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Background/aims: Auscultation of bowel sounds is a traditional technique for evaluating patients with abdominal symptoms. It is, however, subjective and qualitative method in general. Recently, analysis of bowel sounds becomes possible. We analyzed bowel sounds in healthy volunteers and measured platelet depleted plasma 5-hydroxytryptamine (5-HT) that may be associated with postprandial symptoms in irritable bowel syndrome., Methods: We recorded both fasting and postprandial bowel sounds for 30 minutes in 16 healthy volunteers with a sensitive electronic stethoscope attached to a digital recorder. The files were saved in computer as wav files and analyzed with a specialized program. Blood samples were also taken before and 1 hour after meal for 5-HT analysis., Results: Meal challenge made no statistically significant changes in the 5-HT concentrations and all the sound parameters including sound to sound interval, sounds/minute, average of sound amplitudes, sound length, percentage of bowel sounds representing sound clustering and dominant frequency of sounds., Conclusions: Postprandial changes in bowel sounds and plasma 5-HT were insignificant in healthy Korean volunteers.

Background/aims: The clinical usefulness of urinary trypsinogen-2 dipstick test is still in controversy. We evaluated the usefulness of urinary trypsinogen-2 dipstick test in patients with acute pancreatitis., Methods: Urinary trypsinogen-2 dipstick test was prospectively performed in 50 patients with acute pancreatitis, 50 patients with non-pancreatic abdominal pain, and 50 healthy controls., Results: On admission, urinary trypsinogen-2 dipstick test was positive in 36 of 50 patients with acute pancreatitis (sensitivity, 72%) and in 4 of 50 patients with non-pancreatic abdominal pain (specificity, 92%). On the other hand, it was all negative in controls. The sensitivity and specificity of serum lipase were 78% and 94%, respectively. At 24 hours after admission, the positive rate of urinary trypsinogen-2 dipstick test rose from 72% to 94% (p=0.02). The results of urinary trypsinogen-2 dipstick test was positive in 14 of 15 patients with severe pancreatitis and 22 of 35 patients with mild pancreatitis according to the criteria by Atlanta International Symposium, 1992., Conclusions: Urinary trypsinogen-2 dipstick test is comparable to serum lipase in diagnosing acute pancreatitis. Delayed measurement and severe pancreatitis are more likely to yield positive results with urinary trypsinogen-2 dipstick test. Thus, we suggest that the cut-off value of urinary trypsinogen-2 dipstick test should be lowered to increase its sensitivity.

The total culturable virus assay (TCVA) and an integrated cell culture-PCR (ICC-PCR) were compared in parallel to evaluate their detection reliability. Source, finished, and tap water samples from three drinking water treatment plant systems were analyzed by TCVA, and every cell culture dish was subsequently examined by reverse transcription (RT) multiplex PCR using enterovirus- and adenovirus-specific primers. Twenty-seven of 180 (15%) inoculated dishes exhibited cytopathic effects (CPE). Virus concentrations for source water ranged from 3.3 to 21.0 most probable numbers of infectious units (MPN) per 100 liters. No finished or tap water samples were positive. On the other hand, 38 (21%) of the dishes were positive in multiplex ICC-PCR. Virus concentrations ranged from 4.5 to 10.2 MPN/100 liters for source water and 0 to 0.9 MPN/100 liters for finished and tap water. In spite of its superior sensitivity, the ICC-PCR assay resulted in lower virus concentration values than the TCVA for two of the source water sites. Retest of the CPE-positive dishes using reovirus-specific RT-PCR revealed that 24 of the 27 (89%) dishes were also positive for reoviruses. These observations suggested that the detection reliability of ICC-PCR is restricted by the primer sets that are integrated in the reaction mixture. The observation of an uneven distribution of PCR-positive culture dishes in a given sample raises an additional caution that simple extrapolation of the ICC-PCR result from the analysis of a limited fraction of collected samples should be avoided to minimize possible over- and underestimation of the amount of virus.

A series of N-4-methansulfonamidobenzyl-N'-2-substituted-4-tert-butylbenzyl thioureas were prepared for the study of their agonistic/antagonistic activities to the vanilloid receptor in rat DRG neurons. Their structure-activity relationship reveals that there is a space for another hydrophobic binding interaction around 2-position in 4-tert-butylbenzyl region. Among the prepared derivatives, 6n show the highest antagonistic activity against the vanilloid receptor (IC(50)=15 nM).

Objectives: The association of trimethoprim-resistant dfr genes with integrons was investigated in urinary Escherichia coli isolates in Korea from the last two decades., Methods: Of 623 E. coli isolates from urine specimens, 421 trimethoprim-resistant isolates were studied for dfr genes associated with integrons. Integrase genes were amplified and the PCR products restricted using HinfI to classify integron types. Gene cassette regions for the class 1 and class 2 integrons were amplified and sequenced. PFGE was performed to determine the epidemiological relationship of E. coli isolates., Results: The carriage of class 1 integrons was found to be significantly higher in trimethoprim-resistant isolates (69%) than in trimethoprim-susceptible isolates (19%). Among the trimethoprim-resistant isolates, the frequency of dfr genes associated with class 1 integrons increased sharply from 10% of the isolates during 1980-1985 to 53% during 1996-1997 and to 46% during 2001-2002. Five different dfr cassettes--dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17--were identified among the urinary E. coli isolates from the last two decades; dfrA12 was the most prevalent during 1980-1985 and dfrA17 during 1996-1997 and 2001-2002. The majority of dfr genes associated with class 1 integrons were conjugally transferable to recipient E. coli strains. The E. coli isolates that carried dfrA17 associated with class 1 integrons were found to be phylogenetically unrelated, indicating that dfrA17 was widely distributed in the different clones of E. coli., Conclusion: Class 1 integrons were found to be an important genetic element of resistance to trimethoprim among urinary E. coli in Korea, and the prevalence of dfrA17 was mainly due to the horizontal transfer of class 1 integrons through conjugative plasmids.

Background/aims: The blended current is usually used for endoscopic sphincterotomy (EST) to minimize bleeding. The pure cutting current may induce less edema of the ampulla and therefore result in less injury to the pancreas theoretically. The aim of this study was to evaluate effects of electric currents used on the development of serum pancreatic enzyme evaluation, clinical pancreatitis or bleeding after EST., Methods: One hundred and eighteen consecutive patients who underwent EST with standard papillotome alone for the treatment of choledocholithiasis were reviewed. All EST had been performed by two endoscopists whose experience on EST was similar: one uses 'blended current' (BC group, n=74), while the other uses 'pure cutting current' (PC group, n=44)., Results: Baseline clinical, laboratory, and procedural parameters were similar in both groups. The incidences of hyperamylasemia and hyperlipasemia were similar between two groups. There was no significant difference in the incidence of clinical pancreatitis between two groups (BC 6.8% vs PC 0.0%, p=0.1557). All episodes of pancreatitis were mild. No episodes of significant bleeding occurred after EST. The incidences of sepsis, cholangitis and perforation were also not different between two groups., Conclusions: Development of complications after standard EST such as hyperamylasemia, clinical pancreatitis, and bleeding may not depend on the electric current used.

5-Aryl-2,2-dialkyl-4-phenyl-3(2H)furanone derivatives were studied as a novel class of selective cyclooxygenase-2 inhibitors with regard to synthesis, in vitro SAR, antiinflammatory activities, pharmacokinetic considerations, and gastric safety. 1f, a representative compound for methyl sulfone derivatives, showed a COX-2 IC(50) comparable to that of rofecoxib. In case of 20b, a representative compound for sulfonamide derivatives, a potent antiinflammatory ED(50) of 0.1 mg kg(-1) day(-1) was observed against adjuvant-induced arthritis by a preventive model, positioning 20b as one of the most potent COX-2 inhibitors ever reported. Furthermore, 20b showed strong analgesic activity as indicated by its ED(50) of 0.25 mg/kg against carrageenan-induced thermal hyperalgesia in the Sprague-Dawley rat. 3(2H)Furanone derivatives showed due gastric safety profiles as selective COX-2 inhibitors upon 7-day repeat dosing. A highly potent COX-2 inhibitor of the 3(2H)furanone scaffold could be considered suitable for a future generation COX-2 selective arthritis medication with improved safety profiles.

Gene expression and RNA interference phenotypes were investigated for a Caenorhabditis elegans homologue (Ce-RCQ-5) of human RecQ5 protein. Expression of the mRNA was observed by in situ hybridization from earliest embryogenesis and gradually decreased during late embryogenesis. Ce-RCQ-5 was immuno-localized in the nuclei of embryos, germ cells, and oocytes and also in the nuclei of various somatic cells of larvae and adults. Despite ubiquitous expression in postembryonic cells, RCQ-5 protein expression was highest in intestinal cells, which was confirmed by tagging the gene expression with green fluorescence protein. When endogenous Ce-rcq-5 gene expression was inhibited by RNA interference, no clear phenotypes were observed during development. However, C. elegans life span was reduced by 37% due to RNA interference of rcq-5 gene, suggesting its possible role in maintenance of genomic stability, as has been ascribed to other RecQ family DNA helicases. In addition, C. elegans became significantly more sensitive to ionizing radiation after inhibition of rcq-5 gene expression, indicating an involvement of C. elegans RCQ-5 in a cellular response to DNA damage, possibly in DNA repair.

Gene cassettes of class 1 integrons in Escherichia coli isolates from urine specimens collected in Korea during the last 2 decades were characterized. intI1 was detected in 54% of the isolates, yet gene cassette regions were amplified in only 43% of the isolates. intI2 was detected in 29 (5%) isolates, and no intI3 was detected in this study. Twenty-one different genes, including genes encoding resistance to antibiotics, an alcohol dehydrogenase gene (adhE), and unknown genes, were detected. The genes most commonly found in class 1 integrons were those for aminoglycoside and trimethoprim resistance. The occurrence of aminoglycoside resistance genes in class 1 integrons decreased, and the presence of dfr genes increased rapidly, during the last 2 decades. Single-gene cassettes were predominant during the 1980s, while multigene cassettes predominated from the 1990s on. The aadA1, aadA2, and blaP1-aadA2 gene cassettes were frequently found in isolates from the 1980s but were not detected in isolates recovered since 2000. dfrA12-aadA2 and dfrA17-aadA5 were the most prevalent gene cassettes among isolates recovered from the 1990s on. In conclusion, class 1 integrons would appear to be responsible for resistance to antibiotics commonly used to treat urinary tract infections, and selection of a specific gene cassette was found to occur over the course of time.

The resistance to ampicillin and nalidixic acid in Shigella sonnei isolates obtained in Korea during the period 1998 to 2000 was characterized. Recently (J. Y. Oh, H. S. Yu, S. K. Kim, S. Y. Seol, D. T. Cho, and J. C. Lee, J. Clin. Microbiol. 41:421-423, 2003) ampicillin and nalidixic acid resistance was found in 49 and 70%, respectively, of the 67 S. sonnei isolates obtained during this period. We analyzed 138 S. sonnei isolates collected during the same period. Ampicillin and nalidixic acid resistance was found in 30 and 86% of the isolates, respectively. The ampicillin resistance was mediated by a TEM-1 beta-lactamase, and TEM-52 extended-spectrum beta-lactamase was identified in one sporadic S. sonnei isolate from 1999. bla(TEM-1) and bla(TEM-52) were located in conjugative R-plasmids. Tn3 was detected in 41% of the ampicillin-resistant isolates. The R-plasmids from the transconjugants that transferred resistance to ampicillin exhibited different restriction fragment length polymorphism patterns, and a bla(TEM-1) probe was hybridized with the different fragments. The nalidixic acid resistance was exclusively associated with an amino acid substitution, Ser83-->Leu (TCG-->TTG), in gyrA. These findings indicate that the genetically related S. sonnei strains readily acquire resistance to ampicillin, streptomycin, trimethoprim, and sulfamethoxazole but not nalidixic acid through conjugative R-plasmids from difference sources when confronted by antibiotic selective pressures.

A series of acyclic phenethylthiocarbamate derivatives have been synthesized, and their antagonist effect against vanilloid receptor tested. Chain branching led to a significant change in antagonist activity of the parent molecule. Ethyl-branched 1e showed a 6.3 microM of IC(50) value in 45Ca(2+)-influx assay.

A 10-day old male calf exhibited multiple congenital anomalies of the urinary and gastrointestinal tracts, including renal fusion (horseshoe kidney), ureteral fusion, rectovesicular fistula, and atresia ani. In horseshoe kidney, the organs were fused together at the caudal poles. The left kidney and cranial half of right kidney were shrunken, while the remaining lobules were hypertrophic. Ureters were fused cranially and bifurcated caudally. The terminal rectum was narrowed and connected with the bladder. The anus was imperforate. The cause of these anomalies could not be determined.

Four pyrrolidine derivatives were prepared by the formation of a 5-membered ring based on capsazepine. Among them, the two carbon extended derivatives, 4a (IC(50)=55 microM) and 4b (IC(50)=3 microM), both showed different levels of antagonist activity against the vanilloid receptor in a (45)Ca(2+)-influx assay.

The characteristics of ammonia removal by two types of biofilter (a standard biofilter with vertical gas flow and a modified biofilter with horizontal gas flow) were investigated. A mixture of organic materials such as compost, bark, and peat was used as the biofilter media based on the small-scale column test for media selection. Complete removal capacity, defined as the maximum inlet load of ammonia that was completely removed, was obtained. The modified biofilter showed complete removal up to 1.0 g N/kg dry material/day. However, the removal capacity of the standard biofilter started to deviate from complete removal around 0.4 g N/kg dry material/day, indicating that the modified biofilter system has higher removal efficiency than the standard upflow one. In kinetic analysis of the biological removal of ammonia in each biofilter system, the maximum removal rate, Vm, was 0.93 g N/kg dry material/day and the saturation constant, Ks, was 32.55 ppm in the standard biofilter. On the other hand, the values of Vm and Ks were 1.66 g N/kg dry material/day and 74.25 ppm, respectively, in the modified biofilter system.

Dumbbell-shaped molecules consisting of three biphenyls connected through vinyl linkages as a conjugated rod segment and aliphatic polyether dendritic wedges with different cross-sections (i.e., dibranch (1), tetrabranch (2) and hexabranch (3)) were synthesized and characterized. The molecular dumbbells self-assemble into discrete bundles that organize into three-dimensional superlattices. Molecule 1, based on a dibranched dendritic wedge, organizes into primitive monoclinic-crystalline and body-centered, tetragonal liquid crystalline structures, while molecules 2 and 3, based on tetra- and hexabranched dendritic wedges, respectively, form only body-centered, tetragonal liquid crystalline structures. X-ray diffraction experiments and density measurements showed that the rod-bundle cross-sectional area decreases with increasing cross-section of the dendritic wedges. The influences of supramolecular structure on the bulk-state optical properties were investigated by measuring the UV/Vis absorption and steady state fluorescence spectroscopies. As the cross-section of the dendritic wedge of the molecule increases, the absorption and emission maxima shift to higher energy. This can be attributed to a quantum size effect of the three-dimensionally confined nanostructure.

The aim of this study was to determine whether formalin reliably provokes a paw edema and pain behavior. The paw of male Sprague-Dawley rats were injected with 100 microliter of formalin with 2.5% (F2.5), 5% (F5), and 10% (F10) concentrations. Following the formalin (n=8) or saline (control, n=6) injection, the flinching or licking of the paw was recorded for the phase 1 response (0-5 min after injection) and phase 2 response (20-60 min). The formalin-induced paw edema was assessed by measuring the diameters of the injected paws at 4 hr after injection. As for flinching, phase 1 and 2 of all three groups showed higher frequency than those of the control group (p<0.05). As for licking, phase 1 cumulative time of the F2.5 and F10 groups, and phase 2 cumulative time of the F2.5 and F5 groups showed a longer duration than those in the control group (p<0.05). The diameters of the paw in the F10 group were significantly larger than those in the control group (p<0.05). Flinching behavior was more reliably expressed the biphasic response than licking response at all formalin concentrations. Peak of the licking was reached at 2.5% and that of flinching was reached at 5%, whereas the paw edema peaked at 10% concentration. This suggests that there may be some dissociation of nociception from the edema formation.

The 3' noncoding region (NCR) of Japanese encephalitis (JE) viruses isolated in Korea and Nakayama-NIH strain have been sequenced and compared with the 3' NCR sequences of other JE isolates reported previously. Sequence alignment of about 60 nucleotides (based on consensus sequence number) immediately downstream of the open reading frame (ORF) stop codon in the 3' NCR of the Korean isolates showed high degree of sequence variation and deletion; thus, this region was termed as the variable region. However, in the predicted RNA secondary structures, a similar type loop exists at the 5'-terminus of the 3' NCR of JE viruses, despite low level of sequence homology (22%) and deletion in the variable region. The phylogenetic tree based on the 3' NCR sequences of JE viruses including the variable region showed a similar pattern to that based on envelope genes; in that, there are two genetically different types of JE viruses in Korea. Therefore, the variable region would be a useful genetic marker for JE viruses.

A new guaiane type sesquiterpene was isolated from the fruit of Torilis japonica (Umbelliferae). Based on NMR, IR and mass spectroscopy its structure was confirmed as deangeloyloxy torilin, 1beta, 7alpha, 10alphaH-11-acetoxy-guaia-4-en-3-one (1). This is the first report showing that this compound can be isolated from Torilis japonica.

We determined the full-length genome sequence of Japanese encephalitis virus (JEV) K94P05 isolated in Korea. Sequence analysis showed that the 10,963-nucleotide-long RNA genome of K94P05 was 13 or 14 nucleotides shorter than the genome of other JEV isolates because of a deletion in the 3' noncoding region of K94P05. Compared with sequences of other JEV isolates, the full-length nucleotide sequence showed 89.0-89.6% homology, and the deduced amino acid sequence showed between 96.4-97.3% homology. A region of approximately 60 nucleotides immediately downstream of the open reading frame stop codon of K94P05 showed high sequence variability as compared with other JEV isolates. K94P05 formed a distinct group within a phylogenetic tree established with the full-length genome sequences. Cross-neutralization studies showed that polyclonal antibodies to Korean isolates were 3 times better at neutralizing the Korean isolates than antibodies to Nakayama-NIH. These findings suggest that Korean JEV K94P05 is genetically and antigenically distinct from other Asian JEV isolates.

To evaluate risk factors for infection and the role of C-reactive protein (CRP) in the diagnosis of infection, a retrospective case control study was performed among Korean systemic lupus erythematosus patients. Of 120 proven infections, 31 episodes (25.8%) occurred in patients taking no corticosteroids (CS). The risk of infection was lower in patients taking low-dose CS (< 300 mg prednisolone/month) than no CS (odds ratio (OR) 0.36). In patients receiving high-dose CS (> 1000 mg prednisolone/month), however, the risk increased (OR 2.9). In patients taking no CS, disease activity manifested as increased SLEDAI, anemia and active urinary sediment, was associated with infection. The CRP was higher in the patients with infection than controls and the CRP levels over 50 mg/l were observed only in infection. These results suggest that CS have a bimodal influence on infection depending on dose. Disease activity is an important risk factor for infection in patients taking no CS. Finally, CRP levels greater than 50 mg/l suggest the presence of infection.

Little is known about the efficacy and safety of different formulations of omeprazole-based triple therapy regimens for the treatment of Helicobacter pylori-positive peptic ulcer. We compared the efficacy and safety of two formulations of omeprazole used in triple therapies in patients with H. pylori-positive active peptic ulcer. Seventy-four patients with endoscopically proven H. pylori-positive active peptic ulcer were randomized to two groups, each with 37 patients, to receive either OAC-I (6 weeks of "A" formulation of omeprazole [20 mg twice daily] plus 2 weeks of amoxicillin [1.0 g twice daily] and clarithromycin [500 mg twice daily] or OAC-II (6 weeks of "B" formulation of omeprazole [20 mg twice daily] plus 2 weeks of the same antibiotics. The H. pylori and ulcer healing status were assessed at the baseline and at the 6-week endpoint of therapy. Gastrointestinal symptoms, documentation of adverse events, and standard laboratory examinations were assessed at each visit. Eradication of H. pylori (intention to treat [n = 74]/per protocol [n = 66]) and healing of the ulcer were successful in 83.8%/96.9% and 93.8%, respectively, of the OAC-I group patients, and in 91.9%/100% and 97.1%, respectively, of the OAC-II group patients (P = 0.477; P = 0.608). The OAC-I group experienced rapid resolution of symptoms, but no significant differences were found between the two groups for number of days taken for resolution of gastrointestinal symptoms, adverse events, and laboratory findings. The two different formulations of omeprazole used in triple therapy regimens produced similar efficacy and safety results after 6 weeks of treatment in patients with H. pylori-positive active peptic ulcer.

The kinetic characteristics of cellulase and beta-glucosidase during hydrolysis were determined. The kinetic parameters were found to reproduce experimental data satisfactorily and could be used in a simultaneous saccharification and fermentation (SSF) system by coupling with a fermentation model. The effects of temperature on yeast growth and ethanol production were investigated in batch cultures. In the range of 35-45 degrees C, using a mathematical model and a computer simulation package, the kinetic parameters at each temperature were estimated. The appropriate forms of the model equation for the SSF considering the effects of temperature were developed, and the temperature profile for maximizing the ethanol production was also obtained. Briefly, the optimum temperature profile began at a low temperature of 35 degrees C, which allows the propagation of cells. Up to 10 h, the operating temperature increased rapidly to 39 degrees C, and then decreased slowly to 36 degrees C. In this nonisothermal SSF system with the above temperature profile, a maximum ethanol production of 14.87 g/L was obtained.

Secondary amyloidosis is an occasional complication of ankylosing spondylitis (AS) and in most cases renal amyloidosis presents with proteinuria, nephrotic syndrome and decreased renal function. We describe a 32-year-old male patient with AS manifested by frequent diarrhea, intermittent abdominal pain and low serum albumin levels. He has suffered from severe inflammatory back pain for 14 years with multiple peripheral joint involvement. Protein-losing enteropathy due to gastrointestinal amyloidosis was diagnosed with 99mTc-human albumin scintigraphy, fecal alpha-1 antitrypsin clearance and colonoscopic biopsy with Congo red staining. Somatostatin analogue octreotide and prednisolone were introduced with successful result.

A new bisabolene epoxide was isolated from the flower buds of Tussilago farfara, and the structure was determined by spectroscopic methods to be 1alpha,5alpha-bisacetoxy-8-angeloyloxy-3beta, 4beta-epoxy-bisabola-7(14),10-dien-2-one (1). Compound 1 showed inhibition of nitric oxide synthesis in lipopolysaccharide-activated macrophages.

In activated macrophages the inducible form of nitric oxide synthase (i-NOS) generates high amounts of toxic mediator, nitric oxide (NO) which contributes to the circulatory failure associated with septic shock. A sesquiterpene lactone compound (yomogin) isolated from medicinal plant Artemisia princeps Pampan inhibited the production of NO in LPS-activated RAW 264.7 cells by suppressing i-NOS enzyme expression. Thus, yomogin may be a useful candidate for the development of new drugs to treat endotoxemia and inflammation accompanied by the overproduction of NO.

In our studies of murine coronavirus transcription, we continue to use defective interfering (DI) RNAs of mouse hepatitis virus (MHV) in which we insert a transcription consensus sequence in order to mimic subgenomic RNA synthesis from the nondefective genome. Using our subgenomic DI system, we have studied the effects of sequences flanking the MHV transcription consensus sequence on subgenomic RNA transcription. We obtained the following results. (i) Insertion of a 12-nucleotide-long sequence including the UCUAAAC transcription consensus sequence at different locations of the DI RNA resulted in different efficiencies of subgenomic DI RNA synthesis. (ii) Differences in the amount of subgenomic DI RNA were defined by the sequences that flanked the 12-nucleotide-long sequence and were not affected by the location of the 12-nucleotide-long sequence on the DI RNA. (iii) Naturally occurring flanking sequences of intergenic sequences at gene 6-7, but not at genes 1-2 and 2-3, contained a transcription suppressive element(s). (iv) Each of three naturally occurring flanking sequences of an MHV genomic cryptic transcription consensus sequence from MHV gene 1 also contained a transcription suppressive element(s). These data showed that sequences flanking the transcription consensus sequence affected MHV transcription.

Adult macaques do not develop disease after infection with a nef deletion mutant of the simian immunodeficiency virus (SIV) and are protected against challenge with pathogenic virus. This finding led to the proposal to use nef-deleted viruses as live, attenuated vaccines to prevent human acquired immunodeficiency syndrome (AIDS). In contrast, neonatal macaques developed persistently high levels of viremia after oral exposure to and SIV nef, vpr, and negative regulatory element (NRE) deletion mutant. Severe hemolytic anemia, thrombocytopenia, and CD4+ T cell depletion were observed, indicating that neither nef nor vpr determine pathogenicity in neonates. Because such constructs have retained their pathogenic potential, they should not be used as candidate live, attenuated virus vaccines against human AIDS.

Coronavirus subgenomic mRNA possesses a 5'-end leader sequence which is derived from the 5' end of genomic RNA and is linked to the mRNA body sequence. This study examined whether coronavirus transcription involves a discontinuous transcription step; the possibility that a leader sequence from mouse hepatitis virus (MHV) genomic RNA could be used for MHV subgenomic defective interfering (DI) RNA transcription was examined. This was tested by using helper viruses and DI RNAs that were easily distinguishable. MHV JHM variant JHM(2), which synthesizes a subgenomic mRNA encoding the HE gene, and variant JHM(3-9), which does not synthesize this mRNA, were used. An MHV DI RNA, DI(J3-9), was constructed to contain a JHM(3-9)-derived leader sequence and an inserted intergenic region derived from the region preceding the MHV JHM HE gene. DI(J3-9) replicated efficiently in JHM(2)- or JHM(3-9)-infected cells, whereas synthesis of subgenomic DI RNAs was observed only in JHM(2)-infected cells. Sequence analyses demonstrated that the 5' regions of both helper virus genomic RNAs and genomic DI RNAs maintained their original sequences in DI RNA-replicating cells, indicating that the genomic leader sequences derived from JHM(2) functioned for subgenomic DI RNA transcription. Replication and transcription of DI(J3-9) were observed in cells infected with an MHV A59 strain whose leader sequence was similar to that of JHM(2), except for one nucleotide substitution within the leader sequence. The 5' region of the helper virus genomic RNA and that of the DI RNA were the same as their original structures in virus-infected cells, and the leader sequence of DI(J3-9) subgenomic DI RNA contained the MHV A59-derived leader sequence. The leader sequence of subgenomic DI RNA was derived from that of helper virus; therefore, the genomic leader sequence had a trans-acting property indicative of a discontinuous step in coronavirus transcription.

Mouse hepatitis virus (MHV) defective interfering (DI) RNA was used to determine the cis-acting sequences required for MHV RNA replication. A 2.2-kb-long cDNA clone of the MHV DI RNA DIssE was used to test the effect of deletions throughout the DI RNA on replication in DI RNA-transfected, MHV-infected cells. Data from a series of deletion mutants demonstrated that about 470 nucleotides at the 5' terminus, 460 nucleotides at the 3' terminus, and about 135 nucleotides in an internal position approximately 0.9 kb from the 5' end of DI RNA were necessary for DI RNA replication. These data suggested that cis-acting sequences which were necessary for MHV RNA replication required not only terminal sequences but also an internal sequence present at about 3.2 kb from the 5' end of the genome.

Previously, we established a system whereby an intergenic region from mouse hepatitis virus (MHV) inserted into an MHV defective interfering (DI) RNA led to transcription of a subgenomic DI RNA in helper virus-infected cells. By using this system, the duration of a primary transcription initiation activity which transcribes subgenomic-size RNAs from the genomic-size RNA template in MHV-infected cells was examined. Efficient DI genomic and subgenomic RNA synthesis was observed when the DI RNA was transfected at 1, 3, 3.5, 5, and 6 h postinfection, indicating that all activities which are necessary for MHV RNA synthesis are present continuously during the first 6 h of infection. The effect of subgenomic DI RNA synthesis on DI genomic RNA replication was then examined. Replication efficiency of the DI genomic RNA which synthesized the subgenomic RNA was approximately 70% lower than that of DI genomic RNA which did not synthesize the subgenomic DI RNA in MHV-infected cells. Cotransfection of two different-size DI RNAs demonstrated that replication of the larger DI RNA was strongly inhibited by replication of the smaller genomic DI RNA. Cotransfection of two DI RNA species of the same length into MHV-infected cells demonstrated that reduced replication of the genomic DI RNA which synthesizes the subgenomic RNA did not affect the replication of cotransfected DI RNA, demonstrating that the reduction in DI genomic RNA replication works only in cis, not in trans. Therefore, the previously proposed hypothesis that coronavirus, subgenomic RNA synthesis may inhibit the replication of genomic RNA by competing for a limited amount of virus-derived factors seems unlikely. Possible mechanisms of coronavirus transcription are discussed.

A novel concept of membrane bioreactor in which living cells are sandwiched between ultrafiltration (UF) and reverse osmosis (RO) membranes was applied for lactose fermentation to ethanol by genetically engineered yeast cells. The productivity of the Lactophile 13B strains was higher than that of the Lactophile 13D strains. In both cases performance data similar to those for glucose fermentation to ethanol by Saccharomyces cerevisiae were obtained. However, the operational stability of recombinant yeast cells was improved in the new bioreactor in comparison to the stability of these cells in a shake flask.

A bioreactor in which living yeast cells are sandwiched between an ultrafiltration membrane and a reverse osmosis membrane was constructed, and experiments were performed for the conversion of substrate glucose to product ethanol. A set of equations that include both transport through a series of barrier layers and bioreaction rate were developed to predict the performance of the sandwich bioreactor. The above equations were solved by using numerical values for the transport parameter and the bioreaction rate constant, and the results are compared with the experimental data.