RESUMO
Neonatal screening is one of the crucial parts of the tertiary prevention strategy to reduce congenital disability. Traditional neonatal screening, mainly focusing on genetic metabolic diseases, has limitations in disease types and requires genetic testing for further validation and accurate typing. Currently, conducting genetic screening based on biochemical metabolite screening has become the trend in neonatal screening. This article synthesizes the current state of neonatal genome screening at home and abroad. Herein, the comprehensive concepts of "SNV Plus" (single nucleotide variation plus) and "CNV Plus" (copy number variation plus) have been proposed to develop a new technology that can detect the gene structure of SNV and CNV simultaneously and improve the level of neonatal genome screening based on characteristics of the pathogenic gene structure.
Assuntos
Variações do Número de Cópias de DNA , Triagem Neonatal , Humanos , Recém-Nascido , NucleotídeosRESUMO
ABSTRACT: Objective To discuss the application of 3D laser scanner and computer technology in restoration of the accident scene and reconstruction of the accident process, as well as identification of the driver-passenger relationship. Methods The scene of a traffic accident, the accident vehicle and the vehicle of the same type as accident vehicle were scanned using 3D laser scanner. The accident scene, traces and accident vehicle were integrated using computer technology to restore the accident scene, and the accident process was reconstructed and analyzed by combining the characteristics of the body injuries. Results By restoring the accident scene and reconstructing the accident process with 3D laser scanner, it was determined that Wu was in the driving seat at the time of the accident. Conclusion It is more objective and scientific to use 3D laser scanning technology to restore the accident scene, reconstruct the accident process and analyze the moving track of the driver and passengers in the vehicle. It will help to improve the accuracy of forensic identification of road traffic accidents.
Assuntos
Acidentes de Trânsito , Simulação por ComputadorRESUMO
ABSTRACT: "Karoshi" originates from Japan's economic take-off period in the 1960s and 1970s. It is generally believed that overwork lead to the accumulation of fatigue, which triggers the outbreak of potential diseases, and results in sudden death. Karoshi causes great harm to both the community and families because it occurs primarily in 30 to 60 year old young adults. Japan put Karoshi into the category of industrial injury for the first time in 2001 and started to undertake a series of studies in the sociological and pathological fields. However, there is a tremendous gap in the forensic pathological diagnosis domain. In China, research on Karoshi started from the 1990s and is closely related to the reform and opening up policy as well as economic development. According to the incomplete statistics, 600 thousand people die from overwork each year in China, the highest in the world. Karoshi has become one of the most serious social problems in China at the present stage, thus a systematic study in the sociology and forensic pathology fields is urgently required. This paper summarizes the past and present status of Karoshi, and puts forward the problems that need attention during the judicial expertise of Karoshi from forensic pathology perspective.
Assuntos
Morte Súbita/etiologia , Patologia Legal , Estresse Ocupacional/epidemiologia , Adulto , China , Fadiga/epidemiologia , Humanos , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
ABSTRACT: Objective To study the effect of overwork stress response on the expression of connexin 43ï¼Cx43ï¼ and connexin 45ï¼Cx45ï¼ in cardiomyocytes and on cardiac function. Methods The experimental animals were divided into control group, overworked 1-month group and overworked 2-month group. A overworked rat model was established by forcing swimming of overworked group. The expressions of Cx43 and Cx45 in myocardial tissues of experimental animals were detected by Western blotting, while the corresponding myocardial tissues were stained with hematoxylin-eosin ï¼HEï¼ staining and Masson's staining, then histologically observed. Results Western blotting results showed that, compared with the control group, Cx43 expression in myocardial tissues of overworked rats decreased while Cx45 expression increased. HE staining and Masson's staining results showed that hypertrophy, rupture and interstitial fiber tissue hyperplasia were observed in myocardial fibers of overworked rats. Conclusion Overwork stress response may affect cardiac function as an independent factor and may even cause heart failure or arrhythmias and lead to death.
Assuntos
Arritmias Cardíacas/etiologia , Conexina 43/metabolismo , Conexinas/metabolismo , Insuficiência Cardíaca , Miócitos Cardíacos/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Miocárdio , RatosRESUMO
Objective: There is no standard method for esophageal remnant gastric reconstruction for proximal gastrectomy. Reflux esophagitis caused by esophagogastrostomy remains a difficult surgical problem. To report the preliminary surgical results of novel esophagus-conical remnant gastric side overlap anastomosis (CGEO) , with particular emphasis on postoperative esophageal reflux. Methods: In June 2022, we developed a novel CGEO for laparoscopic proximal gastrectomy on two patients with Siewert type II esophagogastric junction adenocarcinoma. Surgical procedures for CGEO: (1) Laparoscopic proximal gastrectomy and preparation of conically shaped gastric remnant; (2) Determining anastomotic site of residual stomach and esophagus; (3) Side-to-side anastomosis of right esophageal wall to anterior of conical gastric remnant; (4) Valvuloplasty of esophageal stump. Results: Case 1 was a 71-year-old man with an operation time of 305 minutes and was successfully discharged from the hospital on the 9th day after surgery, and the postoperative pathology was T3N0M0. Case 2 was an 82-year-old man with an operation time of 325 minutes. He was discharged on the 10th day after surgery. In both cases, only mild esophageal mucosal changes were seen in gastroscopy, there were no obvious symptoms of esophageal reflux. There was also no significant weight change at half a year after operation. Conclusion: CGEO is moderately safe in radical surgery for proximal gastric cancer, and may have a preventive effect on the occurrence of postoperative esophageal reflux, but long-term results need to be confirmed by further studies with follow-up.
Assuntos
Esofagite Péptica , Refluxo Gastroesofágico , Neoplasias Gástricas , Masculino , Humanos , Idoso , Idoso de 80 Anos ou mais , Neoplasias Gástricas/cirurgia , Anastomose Cirúrgica , GastrectomiaRESUMO
Objective: To analyze the clinical and genetic characteristics of 33 children with congenital lipoid adrenal hyperplasia (CLAH) caused by StAR gene defects. Methods: The clinical, biochemical, genetic, and follow-up (until December 2021) data of 33 children diagnosed with CLAH from 2006 to 2021 were retrospectively analyzed in Xinhua Hospital, Shanghai Jiao Tong University School of Medicine. Results: Of the 33 children with CLAH, 17 had a karyotype of 46, XX and 16 had a karyotype of 46, XY; 31 were female and 2 were male by social gender. Classic type and non-classic type were found in 30 and 3 children respectively. The age at diagnosis was 9.0 (3.0, 34.5) months. All the 30 cases with classic CLAH presented within the first year of life with skin hyperpigmentation (28 cases, 93%), vomiting and(or) diarrhea (19 cases, 63%), no increase in body weight (8 cases, 27%), elevated adrenocorticotropic hormone levels (21cases (70%)>275 pmol/L), decreased cortisol levels (47 (31,126) nmol/L), hyponatremia ((126±13) mmol/L), hyperkalemia ((5.7±1.1) mmol/L), and normal 17α-hydroxyprogesterone levels (30 cases, 100%). All these with classic CLAH exhibited female external genitalia. Three children with non-classic CLAH (including 2 cases of 46, XY and 1 case of 46, XX) also showed signs and symptoms of adrenal insufficiency, but 2 of them had an age of onset later than 1 year of age, including 1 case of 46, XY with male external genitalia and 1 case of 46, XX with female external genitalia. The other 46, XY patient with non-classic CLAH presented with adrenal insufficiency at 2 months of age, showing micropenis and hypospadias. In the 17 females with 46, XX, 4 older than 10 years of age showed spontaneous pubertal development. A total of 25 StAR gene pathogenic variants were identified in 33 patients, with p.Q258* (18/66, 27%), p.K236Tfs*47 (8/66, 12%) and p.Q77* (6/66, 9%) being the common variantion. Six novel variants were found, including c.358T>G, c.713_714del, c.125del, c.745-1G>A, c.179-2A>C, and exon 1 deletion. Conclusions: Patients with classic CLAH typically present with signs and symptoms of primary adrenal insufficiency in the early infancy period and female external genitalia. p.Q258*, p.K236Tfs*47 and p.Q77* are common variants in CLAH patients.
Assuntos
Hiperplasia Suprarrenal Congênita , Insuficiência Adrenal , Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/genética , Hormônio Adrenocorticotrópico , Pré-Escolar , China , Transtorno 46,XY do Desenvolvimento Sexual , Feminino , Humanos , Hidrocortisona , Hidroxiprogesteronas , Hiperplasia , Lactente , Masculino , Mutação , Fosfoproteínas/genética , Estudos RetrospectivosRESUMO
Objective: To summarize the clinical manifestation and genetic characteristics of Chinese patients with achondroplasia (ACH) which is caused by pathogenic variants fibroblast growth factor receptor 3 (FGFR3) gene and establish the reference value of height centiles and height for age growth curve of patients for a more practical, simple and useful growth evaluation tool in China. Methods: Through a nationwide cross-sectional survey in China from July 2019 to January 2020 designed by Department of Pediatric Endocrinology and Genetics, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 210 subjects (110 boys, 100 girls), who harbored the pathogenic variant of FGFR3 gene and were diagnosed with achondroplasia, were recruited. The clinical and genetic data of enrolled subjects were collected and analyzed to explore the clinical genetic characteristics of Chinese ACH patients. Furthermore, according to the data of height (body length under 2 years old) of boy and girl subjects aged 0-12 years, centiles and height for age growth curve of achondroplasia were calculated and established by LMS method respectively. Results: The characteristic clinical manifestations of 210 Chinese patients (0-14 years old) were disproportionate short stature (206/210, 98.1%), macrocephaly and characteristic facial features (205/210, 97.6%), trident configuration of the hands (191/210, 90.9%), limbs deformity (156/210, 74.3%), together with normal intelligence. Up to 81.9% (172/210) of patients have different complications, and the kyphosis (121/210, 57.6%) and narrow thoracic (79/210, 37.6%) are common complications. Besides, up to 98.6% (207/210) of patients harbored hotspot variants of FGFR3 gene which cause G380R amino acid substitutions. It is notable that the growth pattern of boy and girl patients (0-12 years old) is obviously different from the normal children (t=9.849, 9.596, P<0.01) respectively. The height different between ACH patients and normal children gradually widened with age. The average height of the boy (49.2 cm) and girl patients(48.4 cm) of achondroplasia at birth was -1.22 s and -2 s, however, at the age of twelve, the average height of the boy(113.7 cm) and girl patients(112.4 cm) of achondroplasia was -5.23 s and -6.15 s compared to currently standard reference height for age growth curve of normal children in China, respectively. Conclusions: The results of our study demonstrated that in China disproportionate short stature, macrocephaly and characteristic facial features were typical manifestations of ACH patients, and that up to 98.6% of patients harbored hotspot variants of FGFR3 gene. In addition, the reference value of height centiles and height for age growth curve of ACH patients we establish will be a valuable tool for evaluating the growth pattern, monitoring factors affecting growth, estimating ultimate height, and assessing the curative effect of growth-promoting treatments in Chinese patients with achondroplasia.
Assuntos
Acondroplasia/genética , Adolescente , Estatura , Criança , Pré-Escolar , China , Estudos Transversais , Feminino , Gráficos de Crescimento , Humanos , Lactente , Recém-Nascido , Masculino , Valores de ReferênciaRESUMO
Thermal properties of a lowly Nd(3+)-doped disordered Nd:CNGG crystal have been symmetrically investigated. At room temperature, the specific heat, thermal diffusion coefficient and density were determined to be 0.595 J/g.K, 1.223 mm(2)/s and 4.718 g/cm(3), corresponding the thermal conductivity of 3.43 W/m.K. By measuring the thermal lens at different pump power, the thermal-optical coefficient was calculated to be 9.2x10(-6) K(-1) for the first time to our knowledge. All the thermal properties recovered that this material can be used in the moderate and even high pump power.
Assuntos
Lasers de Estado Sólido , Lentes , Desenho Assistido por Computador , Cristalização/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The trimeric protein hemagglutinin (HA) of the influenza viral envelope is essential for cell entry. To investigate the interaction of HA with membranes, two 40-residue, cysteine-substituted peptides comprising the loop region and the first part of the coiled-coil stem were synthesized and modified with a nitroxide spin label. Electron paramagnetic resonance analysis revealed that the peptide inserts reversibly into phospholipid vesicles under endosomal pH conditions. This result suggests that some or all of the long coiled-coil trimer of HA may insert into membranes, which could bring the viral and cell membranes closer together and facilitate fusion.
Assuntos
Hemaglutininas Virais/metabolismo , Bicamadas Lipídicas/metabolismo , Fusão de Membrana , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Espectroscopia de Ressonância de Spin Eletrônica , Endocitose , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Orthomyxoviridae/fisiologia , Conformação Proteica , Estrutura Secundária de Proteína , Temperatura , Proteínas do Envelope Viral/químicaRESUMO
Objective: To investigate clinical, molecular genetic characteristics, and treatment outcomes of 3 children with sitosterolemia. Methods: Three cases of children presented with multiple xanthomas during June 2016 to June 2017 were included. The clinical manifestations, laboratory examinations and follow-up data were retrospectively analyzed. DNA was extracted from peripheral blood and analyzed with whole exome sequencing(WES). All the detected variants were confirmed by Sanger sequencing. Plasma plant sterol concentrations were measured by gas chromatography-mass spectrometry. Results: Three cases of children including 1 boy and 2 girls presented with multiple linear and intertriginous xanthomas around skin of the joint areas at the age from 15 months to 6 years and 2 months. Total cholesterol of the 3 cases was elevated to 14.45, 15.47 and 15.85 mmol/L (3.36-6.46), and low density lipoprotein cholesterol was 9.02, 13.54 and 12.47 mmol/L (< 3.36) respectively. Genetic analysis with WES revealed that 2 cases carried compound heterozygous variants in ABCG5 gene, 1 case carried compound heterozygous variants in ABCG8 gene. Two reported variants (p. N437K, p.R446X) and one novel variant (p.Q251X) of ABCG5 were identified in case 2 and 3. Two novel ABCG8 variants (p.R263Q, c.1528_1530delATC) were found in case 1. All these children had extremely high plasma plant sterol levels, thus the diagnosis of sitosterolemia was confirming. The campesterol level was 111.35, 102.86 and 58.91 µmol/L(0.01-10.00), the stigmasterol was 14.97, 29.43 and 17.79 µmol/L (0.10-8.50) and the sitosterol was 231.20, 177.66 and 114.20 µmol/L (1.00-15.00) respectively. The total serum cholesterol levels of three children decreased to nomal after the patients were placed on the low plant sterol/low cholesterol diet. The xanthomas regressed gradually, and almost disappeared after 8 months of treatment in case 1 and 3. Conclusions: Children with sitosterolemia presented with skin xanthomas around the joint areas. The level of total cholesterol, low density lipoprotein cholesterol and plant sterols increased obviously. One novel variant (p.Q251X) of ABCG5 and 2 novel variants (p.R263Q, c.1528_1530delATC) of ABCG8 were identified. Children with sitosterolemia responded well to a low plant sterols/low cholesterol diet.
Assuntos
Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Hipercolesterolemia , Enteropatias , Erros Inatos do Metabolismo Lipídico , Lipoproteínas , Fitosteróis/efeitos adversos , Xantomatose , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP , Criança , Colesterol , Feminino , Humanos , Hipercolesterolemia/diagnóstico , Hipercolesterolemia/genética , Hipercolesterolemia/terapia , Enteropatias/diagnóstico , Enteropatias/genética , Enteropatias/terapia , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/terapia , Lipoproteínas/metabolismo , Masculino , Fitosteróis/genética , Estudos Retrospectivos , Xantomatose/etiologiaRESUMO
Objective: To investigate the clinical and laboratory features of three children with late-onset type â ¡ glycogen storage disease(GSD) who presented with hypertrophic cardiomyopathy and to analyze the effect of five mutations identified on the acid-α-glucosidase (GAA) activity and stability. Method: Three cases of children with muscle weakness were included in this study.GAA activity was analyzed in Dried Blood Spot of the patients.DNA was extracted from peripheral blood in all the patients and their parents and subjected to polymerase chain reaction and directly sequencing of GAA gene.Five mutant pcDNA3.1-myc-his-GAA expression plasmids(p.G478R, p.P361L, p.P266S, p.Q323X, p.R672Q) were constructed and transient instantaneously transfected into 293T cells to analyze the enzyme activity and stability of GAA. Result: All the three children had the onset of disease at 3 years or 1.5 years of age.They presented with developmental delay, muscle weakness and hypertrophic cardiomyopathy.GAA activity of 3 patients was 2.65, 3.55 and 1.51 pmol(punch·h)(8.00-98.02)respectively. Genetic analysis found 5 mutations (p.G478R, p. P361L, p. P266S, p. Q323X, p. R672Q), and all of these 3 cases had clinical manifestations and were diagnosed as late-onset type â ¡ glycogen storage disease.Five mutant pcDNA3.1-myc-his-GAA expression plasmids were transfected into 293T cells.Five mutant enzyme activities were found to be only 9.9%-22.5% of the wild-type enzyme activity and the protein expression of the five mutants was 32.0%-63.9% compared with the wild type. Conclusion: This study reports 3 children with late-onset GSD â ¡ accompanied by hypertrophic cardiomyopathy and compensatory stage of cardiac function in addition to limb muscle weakness.Five pathogenic mutations were identified, and these 5 mutations result in decreased GAA activity and GAA expression by in vitro functional analysis.
Assuntos
Doença de Depósito de Glicogênio Tipo II/genética , Mutação , alfa-Glucosidases/genética , Idade de Início , Cardiomiopatia Hipertrófica/complicações , Criança , DNA , Testes Genéticos , Glucana 1,4-alfa-Glucosidase , Doença de Depósito de Glicogênio Tipo II/complicações , Humanos , Debilidade Muscular , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
OBJECTIVE: To analyze the phenotype-genotype correlation of 21-hydroxylase deficiency (21-OHD) patients found by neonatal screening, and to investigate the characteristics of gene frequency of these patients. METHOD: Clinical and biochemical data of 66 21-OHD patients diagnosed by neonatal screening in department of pediatric endocrinology and genetics and neonatal screening center of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from 2009 to 2014 were retrospectively analyzed. Point mutations of CYP21A2 gene were analyzed by Sanger sequencing, and large gene deletions were detected by multiplex ligation-dependent probe amplification (MLPA). Then the correlation between phenotypes and genotypes of these patients were analyzed. RESULT: (1) Forty-one out of 66 patients who presented adrenal crisis or other signs of salt loss at age range from 4 days to 2 months were classified as salt-wasting forms. The remaining 25 patients did not present any signs of salt loss at preliminary diagnosis (12 days-2 months). (2) Definite mutations of CYP21A2 gene on two alleles were found in all 66 patients (132 alleles). A total of thirteen types of different point mutations (98/132, 74.2%), large gene deletions (24/132, 18.2%) and clusters of point mutations (10/132, 7.6%) were found. The most frequent point mutations were I2G, p. I173N, p. R357W, p. G111Vfs*21 and p. Q319*, accounting for 65.2% of alleles. (3) Phenotype and genotype correlation analysis was performed in 41 21-OHD patients with salt wasting forms. Predicted phenotypes according to genotypes in 36 (87.8%) of the 41 patients were consistent with their actual phenotypes. In 4 out of the 41 patients, the actual phenotypes were different from predicted phenotypes according to their genotypes. And in one patient, prediction of phenotype could not be made based on genotype as carrying an unknown function mutation on one allele. CONCLUSION: Adrenal crisis or other signs of salt loss were found in 62% of 21-OHD patients at age range from 4 days to 2 months. In 66 Chinese 21-OHD children, total mutation frequency of I2G, p. I173N, p. R357W, p. G111Vfs*21 and p. Q319* accounted for 65.2% of alleles. In 87.8% of patients with salt wasting forms, predicted phenotypes according to genotypes were consistent with their actual phenotypes.
Assuntos
Hiperplasia Suprarrenal Congênita , Genótipo , Fenótipo , Alelos , Povo Asiático , China , Feminino , Deleção de Genes , Frequência do Gene , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase Multiplex , Mutação , Triagem Neonatal , Mutação Puntual , Esteroide 21-HidroxilaseRESUMO
OBJECTIVE: To detect large genomic deletions or duplications of ornithine transcarbamylase (OTC) gene by multiplex ligation-dependent probe amplification (MLPA). METHOD: Thirty cases of suspected OTC deficiency (OTCD) patients based on tandem-mass spectrum results were recruited in Xinhua Hospital from 2012 to 2014, among whom 13 were male and 17 were female. Sanger sequencing of OTC gene revealed mutations in 23 cases. MLPA was performed in the patients whose previous Sanger sequencing failed to detect any disease-causing mutation. The samples were treated via the steps of DNA degeneration, the probe hybridization, connecting the hybridization probe, PCR amplification and capillary electrophoresis. The data were analyzed using Coffalyser software. RESULT: Abnormal MLPA results were found in 5 patients without mutation detected in previous Sanger sequencing. Patient 1, a 9-year old girl, had a heterozygous deletion of Exon 2-4. Patient 2, a male newborn, died 10 days after birth. The examination of the mother's sample by MLPA revealed a heterozygous duplication of exon 2-6. Patient 3, a 10-day old boy, was found to harbor a hemizygous deletion of exon 7-10. Patient 4, a 2-year old girl, harbored a heterozygous deletion of exon 1-4. The fifth patient died at the age of 6 years, and his mother carried a heterozygous duplication of exon 1-4. CONCLUSION: MLPA can be helpful in detecting the OTC gene defects, particularly for OTCD patients without mutation detected by Sanger sequencing.
Assuntos
Reação em Cadeia da Polimerase Multiplex , Doença da Deficiência de Ornitina Carbomoiltransferase/diagnóstico , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Éxons , Feminino , Heterozigoto , Humanos , Recém-Nascido , Masculino , Mutação , Deleção de SequênciaRESUMO
OBJECTIVE: To explore the clinical and genetic characteristics of an infant with isolated 17, 20-lyase deficiency. METHOD: The clinical, biochemical and genetic characteristics were analyzed in an 8-month-old infant with 46, XY gonadal dysgenesis who presented predominantly the female external genitalia. RESULT: The infant was referred because of"masses in bilateral inguinal region and 46, XY gonadal dysgenesis". He was normotensive. Laboratory tests revealed elevated levels of progesterone and 17-hydroxyprogesterone. The detailed parameters are as follows: progesterone 29.35(reference range 0.09-1.0)nmol/L, 17-hydroxyprogesterone 10.9(reference range 0.6-2.6)nmol/L, testosterone 0.7(reference range 0.1-3.1)nmol/L, dehydroepiandrosterone sulfate <0.15(reference range 0.80-5.6)mg/L, androstenedione <0.3 (reference range 0.6-3.1) µg/L, luteinizing hormone 6.6(reference range 0.6-1.7)U/L, follicle stimulating hormone 1.8 (reference range 0.5-3.7)U/L, estradiol 37.66(reference range 73.4-146.8)pmol/L. The patient had normal levels of serum sodium, potassium, corticosteroid and plasma adrenocorticotropic hormone. Genomic DNA was extracted from the leukocytes of peripheral blood of the patient and subjected to next generation sequencing (NGS) for testing more than 200 sexual development related genes. Sanger sequencing was used to confirm the results of NGS. Genetic analysis revealed that the patient harbored compound heterozygous mutations of c. 1226C>G (p.Pro409Arg, P409R) and c. 707T>G (p.Val236Gly, V236G) in CYP17A1 gene derived from paternal and maternal allele. V236G was a novel mutation predicted to be pathogenic. The infant was diagnosed as isolated 17, 20-lyase deficiency combined with clinical and molecular characteristics of CYP17A1 gene. CONCLUSION: We have identified the compound heterozygous mutations of P409R and V236G in the CYP17A1 gene in one infant with isolated 17, 20-lyase deficiency. He presented with 46, XY gonadal dysgenesis, normal blood pressure and elevated concentration of progesterone and 17-hydroxyprogesterone.
Assuntos
Hiperplasia Suprarrenal Congênita , Disgenesia Gonadal 46 XY , Hormônio Luteinizante , Testículo/anormalidades , 17-alfa-Hidroxiprogesterona , Androstenodiona , Estradiol , Hormônio Foliculoestimulante , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Liases , Masculino , Mutação , Progesterona , Esteroide 17-alfa-Hidroxilase , TestosteronaRESUMO
The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP(H) and NAD(H)-dependent malate and lactate dehydrogenase reactions.
Assuntos
L-Lactato Desidrogenase/química , Malato Desidrogenase/química , Mathanococcus/enzimologia , Regulação Alostérica , Sequência de Aminoácidos , Arginina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Genes Arqueais/genética , Glicina/metabolismo , Ligação de Hidrogênio , L-Lactato Desidrogenase/classificação , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/classificação , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Mathanococcus/genética , Modelos Moleculares , Dados de Sequência Molecular , NAD/metabolismo , NADP/metabolismo , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Superoxide dismutase (SOD) from Aquifex pyrophilus, a hyperthermophilic bacterium, is an extremely heat-stable enzyme that maintains about 70% of its activity after heat treatment for 60 minutes at 100 degrees C. To understand the molecular basis of thermostability of this enzyme, we have determined the crystal structure of A. pyrophilus superoxide dismutase (Ap SOD), an Fe containing homotetrameric enzyme, at 1.9 A resolution, and compared it with SOD structures from a mesophile and a thermophile, and other enzyme structures from other hyperthermophiles. The structure has been refined to a crystallographic R-factor (I > 2sigma) of 17.0% and R-free (I > 2sigma) of 19.9%. While the overall structure of the Ap SOD monomer is similar to the other SODs, significant conformational differences are observed in a highly variable loop region and the C-terminal helix. The conformational differences in these regions alter the subunit arrangement of this enzyme and generate a very compact tetramer. Structural comparisons of three SODs have revealed that Ap SOD has some stabilizing features at both the tertiary and the quaternary structural level: The Ap SOD monomer contains a large number of ion-pairs and the Ap SOD tetramer has a dramatically increased buried surface area per monomer. Comparisons of the Ap SOD structure with that of other known enzymes from hyperthermophiles reveal that the increased number of intrasubunit ion-pairs is a common feature.
Assuntos
Bactérias Aeróbias Gram-Negativas/enzimologia , Temperatura Alta , Superóxido Dismutase/química , Sequência de Aminoácidos , Aminoácidos/química , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , Estabilidade Enzimática , Ferro/metabolismo , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Conformação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Superóxido Dismutase/metabolismo , Propriedades de Superfície , Thermus thermophilus/enzimologiaRESUMO
Large crystals of probable amylase/protease inhibitor-B have been grown at room temperature from ammonium sulfate solution. The crystals grow within five days to dimensions of 0.6 mm x 0.6 mm x 0.6 mm. They diffract to at least 1.7 A upon exposure to synchrotron X-rays. The crystals belong to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = 38.02 A and c = 98.98 A. The presence of one molecule per asymmetric unit gives the unit cell volume per protein mass (Vm) of 1.99 A3/Da and the solvent fraction of 38.2% by volume. X-ray data have been collected to 2.0 A Bragg spacing from native crystals.
Assuntos
Oryza/química , Proteínas de Plantas/química , Cristalização , Inibidores de Proteases/química , Difração de Raios X , alfa-Amilases/antagonistas & inibidoresRESUMO
The soybean Rsv1 gene for resistance to soybean mosaic virus (SMV; Potyvirus) has previously been described as a single-locus multi-allelic gene mapping to molecular linkage group (MLG) F. Various Rsv1 alleles condition different responses to the seven (G1-G7) described strains of SMV, including extreme resistance, localized and systemic necrosis, and mosaic symptoms. We describe the cloning of a cluster of NBS-LRR resistance gene candidates from MLG F of the virus-resistant soybean line PI96983 and demonstrate that multiple genes within this cluster interact to condition unique responses to SMV strains. In addition to cloning 3gG2, a strong candidate for the major Rsv1 resistance gene from PI96983, we describe various unique resistant and necrotic reactions coincident with the presence or absence of other members of this gene cluster. Responses of recombinant lines from a high-resolution mapping population of PI96983 (resistant) x Lee 68 (susceptible) demonstrate that more than one gene in this region of the PI96983 chromosome conditions resistance and/or necrosis to SMV. In addition, the soybean cultivars Marshall and Ogden, which carry other previously described Rsv1 alleles, are shown to possess the 3gG2 gene in a NBS-LRR gene cluster background distinct from PI96983. These observations suggest that two or more related non-TIR-NBS-LRR gene products are likely involved in the allelic response of several Rsv1-containing lines to SMV.
Assuntos
Genes de Plantas , Glycine max/genética , Glycine max/virologia , Potyvirus/patogenicidade , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , DNA de Plantas/genética , Variação Genética , Dados de Sequência Molecular , Família Multigênica , Nucleotídeos/metabolismo , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/virologia , Recombinação Genética , Glycine max/metabolismoRESUMO
Opioid-induced rewarding and motorstimulant effects are mediated by an increased activity of the ventral tegmental area (VTA) dopamine (DA) neurons. The excitatory mechanism of opioids on VTA-DA neurons has been proposed to be due to the depression of GABAergic synaptic transmission in VTA-DA neurons. However, how opioids depress GABAergic synaptic transmission in VTA-DA neurons remain to be studied. In the present study, we explored the mechanism of the inhibitory effect of [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) on GABAergic synaptic transmission in VTA-DA neurons using multiple approaches and techniques. Our results showed that (1) DAMGO inhibits GABAergic inputs in VTA-DA neurons at presynaptic sites; (2) effect of DAMGO on GABAergic inputs in VTA-DA neurons is inhibited by potassium channel blocker 4-aminopyridine (4-AP) and Gi protein inhibitor N-ethylmaleimide (NEM); (3) phospholipase A2 (PLA2) does not mediate the effect of DAMGO on GABAergic inputs in VTA-DA neurons, but mediates it in the periaqueductal gray (PAG); (4) multiple downstream signaling molecules of µ receptors do not mediate the effect of DAMGO on GABAergic inputs in VTA-DA neurons. These results suggest that DAMGO depresses inhibitory synaptic transmission via µ receptor-Gi protein-Kv channel pathway in VTA-DA neurons, but via µ receptor-PLA2 pathway in PAG neurons.