RESUMO
This trial evaluated the individual and interactional effects of diet and type of pregnancy (twin or single) on plasma metabolic response in ewes and their lambs from late pre-partum to late post-partum. Thus, a flock of 18 Ile de France breed sheep, consisting of 8 twin-bearing and 10 single-bearing ewes, were allocated to one of two groups according to their diet, either based on ad libitum naturalized pasture hay (NPH) or red clover hay (RCH), from d 45 pre-partum to d 60 post-partum. Plasma samples were collected at different times to determine albumin, cholesterol, total protein and urea, plus glucose and ß-hydroxybutyrate (BHB) concentration in ewes. The data was processed using the lme4 package for R, and SPSS Statistics 23.0 for Windows. The results showed that both diet and type of pregnancy influenced the metabolic profile in ewes, showing an inverse relationship between single- and twin-bearing ewes regarding glucose and especially BHB proportions from pre-partum to birth. During post-partum, higher urea concentrations were observed in twin- and single-bearing ewes fed RCH in contrast to those fed NPH, as a result of the higher-quality forage offered to ewes. Regarding lambs, the diet and type of pregnancy influenced the total protein and urea levels, where an inverse relationship at birth and early post-partum between albumin and cholesterol vs. total protein and urea was detected, reflecting a trend (P value between 0.06 and 0.07) to a better performance by groups of single lambs, especially those from single-bearing ewes fed RCH. Finally, under the conditions of this study, the maternal diet and type of pregnancy influenced the plasma metabolic response in ewes and their lambs, affecting the lamb performance especially at birth.
Assuntos
Ração Animal/análise , Dieta/veterinária , Fenômenos Fisiológicos da Nutrição Materna , Prenhez , Gravidez Múltipla , Ovinos/sangue , Animais , Feminino , Humanos , Gravidez , Ovinos/fisiologiaAssuntos
Antibacterianos/farmacologia , Doenças dos Peixes/tratamento farmacológico , Testes de Sensibilidade Microbiana/métodos , Oxitetraciclina/farmacologia , Piscirickettsia/efeitos dos fármacos , Infecções por Piscirickettsiaceae/veterinária , Tianfenicol/análogos & derivados , Animais , Doenças dos Peixes/microbiologia , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Oncorhynchus mykiss , Infecções por Piscirickettsiaceae/tratamento farmacológico , Infecções por Piscirickettsiaceae/microbiologia , Reação em Cadeia da Polimerase/veterinária , Salmo salar , Tianfenicol/farmacologiaRESUMO
Piscirickettsiosis or salmonid rickettsial septicaemia (SRS) caused by Piscirickettsia salmonis constitutes one of the main problems in farmed salmonid and marine fishes. Since the first reports of the disease, it has been successfully isolated and maintained in eukaryotic cell--culture systems, but these systems are time-consuming, the media are costly, and eliminating heavily contaminated host cell debris is difficult. In this report, we describe a marine-based broth supplemented with L-cysteine, named AUSTRAL-SRS broth, that facilitates superior growth of P. salmonis strains. Strains reached an optical density of approximately 1.8 when absorbance was measured at 600 nm after 6 d incubation at 18°C. Several passages (n = 6) did not alter the culture kinetics. We report for the first time the purification of DNA, lipopolysaccharide (LPS) and whole membrane protein obtained from P. salmonis grown in this liquid medium, and thus provide a suitable platform to simplify the preparation of P. salmonis cells for genetic and serological studies. Moreover, the results of the cytopathic effect test showed that P. salmonis grown in AUSTRAL-SRS broth maintained their virulence properties, inducing apoptosis after 3 d. This makes the medium a good candidate for the successful growth of P. salmonis and an excellent basis for the development of low cost vaccines.
Assuntos
Técnicas Bacteriológicas , Meios de Cultura/química , Piscirickettsia/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Cisteína/química , Regulação Bacteriana da Expressão Gênica/fisiologia , Rim Cefálico/citologia , Salmão , Fatores de TempoRESUMO
BACKGROUND: Tacrolimus (FK506) is a macrolide immunosuppressant drug from the calcineurin inhibitor family, widely used in solid organ and islet cell transplantation, but produces significant side-effects. OBJECTIVE: This study examined the effect of FK506 on interleukin-2 (IL-2) and insulin secretion, establishing a novel characteristic of this drug that could explain its diverse adverse effects, and developed an experimental model for the simultaneous analysis of mRNA expression and protein secretion affected by this drug. METHODS: The IL-2 levels when tacrolimus was administered were analysed by Western blot, immunocytochemistry and RT-PCR in a T lymphocyte cellular line (Jurkat) 24 h post-stimulation. The insulin levels when tacrolimus was administered were analysed 4 h after stimulation of glucose-induced insulin secretion in a pancreatic cellular line (MIN6). RESULTS: The previously published information describes tacrolimus as only capable of partially blocking IL-2 mRNA expression. In our hands, the cellular content of IL-2 is almost undetectable in stimulated Jurkat cells and can be detected in cellular extracts only when the secretory pathway is blocked by brefeldin A (BFA). BFA added 2 h after the beginning of stimulation was able to inhibit IL-2 secretion, without affecting IL-2 mRNA expression. Therefore BFA utilization allowed us to establish a model to analyze the effect on IL-2 secretion, separately from its expression. Tacrolimus added before stimulation inhibits only IL-2 synthesis, but blocks IL-2 protein secretion when added 2 h after stimulation. Similarly, tacrolimus is also capable of blocking the glucose-stimulated secretion of insulin by MIN6 cells. An increase of the intracellular content can be detected concomitantly with a decrease of the hormone measured in the culture medium. CONCLUSIONS: Results of this study indicate that tacrolimus possesses another important effect in addition to the inhibition of IL-2 gene transcription, namely the ability to act as a general inhibitor of the protein secretory pathway. These results strongly suggest that the diabetogenic effect of the immune suppressant FK506 could be caused by the blockade of insulin secretion. This novel effect also provides an explanation for other side-effects observed in immunosuppressive treatment.
Assuntos
Terapia de Imunossupressão , Imunossupressores/efeitos adversos , Imunossupressores/farmacologia , Proteínas/metabolismo , Via Secretória/efeitos dos fármacos , Tacrolimo/efeitos adversos , Tacrolimo/farmacologia , Animais , Brefeldina A/farmacologia , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Glucose/farmacologia , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Fito-Hemaglutininas/farmacologia , Proteínas/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.