Biomedical subjects

J Chun

Publications and source records attributed to J Chun.

At least 91 records · Page 5Linked to original sources

PubMed1999

Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus.

Vibrio cholerae identification based on molecular sequence data has been hampered by a lack of sequence variation from the closely related Vibrio mimicus. The two species share many genes coding for proteins, such as ctxAB, and show almost identical 16S DNA coding for rRNA (rDNA) sequences. Primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rDNAs were used to amplify the 16S-23S rRNA intergenic spacer regions of V. cholerae and V. mimicus. Two major (ca. 580 and 500 bp) and one minor (ca. 750 bp) amplicons were consistently generated for both species, and their sequences were determined. The largest fragment contains three tRNA genes (tDNAs) coding for tRNAGlu, tRNALys, and tRNAVal, which has not previously been found in bacteria examined to date. The 580-bp amplicon contained tDNAIle and tDNAAla, whereas the 500-bp fragment had single tDNA coding either tRNAGlu or tRNAAla. Little variation, i.e., 0 to 0.4%, was found among V. cholerae O1 classical, O1 El Tor, and O139 epidemic strains. Slightly more variation was found against the non-O1/non-O139 serotypes (ca. 1% difference) and V. mimicus (2 to 3% difference). A pair of oligonucleotide primers were designed, based on the region differentiating all of V. cholerae strains from V. mimicus. The PCR system developed was subsequently evaluated by using representatives of V. cholerae from environmental and clinical sources, and of other taxa, including V. mimicus. This study provides the first molecular tool for identifying the species V. cholerae.

Base Sequence↗

PubMed1999

Lysophospholipid receptors: implications for neural signaling.

Lysophospholipids (LPs) such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) represent quantitatively minor phospholipid species that nonetheless are capable of acting as extracellular signals. As an organ system dominated by lipids, the nervous system would seem a likely benefactor of this form of intercellular signaling. A major difficulty in determining the neurobiological importance of these lipids, however, has been a lack of cloned receptors. The unavailability, indeed, uncertain existence, of these receptors has been particularly problematic because of the absence of specific, competitive antagonists to block function. Further, these lipids have detergent-like chemical structures, raising the explanation that any observed effects of exogenously applied lysophospholipids could be due to nonspecific membrane perturbations. During studies of G-protein coupled receptor (GPCR) genes involved with cerebral cortical neurogenesis, the first lysophospholipid receptor gene (lpA1/vzg-1) was isolated (Hecht et al., J. Cell Biol., 135, 1071, 1996), implicating receptor-mediated lysophospholipid signaling as potentially important components of nervous system development and function. Expression studies indicated roles in neurogenesis, cortical development, and effects on glia, particularly oligodendrocyte and Schwann cell development. Reviewed here are the molecular biology of LP receptors, relevant aspects of intracellular signaling, and their possible roles in the nervous system.

Animals↗

PubMed1998

Lysophosphatidic acid receptor gene vzg-1/lpA1/edg-2 is expressed by mature oligodendrocytes during myelination in the postnatal murine brain.

The growth-factor-like phospholipid lysophosphatidic acid (LPA) mediates a wide variety of biological functions. We recently reported the cloning of the first G-protein-coupled receptor for LPA, called ventricular zone gene-1 (vzg-1/lpA1/edg-2) because its embryonic central nervous system (CNS) expression is restricted to the neocortical ventricular zone (Hecht et al. [1996] J. Cell Biol. 135:1071-1083). Vzg-1 neural expression diminishes at the end of the cortical neurogenetic period, just before birth. Here, we have investigated the subsequent reappearance of vzg-1 expression in the postnatal murine brain, by using in situ hybridization and northern blot analyses. Vzg-1 expression was undetectable by in situ hybridization at birth, but reappeared in the hindbrain during the 1st postnatal week. Subsequently, expression expanded from caudal to rostral, with peak expression observed around postnatal day 18. At all postnatal ages, vzg-1 expression was concentrated in and around developing white matter tracts, and its expansion, peak, and subsequent downregulation closely paralleled the progress of myelination. Double-label in situ hybridization studies demonstrated that vzg-1-expressing cells co-expressed mRNA encoding proteolipid protein (PLP), a mature oligodendrocyte marker, but not glial fibrillary acidic protein (GFAP), an astrocyte marker. Consistent with this, vzg-1 mRNA expression was reduced by 40% in the brains of jimpy mice, which exhibit aberrant oligodendrocyte differentiation and cell death. Together with our characterization of vzg-1 during cortical neurogenesis, these data suggest distinct pre- and postnatal roles for LPA in the development of neurons and oligodendrocytes and implicate lysophospholipid signaling as a potential regulator of myelination.

Animals↗

PubMed1998

Complete cDNA sequence, genomic structure, and chromosomal localization of the LPA receptor gene, lpA1/vzg-1/Gpcr26.

The lpA1/Gpcr26 locus encodes the first cloned and identified G-protein-coupled receptor that specifically interacts with lysophosphatidic acid. A murine full-length cDNA of size consistent with that seen on Northern blots (3.7 kb) was determined using 3' rapid amplification of cDNA ends. Analysis of genomic clones revealed that the gene is divided into five exons, with one intron inserted in the coding region for transmembrane domain VI and one exon encoding the divergent 5' sequence in another published cDNA clone variant (orphan receptor mrec1.3). This structure differs from the intronless coding region for a homologous receptor, Edg1, but is identical to another more similar orphan receptor (lpA2) that has been deposited with GenBank. Using backcross analysis, both exons 1 and 4 mapped to a proximal region of murine Chromosome 4 indistinguishable from the vacillans gene. Exon 4 also mapped to a second locus on proximal Chromosome 6 in Mus spretus, and this partial duplication was confirmed by Southern blot. The genomic structure indicates a distinct, divergent evolutionary lineage for the vzg-1/lpA1 subfamily of receptors compared to those of homologous orphan receptor genes.

Amino Acid Sequence↗

PubMed1998

Programmed cell death is a universal feature of embryonic and postnatal neuroproliferative regions throughout the central nervous system.

During central nervous system (CNS) development, programmed cell death (PCD) has been viewed traditionally as a fate reserved for differentiating neurons that are in the process of making synaptic connections. Recent studies in the embryonic cerebral cortex (Blaschke et al. [1996] Development 122:1165-1174), however, have shown that many neuroblasts in the proliferative ventricular zone undergo PCD as well and that this likely represents a novel form distinct from that found in regions of postmitotic neurons. To determine the commonality of this form of PCD throughout the CNS, the prevalence of dying cells identified by in situ end labeling plus (ISEL +; Blaschke et al. [1996]) was determined within populations of proliferating neuroblasts that were identified by rapid bromodeoxyuridine incorporation. Based on this approach, dying cells were observed to be a common feature of all proliferative neuroblast populations examined. In addition, when ISEL+ was combined with in situ hybridization for postmitotic neural gene-1 (png-1; Weiner and Chun [1997] J. Comp. Neurol. 381:130-142), which identifies newly postmitotic neurons, a positive correlation was found between the start of differentiation and the onset of PCD. These data indicate that PCD in neuroblast proliferative zones is a universal feature of nervous system development. Moreover, cell death represents a prominent cell fate that may be linked to mechanisms of differentiation.

Aging↗

PubMed1998

A single receptor encoded by vzg-1/lpA1/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid.

Extracellular lysophosphatidic acid (LPA) produces diverse cellular responses in many cell types. Recent reports of several molecularly distinct G protein-coupled receptors have raised the possibility that the responses to LPA stimulation could be mediated by the combination of several uni-functional receptors. To address this issue, we analyzed one receptor encoded by ventricular zone gene-1 (vzg-1) (also referred to as lpA1/edg-2) by using heterologous expression in a neuronal and nonneuronal cell line. VZG-1 expression was necessary and sufficient in mediating multiple effects of LPA: [3H]-LPA binding, G protein activation, stress fiber formation, neurite retraction, serum response element activation, and increased DNA synthesis. These results demonstrate that a single receptor, encoded by vzg-1, can activate multiple LPA-dependent responses in cells from distinct tissue lineages.

Animals↗

PubMed1998

Phylogenetic analysis of the nef gene reveals a distinctive monophyletic clade in Korean HIV-1 cases.

To study the genetic variation of the HIV-1 strains prevalent in South Korea, we analyzed the nef sequences derived from 46 HIV-1-positive individuals living in various geographic regions in Korea. Phylogenetic analysis revealed four subtypes of HIV-1: A (3 patients), B (41 patients), D (1 patient), and a type that could not be clearly classified to any known subtype (1 patient). Thirty-five of the 41 Korean subtype B isolates formed a distinct monophyletic clade that is not related to any of the international sequences from the Los Alamos Database or GenBank as of June 1997. Indeed, the presence of unique conserved sequences was identified among the Korean isolates in this Korean subtype B group. The variations in the nucleotide sequences of a majority (32 of 35) subtype B samples within the Korean clade were 1.9% to 8.8%, and amino acid sequences varied from 3.9% to 15.5%. These results suggest that HIV-1 strains currently present in South Korea might have originated from a few sources or might be developing through a certain selective pressure. This is the first report on the molecular nature of the HIV-1 infection present in South Korea.

Adolescent↗

PubMed1998

Inactivation of cytochrome P450 2E1 by tert-butylisothiocyanate.

Several naturally occurring and synthethic isothiocyanates were evaluated for their ability to inactivate the major ethanol-inducible hepatic cytochrome P450 2E1. Of the compounds tested, tert-butylisothiocyanate (tBITC) was found to be the most selective inactivator of the 2E1 p-nitrophenol hydroxylation activity. tBITC was more specific for inactivating P450 2E1 activity than for rat P450 1A1, 1A2, 3A2, and 2B1, or the human cytochromes P450 3A4 and 2B6. The kinetics of inactivation of P450 2E1 by tBITC were characterized. P450 2E1, either in rat liver microsomes or in a purified reconstituted system containing the bacterially expressed rabbit cytochrome, was inactivated by tBITC in a mechanism-based manner. The loss of activity followed pseudo-first-order kinetics and was NADPH- and tBITC-dependent. The maximal rates for inactivation of P450 2E1 in microsomes or for the purified P450 2E1 at 30 degrees C were 0.72 and 0.27 min-1 and the apparent KI values were 11 and 7.6 microM, respectively. When cytochrome b5 was co-reconstituted with P450 2E1, the apparent KI for P450 2E1 inactivation by tBITC was similar to that seen in microsomes (14 microM). P450 2E1 T303A was also inactivated by tBITC with kinetic constants similar to that of the wild type enzyme. Co-incubations with an alternate substrate protected P450 2E1 from inactivation by tBITC. The extent of P450 2E1 inactivation by tBITC resulted in a comparable loss of the ability of the enzyme to form a reduced CO complex.

Animals↗

PubMed1998

Onset of apoptotic DNA fragmentation can precede cell elimination by days in the small intestinal villus.

DNA fragmentation is a hallmark of apoptosis, and has been viewed as a short-lived process (<hour) that immediately precedes cell elimination. However, use of sensitive techniques like in situ end-labelling plus (ISEL+) has indicated that the period between the initiation of detectable fragmentation and cell elimination could be longer (days). To address this possibility, we used a model system of cell death and replacement, the murine small intestinal villus. Pulses of 5-bromo-2'-deoxyuridine were used to follow quantitatively cohorts of cells from their generation in the crypts to their elimination at the villus tips, resulting in a temporal 'yard-stick' where position on the villus indicated time before cell elimination; these data allowed a mathematical description of cell movement and clearance. Combining these data with ISEL+ quantitation, enterocytes were found to commence and maintain DNA fragmentation 2 - 3 days before elimination, a phenomenon that likely has relevance to studies on apoptosis also in other systems.

Animals↗

PubMed1998

Nocardia crassostreae sp. nov., the causal agent of nocardiosis in Pacific oysters.

Seven strains of bacteria were isolated from Pacific oysters, Crassostrea gigas, with a focal or systemic disease. The strains were aerobic, Gram-positive, acid-fast, produced a mycelium which fragmented into irregular rod-like elements, had a peptidoglycan containing meso-diaminopimelic acid, arabinose and galactose as major sugars, mycolic acids with 46-58 carbon atoms and G + C-rich DNA. All of these properties are consistent with the classification of the organisms in the genus Nocardia. A partial sequence of the 16S rRNA gene of isolate NB4H was determined following isolation and cloning of the PCR-amplified gene. The sequence was aligned with those of representative mycolic-acid-containing taxa and a phylogenetic tree was generated using the neighbour-joining method. It was evident from the phylogenetic tree that the three strains tested, RB1, OB3P and NB4H, were identical and belonged to the Nocardia otitidiscaviarum rRNA sub-group. The biochemical, chemical, morphological and physiological properties of the isolates were also essentially identical and served to distinguish them from representative nocardiae. It is, therefore, proposed that the strains isolated from the diseased Pacific oysters be assigned to a new species, Nocardia crassostreae. The type strain is NB4H (= ATCC 700418).

Animals↗

PubMed1998

Nocardia flavorosea sp. nov.

An actinomycete strain, 'Nocardai flavorosea' JCM 3332, was found to have properties consistent with its classification in the genus Nocardia. An almost complete gene sequence of the 16S rDNA of the strain was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for nocardiae and phylogenetic trees were inferred using four tree-making algorithms. The organisms consistently formed a distinct clade with the type strain of Nocardia carnea. However, DNA relatedness experiments showed that the strain and N. carnea DSM 43397T belonged to two distinct genomic species. The organism was also distinguished from representative of all of the validly described species of Nocardia using a combination of phenotypic properties. These genotypic and phenotypic data show that the strain merits recognition as a new species of the genus Nocardia. The name proposed for the new species of is Nocardia flavorosea sp. nov. The type strain is JCM 3332T.

Base Sequence↗

PubMed1997

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain.

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

A Kinase Anchor Proteins↗

PubMed1997

Png-1, a nervous system-specific zinc finger gene, identifies regions containing postmitotic neurons during mammalian embryonic development.

To identify genes associated with early postmitotic cortical neurons, gene fragments were examined for expression in postmitotic, but not proliferative, zones of the embryonic murine cortex. Through this approach, a novel member of the zinc finger gene family, containing 6 C2HC fingers, was isolated and named postmitotic neural gene-1, or png-1. Embryonic png-1 expression was: 1) nervous system-specific; 2) restricted to zones containing postmitotic neurons; and 3) detected in all developing neural structures examined. In the cortex, png-1 expression was first observed on embryonic day 11, correlating temporally and spatially with the known generation of the first cortical neurons. Gradients of png-1 expression throughout the embryonic central nervous system further correlated temporally and spatially with known gradients of neuron production. With development, expression remained restricted to postmitotic zones, including those containing newly-postmitotic neurons. Png-1 was also detected within two days of neural retinoic acid induction in P19 cells, and expression increased with further neuronal differentiation. These data implicate png-1 as one of the earliest molecular markers for postmitotic neuronal regions and suggest a function as a panneural transcription factor associated with neuronal differentiation.

Amino Acid Sequence↗

PubMed1997

Maternally derived immunoglobulin light chain is present in the fetal mammalian CNS.

Toward identifying molecules involved in cell-cell interactions during cerebral cortical development, we have investigated the nature of immunoglobulin-like immunoreactivity (Ig-ir) in the murine cortex. Immunohistochemistry using several antisera recognizing IgG revealed intense immunoreactivity in the subplate and marginal zone of embryonic day 16 cortex, as well as in the hindbrain and spinal cord, particularly within ventral fiber tracts. In three independently derived mouse strains lacking the recombination activating genes RAG-1 or RAG-2, which are essential for Ig production, Ig-ir was absent from the fetal CNS. Western blot analyses of wild-type brains from embryonic day 12 through birth identified a 25 kDa protein that co-migrated with Ig light chain and was absent from RAG-1 or RAG-2 -/- brain samples. This result could be replicated with an antiserum specific for Ig kappa light chain, but not with antisera specific for Ig gamma or mu heavy chain. No Ig-ir was detected in the brains of RAG-1 +/- embryos carried by a -/- female, suggesting a maternal source of the immunoreactive molecule. In confirmation of this, Ig-ir could be partially reproduced by intraperitoneal injection of pregnant RAG-1 -/- females with normal mouse serum. We conclude that maternally derived Ig light chain is present in the fetal murine CNS. This may represent a novel maternal contribution to fetal neural development and implicates Ig molecules as potential mediators of cortical developmental events.

Animals↗

PubMed1997

Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits.

Compartmentalization of cAMP-dependent protein kinase is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). All of the anchoring proteins identified previously target the kinase by tethering the type II regulatory subunit. Here we report the cloning and characterization of a novel anchoring protein, D-AKAP1, that interacts with the N terminus of both type I and type II regulatory subunits. A novel cDNA encoding a 125-amino acid fragment of D-AKAP1 was isolated from a two-hybrid screen and shown to interact specifically with the type I regulatory subunit. Although a single message of 3.8 kilobase pairs was detected for D-AKAP1 in all embryonic stages and in most adult tissues, cDNA cloning revealed the possibility of at least four splice variants. All four isoforms contain a core of 526 amino acids, which includes the R binding fragment, and may be expressed in a tissue-specific manner. This core sequence was homologous to S-AKAP84, including a mitochondrial signal sequence near the amino terminus (Lin, R. Y., Moss, S. B., and Rubin, C. S. (1995) J. Biol. Chem. 270, 27804-27811). D-AKAP1 and the type I regulatory subunit appeared to have overlapping expression patterns in muscle and olfactory epithelium by in situ hybridization. These results raise a novel possibility that the type I regulatory subunit may be anchored via anchoring proteins.

A Kinase Anchor Proteins↗

PubMed1997

Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of beta 1 integrin.

HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX). The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations. There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) further increases the relative content of F-actin and the rate and extent to which the cells spread. Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content. The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation. In addition, PKC epsilon, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin. Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed by TPA treatment. This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading. PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plasma membrane domain exposed to the culture medium and with an intracellular membrane fraction. In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of beta 1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole. The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading. One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading.

Actins↗

PubMed1997

Long-term identification of streptomycetes using pyrolysis mass spectrometry and artificial neural networks.

Sixteen reference strains and thirteen fresh isolates of three putatively novel Streptomyces species were examined six times over twenty months using pyrolysis mass spectrometry to examine the long-term reproducibility of the procedure. The reference strains and new isolates were correctly identified using information in each of the datasets and operational fingerprinting, but direct statistical comparison of the datasets for strain identification was unsuccessful between datasets. Artificial neural networks were also used to identify the strains held in the datasets. Neural networks trained with pyrolysis mass spectra from a single dataset were found to successfully identify the reference strains and fresh isolates in that dataset but were unable to identify many of the strains in the other datasets. However, a neural network trained on representative pyrolysis mass spectra from each of the first three datasets were found to identify the reference strains and fresh isolates in those three datasets and in the three subsequent datasets. Therefore, artificial neural network analysis of pyrolysis mass spectrometric data can provide a rapid, cost-effective, accurate and long-term reproducible way of identifying and typing microorganisms.

Humans↗

PubMed1997

Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends.

Apoptosis is a form of programmed cell death (PCD) characterized by morphological changes and stereotypical DNA degradation described as a nucleosomal ;ladder'. However, nucleosomal ladders have only been clearly demonstrated in vertebrate tissues when large numbers of cells die in synchrony. Their absence may be explained by asynchronous death under physiological conditions, or by distinct molecular mechanisms. In this study, nucleosomal ladders were revealed by a ligation-mediated polymerase chain reaction (LMPCR), that amplifies DNA fragments with blunt, 5' phosphorylated ends. Numerous tissues from different organisms were examined which demonstrated that nucleosomal ladders (a) accompany physiological cell death in mammalian tissues where previously DNA fragmentation has not been detected; (b) are produced during invertebrate cell death; (c) are invariably generated via the production of blunt, 5' phosphorylated double strand breaks. These results suggest that PCD in multicellular organisms consistently involves apoptotic mechanisms and that the endonuclease activity is evolutionarily conserved.

Journal Article↗