Subcellular topology of rat liver methionyl-, leucyl-, and arginyl-tRNA synthetases.

We have investigated the distribution of methionyl-, leucyl-, and arginyl- tRNA synthetases in primary liver fractions obtained by differential centrifugation of homogenates in isotonic sucrose: 78-93% of synthetase activities are recovered in the cytosolic fraction. Microsomes contain only 4.8%, 19.4%, and 6.4% of the methionyl-, leucyl-, and arginyl-tRNA synthetases activities, respectively. This proportion increases up to 11.3%, 26.1%, and 20.7%, respectively, when the homogenization medium is supplemented with 5 mM Mg2+ and 25 mM K+. The presence of protease inhibitors in the homogenization medium does not increase the proportion of synthetases recovered in microsomes. After subfractionation of microsomes by isopycnic centrifugation, the distributions of the 3 synthetases display a second peak overlapping that of at a density of 1.12. In addition, methionyl- and leucyl- tRNA synthetases display a second peak overlapping that of RNA. This suggests that a small proportion of these synthetases (0.7% and 5.71% of total activities, respectively) bind to the d domain of the ER. The Golgi complex, the plasma membranes, and the peroxisomes lack aminoacyl-tRNA synthetase activity. The 3 synthetases are readily detached from membranes when intact microsomes are washed with 250 mM sucrose alone or containing 5 mM PPi, or 320 mM KCl. The binding of methionyl-tRNA synthetases to microsomes was measured in vitro, at 4 degrees C, with a sample of the cytosolic fraction as a source of synthetase. Microsomes stripped of their bound polysomes display a binding capacity that is not significantly different from that of unstripped microsomes. Even in the presence of cations, the amount of synthetase bound to the membranes remained low by comparison with the cytosolic content

A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum.

We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane-bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na-pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6-phosphatase with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also contains the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co- and post-translationally the signal peptide of pre-secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain

Determination by Fourier Transform Raman Spectroscopy of Conjugated Linoleic Acid in I(2)-Photoisomerized Soybean Oil.

The potential of Fourier transform (FT)-Raman spectroscopy to quantify the total conjugated linoleic acid (CLA) content was evaluated to find a technique for the routine control of CLA synthesis by chemical procedures. The calibration and validation samples were obtained by photoisomerization of linoleic acid contained in soybean oil. The catalyst was iodine (I(2)), and the light source was the green line (514.5 nm) of an argon ion laser. The criteria to select the best partial least-squares (PLS) calibration model were a low standard error of prediction (SEP), a high correlation coefficient (R), and the selection of relevant variables of the Raman spectrum to reduce spectral interferences. The total CLA content of the 22 samples ranged from 0.05 to 3.28% of total lipids. The best PLS calibration model was obtained with three optimal factors, a SEP of 0.22, and a R of 0.97. This calibration model was obtained after baseline correction of the CC stretching region (1642-1680 cm(-1)), which contained sufficient spectral information for reliable CLA quantification

Study of bioconversion of conjugated linolenic acid (CLNA) of Ricinodendron heudelotii (Bail.) seed in male rats into conjugated linoleic acid (CLA) using UV-Vis spectrometry and gas chromatography

The present study evaluates the efficiency of conversion of CLNA (conjugated linolenic acids; α-eleostearic acid) of Ricinodendron heudelotii to CLA (conjugated linoleic acids; rumenic acid) in the liver, small bowel and serum of male rats using ultraviolet spectrometry (UV) and Gas Chromatography (GC) techniques. One milliliter of oil was orally administrated to 30 rats during 24 h. The spectra of R. heudelotii oil, linoleic acid, rumenic acid and α-eleostearic acid (α-ESA) were determined. The concentrations of α-ESA and CLA were determined after 0, 3, 6, 12 and 24 h of oral administration of oil in liver, small bowel and serum. Linoleic acid has a peak at 215 nm and rumenic acid at 232 nm. Spectra of standard α-eleostearic acid and R. heudelotii oil were bunk with a characteristic peak at 270 nm. In the serum, we did not find any trace of fatty acid with either UV or GC. UV and GC methods showed that α-ESA was gradually converted in rumenic acid in the liver and small bowel. The liver had a faster and higher enzyme activity than the small bowel. These results showed that the conversion of CLNA to CLA in rats using UV spectrometry is the most method to use. © 2012 Academic Journals Inc

Influence of the diet structure on ruminal biohydrogenation and milk fatty acid composition of cows fed extruded linseed

This experiment studied the influence of the diet structure value (SV) on ruminal biohydrogenation and milk fatty acid (FA) responses in cows fed heterogeneous basal diets equally supplemented with FA. Eight lactating Holstein cows were used in a replicated 4×4 Latin square design with four dietary treatments and four 21-day periods. The iso-fat, iso-18:2 n−6 and iso-18:3 n−3 diets were formulated to display three different SV, using different sources and proportions of forages, energy and nitrogen concentrates. The four diets contained maize silage as the main forage (SV1.2 diet), grass hay as the main forage (SV2.0 diet), maize silage and grass hay in a 4:1 ratio (SV1.6M diet) or maize silage and grass hay in a 1:1 ratio (SV1.6H diet). The diets also contained soya bean meal and/or urea as additional sources of nitrogen, sugar beet pulp and barley in a 1:1 ratio as additional source of energy, extruded linseed as supplemental 18:3 n−3, a mineral and vitamin mix and a vitamin E preparation. Wheat straw was added to the diets as additional structure source, except for the SV2.0 diet. Soya bean oil was added to the diets as supplemental 18:2 n−6 to adjust the diets for this FA, except for the SV1.2 diet. The diets were distributed as a restricted total mixed ration. The various C18 FA expressed as 100g of total C18 FA in milk fat are relevant indicators of ruminal biohydrogenation since duodenal concentrations of C18 FA follow similar changes as those in milk fat, and since these ratios only take into account FA involved in ruminal biohydrogenation. All the various C18 FA to total C18 FA in milk fat differed among diets (P<0.05). Milk 18:2 n−6+18:3 n−3/total C18 FA and total trans-C18 FA/total C18 FA decreased from SV1.2 to SV2.0 diets, whereas 18:0/total C18 FA increased from SV1.2 to SV2.0 diets. Subsequently, transfer efficiencies of 18:2 n−6 and 18:3 n−3 from diet to milk were higher for the SV1.2 diet than for the other diets (P<0.05). These results confirm the hypothesis that ruminal biohydrogenation is more complete with higher diet SV, which is consistent with results from other published experiments where high forage diets or grass silage compared to maize silage-based diets were used. This experiment showed that the concept of diet SV is a valid tool characterizing heterogeneous basal diets differing in sources and proportions of forages and concentrates

Influence of an increase in diet structure on milk conjugated linoleic acid content of cows fed extruded linseed

This experiment studied the effect of a modest difference in diet structure value (SO on milk conjugated linoleic acid (CLA) contents of cows fed diets supplemented with extruded linseed, in situations where the diets provided enough SV and therefore did not induce milk fat depression. Six lactating Holstein cows were used in a crossover design with two treatments ('SV 1.50' and 'SV 1.73') and two periods of 21 days. The 'SV 1.50' diet contained 59% maize silage, 13% soya bean meal, 13% sugar beet pulp and 14% Nutex Compact (containing 56% extruded linseed) (dry matter (DM) basis) and was offered as a restricted total mixed ration. For the 'SV 1.73' diet, 8% wheat straw (DM basis) was added to the 'SV 1.50' diet as an additional structure source. The two diets had a forage-to-concentrate ratio of 59:41 and 62:38. The inclusion of straw in the diet resulted in an additional intake of NDF (+ 1110 g/day), which accounted for 90% of the additional intake of OM, whereas additional intakes of the other nutrients were minor Milk yield and composition did not differ among treatments. The inclusion of straw in the diet did not affect the milk levels of t 10-18:1, 18:2n-6, c9-16:1, c9-18:1, c11-18:1, 6:0, 8:0, 20:4 and 20:5. It decreased the milk levels of c9,t11-CLA (2.13% v. 3.03% of fatty acids (FA) reported, P < 0.001), t11-18:1 (4.99% v. 7.10% of FA reported, P < 0.001), 18:3n-3, t9-16:1 and t9-18:1, while it increased the milk levels of 6:0-14:0 (20.90% v. 19.69% of FA reported, P < 0.01), 16:0 (26.55% v. 25.25% of FA reported, P < 0.01), 18:0 (13.54% v. 12.59% of FA reported, P < 0.001), 17:0, 20:0 and 22:5. Regarding the ratio between FA, the inclusion of straw increased the 18:0/total C18 FA ratio (37.74% v. 32.07%, P < 0.001), whereas it decreased the total trans-C18 FA/total C18 FA ratio (15.46% v. 20.34%, P < 0.001), the t11-18:1/total C18 FA ratio (13.70% v. 17.95%, P < 0.01) and the c9,t11-CLA/total C18 FA ratio (5.82% v. 7.64%, P < 0.001). We conclude from this experiment that even a modest increase in SV to a diet supplemented with extruded linseed, yet already providing enough SV, alters the rumen lipid metabolism and, hence, CLA levels in milk fat

The tum- antigens P91A and P198 derive from proteins located in the cytosolic compartment of cells.

To characterize the proteins P91Ap and P198p, of which mutants generate the tum- antigens P91A and P198, respectively, rabbit antisera were raised with ovalbumin-coupled synthetic peptides that correspond to their respective C terminus. In immunoadsorption tests using immobilized protein A the antisera recognized the translation products synthesized by rabbit reticulocyte lysates programmed with the SP6 polymerase transcripts of the P91A and P198 cDNA. The presence of the two proteins was demonstrated by SDS-PAGE and immunoblotting in all the mouse cells and organs examined. P91Ap is a constituent of the cytosol; despite a remarkable homology to the Drosophila diphenol oxidase DOX-A2, it separates from murine catechol oxidase activity in rate zonal sedimentation analysis. P198p is a ribosomal constituent, or a factor firmly linked to both the free and membrane-bound ribosomes. These subcellular localizations strengthen other evidence that the antigens presented to T lymphocytes by class I products of the major histocompatibility complex derive from proteins of the cytosol, or in direct contact with it