Biomedical subjects

B S Kwon

Publications and source records attributed to B S Kwon.

At least 91 records · Page 5Linked to original sources

PubMed1991

A melanocyte-specific gene, Pmel 17, maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on human chromosome 12.

Melanocytes preferentially express an mRNA species, Pmel 17, whose protein product cross-reacts with anti-tyrosinase antibodies and whose expression correlates with the melanin content. We have now analyzed the deduced protein structure and mapped its chromosomal location in mouse and human. The amino acid sequence deduced from the nucleotide sequence of the Pmel 17 cDNA showed that the protein is composed of 645 amino acids with a molecular weight of 68,600. The Pmel 17 protein contains a putative leader sequence and a potential membrane anchor segment, which indicates that this may be a membrane-associated protein in melanocytes. The deduced protein contains five potential N-glycosylation sites and relatively high levels of serine and threonine. Three repeats of a 26-amino acid motif appear in the middle of the molecule. The human Pmel 17 gene, designated D12S53E, maps to chromosome 12, region 12pter-q21; and the mouse homologue, designated D12S53Eh, maps to the distal region of mouse chromosome 10, a region also known to carry the coat color locus si (silver).

Amino Acid Sequence↗

PubMed1991

Macrophage inflammatory protein (MIP)-1 beta abrogates the capacity of MIP-1 alpha to suppress myeloid progenitor cell growth.

The effects of recombinant murine macrophage inflammatory protein (MIP)-1 beta and MIP-2 on the suppressive activity of MIP-1 alpha were tested using colony formation by human and murine bone marrow burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte erythroid macrophage, megakaryocyte (CFU-GEMM), and colony-forming unit-granulocyte macrophage (CFU-GM) progenitor cells. MIP-1 beta, but not MIP-2, when added with MIP-1 alpha to cells, blocked the suppressive effects of MIP-1 alpha on both human and murine BFU-E, CFU-GEMM, and CFU-GM colony formation. Similar results were observed regardless of the early acting cytokines used: human rGM-CSF plus human rIL-3, and two recently described potent cytokines, a genetically engineered human rGM-CSF/IL-3 fusion protein and MGF, a c-kit ligand. The more potent the stimuli, the greater the suppressive activity noted. Pulse treatment of hu bone marrow cells with MIP-1 alpha at 4 degrees C for 1 h was as effective in inhibiting colony formation as continuous exposure of cells to MIP-1 alpha, and the pulsing effect with MIP-1 alpha could not be overcome by subsequent exposure of cells to MIP-1 beta. Also, pulse exposure of cells to MIP-1 beta blocked the activity of subsequently added MIP-1 alpha. For specificity, the action of a nonrelated myelosuppressive factor H-ferritin, was compared. MIP-1 alpha and H-ferritin were shown to act on similar target populations of early BFU-E, CFU-GEMM, and CFU-GM. MIP-1 beta did not block the suppressive activity of H-ferritin. Also, hemin and an inactive recombinant human H-ferritin mutein counteracted the suppressive effects of the wildtype H-ferritin molecule, but did not block the suppressive effects of MIP-1 alpha. These results show that MIP-1 beta's ability to block the action of MIP-1 alpha is specific. In addition, the results suggest that MIP-1 alpha and MIP-beta can, through rapid action, modulate early myeloid progenitor cell proliferation.

Bone Marrow↗

PubMed1991

Mapping the human CAS2 gene, the homologue of the mouse brown (b) locus, to human chromosome 9p22-pter.

Melanin biosynthesis is a multistep process with the first step being the conversion of L-tyrosine to L-Dopa catalyzed by the enzyme tyrosinase. The enzymes which catalyze the other steps of melanogenesis are not known. One murine pigmentation gene, the brown (b) locus, when mutated, leads to a brown or hypopigmented coat. The b-locus protein has been shown to display catalase activity. The human b-locus, therefore, is designated as CAS2. We used the mouse b-locus cDNA to isolate the human homologue, which in turn, was used to map the CAS2 locus to a human chromosome. The potential CAS2 protein codes for 527 amino acids containing a putative signal sequence and transmembrane domain. The CAS2 protein has primary and probably secondary structures similar to human tyrosinase. The CAS2 was mapped to human Chromosome 9 by somatic cell hybridization and, more specifically, to 9p22-pter by in situ hybridization. The assignment of CAS2 on the human Chromosome 9 extends this region of known homology on mouse Chromosome 4.

Amino Acid Sequence↗

PubMed1991

A single base insertion in the putative transmembrane domain of the tyrosinase gene as a cause for tyrosinase-negative oculocutaneous albinism.

We have determined a molecular defect to be the likely basis for inactivity of the tyrosinase (EC 1.14.18.1) from a patient with tyrosinase-negative oculocutaneous albinism. A single base (thymine) was inserted in exon 5 of the tyrosinase gene following codon 471 in the putative transmembrane coding region. This insertion caused a shift in the reading frame of 19 amino acids at the 3' end and introduced a premature termination signal that would be expected to truncate the protein by 21 amino acids at the carboxyl terminus. The albino tyrosinase was not recognized by antibodies directed to the carboxyl terminus of tyrosinase. Furthermore, as shown by gel electrophoresis of the immunoprecipitated protein, the tyrosinase was approximately 3 kDa smaller than normal. Similar immunoprecipitation data were obtained when cloned normal and mutant tyrosinases were expressed in COS-1 cells.

Albinism, Oculocutaneous↗

PubMed1991

Structure of the mouse pore-forming protein (perforin) gene: analysis of transcription initiation site, 5' flanking sequence, and alternative splicing of 5' untranslated regions.

We studied the 5' untranslated regions (UTRs) of the mouse lymphocyte pore-forming protein (PFP, perforin, and cytolysin). 5' UTRs were determined by primer extension analysis, sequencing PFP cDNA clone PFP-7, ribonuclease protection assays, and amplification of poly(A)+ RNA of cytolytic T lymphocyte using polymerase chain reaction (PCR). Two alternatively spliced 5' UTRs, designated type I and type II, of 222 and 115 bp, respectively, were found associated with PFP. Type II is identical to type I, except for being 107 bp shorter in the second exon. This deletion was generated by the use of alternative acceptor splice sites. The mouse PFP gene (Pfp) encodes three exons, is separated by two small introns, and spans a chromosomal region of approximately 7 kb. The first exon contains 79 bp of 5' UTR, the second exon contains 143 or 36 bp of 5' UTR (type I or type II UTR, respectively) plus the NH2-terminal region of the mouse PFP, and the third exon contains the rest of the COOH-terminal mouse PFP. The organization of the mouse Pfp is similar to that of the human gene. Moreover, the 5' flanking sequence of the mouse Pfp is highly homologous to that of the human Pfp. In contrast to the human sequence, the more immediate 5' flanking sequence of mouse Pfp contains two tandem "TATA" box-related elements and a GC box, but lacks a typical CAAT box-related sequence. Several other enhancer elements were found further upstream, including cAMP-, phorbol ester-, interferon-gamma-, and UV-responsive elements, and PU box-like and NFkB binding site-like elements. In addition, we found a nuclear inhibitory protein-like element, a transcriptional silencer, and a pair of purine-rich sequence motifs that were found in other T cell-specific genes, and three repeats of GGCCTG that may be a variation of a highly repetitious GCCCTG consensus sequence found in human Pfp.

Animals↗

PubMed1991

Demonstration of YAC target cell lysis by murine granulated metrial gland cells.

Granulated metrial gland (GMG) cells are a consistently observed but poorly understood feature of the murine uterus during successful pregnancy. From morphological studies and antibody phenotyping it has been suggested that GMG cells may be members of the natural killer (NK) cell lineage. However, lysis of murine NK cell targets by GMG cells has not been observed although lysis of freshly dissociated trophoblast cells by GMG cells has been recorded using timelapse video. We failed to demonstrate significant interactions between migrating GMG cells, collected from explant cultures under previously reported cultures conditions, and YAC target cells. However, YAC cell lysis did occur if hrIL-2 was present throughout the periods of explant culture and lysis assay. Furthermore, lysis was enhanced if the pregnant females were treated with the interferon inducer poly I.C. 24 hr before metrial gland collection. GMG cells expressed perforin and serine protease mRNA. Consistent with the lysis experiments, expression of these genes was enhanced when the cells were incubated with hrIL-2. Our data provide further support for a relationship between GMG cells and NK cells, but do not establish a relationship of identity since hrIL-2, a growth factor sufficient for the culture of NK cells, cannot support growth or prolong survival of GMG cells.

Animals↗

PubMed1991

Rapid loss of perforin and serine protease RNA in cytotoxic lymphocytes exposed to sensitive targets.

We have previously reported that cytotoxic lymphocytes, when exposed to sensitive target cells, temporarily lose their lytic potential. The mechanism leading to this loss of lytic activity is still unknown but it is reversible and the lytic potency can be recovered when the effector cells are incubated with interleukin-2 (IL-2) for 12-14 hr. In this study, we have investigated the regulation of RNA coding for perforin and for two serine proteases, HSP1 and HSP2, in cytotoxic lymphocytes exposed to sensitive targets. Perforin and the two serine proteases are contained in granules of major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxic lymphocytes, but their exact role in the lytic mechanism is still debated. Here we used four different human cytotoxic lymphocytes (CTL) as effector cells: an MHC-restricted CTL (SG-CTL), a non-MHC-restricted CTL (IE6), a natural killer (NK)-like cell line (3.3) and lymphokine-activated killer (LAK) cells. In all effector cells we observed a rapid loss of perforin and of serine protease RNAs within 5 min following the addition of sensitive targets. The effector cells recovered the RNA messages as early as 30 min, although the kinetics of recovery was faster with CTL than with NK-like or LAK effector cells. When we exposed the effector cells to resistant targets we did not detect any loss of perforin or serine protease RNAs. Incubation of the effector cells with cycloheximide, prior to the addition of sensitive targets, did not block message loss, indicating that de novo protein synthesis was not required in this process. Cycloheximide treatment, however, inhibited the recovery of perforin and serine protease RNAs. Taken together, our results indicate that the target-mediated loss of lytic activity in cytotoxic lymphocytes may be a consequence of the down-regulation of perforin or of serine protease transcripts, or both.

Blotting, Northern↗

PubMed1990

Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice.

Albinism, due to a lack of melanin pigment, is one of the oldest known mutations in mice. Tyrosinase (monophenol oxygenase, EC 1.14.18.1) is the first enzyme in the pathway for melanin synthesis, and the gene encoding this enzyme has been mapped to the mouse albino (c) locus. We have used mouse tyrosinase cDNA clones and genomic sequencing to study the albino mutation in laboratory mice. Within the tyrosinase gene coding sequences, a G to C transversion at nucleotide 308, causing a cysteine to serine mutation at amino acid 103, is sufficient to abrogate pigment production in transgenic mice. This same base pair change is fully conserved in classical albino strains of laboratory mice. These results indicate that a conserved mutation in the tyrosinase coding sequences is responsible for the classical albino mutation in laboratory mice, and also that most albino laboratory mouse strains have been derived from a common ancestor.

Albinism↗

PubMed1990

Enhancing and suppressing effects of recombinant murine macrophage inflammatory proteins on colony formation in vitro by bone marrow myeloid progenitor cells.

Purified recombinant (r) macrophage inflammatory proteins (MIPs) 1 alpha, 1 beta, and 2 were assessed for effects on murine (mu) and human (hu) marrow colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) colonies. Recombinant MIP-1 alpha, -1 beta, and -2 enhanced muCFU-GM colonies above that stimulated with 10 to 100 U natural mu macrophage-colony-stimulating factor (M-CSF) or rmuGM-CSF, with enhancement seen on huCFU-GM colony formation stimulated with suboptimal rhuM-CSF or rhuGM-CSF; effects were neutralized by respective MIP-specific antibodies. Macrophage inflammatory proteins had no effects on mu or huBFU-E colonies stimulated with erythropoietin (Epo). However, natural MIP-1 and rMIP-1 alpha, but not rMIP-1 beta or -2, suppressed muCFU-GM stimulated with pokeweed mitogen spleen-conditioned medium (PWMSCM), huCFU-GM stimulated with optimal rhuGM-CSF plus rhu interleukin-3 (IL-3), muBFU-E and multipotential progenitors (CFU-GEMM) stimulated with Epo plus PWMSCM, and huBFU-E and CFU-GEMM stimulated with Epo plus rhuIL-3 or rhuGM-CSF. The suppressive effects of natural MIP-1 and rMIP-1 alpha were also apparent on a population of BFU-E, CFU-GEMM, and CFU-GM present in cell-sorted fractions of human bone marrow (CD34 HLA-DR+) highly enriched for progenitors with cloning efficiencies of 42% to 75%. These results, along with our previous studies, suggest that MIP-1 alpha, -1 beta, and -2 may have direct myelopoietic enhancing activity for mature progenitors, while MIP-1 alpha may have direct suppressing activity for more immature progenitors.

Animals↗

PubMed1990

Induction of perforin and serine esterases in a murine cytotoxic T lymphocyte clone.

The expression of perforin and serine esterase (SE) activities and genes was examined in a murine cytotoxic T lymphocyte line (R8i) that does not require exogenous IL-2 for proliferation. Although perforin (hemolytic) activity was detected in unstimulated R8i, it was induced 2- to 14-fold in the presence of IL-2, IL-3, IL-4, and IL-6, and to a lesser degree (less than 4-fold) by TNF and IFN-gamma. A transient induction was also observed at the mRNA level. Peak perforin protein and mRNA levels were reached within 24 h and started to decline 48 h after stimulation. A trypsinlike SE activity which cleaves the chromogenic substrate N, alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester was also induced 2- to 4-fold in the presence of the various IL tested. At the mRNA level, the message for SE SE1/granzyme A/Hanukah factor was absent from R8i whereas SE2/granzyme B/CTLA-1 increased by greater than 3-fold in the presence of IL-2, IL-3, IL-4, and IL-6 and occurred with the same kinetics and pattern as perforin. The induction response occurred without any enhancement of cell proliferation, suggesting that the cytokines tested may provide a direct differentiation signal to CTL. The induction response was abrogated effectively by inhibitors of protein (cycloheximide or emetine) and RNA (actinomycin D) syntheses. These findings suggest that the various IL may provide both a growth signal and a differentiation signal to CTL, resulting in the direct activation of perforin and SE genes.

Animals↗

PubMed1990

Genomic organization of the mouse pore-forming protein (perforin) gene and localization to chromosome 10. Similarities to and differences from C9.

Genomic clones encompassing the entire coding region of the mouse lymphocyte pore-forming protein gene (Pfp) have been isolated and used to determine its intron-exon organization. In contrast to C9, Pfp has a simple structure, consisting of only three exons (two of which encode polypeptide), a large 5' intron, and a single, smaller intron that is situated approximately one-third of the way through the protein-coding portions of the gene. The regions encoding the homologous domains of PFP and C9 are encoded on exons 7, 8, 9, and 10 of C9, but form only approximately half of the open reading frame of exon III in Pfp. Although encoding polypeptides with related functions, the two genes possess such sharply contrasting structures as to suggest that their analogous regions may have risen independently, by a process of convergent evolution. Using a panel of somatic cell hybrid cell lines, Pfp has been mapped to chromosome 10.

Amino Acid Sequence↗

PubMed1990

Expression of mRNAs for pore-forming protein and two serine esterases in murine primary and cloned effector lymphocytes.

The cDNAs encoding several proteins present in the granules of cytolytic effector lymphocytes have now been cloned. These include the cytolytic pore-forming protein (PFP) or perforin, and at least six serine esterases (SE), also called granzymes. The cDNA probes for PFP, SE-1, and SE-2 are used here to study the expression of these proteins in murine primary effector lymphocytes. Among the stimuli effective in inducing the expression of PFP, SE-1, and SE-2 were recombinant interleukin-2, the lectin concanavalin A in the presence of phorbol esters, and allogeneic cells in mixed lymphocyte cultures. Some correlation was seen between the levels of PFP and SE mRNAs and cytotoxicity measured in a standard 51Cr release assay. We also examined a panel of 13 cloned cytotoxic T lymphocyte (CTL) lines and found that mRNAs for PFP and SE-2 were expressed in all CTL lines, including some that were previously considered not to produce PFP. Twelve of the 13 CTL lines also proved to possess the mRNA for SE-1. One thymoma cell line, TIMI.4, did not express mRNA for PFP, although it expressed mRNA for SE-1 and SE-2.

Animals↗

PubMed1990

Genetic and molecular analysis of chlorambucil-induced germ-line mutations in the mouse.

Eighteen variants recovered from specific locus mutation rate experiments involving the mutagen chlorambucil were subjected to several genetic and molecular analyses. Most mutations were found to be homozygous lethal. Because lethality is often presumptive evidence for multilocus-deletion events, 10 mutations were analyzed by Southern blot analysis with probes at, or closely linked to, several of the specific locus test markers, namely, albino (c), brown (b), and dilute (d). All eight mutations (two c; three b; two d; and one dilute-short ear [Df(d se)]) that arose in post-spermatogonial germ cells were deleted for DNA sequences. No evidence for deletion of two d-se region probes was obtained for the remaining two d mutations that arose in stem-cell spermatogonia. Six of the primary mutants also produced low litter sizes ("semisterility"). Karyotypic analysis has, to date, confirmed the presence of reciprocal translocations in four of the six. The high frequency of deletions and translocations among the mutations induced in post-spermatogonial stages by chlorambucil, combined with its overall high efficiency in inducing mutations in these stages, should make chlorambucil mutagenesis useful for generating experimentally valuable germ-line deletions throughout the mouse genome.

Animals↗

PubMed1989

Molecular basis of mouse Himalayan mutation.

Many different coat-colors result from the c-locus mutation in the mouse. One of these interesting mutants is a Himalayan, which produces temperature sensitive tyrosinase, and the basis of this sensitivity remains unknown. We cultured Himalayan mouse melanocytes from the skin and constructed a cDNA library; then, we isolated the Himalayan tyrosinase cDNAs and determined the nucleotide sequence. The tyrosinase gene in the Himalayan mouse contains an A----G change at nucleotide 1259 that alters a histidine residue to an arginine residue at amino acid 420. This histidine residue and the surrounding amino acids are conserved in their evolution from mouse to human. Interestingly, the residue with its surrounding eight amino acids are aligned between mouse b-protein and human tyrosinase. These results indicate the possibility that the altered residue at amino acid 420 of mouse tyrosinase may be important in stabilization of the tyrosinase molecule, or in interaction with other molecules, such as tyrosinase inhibitors.

Albinism↗

PubMed1989

The structure of the mouse lymphocyte pore-forming protein perforin.

Purified murine lymphocyte pore-forming protein (PFP or perforin) was partially sequenced. Oligonucleotides synthesized on the basis of this sequence information were used to screen a murine cytotoxic T lymphocyte (CTL) cDNA library. Seven clones were obtained, two of which were sequenced, providing full-length sequence information on PFP. Murine PFP (534 a.a.) is 68% identical to human PFP. Hydropathic analysis revealed a predominantly hydrophilic protein with some hydrophobic domains, including a region (a.a. 191-251) that could contain putative membrane-spanning domains. PFP is approx. 20% identical to human C7, C8 and C9 within a region encompassing 270 a.a., confirming previous immunological cross-reactivity studies. Northern blot analysis showed that expression of PFP but not of a serine esterase transcript is enhanced in a CTL line by antigen receptor-stimulation. Southern blot analysis of mouse genomic DNA indicated that PFP is encoded as a single-copy gene with the coding region contained within 10 kilobases of genomic DNA.

Amino Acid Sequence↗

PubMed1989

Establishment and partial characterization of SV40 virus-immortalized hepatocyte lines of normal and lethal mutant mice carrying a deletion on chromosome 7.

Deletions in chromosome 7 of the mouse have been shown to cause failure of expression of various hepatocyte-specific genes in newborn deletion homozygotes, including the gene encoding tyrosine amino transferase (TAT) (EC 2.6.1.5) (Gluecksohn-Waelsch, 1979). Primary liver cultures of newborn albino deletion mutant mice (c14CoS/c14CoS) and of phenotypically normal mice (c14CoS/cch or cch/cch) were infected with SV40 virus and multiplying hepatocytes selected in arginine-deficient medium containing epidermal growth factor (EGF), insulin, and hydrocortisone (HC). Resulting normal (NMH-ch) and mutant (NMH-m14) hepatocyte lines expressing integrated viral transforming sequences did not senesce, they multiplied autonomously of EGF in medium with insulin plus HC, and they retained hepatocyte-specific functions. Both lines synthesized arginine and contained albumin and alpha-fetoprotein (AFP) mRNAs. TAT-specific mRNA was detected in normal but not in mutant hepatocyte lines. A fragment of the mouse tyrosinase gene, known to map at the albino locus (c) within the region deleted in the c14CoS mutant, hybridized with a 2.5 kb EcoRI fragment of normal NMH-ch DNA, whereas this fragment was undetectable in mutant NMH-m14 DNA. These immortalized hepatocyte lines reflect important properties of normal and mutant liver tissues from which they were derived. The deletion mutant mouse cell lines may be useful for complementation studies involving sequences corresponding to the deletions that encode regulatory gene(s) involved in the control of inducible expression of certain hepatocyte-specific genes such as TAT.

Animals↗

PubMed1989

Expression characteristics of two potential T cell mediator genes.

T lymphocyte subset-specific cDNA clones were recently isolated by a modified differential screening procedure. The expression patterns of two of these cDNAs, designated as 4-1BB and L2G25B, were studied in greater detail. Nucleotide sequence comparison revealed that 4-1BB was not previously recognized. Although the L2G25B sequence had been recognized recently, the function of the encoded molecule has yet to be well studied. The transcripts of the two cDNAs were inducible by concanavalin A in mouse spleen cells, cloned helper T cells (L2), cloned cytolytic T cells (L3), and cytolytic T cell hybridomas. They were also inducible with stimulation through antigen receptor (TCR), with immobilized anti-TCR antibody in cloned T cells L2, dB45, and L3. Concanavalin A inducibility was inhibited by cyclosporin A. They were not inducible by IL-2 stimulation. The expression patterns of these transcripts were similar to those of IFN-gamma, except that the level of transcripts of the two cDNAs was at least fivefold lower than that of IFN-gamma, and the peak level of expression occurred earlier. These data suggest that L2G25B and 4-1BB may represent new T cell mediators.

Animals↗

PubMed1989

Identification and production of monoclonal antibodies against a discriminating protein molecule between B and D variants of encephalomyocarditis virus.

The protein profiles of two EMC virus variants B and D in the infected L929 fibroblast lysates were examined using one- and two-dimensional gel electrophoresis. Both variants yielded a protein molecule of similar molecular weight but differing in its isoelectric point (pI). The B variant lysate yielded a molecule with pI congruent to 7.5 whereas the same molecule from the D lysate focused at pI congruent to 5.2. A monoclonal antibody (MCA) produced against pI congruent to 5.2 fraction of the D variant successfully detected viral antigens in the D variant infected fibroblasts with only background cross reactivity with the B variant infected cells. This MCA also detected D viral antigen(s) in organ sections obtained from D but not from B variant infected mice. This study therefore suggests a clear shift in the pI of a 23 kD protein molecule serving as a possible discriminating antigen between the B and D variants of EMC virus.

Animals↗