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INTRODUCTION: There are high chances of post-hysterectomy vault prolapse (PHVP) if the vault is not well supported after vaginal hysterectomy in cases of pelvic organ prolapse (POP). High uterosacral ligament suspension (HUSLS) and McCall's culdoplasty are the well-recommended modalities to suspend the vault after vaginal hysterectomy. As both the procedures are accessible to non-urologic gynaecologists, the study was planned in cases of POP. Objective: The study was conducted to compare the anatomic and functional outcomes of patients undergoing vaginal HUSLS vs. McCall's culdoplasty at the time of vaginal hysterectomy. MATERIALS AND METHODS: This prospective interventional study was done in a tertiary care hospital. A total of 80 patients were included and divided into two groups of 40 patients each. In one group, patients underwent high uterosacral ligament suspension and in the second group, McCall's culdoplasty was done for vault suspension. All procedures were done by two trained surgeons. The effectiveness of both the procedures was assessed by preoperative and postoperative pelvic organ prolapse quantification (POP-Q) (up to two years). Patients were followed for two years to see for any postoperative problem/recurrence. RESULTS: Vault suspension by HUSLS showed better results than McCall's culdoplasty, in terms of POP-Q point C, perineal body (PB), genital hiatus (GH) and total vaginal length (TVL) as compared to McCall's culdoplasty. CONCLUSION: The anatomical correction is much better with HUSLS, which suspends the vault in the normal vaginal axis. However, it takes longer compared to McCall's culdoplasty, so the procedure should be individualised and performed with several precautions.
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AIM: To evaluate the efficacy and safety of ART in prevention of mother-to-child transmission of HIV. METHODS: All pregnant females who were tested and found positive for HIV from April 2015 to March 2017 were included in this study and started on TLE regimen (Tenofovir 300 mg, Lamivudine 300 mg, Efavirenz 600 mg). After delivery these newborns were given syrup Nevirapine 2 mg/kg for 6 weeks of life if mother was started on ART before 12 weeks of gestation and was continued upto 12 weeks if mother was started on ART later than 12 weeks of gestation. Infants were tested with PCR and Rapid test for HIV was done at 6 weeks, 6 months, 12 months, and 18 months of life. RESULT: Out of 66 HIV-positive mothers (26 diagnosed in our institute and 40 referred patients), 53 delivered in our hospital. Out of these 53 deliveries, 3 were stillbirth, so 50 babies were registered. Out of 50 registered babies, 2 babies died in follow-up (one at the age of 12 months due to malnutrition and other at the age of 4 months due to diarrhea). Five babies were referred to nearby ART centers. So, we followed 43 babies out of which two were found to be positive for HIV (transmission rate of HIV was 4.6%). CONCLUSION: The risk of transmission of HIV from mother-to-child had declined with the use of combination ART, and the emergence of drug resistance was reduced. The ART used during pregnancy appears to be well tolerated and safe.
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Dens invaginatus, also known as dens in dente, is a rare anomaly affecting human dentition. The condition results in invagination of an amelodental structure within the pulp. This case report discusses the current management protocol of dens invaginatus using a minimally invasive and nonsurgical treatment option. As with most conditions, early diagnosis and preventive measures help minimize complications in dens invaginatus cases.
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A compelling case for promoting male circumcision (MC) as an intervention for reducing the risk of heterosexually acquired HIV infection was made by dissemination of the results of three studies in Africa. The WHO/UNAIDS recommendation for MC for countries like India, where the epidemic in concentrated in high-risk groups, advocates MC for specific population groups such as men at higher risk for HIV acquisition. A multicentre qualitative study was conducted in four geographically distinct districts (Belgaum, Kolkata, Meerut and Mumbai) in India during June 2009 to June 2011. Two categories of health care providers: Registered Healthcare Providers (RHCPs) and traditional circumcisers were interviewed by trained research staff who had received master's level education using interview guides with probes and open-ended questions. Respondents were selected using purposive sampling. A comparative analysis of the perspectives of the RHCP vs. traditional circumcisers is presented. Representatives of both categories of providers expressed the need for Indian data on MC. Providers feared that promoting circumcision might jeopardize/undermine the progress already made in the field of condom promotion. Reservation was expressed regarding its adoption by Hindus. Behavioural disinhibition was perceived as an important limitation. A contrast in the practice of circumcision was apparent between the traditional and the trained providers. MC should be mentioned as a part of comprehensive HIV prevention services in India that includes HIV counselling and testing, condom distribution and diagnosis and treatment of sexually transmitted infections. It should become an issue of informed personal choice rather than ethnic identity.
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Actitud del Personal de Salud , Circuncisión Masculina/estadística & datos numéricos , Infecciones por VIH/prevención & control , Pautas de la Práctica en Medicina , Adulto , Humanos , India , Masculino , Enfermedades de Transmisión Sexual/prevención & controlRESUMEN
Cell-cell adhesion is paramount in providing and maintaining multicellular structure and signal transmission between cells. In the skin, disruption to desmosomal regulated intercellular connectivity may lead to disorders of keratinization and hyperproliferative disease including cancer. Recently we showed transgenic mice overexpressing desmoglein 2 (Dsg2) in the epidermis develop hyperplasia. Following microarray and gene network analysis, we demonstrate that Dsg2 caused a profound change in the transcriptome of keratinocytes in vivo and altered a number of genes important in epithelial dysplasia including: calcium-binding proteins (S100A8 and S100A9), members of the cyclin protein family, and the cysteine protease inhibitor cystatin A (CSTA). CSTA is deregulated in several skin cancers, including squamous cell carcinomas (SCC) and loss of function mutations lead to recessive skin fragility disorders. The microarray results were confirmed by qPCR, immunoblotting, and immunohistochemistry. CSTA was detected at high level throughout the newborn mouse epidermis but dramatically decreased with development and was detected predominantly in the differentiated layers. In human keratinocytes, knockdown of Dsg2 by siRNA or shRNA reduced CSTA expression. Furthermore, siRNA knockdown of CSTA resulted in cytoplasmic localization of Dsg2, perturbed cytokeratin 14 staining and reduced levels of desmoplakin in response to mechanical stretching. Both knockdown of either Dsg2 or CSTA induced loss of cell adhesion in a dispase-based assay and the effect was synergistic. Our findings here offer a novel pathway of CSTA regulation involving Dsg2 and a potential crosstalk between Dsg2 and CSTA that modulates cell adhesion. These results further support the recent human genetic findings that loss of function mutations in the CSTA gene result in skin fragility due to impaired cell-cell adhesion: autosomal-recessive exfoliative ichthyosis or acral peeling skin syndrome.
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Ciclo Celular , Cistatina A/metabolismo , Desmogleína 2/metabolismo , Redes Reguladoras de Genes , Animales , Adhesión Celular , Línea Celular Tumoral , Cistatina A/genética , Desmogleína 2/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , TranscriptomaRESUMEN
BACKGROUND: Although male circumcision (MC) is recommended as an HIV prevention option, the religious, cultural and biomedical dimensions of its feasibility, acceptability and practice in India have not been explored till date. This study explores beliefs, experiences and understanding of the community and healthcare providers (HCPs) about adult MC as an HIV prevention option in India. METHODS: This qualitative study covered 134 in-depth interviews from Belgaum, Kolkata, Meerut and Mumbai cities of India. Of these, 62 respondents were the members of circumcising (CC)/non-circumcising communities (NCC); including medically and traditionally circumcised men, parents of circumcised children, spouses of circumcised men, and religious clerics. Additionally, 58 registered healthcare providers (RHCPs) such as general and pediatric surgeons, pediatricians, skin and venereal disease specialists, general practitioners, and operation theatre nurses were interviewed. Fourteen traditional circumcisers were also interviewed. The data were coded and analyzed in QSR NUD*IST ver. 6.0. The study has not explored the participants' views about neonatal versus adult circumcision. RESULTS: Members of CC/NCC, traditional circumcisers and RCHPs expressed sharp religious sensitivities around the issue of MC. Six themes emerged: Male circumcision as the religious rite; Multiple meanings of MC: MC for 'religious identity/privilege/sacrifice' or 'hygiene'; MC inflicts pain and cost; Medical indications outweigh faith; Hesitation exists in accepting 'foreign' evidence supporting MC; and communication is the key for acceptance of MCs. Medical indications could make members of NCC accept MC following appropriate counseling. Majority of the RHCPs demanded local in-country evidence. CONCLUSION: HCPs must educate high-risk groups regarding the preventive and therapeutic role of MC. Communities need to discuss and create new social norms about male circumcision for better societal acceptance especially among the NCC. Feasibility studies on MC as an individual specific option for the high risk groups in health care setting needs to be explored.
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Circuncisión Masculina , Conocimientos, Actitudes y Práctica en Salud , Personal de Salud , Investigación Cualitativa , Características de la Residencia , Adulto , Circuncisión Masculina/efectos adversos , Circuncisión Masculina/economía , Comunicación , Humanos , Higiene , India , Masculino , Dolor/etiología , ReligiónRESUMEN
INTRODUCTION: Cervical intraepithelial neoplasia and cervical cancer remain important health problems. Cervical cytology by Papanicolaou (Pap) smears is an effective means of screening for cervical premalignant and malignant conditions. AIM: The aim of this study was to assess the prevalence of cervical dysplasia in pre- and postmenopausal women in western Uttar Pradesh and to find out risk factors as far as possible. MATERIALS AND METHODS: A total of 4,703 cases were enrolled, cervical scrape smears were collected and stained using Papanicolaou's method and hematoxylin and eosin stain. The emphasis was put on epithelial abnormalities and smears were classified according to The Bethesda System 2001. RESULTS: 81.06% (3812) smears were satisfactory according to The Bethesda System. Maximum numbers of cases (40.37%) were in age group 30-39 years. The epithelial abnormalities constituted 3.23% of all cases. Low-grade squamous intraepithelial lesion (LSIL) formed the largest number (1.36%), while high-grade squamous intraepithelial lesion (HSIL) formed 0.91%. Eleven cases of squamous cell carcinoma (SCC) were detected. The study has shown a relatively high prevalence of epithelial abnormalities in cervical smears with increasing age, parity, early age at first coitus (<20 year), and lower socioeconomic status in symptomatic women with clinical lesions on per speculum examination. CONCLUSION: Epithelial abnormalities of cervix are not uncommon in our setup and are associated with early age at marriage and parity.
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OBJECTIVE: To compare reduction in incidence of feed intolerance in neonates born with meconium stained amniotic fluid (MSAF) by use of gastric lavage to those who did not receive lavage. METHODS: This Randomized controlled trial was conducted in all vigorous newborns delivered through MSAF, with birth weight ≥1800 g and gestation ≥34 wk. In the lavage group, gastric lavage with 10 ml/kg of normal saline was done. RESULTS: Twelve neonates in the lavage group (n = 124) developed feed intolerance compared to 16 neonates in control group (n = 120), (p = .309; OR 0.69; 95%CI 0.27-1.58). No difference in any other morbidity was noted. CONCLUSIONS: Gastric lavage in neonates with MSAF does not reduce feed intolerance, irrespective of thickness of MSAF and it confers no advantages.
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Líquido Amniótico , Lavado Gástrico , Meconio , Náusea/prevención & control , Vómitos/prevención & control , Femenino , Humanos , Recién Nacido , Masculino , Náusea/etiología , Resultado del Tratamiento , Vómitos/etiologíaRESUMEN
OBJECTIVE: The objective of the study was to investigate the effect of umbilical cord milking as compared with early cord clamping on hematological parameters at 6 weeks of age among term and near term neonates. STUDY DESIGN: This was a randomized control trial. Eligible neonates (>35 weeks' gestation) were randomized in intervention and control groups (100 each). Neonates of both groups got early cord clamping (within 30 seconds). The cord of the experimental group was milked after cutting and clamping at 25 cm from the umbilicus, whereas in control group cord was clamped near (2-3 cm) the umbilicus and not milked. Both groups got similar routine care. Unpaired Student t and Fisher exact tests were used for statistical analysis. RESULTS: Baseline characteristics were comparable in the 2 groups. Mean hemoglobin (Hgb) (11.9 [1.5] g/dL and mean serum ferritin 355.9 [182.6] µg/L) were significantly higher in the intervention group as compared with the control group (10.8 [0.9] g/dL and 177.5 [135.8] µg/L), respectively, at 6 weeks of age. The mean Hgb and hematocrit at 12 hours and 48 hours was significantly higher in intervention group (P = .0001). The mean blood pressure at 30 minutes, 12 hours, and 48 hours after birth was significantly higher but within normal range. No significant difference was observed in the heart rate, respiratory rate, polycythemia, serum bilirubin, and need of phototherapy in the 2 groups. CONCLUSION: Umbilical cord milking is a safe procedure and it improved Hgb and iron status at 6 weeks of life among term and near term neonates.
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Ferritinas/sangre , Sangre Fetal/trasplante , Hemoglobinas/análisis , Recien Nacido Prematuro/sangre , Adolescente , Adulto , Anemia/prevención & control , Constricción , Femenino , Estudios de Seguimiento , Hematócrito , Humanos , Recién Nacido , Masculino , Periodo Posparto , Factores de Tiempo , Cordón Umbilical , Adulto JovenRESUMEN
BACKGROUND: This study determine oxidative stress and survival prospectively in advanced stage non small cell lung cancer (NSCLC) patients following cisplatin based combination chemotherapy. MATERIALS AND METHODS: The oxidative stress levels (LPO, NO, GSH and SOD) of 144 control subjects and 203 advanced stage (IIIA/IIIB/IV) newly diagnosed NSCLC patients were assessed at pre-treatment (day '0'), and after the 3rd and 6th cycles of chemotherapy. Groups were compared by repeated measures ANOVA while comparison of survival curves was conduced by Kaplan-Meier methods. RESULTS: The pre-treatment mean levels of LPO and NO in patients were significantly (P<0.01) higher while GSH and SOD were significantly (P<0.01) lower as compared to control. The oxidative stress was elevated more significantly (P<0.01) after the chemotherapy and was more evident in higher stage than lower stage patients. The two year overall survival (%) of stage IV patients was significantly lower (P<0.05) as compared to stage III A and III B. The proportional mortality was also maximal in stage IV patients (37.0%) followed by stage III B (31.7%) and III A (20.0%). CONCLUSION: Cisplatin based combination chemotherapy induces oxidative stress in NSCLC patients.
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Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Grandes/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Grandes/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/patología , Estudios de Casos y Controles , Cisplatino/administración & dosificación , Etopósido/administración & dosificación , Femenino , Estudios de Seguimiento , Humanos , Peroxidación de Lípido/efectos de los fármacos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Superóxido Dismutasa/metabolismo , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
UNLABELLED: Levels of lipid peroxidation, nitric oxide and glutathione, as well as superoxide dismutase activity were evaluated in non-small cell lung cancer patients before and after chemotherapy. Oxidative stress was shown to influence treatment efficacy and survival of these patients. BACKGROUND AND OBJECTIVE: The aim of this study was to assess the level of oxidative stress after chemotherapy in non-small cell lung cancer patients, and its association with treatment response and survival. METHODS: Two hundred and three previously untreated non-small cell lung cancer patients and 150 healthy subjects were selected for the study. Patients received cisplatin+etoposide, and were followed for up to six cycles, for evaluation of oxidative stress. Blood levels of lipid peroxidation products (LPO), glutathione (GSH) and nitric oxide (NO), and superoxide dismutase (SOD) activity were measured at day 0 and after the third and sixth cycles of chemotherapy. Response and survival were measured at the end of follow up. Survival was estimated by the Kaplan-Meier method using the log-rank test. RESULTS: In the patients, pretreatment levels of LPO and NO were low, while GSH and SOD levels were high compared with those after the third and sixth cycles of chemotherapy. Among the 203 patients, there were 51 deaths, 82 non-responders and 70 responders at the end of the sixth cycle. Overall mean survival was higher among responders than non-responders (24.6 vs 21.2 weeks, P<0.01). The hazard ratio was 2.4 (95% CI: 1.3-3.77). Pretreatment levels of oxidative stress were similar among responders and non-responders (P>0.05). After the third and sixth cycles of chemotherapy, LPO and NO levels were low (P<0.05 or P<0.01) and GSH levels and SOD activity were high (P<0.05 or P<0.01) in responders as compared with non-responders. CONCLUSIONS: In lung cancer patients, oxidative stress increased and anti-oxidant enzymes decreased as the disease progressed. Chemotherapy may suppress oxidative stress and decreased anti-oxidant enzyme activity in responders as compared with non-responders. These effects may contribute to improved survival among responders.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/fisiopatología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/fisiopatología , Estrés Oxidativo/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Estudios de Casos y Controles , Cisplatino/administración & dosificación , Etopósido/administración & dosificación , Femenino , Glutatión/sangre , Humanos , Estimación de Kaplan-Meier , Peroxidación de Lípido/fisiología , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Óxido Nítrico/sangre , Superóxido Dismutasa/sangre , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Angiogenesis plays an important role in cervical cancer progression. Currently among several factors known to promote angiogenesis, vascular endothelial growth factor (VEGF) is most important. To evaluate the effect of treatment on VEGF levels and their correlation with other predictive factors, pre-and post treatment levels of VEGF were estimated in cervical cancer patients. 110 cases of frank cancer and 50 controls were enrolled for the present study: 18 in Stage I, 32 in Stage II, 48 in Stage III, and 12 in Stage IV. Serum VEGF levels were estimated by ELISA in patients on the day of recruitment and post treatment follow-up at a fixed time interval of 6-8 weeks. VEGF levels were highly significant among patients as compared to controls (p = 0.001). The pre-treatment VEGF levels among different stages of the disease were marginally insignificant (p = 0.07). However, they were significantly different for (i) various grades (p < 0.001), (ii) tumor size (p = 0.026), and (iii) smoking habits (p = 0.018). Post treatment levels were highly significant, as compared to pre-treatment values (p = 0.001). The pre-treatment and post-treatment VEGF levels were associated with (i) disease stage (p = 0.002), (ii) grade (p = 0.001), and (iii) tumor size (p = 0.001). In conclusion, VEGF is a potent angiogenic factor and can be considered as an effective prognostic marker in cervical cancer.
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Biomarcadores de Tumor/sangre , Neoplasias del Cuello Uterino/sangre , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/radioterapia , Factor A de Crecimiento Endotelial Vascular/sangre , Análisis de Varianza , Braquiterapia , Cisplatino/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , India , Estadificación de Neoplasias/estadística & datos numéricos , Estadísticas no Paramétricas , Neoplasias del Cuello Uterino/patologíaRESUMEN
Cigarette smoking is a well known environmental risk factor for lung cancer; furthermore it can also enhance lung carcinogenesis by free radical mediated reactions. In addition smoking affects the rates of metabolism of several drugs and may contribute to poor cancer survival. The purpose of the present work, therefore, was to see the relationship of different smoking intensities with oxidative stress and survival after platinum based chemotherapy in non-small cell lung cancer (NSCLC). The oxidative stress levels (LPO, NO, SOD, and GSH) of 144 control subjects and 203 advanced stage NSCLC patients were assessed at day '0', after the 3rd and 6th cycle of chemotherapy. Pack year (PY) was stratified in groups (1-20, 21-50, > 50) for further analysis. Groups were compared using repeated measured ANOVA, while survival curves were compared by Kaplan-Meier methods. Oxidative stress levels of smokers were significantly high (p < 0.01 or p < 0.05) as compared to non-smoker at pretreatment, after the 3rd cycle and 6th cycle of chemotherapy but not well correlated with the PY exposures. Overall mean survival of smoker patients were significantly low when compared to non-smokers. The survival of > 50 PY group was significantly lowered (p < 0.01) as compared to others PY groups, indicating that survival after chemotherapy in smoker NSCLC patients may be dependent on their PY exposures. In conclusion, smoking is a bad prognostic factor in lung cancer therapy, besides its role in oxidative stress, and poor survival. Therefore, this factor can be used in patient selection for chemoprevention.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Fumar/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Platino (Metal)/uso terapéutico , PronósticoRESUMEN
AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK beta subunit acts as a scaffold for the alpha catalytic and gamma regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK beta glycogen binding domain in complex with beta-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. beta-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two beta-cyclodextrin glucose units and a leucine residue piercing the beta-cyclodextrin ring. Mutation of key beta-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate's helical surface.
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Glucógeno/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Conformación de Carbohidratos , Dominio Catalítico , Cristalografía por Rayos X , Glucanos/farmacología , Glucosa/química , Glucógeno/química , Glucógeno/genética , Leucina/química , Hígado/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/aislamiento & purificación , Mutagénesis Sitio-Dirigida , Mutación , Oligosacáridos/farmacología , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Espectrometría Raman , Triptófano/química , Agua/química , beta-Ciclodextrinas/química , beta-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/farmacologíaRESUMEN
AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic alpha subunit and two regulatory subunits, beta and gamma. Mutations in the gamma subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the beta subunit. Here, the crystallization of GBD in the presence of beta-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein.
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Glucógeno/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas Activadas por AMP , Animales , Sitios de Unión , Clonación Molecular , Cristalización , Cartilla de ADN , Complejos Multienzimáticos , Fragmentos de Péptidos/química , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Subunidades de Proteína/química , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Selenometionina , Difracción de Rayos XRESUMEN
AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients, as well as hormones, including adiponectin and leptin. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK. Mammalian AMPK is an alphabetagamma heterotrimer with multiple isoforms of each subunit comprising alpha1, alpha2, beta1, beta2, gamma1, gamma2, and gamma3, which have varying tissue and subcellular expression. Mutations in the AMPK gamma subunit cause glycogen storage disease in humans, but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK beta subunit contains a functional glycogen binding domain (beta-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished beta-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK beta1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.