Effect of diet and type of pregnancy on plasma metabolic response in sheep and its further effect on lamb performance.

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.

Broth medium for the successful culture of the fish pathogen Piscirickettsia salmonis .

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.

Tacrolimus causes a blockage of protein secretion which reinforces its immunosuppressive activity and also explains some of its toxic side-effects.

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.

The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition.

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.

Increased uptake and accumulation of vitamin C in human immunodeficiency virus 1-infected hematopoietic cell lines.

Vitamin C (ascorbic acid) is required for normal host defense and functions importantly in cellular redox systems. To define the interrelationship between human immunodeficiency virus (HIV) infection and vitamin C flux at the cellular level, we analyzed vitamin C uptake and its effects on virus production and cellular proliferation in HIV-infected and uninfected human lymphoid, myeloid, and mononuclear phagocyte cell lines. Chronic or acute infection of these cell lines by HIV-1 led to increased expression of glucose transporter 1, associated with increased transport and accumulation of vitamin C. Infected cells also showed increased transport of glucose analogs. Exposure to vitamin C had a complex effect on cell proliferation and viral production. Low concentrations of vitamin C increased or decreased cell proliferation depending on the cell line and either had no effect or caused increased viral production. Exposure to high concentrations of vitamin C preferentially decreased the proliferation and survival of the HIV-infected cells and caused decreased viral production. These findings indicate that HIV infection in lymphocytic, monocytic, and myeloid cell lines leads to increased expression of glucose transporter 1 and consequent increased cellular vitamin C uptake. High concentrations of vitamin C were preferentially toxic to HIV-infected host defense cell lines in vitro.

Genistein is a natural inhibitor of hexose and dehydroascorbic acid transport through the glucose transporter, GLUT1.

Genistein is a dietary-derived plant product that inhibits the activity of protein-tyrosine kinases. We show here that it is a potent inhibitor of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, genistein inhibited the transport of dehydroascorbic acid, deoxyglucose, and methylglucose in a dose-dependent manner. Transport was not affected by daidzein, an inactive genistein analog that does not inhibit protein-tyrosine kinase activity, or by the general protein kinase inhibitor staurosporine. Genistein inhibited the uptake of deoxyglucose and dehydroascorbic acid in Chinese hamster ovary (CHO) cells overexpressing GLUT1 in a similar dose-dependent manner. Genistein also inhibited the uptake of deoxyglucose in human erythrocytes indicating that its effect on glucose transporter function is cell-independent. The inhibitory action of genistein on transport was instantaneous, with no additional effect observed in cells preincubated with it for various periods of time. Genistein did not alter the uptake of leucine by HL-60 cells, indicating that its inhibitory effect was specific for the glucose transporters. The inhibitory effect of genistein was of the competitive type, with a Ki of approximately 12 microM for inhibition of the transport of both methylglucose and deoxyglucose. Binding studies showed that genistein inhibited glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts in a competitive manner, with a Ki of 7 microM. These data indicate that genistein inhibits the transport of dehydroascorbic acid and hexoses by directly interacting with the hexose transporter GLUT1 and interfering with its transport activity, rather than as a consequence of its known ability to inhibit protein-tyrosine kinases. These observations indicate that some of the many effects of genistein on cellular physiology may be related to its ability to disrupt the normal cellular flux of substrates through GLUT1, a hexose transporter universally expressed in cells, and is responsible for the basal uptake of glucose.