The present investigation aimed to expand the knowledge of bioaccessibility of carotenoids, tocopherols, and tocotrienols from cereal products such as pasta. Because most of the published approaches assessing the bioaccessibility of lipophilic micronutrients dealt with fruits and vegetables, a prevalent in vitro digestion procedure was modified. Additionally, several digestion parameters were evaluated with regard to their impact on the bioaccessibility of carotenoids and vitamin E from pasta. Overall, the estimated values were highly dependent on the amount of bile extract present in the digestive medium and to a lesser extent on the simulated gastric pH and the incubation time with digestive enzymes. The bioaccessibility of carotenoids and vitamin E from durum wheat pasta was quite high (71 ± 5 and 70 ± 4%, respectively), whereas these micronutrients were considerably less accessible from pasta containing 10% eggs (57 ± 1 and 49 ± 5%, respectively).

Carotenoids and vitamin E in oils from the market -6 rapeseed and 6 sunflower oils, half of each cold pressed and refined -and in the oils of rape, sunflower, flax and safflower as well as the respective seeds and press cakes from a local oil mill were quantified by HPLC. Furthermore, a photometric determination of carotenoid content was tested and checked against the chromatographic method. In the cold pressed oils minor amounts of xanthophylls (all-E)-lutein and (all-E)-zeaxanthin were determined. With exception of traces of (all-E)-b-carotene in cold-pressed rapeseed oil this provitamin A active compound did not occur. Cold pressed rapeseed oils contained 0.5-1.5 mg total carotenoids/100 g which was manifold the content of the further oils. Vitamin E was found in all vegetable oils at plant-typic tocopherol patterns. The photometric determination of carotenoids resulted in significantly higher concentrations compared to the HPLC. This overestimation bases on the carotenoid pattern which was validated by comparison with known high-carotenoid materials, i.e. maize flour with an abundant amount of xanthophylls and carrots with an abundant amount of carotenes.

Reverse transcriptase (RT) isolated from Rous sarcoma virus (RSV) consists of heterodimeric RT␣␤, RT␣, and RT␤. The ␣ subunit (63 kDa) contains an N-terminal polymerase and a C-terminal RNase H domain. The N terminus of ␤ (95 kDa) corresponds to ␣ with the integrase domain attached to the C terminus (32 kDa). We have constructed baculoviruses expressing the genes for ␣ or ␤ or the entire pol (99 kDa). Infection of insect cells with recombinant virus yielded highly active and soluble RSV RT enzymes that could be purified to >90% homogeneity. HPLC gel filtration showed that ␣ is a dimeric enzyme that can be partially monomerized upon the addition of 45% Me 2 SO. DNA synthesis on DNA-DNA and DNA-RNA primer-templates in the presence of competitor substrates revealed that ␣␤ and ␤ as well as ␣ are processive polymerases. However, the affinity of ␤ and ␣␤ for primer-template substrates appears to be higher than that of ␣. All RSV enzymes investigated have the potential to displace RNA-RNA duplexes more efficiently than human immunodeficiency virus type 1 RT. Unlike human immunodeficiency virus type 1 RT, RSV RTs can catalyze an initial RNase H endonucleolytic cleavage of the RNA template but not a 3 3 5 directed processing activity.

The genes encoding the ␣ (63-kDa) and ␤ (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV ␣␤ and ␣Pol RTs within which one subunit was selectively mutated. When ␣␤ heterodimers contained the Asp 1813Asn mutation in their ␤ subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their ␣ subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in ␣␤ heterodimers mutated in the ␤ subunit but was lost when the mutation was present in the ␣ subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the ␣ subunit of the heterodimeric RSV RT ␣␤. Reverse transcriptase (RT) of Rous sarcoma virus (RSV) is a component of the Gag-Pol precursor protein.Pol is composed of polymerase, RNase H, and integrase domains and an additional short 4.1-kDa protein located at the C terminus of the protein. Pol is processed into polypeptides of various lengths by the viral protease; its ␣ polypeptide (63 kDa) contains the polymerase and RNase H domains, and its ␤ polypeptide (95 kDa) consists of the polymerase, RNase H, and integrase domains but lacks the C-terminal 4.1-kDa protein (1,7,8,17,28,30). In addition, the integrase domain (32 kDa) is also present and active as a separate enzyme (9, 30). Three forms of RT have been isolated from avian sarcoma and leukosis viruses (ASLV): ␣, ␤, and ␣␤, with the major form being the heterodimer (8,11,16). We have shown previously that the different forms of RSV RT can be expressed and purified from insect cells using the baculovirus expression system (37). In order to examine the subunit organization of RSV RT, the technique of subunit-selective mutagenesis was used (13,21) to analyze the effect of RSV RTs carrying a mutation in only one of the two subunits constituting the ␣␤ or ␣Pol heterodimer.It has been shown previously by biochemical and crystallographic data that heterodimeric human immunodeficiency virus type 1 (HIV-1) RT p66-p51 reveals an asymmetric subunit organization. The polymerase active site is present only in the larger p66 subunit of the heterodimer (13,14,19,36), while the p51 subunit, which is identical in sequence to p66 but lacks the RNase H domain, is not directly involved in catalysis (21). However, p51 has to fulfill an important stabilizing function since the monomeric subunits are inactive (26,27). Recent kinetic analyses we performed with homodimeric p51 RT from equine infectious anemia virus (EIAV) lacking the RNase H domain indicated an asymmetric subunit organization similar to that of heterodimeric p66-p51 RT, with the polymera...