RESUMO
PROBLEM: Clinical significance of endometrial and peripheral blood natural killer (NK) and regulatory T cells (Tregs) during frozen embryo transfer (FET) cycles has not been well characterized. DESIGN: Retrospective cohort study. METHOD OF STUDY: Endometrial tissue was collected from infertility patients prior to a frozen embryo transfer cycle as part of an endometrial receptivity analysis (ERA® ) biopsy or endometrial scratch test. Uterine NK (uNK) and Treg cell density was compared based on pregnancy status in the subsequent frozen embryo transfer cycle. Peripheral blood was also collected from a separate cohort of patients undergoing frozen embryo transfer. Treg cell density was compared by the presence or the absence of a clinical pregnancy in each phase of the cycle. RESULTS: In the 33 luteal phase biopsies there were more endometrial Tregs, similar uNK and a trend toward lower CD16+ uNK cells in women with a future ongoing clinical pregnancy compared to non-pregnant women. There were no differences in uNK and Treg density in natural scratch cycles vs programmed cycles or in non-receptive vs receptive endometrium (ERA® cycles). In the peripheral blood analysis, the pregnant group had higher peripheral blood Tregs on the day of serum ß-hCG time point when compared to the non-pregnant group. CONCLUSION: Higher levels of endometrial Tregs and lower levels of CD16+ uNK cells are positive prognostic factors for infertile women prior to frozen embryo transfer. Our work on phenotypic and proportional analyses of endometrial immune cells may complement the ERA® in predicting improved pregnancy rates in patients with implantation failure.
Assuntos
Transferência Embrionária , Endométrio/imunologia , Infertilidade Feminina/imunologia , Células Matadoras Naturais/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Biópsia , Criopreservação , Endométrio/patologia , Feminino , Humanos , Infertilidade Feminina/diagnóstico , Infertilidade Feminina/patologia , Infertilidade Feminina/terapia , Gravidez , Estudos RetrospectivosRESUMO
Since 1978, in the first decades of in vitro fertilization (IVF), the use of ovarian hyperstimulation allowed for the development and transfer of multiple embryos. As IVF technology improved, the number of multiple pregnancies increased, which led to gradual reduction in the number of embryos that were transferred. Embryo freezing (vitrification) was recommended to allow subsequent transfer if the fresh cycle was unsuccessful. However, experimentation has continued to improve pregnancy outcomes. We discuss here the significance of frozen embryo transfer cycle and the impact of uterine and peripheral immunity dominated by NK cells and regulatory T cells and human chorionic gonadotropin on pregnancy outcome in this innovative mode of IVF therapy.
Assuntos
Transferência Embrionária/métodos , Resultado da Gravidez , Adjuvantes Imunológicos/uso terapêutico , Animais , Gonadotropina Coriônica/uso terapêutico , Criopreservação , Embrião de Mamíferos , Feminino , Humanos , Células Matadoras Naturais/imunologia , Gravidez , Reprodução/imunologia , Linfócitos T Reguladores/imunologia , Útero/anatomia & histologia , Útero/imunologiaRESUMO
OBJECTIVE: To examine whether Society for Assisted Reproductive Technology (SART) member in vitro fertilization (IVF) centers adhere to the Society's new advertising policy, updated in January 2018, and evaluate other services advertised by region, insurance mandate and university affiliation status. Historically, a large percentage of IVF clinics have not adhered to SART guidelines for IVF clinic website advertising and have had variability in how financial incentives and other noncore fertility services are advertised. DESIGN: Cross-sectional study. SETTING: Not applicable. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Adherence of SART participating websites to objective criteria from the 2018 SART advertising guidelines. RESULT(S): All 361 SART participating clinic websites were evaluated. Approximately one third of clinics reported success rate statistics directly on their websites, but only 52.6% of those clinics reported current statistics. Similarly, only 67.5% of SART member clinics included the required disclaimer statement regarding their outcome statistics. Only 10.5% of websites were wholly compliant with SART guidelines regarding presentation of supplemental data. There were no significant differences between academic and nonacademic centers, programs in mandated versus nonmandated states, or East versus West Coast clinics in any of these areas. CONCLUSION(S): Many of the SART member websites failed to adhere to core guidelines surrounding reporting IVF clinic success rates. Consideration for additional education and streamlining as well as simplifying success rate advertising guidelines is recommended.
Assuntos
Publicidade/normas , Clínicas de Fertilização , Fidelidade a Diretrizes , Técnicas de Reprodução Assistida , Sociedades Médicas/normas , Instituições de Assistência Ambulatorial/economia , Instituições de Assistência Ambulatorial/organização & administração , Instituições de Assistência Ambulatorial/normas , Instituições de Assistência Ambulatorial/estatística & dados numéricos , Estudos Transversais , Feminino , Clínicas de Fertilização/economia , Clínicas de Fertilização/organização & administração , Clínicas de Fertilização/normas , Clínicas de Fertilização/estatística & dados numéricos , Fertilização in vitro/economia , Fertilização in vitro/normas , Fertilização in vitro/estatística & dados numéricos , Fidelidade a Diretrizes/estatística & dados numéricos , Fidelidade a Diretrizes/tendências , Humanos , Internet/economia , Internet/normas , Internet/estatística & dados numéricos , Gravidez , Taxa de Gravidez , Técnicas de Reprodução Assistida/economia , Técnicas de Reprodução Assistida/normas , Técnicas de Reprodução Assistida/estatística & dados numéricos , Sociedades Médicas/organização & administração , Sociedades Médicas/estatística & dados numéricos , Estados UnidosRESUMO
PROBLEM: To evaluate pregnancy-compatible phenotypic and functional changes in peripheral blood natural killer (pNK) cells during frozen embryo transfer (FET) cycles. METHOD OF STUDY: Peripheral blood was collected from patients undergoing frozen embryo transfer cycles at three separate time points in the cycle. pNK cell phenotype was analyzed by flow cytometry. Impact of pregnancy status on pNK cell cytotoxicity was characterized by two methods: (1) a three-dimensional endovascular tube formation approach and (2) a NK cell-specific K562 cell kill assay. RESULTS: A total of 35 patients were enrolled, 15 with clinical pregnancies and 20 with negative serum ß-hCG levels. Overall percentage of CD45+ CD3- CD56+ pNK cell did not change during the FET cycle. Pregnancy resulted in an increase in CD45+ CD3- CD56+ pNK cell population on the day of serum ß-hCG. pNK cells from non-pregnant patients caused significant tube disruption when compared to pregnant patients. Addition of serum from pregnant women reduced the tube disruption by pNK cells from non-pregnant patients. pNK cells from pregnant patients showed significantly lower cytotoxicity toward K562 cells in serum-free conditions. The addition of pregnancy serum decreased non-pregnant pNK cell cytotoxicity. Pregnancy status had no impact on VEGF-A and VEGF-C serum levels. Recombinant hCG added to non-pregnant serum resulted in a significant reduction in non-pregnant pNK cell-mediated K562 cell kill. CONCLUSION: There was no difference in pNK cell populations based on timing of the FET cycle. However, pregnancy increased the percentage of CD45+ CD3- CD56+ pNK cells. Additionally, pNK cells from pregnant women have reduced cytotoxicity and this is possibly mediated by hCG.
Assuntos
Gonadotropina Coriônica/imunologia , Transferência Embrionária , Células Matadoras Naturais/imunologia , Gravidez/imunologia , Adulto , Antígenos CD/imunologia , Linhagem Celular , Sobrevivência Celular , Gonadotropina Coriônica/sangue , Criopreservação , Embrião de Mamíferos , Feminino , Humanos , Fenótipo , Gravidez/sangueRESUMO
PURPOSE: To explore the effects of traditional vs. intracytoplasmic sperm injection (ICSI) insemination method on the outcome of high-quality blastocyst development in a split sibling oocyte cohort. METHODS: In this retrospective cohort study, we analyzed 62 ICSI/IVF split cycles. Sibling oocytes were randomly assigned to ICSI or IVF insemination. Two hundred thirty-four ICSI-only cycles and 152 IVF-only cycles were also analyzed for comparison. Blastocysts were graded by Gardner's embryo grading and were considered a high-quality blastocyst if 3BB or better (Gardner 1999). RESULTS: In the ICSI/IVF split group, (1) ICSI oocytes had a higher fertilization rate per oocyte allocated (73% vs 62%, p < 0.001), (2) more high-quality day 2 embryos (69% vs 55%, p < 0.005), (3) ICSI oocytes had a lower blastulation rate per 2PN (46% vs 54%, p < 0.05), but a higher blastulation rate when calculated per oocyte allocated (40% vs 32%, p < 0.05). The ICSI-only group had a lower fertilization rate (65% vs 70%, p < 0.001) but more high-quality day 2 embryos in comparison to the IVF-only group (68% vs 64%, p < .05). The total high-quality blastulation rate was higher for the IVF-only group per 2PN (49% vs 43%, p < 0.05) and per oocyte retrieved (34% vs 28%, p < 0.05). CONCLUSIONS: This distinctive IVF/ICSI sibling oocyte split design demonstrated a higher-quality blastulation rate in the IVF group compared to the ICSI group when calculated per 2PN, but not per oocyte allocated to each insemination procedure.
Assuntos
Blastocisto/fisiologia , Fertilização in vitro/métodos , Adulto , Transferência Embrionária , Feminino , Humanos , Masculino , Recuperação de Oócitos , Gravidez , Taxa de Gravidez , Projetos de Pesquisa , Estudos Retrospectivos , Injeções de Esperma Intracitoplásmicas/métodosRESUMO
The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional atg7 knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients. Abbreviation:atg7: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1.
Assuntos
Autofagia , Hipóxia , Lisossomos/metabolismo , Pré-Eclâmpsia/metabolismo , Proteoma/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Catepsina D/biossíntese , Linhagem Celular , Citoplasma/metabolismo , Feminino , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/biossíntese , Proteínas de Membrana Lisossomal/biossíntese , Camundongos , Camundongos Knockout , Placenta/metabolismo , Gravidez , Prenhez , Proteína Sequestossoma-1/biossíntese , Trofoblastos/metabolismoRESUMO
Systemic manifestation of preeclampsia (PE) is associated with circulating factors, including inflammatory cytokines and damage-associated molecular patterns (DAMPs), or alarmins. However, it is unclear whether the placenta directly contributes to the increased levels of these inflammatory triggers. Here, we demonstrate that pyroptosis, a unique inflammatory cell death pathway, occurs in the placenta predominantly from early onset PE, as evidenced by elevated levels of active caspase-1 and its substrate or cleaved products, gasdermin D (GSDMD), IL-1ß, and IL-18. Using cellular models mimicking pathophysiological conditions (e.g., autophagy deficiency, hypoxia, and endoplasmic reticulum (ER) stress), we observed that pyroptosis could be induced in autophagy-deficient human trophoblasts treated with sera from PE patients as well as in primary human trophoblasts exposed to hypoxia. Exposure to hypoxia elicits excessive unfolded protein response (UPR) and ER stress and activation of the NOD-like receptor pyrin-containing 3 (NLRP3) inflammasome in primary human trophoblasts. Thioredoxin-interacting protein (TXNIP), a marker for hyperactivated UPR and a crucial signaling molecule linked to NLRP3 inflammasome activation, is significantly increased in hypoxia-treated trophoblasts. No evidence was observed for necroptosis-associated events. Importantly, these molecular events in hypoxia-treated human trophoblasts are significantly observed in placental tissue from women with early onset PE. Taken together, we propose that placental pyroptosis is a key event that induces the release of factors into maternal circulation that possibly contribute to severe sterile inflammation and early onset PE pathology.
Assuntos
Proteínas de Transporte/genética , Inflamação/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Pré-Eclâmpsia/genética , Adulto , Hipóxia Celular/genética , Estresse do Retículo Endoplasmático/genética , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ligação a Fosfato/genética , Placenta/metabolismo , Placenta/patologia , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Piroptose/genética , Transdução de Sinais , Trofoblastos/metabolismo , Trofoblastos/patologia , Resposta a Proteínas não Dobradas/genéticaRESUMO
BACKGROUND: This study sought to clarify the roles of Anti-müllerian hormone (AMH) and follicle stimulating hormone (FSH) in predicting live birth, especially in patients with discordant AMH and FSH. A large IVF data set provided by eIVF®, consisting of 13,964 cycles with AMH, FSH, age, BMI, and birth outcomes were evaluated. Patients were categorized into four groups: Good prognosis group (AMH ≥1 ng/ml; FSH < 10 mIU/ml), Poor prognosis group (AMH < 1 ng/ml; FSH ≥10 mIU/ml), Reassuring AMH group (AMH ≥1 ng/ml; FSH ≥10 mIU/ml), and Reassuring FSH group (AMH < 1 ng/ml; FSH < 10 mIU/ml). The interaction between AMH, FSH, and their impact on live birth rate among these four groups was evaluated using Generalized Additive Mixed Modeling (GAMM). RESULTS: Analysis revealed a nonlinear relationship of AMH and FSH with live birth rate among all ages. Among the four groups, the good prognosis group had the highest live birth rate while the poor prognosis group had the lowest live birth rate (29.3% vs 13.1%, p < 0.005). In the discordant groups, the live birth rate of the reassuring AMH group was significantly higher than the reassuring FSH group (22.8% vs 15.6%, p < 0.005). CONCLUSIONS: Although both FSH and AMH are widely use to assess the ovarian reserve in women undergoing evaluation for infertility, AMH appears to be superior to FSH among all age groups. This is particularly important for patients with discordant AMH and FSH where reassuring AMH is a better clinical predictor of cycle success.
Assuntos
Hormônio Antimülleriano/sangue , Fertilização in vitro/normas , Hormônio Foliculoestimulante/sangue , Nascido Vivo , Indução da Ovulação/normas , Adulto , Fatores Etários , Feminino , Fertilização in vitro/estatística & dados numéricos , Humanos , Reserva Ovariana/fisiologia , Indução da Ovulação/estatística & dados numéricos , Valor Preditivo dos Testes , Gravidez , Resultado da Gravidez , Prognóstico , Medicina Reprodutiva/normas , Medicina Reprodutiva/estatística & dados numéricos , Estudos Retrospectivos , Estados UnidosAssuntos
Preservação da Fertilidade/legislação & jurisprudência , Cobertura do Seguro/legislação & jurisprudência , Seguro Saúde/legislação & jurisprudência , Programas Obrigatórios/legislação & jurisprudência , Adulto , Custos e Análise de Custo , Feminino , Preservação da Fertilidade/economia , Humanos , Infertilidade , Masculino , Técnicas de Reprodução Assistida/economia , Rhode Island , Planos Governamentais de Saúde/legislação & jurisprudência , Estados UnidosRESUMO
The decision on the part of obstetrics and gynecology residents to opt in or out of abortion training is, for many, a complex one. Although the public debate surrounding abortion can be filled with polarizing rhetoric, residents often discover that the boundaries between pro-choice and pro-life beliefs are not so neatly divided. We present narratives from four residents, training at a 32-resident program in the Northeast, who have a range of views surrounding abortion. Their stories reveal how some struggle with the real-life experience of providing abortions, while others feel angst over lacking the skills to terminate a life-threatening pregnancy. These residents have found that close relationships with coworkers from all sides of this issue, along with a residency program that encourages open conversation, have fostered understanding. Their narratives demonstrate that reasonable providers can disagree fundamentally and still work effectively with one another and that the close relationships formed in residency can allow both sides to see beyond the black and white of the public abortion debate. Our objectives in this commentary are to encourage a more nuanced discussion of abortion among obstetrician-gynecologists, to describe the aspects of our residency program that facilitate open dialogue and respect across diverse viewpoints, and to demonstrate that the clear distinction between being pro-life and pro-choice often breaks down when one is immediately responsible for the care of pregnant women.
Assuntos
Aborto Induzido/psicologia , Atitude do Pessoal de Saúde , Ginecologia/educação , Internato e Residência , Relações Interprofissionais , Obstetrícia/educação , Médicos/psicologia , Aborto Induzido/ética , Feminino , Ginecologia/ética , Humanos , Internato e Residência/ética , Relações Interprofissionais/ética , Obstetrícia/ética , Médicos/ética , Gravidez , Estados UnidosRESUMO
Hypertensive disorders of pregnancy remain among the leading causes of maternal morbidity and mortality. The onset of headaches in patients with hypertensive disorders of pregnancy has been considered as a premonitory symptom for eclampsia and other adverse maternal outcomes. Headaches are very common symptoms during pregnancy and the postpartum period with a reported incidence of 39%; however, headache is absent in 30-50% of women before the onset of eclampsia and is a poor predictor of eclampsia and adverse maternal outcomes. If included in the definition of cerebral or visual disturbances, headache may be considered a symptom of preeclampsia, a diagnostic feature of preeclampsia with severe features, a premonitory symptom of eclampsia, and an indication for delivery. Inclusion of this nonspecific symptom in the diagnosis and management of hypertensive disorders of pregnancy in the absence of an evidence basis may lead to unintended consequences including excessive testing, visits to outpatient offices or emergency departments, additional hospitalization, and iatrogenic preterm delivery without proven benefit. If a cerebral disturbance such as severe or persistent headache presents for the first time during pregnancy or postpartum, an evaluation should be performed that considers a broad differential diagnosis, including but not limited to hypertensive disorders of pregnancy, and the diagnostic evaluation is similar to that in nonpregnant adults. This commentary draws attention to the implications of considering the cerebral disturbance of headache as a symptom that portends adverse pregnancy outcome in the current recommendations for diagnosing and managing hypertensive disorders of pregnancy.
Assuntos
Eclampsia , Cefaleia , Hipertensão , Pré-Eclâmpsia , Complicações Cardiovasculares na Gravidez , Adulto , Gerenciamento Clínico , Eclampsia/diagnóstico , Eclampsia/etiologia , Eclampsia/prevenção & controle , Feminino , Cefaleia/classificação , Cefaleia/diagnóstico , Cefaleia/epidemiologia , Cefaleia/terapia , Humanos , Hipertensão/complicações , Hipertensão/diagnóstico , Hipertensão/terapia , Neuroimagem/métodos , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/prevenção & controle , Gravidez , Complicações Cardiovasculares na Gravidez/diagnóstico , Complicações Cardiovasculares na Gravidez/terapia , Resultado da Gravidez , Prognóstico , Medição de Risco , Fatores de RiscoRESUMO
Heme oxygenase 1 (HO-1) uses molecular oxygen and electrons from NADPH cytochrome P450 reductase to convert heme to CO, ferrous iron, and biliverdin (BV). Enzymatic studies with the purified 30-kDa form of HO-1 routinely use a coupled assay containing biliverdin reductase (BVR), which converts BV to bilirubin (BR). BVR is believed to be required for optimal HO-1 activity. The goal of this study was to determine whether HO-1 activity could be monitored directly by following BV generation or iron release (using the ferrous iron chelator, ferrozine) in the absence of BVR. Using assays for each of the three end products, we found that HO-1 activity was stimulated in the presence of catalase and comparable rates were measured with each assay. Absorbance scans revealed characteristic spectra for BR, BV, and/or the ferrozine-iron complex. The optimal conditions were slightly different for the direct and coupled assays. BSA activated the coupled but inhibited the direct assays, and the assays had different pH optima. By measuring the activity of BVR directly using BV as a substrate, these differences were attributed to different enzymatic properties of BVR and HO-1. Thus, BVR is not needed to measure the activity of HO-1 when catalase is present. In fact, the factors affecting catalysis by HO-1 are better understood using the direct assays because the coupled assay can be influenced by properties of BVR.
Assuntos
Heme Oxigenase-1/química , Heme/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Animais , Bilirrubina/análise , Bilirrubina/química , Bilirrubina/metabolismo , Biliverdina/análise , Biliverdina/química , Biliverdina/metabolismo , Sítios de Ligação , Catalase/química , Catalase/metabolismo , Catálise , Ferrozina/química , Heme Oxigenase-1/metabolismo , Humanos , Concentração de Íons de Hidrogênio , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , CoelhosRESUMO
Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K(m) for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025-0.05 microM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.
Assuntos
Heme Oxigenase (Desciclizante)/metabolismo , Proteínas de Membrana/metabolismo , Animais , Sequência de Bases , Catalase/metabolismo , Primers do DNA , Humanos , Lipossomos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.