RESUMO
Developmental programming, which proposes that "insults" or "stressors" during intrauterine or postnatal development can have not only immediate but also long-term consequences for healthy and productivity, has emerged as a major biological principle, and based on studies in many animal species also seems to be a universal phenomenon. In eutherians, the placenta appears to be programmed during its development, which has consequences for fetal growth and development throughout pregnancy, and likewise has long-term consequences for postnatal development, leading to programming of organ function of the offspring even into adulthood. This review summarizes our current understanding of the placenta's role in developmental programming, the mechanisms involved, and the challenges remaining.
Assuntos
Desenvolvimento Fetal , Placenta , Gravidez , Feminino , AnimaisRESUMO
Wheat leaves contain two isoproteins of the photosynthetic ferredoxin:NADP(+) reductase (pFNRI and pFNRII). Truncated forms of both enzymes have been detected in vivo, but only pFNRII displays N-terminal length-dependent changes in activity. To investigate the impact of N-terminal truncation on interaction with ferredoxin (Fd), recombinant pFNRII proteins, differing by deletions of up to 25 amino acids, were generated. During purification of the isoproteins found in vivo, the longer forms of pFNRII bound more strongly to a Fd affinity column than did the shorter forms, pFNRII(ISKK) and pFNRII[N-2](KKQD). Further truncation of the N-termini resulted in a pFNRII protein which failed to bind to a Fd column. Similar k(cat) values (104-140 s(-1)) for cytochrome c reduction were measured for all but the most truncated pFNRII[N-5](DEGV), which had a k(cat) of 38 s(-1). Stopped-flow kinetic studies, examining the impact of truncation on electron flow between mutant pFNRII proteins and Fd, showed there was a variation in k(obs) from 76 to 265 s(-1) dependent on the pFNRII partner. To analyze the sites which contribute to Fd binding at the pFNRII N-terminal, three mutants were generated, in which a single or double lysine residue was changed to glutamine within the in vivo N-terminal truncation region. The mutations affected binding of pFNRII to the Fd column. Based on activity measurements, the double lysine residue change resulted in a pFNRII enzyme with decreased Fd affinity. The results highlight the importance of this flexible N-terminal region of the pFNRII protein in binding the Fd partner.
Assuntos
Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/química , Folhas de Planta/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Triticum/enzimologia , Sítios de Ligação , Ferredoxina-NADP Redutase/genética , Ferredoxinas/metabolismo , Cinética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Triticum/metabolismoRESUMO
We hypothesized that both day of gestation and maternal nutrition would alter the relative mRNA expression of neutral and acid AA transporters , , , , and . Crossbred Angus heifers ( = 49) were synchronized, bred via AI, assigned to nutritional treatment (100% of NRC requirements for 0.45 kg/d gain [control heifers {CON}] and 60% of CON [restricted heifers {RES}]), and ovariohysterectomized on d 16, 34, or 50 of gestation ( = 6 to 9/d). Nonbred, nonpregnant (NB-NP) controls were ovariohysterectomized on d 16 of the estrous cycle ( = 6) after synchronization. The resulting arrangement was a 2 × 3 factorial + 1 (CON vs. RES × d 16, 34, or 50 + NB-NP controls). Tissues collected included caruncular endometrium (CAR), intercaruncular endometrium (ICAR), fetal membranes (FM; chorioallantois; d 16 and 34), cotyledonary placenta (COT; d 50 only), intercotyledonary placenta (ICOT; d 50 only), and amnion (AMN; d 50 only]). Relative expression of , , , , and was determined for each tissue using NB-NP CAR and NB-NP ICAR tissues for the baseline; for FM, endometrium from NB-NP controls served as the baseline. In CAR, no day × treatment interaction was observed ( > 0.05). However, day of gestation affected relative expression of , where expression on d 16 was greater ( < 0.01) than expression on d 34 and 50. Additionally, relative expression of and was greater ( ≤ 0.05) in pregnant heifers compared with NB-NP heifers. For ICAR, was influenced by a day × treatment interaction ( < 0.01), where expression in d 16 RES was greater ( ≤ 0.05) than that of any other day or nutritional treatment. Furthermore, expression in d 16 CON was greater ( ≤ 0.05) than that in d 50 RES, with those in d 34 CON and RES and d 50 CON being intermediate. In addition, was affected by day of gestation, where expression on d 16 was greater ( < 0.01) than that on d 34 and 50. A day × treatment interaction was not observed ( > 0.05) in FM; however, expression on d 34 was greater ( = 0.02) than on d 50, with that on d 16 being intermediate. Day of gestation also affected expression of , where expression on d 34 and 50 was greater ( < 0.01) than that on d 16. These data support our hypothesis in that both day of gestation and maternal nutrition affected the relative mRNA expression of AA transporter in ICAR, whereas day of gestation has a greater effect on the relative mRNA expression of other neutral and acidic AA transporters in the various tissues studied.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/genética , Bovinos/fisiologia , Animais , Cruzamento , Bovinos/genética , Endométrio/fisiologia , Ciclo Estral , Feminino , Fenômenos Fisiológicos da Nutrição Materna , Placenta/fisiologia , Gravidez , RNA Mensageiro/genética , Útero/fisiologiaRESUMO
We hypothesized that maternal nutrition and day of gestation would impact utero-placental mRNA expression of the nutrient transporters , , , , and in beef heifers. Crossbred Angus heifers (n = 49) were estrous synchronized, bred via AI, assigned to nutritional treatment (CON = 100% of NRC requirements for 0.45 kg/d gain and RES = 60% of CON) and ovariohysterectomized on d 16, 34, or 50 of gestation (n = 6 to 9/d); Non-bred, non-pregnant (NB-NP) controls were fed the CON diet, not bred, and were ovariohysterectomized on d 16 of the synchronized estrous cycle = 6). The resulting arrangement of treatments was a 2 × 3 factorial + 1 (CON vs. RES × d 16, 34, or 50 + NB-NP controls). Caruncle (CAR), intercaruncular endometrium (ICAR), and fetal membranes (FM [chorioallantois]), were obtained from the pregnant uterine horn (the uterine horn containing the conceptus) immediately after ovariohysterectomy. On d 50 cotyledons (COT), intercotyledonary placenta (ICOT) and amnion (AMN) were also collected. Relative expression of nutrient transporters was determined for each tissue utilizing NB-NP-CAR and NB-NP-ICAR tissues as the baseline. For FM, NB-NP endometrium served as the baseline. There was no interaction of day × treatment ( ≥ 0.20) for any genes in CAR. However, CAR expression of was greater ( < 0.01) on d 16 compared with d 34 and 50, and , , and were greater ( ≤ 0.05) on d 34 compared with d 16 and 50. In ICAR, was the only gene to be influenced by the day × treatment interaction ( = 0.01), being greater in d 50 CON compared with d 34 CON and d 16 and 50 RES. In ICAR, expression of was greater ( < 0.01) on d 16 compared with d 34, and expression of was greater ( < 0.01) on d 34 and 50 compared with d 16. In FM, expression of was greater ( = 0.04) on d 16 compared with d 50 of gestation, and expression of was greater ( < 0.01) on d 34 and 50 compared with d 16. On d 50, expression of , , and expression were all greater ( < 0.05) in AMN compared with COT and ICOT, and expression of was greater ( < 0.01) in ICOT compared with COT and AMN. These data indicate that day was a more influential factor for mRNA expression of utero-placental glucose and cationic AA transporters than maternal nutritional status in heifers during early pregnancy.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Bovinos/fisiologia , Frutose/metabolismo , Glucose/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Animais , Cruzamento , Dieta/veterinária , Endométrio/metabolismo , Ciclo Estral/metabolismo , Feminino , Placenta/metabolismo , Gravidez , Útero/metabolismoRESUMO
We hypothesized that maternal nutrient restriction starting at the time of breeding would influence placental vascular development and gene expression of angiogenic factors during the first 50 d of gestation in beef heifers. Commercial Angus crossbred heifers (n = 49) were maintained on a total mixed ration and supplemented with dried distillers grains with solubles. All heifers were subject to 5-d CO-Synch + CIDR estrous synchronization protocol, AI to a single Angus sire, and randomly assigned to dietary treatments. One half were assigned to control diet (CON) targeted to gain 0.45 kg/d and the remaining half were assigned to restricted diet (RES), which received 60% of CON. Heifers were subjected to ovariohysterectomy on d 16, 34, or 50 of gestation. Utero-placental tissues were obtained from the uterine horns ipsilateral and contralateral to the corpus luteum and separated into maternal caruncle (CAR); maternal endometrium, inter-caruncle (ICAR), and fetal membranes (FM). After collection, all tissues were snap frozen and stored at -80°C. There were no treatment × stage of gestation interactions (P >0.13) on the mRNA expression of vascular endothelial growth factor (VEGF) or endothelial nitric oxide synthase (eNOS). Heifers on CON treatment had greater (P = 0.03) expression of VEGF compared with RES heifers in NP-ICAR. On d 50 expression of eNOS was increased (P = 0.05) compared with d 16 in P-CAR. Expression of eNOS mRNA was decreased (P = 0.04) on d 16 compared with d 34 and 50 in CON heifer. Gene expression of eNOS was increased (P < 0.001) in the pregnant uterine horn compared with the NP uterine horn on d 34 and 50. Expression of eNOS was also increased (P < 0.003) on d 34 and 50 in the pregnant uterine horn compared with FM. There was a maternal nutritional plane × stage of gestation interaction (P = 0.01) on the vascular ratio (vascular volume/tissue volume) in maternal tissues. The RES heifers had a greater vascular ratio on d 16 compared with d 34 and 50; whereas, CON heifers had a greater vascular ratio on d 34 compared with d 16 and 50. In the NP uterine horn, there was also an increase (P = 0.02) in vascular volume of FM from CON heifers compared with FM from RES heifers. We conclude that maternal nutrient restriction did alter both vascularity and mRNA expression of angiogenic factor in utero-placental tissues during the establishment of pregnancy in first parity beef heifers.
RESUMO
We hypothesized that the endogenous retroviruses [ERV: syncytin-Rum1 and (BERV-K1)], and pregnancy hormones [interferon-τ (IFN-τ), and pregnancy associated glycoprotein-1 (PAG-1)] would be differentially expressed whereas progesterone and insulin concentrations in maternal blood would remain steady during early gestation. To test this hypothesis Angus crossbred heifers (n = 46; â¼15 mo of age; BW = 363 ± 35 kg) were fed native grass hay, supplemented with cracked corn to gain 0.3 kg/d, and given ad libitum access to water. All heifers were subjected to a 5-d CO-Synch + CIDR estrous synchronization protocol and AI (breeding = d 0). Ovariohysterectomies were performed on d 16, 22, 28, 34, 40, and 50 of gestation and at d 16 of the estrous cycle for non-pregnant (NP) controls. Utero-placental tissues [maternal caruncle (CAR); maternal intercaruncular endometrium (ICAR); and fetal membranes, (FM, chorion on d 16, chorioallantois on d 22 to 50)] were collected from the uterine horn ipsilateral to the corpus luteum (CL). Tissues were flash frozen and stored at -80°C. Expression of mRNA was evaluated using qPCR. In CAR, syncytin-Rum1 expression was greater (P < 0.01) on d 50 (81.5-fold) compared with NP controls or any other day of early pregnancy. In contrast, syncytin-Rum1 expression in I-CAR only tended (P = 0.09) to change across days of early pregnancy and did not differ (P = 0.27) in FM tissues. In CAR, the expression of BERV-K1 was not different (P > 0.79) at d 16 and 22, was intermediate at d 28, 34, and 40, and was greatest on d 50 (108-fold increase compared with NP). Expression of BERV-K1 in FM was increased (P < 0.01) on d 28, 34, and 50 compared with NP controls, but at d 40 did not differ from NP controls. The mRNA expression of IFN-τ in FM at d 22 was greater (P < 0.01) than all other days of gestation. In CAR, expression of PAG-1 increased (P < 0.001) dramatically on d 40 (20,000-fold) and d 50 (86,000-fold) compared with NP heifers (P < 0.01). In ICAR, expression of PAG-1 was greater (P < 0.05) on d 28 and 40 (fold increases of 113 and 102, respectively, compared with NP). Insulin concentrations were not different (P = 0.53) but progesterone was greater (P < 0.01) on d 16, 22, 28, 34, and 40 compared with d 50 of gestation. These data confirm differential ERV, IFN-τ, and PAG-1 gene expression during critical time points of early gestation in utero-placental tissues.
RESUMO
We hypothesized that a standing flank ovariohysterectomy procedure could be developed in beef heifers that would provide high quality tissues for addressing critical questions during early pregnancy, while concomitantly keeping livestock stewardship a high priority. To test the hypothesis, we: 1) developed a standing flank ovariohysterectomy procedure for use in beef heifers, and 2) implemented this procedure in a cohort of heifers up to d 50 of pregnancy for tissue collections, documentation of post-surgical recovery, and assessment of feedlot finishing performance. Ovariectomy and cesarean section protocols are well established in research and veterinary medicine and were used as starting points for procedural development. Crossbred Angus heifers ( = 46; â¼ 15 mo of age; BW = 362.3 ± 34.7 kg) were used to develop this new surgical tissue collection technique. Heifers were subjected to the 5-d CO-Synch + CIDR estrous synchronization protocol so ovariohysterectomy occurred at d 16, 22, 28, 34, 40, and 50 of gestation. Key aspects of the standing flank ovariohysterectomy technique included 1) use of local anesthetic for a standing flank incision, 2) locate the uterine and ovarian arteries via blind palpation and ligate them through the broad ligament via an improved clinch knot, 3) cut the ovaries and uterus free from the broad ligament, 4) ligate the cervix and uterine branch of the vaginal artery, and 5) cut through the cervix and remove the reproductive tract. Surgical times, from skin incision to placement of the last suture, were influenced ( = 0.04) by stage of gestation. In pregnant heifers, time decreased from d 22 (120.0 ± 12.0 min) of gestation to d 40 (79.5 ± 12.0 min) of gestation; then increased at d 50 (90.5 ± 14.7 min) of gestation. Using this procedure, we obtained uterine, placental, and embryo/fetal tissues that had experienced limited hypoxia, little or no trauma, and thus were excellent quality for scientific study. All heifers recovered from surgery quickly and were moved to a finishing period. During the finishing period, ovariohysterectomized heifers had a DMI of 13.8 kg, gained 1.99 ± 0.35 kg/d, and had a G:F of 0.145 over 132-d. The standing flank ovariohysterectomy technique represents a new and viable model to economically obtain high quality tissues for investigating critical biological mechanisms during early pregnancy in beef heifers.
Assuntos
Bovinos/cirurgia , Histerectomia/veterinária , Ovariectomia/veterinária , Prenhez , Animais , Bovinos/fisiologia , Feminino , Histerectomia/métodos , Ovariectomia/métodos , Ovário , Gravidez , ProgesteronaRESUMO
Glucose transporter solute carrier family 2 member 14 () is a duplicon of glucose transporter solute carrier family 2 member 3 () with a 95% shared homology to and has not previously been isolated in ruminant uteroplacental tissues. The transporter has been previously isolated in Holstein heifer uterine epithelium but not in ovine epithelium. We hypothesized that and its duplicon would be found in bovine uteroplacental tissues and that maternal nutrition and day of gestation would impact mRNA expression of and . Crossbred Angus heifers ( = 49) were estrus synchronized, bred via AI, and assigned to nutritional treatment (CON = 100% of requirements to gain 0.45 kg/d; RES = 60% of CON) at breeding. Ovariohysterectomy was performed on d 16, 34, or 50 of gestation ( = 6 to 9/d); nonpregnant (NP) controls were not bred and ovariohysterectomized on d 16 of the synchronized estrous cycle ( = 6). The resulting treatment arrangement was a 2 × 3 factorial + 1. Uteroplacental tissues (caruncle, CAR; intercaruncular endometrium, ICAR; and fetal membrane [chorioallantois], FM) were obtained from the pregnant uterine horn immediately after ovariohysterectomy. For NP controls, only CAR and ICAR were obtained. There were no day × treatment interactions for or gene expression in CAR, ICAR, or FM. Expression of in CAR was greater ( = 0.03) on d 50 compared with d 16. In ICAR, was greatest ( = 0.02) on d 50 compared with d 16 and 34 of gestation. In FM, was greater ( = 0.04) on d 16 compared with d 50. Expression of was greater ( = 0.05) in pregnant compared with nonpregnant heifers. Additionally, expression of was greater ( = 0.01) on d 34 and 50 compared with d 16. Expression of in CAR was greater ( = 0.03) on d 50 compared to d 16 and 34. In CAR, tended ( = 0.07) to be greater on d 34 and 50 than on d 16 and was greater ( = 0.02) on d 50 than on d 34. There was no effect of treatment for either or in CAR, ICAR, or FM. These data demonstrate that glucose transporters and are expressed in beef heifer uteroplacental tissues and that they are expressed differentially by day of gestation in bovine uteroplacental tissues.
Assuntos
Bovinos/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Prenhez , Animais , Sequência de Bases , Cruzamento , Bovinos/fisiologia , Endométrio/fisiologia , Sincronização do Estro , Feminino , Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Placenta/fisiologia , Gravidez , Fenômenos Fisiológicos da Nutrição Pré-Natal , Alinhamento de Sequência/veterinária , Útero/fisiologiaRESUMO
During early gestation, nutrients are transported to the developing embryo via transporters in the uterine endometrium and chorioallantois. In the present study, we examined glucose transporters and and the cationic AA transporters , , and to test the hypotheses that 1) relative mRNA expression of transporters would be different among uteroplacental tissue type as gestation progresses and 2) concentrations of glucose and cationic AA would be different among target sites (placental compartments, serum, and histotrophic) and days of gestation. To test these hypotheses, crossbred Angus heifers ( = 46) were synchronized, bred via AI, and then ovariohysterectomized on d 16, 22, 28, 34, 40, or 50 of gestation (5 to 9/d) or not bred and ovariohysterectomized on d 16 of the synchronized estrous cycle ( = 7) to serve as nonpregnant (NP) controls. Uteroplacental tissues (maternal caruncle [CAR], intercaruncular endometrium [ICAR], and fetal membranes [FM; chorioallantois, d 22 and later]) were collected from the uterine horn ipsilateral to the corpus luteum immediately following ovariohysterectomy. Relative mRNA expression of the glucose transporters and cationic AA transporters was determined for each tissue from d 16 to 50 of gestation and from NP controls. Chorioallantoic, amniotic, and plasma fluids were collected from heifers on d 40 and 50 of gestation to determine concentrations of glucose and cationic AA. Expression of and showed a tendency ( < 0.10) toward being greater in d 16 ICAR and d 34 ICAR, respectively. Day × tissue interactions ( < 0.05) were present for , , and . Expression of was greater in d 50 CAR, expression of was greater on d 34 in ICAR, and expression of was greater in CAR tissue on d 34 compared with all other tissues and days of gestation. Glucose concentrations tended ( = 0.10) to be impacted by a day × fluid interaction. A day × fluid interaction ( = 0.01) for arginine concentration was observed, with greater concentrations in allantoic fluid on d 40 compared with all other days and fluid types. These data support our hypothesis that glucose and cationic AA transporters differ in their level of mRNA expression due to day of gestation and uteroplacental tissue type. In addition, concentrations of nutrients were differentially impacted by day, target site, and/or their respective interaction.
Assuntos
Aminoácidos/metabolismo , Bovinos/fisiologia , Glucose/metabolismo , Proteínas de Membrana Transportadoras/genética , Placenta/metabolismo , Útero/metabolismo , Animais , Transporte Biológico , Bovinos/embriologia , Endométrio/metabolismo , Ciclo Estral , Feminino , Alimentos , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Proteínas de Membrana Transportadoras/metabolismo , GravidezRESUMO
Endogenous retroviral gene elements have been implicated in development and formation of the feto-maternal interface. A variant of the syncytin endogenous retroviral envelope gene family, , was recently found in ruminants. We hypothesized that mRNA would be differentially expressed in utero-placental tissues and would fluctuate during key time points of early gestation in beef heifers. Commercial Angus crossbred heifers ( = 46; â¼15 mo of age; BW = 362.3 ± 34.7kg) housed in 6-animal pens were fed daily with native grass hay and supplemented with cracked corn to gain 0.3 kg/d. The heifers were estrus synchronized, artificially inseminated, (d of breeding= d 0) and ovariohysterectomized on d 16, 22, 28, 34, 40, and 50 ( = 9, 6, 6, 7, 6, and 5, respectively) of gestation and at d 16 of the estrous cycle for non-bred, non-pregnant controls (NP; = 7). Harvested tissues were separated into maternal caruncle (CAR), intercarunclar endometrium (ICAR), and fetal membranes, (FM; chorioallantois, d 22 and later). All tissues were obtained from the ipsilateral uterine horn to the CL. Statistical analyses were conducted via the GLM procedure of SAS. Maternal CAR expression of was greater ( = 0.003) on d 50 by 81.5-fold compared to NP controls. At d 50 expression of in CAR was 190.3-fold greater than ( < 0.0001) ICAR. Fetal membranes had greater ( < 0.002) expression of from d 22 until d 50 of gestation compared to maternal ICAR (d 16 not analyzed). Expression of in FM was greater ( < 0.004) than in CAR until d 40 of gestation. Therefore, we conclude that is differentially expressed in utero-placental tissues and may be involved in the establishment of pregnancy. The expression of in maternal tissues is completely novel and indicates unique functions of syncytin in ruminant pregnancy.
Assuntos
Bovinos/fisiologia , Suplementos Nutricionais , Produtos do Gene env/metabolismo , Proteínas da Gravidez/metabolismo , Animais , Cruzamento , Bovinos/genética , Retrovirus Endógenos , Ciclo Estral , Sincronização do Estro , Feminino , Produtos do Gene env/genética , Inseminação Artificial , Placenta/fisiologia , Folhas de Planta , Poaceae , Gravidez , Proteínas da Gravidez/genética , Carne Vermelha , Sementes , Zea maysRESUMO
PURPOSE: Proliferation of neural precursors adjacent to the granule cell layer of the dentate gyrus has been identified in previous epilepsy models. Convincingly demonstrating that seizure activity is the stimulant for neurogenesis, rather than neuronal death or other insults inherent to seizure models, is difficult. To address this we derived a rapid electrical amygdala kindling model in mice known to be resistant to seizure-induced neuronal death as an experimental model of focal seizures and to analyze subsequent neurogenesis. METHODS: Mice were implanted with bipolar electrodes in the left amygdala and given electrical stimulation (3 s, 100 Hz, 1 ms monophasic square wave pulses every 5 min, 40 in total) while being observed and graded for the development of seizures. Neurogenesis in the hippocampus was assessed by counting bromodeoxyuridine-immunoreactive cells co-labeled for astrocyte (glial fibrillary acidic protein) and neuronal nuclear markers. RESULTS: Bromodeoxyuridine-reactive cell numbers were three-fold higher in stimulated mice compared with controls at 1 week in the subgranular region and at three weeks extensive co-labeling with neuronal nuclear was noted in cells which had migrated into the body of the granule cell layer, while mice receiving stimulation but failing to kindle did not differ significantly from controls. No increase in neuronal death was detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling, Fluorojade or fluorescent examination of hematoxylin and eosin-stained sections in any inter-group comparison. CONCLUSIONS: We propose that this kindling paradigm, not previously applied to mice, demonstrates more convincingly than previously the surge in neurogenesis in response to seizures, and the effects of seizures alone in regard to neuronal injury and regeneration.
Assuntos
Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Excitação Neurológica/fisiologia , Neurônios/patologia , Convulsões/patologia , Animais , Antimetabólitos , Comportamento Animal/fisiologia , Bromodesoxiuridina , Morte Celular , Proliferação de Células , Grânulos Citoplasmáticos/fisiologia , Giro Denteado/patologia , Eletroencefalografia , Genótipo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Mitose/fisiologia , Degeneração Neural/patologiaRESUMO
The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.
Assuntos
Genômica/organização & administração , Mycobacterium tuberculosis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Genoma Bacteriano , Humanos , Cooperação Internacional , Dados de Sequência Molecular , Mycobacterium tuberculosis/metabolismo , Conformação Proteica , Alinhamento de SequênciaRESUMO
The present study was designed to determine whether neurons within cardiovascular control nuclei of the rat brainstem that become activated following a hypotensive insult also possess the capacity to utilize neuropeptide Y. Adult male Wistar-Kyoto rats were injected with glyceryl trinitrate (10 mg/kg, i.p.) or vehicle, and 4 h later anaesthetized (pentobarbitone, 60 mg/kg, i.p.) and transcardially perfused. The brains were removed and processed by standard two-colour peroxidase immunohistochemistry. Activated cells were determined by incubation with a primary antibody to Fos protein, which was followed by a second incubation with a primary antibody to neuropeptide Y for double labelling of Fos-positive cells. Compared to vehicle, glyceryl trinitrate-induced hypotension caused a marked induction of Fos protein in the caudal one-third of the nucleus tractus solitarius (bregma -14 to -13.3 mm), which tailed off rapidly in more rostral sections. Following hypotension, significant populations of activated cells were also observed in the rostral and caudal ventrolateral medulla. In the caudal nucleus tractus solitarius and the posterior part of the medial nucleus tractus solitarius, respectively, 15 of 104 and 40 of 120 Fos-positive cells exhibited cytoplasmic neuropeptide Y immunoreactivity following hypotension, compared to seven of 40 and 15 of 40 in vehicle-treated rats, indicating a significant (two- to three-fold) increase in double-labelled cells following systemic glyceryl trinitrate (P < 0.05, unpaired t-test). In contrast, in the anterior part of the medial nucleus tractus solitarius, the number of double-labelled cells did not change following hypotension. An increase in double-labelled cells was also observed in the rostral ventrolateral medulla (2.5-fold increase compared to vehicle) and caudal ventrolateral medulla (5.8-fold increase compared to vehicle) following hypotension. These data indicate that, in the rat, neuropeptide Y-containing neurons are involved in the central response to a hypotensive challenge. The primary regions where neuropeptide Y-containing neurons appear to be activated are the caudal one-third of the nucleus tractus solitarius and the caudal ventrolateral medulla/rostral ventrolateral medulla, which are key nuclei associated with the integration of the baroreceptor heart rate reflex and sympathetic vasomotor outflow.
Assuntos
Hipotensão/fisiopatologia , Bulbo/fisiopatologia , Neurônios/fisiologia , Neuropeptídeo Y/fisiologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Bulbo/citologia , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Nitroglicerina/farmacologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Ratos , Ratos Endogâmicos WKY , Núcleo Solitário/citologia , Núcleo Solitário/fisiologia , Vasodilatadores/farmacologiaRESUMO
Neurones containing neuropeptide Y (NPY) may participate in central cardiovascular control by tonically influencing barosensitive neurones within the nucleus tractus solitarius. The present study has employed both in situ hybridisation histochemistry and receptor autoradiography, to visualise the expression of prepro-NPY mRNA in the forebrain and to determine the NPY receptor subtype(s) in the brainstem, respectively. Prepro-NPY gene expression was visualised in the hypothalamus, cortex, dentate gyrus and lateral reticular thalamus from age-matched spontaneously hypertensive rats (SHR) and normotensive Don Ryu rats (DRY) and Wistar Kyoto rats (WKY). Quantitative densitometry revealed an increase in the NPY transcript in the arcuate nucleus of SHR rats compared to their normotensive counterparts. Autoradiography using [125I]Bolton-Hunter-NPY (BH-NPY, 15 pM) demonstrated NPY binding sites in the area postrema, the commissural nucleus tractus solitarius (cNTS) and the inferior olivary complex. NPY (1 microM) and peptide YY (1 microM), but not [Leu31,Pro34]NPY (10-100 nM), fully inhibited the binding of [125I]BH-NPY. These results indicate that NPY receptors of the Y2 subtype predominate in the dorsal vagal complex. Unilateral nodose ganglionectomy resulted in a partial loss of NPY binding sites in the commissural NTS, but not the area postrema, suggesting that a proportion of binding sites (Y2 subtype) are present on central vagal terminals. While all three rat strains appear to have the same relative proportions of NPY receptor subtypes in the brainstem, the relevance of the differential NPY gene expression in the arcuate nucleus regarding central cardiovascular control mechanisms and/or the pathogenesis of hypertension remains to be elucidated.
Assuntos
Encéfalo/metabolismo , Expressão Gênica , Hipertensão/metabolismo , Neurônios/metabolismo , Neuropeptídeo Y/biossíntese , Receptores de Neuropeptídeo Y/biossíntese , Animais , Autorradiografia , Sequência de Bases , Hibridização In Situ , Radioisótopos do Iodo , Masculino , Dados de Sequência Molecular , Gânglio Nodoso/fisiologia , Sondas de Oligonucleotídeos , Oligonucleotídeos Antissenso , Precursores de Proteínas/biossíntese , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos , Ratos Endogâmicos WKY , Valores de Referência , Núcleo Solitário/metabolismoRESUMO
The present study has employed immunocytochemistry on free-floating sections of adult rat medulla oblongata to characterise the distribution of nitric oxide synthase- (NOS), adenosine deaminase- (ADA) and neuropeptide Y- (NPY) immunoreactivity (IR) throughout the entire rostro-caudal axis of the nucleus tractus solitarius (NTS). In addition, unilateral nodose ganglionectomy was performed in a group of rats to determine whether any observed immunoreactivity was associated with central vagal afferent terminals. NOS-IR was found throughout the entire NTS, in cells, and both varicose and non-varicose fibres. Furthermore, unilateral nodose ganglionectomy resulted in a clear reduction in NOS-IR (visualised with diaminobenzidine) in a highly restricted portion of the ipsilateral medial NTS. Similarly, ADA- and NPY-containing cells, fibres and terminals were also found throughout the adult rat NTS. However, following unilateral nodose ganglionectomy, there was no apparent reduction in either ADA-IR or NPY-IR on the denervated side of the NTS. These data indicate a role for nitric oxide, purines and neuropeptide Y as neuromodulators within the rat NTS, although only nitric oxide appears to be primarily associated with vagal afferent input. Adenosine deaminase and neuropeptide Y-containing neurons appear to be predominantly postsynaptic to vagal input, although their possible association with vagal afferents cannot be completely excluded.
Assuntos
Adenosina Desaminase/análise , Ganglionectomia , Neuropeptídeo Y/análise , Óxido Nítrico Sintase/análise , Núcleo Solitário/química , Animais , Imuno-Histoquímica , Masculino , Óxido Nítrico Sintase Tipo I , Ratos , Ratos Endogâmicos WKYRESUMO
The inferior vagal ganglia contain the cell bodies of centrally projecting vagal afferent neurones. Using in situ hybridization-histochemistry with a combination of two antisense neuropeptide Y (NPY) oligonucleotides, we have demonstrated that a population of human inferior vagal perikarya express mRNA encoding prepro-NPY, the precursor of NPY. In vitro receptor autoradiography, using both [125I]Bolton Hunter-NPY ([125I]BH-NPY, 15 pM) and [125I]peptide YY ([125I]PYY, 25 pM), enabled visualization of NPY binding sites. Competition binding with NPY (1 microM), PYY (1 microM) and [Leu31,Pro34]NPY (100 nM), suggest that both Y1 and Y2 receptor subtypes are present on human vagal afferent neurones. These observations suggest a potential role for NPY in neuromodulation of vagal transmission in humans.
Assuntos
Neuropeptídeo Y/metabolismo , Neuropeptídeo Y/farmacologia , Precursores de Proteínas/metabolismo , Receptores de Neuropeptídeos/efeitos dos fármacos , Succinimidas/farmacologia , Nervo Vago/efeitos dos fármacos , Autorradiografia , Feminino , Histocitoquímica , Humanos , Hibridização In Situ , Técnicas In Vitro , Masculino , RNA Mensageiro/metabolismoAssuntos
Proteínas de Bactérias , Sistema Enzimático do Citocromo P-450/metabolismo , Oxigenases de Função Mista/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Transporte de Elétrons , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Químicos , Mutagênese Sítio-Dirigida , NADPH-Ferri-Hemoproteína Redutase , Conformação Proteica , Especificidade por SubstratoRESUMO
Flavocytochrome P450 (cytochrome P450) BM3 is an intensively studied model system within the P450 enzyme superfamily, and is a natural fusion of a P450 to its P450 reductase redox partner. The fusion arrangement enables efficient electron transfer within the enzyme and a catalytic efficiency that cannot be matched in P450 systems from higher organisms. P450 BM3's potential for industrially relevant chemical transformations is now recognized, and variants with biotechnological applications have been constructed. Simultaneously, structural and mechanistic studies continue to reveal the intricate mechanistic details of this enzyme, including its dimeric organization and the relevance of this quaternary structure to catalysis. Homologues of BM3 have been found in several bacteria and fungi, indicating important physiological functions in these microbes and enabling first insights into evolution of the enzyme family. This short paper deals with recent developments in our understanding of structure, function, evolution and biotechnological applications of this important P450 system.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas de Bactérias/química , Biotecnologia/métodos , Catálise , Sistema Enzimático do Citocromo P-450/química , Evolução Molecular , Oxigenases de Função Mista/química , Modelos Moleculares , NADPH-Ferri-Hemoproteína Redutase , Filogenia , Conformação ProteicaRESUMO
An extraordinary array of P450 (cytochrome P450) enzymes are encoded on the genome of the human pathogen Mycobacterium tuberculosis (Mtb) and in related mycobacteria and actinobacteria. These include the first characterized sterol 14alpha-demethylase P450 (CYP51), a known target for azole and triazole drugs in yeasts and fungi. To date, only two Mtb P450s have been characterized in detail: CYP51 and CYP121. The CYP121 P450 shows structural relationships with P450 enzymes involved in synthesis of polyketide antibiotics. Both P450s exhibit tight binding to a range of azole drugs (e.g. clotrimazole and fluconazole) and the same drugs also have potent effects on growth of mycobacteria (but not of e.g. Escherichia coli). Atomic structures are available for both Mtb CYP51 and CYP121, revealing modes of azole binding and intriguing mechanistic and structural aspects. This paper reviews our current knowledge of these and the other P450 systems in Mtb including recent data relating to the reversible conversion of the CYP51 enzyme between P450 (thiolate-co-ordinated) and P420 (thiol-co-ordinated) species on reduction of the haem iron in the absence of a P450 substrate. The accessory flavoprotein and iron-sulfur proteins required to drive P450 catalysis are also discussed, providing an overview of the current state of knowledge of Mtb P450 redox systems.
Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Mycobacterium tuberculosis/enzimologia , Azóis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/patogenicidade , Oxirredução , Ligação Proteica , Conformação ProteicaRESUMO
Since its discovery in the 1980s, the fatty acid hydroxylase flavocytochrome P450 (cytochrome P450) BM3 (CYP102A1) from Bacillus megaterium has been adopted as a paradigm for the understanding of structure and mechanism in the P450 superfamily of enzymes. P450 BM3 was the first P450 discovered as a fusion to its redox partner--a eukaryotic-like diflavin reductase. This fact fuelled the interest in soluble P450 BM3 as a model for the mammalian hepatic P450 enzymes, which operate a similar electron transport chain using separate, membrane-embedded P450 and reductase enzymes. Structures of each of the component domains of P450 BM3 have now been resolved and detailed protein engineering and molecular enzymology studies have established roles for several amino acids in, e.g. substrate binding, coenzyme selectivity and catalysis. The potential of P450 BM3 for biotechnological applications has also been recognized, with variants capable of industrially important transformations generated using rational mutagenesis and forced evolution techniques. This paper focuses on recent developments in our understanding of structure and mechanism of this important enzyme and highlights important problems still to be resolved.