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J Kim

Publications and source records attributed to J Kim.

At least 919 records · Page 51Linked to original sources
PubMed1995

The intracellular assembly of antigenic-peptide-class II complexes.

The immune system employs remarkable strategies to ensure that foreign antigens, from the most complex pathogens to the simplest proteins, are displayed on the surfaces of cells which are targets of T lymphocyte recognition. At the heart of these strategies is the molecular transformation of a soluble protein antigen to a complex of a small peptide containing the antigenic determinant bound to a cell surface Major Histocompatibility Complex class I or class II protein. This process is termed antigen presentation. Progress in a variety of laboratories over the last several years has yielded a wealth of information about the molecular mechanisms underlying antigen presentation, providing potential new approaches to vaccine design. Here we describe recent studies in our laboratory aimed at elucidating the intracellular site in B lymphocytes in which antigenic peptide-class II complexes are assembled for recognition by helper T cells and the regulation of this assembly process. Our results suggest that processed antigen-class II complexes are assembled in a unique compartment in the endocytic route which contains all the necessary cellular and molecular machinery for assembly and that B cells regulate the assembly process in response to external and internal signals.

Antigen-Presenting Cells↗
PubMed1994

Toxic shock syndrome toxin-1 complexed with a class II major histocompatibility molecule HLA-DR1.

The three-dimensional structure of a Staphylococcus aureus superantigen, toxic shock syndrome toxin-1 (TSST-1), complexed with a human class II major histocompatibility molecule (DR1), was determined by x-ray crystallography. The TSST-1 binding site on DR1 overlaps that of the superantigen S. aureus enterotoxin B (SEB), but the two binding modes differ. Whereas SEB binds primarily off one edge of the peptide binding site of DR1, TSST-1 extends over almost one-half of the binding site and contacts both the flanking alpha helices of the histocompatibility antigen and the bound peptide. This difference suggests that the T cell receptor (TCR) would bind to TSST-1:DR1 very differently than to DR1:peptide or SEB:DR1. It also suggests that TSST-1 binding may be dependent on the peptide, though less so than TCR binding, providing a possible explanation for the inability of TSST-1 to competitively block SEB binding to all DR1 molecules on cells (even though the binding sites of TSST-1 and SEB on DR1 overlap almost completely) and suggesting the possibility that T cell activation by superantigen could be directed by peptide antigen.

Bacterial Toxins↗
PubMed1994

Phosphorylation, high ionic strength, and calmodulin reverse the binding of MARCKS to phospholipid vesicles.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major cellular substrate of protein kinase C (PKC), and PKC phosphorylation produces translocation of MARCKS from membrane to cytoplasm in many cells. Our working hypothesis is that binding of MARCKS to biological membranes requires both hydrophobic insertion of its myristoyl chain into the lipid bilayer and electrostatic interaction of its basic domain with acidic lipids. We tested this hypothesis by measuring the binding of murine MARCKS to large unilamellar phospholipid vesicles (LUVs). We estimated the partition coefficient of the myristoyl moiety of MARCKS (KH) by measuring the binding of MARCKS to electrically neutral LUVs (KH = 3 x 10(3) M-1). We examined the effect of electrostatic interactions by measuring the binding of MARCKS to LUVs containing 20% acidic lipid and obtained four results. First, incorporating 20% acidic lipid into the LUVs increased binding of MARCKS about 100-fold. Second, PKC phosphorylation, which added 3 negatively charged phosphate groups to the basic domain, reduced 20-fold the binding of MARCKS to these negatively charged vesicles. Third, increasing the KCl concentration from 0.1 to 0.5 M reduced the binding 15-fold. Fourth, Ca(2+)-calmodulin reduced the binding 20-fold. We present a simple theoretical model that explains these results, which are all consistent with the working hypothesis.

Animals↗
PubMed1994

The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure.

The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.

Adenosine Monophosphate↗
PubMed1994

Exciton splitting in phycoerythrin 545.

Phycoerythrin 545 is a biliprotein having a polypeptide structure of alpha 2 beta 2, and each alpha and beta polypeptide has chromophores. Circular dichroism (CD) and absorption spectroscopy in the visible region together with various biochemical protocols have been used to study these chromophores. The CD spectrum exhibits overlapping positive and negative bands. Exciton splitting between closely-spaced pairs of chromophores produces a CD spectrum that has positive and negative bands of equal rotational strengths, a conservative spectrum. Alternatively, any positive or negative band could arise from a single chromophore. The results of this study demonstrate that exciton splitting is the likely cause of the negative and corresponding positive bands. The CD spectra of the separated alpha and beta polypeptides, under conditions where the polypeptide structure is denatured, have no negative bands. When the polypeptides are allowed to refold individually, the chromophores on the beta polypeptide regain a combination of negative and positive CD bands. The spectrum of the alpha polypeptide shows no evidence of exciton splitting under these refolding conditions. In another approach, urea is added to the protein in low concentrations, which result in changes in the conformation and perhaps association of the protein. A difference CD spectrum of native protein minus protein in 0.8 M urea shows a spectrum characteristic of exciton splitting. Moreover, the remaining CD spectra in 0.8, 1.6, or 2.4 M urea still show the possibility of further exciton splitting, but a slightly different wavelengths from the spectrum that is deleted by 0.8 M urea. This finding may suggest that there are two types of exciton splitting.

Circular Dichroism↗
PubMed1994

A strategy for the analysis of gene expression during neural development.

The delineation of cis-acting elements regulating stage-specific gene expression has played a critical role in the definition of the transcriptional circuitry regulating differentiation. In complex tissues such as the central nervous system, differentiation often requires temporally and spatially dynamic epigenetic cues that cannot be reproduced in cell culture. In these cases, identification of critical regulatory sequences and subsequent characterization of cognate transcription factors have been limited by lack of a rapid and efficient assay system for gene expression. We report a methodology that combines particle-bombardment transfection procedures and organotypic slice culture techniques to provide an acute assay system for transcriptional control in the developing central nervous system. Using this system, we demonstrate that cell-specific regulation of the brain lipid-binding protein (BLBP) gene in Bergmann glia, astrocytes, and migrating granule cells is conferred by 1.7 kb of 5' flanking sequences and that Purkinje cell-specific expression of the calbindin D28k gene in cerebellar cortex can be achieved with 1.1 kb of flanking DNA. Nearly 100% cotransfection of multiple DNAs can be achieved, allowing the design of precisely internally controlled experiments and providing the potential for rapid and efficient genetic analysis of gene function in single cells in a wild-type environment.

Aging↗
PubMed1994

Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding.

An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end of a hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 37 degrees C. For the interface (/) in [formula: see text] additional free energy change, delta delta G 37 degrees, for binding is roughly the nearest-neighbor delta G 37 degrees for propagation of an uninterrupted helix of equivalent sequence, CGGC. When X and Z are omitted, the delta delta 37 degrees is even more favorable by approximately 1 kcal/mol (1 cal = 4.184J). On average, predictions of 11 RNA secondary structures improve from 67 to 74% accuracy by inclusion of similar stacking contributions.

Animals↗
PubMed1994

Higher autoantibody levels and recognition of a linear NH2-terminal epitope in the autoantigen GAD65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus.

The smaller form of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in two human diseases that affect its principal sites of expression. Thus, destruction of pancreatic beta cells, which results in insulin-dependent diabetes mellitus (IDDM), and impairment of GABA-ergic synaptic transmission in Stiff-Man syndrome (SMS) are both characterized by circulating autoantibodies to GAD65. Anti-GAD65 autoantibodies in IDDM are predominantly directed to conformational epitopes. Here we report the characterization of humoral autoimmune responses to GAD65 in 35 SMS patients, of whom 13 (37%) also had IDDM. All SMS patients immunoprecipitated native GAD65 and the main titers were orders of magnitude higher than in IDDM patients. Furthermore, in contrast to the situation in IDDM, autoantibodies in 35 of 35 (100%) of SMS patients recognized denatured GAD65 on Western blots. Two major patterns of epitope specificity were identified on Western blots. The first pattern, detected in 25 of 35 SMS patients (71%), of whom 11 had IDDM (44%), was predominantly reactive with a linear NH2-terminal epitope residing in the first eight amino acids of GAD65. Nine of nine individuals who were HLA-haplotyped in this group carried an IDDM susceptibility haplotype and HLA-DR3, DQw2 was particularly abundant. The second pattern, detected in 10 of 35 patients (29%) of whom two had IDDM (20%), included reactivity with the NH2-terminal epitope plus strong reactivity with one or more additional epitope(s) residing COOH-terminal to amino acid 101. The second epitope pattern may represent epitope spreading in the GAD65 molecule, but may also include some cases of epitope recognition associated with IDDM resistant HLA-haplotypes. The principal NH2-terminal linear epitope in GAD65 distinguishes the reactivity of SMS and IDDM autoantibodies and may be a determinant of pathogenicity for GABA-ergic neurons. The greater magnitude and distinct specificity of the humoral response to GAD65 in SMS may reflect a biased involvement of the T helper cell type 2 (Th2) subset of CD4+ T cells and antibody responses, whereas IDDM is likely mediated by the Th1 subset of CD4+ T cells and cytotoxic T cell responses.

Adult↗
PubMed1994

Multiple sets of basal factors initiate transcription by RNA polymerase II.

The minimal requirements for transcription initiation from supercoiled templates were determined for the two major forms of TATA-binding factors found in cell extracts, the 300-kDa B-TFIID and the 1000-kDa D-TFIID complexes. As had been observed for the TATA-binding protein (TBP) subunit (Parvin and Sharp, 1993), transcription from the IgH promoter minimally requires TFIID activity plus TFIIB and RNA polymerase II. This minimal reaction is only active on negatively supercoiled template DNA. In contrast, the supercoiled templates encoding the adenovirus major late promoter (MLP), or several other promoters, require the addition of TFIIF to the minimal reaction. Further addition of TFIIE and TFIIH boosts the level of transcription from these latter promoters but is not required. In contrast to the complete reaction on linear template, transcription from supercoiled IgH or MLP templates does not require the hydrolysis of the beta-gamma bond of ATP. Fourteen different core promoters were compared in complete and minimal basal transcription reactions reconstituted with one of the three TATA activities: TBP, B-TFIID, and D-TFIID. Of these 14 promoters, only the IgH was active in the absence of TFIIF, and the other promoters demonstrated different levels of transcription depending on which basal factors were present in reaction. It is proposed that a significant level of basal transcription only requires a minimal set of factors, and stimulation by upstream activators may in part be mediated by the inclusion of additional basal factors into the initiation reaction.

Animals↗
PubMed1994

Synthesis and turnover of photosystem II reaction center protein D1. Ribosome pausing increases during chloroplast development.

The chloroplast photosystem II reaction center protein D1 contains five membrane-spanning helices and binds chlorophyll, carotenoid, quinone, iron, and probably manganese. Turnover of pulse-labeled D1 in isolated plastids was found to involve cleavage between helix IV and helix V, which releases a 23-kDa N-terminal peptide and two C-terminal peptides of 10 and 8 kDa. Ribosomes pause at specific sites during translation of D1, which results in the accumulation of D1 translation intermediates. Pulse-labeling assays followed by polysome isolation and immunoprecipitation identified paused D1 translation intermediates of 9, 12.5, 15-18, 20, 21, 24, and 28-32 kDa. Ribosome pausing was not altered when dark-grown seedlings were illuminated for up to 1 h, even though this treatment stimulated accumulation of chlorophyll and D1. However, illumination of plants for 16-72 h resulted in increased ribosome pausing and the build-up of D1 translation intermediates. We hypothesize that ribosome pausing during synthesis of D1 improves the efficiency of chlorophyll binding of D1 nascent chains and enhances accumulation of D1 in mature chloroplasts, which have reduced rates of chlorophyll biosynthesis.

Amino Acid Sequence↗
PubMed1994

[The role of beta 2-adrenoceptor on the pathogenesis of insulin resistance in essential hypertension].

The role of beta 2-adrenoceptor on the pathogenesis of insulin resistance in essential hypertension (EH) was explored. After the measurement of blood pressure in 15 EH patients and 8 control subjects, EH patients were divided into two groups by the elevation of plasma NE (delta NE) 5 min after standing: 7 normoadrenergic EH (delta NE < 140 pg/ml) and 8 hyperadrenergic EH (delta NE > or = 140 pg/ml). On the morning after a 12-h overnight fast, regular insulin (0.1 U/kg) was injected intravenously, and glucose disappearance rate (GDR) was measured and used as an index of insulin sensitivity. On the following day, the test was reinvestigated following the administration of mabuterol, a beta 2 agonist. Plasma growth hormone (GH), cortisol, norepinephrine (NE) and epinephrine (Epi) were measured before and after the mabuterol administration. Although there were no significant differences of basal GDR among these three groups, mabuterol induced a considerable decrease in GDR in EH patients but not in control subjects. There was no significant difference in the decrease of GDR between normo- and hyperadrenergic EH. The decrease in GDR tended to correlate with the mean blood pressure at rest in EH but not in normal subjects. Plasma glucose and serum insulin in EH patients were increased more than in normal subjects. Plasma GH, cortisol and Epi were not elevated by mabuterol, but plasma NE increased in each group, significantly in hyperadrenergic EH. There was no correlationship between the increase in plasma NE and the decrease in GDR after mabuterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1994

Differentiating macular holes from macular pseudoholes.

Surgical treatments of macular holes have become increasingly effective in inducing resolution of the cuff of surrounding subretinal fluid, resulting in increased vision in many patients. However, for many conditions that mimic a macular hole, either surgery is not indicated or different surgical manipulations are necessary. Differentiating macular holes from some forms of macular pseudoholes can be difficult or impossible based solely on clinical examination. Adjunctive tests that may enhance the accuracy of diagnosis are either not feasible or not available to most clinical practices. We evaluated three clinic-based tests for their value in allowing the differentiation between macular holes and macular pseudoholes: Amsler grid testing, Watzke-Allen sign, and laser aiming beam test. These tests were evaluated in three groups of clinically defined patients: those with full-thickness macular holes, those with macular pseudoholes, and those who had previously undergone successful macular hole treatment. Although the Amsler grid testing was sensitive in correlating with clinically defined macular holes, it was not specific. The Watzke-Allen sign and, to a greater extent, the laser aiming beam test were extremely sensitive and specific in correlating clinically defined full-thickness macular holes and pseudoholes. These tests improve the accuracy of diagnosis of full-thickness macular holes.

Aged↗
PubMed1994

Rodent BC1 RNA gene as a master gene for ID element amplification.

ID elements are short interspersed repetitive DNA elements (SINEs) which have amplified in rodent genomes via retroposition, a process involving an RNA intermediate. BC1, an abundant ID-related transcript, is transcribed from a conserved, single-copy gene in rodents. The gene encoding BC1 RNA represents one of the earliest and possibly the first ID-containing sequence. Comparison of consensus sequences of each rodent ID with its corresponding BC1 RNA gene showed that the variations of BC1 RNA within rodents corresponded to specific changes within the ID consensus sequence for each rodent species. This supports the hypothesis that the BC1 gene is a master gene responsible for the amplification and evolution of ID elements. The rat ID family consists of at least four subfamilies, with the oldest subfamily having been derived from the BC1 RNA. The other three subfamilies appear to have been derived from a new master gene(s), which has been responsible for the large increase in ID element copy number within the rat genome. We have found that the guinea pig genome contains two copies of the BC1 gene, apparently the result of a DNA-mediated duplication event. Both of these guinea pig BC1 genes have a conserved TATA-like element in the 5' flanking region and have contributed to guinea pig ID amplifications.

Animals↗