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Biomedical subjects

W J Park

Publications and source records attributed to W J Park.

At least 19 recordsLinked to original sources
PubMed2006

Auxin biosynthesis in maize.

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.

Acetonitriles↗
PubMed2001

Interaction of HRC (histidine-rich Ca(2+)-binding protein) and triadin in the lumen of sarcoplasmic reticulum.

HRC (histidine-rich Ca(2+) binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca(2+) with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca(2+)-sensitive. Our data suggest that HRC may play a role in the regulation of Ca(2+) release from the sarcoplasmic reticulum by interaction with triadin.

Amino Acid Sequence↗
PubMed2001

Assessment of substrate specificity of hepatitis G virus NS3 protease by a genetic method.

The RNA genome of hepatitis G virus (HGV) encodes a large polyprotein that is processed to mature proteins by viral-encoded proteases. The HGV NS3 protease is responsible for the cleavage of the HGV polyprotein at four different locations. No conserved sequence motif has been identified for the cleavage sites of the NS3 protease. To determine the substrate specificity of the NS3 protease, amino acid sequences cleaved by the NS3 protease were obtained from randomized sequence libraries by using a screening method referred to as GASP (Genetic Assay for Site-specific Proteolysis). Based on statistical analyses of the obtained cleavable sequences, a consensus substrate sequence was deduced: Gln-Glu-Thr-Leu-Val downward arrow Ser, with the scissile bond located between Val and Ser. The relevance of this peptide as a cleavable substrate was further supported by molecular modeling of the NS3 protease. Our result would provide an insight on the molecular activity of the NS3 protease and may be useful for the design of substrate-based inhibitors.

Base Sequence↗
PubMed2001

An improved strategy for a genetic assay for site-specific proteolysis.

We have previously reported a genetic assay that is suitable for the study of substrate specificity of a protease in vivo, and herein present a simplified version of the method. In this procedure, expressed in Saccharomyces cerevisiae by using the constitutive alcohol dehydrogenase promoter is a fusion protein in which a transcription factor is linked to the intracellular domain of an integral membrane protein by a protease substrate sequence. Following this, a protease is expressed by using the inducible GAL promoter in the same yeast cells. The cleavage of the substrate sequence by the specific protease results in the release of the transcription factor and subsequent activation of reporter genes in nucleus. Since the expression of a protease is strictly under the control of the inducible GAL promoter, false substrate sequences that are cleaved by endogenous yeast proteases can be easily recognized and eliminated from further characterization. This suggests that the modified strategy provides an efficient tool for the analysis of substrate sequences of a protease in vivo.

Endopeptidases↗
PubMed2001

Auxin-induced elongation of short maize coleoptile segments is supported by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one.

Endogenous extractable factors associated with auxin action in plant tissues were investigated, especially their effects on elongation of 1-mm coleoptile segments of maize (Zea mays L.), in the presence of saturating 10 microM indole-3-acetic acid (IAA). The relative growth response, to auxin alone, was much smaller in segments shorter than 2-3 mm compared to 10-mm segments. Fusicoccin-induced elongation, however, was less affected by shortening the segments. A reduced auxin response may result from the depletion through cut surfaces of a substance required for IAA-mediated growth. Sucrose, phenolics like flavonoids, and vitamins were ruled out as the causal factors. A partially purified methanol extract of maize coleoptiles supported longterm, auxin-controlled elongation. The active material was also found among substances bleeding from scrubbed maize coleoptiles. The active factor from maize was further purified by HPLC and characterised by the UV spectrum and its pH shift. This factor was identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by mass spectroscopy. Activity tests confirmed that pure DIMBOA from other sources sustained auxin-induced elongation of short maize coleoptile segments. However, DIMBOA only partially restored the activity lost from short segments. This indicates that an additional factor, other than DIMBOA, is required. Extracts from Avena or Cucurbita did not contain the factor DIMBOA; it was active on maize elongation, but not on Avena coleoptiles or Cucurbita hypocotyls. This narrow specificity and the lack of DIMBOA action in short-term tests with maize indicate that DIMBOA is not the general auxin cofactor but may specifically "spare" the co-auxin in maize.

Benzoxazines↗
PubMed2001

Cooperative action of SLR1 and SLR2 is required for lateral root-specific cell elongation in maize.

Lateral roots play an important role in water and nutrient uptake largely by increasing the root surface area. In an effort to characterize lateral root development in maize (Zea mays), we have isolated from Mutator (Mu) transposon stocks and characterized two nonallelic monogenic recessive mutants: slr1 and slr2 (short lateral roots1 and 2), which display short lateral roots as a result of impaired root cell elongation. The defects in both mutants act specifically during early postembryonic root development, affecting only the lateral roots emerging from the embryonic primary and seminal roots but not from the postembryonic nodal roots. These mutations have no major influence on the aboveground performance of the affected plants. The double mutant slr1; slr2 displays a strikingly different phenotype than the single mutants. The defect in slr1; slr2 does not only influence lateral root specific cell elongation, but also leads to disarranged cellular patterns in the primary and seminal roots. However, the phase-specific nature of the single mutants is retained in the double mutant, indicating that the two loci cooperate in the wild type to maintain the lateral root specificity during a short time of early root development.

Genes, Plant↗
PubMed2001

New E. coli cloning vector using a cellulase gene (celA) as a screening marker.

The extracellular endoglucanase A gene of Clostridium thermocellum (celA) was used as a screening marker for E. coli cloning vector A 1.4-kb EcoRI fragment containing celA from pTvec/celA was isolated and cloned into a pUC18 deleting beta-galactosidase gene fragment. The constructed vectors, pCEL1, pCEL10, pCEL11, and pCEL20, have different multiple cloning sites within celA. If the cellulase, CelA, is inactivated by insertion of a foreign DNA fragment into multiple cloning sites, the recombinant transformants show no clear halos on an agar plate containing cellulose. This process overcomes the ambiguity of color screening in the X-gal/beta-galactosidase system, and over 90% of the recombinant transformants with no halos have foreign DNA inserts. Several E. coli strains were transformed successfully with pCEL series vectors regardless of mutation for alpha-complementation. Because E. coli strains do not have a cellulase gene, a vector using a cellulase gene screening marker can be used in any E. coli strain without limit. The new cloning system is very efficient, convenient, and cost effective.

Base Sequence↗
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

In vivo determination of substrate specificity of hepatitis C virus NS3 protease: genetic assay for site-specific proteolysis.

Hepatitis C virus (HCV) NS3 protease is responsible for the processing of the viral polyprotein and is considered as a primary target for the development of anti-HCV therapy. We have developed a genetic method in yeast to screen for good substrate sequences of the NS3 protease. A library of fusion proteins was constructed with a transcription factor, GAL4, linked to the intracellular domain of an integral membrane protein, STE2, by a randomized protease substrate sequence. In yeast cells expressing NS3 protease, the substrate sequences in the fusion proteins were specifically recognized and cleaved. This cleavage resulted in the release of GAL4 from the cytoplasmic membrane and the subsequent activation of reporter genes by GAL4, which was detected by the growth of yeast cells on selective media. Based on the analysis of 69 isolated substrate sequences, a consensus sequence was deduced: (Glu/Asp)-X-Val-Val-(Leu/Pro)-Cys / (Ser/Ala), with the scissile bond being located between Cys and Ser or Ala and X not being determined. This is largely consistent with the previous results obtained by biochemical methods. An oligopeptide containing the deduced sequence was highly efficiently cleaved in vitro by the purified NS3 protease. These data demonstrated that the present genetic method could be used as an efficient tool for the in vivo determination of substrate specificity of proteases.

Amino Acids↗
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

Anchor epicanthoplasty combined with out-fold type double eyelidplasty for Asians: do we have to make an additional scar to correct the Asian epicanthal fold?

The epicanthal fold along with a lack of a superior palpebral fold, excessive fat, and laxity of pretarsal skin represent the ethnic characteristics and a traditional sense of beauty in the Asian upper eyelid. But, too prominent an epicanthal fold may ruin an otherwise beautiful eye; furthermore, it becomes a restriction that makes the out-fold type double eyelidplasty, one of the two main types of double eyelidplasty, impossible. If a double eyelid as an out-fold type is desired, a concomitant epicanthoplasty should be performed with the possibility of hypertrophic scarring of the medial canthal area in Asians. To address the Asian epicanthal fold without danger of hypertrophic scarring, the authors developed an anchor epicanthoplasty technique that leaves no additional scar when combined with a double eyelidplasty. This technique is based on the concept of trimming of muscle and soft tissue under the Asian epicanthal fold and downward medial advancement and anchoring of the medial canthal skin to the deep tissue. The technique consists of five procedures based on the assumed causes of the Asian epicanthal fold: (1) augmentation rhinoplasty, (2) downward medial advancement of the medial upper lid skin, (3) removal of the superficial insertion of the medial canthal ligament and selective removal of the orbicularis oculi muscle, (4) subcutaneous contouring of the thick nasal skin, and (5) anchoring of the medial end of the incision to the deep tissue. During the past 12 years (1988 to 1999), 67 anchor epicanthoplasty procedures have been performed. Twenty-eight cases were followed up for more than 3 months, and all of the patients were satisfied with the results. There were only a few minor complications, which could be corrected with minimal revision. As an ancillary procedure to a double eyelidplasty, this anchor epicanthoplasty can reduce the Asian epicanthal fold and make a double fold as an out-fold type without an additional scar. In terms of hypertrophic scarring and compatibility with out-fold type double eyelidplasty, this anchor epicanthoplasty is the best method for correcting Asian epicanthal fold compared with other preexisting procedures. Other advantages of this technique are a wide range of applications and no compromise of medial, canthal skin to interfere with other epicanthoplasty techniques. Some disadvantages of this technique are technical difficulty and the possibility of active bleeding.

Adolescent↗
PubMed2000

Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans.

Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.

Animals↗
PubMed2000

Calsequestrin, a calcium sequestering protein localized at the sarcoplasmic reticulum, is not essential for body-wall muscle function in Caenorhabditis elegans.

Calsequestrin is the major calcium-binding protein of cardiac and skeletal muscles whose function is to sequester Ca(2+ )in the lumen of the sarcoplasmic reticulum (SR). Here we describe the identification and functional characterization of a C. elegans calsequestrin gene (csq-1). CSQ-1 shows moderate similarity (50% similarity, 30% identity) to rabbit skeletal calsequestrin. Unlike mammals, which have two different genes encoding cardiac and fast-twitch skeletal muscle isoforms, csq-1 is the only calsequestrin gene in the C. elegans genome. We show that csq-1 is highly expressed in the body-wall muscles, beginning in mid-embryogenesis and maintained through the adult stage. In body-wall muscle cells, CSQ-1 is localized to sarcoplasmic membranes surrounding sarcomeric structures, in the regions where ryanodine receptors (UNC-68) are located. Mutation in UNC-68 affects CSQ-1 localization, suggesting that the two possibly interact in vivo. Genetic analyses of chromosomal deficiency mutants deleting csq-1 show that CSQ-1 is not essential for initiation of embryonic muscle formation and contraction. Furthermore, double-stranded RNA injection resulted in animals completely lacking CSQ-1 in body-wall muscles with no observable defects in locomotion. These findings suggest that although CSQ-1 is one of the major calcium-binding proteins in the body-wall muscles of C. elegans, it is not essential for body-wall muscle formation and contraction.

Amino Acid Sequence↗
PubMed1999

Protection of mice against P. aeruginosa infections by large-scale affinity-purified human IgG specific to P. aeruginosa outer membrane proteins.

In order to develop an effective means to treat Pseudomonas aeruginosa infections, we designed a large-scale process for purification of human IgG specific to P. aeruginosa outer membrane proteins (Oprs) from normal human sera. The process we developed includes affinity column chromatography using P. aeruginosa Oprs as ligands, protein A column chromatography and ultrafiltration, which enriched P. aeruginosa Oprs-specific IgG antibody by 500-fold. The purified anti-Oprs IgG was specific to the Oprs as confirmed by an ELISA competition assay and retained opsonophagocytic-killing capacity. In vivo protective efficacy of anti-Oprs IgG was evaluated by passive protection assays in mice where the 50% protective dose of anti-Oprs IgG against P. aeruginosa infections was 41 microg/kg, which was 20 times lower than that of normal serum IgG. When administered to mice 3 h after bacterial challenge, only anti-Oprs IgG afforded protection. These data demonstrate the feasibility of use of the purification process in producing functionally active target-specific human antibodies for clinical use and provide a rationale for use of anti-Oprs IgG as a valuable adjunct to treat P. aeruginosa infections.

Animals↗
PubMed1999

Selection of Drosophila genes encoding secreted and membrane proteins.

With the use of a yeast-based signal sequence trap (YSST) method, we screened a Drosophila cDNA library to isolate genes encoding secreted and membrane proteins. Of the 136 unique cDNA clones sequenced, 11 clones (8.1%) are identical to previously known Drosophila genes, 18 clones (13.2%) are homologous to other genes identified in various organisms, and 91 clones (66.9%) are novel. Most of these genes are secreted or membrane proteins, or appear to contain putative signal sequences at their amino termini. This indicates that YSST is an effective tool for the isolation and analysis of Drosophila genes that play roles in intercellular communication.

Animals↗
PubMed1999

HRC (histidine-rich Ca2+ binding protein) resides in the lumen of sarcoplasmic reticulum as a multimer.

HRC (histidine-rich Ca2+ binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca2+ with low affinity and high capacity that is reminiscent of calsequestrin. The physiological role of HRC is largely unknown. In this study, we show that HRC exists as a multimeric complex (probably larger than a pentamer) under physiological conditions. At higher Ca2+ concentrations, the complex appeared to dissociate into dimers or trimers that form a more relaxed structure. This is in striking contrast to the characteristics of calsequestrin. An earlier immuno-electron microscopic study showed that HRC resides in the lumen of the sarcoplasmic reticulum (SR), but this conclusion has been challenged by other data. By tryptic digestion and biotinylation of SR vesicles, we provide compelling evidence showing that HRC is indeed present in the lumen of the SR.

Animals↗
PubMed1999

The inturned protein of Drosophila melanogaster is a cytoplasmic protein located at the cell periphery in wing cells.

The inturned (in) gene is a component of the frizzled (fz) signaling pathway that controls the polarity of hairs and bristles in the epidermis of Drosophila. It appears to act downstream of fz, which encodes a putative receptor for a tissue polarity signal. The in gene encodes a novel protein that had been suggested to contain two potential transmembrane domains. It has been suggested that the In protein interacts with the actin cytoskeleton to regulate the formation of the pupal wing prehairs that become adult hairs. The initiation of prehairs is normally restricted to the vicinity of the distal most vertex along the apical surface of the pupal wing cells. In an in mutant, prehairs initate at a variety of locations along the apical cell periphery. We have used immunofluorescence to study the subcellular localization of the In protein. When expressed in cultured cells, we found that In is a cytoplasmic protein. However, we found that it is localized in the vicinity of plasma membrane and the cortical actin cytoskeleton of Drosophila wing disc and pupal wing cells. Thus, in wing cells the In protein is localized to the region of the cell where it appears to function. This subcellular localization presumably requires the function of other proteins and may represent a regulatory mechanism. Our data suggest that fz does not play a major role in the subcellular localization of In. The In protein is notably insoluble in buffers containing high salt and nonionic detergents. This lack of solubility is significantly reduced in fz and mwh mutants, implying that it may be related to the mechanism of in function.

Animals↗