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Biomedical subjects

S W Kim

Publications and source records attributed to S W Kim.

At least 19 recordsLinked to original sources

Regulation of adipogenesis by a transcriptional repressor that modulates MAPK activation.

Mitogen-activated protein kinase (MAPK) is required for cell growth and cell differentiation. In adipogenesis, MAPK activation opposes the differentiation process. The regulatory mechanisms or the cellular factors that regulate the switch between growth and differentiation in the adipogenic lineage have been largely unelucidated. We show here that AEBP1, a transcriptional repressor that is down-regulated during adipogenesis, complexes and protects MAPK from its specific phosphatase in mammalian cells. We further show evidence that the modulation of MAPK activation by AEBP1 is a biologically relevant process in adipogenesis. Our results suggest that modulation of MAPK activation by the protective effect of AEBP1 may constitute a critical part in the determination between cell growth and differentiation in the adipogenic lineage. The proposed mode of action by which a transcription factor regulates MAPK activation is novel.

3T3 Cells↗

Effects of collagen IV and laminin on the reconstruction of human oral mucosa.

To investigate the effects of basement membrane proteins on the reconstruction of mucosa equivalent, oral mucosa substitute were cultured on (1) type I collagen gels, (2) type IV collagen-coated type I collagen gels, (3) laminin-coated type I collagen gels, and (4) type I collagen gels containing both type IV collagen and laminin. H/E and PAS staining showed that the characteristics of the oral mucosa were preserved under all the experimental conditions. However, the basal keratinocytes appeared cuboidal when the type I collagen gels were coated with type IV collagen plus laminin. The expression of the differentiation markers was similar, but weak staining of filaggrin, K13, and involucrin was observed with the type IV collagen plus laminin coating. Furthermore, electron microscopy revealed that the size of the basal keratinocytes was relatively small and uniform when both type IV collagen and laminin were used. These findings suggested that these two major basement membrane proteins are important in the process of differentiation in mucosal keratinocytes.

Animals↗

Prophylactic efficacy of a new gentamicin-releasing urethral catheter in short-term catheterized rabbits.

OBJECTIVE: To describe an indwelling urethral catheter coated with gentamicin sulphate on the inner and outer surface of the catheter, and to evaluate the efficacy and safety of this catheter in preventing catheter-associated infections in rabbits. Materials and methods Sixty rabbits were divided equally into control and experimental groups which were then subdivided equally according to the duration of catheterization (1, 3 and 5 days). Silicone-treated latex catheters were used in the control group and gentamicin-releasing catheters in the experimental group. Urine samples and surface swabs from the catheter were cultured for bacteriological assessment, and the catheter surface examined by scanning electron microscopy to structurally analyse the biofilms. RESULTS: The gentamicin-releasing catheter reduced the incidence of bacteriuria (defined as > or = 100 c.f.u./mL) after both 3 and 5 days of catheterization (eight and 10 rabbits, respectively, for the control catheter, vs two and four rabbits for the gentamicin-releasing catheter, P < 0.05). The surfaces of the gentamicin-releasing catheter were colonized less often than those of the control catheter after both 3 and 5 days (eight and 10, respectively, for the control, vs one and four for the gentamicin-releasing catheter, P < 0.05). Scanning electron microscopy showed the formation of bacterial biofilm throughout the 3-day and 5-day control catheters, but deterioration of the bacterial biofilm was visible on the surface of the gentamicin-releasing catheters. CONCLUSION: This new gentamicin-releasing catheter produced an antibacterial barrier which inhibited catheter-associated urinary tract infection with no toxicity for at least 5 days. These in vivo studies suggest that this new catheter may be useful for controlling infection, with systemic and local safety, in patients undergoing short-term indwelling urethral catheterization.

Animals↗

Microtubule- and dynein-mediated movement of Orientia tsutsugamushi to the microtubule organizing center.

The host cell microfilaments and microtubules (MTs) are known to play a critical role in the life cycles of several pathogenic intracellular microbes by providing for successful invasion and promoting movement of the pathogen once inside the host cell cytoplasm. Orientia tsutsugamushi, an obligate intracellular bacterium, enters host cells by induced phagocytosis, escapes to the cytosol, and then replicates in the cytosol. ECV304 cells infected with O. tsutsugamushi revealed the colocalization of the MT organizing center (MTOC) and cytosolic orientiae by indirect immunofluorescence assay. Using immunofluorescence microscopy in the presence and absence of MT-depolymerizing agents (colchicine and nocodazole), it was shown that the cytosolic oriential movement was mediated by MTs. By transfection study (overexpression of dynamitin [also called p50], which is known to associate with dynein-dependent movement), the movement of O. tsutsugamushi to the MTOC was also mediated by dynein, the minus-end-directed MT-related motor. Although the significance of this movement in the life cycle of O. tsutsugamushi was not proven, we propose that the cytosolic O. tsutsugamushi bacteria use MTs and dyneins to propel themselves from the cell periphery to the MTOC.

Cell Line↗

Clinicopathologic and genotypic study of extranodal nasal-type natural killer/T-cell lymphoma and natural killer precursor lymphoma among Koreans.

BACKGROUND: This study aimed to define genotypic profile and to describe the clinicopathologic features of nasal-type natural killer (NK)/T-cell lymphoma of nasal and extranasal origin and NK precursor lymphoma. METHODS: NK/T-cell lymphomas from the upper aerodigestive tract (n = 45), skin (n = 2), gastrointestinal tract (n = 3), and soft tissue (n = 2) and NK precursor neoplasms (n = 3) were studied. Immunophenotype was analyzed by immunohistochemistry and flow cytometry. In situ hybridization with EBER 1/2 RNA probes was performed. T-Cell Receptor (TCR)-gamma gene rearrangement was analyzed by seminested polymerase chain reaction with heteroduplex analysis. Overall survival rate was correlated with clinicopathologic parameters and compared by Wilcoxon test. RESULTS: Clonal TCR-gamma gene rearrangement was detected in 3 of 31 upper aerodigestive and 1 of 2 skin tumors. When immunostained using paraffin embedded tissue, 6 upper aerodigestive lymphomas were negative for CD56 in which 4 cases lacked clonal TCR gene rearrangement. Epstein-Barr virus (EBV) mRNA was detected in 33 upper aerodigestive tumors including 26 of 29 nasal tumors (90%), and 7 of 10 extranasal tumors (70%). There was no histologic, immunophenotypic, or genotypic differences according to the lineage and EBV association in upper aerodigestive lymphomas. Among the patients with upper aerodigestive tumors, overall 1-year survival rate was 41%, and correlated well with the stage (P < 0.05) but not with the size of tumor cells, EBV status, and lineage (P > 0.05). Median survival rate of lymphomas from other sites excluding upper aerodigestive tract was not significantly different from that of upper aerodigestive lymphomas with same stage (P > 0.05). Unlike nasal-type NK/T-cell lymphomas, NK precursor lymphoma involved the bone marrow and lymph nodes at initial presentation or in the course of disease. Tumor cells were positive for TdT in all and myeloid markers in two. TCR gene rearrangement was germ line. CONCLUSIONS: Most upper aerodigestive nasal-type NK/T-cell lymphomas among Koreans are genotypically of NK derivation and few belong to T lineage. Presence or absence of EBV has no significant correlation with the histologic changes and the lineage of these lymphomas.

Adult↗

Self-assembled hydrogel nanoparticles from curdlan derivatives: characterization, anti-cancer drug release and interaction with a hepatoma cell line (HepG2).

Self-assembled hydrogel nanoparticles were synthesized from carboxymethylated (CM)-curdlan, substituted with a sulfonylurea (SU) as a hydrophobic moiety for self-assembly. The degree of SU substitution was 2.4, 5.6, or 7.2 SU groups per hundred anhydroglucose units of curdlan. The physicochemical properties of the self-assembled hydrogel nanoparticles (DS 2.4, DS 5.6, and DS 7.2) in aqueous media were characterized by dynamic light scattering, transmission electron microscopy, and fluorescence spectroscopy. The mean diameter of all samples was less than 300 nm with a unimodal size distribution. The critical aggregation concentrations (CAC) of self-assembled hydrogel nanoparticles in distilled water were 4.2 x 10(-2), 3.1 x 10(-2) and 1.9 x 10(-2) mg/ml for DS 2.4, 5.6 and 7.2, respectively. The loading and release of all-trans retinoic acid (ATRA) was studied. The ATRA loading efficiencies and loading contents of CM-curdlan/SU nanoparticles increased as the degree of SU substitution increased. The ATRA release rate was controlled by the degree of substitution and drug-loading. For specific interaction with a hepatic carcinoma cell line (HepG2), CM-curdlan was additionally conjugated with lactobionic acid (LBA; galactose moiety) (5.5 LBA molecules per hundred glucose units). HepG2 was strongly luminated by ligand-receptor interactions with fluorescence-labeled LBA/CM-curdlan/SU hydrogel nanoparticles. The luminescence was not observed for other control cases. It is concluded that LBA/CM-curdlan/SU hydrogel nanoparticles are a useful drug carrier for the treatment of liver cancer, because of the potential immunological enhancement activities of CM-curdlan in the body, the ligand-receptor mediated specific interactions, and the controlled release of the anti-cancer drug.

Animals↗

Hematopoietic-specific beta 1 tubulin participates in a pathway of platelet biogenesis dependent on the transcription factor NF-E2.

The cellular and molecular bases of platelet release by terminally differentiated megakaryocytes represent important questions in cell biology and hematopoiesis. Mice lacking the transcription factor NF-E2 show profound thrombocytopenia, and their megakaryocytes fail to produce proplatelets, the microtubule-based precursors of blood platelets. Using mRNA subtraction between normal and NF-E2-deficient megakaryocytes, cDNA was isolated encoding beta1 tubulin, the most divergent beta tubulin isoform. In NF-E2-deficient megakaryocytes, beta1 tubulin mRNA and protein are virtually absent. The expression of beta1 tubulin is exquisitely restricted to platelets and megakaryocytes, where it appears late in differentiation and localizes to microtubule shafts and coils within proplatelets. Restoring NF-E2 activity in a megakaryoblastic cell line or in NF-E2-deficient primary megakaryocytes rescues the expression of beta1 tubulin. Re-expressing beta1 tubulin in isolation does not, however, restore proplatelet formation in the defective megakaryocytes, indicating that other critical factors are required; indeed, other genes identified by mRNA subtraction also encode structural and regulatory components of the cytoskeleton. These findings provide critical mechanistic links between NF-E2, platelet formation, and selected microtubule proteins, and they also provide novel molecular insights into thrombopoiesis. (Blood. 2000;96:1366-1373)

Animals↗

The role of RhoA in the germinal vesicle breakdown of mouse oocytes.

We have investigated a new role of RhoA in the germinal vesicle breakdown (GVBD) of mouse oocytes. First, RhoA was identified by immunostaining and ADP-ribosylation in germinal vesicle (GV) stage-oocytes. RhoA was mainly localized in the ooplasmic area, but rarely detected in germinal vesicle. Incubation of oocyte extract with C3 transferase induced a strong ADP-ribosylation at about 25 kDa. Incubation of GV-stage oocytes in culture medium induced the spontaneous maturation to GVBD by about 78 and 87% of total oocytes at 1 and 3 h, respectively. However, microinjection of C3 transferase into GV-stage oocytes significantly inhibited GVBD at 1 (GVBD = 29%) and 3 h (GVBD = 49%). To study the role of reactive oxygen species (ROS) in the oocyte maturation, the level of intra-oocyte ROS was measured using a ROS-specific fluorescent dye H(2)DCFDA during the oocyte maturation. Spontaneous maturation of GV-stage oocytes induced a significant increase of ROS at 3 h by about twofold over the control level and then the increased level was maintained until 6 h. However, microinjection of C3 transferase inhibited the production of intra-oocyte ROS. Incubation with ROS scavengers, N-acetyl-l-cysteine and catalase, blocked the ROS increase. The ROS scavengers also significantly inhibited GVBD, as did C3 transferase. Thus, it was proposed that RhoA was involved in the GVBD, possibly by the production of ROS in mouse oocytes.

ADP Ribose Transferases↗

Effect of decreasing afferent vagal activity with ondansetron on symptoms of bulimia nervosa: a randomised, double-blind trial.

BACKGROUND: Several lines of evidence have led us to postulate that afferent vagal hyperactivity could be an important factor in the pathophysiology of the eating disorder bulimia nervosa. Ondansetron is a peripherally active antagonist of the serotonin receptor 5-HT3, and is marketed for prevention of vagally-mediated emesis caused by cancer chemotherapeutic agents. We investigated the effects of ondansetron on bulimic behaviours in patients with severe and chronic bulimia nervosa in a randomised, double-blind, placebo-controlled study. METHODS: We enrolled patients with severe bulimia nervosa (at least seven coupled binge/vomit episodes per week). The patients were otherwise healthy, their weight was normal, and they were not receiving medical or psychiatric treatment. During the first week of the study, patients recorded all eating-behaviour events to establish a baseline. In the second week, all patients received placebo, but were told that they were receiving either placebo or active drug. At the end of this single-blind phase, patients were randomly assigned placebo or ondansetron (24 mg daily) for a further 4 weeks. The primary outcome measure was the number of binge/vomit episodes per week. Data were analysed by intention to treat. FINDINGS: 29 patients met the inclusion criteria, of whom 28 completed the baseline study, and 26 completed the single-blind placebo week. 12 patients were assigned placebo, and 14 ondansetron; one patient in the ondansetron group dropped out owing to accidental injury. During the 4th week of double-blind treatment, mean binge/vomit frequencies were 13.2 per week (SD 11.6) in the placebo group, versus 6.5 per week (3.9) in the ondansetron group (estimated difference 6.8 [95% CI 4.0-9.5]; p<0.0001). The ondansetron group also showed significant improvement, compared with the placebo group, in two secondary indicators of disease severity. The amount of time spent engaging in bulimic behaviours was decreased on average by 7.6 h per week in the ondansetron group, compared with 2.3 h in the placebo group (estimated difference 5.1 [0.6-9.7]). Similarly, the number of normal meals and snacks increased on average by 4.3 normal eating episodes without vomiting per week in the ondansetron group, compared with 0.2 in the placebo group (estimated difference 4.1 [1.0-7.2]). INTERPRETATION: The decrease in binge-eating and vomiting under ondansetron treatment was not achieved by compensatory changes in eating behaviour such as by a smaller number of binges of longer duration, or by not eating, or by binge-eating without vomiting. Instead, our findings indicate a normalisation of the physiological mechanism(s) controlling meal termination and satiation. Since meal termination and satiety are mainly vagally mediated functions, since binge-eating and vomiting produce intense stimulation of vagal afferent fibres, and since ondansetron and other 5-HT3 antagonists decrease afferent vagal activity, the symptom improvement may result from a pharmacological correction of abnormal vagal neurotransmission.

Adolescent↗

Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis.

We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg(2074), Asp(2098), Arg(2171), Arg(2174), and Glu(2189) are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg(2074), Asp(2098), Arg(2171), and Arg(2174)) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys(2060) through Glu(2069) in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His(2128) through Lys(2137). The PS-binding site in the factor V C2-domain includes amino acid residues Trp(2063) and Trp(2064). This site overlaps with the epitope for monoclonal antibody HV-1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.

Alanine↗

Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

Delta 5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta 5-3-ketosteroids at a rate approaching the diffusion limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis. The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, Asp-99 COOH...Tyr-14 OH...C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca. 2.4 A, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate.

Asparagine↗

Drug release from biodegradable injectable thermosensitive hydrogel of PEG-PLGA-PEG triblock copolymers.

An aqueous solution of newly developed low-molecular-weight PEG-PLGA-PEG triblock copolymers with a specific composition is a free flowing sol at room temperature but becomes a gel at body temperature. Two model drugs, ketoprofen and spironolatone, which have different hydrophobicities, were released from the PEG-PLGA-PEG triblock copolymer hydrogel formed in situ by injecting the solutions into a 37 degrees C aqueous environment. Ketoprofen (a model hydrophilic drug) was released over 2 weeks with a first-order release profile, while spironolactone (a model hydrophobic drug) was released over 2 months with an S-shaped release profile. The release profiles were simulated by models considering degradation and diffusion, and were better described by a model assuming a core-shell structure of the gel.

Anti-Inflammatory Agents, Non-Steroidal↗

In situ gelation of PEG-PLGA-PEG triblock copolymer aqueous solutions and degradation thereof.

Aqueous solutions of poly(ethylene glycol-b-[DL-lactic acid-co-glycolic acid]-b-ethylene glycol) (PEG-PLGA-PEG) triblock copolymers form a free-flowing sol at room temperature and become a gel at body temperature. In this study, in situ gel formation was investigated in rats. Upon subcutaneous injection of 33 wt % aqueous solutions of PEG-PLGA-PEG triblock copolymer into rats, transparent gels were observed. The gel showed good mechanical strength and the integrity of gels persisted longer than 1 month. The gel underwent degradation by hydrolysis and turned opaque. Degradation study showed preferential mass loss of PEG-rich segment from the in situ formed gel. Number average molecular weight determined by gel permeation chromatography decreased from 3300 to 1900 and 30% mass loss was observed over 1 month.

Animals↗

Thermoreversible copolymer gels for extracellular matrix.

To improve the properties of a reversible synthetic extracellular matrix based on a thermally reversible polymer, copolymers of N-isopropylacrylamide and acrylic acid were prepared in benzene with varying contents of acrylic acid (0 to 3%) and the thermal properties were evaluated. The poly(N-isopropylacrylamide) and copolymers made with acrylic acid had molecular weights from 0.8 to 1.7 x10(6) D. Differential scanning calorimetry (DSC) showed the high-molecular-weight acrylic acid copolymers had similar onset temperatures to the homopolymers, but the peak width was considerably increased with increasing acrylic acid content. DSC and cloud point measurements showed that polymers with 0 to 3% acrylic acid exhibit a lower critical solution temperature (LCST) transition between 30 degrees and 37 degrees C. In swelling studies, the homopolymer showed significant syneresis at temperatures above 31 degrees C. Copolymers with 1 and 1.5% showed syneresis beginning at 32 degrees and 37 degrees C, respectively. At 37 degrees C the copolymers with 1.5-3% acrylic acid showed little or no syneresis. Due to the high water content and a transition near physiologic conditions (below 37 degrees C), the polymers with 1.5-2.0% acrylic acid exhibited properties that would be useful in the development of a refillable synthetic extracellular matrix. Such a matrix could be applied to several cell types, including islets of Langerhans, for a biohybrid artificial pancreas.

Acrylamides↗

C-terminal variations in beta-thymosin family members specify functional differences in actin-binding properties.

Mammalian cells express several isoforms of beta-thymosin, a major actin monomer sequestering factor, including thymosins beta4, beta10, and beta15. Differences in actin-binding properties of different beta-thymosin family members have not been investigated. We find that thymosin beta15 binds actin with a 2.4-fold higher affinity than does thymosin beta4. Mutational analysis was performed to determine the amino acid differences in thymosin beta15 that specify its increased actin-affinity. Previous work with thymosin beta4 identified an alpha-helical domain, as well as a conserved central motif, as crucial for actin binding. Mutational analysis confirms that these domains are also vital for actin binding in thymosin beta15, but that differences in these domains are not responsible for the variation in actin-binding properties between thymosins beta4 and beta15. Truncation of the unique C-terminal residues in thymosin beta15 inhibits actin binding, suggesting that this domain also has an important role in mediating actin-binding affinity. Replacement of the 10 C-terminal amino acids of thymosin beta15 with those of thymosin beta4 did, however, reduce the actin-binding affinity of the hybrid relative to thymosin beta15. Similarly, replacement of the thymosin beta4 C-terminal amino acids with those of thymosin beta15 led to increased actin binding. We conclude that functional differences between closely related beta-thymosin family members are, in part, specified by the C-terminal variability between these isoforms. Such differences may have consequences for situations where beta-thymosins are differentially expressed as in embryonic development and in cancer.

Actins↗

K-ras oncogene mutation in cancer and precancerous lesions of the gallbladder.

BACKGROUND AND OBJECTIVES: To determine whether K-ras mutation plays any role in the development and progression of gallbladder cancer, or has any clinical or pathological significance in gallbladder cancer patients, we investigated the presence and incidence of this mutation in the normal mucosa, and precancerous and cancerous lesions of the gallbladder. METHODS: DNA was obtained from normal mucosa, dysplastic mucosa, primary cancer tissues, and metastatic lymph nodes that were identified and microdissected from the paraffin blocks of 20 gallbladder cancer cases. K-ras codon 12 mutations were investigated using a modified two-step polymerase chain reaction and the restriction fragment length polymorphism method, and by direct sequencing with an automated sequencer. RESULTS: K-ras mutations were detected in the tissues of 10 out of the 20 patients. A mutation was present in the dysplastic epithelium associated with the primary carcinoma in 3 out of 12 specimens, in metastatic carcinoma in 1 out of 5 patients, and in primary carcinoma in 8 out of 20 patients. Mutation was found only once in the dysplastic, noncancerous epithelium, and only once in a metastatic tumor although not detectable in the primary cancer. Direct sequencing showed that the mutations were G to C substitutions (GGT-->CGT) at the first site of codon 12, except in two cases (GGT-->TGT). There were no correlations between K-ras mutations and clinicopathological factors. CONCLUSIONS: K-ras mutations were detected in half of the gallbladder cancer cases. We suggest that K-ras mutation may play a role in the development of premalignant lesions or early carcinogenesis in some gallbladder cancers. We were unable to find any evidence that K-ras mutation plays any role in tumor progression or metastasis, or that it has any clinicopathological significance.

Adult↗

Clinicopathologic features of the intraductal growth type of peripheral cholangiocarcinoma.

The clinicopathologic features of the intraductal growth (IG) type of peripheral cholangiocarcinoma (PCC) were examined retrospectively. Out of 112 patients who underwent surgery for PCC at Seoul National University Hospital, Seoul, Korea, between January 1980 and December 1997, 16 were classified as having the IG type. Thirteen were men and 3 were women. Their ages ranged from 38 to 73 years with a mean age of 55.9 years. Abdominal pain was the most common symptom and jaundice was found in 18.8%. Five patients had associated clonorchiasis and 6 patients experienced hepatolithiasis. Tumor sizes ranged from 0.5 cm to 14.0 cm with a mean of 4.3 cm. The tumor was located in the right lobe in 7 cases and the left lobe in 9 cases. Thirteen patients underwent major hepatic resection, and 3 underwent minor resection, involving a subsegmentectomy. Mucin was found in the bile in 4 cases. Pathology showed papillary adenocarcinoma with a background of adenomatous hyperplasia, and the absence of lymph node metastasis in all cases. The tumors were confined to the mucosa in 5 cases. Out of the 16 hepatic resections, 1 was palliative due to a positive margin. In 2 of the patients who underwent minor resections, recurrences developed, and 1 of these died 56 months after resection. Out of the 16 patients, 15 remain alive, ranging from 1 to 13 years, postoperatively. In conclusion, the IG type of PCC should be distinguished from other types of PCC because a favorable prognosis can be expected after complete surgical resection.

Adult↗

Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria.

Multicopy plasmids are often chosen for the expression of recombinant genes in Escherichia coli. The high copy number is generally desired for maximum gene expression; however, the metabolic burden effects that usually result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications. In this study, low-copy mini-F plasmids were compared to high-copy pMB1-based plasmids for production of two metabolites in E. coli: polyphosphate (polyP) and lycopene derived from isopentenyl diphosphate (IPP). The stationary-phase accumulation of polyP on a per cell basis was enhanced approximately 80% when either high- or low-copy plasmids were used, from 120 micromol/g DCW without augmented polyP kinase (PPK) activity to approximately 220 micromol/g DCW. The cell density of the high-copy plasmid-containing culture at stationary phase was approximately 24% lower than the low-copy culture and 30% lower than the control culture. This difference in cell density is likely a metabolic burden effect and resulted in a lower overall product concentration for the high-copy culture (approximately 130 micromol/L culture) relative to the low-copy culture (approximately 160 micromol/L culture). When the gene for DXP (1-deoxy-D-xylulose 5-phosphate) synthase, the first enzyme in the IPP mevalonate-independent biosynthetic pathway, was expressed from the tac promoter on multicopy and low-copy plasmids, lycopene production was enhanced two- to threefold over that found in cells expressing the chromosomal copy only. Cell growth and lycopene production decreased substantially when isopropyl beta-D-thiogalactosidase (IPTG) was added to the high-copy plasmid-containing culture, suggesting that overexpression of DXP synthase was a significant metabolic burden. In the low-copy plasmid-containing culture, no differences in cell growth or lycopene production were observed with any IPTG concentrations. When dxs was placed under the control of the arabinose-inducible promoter (P(BAD)) on the low-copy plasmid, the amount of lycopene produced was proportional to the arabinose concentration and no significant changes in cell growth resulted. These results suggest that low-copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism.

Acid Anhydride Hydrolases↗