A transformation system mediated by Agrobacterium tumefaciens is routinely used for the genetic engineering of plants. Here, we report an efficient and stable method for transformation of heterogeneous genes into an industrial Cephalosporium acremonium by using a similar transformation system established in plants. Both the phleomycin-resistant gene and vgb gene were used as screening markers to confirm the success of transformation by either Southern hybridization or PCR amplification. It was found that acetosyringone (AS) was necessary only for protoplast transformation and the heterogeneous genes transferred were integrated into the genome of C. acremonium. The transformation efficiency obtained with this system was much higher than the conventional techniques used for transformation of C. acremonium.

Bacillus sp. JB-99, when grown in a chemically defined medium containing lactose as a carbon source, yielded 3,860 U/ml extracellular $\beta$-mannanase, which was high compared to other examined carbon sources. Among the nitrogen sources, yeast extract enhanced the enzyme activity. The enzyme production was growth-associated. The enzyme was optimally active at $65^{\circ}C$, pH 10, and had a half-life of 190 min at $65^{\circ}C$. N-Bromosuccinamide and $AgNO_3,\;CuSO_4$, and $HgCl_2$ strongly inhibited the enzyme, whereas $Ca^{2+}$ stimulated the enzyme activity. The $\alpha$-galactosidase enzyme production was not found in any of the enzyme assays.

Defects in the mitotic spindle or in the attachment of chromosomes to the spindle are believed to release an activated form of spindle checkpoint complex that inhibits APC-dependent ubiquitination and subsequently arrests the cell cycle at metaphase. When the spindle assembly is disrupted, the fission yeast mitotic arrest deficient (mad) mutants fail to arrest and rapidly lose viability. To enhance our understanding of the molecular mechanisms for the pathway of checkpoint function, the functional characterizations of Mad 1 p from Schizosaccharomyces pombe involved in this process have been carried out. Yeast two-hybrid and various deletion analyses of S. pombe Mad1 p reveal that the C terminus of Mad1p is critical for the binding of Mad2p and maintenance of Mad 1 p-Mad2p interaction. In addition, it was found. that the Mad1p region (residues 206-356) is essential for Mad1p-other checkpoint components. Mad1p truncating this region is sufficient to bind Mad2p but abolishes the checkpoint function, indicating that the checkpoint function is necessary for interaction of Mad 1 p-other checkpoint components. The possible functions of S. pombe Mad1p at the cell cycle checkpoint are discussed.

An efficient method of selecting an antagonistic strain for use as a biological control agent strain was developed. In this improved method, the surface tension reduction potential of an isolate was included in the 'decision factor,' in addition to two other factors; the growth rate and pathogen inhibition. By using a statistically designed method, an isolate from the soil was selected and identified as Bacillus sp. GB 16. In the pot test, this strain showed the best performance among the isolated strains. The lowest disease incidence rate and fastest seed growth were observed when the Bacillus sp. GB 16 was used. The action of the surface tension reducing component was assumed to enhance the wetting, spreading, and residing of the antagonistic strain in the rhizosphere. This result showed that the improved selection method was quite effective in selecting the best antagonistic strain for the biological control of soilborne infectious plant pathogens.

A novel recombinant baculovirus, Bactrus, was constructed by the insertion of the Bacillus thuringiensis cry1Ac gene between two polyhedrin genes of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of the polyhedrin gene promoter. Polyhedra produced by Bactrus in insect cells were incorporated with 130 kDa of polyhedrin-Cry1Ac-polyhedrin fusion protein, and 30 kDa of intact polyhedrin, resulting from a homologous recombination between two polyhedrin genes, was also expressed. The insecticidal activity of Bactrus against Spodoptera exigua larvae was similar to that of AcNPV, but it showed significantly higher toxicity towards Plutella xylostella larvae in comparison with that of AcNPV. The expression level of fusion protein and the insecticidal activity of recombinant polyhedra produced by the Bactrus against P. xylostella larvae were decreased after serial passages. In conclusion, the Bactrus had improved insecticidal activity and returned to wild-type AcNPV after several passages.

The role of an effective microbial species is critical to the successful application of biological processes to remove sulfur compounds. A bacterial strain was isolated from the soil of a malodorous site and identified as Burkholderia spp. This isolate was able to oxidize thiosulfate to sulfate, with simultaneous pH decrease and accumulation of elemental sulfur. The specific growth rate and the sulfate oxidation rate using the thiosulfate basal medium were $0.003 h^{-1}\;and\;3.7 h^{-1}$, respectively. The isolated strain was mixotrophic, and supplementation of $0.2\%$ (w/v) of yeast extract to the thiosulfate-basal medium increased the specific growth rate by 50-fold. However, the rate of sulfate oxidation was more than ten times higher without yeast extract. The isolate grew best at pH 7.0 and $30^{\circ}C$, and the sulfate oxidation rate was the highest at 0.12 M sodium thiosulfate. In an upflow biofilter, the isolated strain was able to degrade $H_2S\;with\;88\%$ efficiency at 8 ppm and 121/h of incoming gas concentration and flow rate, respectively. The cell density at the bottom of the column reached $3.2{\times}10^8$ CFU/(g bead) at a gas flow rate of 121/h.

Cytosolic sialidase (Neu2), a member of the sialidase family that is responsible for hydrolysis of sialic acid from the terminal position of sialoglycoconjugates, is poorly expressed in skeletal muscle and not detected in any other adult tissues. Thus, we isolated Neu2 cDNA using splicing by overlap extension (SOEing). In order to further characterize this enzyme, a His-tagged derivative was expressed in the bacterial expression system and purified by $Ni^{2+}$-affinity chromatography. A recombinant product of approximately 42 kDa had sialidase activity toward 4-methyl-umbelliferyl-$\alpha$-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH and temperature of the recombinant Neu2 for 4MU-NeuAc was 6.0 and $37.5^{\circ}C$, respectively. The metal ions, such as $Cu^{2+}\;and\;Cd^{2+}$, showed strong inhibitory effect on the activity of the enzyme. The enzyme efficiently hydrolyzed the gangliosides GM3 and GD3 and had relatively low activities on ganglioside GD1a and GD1b, $\alpha$2-3 sialyllactose, and sialylated glycoproteins such as fetuin, transferrin, and orsomucoid, but had hardly any activities on $\alpha$2-6 sialyllactose and ganglioside GM1 and GM2. We concluded that the recombinant Neu2 has a sialidase activity toward glycoproteins as well as gangliosides.

To elucidate the question of whether biofilm formed by the intercellular adhesion (ica) gene cluster has influences on antibiotic resistance in Staphylococcus epidermidis, we compared 124 skin strains with strains isolated from 50 blood cultures that cause septicemic diseases. The results revealed that the blood culture isolates were more resistant to the antibiotics tested than the saprophytic isolates. Moreover, antibiotic multiresistance was more prevalent in the clinical isolates. In the blood culture isolates, $46\%$ of the strains were resistant to three or more antibiotics, whereas only $12\%$ of the saprophytic isolates were resistant to three or more antibiotics. Interestingly, these characteristics were highly correlated with the biofilm formed by the ica gene cluster. In biofilm-producing strains, $84\%$ of the blood culture isolates and $44\%$ of the saprophytic isolates were antibiotic multiresistant, whereas only $22\%=;and\;9\%$, respectively, were antibiotic multiresistant in biofilm-nonproducing strains. Additionally, in the biofilm-producing ica-positive strains, $89\%$ of the blood culture isolates and $57\%$ of the saprophytic isolates were antibiotic multiresistant. However, the rate of the antibiotic multiresistance in the ica-negative strains was very low, thus indicating that the biofim formed by the lea gene cluster in S. epidermidis is an important pathogenic factor in association with the antibiotic multiresistance.

Myxobacterium sp. HK1, isolated from Korean soil, degrades cellulose, differentiates to fruiting body, and its 16s rDNA has $95\%$ similarity to Polyangium sp. An anticancer molecule, CDMHK, was identified from culture broth of Myxobacterium sp. HK1, and purified by Diaion HP20, Silica gel, Sephadex LH-20 chromatography, and preparative HPLC using an YMC OSD-A C18 column. The molecular structure and formula were determined to be $C_{l2}H_{l9}N_3O_2$ (M.W 237) by MS spectrometry, 300 MHz $^{1}H\;and\;^{13}C$ NMR. The CDMHK was not active against Escherichia coli, Staphylococcus aureus, and Candida albicans. However, this molecule inhibited the growth of various cancer cell lines. The $ED_{50}$ values of CDMHK were determined to be 0.147, 0.086, 0.18, 0.166, and 0.142 $\mu$g/ml against A549, SK-OV-3, SK-MEL-2, VF498, and HCTl5 cancer cell lines, respectively. In addition, the CDMHK was able to induce apoptosis of the CCRF-CEM cancer cell line, evidenced by DNA fragmentation assay and DAPI staining.

Bifidobacterium has been previously shown to potentiate immune function, which was mediated through the stimulation of cytokine production by macrophage. This study was performed to further characterize the effective component of Bifidobacterium by measuring the level of interleukin (IL)-6 cytokine using the RAW 264.7 murine cell line as a macrophage model. RAW 264.7 cells were cultured for 24 h in the presence of whole cells (WCs), cell walls (CWs), and cell-free extracts (CFEs) from various strains of Bifidobacterium and other lactic acid bacteria at various concentrations. The most effective component was different depending on the strains and the concentrations used. When tested with each cell fraction from Bifidobacterium sp. BGN4, heat treatment of the cell fractions lowered the production of IL-6. Synergistic effect was obtained, especially when CWs and CFEs were combined. Sonicated WCs stimulated IL-6 production more than intact WCs. The in vitro approaches employed here should be useful in further characterization of the effects of Bifidobacterium on gastrointestinal and systemic immunity.

A phytase from Aeromonas sp. LIK 1-5 was partially purified by ammonium sulfate precipitation and DEAE-Sephacel column chromatography. Its molecular weight was 44 kDa according to SDS-PAGE gel. Enzyme activity was optimal at pH 7 and at $50^{\circ}C$. The purified enzyme was strongly inhibited by 2 mM EDTA, $Zn^{2+},\;Co^{2+},\;or\;Mn^{2+}$, and activated by 2 mM $Ca^{2+}$. The K_m value for sodium phytate was 0.23 mM, and the enzyme was resistant to trypsin. The N-terminal amino acid sequence of the phytase was similar to that of other known alkaline phytases. The phytase was specific for ATP and sodium phytate, which is different from other known alkaline phytases. Based on the substrate specificity, the phytase may therefore be a novel alkaline phytase.

The ccpA gene encoding catabolite control protein A (CcpA) of Leuconostoc mesenteroides SYl, a strain isolated from kimchi, was cloned, sequenced, analyzed for transcript, and overexpressed in Escherichia coli. The ccpA ORF (open reading frame) is 1,011 bp in size, which can encode a protein of 336 amino acid residues with a molecular mass of 36,739 Da. The transcription start site was mapped at a position 49 nucleotides upstream of the start codon, and promoter sequences were also identified. The putative cre site overlapped with the -35 promoter sequence. The deduced amino acid sequence of the CcpA contained the helix-turn-helix motif found in many DNA-binding regulatory proteins. CcpA from 1. mesenteroides SY1 had $54.6\%$ identity with CcpA from Lactobacillus casei. The Northern blot experiment showed that ccpA was transcribed as a single 1.1 kb transcript, and transcription was repressed when grown on media containing glucose. CcpA was overproduced in E. coli BL21(DE3) cells using the pET expression vector, and purified to an apparent homogeneity. Gel Mobility Shift Assay with purified CcpA and a DNA fragment containing the ere sequence of the $\alpha$-galactosidase gene (aga) from L. mesenteroides SY1 revealed that CcpA bound specifically to the cre site of aga.

The signaling mechanism underlying aburatubolactam C-induced FasL upregulation was investigated in human Jurkat T cells. After treatment with aburatubolactam C, the src-family PTKs $p56^{lck}\;and\;p59^{fyn}$, and MAP kinases ERK2 and JNK1, were activated prior to FasL upregulation; Both $p56^{lck}\;and\;p59^{fyn}$ were directly activated 2.4- and 2.2-fold, respectively, in vitro by aburatubolactam C. The aburatubolactam C-induced cellular changes, including the activation of ERK2 and INK1, and FasL upregulation, were completely prevented by the PTK inhibitor genistein. The activation of protein kinase C (PKC$\delta,\;\epsilon\;and\;\mu$ was also induced following aburatubolactam C treatment. Although the activation of $p56^{lck}$ and tyrosine phosphorylation of the cellular proteins were not blocked by the PKC inhibitor GFl09203X, the activation of ERK2 was completely abrogated, along with a detectably enhanced JNK1 activation; FasL upregulation, and apoptosis. However, the FasL upregulation and apoptosis were significantly inhibited by the PKC activator PMA, with a remarkable increase in the ERK2 activation. The cytotoxic effect of aburatubolactam C was reduced in the presence of the anti-Fas neutralizing antibody ZB-4. Although ectopic expression of Bcl-2 failed to completely block the cytotoxicity of aburatubolactam C, it was clearly suppressed. The c-Fos mRNA expression was upregulated in a biphasic manner, where the second phasic expression overlapped with the FasL upregulation. Accordingly, these results demonstrate that aburatubolactam C-induced apoptosis is exerted, at least in part, by FasL upregulation dictated by activation of the PTK ($p56^{lck}\;and\;p59^{fyn}$) /JNKI pathway, which is negatively affected by the concurrent activation of the PKC/ERK2 pathway proximal to PTK activation.

Expression in yeast may prove more amenable to generating large amounts of viral antigens for a vaccine candidate. We, therefore, cloned the gene encoding the Hepatitis C virus (HCV) structural proteins (C-El-E2, c740) fused in-frame with, and immediately 3' to, the chicken-lysozyme signal peptide (C-SIG) gene and under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. In yeast, the HCV structural proteins were expressed in two different forms: a processed and a nonprocessed aggregated form. Biophysical characterization by sucrose linear gradient centrifugation revealed that both forms were present in the same fractions with a buoyant density of 1.127-1.176 g/$cm^3$. These findings suggest that the efficient synthesis of HCV structural proteins in yeast may be an important tool to study virus assembly and may lead to the development of an HCV vaccine.

Ammonia-oxidizing bacteria (AOB) were enriched by repeating fed-batch cultivations in an AOB-selective medium of activated sludges from a domestic wastewater treatment plant. Enriched culture showed strong capabilities of ammonia oxidation [0.810 mg $NH_4^+$-N/mg mixed liquor suspended solids (MLSS)$\cdot$day] as well as $NO_x^-$-N production (0.617 mg $NO_x^-$-N/ mg MLSS$\cdot$day). Degree of enrichment was examined through fluorescent in situ hybridization (FISH) analyses using an AOB-specific Cy3-labeled oligonucleotide probe (NSOl90) and terminal-restriction fragment length polymorphism (T-RFLP) analyses. FISH analyses confirmed that the fraction of AOB among 4',6-diamidino-2-phenylindole (DAPI)-stained cells increased from about less than $0.001\%$ to approximately $42\%$ after enrichment of AOB, and T-RFLP analyses showed that bacterial community became simpler as enrichment was continued. When the enriched culture of AOB was added (150 mg/l as dry suspended solid) to the normal activated sludge (3,000 mg/l as dry suspended solid), nitrification efficiencies were improved from 0.020 mg $NO_x^-$-N/mg MLSS$\cdot$day to 0.041 mg $NO_x^-$-N/mg MLSS$\cdot$day in a synthetic wastewater and also from 0.0007 mg $NO_x^-$-N/mg MLSS$\cdot$day to 0.0918 mg $NO_x^-$-N/mg MLSS$\cdot$day in a real domestic wastewater. Therefore, it is expected that this enrichment method could be used for improving efficiency of nitrification in wastewater treatment plants.

The biomass of the brown seaweed, Ecklonia, was used to remove Cr(VI) from wastewater. Previously, Cr(VI) was removed through its reduction to Cr(III) when brought into contact with the biomass. In this study, the effects of ionic strength, background electrolytes, and Cr(III), Ni(II), Zn(II), and Fe(III) on the Cr(VI) reduction were examined. An increased ionic strength inhibited the Cr(VI) reduction. The presence of other heavy metals, such as Cr(III), Ni(II), or Zn(II), only slightly affected the Cr(VI) reduction, while Fe(III) enhanced the reduction. Although the above various parameters could affect the reduction rate of Cr(VI) by Ecklonia biomass, these effects were relatively smaller than those of pH and temperature. In addition, the previously derived rate equation was found to be applicable over a range of ionic strengths and with different background electrolytes. In conclusion, Ecklonia, bioniass may be a good candidate as a biosorbent for the removal of Cr(VI) from wastewaters containing various other impurities, and scale-up to a practical process may be accomplished using the previously derived rate equation.

Mutation and its pilot-scale fermentation were conducted for the production of teicoplanin from Actinoplanes teichomyceticus. The fermentation medium was optimized by replacement and Plackett-Burman experimental design. A maximum production of 1,500 mg/l teicoplanin was obtained by pilot-scale fermentation in an optimized medium containing (g/l): 30 g maltodextrin, 5 g glucose, 5 g yeast extract, 5 g soybean meal, 0.5 g $MgSO_4{\cdot}7H_2O$, 0.1 g NaCl, 0.1 g $CaCl_2{\cdot}2H_2O$, and 50 g Diaion HP-20. The production of teicoplanin was improved 3-fold from the parental strain by mutation, media optimization, and fermentation, and laboratory-scale fermentation was successfully demonstrated in a pilot-scale fermenter for the industrial production of teicoplanin.

Two novel $\beta$-glucuronidases, which metabolize glycyrrhizin (GL) to 18$\beta$-glycyrrhetinic acid-3-O-$\beta$-D-glucuronide (GAMG), were purified from Streptococcus LJ-22 isolated from human intestinal microflora. $\beta$-Glucuronidases I and II were purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, butyl toyopearl, Q-Sepharose, hydroxyapatite Ultrogel, and GL-attached Sepharose column chromatographies, with the final specific activities of 137 and 190 nmole/min/mg, respectively. The molecular sizes of both $\beta$-glucuronidases were found to be 140 kDa by gel filtration, and they consisted of two identical subunits (M.W. 67 kDa by SDS-PAGE). $\beta$-Glucuronidases I and II showed optimal activity at pH 7.0 and pH 6.5, respectively. Both purified enzymes were potently inhibited by $Cu^{2+}$ and PCMS, and had maximum activity on glycyrrhizin, but did not hydrolyze p-nitrophenyl-$\beta$-glucuronides, baicalin, or GAMG These findings suggest that the biochemical properties and substrate specificities of these enzymes are different from those of the previously purified $\beta$-glucuronidases. This is the first reported purification of sugar (not aglycone)-recognizing $\beta$-glucuronidases from intestinal bacteria.

Leuconostoc mesenteroides SY1, an isolate from kimchi, was able to ferment $\alpha$-galactosides, such as melibiose and raffinose. $\alpha$-Galactosidase ($\alpha$-Gal) activity was higher in cells grown on melibiose and raffinose than cells grown on galactose, sucrose, and fructose. $\alpha$-Gal activity was not detected in cells grown on glucose, indicating the operation of carbon catabolite repression (CCR). A 6 kb DNA fragment was PCR amplified using a primer set based on the nucleotide sequence of a putative $\alpha$-galactosidase gene (aga) from L. mesenteroides ATCC 8293. Nucleotide sequencing of the 6 kb fragment confirmed the presence of aga and other genes involved in the galactosides utilization, and the gene order was galR (transcriptional regulator)-aga-gaIK (galactokinase)-gaIT (galactose-1-phosphate uridylyltransferase). Northern blotting experiment showed that aga, gaIK, and gaIT constituted the same operon, that the transcription was induced by galactosides, such as melibiose and raffinose, whereas gaIR was independently transcribed as a monocistronic gene, and that the level of transcription was fairly constant. The aga was overexpressed in E. coli BL21 (DE3) using pET26b(+) vector, and $\alpha$-Gal was accumulated in E. coli as an inclusion body.

Gibberellins (GAs) play an important role in plant growth and development. Fifteen fungi were isolated from Physalis alkekengi var francheti plant roots, and among them, four isolates showed GA-production activity. A bioassay using waito-c rice was carried out with the culture fluid of the GA-producing fungi. The GA-producing fungi were cultured for 7 days in Czapek's liquid medium at $30^{\circ}C$, 120 rpm, under dark conditions. The culture broth was concentrated 30-fold and 10 ${\mu}l$ of that concentrate was applied to 2-leaf rice sprouts. The height of the rice seedlings treated with the culture fluid of isolate PA08 was 26 cm high, while that of the seedlings treated with the wild-type Gibberella fujikuroi was 13 cm high. As such, the plant growth-promoting activity exhibited by isolate PA08 was 2 times stronger than that exhibited by the wild-type G fujikuroi. The amounts of $GA_l,\;GA_3,\;GA_4,\;GA_7,\;GA_9,\;GA_{20}$, and $GA_{24}$ in the medium were measured using gas chromatography-mass spectrometry (GC-MS), and the quantities produced by isolate PA08 were 4.85 ng/ml, 4.79 ng/ml, 17.30 ng/ml, 6.01 ng/ml, 16.61 ng/ ml, 0.08 ng/ml, and 17.30 ng/ml, respectively. Isolate PA08 was also identified as Fusarium proliferatum KGL0401 by a genetic analysis of the nucleotide sequences of the internal transcribed spacer region of the ribosomal DNA.

In order to extend the shelf-life of packaged or coated foods, an antibacterial edible film was developed from soybean meal that had been fermented with Bacillus subtilis under the optimum condition of pH 7.0-7.5 and $33^{\circ}C$ for 33 h. The water vapor permeability of the fermented film ($86.0 mg/cm^2{\cdot}h$) was higher than those of normal soybean films ($66.9 mg/cm^2{\cdot}h$). Protein solubility of the fermented film was also higher than ordinary soy protein film at the pH range of 3 -10. The fermented soybean film had higher tensile strength and lower $\%$ elongation (elongation rate) than the ordinary soybean film, mainly because partial hydrolysis of proteins in the soybean film occurred during fermentation. Antimicrobial properties of the fermented film on foodstuffs were measured by placing the films on surime, jerked beef, and mashed sausage media; containing $10^2-10^3$ CFU/plate of foodborne pathogenic bacteria, and showed significantly higher inhibitory effects on the growths of all the indicating bacteria. The film could be used as a packaging material in the food industry. However, before direct application of the fermented film to the commercial food industry, its poor mechanical and antibacterial properties need to be improved.

This study was undertaken to evaluate the effect of bacteria-derived $\beta$-glucan fiber on serum lipids, adiposity and uncoupling protein (UCP) expression in rats. In order to induce obesity, Sprague-Dawley weanling male rats were allowed free access to AIN-76A diet until 4 weeks of age, and fed high-fat diet (beef tallow, $40\%$ of calories as fat) for 6 weeks until 10 weeks of age. Rats were then fed with $0\%$ thigh- fat control group), $1\%$, or $5\%$ bacterial ~-glucan supplemented high-fat diets (w/w) for another 6 weeks. For comparison, normal control group was fed with AIN-76 diet $11.7\%$ fat). Supplementation with bacterial $\beta$-glucan resulted in a significant reduction of high-fat-induced white fat (i.e., visceral and peritoneal fat) development, adipocyte hypertrophy, and development of hyperinsulinemia and hyperleptinemia. Serum triglyceride, total cholesterol, and free fatty acid levels were greatly reduced, but, HDL-cholesterol concentrations were increased by bacterial $\beta$-glucan supplementation. Serum leptin level was lower in the $\beta$-glucan groups than in the high-fat group. The expression of UCPs (UCP1, UCP2, and UCP3) in brown adipose tissue (BAT) were significantly increased by $5\%$ bacterial $\beta$-glucan-containing diet. This study suggests that the anti-obesity effect of $5\%$ bacterial $\beta$-glucan is attributed to upregulation of UCPs and inefficient energy utilization.

Low-temperature adaptation and cryoprotection were studied in Leuconostoc mesenteroides SYl, a strain isolated from Kimchi. L. mesenteroides SY1 cells grown in exponential growth phase at $30^{\circ}C$ were exposed to $15^{\circ}C,\;10^{\circ}C$, and $5^{\circ}C$ for 2, 4, and 6 h, respectively, and then frozen at $- 70^{\circ}C$ for 24 h. Survival ratio was measured after the cells were thawed. The freezing-thawing cycles were repeated four times. Preadapted cells survived better than non-adapted control cells, and the highest survival ratio ($96\%$) was observed for cells preadapted for 2 h at $5^{\circ}C$, whereas control cells showed only $22\%$. The 2D gel showed that two proteins (spots A and B) were induced in cells preadapted at lower temperatures. Spots A and B have the same molecular weight (7 kDa), but the pI was 4.6 for spot A and 4.3 for spot B. The first 29 and 15 amino acid sequences from spots A and B were determined, and they were identical, except for one amino acid. A csp gene was cloned, and nucleotide sequencing confirmed that the gene encoded spot A cold shock protein.

Brain-derived neurotrophic factor (BDNF) is a small secretory protein and a member of the nerve growth factor (NGF) gene family. We cloned the flounder BDNF gene from a flounder brain cDNA library. The nucleotide sequence of the cloned gene showed an open reading frame (ORF) consisting of 810 bp, corresponding to 269 amino acid residues. The tissue distribution of flounder BDNF was determined by reverse transcription-polymerase chain reaction (RT-PCR) in brain, embryo, and muscle tissues. To express fBDNF using a eukaryotic expression system, we constructed the vector mpCTV-BDNF containing the fBDNF gene and transformed this vector into Chlorella ellipsoidea. Stable integration of introduced DNA was confirmed by PCR analysis of genomic DNA, and mRNA expression in C. ellipsoidae was confirmed by RT-PCR analysis.

We constructed a defined physical and genetic map of H. pylori strain 51, previously isolated from a Korean patient with a duodenal ulcer, by combining a restriction analysis by pulse-field gel electrophoresis with the construction of a BAC library. A Notl-digest of H. pylori strain 51 genome yielded seven fragments, from which the genomic size was estimated to be 1,698$\pm$24 kb. The BAC library was constructed from 50 to 200 kb fragments of HindIII-digested genomic DNA. From 700 BAC clones, an ordered overlapping maxi-set of 82 BAC clones was assembled that covered the entire genome. The positions of 15 genes were localized in the strain 51 genome with 4-22 kb of resolution and were compared with their orthologues in strain 26695 and strain J99. The arrangement of the 15 genes was identical in strain 51 and strain J99, except for flaA and hpaA. The plasticity zone of strain 51, like that of strain J99, was located in the single region, and was shorter than those of strain 26695 and strain J99. The strain 51 plasticity zone consisted of ORFs common only to strain 51 and J99 or to strain 51 and 26695, as well as strain 51-specific ORFs. Three genetic translocations and/or inversions were found between orthologue ORFs in strain 51 and strain J99. These results show that the chromosomal organization of strain 51 differs from Western strains such as strain 26695 and strain J99.

The bioavailability of Ca is currently one of the most important topics in nutrition research and is correlated with gastrointestinal solubility. Thus, to increase the solubility of calcium, this study was undertaken to examine the effect of $\gamma$-PGA on intestinal Ca solubility. The calcium solubility increased when the amount of $\gamma$-PGA was increased, due to the inhibition of the formation of an insoluble Ca complex with phosphate. Therefore, when $\gamma$-PGA-500 (avg. MW 5,000 kDa) was added at 0.5 mg/ml, $75\%$ of the total Ca was soluble. The amount of soluble Ca uptake in the small intestine was investigated using Balb/c mice as an animal model system. The soluble Ca uptake in the mice orally administered with $\gamma$-PGA-500 (avg. MW 5,000 kDa) was significantly higher than that in the $\gamma$-PGA-l00 (avg. MW 1,000 kDa)-administered mice (P<0.05). Accordingly, these results strongly support the notion that the molecular size of $\gamma$-PGA is correlated with Ca solubility. The effects of other factors, such as casein phosphopeptide and vitamin D, on intestinal Ca absorption have also previously been investigated. Therefore, it is hoped that the present observations will help clarify the role of $\gamma$-PGA in Ca solubility and its industrial application as an additive.

The stimulatory effects of exogenous salicylate as a pathway inducer on phenanthrene biodegradation were investigated using Burkholderia cepacia PM07. The phenanthrene degradation rate was greatly enhanced by increasing the salicylate additions, and the maximum rate was 19.6 mg $I^{-1}\;d^{-1}$ with the addition of 200 mg $I^{-1}$ of salicylate, 3.5 times higher than that (5.6 mg $I^{-1}\;d^{-1}$) without the addition of salicylate. The degradation rate was decreased at higher concentrations of salicylate (above 500 mg$I^{-1}$), and cell growth was significantly inhibited. The phenanthrene degradation was not affected by increasing glucose up to 2 g $I^{-1}$, although dramatic microbial growth was obtained. The stimulatory effect of exogenous salicylate decreased in the presence of glucose. After the addition of 200 mg $I^{-1}$ of salicylate, approximately $60\%$ of the initial phenanthrene (50 mg $I^{-1}$) was degraded after 96 h. However, with extra addition of 200 mg $I^{-1}$ of glucose, the phenanthrene degradation rate decreased, and only $18.5\%$ of the initial phenanthrene was degraded.

H. pylori is known to cause severe gastric diseases, including peptic ulcers and gastric cancers, and a link has also been suggested with iron-deficiency anemia (IDA). However, little is known about the pathogenesis of H. pylori-associated IDA. In the present study, to determine whether H. pylori strains are correlated with the prevalence of IDA, we analyzed and compared the sequences of the napA genes encoding a bacterioferritin-like protein in H. pylori strains. A total of 20 H. pylori strains were isolated from antral biopsies of patients with and without IDA, and the napA genes amplified from the genomic DNA were sequenced. A comparison of the deduced amino acid sequences for NapA revealed two sites with major variations. At residue 70, five out of the 12 non-IDA strains ($41.7\%$) contained serine, while only one of the 8 IDA strains ($12.5\%$) contained serine, indicating a significantly higher frequency of serine in the non-IDA strains. In addition, the NapA proteins from all 17 Western strains available on Web sites were found to contain serine residues at this position. Meanwhile, the other major variation was located at residue 73, where all eight IDA strains ($100\%$) contained leucine, while this was only true for eight of the 12 non-IDA strains ($66.7\%$). Therefore, these results indicated that the strains within each group were more genetically related to each other than to strains in the other group. When the expression level of the napA genes in the H. pylori strains was measured using RT-PCR, no significant difference was observed between the two groups, suggesting a similar intensity for the inflammatory responses induced by the NapA protein among the strains. Consequently, when taken together, the present data suggest that the occurrence of H. pylori-associated IDA may be partly determined by the infecting H. pylori strain, and the non-IDA strains are more closely related to Western strains than the IDA strains.

Red sea bream iridovirus (RSIV) obtained from infected rock bream was propagated by Bluegill fry-2 (BF-2) cell culture. The virus titer was determined as $10^{5.5}\;TCID_{50}/ml$ on confluent BF-2 cell monolayers. The integrin binding site of ORF 055L of infectious spleen and kidney necrosis virus (ISKNV) was selected for the construction of a primer to obtain the RSIV ORF 055L gene. The genes were amplified using RSIV gene lyzate by PCR. The homologies of the ORF 055L sequence of RSIV with ISKNV and rock bream iridovirus (RBIV) were approximately $96\%$ and $100\%$, respectively. DNA vaccine was constructed by cloning the ORF 055L of RSN into pcDNA 3.1 (+), containing a cytomegalovirus (CMV) promoter. For antibody production, pcDNA-055 DNA vaccine was injected to BALB/c mice. The production of antibodies against pcDNA-055 DNA vaccine was confirmed by the Western blot analysis. The antibodies produced by the pcDNA-055 DNA vaccine showed efficacy to neutralize the RSIV in the neutralization test in BF-2 cell culture.

Using carotenoid genes of Erwinia herbicola, metabolic engineering was carried out for lycopene production with the pAC-LYCO4 plasmid, which was composed of a chromosomal DNA fragment of E. herbicola containing the crtE, crtB, and crtI genes under the control of the tetracycline promoter and the ipi gene of Haematococcus pluvialis with the trc promoter. Plasmid pAC-LYCm4 was constructed for efficient expression of the four exogenous genes using a strong RBS sequence and the same tetracycline promoter. The optimized expression construct of pAC-LYCm4 increased Iycopene production three times as compared with pAC-LYCO4. pAC-LYCm5 containing ispA behind the four exogenous genes was constructed. There was no significant difference in Iycopene production and cell growth between pAC-LYCm4 and pAC-LYCm5. FPP synthase encoded by ispA was not rate-limiting for Iycopene production. Each gene of crtE, crtB, crtI, and ipi was overexpressed, using pBAD-crtE, pBAD-crtIB, and pBAD-ipiHPI, in addition to their expression from pAC-LYCm4. However, there was no increase oflycopene production with the additional overexpression of each exogenous gene. The four exogenous genes appeared to be not rate-limiting in cells harboring pAC-LYCm4. When pDdxs, pBAD24 containing dxs, was introduced into cells harboring lycopene synthetic plasmids, lycopene production of pAC-LYCO4, pAC-LYCm4, and pAC-LYCm5 was increased by 4.7-, 2.2-, and 2.2-fold, respectively. Lycopene production of pBAD-DXm4 containing crtE, crtB, crtI, ipi, and dxs was 5.2 mg/g dry cell weight with $0.2\%$ arabinose, which was 8.7-fold higher than that of the initial strain with pAC-LYC04. Therefore, the present study showed that proper regulation of a metabolically engineered pathway is important for Iycopene production.

To evaluate the hepatoprotective activity of lactic acid bacteria, their effects on tert-butylperoxide (t-BHP)-induced hepatotoxicity in mice were measured. When lactic acid bacteria at doses of 0.5 and 2 g (wet weight)/kg were orally administered to mice with t-BHP-induced liver injury, these bacteria significantly inhibited the increase of plasma alanine aminotransferase and aspartate aminotransferase activities by $17-57\%$ and $57-66\%$ of the t-BHP control group, respectively. However, these lactic acid bacteria did not protect cytotoxicity induced by t-BHP against HepG2 cells. The inhibitory effects of these lactic acid bacteria at a dose of 15 g/kg were comparable with that of diphenyl dimethyl bicarboxylate at a dose of 0.2 g/kg, which has been used as a commercial hepatoprotective agent. Among these lactic acid Jacteria, Bifidobacterium longum HY8001 exhibited the most potent hepatoprotective effect. These orally administered lactic acid bacteria inhibited liver lipid peroxidation on t-BHP-induced hepatotoxicity of mice. We suggest that lactic acid bacteria may be an effective agent against liver injury.

In the course of screening a melanin biosynthesis inhibitor, terrein, 4,5-dihydroxy-3-propenyl-2-cyclopenten-l-one, was isolated from Penicillium sp. Terrein was found to have a strong inhibitory activity on melanin formation in B 16 melanoma and melanocyte Mel-Ab cells.

In the course of searching for potent chitin synthase 1 inhibitors from natural resources, Streptomyces sp. A6705 was found to exhibit potent inhibitory activity against the chitin synthase 1 from C. albicans (CaCHS1p). As a result, the inhibitor was isolated and identified using a series of chromatographies. Through chemical analyses with UV spectrophotometry, MS spectrometry, and various NMR techniques, the inhibitor was identified as N,N-bis(2-phenylethyl)urea. The compound exhibited strong inhibitory activity against the chitin synthase 1 from C. albicans with an $IC_{50}$ of 14 ${\mu}g$/ml, representing a similar inhibitory activity to that of the well-known chitin synthase inhibitor, polyoxin D ($IC_{50}$ 15 ${\mu}g$/ml). However, the compound showed no inhibitory activity against the chitin synthase 2 of Saccharomyces cerevisiae up to 280 ${\mu}g$/ml, which is structurally and functionally analogous to CaCHS 1 p. In addition, the compound exhibited weak antifungal activities against Cryptococcus neoformans and Rhizoctonis solani.

Peptide assimilation by Helicobacter pylori was investigated using L-phenylalanyl-3-thia-phenylalanine (PSP) as a detector peptide; the release of thiophenol upon enzymatic hydrolysis of PSP was spectrophotometrically detected with the aid of 5,5'-dithiobis[2-nitrobenzoic acid] (DTNB). By adding PSP to whole-cell suspension, thiophenol was produced progressively, resembling that found in Esherichia coli or Staphylococcus aureus. Interestingly, the rate of thiophenol production by H pylori in particular was markedly reduced when cells were pretreated with trypsin, indicating surface exhibition of peptidase. According to the competitive spectrophotometry using alanyl-peptides, H pylori did not appear to assimilate PSP through the peptide transport system. No discernible PSP assimilation could be ascertained in H pylori cells, unless provided with some additives necessary for peptidase activity, such as $Ni^{2+}\;or\;Mg^{2+}$ and an appropriate concentration of potassium or ammonium salts. These observations strongly suggest that, regardless of a presumptive peptide transport system, peptide assimilation of H. plori appears to be highly dependent upon milieu conditions, due to unique peptidase exhibition on the cell surface.

Inoculation of the carrier-based mixed bioinoculants af N-fixer (Azospirillum lipoferum strain Az204/Rhizobium strain BMBS P47) and phosphate-solubilizing bacterium (Bacillus megaterium var phosphaticum strain Pb 1) promoted growth and yield of pearl millet and blackgram under pot-culture conditions. The mixed inoculant of Az204 and Pb 1 enhanced germination, seedling vigor, plant height, and seed weight, and resulted in $6\%$ increase in grain yield of pearl millet. Likewise, the mixed inoculant of BMBS P47 and Pb1 increased growth, nodulation, and yield in blackgram. The rhizosphere soil enzyme activities, including nitrogenase, urease, and phosphatase, in both pearl millet and blackgram were significantly increased by the inoculation of the mixed inoculant, compared to that of the individual inoculants. The results clearly indicate the beneficial effect of co-culturing the N-fixer and P-solubilizer in inoculants production.

In the course of screening for selective growth inhibitors against the mycelial form of Candida albicans, we isolated a Streptomyces sp. A6792 from soils. The inhibitor was isolated from the above bacterium and identified through several spectral analyses with UV and mass spectrophotometries, and various NMR. The compound was determined to be a macrocyclic dilactone antibiotic, IKD-8344 (molecular weight: 844, molecular formula: $C_{48}H_{76}O_{12}$). The compound selectively inhibited the growth of mycelial form of C. albicans with an MIC of 6.25 ${\mu}g/ml$. It also exhibited strong inhibitory effect preferentially on the mycelial form of various Candida spp. including C. krusei, C. tropicalis, and C. lusitaniae, with MICs ranging from 1.56 to 25 ${\mu}g$/ml. Furthermore, the compound showed no significant toxicity against SPF ICR mice up to 60 mg/kg. These results suggest that IKD-8344 is a useful lead compound for the development of novel antifungal agents, based on the preferential growth inhibition against Candida spp.