RESUMO
We previously established a novel mouse model for human aging and identified the genetic foundation responsible for it. A defect in expression of a novel gene, termed klotho (kl), leads to a syndrome resembling human aging in mice. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to beta-glucosidases. In this report, we present the entire mouse kl gene organization. The mouse kl gene spans about 50 kilobases and consists of five exons. The promoter region lacks a TATA-box and contains four potential binding sites for SP1. We further show that two kl gene transcripts encoding membrane or secreted protein are generated through alternative transcriptional termination. These findings provide fundamental information for further study of the kl gene which may regulate aging in vivo.
Assuntos
Proteínas de Membrana/genética , RNA Mensageiro/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Glucuronidase , Humanos , Proteínas Klotho , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transcrição GênicaRESUMO
Leukemic cells from 27 adult patients with acute myelocytic leukemia (AML) were investigated to determine the cell surface inducibility of interleukin-2 receptor (IL-2R) in in vitro culture with and without interleukin-1 beta (IL-1 beta). IL-2R alpha chain (IL-2R alpha) was induced on leukemic cells in 11 of 27 cases. Most of the cases could induce IL-2R alpha spontaneously without IL-1 beta, while the IL-2R beta chain (IL-2R beta) did not appear on leukemic cells from any of the cases tested. AML cases expressing CD7 or HLA-DR antigen could induce IL-2R alpha more frequently than any other type of AML. Among interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF) and G-CSF, IL-3 showed a more prominent effect on DNA synthesis in IL-2R alpha inducible cases than in its uninducible cases. These results suggest that IL-2R alpha but not IL-2R beta was easily inducible on AML cells with immature characteristics.
Assuntos
Leucemia Mieloide/metabolismo , Receptores de Interleucina-2/metabolismo , Doença Aguda , Adulto , Divisão Celular , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunofenotipagem , Técnicas In Vitro , Interleucina-1/farmacologia , Interleucina-3/farmacologia , Leucemia Mieloide/imunologia , Leucemia Mieloide/patologia , Receptores de Interleucina-2/química , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Fourteen patients with lymphoid and mixed blast crisis (BC) of chronic myelocytic leukemia were studied by immunophenotyping and genotyping. Rearrangements of immunoglobulin heavy chain (IgH), T-cell receptor (TcR) gamma and TcR beta genes were detected in all 14, in nine and in four patients, respectively. Interestingly, more than two rearranged bands of IgH gene in three lymphoid BC and two rearranged bands with germ line band in 1 biphenotypic BC indicated the genetic heterogeneity of the blasts. Some blastic transformations are thought to occur at a more immature stage of hematopoietic differentiation than that indicated by the phenotype and genotype of BC cells.
Assuntos
Crise Blástica/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Antígenos de Diferenciação/análise , Southern Blotting , Rearranjo Gênico , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T/genética , Genes de Imunoglobulinas/genética , Genótipo , Humanos , Masculino , FenótipoRESUMO
In 43 cases of various B-cell lineage tumors, precise gene structures of rearranged immunoglobulin heavy chain (IgH) were studied. By Southern-blot analysis of D upstream (5'D) gene of IgH, biallelic rearrangement structures, D-J or V-D-J, were determined and consequently maturational stage specific IgH rearrangement patterns were investigated. B-precursor ALL cases (especially stage IV of Nadler's criteria) have V-D-J rearranged IgH genes on both alleles. In contrast, most of the mature B-cell malignancies, excluding multiple myeloma, have IgH genotype of D-J/V-D-J. In addition, in case of D-J/V-D-J, the D gene used in D-J joining has been speculated by Southern-blot of D genes. So, these approaches for inquiring precise structures of rearranged IgH genes are supposed to provide new information of lymphocyte differentiation and leukemogenesis.
Assuntos
DNA de Neoplasias/análise , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Leucemia Linfocítica Crônica de Células B/genética , Linfoma não Hodgkin/genética , Mieloma Múltiplo/genética , Variação Antigênica/genética , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , HumanosRESUMO
Leukemic cells from 21 to 197 adult patients with de novo acute myelocytic leukemia (AML) were positive for IL-2R alpha chain (IL-2R alpha), whereas IL-2R beta chain (IL-2R beta), which is responsible for IL-2 signal transduction, was not found on leukemic cells from any of these cases tested. The expression of IL-2R alpha was closely associated with that of adhesion molecules CD4, CD11b and CD22, and endopeptidase CD10. None of the IL-2R alpha (+) AML cells responded to recombinant human IL-2. These data suggest that IL-2R alpha on AML cells may not be involved in cellular proliferation as one of growth factor receptors but may have a role in the control of cell-to-cell interactions.
Assuntos
Comunicação Celular , Leucemia Mieloide Aguda/patologia , Receptores de Interleucina-2/fisiologia , Adulto , Antígenos CD/análise , Divisão Celular/efeitos dos fármacos , Humanos , Interleucina-2/farmacologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Receptores de Interleucina-2/análise , Proteínas Recombinantes/farmacologiaRESUMO
In the past decade, there have been remarkable advances in tissue engineering technology toward the goal of creating organoids in vitro from cells and cellular scaffolding. Indeed, tissue-engineered organoids such as skin and cartilage, each with comparatively simple architectures, are presently at the clinical stage. However, conventional tissue engineering techniques have not allowed for the reconstruction of an organoid that mimics an organ of complex architecture of abundant vascular networks. We established a method for organ engineering that can remodel a rat liver into a reconstructed organoid without separating the majority of liver cells by a continuous three-step perfusion. The liver was perfused through its vascular system with a buffered balanced salt solution to cleanse blood from the organ, with a collagenase/dispase medium to deconstruct cellular scaffolds, and with a culture medium containing collagen type I to reorganize the multicellular architecture. The reconstructed organoid was then prepared by excising the perfused liver from the rat and culturing it at 37 degrees C for 2 h. Histologically healthy parenchymal hepatocytes expressing albumin were observed in the excised organoid even after culture for 3 weeks. Furthermore, a fibroblast-implanted organoid was prepared by using a culture medium containing suspended fibroblasts in the third step of the perfusion procedure, demonstrating the efficacy of heterogeneous cells for the reconstruction of an organoid. This method may be applicable to the formation of organoids from other organs, such as kidney and spleen, each of which have abundant capillaries, and therefore the method provides a novel concept for the development of lab-grown organs, i. e., organ engineering.
Assuntos
Fígado/fisiologia , Técnicas de Cultura de Órgãos , Animais , Biotecnologia , Colagenases , Técnicas de Cultura , Endopeptidases , Regeneração Hepática , Masculino , Técnicas de Cultura de Órgãos/métodos , Organoides/fisiologia , Perfusão , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
INTRODUCTION: In situ hybridization of whole mouse fetuses and their tibias with a stem cell growth factor (SCGF) antisense probe demonstrated specific expression of SCGF mRNA around skeletal tissues, particularly in bone marrow cells, proliferating chondrocytes, the perichondrium and periosteum, but little expression in resting or hypertrophic chondrocytes. METHODS: Recombinant human (rh) SCGF-alpha was purified from a conditioned medium of SCGF-alpha gene-transfected CHO cells. The molecular mass of rhSCGF-alpha, 45 kDa, was shifted down to 40 kDa by digestion with endo-O-glycosidase and sialidase, suggesting O-glycosylation of rhSCGF-alpha with sialic acids. RESULTS: For human bone marrow CD34+Lin- cells, rhSCGF-alpha alone did not stimulate colony-formation, but small cluster-formation (10.3 +/- 2.5/1 x 10(3) CD34+Lin- cells). It promoted growth of erythroid and granulocyte/macrophage (GM) colonies in the primary culture with erythropoietin and GM colony-stimulating factor (CSF) or G-CSF, respectively, and further supported GM progenitor cells in a short-term liquid culture. In contrast, rhSCGF-alpha suppressed stem cell factor (SCF)-stimulated erythroid bursts, indicating some competitive interaction between SCGF and SCF. rhSCGF-alpha was synergistic with interleukin-3 and the flt3 ligand to enhance GM colony-growth, but not synergistic with those inducing ex vivo expansion of GM progenitor cells. CONCLUSION: SCGF is selectively produced by osseous and hematopoietic stromal cells, and can mediate their proliferative activity on primitive hematopoietic progenitor cells.
Assuntos
Fatores de Crescimento de Células Hematopoéticas/genética , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Lectinas Tipo C , Lectinas/genética , Lectinas/farmacologia , Animais , Técnicas de Cultura de Células , Divisão Celular/efeitos dos fármacos , Clonagem Molecular/métodos , Cricetinae , Citocinas/farmacologia , Interações Medicamentosas , Feto/química , Hematopoese/efeitos dos fármacos , Fatores de Crescimento de Células Hematopoéticas/isolamento & purificação , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Hibridização In Situ , Camundongos , RNA Mensageiro/metabolismo , Fator de Células-Tronco/farmacologia , Distribuição TecidualRESUMO
We report a case of a 72-year-old man with large granular lymphocyte (LGL) leukemia. Immunophenotypical analysis of the abnormal cells showed the following results: CD 2+, CD 3+, CD 4+, CD 8-, CD 11+, CD 16-, Leu 7+. These cells had natural killer (NK) activity, responded to PHA and recombinant interleukin-2 (rIL-2), and showed neither helper nor suppressor function in B-cell differentiation. Molecular genetical analysis showed monoclonal rearrangement of T-cell receptor beta-chain gene, indicating they are of T-cell origin. These findings provide information on biological characteristics of normal CD 4+, Leu 7+ cells.
Assuntos
Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação/análise , Leucemia Linfoide/imunologia , Idoso , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Leucemia Linfoide/patologia , Masculino , FenótipoRESUMO
An 18 years old female was admitted to hospital due to pancytopenia on May 25, 1987 and found to have petechiae, mild hepatomegaly and severe splenomegaly. The bone marrow was found to contain 12% of blast cells. Splenectomy was performed followed by CHOP therapy. In September, 1987 the peripheral blood was found to contain tumor cells, which turned out to be resistant to various combination chemotherapies. The patient died on August 21, 1988. The phenotype of tumor cells in this case was CD2+ CD7+ CD3+ CD4- CD8- WT31-. Genetic analysis detected rearrangement of the beta and gamma chain of TcR but not transcription or translation of the beta chain of TcR, while the antibodies of delta TCS 1 and TcR delta 1 to the delta chain of TcR were positive. From this fact, the present case was considered to be the malignant counterpart of normal CD3+ WT31- double negative T cells. The reactivity of this tumor cells to IL-2 and IL-1 beta suggested the association of the IL-2R beta chain.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfoma/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Adolescente , Complexo CD3 , Feminino , Humanos , Fenótipo , Receptores de Antígenos de Linfócitos T gama-deltaRESUMO
We report a 35-year-old male with leukemic change and gastric involvement of Burkitt's lymphoma. A monoclonal immunoglobulin (Ig), IgM-kappa, was detected in the serum by means of immunoelectrophoresis. Immunophenotypical analysis showed that the neoplastic cells were CD20+, OKIal+, CD10-, CD21-, surface Ig+ (M-kappa), and cytoplasmic Ig-. The neoplastic cells did not secret Ig by using of protein A plaque forming cell assay. Active transcription of Ig heavy chain genes was not detected by cell dot analysis of the neoplastic cells. These findings support the possibility that the presence of the monoclonal Ig in the serum does not result from secretion of Ig from the neoplastic cells. The shedding of surface Ig from the neoplastic cells might result in the occurrence of monoclonal Ig in the serum.
Assuntos
Linfoma de Burkitt/complicações , Neoplasias Gástricas/complicações , Macroglobulinemia de Waldenstrom/etiologia , Adulto , Linfoma de Burkitt/imunologia , Humanos , Imunoglobulina M/metabolismo , Cadeias kappa de Imunoglobulina/metabolismo , Masculino , Neoplasias Gástricas/imunologia , Macroglobulinemia de Waldenstrom/imunologiaRESUMO
A 56-year-old male presented with fever of unknown origin and subacute dementia which progressed to death with seizure, coma and acute deterioration of general conditions. He had splenomegaly but not skin eruption or lymph node swelling. Autopsy findings showed that mononuclear tumor cells were widespread within the lumens of small blood vessels, indicating the features of neoplastic angioendotheliosis. The involved organs were shown to be brain, lung, adrenal grand, testis, bone marrow, heart and thyroid gland. To determine the origin of tumor cells, an immunohistochemical study was carried out using a panel of monoclonal antibodies. The results indicated that the tumor cells were of B-lymphocyte origin. These findings support the possibility that neoplastic angioendotheliosis is a lymphoma with proliferation in small blood vessels throughout the body.
Assuntos
Linfócitos B/patologia , Hemangioendotelioma/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Hemangioendotelioma/irrigação sanguínea , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Primárias MúltiplasRESUMO
Uricase from Cellulomonas flavigena SK-4 is an industrially useful enzyme for commercial formulations of hair coloring. The uricase production by recombinant Escherichia coli strain with a high cell density cultivation technique was described. Of three kinds of media, synthetic media with the feeding of a high concentration of glucose solution were suitable for high cell density cultivation. As for feeding, both biomass concentration and uricase productivity were increased by about two (61.2 g dry cell weight (DCW)/liter) and three times (1037 U/ml broth), respectively, in 24 h by continuous supply. In the case of feeding by a DO-stat method, however, cell concentration was comparable to continuous glucose supply but uricase activity was reduced. By supplying pure oxygen to compensate for oxygen limitation during cultivation, the highest values of 77.4g DCW/liter and 1113 U/ml broth of the uricase activity were achieved with the total cultivation time of 15 h.
Assuntos
Escherichia coli/enzimologia , Proteínas Fúngicas/biossíntese , Urato Oxidase/biossíntese , Actinomycetales/enzimologia , Técnicas Bacteriológicas , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Glucose/farmacologia , Oxigênio/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Urato Oxidase/genética , Urato Oxidase/metabolismoRESUMO
A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 +/- 4.6 microM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a low K(m) phytase from A. niger SK-57 and a high K(m) phytase from Aspergillus ficuum was recognized.
Assuntos
6-Fitase/metabolismo , Aspergillus niger/enzimologia , Ácido Fítico/metabolismo , Concentração de Íons de Hidrogênio , Cinética , FosforilaçãoRESUMO
The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by L-lysine, was cloned from an L-threonine- and L-lysine-coproducing mutant of the obligate methylotroph Methylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapA mutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an L-threonine producing mutant, elevated the specific activity of DDPS 20-fold and L-lysine production 2- to 3-fold with concomitant reduction of L-threonine in test tube cultures. AL119 containing the dapA gene produced 8 g of L-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M(r) of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition by L-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector, L-lysine.
Assuntos
Hidroliases/genética , Lisina/metabolismo , Metanol/metabolismo , Methylobacillus/enzimologia , Mutação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Meios de Cultura , Hidroliases/química , Hidroliases/metabolismo , Methylobacillus/genética , Methylobacillus/crescimento & desenvolvimento , Dados de Sequência Molecular , Plasmídeos/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
L-Glutamic acid (Glu) hyper-producing mutants were isolated from Methylobacillus glycogenes ATCC 21276 and ATCC 21371 during the screening of amino acid auxotrophs. iA111, derived from ATCC 21276, and 1009 (Phe(-)), derived from ATCC 21371, produced about 10 times as much Glu as the wild type strains. The highest producer, RV3, a phenylalanine auxotrophic revertant of 1009, produced 38.8 g/liters of Glu in 84h in a 5-liter jar fermentor. iA111 and 1009 were mutagenized, and L-threonine (Thr) producing mutants were isolated among Thr and L-Iysine (Lys) analog-resistant strains. The highest Thr producer derived from iA111, AL119 (AHV + Lys(R)), produced 11.0g/liters of Thr in 72h in a 5-liter jar fermentor. AL119 was further mutagenized, and a Thr and Lys producer, DHL 122 (DHL(R)), was isolated. DHL 122 co-produced 3.1 g/liters of Lys and 5.6 g/liters of Thr in 72 h in a 5-liter jar fermentor.
RESUMO
We constructed recombinant plasmids carrying the genes coding for the L-threonine biosynthetic enzymes, the hom gene, the hom-thrC genes, and the thrB genes, of a gram-negative obligate methylotroph, Methylobacillus glycogenes, and examined the effects of them on the production of L-threonine from methanol. The hom gene, which encodes the homoserine dehydrogenase, and the hom-thrC genes, containing the gene coding for threonine synthase together with the hom gene, were cloned from a wild-type strain, and the thrB gene encoding the desensitized homoserine kinase was cloned from an L-threonine-producing mutant, ATR80. The recombinant plasmids were transferred into ATR80 and its L-isoleucine auxotroph, A513, by conjugation. Amplification of the genes coding for the L-threonine biosynthetic enzymes elevated the activities of the L-threonine biosynthetic enzymes of the transconjugants 10- to 30-fold over those of the strains containing only vectors. The L-threonine production from methanol in test-tube cultivation was increased about 30% and 40% by the amplification of the hom gene and the hom-thrC gene respectively, and it was slightly increased by that of the thrB gene. The effects of gene amplification were confirmed by the cultivation in 5-1 jar fermentors. The best producer, an A513 transconjugant containing the plasmid carrying the hom-thrC genes, produced 16.3 g/l L-threonine for 72 h.
Assuntos
Carbono-Oxigênio Liases , Genes Bacterianos , Methylococcaceae/genética , Methylococcaceae/metabolismo , Treonina/biossíntese , Biotecnologia , Mapeamento Cromossômico , Clonagem Molecular , Amplificação de Genes , Homosserina Desidrogenase/genética , Liases/genética , Metanol/metabolismo , Plasmídeos/genética , Transformação GenéticaRESUMO
Cyclophilin 40 (CyP4O) is a recently identified member of the cyclophilin family that may be a component of unactivated steroid receptor complexes. It consists of an N-half portion that is highly homologous to cyclophilin A and has peptidyl prolyl isomerase (PPIase) activity, and a C-half portion that resembles the C-terminal portion of FKBP52 (FK506 binding protein 52), another component of unactivated steroid receptor complexes. To better understand the structure and functional characteristics of this new class of cyclophilin, we have raised monoclonal antibodies against the C-half portion of human CyP4O. Immunostaining with the antibodies showed its preferential localization in cytoplasm. One antibody cross-reacted with a 45 kDa protein in yeast, suggesting high conservation throughout evolution. A CyP4O-associated protein was isolated from rabbit reticulocyte lysate by means of an affinity resin, and was identified as hsp90. The C-half portion of CyP4O was necessary and sufficient for the interaction.
Assuntos
Isomerases de Aminoácido/metabolismo , Proteínas de Transporte/metabolismo , Ciclofilinas , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/metabolismo , Peptidilprolil Isomerase , Isomerases de Aminoácido/química , Isomerases de Aminoácido/imunologia , Animais , Anticorpos Monoclonais , Western Blotting , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Células Cultivadas , Sequência Conservada , Peptidil-Prolil Isomerase F , Citoplasma/química , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , Imunofluorescência , Proteínas Fúngicas/metabolismo , Humanos , Rim/metabolismo , Microscopia de Fluorescência , Ligação Proteica , Coelhos , Receptores de Esteroides , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reticulócitos/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Suínos , Proteínas de Ligação a TacrolimoRESUMO
We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.
Assuntos
Carbono-Oxigênio Liases , Genes Bacterianos , Homosserina Desidrogenase/genética , Liases/genética , Methylococcaceae/enzimologia , Methylococcaceae/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Sondas de DNA , DNA Bacteriano/genética , Escherichia coli/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
The kinase insert domain-containing receptor (KDR) for vascular endothelial growth factor (VEGF) has been shown to be involved in vasculogenesis and angiogenesis. This receptor is characterized by seven immunoglobulin (Ig)-like domains within its extracellular region. To identify the domains involved in VEGF binding, we constructed various deletion mutants of the extracellular region fused with the crystallizable fragment portion of an IgG and then examined the binding affinity with VEGF by means of the BIAcore biosensor assay. Deletion of the COOH-terminal two or three Ig-like domains out of a total of seven affected ligand dissociation rather than association. Further deletion of the fourth domain caused a drastic decrease in the association rate. Binding ability was abolished completely with removal of the third domain. The mutant KDR proteins lacking the NH2-terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants having this domain. Deletion of the first two NH2-terminal Ig-like domains caused a drastic reduction in the association rate, but affinity to VEGF was retained. These results suggest that the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule. The first Ig-like domain may regulate the ligand binding.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Sítios de Ligação , Técnicas Biossensoriais , Humanos , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Immunophenotypes and genotypes were analyzed for a case of chronic myelocytic leukemia in blast crisis (BC). In the early stage of BC (early BC), the blasts consisted of lymphoid-myeloid cells, but in the later stage of BC (late BC), they were myeloid cells, morphologically and phenotypically. On Southern blot of DNAs in early BC, a single rearranged fragment of immunoglobulin heavy chain (IgH) genes was detected, whereas IgH genes were in germline configuration in both initial chronic phase and late BC. A clone which had a rearranged IgH gene in early BC, was considered to have co-existed with a clone which had the germline IgH gene. Analysis for bcr genes confirmed that the chronic phase as well as early and late BC were of the same clonal origin. Phenotypic and immunogenotypic analyses, however, revealed that at least two secondary clones emerged from the primary clone. The therapeutic effect against lymphoid population among mixed crisis cells could be evuluated not only phenotypically but also genotypically.