RESUMO
Sublethal stress treatment has been reported to enhance gametes' performance in subsequent procedures, such as cryopreservation. The aim of the present study was to evaluate the effect of different equilibration times between the termination of a sublethal hydrostatic pressure (HP) stress treatment and the initiation of vitrification on the post-thaw survival, continued in vitro development, hatching rate and gene expression of selected candidate genes of in vitro-produced (IVP) expanded bovine blastocysts. Day 7 IVP blastocysts were subjected to 600 bar pressure for 60 min at 32°C. Immediately after pressure treatment (HP0h) or after 1 or 2h incubation (HP1h and HP2h groups, respectively), embryos were either vitrified and warmed using the open pulled straw method, followed by 72 h in vitro culture or were stored at -80°C until gene expression analysis. Re-expansion and hatching rates after vitrification-warming were significantly (P<0.05) higher in the HP0h (88 and 76%, respectively) and HP1h (90 and 75%, respectively) groups than in the untreated (82 and 63%, respectively) and HP2h groups (79 and 70%, respectively). Moreover, the HP1h group showed further improvement in the speed of re-expansion and resumption of normal in vitro development. Cumulative analysis of all genes (SC4MOL, HSP1A1A, SOD2 and GPX4) revealed a similar pattern of expression, with a tendency for peak transcript abundance 1h after HP treatment. Application of HP stress treatment was found to be efficient in increasing the in vitro developmental competence of vitrified bovine embryos.
Assuntos
Blastocisto , Desenvolvimento Embrionário/fisiologia , Expressão Gênica , Estresse Mecânico , Vitrificação , Animais , Blastocisto/metabolismo , Blastocisto/fisiologia , Bovinos/embriologia , Sobrevivência Celular , Células Cultivadas , Criopreservação/métodos , Técnicas de Cultura Embrionária , Feminino , Fertilização in vitro , Pressão Hidrostática , MasculinoRESUMO
IFN-gamma mRNA expression was evaluated in nonstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected and seronegative individuals using quantitative competitive and semiquantitative RT-PCR and the sensitivity of these methods was compared. A significant correlation was found between quantitative competitive and semiquantitative RT-PCR in samples of both HIV-seronegative (P = 0.004) and HIV-infected individuals (P = 0.0004). PBMC from HIV-infected individuals presented a remarkable increase of IFN-gamma mRNA expression, as determined by both types of RT-PCR methods. Semiquantitative RT-PCR even without an internal standard is also acceptable for measuring cytokine mRNA expression, but less reliable if small amounts are quantified. Moreover, we found that increased IFN-gamma mRNA expression is independent of CD4+ cell count in AIDS-free HIV-infected patients.
Assuntos
Infecções por HIV/sangue , Interferon gama/sangue , Leucócitos Mononucleares/metabolismo , RNA Mensageiro/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome da Imunodeficiência Adquirida/sangue , Síndrome da Imunodeficiência Adquirida/imunologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos , Infecções por HIV/imunologia , Soronegatividade para HIV , Humanos , Estudos Longitudinais , Sensibilidade e EspecificidadeRESUMO
IFN-gamma mRNA expression was evaluated in nonstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected and seronegative individuals using quantitative competitive and semiquantitative RT-PCR and the sensitivity of these methods was compared. A significant correlation was found between quantitative competitive and semiquantitative RT-PCR in samples of both HIV-seronegative (P = 0.004) and HIV-infected individuals (P = 0.0004). PBMC from HIV-infected individuals presented a remarkable increase of IFN-gamma mRNA expression, as determined by both types of RT-PCR methods. Semiquantitative RT-PCR even without an internal standard is also acceptable for measuring cytokine mRNA expression, but less reliable if small amounts are quantified. Moreover, we found that increased IFN-gammamRNA expression is independent of CD4+ cell count in AIDS-free HIV-infected patients
Assuntos
Humanos , Infecções por HIV , Interferon gama , Leucócitos Mononucleares , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/metabolismo , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos , Infecções por HIV , Soronegatividade para HIV , Interferon gama , Estudos Longitudinais , RNA Mensageiro , Sensibilidade e EspecificidadeRESUMO
We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphatidylinositol (GPI)-anchoring and intracellular transport of the yeast Gas1 protein. No effect on anchor attachment was demonstrable, but a selective delay in transport from the endoplasmic reticulum to the Golgi complex was observed. The compound also blocked remodeling of GPI-anchors from their base-sensitive to base-resistant forms. A recessive mutation was found that caused resistance to the drug, restored transport of Gas1p, but did not restore ceramide biosynthesis in the presence of the inhibitor. Our results suggest that intracellular transport of GPI-anchored proteins is stimulated by new ceramide synthesis. The role of ceramide may be direct or may be through its use in the remodeling of GPI-anchored proteins other than Gas1p. The need for ceramide can be overcome in the mutant strain.
Assuntos
Ceramidas/metabolismo , Proteínas Fúngicas/metabolismo , Glicosilfosfatidilinositóis/química , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ceramidas/fisiologia , Retículo Endoplasmático/metabolismoRESUMO
We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphatidylinositol (GPI)-anchoring and intracellular transport of the yeast Gas 1 protein. No effect on anchor attachment was demonstrable, but a selective delay in transport from the endoplasmic reticulum to the Golgi complex was observed. The compound also blocked remodeling of GPI-anchors from their base-sensitive to base-resistant forms. A recessive mutation was found that caused resistance to the drug, restored transport of Gas 1p, but did not restore ceramide biosynthesis in the presence of the inhibitor. Our results suggest that intracellular transport of GPI-anchored proteins is stimulated by new ceramide synthesis. The role of ceramide may be direct or may be through its use in the remodeling of GPI-anchored proteins other than Gas 1p. The need for ceramide can be overcome in the mutant strain
Assuntos
Ceramidas/biossíntese , Fosfatidilinositóis/metabolismo , Glicolipídeos/metabolismo , Leveduras , Sequência de Aminoácidos , Retículo Endoplasmático , Saccharomyces cerevisiaeRESUMO
Various types of fish-poisoning mainly occur in tropical areas. Saxitoxin, tetrodotoxin, scombrotoxin and clupeotoxin are heat labile while ciguatoxin, in contrast, is heat stable thus it may be the source of severe poisoning even in case of thorough cooking and baking. In December 1987 a mass alimentary poisoning occurred following ingestion of fish meat in a group returning from Cuba. The most severe case was admitted to our department with symptoms of extended paresis. Diagnosis of ciguatoxin poisoning was established based upon the history, clinical features and laboratory results. Nowadays exotic, remote "Fish-poisoning" cases have to be taken into consideration even in our country.