Effects of pelvic floor muscle exercise on faecal incontinence in rectal cancer patients after stoma closure.

The purpose of this study was to examine the effects of pelvic floor muscle exercise (PFME) on the faecal incontinence (FI) of rectal cancer patients following stoma closure. Participants were randomly distributed into an exercise group (n = 27) and non-exercise group (n = 26). An experimental design and longitudinal approach were implemented for data collection. Baseline data were collected at 1 day before discharge, and then PFME was taught before the patients were discharged from the hospital. We collected data and followed up with the patients at their pre-discharge visit and at 1, 2, 3, 6 and 9 months after discharge. The Cleveland Clinic Faecal Incontinence (CCI) score was used to measure patient outcome. PFME proved to effectively decrease the degree of FI in stoma closure recipients. The FI score of the exercise group significantly decreased from 8.37 to 2.27 after PFME compared with that of the non-exercise group (from 8.54 to 2.58). The generalised estimation equation tests showed that both group and time were significantly different. The tests also indicated that although PFME appeared to hasten the decline of incontinence, this effect was no longer detectable at 9 months; thus, it may be an effective intervention for FI when implemented up to half a year after discharge.

Effects of herpes simplex virus type 1 infection on immune functions of human neutrophils.

BACKGROUND AND OBJECTIVE: Herpesviruses may play roles in the development of periodontal diseases. This study analyzed the effects of herpes simplex virus type 1 (HSV-1) infection on neutrophil function. The effects of lipopolysaccharide (LPS) from the periodontal pathogen, Porphyromonas gingivalis, during HSV-1 infection were also determined. MATERIAL AND METHODS: Purified HSV-1 was pretreated with buffer containing no serum, with HSV-1 immunoglobulin G (IgG)-positive serum (HSV-1 antiserum) or with control serum. Neutrophils were mock-infected or infected with the pretreated HSV-1. Viral binding and phagosome formation were detected using immunostaining. Intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescin diacetate and fluorometry. Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) were detected using enzyme immunoassays. Release of matrix metalloproteinase-9 (MMP-9) was examined using gelatin zymography. Phosphorylation of Akt/glycogen synthase kinase-3 (GSK-3) was determined using western blotting. RESULTS: HSV-1 bound directly to neutrophils and enhanced the release of MMP-9. HSV-1 immune complexes, formed in the HSV-1 antiserum, bound neutrophils and induced the formation of early phagosome more effectively than did HSV-1 alone. The relative levels of ROS and phosphorylation of Akt/GSK-3 were increased significantly in neutrophils after infection with HSV-1 immune complexes. Infection with HSV-1 and HSV-1 immune complexes also stimulated the production of inflammatory mediators, LTB(4) and IL-8. Moreover, LPS enhanced the HSV-1-stimulatory production of IL-8. CONCLUSION: This study demonstrated differences in neutrophils infected with HSV-1 alone or with HSV-1 immune complexes, suggesting that opsonization of HSV-1 might enhance its effects on neutrophils. The in vitro findings suggest that HSV-1 infection may induce the inflammatory response and affect periodontal health.

Effects of areca nut extract on the apoptosis pathways in human neutrophils.

BACKGROUND AND OBJECTIVE: Areca nut, a major component in area quid, possesses genotoxic and carcinogenic activities. Areca nut extract (ANE) may affect the defensive functions of neutrophils. Recent studies suggest that areca nut chewing is associated with a higher prevalence of periodontal disease as a result of the detrimental effects of ANE on the host defense system. This study examined the effects of ANE on the apoptosis pathways in human neutrophils. MATERIAL AND METHODS: Apoptosis/necrosis of neutrophils was determined using flow cytometry. Proteins involved in the apoptosis pathway were determined using western blotting analysis. RESULTS: The results indicated that ANE reduced early apoptosis, but increased the primary necrosis of neutrophils. ANE may arrest neutrophils in the G0/G1 phase and reduce the apoptotic hypodiploid DNA contents. The levels of cleaved forms of poly(ADP-ribose) polymerase, and of caspase-3 and caspase-8 were decreased by treatment with ANE. Moreover, glycogen synthase kinase-3 alpha/beta may be involved in the ANE-modulated effects of neutrophils. CONCLUSION: Areca nut may regulate death pathways in neutrophils. This may be one mechanism by which areca nut compromises the periodontal health of areca nut chewers.

Areca nut extracts increased expression of inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8, in peripheral blood mononuclear cells.

BACKGROUND AND OBJECTIVE: Cytokines represent a central role in inflammatory tissue destruction and regulate the immune responses that may govern the progression of periodontal diseases. This study investigated the effects of areca nut extracts on the expression of inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8 in peripheral blood mononuclear cells. The role of oxidative stress of areca nut extracts was also examined using curcumin. MATERIAL AND METHODS: The expression of cytokines in peripheral blood mononuclear cells treated with extracts of ripe areca nut or extracts of tender areca nut was analyzed using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. RESULTS: Both extracts of ripe areca nut (< or = 40 microg/mL) and extracts of tender areca nut significantly enhanced the production of tumor necrosis factor-alpha and interleukin-1beta in peripheral blood mononuclear cells in a dose-dependent and time-dependent manner. The kinetics of mRNA expression of both cytokines was also enhanced by areca nut extracts. The stimulatory effects of areca nut extracts on the secretion of tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8 and on the mRNA expression of tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 at 4 h of incubation were reduced by curcumin (20-50 microm). However, the level of interleukin-8 transcripts was not affected by curcumin. Moreover, interleukin-1beta induction by extracts of tender areca nut, but not by extracts of ripe areca nut, was weakened by 10 microm curcumin. The inhibitory effects of curcumin may vary with different cytokines and with different areca nut extract treatments. CONCLUSION: The complex cytokine profile induced by areca nut extracts-treated peripheral blood mononuclear cells implied the possibility of enhanced local inflammation and altered immune functions by the areca chewing habit. The inhibitory effects of curcumin on cytokine expression suggested that oxidative stress might be involved in areca nut extracts-associated immune alteration.

Areca nut extracts-activated secretion of leukotriene B4, and phosphorylation of p38 mitogen-activated protein kinase and elevated intracellular calcium concentrations in human polymorphonuclear leukocytes.

BACKGROUND AND OBJECTIVE: Polymorphonuclear leukocytes are the major source of leukotriene B4, which is synthesized via the 5-lipoxygenase pathway. Activation of the 5-lipoxygenase pathway is regulated by intracellular calcium and the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The impact of areca nut extracts on the biosynthesis of leukotriene B4 by human polymorphonuclear leukocytes was evaluated, and some of the possible mechanisms underlying the responses were examined. MATERIAL AND METHODS: Polymorphonuclear leukocytes were treated with various concentrations of areca nut extracts. The concentrations of leukotriene B4 released into the supernatants were evaluated using enzyme immunoassay. The phosphorylation of p38 MAPK was monitored using immunoblotting, and the cytosolic calcium kinetics were assessed fluorometrically using Fura-2. RESULTS: Exposure of polymorphonuclear leukocytes to areca nut extracts led to a dose-dependent increase in the production of leukotriene B4, with levels peaking at 30 min and decreasing thereafter. Areca nut extracts enhanced the phosphorylation of p38 MAPK, an enzyme known to activate 5-lipoxygenase. Incubation with areca nut extracts also resulted in a rapid elevation of intracellular calcium concentrations in polymorphonuclear leukocytes. The induction of leukotriene B4 by areca nut extracts was suppressed with the p38 MAPK inhibitor, SB203580, or with the intracellular calcium chelator, BAPTA-AM. CONCLUSION: The interaction of areca nut extracts with polymorphonuclear leukocytes activated the arachidonic acid metabolic cascade. Incubation of polymorphonuclear leukocytes with areca nut extracts resulted in the activation of intracellular events, such as phosphorylation of p38 MAPK and Ca2+ mobilization, involved in the release of pro-inflammatory lipid mediators. The results of this study emphasize the potential importance of polymorphonuclear leukocytes as a source of leukotriene B4, which may modulate the inflammatory response in areca chewers.

Areca nut extracts reduce the intracellular reactive oxygen species and release of myeloperoxidase by human polymorphonuclear leukocytes.

BACKGROUND AND OBJECTIVE: Polymorphonuclear leukocytes (PMN) represent the first line of host defense. Areca nut extract inhibits the bactericidal activity of, and the release of superoxide anion (O2- ) by, PMN. This study investigated the effects of areca nut extract on the intracellular production of reactive oxygen species (ROS) and on the extracellular release of lysosomal enzyme, myeloperoxidase (MPO), by PMN. The effects of arecoline, a principal component of areca nut, were also examined. MATERIAL AND METHODS: Human PMN were treated with various concentrations of areca nut extract or arecoline followed by treatment with Hanks' balanced salt solution, with or without cytochalasin B and fMet-Leu-Phe (CB/fMLP). The viability of PMN was determined using propidium iodide staining and flow cytometry. The presence of intracellular ROS was determined using 2',7'-dichlorofluorescin diacetate and fluorometry. MPO release was determined using a substrate assay. RESULTS: Areca nut extract (25 and 50 microg/ml) significantly decreased the viability of PMN. The intracellular levels of ROS and the extracellular release of MPO were induced in PMN by CB/fMLP. Exposure of PMN to areca nut extract (up to 25 microg/ml) or to arecoline (up to 2 mg/ml) did not directly affect the levels of ROS and MPO activity. However, under conditions that did not affect the viability of PMN, the ability of CB/fMLP to trigger production of intracellular ROS and release of MPO in human PMN was significantly suppressed by areca nut extract and arecoline. CONCLUSION: Areca nut impaired the activation of PMN by CB/fMLP that might decrease the effectiveness of PMN in the host defense. Alternatively, exposure of PMN to areca nut extract could decrease the capacity of PMN to damage tissues.

Cytotoxic effects of dental resin liquids on primary gingival fibroblasts and periodontal ligament cells in vitro.

Cytotoxic effects of resin liquids of three in situ relining dental polymers, Alike, Kooliner, and Tokuso Rebase, and their major components, methyl methacrylate (MMA), isobutyl methacrylate (IBMA), and 1,6-hexanediol dimethacrylate (1,6-HDMA) were investigated. The concentrations of major monomers in these resin liquids were determined by high-performance liquid chromatography. Cellular viability of human gingival fibroblasts (GF) and periodontal ligament (PDL) cells were evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay. Moreover, patterns of cell death were analysed using annexin V/propidium iodide staining with flow cytometry. The results indicated that Alike liquid contained 91.3% MMA, Kooliner liquid contained 94.5% IBMA, and Tokuso Rebase liquid contained 65.8% 1,6-HDMA. All materials examined had cytotoxic effects on GF and PDL cells in dose-dependent manners. Tokuso Rebase liquid appeared to be the most cytotoxic among the various resin liquids examined. The effects of Kooliner and Tokuso Rebase liquids may have resulted from IBMA and 1,6-HDMA, respectively. Furthermore, the majority of treated cells died from necrosis; whereas a small portion of cells died from apoptosis. In conclusion, the results demonstrated that these liquid forms of dental polymers and their major monomers cause cytotoxic reactions. The direct relining procedure that cures these materials in situ should be used cautiously.

Expression and roles of herpesvirus entry mediators A and C in cells of oral origin.

The roles of viral glycoprotein D (gD) and cellular herpesvirus entry mediators A (HveA) and C (HveC) in herpes simplex virus entry into oral cells were determined. Studies with purified truncated forms of gD-1, HveA and HveC indicated that these molecules may be involved in herpes simplex virus entry into oral cells. Moreover, HveA was expressed similarly in primary cultures of gingival keratinocytes and fibroblasts, whereas HveC was expressed at higher levels in gingival keratinocytes, as determined by RT-PCR and immunocytochemical staining. Further analysis using immunohistochemistry demonstrated that both HveA and HveC were expressed in epithelial cells, fibroblasts and vascular endothelial cells in gingival tissues. However, only HveC was detected in nerve fibers. Also, HveA was detected throughout the epidermis, whereas HveC was pronounced in the strata basale and spinosum. In conclusion, this study characterized HveA and HveC, molecules that may participate in entry of herpes simplex virus into oral cells.

Association between betel quid chewing, periodontal status and periodontal pathogens.

The present investigation examined whether an association exists between betel quid chewing and signs of periodontal disease and determined the prevalence of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by polymerase chain reaction. The periodontal status of 34 betel quid chewers and 32 non-betel quid chewers were compared. A significantly higher prevalence of bleeding on probing was found in betel quid chewers than non-chewers among the subjects with higher plaque level, greater gingival inflammation, deeper probing depth or greater attachment loss. Also, the results suggested that betel quid chewers may harbor higher levels of infection with A. actinomycetemcomitans and P. gingivalis than non-betel quid chewers. The association persists after adjusting for severity of the clinical parameters. In conclusion, betel quid chewing was associated with a higher prevalence of bleeding on probing where higher clinical levels of disease existed, and with a likelihood of subgingival infection with A. actinomycetemcomitans and P. gingivalis.

High-order MS_CMAC neural network.

A macro structure cerebellar model articulation controller (CMAC) or MS_CMAC was developed by connecting several one-dimensional (1-D) CMACs as a tree structure, which decomposes a multidimensional problem into a set of 1-D subproblems, to reduce the computational complexity in multidimensional CMAC. Additionally, a trapezium scheme is proposed to assist MS_CMAC to model nonlinear systems. However, this trapezium scheme cannot perform a real smooth interpolation, and its working parameters are obtained through cross-validation. A quadratic splines scheme is developed herein to replace the trapezium scheme in MS_CMAC, named high-order MS_CMAC (HMS_CMAC). The quadratic splines scheme systematically transforms the stepwise weight contents of CMACs in MS_CMAC into smooth weight contents to perform the smooth outputs. Test results affirm that the HMS_CMAC has acceptable generalization in continuous function-mapping problems for nonoverlapping association in training instances. Nonoverlapping association in training instances not only significantly reduces the number of training instances needed, but also requires only one learning cycle in the learning stage.

Effects of areca nut extracts on the functions of human neutrophils in vitro.

Aqueous extracts of ripe areca nut without husk (ripe ANE) and fresh and tender areca nut with husk (tender ANE) were examined for their effects on the defensive functions of human neutrophils. Exposure of peripheral blood neutrophils to ripe ANE and tender ANE inhibited their bactericidal activity against oral pathogens, including Actinobacillus actinomycetemcomitans and Streptococcus mutans, in a dose-dependent manner. At the concentrations tested, ripe and tender ANEs did not significantly affect the viability of neutrophils as verified by their ability to exclude trypan blue dye. However, both ANEs inhibited the production of bactericidal superoxide anion by neutrophils as measured by cytochrome c reduction. Moreover, the ripe ANE inhibited neutrophils more effectively than did tender ANE. Arecoline, a major alkaloid of areca nut, only exhibited an inhibitory effect on the functions of neutrophils when high concentrations were used. Therefore, arecoline could not be used to explain the inhibitory effects observed for ANEs. In conclusion, our results demonstrated that ripe and tender ANEs reduced the antibacterial activity and the superoxide anion production of neutrophils. This effect may contribute to a less efficient elimination of bacteria from the periodontal environment. Inhibition of the antimicrobial functions of neutrophils may alter the microbial ecology of the oral cavity, and this may be one possible mechanism by which areca nut compromises the oral health of users of areca nut products.

Recurrent lacrimal sac papilloma: case report.

Tumors of the lacrimal sac are rare. Benign papillomas comprise approximately 40% of all neoplasms of the lacrimal drainage system. They often present insidiously with symptoms of dacryostenosis or dacryocystitis. Recurrent bouts of dacryocystitis and nasolacrimal duct obstruction were reported in a 35-year-old man over a period of 13 years. A medial canthal mass was noted in the 6th year after the onset of symptoms. A tumor was discovered incidentally during surgical intervention for presumed dacryostenosis. Surgical removal of the tumor and dacryocystorhinostomy were performed. The histopathologic report turned out to be benign papiloma. Local recurrences occurred during the follow-up period. In addition to surgical excision, we applied cryotherapy and CO2 laser to prevent further recurrence. This case we presented the characteristic recurrence of lacrimal sac papilloma and implied the possibility of tumor occurrence in a patient with recurrent dacryocystitis. We must bear in mind that a recurrent dacryocystitis may be a presentation of a lacrimal sac tumor, because early diagnosis and aggressive treatment can prevent recurrence and result in a cure.

Analysis of the steps involved in Dengue virus entry into host cells.

The initial steps of dengue viral entry have been divided into adsorption and penetration using acid glycine treatment to inactivate extracellular virus after attachment to baby hamster kidney (BHK) cells but prior to penetration. First, we showed that virus infection was accomplished within 2 h after adsorption. Second, the assay was used to examine the properties of dengue envelope E protein-specific monoclonal antibodies (MAbs), lectins, and heparin. We found that three MAbs, 17-2, 46-9, and 51-3, may neutralize dengue 2 virus (DEN-2) through inhibition of not only viral attachment but also of penetration. However, one MAb, 56-3.1, interfered specifically with attachment. Therefore, the functional domains of E protein involved in attachment and penetration may be different. Moreover, studies with lectins indicated that carbohydrates, especially alpha-mannose residues, present on the virion glycoproteins may contribute to binding and penetration of the virus into BHK and mosquito C6/36 cells. Finally, virus infectivity was inhibited by heparin through its blocking effects at both virus attachment and penetration. This suggests that cell surface heparan sulfate functions in both viral attachment and penetration of DEN-2 virus. In conclusion, our results further elucidated some aspects of the dengue virus entry process.

Sequences regulating poly(A) site selection within the adenovirus major late transcription unit influence the interaction of constitutive processing factors with the pre-mRNA.

The adenovirus major late transcription unit (MLTU) encodes five families of mRNAs, L1 to L5, each distinguished by a unique poly(A) site. Use of the promoter-proximal L1 poly(A) site predominates during early infection, whereas poly(A) site choice shifts to the promoter-distal sites during late infection. A mini-MLTU containing only the L1 and L3 poly(A) sites has been shown to reproduce this processing switch. In vivo analysis has revealed that sequences extending 5' and 3' of the L1 core poly(A) site are required for efficient processing as well as for regulated expression. By replacement of the L1 core poly(A) site with that of the ground squirrel hepatitis virus poly(A) site, we now demonstrate that the L1 flanking sequences can enhance the processing of a heterologous poly(A). Upon recombination of the chimeric L1-ground squirrel hepatitis virus poly(A) site onto the viral chromosome, the L1 flanking sequences were also found to be sufficient to reproduce the processing switch during the course of viral infection. Subsequent in vitro analysis has shown that the L1 flanking sequences function to enhance the stability of binding of cleavage and polyadenylation specificity factor to the core poly(A) site. The impact of L1 flanking sequences on the binding of cleavage and polyadenylation specificity factor suggests that the regulation of the MLTU poly(A) site selection is mediated by the interaction of constitutive processing factors.

The interaction of glycoprotein C of herpes simplex virus types 1 and 2 with the alternative complement pathway.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) binds the human complement fragment C3b, but the two proteins differ in their ability to bind C3b on infected cell surfaces. In addition, gC-1, but not gC-2, accelerates the decay of the alternative pathway C3 convertase, thereby affecting later steps of the complement cascade. Previously, we constructed linker insertion and deletion mutants of gC-1 and gC-2 and used transient transfection to express mutant proteins in uninfected cells. In spite of the differences between gC-1 and gC-2, C3b binding was localized to residues within the central portion of both proteins, encompassing the first four cysteines. For gC-1, deletion mutants lacking amino acids 33 to 123 or 367 to 469 or lacking both regions still bound C3b. We recombined these deleted forms of gC-1 into gC-39, an HSV-1 strain lacking the gC gene. The altered forms of gC-1 were incorporated into virions, expressed on the surface of infected cells, and bound C3b. We used these proteins to investigate the structural basis for the inhibitory action of gC-1 on the complement cascade. We found that gC-1 does not inhibit formation of the alternative pathway C3 convertase. This convertase is stabilized by the serum protein properdin. Purified gC-1, but not gC-2, inhibits the binding of properdin to C3b, suggesting that this destabilizes the convertase. The mutant lacking amino acids 367 to 449 was able to inhibit properdin binding to a limited extent when present at high concentrations, although it bound to C3b more weakly than wild-type gC. In contrast, the protein lacking amino acids 33 to 123 was unable to inhibit properdin binding to C3b. Thus, gC-1 contains two structural domains, one for C3b binding, residues 124 to 366, and another, residues 33 to 133, which interferes with properdin binding to C3b.

A parallel genetic/neural network learning algorithm for MIMD shared memory machines.

A new algorithm is presented for training of multilayer feedforward neural networks by integrating a genetic algorithm with an adaptive conjugate gradient neural network learning algorithm. The parallel hybrid learning algorithm has been implemented in C on an MIMD shared memory machine (Cray Y-MP8/864 supercomputer). It has been applied to two different domains, engineering design and image recognition. The performance of the algorithm has been evaluated by applying it to three examples. The superior convergence property of the parallel hybrid neural network learning algorithm presented in this paper is demonstrated.

Limited joint mobility of the hand: prevalence and relation to chronic complications in non-insulin-dependent diabetes mellitus patients.

A total of 170 non-insulin-dependent diabetes mellitus (NIDDM) patients admitted to Kaohsiung Chang Gung Memorial Hospital during the period from January 1988 to December 1989 were examined for the presence of cheiroarthropathy. Fifty-four percent (92 patients) had limited joint mobility (LJM) of the hand, a prevalence significantly higher than that of non-diabetic controls (7%). Duration of diabetes was positively related to the severity of LJM (p = 0.03, chi-square contingency table). Chronic diabetic complications such as cataracts (p = 0.04) and overt proteinuria (p = 0.03) were also associated with the presence of LJM, and diabetic retinopathy (p = 0.007); vision-threatening retinopathy was strongly associated with the presence of LJM (p = 0.0002). The sex ratio, HbA1c and creatinine clearance rates did not show significant differences between patients with and without LJM. In conclusion, the occurrence of LJM of the hand in NIDDM patients observed at this hospital is common and is often accompanied by other chronic diabetic complications. Its presence should alert physicians to other associated diabetic complications with increased morbidity and mortality.

Structural basis of C3b binding by glycoprotein C of herpes simplex virus.

Glycoproteins C (gC) from herpes simplex virus type 1 (HSV-1) and HSV-2, gC-1 and gC-2, bind the human complement fragment C3b, although the two glycoproteins differ in their abilities to act as C3b receptors on infected cells and in their effects on the alternative complement pathway. Previously, we identified three regions of gC-2 (I, II, and III) which are important for C3b binding. In this study, our goal was to identify C3b-binding sites on gC-1 and to continue our analysis of gC-2. We constructed a large panel of mutants by using the cloned gC-1 and gC-2 genes. Most of the mutant proteins were transported to the surface of transiently transfected L cells and reacted with one or more monoclonal antibodies to discontinuous epitopes. By using 31 linker insertion mutants spread across the coding region of gC-1, we identified four regions in the ectodomain of gC-1 which are important for C3b binding, three of which are similar in position to C3b-binding regions I, II, and III of gC-2. Region III shares some similarities with the short consensus repeat found in CR1, the human complement receptor. These were, in part, the targets for construction of 20 single amino acid changes in region III of gC-1 and gC-2. These mutants identified similarities and differences in the C3b-binding properties of gC-1 and gC-2 and suggest that the amino half of region III is more important for C3b binding. However, our results do not support the concept of a structural relationship between the short consensus repeat of CR1 and gC, since mutations of some of the conserved residues, including three of four cysteines in region III, had no effect on C3b binding. Finally, we constructed four deletion mutants of gC-1, including one which lacked residues 33 to 123, as well as residues 367 to 449. This severely truncated molecule, lacking four cysteines and five potential N-linked glycosylation sites, was transported to the cell surface and retained its ability to bind monoclonal antibodies as well as C3b. Thus, the four distinct C3b-binding regions of gC-1 and several epitopes within two different antigenic sites are localized within residues 124 to 366.

The 9-kDa hydrophobic protein encoded at the 3' end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated.

The open reading frame potentially encoding a 78 amino acid, 9101 Da hydrophobic protein (HP) and, mapping at the 3' end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome, was shown to be expressed during virus replication. The cloned HP gene was placed in a plasmid under control of the T7 RNA polymerase promoter and in vitro translation of transcripts generated in vitro yielded a 9.1-kDa protein that was immunoprecipitable with porcine hyperimmune anti-TGEV serum. Antiserum raised in rabbits against a 31 amino acid synthetic polypeptide that represented the central hydrophilic region of HP specifically immunoprecipitated HP from TGEV-infected cells. HP was further shown to become associated with microsomal membranes during synthesis in vitro and was found to be closely associated with the endoplasmic reticulum and cell surface membranes in infected cells. The intracellular location of HP suggests that it may play a role in the membrane association of replication complexes or in virion assembly.