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Associative learning involves complex interactions of multiple cognitive factors. While adult subjects can articulate these factors verbally, for model animals such as macaques, we rely on behavioral outputs. In our study, we used pupillary responses as an alternative measure to capture these underlying cognitive changes. We recorded the dynamic changes in the pupils of three male macaques when they learned the associations between visual stimuli and reward sizes under the classical Pavlovian experimental paradigm. We found that during the long-term learning process, the gradual changes in the pupillary response reflect the changes in the cognitive state of the animals. The pupillary response can be explained by a linear combination of components corresponding to multiple cognitive factors. These components reflect the impact of visual stimuli on the pupils, the prediction of reward values associated with the visual stimuli, and the macaques' understanding of the current experimental reward rules. The changing patterns of these factors during interday and intraday learning clearly demonstrate the enhancement of current reward-stimulus association and the weakening of previous reward-stimulus association. Our study shows that the dynamic response of pupils can serve as an objective indicator to characterize the psychological changes of animals, understand their learning process, and provide important tools for exploring animal behavior during the learning process.
Assuntos
Aprendizagem por Associação , Cognição , Condicionamento Clássico , Pupila , Recompensa , Animais , Masculino , Aprendizagem por Associação/fisiologia , Pupila/fisiologia , Condicionamento Clássico/fisiologia , Cognição/fisiologia , Estimulação Luminosa/métodos , Macaca mulatta , Reflexo Pupilar/fisiologiaRESUMO
Nitrogen (N) is a critical factor for crop growth and yield. Improving N use efficiency (NUE) in agricultural systems is crucial for sustainable food production. However, the underlying regulation of N uptake and utilization in crops is not well known. Here, we identified OsSNAC1 (stress-responsive NAC 1) as an upstream regulator of OsNRT2.1 (nitrate transporter 2.1) in rice (Oryza sativa) by yeast 1-hybridization screening. OsSNAC1 was mainly expressed in roots and shoots and induced by N deficiency. We observed similar expression patterns of OsSNAC1, OsNRT2.1/2.2, and OsNRT1.1A/B in response to NO3- supply. Overexpression of OsSNAC1 resulted in increased concentrations of free NO3- in roots and shoots, as well as higher N uptake, higher NUE, and N use index (NUI) in rice plants, which conferred increased plant biomass and grain yield. On the contrary, mutations in OsSNAC1 resulted in decreased N uptake and lower NUI, which inhibited plant growth and yield. OsSNAC1 overexpression significantly upregulated OsNRT2.1/2.2 and OsNRT1.1A/B expression, while the mutation in OsSNAC1 significantly downregulated OsNRT2.1/2.2 and OsNRT1.1A/B expression. Y1H, transient co-expression, and ChIP assays showed OsSNAC1 directly binds to the upstream promoter regions of OsNRT2.1/2.2 and OsNRT1.1A/1.1B. In conclusion, we identified a NAC transcription factor in rice, OsSNAC1, with a positive role in regulating NO3- uptake through direct binding to the upstream promoter regions of OsNRT2.1/2.2 and OsNRT1.1A/1.1B and activating their expression. Our results provide a potential genetic approach for improving crop NUE in agriculture.
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Transportadores de Nitrato , Oryza , Oryza/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Expressão Gênica , Nitratos/metabolismoRESUMO
China is a country with one of the most species-rich reptile faunas in the world. However, nearly a quarter of Chinese lizard species assessed by the China Biodiversity Red List are threatened. Nevertheless, to date, no study has explicitly examined the pattern and processes of extinction and threat in Chinese lizards. In this study, we conducted the first comparative phylogenetic analysis of extinction risk in Chinese lizards. We addressed the following 3 questions: (1) What is the pattern of extinction and threat in Chinese lizards? (2) Which species traits and extrinsic factors are related to their extinction risk? (3) How can we protect Chinese lizards based on our results? We collected data on 10 species traits (body size [BS], clutch size, geographic range size, activity time, reproductive mode, habitat specialization [HS], habitat use, leg development, maximum elevation, and elevation range) and 7 extrinsic factors (mean annual precipitation (MAP), mean annual temperature, mean annual solar insolation, normalized difference vegetation index (NDVI), human footprint, human population density, and human exploitation). After phylogenetic correction, these variables were used separately and in combination to assess their associations with extinction risk. We found that Chinese lizards with a small geographic range, large BS, high HS, and living in high MAP areas were vulnerable to extinction. Conservation priority should thus be given to species with the above extinction-prone traits so as to effectively protect Chinese lizards. Preventing future habitat destruction should also be a primary focus of management efforts because species with small range size and high HS are particularly vulnerable to habitat loss.
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Recent developments in the instrumentation and data analysis of synchrotron small-angle X-ray scattering (SAXS) on biomolecules in solution have made biological SAXS (BioSAXS) a mature and popular tool in structural biology. This article reports on an advanced endstation developed at beamline 13A of the 3.0â GeV Taiwan Photon Source for biological small- and wide-angle X-ray scattering (SAXS-WAXS or SWAXS). The endstation features an in-vacuum SWAXS detection system comprising two mobile area detectors (Eiger X 9M/1M) and an online size-exclusion chromatography system incorporating several optical probes including a UV-Vis absorption spectrometer and refractometer. The instrumentation and automation allow simultaneous SAXS-WAXS data collection and data reduction for high-throughput biomolecular conformation and composition determinations. The performance of the endstation is illustrated with the SWAXS data collected for several model proteins in solution, covering a scattering vector magnitude q across three orders of magnitude. The crystal-model fittings to the data in the q range â¼0.005-2.0â Å-1 indicate high similarity of the solution structures of the proteins to their crystalline forms, except for some subtle hydration-dependent local details. These results open up new horizons of SWAXS in studying correlated local and global structures of biomolecules in solution.
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The optical design and performance of the recently opened 13A biological small-angle X-ray scattering (SAXS) beamline at the 3.0â GeV Taiwan Photon Source of the National Synchrotron Radiation Research Center are reported. The beamline is designed for studies of biological structures and kinetics in a wide range of length and time scales, from angstrom to micrometre and from microsecond to minutes. A 4â m IU24 undulator of the beamline provides high-flux X-rays in the energy range 4.0-23.0â keV. MoB4C double-multilayer and Si(111) double-crystal monochromators (DMM/DCM) are combined on the same rotating platform for a smooth rotation transition from a high-flux beam of â¼4 × 1014â photonsâ s-1 to a high-energy-resolution beam of ΔE/E ≃ 1.5 × 10-4; both modes share a constant beam exit. With a set of Kirkpatrick-Baez (KB) mirrors, the X-ray beam is focused to the farthest SAXS detector position, 52â m from the source. A downstream four-bounce crystal collimator, comprising two sets of Si(311) double crystals arranged in a dispersive configuration, optionally collimate the DCM (vertically diffracted) beam in the horizontal direction for ultra-SAXS with a minimum scattering vector q down to 0.0004â Å-1, which allows resolving ordered d-spacing up to 1â µm. A microbeam, of 10-50â µm beam size, is tailored by a combined set of high-heat-load slits followed by micrometre-precision slits situated at the front-end 15.5â m position. The second set of KB mirrors then focus the beam to the 40â m sample position, with a demagnification ratio of â¼1.5. A detecting system comprising two in-vacuum X-ray pixel detectors is installed to perform synchronized small- and wide-angle X-ray scattering data collections. The observed beamline performance proves the feasibility of having compound features of high flux, microbeam and ultra-SAXS in one beamline.
Assuntos
Fótons , Síncrotrons , Espalhamento a Baixo Ângulo , Taiwan , Difração de Raios X , Raios XRESUMO
[Figure: see text].
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Anticolesterolemiantes/farmacologia , Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Benzamidas/farmacologia , Colesterol/metabolismo , Macrófagos/efeitos dos fármacos , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Modelos Animais de Doenças , Feminino , Células Hep G2 , Humanos , Eliminação Intestinal/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macrófagos/metabolismo , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Células RAW 264.7 , Receptores de Quinase C Ativada/genética , Receptores de Quinase C Ativada/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: Circulating progenitor cells possess vasculogenesis property and participate in repair of vascular injury. Cx (connexin) 43-a transmembrane protein constituting gap junctions-is involved in vascular pathology. However, the role of Cx43 in smooth muscle progenitor cells (SPCs) remained unclear. Approach and Results: Human SPCs cultured from CD34+ peripheral blood mononuclear cells expressed smooth muscle cell markers, such as smooth muscle MHC (myosin heavy chain), nonmuscle MHC, calponin, and CD140B, and Cx43 was the most abundant Cx isoform. To evaluate the role of Cx43 in SPCs, short interference RNA was used to knock down Cx43 expression. Cellular activities of SPCs were reduced by Cx43 downregulation. In addition, Cx43 downregulation attenuated angiogenic potential of SPCs in hind limb ischemia mice. Protein array and ELISA of the supernatant from SPCs showed that IL (interleukin)-6, IL-8, and HGF (hepatocyte growth factor) were reduced by Cx43 downregulation. Simultaneously, Cx43 downregulation reduced the phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Akt (protein kinase B) pathway and reactivation of NF-κB and Akt using betulinic acid, and SC79 could restore the secretion of growth factors and cytokines. Moreover, FAK (focal adhesion kinase)-Src (proto-oncogene tyrosine-protein kinase Src) activation was increased by Cx43 downregulation, and inactivation of Akt-NF-κB could be restored by Src inhibitor (PP2), indicating that Akt-NF-κB inactivated by Cx43 downregulation arose from FAK-Src activation. Finally, the depressed cellular activities and secretion of SPCs after Cx43 downregulation were restored by FAK inhibitor PF-562271 or PP2. CONCLUSIONS: SPCs possess angiogenic potential to repair ischemic tissue mainly through paracrine effects. Gap junction protein Cx43 plays an important role in regulating cellular function and paracrine effects of SPCs through FAK-Src axis.
Assuntos
Conexina 43/metabolismo , Isquemia/cirurgia , Músculo Esquelético/irrigação sanguínea , Miócitos de Músculo Liso/transplante , NF-kappa B/metabolismo , Neovascularização Fisiológica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Conexina 43/genética , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Membro Posterior , Humanos , Mediadores da Inflamação/metabolismo , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Miócitos de Músculo Liso/metabolismo , Fosforilação , Proto-Oncogene Mas , Interferência de RNA , Fluxo Sanguíneo Regional , Transdução de Sinais , Transplante de Células-TroncoRESUMO
AbstractSome species show high rates of reproductive failure, which is puzzling because natural selection works against such failure in every generation. Hatching failure is common in both captive and wild zebra finches (Taeniopygia guttata), yet little is known about its proximate causes. Here we analyze data on reproductive performance (the fate of >23,000 eggs) based on up to 14 years of breeding of four captive zebra finch populations. We find that virtually all aspects of reproductive performance are negatively affected by inbreeding (mean r=-0.117); by an early-starting, age-related decline (mean r=-0.132); and by poor early-life nutrition (mean r=-0.058). However, these effects together explain only about 3% of the variance in infertility, offspring mortality, fecundity, and fitness. In contrast, individual repeatability of different fitness components varied between 15% and 50%. As expected, we found relatively low heritability in fitness components (median: 7% of phenotypic variation and 29% of individually repeatable variation). Yet some of the heritable variation in fitness appears to be maintained by antagonistic pleiotropy (negative genetic correlations) between male fitness traits and female and offspring fitness traits. The large amount of unexplained variation suggests a potentially important role of local dominance and epistasis, including the possibility of segregating genetic incompatibilities.
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Tentilhões/fisiologia , Infertilidade/genética , Envelhecimento , Fenômenos Fisiológicos da Nutrição Animal , Animais , Feminino , Tentilhões/embriologia , Tentilhões/genética , Endogamia , Masculino , Reprodução/genética , Reprodução/fisiologiaRESUMO
Diamide resistant phenotypes have evolved in the field and the resistance has been attributed to target-site mutations in some lepidopteran pests. In this study, we documented the resistance status of Chilo suppressalis to chlorantraniliprole during 2016-2018 in seven provinces of China. To investigate the possible role of target-site mutations as known from lepidopterans, we sequenced respective domains of the RyR gene of C. suppressalis with different levels of diamide resistance. The results revealed that I4758M (corresponding to I4790M in P. xylostella), Y4667D/C (numbered according to C. suppressalis), G4915E (corresponding to G4946E in P. xylostella), and one novel Y4891F (numbered according to C. suppressalis) RyR target-site mutations were present. The contribution of these mutations was further investigated by diamide toxicity bioassays with eight genome modified Drosophila melanogaster lines. The study showed that genome modified flies bearing the Y4667D mutation (corresponding to the Y4667D and I4758M simultaneous mutation in C. suppressalis) exhibited high resistance ratios to chlorantraniliprole (1542.8-fold), cyantraniliprole (487.9-fold) and tetrachlorantraniliprole (290.1-fold). The M4758I and G4915E simultaneous mutations (corresponding to single G4915E mutation in C. suppressalis) showed high resistance ratios to chlorantraniliprole (153.1-fold) and cyantraniliprole (323.5-fold), and relatively low resistance to flubendiamide (28.9-fold) and tetrachlorantraniliprole (25.2-fold). These findings suggest that multiple point mutations in RyR confer diamide resistance of C. suppressalis. The results contribute to a better understanding of insect diamide resistance mechanisms and provide insights on the impact of RyR target-site mutations in insects.
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Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Mariposas/genética , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Sequência de Aminoácidos , Animais , Benzamidas/farmacologia , Sistemas CRISPR-Cas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Mariposas/efeitos dos fármacos , Mariposas/metabolismo , Pirazóis/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Alinhamento de Sequência , Sulfonas/farmacologia , ortoaminobenzoatos/farmacologiaRESUMO
Fat-soluble vitamins (FSVs), A, D, and E, are components of prenatal vitamin care. Previously, limited evidence existed to explain on a molecular level how maternal FSV supplementation affects the fetus during pregnancy. We developed a simplified LC-MS/MS method to simultaneously detect FSVs in maternal plasma (MP) and amniotic fluid (AF); we used this approach to investigate the correlation between FSV levels in these two matrices. With this method, we circumvented frequently used liquid-liquid extraction or solid-phase extraction methods and, instead, used simple protein precipitation with acetonitrile for sample preparation. This method displayed satisfactory linearity, intra- and inter-day imprecision, and accuracy. We validated the consistency with standard reference material 972a and 968f certification. In analysis of MP and AF samples from 50 pregnant women in the second trimester, concentrations of retinol, 25-hydroxyvitamin D3 [25(OH)D3], and α-tocopherol (reflecting vitamins A, D, and E, respectively) were lower in AF than in MP. Significant positive correlations existed between MP and AF for 25(OH)D3 (r = 0.667; P < 0.001) and retinol (r = 0.393; P = 0.005), but not for α-tocopherol (r = 0.145, P > 0.05). This novel LC-MS/MS method shows prominent applicability for FSV detection and the observed correlations contribute to research on fetal development.
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Líquido Amniótico/química , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Vitaminas/análise , Vitaminas/química , Adulto , Ácidos Graxos/química , Feminino , Humanos , Masculino , Solubilidade , Vitaminas/sangue , Adulto JovemRESUMO
BACKGROUND: Clinical recommendations to limit gestational weight gain (GWG) imply high GWG is causally related to adverse outcomes in mother or offspring, but GWG is the sum of several inter-related complex phenotypes (maternal fat deposition and vascular expansion, placenta, amniotic fluid and fetal growth). Understanding the genetic contribution to GWG could help clarify the potential effect of its different components on maternal and offspring health. Here we explore the genetic contribution to total, early and late GWG. PARTICIPANTS AND METHODS: A genome-wide association study was used to identify maternal and fetal variants contributing to GWG in up to 10 543 mothers and 16 317 offspring of European origin, with replication in 10 660 mothers and 7561 offspring. Additional analyses determined the proportion of variability in GWG from maternal and fetal common genetic variants and the overlap of established genome-wide significant variants for phenotypes relevant to GWG (for example, maternal body mass index (BMI) and glucose, birth weight). RESULTS: Approximately 20% of the variability in GWG was tagged by common maternal genetic variants, and the fetal genome made a surprisingly minor contribution to explain variation in GWG. Variants near the pregnancy-specific beta-1 glycoprotein 5 (PSG5) gene reached genome-wide significance (P=1.71 × 10-8) for total GWG in the offspring genome, but did not replicate. Some established variants associated with increased BMI, fasting glucose and type 2 diabetes were associated with lower early, and higher later GWG. Maternal variants related to higher systolic blood pressure were related to lower late GWG. Established maternal and fetal birth weight variants were largely unrelated to GWG. CONCLUSIONS: We found a modest contribution of maternal common variants to GWG and some overlap of maternal BMI, glucose and type 2 diabetes variants with GWG. These findings suggest that associations between GWG and later offspring/maternal outcomes may be due to the relationship of maternal BMI and diabetes with GWG.
Assuntos
Feto/fisiologia , Ganho de Peso na Gestação/genética , Gravidez/genética , Feminino , Estudo de Associação Genômica Ampla , Ganho de Peso na Gestação/fisiologia , Humanos , Gravidez/fisiologia , Gravidez/estatística & dados numéricosRESUMO
Dromedary camels (Camelus dromedarius) are an important protein source for people in semi-arid and arid regions of Africa. In Kenya, camel populations have grown dramatically in the past few decades resulting in the potential for increased disease transmission between humans and camels. An estimated four million Kenyans drink unpasteurized camel milk, which poses a disease risk. We evaluated the seroprevalence of a significant zoonotic pathogen, Coxiella burnetii (Q fever), among 334 camels from nine herds in Laikipia County, Kenya. Serum testing revealed 18.6% positive seroprevalence of Coxiella burnetii (n = 344). Increasing camel age was positively associated with C. burnetii seroprevalence (OR = 5.36). Our study confirmed that camels living in Laikipia County, Kenya, have been exposed to the zoonotic pathogen, C. burnetii. Further research to evaluate the role of camels in disease transmission to other livestock, wildlife and humans in Kenya should be conducted.
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Camelus/sangue , Coxiella burnetii , Febre Q/veterinária , Animais , Camelus/microbiologia , Feminino , Quênia/epidemiologia , Masculino , Febre Q/epidemiologia , Estudos SoroepidemiológicosRESUMO
Spectral properties of sea foam greatly affect ocean color remote sensing and aerosol optical thickness retrieval from satellite observation. This paper presents a combined Mie theory and Monte Carlo method to investigate visible and near-infrared spectral reflectance and bidirectional reflectance distribution function (BRDF) of sea foam layers. A three-layer model of the sea foam is developed in which each layer is composed of large air bubbles coated with pure water. A pseudo-continuous model and Mie theory for coated spheres is used to determine the effective radiative properties of sea foam. The one-dimensional Cox-Munk surface roughness model is used to calculate the slope density functions of the wind-blown ocean surface. A Monte Carlo method is used to solve the radiative transfer equation. Effects of foam layer thickness, bubble size, wind speed, solar zenith angle, and wavelength on the spectral reflectance and BRDF are investigated. Comparisons between previous theoretical results and experimental data demonstrate the feasibility of our proposed method. Sea foam can significantly increase the spectral reflectance and BRDF of the sea surface. The absorption coefficient of seawater near the surface is not the only parameter that influences the spectral reflectance. Meanwhile, the effects of bubble size, foam layer thickness, and solar zenith angle also cannot be obviously neglected.
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The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo.
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Anticolesterolemiantes/farmacologia , Azetidinas/farmacologia , Colesterol/metabolismo , Intestino Delgado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Membrana Celular/metabolismo , Endocitose , Ezetimiba , Absorção Intestinal/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismo , Esterol O-Aciltransferase 2RESUMO
An 8-wk experiment was conducted to evaluate the effect of replacing fish meal (FM) with soy protein isolate (SPI) on the growth, digestive enzyme activity and serum biochemical parameters of juvenile Amur sturgeon (Acipenser schrenckii). SPI was used to replace 0, 25, 50, 62.5, 75, 87.5, 100% of dietary FM and 100% replacement supplemented crystalline amino acid. Healthy sturgeon with an average initial weight of 26.38±0.24 g were randomly assigned to 24 aquaria (8 treatments with triplicates each) at an initial stocking density of 11 fish per aquarium and cultured for 8 wks. The results showed that 75.00% or more substitution resulted in a poor weight gain rate, feed conversion ratio and survival rate compared to that of fish fed the control diet (p<0.05), whereas no significant differences were observed between diets of 25.00% to 62.50% substitution. Protease, lipase and amylase activity in foregut, mid-gut and hindgut were significantly (p<0.05) decreased by diets where SPI replacement levels were 62.50% or more. Levels of serum total protein (TP) and globulin decreased significantly from 21.03, 10.34 to 14.05, 5.63 g/L with the increasing dietary SPI (p<0.05), but alkaline phosphatase activity significantly increased (p<0.05). In addition, supplemental crystalline amino acid in the FM absence diet did not improve growth performance, intestine digestive enzyme activities and serum biochemical parameters. In conclusion, the results from this study showed adverse effects of inclusion of SPI in diets on growth performance, feed utilization and serum biochemical parameters in juvenile Amur sturgeon. Based on WGR and replacement ratio presented in this report, a 57.64% replacement level was recommended.
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BACKGROUND: Prostaglandins (PG) play an important role in cutaneous homeostasis. Among other skin cells, human sebocytes express cyclooxygenases and can produce PGE(2). Various prostanoid receptors have been demonstrated in epidermis and hair follicles, while limited data are available regarding their expression in sebaceous glands. In addition, the interaction between PGE(2) and androgenesis remains largely unclear. OBJECTIVES: To examine the expression of PGE(2) receptor (EP) and PGF(2alpha) receptor (FP) in human sebocytes and the influence of PGE(2) or PGF(2alpha) on testosterone production. METHODS: A reverse transcription-polymerase chain reaction study was used to detect the expression of EP subtypes and FP. A testosterone radioimmunoassay was used to measure the amount of testosterone in the supernatant of cultured SZ95 sebocytes treated with PGE(2) or PGF(2alpha) alone or in the presence of various androgen precursor substrates. RESULTS: SZ95 sebocytes expressed mainly EP2 and EP4 but not EP3 or FP. Testosterone production was not induced by PGE(2) or PGF(2alpha), alone or in the presence of cholesterol. PGE(2) did not affect androgenesis in cultured sebocytes. CONCLUSIONS: The expression patterns of prostanoid receptors differ between sebocytes, hair follicles and epidermis. The effects of PGE(2) and PGF(2alpha) on the proliferation, lipogenesis and inflammation of sebocytes appear not to be associated with androgenesis.
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Dinoprostona/farmacologia , Receptores de Prostaglandina E/metabolismo , Glândulas Sebáceas/metabolismo , Testosterona/metabolismo , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Humanos , Radioimunoensaio , Receptores de Prostaglandina E/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Sebáceas/citologia , Glândulas Sebáceas/efeitos dos fármacosRESUMO
The effects of corticosterone, a natural glucocorticoid of rat, on the acetylcholine (ACh)-induced current (I(ACh)) were studied in pheochromocytoma (PC12) cells by using whole-cell clamp technique. The I(ACh) proved to be generated through neuronal nicotinic receptor. ACh (30 microM) induced an inward current at a holding potential of -80 mV. When cells were preincubated with corticosterone (0.1-100 microM) for 4 min, an inhibitory effect of corticosterone on the peak of I(ACh) was found. This effect was reversible, concentration-dependent, and voltage-independent. Intracellular application of corticosterone through the patch electrode did not affect the I(ACh). Extracellular application of 10 microM corticosterone neither shifted the dose-response curve of the peak I(ACh) to the right (dissociation constant (K(d)) = 16.5 microM) nor affected its coefficient (1.8) but inhibited the curve amplitudes by approximately 49% in the cells pretreated with corticosterone for 4 min. Bovine serum albumin-conjugated corticosterone (0.1-10 microM) had the inhibition similar to corticosterone. The inhibitor of transcription, actinomycin D (10 microM), and the protein synthesis inhibitor, cycloheximide (50 microM), had no effect on the inhibition induced by corticosterone on I(ACh). These results suggest that corticosterone has rapid inhibitory effect on I(ACh) in PC12 cells, which is mediated by a nongenomic mechanism. It indicates that corticosterone binds to the specific site on the outer cell membrane, probably on the neuronal nicotinic receptor-coupled channel, and inhibits the I(ACh) in a noncompetitive manner, thus controlling the immediate catecholamine release from the sympathetic cells.
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Corticosterona/farmacologia , Neurônios/efeitos dos fármacos , Células PC12/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Acetilcolina/antagonistas & inibidores , Acetilcolina/farmacologia , Animais , Colesterol/farmacologia , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Células PC12/metabolismo , Técnicas de Patch-Clamp , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Receptores Nicotínicos/metabolismo , Soroalbumina Bovina/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Brain-derived neurotrophic factor (BDNF) may play an important role in the modulation of N-methyl-d-asparate (NMDA) receptor function. To elucidate the underlying mechanisms, whole-cell patch-clamp recording was used to assess the effect of BDNF on the responses of cultured hippocampal neurons to the glutamate receptor agonist NMDA. We found that peak amplitude of NMDA-evoked currents in cultured hippocampal pyramidal neurons at Day 18 in vitro decreased significantly compared to that of NMDA currents at Day 10 or 14. Interestingly, NMDA-evoked currents were greatly enhanced by BDNF (50 ng/ml) in cultured neurons at Day 18, but not at Day 10 or 14. Treatment with Rp-cAMP abolished the potentiating effects of BDNF on NMDA current. Elevating the amount of cytosolic cAMP by preincubation with forskolin or Sp-cAMP also enhanced NMDA currents as effectively as BDNF in 18-day-old hippocampal neurons. Measurement of the cellular content of cAMP by RIA indicated that cultured hippocampal neurons showed decreased basal cAMP levels at the time NMDA currents were decreased and BDNF increased the decreased cAMP levels. Taken together, these results suggest that BDNF may restore decreased NMDA receptor activity in cultured hippocampal neurons by the cAMP pathway.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Hipocampo/metabolismo , Células Piramidais/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Líquido Intracelular/metabolismo , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Tionucleotídeos/farmacologia , Fatores de TempoRESUMO
The total unbound calmodulin (i.e., not bound to target proteins) level in living smooth muscle cells from the ferret portal vein was monitored with a low-affinity, calmodulin-binding peptide tagged with an environmentally sensitive fluorophore. GS17C, a previously characterized peptide, from the calmodulin-binding domain of caldesmon was tagged with iodoacetyl nitrobenz-2-oxa-1,3-diazole (NBD) or, as a negative control, with iodoacetylfluorescein isothiocyanate. Increases in NBD-GS17C fluorescence were detected by using confocal microscopy when chemically loaded cells were stimulated with solutions of elevated [K(+)] or the calcium ionophore 4-bromoA-23187 to elicit increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) quantified by fura 2. Increases in peptide fluorescence were detected in response to a phorbol ester in the absence of changes in [Ca(2+)](i). These changes were blocked by the addition of the calmodulin antagonist calmidazolium. These results suggest that the total unbound intracellular calmodulin levels may be sufficient to regulate the activity of caldesmon and, furthermore, that phosphorylation of protein kinase C substrates may increase the level of available calmodulin in living smooth muscle cells.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Músculo Liso Vascular/enzimologia , Proteínas Quinases/metabolismo , 4-Cloro-7-nitrobenzofurazano , Animais , Proteínas de Ligação a Calmodulina/metabolismo , Furões , Indicadores e Reagentes , Músculo Liso Vascular/citologia , Veia Porta/citologia , Veia Porta/metabolismoRESUMO
We show that calmodulin (CaM) has an extended conformation in its complexes with sequences from the smooth muscle thin filament protein caldesmon (CaD) by using small-angle X-ray and neutron scattering with contrast variation. The CaD sequences used in these experiments were a C-terminal fragment, 22kCaD, and a smaller peptide sequence within this fragment, MG56C. Each of these sequences contains the CaM-binding sites A and B previously shown to interact with the C- and N-terminal lobes of CaM, respectively [Wang et al. (1997) Biochemistry 36, 15026]. By modeling the scattering data, we show that the majority of the MG56C sequence binds to the N-terminal domain of CaM. FTIR data on CaM complexed with 22kCaD or with MG56C peptide show the 22kCaD sequence contains unordered, helix, and extended structures, and that the extended structures reside primarily in the MG56C portion of the sequence. There are small changes in secondary structure, involving approximately 12 residues, induced by CaM binding to CaD. These changes involve a net decrease in extended structures accompanied by an increase in alpha-helix, and they occur within the CaM and/or in the MG56C sequence.