Studies on the transport mechanism of 5-methyltetrahydrofolic acid in freshly isolated hepatocytes: effect of ethanol.

The effect of ethanol on the transport of 5-methyltetrahydrofolate in freshly isolated hepatocytes in vitro resulted in about a 30% increase in accumulation of substrate. It was shown that this was not due to differences in metabolism, nor to an inhibition of efflux. Preincubation with 40 mm ethanol for 45 min resulted in a significantly increased rate of entry of 5-methyltetrahydrofolate into the cells. The stimulatory effect was specific to 5-methyltetrahydrofolate since ethanol inhibited uptake of folate and methotrexate. The increased uptake was due to metabolism of ethanol as shown by studies with pyrazole. Also, the n-alkanols, propanol through pentanol, and sorbitol but not methanol were stimulatory. Anaerobiosis and sodium azide stimulated uptake of 5-methyl-tetrahydrofolate but were inhibitory to methotrexate uptake. These data, taken together, suggest that the ethanol effect is due to increased entry of 5-CH₃-H₄PteGlu into the cells possibly as the result of an increased cellular $\text{NADH}\text{NAD}$ ratio.     

Herpes-Simplex-virus-specific, H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2k) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected taraget cells expressing the Dd alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2Dk determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for Dd alloantigen could be positively selected on H-2d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing Dd alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

Translation of poly-A RNA from rat liver in vitro. Evidence for a high molecular weight subunit of tyrosine aminotransferase

Poly-A RNA extracted from the rat liver was translated in a cell-free wheat germ system and a rabbit reticulocyte lysate. The subunit of tryptophan pyrrolase precipitated by specific antiserum after synthesis in vitro has the same molecular weight as the corresponding subunit derived from the rat liver. With specific antiserum prepared against tyrosine aminotransferase, however, a radioactive protein from both the in vitro assays was precipitated with an about 5% higher molecular weight than the tyrosine aminotransferase subunit precipitated from rat liver. The immunological evidence and the comparison of the specific peptide patterns prepared by cyanogen bromide treatment showed that the in vitro product corresponds to tyrosine aminotransferase. Various concentrations of potassium or spermidine used in the wheat germ translation system did not alter the size of the enzyme subunit synthesized. The run of the tyrosine aminotransferase purified form the rat liver in the SDS-polyacrylamide gel electrophoresis was not influenced by treatment with Escherichia coli alkaline phosphatase. The possibility is discussed that the larger enzyme synthesized in vitro represents a precursor molecule which is cleaved proteolytically in vivo.     

Conformation of sequential and random lysine–phenylalanine copolypeptides

The sequential copolypeptides (Lys-Phe-Lys)_n and (Lys₂-Phe-Lys)_n and a series of related random copolypeptides were investigated with respect of their ability to adopt the α-helix or β-conformation. Conformational transitions were induced by increasing the pH or by addition of NaClO₄ or methanol and were observed by recording the CD spectra. In contrast to the respective alternating copolypeptide (Phe-Lys)_n with its strong tendency for the β-structure reported previously, (Lys-Phe-Lys)_n can adopt either secondary structure, whereas (Lys₂-Phe-Lys)_n strongly favors the α-helix. Together with the random copolypeptides, whose composition varied from 20 to 50 mol % phenylalanine and whose average molecular weights ranged from 10,000 to 90,000, the influence of the phenylalanine content and of the chain length on conformational stability and the rotatory strength of the respective secondary structures were elaborated.