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I Hwang

Publications and source records attributed to I Hwang.

At least 55 records · Page 3Linked to original sources
PubMed1999

The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase.

The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.

Amino Acid Sequence↗
PubMed1999

Essential components of the Ti plasmid trb system, a type IV macromolecular transporter.

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.

Agrobacterium tumefaciens↗
PubMed1998

A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes.

Dynamin, a GTP-binding protein found in rat brain, plays a role in endocytosis. Suborganellar fractionation studies of Arabidopsis leaf tissue revealed that a dynamin-like protein, ADL1, is localized in the thylakoid membranes of chloroplasts. This notion was supported further by in vivo targeting experiments using an ADL1-green fluorescent fusion protein and immunogold labeling with the anti-ADL1 antibody. Transgenic plants harboring various deletion mutant genes of ADL1 had a yellow leaf phenotype where the cells had very few chloroplasts. In addition, the remaining chloroplasts appeared morphologically not fully developed. The detailed structure of the chloroplasts revealed by electron microscopy showed a greatly reduced amount of thylakoid membranes. Also, the level of thylakoid membrane proteins such as the light-harvesting complex II and CP29 was greatly reduced in these transgenic plants. When we examined the expression of the ADL1 deletion mutant genes, these genes were highly expressed at the transcriptional level. However, the mutant ADL1s were not detectable at the protein level by Western blot analysis. Moreover, the endogenous ADL1 protein level was greatly reduced in these transgenic plants, probably due to a post-transcriptional silencing effect of the transgenes. We propose, therefore, that ADLl is involved in the biogenesis of thylakoid membranes.

Arabidopsis↗
PubMed1998

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain.

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Amino Acid Sequence↗
PubMed1998

Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15.

Proliferation of memory-phenotype (CD44hi) CD8+ cells induced by infectious agents can be mimicked by injection of type I interferon (IFN I) and by IFN I-inducing agents such as lipopolysaccharide and Poly I:C; such proliferation does not affect naive T cells and appears to be TCR independent. Since IFN I inhibits proliferation in vitro, IFN I-induced proliferation of CD8+ cells in vivo presumably occurs indirectly through production of secondary cytokines, e.g., interleukin-2 (IL-2) or IL-15. We show here that, unlike IL-2, IL-15 closely mimics the effects of IFN I in causing strong and selective stimulation of memory-phenotype CD44hi CD8+ (but not CD4+) cells in vivo; similar specificity applies to purified T cells in vitro and correlates with much higher expression of IL-2Rbeta on CD8+ cells than on CD4+ cells.

Animals↗
PubMed1998

Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress.

Plant responses to high salt stress have been studied for several decades. However, the molecular mechanisms underlying these responses still elude us. In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis. Here, we report the cloning of a cDNA encoding a novel Ca2+-binding protein, named AtCP1, which shares sequence similarities with calmodulins. AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca2+-binding loops of the EF hands of calmodulin. However, unlike calmodulin, AtCP1 appears to have only three Ca2+-binding loops. We examined Ca2+ binding of the protein by a Ca2+-dependent electrophoretic mobility shift assay. A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca2+-dependent electrophoretic mobility shift. To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out. The AtCP1 gene had a tissue-specific expression pattern: high levels of expression in flower and root tissues and nearly undetectable levels in leaves and siliques. Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment. Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids.

Dynamin-related proteins are high molecular weight GTPase proteins found in a variety of eukaryotic cells from yeast to human. They are involved in diverse biological processes that include endocytosis in animal cells and vacuolar protein sorting in yeast. We isolated a new gene, ADL2, that encodes a dynamin-like protein in Arabidopsis. The ADL2 cDNA is 2.68 kb in size and has an open reading frame for 809 amino acid residues with a calculated molecular mass of 90 kDa. Sequence analysis of ADL2 revealed a high degree of amino acid sequence similarity to other members of the dynamin superfamily. Among those members ADL2 was most closely related to Dnm1p of yeast and thus appears to be a member of the Vps1p subfamily. Expression studies showed that the ADL2 gene is widely expressed in various tissues with highest expression in flower tissues. In vivo targeting experiments showed that ADL2:smGFP fusion protein is localized to chloroplasts in soybean photoautroph cells. In addition experiments with deletion constructs revealed that the N-terminal 35 amino acid residues were sufficient to direct the smGFP into chloroplasts in tobacco protoplasts when expressed as a fusion protein.

Amino Acid Sequence↗
PubMed1998

Sequence of a cDNA encoding mouse ribosomal protein S14.

An involution-induced clone was identified by differential screening from a cDNA library of mouse mammary gland. The clone was identified as full-length cDNA encoding the 40S subunit of ribosomal protein S14 (rps14). Comparison of the deduced amino acid sequence to sequences of rps14 from humans, hamsters, and rats showed a conservation.

Amino Acid Sequence↗
PubMed1998

Recanalization of chronic peripheral arterial occlusions by alternating intra-arterial rt-PA and PGE1.

BACKGROUND: Recanalization of femoral artery occlusions is difficult and different forms of intra-arterial lysis or mechanical procedures have been described. We investigated the effectiveness of a prolonged intra-arterial lysis with rt-pa in combination with PGE1 followed by angioplasty. PATIENTS AND METHODS: 43 patients (age 60.4 +/- 14.3 years) with peripheral arterial occlusions older than 3 months and longer than 10 cm were treated with intra-arterial rt-pa (3 mg in 3 h) followed by PGE1 (2.1 ml/h for 3 h, concentration: 20 micrograms/50 ml NaCl) in alternating order. Treatment times ranged from 1 to 7 days (2.9 +/- 1.6). Doses of 26.5 +/- 21.9 mg rt-PA and 20.4 +/- 16 micrograms PGE1 were used. If necessary, angioplasty was performed after a wire was passed through the lumen. RESULTS: Recanalization was achieved in 47 out of 85 arterial segments with reocclusion in 9 segments. The arterial perfusion deteriorated once due to peripheral embolism. Other adverse effects included one case of retroperitoneal and one case of intracrural bleeding and one aneurysma spurium. CONCLUSION: In chronic arterial occlusions which do not respond to conventional angioplasty and/or short term fibrinolysis recanalization may be achieved in about 50% by means of prolonged alternating application of rt-pa and PGE1.

Adult↗
PubMed1998

Adrenocortical SPECT using iodine-131 NP-59.

Adrenal scintigraphy with 131I-labeled 6-beta-iodomethyl-19-norcholesterol (NP-59) is a technically demanding and complex procedure. However, it can provide crucial and unique information about the functional status of the adrenal glands and guide the appropriate therapeutic management of patients with biochemically proven disease. Since the introduction of this new investigational drug, scintigraphic imaging has been performed using conventional planar techniques. We present an interesting case of primary aldosteronism in which planar scintigraphy and SPECT were combined in an attempt to increase the sensitivity of the study. SPECT revealed scintigraphic evidence of bilateral adrenocortical hyperplasia. Interestingly, the CT scan of this patient showed only an equivocal abnormality in the left adrenal gland, suggestive of an adenoma.

Adosterol↗
PubMed1997

The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin.

In higher plant cytokinesis, plasma membrane and cell wall originate by vesicle fusion in the plane of cell division. The Arabidopsis KNOLLE gene, which is required for cytokinesis, encodes a protein related to vesicle-docking syntaxins. We have raised specific rabbit antiserum against purified recombinant KNOLLE protein to show biochemically and by immunoelectron microscopy that KNOLLE protein is membrane associated. Using immunofluorescence microscopy, KNOLLE protein was found to be specifically expressed during mitosis and, unlike the plasma membrane H+-ATPase, to localize to the plane of division during cytokinesis. Arabidopsis dynamin-like protein ADL1 accumulates at the plane of cell plate formation in knolle mutant cells as in wild-type cells, suggesting that cytokinetic vesicle traffic is not affected. Furthermore, electron microscopic analysis indicates that vesicle fusion is impaired. KNOLLE protein was detected in mitotically dividing cells of various parts of the developing plant, including seedling root, inflorescence meristem, floral meristems and ovules, and the cellularizing endosperm, but not during cytokinesis after the male second meiotic division. Thus, KNOLLE is the first syntaxin-like protein that appears to be involved specifically in cytokinetic vesicle fusion.

Animals↗
PubMed1997

EGF inhibits expression of WDNM1 and sulfated glycoprotein-2 genes in mammary epithelial cells.

We have previously shown that expressions of ferritin heavy chain (FHC), WDNM1, and sulfated glycoprotein-2 (SGP-2) genes are induced at an involution stage of mammary gland. Here we studied the effect of lactogenic hormones and EGF on the expression of involution-induced genes in HC11 mammary epithelial cells. Insulin, dexamethasone, prolactin, and its combinations did not affect expression of the genes. When cells were cultured in growth medium containing EGF, expression of WDNM1 and SGP-2 genes was strongly inhibited in a dose- and time- dependent manner, whereas expression of FHC gene was not influenced by EGF. Results demonstrate that EGF inhibits expression of WDNM1 and SGP-2 genes in mammary epithelial cells.

Animals↗
PubMed1997

Isolation of molecular markers for salt stress responses in Arabidopsis thaliana.

Characterization of many osmotic stress-induced genes has greatly contributed to the understanding of the physiological responses of plant cells to osmotic stress at the molecular level. In this study we constructed a subtraction library and generated 15 salt stress-inducible ESTs from this library to use as molecular markers that reflect the cellular responses to salt stress responses in Arabidopsis. The sequence analysis showed that 5 salt stress-inducible ESTs were identical to previously identified genes in Arabidopsis, 6 cDNAs were homologous to known genes found in plants as well as yeast, and 4 cDNAs were new genes. To confirm that expression of these clones are induced by salt stress, we carried out Northern blot analysis. When we examined for 15 cDNA clones, they were indeed induced by NaCl treatment. The induction level was variable among these genes ranging from approximately 2-fold to more than 50-fold. Also, Northern blot analysis revealed that these genes can be divided into three different induction patterns: early induction, late induction, and continuous induction.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of an antifungal PR-5 protein from pumpkin leaves.

A 28-kDa antifungal PR-5 protein (PLTP) was purified from pumpkin leaves to homogeneity by using ammonium sulfate fractionation, a regenerated chitin column, and reversed-phase column chromatographies on butyl-Toyopearl and HPLC C18 columns. Analysis of 14 N-terminal amino acid sequences of PLTP shows 100% sequence identity to those of two PR-5 proteins, NP24 from tomatoes and AP24 from tobacco. The identical sequence also exhibited high amino acid sequence homology to that of an osmotin-like protein (OLP; 71%) from tobacco cells and thaumatin (64%), a sweet-tasting protein of Thaumatococcus danielli Bench. When the PLTP was immuno-blotted with antiserum raised against the tobacco OLP, the OLP antibody specifically cross-reacted with the PLTP, suggesting that they share several common epitopes in their tertiary structure of the proteins. The purified PLTP rapidly lyzed hyphal tips of Neurospora crassa at a concentration greater than 200 nM and significantly inhibited the fungal growth of Fusarium oxysporum in an agar-disc plate at a concentration greater than 2 microM. It also shows a synergistic effect with nikkomycin, a chitin synthase inhibitor, for the growth inhibition of Candida albicans.

Amino Acid Sequence↗
PubMed1997

The Bop gene adjacent to the mouse CD8b gene encodes distinct zinc-finger proteins expressed in CTLs and in muscle.

The Bop gene (for CD8b opposite) is located immediately upstream of the mouse CD8b gene. Expression of Bop gene transcripts was previously observed in several long term CTL lines and in thymus. The present studies demonstrate that expression of the Bop gene in lymphocytes appears to be confined to CD8-positive cells, and that Bop gene expression is inducible by Con A. They further show that a single Bop gene encodes protein products with distinct amino-terminal sequences that are expressed in CTLs (t-BOP) and in cardiac and skeletal muscle (skm-BOP), as well as what appears to be a noncoding cDNA (t-ncb) expressed only in CTLs. The t-BOP and t-ncb cDNAs in CTLs result from alternative splicing of a single primary transcript, whereas the Bop transcripts expressed in CTLs and in muscle appear to be transcribed from different promoters. The BOP proteins expressed in CTLs and muscle contain zinc finger-like motifs with homology to those of the ETO/MTG8 proto-oncogene and several other proteins of interest. Western blot analysis with a hamster anti-BOP mAb have detected the BOP protein in muscle cells and in COS 7 cells transfected with Bop cDNA constructs.

Amino Acid Sequence↗
PubMed1997

Differential expression of two functional serine/threonine protein kinases from soybean that have an unusual acidic domain at the carboxy terminus.

Two soybean cDNA clones, SPK-3 and SPK-4, encoding putative protein kinases were isolated and characterized. Both cDNAs encoded approximately 40-kDa serine/threonine kinases with unusual stretches of acidic amino acids in their carboxy-terminal regions, which are highly homologous to PKABA1 from wheat and ASKs from Arabidopsis. These kinases are encoded by one- or two-copy genes in the soybean genome. Notably, SPK-3 and -4 showed different patterns of expression in various soybean tissues. SPK-3 is highly expressed in dividing and elongating tissues of young seedlings but relatively weakly in tissues of mature plants. In contrast, SPK-4 showed relatively high and constitutive expression in all the tissues examined except for leaf tissues of mature plants. Although various stressors, such as dehydration and high salinity, increased the expression of both genes, the induction kinetics were different. The two genes also differed in their response to abscisic acid (ABA). SPK-3 was induced but SPK-4 was not affected by exogenously supplied abscisic acid. In accordance with these expression data analysis of the activity of a chimeric SPK-3 promoter::beta-glucuronidase (GUS) reporter gene by transient expression in tobacco leaves confirmed the inducibility of SPK-3 by salt and ABA. Polyclonal antibodies raised against a recombinant SPK-4 protein produced in Escherichia coli specifically recognized both recombinant SPK-3 and -4 proteins. Kinase assays using affinity-purified SPK-4/ antibody complexes with crude soybean extracts as substrate identified specific phosphorylation of two 41 and 170 kDa soybean proteins that were phosphorylated on serine residues. Taken together, our results suggest that SPK-3, and/or SPK-4 are functional serine protein kinase(s). Furthermore, SPK-3 and -4 may play different roles in the transduction of various environmental stresses.

Amino Acid Sequence↗
PubMed1997

A dynamin-like protein, ADL1, is present in membranes as a high-molecular-mass complex in Arabidopsis thaliana.

Dynamin, a GTP-binding protein, is involved in endocytosis in animal cells. We found that a dynamin-like protein, ADL1, is present in multiple forms in Arabidopsis leaf tissue. Subcellular fractionation experiments, together with gel-filtration and nondenaturing-gel electrophoresis revealed that most of ADL1 is present as a high-molecular-mass complex of 400 to 600 kD in the membrane or pellet fraction, whereas ADL1 is present in the soluble fraction as a monomer. The subcellular distribution of ADL1 is affected by various agents such as Ca2+, cyclosporin A, GTP, and ATP. Ca2+ increases the amount of ADL1 present in the membrane fraction, whereas cyclosporin A inhibits the membrane association. Furthermore, Ca2+ and GTP change the migration pattern of ADL1 in nondenaturing polyacrylamide gels, indicating that these chemicals influence either the complex formation and/or the conformation of the ADL1 complex. Our results demonstrate that ADL1 has characteristics that are similar to Dynamin I, which is found in animal cells. Therefore, it is possible that ADL1 is also involved in biological processes that require vesicle formation.

Adenosine Triphosphate↗