RESUMO
ATP11C, a member of the P4-ATPase family, translocates phosphatidylserine and phosphatidylethanolamine at the plasma membrane. We previously revealed that its C-terminal splice variant ATP11C-b exhibits polarized localization in motile cell lines, such as MDA-MB-231 and Ba/F3. In the present study, we found that the C-terminal cytoplasmic region of ATP11C-b interacts specifically with ezrin. Notably, the LLxY motif in the ATP11C-b C-terminal region is crucial for its interaction with ezrin as well as its polarized localization on the plasma membrane. A constitutively active, C-terminal phosphomimetic mutant of ezrin was colocalized with ATP11C-b in polarized motile cells. ATP11C-b was partially mislocalized in cells depleted of ezrin alone, and exhibited greater mislocalization in cells simultaneously depleted of the family members ezrin, radixin and moesin (ERM), suggesting that ERM proteins, particularly ezrin, contribute to the polarized localization of ATP11C-b. Furthermore, Atp11c knockout resulted in C-terminally phosphorylated ERM protein mislocalization, which was restored by exogenous expression of ATP11C-b but not ATP11C-a. These observations together indicate that the polarized localizations of ATP11C-b and the active form of ezrin to the plasma membrane are interdependently stabilized.
Assuntos
Adenosina Trifosfatases , Polaridade Celular , Membrana Celular , Citoplasma , Proteínas do Citoesqueleto , FosfoproteínasRESUMO
Mammalian syntaxin 17 (Stx17) has several roles in processes other than membrane fusion, including in mitochondrial division, autophagosome formation and lipid droplet expansion. In contrast to conventional syntaxins, Stx17 has a long C-terminal hydrophobic region with a hairpin-like structure flanked by a basic amino acid-enriched C-terminal tail. Although Stx17 is one of the six ancient SNAREs and is present in diverse eukaryotic organisms, it has been lost in multiple lineages during evolution. In the present study, we compared the localization and function of fly and nematode Stx17s expressed in HeLa cells with those of human Stx17. We found that fly Stx17 predominantly localizes to the cytosol and mediates autophagy, but not mitochondrial division. Nematode Stx17, on the other hand, is predominantly present in mitochondria and facilitates mitochondrial division, but is irrelevant to autophagy. These differences are likely due to different structures in the C-terminal tail. Non-participation of fly Stx17 and nematode Stx17 in mitochondrial division and autophagy, respectively, was demonstrated in individual organisms. Our results provide an insight into the evolution of Stx17 in metazoa. This article has an associated First Person interview with the first author of the paper.
Assuntos
Fusão de Membrana , Proteínas SNARE , Animais , Autofagia , Células HeLa , Humanos , Proteínas Qa-SNARE/genéticaRESUMO
OBJECTIVES: This study aimed to clarify the relationship between the oral assessment guide (OAG), a simple method for assessing oral function and poor nutrition in gastric cancer patients and investigate the reduction of oral mucositis through appropriate oral function management. SUBJECTS AND METHODS: Gastric cancer patients who underwent chemotherapy at the Nagoya Ekisaikai Hospital between January 2015 and December 2020 were evaluated. The prognostic nutritional index (PNI), as the objective variable, was used to assess nutritional status. The explanatory variables were sex, age, smoking status, body mass index (BMI), number of remaining teeth, cancer stage, albumin level, C-reactive protein level, white blood cell count and the OAG score. RESULTS: PNI was significantly associated with age, number of remaining teeth, cancer stage and the OAG score (p < 0.05) among the 217 patients. There were significant differences in age, BMI, cancer stage and the OAG score among the patients. An abnormal BMI and an advanced cancer stage were more common in older patients, and abnormal OAG scores were associated with a lower PNI. CONCLUSIONS: For gastric cancer patients undergoing postoperative chemotherapy, worse oral functional status is associated with worse PNI and nutritional status.
Assuntos
Estado Nutricional , Neoplasias Gástricas , Humanos , Idoso , Neoplasias Gástricas/complicações , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/cirurgia , Prognóstico , Estudos Retrospectivos , Avaliação NutricionalRESUMO
PGAM5, a mitochondrial protein phosphatase that is genetically and biochemically linked to PINK1, facilitates mitochondrial division by dephosphorylating the mitochondrial fission factor Drp1. At the onset of mitophagy, PGAM5 is cleaved by PARL, a rhomboid protease that degrades PINK1 in healthy cells, and the cleaved form facilitates the engulfment of damaged mitochondria by autophagosomes by dephosphorylating the mitophagy receptor FUNDC1. Here, we show that the function and localization of PGAM5 are regulated by syntaxin 17 (Stx17), a mitochondria-associated membrane/mitochondria protein implicated in mitochondrial dynamics in fed cells and autophagy in starved cells. In healthy cells, loss of Stx17 causes PGAM5 aggregation within mitochondria and thereby failure of the dephosphorylation of Drp1, leading to mitochondrial elongation. In Parkin-mediated mitophagy, Stx17 is prerequisite for PGAM5 to interact with FUNDC1. Our results reveal that the Stx17-PGAM5 axis plays pivotal roles in mitochondrial division and PINK1/Parkin-mediated mitophagy.
Assuntos
Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Mitofagia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Qa-SNARE/metabolismo , Transdução de Sinais , Autofagossomos/metabolismo , Dinaminas , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/genética , Fosfoproteínas Fosfatases/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteólise , Proteínas Qa-SNARE/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
ATP11C, a member of the P4-ATPase family, is a major phosphatidylserine (PS)-flippase located at the plasma membrane. ATP11C deficiency causes a defect in B-cell maturation, anemia and hyperbilirubinemia. Although there are several alternatively spliced variants derived from the ATP11C gene, the functional differences between them have not been considered. Here, we compared and characterized three C-terminal spliced forms (we designated as ATP11C-a, ATP11C-b and ATP11C-c), with respect to their expression patterns in cell types and tissues, and their subcellular localizations. We had previously shown that the C-terminus of ATP11C-a is critical for endocytosis upon PKC activation. Here, we found that ATP11C-b and ATP11C-c did not undergo endocytosis upon PKC activation. Importantly, we also found that ATP11C-b localized to a limited region of the plasma membrane in polarized cells, whereas ATP11C-a was distributed on the entire plasma membrane in both polarized and non-polarized cells. Moreover, we successfully identified LLXY residues within the ATP11C-b C-terminus as a critical motif for the polarized localization. These results suggest that the ATP11C-b regulates PS distribution in distinct regions of the plasma membrane in polarized cells.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Células 3T3-L1 , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Endocitose , Ativação Enzimática , Células HCT116 , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Camundongos , Isoformas de Proteínas , Proteína Quinase C/metabolismo , Células RAW 264.7RESUMO
In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.
Assuntos
Inibidores de Histona Desacetilases/química , Técnicas de Transferência Nuclear/instrumentação , Oócitos/química , Animais , Inibidores de Histona Desacetilases/classificação , Camundongos , Peptídeos Cíclicos/químicaRESUMO
Vitamin A levels in fattening Japanese Black cattle affect meat quality; therefore, it is important to monitor serum retinol concentrations. To simplify and accelerate the evaluation of serum retinol concentrations in cattle, we developed a new predictive method using excitation-emission matrix (EEM) fluorescence spectrophotometry. For analytical comparison, the concentration of serum retinol was also measured using the conventional HPLC method. We examined excitation (Ex) and emission (Em) wavelengths of cattle serum, which were 250-450 and 250-600 nm, respectively. Parallel factor analysis separated four components from EEM data, one of which was related to retinol. Next, a partial least square regression model was created using the obtained EEMs as explanatory variables and accrual measurement values as objective variables. The determination coefficient value (R2), root mean squared error of prediction (RMSEP), and the ratio of performance to deviation (RPD) of the model were determined. A comparison with reference values found that R2, RMSEP, and RPD of the calibration model were 0.95, 6.4 IU/dl, and 4.2, respectively. This implies that EEM can estimate the serum retinol concentration with high accuracy. Additionally, the fluorescent peaks that contributed to the calibration, which were extracted from the regression coefficient and variable importance in projection plots, were Ex/Em = 320/390 and 330/520 nm. Thus, we assume that this method observes not only free retinol, but also retinol-binding protein. In conclusion, multidimensional fluorescence analysis can accurately and quickly determine serum retinol concentrations in fattening cattle.
Assuntos
Análise Química do Sangue/métodos , Espectrometria de Fluorescência , Vitamina A/sangue , Animais , Bovinos , Análise dos Mínimos QuadradosRESUMO
The formation and the chemical characterization of single atoms of dubnium (Db, element 105), in the form of its volatile oxychloride, was investigated using the on-line gas phase chromatography technique, in the temperature range 350-600 °C. Under the exactly same chemical conditions, comparative studies with the lighter homologues of Groupâ 5 in the Periodic Table clearly indicate the volatility sequence being NbOCl3 > TaOCl3 ≥ DbOCl3 . From the obtained experimental results, thermochemical data for DbOCl3 were derived. The present study delivers reliable experimental information for theoretical calculations on chemical properties of transactinides.
RESUMO
In fed cells, syntaxin 17 (Stx17) is associated with microtubules at the endoplasmic reticulum-mitochondria interface and promotes mitochondrial fission by determining the localization and function of the mitochondrial fission factor Drp1. Upon starvation, Stx17 dissociates from microtubules and Drp1, and binds to Atg14L, a subunit of the phosphatidylinositol 3-kinase complex, to facilitate phosphatidylinositol 3-phosphate production and thereby autophagosome formation, but the mechanism underlying this phenomenon remains unknown. Here we identify MAP1B-LC1 (microtubule-associated protein 1B-light chain 1) as a critical regulator of Stx17 function. Depletion of MAP1B-LC1 causes Stx17-dependent autophagosome accumulation even under nutrient-rich conditions, whereas its overexpression blocks starvation-induced autophagosome formation. MAP1B-LC1 links microtubules and Stx17 in fed cells, and starvation causes the dephosphorylation of MAP1B-LC1 at Thr217, allowing Stx17 to dissociate from MAP1B-LC1 and bind to Atg14L. Our results reveal the mechanism by which Stx17 changes its binding partners in response to nutrient status.
Assuntos
Autofagossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Qa-SNARE/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Mitocôndrias/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismoRESUMO
Highly enantioselective organocatalytic construction of spirochromans containing a tetrasubstituted stereocenter was developed. Intramolecular oxy-Michael addition was catalyzed with a bifunctional cinchona alkaloid thiourea catalyst. A variety of spirochroman compounds containing a tetrasubstituted stereocenter were obtained with excellent enantioselectivities of up to 99% enantiomeric excess. The reaction was applied to the asymmetric formal synthesis of (-)-(R)-cordiachromene.
RESUMO
Androgen receptor (AR) has a key role in the development and progression of prostate cancer, and AR antagonists are used for its remedy. Recently, carborane derivatives, which are carbon-containing boron clusters have attracted attention as new AR ligands. Here we determined the force field parameters of 10-vertex and 12-vertex p-carborane to facilitate in silico drug design of boron clusters. Then, molecular dynamics (MD) simulations of complexes of AR-carborane derivatives were performed to evaluate the parameters and investigate the influences of carborane derivatives on the three-dimensional structure of AR. Energy profiles were obtained using quantum chemical calculations, and the force-field parameters were determined by curve fitting of the energy profiles. The results of MD simulations indicated that binding of the antagonist-BA341 changed some hydrogen-bond formations involved in the structure and location of helix 12. Those results were consistent with previously reported data. The determined parameters are also useful for refining the structure of the carborane-receptor complex obtained by docking simulations and development of new ligands with carborane cages not only for AR but also for various receptors.
Assuntos
Antagonistas de Receptores de Andrógenos/química , Compostos de Boro/química , Sistemas de Liberação de Medicamentos/métodos , Simulação de Dinâmica Molecular , Receptores Androgênicos/química , Antagonistas de Receptores de Andrógenos/administração & dosagem , Antagonistas de Receptores de Andrógenos/metabolismo , Compostos de Boro/administração & dosagem , Compostos de Boro/metabolismo , Estrutura Secundária de Proteína , Receptores Androgênicos/metabolismo , Relação Estrutura-AtividadeRESUMO
Avian glucose transporters (GLUT) responsible for insulin-responsive glucose uptake into adipocytes remain poorly characterized. We aimed to identify the insulin-responsive GLUT using primary culture of chicken adipocytes. Acute stimulation with 1⯵M insulin for 20â¯min increased 2-deoxyglucose uptake, AKT protein phosphorylation, and GLUT1 protein levels on the plasma membrane of the chicken adipocytes, whereas pretreatment with 10⯵M triciribine, an AKT inhibitor, canceled these effects. Furthermore, the insulin stimulation did not affect GLUT12 protein levels on the plasma membrane of the chicken adipocytes. Our results suggest that GLUT1 is an insulin-responsive GLUT in chicken adipocytes.
Assuntos
Adipócitos/metabolismo , Membrana Celular/metabolismo , Galinhas/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glucose/metabolismo , Insulina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adipócitos/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Desoxiglucose/metabolismo , Masculino , Fosforilação/efeitos dos fármacosRESUMO
The common marmoset (Callithrix jacchus) represents a promising nonhuman primate model for the study of human diseases because of its small size, ease of handling, and availability of gene-modified animals. Here, we aimed to devise reproductive technology for marmoset spermatid injection using immature males for a possible rapid generational turnover. Spermatids at each step could be identified easily by their morphology under differential interference microscopy: thus, early round spermatids had a round nucleus with a few nucleolus-like structures and abundant cytoplasm, as in other mammals. The spermatids acquired oocyte-activating capacity at the late round spermatid stage, as confirmed by the resumption of meiosis and Ca2+ oscillations upon injection into mouse oocytes. The spermatids could be cryopreserved efficiently with a simple medium containing glycerol and CELL BANKER®. Late round or elongated spermatids first appeared at 10-12 months of age, 6-8 months before sexual maturation. Marmoset oocytes microinjected with frozen-thawed late round or elongated spermatids retrieved from a 12-month-old male marmoset developed to the 8-cell stage without the need for artificial oocyte activation stimulation. Thus, it might be possible to shorten the intergeneration time by spermatid injection, from 2 years (by natural mating) to 13-15 months including gestation.
Assuntos
Sinalização do Cálcio , Núcleo Celular/metabolismo , Criopreservação , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Espermátides/metabolismo , Animais , Callithrix , Feminino , Masculino , Camundongos , Microinjeções , Oócitos/citologia , Espermátides/citologiaRESUMO
Background: Although both the presence and progression over time of vascular calcification have been shown to independently predict cardiovascular disease and mortality in chronic dialysis patients, the impact of the pattern of accumulation of abdominal aortic calcification on mortality has not yet been investigated. Methods: We conducted a longitudinal study at a dialysis hospital in Hokkaido, Japan from 2005 to 2014. An abdominal calcification index (ACI) was generated for 396 patients from their annual abdominal computed tomography (CT) scans. The trajectories of ACIs during the first 2 years were classified using group-based trajectory modeling into four groups; stable (29.0%), slow increase (29.2%), rapid nonlinear increase (24.4%) and advanced with slow increase (17.4%). Incidence rates by group of all-cause mortality during the follow-up period (mean of 4.5 years) were investigated using the Cox proportional hazard model. Results: Compared with the stable trajectory, both the rapid nonlinear increase and the advanced with slow increase trajectories were associated with an increased risk of death [adjusted hazard ratios (HR) 1.91; 95% confidence interval (CI) 1.02-3.58 and adjusted HR 2.79; 95% CI: 1.44-5.11, respectively]. Sensitivity analyses indicated that ACI trajectories were associated with subsequent mortality, while ACI at individual time points was not. Conclusions: Chronic hemodialysis patients with a trajectory of longitudinal high or rapid accumulation of vascular calcification over time were at a higher risk of death. Individual trajectories of vascular calcification may be suggested to allow for more accurate mortality risk calculations than one-time assessment.
Assuntos
Aorta Abdominal/patologia , Doenças Cardiovasculares/mortalidade , Diálise Renal/mortalidade , Calcificação Vascular/mortalidade , Doenças Cardiovasculares/etiologia , Progressão da Doença , Feminino , Humanos , Incidência , Japão/epidemiologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prognóstico , Diálise Renal/efeitos adversos , Fatores de Risco , Taxa de Sobrevida , Tomografia Computadorizada por Raios X/métodos , Calcificação Vascular/epidemiologia , Calcificação Vascular/etiologiaRESUMO
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways.
Assuntos
Endocitose/fisiologia , Receptores ErbB/metabolismo , Transporte Proteico/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Membrana Celular/metabolismo , Endossomos/metabolismo , Células Hep G2 , Humanos , Fusão de Membrana/fisiologia , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno , Transferrina/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
1. Little is known about drug metabolism in carnivores. Although the domestic cat (Felis catus) is an obligate carnivore and is the most common companion animal, usage and dosage of many drugs are determined according to information obtained from humans and dogs. We determined the complete cDNA sequence of CYP2B6 from the feline lung. 2. Feline CYP2B6 consists of 494 deduced amino acids, showing highest identity with the dog CYP2B ortholog, followed by those of horse, pig, primate and human. 3. Feline CYP2B6 transcripts were expressed predominantly in the lung and slightly in the small intestine but not in the liver without significant sex-dependent differences. Western blot analysis with an anti-human CYP2B6 antibody confirmed the presence of CYP2B protein in the lung but not in the liver. 4. Feline CYP2B6 proteins heterologously expressed in Escherichia coli metabolized several substrates specific to human CYP2B6, including 7-ethoxy-4-(trifluoromethyl) coumarin (EFC). The metabolic activity was strongly inhibited by medetomidine and atipamezole, potent inhibitors of canine CYP2B11 (now officially CYP2B6) as well as by ticlopidine and sertraline, inhibitors selective to human CYP2B6. 5. The results suggest that feline CYP2B6 is a functional CYP2B ortholog that plays a role in the local defense mechanism in the cat respiratory system and intestine.
Assuntos
Citocromo P-450 CYP2B6/genética , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Gatos , Citocromo P-450 CYP2B6/metabolismo , DNA Complementar/metabolismo , Cães , HumanosRESUMO
Adrenaline changes expression of the genes encoding peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), which is known as a regulator of muscle size, and atrogin-1/muscle atrophy F-box (MAFbx), which is a muscle-specific ubiquitin ligase. However, the subtype of ß-adrenergic receptor (ß-AR) involved in regulating these genes in skeletal muscle is not yet well defined. In this study, the effects of intraperitoneal injection of adrenaline and three ß1-3-AR selective agonists on chick skeletal muscle metabolism were examined, to evaluate the functions of ß-AR subtypes. Adrenaline decreased atrogin-1/MAFbx mRNA levels accompanied by an increase in PGC-1α mRNA and protein levels. However, among the three selective agonists, only the ß1-AR agonist, dobutamine, increased PGC-1α mRNA and protein levels, while the ß2-AR agonist, clenbuterol, suppressed atrogin-1/MAFbx mRNA levels. In addition, preinjection of the ß1-AR antagonist, acebutolol, and the ß2-AR antagonist, butoxamine, inhibited the adrenaline-induced increase in PGC-1α mRNA levels and the decrease in atrogin-1/MAFbx mRNA levels, respectively. Compared with adrenaline administration, the ß3-AR agonist, BRL37344, decreased PGC-1α mRNA levels and increased atrogin-1/MAFbx mRNA levels. These results suggest that, in chick skeletal muscle, PGC-1α is induced via the ß1-AR, while atrogin-1/MAFbx is suppressed via the ß2-AR.
Assuntos
Regulação da Expressão Gênica , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Galinhas , Masculino , Proteínas Musculares/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , RNA Mensageiro/metabolismoRESUMO
D1Pas1 is a mouse autosomal DEAD-box RNA helicase expressed predominantly in the testis. To assess its possible function, we generated D1Pas1-deficient mice using embryonic stem cells with a targeted D1Pas1 allele. Deletion of D1Pas1 did not cause noticeable embryonic defects or death, indicating that D1Pas1 is not essential for embryogenesis. Whereas homozygous knockout female mice showed normal reproductive performance, homozygous knockout male mice were completely sterile. The seminiferous epithelium of D1Pas1-deficient males contained no spermatids or spermatozoa because of spermatogenic arrest at the late pachytene stage. Upregulation of retrotransposons such as LINE-1 was not found in D1Pas1-deficient males, unlike males lacking Mvh, another testicular DEAD-box RNA helicase. Meiotic chromosome behavior in developing spermatocytes of D1Pas1-deficient males was indistinguishable from that in wild-type males, at least until synaptonemal complex formation. Thus, mouse D1Pas1 is the first-identified DEAD-box RNA helicase that plays critical roles in the final step of the first meiotic prophase in male germ cells.
Assuntos
RNA Helicases DEAD-box/genética , Meiose , Espermatogênese , Animais , RNA Helicases DEAD-box/metabolismo , Feminino , Técnicas de Inativação de Genes , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retroelementos , Espermatócitos/citologia , Espermatócitos/metabolismoRESUMO
Producing many mature oocytes is of great importance for assisted reproductive technologies. In mice, superovulation by consecutive injections of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) has been the gold standard for oocyte collection. However, the yield of mature oocytes by this regimen can fluctuate according to the stage of the estrous cycle, strain, and age. Therefore, our objective was to develop a high-yield superovulation protocol to collect higher numbers of oocytes from adult female mice of different strains and ages. First, we aimed to synchronize the estrous cycle using C57BL/6 (B6) female mice. Most (93%) were synchronized to metestrus after two daily injections of progesterone. Second, we found that with the injection of anti-inhibin serum (AIS) instead of eCG, the mean number of ovulated oocytes almost doubled (21 vs. 41 per mouse). Third, by combining estrous cycle synchronization with two AIS injections, we obtained 62 oocytes per mouse, about three times that with the eCG-hCG protocol. Importantly, this approach increased the proportion of mice that ovulated >25 oocytes from about 40% (eCG-hCG) to 90%. The same protocol was also effective in other inbred (BALB/cA), outbred (ICR), and hybrid (B6D2F1) strains. In addition, B6 female mice aged over 1 yr ovulated 1.8-fold more oocytes by this protocol. Thus, estrous cycle synchronization followed by AIS-hCG yielded a broadly applicable, highly efficient superovulation. This protocol should promote the effective use of invaluable female mouse strains and decrease the numbers of animals euthanized.
Assuntos
Anticorpos Bloqueadores/farmacologia , Sincronização do Estro/efeitos dos fármacos , Inibinas/antagonistas & inibidores , Inibinas/imunologia , Superovulação/efeitos dos fármacos , Envelhecimento , Animais , Gonadotropina Coriônica/farmacologia , Feminino , Fertilização in vitro/métodos , Hormônio Foliculoestimulante/sangue , Metestro/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Oócitos , Gravidez , Progesterona/farmacologia , Zona Pelúcida/efeitos dos fármacosRESUMO
The highly enantioselective organocatalytic construction of spiroindanes containing an all-carbon quaternary stereocenter by intramolecular Friedel-Crafts-type 1,4-addition is described. The reaction was catalyzed by a cinchonidine-based primary amine and accelerated by water and p-bromophenol. A variety of spiro compounds containing quaternary stereocenters were obtained with excellent enantioselectivity (up to 95 %â ee). The reaction was applied to the asymmetric formal synthesis of the spirocyclic natural products (-)-cannabispirenonesâ A and B.