The laser method for efficient introduction of foreign DNA into cultured cells

A minute hole upon a cultured cell, perforated with a finely focused laser beam, was found to repair itself within a short period of time. The procedure constitutes a new way of introducing exogenous gene materials dissolved in medium into cells. The 'laser-aided' DNA transfection is better than the existing methods because it allows the treatment of a large number of cells in a shorter time, and an improved success rate.     

Structural homology of lens crystallins. II. Homology expressed by correlation coefficients and hydropathy profiles

Bovine lens alpha A- and alpha B-crystallin polypeptides show extensive sequence homology with each other, but apparently none with beta Bp- and gamma 2-crystallin. Despite only 30% sequence homology, the latter two proteins are assumed to have a strong correspondence in tertiary structure, consisting of four structurally similar folding units of antiparallel beta-sheet. We have tested for internal structural repeats in all crystallins, and structural homology between crystallins, by comparing various physical properties of the amino acid residues, such as bulkiness and propensity to form beta-sheet and beta-turn structure. Two procedures used a combination of five physical parameters to calculate correlation coefficients. The 4-fold structural repeat in gamma 2-crystallin and the internal duplication in beta Bp-crystallin were readily detectable, as was also the strong structural homology between corresponding folding units in beta Bp- and gamma 2-crystallin. However, for alpha-crystallin polypeptides, no conclusive support was obtained for either a four-unit or a six-unit folding, the two models previously considered by us. The third procedure compared smoothened hydropathy plots, representing hydrophilic and hydrophobic regions along the polypeptide sequences. Hydropathy profiles were found to show strong correspondence, particularly between alpha B-crystallin and beta Bp-crystallin. These observations support a similar 4-fold folding pattern for all bovine crystallins. A possible role in subunit interactions of the N-terminal folding unit, which has hydrophobic surface characteristics in both alpha- and beta-crystallin polypeptides, is proposed.     

Synthesis of 5-deoxy-5-fluorosporaricin A

The structures of the O-specific side-chains in the lipopolysaccharides of Salmonella greenside, group Z, and Salmonella adelaide, group O, have been investigated. The former proved to be identical with that of Escherichia coli O 55. The latter, which was more extensively studied, was composed of repeating units having the structure

in which Col is colitose (3,6-dideoxy-l-xylo-hexose). This was also shown to be the biological repeating-unit. The same structure has been proposed for the O-antigen of E. coli O 111. The biological repeating-unit for the S. greenside O-antigen was also defined. The structural studies also confirmed that both lipopolysaccharides contain the hexose region typical for the Salmonella core.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity: I. Suppression of cutaneous delayed-type hypersensitivity by migration inhibitory factor

The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent column by using anti-guinea pig MIF antiserum. Suppression of cutaneous delayedtype hypersensitivity was achieved by intraperitoneal injection of the MIF fraction into the animals bearing macrophage-rich peritoneal exudates. Skin reactions induced by phytohemagglutinin (PHA) were also suppressed in these animals. Reactivity to skin reactive factor (SRF) was suppressed in these animals as well. The sera obtained from these animals exhibited the inhibitory activity against production of lymphokines from sensitized lymphocytes.     

Intracellular location of small circular DNA complexes in mammalian cell lines

For determination of the cellular location of small polydisperse circular DNA complexes, rat myoblastic L6 cells, HeLa cells, and mouse L cells were enucleated and processed by the micapress-adsorption method for electron microscopy (H. Yamagishi, T. Kunisada, and T. Tsuda, 1982, Plasmid8, 299–306). Small circular DNA complexes from intact cells showed a heterogeneous size distribution of from 0.1 to more than 2 μm with a mean contour length of 0.6 to 0.8 μm, like that of covalently closed circular DNAs. Cells contained 400 to 1200 copies. The size distribution in the cytoplasts was narrow and the number-average length was 0.3 to 0.4 μm, whereas that in L6 karyoplasts was wide and the average length was 0.9 μm. The longer circular complexes appeared to be absent from the cytoplasts. The origin and biological functions of these complexes are discussed in relation to the cellular locations of the complexes.     

Homology in protein sequences expressed by correlation coefficients

Internal homologies in an amino acid sequence of a protein and in amino acid sequences of two different proteins are examined, using correlation coefficients calculated from the sequences when residues are replaced by various quantitative properties of the amino acids such as hydrophobicity. To improve the signal-noise ratio the average correlation coefficient is used to detect homology because the correlation depends on the property considered. In this way, any sequence repetition in a protein and the extent of the similarity and difference among proteins can be estimated quantitatively. The procedure was applied first to the sequences of proteins which have been assumed on other grounds to contain some internal sequence repetitions, α-tropomyosin from rabbit skeletal muscle, calmodulin from bovine brain, troponin C from skeletal and cardiac muscle, and then to the sequences of calcium binding proteins, calmodulin, troponin C, and L2 light chain of myosin. The results show that α-tropomyosin has a markedly periodic sequence at intervals of multiples of seven residues throughout the whole sequence, and calmodulin and skeletal troponin C contain two homologous sequences, the homology of troponin C being weaker than that of calmodulin. Candidates for the calcium binding regions of both troponin C, calmodulin, and L2 light chain are the homologous parts having a high average correlation coefficient (about 0·5) with respect to the sequences of the CD and EF hand regions of carp parvalbumin. The procedure may be a useful method for searching for homologous segments in amino acid sequences.     

WHT/Ht mouse contains plasma cells in the thymus.

Numerous plasma cells and B cells were present in the thymuses of aged WHT/Ht mice. Plasma cells containing IgG and the follicle-like structure were present in the cortex and medulla, respectively. In these respects, WHT/Ht strain of mice is unique and interesting for studies on lymphocyte development.     

A saponin from callus tissue of Stauntonia hexaphylla.

A new 30-nortriterpenoid saponin was isolated from the callus tissues of Stauntonia hexaphylla. The structure of the saponin (tentatively named mubenoside A) was elucidated as 3 beta,20 alpha-dihydroxy-30-nor-olean-12-en-28-oic acid 3-O-[beta-D-xylopyranosyl(1----2)-alpha-L-arabinopyranosyl(1----3) ]-beta-D- glucopyranoside by means of spectral experiments.     

Effects of transforming growth factor-beta on normal clonal bone cell populations.

Although transforming growth factor-beta (TGF-beta) has been implicated in the local regulation of bone growth and remodelling, its specific effects on different subpopulations of bone cells have not been elucidated. Cells derived from bone are known to be heterogeneous and include both cells of different lineages and osteoblastic populations with different levels of expression of osteoblast-associated properties. Consequently, we have isolated clonal populations of bone cells to examine more precisely the effects of TGF-beta on individual subpopulations. Several clonal populations were isolated by limiting dilution from cells derived from 21-day-old fetal rat calvaria. Two of these clones, RCA 11 and RCB 2, were used here. While the two clones responded similarly to parathyroid hormone (PTH) and isoproterenol (ISP) with increases in intracellular cAMP, prostaglandin E2 (PGE2) elicited a 10-fold higher response in RCB 2 cells compared with RCA 11. RCB 2 cells expressed a 10-fold higher alkaline phosphatase activity compared with RCA 11. Both clones synthesized a variety of bone matrix associated proteins, but only RCA 11 synthesized SPP-1 (osteopontin) constitutively. TGF-beta stimulated growth of RCB 2 cells after 24 and 48 h of treatment, but had no effect on growth of RCA 11. TGF-beta supported anchorage-independent growth of RCB 2 cells, but not that of RCA 11. A 24-h exposure to TGF-beta decreased cAMP responsiveness to PTH and ISP slightly in both clones, but had no effect on PGE2 responses. Significant reductions in alkaline phosphatase activity were seen in both clones after 24- and 48-h treatments with TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)