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Biomedical subjects

D K Kim

Publications and source records attributed to D K Kim.

At least 91 records · Page 5Linked to original sources
PubMed2000

Comparison of two immunization schedules for a Pseudomonas aeruginosa outer membrane proteins vaccine in burn patients.

The aim of the present study was to compare two immunization schedules for a Pseudomonas aeruginosa outer membrane proteins (OMPs) vaccine in burn patients. In a double-blind, randomized and placebo-controlled clinical trial, 95 adult patients with burn injuries in 10% or greater of total body surface area were randomly allocated to either placebo or immunization groups. Three doses of the vaccine (0.5 or 1.0 mg) were administered intramuscularly at either 3- or 7-day intervals. The vaccine was well tolerated, and no severe adverse reactions were observed in any of the vaccinees. After three immunizations, 88 patients were available for evaluation of serum antibody titers. Elevation of OMPs-specific antibody titers in the immunization groups was significantly higher as compared with the placebo group, and the highest antibody response was obtained by immunization with 1.0-mg doses at 3-day intervals. Conventional blood culture, tissue culture of wound biopsy specimens and a nested polymerase chain reaction (PCR) assay of blood specimens were performed to determine the protective efficacy. The results of the nested PCR indicated that the overall detection rate of P. aeruginosa in blood was significantly lower among immunized patients than placebo patients (6.1 vs. 40.0%, P<0.001). Based on these results, we concluded that the P. aeruginosa OMPs vaccine is safe and highly immunogenic in burn patients, especially with 1.0-mg doses at 3-day intervals, and may be effective in conferring protection against P. aeruginosa bacteremia in burn patients.

Adult↗
PubMed2000

A new member of the HCO3(-) transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney.

The kidneys play pivotal roles in acid-base homeostasis, and the acid-secreting (alpha-type) and bicarbonate-secreting (beta-type) intercalated cells in the collecting ducts are major sites for the final modulation of urinary acid secretion. Since the H(+)-ATPase and anion exchanger activities in these two types of intercalated cells exhibit opposite polarities, it has been suggested that the alpha- and beta-intercalated cells are interchangeable via a cell polarity change. Immunohistological studies, however, have failed to confirm that the apical anion exchanger of beta-intercalated cells is the band 3 protein localized to the basolateral membrane of alpha-intercalated cells. In the present study, we show the evidence that a novel member of the anion exchanger and sodium bicarbonate cotransporter superfamily is an apical anion exchanger of beta-intercalated cells. Cloned cDNA from the beta-intercalated cells shows about 30% homology with anion exchanger types 1-3, and functional expression of this protein in COS-7 cells and Xenopus oocytes showed sodium-independent and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive anion exchanger activity. Furthermore, immunohistological studies revealed that this novel anion exchanger is present on the apical membrane of beta-intercalated cells, although some beta-intercalated cells were negative for AE4 staining. We conclude that our newly cloned transporter is an apical anion exchanger of the beta-intercalated cells, whereas our data do not exclude the possibility that there may be another form of anion exchanger in these cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Expression of a system L neutral amino acid transporter at the blood-brain barrier.

Amino acid transport system L has been proposed to be one of the major nutrient transport systems at the blood-brain barrier. Using immunohistochemical analyses, a system L transporter LAT1 was shown to be expressed in the brain capillary endothelial cells in rats. Because LAT1 was coexpressed with 4F2 heavy chain which brings LAT1 to the plasma membrane, LAT1 is proposed to be functional in the plasma membrane of brain capillary endothelial cells. Both LAT1 and 4F2hc immunoreactivities were detected in a double line appearance surrounding endothelial cell nuclei, suggesting both proteins are present in the luminal and abluminal membranes. LAT1 is, thus, a blood-brain barrier system L transporter responsible for the permeation of aromatic or branched-chain amino acids and amino acid-related drugs such as L-DOPA.

Amino Acid Transport Systems↗
PubMed2000

FLIP is constitutively hyperexpressed in Fas-resistant U266 myeloma cells, but is not induced by IL-6 in Fas-sensitive RPM18226 cells.

Despite the expression of Fas, some clones of myeloma cells are resistant to Fas-mediated apoptosis. To define a cellular factor involved in the resistance, we performed a comparative study using two clones of myeloma cells, RPM18226 and U266. These cells were reported to express cell surface Fas at similar levels, but only RPM18226 cells lost their viability upon anti-Fas treatment. The resistance of U266 cells to anti-Fas did not appear to reflect dysregulation of Bcl-2, Bcl-X(L), and Bax, because these proteins were expressed in both RPM18226 and U266 cells to similar levels. Moreover, levels of those proteins were not significantly altered by treating RPM18226 cells with IL-6, a cytokine which suppresses the Fas-mediated death of RPM18226 cells. Interestingly, mRNA levels of FLIP(L), an endogenous inhibitor of Fas signaling, were constitutively elevated in U266 cells. Consistent with this observation, U266 cells expressed both FLIPL protein and its truncated 43 kDa product which is seen in FLIP(L)-overexpressing cells. The truncated form of FLIP(L) protein was not detected in RPM18226. Moreover, the levels of truncated FLIP(L) in U266 cells were considerably higher than those of pro-FLIP(L) in RPM18226. The overall data indicate that FLIPL is constitutively hyperexpressed in U266 cells. However, IL-6 failed to enhance the protein levels of FLIP molecules in either of the tested cells. It appears, therefore, that FLIP(L) plays a role in the intrinsic resistance of U266 cells to the apoptotic action of Fas, but is not involved in the protective action of IL-6.

Antibodies↗
PubMed2000

Successful pregnancy after transfer of rabbit blastocysts grown in vitro from single-cell zygotes.

To establish successful pregnancy in rabbits after the transfer of blastocysts cultured in vitro for 72 h, pregnancy rates were compared according to synchronization methods of recipient and embryo transfer sites. Also, the effect of RDH (1:1:1 mixture of RPMI, DMEM and Ham's F10) medium with additives such as BSA and taurine was evaluated for developmental capacity and cell number. Developmental capacity and cell number were considered important for implantation. When we evaluated the relative survival of rabbit one-cell embryos after culture in Ham's F10, in RD or in RDH for 72 h, embryos cultured in RDH and RD developed much better than in Ham's F10. When the effects of BSA and taurine in RDH medium were tested for rabbit embryo development, BSA or taurine promoted transition to the blastocyst stage and increased cell numbers of cultured embryos in RDH medium. The BSA and taurine together in RDH medium had a synergistic effect on embryo development. By transferring cultured blastocysts to the oviduct of the recipient doe synchronized one day behind the donor, live-born pups were obtained successfully. These results demonstrated that rabbit blastocysts can develop to normal pups after in vitro culture and embryo transfer.

Animals↗
PubMed2000

Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene.

Tumor necrosis factor-related, activation-induced cytokine (TRANCE), a tumor necrosis factor family member, mediates survival of dendritic cells in the immune system and is required for osteoclast differentiation and activation in the skeleton. We report the skeletal phenotype of TRANCE-deficient mice and its rescue by the TRANCE transgene specifically expressed in lymphocytes. TRANCE-deficient mice showed severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and exhibited profound growth retardation at several skeletal sites, including the limbs, skull, and vertebrae. These mice had marked chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes. Transgenic overexpression of TRANCE in lymphocytes of TRANCE-deficient mice rescued osteoclast development in two locations in growing long bones: excavation of marrow cavities permitting hematopoiesis in the marrow spaces, and remodeling of osteopetrotic woven bone in the shafts of long bones into histologically normal lamellar bone. However, osteoclasts in these mice failed to appear at the chondroosseous junction and the metaphyseal periosteum of long bones, nor were they present in tooth eruption pathways. These defects resulted in sclerotic metaphyses with persistence of club-shaped long bones and unerupted teeth, and the growth plate defects were largely unimproved by the TRANCE transgene. Thus, TRANCE-mediated regulation of the skeleton is complex, and impacts chondrocyte differentiation and osteoclast formation in a manner that likely requires local delivery of TRANCE.

Animals↗
PubMed2000

Apoptotic activity of ursolic acid may correlate with the inhibition of initiation of DNA replication.

Ursolic acid (UA), a pentacyclic triterpene acid, has been reported to exhibit anti-tumor activity. In this study, we investigated the pro-apoptotic effect of UA on HepG2 human hepatoblastoma cells. Treatment with UA decreased the viability of HepG2 cells in a concentration- and time-dependent manner. Furthermore, 30 microM of UA induced DNA fragmentation and subdiploid cells and enhanced the release of cytochrome c and the activation of caspase-3. These results suggest that UA induces cell death through apoptosis, which may be mediated by cytochrome c-dependent caspase-3 activation. In addition, cell-cycle analysis revealed that UA-treated cells were arrested predominantly in the G(0) and G(1) phases with a concomitant decrease in the cell population of S phase. Moreover, expression of p21(WAF1), a cell-cycle regulator, was increased by UA, indicating that p21(WAF1) might mediate UA-induced cell-cycle arrest. However, UA markedly inhibited SV40 DNA replication in the initiation stage in vitro and significantly reduced the DNA cleaving of topoisomerase I and the ssDNA binding activity of replication protein A. These results indicate that the inhibition of DNA replication by UA may result from blockade of the establishment of the replication fork during initiation stage, consequently contributing to UA-induced cell-cycle arrest. Taken together, we suggest that UA-induced cell-cycle arrest may be mediated by inhibition of DNA replication and the increase of p21(WAF1) expression, which induces the release of cytochrome c and the activation of caspase-3, leading to apoptosis of HepG2 cells.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Transport properties of a system y+L neutral and basic amino acid transporter. Insights into the mechanisms of substrate recognition.

The properties of system y(+)L-mediated transport were investigated on rat system y(+)L transporter, ry(+)LAT1, coexpressed with the heavy chain of cell surface antigen 4F2 in Xenopus oocytes. ry(+)LAT1-mediated transport of basic amino acids was Na(+)-independent, whereas that of neutral amino acids, although not completely, was dependent on Na(+), as is typical of system y(+)L-mediated transport. In the absence of Na(+), lowering of pH increased leucine transport, without affecting lysine transport. Therefore, it is proposed that H(+), besides Na(+) and Li(+), is capable of supporting neutral amino acid transport. Na(+) and H(+) augmented leucine transport by decreasing the apparent K(m) values, without affecting the V(max) values. We demonstrate that although ry(+)LAT1-mediated transport of [(14)C]l-leucine was accompanied by the cotransport of (22)Na(+), that of [(14)C]l-lysine was not. The Na(+) to leucine coupling ratio was determined to be 1:1 in the presence of high concentrations of Na(+). ry(+)LAT1-mediated leucine transport, but not lysine transport, induced intracellular acidification in Chinese hamster ovary cells coexpressing ry(+)LAT1 and 4F2 heavy chain in the absence of Na(+), but not in the presence of physiological concentrations of Na(+), indicating that cotransport of H(+) with leucine occurred in the absence of Na(+). Therefore, for the substrate recognition by ry(+)LAT1, the positive charge on basic amino acid side chains or that conferred by inorganic monovalent cations such as Na(+) and H(+), which are cotransported with neutral amino acids, is presumed to be required. We further demonstrate that ry(+)LAT1, due to its peculiar cation dependence, mediates a heteroexchange, wherein the influx of substrate amino acids is accompanied by the efflux of basic amino acids.

Amino Acid Transport Systems, Basic↗
PubMed2000

Cloning and characterization of a human brain Na(+)-independent transporter for small neutral amino acids that transports D-serine with high affinity.

We isolated a cDNA for the human homologue of system asc transporter Asc-1 from human brain. The encoded protein designated as hAsc-1 (human Asc-1) exhibited 91 % sequence identity to mouse Asc-1. Consistent with mouse Asc-1, hAsc-1 required 4F2 heavy chain for its functional expression in Xenopus oocytes. hAsc-1 exhibited the properties of amino acid transport system asc which transports small neutral amino acids in a Na(+)-independent manner. hAsc-1 transported D-serine at high affinity with a K(m) value of 22.8 microM. In brain, 2.0 kb mRNA was highly expressed. hAsc-1 gene was mapped to human chromosome 19, region q12-q13.1. Because of the high-affinity transport with the K(m) value close to the physiological concentration of D-serine, together with the high levels of expression in brain, hAsc-1 is proposed to play significant roles in the D-serine mobilization in brain.

Amino Acid Transport System y+↗
PubMed2000

Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function.

As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen. Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding. mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm. Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX. When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced. TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1. Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress. To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells. When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells. In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated. Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity.

3T3 Cells↗
PubMed2000

Improved expression of vascular endothelial growth factor by naked DNA in mouse skeletal muscles: implication for gene therapy of ischemic diseases.

We have constructed an expression vector, pCK, that is able to drive high levels of gene expression in the skeletal muscles of mice. pCK contains not only the full length immediate-early (IE) promoter of human cytomegalovirus but also its entire 5' untranslated region upstream from the start codon of the IE gene. In addition, pCK contains the kanamycin resistance gene, but lacks nucleotide sequences unnecessary for its function as a gene delivery vector, allowing the plasmid size to be 3.7 kb. pCK produced significantly higher levels of vascular endothelial growth factor 165 both in vitro and in vivo than the control vector, the structure of which is similar to naked DNA vectors employed in previous gene therapy trials. pCK would not only significantly increase the therapeutic effects of naked DNA gene therapy but also dramatically cut down the costs for production and treatment.

Animals↗
PubMed2000

Caspase-dependent and -independent events in apoptosis induced by hydrogen peroxide.

To define the role of caspase-3 in H2O2-induced apoptosis, we introduced caspase-3 cDNA into MCF-7 breast carcinoma cells that otherwise lack caspase-3 expression. H2O2 treatment induced DNA fragmentation and nuclear condensation in the caspase-3-expressing cells, but not in the caspase-3-deficient cells. This indicated that caspase-3 is essential for nuclear events. However, H2O2 induced an externalization of membrane phosphatidylserine (PS) and cell death regardless of caspase-3 expression. These events were not suppressed by Ac-DEVD-CHO and Z-VAD-fmk, which inhibit DEVD-specific caspases and a broad spectrum of caspases, respectively. In Jurkat T cells, these inhibitors abolished H2O2-induced PS relocalization, but not cell death. Therefore, caspases appear to be dispensable for lethality by H2O2, but required for PS redistribution in a cell-type-specific manner.

Annexins↗
PubMed2000

Ectopic expression of active processed form of atrial natriuretic peptide in skeletal myoblasts.

Atrial natriuretic peptide (ANP) is a cardiac hormone that elicits a profound diuresis, natriuresis, and hypotension. As a preliminary study toward ANP gene therapy of cardiovascular disorders, we have cloned a cDNA for mouse preproANP and carried out expression studies in muscle cells. The expression cassette, which was flanked by ITRs from AAV-2, consisted of HCMV IE enhancer/promoter, preproANP gene, and polyadenylation signal from bovine growth hormone. We transfected this expression vector into primary skeletal myoblasts and examined the following points: (1) secretion of immunoreactive ANP, (2) biological activity, and (3) nature of secreted ANP(s). The conditioned media from cells transfected with ANP vector had significantly higher levels of irANP in comparison to mock control. The secreted irANP had biological activity as confirmed by the elevated level of intracellular cGMP in human umbilical vein endothelial cells. Reverse-phase HPLC analysis showed that the processed form of ANP was the predominant form. These results demonstrate that preproANP gene could be ectopically expressed and correctly processed in skeletal myoblasts, which has implications for development of muscle-based ANP gene therapy.

Amino Acid Sequence↗
PubMed2000

Involvement of p27(kip1) in ceramide-mediated apoptosis in HL-60 cells.

Ceramide acts as a mediator of apoptosis in various cell lines, but little is known regarding the molecular mechanism linked to the cell cycle. In the present study, we examined the expression of p27(kip1) and its relationship to apoptosis induced by ceramide. We demonstrated that treatment of HL-60 cells with C6-ceramide resulted in G1 phase elevation followed by apoptotic cleavage associated with increase in the level of cdk inhibitor p27(kip1). Ceramide inhibited the kinase activities of cdk2 and cdk4 within 24 h of treatment. Ceramide-induced inhibition of cdk2 and cdk4 kinase activities was accompanied by increase of p27(kip1) in the cdks complexes. In addition, we have shown that both the cell death and expression of p27(kip1) protein induced by ceramide were significantly decreased in HL-60 cells overexpressing bcl-2. Furthermore, ceramide induced a significant increase in Bax protein expression coincided with increase in p27(kip1) protein level. These findings indicate that p27(kip1) may play important roles in mediating ceramide-induced apoptosis and its expression can be regulated by Bax and Bcl-2.

Apoptosis↗
PubMed2000

Identification and characterization of a Na(+)-independent neutral amino acid transporter that associates with the 4F2 heavy chain and exhibits substrate selectivity for small neutral D- and L-amino acids.

A cDNA was isolated from the mouse brain that encodes a novel Na(+)-independent neutral amino acid transporter. The encoded protein, designated as Asc-1 (asc-type amino acid transporter 1), was found to be structurally related to recently identified mammalian amino acid transporters for the transport systems L, y(+)L, x(C)(-), and b(0,+), which are linked, via a disulfide bond, to the type II membrane glycoproteins, 4F2 heavy chain (4F2hc), or rBAT (related to b(0,+) amino acid transporter). Asc-1 required 4F2hc for its functional expression. In Western blot analysis in the nonreducing condition, a 118-kDa band, which seems to correspond to the heterodimeric complex of Asc-1 and 4F2hc, was detected in the mouse brain. The band shifted to 33 kDa in the reducing condition, confirming that Asc-1 and 4F2hc are linked via a disulfide bond. Asc-1-mediated transport was not dependent on the presence of Na(+) or Cl(-). Although Asc-1 showed a high sequence homology (66% identity at the amino acid level) to the Na(+)-independent broad scope neutral amino acid transporter LAT2 (Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., and Kanai, Y. (1999) J. Biol. Chem. 274, 19745-19751), Asc-1 also exhibited distinctive substrate selectivity and transport properties. Asc-1 preferred small neutral amino acids such as Gly, L-Ala, L-Ser, L-Thr, and L-Cys, and alpha-aminoisobutyric acid as substrates. Asc-1 also transported D-isomers of the small neutral amino acids, in particular D-Ser, a putative endogenous modulator of N-methyl-D-aspartate-type glutamate receptors, with high affinity. Asc-1 operated preferentially, although not exclusively, in an exchange mode. Asc-1 mRNA was detected in the brain, lung, small intestine, and placenta. The functional properties of Asc-1 seem to be consistent with those of a transporter subserving the Na(+)-independent small neutral amino acid transport system asc.

Amino Acid Sequence↗
PubMed2000

Immunization of burn-patients with a Pseudomonas aeruginosa outer membrane protein vaccine elicits antibodies with protective efficacy.

The aim of this study was to determine whether the antibodies raised in burn patients by active immunization with a Pseudomonas aeruginosa OMPs vaccine have a protective efficacy against infection with P. aeruginosa. The binding patterns with P. aeruginosa OMPs of immunized burn patient sera were similar to the sera of immunized healthy humans as determined by immunoblot and immunoprecipitation analyses. The sera pooled from immunized burn patients after three immunizations showed a significantly higher opsonophagocytic-killing activity than the corresponding pre-immune sera, while the sera from unimmunized patients collected at the same day did not. Passive immunization of mice with post-immune sera of burn patients significantly enhanced the survival rate upon a lethal challenge with P. aeruginosa compared to the pre-immune sera, indicating the protective ability of the antibodies induced in burn patients by immunization. These results suggest that anti-P. aeruginosa OMPs antibodies elicited in burn patients by active immunization are protective against infection with P. aeruginosa, and provide a rational for further development of the vaccine for prevention against P. aeruginosa infection in burn patients.

Animals↗
PubMed2000

Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta.

A cDNA encoding a novel multispecific organic anion transporter, OAT4, was isolated from a human kidney cDNA library. The OAT4 cDNA consisted of 2210 base pairs that encoded a 550-amino acid residue protein with 12 putative membrane-spanning domains. The amino acid sequence of OAT4 showed 38 to 44% identity to those of other members of the OAT family. Northern blot analysis revealed that OAT4 mRNA is abundantly expressed in the placenta as well as in the kidney. When expressed in Xenopus oocytes, OAT4 mediated the high affinity transport of estrone sulfate (K(m) = 1.01 microM) and dehydroepiandrosterone sulfate (K(m) = 0.63 microM) in a sodium-independent manner. OAT4 also mediated the transport of ochratoxin A. OAT4-mediated transport of estrone sulfate was inhibited by several sulfate conjugates, such as p-nitrophenyl sulfate, alpha-naphthyl sulfate, beta-estradiol sulfate, and 4-methylumbelliferyl sulfate. By contrast, glucuronide conjugates showed little or no inhibitory effect on the OAT4-mediated transport of estrone sulfate. OAT4 interacted with chemically heterogeneous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, sulfobromophthalein, penicillin G, and bile salts, whereas tetraethylammonium, an organic cation, did not. OAT4 is the first member of the multispecific organic anion transporter family, which is expressed abundantly in the placenta. OAT4 might be responsible for the elimination and detoxification of harmful anionic substances from the fetus.

Amino Acid Sequence↗