Ontogeny of diversity in the receptor repertoire of murine cytotoxic lymphocytes. I. Comparison of recognition patterns of activated T cells of B10.D2 and B10.BR mice of different ages and analysis of changes in F1 hybrid and bone marrow radiation chimeras

Cytotoxic T lymphocyte precursors (CTLp) from B10.D₂, B10.BR, and (B10.D₂ × B10.BR)F₁ mice of different ages have been activated by irradiated “wild-type” H2K^b antigens (from B10.A(3R) mice) under limiting dilution conditions such that cytotoxic cells in responder wells represent the progeny of a single CTLp. After expansion in the presence of IL₂ and irradiated C57B1/6Kha spleen cells the contents of each well were divided into equal aliquots and tested for lysis with a panel of selected H2K^b mutant targets. As has been observed for the murine B-cell repertoire, there seems to be substantially more homogeneity in the neonatal allo-T-cell repertoire than in the adult mouse. Furthermore, while the adult F₁ repertoire is markedly distinct from that expressed by either parental T-lymphocyte pool, the neonatal repertoire apparently reflects a relatively accurate composite of each parental population, codominantly expressed. These data, combined with studies of adult bone marrow radiation chimeras, suggest that during development of the adult T-lymphocyte repertoire from the initially expressed restricted (germ-line?) recognition specificities, somatic diversification driven by environmental (MHC?) antigenic determinants occurs. In addition to this ontogenetic development, during senescence another “regulation” of the repertoire becomes apparent, and once more the heterogeneity of recognition specificities is diminished. Nevertheless, the homogeneity seen in aged mice does not represent a simple return to the expression of the limited number of allo-specificities encoded in the neonatal repertoire.     

Comparative study of kinetoplast DNA in culture, blood and intracellular forms of Trypanosoma cruzi.

A comparative study has been made on the kinetoplast DNA (kDNA) from I in culture epimastigote, blood trypomastigote and intracellular amastigote stages of Trypanosoma cruzi. The basic properties of the kDNA in all 3 forms were identical. Thus the DNA was in the form of networks of density 1.698-9 g/cm3 and with sedimentation coefficients (S20w) of approximately 5500, the networks being composed of large complexes of minicircular and apparently linear molecules, the former having contour lengths of 0.45 MICROMETer. Several differences were noted. The ultrastructural arrangement of the kDNA in the kinetoplast of the blood stage consisted of three to four double rows of DNA as compared to one double layered row in the other two stages. There was proportionately more kDNA in the blood stages, suggesting that, since the networks have apparently the same size (see above), more than one is present. DNA loops situated at the periphery of the kDNA networks were observed in higher proportion in blood and intracellular forms. Dimeric and oligomeric circles were present in the kDNA of the blood and intracellular stages in much greater proportion than in culture epimastigote stages. Few large circular molecules, heterogeneous in size, were also observed in intracellular blood stages. There were some differences, mainly quantitative, in the gel electrophoresis patterns after endonuclease digestion.     

Cold resistance of the brain during hibernation. Temperature sensitivity of the partial reactions of the Na+, K+-ATPase.

The regulatory effect of calcium added in vitro on 25-hydroxycholecalciferol metabolism was studied in kidney mitochondria and in renal tubules from vitamin D-deficient chicks. The addition of calcium (0.05 – 0.2 mm) to mitochondrial suspensions prepared with calcium-chelating agents caused a marked and dose-related stimulation of 1-hydroxylation. A sharp decline in the activity was induced by higher concentrations of calcium (0.3 – 0.7 mm). A similar but less striking biphasic effect of calcium on 1-hydroxylation was observed in mitochondria prepared in the absence of calcium chelating agents. The effect of calcium was not a consequence of accelerated mitochondrial translocation of either exogenous NADP or Mg²⁺ but was related to mitochondrial calcium content. The addition of inhibitors of the calcium uptake, i.e., LaCl₃ or ruthenium red, or a calcium ionophore (A 23187) significantly inhibited the calcium-induced stimulation of the 1-hydroxylation reaction. Similar calcium effects were also observed in renal tubules isolated from intact, but not from parathyroidectomized, vitamin D-deficient chicks. These data strongly suggest that mitochondrial calcium plays an important role in the regulation of 1-hydroxylase activity in kidney.     

Analysis of subpopulations of glass-adherent mouse skin cells controlling resistance/susceptibility to infection with Leishmania tropica,and correlation with the development of independent proliferative signals to Lyt-1+/Lyt-2+ T lymphocytes

A comparison has been made between the course of Leishmania tropica infection of BALB/ c, CBA, and (BALB/c × CBA)F₁ mice in vivo and the growth of the parasite in isolated adherent skin cells in vitro. The susceptible phenotype of the BALB/c mouse was reflected in an innate susceptibility of a discrete subpopulation of adherent skin cells to permit extensive and prolonged growth and replication of the parasite in tissue culture. When cells infected in culture were used to stimulate proliferation of immune lymphocytes from “cured” mice, the skin cells of susceptible BALB/c mice were deficient in their ability to induce proliferation of lymphocytes of BALB/c, CBA, or BCF₁ origin (all immunized in the appropriate bone marrow reconstituted irradiated BCF₁ hosts). In contrast, these skin cells were able to induce proliferation of immune lymphocytes if the L. tropica antigen source used was a soluble excreted extract (EF), rather than that produced by a live parasite infection. Stimulation of naive lymphocytes using an infected adherent skin cell population from BALB/c mice was found to produce a cell population(s) (Thy-1.2⁺, Lyt-2⁺ and including some Lyt-1⁺ cells) able to inhibit subsequent sensitization of normal BCF₁ lymph node cells by L. tropica antigens. The susceptibility of the BALB/c mouse in vivo thus may be attributable to the early contact of T-lymphocyte subsets in BALB/c mice with the high-antigen load maintained in this discrete skin cell population. These particular skin cells were also found to express low levels of Ia antigens.     

The acquisition of resistance to ecdysteroids in cultured Drosophila cells

Drosophila melanogaster K_c cells become refractory toward ecdysteroids after 4 days of exposure to the molting hormone, 20-OH-ecdysone. Associated with the appearance of hormonal insensitivity is a loss of ecdysteroid receptors. Hormone-resistant cells maintain a low level of receptor that is indistinguishable from that of responsive, hormonally naive cells. After extended periods in culture, ecdysteroid receptor content in previously exposed cells returns to that of naive control cells. The reappearance of receptor is coincident with the resumption of hormonally induced growth inhibition.     

Macrophage heterogeneity and Ir-gene control as factors involved in the immune response of guinea pigs to infection with Leishmania enrietti

Macrophages from strains 2/N, 13/N, and (2 × 13)F₁, guinea pigs have been fractionated by velocity sedimentation and the various subpopulations used as target cells for infection by Leishmania enrietti. The data presented show: (i) that the ability of infected macrophages to support the subsequent growth and replication of the parasite varies according to the cell subpopulation examined, (ii) that different subpopulations differ in their capacity to promote lymphocyte proliferation from lymph node cells of a guinea pig which have recovered from a primary lesion, (iii) that lymphocyte proliferation depends upon presentation of leishmanial antigens in the context of products of the original I-region-coded genes present during the initial (in vivo) infection and, (iv) that in immune animals, changes occur in terms of the ability of the macrophages to promote lymphocyte proliferation, but not apparently in terms of their ability to support parasite growth in vitro.     

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.     

A new approach to the characterization of the B and A forms of DNA by I.R. spectroscopy

The RNA conformational changes of B, A and C forms are reflected in the infrared absorption spectra in the region of 800 cm^?1 to 900 cm^?1 and allow one to investigate unoriented samples. The transition to the A form is characterized by the appearence of bands at about 870 cm^?1 and at 813 cm^?1 whereas the B and the C forms exhibit a band at 837 cm^?1, these bands undoubtedly arise from phosphate diester stretching vibrations and yield information about backbone conformation. The presence of these infrared bands provides a criterion for testing the simultaneous presence of two coexisting forms of DNA. It represents a useful method for structural studies of nucleic acid complexes such as protein-DNA for which it is difficult to obtain orientation.     

Excision repair of ultraviolet radiation damage in toluene treated Escherichia coli

ATP independent excision repair of UV damage has been studied in $\text{E. coli}$ made permeable to nucleotides by treatment with toluene. In using this system, separation of the first step from the subsequent steps in the repair process is achieved. It was found that completion of repair is observed only in strains that have normal levels of DNA polymerase I.     

Cross-linking of fibronectin to sulfated proteoglycans at the cell surface.

Fibronectin is a major surface protein of normal animal cells but is absent from many transformed cells. Addition of fibronectin to transformed cells causes increased cell substrate adhesion and changes in the morphology and cytoskeleton of the cells. We have coupled fibronectin to photoactivable chemical cross-linkers and have added it to cells to identify those molecules to which it binds. In this way, fibronectin can be cross-linked to sulfated proteoglycans at the cell surface. The cross-linking is specific for fibronectin. The fibronectin-proteoglycan complex is sensitive to chondroitinase ABC and AC and to trypsin. Addition of fibronectin also affects binding of hyaluronic acid to the cells. These results suggest that fibronectin interacts with proteoglycans at the cell surface. The existence of such interactions may have implications for the role of fibronectin and proteoglycans in cell adhesion.     

Resistance and susceptibility to infection in inbred murine strains. II. Variations in the effect of treatment with thymosin fraction 5 on the release of lymphokines in vivo

Of nine inbred murine strains sensitized intravenously with killed lyophilized Candida albicans and challenged 3 weeks later with a C. albicans filtrate, four strains were low responders and five were high responders in the in vivo release of migration inhibitory factor (MIF) and gamma interferon (IFN-gamma). An identical distribution of high- and low-responder strains occurred in response to sensitization with Mycobacterium bovis BCG and subsequent challenge with old tuberculin. Treatment of the murine strains with thymosin fraction 5 prior to sensitization and challenge had different effects: (a) the high-responder strains had a decrease in their release in vivo of the two lymphokines; (b) three of five of the low-responder strains had a striking increase in the in vivo release of MIF and IFN-gamma; and (c) one low-responder strain did not have its response altered. A parallelism existed between the capacity of a murine strain to release the two lymphokines in vivo on stimulation with C. albicans antigens and the capacity of that strain to resist intravenous infection with living C. albicans.     

Rotational diffusion of Escherichia coli ribosomes. II. - Ribosome attachment to polyurydilic acid.

Ribosome attachment to poly(U) has been studied by following the rotational diffusion of polyribosomes in solution. On the average, 13-17 and 50 nucleotides are found to be associated with 30S and 70S ribosome respectively. For an equal length of poly(U), the number of particles in a 30S polysome is four times that in a 70S polysome. The results are consistent with a structure of the polysome in which individual ribosomes are in close contact.