RESUMO
PURPOSE: The most common sites of low-energy trauma fractures are the femur, vertebra, humerus, and forearm. Ankle fractures have significant morbidity and high costs for surgical procedure. Forearm fractures are common nonvertebral fractures. Forearm fractures are classified as fragility fractures and predictive for fractures at other sites, although do not allow osteoporosis diagnosis. It is controversial whether ankle fractures are osteoporosis fractures. METHODS: Retrospective observational study, with secular trend analysis, in patients over 50 years old admitted in the Brazilian Public Health System, from 2004 to 2013. We collected hospitalization data according to the ICD-10 for low-trauma ankle and forearm fractures. Fracture rate was calculated according to gender, age, and geographic region, performed linear regression analysis, and estimated fracture rates for 2030. Comparison of ankle and forearm rates was also performed, grouping them in 3-year block. ANOVA test was used to compare each block. RESULTS: Ankle fracture rate was 21.39 fractures per 100,000 inhabitants, 23.98 in females and 18.49 in males. Fracture rates were higher in the South and Southeast regions. In absolute numbers, although ankle fracture rate increased with age, there was a significant decrease in the population over 80 years old. Data showed stabilization in ankle fractures from 2004 to 2013, in women and men. In 3-year block analysis, men had higher ankle fracture rates than forearm. However, in women, forearm rates were higher than ankle. CONCLUSION: Our data suggest that ankle fractures in men would be considered as a sentinel fracture with a similar clinical impact of forearm fracture.
Assuntos
Fraturas do Tornozelo , Traumatismos do Antebraço , Fraturas Ósseas , Fraturas por Osteoporose , Idoso , Idoso de 80 Anos ou mais , Tornozelo , Fraturas do Tornozelo/epidemiologia , Brasil/epidemiologia , Feminino , Traumatismos do Antebraço/epidemiologia , Fraturas Ósseas/epidemiologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Fraturas por Osteoporose/diagnóstico , Fraturas por Osteoporose/epidemiologia , Estudos RetrospectivosRESUMO
Endostatin (ES) inhibits angiogenesis, reducing tumor growth in animal models. However, it has low therapeutic effect in human clinical trials. BAX is a member of the BCL-2 family of proteins; its proapoptotic (BH3) domain interacts with other members of the family in the cytoplasm, to induce apoptosis. Here, we fused the BAX BH3 domain with murine ES, to enhance ES potency. Endothelial cells specifically internalize the fusion protein ES-BAX. The presence of the BAX domain enhances endothelial cell death by apoptosis by 1.8-fold and diminishes microvessel outgrowth in the rat aortic ring assay by 6.5-fold. Daily injections of 15 µg of ES-BAX/g in tumor-bearing mice reduce tumor weight by 86.9% as compared with ES-treated animals. Co-immunoprecipitation assays confirmed that ES-BAX interacts with members of the BCL-2 family. Also, ES interacts with BCL-2, BCL-XL, and BAK in endothelial cell lysates, suggesting a potential new mechanism for the apoptosis induction by ES. The superiority of the ES-BAX antiangiogenic effect indicates that this fusion protein could be a promising therapeutic alternative to treat cancer.
Assuntos
Inibidores da Angiogênese/toxicidade , Apoptose/efeitos dos fármacos , Endostatinas/toxicidade , Proteína X Associada a bcl-2/metabolismo , Sequência de Aminoácidos , Inibidores da Angiogênese/uso terapêutico , Animais , Linhagem Celular Tumoral , Endostatinas/genética , Endostatinas/uso terapêutico , Escherichia coli/metabolismo , Neoplasias Renais/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes de Fusão/toxicidade , Transplante Homólogo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genéticaRESUMO
C-type lectins are animal proteins that contain at least one carbohydrate recognition domain (CRD) capable of mediating sugar and calcium binding. Carbohydrate recognition is directly required for some biological functions, including the innate immune response. We cloned two novel C-type lectin (CTL) precursors from the commercial marine shrimp Litopenaeus vannamei. The cloned cDNAs encompass ORFs of 1044 nucleotides and encode highly similar two-domain polypeptides of 347 residues. The predicted proteins, LvCTL-br1 and -br2, contain the consensus triad that recognizes galactose (-GlnProAsp-) in CRD1 but also contain a mutated mannose-binding site (-GluProAsn-) in the second domain (CRD2). Phylogenetic analysis of LvCTL-br1 and -br2 and hundreds of CTL-like domain-containing proteins have allowed grouping of penaeid shrimp CTLs into three functional clusters. Reverse transcription coupled to PCR indicated that LvCTL-br1 expression is induced in shrimp gills upon IHHNV infection. Computational molecular modeling of LvCTL-br1 and -br2 revealed that three amino acid substitutions in CRD1 occur near the sugar binding site. Also, the 3-D models show a long loop of LvCTL-br1 CRD2 that might accommodate complex sugars. The structural data, evolutionary history and functional analysis support the hypothesis that gene duplication and accelerated evolution have caused functional diversification of penaeid shrimp C-type lectins.
Assuntos
Lectinas Tipo C/genética , Lectinas de Ligação a Manose/genética , Mutação , Penaeidae/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Evolução Molecular , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Gyroxin is one of main serine proteases of Crotalus durissus terrificus venom, representing about 2% of the protein content in the crude venom. It is a 33 kDa glycoprotein with 3.8% by weight of sugar moiety. This toxin induces hemotoxicity in mice and a neurological condition called barrel rotation syndrome. In the present work, we report the molecular cloning of five new nucleotide sequences from a cDNA library of the venom glands of a single specimen of C. d. terrificus. These sequences have been analyzed in silico with respect to their cDNA organization and similarity with other snake venom serine proteases (SVSPs). We also describe a rapid and efficient method for screening vectors for mammalian cell expression, based on the fact that SVSPs are difficult-to-express toxins due to the presence of several disulfide bonds and glycosylation in their structures. Thus, one of the Gyroxin cDNAs was subcloned into pSectag2 HygroA and pED vectors and used to transfect COS-7 cells. Expression of the functional recombinant Gyroxin isoform was achieved with this cell line with esterase activity in the conditioned culture medium, as revealed by immunoblot of secreted protein and standard anti-crotalic serum from Butantan Institute.
Assuntos
Venenos de Crotalídeos/biossíntese , DNA Complementar/biossíntese , Glândulas Exócrinas/química , Serina Endopeptidases/biossíntese , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Clonagem Molecular , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/genética , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Esterases/química , Esterases/metabolismo , Glândulas Exócrinas/enzimologia , Biblioteca Gênica , Vetores Genéticos , Camundongos , Peso Molecular , Plasmídeos/genética , Proteínas Recombinantes/genética , Serina Endopeptidases/genéticaRESUMO
The phospholipase A2 superfamily encompasses 15 groups that are classified into: secreted PLA2 (sPLA2); cytosolic PLA2 (cPLA2); Ca2+-independent intracellular PLA2 (iPLA2); platelet-activating factor acetylhydrolase (PAF-AH); and lysosomal PLA2. Currently, approximately 700 PLA2 sequences are known, of which 200 are obtained from the venom gland of Crotalinae snakes. However, thus far, little information is available on cloning, purification and structural characterization of PLA2 from Crotalus durisssus cascavela venom gland. In the present work, we report the molecular cloning of a novel svPLA2 from C. d. cascavella (Cdc), a predominant rattlesnake subspecies in northeastern Brazil. The Cdc svPLA2 cDNA precursor is 689 nucleotides long and encodes a protein of 138 amino acid residues, with a calculated molecular mass of approximately 13,847 Da and an estimated isoelectric point of 5.14. Phylogenetic analysis of Crotalinae PLA2 reveals that Cdc PLA2 clustered with other acidic type IIA PLA2 homologues is also present in the venom of North American rattlesnakes. Hitherto, this study presents a novel PLA2 cDNA precursor from C. d. cascavella and data reported herein will be useful for further steps in svPLA2 purification and analysis.(AU)
Assuntos
Animais , Masculino , Clonagem Molecular , Venenos de Crotalídeos , Fosfolipases A2RESUMO
The phospholipase A2 superfamily encompasses 15 groups that are classified into: secreted PLA2 (sPLA2); cytosolic PLA2 (cPLA2); Ca2+-independent intracellular PLA2 (iPLA2); platelet-activating factor acetylhydrolase (PAF-AH); and lysosomal PLA2. Currently, approximately 700 PLA2 sequences are known, of which 200 are obtained from the venom gland of Crotalinae snakes. However, thus far, little information is available on cloning, purification and structural characterization of PLA2 from Crotalus durisssus cascavela venom gland. In the present work, we report the molecular cloning of a novel svPLA2 from C. d. cascavella (Cdc), a predominant rattlesnake subspecies in northeastern Brazil. The Cdc svPLA2 cDNA precursor is 689 nucleotides long and encodes a protein of 138 amino acid residues, with a calculated molecular mass of approximately 13,847 Da and an estimated isoelectric point of 5.14. Phylogenetic analysis of Crotalinae PLA2 reveals that Cdc PLA2 clustered with other acidic type IIA PLA2 homologues is also present in the venom of North American rattlesnakes. Hitherto, this study presents a novel PLA2 cDNA precursor from C. d. cascavella and data reported herein will be useful for further steps in svPLA2 purification and analysis.
Assuntos
Animais , Masculino , Clonagem Molecular , Venenos de CrotalídeosRESUMO
Snake venom metalloproteases encompass a large family of toxins, with approximately 200 members already catalogued, which exhibit a diversity of structures and biological functions. From this relatively large number, only a dozen examples of apoptosis-inducing metalloproteases, like VAP1 and 2 from the venom of Crotalus atrox, are known. Since most VAP1-like toxins ever characterized were purified from the venom of Viperidae species inhabiting diverse places on earth, we investigate the expression of VAP-like metalloproteases in the venom gland of three representative pit vipers of the Brazilian territory. By molecular cloning and quantitative real-time polymerase chain reaction, using as calibrator gene the Crotalus durissus terrificus homolog of VAP1, named crotastatin, it is reported here that VAP1/crotastatin-like homologues in the venom gland of Bothrops atrox, C. d. cascavella and Lachesis m. rhombeata are expressed at different levels. Hence, batroxstatins, the crotastatin-like precursors from B. atrox, are expressed 87 times more than crotastatin-1, from C. d. cascavella, and 7.5-fold that lachestatins, from L. m. rhombeata. Moreover, in silico structural analysis of amino acid sequences indicates that batroxstatin-2, crotastatins and lachestatin-1 and -2 which share the archetypal motifs and metal- binding sites of VAP1, are subgrouped in a branch that comprises some apoptosis-inducing toxins.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Venenos de Crotalídeos/genética , Crotalus/genética , Metaloendopeptidases/genética , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Simulação por Computador , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Crotalus/metabolismo , Expressão Gênica , Biblioteca Gênica , Modelos Lineares , Metaloendopeptidases/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo Genético , RNA Mensageiro/análise , Alinhamento de SequênciaRESUMO
Crotamine is a cationic peptide (4.9 kDa, pI 9.5) of South American rattlesnake, Crotalus durissus terrificus' venom. Its presence varies according to the subspecies or the geographical locality of a given species. At the genomic level, we observed the presence of 1.8 kb gene, Crt-p1, in crotamine-positive specimens and its absence in crotamine-negative ones. In this work, we described a crotamine-related 2.5 kb gene, crotasin (Cts-p2), isolated from crotamine-negative specimens. Reverse transcription coupled to polymerase chain reaction indicates that Cts-p2 is abundantly expressed in several snake tissues, but scarcely expressed in the venom gland. The genome of crotamine-positive specimen contains both Crt-p1 and Cts-p2 genes. The present data suggest that both crotamine and crotasin have evolved by duplication of a common ancestor gene, and the conservation of their three disulfide bonds indicates that they might adopt the same fold as beta-defensin. The physiological function of the crotasin is not yet known.
Assuntos
Venenos de Crotalídeos/genética , Crotalus/genética , Perfilação da Expressão Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Brasil , Primers do DNA , Evolução Molecular , Componentes do Gene , Biblioteca Genômica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Eukaryote genomes are endowed with varying quantities of repeated DNA families. These families show different patterns of conservation among species, copy numbers, chromosomal distribution, and transcription. Characterization of repeated DNA sequences could help to understand the genome anatomy and organization or be used in molecular systematics and molecular evolution studies. We describe here a repetitive DNA sequence of the HindIII family present in the genome of the rattlesnake Crotalus durissus terrificus. In Brazil, the family Crotalus is comprised only by one species durissus, which include several subspecies. The number and distribution of these subspecies are controversial. In the present study, the genomic DNA of a female rattlesnake was digested with HindIII resulting in a strong 1.9 Kb band. A partial genomic library was constructed from the 1.9 Kb DNAs rescued from the agarose gel after HindIII digestion and ligated to the vector pGEM3Zf(+) (Promega). Analysis of 69 clones, 44 hybridized with the 1.9 Kb probe isolated from one of the clones-clone 76, indicating that the DNA isolated from this clone should represent the 1.9 Kb HindIII fragment. This 1.9 Kb HindIII DNA was completely characterized by sequencing.