RESUMO
PURPOSE: To determine the phenotypic and biochemical characteristics of the p.E139K missense variant in tissue inhibitor of metalloproteinase 3 (TIMP3) associated with Sorsby fundus dystrophy (SFD). METHODS: The coding regions and adjacent intronic sequence of TIMP3 were amplified by polymerase chain reaction and then analyzed by bidirectional sequencing. Allele-specific PCR was used to determine the minimum allele frequency of the mutant allele in ethnically matched controls. Clinical examination and imaging of affected individuals with color fundus photography, scanning laser ophthalmoscope (fundal autofluorescence), and optical coherence tomography was performed. A mutant construct of the TIMP3 protein was created and expressed in human retinal pigment epithelium (ARPE19) cells, which were then assayed for oligomerization and intrinsic matrix metalloproteinase (MMP) inhibitory activity. RESULTS: Three affected individuals from a family of Welsh origin each harbored one allele of the TIMP3 missense variant c.415 G>A, (p.E139K), which was not identified in 534 ethnically matched control chromosomes and thus presumed pathogenic. The mutant protein was shown to dimerize in culture cells and retain its MMP inhibitory activity. Retinal examination was variable between eyes of affected individuals and between family members. Drusen-like deposits were common to all three affected individuals and yellow subretinal deposits, exudative maculopathy, and geographic atrophy were also observed. Optical coherence tomography (OCT) images of affected individuals demonstrated hyperreflectivity of the RPE-photoreceptor-choroid complex. CONCLUSIONS: The TIMP3 p.E139K mutation is another cause of SFD. It is the second TIMP3 sequence variant reported that does not affect the number of cysteine residues in the mutant protein yet dimerizes in vitro. The clinical presentation of this family is in keeping with previous clinical reports of this disorder.
Assuntos
Mutação de Sentido Incorreto , Doenças Retinianas/genética , Doenças Retinianas/fisiopatologia , Inibidor Tecidual de Metaloproteinase-3/genética , Feminino , Fundo de Olho , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Reação em Cadeia da Polimerase , Doenças Retinianas/metabolismo , Análise de Sequência de DNA , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Tomografia de Coerência ÓpticaRESUMO
OBJECTIVE: To determine whether oncostatin M (OSM) + tumour necrosis factor alpha (TNFalpha) induces aggrecanase activity in chondrocyte membranes, to determine the effects of transforming growth factor beta1 (TGFbeta1), interleukin 4 (IL4), and tissue inhibitor of metalloproteinases (TIMPs) on this activity, and to determine whether this activity is due to a known ADAMTS aggrecanase. METHODS: Aggrecanase activity and ability of agents to prevent membrane associated aggrecanase activity were assessed by Western blotting. Expression of known aggrecanases was measured by real time polymerase chain reaction in bovine nasal and human articular chondrocytes. RESULTS: Chondrocyte membrane associated aggrecanase activity and increased mRNA expression of ADAMTS-1, -4, -5, and -9, but not ADAMTS-4 or -15, were enhanced after stimulation by OSM+TNFalpha in bovine chondrocytes. This activity was inhibited by TIMP-3. In human chondrocytes, OSM+TNFalpha also enhanced ADAMTS-1 and -4 expression, but not that of other ADAMTSs. TNFalpha alone induced ADAMTS-9 expression, whereas OSM addition caused suppression. Both TGFbeta1 and IL4 blocked membrane associated aggrecanase activity and decreased OSM+TNFalpha-induced expression of ADAMTS-9 in bovine and human chondrocytes. IL4 down regulated ADAMTS-4 mRNA, whereas TGFbeta1 increased this expression in both bovine and human chondrocytes. CONCLUSIONS: OSM+TNFalpha up regulates membrane associated aggrecanase activity and several ADAMTS aggrecanase mRNAs in chondrocytes. The chondroprotective effects of IL4 and TIMP-3 suggest that they may have therapeutic benefit for aggrecanolysis, whereas the differential inhibitory effects of TGFbeta1 may limit its therapeutic potential. Induced membrane associated aggrecanase activity is distinct from known soluble ADAMTS aggrecanases and merits further investigation.
Assuntos
Condrócitos/efeitos dos fármacos , Endopeptidases/metabolismo , Peptídeos/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Western Blotting , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Células Cultivadas , Condrócitos/enzimologia , Endopeptidases/genética , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-4/farmacologia , Oncostatina M , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Inibidor Tecidual de Metaloproteinase-3/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
During bone formation, there are numerous pivotal changes in the interrelationships between osteoblasts and molecules of the extracellular matrix (ECM). Consequently, the mechanisms that underlie the temporal and spatial distribution of ECM molecules in bone are of considerable interest in understanding its formation. A subfamily of a disintegrin and metalloproteinase (ADAMs) has been identified, which contain thrombospondin-like motifs (ADAMTS), and can break down several ECM molecules. Using reversed transcribed PCR, we identified ADAMTS-1, -4 and -5 mRNA expression in cultures of rat osteoblasts treated with ascorbic acid, beta-glycerophosphate and dexamethasone, molecules known to drive osteoblast differentiation. Of these, ADAMTS-1 followed most closely the osteogenic marker osteocalcin during in vitro mineralisation. Consequently, we studied, in detail, protein expression of ADAMTS-1 during in vitro osteogenesis together with ADAMTS-1 immunohistochemistry staining of sections from 2- and 10-day-old rat femur. Western analysis of osteoblast proteins showed ADAMTS-1 products that correspond well with both full-length and furin-processed species. In the ECM laid down by osteoblasts, only the mature secreted protein (approximately 90 kDa) and its accumulation during the later stages of osteogenesis in vitro were noticed. Furthermore, immunostaining with an antibody recognising ADAMTS-1 demonstrated strong expression around mineralised nodules and intense focal staining of putative new areas of nodule formation in vitro. Finally, immunohistochemistry of 2- and 10-day-old rat femur localised ADAMTS-1 protein to regions associated with osteogenesis. These data show that ADAMTS-1 protein accumulates in osteoblast ECM during differentiation. Furthermore, the focalised expression of ADAMTS-1 in regions of osteogenesis, both in vitro and in vivo, implicates this multifunctional protein to be involved in mineralised nodule and bone formation.
Assuntos
Desintegrinas/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Metaloendopeptidases/biossíntese , Osteoblastos/enzimologia , Osteogênese/fisiologia , Regulação para Cima/fisiologia , Proteínas ADAM , Proteína ADAMTS1 , Animais , Células Cultivadas , Desintegrinas/genética , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Metaloendopeptidases/genética , Proteoglicanas/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
In this study, we identified the expression and the regulation of ADAM members (a disintegrin and metalloprotease) at both gene and protein levels during human osteoclast differentiation and activity. Human peripheral blood monocytes (HPBMC) treated with M-CSF and RANKL were used as an in vitro fusion model. In parallel, we used human osteoclastoma (OCL) tumor as a source of mature osteoclasts, and human osteoblastic cells as a control representing nonfusing and non-resorbing bone cells. RT-PCR using ADAM-specific primers enabled us to identify the expression of ADAM 8, 9, 10, 15, 17, and 28 in both osteoclasts and osteoblasts. Using primers specific for each ADAM 12 isoform (L and S), we observed a strong signal for both forms (ADAM 12L and ADAM 12S) in osteoblastic cells, while only ADAM 12S was detectable in HPBMC-derived osteoclasts and osteoclastoma. Gene regulation was studied using real-time PCR analysis performed during HPBMC differentiation; this showed a progressive increase of ADAM 12 mRNA level from day 1 to 8 of the culture, while at around day 9, ADAM 12 mRNA level decreased 2-fold. We also showed that ADAM 8, ADAM 17, and ADAM 28 decreased according to the stage of HPBMC differentiation or fusion. ADAM 10 was unaltered during cell fusion. However, confocal immunolocalization showed that ADAM 10 protein re-localized from the nuclei and cytoplasm to the plasma membrane during culture and to the ruffled border in resorbing cells. The same re-localization process was observed using an ADAM 12S-specific antibody during HPBMC differentiation. Between days 12 and 14, ADAM 12 co-localized with the F-actin ring, and at day 15, a strong signal was also present in ruffled border or sealing zone area of osteoclasts. Our results describe the expression and regulation of various ADAMs in human bone cells and the selective expression of ADAM 12L in osteoblasts. Our gene regulation and protein localization studies suggest a function for ADAM 10 and ADAM 12S in the formation of osteoclasts from HPBMC and resorption activity.
Assuntos
Desintegrinas/biossíntese , Metaloendopeptidases/biossíntese , Osteoclastos/metabolismo , Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular Tumoral , Desintegrinas/genética , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Fator Estimulador de Colônias de Macrófagos/farmacologia , Glicoproteínas de Membrana/farmacologia , Metaloendopeptidases/genética , Monócitos/citologia , Monócitos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Ligante RANK , RNA Mensageiro/biossíntese , Receptor Ativador de Fator Nuclear kappa-BRESUMO
AIMS: To describe the phenotype in three family members affected by a novel mutation in the gene coding for the enzyme tissue inhibitor of metalloproteinase-3 (TIMP-3). METHODS: Three members of the same family were seen with a history of nyctalopia and visual loss due to maculopathy. Clinical features were consistent with Sorsby's fundus dystrophy. Exon 5 of the gene coding for TIMP-3 was amplified by the polymerase chain reaction, single strand conformation polymorphism analysis undertaken and exon 5 amplicons were directly sequenced. RESULTS: Onset of symptoms was in the third to fourth decade. Five of six eyes had geographic macular atrophy rather than neovascularisation as a cause for central visual loss. Peripheral retinal pigmentary disturbances were present. Scotopic ERGs were abnormal in all three. Mutation analysis showed a G-->T transversion in all three resulting in a premature termination codon, E139X, deleting most of the carboxy terminal domain of TIMP-3. CONCLUSIONS: The patients described had a form of Sorsby's fundus dystrophy which fell at the severe end of the spectrum of this disease. Postulated disease mechanisms include deposition of dimerised TIMP-3 protein.
Assuntos
Códon sem Sentido , Degeneração Macular/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Adulto , Idoso , Eletrorretinografia , Angiofluoresceinografia , Humanos , Pessoa de Meia-Idade , FenótipoRESUMO
Sorsby's fundus dystrophy (SFD) is a dominantly inherited degenerative disease of the retina that leads to loss of vision in middle age. It has been shown to be caused by mutations in the gene for tissue inhibitor of metalloproteinases-3 (TIMP-3). Five different mutations have previously been identified, all introducing an extra cysteine residue into exon 5 (which forms part of the C-terminal domain) of the TIMP-3 molecule; however, the significance of these mutations to the disease phenotype was unknown. In this report, we describe the expression of several of these mutated genes, together with a previously unreported novel TIMP-3 mutation from a family with SFD that results in truncation of most of the C-terminal domain of the molecule. Despite these differences, all of these molecules are expressed and exhibit characteristics of the normal protein, including inhibition of metalloproteinases and binding to the extracellular matrix. However, unlike wild-type TIMP-3, they all form dimers. These observations, together with the recent finding that expression of TIMP-3 is increased, rather than decreased, in eyes from patients with SFD, provides compelling evidence that dimerized TIMP-3 plays an active role in the disease process by accumulating in the eye. Increased expression of TIMP-3 is also observed in other degenerative retinal diseases, including the more severe forms of age-related macular degeneration, the most common cause of blindness in the elderly in developed countries. We hypothesize that overexpression of TIMP-3 may prove to be a critical step in the progression of a variety of degenerative retinopathies.
Assuntos
Degeneração Macular/genética , Mutação , Inibidor Tecidual de Metaloproteinase-3/genética , Animais , Células COS , Feminino , Humanos , Masculino , Linhagem , Fenótipo , Polimorfismo Conformacional de Fita Simples , Conformação Proteica , Inibidor Tecidual de Metaloproteinase-3/químicaRESUMO
In Drosophila melanogaster, the role of the metallodisintegrin, Kuzbanian (kuz), is thought to involve activation of the Drosophila Notch receptor that plays a role in cell-fate determination during neurogenesis and myoblast differentiation. To understand the possible function(s) of a-disintegrin and metalloproteinase (ADAM10), the mammalian ortholog of kuz, in the skeleton, we studied its expression as well as the messenger RNA (mRNA) encoding one candidate substrate, the mammalian Notch2 receptor in bone, bone cells, and cartilage. In sections of neonatal rat tibiae, ADAM10 is expressed in specific regions of articular cartilage and metaphyseal bone. Expression of ADAM10 in articular cartilage occurs predominantly in superficial chondrocytes and becomes more sporadic with increasing distance from the articular surface. In bone, ADAM10 is expressed by periosteal cells, osteoblasts, and osteocytes at locations of active bone formation. Osteoclasts did not express ADAM10. Notch2 mRNA expression was not detectable in superficial chondrocytes. However it colocalized at all sites of ADAM10 expression in bone cells. In vitro, both primary human osteoblasts and osteoblast cell lines expressed a single 4.5 kb and 7.5 kb transcript of ADAM10 and the Notch2 receptor homolog, respectively. Subcellular localization of the ADAM10 protein in MG-63 cells was determined using immunofluorescent techniques. These observations showed clearly that the ADAM10 protein was expressed in the trans-Golgi network and on the plasma membrane. Western blot analysis of fractionated cells showed that, in the plasma membrane fraction, the previously characterized 58 kDa and 56 kDa isoforms were present, whereas, in the trans-Golgi network, the ADAM10 protein was present in several additional bands, possibly indicative of further interdomain processing of the ADAM10 protein. The metallodisintegrins (ADAMs) have several putative functions, including modulation of cell adhesion, membrane-associated proteolysis, and cell-cell signaling. These observations suggest that, in bone but not cartilage, ADAM10 has catalytic activity within the transGolgi network and may play a role in the activation of Notch receptor homologs. This implicates ADAM10 in cell-fate determination of osteoblast progenitor cells, possibly during skeletal development and normal bone remodeling. Plasma-membrane-associated ADAM10 may confer alternative functions.
Assuntos
Osso e Ossos/química , Desintegrinas/análise , Proteínas de Membrana/análise , Metaloendopeptidases/análise , Receptores de Superfície Celular/análise , Sequência de Aminoácidos , Animais , Western Blotting , Catálise , Linhagem Celular , Separação Celular , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Osteoblastos/química , Ratos , Receptores Notch , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/químicaRESUMO
During reproduction, female teleost fish display increased plasma estrogen and greatly increased total plasma calcium concentrations; the main source of this calcium seems to be the scale. Osteonectin, a collagen-binding glycoprotein, is a major noncollagenous constituent of mammalian bone and is a product mainly of the osteoblasts. RT-PCT has been applied to clone and sequence part of the osteonectin gene from the goldfish, Carassius auratus. The use of a goldfish scale cell line (GFS) and a specific probe to goldfish osteonectin mRNA has allowed the study of the potential effects of estrogen and other calcitropic hormones on the cells derived from the scales. Osteonectin mRNA was detected in teleost bone, scale, and GFS cells by Northern blot analysis, hybridising to a transcript of approximately 1.6 kb. Expression of osteonectin mRNA was markedly down-regulated by 17beta-estradiol (10(-8) to 10(-11) M) in a dose-dependent fashion but was unaffected by calcitriol, TGFbeta, IL-1beta, calcitonin, and PTHrP. Down-regulation of osteonectin by estrogen is further evidence that estrogen participates in calcium homeostasis during vitellogenesis, acting directly on the cells responsible for matrix and mineral fluxes in scales.
Assuntos
Estrogênios/fisiologia , Carpa Dourada/genética , Osteonectina/genética , Sequência de Aminoácidos , Animais , Northern Blotting/veterinária , Osso e Ossos/química , Calcitriol/farmacologia , Bovinos , Galinhas , Clonagem Molecular , DNA Complementar/química , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica , Carpa Dourada/fisiologia , Humanos , Interleucina-1/farmacologia , Dados de Sequência Molecular , Proteína Relacionada ao Hormônio Paratireóideo , Reação em Cadeia da Polimerase/veterinária , Proteínas/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Xenopus laevisRESUMO
A transient COS-7 cell expression system was used to investigate the functional domain arrangement of tissue inhibitor of metalloproteinases-3 (TIMP-3), specifically to assess the contribution of the amino- and carboxyl-terminal domains of the molecule to its matrix metalloproteinase (MMP) inhibitory and extracellular matrix (ECM) binding properties. Wild type TIMP-3 was entirely localized to the ECM in both its glycosylated (27 kDa) and unglycosylated (24 kDa) forms. A COOH-terminally truncated TIMP-3 molecule was found to be a non-ECM bound MMP inhibitor, whereas a chimeric TIMP molecule, consisting of the NH2-terminal domain of TIMP-2 fused to the COOH-terminal domain of TIMP-3, displayed ECM binding, albeit with a lower affinity than the wild type TIMP-3 molecule. Thus the functional domain arrangement of TIMP-3 is analogous to that seen in TIMP-1 and -2, namely that the NH2-terminal domain is responsible for MMP inhibition whereas the COOH-terminal domain is most important in mediating the specific functions of the molecule. A mutant TIMP-3 in which serine 181 was changed to a cysteine, found in Sorsby's fundus dystrophy, a hereditary macular degenerative disease, was also expressed in COS-7 cells. This gave rise to an additional 48-kDa species (possibly a TIMP-3 dimer) that retained its ability to inhibit MMPs and localize to the ECM. These data favor the hypothesis that the TIMP-3 mutations seen in Sorsby's fundus dystrophy contribute to disease progression by accumulation of mutant protein rather than by the loss of functional TIMP-3.
Assuntos
Degeneração Macular/genética , Mutação , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Animais , Células COS , Humanos , Proteínas Recombinantes de Fusão/genética , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-3/genética , TransfecçãoRESUMO
The metallo-disintegrins (ADAMs) are a family of mammalian proteins with significant amino acid sequence identity and a domain organisation similar to the snake venom metalloproteinases (reprolysins). They have been implicated in a wide variety of processes such as cell-cell and cell matrix adhesion and proteolysis of the extracellular matrix in a wide variety of cell types. They may also be involved in events such as the processing of plasma membrane proteins, proteolysis in the secretory pathway and pro-cytokine conversion processes. Due to the close relationship of the ADAM proteins with snake venom enzymes which have been demonstrated to be type IV collagenases, we investigated whether purified bovine ADAM10 could cleave basement membrane type IV collagen. We show here that ADAM10 purified from bovine kidney can cleave a basement membrane collagen type IV preparation as assessed by SDS-PAGE analysis and novel epitope recognition with a specific antibody to type IV collagen. The demonstration that a metallo-disintegrin displays a type IV collagenase activity may be relevant to tumour metastasis and may have general relevance to extracellular re-modeling in renal pathology and a variety of other pathological states where compromise of the basement membrane is involved.
Assuntos
Colágeno/metabolismo , Colagenases/metabolismo , Rim/enzimologia , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Proteínas ADAM , Animais , Membrana Basal/química , Western Blotting , Bovinos , Desintegrinas/metabolismo , Proteína Básica da Mielina/metabolismo , Especificidade por SubstratoRESUMO
ADAMs (A disintegrin and metalloproteinase) are a recently discovered family of proteins with significant primary sequence similarity to the reprolysin family of snake venomases. These ADAMs closest known homologues are the type III reprolysin enzymes which have been demonstrated to be, among other things potent type IV collagenases. ADAMs are putative membrane linked proteins with several domains including a metalloproteinase domain, a potential integrin binding domain, a cysteine rich sequence and an EGF like sequence. They have been implicated in a wide variety of functions including basement membrane degradation and cell-cell and cell-matrix interactions. We have used RT-PCR and Northern blotting to characterise the expression of members of this family in cells derived from a variety of haematological malignancies including leukaemia (HL60 and Jurkat), erythroleukaemia (K562), lymphoma (U937 and Cupillo) and myeloma (U266B1). We find clear expression of four members of this novel family of proteins but note differences in the expression levels of each member. The ADAMs known as MADM (ADAM10), MCMP (ADAM12, MDC9) and Metargidin (ADAM15) which all possess potentially active metalloproteinase domains are expressed in all these cell types to significant levels. The putative tumour suppressor gene MDC (ADAM11) is expressed at very low levels in all cells examined. As ADAMs may have both potential metalloproteinase activity and adhesive domains we wish to explore the role of these proteins with regard to pathophysiology of haematological malignancy such as egression of leukaemic cells from the bone marrow.
Assuntos
Regulação Enzimológica da Expressão Gênica , Leucemia/enzimologia , Linfoma/enzimologia , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Northern Blotting , Clonagem Molecular , Primers do DNA , DNA Complementar , Humanos , Integrinas/química , Integrinas/genética , Proteínas de Membrana/química , Metaloendopeptidases/química , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Using resting chondrocytes derived from human articular femoral head and a conditionally immortalised human articular chondrocyte cell line we have studied the expression of members of the novel metalloproteinase/disintegrin family termed ADAM. Using RT-PCR we can detect the expression of ADAM-12 a novel family member isolated from myeloma cells [1]. We also find expression of ADAM 10 a functional metalloproteinase/disintegrin first isolated from bovine brain and ADAM-15 a metallodisintegrin isolated from mammary derived epithelial cells. Northern blotting was used to confirm expression. One main transcript is visible for ADAM-12 whereas both ADAM-10 and ADAM-15 have multiple transcripts indicating possible RNA variants potentially derived from alternative splicing or alternative use of polyadenylation sites. Since chondrocytes are proposed as an important source of metalloproteinase enzymes involved in joint pathology the potential relevance of the expression of these molecules to connective tissue disorders is discussed.
Assuntos
Cartilagem Articular/enzimologia , Proteínas de Membrana/biossíntese , Metaloendopeptidases/biossíntese , Transcrição Gênica , Proteínas ADAM , Proteína ADAM10 , Proteína ADAM12 , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Sequência de Bases , Encéfalo/enzimologia , Cartilagem Articular/citologia , Bovinos , Linhagem Celular Transformada , Membrana Celular/enzimologia , Células Cultivadas , Primers do DNA , Feminino , Humanos , Proteínas de Membrana/química , Metaloendopeptidases/química , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
We have isolated a novel cDNA from human myeloma cells encoding a member of the reprolysin family of metalloproteinases. Derived amino acid sequence predicts a protein of approx. 76 kDa. The open reading frame predicts the presence of a leader peptide, a pro-peptide with a 'cysteine switch', a metalloproteinase domain, a disintegrin-like domain, a cysteine-rich domain, an epidermal growth factor-like domain and a putative transmembrane sequence. Expression of the mRNA for this metalloproteinase has been demonstrated in human myeloma cells.
Assuntos
Metaloendopeptidases/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Linhagem Celular , Membrana Celular/enzimologia , Clonagem Molecular , Primers do DNA , Desintegrinas/química , Feminino , Biblioteca Gênica , Humanos , Metaloendopeptidases/química , Dados de Sequência Molecular , Mieloma Múltiplo , Placenta/enzimologia , Reação em Cadeia da Polimerase , Gravidez , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
This paper reviews the range of health surveillance activities which can be utilized in the workplace by occupational health professionals for assessing fitness for work and contributing to the prevention of occupational illness and promotion of good health. The systematic approach described categorizes health surveillance procedures into occupational or non-occupational, risk-based or unfocused, and as primary, secondary or tertiary preventive measures. All categories of health surveillance are currently being practised to some extent, but the type of surveillance may not match the needs of the workplace in some situation. In order to aid health professionals in deciding which procedures should be implemented, recommendations based on an assessment of health risks are made. The key proposal is to establish a minimum level of periodic health surveillance for all workers based on a targeted lifestyle health risk assessment and a structured health questionnaire. Additional procedures can then be added sequentially as appropriate to manage any health risks in the workplace. The role of the unfocused periodic general medical examination is discussed in the context of the systematic approach and allows occupational professionals to critically appraise its usefulness.
Assuntos
Indicadores Básicos de Saúde , Doenças Profissionais/epidemiologia , Saúde Ocupacional , Vigilância da População , Promoção da Saúde , Humanos , Estilo de Vida , Local de TrabalhoRESUMO
The coding sequence for rat testis thimet oligopeptidase (TOP) (EC 3.4.24.15) was placed under the control of the T7 polymerase/promoter system. Cultures of Escherichia coli transfected with the resulting plasmid expressed the enzyme as a soluble cytoplasmic protein. Medium-scale cultures allowed isolation of the enzyme in quantities of tens of milligrams. The availability of the recombinant enzyme permitted the determination of such chemical properties as epsilon 280 (48,960), zinc content (2 atom/molecule) and available thiol content (8-10/molecule) for TOP. The recombinant enzyme showed the catalytic activities previously reported for the naturally occurring enzyme, so that we can now conclude with confidence that these are all due to TOP and there is no need to postulate the existence of separate 'Pz-peptidase' or 'endo-oligopeptidase A' enzymes.
Assuntos
Metaloendopeptidases/metabolismo , Alquilantes/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Catálise , Clonagem Molecular , Escherichia coli , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Metaloendopeptidases/genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Compostos de Sulfidrila/análise , Zinco/análiseRESUMO
The deduced amino acid sequence of pig liver soluble angiotensin II-binding protein [Sugiura, Hagiwara and Hirose (1992) J. Biol. Chem. 267, 18067-18072] is similar over most of its length to that reported for rat testis thimet oligopeptidase (EC 3.4.24.15) by Pierotti, Dong, Glucksman, Orlowski and Roberts [(1990) (Biochemistry 29, 10323-10329]. We have found that homogeneous rat testis thimet oligopeptidase binds angiotensin II with the same distinctive characteristics as the pig liver protein. Analysis of the nucleotide sequences reported for the two proteins pointed to the likelihood that sequencing errors had caused two segments of the amino acid sequence of the rat protein to be translated out of frame, and re-sequencing of selected parts of the clone (kindly provided by the previous authors) confirmed this. The revised deduced amino acid sequence of rat thimet oligopeptidase contains 687 residues, representing a protein of 78,308 Da, and is more closely related to those of the pig liver protein and other known homologues of thimet oligopeptidase than that described previously.
Assuntos
Angiotensina II/metabolismo , Metaloendopeptidases/genética , Receptores de Angiotensina/genética , Testículo/enzimologia , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Fígado/enzimologia , Masculino , Dados de Sequência Molecular , Ratos , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solubilidade , SuínosRESUMO
The linked structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been altered by site-directed mutagenesis and placed under the control of an inducible phage-T7-specific plasmid promoter in Escherichia coli. Conditions have been found under which both alpha- and beta-subunits are produced in soluble form, in near 1:1 ratio, and assemble to form apo-mutase totalling about 5% of the total cellular protein. Methylmalonyl-CoA mutase purified from these cells could be readily converted into the holoenzyme by addition of adenosylcobalamin. The active holoenzyme apparently crystallizes in the same space group as an inactive corrinoid-containing form of the enzyme obtained previously.
Assuntos
Cobamidas/farmacologia , Escherichia coli/enzimologia , Isomerases/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Propionibacterium/enzimologia , Sequência de Bases , Clonagem Molecular , Cristalização , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/isolamento & purificação , Dados de Sequência Molecular , Mutação , Plasmídeos , Propionibacterium/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been cloned, with the use of synthetic oligonucleotides as primary hybridization probes. The genes are closely linked and are transcribed in the same direction. Nucleotide sequence analysis of 4.5 kb of DNA encompassing both genes allowed us to infer the complete amino acid sequence of the two subunits: the beta-subunit is the product of the upstream gene, and consists of 638 amino acid residues (Mr 69465) and the alpha-subunit consists of 728 amino acid residues (Mr 80,147). There is a very close structural homology between the two subunits, reflecting the probable duplication of a common ancestral gene. A sequence present only in the alpha-subunit is significantly homologous to a portion of the sequence of the methylmalonyl-CoA-binding subunit of transcarboxylase from P. shermanii [Samols, Thornton, Murtif, Kumar, Haase & Wood (1988) J. Biol. Chem. 263, 6461-6464], and this homologous region may form part of the CoA ester-binding site in both enzymes.
Assuntos
Genes Bacterianos , Isomerases/genética , Metilmalonil-CoA Mutase/genética , Propionibacterium/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Genes , Dados de Sequência Molecular , Propionibacterium/enzimologia , Homologia de Sequência do Ácido NucleicoRESUMO
After the accidental ascent of a diving bell from 80 m, one diver died from pulmonary barotrauma and the other-though grossly ill-survived. After recompression therapy, this diver was tetraplegic with evidence of patchy microcirculatory damage of brain, cord, liver, kidneys, and gut. All systems eventually returned to normal, except the spinal cord, mainly because of the post-recompression phase of management, in which pharmacological doses of steroids, hyperbaric oxygen, and dextran were used. Although function returned in the upper limbs, the diver remained paraplegic.