We investigated the growth-inhibitory mechanism of Helicobacter pylori by omeprazole (OMP) and its activated sulfenamide (OAS). Using dithiothreitol (DTT) and 5,5'-dithio-bis[2-nitrobenzoic acid] (DTNB; Ellman's reagent), we first determined the relationship between the binding capacity of these compounds to H. pylori membrane and its significance to membrane P-type ATPase activity. After incubation of the intact H. pylori cells with either OMP or OAS, the residual quantity of free SH-groups on the cell membrane was measured, and, the resulting values were plotted as a function of time. From this experiment, we found that there was a considerable difference in the membrane-binding rates between OMP and OAS. At neutral pH, the disulfide bond formation on H. pylori membrane was completed within 2 min of incubation of the intact cells with OAS. By OMP, however, it was gradually formed, exceeding 10 min of incubation for completion, whereby, the extent of P-type ATPase inhibition appeared to be proportional to the disulfide forming rate. From this data, it was suggested that the disulfide formation might directly affect enzyme activity. Since OMP per se cannot yield a disulfide bond with cysteine, it is predicted that the enzyme inactivation must be caused by the OAS form. Accordingly, we postulated that, under the neutral pH, OMP could be converted to OAS in the course of transport. By extrapolating the inhibitory slopes, we could evaluate K₁ values, relating to their minimal inhibitory concentrations (MICs) for H. pylori growth. In these MIC ranges, H. pylori uptake or vesicular export of nutrients such as peptides were totally prohibited, but their effect in Escherichia coli were negligible. From these observations, we strongly suggest that the P-type ATPase activity is essential for the survival of H. pylori cells in particular.

Ultrasonic wave technology was employed to improve filtration performance and ethanol production in a bioreactor equipped with an internal ceramic-membrane filter module. The filtration performance was found to depend on the power and the pattern of ultrasonic wave irradiation. Under the optimized conditions (irradiation time: 25 see, period: 5 min, and ultrasonic power: 60 W), the flux was improved with the periodic-pause method by 200-700％ compared with the control (with no irradiation), while the improvement was only 30 to 90％ without the periodic-pause method. The final ethanol concentration also increased slightly. However, in a more severe condition (irradiation time: 2.5 min, period: 5 min, and ultrasonic power: 110 W), the irradiation of ultrasonic waves was observed to disturb cell integrity and viability, and thus to decrease ethanol production.

A catechol 2,3-dioxygenase (C23O) was purified to apparent homogeneity from Pseudomonas putida SU10 through several purification steps consisting of ammonium sulfate precipitation and chromatographies on DEAE 5PW, Superdex S-200, and Resource-Q. Gel filtration indicated a molecular mass under nondenaturing conditions of about 130 kDa. The enzyme has a subunit of 34 kDa as was determined by SDS-PAGE. These results suggest that the native enzyme is composed of four identical subunits. The N-terminal amino acid sequence (30 residues) of the enzyme has been determined and exhibits high identity with other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 4-methylcatechol cleaved at a higher rate than catechol or 3-methylcatechol. $K_m$ values of the enzyme for these substrates are between 3.5 and 5.7 M.

A 6.9-kb DNA fragment including the minimal Bacillus pasteurii urease gene cluster was subcloned into a high-copy-number plasmid vector, pUC19, and the recombinant B. pasteurii urease was overexpressed in Escherichia coli. The recombinant urease was purified 25.9-fold by using combinations of anion-exchange and gel-filtration chromatography followed by Mono-Q chromatography on a FPLC. N-terminal peptide sequencing analyses revealed that two distinct smaller peptide bands resolved on a 10-18％ gradient SDS-PAGE corresponded to UreA and UreB peptides, respectively. It was also shown that the ureB gene was translated from a GUG codon and the first methionine residue was post-translationally cleaved off. The native molecular weight of the recombinant urease was 176,000 and 2 nickel atoms were present per catalytic unit. pH stability studies of the purified enzyme showed that the recombinant Bacillus pasteurii urease is stable in alkaline pH range, which is similar to the enzyme of the evolutionarily related bacterium, Sporosarcina ureae.

The combined activities of dextranase and amylase(DXAMase) from Lipomyces starkeyi KSM 22 produced from starch fermentation inhibited or prevented dental plaque formation. The activities were stable in commercial mouthwash products; DXAMase activity retained over 93％ of original activity after 6 months at 23℃. We examined the effects of enzyme inhibitors and active ingredients in mouthwash on DXAMase activity. The DXAMase was stable with 0.29％(w/v) EDTA, 20％ (v/v) ethanol, 0.05％ (w/v) fluoride, and 0.05％ (w/v) SDS. Among the active ingredients of mouthwash, sodium benzoate (up to 1 ％, w/v) had no inhibitory effect on either dextranase or amylase activity. In the case of cetylpyridinium chloride, the addition of 0.05％ (w/v) inhibited 6％ of dextranase activity and 13％ of amylase activity. Propylene glycol (up to 1％, w/v) showed no inhibitory effect on either enzyme activity. DXAMase (5 IU/㎖) in mouthwash could remove pre-formed films of glucan-bound S. mutans cells. The addition of 0.1 IU/㎖ DXAMase in mouthwash prevented the formation of insoluble-glucan. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent.

A three phase fluidized bed bioreactor immobilized with Thiobacillus sp. IW was tested to remove hydrogen sulfide and methylmercaptan with high loading rate. In a single gas treatment, the bioreactor removed 92- 98％ of hydrogen sulfide with loading rate of 15- 66 g/l/h and removed 87-98％ of methylmercaptan with loading rate of 14-60 gl/sup -1/h/sup -1/. In the mixed gas treatment, the removal efficiencies of hydrogen sulfide and methylmercaptan maintained at 89-99％ for various inlet loading rates and were not affected by the inlet loading ratio of both gases in low loading rates. When the inlet concentration of methylmercaptan increased 3.8 times and was maintained for 30 h to observe the response of the bioreactor to sudden environmental change, the removal efficiency of methylmercaptan was maintained at an average of 91％.

The intracellular superoxide dismutase (SOD) from Staphylococcus sciuri was isolated to homogeneity by continuous steps, including ammonium sulfate fractionation, DEAE-ion-exchange chromatography, gel filtration, and phenyl hydrophobic gel chromatography. Pure SOD had a specific activity of 4,625 U/mg and was purified 158-fold with a yield of 31 % from a cell free extract. The molecular weight of the purified SOD was determined to be approximately 35.5 kDa by gel filtration and the enzyme was also shown to be composed of dimeric subunits on denaturing SDS-PAGE. The enzyme activity remained stable at pH 5 to 11 and also to heat treatment of up to $50^{\circ}C$ at pH 7.8, with 80% relative activity. The enzyme was insensitive to cyanide, hydrogen peroxide, and azide, indicating that it is a manganese-containing SOD. The EPR spectrum showed the enzyme containing manganese as a cofactor.

In order to determine the functional region of the antifungal protein (AFP) isolated from Aspergillus giganteus responsible for growth inhibitory activity and the promotion of phospholipid vesicle aggregation, overlapping peptides covering the complete sequence of AFP were synthesized. The antibiotic activity against bacterial, fungal, and tumor cells, and the vesicle-aggregation activity of the synthetic peptides were investigated. The AFP functional sequence responsible for antibiotic and vesicle-aggregation activity was determined to be located within the region between AFP residues 19 to 32. AFP (19-32) exhibited an a-helical conformation in a cell membrane-like environment. AFP (19-32) displayed potent antibiotic activity against bacterial, fungal, and tumor cells without peptide toxicity as indicated by hemolysis. Accordingly, AFP (19-32) could be used as a good model for the design of effective antibiotic agents with powerful antibiotic activity yet without any cytotoxic effects against the host organism.

Lactic acid bacteria were isolated from Kimchi and screened for bacteriocin. A total of 99 strains showed antimicrobial activity when grown on solid media, yet only 10 showed antimicrobial activity in liquid media. Strain H-559, identified as Lactococcus lactis subsp. lactis, exhibited the strongest inhibitory activity and was active against pathogenic bacteria including Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus as well as other lactic acid bacteria. The antimicrobial substance produced by L. lactis subsp. lactis H-559 was confirmed to be a bacteriocin by the treatment of $\alpha$-chymotrypsin, and protease type Ⅸ and ⅩIV. The bacteriocin activity remained stable between pH 2.0 and pH 11.0 and during heating for 10 min at $100^{\circ}C$. The bacteriocin production started in the exponential phase and stopped in the stationary phase. L. lactis subsp. lactis H-559 showed the highest bacteriocin activity at a culture temperature of $25^{\circ}C$, and an inverse relationship between the bacteriocin productivity and mean growth rate at different culture temperatures was observed. The mean growth rate and bacteriocin productivity of L. lactis subsp. lactis H-559 increased as the initial pH of the media increased.

The high density mixed mode adsorbent known by the trade name of Mimo-AD was used to purify lysozyme directly from the hen egg white (HEW). The homogenized hen egg white was treated with the adsorbent in a stirred vessel for lysozyme adsorption, and then the adsorbent, easily separated from the HEW by sedimentation, was packed into a column. The remaining HEW and contaminant proteins were removed by washing with pH 11 distilled water in an expanded-bed state, and subsequently the elution was performed with pH 12 distilled water in a packed-bed state. By this simple and rapid adsorption, washing, and elution procedure, lysozyme was purified to＞95％ with an overall recovery yield of 66％. This process offers a great potential for industrial application by allowing the extraction of lysozyme while retaining the commercial value of HEW.

Simultaneous saccharification and fermentation (SSF) experiments were carried out with a lignocellulosic biomass. The effects of temperature on enzymatic saccharification and the ethanol fermentation were also investigated. The batch SSF process gave a final ethanol concentration of 10.44 g/l and equivalent glucose yield of 0.55 g/g, which was increased by 67％ or higher over the saccharification at 42℃. The optimal operating condition was found to vary in several parameters, such as the transmembrane pressure, permeation rate, and separation coefficient, related to the SSF combined with membrane system (semi-batch system). When the fermentation was operated in a semi-batch mode, the efficiency of the enzymes and yeast lasted three times longer than in a batch mode.

A cost-effective mass production method for a strong antimicrobial peptide, buforin II, which was isolated from the stomach of Bufo bufo gargarizans, has been developed. This method is based on the neutralization of the positive charge of buforin II by fusion with a cysteine-rich acidic peptide (CAP) to avoid any lethal effect on the host. The neutralized fusion peptide was multimerized and expressed in Escherichia coli as tandem repeats to increase the production yield. Multimers of the CAP-buforin II fusion peptide were successfully expressed at high levels in E. coli as inclusion bodies. More than 100mg of pure buforin II was obtained per 11 of E. coli culture after cleaving the multimeric polypeptide with CNBr. The buforin II obtained from the recombinant E. coli had antimicrobial activity identical to that of natural buforin II. The proposed expression system can provide a cost-effective mass production method for both antimicrobial peptides and other host-lethal basic proteins.

Human T-cell leukemia virus Type-I (HTLV-I) is etiologically associated with rare adult T-cell leukemia, a malignant T-cell disorder. cDNAs encoding p24 (gag), gp21(env), and pXII of HTLV-I were amplified by polymerase chain reaction (PCR) using the genomic DNA extracted from HUT102 cell line as a template. The amplified cDNAs were cloned into the Escherichia coli expression vectors and over-expression of the recombinant proteins were achieved by adding IPTG into the culture media in order to induce the promoter. The molecular weights of the recombinant p24, gp21, and pXII, determined by SDS-PAGE, were found to be approximately 28 kDa, 23 kDa, and 15 kDa, respectively. Reactivity of the recombinant proteins with human sera was tested by the immunoblot assay. The gp21 and p24 reacted against the sera obtained from HTLV-I-infected individuals but not against the sera obtained from normal persons. These results suggest that the recombinant proteins expressed in E. coli were recognized by antibodies in sera from HTLV-I infected patients. These recombinant proteins would be applicable for detecting the presence of antibodies against HTLV-I in human blood samples.

A new baculovirus transfer vector (pPGP404) was constructed to increase the expression level of human thrombopoietin (hTPO) in insect cells. In pPGP404, hTPO was cloned next to the AcNPV polyhedrin-gp64 dual promoter and the leader sequence of hTPO was substituted with that of gp64. A recombinant baculovirus, AcPGP404, was constructed by using pPGP404 as a transfer vector. hTPO was expressed in AcPGP404-infected TN5 cells and it was observed that the expression levels of hTPO in TN5 cells increased three-fold ($6.0 {\mu}g/ml^{-1}$) compared to the level expressed under the control of the polyhedrin single promoter. These results indicate that the polyhedrin-gp64 dual promoter system would be useful for expression in large quantities of recombinant proteins in insect cells.

It was shown that brain-derived neurotrophic factors (BDNFs) secreted from human neuroblastoma cells can significantly improve the growth of the neurites of PC12 nerve cells. The addition of purified BDNFs elongated the neurites of PC 12 nerve cells two to three times more than the case where the addition was not made. The perfusion rate strongly affected the change of the size of human neuroblastoma cells because the cell size decreased as the perfusion rate increased. This could also influence the productivity of BDNF from the cells. It is also important to note that the BDNF production was decreased when the cell size was reduced. BDNF production rate also decreased at a fast perfusion rate in a smaller cell size. At the relatively fast perfusion rate of 18 ml/h, the ratio of apoptotic to necrotic cells dramatically decreased, which possibly caused the decrease of BDNF production. It has been proven that the secretion of BDNF from human neuroblastoma cells was a partially growth-related process by yielding 6.2$\times l0^{-8}/g$ of BDNF/cell/h of growth related parameter and $0.48{\times}l0^{-9}/g$ of BDNF/cell/h of nongrowth-related parameter in a growth kinetic model. In addition, it was also found that the perfusion rate played a very important role in controlling the cell death mechanism.

Susceptibility of 61 strains of Helicobacter pylori to metronidazole was examined by both the disk diffusion method using a cut-off of 15㎜ for resistance and the E test with a cut-off of 8㎎/l. The MIC/sub 50/ and MIC/sub 90/ by the E test were 2 ㎎/l and 256㎎/l, respectively. Metronidazole resistance was found in 22 (36％) out of the 61 H. pylori strains by the E test and in three additional strains by the disk diffusion method. Amongst the latter three isolates, the MICs by the E test were 4 ㎎/l, 6㎎/l, and 6㎎/l, respectively. These figures are one log₂ or half log₂ dilution lower than the cut-off of 8㎎/l recommended as resistance for the E test. All 22 metronidazole resistant H. pylori isolates by the E test that were subjected to random amplified polymorphic DNA (RAPD) fingerprinting showed different DNA fingerprints. Interestingly, >90％ of resistant isolates possess two common DNA bands of 0.4 and 0.9 kb. This study demonstrates that the results of the disk diffusion method for testing H. pylori susceptibility to metronidazole correlates well with that of the E test. The criteria for interpretation need to be internationally standardized so that the results from different centers can be compared.

More innovative molecular markers were investigated for rapid and consistent differentiation of Metarhizium anisopliae var. majus from M. anisopliae var. anisopliae. A total of 28 isolates were obtained from various countries and hosts: 13 isolates of M. anisopliae var. anisopliae, 12 isolates of M. anisopliae var. majus, and 3 isolates of M. anisopliae collected in Korea. This study involved restriction enzyme digestions of a PCR product amplified from nuclear internally transcribed spacer (ITS) and a portion of the 28S rDNA regions. Among 11 different restriction enzymes used in this study, MboⅠ digestion particularly produced a restriction pattern that had characteristics of M. anisopliae var. majus. This restriction pattern was consistent in all isolates of M. anisopliae var. majus regardless of their geographic origins and insect hosts. Mapping experiments revealed that MboⅠ sites of M. anisopliae var. majus are identical to those of M. anisopliae var. anisopliae with an exception for the presence of an additional site in the PCR product. Results from this study provide an additional method for identification and differentiation of isolates of these two varieties of M. anisopliae for use in the field and laboratory experiments.

A novel simultaneous saccharification and fermentation (SSF) process from the microcrystalline cellulose to ethanol was developed by using $\delta$-integrated recombinant cellulolytic Saccharomyces cerevisiae L2612$L2612\deltaGC$, which can utilize cellulose as carbon and energy sources. The optimum amount of enzymes needed for the efficient conversion of cellulose to ethanol at $30^{\circ}C$ was determined with commercial cellulolytic enzymes. By fed-batch cultivation, the heterologous cellulolytic enzymes were accumulated up to 42.67% of the total cellulase and 29% of the $\beta$-glucosidase needed for the efficient SSF process. When this $\delta$-integrated recombinant yeast was applied to the successive SSF step for ethanol production, 20.35 g/l of ethanol was produced after 12 h from 50 g/l of microcrystalline cellulose. By using this novel SSF process, a considerable amount of commercial enzymes was reduced.

Tumor cells may alter the expression of proteins involved in antigen processing and presentation, allowing them to avoid recognition and elimination by cytotoxic T cells. In order to investigate whether the major histocompatibility complex (MHC) class I-mediated antigen processing machinery is preserved in human lung cancer cell lines, we examined the expression of multiple components of the MHC class I antigen processing pathway, including transporter associated with antigen processing (TAP), $\beta_2$-microglobulin, MHC class I molecules, and chaperones which have not been previously examined in this context. Row cytometry analysis showed that the cell surface expression of MHC class I molecules was downregulated in all of the cell lines. While some cell lines showed no detectable expression of MHC class I molecules, pulse-chase experiments showed that MHC class I molecules were synthesized in the other cell lines but not transported from the endoplasmic reticulum to the cell surface. Low or nondetectable levels of TAP1 and/or TAP2 expression were demonstrated by Western blot analysis in all of the cell lines, representing a variety of lung tissue types. In some cases, this was accompanied by loss of tapasin expression. Our findings suggest that downregulation of antigen processing may be one of the strategies used by tumors to escape immune surveillance. This study provides further information for designing the potential therapeutic applications such as immunotherapy and gene therapy against cancers.

Enterobacter sp. B54 inhibited growth of the fungus Phytophthora capsici on potato dextrose agar (PDA). Three mutants with antifungal activities (denoted M54-47, M54-113, and M54-329) which were lost or increased, through Pl::Tn5 lac mutagenesis, were used to isolate genes responsible for fungal inhibition on PDA. Two clones were selected from the partially EcoR1-digested genomic library of the wild-type strain by probing with genomic flanking sequences of each mutant. We have isolated a 20-kb EcoR1 genomic DNA fragment from this strain that contains genes involved in hyphal growth inhibition of P. capsici on PDA. Subcloning and expression analysis of the above DNA fragment identified a 8-kb region which was necessary for antifungal activities. A 8-kb HindⅢDNA fragment covers three genomic loci inserted by Tn5 lac in each mutant. This suggested that all genes which are related to antifungal activities might be clustered in simple forms of at least 5-8 kb sizes.

The effect of pectinase on menthol production by Mentha piperita in shake flasks was investigated. The optimum concentration of pectinase and period of elicitation for menthol production were 15 U/1 and 9 days, respectively. Pectinase elicitation at 15 U/1 for 9 days using a Lin-Staba medium with 2,4-dichlorophenoxyacetic acid (2,4-D) enhanced menthol production 37-fold (211.5 ㎎ menthol/l) with the specific menthol concentration (menthol concentration per unit weight of cells) of 27.5 ㎎/g dry cell weight (DCW). Our results also indicate that pectinase elicitation may activate the conversion of pulegone to menthol.

The binding characteristics of Anagrapha falcifera nuclear polyhedrosis virus (AtNPV) to Spodoptera frugiperda 21 (Sf21) cells were investigated. The cells displayed an affinity of 4.7×10/sup 10/M/sup -1/ with about 3,300 binding sites per cell. The biochemical nature of the AfNPV-binding sites on the cell surface was also partially identified. Our findings suggest that the binding-site moiety has a glycoprotein component, but that the direct involvement of oligosacccharides containing N-acetylglucosamine or sialic acid residues in binding is unlikely, and that AfNPV entry into Sf21 cells may be via receptor-mediated endocytosis.

A fuel cell type biosensor for lactate was developed using a metal-reducing bacterium, Shewanella putrefaciens IR-1. Under the operational conditions, the bacterial cell suspension generated the current without an electrochemical mediator in the presence of lactate. The current was proportional to the lactate concentration up to 30 mM.

The protective role of Bifidobacterium spp. (B. breve K-110, B. breve K-111, and B. infantis K-525) isolated from the fecal samples of healthy Koreans was investigated on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci(ACF) formation in mouse colon. In mice fed normal diet with DMH treatment, an average of 68.5 ACF/colon was formed, whereas in mice administered with B. breve K-110, B. breve K-111, and B. infantis K-525, the numbers of DMH-induced ACF decreased to 7.2, 10.9, and 6.6 ACF/ colon, respectively. The mean number of crypts/focus was not significantly altered. Fecal harmful enzymes, such as β-glucuronidase, tryptophanase, and urease, were effectively inhibited during the administration of these bifidobacteria to mice. These results suggest that bifidobacteria could prevent colon cancer.

AL072 is a potent anti-Legionella antibiotic produced by Streptomyces strain AL91. The minimum inhibitory concentration (MIC) of AL072 against Legionella pneumophila was 0.2$\mu$g/ml. Bacterial growth was rapidly inhibited at the dose range between the MIC and 20 times of the MIC when the antibiotic was added at the mid-exponential phase. Ultrastructural changes in L. pneumophila were observed upon treatment with AL072. Under electron microscopical observation, the organisms treated with AL072 exhibited characteristic morphological changes in the cellular outer coat. Also irregular morphological changes, such as the formation of filamentous materials in the cytoplasm, an increase in the size and number of cytoplasmic vacuoles, the extruding of cytoplasmic contents, the formation of spheroplast and ghost cells, and blebbings in the cell wall were observed. Furthermore, immunoelectron microscopical observation of the group treated with the MIC showed that the immune complex attached mainly to the cell wall. The results of these experiments indicate that AL072, like the inhibitors of cell wall synthesis, act selectively on the cell wall of L. pneumophila.

Constructs, encoding a single-chain variable fragment of a catalytic antibody 4f4f (scFv-4f4f) with glycosidase activity, were made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding a linker (GGGGS). Using three different plasmid systems, single-chain antibodies were expressed separately in Escherichia coli, demonstrating significant differences in the expression level and amounts in soluble form of the recombinant protein. The protein expression from pET3a-scFv-4f4f was up to 20% of the total soluble proteins and, more importantly, the proteins were mostly found in a soluble form. An SDS-PAGE analysis of the purified single-chain proteins, yielding higher than 5mg from a 1-1 culture, showed a single band corresponding to its molecular weight of 29,100. A preliminary study shows that the expressed scFv-4f4f is catalytically active. The catalytic parameters for the hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside by scFv-4f4f are being investigated.