RESUMO
Williams-Beuren syndrome (WBS) is most often caused by hemizygous deletion of a 1.5-Mb interval encompassing at least 17 genes at 7q11.23 (refs. 1,2). As with many other haploinsufficiency diseases, the mechanism underlying the WBS deletion is thought to be unequal meiotic recombination, probably mediated by the highly homologous DNA that flanks the commonly deleted region. Here, we report the use of interphase fluorescence in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE) to identify a genomic polymorphism in families with WBS, consisting of an inversion of the WBS region. We have observed that the inversion is hemizygous in 3 of 11 (27%) atypical affected individuals who show a subset of the WBS phenotypic spectrum but do not carry the typical WBS microdeletion. Two of these individuals also have a parent who carries the inversion. In addition, in 4 of 12 (33%) families with a proband carrying the WBS deletion, we observed the inversion exclusively in the parent transmitting the disease-related chromosome. These results suggest the presence of a newly identified genomic variant within the population that may be associated with the disease. It may result in predisposition to primarily WBS-causing microdeletions, but may also cause translocations and inversions.
Assuntos
Inversão Cromossômica , Polimorfismo Genético/genética , Síndrome de Williams/genética , Adolescente , Cromossomos Humanos Par 7/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Genótipo , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Mapeamento Físico do CromossomoRESUMO
The 22q13.3 deletion syndrome results from loss of terminal segments of varying sizes at 22qter. Few genotype-phenotype correlations have been found but all patients have mental retardation and severe delay, or absence of, expressive speech. We carried out clinical and molecular characterization of 13 patients. Developmental delay and speech abnormalities were common to all and comparable in frequency and severity to previously reported cases. Array-based comparative genomic hybridization showed the deletions to vary from 95 kb to 8.5 Mb. We also carried out high-resolution 244K array comparative genomic hybridization in 10 of 13 patients, that defined the proximal and distal breakpoints of each deletion and helped determine the size, extent, and gene content within the deletion. Two patients had a smaller 95 kb terminal deletion with breakpoints within the SHANK3 gene while three other patients had a similar 5.5 Mb deletion implying the recurrent nature of these deletions. The two largest deletions were found in patients with ring chromosome 22. No correlation could be made with deletion size and phenotype although complete/partial SHANK3 was deleted in all patients. There are very few reports on array comparative genomic hybridization analysis on patients with the 22q13.3 deletion syndrome, and we aim to accurately characterize these patients both clinically and at the molecular level, to pave the way for further genotype-phenotype correlations. (c) 2010 Wiley-Liss, Inc.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 22/genética , Anormalidades Múltiplas/genética , Adolescente , Transtorno Autístico/genética , Proteínas de Transporte/genética , Criança , Pré-Escolar , Hibridização Genômica Comparativa , Deficiências do Desenvolvimento/genética , Feminino , Estudos de Associação Genética , Humanos , Transtornos do Desenvolvimento da Linguagem/genética , Masculino , Proteínas do Tecido Nervoso , Fenótipo , Síndrome , Adulto JovemRESUMO
The components of the Escherichia coli chemosensory system have been identified and their activities characterized, but how sensory information is processed to give an integrated response remains an open question.
Assuntos
Células Quimiorreceptoras/fisiologia , Quimiotaxia , Escherichia coli/fisiologia , Proteínas de Bactérias , Escherichia coli/efeitos dos fármacos , Modelos BiológicosRESUMO
Acyl-CoA dehydrogenase 9 (ACAD9), linked to chromosome 3q21.3, is one of a family of multimeric mitochondrial flavoenzymes that catalyze the degradation of fatty acyl-CoA from the carnitine shuttle via ß-oxidation (He et al. 2007). ACAD9, specifically, is implicated in the processing of palmitoyl-CoA and long-chain unsaturated substrates, but unlike other acyl-CoA dehydrogenases (ACADs), it has a significant role in mitochondrial complex I assembly (Nouws et al. 2010 & 2014). Mutations in this enzyme typically cause mitochondrial complex I deficiency, as well as a mild defect in long chain fatty acid metabolism (Haack et al. 2010, Kirby et al. 2004, Mcfarland et al. 2003, Nouws et al. 2010 & 2014). The clinical phenotype of ACAD9 deficiency and the associated mitochondrial complex I deficiency reflect this unique duality, and symptoms are variable in severity and onset. Patients classically present with cardiac dysfunction due to hypertrophic cardiomyopathy. Other common features include Leigh syndrome, macrocephaly, and liver disease (Robinson et al. 1998). We report the case of an 11-month old girl presenting with microcephaly, dystonia, and lactic acidosis, concerning for a mitochondrial disorder, but atypical for ACAD9 deficiency. Muscle biopsy showed mitochondrial proliferation, but normal mitochondrial complex I activity. The diagnosis of ACAD9 deficiency was not initially considered, due both to these findings and to her atypical presentation. Biochemical assay for ACAD9 deficiency is not clinically available. Family trio-based whole exome sequencing (WES) identified 2 compound heterozygous mutations in the ACAD9 gene. This discovery led to optimized treatment of her mitochondrial dysfunction, and supplementation with riboflavin, resulting in clinical improvement. There have been fewer than 25 reported cases of ACAD9 deficiency in the literature to date. We review these and compare them to the unique features of our patient. ACAD9 deficiency should be considered in the differential diagnosis of patients with lactic acidosis, seizures, and other symptoms of mitochondrial disease, including those with normal mitochondrial enzyme activities. This case demonstrates the utility of WES, in conjunction with biochemical testing, for the appropriate diagnosis and treatment of disorders of energy metabolism.
RESUMO
Chromosome 15q11q13 is among the least stable regions in the genome due to its highly complex genomic architecture. Low copy repeat elements at 15q13.3 facilitate recurrent copy number variants (CNVs), with deletions established as pathogenic and CHRNA7 implicated as a candidate gene. However, the pathogenicity of duplications of CHRNA7 is unclear, as they are found in affected probands as well as in reportedly healthy parents and unaffected control individuals. We evaluated 18 children with microduplications involving CHRNA7, identified by clinical chromosome microarray analysis (CMA). Comprehensive phenotyping revealed high prevalence of developmental delay/intellectual disability, autism spectrum disorder, and attention deficit/hyperactivity disorder. As CHRNA7 duplications are the most common CNVs identified by clinical CMA, this study provides anticipatory guidance for those involved with care of affected individuals.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Espectro Autista/genética , Variações do Número de Cópias de DNA/genética , Deficiências do Desenvolvimento/genética , Fenótipo , Receptor Nicotínico de Acetilcolina alfa7/genética , Criança , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Análise em Microsséries , LinhagemRESUMO
Signal transduction in microorganisms and plants is often mediated by His-Asp phosphorelay systems. Two conserved families of proteins are centrally involved: histidine protein kinases and phospho-aspartyl response regulators. The kinases generally function in association with sensory elements that regulate their activities in response to environmental signals. A sequence analysis with 348 histidine kinase domains reveals that this family consists of distinct subgroups. A comparative sequence analysis with 298 available receiver domain sequences of cognate response regulators demonstrates a significant correlation between kinase and regulator subfamilies. These findings suggest that different subclasses of His-Asp phosphorelay systems have evolved independently of one another.
Assuntos
Proteínas Quinases/fisiologia , Sequência de Aminoácidos , Archaea/enzimologia , Archaea/genética , Bactérias/enzimologia , Bactérias/genética , Evolução Molecular , Histidina Quinase , Dados de Sequência Molecular , Proteínas Quinases/classificação , Proteínas Quinases/genética , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Controversy exists over the distinction between cardio-facio-cutaneous (CFC) syndrome and Noonan syndrome (NS). Several authors have suggested that they are different phenotypes of the same condition. We present the cases of two patients with CFC syndrome to show that it is a distinct condition with a unique combination of findings and a more complex natural history. These patients, both girls, were born with signs of fetal edema following pregnancies complicated by polyhydramnios. Each has short stature with relative macrocephaly; fuzzy, sparse hair; and the typical craniofacial features, including a square forehead. Both have heart abnormalities, failure to thrive, and severe feeding problems requiring gastrostomy. They are markedly hypotonic and developmentally delayed. They show signs of frequent eyelid fluttering and have oral aversion, tactile hypersensitivity, and sensory integration abnormalities. Keratosis pilaris, the characteristic skin symptom, is also present in both patients. In a review we identified 56 cases of CFC syndrome. We scored these cases by 10 clinical criteria and identified a subset with a specific, severe phenotype distinct from that of NS. The serious neurologic and gastrointestinal complications, in addition to the skin abnormalities and characteristic facies in this group, clearly separate these patients from the mildly affected ones, most of whom appear to have NS or another syndrome. We discuss the differences between the severe CFC phenotype and those of overlapping conditions. We set forth stringent diagnostic criteria for CFC syndrome, the initial step toward identifying a molecular basis for this condition.
Assuntos
Anormalidades do Sistema Digestório , Expressão Facial , Cardiopatias Congênitas/diagnóstico , Doenças do Sistema Nervoso/diagnóstico , Anormalidades da Pele/diagnóstico , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico , Diagnóstico Diferencial , Pálpebras/anormalidades , Fácies , Feminino , Humanos , Hidropisia Fetal , Ceratose/diagnóstico , Hipotonia Muscular/diagnóstico , Fenótipo , SíndromeRESUMO
We report on a two-generation family exhibiting dominant inheritance of complex polysyndactyly associated with hypothalamic hamartoma. These individuals have some manifestations of Pallister-Hall syndrome (PHS), but their phenotype is milder. The proposita is a 16-year-old girl with polysyndactyly of the hands and feet, short stature, and a large hypothalamic hamartoma. Her brother and father also have polysyndactyly and a hypothalamic mass on MRI scan. All three have normal appearance and intelligence, with normal pituitary function. Several other paternal relatives have polysyndactyly as well. We propose that this family may represent a clinically and perhaps genetically distinct entity from PHS, based on normal survival, normal intelligence, lack of endocrine dysfunction or facial anomalies, and few other structural malformations. Linkage analysis is in progress to determine whether this represents a benign form of PHS or a genetically separate condition. The phenotypic differences between these cases and classic PHS have important prognostic and recurrence risk implications.
Assuntos
Dedos/anormalidades , Hamartoma/diagnóstico , Doenças Hipotalâmicas/diagnóstico , Polidactilia/genética , Sindactilia/genética , Dedos do Pé/anormalidades , Adolescente , Adulto , Feminino , Hamartoma/complicações , Deformidades da Mão/genética , Humanos , Doenças Hipotalâmicas/complicações , Masculino , Linhagem , Fenótipo , SíndromeRESUMO
"Epidermal nevus syndrome" ("ENS") is a neurocutaneous disorder in which epidermal nevi are associated with other abnormalities, most commonly of the skeletal and central nervous systems. We present two cases of epidermal nevus syndrome (ENS) with very different clinical findings. The first case is a newborn with multiple linear epidermal nevi of the trunk and limbs, and several other anomalies, including bony duplications of the lower limbs and hypoplastic left heart syndrome. The second patient, a 6-year-old boy, has a linear nevus sebaceous of the scalp with severe CNS involvement, including generalized seizures, moderate mental retardation, microcephaly, and a left hemiparesis. He also has genitourinary, cardiac, and skeletal defects. These two patients exhibit several abnormalities not previously recognized and illustrate the wide clinical spectrum of "epidermal nevus syndrome." We present a review of the clinical findings in 74 cases of "ENS." Correlation was noted between the presence of skin lesions located on the head and CNS involvement. The wide clinical spectrum of "ENS" as illustrated by these two patients suggests that "ENS" is a causally heterogeneous group of disorders.
Assuntos
Anormalidades Múltiplas , Sistema Nervoso Central/anormalidades , Cardiopatias Congênitas , Nevo Pigmentado/congênito , Neoplasias Cutâneas/congênito , Criança , Anormalidades do Olho , Deformidades Congênitas do Pé , Humanos , Recém-Nascido , Masculino , SíndromeRESUMO
Deletion of 7p results in a wide spectrum of congenital abnormalities and minor facial and hand anomalies, often including craniosynostosis. We report on the oldest recognized patient with this disorder, a 24-year-old woman with an interstitial deletion from p15.3-p21.2 or p21.3. The manifestations in this patient are milder than those of previously described patients, and include borderline mental retardation, short stature, minor facial anomalies, and several skeletal changes. The absence of craniosynostosis in this patient is noteworthy, given previous suggestions that there is a specific locus for this finding in the 7p region. Twelve cases of 7p deletion, in which the missing segment overlaps that of the current case, are reviewed. This case delineates a broader spectrum for patients with 7p deletion syndrome.
Assuntos
Anormalidades Múltiplas/genética , Deleção Cromossômica , Cromossomos Humanos Par 7 , Adulto , Criança , Pré-Escolar , Bandeamento Cromossômico , Mapeamento Cromossômico , Craniossinostoses/genética , Feminino , Deformidades Congênitas da Mão/genética , Humanos , Deficiência Intelectual/genética , Cariotipagem , Masculino , SíndromeRESUMO
A 9-month-old boy with pre- and post-natal growth retardation, microcephaly, plagiocephaly, and several minor anomalies had the initial karyotype: 46,XY,der(1)t(1;?) (p36.1;?). Further analysis showed that the der(1) was derived from an unfavorable segregation of a maternal complex chromosome rearrangement, i.e., 46,XX,der(1)t(1;?) (p36.1;?), der(4)t(4;?)(q?;?). Whole chromosome fluorescence in situ hybridization (FISH) and chromosome microdissection were used to clarify the maternal karyotype as: 46,XX,der(1)t(1;4)(4qter-->4q33::1p36.13-->1qter),der( 4)t(1;4)inv(4)(4pter-->4q31.3::1p36.33-->1p36.13::4q33 -->4q31.3::1p36.33-->1pter). Therefore, the karyotype of the boy actually was 46,XY,der(1)t(1;4) (p36.13;q33). Clinical comparison of the patient's clinical findings showed similarities to individuals with partial del(1p) and dup(4q). To our knowledge the above cytogenetic abnormalities have not been described previously. This case further demonstrates the advantages of chromosome microdissection and FISH in the identification of anomalous chromosome regions and breakpoints.
Assuntos
Anormalidades Múltiplas/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 4/genética , Anormalidades Craniofaciais/genética , Doenças Fetais/genética , Translocação Genética , Bandeamento Cromossômico , Mapeamento Cromossômico , Cromossomos Humanos Par 1/ultraestrutura , Cromossomos Humanos Par 4/ultraestrutura , Sondas de DNA , Feminino , Transtornos do Crescimento/genética , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Cariotipagem , Masculino , Metáfase , Linhagem , Gravidez , Diagnóstico Pré-Natal , SíndromeRESUMO
Different penicillin-binding proteins PBPs are affected in cefotaxime-resistant laboratory mutants compared to piperacillin-resistant mutants. PBP2x acts as the primary PBP target in cefotaxime-resistant mutants, whereas PBP2b is the primary target in piperacillin-resistant mutants. Depending on the mutations in PBP2x, it functions as a resistance determinant for cefotaxime only, or for penicillins as well. Mutations in PBP2x of laboratory mutants are found exclusively in the penicillin-binding domain that contains three homology boxes common to all penicillin-interacting enzymes. Most mutations relevant for resistance occur close to the SXN or the KT/SG box, or at the C-terminal end of the penicillin-binding domain, similar to mutations described in PBP2b of laboratory mutants. Amino acid alterations occur at similar sites also in PBP2x of beta-lactam-resistant clinical isolates and most of these proteins also contain changes in the SXXK box with the active site serine, suggesting that these alterations may be critical for resistance development in clinical isolates.
Assuntos
Aminoaciltransferases , Proteínas de Bactérias , Proteínas de Transporte/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Resistência às Penicilinas/genética , Peptidil Transferases , Streptococcus pneumoniae/genética , Cefotaxima/farmacologia , Resistência às Cefalosporinas , Cefalosporinas/farmacologia , Mutação , Proteínas de Ligação às Penicilinas , Penicilinas/farmacologia , Piperacilina/farmacologia , Reação em Cadeia da Polimerase , Transformação BacterianaRESUMO
Laboratory mutants of Streptococcus pneumoniae resistant to either cefotaxime or piperacillin reveal defects in competence development independent of the selective beta-lactam. A resistance determinant ciaH encoding a putative histidine kinase of a two-component signal-transducing system that is also involved in competence regulation was recently identified in cefotaxime-resistant mutants. We show now that the CiaH protein can be phosphorylated by ATP in vitro, and that it also phosphorylates the cognate response regulator CiaR. The mutant C306 containing the CiaH mutation Thr-230-Pro is completely noncompetent. It does not release competence-inducing activity (competence factor) into the medium nor can such an activity be released from the cells. Competence in C306 cannot be induced upon addition of external competence factor, in contrast to the competence-defective piperacillin-resistant mutants P506 and P408. A novel resistance determinant cpoA specific for piperacillin was identified in piperacillin-resistant mutants. CpoA is responsible for the competence defect in P506 but not in P408. The results document a tight link between the action of beta-lactams and competence development in the pneumococcus and confirm that the two beta-lactams piperacillin and cefotaxime act via different primary targets.
Assuntos
Antibacterianos/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Resistência beta-Lactâmica/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Histidina Quinase , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Streptococcus pneumoniae/enzimologia , beta-LactamasRESUMO
The potency of the putatively alpha(1B)-adrenoceptor selective drug, 1-[biphenyl-2-yloxy]-4-imino-4-piperidin-1-yl-butan-2-ol (AH11110A), to antagonize contraction upon stimulation of alpha(1A)-adrenoceptors in rat vas deferens and rat perfused kidney, alpha(1B)-adrenoceptors in guinea-pig spleen, mouse spleen and rabbit aorta, and alpha(1D)-adrenoceptors in rat aorta and pulmonary artery was evaluated and compared to that of a number of subtype-discriminating antagonists. N-[3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-2-phenyl-4H-1-benzopyran-8-carboxamide (Rec 15/2739) and (+/-)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione (B8805-033) were confirmed as selective for alpha(1A)-adrenoceptors, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY 7378), 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]-8-azaspiro[4.5]decane-7,9-dione (MDL 73005EF), and cystazosin were found to be selective for alpha(1D)-adrenoceptors, whereas spiperone was weakly selective for alpha(1B)-over alpha(1A)-adrenoceptors. However, from the functional affinity profile obtained for AH11110A at alpha(1A)-adrenoceptors (pA(2)=6.41 in rat vas deferens), alpha(1B)-adrenoceptors (pA(2)=5.40-6.54) and alpha(1D)-adrenoceptors (pA(2)=5.47-5.48), the affinity and presumed selectivity previously obtained for AH11110A in radioligand binding studies at native alpha(1B)- and cloned alpha(1b)-adrenoceptors (pK(i)=7.10-7.73) could not be confirmed. Additionally, AH11110A enhanced the general contractility of rat vas deferens, produced a bell-shaped dose-response curve of vasodilation in perfused rat kidney, and its antagonism in most other tissues was not simply competitive. The affinity of AH11110A for prejunctional alpha(2)-adrenoceptors in rabbit vas deferens (pA(2)=5.44) was not much lower than that displayed for alpha(1)-adrenoceptor subtypes, revealing that AH11110A, besides alpha(1)-adrenoceptors, also interacts with alpha(2)-adrenoceptors, and thus may be unsuitable for alpha-adrenoceptor subtype characterization, at least in smooth muscle containing functional studies.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Agonistas alfa-Adrenérgicos/química , Antagonistas Adrenérgicos alfa/química , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Relação Dose-Resposta a Droga , Cobaias , Iminas/farmacologia , Rim/efeitos dos fármacos , Rim/fisiologia , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Piperidinas/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Coelhos , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos alfa 2/fisiologia , Baço/efeitos dos fármacos , Baço/fisiologia , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/fisiologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
The muscarinic receptor mediating contraction of the guinea-pig isolated gallbladder, currently being disputed to belong either to the M3 or M4 subtype, was characterized by subtype-preferring agonists and discriminating antagonists. Highly significant correlations of agonist potencies to contract the gallbladder, e.g., arecaidine propargyl ester, oxotremorine, 5-methylfurtrethonium > arecoline, arecaidine 2-butyne-1,4-diyl bisester > (R)-nipecotic acid ethyl ester > 4-[[N-(4-chlorophenyl)carbamyl]oxy]-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343), (S)-nipecotic acid ethyl ester > 4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyltrimethylammonium chloride (McN-A-343) were found with muscarinic M3 receptors mediating contraction of the guinea-pig ileum and vasodilation in rat perfused kidney. Functional affinities at guinea-pig gallbladder muscarinic receptors of antagonists known to distinguish between native or cloned muscarinic M3/m3 and M4/m4 receptors, e.g., himbacine, methoctramine, mefurtramine, tripitramine, idaverine, zamifenacin and 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyr ido(2,3-b)(1,4)benzodiazepin-6-one (AQ-RA 741), were consistent with those at guinea-pig ileal muscarinic M3 receptors but not with published data at recently defined muscarinic M4 receptors in rabbit anococcygeus muscle or at muscarinic M1 and M2 receptors in rabbit vas deferens. Antagonist affinities at guinea-pig gallbladder correlated also best with published binding data on native or cloned muscarinic M3/m3 receptors but not with those for muscarinic M4/m4 receptors. The agonist potencies and antagonist affinities suggest that smooth muscle contraction elicited by muscarinic stimuli in guinea-pig gallbladder is mediated by functional muscarinic M3 receptors.
Assuntos
Vesícula Biliar/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Cobaias , Íleo/efeitos dos fármacos , Técnicas In Vitro , Rim/efeitos dos fármacos , Modelos Lineares , Masculino , Coelhos , Ratos , Ducto Deferente/efeitos dos fármacosRESUMO
The affinity for functional alpha1-adrenoceptor subtypes of buspirone in comparison with its close structural analogs and selective alpha1D-adrenoceptor antagonists, BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]dec ane-7,9-dione) and MDL 73005EF (8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]-8-azaspiro+ ++[4.5]decane-7,9-dione), was determined, namely at subtype A in rat vas deferens and perfused kidney, at subtype B in guinea-pig and mouse spleen, at subtype L in rabbit spleen, and at subtype D in rat aorta and pulmonary artery against noradrenaline-evoked contractions. BMY 7378 and MDL 73005EF were confirmed as 30- and 20-fold selective antagonists, respectively, for alpha1D- over both alpha1A- and alpha1B-adrenoceptors. Buspirone was a weak antagonist without intrinsic activity at alpha1A-adrenoceptors in rat vas deferens (pA2 = 6.12), at alpha1B-adrenoceptors in guinea-pig and mouse spleen (pA2 = 5.54 and 5.59) and at alpha1L-adrenoceptors in rabbit spleen (pA2 = 4.99), but caused partial vasoconstriction in rat kidney that was attenuable by the subtype D-selective adrenoceptor antagonist BMY 7378, but hardly by the subtype A-selective adrenoceptor antagonist B8805-033 ((+/-)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-be nzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedion e), confirming the additional presence of alpha1D-adrenoceptors mediating rat renal vasoconstriction. Buspirone behaved as a partial agonist at alpha1D-adrenoceptors in rat aorta (pD2 = 6.77, intrinsic activity (i.a.)= 0.40) and pulmonary artery (pD2 = 7.16, i.a. = 0.59). With buspirone as agonist in these tissues, the pA2 values of subtype-discriminating antagonists were consistent with their alpha1D-adrenoceptor affinity determined in rat aorta against noradrenaline and with published binding data on cloned alpha1d-adrenoceptors. The results provide pharmacological evidence that (1) in functional preparations for the A subtype, like rat vas deferens and perfused kidney, for the B subtype, like guinea-pig and mouse spleen, and for the L subtype, like rabbit spleen, buspirone is a weak antagonist without intrinsic activity, but (2) behaves as a partial agonist in rat aorta and pulmonary artery as models for the D subtype and (3) detects an additional vasoconstrictor alpha1D-adrenoceptor in rat kidney. Buspirone, like its close analogs BMY 7378 and MDL 73005EF, thus might also be a useful tool for functionally discriminating alpha1D- from alpha1A-, alpha1B- and alpha1L-adrenoceptors in various tissues.
Assuntos
Agonistas Adrenérgicos/farmacologia , Antagonistas Adrenérgicos/farmacologia , Buspirona/farmacologia , Receptores Adrenérgicos/metabolismo , Animais , Aorta Torácica , Dioxinas/farmacologia , Cobaias , Técnicas In Vitro , Rim/efeitos dos fármacos , Rim/metabolismo , Modelos Lineares , Masculino , Camundongos , Piperazinas/farmacologia , Artéria Pulmonar , Coelhos , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/metabolismo , Compostos de Espiro/farmacologia , Baço/efeitos dos fármacos , Baço/metabolismo , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
High-level resistance to beta-lactam antibiotics in Streptococcus pneumoniae is mediated by successive alterations in essential penicillin-binding proteins (PBPs). In the present work, single amino acid changes in S. pneumoniae PBP 2x and PBP 2b that result in reduced affinity for the antibiotic and that confer first-level beta-lactam resistance are defined. Point mutations in the PBP genes were generated by PCR-derived mutagenesis. Those conferring maximal resistance to either cefotaxime (pbp2x) or piperacillin (pbp2b) were obtained after transformation of the susceptible laboratory strain R6 with the PCR-amplified PBP genes and selection on agar with various concentrations of the antibiotic. In the case of PBP 2x, transformants for which the cefotaxime MIC was 0.16 microgram/ml contained the substitution of a Thr for an Ala at position 550 (Thr550-->Ala), close to the PBP homology box Lys547SerGly, a mutation frequently observed in laboratory mutants and in a high-level cefotaxime-resistant clinical isolate as well. After further selection, transformants resisting 0.3 microgram of cefotaxime per ml were obtained; they contained the substitution Gly550 as the result of two mutations in the same codon. In PBP 2b, Thr446-->Ala, adjacent to another homology box Ser443SerAsn, was the mutation selected with piperacillin. This substitution has been described in all clinical isolates with a low-affinity PBP 2b but was distinct from point mutations found in laboratory mutants. Both pbp2b with the single mutation and a mosaic pbp2b of a clinical isolate conferred a twofold increase in piperacillin resistance. Attempts to select PBP 2b variants at higher piperacillin concentrations were unsuccessful. The mutated PBP 2b also markedly reduced the lytic response to piperacillin, suggesting that such a mutation is an important step in resistance development in clinical isolates.
Assuntos
Aminoaciltransferases , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Peptidil Transferases , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Resistência beta-Lactâmica/genética , beta-Lactamas/farmacologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , Cefotaxima/farmacologia , Relação Dose-Resposta a Droga , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas , Piperacilina/metabolismo , Reação em Cadeia da Polimerase , Streptococcus pneumoniae/metabolismoRESUMO
Piperacillin resistance in Streptococcus pneumoniae was mediated by mutations in a novel gene, cpoA, that also confer transformation deficiency and a decrease in penicillin-binding protein la. cpoA is part of an operon located downstream of the primary sigma factor of S. pneumoniae. The deduced protein, CpoA, and the peptide encoded by the adjacent 3' open reading frame contained domains homologous to glycosyltransferases of procaryotes and eucaryotes that act on membrane-associated substrates, such as enzymes functioning in lipopolysaccharide core biosynthesis of gram-negative bacteria, RodD of Bacillus subtilis, which is involved in teichoic acid biosynthesis, and the human PIG-A protein, which is required for early steps of glycosylphosphatidylinositol anchor biosynthesis. This suggests that the cpo operon has a similar function related to cell surface components.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte , Glicosiltransferases/genética , Muramilpentapeptídeo Carboxipeptidase , Resistência às Penicilinas/genética , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Genes Bacterianos , Hexosiltransferases/genética , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Mutagênese Insercional , Mutação , Óperon , Proteínas de Ligação às Penicilinas , Peptidil Transferases/genética , Fenótipo , Plasmídeos/isolamento & purificação , Análise de Sequência de DNA , Especificidade da Espécie , Transcrição GênicaRESUMO
In Streptococcus pneumoniae, alterations in penicillin-binding protein 2b (PBP 2b) that reduce the affinity for penicillin binding are observed during development of beta-lactam resistance. The development of resistance was now studied in three independently obtained piperacillin-resistant laboratory mutants isolated after several selection steps on increasing concentrations of the antibiotic. The mutants differed from the clinical isolates in major aspects: first-level resistance could not be correlated with alterations in the known PBP genes, and the first PBP altered was PBP 2b. The point mutations occurring in the PBP 2b genes were characterized. Each mutant contained one single point mutation in the PBP 2b gene. In one mutant, this resulted in a mutation of Gly-617 to Ala within one of the homology boxes common to all PBPs, and in the other two cases, the same Gly-to-Asp substitution at the end of the penicillin-binding domain had occurred. The sites affected were homologous to those determined previously in the S. pneumoniae PBP 2x of mutants resistant to cefotaxime, indicating that, in both PBPs, similar sites are important for interaction with the respective beta-lactams.