The polymorphisms of interleukin 17A (IL17A) gene and its association with pediatric asthma in Taiwanese population.

BACKGROUND: The interleukin 17A (IL17A) gene, located on chromosome 6p and linked to asthma phenotype, is a highly potential candidate gene conferring asthma susceptibility. The purpose of this study was to investigate the genetic association between single nucleotide polymorphisms (SNPs) of IL17A and asthma in Taiwanese children. METHODS: We selected and performed genotyping on nine SNPs that encompass the genomic region of IL17A in Taiwanese children with or without asthma. A total of 1939 subjects containing 1027 subjects in testing group and 931 subjects in validation group were recruited in this study. RESULTS: After Bonferroni correction, SNP rs8193036 was found to have a weak association (P = 0.0074 x 9 = 0.066) in genotype frequency test. This association was confirmed by validation group. Logistic regression adjusted allergy comorbidity and gender showed a slightly weaker association. CONCLUSIONS: The results indicated an independent role of IL17A promoter polymorphism rs8193036 in the association with pediatric asthma in Taiwanese population.

The genetic differences with whole genome linkage disequilibrium mapping between responder and non-responder in interferon-alpha and ribavirin combined therapy for chronic hepatitis C patients.

Interferon-alpha and ribavirin combined therapy has been a mainstream treatment for hepatitis C infection. The efficacy of this combined treatment is around 30% to 60%, and the factors affecting the responsiveness are still poorly defined. Our study is intended to investigate the genetic differences between responder and non-responder patients. The genome-wide linkage disequilibrium screening for loci associated with genetic difference between two patient groups was conducted by using 382 autosomal short tandem repeat (STR) markers involving 92 patients. We have identified 19 STR markers displaying different allele frequencies between the two patient groups. In addition, based on their genomic location and biological function, we selected the CD81 and IL15 genes to perform single nucleotide polymorphism genotyping. In conclusion, this study may provide a new approach for identifying the associated polymorphisms and the susceptible loci for interferon-alpha and ribavirin combined therapy in patients with chronic hepatitis C.

Antioxidants attenuate multiple phases of formalin-induced nociceptive response in mice.

An emerging theme in the study of the pathophysiology of chronic and persistent pain is the role of pro-oxidant substances. Reactive oxygen species (ROS) have been implicated in contributing to and/or maintaining conditions of chronic pain. Recent pre-clinical reports suggest that antioxidants are effective analgesics in neuropathic and inflammatory pain models. The present study extends this work by examining the effect of three antioxidants on tissue injury-induced nociception. C57BL6 mice (20-25 g) were pretreated with either phenyl-N-tert-butylnitrone (PBN; 50 mg/kg, i.p.), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy (TEMPOL; 200 or 50 mg/kg, i.p.), N-acetyl-L-cysteine (NAC; 200 or 100mg/kg, i.p.), or vehicle (0.5 ml/100 g), 5 min before intraplantar formalin (10%, 20 microl) injection. Nociceptive responding, indicated by licking or biting the affected hindlimb, was quantified for 30 min after formalin injection. Each drug was effective in attenuating two or more phases (acute, quiescent, and tonic) of the formalin response. To assess putative site of action, intrathecal TEMPOL (380 nmol/5 microl, i.t.) was given 5 min before intraplantar formalin. Intrathecal TEMPOL produced a 83% reduction in nociceptive responding in the tonic phase, but no significant attenuation of the acute phase response. To confirm that the antioxidant property of intrathecal TEMPOL was responsible for its analgesic effect on the formalin-induced pain response, intrathecal TEMPOL was coadministered with the free radical donor tert-butylhydroperoxide (tert-BuOOH). Tert-BuOOH coadminstration reversed the TEMPOL-induced analgesia in the tonic intraplantar formalin response reduction. The data suggest that pro-oxidant species may be important mediators of tissue injury-induced algesia in rodents, and that a spinal site of action is implicated in the tonic response.

Selection of monoclonal antibodies for probing of functional intermediates in incision of UV-irradiated DNA by Uvr(A)BC endonuclease from Escherichia coli.

Monoclonal antibodies (mAbs) were generated that recognize UvrA and UvrB proteins. These proteins are components of the Uvr(A)BC endonuclease, which initiates nucleotide excision repair in Escherichia coli. mAbs, which can be used for probing of structural intermediates of Uvr(A)BC endonuclease functioning, were selected for their ability to: (i) recognize different epitopes; (ii) have a high-affinity for native antigenic protein; (iii) preserve functionality of the Uvr protein in immunocomplex. The adherence of anti-Uvr mAbs with these criteria was verified by additivity and competition tests, and by their influence on the ATPase activities of UvrA and UvrB*, the functionally active proteolytic fragment of UvrB. Two out of twelve anti-UvrA and seven out of thirteen anti-UvrB/anti-UvrB* hybridoma lines were shown to satisfy these criteria. Recognition of UvrA and UvrB deletion mutant proteins by mAbs was used to map their epitopes. Epitopes of A2D1 and A2B1 mAbs were mapped to regions of amino acids 230-281 and 560-680 of UvrA, respectively. Epitopes of anti-UvrB/UvrB* mAbs were assigned to the following amino acid regions of UvrB: B2A1, 8-61; B2C5 and B*2E3, 171-278; B2E2, 631-673; B3C1, 1-7 and/or 62-170; B*2B9, 473-630; B*3E11, 379-472. The ability of selected mAbs to neutralize the incision function of Uvr(A)BC was analyzed. The results are discussed in terms of the applicability of these mAbs to probe the structures of intermediates in the functioning of Uvr(A)BC.

Demonstration of the presence of protease-cutting site in the spacer of hepatitis B viral Pol protein.

Molecular genetic studies have revealed that the human hepatitis B viral (HBV) Pol protein, a polypeptide of about 94 kDa, contains four domains. These are the 5'-terminal protein, spacer, RNA reverse transcriptase/DNA polymerase, and RNase H, respectively, from the amino (N) to carboxy (C) terminus. No evidence indicates as yet the involvement of a specific protease in cleaving the Pol protein or the presence of protease-cutting sites in the Pol protein. An in vitro-translated Pol protein was shown to be cleaved by purified thrombin but not in the presence of its inhibitor, hirudin. Two thrombin-cutting sites, spanning 194 amino acids, were then deduced by thrombin digestion of Pol protein with various lengths of C-terminal deletion. These two putative cutting sites, one located in the spacer region and the other in the beginning of the polymerase region, were found to be conserved at similar positions in the Pol protein of all hepadnaviruses. By using a novel method called the LacZ localization assay (LLA), it was demonstrated that a tripartite fusion protein containing the nucleus localization sequence (NLS) of SV40 large T Ag, the putative thrombin cutting sequence (Ile-Arg-Ile-Pro-Arg320-Thr) of HBV Pol protein and the full length beta-galactosidase of E. coli, exhibited a lower percentage (approximately 53%) of targeting into the nucleus of transfected hepatoma cells when compared with a similar tripartite protein containing a single mutation (Arg320 residue into Trp320) of HBV Pol protein (approximately 78%) or with a bipartite protein of SV40 NLS-beta-galactosidase (approximately 90%). These results indicate that the putative thrombin-cutting site in the spacer region of HBV Pol protein could be cleaved by a cellular protease resulting in the separation of NLS sequence from the beta-galactosidase and rendering a lower frequency of X-gal staining in the nucleus.

Prophylactic anticonvulsants for prevention of immediate and early postcraniotomy seizures.

Phenytoin (15 mg/kg) was administered intravenously to 189 patients shortly before their intracranial, supratentorial surgery was completed. Intravenous phenytoin of 5-6 mg/kg/day in three divided doses was administered daily for the first 3 postoperative days. Therapeutic serum levels (10-20 micrograms/mL) were achieved in 113 (59.8%) patients. An equally constituted, randomized control group of 185 patients received a placebo under identical conditions. The group receiving phenytoin had only one immediate and two early postoperative seizures. The 185 controls had four immediate and nine early postoperative seizures. None of the follow-up computed tomography scans of the patients with seizures showed postoperative hematoma. One patient had a significant tension pneumocranium, a possible cause of postoperative seizures. To avoid a decrease in the serum anticonvulsant level due to intraoperative blood loss, it is suggested that for patients who need an urgent or emergent craniotomy, prophylatic anticonvulsant medication should be given at least 20 minutes before completion of wound closure.

[Community-based cervical cancer screening in seven townships in Taiwan].

Cervical cancer is the leading malignant neoplasm in women in Taiwan. In order to compare the validity of various cervical neoplasia screening methods, estimate the prevalence of low- and high-grade squamous intraepithelial lesions (LSIL and HSIL), and identify risk factors for LSIL and HSIL, a community-based cervical neoplasia screening program was implemented in Sanchi, Chutung, Potzu, Kaohsu, Makung, Huhsi, and Paihsa townships, Taiwan. Both cervical smears and cervicograms were used for the screening of cervical neoplasia. Subjects who had positive cervical smears, cervicogram, or both, were further confirmed by colposcopy-guided biopsy. A total of 10,628 married women aged 30 to 64 years were recruited from seven study townships which gave a response rate of 25.2%. Among 667 subjects who screened positive, 555 (82%) underwent colposcopy-guided biopsy. The age-adjusted prevalence was 3.4% for LSIL and 1.7% for HSIL. The biopsy-confirmed rates for cervical smear-detected LSIL and HSIL were 62.8% and 80.6%, respectively; while 56.6% of minor lesions and 22.2% of major lesions identified by cervicogram were biopsy-confirmed as LSIL and HSIL, respectively. The sensitivity of detecting LSIL was higher for cervicograms (79.3%) than for cervical smears (16.7%), and cervicograms had a lower sensitivity in detecting HSIL (48.4%) than cervical smears (90.0%). Multiple logistic regression analysis showed a striking geographical variation in prevalence of LSIL and HSIL. The prevalence of LSIL decreased with the increase in age, and increased with the duration of taking oral contraceptives. The prevalence of HSIL increased with the parity and the duration of taking oral contraceptives and was also significantly associated with the history of cervical cancer among mother and sisters. It is suggested that improvements in the participation rate of cervical neoplasia screening would promote women's health in Taiwan.

Single nucleotide polymorphisms and haplotype of MD-1 gene associated with high serum IgE phenotype with mite-sensitive allergy in Taiwanese children.

MD-1 (myeloid differentiation 1; also known as Ly86, lymphocyte antigen 86), interacting with RP105, plays an important role in Toll-like receptor 4 (TLR4) signalling pathway. It has been suggested to be involved in the pathological mechanism of inflammation and atopic diseases. The purpose of this study was to investigate the genetic association between single nucleotide polymorphisms (SNPs) of MD-1 promoter and coding region and mite-sensitive allergy in Taiwanese children. We conducted a case-control study on 237 controls and 281 allergic patients sensitive to Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) by genotyping 35 SNPs in MD-1 gene region. In the promoter region we identified three SNPs, rs1334710, rs4959389, and rs977785 that are associated with mite-sensitive allergy in Taiwanese children. The P-values ranged from 0.0150 to 0.009. The haplotypes including promoter region were also associated with mite-sensitive allergy. Our results suggested that MD-1 could be a susceptible gene for mite-sensitive allergy in Taiwanese children.

Evidence for involvement of a ribosomal leaky scanning mechanism in the translation of the hepatitis B virus pol gene from the viral pregenome RNA.

In retroviruses, the pol gene is expressed in the form of a gag-pol fusion protein by the mechanism of ribosomal frameshifting. In studies of the possible mechanism of hepadnaviral pol protein synthesis, recent results have ruled out core-pol fusion protein synthesis by ribosomal frameshifting. In this study, an in vitro transcription and translation coupling system was used to demonstrate that the HBV core and pol proteins could be synthesized independently using the pregenome RNA template. The result has led us to design experiments to distinguish between the involvement of a termination-reinitiation, internal initiation, or leaky scanning mechanism in the pol protein synthesis. In vitro experiments were then carried out to measure the amount of pol proteins being synthesized from (i) the preC mRNA, which contained an extra AUG and seven more nucleotides at the 5'-end in comparison with the pregenome RNA; (ii) the pregenome RNA in the presence of various amounts of antisense RNA annealing to the 5'-end of the pregenome RNA; and (iii) the pregenome RNA with an additional hairpin structure located upstream of the C gene. Results indicated that the synthesis of both core and pol proteins was concomitantly reduced in these three conditions, which suggested that leaky scanning is the most probable mechanism for pol protein synthesis in vitro. To further verify the mechanism in vivo, experiments were performed to assay the activity of DNA polymerase in virions, which were obtained from hepatoma cells transfected by plasmids containing either a wild-type sequence (5'-GGCATGG-3') or an optimal initiation context (5'-ACCATGG-3') of the C gene. Transfection results showed that the plasmid-containing mutations of the C gene significantly decreased the DNA polymerase activity in virions. This observation supports our hypothesis that the leaky scanning model is involved in the synthesis of pol protein.

DNA damage-dependent recruitment of nucleotide excision repair and transcription proteins to Escherichia coli inner membranes.

The entire process of nucleotide excision repair (NER) in Escherichia coli has been reconstituted in vitro from purified proteins and defined DNA substrates. However, how this system is organized in vivo in unclear. We report here the isolation and characterization of macromolecular assemblies containing NER and transcription proteins from E. coli. This ensemble consists of at least 17 proteins. They are recruited, as a consequence of DNA damage induced by UV irradiation, to the inner membrane. The UV-induced 6-4 photoproducts are also relocated to the inner membrane following UV-irradiation of the cells. This recruitment process is dependent on the uvrA, uvrC and recA gene products. These results suggest that at least part of the repair process may associate with the inner membrane and also provide insights into understanding the cellular organization of repair processes.

Rab3A delayed catecholamine secretion from bovine adrenal chromaffin cells.

To study the role of Rab3A in regulated secretion, recombinant Rab3A protein plus various guanine nucleotides were perfused into bovine adrenal chromaffin cells via a patch pipette, and depolarization-evoked catecholamine secretion from individual cells was measured by amperometry. Rab3A plus GTP, GDP, and GTP gamma S all delayed the occurrence of exocytosis while Rab3A, GTP, or GDP alone had no effect. The delay of evoked-secretion is specific for Rab3A plus guanine nucleotides, since treatments with GTP plus BSA or GDP beta S plus Rab3A had no effect on the appearance of secretion events. Rab3A plus GTP increased the frequency of the exocytosis of smaller packets of catecholamine. These results are consistent with the notion that an excess of Rab3A in the cytoplasm depletes the regulatory proteins responsible for regulating the interconversion between GTP- and GDP-bound forms of Rab3A and that Rab3A is involved in the final steps of regulated exocytosis.

Characterization of Rab3A, Rab3B and Rab3C: different biochemical properties and intracellular localization in bovine chromaffin cells.

In this study we examined the biochemical properties and subcellular localization of Rab3A, Rab3B and Rab3C in bovine adrenal chromaffin cells. The Kd for guanosine 5'-[gamma-thio]triphosphate (GTP[S]) of the three Rab3 proteins was 15, 2700 and 204 nM for Rab3A, Rab3B and Rab3C respectively. The intrinsic GTPase activity of the three Rab3 proteins seemed similar and was increased approx. 3-fold by bovine chromaffin cell lysate. Truncation of the C-terminal 31 amino acid residues decreased the binding affinity for GTP[S] of the three Rab3 proteins. When the C-terminus of Rab3C was replaced with that of Rab3A, the binding affinity of Rab3C for GTP[S] was decreased, but the replacement did not affect the affinity of Rab3B for GTP[S]. Immunostaining experiments showed that Rab3A, Rab3B and Rab3C are localized separately within chromaffin cells. Anti-Rab3A and anti-Rab3C antibodies stained vesicle-like structures, whereas anti-Rab3B antibody distinctly stained the plasma membrane. In summary, bovine chromaffin cells express the three Rab3 proteins but the subcellular localization and biochemical properties of the three Rab3 proteins are distinct.

Cytotoxic effects of anthrax lethal toxin on macrophage-like cell line J774A.1.

The cytotoxic effects of anthrax lethal toxin purified from an avirulent strain were examined on mouse macrophage-like J774A.1 cells. Cell death induced by high concentration of purified lethal toxin had the characteristics of necrosis. At lower concentrations, the toxin caused no morphological change and most of the cells were viable. Interestingly, apoptotic cells were observed when the cells were preincubated with a serine/threonine phosphatase inhibitor, calyculin A, and then exposed to a toxin concentration of 0.1 microg/ml. This is the first report that lethal toxin of the anthrax bacillus can induce both necrosis and apoptosis and that protein phosphatases are implicated in the regulation of bacterial toxin-induced apoptosis.

Persistence of Japanese encephalitis virus is associated with abnormal expression of the nonstructural protein NS1 in host cells.

Persistent infection with Japanese encephalitis virus (JEV) was established in murine neuroblastoma N18 cells, and the persistency has been maintained in cell culture for over 6 months. From the persistently infected cells, a clone named C2-2 was selected and expanded to form a stable cell line. The vast majority of C2-2 cells showed viral protein staining by immunofluorescence and continuously produced low levels of virus (10(3) to 10(4) PFU/ml) without marked cytopathic effects or cyclic variations. In addition to the wild-type viral proteins, truncated forms of the viral nonstructural protein 1 (NS1) as well as its derivative NS1' were produced in C2-2 cells. Both truncated NS1 and NS1' contain deletions at their N-termini; however, the analyses by RT-PCR and direct sequencing of the viral RNA failed to detect any truncations or mutations within the NS1 region, suggesting that NS1 truncation was a result of a unique posttranslational proteolytic cleavage of NS1 in the persistently infected cells. Similar but not identical truncation of NS1 was also observed in two other persistently infected cell lines established in Vero and DBT (murine astrocytoma) cells. However, viruses released from C2-2 cells did not produce truncated NS1 upon infection of N18 cells, suggesting that NS1 truncations were the result of virus-cell interaction in persistently infected cells. These data indicate a strong association between abnormal NS1 expression and JEV persistency. A probable involvement of dysfunctional NS1 in the establishment and/or maintenance of JEV persistency in tissue culture is discussed.

Generation and characterization of organ-tropism mutants of Japanese encephalitis virus in vivo and in vitro.

Using gamma-ray irradiation, a pair of virulent (RP-9) and attenuated (RP-2ms) variants of Japanese encephalitis virus (JEV) were generated from a Taiwanese isolate, NT109. The two variants differed in plaque morphology, virus adsorption, and growth properties in BHK-21 cells: (i) RP-2ms produced smaller plaques than RP-9; (ii) RP-2ms adsorbed less efficiently to host cells but yielded a higher virus titer (burst size); and (iii) RP-2ms virions were mostly accumulated intracellularly, whereas RP-9 was released extracellularly. In addition, in an in vitro binding assay, the envelope (E) protein of RP-9, but not that of RP-2ms, bound specifically to a cellular protein of 57-kDa derived from BHK-21 cells. When injected into mice intracerebrally, RP-2ms was much less virulent than RP-9, with 50% lethal doses of > 10(7) and 0.4 plaque forming units, respectively. Moreover, when inoculated intraperitoneally, their organ tropism differed in that the main target organ for RP-2ms was liver, whereas that for RP-9 was brain. These results suggest that RP-2ms was less neurovirulent and less neuroinvasive from peripheral routes. Molecular analysis of the virus structural proteins detected only two differences between RP-9 and RP-2ms: one in E protein, Glu-138 in RP-9 and Lys-138 in RP-2ms, and the other in prM, Tyr-43 in RP-9 and His-43 in RP-2ms. Since the N-terminal 92 amino acids of prM are cleaved and not present in mature JEV virions, the single-amino-acid change of the E protein at position 138 may account for the difference between the mutants in the in vitro binding assay. Such mutation in E protein, or perhaps in conjunction with the prM mutation, may be responsible, in part, for the phenotypic differences observed in vitro and in vivo between the two mutants.