Mouse targets undergo double-strand DNA fragmentation when exposed to syngeneic or xenogeneic LAK cells whereas human targets undergo single-strand breaks

A number of recent studies have shown that mouse target cells (TC) of hematopoietic origin, when exposed to cytotoxic lymphocytes, undergo double-stranded DNA fragmentation. The cause and relevance of the fragmentation remain controversial. In this study we generated a number of mouse (M-LAK) and human LAK (H-LAK) cells and exposed them to a variety of mouse and human TC. YAC and SP/2, 2 mouse TC underwent rapid and extensive fragmentation when lysed by either human or mouse LAK whereas K562 and Daudi, 2 human TC, under the same conditions did not. All 4 TC, however, were killed quite efficiently. Next we labeled TC with 125I-deoxyuridine, exposed them to LAK cells for up to 18 h and loaded the LAK:TC mixtures over an alkaline linear sucrose gradient. After lysing the cells with a lysis buffer containing Triton X-100 we showed that K562 that had been in contact with LAK cells for more than 1 h exhibited single-strand nicks. However, whereas double-strand fragmentation preceded chromium release (lytic activity), the appearance of single-strand nicks did not. Finally, protein synthesis was not required for either type of fragmentation. In summary, we have demonstrated that: (1) the ability to undergo DNA fragmentation is a property of the TC rather than the effector cells that mediated their death, and (2) K562 and Daudi, 2 human TC, undergo single-strand nicks when lysed by LAK cells whereas SP/2 and YAC, 2 mouse TC undergo double-strand fragmentation when exposed to the same syngeneic or xenogeneic effector cells.     

Enantioselective synthesis of (R)- and (S)-2-methyl-[3,3,2-2H3] alanines

The synthesis of optically pure (R)- and (S)-2-methyl-[3,3,3-2H3] alanines of biological interest is described. The stereochemistry of the reaction of the lithio derivative of (R)-(-)-2,5-dimethoxy-3-benzyl-3-methyl-3,6-dihydropyrazine with alkyl and deuterated alkyl iodides is discussed. The configuration of the newly formed center of chirality in (R)- and (S)-2-methyl-[3,3,3-2H3] alanines is derived from 1H NMR.     

Mitotic Frequency Determinations and Micro-Photometric Feulgen Dye Measurements in Root Tips

Treating a Feulgen stained (10 min. acetic-alcohol fixation, 8 min. hydrolysis) onion root tip with 5% aqueous pectinase for 6 hours causes it to disintegrate on shaking in 1 ml. water into a suspension of stained cells, either individual or in small groups. Vicia faba and pea root tips require 15 minutes hydrolysis and 12 hours pectinase treatment. Absence of cell destruction allows absolute cell-number determination by Brown and Rickless' counting chamber technic. By centrif uging the cells down, resuspending them in 2 drops of a Karo syrup-phosphate buffer mixture (2 parts Karo to 1 part 0.5M, pH7 buffer) and mounting a small drop of the now concentrated suspension, a semipermanent slide containing a concentration of well-spread, randomized cells suitable for rapid mitotic frequency determination is obtained. Scoring about 1,000 cells as to nuclear stage gives a representative, workable statistical sample and takes only 20-30 minutes if interphases are recorded on a mechanical tally. Permanent slides can be prepared by carrying the intact, stained, pectinase-treated root through a water wash, 70%, 95%, and absolute alcohol, shaking and performing the centrifuge procedure with Diaphane in place of the Karo mixture. Although the pectinase treatment appears to introduce some error, the Diaphane mounts are adequate for conventional microphotometric Feulgen dye (DNA equivalent) determinations. The three dimensional character of the cells is retained in all preparations.