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Biomedical subjects

J Kim

Publications and source records attributed to J Kim.

At least 865 records · Page 48Linked to original sources
PubMed1995

A dominant negative bone morphogenetic protein 4 receptor causes neuralization in Xenopus ectoderm.

Injection of DN-BR mRNA encoding a dominant negative type I receptor for bone morphogenetic protein 4 (BMP4) converted prospective ectoderm into neural tissue in Xenopus animal cap explants, in the absence of expression of mesodermal marker genes. The injected caps expressed a general neural marker NCAM and the forebrain marker opsin. Coinjection of wild-type BMP4 receptor mRNA completely reversed the neuralization by DN-BR. No expression of known neuralizing factors, i.e., noggin and follistatin, was detected in the DN-BR-injected animal caps. Furthermore, neuralization elicited by noggin or 3m, a LIM domain mutant of Xlim-1, was substantially inhibited by co-injection of BMP4 mRNA. Since BMP4 is expressed in the prospective ectoderm during gastrulation, our results suggest that the ventralizing factor BMP4 acts also as a physiological inhibitor of neuralization in the development of Xenopus ectoderm.

Animals↗
PubMed1995

Transcription and processing of the rodent ID repeat family in germline and somatic cells.

ID elements comprise a rodent SINE (short interspersed DNA repetitive element) family that has amplified by retroposition of a few master genes. In order to understand the important factors of SINE amplification, we investigated the transcription of rat ID elements. Three different size classes of ID transcripts, BC1, BC2 and T3, have been detected in various rat tissues, including brain and testes. We have analysed the nucleotide sequences of testes- and brain-derived ID transcripts isolated by size-fractionation, C-tailing and RACE. Nucleotide sequence variation of testes ID transcripts demonstrated derivation from different loci. However, the transcripts represent a preferred set of ID elements that closely match the subfamily consensus sequences. The small ID transcripts, T3, are not comprised of primary transcripts, but are instead processed polyA-transcripts generated from many different loci. These truncated transcripts would be expected to be retroposition-incompetent forms. Therefore, the amplification of ID elements is likely to be regulated at multiple steps of retroposition, which include transcription and processing. Although brain ID transcripts showed a similar pattern, with the addition of very high levels of transcription from the BC1 locus, we also found evidence that a single locus dominated the production of brain BC2 RNA species. BC1 RNA is highly stable in both germ line and brain cells, based on the low level of detection of the processing product, T3. This stability of BC1 RNA might have been a contributing factor in its role as a master gene for ID amplification.

Animals↗
PubMed1995

Implication of mammalian ribosomal protein S3 in the processing of DNA damage.

A human apurinic/apyrimidinic endonuclease activity, called AP endonuclease I, is missing from or altered specifically in cells cultured from Xeroderma pigmentosum group-D individuals (XP-D cells) (Kuhnlein, U., Lee, B., Penhoet, E. E., and Linn, S. (1978) Nucleic Acids Res. 5,951-960). We have now observed that another nuclease activity, UV endonuclease III, is similarly not detected in XP-D cells and is inseparable from the AP endonuclease I activity. This activity preferentially cleaves the phosphodiester backbone of heavily ultraviolet-irradiated DNA at unknown lesions as well as at one of the phosphodiester bonds within a cyclobutane pyrimidine dimer. The nuclease activities have been purified from mouse cells to yield a peptide of M(r) = 32,000, whose sequence indicates identity with ribosomal protein S3. The nuclease activities all cross-react with immunopurified antibody directed against authentic rat ribosomal protein S3, and, upon expression in Escherichia coli of a cloned rat cDNA for ribosomal protein S3, each of the activities was recovered and was indistinguishable from those of the mammalian UV endonuclease III. Moreover, the protein expressed in E. coli and its activities cross-react with the rat protein antibody. Ribosomal protein S3 contains a potential nuclear localization signal, and the protein isolated as a nuclease also has a glycosylation pattern consistent with a nuclear localization as determined by lectin binding. The unexpected role of a ribosomal protein in DNA damage processing and the unexplained inability to detect the nuclease activities in extracts from XP-D cells are discussed.

Amino Acid Sequence↗
PubMed1995

Site-directed mutagenesis identifies residues involved in ligand recognition in the human A2a adenosine receptor.

The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5-7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nM) and [3H]XAC (4 nM), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5'. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were > 30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5'-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.

Amino Acid Sequence↗
PubMed1995

Activation of the TFIID-TFIIA complex with HMG-2.

The nonhistone chromosomal protein HMG-2 was identified as a factor necessary for activation in a defined transcription reaction in vitro containing RNA polymerase II and purified factors. Activation occurred on all promoters assayed except that of the immunoglobulin IgH gene. TFIIA was required for stimulated levels of transcription. The activation process depended on the presence of TAFs in the TFIID complex and generated a preinitiation complex from which TFIIB dissociated more slowly. However, titration of TFIIB over three orders of magnitude did not obviate the requirement of activator and HMG-2 to achieve stimulated levels of transcription. Analysis of the activated reaction identified the TFIID-TFIIA complex as the first stage of modification during activation. These results suggest that activation can occur solely in the presence of the basal factors, activator protein, and an "architectural" HMG factor, which probably stabilizes an activated conformation of the TFIID-TFIIA-promoter complex.

Base Sequence↗
PubMed1995

A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.

Bone Neoplasms↗
PubMed1995

Tolerance induced by direct inoculation of donor antigen into the thymus in low and high responder rodents.

A novel approach to tolerance induction in rats was recently described where donor antigen is inoculated directly into the thymus along with a brief period of immunosuppression in a pretransplant strategy. To develop a strategy that has more clinical appeal, we evaluated the timing of donor antigen inoculation in relation to allografting, the use of frozen bone marrow as the antigen, and the dose response of purified T cells as the antigen in a low responder heterotopic heart allograft combination. Additionally, the success of this pretransplant strategy in different low and high responder strain combinations was defined. In tolerant low responder animals we evaluated in vitro and in vivo cellular immunity. Tolerant host strain naive CD8+ T cell responses to donor and third party stimulators were compared to determine if tolerance is related to the strength of the response of the T cell subsets to donor antigen. Frozen bone marrow can induce tolerance in a low responder combination. Additionally, the dose of purified T cells necessary for tolerance induction was 5 x 10(5) cells. The pretransplant strategy was successful in two low responder strain combinations, Lewis into Wistar Furth and Lewis into DA, but unsuccessful in all high responder strain combinations evaluated. Low responder animals unresponsive to donor heart allografts demonstrate intact cell-mediated immunity and donor-specific tolerance in vivo by rejecting third party but not second donor strain hearts. The in vitro responses of tolerant animals demonstrated donor-specific suppression of the MHC class II response but an intact (normal) response to third party stimulators by proliferation assays and IFN-gamma production, suggesting suppression at the CD4+ T cell subset level.

Animals↗
PubMed1995

Galactose-1-phosphate uridylyltransferase from Escherichia coli, a zinc and iron metalloenzyme.

Galactose-1-P uridylyltransferase purified from Escherichia coli cells grown in enriched medium contains approximately 1.2 mol of tightly bound zinc/mol of subunits as well as variable amounts of iron, up to 0.7 mol/mol of subunits, and no detectable Ca, Cd, Cu, Mo, Ni, Co, Mn, As, Pb, or Se. The chelators, 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline sulfonate, and 2,2'-bipyridyl remove metal ions from the enzyme and allow the importance of zinc and iron to be evaluated. Dialysis of this enzyme against 2 mM 1,10-phenanthroline, 8-hydroxyquinoline sulfonate, and 2,2'-bipyridyl at millimolar concentrations slowly removes both zinc and iron from the enzyme (t1/2 = 4 days at 24 degrees C) with concomitant loss of enzymatic activity. In chelation experiments utilizing 1,10-phenanthroline, residual enzymatic activity was found to be proportional to the zinc content, to the iron content, and to the sum of zinc and iron. UDP-glucose (0.35 mM) protects the enzyme against loss of metal ions and activity in the presence of 1,10-phenanthroline, whereas glucose-1-P at 70 mM (400 x Km) fails to protect. The enzyme purified from cells grown on a minimal medium containing inorganic salts and glucose supplemented with either ZnSO4 or FeSO4 shows approximately the same level of enzymatic activity as the enzyme from cells grown on enriched medium. These experiments showed that enzymatic activity is supported by either iron or zinc associated with two sites in the enzyme. Enzyme depleted of metal ions by chelators can be partially reactivated by addition of ZnSO4.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1995

Interleukin-8 receptor beta. The role of the carboxyl terminus in signal transduction.

Two interleukin-8 (IL-8) receptors, alpha and beta, have been identified and cloned. Both receptors are thought to transduce signals by coupling to GTP-binding proteins. The aim of this study is to determine whether the carboxyl terminus (C') of IL-8 receptor beta (IL-8R beta) is involved in signaling in response to IL-8. We have constructed a number of IL-8R beta genes that encode truncated forms of the IL-8R beta. The deletions consisted of amino acids 349-355, 336-355, 325-355, and 317-355 (termed beta 2, beta 3, beta 4, and beta 5, respectively). 293 human embryonic kidney cells were transfected with the wild type IL-8R beta (beta 1) and with these mutants. Cells transfected with the mutated receptors expressed the receptors and bound IL-8 with the same high affinity as cells transfected with the wild type receptor. The capacity of the mutated receptors to convey functional signals was evaluated by comparing the chemotaxis index of cells expressing the C'-truncated receptors to the index of cells expressing the wild type receptor. The results indicate that while cells expressing beta 1, beta 2, beta 3, and beta 4 were chemoattracted in response to IL-8, cells expressing beta 5 did not migrate in response to IL-8 stimulation. Therefore, the data suggest that amino acids 317-324 are involved in signaling by IL-8R beta.

Chemotaxis↗
PubMed1995

A thermostable mutation located at the hydrophobic core of alpha 1-antitrypsin suppresses the folding defect of the Z-type variant.

A thermostable mutation, F51L, at the hydrophobic core of human alpha 1-antitrypsin (alpha 1AT) increased the conformational stability of the molecule by decreasing the unfolding rate significantly without altering the refolding rate. The mutation specifically influenced the transition between the native state and a compact intermediate, which retained approximately 70% of the far-UV CD signal, but which had most of the fluorescence signal already dequenched. The mutant alpha 1AT protein was more resistant than the wild-type protein to the insertion of the tetradecapeptide mimicking the sequence of the reactive center loop, indicating that the mutation increases the closing of the central beta-sheet, the A-sheet, in the native state. The F51L mutation enhanced the folding efficiency of the Z-type (E342K) genetic variation, which causes aggregation of the molecule in the liver. It has been shown previously that the aggregation of the Z protein occurs via loop-sheet polymerization, in which the reactive center loop of one molecule is inserted into the opening of the A-sheet of another molecule. Our results strongly suggest that the hydrophobic core of alpha 1AT regulates the opening-closing of the A-sheet and that certain genetic variations that cause opening of the A-sheet can be corrected by inserting an additional stable mutation into the hydrophobic core.

Enzyme Stability↗
PubMed1995

Contribution of L- and N-type calcium currents to exocytosis in rat adrenal medullary chromaffin cells.

The excitation-secretion coupling process requires Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) but the contribution of L-type or N-type VDCC during the secretion from adrenal chromaffin cell is still on debate. In this study we explored the contribution of each VDCC to exocytosis in single rat adrenal chromaffin cells. Chromaffin cells were voltage-clamped clamped in the whole-cell recording mode. Ca2+ inward current (ICa) was elicited by depolarization from -70 mV to +10 mV and the change in cell membrane capacitance (Cm) was monitored as an indicator of the resultant exocytosis. The increase in Cm had positive correlation with the amount of ICa and replacing the internal Ca2+ buffer to high EGTA (5 mM) decreased the sensitivity of Cm increase to Ca2+ influx. After blockage of ICa with 100 microM Cd2+, there was no increase in Cm following membrane potential depolarization while INa was intact. To clarify the contribution of each type of VDCC to induce exocytosis during membrane potential depolarization, L- and N-type ICa were blocked selectively by Ca2+ channel antagonists. After blockage of L-type ICa with nicardipine (1 microM), ICa was blocked to 35 +/- 6.2% (mean +/- standard error) of control and the resultant change in Cm was reduced to 38 +/- 4.6% of control. Bay K-8644 (1 microM) enhanced ICa and the similar proportion of Cm was increased by this L-type VDCC agonist. On the other hand omega-conotoxin GVIA (1 microM), an N-type VDCC antagonist, blocked ICa to 60 +/- 4.3% of control and reduced the change in Cm to 58 +/- 3.9% of control.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1995

Presence of both A1 and A2a adenosine receptors in human cells and their interaction.

We have obtained pharmacological evidence for the expression of both A1- (inhibitory) and A2a- (stimulatory) adenosine receptors in cultured human foreskin fibroblasts and lung fibroblasts. The A1 receptors were sufficiently abundant in foreskin fibroblasts so that binding studies with a radioactively labeled specific ligand confirmed their existence. Both receptors were activated during the stimulation of cAMP accumulation and DNA synthesis by adenosine.

Adenosine↗
PubMed1995

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, on agonist-stimulated phasic myometrial contractions.

Phasic myometrial contractions utilize mechanisms involving the cycling of calcium into and out of intracellular calcium stores. These studies were performed to determine the effects of 2,5-di(tert-butyl)-1,4-hydroquinone (tBHQ), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, on in vitro isometric myometrial contractions. These studies demonstrated that low concentrations of tBHQ (eg. 10 microM) appear to inhibit intracellular calcium cycling, whereas higher concentrations also inhibit extracellular calcium influx. These combined tBHQ effects markedly suppressed myometrial contractions stimulated in response to various agonists including oxytocin, PGF2 alpha, KCl, ionomycin, and Bay K 8644.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Overexpression of IL-10 in atopic dermatitis. Contrasting cytokine patterns with delayed-type hypersensitivity reactions.

The skin lesions of patients with atopic dermatitis provide a model to study immunoregulation in human allergy. To determine the local cytokine pattern of cells present (both endogenous and recruited) at the site of disease, we extracted RNA from skin biopsy specimens from patients with atopic dermatitis, allergic contract dermatitis, and positive tuberculin reactions and used PCR to assay for cytokine mRNA. cDNAs were normalized to the intensity of the CD3 delta PCR product as a marker of T cell mRNA. We found overexpression of IL-10 mRNA in atopic dermatitis lesions, in comparison with allergic contact dermatitis lesions and tuberculin reactions. In contrast, IL-4 mRNA was most strongly expressed in allergic contact dermatitis lesions and IFN-gamma mRNA was the predominant cytokine in tuberculin reactions. Using an anti-IL-10 mAb with immunoperoxidase, we localized IL-10 protein to large mononuclear cells in the dermal infiltrate of atopic lesions. After immunomagnetic sorting of mononuclear cell populations from PBMC of atopic dermatitis subjects, IL-10 mRNA as measured by PCR was found to be strongly expressed in CD14+ cells. Spontaneous release of IL-10 from PBMC-derived adherent cells was greater in atopic dermatitis donors than normal controls. We therefore renormalized skin biopsy cDNA according to the level of beta-actin PCR product, as a marker of total cellular mRNA, and found by PCR that IL-10 was nevertheless greatest in atopic dermatitis subjects. We conclude that the relative overexpression of IL-10 in atopic dermatitis greatest in atopic dermatitis subjects. We conclude that the relative overexpression of IL-10 in atopic dermatitis may contribute to the up-regulation of humoral responses and the down-regulation of Th1 responses.

Adult↗
PubMed1995

Intrinsically bent DNA in a eukaryotic transcription factor recognition sequence potentiates transcription activation.

Many eukaryotic transcription factors induce DNA bending on binding to their recognition sequences. DNA bending could play a structural role by altering contacts between the protein and DNA. Alternatively, DNA bending could play a more direct role in transcription activation. To distinguish between these possibilities, we inserted two to eight copies of the intrinsic bending sequence, AAAAAACGTG, into a minimal promoter containing only a TATA box. The intrinsic DNA bending sequence was a potent activator of transcription in both in vivo transfection experiments and in a cell-free transcription system. A protein binds to the intrinsic bending sequence with high specificity in gel mobility shift assays and was required for its transcription in cell-free extracts. The intercalator, distamycin, which eliminates the ability of the sequence to bend, specifically reduced its transcription by about 60%. Mutations in the sequence which abolished DNA bending reduced transcription by approximately 70% in vivo. Competition gel mobility shift assays showed that the transcription factor bound equally well to mutants in which DNA bending was abolished and to the intrinsic bending sequence. These data indicate that DNA bending can play a direct role in the activation of eukaryotic transcription.

Animals↗