Stimulation of mucosal uptake of selenium from selenite byl-cysteine in sheep small intestine

The influence of cysteine (Cys) on mucosal uptake of 75Se-labeled selenite in sheep midjejunum was investigated using a short-term uptake technique. L-Cys (concn.: 1.0 mmol/L) significantly stimulated uptake of Se from selenite (concn.: 10 mumols/L). The stimulatory effect of L-Cys on mucosal uptake of Se from selenite was Na(+)- and pH-dependent. In the absence of Na+, or at an acidic pH (5.0), the stimulatory effect of L-Cys was abolished. L-alanine and L-lysine, but not L-glutamic acid inhibited uptake of Se from selenite in the presence of L-Cys. Preincubation of mucosal preparations with 10 mmol/L L-Cys produced enhanced mucosal uptake of Se from selenite. It is concluded from these results that L-Cys stimulates absorption of Se from selenite probably by generation of selenodicysteine and maybe cysteine selenopersulfide that are subsequently transported across the intestinal brush border membrane by Na(+)-dependent amino acid carriers. Furthermore, intracellular generation of selenodicysteine might contribute to the uptake of Se from selenite by maintaining the concentration gradient for diffusive uptake of selenite.     

Detection of prion protein particles in blood plasma of scrapie infected sheep

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.     

In vitro studies on methyl mercury distribution in human blood

Studies comparing the methyl mercury (mHg) content of maternal and newborn blood have shown increased levels in the newborn. This has been attributed to facilitated transplacental diffusion because of high fetal hematocrit (Hct). This study shows the converse, that the diffusion of mHg diminishes progressively with increasing Hct. The diffusion of m203Hg across a Millipore membrane (0.45 microns) separating compartments A and B of a diffusion cell was studied. When both compartments contained saline or plasma alone, equilibration from A to B occurred in 5 h. Introduction of human red blood cells (RBC) in saline (Hct 20%) into B resulted in a twofold increase in diffusion of mHg when compared to saline alone. Increasing Hct in saline in compartment B resulted in a progressive decrease in diffusion (r = -0.95, P less than 0.001). The presence of RBC in plasma (Hct 20%) in B resulted in a 70% decrease in diffusion; with increasing Hct, diffusion was further reduced (r = -0.95, P less than 0.001). Direct addition of mHg to RBC in saline resulted in 98% RBC uptake. Increasing concentrations of plasma (at a constant Hct) resulted in a progressive decrease in RBC uptake. In undiluted plasma at Hct 14%, RBC uptake of mHg was 35%. Plasma electrophoresis showed that much of the mHg was associated with a high-molecular-weight lipoprotein fraction. Plasma components appear to be important in the distribution of mHg in blood, and may be a factor in the relatively higher blood levels in the fetus.     

Progesterone and 20 alpha-dihydroprogesterone in sheep: a model of their distribution and metabolism.

The rates of metabolism of progesterone and 20alpha-dihydroprogesterone (20alpha-diHP) in sheep have been measured during and after the infusion of tracer amounts of [3H]progesterone. There were significant differences in the blood concentration of [3H]progesterone between experiments, but these were not attributable to the stage of gestation or to the difference between pregnant and non-pregnant animals. The mean (+/- S.E.M.) metabolic clearance rate of progesterone was 3-277 +/- 0-239 litres blood/min. The simplest model of the distribution of progesterone was one containing two pools, V1[P] and V2[P], where [p] is the blood concentration of progesterone, and in 23 experiments on 7 sheep the mean pool dimensions were 7-8[P] mug and 70[P] mug, respectively. This model was developed to include the formation of 20alpha-diHP from progesterone. Progesterone appeared to be the major source of 20alpha-diHP, though this did not seem to be an obligatory metabolite. The parameters obtained gave comparably low residual deviations for both labelled steroids and were consistent with other observations made on progesterone clearance.     

Interactions of cadmium and selenium in rat plasma in vivo and in vitro.

75Se and 109Cd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO32- doses and times upt to 24 h after the simultaneous subcutaneous administration of SeO32- markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 mumol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO32- does not interact directly with Cd and plasma proteins. It is metabolized by erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.     

In vitro thymidine labelling in human and porcine growth plates

An in vitro technique has been used to label dividing cells in the growth plates of human bones with tritiated thymidine. The patterns of labelling in autoradiographs of human plates are described and values given for the labelling index, the number of cells in the proliferation zones and the heights of hypertrophied cells. In two of the four subjects no labelled cells were found in the growth plates and possible causes for these failures are discussed. In vitro labelling data on five porcine growth plates are also presented for comparison with the human data. In both structure and cell kinetics the epiphyseal cartilage plate in the pig is intermediate between the human and rodent plates.     

Comparison of distribution and metabolism between tellurium and selenium in rats

Tellurium (Te) has shown recent increase in use as a component of optical magnetic disks having phase-change property, such as digital versatile disk-random access memory (DVD-RAM) and DVD-rewritable (DVD-RW). However, the toxicity and metabolic pathway of Te remain unclear despite its being known as a non-essential and harmful metalloid. This study was performed to gain an insight into Te metabolism in the body. The mechanism for the distinction of Te from selenium (Se), an essential metalloid belonging to the same group as Te, was also clarified. Rats were given drinking water containing tellurite and ⁸²Se-labeled selenite at the same concentration, and the concentrations of these metalloids in organs, body fluid and excreta were determined 2 days later. The results demonstrate that urinary and fecal excretion of Te was, respectively, lower and higher than that of exogenous (labeled) Se, suggesting that Te was less absorbed than Se. The ingested Te was transformed, i.e., methylated in organs and effluxed into bloodstream, and the effluxed Te was highly accumulated in rat red blood cells (RBCs) in the form of dimethylated Te. In contrast, Se was not accumulated in RBCs. Finally, Te was excreted in urine as trimethyltelluronium and might be exhaled as dimethyltelluride. The results suggest that the metabolism of Te was distinct from that of Se in rats.     

In vitro interaction of fenretinide with plasma retinol-binding protein and its functional consequences.

The synthetic retinoid fenretinide (4-HPR; N-[4-hydroxyphenyl] all-trans-retinamide) interacts with plasma apo-retinol-binding protein (RBP) to form a tight complex (K'd approximately 0.2 microM) which does not exhibit binding affinity to transthyretin (TTR). Therefore, a substantial modification of the retinol hydroxyl group does not appear to affect the interaction with RBP but does drastically interfere with the protein-protein recognition. The remarkable early reduction in plasma retinol level induced by fenretinide administration may be associated with the high binding affinity of this retinoid to RBP and to its interference with the RBP-TTR complex formation.     

In vitro synthesis of glutathione peroxidase from selenite. Translational incorporation of selenocysteine

The synthesis of glutathione peroxidase from [75Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [75Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [3H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and 75Se was incorporated from [75Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the 75Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [75Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase.     

In vitro culture of sheep oocytes matured and fertilized in vitro

Sheep oocytes that matured and fertilized in vitro were cultured to evaluate their cleavage to the 8- to 16- cell stage and further development in five different media as follows: 1) CPMW (TCM199 + 20% ewe serum + 0.4% BSA), 2) Ham's F-10 + 10% ewe serum, 3) Brinster's pyruvate medium + 0.1% glucose (BPM-G), 4) co-culture with sheep oviduct epithelial cells in TCM199 + 10% fetal calf serum, and 5) co-culture with sheep granulosa cells in the same medium as 4. The culture duration was 4 or 7 d for 8- to 16-cell or further development. The proportions of 8- to 16-cell eggs were 1) 16% (8 49 ), 2) 25% (12 49 ), 3) 52% (58 112 ), 4) 63% (105 167 ) and 5) 45% (27 60 ). The co-culture with sheep oviduct cells resulted in a significantly (P < 0.05) higher rate of cleavage than the other media, except BPM-G. The proportion of noncompacted morula (35%, 24 68 ) was also significantly (P < 0.05) higher in the co-culture of sheep oviduct cells than the other media. The 8- to 16-cell eggs produced by BPM-G (n=38) and the co-culture with sheep oviduct cells (n=42) were transferred into the uterus of recipient ewes, but no elongated blastocysts were obtained 13 d later. On the other hand, 8 out of 55 one-cell eggs (15 to 18 h after in vitro insemination) transferred to the oviduct of recipient ewes were elongated blastocysts (24% of 34 recovered eggs). The data show that the co-culture of in vitro fertilized eggs with sheep oviduct epithelial cells could support development of 8- to 16-cell embryos or early morula, but their viability is still questionable.     

Influences of heparin on ACTH distribution and immunoreactivity in plasma of the rat. In vivo and in vitro studies

1. Blood samples from non-pregnant female rats were incubated in vitro with porcine 125I-ACTH, and the corresponding plasmas were chromatographed on fine Sephadex G 50. When heparin was added in vivo or in vitro, almost all the radioactivity appeared in the void volume of the columns; the same was observed when labelled ACTH was added to heparin-containing saline. In contrast, when NaCl instead of heparin was added to the blood in vivo as well as in vitro, almost all the plasma radioactivity was eluted later, with 125I-ACTH. 2. When labelled ACTH was i.v. administered to pregnant females, it was eluted in the void volume in the presence of heparin, and further down in its absence. 3. The same plasma samples from non-stressed or ether-stressed females were radioimmunoassayed for ACTH, with and without heparin. The degradation of ACTH was greater in the presence of heparin, and plasma ACTH concentration was understimated for low blood levels of heparin (5 UI/ml or less) and in contrast overestimated for high ones (25 or 50 UI/ml). 4. In conclusion, the reported data clearly demonstrated firstly that heparin added to rat blood traps ACTH molecules, promoting the formation of aggregates with apparent height molecular weight; secondly that heparin interferes with the direct radioimmunoassay of ACTH in the plasma.     

In vitro lipid metabolism in the rat pancreas. I. Basal lipid metabolism.

1. The in vitro basal lipid metabolism of rat pancreatic fragments was compared with that in adipose tissue fragments and liver slices. 2. [1-14C]Acetate added to the media was mostly incorporated into palmitic acid and to a lesser extent into oleic acid. In addition, pancreatic tissue exhibited a marked capacity for elongation of polyunsaturated fatty acids by [1-14C]acetate and resulting desaturation when compared to adipose tissue and liver. 3. Data obtained in the presence of [U-14C]glucose, [1-14C]palmitate and 3H20 indicate that acetyl-CoA derived from glucose and from beta-oxidation of fatty acids contributed to de novo lipogenesis. 4. Oxidation of [1-14C]palmitic acid was 9-13 times higher in the pancreas than in adipose tissue or liver when expressed on a wet weight basis. 5. The fatty acid moiety of pancreatic glycerolipids could be derived from de novo synthesis, fatty acids added to the medium, or from fatty acids formed from the hydrolysis of endogenous lipids. The glycerol moiety could be derived either from glucose, or directly from glycerol through participation of glycerol kinase.     

Properties of the cadmium and selenium complex formed in rat plasma in vivo and in vitro.

Following the simultaneous subcutaneous administration of CdCl2 and Na2SeO3 to rats, evidence of a Cd-Se complex was detected in plasma by gel filtration chromatography. A similar complex was found in plasma after incubation of selenite, Cd, rat erythrocytes, and plasma in vitro, and after incubation of H2Se, Cd, and plasma in vitro. No interaction of selenite, selanete, or selenodiglutathione with Cd and plasma in the absence of erythrocytes in vitro was noted. Characterization by gel filtration, ion-exchange chromatography, affinity chromatography, and ammonium sulfate fractionation showed that these Cd-Se complexes are similar. The results support the hypothesis that H2Se or a similarly reduced selenide is the product of selenite metabolism by rat erythrocytes. Hydrogen selenide also altered the distribution of inorganic mercury in rat plasma in vitro in such a way that the apparent molecular weights of the Se-Hg and Cd-Se complexes associated with protein were similar. Hydrogen selenide had no effect upon the distribution of methylmercury in plasma. The stability of the Cd-Se complex in plasma depended upon the integrity of the native protein components, as shown by incubation with Proteinase K. The properties of the complex suggested that it existed in a single form associated with different plasma components under various conditions.     

An estimation of dominance at the locus controlling blood potassium in sheep

1. Potassium concentrations in erythrocytes and plasma were influenced by Ka genotype and breed.
2. Dominance at the Ka locus controlling red cell potassium concentration was incomplete.
3. The lower limit of the difference between heterozygous and homozygous low phenotypes was 4.1 meq per litre.
4. Sheep of the Finnish Landrace breed had relatively high potassium concentrations and the highest gene frequency of the recessive allele, of those examined.