Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Codon Substitution in Evolution and the "Saturation" of Synonymous Changes

A mathematical model for codon substitution is presented, taking into account unequal mutation rates among different nucleotides and purifying selection. This model is constructed by using a 61 X 61 transition probability matrix for the 61 nonterminating codons. Under this model, a computer simulation is conducted to study the numbers of silent (synonymous) and amino acid-altering (nonsynonymous) nucleotide substitutions when the underlying mutation rates among the four kinds of nucleotides are not equal. It is assumed that the substitution rates are constant over evolutionary time, the codon frequencies being in equilibrium, and, thus, the numbers of synonymous and nonsynonymous substitutions both increase linearly with evolutionary time. It is shown that, when the mutation rates are not equal, the estimate of synonymous substitutions obtained by F. Perler, A. Efstratiadis, P. Lomedico, W. Gilbert, R. Kolodner and J. Dodgson's "Percent Corrected Divergence" method increases nonlinearly, although the true number of synonymous substitutions increases linearly. It is, therefore, possible that the "saturation" of synonymous substitutions observed by Perler et al. is due to the inefficiency of their method to detect all synonymous substitutions.     

Changes in Microtubules in Onion Leaf Sheath Cells during Bulb Development

Cortical microtubules are oriented transversely to the cellaxis in leaf sheath cells of onion plants (Allium cepa L. cv.Osaka-Okute) that have not started bulb formation. As the bulbdevelops and the leaf sheath cells swell, the microtubules becomedisoriented and scattered and finally disappear. The microtubuleinhibitors colchicine and cremart [O-ethyl O-(3-methyl-6-nitrophenyl)N-sec-butylphosphorothioamidate]cause swelling of leaf sheath cells and make the basal partof the plant bulbous. The cortical microtubules may have animportant role in regulating bulb development in onion plants. (Received August 21, 1982; Accepted December 6, 1982)     

Clinical Use of a New Mitral Disc Valve

A disc valve of new design was used successfully for the replacement of the mitral valve in patients with rheumatic mitral valve disease. This valve would appear to have the following advantages over the mitral ball valve prosthesis:• Lower left atrial pressure after replacement.• Elimination of the hazard of left ventricular outflow tract obstruction with mitral valve replacement.• Decreased incidence of thromboembolization.• Abolition of possibility of ventricular septal irritation.Despite the better outlook for this valve compared with the ball valve for mitral valve substitution, the mitral valve should always be repaired whenever feasible. Repair is possible in the majority of patients.     

Establishment of five human myeloma cell lines

Summary Five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.     

Fully differentiated Xenopus eye fragments regenerate to form pattern-duplicated visuo-tectal projections

In order to determine if differentiated Xenopus retina is capable of undergoing regeneration and duplicative pattern formation, we devised a new surgical technique for removal of the temporal two-thirds of the retina. In a series of progressively older larval eyes starting with late tailbud stage embryos (stage 38) and extending to limb-bud stage tadpoles (stage 48), nasal one-third-sized eye fragments successfully regenerated to form nearly normal sized eyes over 75% of the time. Histological preparations showed that early wound healing involved the formation of a neuroepithelium at the ventro-temporal region of the fragment. The pigmented retinal epithelium and associated retinal tissue appeared to be involved in this process. Animals from each stage were reared through metamorphosis and electrophysiologic techniques were employed to determine visuo-tectal projections. Seventy percent of stage 38 animals showed evidence of pattern-duplicated projections. Ninety percent of their responding tectal points showed duplicate innervation from two retinal regions. Older animals (stages 44 to 48) showed less duplication. Only 52% of their responding tectal points duplicated (P less than 0.001). Thus, fully differentiated Xenopus retina can undergo regeneration and duplicative pattern formation similar to that shown by embryonic retinal tissue.