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1.
Mol Psychiatry ; 21(5): 594-600, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26952864

RESUMO

Using Icelandic whole-genome sequence data and an imputation approach we searched for rare sequence variants in CHRNA4 and tested them for association with nicotine dependence. We show that carriers of a rare missense variant (allele frequency=0.24%) within CHRNA4, encoding an R336C substitution, have greater risk of nicotine addiction than non-carriers as assessed by the Fagerstrom Test for Nicotine Dependence (P=1.2 × 10(-4)). The variant also confers risk of several serious smoking-related diseases previously shown to be associated with the D398N substitution in CHRNA5. We observed odds ratios (ORs) of 1.7-2.3 for lung cancer (LC; P=4.0 × 10(-4)), chronic obstructive pulmonary disease (COPD; P=9.3 × 10(-4)), peripheral artery disease (PAD; P=0.090) and abdominal aortic aneurysms (AAAs; P=0.12), and the variant associates strongly with the early-onset forms of LC (OR=4.49, P=2.2 × 10(-4)), COPD (OR=3.22, P=2.9 × 10(-4)), PAD (OR=3.47, P=9.2 × 10(-3)) and AAA (OR=6.44, P=6.3 × 10(-3)). Joint analysis of the four smoking-related diseases reveals significant association (P=6.8 × 10(-5)), particularly for early-onset cases (P=2.1 × 10(-7)). Our results are in agreement with functional studies showing that the human α4ß2 isoform of the channel containing R336C has less sensitivity for its agonists than the wild-type form following nicotine incubation.


Assuntos
Predisposição Genética para Doença , Mutação de Sentido Incorreto , Receptores Nicotínicos/genética , Fumar/genética , Tabagismo/complicações , Tabagismo/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aneurisma da Aorta Abdominal/etiologia , Aneurisma da Aorta Abdominal/genética , Feminino , Estudos de Associação Genética , Humanos , Islândia , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/etiologia , Doença Arterial Periférica/genética , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/genética , População Branca/genética , Adulto Jovem
2.
Science ; 269(5224): 688-90, 1995 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-7542803

RESUMO

An immunoglobulin E (IgE)-dependent histamine-releasing factor (HRF) produced by lymphocytes of atopic children and present in biological fluids of allergic patients has been identified and purified. Amino-terminal sequencing revealed extensive homology to a mouse protein, p21, and its human homolog, p23. Both recombinant proteins caused histamine release from the human basophils of a subpopulation of donors, and this release was dependent on IgE. Polyclonal antibodies recognized and removed the biological activity of recombinant and native HRF. HRF identifies a heterogeneity of IgE and is believed to play a prominent role in chronic allergic disease processes.


Assuntos
Biomarcadores Tumorais , Liberação de Histamina , Imunoglobulina E/imunologia , Linfocinas/química , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sequência de Bases , Basófilos/imunologia , Linhagem Celular , Clonagem Molecular , Humanos , Cinética , Linfocinas/imunologia , Linfocinas/isolamento & purificação , Linfocinas/farmacologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/farmacologia , Homologia de Sequência de Aminoácidos , Proteína Tumoral 1 Controlada por Tradução
3.
J Med Genet ; 45(5): 284-9, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18178632

RESUMO

BACKGROUND: Germline CDKN2A mutations have been observed in 20-40% of high risk, melanoma prone families; however, little is known about their prevalence in population based series of melanoma cases and controls. METHODS: We resequenced the CDKN2A gene, including the p14ARF variant and promoter regions, in approximately 703 registry ascertained melanoma cases and 691 population based controls from Iceland, a country in which the incidence of melanoma has increased rapidly. RESULTS: We identified a novel germline variant, G89D, that was strongly associated with increased melanoma risk and appeared to be an Icelandic founder mutation. The G89D variant was present in about 2% of Icelandic invasive cutaneous malignant melanoma cases. Relatives of affected G89D carriers were at significantly increased risk of melanoma, head and neck cancers, and pancreatic carcinoma compared to relatives of other melanoma patients. Nineteen other germline variants were identified, but none conferred an unequivocal risk of melanoma. CONCLUSIONS: This population based study of Icelandic melanoma cases and controls showed a frequency of disease related CDKN2A mutant alleles ranging from 0.7% to 1.0%, thus expanding our knowledge about the frequency of CDKN2A mutations in different populations. In contrast to North America and Australia where a broad spectrum of mutations was observed at a similar frequency, in Iceland, functional CDKN2A mutations consist of only one or two different variants. Additional genetic and/or environmental factors are likely critical for explaining the high incidence rates for melanoma in Iceland. This study adds to the geographic regions for which population based estimates of CDKN2A mutation frequencies are available.


Assuntos
Genes p16 , Mutação em Linhagem Germinativa , Melanoma/epidemiologia , Melanoma/genética , Alelos , Austrália , Estudos de Casos e Controles , Frequência do Gene , Genótipo , Humanos , Islândia/epidemiologia , América do Norte , Grupos Populacionais , Fatores de Risco
4.
Mol Immunol ; 35(8): 459-67, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9798650

RESUMO

The ragweed allergens Amb t 5 and Amb a 5 are among the smallest inhaled protein allergens known, containing a single, immunodominant T-cell epitope. In this study we analyzed the B-cell epitope structure of Amb t 5. The three-dimensional structures of Amb t 5 and Amb a 5 have been determined by NMR spectroscopy, providing a rare opportunity to analyze three-dimensional antigenic sites. Amb t 5 residues likely to be important for antigenicity were identified by examining the surface area of Amb t 5 accessible to a probe of the size of an antibody molecule. After changing these residues to the corresponding Amb a 5 residues, recombinant proteins were purified and tested for loss of antigenic activity. Inhibition radio-immunoassays, using sera from 8 individuals who had received immunotherapy with giant ragweed extract, allowed the mutations to be divided into three groups: (1) mutations that had little or no effect on antibody binding, (2) mutations that caused a loss of antigenic activity to a different degree in different sera and (3) mutations that drastically reduced antigenic activity in all sera tested. This last set of mutations clustered in the third loop of Amb t 5, suggesting that antibody recognition of Amb t 5, like T-cell recognition, is primarily directed towards a single, immunodominant site.


Assuntos
Alérgenos/química , Proteínas de Plantas/química , Pólen/imunologia , Aminoácidos/genética , Formação de Anticorpos , Reações Antígeno-Anticorpo/genética , Antígenos de Plantas , Sítios de Ligação de Anticorpos/genética , Humanos , Mutagênese , Mutação/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética
5.
Mol Immunol ; 35(4): 249-57, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9736341

RESUMO

The protein tyrosine kinase Csk downregulates the activity of the Src family of kinases and has a negative effect on signal transduction through several Src kinase-associated receptors. Because the Src-family kinase Lyn plays a pivotal role in FcepsilonRI-mediated cellular activation, we examined whether Csk is involved in FcepsilonRI signaling events. Using anti-Csk antibodies and recombinant fusion proteins we detected a single tyrosine-phosphorylated protein of 60 kD (herein referred to as 'p60') that associates with the SH2 domain of Csk after stimulation of the FcepsilonRI. p60 phosphorylation reached a maximum within one minute and remained constant while the receptors were aggregated; disaggregation of the receptors resulted in rapid dephosphorylation of p60. The phosphorylation of p60 was only detected after activation by IgE and antigen and not by stimulation with PMA and/or ionomycin. Phosphorylated p60 was associated entirely with the membrane fraction of the cells. A considerable fraction of Csk was associated with the membrane in both unstimulated and stimulated cells, this fraction did not change upon activation. p60 coprecipitated with Csk from both unstimulated and FcepsilonRI stimulated cells and was phosphorylated by the immunocomplex. Total kinase activity of Csk immunoprecipitates increased upon FcepsilonRI stimulation. p60 did not react with antibodies to a number of known signaling molecules, including the recently cloned, GAP-associated protein, p62dok. Our data demonstrate that Csk associates with a membrane-anchored protein complex that is directly involved in FcepsilonRI signal transduction.


Assuntos
Proteínas de Ligação a DNA , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ligação a RNA , Receptores de IgE/fisiologia , Quinases da Família src/fisiologia , Animais , Western Blotting , Proteína Tirosina Quinase CSK , Proteína Oncogênica pp60(v-src)/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Fosforilação , Testes de Precipitina , Transdução de Sinais , Frações Subcelulares/química , Células Tumorais Cultivadas , Domínios de Homologia de src/fisiologia , Quinases da Família src/análise , Quinases da Família src/imunologia
6.
FEBS Lett ; 532(1-2): 247-52, 2002 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-12459499

RESUMO

Germline mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are thought to account for a large portion of familial breast cancer. The increased risk of breast cancer in women carrying such mutations suggests that these proteins play a critical role in the growth regulation of mammary epithelial cells. Another protein, Stat5a, is known to be essential for growth and terminal differentiation of breast epithelial cells. Here we show that Stat5a forms a complex with both BRCA1 and BRCA2 in breast epithelial cells upon stimulation with prolactin. In addition, we show that the activity of Stat5a on the beta-casein promoter is modulated by both BRCA1 and BRCA2. This interaction may be important during the expansion and terminal differentiation of breast epithelial cells, as happens during pregnancy and lactation.


Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Mama/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas do Leite , Transativadores/antagonistas & inibidores , Ativação Transcricional , Animais , Mama/citologia , Neoplasias da Mama/metabolismo , Células COS , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Feminino , Testes de Precipitina , Fator de Transcrição STAT5 , Transativadores/metabolismo
9.
J Bacteriol ; 161(1): 207-11, 1985 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-3155715

RESUMO

We describe the cloning and the DNA sequence of the Escherichia coli supH missense suppressor and of the supD60(Am) suppressor genes. supH is a mutant form of serU which codes for tRNASer2. The supH coding sequence differs from the wild-type sequence by a single nucleotide change which corresponds to the middle position of the anticodon. The CGA anticodon of wild-type tRNA and CUA anticodon of supD tRNA is changed to CAA in supH tRNA, which is expected to recognize the UUG leucine codon. We propose that the supH suppressor causes the insertion of serine in response to this codon. The temperature sensitivity caused by supH may be due to a conformation of the CAA anticodon in the supH tRNASer that is slightly different than that in the corresponding tRNALeu species.


Assuntos
Escherichia coli/genética , Aminoacil-RNA de Transferência/genética , Supressão Genética , Anticódon/genética , Bacteriófago lambda/genética , Sequência de Bases , Clonagem Molecular , Mutação , Temperatura , Transdução Genética
10.
J Bacteriol ; 161(1): 219-22, 1985 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2981802

RESUMO

We describe the cloning and the DNA sequence of an amber suppressor allele of the Escherichia coli leuX (supP) gene. The suppressor allele codes for a tRNA with anticodon CUA, presumably derived by a single base change from a CAA anticodon. The mature coding sequence of the leuX gene is preceded by a putative Pribnow box sequence (TATAAT) and followed by a termination signal. The sequence of the leuX-coded tRNA is compared with the sequences of the four remaining tRNALeu isoacceptors of E. coli and with two tRNALeu species from bacteriophage T4 and T5. The conserved nucleotides in these seven tRNAs recognized by E. coli leucyl-tRNA synthetase are located mainly in the aminoacyl stem and in the D-stem/loop region.


Assuntos
Escherichia coli/genética , Genes Bacterianos , Aminoacil-RNA de Transferência/genética , Supressão Genética , Anticódon , Bacteriófago lambda/genética , Bacteriófagos/genética , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Desoxirribonuclease HindIII , Plasmídeos
11.
J Biol Chem ; 271(16): 9698-703, 1996 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-8621646

RESUMO

The protein-tyrosine kinase Csk is one of the main down-regulators of the Src family of kinases. Csk may be involved in the down-regulation of T cell receptor (TCR) signaling by C-terminal tyrosine phosphorylation of Lck and Fyn; however, it is not known how Csk activity is regulated or how it targets these Src family members. We used Jurkat T cells and normal human T cells to examine proteins that bind to the SH2 domain of Csk. In both Jurkat and normal T cells, the Src homology 2 (SH2) domain of Csk bound constitutively to a tyrosine-phosphorylated protein of 60 kDa (p60). The 60-kDa protein was detected in Csk immunoprecipitates from both unstimulated and CD3-stimulated cells. In addition to p60, a protein of 190 kDa coprecipitated with Csk, and both proteins were phosphorylated on tyrosine residues by the immunocomplex. Small amounts of GTPase-activating protein (GAP) were detected in anti-Csk immunoprecipitates, suggesting that p60 may be a GAP-associated protein. Our data demonstrate that the SH2 domain of Csk specifically associates with at least two tyrosine-phosphorylated proteins in normal human T cells, that this association is independent of TCR/CD3 activation, and that Csk may be a part of a multiprotein complex containing GAP.


Assuntos
Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Linfócitos T/enzimologia , Sequência de Aminoácidos , Proteína Tirosina Quinase CSK , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/biossíntese , Glutationa Transferase/isolamento & purificação , Humanos , Dados de Sequência Molecular , Peso Molecular , Fosfoproteínas/isolamento & purificação , Fosfotirosina , Ligação Proteica , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais Cultivadas , Quinases da Família src
12.
J Immunol ; 150(12): 5391-9, 1993 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-7685794

RESUMO

We have cloned and sequenced Amb a V, an Ambrosia artemisiifolia (short ragweed) pollen allergen that has proved to be particularly useful in the genetic analysis of human immune responsiveness. The amino acid sequence deduced from the cloned cDNA sequence corresponds to the published sequence of the protein, except that the cDNA sequence encodes an extra 10 amino acids at the C-terminus. The expressed 55-residue protein is then presumably cleaved enzymatically at the C-terminal lysine found in the 45-residue protein isolated from the pollen. The cloning and sequencing of Amb a V genomic DNA confirmed the cDNA sequence and showed that the Amb a V gene has no introns. Recombinant Amb a V allergen, expressed in Escherichia coli, bound to IgG and IgE antibodies in all Amb a V-allergic individuals tested and inhibition studies demonstrated that the recombinant protein contains a subset of the antigenic epitopes found on native Amb a V. In addition, recombinant Amb a V released histamine efficiently from basophils from Amb a V-allergic patients. The recombinant Amb a V allergen and mutants of Amb a V should, therefore, be useful in studies of allergen epitopes in humans, as well as providing a diagnostic tool.


Assuntos
Alérgenos/genética , Clonagem Molecular , Proteínas de Plantas/genética , Pólen/imunologia , Proteínas Recombinantes/biossíntese , Alérgenos/biossíntese , Alérgenos/imunologia , Sequência de Aminoácidos , Antígenos de Plantas , Sequência de Bases , DNA/química , Escherichia coli/genética , Liberação de Histamina/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia
13.
J Biol Chem ; 267(29): 21119-23, 1992 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-1400422

RESUMO

The Amb V allergens are small, highly disulfide-bonded ragweed pollen allergens that serve as useful models for understanding the molecular basis of the human immune response. We have produced recombinant Amb a V and Amb t V (from short and giant ragweed pollens, respectively) in Escherichia coli and have compared their structural and functional characteristics to those of the native proteins. Recombinant Amb t V was indistinguishable from native Amb t V as determined by NMR spectroscopy and antibody-binding studies. Whereas inhibition analysis showed that recombinant Amb a V possessed only approximately 50% of the antibody-binding activity of native Amb a V, the two proteins were similarly effective in stimulating Amb a V-specific T-cells. Our results demonstrate that even highly homologous proteins exhibit different abilities to fold into their native three-dimensional conformations and establish the potential and limits of expressing the recombinant Amb V allergens intracellularly in E. coli.


Assuntos
Alérgenos/genética , Proteínas de Plantas/genética , Alérgenos/química , Alérgenos/imunologia , Anticorpos , Antígenos de Plantas , Sequência de Bases , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Pólen , Radioimunoensaio , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia
14.
J Biol Chem ; 266(2): 1229-36, 1991 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-1702434

RESUMO

To determine the structure of Amb a I (previously called antigen E), the major allergen from short ragweed, cDNA from pollen was cloned into lambda gt11 and lambda gt10. One of the three distinct clones isolated from the lambda gt11 library by screening with anti-denatured Amb a I antibodies was used to screen both libraries for other Amb a I sequences. Multiple clones were isolated and sequenced and proved to be highly homologous but nonidentical. The clones could be divided into three groups based on sequence similarity, and in accordance with the International Union of Immunological Societies-approved nomenclature (Marsh, D. G., Goodfriend, L., King, T. P., Lowenstein, H., and Platts-Mills, T. A. E. (1986) Bull. WHO 64, 767-770) they have been designated Amb a I.1, Amb a I.2, and Amb a I.3. Clones within a group have greater than 99% identity, and similarity among groups is 85-90% at the nucleotide level. The amino acid sequence of four peptides (isolated from antigen E obtained from the Research Resources Branch of the National Institutes of Health) containing 132 amino acids was identical to one of the clones (Amb a I.1). The presence of multiple naturally occurring isoelectric forms of Amb a I was demonstrated by two-dimensional gel electrophoresis and Western blotting. Southern blot analysis demonstrates the presence of multiple Amb a I-related sequences in the ragweed genome. Amb a I is therefore not a single molecule but rather a family of closely related proteins.


Assuntos
Alérgenos/genética , Proteínas de Plantas , Pólen/genética , Sequência de Aminoácidos , Antígenos de Plantas , Sequência de Bases , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Eletroforese em Gel Bidimensional , Immunoblotting , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , Homologia de Sequência do Ácido Nucleico
15.
J Immunol ; 146(10): 3380-5, 1991 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-1709193

RESUMO

The relationship between the structure and abundance of an inhaled protein and its potential for causing an allergic response is unknown. This study analyzes Amb a I, a family of related proteins formerly known as Ag E, that comprise the major allergens of short ragweed (Ambrosia artemisiifolia). T cells isolated from ragweed allergic patients were shown to proliferate in response to purified Amb a I.1 protein from pollen in in vitro secondary cultures, demonstrating the presence of T cell stimulatory epitopes in Amb a I.1. Three recombinant forms of Amb a I (Amb a I.1, Amb a I.2, and Amb a I.3) obtained as cDNA derived from pollen mRNA were expressed in bacteria. All three recombinant forms were shown to be specifically recognized by pooled ragweed-allergic human IgE on immunoblots, confirming these gene products are important allergens. An examination of immunoblots probed with sera derived from allergic patients revealed a variation in IgE binding specificity. A minority of patients' IgE exclusively reacted with recombinant Amb a I.1, whereas most patients' IgE reacted with Amb a I.1 as well as Amb a I.2 and Amb a I.3 proteins. A detailed examination of the reactivity of T cells derived from 12 allergic patients to these recombinant Amb a I forms revealed that these allergens are all capable of stimulating T cell proliferation in in vitro assays. It is concluded that the allergic response to ragweed pollen in most allergic patients is composed of a reaction to multiple related Amb a I proteins at both the B and T cell levels.


Assuntos
Alérgenos/imunologia , Imunoglobulina E/imunologia , Rinite Alérgica Sazonal/imunologia , Linfócitos T/imunologia , Alérgenos/análise , Células Cultivadas , Epitopos/análise , Humanos , Proteínas de Plantas/imunologia , Pólen/análise , Proteínas Recombinantes/análise , Rinite Alérgica Sazonal/etiologia , Rinite Alérgica Sazonal/terapia
16.
Br J Cancer ; 83(12): 1715-21, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11104571

RESUMO

Fas ligand (FasL) is expressed on some cancers and may play a role in the immune evasion of the tumour. We used immuno-histochemistry to study the expression of Fas and FasL in tissue samples from breast cancer patients, as well as normal breast tissue. Our results show that Fas and FasL are co-expressed both in normal tissue and in breast tumours. Fas and FasL mRNA were expressed in fresh normal and malignant breast tissue, as well as cultured breast epithelium and breast cancer cell lines. Flow cytometry analysis of live cells failed to detect FasL on the surface of normal or malignant breast cells; however, both stained positive for FasL after permeabilization. Fas was detected on the surface of normal breast cells and T47D and MCF-10A cell lines but only intracellularly in other breast cell lines tested. Neither normal breast epithelium nor breast cell lines induced Fas-dependent apoptosis in Jurkat cells. Finally, 20 tumour samples were stained for apoptosis. Few apoptotic cells were detected and there was no increase in apoptotic cells on the borders between tumour cells and lymphocytes. We conclude that FasL is expressed intracellularly in both normal and malignant breast epithelium and unlikely to be important for the immune evasion of breast tumours.


Assuntos
Apoptose , Neoplasias da Mama/metabolismo , Mama/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor fas/metabolismo , Animais , Western Blotting , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células Cultivadas , Epitélio/metabolismo , Epitélio/patologia , Proteína Ligante Fas , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Células Jurkat , Glicoproteínas de Membrana/genética , RNA/genética , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Receptor fas/genética
17.
J Immunol ; 152(6): 2882-9, 1994 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-7511632

RESUMO

We have purified and characterized the Amb p V allergen (A1 variant) from western ragweed (Ambrosia psilostachya) pollen. This allergen was found to be highly cross-reactive with the Amb a VA1 allergen from short ragweed (A. artemisiifolia) pollen in a competitive double-Ab radioimmunoassay (DARIA) and the two allergens showed concordant allergenic potency in histamine-release experiments. We cloned and sequenced several Amb p V genes from western ragweed pollen and flowers by direct PCR of genomic DNA. The amino acid sequences deduced from the nucleotide sequences indicated the presence of multiple forms of Amb p V that could be broadly classified into two groups: Amb p VA and Amb p VB variants. The sequences of the Amb p VA variants are highly homologous to Amb a V (about 90% identity) and very similar to the protein sequence that we obtained. The Amb p VB variants share approximately 65% amino acid homology with Amb a V and have five to seven cysteine residues as compared with the eight found in Amb a V and Amb t V. Two cysteine residues that form disulfide bonds in other Amb Vs (positions 19 and 43 in Amb a V) are replaced by serine and alanine in the Amb p VB1 and Amb p VB2 variants. We have generated model structures of Amb p VA1, VA2, VA3, and VB1 variants from the nuclear magnetic resonance-derived structure of Amb a VA1 by homology modeling. Comparison of antigenic epitopes predicted for the structures of Amb p V variants and Amb a VA1 explains the observed cross-reactivity of the two ragweed proteins and suggests the epitopes likely to be involved in Ab recognition.


Assuntos
Alérgenos/imunologia , Pólen/imunologia , Alérgenos/genética , Alérgenos/isolamento & purificação , Sequência de Aminoácidos , Antígenos de Plantas , Sequência de Bases , Basófilos/metabolismo , Clonagem Molecular , Reações Cruzadas , Liberação de Histamina , Humanos , Dados de Sequência Molecular , Proteínas de Plantas/imunologia
18.
J Allergy Clin Immunol ; 102(3): 443-8, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9768586

RESUMO

BACKGROUND: Recently, we have obtained evidence for linkage between Der p 1-specific IgE antibodies and markers on chromosome 6p21 (HLA-D region) in a genome-wide screening in Caucasian families recruited as a part of the Collaborative Study on the Genetics of Asthma (CSGA). OBJECTIVE: Specific IgE antibodies toward different Dermatophagoides pteronyssinus (Der p) polypeptides were detected by immunoblotting analysis, and the transmission/disequilibrium test (TDT) was performed between specific IgE responsiveness toward each different Der p polypeptide and markers on chromosome 6p21 to better clarify the genetic contribution of HLA-D genes. METHODS: We studied 299 individuals in 45 Caucasian families participating in the CSGA. Serum samples from 137 individuals that showed elevated specific IgE antibodies toward the Der p crude allergen (> -0.5 log IU/mL) by ACCESS immunoassay were subjected to immunoblotting analysis. TDT was conducted between the presence of specific IgE antibodies toward each of 12 different Der p polypeptides and 4 polymorphic markers on chromosome 6p21. RESULTS: The 196-bp allele of D6S1281 and the 104-bp allele of DQCAR showed significant excess transmission to specific IgE responders toward a particular Der p polypeptide (120 kd, 55 kd, 45 kd, or 37 kd). In contrast, the 200-bp allele of D6S1281 and the 204-bp allele of D6S291 showed significantly decreased transmission to specific IgE responders toward a particular Der p polypeptide (120 kd, 90 kd, 52 kd, or 45 kd). Deviation from the expected 50% transmission in heterozygous parents was statistically significant after correcting for multiple comparisons. CONCLUSION: This study supported our previous findings that genes on chromosome 6p21 (HLA-D region) may influence the expression of Der p-specific IgE responsiveness in this Caucasian population. Our results, however, reveal the complexity of genetic regulations of Der p-specific IgE responsiveness by HLA-D genes, suggesting the strong influence of non-HLA loci and perhaps environmental factors for the development of Der p-specific IgE responsiveness.


Assuntos
Asma/genética , Asma/imunologia , Cromossomos Humanos Par 6 , Glicoproteínas/imunologia , Antígenos HLA-D/genética , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Desequilíbrio de Ligação , Ácaros/imunologia , Alelos , Animais , Especificidade de Anticorpos , Antígenos de Dermatophagoides , Mapeamento Cromossômico , Saúde da Família , Feminino , Ligação Genética , Genótipo , Humanos , Immunoblotting , Masculino , Polimorfismo Genético , População Branca/genética
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