Regulation of 5-hydroxytryptamine-induced calcium mobilization by cAMP-elevating agents in cultured canine tracheal smooth muscle cells

The effects of increases in cellular adenosine 3′5′-cyclic monophosphate (cAMP) on 5-hydroxytryptamine-(5-HT-) induced generation of inositol phosphates (IPs) and increases in intracellular Ca²⁺ ([Ca²⁺]_i) were investigated using canine cultured tracheal smooth muscle cells (TSMCs). Cholera toxin and forskolin induced concentration- and time-dependent cAMP formation with half-maximal effects (−logEC₅₀) produced at concentrations of 7.0 ± 0.5 and 4.9 ± 0.4  respectively. Pretreatment of TSMCs with either forskolin or dibutyryl cAMP inhibited 5-HT-stimulated responses. Even after treatment for 24h, these agents still inhibited the 5-HT-induced Ca²⁺ mobilization. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration response curves of 5-HT. The water-soluble forskolin analogue L-858051 [7-deacetyl-7β-(γ-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated accumulation of IPs. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on this response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-isoquinolinesulphonamide] and HA-1004[N-(2-guanidinoethyl)-5-isoquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IPs. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The AlF₄ ⁻-induced accumulation of IPs was inhibited by forskolin, suggesting that G protein(s) are directly activated by AlF₄ ⁻- and uncoupled from phospholipase C by forskolin treatment. These results suggest that activation of cAMP/PKA might inhibit the 5-HT-stimulated phosphoinositide breakdown and consequently reduce the [Ca²⁺]_i increase or inhibit both responses independently. Received: 14 March 1996/Accepted: 10 April 1996     

Surveillance of respiratory viral infections among pediatric outpatients in northern Taiwan.

BACKGROUND: Viruses are a frequent cause of upper respiratory tract infections in children. Like Taiwan, there were few virological surveillance systems for respiratory viral infections among children in developing countries. MATERIALS AND METHODS: During August 1995 and July 1997, 6-10 throat swab specimens per week were taken from pediatric outpatients with acute, febrile upper respiratory tract infections (URTI). The specimens were randomly obtained by two pediatricians at Chang Gung Children's Hospital and sent for virus isolation and identification. RESULTS: A total of 910 specimens were collected and 365 specimens (40%) were positive for at least 1 virus and included 81 enterovirus, 73 adenovirus, 58 influenza B virus, 54 influenza A virus, 48 cytomegalovirus (CMV), 25 herpes simplex virus-1 (HSV-1), 7 parainfluenza virus, 3 respiratory syncytial virus (RSV) and 16 mixed viruses. Adenovirus and enterovirus were identified throughout the study period. No seasonal variation was noted for adenovirus while enterovirus peaked between May and July and also during September and November. Influenza viruses, both A and B, were identified during two periods, respectively and altogether, influenza viruses could be detected almost throughout the year. An association between the virus type identified and the mean age of patients was found (P-value = 0.0001 by ANOVA test). The mean age of patients infected with influenza viruses, either A or B, was significantly higher than those of patients infected with adenovirus, HSV-1, CMV and enterovirus. CONCLUSION: The results of this study demonstrate that adenovirus and enterovirus are the two most common viruses isolated from pediatric outpatients with acute, febrile URTIs and can be identified throughout the year in northern Taiwan. Influenza viruses also can be identified throughout the year and during the epidemic, a child older than 5 years of age with acute febrile URTI is likely to be a case of influenza.     

Downregulation and abnormal expression of E-cadherin and beta-catenin in nasopharyngeal carcinoma: close association with advanced disease stage and lymph node metastasis

Nasopharyngeal carcinoma (NPC) is predominantly of the undifferentiated histological subtype. Histological differentiation is of limited prognostic significance in NPC. Recent studies have suggested that downregulation of the cadherin-catenin cell adhesion complex may play a crucial role in the initial stage of cancer invasion and metastasis and is associated with poor prognosis in human cancers. Expression of E-cadherin has not been reported previously in NPC, and its prognostic value in NPC is unknown. The purpose of this study was to examine the expression pattern of E-cadherin and its associated partner, beta-catenin, in NPC and their possible applications as prognostic markers to predict the clinical outcome of NPC. Expression of the E-cadherin and beta-catenin was examined by immunohistochemical methods in 74 cases of primary NPC and 17 of their corresponding lymph node metastases. Normal nasopharyngeal epithelium showed strong and homogeneous immunocytochemical staining of E-cadherin and beta-catenin at the cell membranes and intercellular junctions. In contrast, primary NPC showed variable and heterogeneous staining patterns of E-cadherin and beta-catenin. Loss of membranous E-cadherin expression was significantly associated with advanced stages of diseases (P<.001). Eighty percent to ninety percent of NPC in stages IV and V (Ho's staging), respectively, showed a reduced (<35%) membranous staining of E-cadherin compared with normal nasopharyngeal epithelium. Expression of beta-catenin also was downregulated in advanced NPC. Ninety percent to one hundred percent of NPC in stages IV and V (Ho's staging) expressed a reduction (<35%) of imnmunocytochemical staining of beta-catenin. The expression pattern of beta-catenin staining was strongly associated with the expression of E-cadherin (P<.001). Unlike E-cadherin, nuclear staining of beta-catenin expression was observed in some of the primary NPC and lymph node metastasis. Reduced expression of E-cadherin and beta-catenin expression was associated with a shorter survival of NPC patients (P<.001). In advanced NPC patients (stages IV and V), a significant difference in survival was observed in tumors with higher or lower levels of E-cadherin expression (P=.0224, log-rank test). These observations suggests that expression of E-cadherin and beta-catenin may have prognostic values in NPC patients.     

Impact of cadaveric organ donation on Taiwanese donor families during the first 6 months after donation

OBJECTIVE: Organ donation is a complex decision for family members of Asian donors. The impact of cadaveric organ donation on both Chinese and Western donor families has not been well investigated within a cultural framework. The purposes of this study were to follow Chinese family members' appraisal of their decision to donate organs, to explore the possible negative and positive impacts of organ donation on their family life, and to determine what help they expected from healthcare providers during the first 6 months after donation. METHODS: Twenty-two family members (10 men and 12 women) of cadaveric organ donors who signed consent forms at an organ transplant medical center in Taiwan participated in this project and completed in-depth interviews during the sixth month after donation. RESULTS: Participants were 25 to 56 years old (mean = 48.15 +/- 8.31 years). The type of kinship of the participants included the donor's parents, older sister, and spouse. Subjects reported several negative impacts: worry about the donor's afterlife (86%), stress due to controversy among family members over the decision to donate (77%), and stress due to others' devaluation of the donation (45%). Positive impacts reported by the subjects included having a sense of reward for helping others (36%), having an increased appreciation of life (32%), having closer family relationships (23%), and planning to shift life goals to the study of medicine (9%). Subjects expected the transplant team to provide information about organ recipients (73%), to submit the necessary documents so that family members could receive healthcare payments from the insurance company (68%), to help resolve legal proceedings and settlements associated with accidents (64%), and to not overly publicize their decision to donate (64%). CONCLUSIONS: Although all of the subjects reported that organ donation was the right decision, the decision to donate did not protect Taiwanese donor families from negative psychocognitive bereavement. The impacts of organ donation were affected by the subject's social cultural, spiritual, and legal context and the nature of their bereavement.     

An evaluation of behavioral treatments for insomnia in the older adult.

Behavioral treatments were evaluated for their effect on the subjective and objective sleep of older adult insomniacs (N = 53) aged 47 to 76 years. Conditions were support and sleep hygiene, support and sleep hygiene plus progressive relaxation, support and sleep hygiene plus stimulus control, or a measurement control group. The results indicated that all groups, including the measurement control group, were effective in improving the sleep diary assessed awakenings, nap time, and feeling refreshed upon awakening. Subjects at 3 weeks felt less depressed and felt that they had more control over their sleep. Stimulus control was most effective in improving sleep at the posttherapy period. A 2-year follow-up showed that the stimulus control subjects most frequently used the treatment instructions and had shorter sleep latencies and highest sleep quality. Behavioral treatments were found to be effective in improving the perception of sleep among older adult insomniacs.     

The effects of continuous exposure to epidermal growth factor on the spontaneous transformation of cultured rat liver epithelial cells.

A long-term continuous exposure to epidermal growth factor (EGF) enhanced the tumorigenicity of spontaneously transformed cells arising in a clonal population of normal cultured rat liver epithelial cells propagated in a selective growth condition. Lengthy EGF exposure also induced the expression of several phenotypes that differed from the phenotypes of rat liver epithelial cells transformed spontaneously in the absence of EGF. Epidermal growth factor treatment caused consistently an enhancement of the constitutive mRNA expression of transforming growth factor-alpha (TGF-alpha), but not of the EGF receptor and transforming growth factor-beta. The overexpression of TGF-alpha persisted in cell lines derived from tumors formed by the EGF-treated transformed cells. These tumors also exhibited high metastatic incidence and ductal cell differentiation. In contrast, untreated spontaneously transformed cells formed non-metastatic tumors with hepatocellular differentiation. These results suggest that long-term, continuous exposure to EGF/TGF-alpha may modulate the phenotypic expressions of neoplastic transformation.     

Molecular cytogenetic analysis of non-small cell lung carcinoma by spectral karyotyping and comparative genomic hybridization

The overall pattern of chromosomal changes detected by spectral karyotype (SKY) analysis of two cell lines of each major histological subtype of NSCLC, namely squamous cell carcinoma (SQCC) and adenocarcinoma (ADC), indicated a greater degree of chromosomal rearrangement, than was present or predicted by either comparative genomic hybridization (CGH) or G-banding analysis alone. To investigate these observations, CGH was used to screen DNA derived from 8 primary tumors and 15 cell lines. The results indicated that the most frequently gained chromosome arms were 5p (70%), 8q (65%), 15q (52%), 20q (48%), 1q (43%), 19q (39%), 3q (35%), and 11q (35%). Chromosomal losses were less frequently observed, and included 18q (39%), 9 (35%), 6q (30%), 13q (21%), 5q12-q32 (17%), and 19p (17%). Amplifications were found on 2p23-p24, 3q24-q27, 5p, 6cen-p21.1, 6q26, 7p21, 7q31, 8q, 11q13-qter, 20q12-q13.2. Comparison between CGH findings of the two major histological subtypes showed that gains at 1q22-q32.2, 15q, 20q, and losses at 6q, 13q, and 18q was common in ADCs, whereas SQCCs exhibited gains/amplifications at 3q. Distal 8q was gained by CGH in 65% of tumors of both subtypes. Low level MYCC amplification was confirmed by direct fluorescence in situ hybridization (FISH) analysis. The pattern of overall chromosomal changes detected using combinations of molecular cytogenetic analytical methods suggests that it will be easier to detect recurrent subtype-dependent aberrations in NSCLC.     

Rapid detection of beta-globin gene (HBB) mutations coupling heteroduplex and primer-extension analysis by DHPLC

Beta-thalassemia is a common inherited disease, resulting from one or more of a total of more than 200 different mutations in the beta-globin gene (HBB). Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at-risk populations based upon a molecular diagnosis. We have developed a rapid and highly-specific mutation screening test for the diagnosis of beta-thalassemia by coupling heteroduplex and primer-extension analysis based on the denaturing high performance liquid chromatography (DHPLC) system. A total of 161 healthy heterozygous Taiwanese carriers featuring 10 different HBB mutations and 30 patients exhibiting 12 different compound heterozygous or homozygous HBB mutations were subjected to DHPLC. The elution profile for the heteroduplex analysis of DHPLC could be successfully used to identify the common disease-causing mutations of HBB. To further confirm the sequence variants, we developed a technique combining multiplex primer-extension analysis coupled with DHPLC for the genotyping of eight common disease-causing mutations in the HBB gene. Overall, by coupling heteroduplex and primer-extension analysis based upon DHPLC, we were able to unambiguously identify the most-common beta-thalassemia mutations corresponding to more than 99% of HBB alleles among the Taiwanese population. In conclusion, compared to classic approaches to mutation screening for this malady, we suggest that DHPLC is an excellent technique to be applied to the genetic screening of prenatal and postnatal individuals as a part of a diagnosis program for beta-thalassemia and provides a more-efficient, economic, and sensitive means to undertake such a screening program.