RESUMO
AIMS: Pompe disease is an autosomal recessive lysosomal storage disorder resulting from deficiency of acid α-glucosidase (GAA) enzyme. Histopathological hallmarks in skeletal muscle tissue are fibre vacuolization and autophagy. Since 2006, enzyme replacement therapy (ERT) is the only approved treatment with human recombinant GAA alglucosidase alfa. We designed a study to examine ERT-related skeletal muscle changes in 18 modestly to moderately affected late onset Pompe disease (LOPD) patients along with the relationship between morphological/biochemical changes and clinical outcomes. Treatment duration was short-to-long term. METHODS: We examined muscle biopsies from 18 LOPD patients at both histopathological and biochemical level. All patients underwent two muscle biopsies, before and after ERT administration respectively. The study is partially retrospective because the first biopsies were taken before the study was designed, whereas the second biopsy was always performed after at least 6 months of ERT administration. RESULTS: After ERT, 15 out of 18 patients showed improved 6-min walking test (6MWT; P = 0.0007) and most of them achieved respiratory stabilization. Pretreatment muscle biopsies disclosed marked histopathological variability, ranging from an almost normal pattern to a severe vacuolar myopathy. After treatment, we detected morphological improvement in 15 patients and worsening in three patients. Post-ERT GAA enzymatic activity was mildly increased compared with pretreatment levels in all patients. Protein levels of the mature enzyme increased in 14 of the 18 patients (mean increase = +35%; P < 0.05). Additional studies demonstrated an improved autophagic flux after ERT in some patients. CONCLUSIONS: ERT positively modified skeletal muscle pathology as well as motor and respiratory outcomes in the majority of LOPD patients.
Assuntos
Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , alfa-Glucosidases/uso terapêutico , Adulto , Idoso , Terapia de Reposição de Enzimas/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/uso terapêutico , Estudos RetrospectivosRESUMO
OBJECTIVE: To assess the feasibility and utility of contrast-enhanced microcomputed tomography (micro-CT) for identifying structural anomalies in ex-vivo first- and second-trimester human fetuses and isolated fetal hearts. METHODS: Radiopaque iodine staining and micro-CT scanning protocols were first developed in rodent studies and then used to examine routinely fixed whole human fetuses (n = 7, weight 0.1-90 g, gestational age, 7-17 weeks) and isolated fetal hearts (n = 14, weight 0.1-5.2 g, gestational age, 11-22 weeks). Samples were scanned using an isotropic resolution of 18 (and, if necessary, 9 or 35) µm and findings were interpreted jointly by four fetal pathologists, a fetal cardiologist and a radiologist. Samples with gestational ages ≥ 13 weeks also underwent conventional autopsy or dissection. RESULTS: Micro-CT identified all anatomical structures and abnormalities documented by the macroscopic examination. In all seven cases involving fetuses ≤ 13 weeks (four fetuses, three isolated hearts), micro-CT excluded the presence of structural anomalies. In the remaining 14 cases, it provided all the information obtained with invasive autopsy or dissection and in seven of the 14 (two fetuses, five isolated hearts) it furnished additional diagnostic details. CONCLUSIONS: This pilot study confirms the feasibility of postmortem contrast-enhanced micro-CT assessment of structural anomalies in whole small fetuses and fetal hearts. Further study is needed to confirm our findings, particularly in whole fetuses, and to define the extent to which this virtual examination might be used instead of conventional invasive autopsy.
Assuntos
Feto/anormalidades , Microtomografia por Raio-X/métodos , Animais , Autopsia , Meios de Contraste , Estudos de Viabilidade , Feminino , Coração Fetal/anormalidades , Coração Fetal/diagnóstico por imagem , Feto/diagnóstico por imagem , Idade Gestacional , Humanos , Iodetos , Camundongos Endogâmicos C57BL , Projetos Piloto , Gravidez , Segundo Trimestre da Gravidez , Ratos Sprague-DawleyRESUMO
We evaluated how the increase in lung interstitial pressure correlates with the pulmonary vascular response to chronic hypoxia. In control and hypoxic (30 days; 10% O2) Wistar male rats, we measured: pulmonary interstitial pressure (P(ip)), cardiac and haemodynamic parameters by echocardiography, and performed lung morphometry on tissue specimens fixed in situ. In control animals, mean ± sd P(ip), air/tissue volume ratio and capillary vascularity index in the air-blood barrier were -12 ± 2.03 cmH2O, 3.9 and 0.43, respectively. After hypoxia exposure, the corresponding values of these indices in apparently normal lung regions were 2.6 ± 1.7 cmH2O, 3.6, and 0.5, respectively. In oedematous regions, the corresponding values were 12 ± 4 cmH2O, 0.4 and 0.3, respectively. Furthermore, in normal regions, the density of pre-capillary vessels (diameter ~50-200 µm) increased and their thickness/internal diameter ratio decreased, while opposite results were found in oedematous regions. Pulmonary artery pressure increased in chronic hypoxia relative to the control (39.8 ± 5.9 versus 26.2 ± 2.2 mmHg). Heterogeneity in local lung vascular response contributes to developing pulmonary hypertension in chronic hypoxia. In oedematous regions, the decrease in capillary vascularity correlated with the remarkable increase in interstitial pressure and morphometry of the pre-capillary vessels suggested an increase in vascular resistance; the opposite was true in apparently normal regions.
Assuntos
Hipóxia/fisiopatologia , Pulmão/fisiopatologia , Edema Pulmonar/fisiopatologia , Animais , Capilares , Ecocardiografia/métodos , Hemodinâmica , Hipertensão Pulmonar/fisiopatologia , Pulmão/patologia , Masculino , Oxigênio/química , Pressão , Artéria Pulmonar/fisiopatologia , Ratos , Ratos Wistar , Pressão VentricularRESUMO
Three human ovarian carcinoma lines (HOC8) derived from the same patient before (P-HOC8) and after (R-HOC8 and Y-HOC8) cycles of chemotherapy were established i.p. in nude mice. The biological characterization showed that these tumor lines had various features in common. Cytological and histopathological characteristics and the expression of tumor-associated antigens OC125 and MOV18 were maintained in the three variants and were comparable to the patient's primary tumor. The HOC8 variants were aneuploid with a chromosome mode number of 80-81. All three tumor lines grew better i.p. than s.c. in nude mice. After i.p. injection the HOC8 lines produced ascites in all the mice, infiltration of pancreas, liver, diaphragm, and lung metastases. The sensitivity to cisplatin was evaluated for HOC8 lines growing in nude mice and mirrored the clinical development of resistance. Treatment with cisplatin of mice transplanted i.p. with P-HOC8 (obtained before the patient received chemotherapy) resulted in a significant increase in survival time; the R-HOC8 and Y-HOC8 lines (obtained after chemotherapy) were less sensitive. HOC8 xenografts, which represent the course of a single patient's disease, are a useful model for investigating the development of drug resistance in ovarian carcinoma.
Assuntos
Carcinoma/patologia , Neoplasias Ovarianas/patologia , Animais , Ascite/etiologia , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/secundário , Cisplatino/farmacologia , Resistência a Medicamentos , Feminino , Humanos , Cariotipagem , Camundongos , Camundongos Nus , Estadiamento de Neoplasias , Transplante de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologiaRESUMO
PURPOSE: The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in the response to platinum compounds in ovarian carcinoma patients. Because taxanes could induce p53-independent apoptosis, we assessed the relevance of p53 gene status to response in ovarian carcinoma patients receiving paclitaxel and platinum-containing chemotherapy. PATIENTS AND METHODS: Forty-eight previously untreated patients with advanced disease received standard paclitaxel/platinum-based chemotherapy. In tumor specimens collected at the time of initial surgery, before therapy, p53 gene status and expression were examined by single-strand conformation polymorphism, sequence analysis, and immunohistochemical analysis. Microsatellite instability analysis was performed on available samples from 30 patients. RESULTS: Thirty-four (71%) of the 48 patients had a clinical response. Pathologic complete remission was documented in 13 (27%) of 48 patients. p53 mutations were detected in 29 (60%) of 48 tumors. Among the patients with mutant p53 tumors, 25 patients (86%) responded to chemotherapy. Only nine (47%) of 19 patients with wild-type p53 tumors responded to the same treatment. The overall response rate and the complete remission rate were significantly higher among patients with mutant p53 tumors than among patients with wild-type p53 tumors (P: =.008). Most of the tested tumors not associated with complete remission (10 of 12 tumors) were also characterized by microsatellite instability. The complete remission rate was higher among patients with tumors without microsatellite instability (five of seven patients). CONCLUSION: In contrast to the limited efficacy of treatment with paclitaxel in combination with standard platinum doses against wild-type p53 ovarian tumors, patients with mutant p53 ovarian tumors were more responsive to paclitaxel-based chemotherapy. The pattern of response to chemotherapy containing paclitaxel is different from that associated with high-dose cisplatin therapy. Determining p53 mutational status can be useful in predicting therapeutic response to drugs effective in ovarian carcinoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/tratamento farmacológico , Carcinoma/genética , Genes p53/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Pareamento Incorreto de Bases , Carcinoma/patologia , Cisplatino/administração & dosagem , Reparo do DNA , Feminino , Humanos , Repetições de Microssatélites/efeitos dos fármacos , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Análise Multivariada , Mutação de Sentido Incorreto , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Indução de Remissão , Estudos RetrospectivosRESUMO
Paclitaxel resistance has been associated with overexpression of P-glycoprotein and alterations involving tubulin. To investigate the clinical relevance of these in vitro resistance mechanisms, we established 12 human ovarian carcinoma xenografts, using samples from patients before the start of therapy or after paclitaxel treatment. These xenografts showed a wide range of sensitivity to paclitaxel, and in 4 of them, very low levels of multidrug resistance-1 expression were detected. Using quantitative PCR and human specific primers, the expression of five beta-tubulin isotypes was determined. HM40 was the predominant, accounting for 84.7-98.7% of all tubulin; expression of the other four isotypes (Hbeta9, Hbeta4, H5beta, and Hbeta2) was also detected but at lower levels. No correlation could be demonstrated between isotype expression and paclitaxel sensitivity in these 12 xenografts. A similar pattern of beta-tubulin isotype expression was observed in a subset of cell lines from the National Cancer Institute-Anticancer Drug Screen. In these cell lines, however, a significant correlation between increased expression of Hbeta4 isotype and resistance to paclitaxel was found. Taken together, these results suggest that altered expression of specific beta-tubulin isotypes may not play a significant role in paclitaxel sensitivity in vivo and argue against a possible significance in a clinical setting.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/uso terapêutico , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Distribuição Tecidual , Tubulina (Proteína)/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Batimastat (also known as BB-94) is a synthetic matrix metalloproteinase inhibitor that has shown antineoplastic and antiangiogenic activity in various tumor models. In this study, two human ovarian carcinoma (HOC) xenografts (HOC22 and HOC8) were used to investigate the effect of batimastat on the antineoplastic activity of cisplatin. Both xenografts produced ascites and solid lesions in the peritoneal cavity of nude mice. HOC cells were inoculated i.p. in nude mice, and treatment was started at different stages of the disease. Batimastat was administered alone or concurrently with or subsequent to cisplatin therapy. In all of the protocols, the response of HOC xenografts was confirmed by cytological analysis of ascites and histological examination of the organs in the peritoneal cavity. Treatment of nude mice bearing early-stage (3 days after tumor implantation) HOC22 or HOC8 with cisplatin or batimastat alone delayed tumor growth and increased the survival time of the mice, although all animals eventually died. In contrast, treatment with batimastat (60 mg/kg i.p. every other day, for a total of eight injections) concomitantly with cisplatin (4 mg/kg i.v., every 7 days for a total of three injections) completely prevented growth and spread of both xenografts, and all animals were alive and healthy on day 200. The potentiation of cisplatin's activity by batimastat was dose dependent and was observed in the treatment of both advanced (7 days after tumor inoculation) and late-stage (20 days after inoculation) tumor. The administration of batimastat following cisplatin therapy also led to significant improvement in the survival of mice compared to treatment with cisplatin alone. These results suggest a potentiation of the antineoplastic activity of cisplatin by batimastat and support the use of the two agents in combination in the treatment of ovarian cancer patients.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma/tratamento farmacológico , Cisplatino/uso terapêutico , Metaloendopeptidases/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Fenilalanina/análogos & derivados , Inibidores de Proteases/farmacologia , Tiofenos/farmacologia , Animais , Antineoplásicos/administração & dosagem , Carcinoma/mortalidade , Cisplatino/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/mortalidade , Fenilalanina/administração & dosagem , Fenilalanina/farmacologia , Inibidores de Proteases/administração & dosagem , Análise de Sobrevida , Tiofenos/administração & dosagem , Transplante HeterólogoRESUMO
The antitumour activity of paclitaxel (NSC 125973) and docetaxel (RP 56976, NSC 628503) was evaluated and compared against human ovarian carcinoma (HOC) xenografts in nude mice. Paclitaxel and docetaxel were given intravenously (i.v.) at a dose range of 16.6-34.5 mg/kg, once every 4 days for three consecutive doses to nude mice with HOC xenografts, transplanted subcutaneously (s.c.) (HOC18 and HOC22-S) or intraperitoneally (i.p.) (HOC8 and HOC22). Paclitaxel and docetaxel, at the highest dosage, induced complete tumour regression in 80-100% and 67% of mice bearing HOC22-S and HOC18 s.c., respectively. Both drugs cured 100% of mice bearing early stage HOC22 tumour in the peritoneal cavity, while treatment at an advanced stage significantly increased the survival time of all the mice. Both induced a 57% cure rate in mice bearing HOC8 in the peritoneal cavity. Paclitaxel and docetaxel were more effective than cisplatin (4 mg/kg, same dosing regime as above) used as a reference compound. These findings indicate that paclitaxel and docetaxel were highly active on four HOC xenograft models. No significant difference between them was detected in ovarian cancer xenografts.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/análogos & derivados , Paclitaxel/uso terapêutico , Taxoides , Animais , Ascite/tratamento farmacológico , Docetaxel , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/tratamento farmacológico , Transplante HeterólogoRESUMO
OBJECTIVE: To report and evaluate a conservative and individualized treatment policy in a homogeneously selected series of patients affected by pure ovarian immature teratoma. METHODS: This prospective trial, with specific treatment policies according to stage and grade, was planned and started in 1982. The study population consisted of 32 patients affected by pure immature teratoma, with the exclusion of mixed germ cell tumors. Fertility-sparing surgery was performed whenever possible. Surgery alone, with careful follow-up, was adopted for stage I or II according to the International Federation of Gynecology and Obstetrics (FIGO) and grade 1 or 2 tumors. The other patients, with stage III or with grade 3 stage I or II tumors, or those referred at relapse, were treated with platinum-based chemotherapy regimens. RESULTS: Thirty of 32 patients underwent fertility-sparing surgery. Ten of 32 patients received chemotherapy after surgery, either as adjuvant treatment or in the presence of visible tumor. All 32 patients are alive and disease-free, with a median follow-up from surgery of 47 months (range 11-138). In six patients, regardless of the administration of chemotherapy, the tumor either spontaneously differentiated toward mature glia or increased in volume, mimicking progression but still remaining completely mature. Five of six patients wishing to procreate had a total of seven normal pregnancies. CONCLUSIONS: Pure ovarian immature teratoma is a potentially curable disease with a unique natural history. Our data substantiate the hypothesis that low-grade and low-stage tumors do not require chemotherapy, and that a fertility-sparing surgical approach is warranted in all cases.
Assuntos
Neoplasias Ovarianas/terapia , Teratoma/terapia , Adolescente , Adulto , Criança , Terapia Combinada , Feminino , Seguimentos , Humanos , Estadiamento de Neoplasias , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Estudos Prospectivos , Indução de Remissão , Teratoma/sangue , Teratoma/patologia , alfa-Fetoproteínas/análiseRESUMO
The X-ray structures of c-2,t-3-di-tert-butyl-r-1-methylthiiranium 8 BF(4)(-), t-2,t-3-di-tert-butyl-r-1-methylthiiranium ion 10 BF(4)(-), and 2,3-di-tert-butyl-1-methylthiirenium 11 BF(4)(-) have been determined. The DeltaG()(298) values for the rearrangements from the cis and the trans tert-butyl groups of 8 SbCl(6)(-) to thietanium ion (two intramolecular S(N)2 displacements) and for the rearrangement of 11 SbCl(6)(-) to thietium ion (an intramolecular S(N)2-Vin displacement) are linearly correlated with the strengths of the C-S breaking bonds, suggesting that the two mechanisms are, in the absence of steric hindrance, uniquely governed by the nucleofugality of the sulfonium leaving group.
RESUMO
The addition of nitrile oxides to [60]fullerene, leading to isoxazolinofullerenes, can be reversed using reducing agents such as Mo(CO)(6) or DIBALH. Thus, this reaction can be used, in principle, for protection/deprotection of [60]fullerene or for solubilization purposes. The tether-controlled tandem addition of nitrile oxides and azomethine ylides provides mainly cis-1 patterns. The determination of the structure of bisadducts was obtained by NMR spectroscopy with the help of HMQC, HMBC, and NOEDS techniques. The isoxazoline moiety could be removed using Mo(CO)(6) leaving a fulleropyrrolidine derivative.
RESUMO
The isolation and characterization of photocycloadducts with pyrimidine bases from DNA samples irradiated (365 nm) in the presence of four 4',5'-substituted methylangelicins was performed. All these furocoumarins yielded mainly the cis-syn furan-side cycloadduct with thymine. For 4',5'-dimethyl-, 5,4',5'-trimethyl- and 6,4',5'-trimethylangelicin this adduct was accompanied by two pyrone-side adducts (cis-syn and cis-anti), whereas the 4,4',5'-trimethyl derivative gave the furan-side adduct with cytosine. The characterization of the regio- and stereochemistry of the adducts was accomplished by 1H NOE (nuclear Overhauser effect) and 1H-13C HMBC (heteronuclear multiple-bond connectivity) spectroscopies. The formation of different cycloadducts in DNA by the various derivatives highlights the role of the methyl groups in determining the regio- and stereochemistry of the cycloaddition.
Assuntos
DNA/efeitos da radiação , Furocumarinas/química , Animais , DNA/química , Furocumarinas/farmacologia , Humanos , Técnicas In Vitro , Masculino , Modelos Moleculares , Fotoquímica , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Pirimidinas/química , Pirimidinas/efeitos da radiação , EstereoisomerismoRESUMO
A study of dark interaction and photoreaction between 4,6-dimethyltetrahydrobenzoangelicin (THBA) and DNA is described. 4,6-Dimethyltetrahydrobenzoangelicin is a furocoumarin derivative in which 4' and 5' carbons are linked by a four-methylene bridge. In spite of the bulky aliphatic ring, THBA forms a complex with DNA in the dark and, on UVA irradiation, reacts with pyrimidine bases of DNA yielding monoadducts only involving its furan side double bond. Two main photoproducts form: they derive from a C4-cycloaddition to thymine and cytosine, respectively, and account for 56% and 39% of the total photoreaction yield. Both show cis-syn configuration. Two other isomers, one with thymine and one with cytosine, formed with so much lower yield (ca 3 and 1%, respectively) that their structure could not be assigned. Furthermore, in spite of its angular structure, THBA induces a small number of crosslinks in DNA.
Assuntos
DNA/química , DNA/efeitos da radiação , Furocumarinas/química , Escuridão , Luz , Modelos Moleculares , Fotoquímica , Solubilidade , Timina/química , Uracila/químicaRESUMO
Four new cerebrosides 4-6 were isolated from the latex of Euphorbia biglandulosa Desf. and their structures determined. Normal and reverse-phase flash chromatography was effective for the isolation of the cerebrosides, and FAB-MS spectrometry, 1H-NMR, 13C-NMR, DQF-COSY and HMQC experiments and chemical reactions were useful in elucidating their structure. EI-MS of the dimethyl disulfide derivatives of the long chain bases and glucosphingoside-heptaacetates was decisive for the determination of the double bond position on the long chain parts.
Assuntos
Cerebrosídeos/química , Plantas Medicinais/química , Cromatografia , Látex/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
4,4',5'-Trimethyl-1'-thioangelicin (1) and 4,6,4',5'-tetramethyl-1'-thioangelicin (2), two newly synthesised isosters of furocoumarins having a sulfur atom in their five-membered ring, were studied in terms of interactions with DNA, both in the ground state and after UVA light absorption. The compounds were able to intercalate the macromolecule and to photobind efficiently, forming C4-cycloadducts with thymine. The antiproliferative effect of this binding was shown in Ehrlich and HeLa cells and by T2 phage inactivation. Tests on Salmonella typhimurium indicated low mutagenic activity. In particular, compound 1 has photobiological activity comparable with that of 4,6,4'-trimethylangelicin, but is less mutagenic.
Assuntos
Cumarínicos/farmacologia , Furocumarinas/farmacologia , Mutagênicos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Tiofenos/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Cumarínicos/síntese química , Cumarínicos/efeitos da radiação , Reagentes de Ligações Cruzadas , DNA/efeitos dos fármacos , Adutos de DNA , Furocumarinas/síntese química , Furocumarinas/efeitos da radiação , Células HeLa , Humanos , Espectrometria de Massas , Estrutura Molecular , Mutagênese/efeitos dos fármacos , Mutagênese/efeitos da radiação , Mutagênicos/síntese química , Mutagênicos/efeitos da radiação , Myoviridae/efeitos dos fármacos , Myoviridae/genética , Myoviridae/efeitos da radiação , Fotobiologia , Fotoquímica , Fotólise , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/efeitos da radiação , Espécies Reativas de Oxigênio , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/efeitos da radiação , Tiofenos/síntese química , Tiofenos/efeitos da radiaçãoRESUMO
Occasional crossbreeding between free-ranging domestic dogs and wild wolves (Canis lupus) has been detected in some European countries by mitochondrial DNA sequencing and genotyping unlinked microsatellite loci. Maternal and unlinked genomic markers, however, might underestimate the extent of introgressive hybridization, and their impacts on the preservation of wild wolf gene pools. In this study, we genotyped 220 presumed Italian wolves, 85 dogs and 7 known hybrids at 16 microsatellites belonging to four different linkage groups (plus four unlinked microsatellites). Population clustering and individual assignments were performed using a Bayesian procedure implemented in structure 2.1, which models the gametic disequilibrium arising between linked loci during admixtures, aiming to trace hybridization events further back in time and infer the population of origin of chromosomal blocks. Results indicate that (i) linkage disequilibrium was higher in wolves than in dogs; (ii) 11 out of 220 wolves (5.0%) were likely admixed, a proportion that is significantly higher than one admixed genotype in 107 wolves found previously in a study using unlinked markers; (iii) posterior maximum-likelihood estimates of the recombination parameter r revealed that introgression in Italian wolves is not recent, but could have continued for the last 70 (+/- 20) generations, corresponding to approximately 140-210 years. Bayesian clustering showed that, despite some admixture, wolf and dog gene pools remain sharply distinct (the average proportions of membership to wolf and dog clusters were Q(w) = 0.95 and Q(d) = 0.98, respectively), suggesting that hybridization was not frequent, and that introgression in nature is counteracted by behavioural or selective constraints.
Assuntos
Cães/genética , Hibridização Genética/genética , Desequilíbrio de Ligação/genética , Lobos/genética , Alelos , Animais , Teorema de Bayes , Cromossomos de Mamíferos/genética , Análise por Conglomerados , Feminino , Variação Genética , Genética Populacional , Geografia , Itália , Masculino , Repetições de Microssatélites/genéticaRESUMO
The entire mitochondrial DNA control region (mtDNA D-loop) was sequenced in the seven extant species of Alectoris partridges. The D-loop length is very conserved (1155 +/- 2 nucleotides), and substitution rates are lower than for the mitochondrial cytochrome b gene of the same species, on average. Comparative analyses suggest that these D-loops can be divided into three domains, corresponding to the highly variable peripheral domains I and III and to the central conserved domain II of vertebrates (Baker and Marshall 1997). Nevertheless, the first 161 nucleotides of domain I of the Alectoris, immediately flanking the tRNAGlu, evolve at an unusually low rate and show motifs similar to the mammalian extended termination-associated sequences [ETAS1 and ETAS2 (Sbisà et al. 1997)], which can form stable secondary structures. The second part of domain I contains a hypervariable region with two divergent copies of a tandemly repeated sequence described previously in other species of anseriforms and galliforms (Quinn and Wilson 1993; Fumihito et al. 1995). Some of the conserved sequence blocks of mammals can be mapped in the central domain of Alectoris. Domain III is highly variable and has sequences similar to mammalian CSB1. The bidirectional transcription promoter HSP/LSP box of the chicken is partially conserved among the Alectoris. This structural organization can be found in the anseriform and galliform species studied so far, suggesting that strong functional constraints might have controlled the evolution of the D-loop since the origin of Galloanserae. Their conserved organization and slow molecular evolution make D-loops of galliforms appropriate for phylogenetic studies, although homoplasy can be be generated at a few hypervariable sites and at some sites which probably have mutated by strand slippage during DNA replication. Phylogenetic analyses of D-loops of Alectoris are concordant with previously published cytochrome b and allozyme phylogenies (Randi 1996). Alectoris is monophyletic and includes three major clades: (1) basal barbara and melanocephala; (2) intermediate rufa and graeca; and (3) recent philbyi, magna, and chukar. Comparative description of the organization and substitution patterns of the mitochondrial control region can aid in mapping hypervariable sites and avoid some sources of homoplasy in data sets which are to be used in phylogenetic analyses.
Assuntos
Aves/classificação , Aves/genética , DNA Mitocondrial/genética , Evolução Molecular , Filogenia , Animais , Sequência de Bases , Sequência Conservada , DNA Mitocondrial/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
The rock partridge (Alectoris graeca) is distributed in the Alps, Apennines and Balkans, in mountainous areas that were heavily affected by cyclic climate and landscape changes during the last Pleistocene glaciations. Some partridge populations have colonized and expanded their present ranges only after deglaciation and recent deforestation by humans. Other populations have been segregated in land-bridge islands isolated after the rise in level of the Mediterranean Sea. Consequently, partridges from different areas could be genetically differentiated. This study has analysed 436 nucleotides of the domain I of the mitochondrial DNA control-region in 70 rock partridges collected from 14 populations throughout their distribution. Phylogenetic analysis and analysis of molecular variance showed that the Sicilian population is very divergent from the continental populations. The Albanian and central Apennine samples are slightly divergent from the Alpine rock partridges, which cluster in two groups according to their east-west distributions. The mitochondrial DNA sequences suggest the existence of a phylogeographical structuring among rock partridge populations, resulting from genetic divergence in southern refugia and subsequent postglacial colonization of northern mountain areas. Sicilian partridges could have been isolated for approximately 500,000 years, whereas the Albanian-Apennine populations could have been in contact since the last glacial maximum (21,000 yr BP) through the north Adriatic land-bridge. The Alps could have been colonized in two different periods or by two different source populations. The mitochondrial DNA phylogeographical structuring is not completely concordant with the subspecies distribution. These findings suggest that rock partridges should be managed based on the identification of phylogeographical units.