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Biomedical subjects

K Kim

Publications and source records attributed to K Kim.

At least 343 records · Page 19Linked to original sources
PubMed1996

A global odds ratio regression model for bivariate ordered categorical data from ophthalmologic studies.

In typical clinical trials or epidemiologic studies of a bilateral eye disease, the primary outcome data consist of pairs of ordered categorical responses that tend to be highly correlated. In such studies, interest often centres in associating the outcome data with a grouping variable such as the treatment indicator or the exposure status and other person- and eye-specific covariates. In this paper, we propose a latent variable regression model to analyse such bivariate ordered categorical data. We use as a joint distribution for bivariate latent random variables the cross ratio distribution proposed by Plackett, which results in modelling the dependency between the fellow eyes with the global odds ratio. We illustrate the proposed model with data from the Wisconsin Epidemiologic Study of Diabetic Retinopathy, a study that seeks to identify risk factors among younger-onset diabetics.

Age of Onset↗
PubMed1996

Identification of ovarian enhancer-binding factors which bind to ovarian enhancer 1 of the Drosophila genes yp1 and yp2.

It has been reported that three different DNA regions-the fat body enhancer and ovarian enhancers 1 and 2-direct the tissue-specific expression of yp1 and yp2 in Drosophila melanogaster. In the present study, we identified ovarian enhancer 1-specific binding proteins. Electrophoretic mobility shift assay revealed that these proteins are present in the adult ovary, but not in adult testis or fat body. Southwestern blot analysis showed that about 130 kDa and 40 kDa proteins, designated OEF1 and OEF2, respectively, from ovarian nuclear or crude extracts bind specifically to the ovarian enhancer 1. The two proteins were partially purified by streptavidin/agarose-DNA affinity chromatography, and their binding activity was confirmed by electrophoretic mobility shift assay. These ovarian enhancer factors may play an important role in the regulation of transcription of yp1 and yp2 in the ovary.

Animals↗
PubMed1996

Intestinal fatty acid-binding protein: the structure and stability of a helix-less variant.

The structure of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) exhibits a beta-clam topology comprised of two five-stranded antiparallel beta-sheets surrounding a large solvent-filled cavity into which the ligand binds. It also contains two alpha-helices that span residues E15-A32 and join beta-strands A and B. This helical domain is conserved in all proteins of this family for which structures have been determined. In order to assess the structural and functional role of the helical domain, we engineered a variant of I-FABP by deleting residues 15-31 and inserting a Ser-Gly linker after residue 14. Circular dichroism measurements indicated that this I-FABP variant, termed delta 17-SG, has a high beta-sheet content similar to that of the wild-type protein. Two-dimensional NMR spectra of delta 17-SG revealed patterns similar to those observed for wild-type I-FABP, except for the selective absence of resonances and through-space interactions assigned to the helical domain. The delta 17-SG variant was less stable to denaturant than wild-type I-FABP, but the folding-unfolding transition was highly cooperative and reversible. Taking into account the lower stability, the refolding kinetics of delta 17-SG were essentially identical to those of wild-type. We conclude that delta 17-SG is a helix-less, essentially all-beta-sheet variant of I-FABP and that the helical domain is not a required element of the beta-clam topology of I-FABP. In addition, the helical domain does not appear to serve as a nucleation site for the refolding process. As shown in the accompanying paper [Cistola, D. P., Kim, K., Rogl, H., & Frieden, C. (1996) Biochemistry 35, 7559-7565], the helices may function to regulate the kinetics and energetics of ligand binding.

Amino Acid Sequence↗
PubMed1996

Fatty acid interactions with a helix-less variant of intestinal fatty acid-binding protein.

Intestinal fatty acid-binding protein (I-FABP) binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two antiparallel beta-sheets. The structure also contains two short alpha-helices which form a cap over one end of the binding cavity adjacent to the methyl terminus of the fatty acid. In this study, we employed a helix-less variant of I-FABP known as delta 17-SG [Kim, K., Cistola, D.P.,& Frieden, C. (1996) Biochemistry 35, 7553-7558] to investigate the role of the helical region in maintaining the integrity of the binding cavity and mediating the acquisition of ligand. Fluorescence and NMR experiments were used to characterize the energetic, structural, and kinetic properties of fatty acid binding to this variant, and the results were compared and contrasted with those of wild-type I-FABP and a single-site mutant, R106T. Remarkably, oleate bound to delta 17-SG with a dissociation constant of 4.5 microM, a value comparable to that for R106T and approximately 20-100-fold higher than that for wild-type I-FABP. Heteronuclear two-dimensional NMR spectra for [2-13C]palmitate complexed with delta 17-SG revealed a pattern nearly identical to that observed for the wild-type protein, but distinct from that for R106T. In addition, the ionization behavior of bound [1-13C]palmitate and the nearest neighbor patterns for [2-13C]palmitate derived from 13C-filtered NOESY experiments were very similar for delta 17-SG and the wild-type protein. These results implied that the fatty acid-protein interactions characteristic of the carboxyl end of the fatty acid binding cavity in the wild-type protein were essentially intact in the helix-less variant. In contrast, 13C-filtered NOESY spectra of [16-13C]palmitate bound to delta 17-SG indicated that the fatty acid-protein interactions at the methyl end of the binding cavity were disrupted. As determined by stopped-flow fluorescence, the observed ligand association rates for both delta 17-SG and wild-type I-FABP increased with increasing oleate concentration, but only the wild-type protein exhibited a limiting value of 1000 s-1. This rate-limiting process was interpreted as a conformational change involving the helical region that allows the ligand access to the internal cavity. Simulation and fitting of the kinetic results yielded ligand association rates for delta 17-SG and wild-type I-FABP that were comparable. However, the dissociation rate for wild-type protein was 16-fold lower than that for delta 17-SG. We conclude that the alpha-helices of I_FABP are not required to maintain the integrity of the fatty acid binding cavity but may serve to regulate the affinity of fatty acid binding by selectively altering the dissociation rate constant. In this manner, conformational changes involving the alpha-helical domain may help control the transfer of fatty acids within the cell.

Amino Acid Sequence↗
PubMed1996

Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids.

Atomic force microscopy (AFM, also called scanning force microscopy) is proving to be a useful technique for imaging DNA. Thus it is important to push the limits of AFM imaging in order to explore both what types of DNA can be reliably imaged and identified and also what substrates and methods of sample preparation are suitable. The following advances in AFM of DNA are presented here. (i) DNA molecules as short as 25 bases can be seen by AFM. The short single-stranded DNAs imaged here (25 and 50 bases long) appeared globular in the AFM, perhaps because they are all capable of intramolecular base pairing and because the DNAs were in a Mg(ll) buffer, which facilitates intramolecular cross-bridging. (ii) AFM images in air of short double-stranded DNA molecules, 100-200 bp, gave lengths consistent with A-DNA. (iii) AFM images of poly (A) show both short bent lumpy molecules with an apparent persistence length of 40 nm and long straight molecules with an apparent persistence length of 600 nm. For comparison, the apparent persistence length for double-stranded DNA from phX-174 under the same conditions was 80 nm. (iv) Structures believed to be triple- stranded DNA were seen in samples of poly(dA.poly(dT) and poly (dG).poly(dC). These structures were twice as high as double-stranded DNA and the same width. (v) Entire molecules of lambda DNA, approx. 16 micron long, were imaged clearly in overlapping scans. (vi) Plasmid DNA was imaged on oxidized silicon, although less clearly than on mica.

Base Sequence↗
PubMed1996

Adenocarcinoma of duodenum and ampulla of Vater: clinicopathology study and expression of p53, c-neu, TGF-alpha, CEA, and EMA.

Oncogenes, tumor suppressor genes, and growth factors are being explored as to their role in the initiation and progression of most neoplasms, but little information exists on the expression of oncoproteins or growth factors in adenocarcinoma of the duodenum or ampulla of Vater. This report covers expressions of p53, c-neu, TGF-alpha, CEA, and EMA in duodenal adenocarcinoma and ampullary adenocarcinoma, as well as correlations between expressions and tumor stage, histological grade and patient survival. The expression of p53, c-neu, TGF-alpha, CEA, and EMA has been studied in 15 duodenal adenocarcinomas and in eight ampullary adenocarcinomas by avidin-biotin-peroxidase complex indirect immunoperoxidase technique. The positive reaction for p53, c-neu, TGF-alpha, CEA, and EMA in duodenal adenocarcinoma was 20%, 60%, 60%, 73%, and 100%, respectively, and in ampullary adenocarcinoma, 13%, 100%, 50%, 63%, and 100%. Among the duodenal tumors, C-neu and p53 expression was noted more frequently in groups with high histological grades. Patients with c-neu positive duodenal adenocarcinoma had a shorter survival than the patients with c-neu negative duodenal adenocarcinoma (P < 0.01). C-neu product may serve as an unfavorable prognostic indicator in duodenal adenocarcinoma. No statistically significant correlation was found between the expressions of CEA, EMA, p53, and TGF-alpha and patient survival, tumor stage, or histological grade in either duodenal or ampullary adenocarcinomas.

Adenocarcinoma↗
PubMed1996

Short- and long-term clinical effects of primary directional coronary atherectomy for acute myocardial infarction.

We performed primary directional coronary atherectomy (DCA) without antecedent thrombolytic therapy in 21 of 67 patients with acute myocardial infarction within 24 hr of onset between June 1993-March 1994. Reperfusion with primary DCA was successful in 18 patients (85.7%, group D). Results were compared with those of primary balloon angioplasty patients treated between June 1992-May 1993 (group P). Minimum lumen diameter (MLD) values both immediately after reperfusion and in predischarge angiograms were significantly larger in group D than in group P, but were similar in late follow-up angiograms. Although a larger MLD in group D than in group P contributed to the prevention of reocclusion of the coronary artery before discharge in DCA patients, a high rate of restenosis at late follow-up canceled the beneficial effects of primary DCA.

Aged↗
PubMed1996

Differential expression of D-type G1 cyclins during mouse development and liver regeneration in vivo.

D-type G1 cyclins are the primary cell cycle regulators of G1/S transition in eukaryotic cells, and are differentially expressed in a variety of cell lines in vitro. Little is known, however, about the expression patterns of D-type G1 cyclins in normal mouse in vivo. Thus, in the present study, tissue-specific expressions of cyclin D1 and D3 genes were examined in several tissues derived from adult male mice, and stage-specific expression of cyclin genes was studied in brain, liver, and kidney of developing mice from embryonic day 13 to postnatal day 11. Cell cycle-dependent expression of cyclins was also examined in regenerating livers following partial hepatectomy. Our results indicate that (1) cyclins D1 and D3 are expressed in a tissue-specific manner, with cyclin D1 being highly expressed in kidney and D3 in thymus; (2) cyclin D3 mRNA is abundantly expressed in young proliferating tissues and is gradually reduced during development, whereas cyclin D1 mRNA fluctuates during development; and (3) compensatory regeneration of liver induces cyclin D1 gene expression 12 hr after partial hepatectomy, and cyclin D3 gene expression from 36 to 42 hr (at the time of G1/S transition). In conclusion, this study indicates that cyclin D1 and D3 genes are differentially expressed in vivo in a tissue-specific, developmental stage-dependent, and cell cycle-dependent manner.

Animals↗
PubMed1996

Differential expression of laminin chain-specific mRNA transcripts during mouse preimplantation embryo development.

Laminin is the first extracellular matrix protein that has been shown to be synthesized in preimplantation mouse embryos. In the present study, chain-specific expression patterns of laminin mRNAs were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). During preimplantation mouse embryo development, temporal expression patterns of laminin chain mRNAs were somewhat differential: B1 chain mRNA was first detectable at the late two-cell stage and its level was gradually increased by the blastocyst stage. In contrast, B2 and A chain mRNAs first appeared at the morula and blastocyst stages, respectively. At the blastocyst stage, all of the laminin chain mRNAs were highly detected compared to the earlier stages. When embryos were flushed at the morula stage and cultured in vitro, all laminin chain mRNA levels were decreased or not changed in the process of blastocoele expansion. In contrast, in the in vivo condition where embryos at different stages of blastocyst were flushed at different time points, laminin chain mRNA levels were increased as a function of blastocoele expansion. These changes in laminin mRNAs were parallel with its receptors such as integrin alpha 3 and alpha 6. 3-Isobutyl-1-methylxanthine (IBMX), which is known to be a potent activator of blastocoele expansion and regulates cAMP metabolism, upregulated laminin expression (except B1 chain) in blastocysts cultured in vitro. In vitro cultured embryos normally developed up to the late blastocyst, although their development was delayed compared with the in vivo condition where laminin gene expression was gradually increased as the blastocoele expanded. These results indicate that laminin expression may not be involved directly in the regulation of blastocoele expansion. The uterine environment enclosing the preimplantation embryos appears, therefore, to play an important role in the regulation of laminin gene expression during blastocyst development.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Sensitivity-enhanced echo-planar MRI at 1.5T using a 5 x 5 mesh dome resonator.

In this work a 5 x 5 mesh dome resonator that has been optimized for functional brain imaging is presented. The resonator was reduced in length and diameter compared with previous versions to reduce sample losses, thus enhancing the signal-to-noise ratio of the acquired data. In addition, a 5 x 5 mesh design was employed, which offered improved axial homogeneity over an earlier 3 x 3 mesh version. The new resonator exhibited high sensitivity and good homogeneity over the brain volume, permitting analysis of functional activation over large areas of the cerebral cortex. In a direct comparison with a standard clinical head-imaging resonator, the high sensitivity of the 5 x 5 mesh dome resonator resulted in greater statistical confidence in functional activation.

Cerebral Cortex↗
PubMed1996

An efficient expression of human growth hormone (hGH) in the milk of transgenic mice using rat beta-casein/hGH fusion genes.

In order to produce human growth hormone (hGH) in the milk of transgenic mice, two expression vectors for hGH differing in their 3' flanking sequences were constructed by placing the genomic sequences of hGH gene under the control of the rat beta-casein gene promotor. The 3' flanking sequences of the expression constructs were derived from either the hGH gene (pBCN1GH) or the rat beta-casein gene (pBCN2GH). Transgenic lines bearing pBCN1GH expressed hGH more efficiently than those bearing pBCN2GH in the milk (19-5500 micrograms/mL vs 0.7-2 micrograms/mL). In particular, one of the BCN1GH lines expressed hGH as much as 5500 +/- 620 micrograms/mL. Northern blot analysis showed that the transgene expression was specifically confined to the mammary gland and developmentally regulated like the endogenous mouse beta-casein gene in the mammary gland. However, a low level of nonmammary expression was also detected with more sensitive assay methods. In conclusion, the rat beta-casein/hGH fusion gene could direct an efficient production of hGH in a highly tissue-and stage-specific manner in the transgenic mice and the 3' flanking sequences of hGH gene had an important role for the efficient expression.

Animals↗
PubMed1996

The construction of a stable starch-fermenting yeast strain using genetic engineering and rare-mating.

To develop a yeast strain that is able to produce ethanol directly from starch, alpha-amylase cDNA (originated from mouse salivary glands) was introduced into the hyploid Saccharomyces diastiticus cells secreting glucoamylase by using a linearized integrating vector. The integrating vector contains a LEU2 gene and the inside of the LEU2 gene was cut by KpnI to make the linearized vector. One of the transformants exhibited 100% mitotic stability after 100 generations of cell multiplication. To improve its ethanol-fermentability, the haploid transformant was rare-mated with a polyploid industrial strain having no amylase activity. The resulting hybrid RH51 produced 7.5 (w/v) ethanol directly from 20% (w/v) soluble starch and its mitotic stability was 100% at the end of fermentation.

Animals↗
PubMed1996

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos.

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.

Journal Article↗
PubMed1996

Selection of peptides that bind to the HLA-A2.1 molecule by molecular modelling.

Cytotoxic T lymphocytes recognize antigenic peptides in association with major histocompatibility complex class I proteins. Although a large set of class I binding peptides has been described, it is not yet easy to search for potentially antigenic peptides without synthesis of a panel of peptides, and subsequent binding assays. In order to predict HLA-A2.1-restricted antigenic epitopes, a computer model of the HLA-A2.1 molecule was established using X-ray crystallography data. In this model nonameric peptide sequences were aligned. In a molecular dynamics (MD) simulation with two sets of peptides known to be presented by HLA-A2.1, it was important to know the anchor amino acid residue preference and the distance between the anchor residues. We show here that the peptides bound to the HLA-A2.1 model structure possess a side chain of C-terminal anchor residue oriented into the binding groove with different distances between the two anchor residues from 15 to 21A. We also synthesized a set of nonamer peptides containing amino acid sequences of Hepatitis B virus protein that were selected on the basis of previously described HLA-A2.1 specific motifs. When results obtained from the MD simulation were compared with functional binding assays using the TAP-deficient cell line T2, it was evident that the MD simulation method improves prediction of the HLA-A2.1 binding epitope sequence. These results suggest that this approach can provide a way to predict peptide epitopes and search for antigenic regions in sequences in a variety of antigens without screening a large number of synthetic peptides.

Amino Acid Sequence↗
PubMed1996

Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase-thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum.

We have determined the nucleic acid sequences of a gene encoding the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) from bovine and human AIDS isolates of Cryptosporidium parvum. THe DHFR-TS gene was isolated from genomic DNA libraries by hybridization with a probe amplified from C. parvum genomic DNA using generic TS primers in the polymerase chain reaction. Genomic Southern and electrophoretic karyotype analyses reveal C. parvum DHFR-TS is a single-copy gene on a 1200-kb chromosome. The DHFR-TS nucleic acid sequence contains no introns and the single 1563-bp open reading frame encodes a 179 residue N-terminal DHFR domain connected by a 55 amino acid junction peptide to a 287 residue C-terminal TS domain. The sequences of the DHFR-TS gene from the bovine and human C. parvum isolates differ at two positions in the 5'-flanking sequence and at 38 positions in the encoding sequence. These DNA sequence polymorphisms will provide a powerful probe to examine the genotypic diversity and genetic population structure of C. parvum. The two sequences encode identical TS domains which share all except one of the phylogenetically conserved amino acid residues identified among reported TS sequences. The predicted DHFR domain sequences contain nine amino acid differences; these polymorphisms all map to non-active site, surface locations in known DHFR structures. The C. parvum DHFR active site contains novel residues at several positions analogous to those at which point mutations have been shown to produce antifolate resistance in other DHFRs. Thus C. parvum DHFR may be intrinsically resistant ti inhibition by some antifolate DHFR inhibitors which may explain why cryptosporidiosis is refractory to treatment with the clinically common antibacterial and antiprotozoal antifolates.

Amino Acid Sequence↗
PubMed1996

Application of the Roche Amplicor Mycobacterium tuberculosis (PCR) test to specimens other than respiratory secretions.

The ability of the Roche AMPLICOR Mycobacterium tuberculosis (MTB) test to detect M. tuberculosis in specimens other than respiratory secretions was evaluated. A total of 249 specimens from 219 patients were tested. Of these, 12 specimens grew isolates of the M. tuberculosis complex and four grew isolates of the M. avium complex. The AMPLICOR MTB test was positive for 10 of the 12 specimens which grew M. tuberculosis and for three specimens which were culture negative. Two of the latter specimens were from patients with a clinical diagnosis of tuberculosis and with multiple sputum specimens which grew M. tuberculosis. Four specimens grew M. avium complex isolates, and all yielded negative AMPLICOR MTB test results. The sensitivity, specificity, and positive and negative predictive values for the AMPLICOR MTB test were 85.7%, 99.5%, 92.3%, and 99.1%, respectively. Our data indicate that the AMPLICOR MTB test will permit the rapid detection of M. tuberculosis in specimens other than respiratory secretions.

Biopsy↗