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Recent studies have suggested that acid-sensitive background K+ channels such as TASK-1 and TASK-3, members of two-pore domain K+ (K2P) channel family, express and contribute to extracellular acidification-induced responses in dorsal root ganglia (DRG) neurons. However, it has remained to address whether other acid-sensitive background K+ channels are functionally expressed in DRG neurons. Here we characterized biophysical and pharmacological properties of a novel acid-sensitive background K+ channel in DRG neurons isolated from neonatal rats. We recorded an 80-pS K+ channel with a weak inward rectification current-voltage relationship in cell-attached patches in 150mM KCl bath solution. The 80-pS K+ channel was inhibited by extracellular low pH (pHo 6.3). Interestingly, the channel was similar to TASK-2 cloned from mouse and rat in biophysical and pharmacological properties. However, extracellular alkaline condition which activates TASK-2 channel, failed to activate the 80-pS K+ channel. Lidocaine and quinine more inhibited the channel activity of 80-pS K+ channel than that of TASK-2 channel. Our results suggest that the acid-sensitive 80-pS K+ channels may regulate resting membrane potential and may play a critical role in various processes such as cell metabolism, pH, and pain sensation in DRG neurons.

As part of the long-term monitoring projects on Antarctic terrestrial vegetation in relation to global climate change, a lichen floristical survey was conducted around the Korean Antarctic Station (King Sejong Station), which is located on Barton Peninsula, King George Island, in January and February of 2006. Two hundred and twenty-five lichen specimens were collected and sixty-two lichen species in 38 genera were identified by morphological characteristics, chemical constituents, TLC analysis and ITS nucleotide sequence analysis.

Objectives: To characterize a novel ceftazidime-hydrolysing CTX-M mutant, designated CTX-M-54, produced by Klebsiella pneumoniae clinical isolate BDK0419 and to investigate its genetic environment., Methods: Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the double-disc synergy test was carried out. Detection of genes encoding class A beta-lactamases was performed by PCR amplification, and the genetic organization of the blaCTX-M-54 gene was investigated by PCR and sequencing of the regions surrounding this gene. Kinetic parameters were determined from purified CTX-M-54., Results: The strain BDK0419 contained a transferable plasmid with a molecular size of approximately 21 kbp that carries both blaSHV-2a and blaCTX-M-54 beta-lactamase genes, along with two other plasmids. The blaCTX-M-54 gene was flanked upstream by an ISEcp1 insertion sequence and downstream by an IS903-like element. CTX-M-54 had a P167Q substitution within the omega loop region of class A beta-lactamases compared with the sequence of CTX-M-3. The MIC of ceftazidime for K. pneumoniae BDK0419 was 16-fold higher than that of cefotaxime; however, the kinetic parameter of CTX-M-54 against ceftazidime revealed a low catalytic efficiency., Conclusions: This work shows once again that novel CTX-M enzymes with an expanded activity towards ceftazidime through a single amino acid substitution can be identified from clinical isolates. Thus, detection of CTX-M enzymes can no longer be based solely on the resistance phenotypes of clinical isolates towards ceftazidime and cefotaxime.

COS-7 cells originally isolated from monkey kidney and used in many transfection studies were found to express a background K+ channel and therefore, its biophysical and pharmacological properties were examined. In cell-attached patches, a 32-pS K+ channel with a linear current-voltage relationship could be recorded. The open probability was highly voltage-dependent, with greater channel activity at depolarized potentials. The channel was markedly sensitive to changes in extracellular pH (pH(o)), showing a 70+/-10% inhibition by changing the pH(o) from 7.3 to 6.3. Arachidonic acid (5 microM) augmented channel activity 12-fold. Applying negative pressure (-40 mmHg) to the membrane patch also increased channel activity by 4-fold. These results show that COS-7 cells express a K+ channel with unique properties that must be considered when using these cells as transfection system.

Background: The aims of this study were to survey the nation-wide susceptibilities of Klebsiella pneumoniae isolates against ceftazidime and cefotaxime and to determine the prevalence of class A extended-spectrum beta-lactamases (ESBLs)., Methods: During the period of February to July 2004, K. pneumoniae isolates intermediate or resistant to ceftazidime and/or cefotaxime were collected from 12 hospitals in Korea. Antimicrobial susceptibilities were determined by the disk diffusion and the agar dilution methods and ESBL-production was by double-disk synergy test. Ceftazidime or cefotaxime-resistance determinants of the ESBLproducers were transfered to Escherichia coli J53 by transconjugation. Searches for class A ESBL genes were performed by PCR amplication., Results: Among 212 clinical K. pneumoniae isolates, 172 (81%) isolates showed positive results in double-disk synergy test; the most prevalent ESBL was SHV-12 (n=104). Genes encoding ESBLs including SHV-2 (n=6), SHV-2a (n=17), CTX-M-3 (n=18), CTX-M-9 (n=6), CTX-M-12 (n=1), CTX-M- 14 (n=27), CTX-M-15 (n=3), and a novel CTX-M-type beta-lactamases were also detected., Conclusions: It is concluded that diversity of ESBLs in K. pneumoniae isolates are increasing in Korea. CTX-M-12 has never been reported in Asia, and a novel CTX-M-type ESBL has emerged.

Detection of plasmid-mediated (P-M) AmpC beta-lactamase-producing isolates is considered critical for epidemiologic studies and hospital infection control, but the documents of the Clinical and Laboratory Standards Institute do not contain any recommendation for the phenotypic detection. In this study, phenotypic detection methods, cefoxitin-Hodge test and induction test, were evaluated using cefoxitin-resistant Escherichia coli and Klebsiella pneumoniae isolates. The cefoxitin-Hodge test detected all bla(CMY-10), and 97.4% of bla(CMY-2) allele-positive isolates, but only 57.3% of bla(DHA-1) allele-positive isolates. Induction test with an aztreonam and an amoxicillin-clavulanic acid disk was more sensitive than with cefoxitin disk, which detected 86.6% of bla(DHA-1) allele-positive isolates. These phenotypic tests should be useful to screen P-M AmpC beta-lactamase-producing E. coli and K. pneumoniae isolates.

Objectives: To assess the prevalence and genotypes of Ambler class A extended-spectrum beta-lactamases (ESBLs) in Korea., Methods: Clinical isolates of Escherichia coli and Klebsiella pneumoniae collected from 12 Korean hospitals during February-July 2003 were evaluated. Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the putative ESBL-producing strains were tested by the double-disc synergy method. Detection of genes encoding class A beta-lactamases was performed by PCR amplification, and the PCR products were subjected to direct sequencing., Results: The double-disc synergy test showed positive results in 9.3% (23/246) of E. coli and 23.0% (55/239) of K. pneumoniae isolates. The most prevalent types of Ambler class A ESBLs in E. coli isolates were CTX-M-15 (n = 4) and CTX-M-3 (n = 3), and those in K. pneumoniae isolates were SHV-12 (n = 30) and CTX-M-3 (n = 13). Two isolates produced both SHV-12 and GES-3, simultaneously., Conclusions: CTX-M-type and/or SHV-12 ESBL-producing E. coli and K. pneumoniae isolates are spreading, and a GES-type ESBL has emerged in Korea.

APP (beta-amyloid precursor protein) plays an important role in the formation of Alzheimer's Disease (AD), and its proteolytic product, the intracellular domain AID is believed to be involved in this process by promoting cell apoptosis. To further understand the function of AID in the pathology of AD, we utilized AID as a bait to identify AID interacting proteins in a yeast two-hybrid system. One of the positive clones encodes the fragment corresponding to amino acids 90-204 of heterogeneous nuclear ribonucleoprotein D-like JKTBP2. The interaction between JKTBP2(90)-204 and AID was further confirmed by co-immunoprecipitation in the mammalian 293T cells. These results indicate that JKTBP2 may have an important function in AD formation.

A gene for the Alternaria major allergen, Alt a 1, was amplified from 52 species of Alternaria and related genera, and sequence information was used for phylogenetic study. Alt a 1 gene sequences evolved 3.8 times faster and contained 3.5 times more parsimony-informative sites than glyceraldehyde-3-phosphate dehydrogenase (gpd) sequences. Analyses of Alt a 1 gene and gpd exon sequences strongly supported grouping of Alternaria spp. and related taxa into several species-groups described in previous studies, especially the infectoria, alternata, porri, brassicicola, and radicina species-groups and the Embellisia group. The sonchi species-group was newly suggested in this study. Monophyly of the Nimbya group was moderately supported, and monophyly of the Ulocladium group was weakly supported. Relationships among species-groups and among closely related species of the same species-group were not fully resolved. However, higher resolution could be obtained using Alt a 1 sequences or a combined dataset than using gpd sequences alone. Despite high levels of variation in amino acid sequences, results of in silico prediction of protein secondary structure for Alt a 1 demonstrated a high degree of structural similarity for most of the species suggesting a conservation of function.

Accurate identification of Alternaria spp. is dependent upon the production of diagnostic morphological characters under defined cultural conditions and the proper assessment of character variation. This process is often compromised by variation in laboratory facilities and technical expertise. To assist taxon identification and phylogenetic studies, restriction site information from the intergenic spacer (IGS) region of nuclear rDNA was evaluated. Restriction maps were constructed from 15 species of Alternaria and Stemphylium botryosum (telemorph Pleospora herbarum) for 11 restriction enzymes using a new method for restriction mapping based on differential priming of IGS amplicons. IGS fragment size varied among species from 2.2-3.9 kb. Based upon restriction site homology among closely-related and more distantly related species, the IGS region could be divided into conserved and variable domains. The conserved domain was approximately 0.75 kb in size and was located at the 3' end of the IGS region. Restriction site homology within this region was very high, especially among closely related taxa. The remainder of the region comprised the variable domain, which encompassed considerable differences in size and restriction sites among taxa. The presence or absence of restriction sites among taxa was analyzed using methods of neighbor-joining. Phylogenetic relationships based on this method were concordant with those previously resolved based upon other methods and other genomic regions.

The bowel transplantation team at the Catholic Medical Center, Korea, on April 9 2004, accomplished a case of isolated small bowel transplantation (SBT) in a 57 year-old female with short bowel syndrome. The primary surgery was a jejunocolostomy due to mesenteric vein thrombosis, while maintaining 30 cm of the jejunum and colon distal to the splenic flexure. Her renal function was partially unbalanced. During more than 2 years of home TPN, the superior vena cava (VC) and subclavian veins had become occluded, but the inferior VC line remained. SBT was planned due to the repeated life-threatening infections of the last central line. One hundred and fifty centimeter of the distal ileum of the 27 year-old living-related donor, the patient's daughter, was harvested. The graft mesenteric artery and vein were anastomosed to the recipient's inferior mesenteric vessels. A proximal end-to-end jejuno-ileostomy and a distal end-to-side ileo-colostomy of the graft were made, creating a Bishop-Koop enterostomy for graft surveillance. A tube jejunostomy, via a gastrostomy, was established for early feeding and simultaneous gastric drainage. Induction with Daclizumab and immunosuppression consisted of tacrolimus and methylprednisolone, given intravenously, and then mycophenolate mofetil (MMF), enterally from day 3. The patient was discharged on day 42. A CMV infection on day 83 was successfully treated with 3 weeks of gancyclovir therapy. She has been nutritionally independent, with complete oral feeding, and free of rejection until day 170 after the transplantation.

Two novel spore-forming bacteria with broad antimicrobial activity were isolated from roots of Perilla frutescens. The isolates, SD17(T) and SD18, were facultatively anaerobic and showed variable Gram reaction. Growth was observed between 20 and 45 degrees C. DNA G+C content of SD17(T) was 51.7 mol%, and the major fatty acid was anteiso-C(15 : 0) (54.1 %). 16S rRNA gene sequence similarity of SD17(T) ranged from 98.6 to 91.3 % with other Paenibacillus species. The phylogenetic tree showed that isolate SD17(T) formed a significant monophyletic clade with Paenibacillus koreensis KCTC 2393(T) and Paenibacillus ehimensis IFO 15659(T). DNA-DNA relatedness values for strain SD17(T) with Paenibacillus koreensis KCTC 2393(T) and Paenibacillus ehimensis IFO 15659(T) were 17.4 and 19.8 %, respectively. These isolates thus merit species status within Paenibacillus, for which the name Paenibacillus elgii sp. nov. is proposed. The type strain is SD17(T) (=KCTC 10016BP(T)=NBRC 100335(T)).

Characteristics and structures of mt SSU rDNA were investigated for the phylogenetic study of Ganoderma. Phylogenetic information was concentrated mostly in the V1, V4, V5, V6 and V9 variable domains, but informative sites in conserved domains also significantly contributed in resolving phylogenetic relationships between Ganoderma groups. Secondary structure information of variable domains was found to be a useful marker in delineation of phylogenetic groups. Strains of Ganoderma species used in this study were divided into six monophyletic groups. Ganoderma colossus made a distinct basal lineage from other Ganoderma species and Tomophagus, created for G. colosuss, appeared to be a valid genus. Ganoderma applanatum and G. lobatum classified in subgenus Elfvingia made a monophyletic group. Ganoderma tsugae from North America and G. valesiacum from Europe, both living on conifers, were closely related. Ganoderma oregonense and strains labeled G. lucidum from Europe and Canada were grouped with G. tsugae and G. valesiacum. Strains labeled G. lucidum living on hardwoods from the United States and Taiwan were grouped with G. resinaceum, G. pfeifferi and G. subamboinense var. laevisporum, and they all produced chlamydospores. Two strains labeled G. lucidum and three strains labeled G. resinaceum from America were concluded to be conspecific. Strains labeled G. lucidum from Korea and Japan were monophyletic and were distinguished from strains labeled G. lucidum from Europe and North America. Host relationships and the presence of chlamydospores in culture proved to be important characteristics in the systematics as well as the phylogenetic relationships of Ganoderma.

A new semi-selective medium, acidified weak potato-dextrose agar (AWPDA) with Mertect (active ingredient: thiabendazole), was developed for the isolation and enumeration of Alternaria species from samples of soil and plant debris. The medium was selected based on growth inhibition tests against Alternaria and several other commonly encountered saprobic fungi utilizing three antifungal agents, Botran (active ingredient: dichloran), Bayleton (active ingredient: triadimefon), and Mertect, and two basal media, acidified potato-dextrose agar (APDA) and AWPDA. Botran inhibited growth of Rhizopus stolonifer moderately, but had little effect on Cladosporium cladosporoides, Fusarium oxysporum, Penicillium chrysogenum, or Trichoderma harzianum. Bayleton inhibited growth of R. stolonifer and C. cladosporoides severely, and inhibited growth of F. oxysporum, P. chrysogenum, and T. harzianum moderately. Mertect inhibited growth of C. cladosporoides, F. oxysporum, P. chrysogenum, and T. harzianum completely, but had little or moderate effect on R. stolonifer. All three antifungal agents inhibited growth of Alternaria species slightly or moderately. The combination of Bayleton and Mertect inhibited growth of all fungi severely. A comparison of recovery rates of Alternaria from soil and plant debris samples on AWPDA with Mertect and weak potato-dextrose agar (WPDA) revealed that Alternaria spp. accounted for 63.6%-81.0% of recovered fungal isolates on AWPDA with Mertect as compared to 0.6%-2.7% of recovered isolates on WPDA. The AWPDA medium with Mertect exhibited superior selective growth of Alternaria species from samples of soil and plant debris, and will be useful in studies where the recovery and enumeration of Alternaria species is necessary.

Intravascular papillary endothelial hyperplasia (Masson's hemangioma) is a disease characterized by exuberant endothelial proliferation within the lumen of medium-sized veins. In 1923, Masson regarded this disease as a neoplasm inducing endothelial proliferation, however, now it is considered to be a reactive vascular proliferation following traumatic vascular stasis. The lesion has a propensity to occur in the head, neck, fingers, and trunk. Occurrence within the abdominal cavity is known to be very rare, and especially in the liver, there has been no reported case up to date. The authors have experienced intravascular papillary endothelial hyperplasia of the liver in a 69-yr-old woman, and report the case with a review of the literature.

Aim: To investigate the cellular effects of hybrid polar compound hexamethylene bisacetamide (HMBA) on the growth and apoptosis of human hepatocellular carcinoma cells and to provide the molecular mechanism for potential application of HMBA in the treatment of liver cancer., Methods: Effects of HMBA on the growth of human hepatocellular carcinoma SMMC-7721 cells were assayed by MTT chronometry. Apoptosis induced by HMBA was detected by phase-contrast microscopy, flow cytometry, propidium iodide staining and immunocytochemical analysis., Results: The growth of SMMC-7721 cells was significantly inhibited by HMBA, and the growth inhibitory rate was 51.1%, 62.6%, 68.7% and 73.9% respectively after treatment with 5.0, 7.5, 10.0 and 12.5 mmol/L of HMBA. In the cells treated with 10 mmol/L of HMBA for 72 h, the population of cells at sub-G(1) phase significantly increased, and the apoptotic bodies and condensed nuclei were detected. Moreover, treatment of SMMC-7721 cells with 10 mmol/L of HMBA down-regulated the expression of Bcl-2 anti-apoptotic protein, while slightly up-regulated the level of pro-apoptotic protein Bax., Conclusion: Treatment with 10.0 mmol/L of HMBA can significantly inhibit the growth and induce apoptosis of human hepatocellular carcinoma SMMC-7721 cells by decreasing the ratio of Bcl-2 to Bax.

The 5th year KONSAR surveillance in 2001 was based on routine test data at 30 participating hospitals. It was of particular interest to find a trend in the resistances of enterococci to vancomycin, of Enterobacteriaceae to the 3rd generation cephalosporin and fluoroquinolone, and of Pseudomonas aeruginosa and acinetobacters to carbapenem. Resistance rates of Gram-positive cocci were: 70% of Staphylococcus aureus to oxacillin; 88% and 16% of Enterococcus faecium to ampicillin and vancomycin, respectively. Seventy-two percent of pneumococci were nonsusceptible to penicillin. The resistance rates of Enterobacteriaceae were: Escherichia coli, 28% to fluoroquinolone; Klebsiella pneumoniae, 27% to ceftazidime, and 20% to cefoxitin; and Enterobacter cloacae, > or =40% to cefotaxime and ceftazidime. The resistance rates of P. aeruginosa were 21% to ceftazidime, 17% to imipenem, and those of the acinetobacters were > or =61% to ceftazidime, aminoglycosides, fluoroquinolone and cotrimoxazole. Thirty-five percent of non-typhoidal salmonellae were ampicillin resistant, and 66% of Haemophilus influenzae were beta-lactamase producers. Notable changes over the 1997-2001 period were: increases in vancomycin-resistant E. faecium, and amikacin- and fluoroquinolone-resistant acinetobacters. With the increasing prevalence of resistant bacteria, nationwide surveillance has become more important for optimal patient management, for the control of nosocomial infection, and for the conservation of the newer antimicrobial agents.

Changes in intracellular Ca2+ concentration are required for the activation of mammalian oocytes. They are caused mainly by Ca2+ release from the endoplasmic reticulum (ER) via Ins P3 receptors (Ins P3R). Several studies have reported that acetylcholine (ACh) is capable of triggering early activation events in mouse oocytes over-expressed with the m1 muscarinic ACh receptor (m1AChR). Here we examined which subtypes of the mAChR (m1 to m4) are involved in the generation of Ca2+ oscillations in native mouse oocytes. ACh (10 microM) elicited regular Ca2+ oscillations similar to those induced by sperm in their temporal characteristics. The Ca2+ oscillations were abolished by application with atropine, the mAChR inhibitor. Within 1 min after treatment of ACh, intracellular Fluo-3 fluorescence intensity increased from 794+/-119 to 2023+/-755 (increase to 250% of original value), indicating a strong rise of cytosolic Ca2+ concentration. 4-DAMP mustard and Tropicamide, specific antagonists of m3AChR and m4AChR, completely abolished ACh-induced Ca2+ oscillations. In the ovulated oocytes, the expression of m3/m4 AChR was clearly detected by RT-PCR analysis. Furthermore, ACh-induced Ca2+ oscillations were also abolished or decreased by PLC inhibitors (U73122 or D609) and an Ins P3-receptor antagonist (xestospongin C), confirming that ACh generates Ca2+ oscillations via the PLC-Ins P3 (PI) pathway. These results strongly suggest that m3/m4AChR is coupled to the generation of Ca2+ oscillations mainly via the PI pathway in mouse oocytes.

The taxonomic status of Hongia koreensis was investigated by molecular systematic methods. On the basis of 16S rDNA phylogeny, H. koreensis was closely associated with Kribbella flavida and Kribbella sandramycini and formed a monophyletic clade with these species. These three taxa shared over 98% 16S rDNA sequence similarity and many chemotaxonomic properties, which strongly indicates that they belong to the same genus. DNA-DNA pairing was employed to elucidate the genomic relatedness among these taxa. H. koreensis represented a distinct genomic species that can be differentiated from members of the genus Kribbella. Physiological characteristics and phospholipid and cellular fatty acid compositions can be also used to separate H. koreensis from species of the genus Kribbella. On the basis of data presented in this and earlier studies, it is proposed that H. koreensis Lee et al. 2000 should be transferred to the genus Kribbella Park et al. 1999 as Kribbella koreensis comb. nov.

Aim: To investigate the effects of tachyplesin on the cell cycle regulation in human hepatcarcinoma cells., Methods: Effects of tachyplesin on the cell cycle in human hepatocarcinoma SMMC-7721 cells were assayed with flow cytometry. The protein levels of p53, p16, cyclin D1 and CDK4 were assayed by immunocytochemistry. The mRNA levels of p21(WAF1/CIP1) and c-myc genes were examined with in situ hybridization assay., Results: After tachyplesin treatment, the cell cycle arrested at G0/G1 phase, the protein levels of mutant p53, cyclin D1 and CDK4 and the mRNA level of c-myc gene were decreased, whereas the levels of p16 protein and p21(WAF1/CIP1) mRNA increased., Conclusion: Tachyplesin might arrest the cell at G0/G1 phase by upregulating the levels of p16 protein and p21(WAF1/CIP1) mRNA and downregulating the levels of mutant p53, cyclin D1 and CDK4 proteins and c-myc mRNA, and induce the differentiation of human hepatocacinoma cells.

A novel asexual ascomycetous yeast, Candida kunwiensis (SG99-26T = KCTC 17041T = CBS 9067T), was isolated from sweet potato (Ipomoea batatas) flowers in Korea and from the body surface of pollinating bumblebees in Germany. Comparative analysis of the D1/D2 domain of 26S rDNA of all available sequences for ascomycetous yeasts showed that the novel species was phylogenetically related to the genus Metschnikowia, but the sequence similarity was low. Morphologically and physiologically, C. kunwiensis in many ways resembles Metschnikowia pulcherrima, but can be distinguished from this species by its ability to assimilate lactic acid and its inability to produce pulcherrimin.

Aim: To investigate the antitumor activities of tachyplesin on human hepatocellular carcinoma (HCC) cells., Methods: Tachyplesin, isolated from acid extracts of Chinese horseshoe crab (Tachypleus tridentatus) hemocytes, was used to treat the human HCC cell line SMMC-7721. Effects of tachyplesin on the proliferation of SMMC-7721 cells were measured with trypan blue dye exclusion test and HE staining. The morphology and ultrastructure of the cells were examined by light microscopy and transmission electron microscopy, respectively. The activities of gamma-glutamyltransferase (gamma-GT) and tyrosine aminotransferase (TAT) were assayed with biochemical methods. The levels of alpha fetoprotein (alpha-FP), proliferating cell nuclear antigen (PCNA), p21( WAF1/CIP1) and c-myc were examined by immunocytochemistry., Results: After treatment with tachyplesin 3.0 mg/L, the proliferation of SMMC-7721 cells was inhibited significantly, with the cell growth inhibitory rate amounted to 55.57 % and the maximum cell mitotic index declined by 43.68 %. The morphology and ultrastructure underwent restorational alteration. The activity of gamma-GT declined while TAT activity increased obviously, and the levels of alpha-FP and PCNA decreased. Moreover, the expression of p21(WAF1/CIP1) protein was up-regulated and that of c-myc protein was down-regulated., Conclusion: Tachyplesin could effectively inhibit the proliferation of hepatocarcinoma cells, reverse the malignant morphological and ultrastructural characteristics, alter the levels of enzymes and antigens, regulate the expression of differentiation-associated oncogene and tumor suppressor gene, and induce hepatocarcinama cell differentiation.

The taxonomy of soil isolates AP-20(T) and AP-37 was examined. These alkaliphilic organisms grew over a wide pH range (pH 7.0-13.0). The growth rate was higher at pH 8.0-12.0 than at pH 7.0. The G+C composition of these strains averaged 53 mol%. These strains contained MK-7 as the main respiratory quinone and meso-diaminopimelic acid in the cell-wall peptidoglycan. The major cellular fatty acid of the isolates was 12-methyltetradecanoic acid. Levels of 16S rDNA similarity between strain AP-20(T), AP-37 and other Paenibacillus species were 94.2-90.2%. Phylogenetic analysis based on 16S rDNA sequence revealed that strains AP-20(T) and AP-37 formed an evolutionary lineage distinct from other Paenibacillus species. Based on the evaluation of morphological, physiological and chemotaxonomic characteristics, and on 16S rDNA sequence comparison, the new species Paenibacillus daejeonensis sp. nov. is proposed; the type strain is AP-20(T) ( = KCTC 3745(T) = JCM 11236(T)).

There has been a systematic need to seek adequate phylogenetic markers that can be applied in phylogenetic analyses of fungal taxa at various levels. The mitochondrial small subunit ribosomal DNA (mt SSU rDNA) is generally considered to be one of the molecules that are appropriate for phylogenetic analyses at a family level. In order to obtain universal primers for polypores of Hymenomycetes, mt SSU rRNA genes were cloned from Bjerkandera adusta, Ganoderma lucidum, Phlebiopsis gigantea, and Phellinus laevigatus and their sequences were determined. Based on the conserved sequences of cloned genes from polypores and Agrocybe aegerita, PCR primers were designed for amplification and sequencing of mt SSU rDNAs. New primers allowed effective amplification and sequencing of almost full-sized genes from representative species of polypores and related species. Phylogenetic relationships were resolved quite efficiently by mt SSU rDNA sequences, and they proved to be more useful in phylogenetic reconstruction of Ganoderma than nuclear internal transcribed spacer (ITS) rDNA sequences.

In this study, the effects of HMBA on the expression of G0/G1 phase arrest-associated genes in human hepatocarcinoma SMMC-7721 cells were investigated. Immunocytochemistry and in situ hybridization assay revealed that the levels of p21WAF1/CIP1 and p16 proteins and the level of p21WAF1/CIP1 mRNA were increased while the levels of CDK4 and Cyclin D1 proteins and c-myc mRNA were decreased in the cells treated with HMBA. These results showed that HMBA could up-regulate the expression of p21WAF1/CIP1 and p16 genes, down-regulate the activity of Cyclin D1-CDK4 and the transcription of c-myc gene which were necessary for cells entering into S phase, and so arrest the cells in G0/G1 phase, and induce the differentiation of human hepatocarcinoma SMMC-7721 cells.

Background & Objective: The study on antitumor activities of marine bioactive substances is an important field in exploiting marine bioactive substances and antitumor drugs. And the induction of tumor cell differentiation is a new strategy for drug therapy of tumors. So the authors used tachyplesin, a marine bioactive substance, to investigate its effects on the differentiation of human hepatocarcinoma SMMC-7721 cells for further studying its antitumor activities and mechanisms., Methods: Tachyplesin, which was isolated from hemocytes of horseshoe crab (Tachypleus tridentatus), was used to treat human hepatocarcinoma SMMC-7721 cells. Light microscopy and transmission electron microscopy were applied to examine the changes in morphology and ultrastructure of SMMC-7721 cells, respectively. The activities of alkaline phosphatase or the expression of AFP and PCNA were assessed by cytochemistry or immunocytochemistry., Results: In the cells treated with 3.0 micrograms/ml tachyplesin, the morphology and ultrastructure returned to be normal, the activity of alkaline phosphatase was decreased and the levels of AFP and PCNA were downregulated., Conclusion: Tachyplesin might effectively reverse the malignant morphology and ultrastructure, change the activity of enzyme and the levels of antigens associated with hepatocarcinoma cell, and induce the differentiation of the human hepatocarcinoma SMMC-7721 cells.

The root of Paeonia suffruticosa ANDREWS is an important Chinese crude drug used in many traditional prescriptions. 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG), a major component of this crude drug, was found to exhibit in vitro growth-inhibiting effect on human hepatocellular carcinoma cell line, SK-HEP-1 cells. The growth-inhibitory effect was related to the ability of PGG not only to cause a G(0)/G(1) phase arrest but also to suppress the activation of nuclear factor-kappa B. Neither apoptosis nor necrosis was observed in the cells treated with PGG. These findings suggest that PGG could be a candidate for developing a low-toxic anticancer agent.

In this paper, the effects of HMBA on the differentiation of human hepatocarcinoma cell line SMMC-7721 were investigated. After treated with 5 mmol/L HMBA, the proliferation of SMMC-7721 cells was inhibited remarkably, the cell growth inhibitory rate amounted to 64.14%, the cell mitotic index was declined by 53.88%. Light microscopy and transmission electron microscopy showed that the morphology and ultrastructure of the cells treated with HMBA undergone restorational alteration. Cytochemistry and immunocytochemistry assay revealed that the activities of gamma-GT declined and the levels of AFP and PCNA downregulated while the activity of TAT increased significantly after HMBA treatment. In the meantime, flow cytometry analysis showed that HMBA could arrest the cells in G0/G1 phase. The results showed that HMBA could effectively inhibit the proliferation, reverse the malignant morphology and ultrastructure, alter the levels of enzymes and antigens, arrest the cells in G0/G1, and induce the differentiation of human hepatocarcinoma SMMC-7721 cells in vitro.

The effect of mistletoe lectin I (ML-I), an inhibitor of ribosomal protein synthesis, on the in vitro cytotoxicity of a clinically important anticancer drug, paclitaxel, was studied on cultured human hepatocarcinoma SK-Hep1 cells using the microculture tetrazolium test. The interaction between these two agents was analyzed for true synergism using the ED50 isobologram. Synergism was observed in the simultaneous treatment of the cells with ML-I in combination with paclitaxel. In addition, 24-h exposure of the cells to a non-toxic dose of ML-I and lower toxic doses of paclitaxel in combination resulted in apoptotic cell death, as observed by agarose-gel electrophoresis of low-molecular-weight DNA and DNA flow cytometry. Thus, the results presented here indicate the potential clinical usefulness of ML-I combination therapy with paclitaxel.

A novel ascomycetous yeast was isolated from flowers of Lilium sp. and Ipomoea sp. in Korea. The name Metschnikowia koreensis sp. nov. (type strain SG99-34T = CBS 8854T = KCTC 7998T) is proposed for this novel species based on comparative sequence analyses of the D1/D2 domain of 26S rDNA and phenotypic characteristics.

AIM:To investigate the morphological and ultrastructural changes in the human gastric carcinoma cell line BGC-823 after being treated with tachyplesin.METHODS:Tachyplesin was isolated from acid extracts of Chinese horseshoe crab (Tachypleus tridentatus) hemocytes. BGC-823 cells and the cells treated with 2.0mg/L tachyplesin were examined respectively under light microscope, scanning and transmission electron microscope.RESULTS: BGC-823 cells had undergone the restorational alteration in morphology and ultrastructure after tachyplesin treatment. The changes were as follows: the shape of cells was unanimous, the volume enlarged and cells turned to be flat and spread, the nucleo-cytoplasmic ratio lessened and nuclear shape became rather regular, the number of nucleolus reduced and its volume lessened,heter-chromatin decreased while euchromatin increased in nucleus. In the cytoplasm, mitochondria grew in number with consistent structure relatively, Golgi complex turned to be typical and well-developed,rough endoplasmic reticulum increased and polyribosome decreased. The microvilli at cellular surface were rare and the filopodia reduced while lamellipodia increased at the cell edge.CONCLUSION:Tachyplesin could alter the malignant morphological and ultrastructural charact-eristics of human gastric carcinoma cells effectively and have a certain inducing differen-tiation effect on human gastric carcinoma cells.

Oxidative stress is a major cause of neurodegenerative diseases, so the protection of neuronal cells from reactive oxygen species can be beneficial for the prevention and treatment of these diseases. Methanol extract of Orostachys japonicus A. Berger (Crassulaceae), a traditional oriental medical herb, was shown to have a protective effect on H2O2-induced apoptosis in GT1-1 mouse hypothalamic neuronal cell line which was detected by flow cytometry after propidium iodide staining. Among fractions of O. japonicus, chloroform fraction had the highest protective effect, and water fraction had no protective effect suggesting that the active ingredients might be hydrophobic compounds.

Rapid desensitization of the muscarinic K+ current (KACh current) is observed in cell-attached patches with 10 microM acetylcholine in the pipette. When inside-out patches were formed within approximately 1 s after formation of cell-attached patches and GTP was applied to the cytoplasmic side of the membrane, desensitization was not observed, indicating that a cytosolic factor mediated the desensitization. Applying the atrial cytosolic extract directly to the cytoplasmic side of such inside-out patches elicited a rapid desensitization of the KACh current. ATP (1-4 mM) reversed this effect of the cytosol and reverted the KACh channel to the undesensitized state. These effects of ATP and cytosol on the KACh channel could occur in the absence of GTP or in the presence of 100 microM guanosine 5'-O-(3-thiotriphosphate), indicating that G protein was not involved. Treatment of the cytosol with proteases (trypsin, chymotrypsin, bacterial protease) or heat denaturation abolished the effect of the cytosol on the KACh channel kinetics, indicating that the cytosolic factor was a protein. Functional assay of the fractions collected from gel filtration column indicated that the molecular mass of the native protein was 95-130 kDa. We conclude that a large cytosolic protein mediates the rapid desensitization of the KACh channel current via a G protein-independent pathway.

Malus pumila Mill. twigs were collected from September through December and stored at 5 degrees C until the low temperature exotherms of the xylem were determined by differential thermal analysis. During the differential thermal analysis, cooling was interrupted, and temperatures of 5 to -18 degrees C were held for 0.4 to 10 hours before cooling to -50 degrees C was resumed. Control twigs were cooled to -50 degrees C without interruption. Holding the twigs at 1.3 to -5 degrees C shifted the start of the low temperature exotherm from about -20 to -30 degrees C. Slightly higher (2.6 degrees C) and lower (-10 degrees C) temperatures were occasionally effective. The shift began within 20 to 30 minutes and increased progressively to 150 minutes. The acclimation was reversibly inhibited by N(2) atmosphere.

Exudation, (86)Rb transport, and water permeability were examined in excised roots of honey locust (Gleditsia triacanthos L.) treated by removing the tip 2 mm (tip-cut 2 mm) or tip 8 mm of the root, or by adding kinetin, or by both treatments. Tip removal increased the rate of exudation. Kinetin, 5 x 10(-6)m, inhibited exudation and Rb transport in tip-cut 2-mm roots; the inhibition was reversible. Kinetin inhibition of exudation was initially associated with lower K(Rb) transport and later with decreases in both ion transport and water permeability. Exudation was also inhibited at 10(-10) to 10(-7)m kinetin. Exudation from roots with intact tips was not altered by kinetin until after about 24 hours. Light during the exudation period had no significant (95%) influence on rate of exudation during the first 24 hours whether root tips were cut or kinetin applied.The results suggest the involvement of the root tip in regulating exudation in other parts of the root. This regulation might occur through cytokinin control of water permeability and the rate of ion transport.

The low temperature exotherms (LTE) of 1-year-old twigs of Haralson apple (Malus pumila Mill.), shagbark hickory (Carya ovata [Mill.] K. Koch), green ash (Fraxinus pennsylvanica Marsh), honey locust (Gleditsia triacanthos L.), American chestnut (Castanea dentata [Marsh] Borkh.), and red oak (Quercus rubra L.) were determined by differential thermal analysis (DTA). In one type of experiment freezing during a DTA experiment was halted for up to 2.5 hours after part of the supercooled water had frozen at temperatures between -25 and -42 C. Upon resumption of cooling the freezing started within 2 C of the stopping temperature. In a second type of experiment living and dead cells were microscopically observed in the same ray after partial freezing in the DTA apparatus. In another experiment, the LTE persisted even after tangential and radial sectioning of the twig to 0.13 millimeters. In a final experiment the LTE of a single multiseriate ray of red oak had the same shape as the LTE of wood with many uniseriate rays.These experiments confirm that the deep supercooled water in woody xylem or pith freezes in numerous independent events over a span of as much as 20 C. The units which freeze in an event are single cells or small groups of cells. Ice grows very slowly if at all from these units, and water moves very slowly from unfrozen cells to frozen ones. Deep supercooling of ray parenchyma does not require an intact ray.