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J Kim

Publications and source records attributed to J Kim.

At least 613 records · Page 34Linked to original sources
PubMed1998

HIV envelope protein gp120 induces neuropeptide Y receptor-mediated proliferation of vascular smooth muscle cells: relevance to AIDS cardiovascular pathogenesis.

Hyperplasia of vascular smooth muscle cells (VSMCs) occurs during HIV infection, part of a spectrum of HIV-mediated cardiovascular and microvascular pathologies. These changes are not due to direct viral infection but may involve the receptor-mediated action of viral proteins, such as the envelope protein gp120. We sought to identify gp120 receptors which might mediate the vascular smooth muscle cell hyperplasia present in HIV infection. A homology between neuropeptide Y (NPY) and the previously identified receptor-active V2-region of gp120 defined by an octapeptide sequence (Peptide T) related to VIP was noted. Since NPY is mitogenic for VSMCs we therefore determined whether gp120 shares this activity. Rat aortic VSMCs were treated for 24 h with human (h)NPY and gp120 in the presence of 0.5% serum to measure [3H]thymidine incorporation, an index of cell proliferation. NPY increased [3H]thymidine incorporation by 80% after a 24-h treatment in a bimodal fashion, with peak effects at 10(-10) M and 10(-8) M. Gp120 was an even more potent mitogen for VSMCs with peak activity occurring at 10(-12) M. Peptide T was equipotent with gp120, and slightly less efficacious, suggesting that this domain may mediate gp120 effects on VSMCs. When combined, gp120 and NPY acted to antagonize one another, lowering DNA synthesis to basal levels. The profile of pharmacologic inhibition supports a role for NPY receptors since antagonists of Y1 and Y2 subtypes substantially or completely inhibited gp120-mediated VSMC proliferation. This is the first demonstration of the proliferative effects of HIV viral protein gp120 on VSMCs. The effect appears to be mediated via gp120 sequences related to VIP, peptide T, and NPY. These ligands may be competitive inhibitors of binding or gp120 processing. Novel treatments may emerge based upon VIP and NPY receptor antagonists if further work substantiates a role for gp120 in the vascular abnormalities of AIDS.

Amino Acid Sequence↗
PubMed1998

The influence of DNA ploidy of a human tumor cell line on the frequencies of micronuclei or chromosome aberrations after irradiation.

To develop methods for assessing the intrinsic cellular radiosensitivity, it is important to evaluate the relationship between DNA ploidy of cells and frequencies of micronuclei (MN) or chromosome aberrations after irradiation. From the original human fibrosarcoma cell line HT-1080, we isolated two clones which have different chromosome ploidy: clone 5 is pseudodiploid and clone 1 is heteroploid. We examined the radiosensitivity of the two clones using a clonogenic cell survival assay and a cytokinesis-block MN assay, and by scoring chromosome aberrations using the premature chromosome condensation (PCC) method combined with a fluorescence in situ hybridization (FISH) procedure immediately and at 24 h after irradiation. The MN frequency increased according to the irradiation dose in both clones. The MN frequency of clone 1 was significantly higher than that of clone 5 regardless of whether the assay was performed immediately or 24 h after irradiation. However, when the numbers of MN were normalized by the DNA index of each clone, a significant difference in the frequency of MN was not observed. In the PCC and FISH studies, there was a linear relationship between the radiation dose and the initial breaks of chromosome 4, but the breaks of clone 1 were much more frequent than those of clone 5. Twenty-four h after irradiation, the chromosome 4 breaks of clone 1 were observed much more frequently than those of clone 5 at the same radiation dose. When the numbers of chromosome 4 breaks were normalized by the number of chromosome 4 in each clone without radiation, no such difference in the number of breaks was observed. These findings demonstrated that the DNA content or chromosome ploidy influenced the induction of the MN or chromosome aberrations in HT-1080 cells after irradiation.

Cell Survival↗
PubMed1998

Transcriptional regulation of BMP-4 in the Xenopus embryo: analysis of genomic BMP-4 and its promoter.

Recent experiments in the Xenopus embryo suggest that proper regulation of BMP-4 signaling is critical to the dorsal ventral specification of both mesoderm and ectoderm. Regulation of BMP-4 signaling is known to occur extracellularly by direct binding with chordin, noggin, and follistatin, and intracellularly through the antagonistic signal interaction with dorsalizing TGF-beta family member activin. However, tight repressional regulation of BMP transcription may also be required to sustain the dorsal and neural status of the induced cells. Here we demonstrate that the dominant negative mutant of the BMP receptor (DN-BR) or the BMP-4 antagonizers, chordin and noggin, negatively regulate BMP-4 transcription in animal cap explants. We suggest that repression of BMP-4 transcription is important in the maintenance of dorsal fate and that continuous input of BMP-4 signaling is required to sustain the expression of BMP-4 transcription in the maintenance of epidermal/ventral fate. Consistent with this postulation, we found that the promoter region of the isolated BMP-4 genomic DNA includes several consensus binding sites for transcriptional regulators functioning under BMP-4 signaling such as GATA binding and ventralizing homeobox genes. In a functional assay we found that the GATA binding and ventral homeobox proteins can positively modulate BMP-4 promoter activity. We also observed that DN-BR decreases BMP-4 promoter activity. This was likely due to a repression of the above-mentioned transcription factors. The significance of these observations to embryonic patterning is discussed.

Animals↗
PubMed1998

Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay.

Proteases are crucial for cancer metastasis, but due to lack of assays, their role in intravasation has not yet been tested. We have developed a human Alu sequence PCR-based assay to quantitate intravasated cells in an in vivo model. We demonstrated that metalloproteinases (MMPs), and most likely MMP-9, are required for intravasation by showing that marimastat, an inhibitor of MMPs, reduced intravasation by more than 90%, and that only tumor cell lines expressing MMP-9 intravasated. Cells with low surface urokinase plasminogen activator (uPA) and uPA receptor (uPAR) were also incapable of intravasation, despite the presence of high levels of MMP-9. We concluded that breaching of the vascular wall is a rate-limiting step for intravasation, and consequently for metastasis, and that cooperation between uPA/uPAR and MMP-9 is required to complete this step.

Allantois↗
PubMed1998

Choline acetyltransferase expression during a putative developmental waiting period.

The relationship between the cholinergic expression, morphological development, and target cell innervation of olivocochlear (OC) efferent neurons was investigated in the postnatal hamster. Similar to what was found in previous studies, tracer injections into the contralateral cochlea labeled cells bodies retrogradely in periolivary regions and labeled cell bodies only rarely in the lateral superior olive (LSO). Few morphological differences were found among cell bodies labeled between postnatal day 1 (P1) and P30. Tracer injections into the crossed OC bundles within the brainstem anterogradely labeled terminals below the inner hair cells of the cochlea prior to P5 and labeled terminals below outer hair cells after P5, consistent with a period of transient innervation, as hypothesized previously. Within the superior olive, choline acetyltransferase (ChAT) was expressed differentially. In periolivary regions, ChAT was expressed as early as P0. ChAT-immunoreactive cell bodies in periolivary regions were similar morphologically to retrogradely labeled OC neurons. In contrast, within the LSO, ChAT was not expressed until after P2. Consistent with a medical OC projection to the cochlea at early postnatal ages, ChAT immunoreactivity was detected below inner hair cells as early as P2 but was not detected below outer hair cells until after P6. Our results suggest that medial OC neurons not only provide transient connections to inner hair cells but also may express ChAT when they are below inner hair cells. Furthermore, these results raise the possibility that OC neurons may be capable of acetylcholine synthesis and release prior to or simultaneous with their innervation of the cochlea.

Animals↗
PubMed1998

Relation of exocytosis and Ca2+-activated K+ current during Ca2+ release from intracellular stores in individual rat chromaffin cells.

Measurement of the change in cell membrane capacitance (Cm) along with the change in IK(Ca) was used to investigate the effects of bradykinin and caffeine on the secretory process in rat adrenal chromaffin cells. In a Ca2+-free external solution, bradykinin (100 nM) caused a transient increase in Cm with a concurrent change in IK(Ca). Extracellular application of neomycin as an inhibitor of phospholipase C activity reversibly inhibited the bradykinin-activated event, implying an IP3-mediated increase of submembrane-free Ca2+. The increases in Cm and IK(Ca) caused by bradykinin were transient even with the sustained application of bradykinin. Caffeine also caused exocytosis in the Ca2+-free solution, and this was irreversibly blocked by ryanodine (1 microM) in a use-dependent manner. Caffeine-sensitive intracellular Ca2+ stores were also depleted in several seconds and recovered by an influx of external Ca2+. The sequential application of bradykinin and caffeine showed that these are likely to activate Ca2+ release from the same or distinct but rapidly equilibrating intracellular Ca2+ stores. The single cell assay of exocytosis and the increase in IK(Ca) revealed cell-to-cell variability in bradykinin- and caffeine-induced exocytotic response. Our results suggest that Ca2+ release from intracellular stores potentially increases submembrane Ca2+ concentration and modulates simultaneously two submembrane Ca2+-dependent processes, exocytosis and IK(Ca), in rat adrenal chromaffin cells.

Animals↗
PubMed1998

Visceral afferent pathways to the thalamus and olfactory tubercle: behavioral implications.

The goal of this study was to support the hypothesis that visceral signals may integrate and influence behavior by way of direct pathways from the nucleus tractus solitarii (NTS) to the olfactory tubercle and the midline/intralaminar thalamus. An anterograde tracer, biotinylated dextran amine (BDA) was iontophoresed bilaterally into the caudal NTS to optimize terminal labeling. NTS-cortical projections traversed both limbs of the diagonal bands providing heavy innervation, and terminated lightly within layer 3 of the olfactory tubercle. NTS-thalamic projections terminated within anterior and, as previously shown, posterior divisions of nucleus paraventricularis thalami and avoided the adjoining mediodorsal thalamic nucleus. Heretofore unrecognized projections were traced to the parafascicular and reuniens thalamic nuclei, and the peripeduncular nucleus. Control experiments identified the nucleus gracilis as the principal source of ascending projections to ventroposterior lateral, posterior and intralaminar thalamic nuclei. Our data corroborate the supposition that olfactory signals may integrate with visceral stimuli in the striatal compartment of olfactory tubercle. NTS projections encompass thalamic nuclei that project topographically to the prefrontal cortex, hippocampus and ventral (limbic) striatum, regions activated by visceral stimulation. Structural data support the idea that compartments of the non-discriminative thalamus may contribute to perception and behavioral responses to visceral stimulation.

Afferent Pathways↗
PubMed1998

Insights into the mechanism of catalysis by the P-C bond-cleaving enzyme phosphonoacetaldehyde hydrolase derived from gene sequence analysis and mutagenesis.

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and inorganic phosphate. In this study, the genes encoding phosphonatase in Bacillus cereus and in Salmonella typhimurium were cloned for high-level expression in Escherichia coli. The kinetic properties of the purified, recombinant phosphonatases were determined. The Schiff base mechanism known to operate in the B. cereus enzyme was verified for the S. typhimurium enzyme by phosphonoacetaldehyde-sodium borohydride-induced inactivation and by site-directed mutagenesis of the catalytic lysine 53. The protein sequence inferred from the B. cereus phosphonatase gene was determined, and this sequence was used along with that from the S. typhimurium phosphonatase gene sequence to search the primary sequence databases for possible structural homologues. We found that phosphonatase belongs to a novel family of hydrolases which appear to use a highly conserved active site aspartate residue in covalent catalysis. On the basis of this finding and the known stereochemical course of phosphonatase-catalyzed hydrolysis at phosphorus (retention), we propose a mechanism which involves Schiff base formation with lysine 53 followed by phosphoryl transfer to aspartate (at position 11 in the S. typhimurium enzyme and position 12 in the B. cereusphosphonatase) and last hydrolysis at the imine C(1) and acyl phosphate phosphorus.

Amino Acid Sequence↗
PubMed1998

Ultrabithorax regulates genes at several levels of the wing-patterning hierarchy to shape the development of the Drosophila haltere.

Arthropods and vertebrates are constructed of many serially homologous structures whose individual patterns are regulated by Hox genes. The Hox-regulated target genes and developmental pathways that determine the morphological differences between any homologous structures are not known. The differentiation of the Drosophila haltere from the wing through the action of the Ultrabithorax (Ubx) gene is a classic example of Hox regulation of serial homology, although no Ubx-regulated genes in the haltere have been identified previously. Here, we show that Ubx represses the expression of the Wingless (Wg) signaling protein and a subset of Wg- and Decapentaplegic-activated genes such as spalt-related, vestigial, Serum Response Factor, and achaete-scute, whose products regulate morphological features that differ between the wing and haltere. In addition, we found that some genes in the same developmental pathway are independently regulated by Ubx. Our results suggest that Ubx, and Hox genes in general, independently and selectively regulate genes that act at many levels of regulatory hierarchies to shape the differential development of serially homologous structures.

Animals↗
PubMed1998

Reactive astrocytes express nitric oxide synthase in the spinal cord of transgenic mice expressing a human Cu/Zn SOD mutation.

The distribution of the neuronal isoform of nitric oxide synthase (nNOS) in the spinal cord of transgenic mice expressing a mutated human copper/zinc superoxide dismutase gene was enhanced when investigated by immunocytochemistry. Immunocytochemistry showed intensely stained NOS-immunoreactive (IR) glial cells with the appearance of astrocytes in the spinal cord and brain stem of transgenic mice, but none were observed at these sites in control mice. Using antisera directed against GFAP, the specific marker for astrocyte, the glial cells were confirmed by immunocytochemistry to be astrocytes. This immunocytochemical evidence suggests that nitric oxide may mediate glutamate neurotoxicity, and this study provides the first in vivo evidence that nitric oxide may be implicated in the pathologic process of human familial amyotrophic lateral sclerosis.

Animals↗
PubMed1998

Long-term degeneration in the cochlear nerve and cochlear nucleus of the adult chinchilla following acoustic overstimulation.

Adult chinchillas were exposed once to an octave-band noise, centered at 4 kHz, and allowed to survive for 16 days or for 1, 2, 4, and 8 months. Axonal degeneration was mapped in the cochlear nucleus, using the Nauta-Rasmussen silver method, and related to hair cell damage and to loss of myelinated nerve fibers in the osseous spiral lamina of the cochlea. Axonal degeneration in the dorsal cochlear nucleus had already reached a peak by 16 days and disappeared after 1 month. Meanwhile, myelinated nerve fiber degeneration in the cochlea extended basally, followed 2 weeks to 2 months later by spread of axonal degeneration into the corresponding high-frequency region of the ventral cochlear nucleus. Axonal degeneration occurred early in the low-frequency region of the ventral cochlear nucleus, followed 2-4 weeks later by spread of myelinated fiber degeneration into more apical regions of the cochlea. New degeneration of axons in the cochlear nerve and in the ventral cochlear nucleus continued to occur for up to 8 months after stimulation. These findings imply that plastic changes in the central auditory pathways could play a role in the long-term effects of cochlear damage and acoustic overstimulation, possibly leading to a chronic neurodegenerative condition in the ear and in the brain.

Acoustic Stimulation↗
PubMed1998

The desmoplastic small round cell tumor t(11;22) translocation produces EWS/WT1 isoforms with differing oncogenic properties.

Structural alterations of the Wilms' tumor locus (WT1) at 11p13 have been implicated in the etiology of two human cancers--Wilms' tumor (WT), a pediatric renal malignancy, and Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer of the abdominal serosal lining with predilection for male adolescents. Germline mutations within the WT1 tumor suppressor gene predispose to WT and are associated with congenital malformations of the urogenital system, and somatic mutations are associated with initiation of transformation in WTs. In DSRCT, a recurrent translocation, t(11;22)(p13;q12), fuses the amino terminal domain of the EWS1 gene product to three of the four WT1 zinc fingers. Two EWS/WT1 isoforms are generated as a result of an alternative splicing event between zinc fingers III and IV, inserting or removing three amino acids (+/- KTS). We demonstrate that introduction of EWS/WT1(-KTS) into NIH3T3 cells causes their tumorigenic transformation as determined by: formation of transformed foci on a monolayer of cells; anchorage-independent growth; and tumor formation in nude mice. EWS/WT1(+KTS) showed no transforming potential in these assays. These results indicate the oncogenic effect of the t(11;22) translocation is mediated by the EWS/WT1(-KTS) isoform and that fusion of the EWS amino terminal domain to the WT1 DNA binding domain produces a chimeric product showing a gain of function.

3T3 Cells↗
PubMed1998

Topological disposition of Cys 222 in the alpha-subunit of nicotinic acetylcholine receptor analyzed by fluorescence-quenching and electron paramagnetic resonance measurements.

The structure of the nicotinic acetylcholine receptor (AChR) has been studied using a combination of fluorescence quenching and electron paramagnetic resonance (EPR) collision gradient methods. The AChR from Torpedo californica was labeled with a fluorescent probe, N-(1-pyrenyl)maleimide, specific for sulfhydryls in a hydrophobic environment, under conditions of selective labeling of Cys222 in the alpha-subunit. alphaCys222 is located in the postulated M1 transmembrane domain and predicted to be at the center of an alpha-helical secondary structure. The spatial disposition of the acetylcholine receptor-bound pyrene with respect to the membrane bilayer was assessed by fluorescence quenching measurements. Quenching of pyrene fluorescence by spin-labeled fatty acids with the doxyl group at positions C-5 and C-12 revealed that the former was more effective, suggesting that the fluorophore is located closer to the membrane-water interface than to the hydrophobic interior. Power saturation EPR spectroscopy was also used to examine the effect of molecular oxygen and water-soluble paramagnetic reagents on the saturation behavior of a nitroxide spin label, which was specifically attached to the same alphaCys222 residue. Using the gradients of these paramagnetic reagents through the membrane-solution interface, the distance for the nitroxide derivative from the membrane-solution interface was measured to be approximately 7 A from the headgroup region of the phospholipid bilayer, in agreement with fluorescence quenching results. These results suggest that the M1 transmembrane domain of the AChR probably forms an irregular structure, a beta-strand, or an alpha-helical structure that may span the membrane in a way different from a linear alpha-helix.

Animals↗
PubMed1998

Characterization and intracellular localization of the Rok1 protein involved in yeast cell division.

The ROK1 gene is essential for the cell cycle progression in Saccharomyces cerevisiae. ROK1 has been predicted to encode an ATP-dependent RNA helicase of the DEAD-box family. We have analyzed the ROK1 gene expression both at the protein and RNA levels. Polyclonal antibodies were raised against trpE::rok1 hybrid proteins and were affinity purified by using lacZ::rok1 hybrid proteins. Western blot experiments using anti-Rok1 antibodies revealed a single protein band of 64 kDa which is an expected size from the Rok1 amino acid sequence. Indirect immuno-fluorescence showed that the Rok1 protein is localized predominantly to the cytoplasm of the vegetatively growing cells. We have detected immunocross-reactive homologs of Rok1p in Candida albicans and Drosophila melanogaster.

Animals↗
PubMed1998

The DNA binding domains of the WT1 tumor suppressor gene product and chimeric EWS/WT1 oncoprotein are functionally distinct.

The t(11;22)(p13;q12) translocation associated with desmosplastic small round cell tumor results in a chimeric molecule fusing the amino terminal domain (NTD) of the EWS1 gene to three of the four carboxy-terminal zinc fingers of the WT1 tumor suppressor gene. Since the DNA binding domains of WT1 and EWS/WT1 are structurally different, we have assessed the functional consequences of the EWS/WT1 fusion. We find that the EWS/WT1 protein has a higher binding affinity for a given recognition target than the WT1 product. This is unlike other fusion products involving translocation of the NTD of EWS to DNA binding domains in which DNA binding specificity and affinity is not changed. We demonstrate that EWS/WT1 is a nuclear protein and that the NTD of EWS contains (a) nuclear localization signal(s). We also find that the integrity of a domain within the WT1 zinc fingers, responsible for mediating interaction between WT1 and the transcriptional repressor par-4, is disrupted in the EWS/WT1 fusion product. Deletion analysis of the NTD of EWS indicated that integrity of the entire domain was necessary to achieve full transactivation potential.

Animals↗
PubMed1998

Isolation and characterization of the carbon-phosphorus bond-forming enzyme phosphoenolpyruvate mutase from the mollusk Mytilus edulis.

The enzyme phosphoenolpyruvate mutase was purified to homogeneity from the mollusk Mytilus edulis. The subunit size of the native homotetramer was determined to be 34,000 Da. The steady-state kinetic constants for catalysis of the conversion of phosphonopyruvate to phosphoenolpyruvate at pH 7.5 and 25 degrees C were measured at kcat = 34 s-1, phosphonopyruvate Km = 3 microM, and Mg2+ Km = 4 microM. The enzyme displayed a broad specificity for divalent metal ion activation; Co2+, Mn2+, Zn2+, and Ni2+ are activators, whereas Ca2+ is not. Analysis of the pH dependence of the Mg2+-activated mutase-catalyzed reaction of phosphonopyruvate revealed one residue that must be protonated (apparent pKa = 8.3) and a second residue that must be unprotonated (apparent pKa = 7.7) for maximal catalytic activity.

Amino Acid Sequence↗
PubMed1998

Loss of cell adhesion in Xenopus laevis embryos mediated by the cytoplasmic domain of XLerk, an erythropoietin-producing hepatocellular ligand.

The erythropoietin-producing hepatocellular (Eph) family of ligands and receptors has been implicated in the control of axon guidance and the segmental restriction of cells during embryonic development. In this report, we show that ectopic expression of XLerk, a Xenopus homologue of the murine Lerk-2 (ephrin-B1) transmembrane ligand, causes dissociation of Xenopus embryonic blastomeres by the mid-blastula transition. Moreover, a mutant that lacks the extracellular receptor binding domain can induce this phenotype. The carboxyl-terminal 19 amino acids of the cytoplasmic domain of XLerk are necessary but not sufficient to induce cellular dissociation. Basic fibroblast growth factor, but not activin, can rescue both the loss of cell adhesion and mesoderm induction in ectodermal explants expressing XLerk. Collectively, these results show that the cytoplasmic domain of XLerk has a signaling function that is important for cell adhesion, and fibroblast growth factor signaling modulates this function.

Animals↗