs from that of other previously reported antibodies that recognize the tandem repeat region of MUC1. Immunohistochemical experiments indicate that the reactivity of the H9 antibody is similar to that of other antibodies directed against MUC1 core proteins. A new cancer-associated protein detected by a sandwich assay using the H9 antibody as a catcher and the KL-6 antibody as a tracer is designated HK9. Serum HK9 levels showed a high expression level in lung cancer: 51% (19/37 cases) for adenocarcinoma, 39% (11/28 cases) for squamous cell carcinoma, and 67% (10/15 cases) for small cell carcinoma. The HK9 expression in lung cancer increased with cancer progression. These findings suggest monoclonal antibody H9 to be a novel antibody that reacts with an epitope within the tandem repeat region of MUC1, and that the cancer-associated antigen HK9 may have useful tumor-associated properties.