Hydrogen peroxide: an effective treatment for ballast water.
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Animals↗
Biomedical subjects
P L James
Publications and source records attributed to P L James.
ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK)
Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkin's lymphoma, but further insights into its normal structure and function are lacking. Here, we characterize a full-length normal human ALK cDNA and its product, and determine the pattern of expression of its murine homologue in embryonic and adult tissues as a first step toward the functional assessment of the receptor. Analysis of the 6226 bp ALK cDNA identified an open reading frame encoding a 1620-amino acid (aa) protein of predicted mass approximately 177 kDa that is most closely related to leukocyte tyrosine kinase (LTK), the two exhibiting 57% aa identity and 71% similarity over their region of overlap. Biochemical analysis demonstrated that the approximately 177 kDa ALK polypeptide core undergoes co-translational N-linked glycosylation, emerging in its mature form as a 200 kDa single chain receptor. Surface labeling studies indicated that the 200 kDa glycoprotein is exposed at the cell membrane, consistent with the prediction that ALK serves as the receptor for an unidentified ligand(s). In situ hybridization studies revealed Alk expression beginning on embryonic day 11 and persisting into the neonatal and adult periods of development. Alk transcripts were confined to the nervous system and included several thalamic and hypothalamic nuclei; the trigeminal, facial, and acoustic cranial ganglia; the anterior horns of the spinal cord in the region of the developing motor neurons; the sympathetic chain; and the ganglion cells of the gut. Thus, ALK is a novel orphan receptor tyrosine kinase that appears to play an important role in the normal development and function of the nervous system.
Amino Acid Sequence↗
Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation.
Previous studies have shown that exogenous insulin-like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF-II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF-II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF-II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF-II overproduction. Similar to observations with other muscle cell lines, IGF-II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low-serum differentiation medium. Accelerated differentiation of IGF-II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24-48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle-specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein-5 when compared with controls. These results confirm a role for IGF-II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation.
Animals↗
Facile production of native-like kappa-bungarotoxin in yeast: an enhanced system for the production of a neuronal nicotinic acetylcholine receptor probe.
Research on the mammalian central nervous system had been hindered by the limited number and meager supply of naturally occurring toxins that can be used as pharmacological reagents. The kappa-neurotoxins in particular are not found abundantly in nature and are difficult to obtain and isolate in quantities sufficient for research purposes. Here we report the expression and isolation of relatively large quantities of the kappa-neurotoxin, kappa-bungarotoxin, in an active form using a yeast, Pichia pastoris, expression system. The resultant product of the expression system has a short amino-terminal amino acid extension relative to venom-derived kappa-bungarotoxin, but is equivalent to the native toxin in physical and biological properties, as judged by the CD spectra, the ability to form dimers in solution, and the activity on chick ciliary ganglia. The yeast system produces approximately 0.2 mg from a 2 liter culture and the purification takes approximately 2 days. In contrast, E. coli, the only other available expression system for this toxin, produces one-fifth to one-half as much active material from a 5 liter high-density fermentation and the resulting protein takes over a week to purify. No high mol. wt disulfide-bonded aggregates were found in the yeast expression system product, indicating that the product is that of a biologically assisted folding process. This has significant implications not only for the efficient production of native toxin but also for the production of mutant proteins to study the structure-function relationship in these proteins.
Acetylcholine↗
Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism.
The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process stimulated by IGFs. We stably transfected C2 cells with IGFBP-5 cDNAs under control of a constitutively active promoter. Compared with vector-transfected control cells, C2 myoblasts expressing the IGFBP-5 transgene in the sense orientation exhibit increased IGFBP-5 levels in the extracellular matrix during proliferation, and subsequently fail to differentiate normally, as assessed by both morphological and biochemical criteria. Compared to controls, IGFBP-5 sense myoblasts show enhanced survival in low serum medium, remaining viable for at least four weeks in culture. By contrast, myoblasts expressing the IGFBP-5 antisense transcript differentiate prematurely and more extensively than control cells. The inhibition of myogenic differentiation by high level expression of IGFBP-5 could be overcome by exogenous IGFs, with des (1-3) IGF-I, an analogue with decreased affinity for IGFBP-5 but normal affinity for the IGF-I receptor, showing the highest potency. These results are consistent with a model in which IGFBP-5 blocks IGF-stimulated myogenesis, and indicate that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action. Our observations also suggest that IGFBP-5 normally inhibits muscle differentiation, and imply a role for IGFBP-5 in regulating IGF action during myogenic development in vivo.
Animals↗
Rapid activation of insulin-like growth factor binding protein-5 gene transcription during myoblast differentiation.
Insulin-like growth factor binding proteins (IGFBPs) comprise a family of secreted proteins that bind insulin-like growth factors-I and -II (IGF-I and -II) with high affinity and potentially modulate their biological effects. We have demonstrated previously that IGFBP-5, the most conserved of the six known IGFBPs, is expressed in muscle cells in the developing embryo and during the terminal differentiation of several myogenic cell lines. In this study we show that an IGF-I analog that binds minimally to IGFBPs potently enhances the differentiation of the stringently controlled inducible C2 myoblast (C2l) cell line and identify IGFBP-5 as the sole IGFBP secreted during C2l differentiation. We find that induction of IGFBP-5 mRNA and protein is coincident with the onset of myogenin gene expression and occurs secondary to the rapid activation of IGFBP-5 gene transcription. By transient gene transfer experiments we demonstrate that a 1004 base pair segment of the IGFBP-5 promoter is very active in directing expression of the reporter gene luciferase in C2l myoblasts. A promoter fragment containing only 156 nucleotides of 5'-flanking DNA retained more than 70% of maximal activity and mediated at least part of the differentiation-dependent rise in IGFBP-5 gene transcription. Within this active segment are several potential binding sites for muscle-enriched transcription factors. Our results show that induction of IGFBP-5 expression is an early event in the myogenic differentiation of the C2l cell line and suggest that one function of this IGFBP is to modulate IGF-induced differentiation. C2l cells are thus an excellent in vitro model for elucidating the developmental factors that control IGFBP-5 gene transcription and action in skeletal muscle.
Animals↗
Identification of two clusters of mouse insulin-like growth factor binding protein genes on chromosomes 1 and 11.
The genes for insulin-like growth factor binding proteins (IGFBPs) encode secreted proteins that bind insulin-like growth factors I and II with high affinity and modulate their biological activities. In this report we have used interspecific backcross mapping and gene cloning to define the chromosomal locations of 4 mouse Igfbp genes. Igfbp1 and 3 are found in the proximal part of chromosome 1. In the human genome these two loci map within 20 kb of one another on chromosome 7p14-p12, and the genes are organized in a tail-to-tail configuration. Mouse Igfbp2 and 5 colocalize to a proximal region of chromosome 1 that is syntenic with human chromosome 2q33-q36, and the two genes are 5 kb apart in a tail-to-tail orientation. These results suggest an evolutionary scheme in which a primordial IGFBP gene duplicated to form a cluster that was later replicated to create second linkage group.
Animals↗
A highly conserved insulin-like growth factor-binding protein (IGFBP-5) is expressed during myoblast differentiation.
Insulin-like growth factor-binding proteins (IGFBPs) are a family of secreted proteins that bind insulin-like growth factors I and II (IGFs I and II) and are capable of modulating IGF actions on target cells. We have shown previously that C2 myoblasts secrete a single approximately 29-kDa IGFBP during their terminal differentiation (Tollefsen, S. E., Lajara, R., McCusker, R. H., Clemmons, D. R., and Rotwein, P. (1989) J. Biol. Chem. 264, 13810-13817). In this study, we have purified the protein from C2 cell-conditioned media by conventional and IGF-affinity chromatography, cloned its cDNA by PCR-based and traditional library screening, and identified it as mouse IGFBP-5. The resultant 5561 nucleotide cDNA encodes a 252-amino acid mature protein (predicted M(r) approximately 28,400) that is 97% identical to rat and human IGFBP-5. In differentiating C2 myoblasts and in F3 azamyoblasts the > 6-kilobase IGFBP-5 mRNA accumulates concomitantly with induction of myogen mRNA, an early marker of muscle differentiation. Ligand blot analysis shows that IGFBP-5 protein is secreted within 12 h of the onset of differentiation in these cells and that it is the only IGFBP produced in several fusing skeletal muscle cell lines. In vivo, IGFBP-5 transcripts are expressed in a variety of mouse tissues including striated muscle, but, unlike other IGFBPs, it is barely detectable in liver. IGFBP-5 is more conserved than other IGFBPs in mammals; its conserved structure and sequence also extends to non-mammalian vertebrates. Hybridization of a mouse BP5 coding region probe to RNA from several chicken and Xenopus tissues demonstrated similarly sized transcripts in these species. A partial Xenopus cDNA is identical in 38/45 deduced amino acids to the mammalian proteins. Identification of an IGF-binding protein that is produced during myoblast differentiation provides a model system in which to study the potential modulatory role of IGFBPs in development.
Amino Acid Sequence↗
The injuries to JFK.
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Craniocerebral Trauma↗
Functional analysis of the rat insulin-like growth factor I gene and identification of an IGF-I gene promoter.
Insulin-like growth factor I (IGF-I) mediates many of the systemic growth-promoting effects of growth hormone and also functions as a locally acting growth stimulator. In mammals, IGF-I gene expression is complicated, as the gene is transcribed and processed into multiple mRNAs (ranging in length from less than 1 to nearly 7.5 kb) that encode at least two protein precursors. As a step toward understanding the regulation of IGF-I, we report the complete organization of the rat IGF-I gene, including identification of the structural determinants for all IGF-I mRNA species, and an initial functional analysis of its promoters. The gene is composed of 6 exons distributed over nearly 80 kb of chromosomal DNA and is structurally heterogeneous. Several transcription start sites were identified within IGF-I exons 1 and 2, adjacent to presumptive promoters 1 and 2, respectively, and at least three polyadenylation sites were mapped to exon 6. To test promoter function, fusion genes were constructed linking fragments of IGF-I DNA to a reporter plasmid. Chimeric genes containing at least 395 bp of DNA from the 5'-flanking region of exon 1 enhanced luciferase activity after transfection into the IGF-I-producing SK-N-MC cell line, while fusion plasmids containing up to 1,300 bp of DNA from the 5'-flanking region of exon 2 were inactive. Relative levels of IGF-I mRNAs containing exons 1 or 2 varied among different rat tissues, although in response to acute or chronic growth hormone treatment both classes of transcripts were induced coordinately in rat liver. These observations represent the first thorough characterization of a mammalian IGF-I gene, and provide a starting point for defining the mechanisms by which growth hormone and other trophic factors regulate IGF-I gene expression.
Amino Acid Sequence↗
"Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II.
Differentiation of muscle cells to form postmitotic myotubes is usually viewed as being negatively controlled by medium components, sometimes designated "mitogens." However, we have found that a family of mitogenic agents, the insulin-like growth factors (IGFs), are potent stimulators of differentiation in myoblasts which act by inducing expression of the myogenin gene. We show here that this action of the IGFs occurs even when these growth factors are not added to the cell medium; upon transfer to low-serum "differentiation medium," myoblasts begin active expression of the IGF-II gene, at both the mRNA and protein levels. Furthermore, autocrine secretion of IGF-II is essential for the process of terminal differentiation of the cells. These conclusions are based upon four lines of evidence. (1) The rate of spontaneous differentiation in several sublines of myogenic cells correlates with their level of expression of IGF-II. (2) C2 and Sol 8 cells, which secrete high levels of IGF-II, are relatively insensitive to exogenous IGFs, in contrast to L6 lines, which exhibit lower levels of IGF-II gene expression. (3) An antisense oligodeoxyribonucleotide complementary to the first five codons of IGF-II inhibits myogenic differentiation in the absence but not in the presence of exogenous IGF-II. (4) Spontaneous differentiation in response to autocrine IGF-II involves the same mechanism that occurs in cells stimulated by the IGFs, i.e. elevation of expression of the myogenin gene.
Animals↗
Well-differentiated intra-osseous osteosarcoma in the mandible of a six-year-old child.
The case of an osteosarcoma arising in the mandible of a six-year-old child is presented. It was originally misdiagnosed as a desmoplastic fibroma, and after initial curettage continued to grow for twelve years. The differential diagnosis is presented and details of the surgical access and difficulties encountered in the surgical removal of this very large tumour are described. The relationship between pre-existing disease and osteosarcoma of the mandible is discussed, and the difficulties experienced in distinguishing between the fibro-osseous lesions and osteosarcoma of the mandible are highlighted. The case is presented as a well-differentiated, intra-osseous osteosarcoma with subsequent transformation into a high-grade, conventional osteosarcoma.
Child↗
Type II collagen defects in the chondrodysplasias. I. Spondyloepiphyseal dysplasias.
The spondyloepiphyseal dysplasias (SEDs) and spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of skeletal dysplasias (dwarfing disorders) characterized by abnormal epiphyses, with and without varying degrees of metaphyseal irregularities, flattened vertebral bodies, and myopia. To better define the underlying cause of these disorders, we have analyzed the collagens from costal cartilage from several of these patients, using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) of intact chains and cyanogen bromide (CNBr) peptides and amino acid analysis. In almost all of the patients in this study group, the type II collagen exhibited a slower electrophoretic mobility when compared with that in normal controls. The mobility of many, but not all, of the CNBr peptides was also retarded. Peptides near the amino terminus were almost always altered, while the mobility of peptides close to the carboxyl terminus were normal in all but the severely affected cases. Analysis of the CNBr peptides on an HPLC sieving column confirmed that the electrophoretically abnormal peptides were of a higher molecular weight than were control peptides. Amino acid analysis indicated that the abnormal collagens have a higher ratio of hydroxylysine to lysine than does control collagen, suggesting that overmodification may be involved in the altered mobility. Our results are consistent with a defect in the collagen helix that results in overmodification of the molecule from that point toward the amino terminus. We propose that some forms of SED and SEMD are associated with abnormalities in type II collagen that results in delayed helix formation and consequent overmodification of the collagen. Cases of SED fit onto a continuous spectrum of clinical severity that correlates positively with both the extent of alteration and the proximity of the defect to the carboxyl terminus.
Adolescent↗
Subcellular localization of platelet elastase and its retention during the release reaction.
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Blood Platelets↗
A unique elastase in human blood platelets.
Previous investigations suggested that elastolytic activity found in platelets could be due to contamination by neutrophil elastase. In the present study, the lysate of blood platelets free of detectable neutrophils was examined for elastase-like activity using tertiary-butyloxycarbonyl (tBOC)-ala-ala-pro-ala-aminomethyl coumarin (I), tBOC-ala-ala-pro-val-aminomethyl coumarin (II), and succinyl-tri-ala-rho-nitroanilide (SAPNA), and for elastolytic activity using 3H-labeled dog and human lung elastins. The platelet lysate degraded I at a higher rate than II, while the reverse was true of neutrophil elastase. The rate of degradation of I, II, and SAPNA by the lysate increased with reaction time up to 20 min. The rate of I, II, and SAPNA degradation by the lysate was decreased by the presence of 0.5 M NaCl, whereas NaCl greatly potentiated their degradation by neutrophil elastase. Plasma alpha 2-macroglobulin inhibited elastolysis by the platelet lysate, whereas plasma alpha 1-antitrypsin did not. The lysate activity was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, elastatinal, Trasylol, and furoyl-saccharin. The optimum pH for platelet lysate activity was 8.5-9.0, as in other studies using elastin as substrate. The pH 4.5 eluate obtained after incubation of the lysate with dog lung elastin at neutral pH exhibited the same catalytic properties as the activity in the lysate. The different substrate and inhibitor specificities and the failure of IgG specific for neutrophil elastase to remove elastase-like and elastolytic activities from the lysate indicate that a unique elastase occurs in platelets.
Animals↗
Pulsating exophthalmos associated with neurofibromatosis.
A patient with pulsating exophthalmos is presented. He had facial asymmetry, multiple cutaneous café-au-lait spots and a subcutaneous nodule, biopsy of which showed features consistent with neurofibromatosis. Radiographs of the orbit showed a deficiency of the superolateral orbital wall, a feature previously recognized as part of neurofibromatosis. This feature probably occurs as a result of mesodermal dysplasia. The differential diagnosis and management are discussed.
Adult↗
A study of medically qualified dental graduates.
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Adult↗
Multiple neurofibromatosis associated with facial asymmetry.
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Alveolar Process↗