Biomedical subjects

K K Lee

Publications and source records attributed to K K Lee.

At least 73 records · Page 4Linked to original sources

PubMed2000

Production of biologically active human granulocyte colony stimulating factor in the milk of transgenic goat.

We have developed a transgenic female goat harboring goat beta-casein promoter/human granulocyte colony stimulating factor (G-CSF) fusion gene by microinjection into fertilized one-cell goat zygotes. Human G-CSF was produced at levels of up to 50 microg/ml in transgenic goat milk. Its biological activity was equivalent to recombinant human G-CSF expressed from Chinese hamster ovary (CHO) cell when assayed using in vitro HL-60 cell proliferation. Human G-CSF from transgenic goat milk increased the total number of white blood cells in C57BL/6N mice with leucopenia induced by cyclophosphamide (CPA). The secreted human G-CSF was glycosylated although the degree of O-glycosylation was lower compared to CHO cell-derived human G-CSF.

Animals↗

PubMed2000

Virulence of Vibrio parahaemolyticus isolated from cultured small abalone, Haliotis diversicolor supertexta, with withering syndrome.

Outbreaks of mass mortality among cultured small abalone Haliotis diversicolor supertexta with withering syndrome occurred in May and September 1998 in Kao-Hsiung, Taiwan. Bacterial strains CH-1 and B4 were isolated from the haemolymph of the moribund small abalone using tryptic soy agar supplemented with 3% NaCl and/or thiosulphate citrate bile salt sucrose agar. These two strains were characterized and identified as Vibrio parahaemolyticus on the basis of various biochemical tests. The B4 strain and its extracellular products were virulent to small abalone with LD(50) values of 1.6 x 10(5) colony-forming units and 7.58 microg protein g-1 body weight, respectively.

Animals↗

PubMed2000

A comparative evaluation of polytetrafluoroethylene, umbilical vein, and saphenous vein bypass grafts for femoral-popliteal above-knee revascularization: a prospective randomized Department of Veterans Affairs cooperative study.

PURPOSE: Currently, the choice of a vascular prosthesis for a femoral-popliteal above-knee arterial bypass graft is left to the surgeon's preference, because the available information on comparative evaluations is inconclusive. The Department of Veterans Affairs (VA) Cooperative Study 141 was established to identify whether improved patency exists with different bypass graft materials for patients with femoral-popliteal above-knee bypass grafts. METHODS: Between June 1983 and June 1988, 752 patients at 20 VA medical centers were randomized to receive either an externally supported polytetrafluoroethylene (PTFE; N = 265), human umbilical vein (HUV; N = 261), or saphenous vein (SV; N = 226) for an above-knee femoral-popliteal bypass graft. The indication for the bypass grafting operation was limb salvage in 67.5% of the patients. Patients were observed every 3 months for the first year and every 6 months thereafter. All patients were instructed to take aspirin (650 mg) daily for the duration of the study.Doppler-derived ankle-brachial indices (ABIs) were determined preoperatively and serially postoperatively. A bypass graft was considered to be patent when the Doppler-derived postoperative ABI remained significantly improved (more than 0.15 units higher than their preoperative value) and additional objective information, such as angiograms or operations, did not contradict these observations. Patency failure also included bypass grafts that were removed because of an infection or aneurysmal degeneration. Patency rates were compared by using the Kaplan-Meier life table analysis. RESULTS: The cumulative assisted primary patency rates were statistically similar among the different conduit types at 2 years (SV, 81%; HUV, 70%; PTFE, 69%). After 5 years, above-knee SV bypass grafts had a significantly (P </=.01) better patency rate (73%) than HUV bypass grafts (53%), which had a significantly (P </=.01) better patency rate than PTFE bypass grafts (39%). Limb salvage was slightly worse with PTFE conduits. The number of bypass graft thromboses and major amputations within the first 30 days was highest in the HUV group. CONCLUSION: The overall results of this prospective randomized study suggest that the SV should be considered as the bypass graft of choice for femoral-popliteal above-knee reconstruction and that, when a prosthetic bypass graft is used, an HUV should also be considered as an alternative choice to PTFE.

Anastomosis, Surgical↗

PubMed2000

C. elegans nuclear envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing of nuclear envelope breakdown during mitosis.

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes.

Amino Acid Sequence↗

PubMed2000

The Drosophila melanogaster sex determination gene sisA is required in yolk nuclei for midgut formation.

During sex determination, the sisterlessA (sisA) gene functions as one of four X:A numerator elements that set the alternative male or female regulatory states of the switch gene Sex-lethal. In somatic cells, sisA functions specifically in sex determination, but its expression pattern also hints at a role in the yolk cell, a syncytial structure believed to provide energy and nutrients to the developing embryo. Previous studies of sisA have been limited by the lack of a null allele, leaving open the possibility that sisA has additional functions. Here we report the isolation and molecular characterization of four new sisA alleles including two null mutations. Our findings highlight key aspects of sisA structure-function and reveal important qualitative differences between the effects of sisA and the other strong X:A numerator element, sisterlessB, on Sex-lethal expression. We use genetic, expression, clonal, and phenotypic analyses to demonstrate that sisA has an essential function in the yolk nuclei of both sexes. In the absence of sisA, endoderm migration and midgut formation are blocked, suggesting that the yolk cell may have a direct role in larval gut development. To our knowledge, this is the first report of a requirement for the yolk nuclei in Drosophila development.

Alleles↗

PubMed2000

In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer.

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 +/- 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 +/- 2.3%) (P: < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 +/- 2.9, 6.6 +/- 2.3, and 8.1 +/- 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 +/- 10.4 > 24.6 +/- 10.1 > 16.5 +/- 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P: < 0. 05). Nuclear-transferred embryos (9.4 +/- 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 +/- 1.9%) or parthenogenetic embryos (22.4 +/- 7.2%) (P: < 0.01). The mean cell number (28.9 +/- 11.4) of NT-derived blastocysts was smaller than that (38.6 +/- 10.4) of IVF-derived blastocysts (P: < 0. 05) and was similar to that (29.9 +/- 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.

Animals↗

PubMed2000

Screening travelers for hepatitis A antibodies: an observational cost-comparison study of vaccine use.

OBJECTIVES: To measure the seroprevalence of antibodies to hepatitis A virus (anti-HAV) in a health plan population of travelers and to determine whether prevaccination screening for anti-HAV can reduce unnecessary vaccination and thus promote the most effective, economic use of hepatitis A vaccine. DESIGN: Observational, cost-comparison study. SETTING: Central injection clinic of a health maintenance organization medical center. SUBJECTS: Five hundred twenty-seven adults who denied having previous hepatitis A or vaccination. MAIN OUTCOME MEASURES Subgroups with the greatest prevalence of anti-HAV seen between June 1995 and April 1996 for immunizations before traveling to nonindustrialized countries. Relative costs of their screening and immunization. RESULTS: The presence of anti-HAV precluded the need for vaccination in 148 subjects (28.1%). The highest prevalence of anti-HAV (82.7%) was found in subjects born in nonindustrialized countries (62/75), in subjects who had previously traveled to areas of endemic hepatitis A (32.1% [135/420]), and in subjects born before 1945 (29.2% [92/315]). Costs of screening and vaccinating travelers were cheapest if prevaccination antibody sera testing was limited to subjects born in nonindustrialized countries and those born before 1945. CONCLUSIONS: Prevaccination screening of travelers for hepatitis A can be done selectively on the basis of age and country of origin. This strategy could lead to a more economic use of the vaccine and clinic resources.

Adolescent↗

PubMed2000

Myelomatous effusion with poor response to chemotherapy.

While pleural effusion in multiple myeloma is relatively infrequent, myelomatous pleural effusion is extremely rare. We experienced a 61-year-old woman with IgD-lambda multiple myeloma and pleural effusion. The diagnosis was made originally by pleural biopsy, pleural fluid cytology and immunoelectropheresis of pleural fluid. Transient improvement of the pleural effusion was observed after administration of combination chemotherapy of vincristine, melphalan, cyclophosphamide, prednisone (VMCP)/vincristine, cyclophosphamide, adriamycin, prednisone (VCAP). Two months later, myelomatous pleural effusion recurred and no response to salvage therapy was observed. We reviewed the clinical feature of this case and literature concerning myelomatous pleural effusion.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2000

The binding of Proteus mirabilis nonagglutinating fimbriae to ganglio-series asialoglycolipids and lactosyl ceramide.

Proteus mirabilis is a common opportunistic Gram-negative uropathogen that infects the upper urinary tract. We have examined the role of the nonagglutinating fimbriae (NAF) of P. mirabilis in mediating bacterial adhesion to cell surface receptors. Purified NAF of P. mirabilis were demonstrated to bind to a number of glycolipids, including asialo-GM1, asialo-GM2, and lactosyl ceramide (LacCer) in solid-phase binding assays and in thin layer chromatography (TLC) overlay assays. Furthermore, preincubation of the biotinylated NAF (Bt-NAF) with anti-NAF monoclonal antibodies resulted in inhibition of NAF binding to immobilized asialo-GM1, asialo-GM2, and LacCer. In adherence assays, P. mirabilis binding to Madin-Darby canine kidney (MDCK) cells was inhibited by murine anti-asialo-GM1 monoclonal antibodies H2G10 to about 50% of the binding level in the absence of the antibody, specific for the terminal beta-galactopyranosyl residue of asialo-GM1 (Harrison et al. 1998). The results of this study suggest that NAF of P. mirabilis recognize a GalNAc beta 1-4Gal moiety present in the ganglio-series of asialoglycolipids, and that the terminal beta-galactopyranosyl-containing glycoconjugates play a role in NAF-mediated adherence of P. mirabilis to MDCK cells. Similarly to other bacteria, P. mirabilis NAF was also shown to express the LacCer specificity.

Agglutination↗

PubMed1999

Efficient integration of short interspersed element-flanked foreign DNA via homologous recombination.

We investigated whether mouse short interspersed elements (SINEs) could influence the recombination frequency of foreign DNA. Vectors harboring a reporter gene in combinations of SINEs B1 and/or B2 or a portion of long interspersed element-1 were prepared and tested in vitro by a colony assay using HC11 murine mammary epithelial cells and in vivo by microinjection into fertilized mouse eggs. In transfected HC11 cells, the number of colonies surviving G418 selection increased by 3.5-fold compared with control when the reporter was flanked by fused B1-B2 sequences. Similar results were obtained from microinjection study; in fetuses 11.5 days post coitum, transgene positives in control and SINE-flanked vectors were 16 and 53%, respectively. Individual B1- and B2-harboring vectors showed equivalent activities with each other, as determined by the colony assay (2.8-fold versus 3.2-fold compared with control). We determined the contribution of homologous recombination to the SINE-mediated increase in integration frequency through a polymerase chain reaction-based strategy; in more than half of embryos transgenes underwent homologous recombinations involving B1 sequences. These results demonstrate that the SINE sequences can increase the integration rate of foreign DNA and that such an increase is most likely due to the enhancement of homologous recombination.

Animals↗

PubMed1999

Characterization of mouse peroxiredoxin I genomic DNA and its expression.

Peroxiredoxins (Prxs) are a newly defined family of anti-oxidant proteins that have been implicated, via their anti-oxidant activity, in a number of cellular functions, including cell proliferation and differentiation, protection of other proteins from oxidative damage, and intracellular signaling. We isolated genomic DNA sequences of the Prx I genes from the mouse, and characterized their molecular genetic features. Prx I was found to form a small gene family with two and three members; one functional and two pseudogenes. The Prx I-1 gene has splice donor/acceptor site sequences and five or six exons, whereas the Prx I-2 clone has several structural features characteristic of a typical retroposon found to have ORF sequences. We analyzed the expression of pseudogenes, which were not expressed on the transcription levels in the investigated organs. The functional copy of the Prx I-1 gene was expressed abundantly in liver and kidney of the adult, as well as in early developing embryos. This report, together with amino acid/nucleotide sequence similarity between human and mice, provides a basis for speculating on an even earlier event in the evolution of the Prx I gene family, i.e. the Prx I gene was well conserved in human and mice via its anti-oxidant activity.

Amino Acid Sequence↗

PubMed1999

Identification of a novel cis-acting positive element responsible for the cell-specific expression of the NK-1 homeobox gene.

The Drosophila NK-1 homeobox gene belongs to the NK-1 class that includes a large number of vertebrate homeobox genes and is shown to be expressed in specific muscle founder cells and a subset of neuronal cells in the ventral nerve cord during embryogenesis. To determine the cis-acting regulatory elements controlling the cell-specific expression of NK-1, we measured transiently expressed chloramphencol acetyl transferase (CAT) reporter gene activities from transfected C2C12 myoblasts and NG108-15 neuroblastoma cells using various CAT constructs containing different 5' upstream regions of NK-1. From the initial analysis of 3.9 kb of the 5' upstream region, we have found that the regions from -1865 to -476 and from -476 to +100 contained strong negative and positive regulatory elements, respectively. Within the positive cis-acting region an 86-bp DNA fragment (from -435 to -350) was sufficient to activate the reporter gene in C2C12 cells, whereas additional regions (from -157 to -28 and from -510 to -425) were required for optimal activity in NG108-15 cells. Gel shift and DNaseI footprinting assays have defined a plausible binding site for C/EBP, 5'-TTTCGCAAG-3' (-424 to -416), and a novel binding site for unknown factors, 5'-AATTACTCACATCC-3' (-370 to -357). Further mutation analysis has revealed that the novel binding sequence for unknown factors is necessary and sufficient for transcriptional activity for reporter gene expression in C2C12 myoblast cells in an orientation-independent manner.

Animals↗

PubMed1999

gas2 is a multifunctional gene involved in the regulation of apoptosis and chondrogenesis in the developing mouse limb.

The growth-arrest-specific 2 (gas2) gene was initially identified on account of its high level of expression in murine fibroblasts under growth arrest conditions, followed by downregulation upon reentry into the cell cycle (Schneider et al., Cell 54, 787-793, 1988). In this study, the expression patterns of the gas2 gene and the Gas2 peptide were established in the developing limbs of 11.5- to 14. 5-day mouse embryos. It was found that gas2 was expressed in the interdigital tissues, the chondrogenic regions, and the myogenic regions. Low-density limb culture and Brdu incorporation assays revealed that gas2 might play an important role in regulating chondrocyte proliferation and differentiation. Moreover, it might play a similar role during limb myogenesis. In addition to chondrogenesis and myogeneis, gas2 is involved in the execution of the apoptotic program in hindlimb interdigital tissues-by acting as a death substrate for caspase enzymes. TUNEL analysis demonstrated that the interdigital tissues underwent apoptosis between 13.5 and 15.5 days. Exactly at these time points, the C-terminal domain of the Gas2 peptide was cleaved as revealed by Western blot analysis. Moreover, pro-caspase-3 (an enzyme that can process Gas2) was cleaved into its active form in the interdigital tissues. The addition of zVAD-fmk, a caspase enzyme inhibitor, to 12.5-day-old hindlimbs maintained in organ culture revealed that the treatment inhibited interdigital cell death. This inhibition correlated with the absence of the Gas2 peptide and pro-caspase-3 cleavage. The data suggest that Gas2 might be involved in the execution of the apoptotic process.

Animals↗

PubMed1999

Hepatocyte growth factor stimulates chemotactic response in mouse embryonic limb myogenic cells in vitro.

In this study we investigate the influence of Hepatocyte Growth Factor (HGF) on the motility of embryonic forelimb myoblasts. Using Blindwell chemotactic chambers, it was found that HGF at concentrations of 1-50 ng/ml dramatically enhanced the ability of myogenic cells to migrate. This stimulatory effect was elicited in a dose-dependent fashion and the effect was reversed with the addition of HGF neutralizing antibodies. A checkerboard analysis was performed and it revealed that HGF's effect on limb myoblast motility was through both chemokinesis and chemotaxis. HGF was also examined for its ability to stimulate myogenic cell proliferation, using MF20 antibody as the myogenic marker. At all concentrations tested, HGF did not stimulate an overall increase in the numbers of MF20-positive myoblasts in culture. To examine the chemokinetic effect of HGF on cell migration in the limb, cells were isolated from the proximal regions of the limb (areas rich in myogenic cells), exposed to HGF, labeled with DiI and transplanted into 11.5 day mouse forelimbs. After 36 h of culture, it was found that DiI-labeled limb cells, pretreated with HGF, migrated significantly further in the limb than labeled cells that have not been exposed to HGF. The chemotactic effect of HGF was also investigated by implanting beads loaded with and without HGF into the 11.5 day limb. Proximal to the beads, DiI-labeled limb cells were also transplanted. It was found that HGF was able to chemotactically attract and direct the migration of DiI-labeled limb cells. Immunohistological staining was performed with HGF antibodies to determine the distribution of HGF in the 11.5 day mouse forelimb. It was found that HGF was strongly expressed by the apical ectodermal ridge (AER), the ectoderm and the mesenchyme directly beneath the AER. Positive staining was also obtained for the myogenic regions. However, the pattern was heterogeneous--punctuated with myogenic cells expressing and not expressing HGF.

Animals↗

PubMed1999

Electrophoretic characterization of a novel cysteine protease produced by Vibrio harveyi.

Electrophoretic characterization of a novel cysteine protease produced by pathogenic luminous Vibrio harveyi, originally isolated from diseased tiger prawn Penaeus monodon in Taiwan, is demonstrated in the present study using native polyacrylamide gel electrophoresis (native PAGE), sodium dodecyl sulfate-PAGE (SDS-PAGE), crossed immunoelectrophoresis (CIE) and isoelectric focusing (IEF) gels. The protease has a pI of 6.4 and exhibits a fast-migrating feature in native-PAGE and CIE gels indicating that it is a negatively charged protease. The protease electrophoresed as a 22 kDa protein band in native- and SDS-PAGE (in SDS - buffer with or without the presence of 2-mercaptoethanol) while it electrophoresed as a 38 kDa protein band in SDS-PAGE when the samples were boiled for 10 min prior to electrophoresis. The results reveal that the enzyme is an SDS-resistant monomeric protease and its high negative charge is not influenced by SDS (detergent) without boiling the sample. The present results are useful in determining proteins of similar nature to this unique cysteine protease.

Cysteine Endopeptidases↗

PubMed1999

Expression of vascular endothelial growth factor (VEGF) and its receptors during embryonic implantation in the golden hamster (Mesocricetus auratus).

Expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and its receptors (flt-1 and flk-1) during the peri-implantation period (days 3, 4, 5, 6 and 7 post coitus) in the golden hamster was investigated by in situ hybridization, immunohistochemistry and the reverse transcription/polymerase chain reaction (RT-PCR). Three days after mating, in situ hybridization and immunohistochemical staining revealed weak VEGF expression only in the uterine epithelium; this expression was similar to that seen at oestrus. Flt-1 but no flk-1 immunoreactivity was observed. At day 4, the subepithelial stroma and embryo displayed immunoreactivity for VEGF and flt-1, whereas endothelial cells expressed both flt-1 and flk-1. At day 5, immunoreactivity for both VEGF and its receptors was detected in decidual cells and vascular endothelial cells. Only a few embryonic cells expressed VEGF mRNA but strong signals were noted in decidual cells. The patterns of VEGF and VEGF receptor expression were the same in the day-6 and day-7 embryos and decidua, except for an increase in intensity as development progressed. Based on these findings, we conclude that, in addition to its known actions on endometrial angiogenesis and tissue swelling, VEGF may also facilitate the proliferation and differentiation of the endometrium and help to sustain the avascular embryo during this early stage of development.

Animals↗

PubMed1999

Dual challenges of infectious pancreatic necrosis virus and Vibrio carchariae in the grouper, Epinephelus sp.

The grouper industry in Taiwan faces serious threats from various disease problems. The present study investigated dual challenges with infectious pancreatic necrosis virus (IPNV) and Vibrio carchariae in the grouper (Epinephelus sp.). The fish were infected with IPNV for 2 weeks prior to a secondary infection with the bacteria, or vice versa, by either immersion (10(3)-10(4) TCID50 IPNV per ml, 10(6)-10(7) colony forming units (CFU) Vibrio per ml) or by intraperitoneal injection (10(3)-10(4) TCID50 IPNV per g fish or 10(7) CFU Vibrio/g fish) challenges. Mass mortalities occurred in fish infected with IPNV for 2 weeks prior to the infection with the bacteria, or vice versa, in either immersion or intraperitoneal injection challenges. The bacterium could only survive in seawater or brackish water similar to that of cultured groupers.

Animals↗

PubMed1999

Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein.

BACKGROUND/AIMS: Chronic infection with hepatitis B virus is a high-risk factor for hepatocellular carcinoma in humans. The HBV X-protein, a multi-functional viral regulator, has been suspected to play a positive role in hepatocarcinogenesis, as demonstrated by the high incidence of hepatocellular carcinoma in HBx-expressing transgenic mice, although it is still controversial. The aim of this study was to generate transgenic mice expressing the HBV X-gene under authentic promoter control and to test whether the gene products can cause hepatic tumors. METHODS: Three transgenic mouse lines were generated by microinjecting the X-gene construct into hybrid (C57BL/6 x DBA) eggs. Gene expression was tested by protein and mRNA analyses. During an observation period of 18 months, mice were sacrificed and organs subjected to histologic examinations. RESULTS: Grossly defined hepatocellular carcinomas reproducibly were observed in mice expressing the X-protein, which were investigated through six generations from the age of 11 to 18 months. Among 14 transgenic mice investigated from the age of 11 to 18 months, 12 were found to have hepatocellular carcinoma, grossly or microscopically. The lesion of the hepatocellular carcinoma disclosed a significant increase in the proliferating cell nuclear antigen in the nuclei. CONCLUSION: The incidence of hepatocellular carcinoma (86%) in our HBV X transgenic mice may be highly significant, since, except for one case, HBV X-gene transgenic mice produced in other laboratories did not develop liver tumor or any other pathologic phenomena.

Animals↗