Occurrence of B-chromosomes in diploid Allium stracheyi Baker and their elimination in polyploids

Summary In a population of Allium stracheyi Baker (2n=14) growing in Darjeeling both diploids and polyploids occur. The diploids contain B-chromosomes varying from 2–10 in number. Polyploids are conspicuous by absence of B-chromosomes. These in diploids are found in the pollen mother cells and also in the pollen, and some are provided with subterminal constriction.Diploid individuals when brought from Darjeeling to Calcutta (i. e. from temperate to tropical regions) became polyploid within a month and the B-chromosomes were simultaneously lost. In order to confirm this unexpected result, the transfer experiment has been repeated thrice with fresh collections in each case and selection of diploid bulbs after cytological observation. In all cases the result has been the same. In rare cases one or two B-chromosomes were found in the polyploid cells which might represent intermediate steps of the disappearance.B-chromosomes in diploids possibly help the individual to compete with polyploids by enlarging the adaptive capacity.The sudden polyploidisation by transfer from the mountains to the plains might have been the result of a shock due to the temperature difference. The high temperature may be deleterious for the reproduction of B-chromosomes, and their degeneration products possibly contribute to cytoplasmic changes and the spindle disturbances which effect polyploidisation.     

Methionine-oxidized horse heart cytochromec. III. Ascorbate reduction and the methionine-80-sulfur-iron linkage

The ascorbate reduction of the CT-cytochromes—two chemically generated forms of horse heart cytochrome c, F^III and F^II, with both methionines, 80 and 65, as methionine sulfoxides, no iron-sulfur linkage, and potentiometric and physiological oxidoreduction properties distinct from those of the native protein and one another (J. Pande et al., 1987)—has been investigated using a stopped-flow technique. The reaction was monitored at 550 nm, and studies were conducted in 10 mM phosphate +0.17 M NaCl buffer,pH 7.4. Both CT-cytochromes are reduced by triphasic profiles, a faster and an intermediate ascorbate-dependent reaction and a slow, ascorbate-independent process. Both CT-cytochromes contain three molecular forms in slow equilibrium, two reducing directly by reaction with ascorbate and a third through conversion to one of the reducible forms. Like the reaction of the native protein, the ascorbate dependence of both the rapid and the intermediate process is nonlinear, approaching saturation values at high concentrations. The ascorbate profiles of the pseudo-first-order reduction constants are typical of the model for the reduction reaction of the unmodified protein, binding followed by a first-order reduction reaction (Myer et al., 1980; Myer and Kumar, 1984), but with distinct kinetic parameters, the first-order reduction constants and the protein-ascorbate stability constants. It has been concluded that the functional-conformational differences between the two CT-cytochromes are not operational to any significant extent in the reduction reaction with ascorbate. The methionine-80-sulfur-iron linkage of the protein is not a crucial requirement for the ascorbate reduction of the protein. The mechanism of the reaction in the main is also insensitive to the replacement of Met-80-S from heme coordination and/or the associated conformational-oxidoreduction properties of the protein. Of the two aspects of the reaction, the efficiency of the electron-transfer reaction and the stability of the ascorbate dianion-protein complex, the former is dependent on the integrity of the structural-conformational state of the molecule.     

Selection and characterization of an L-thiazolidine-4-carboxylic acid resistant callus culture of Vigna radiata (L.) Wilczek var. radiata

Callus cultures were established from seedling root tips of mungbean (Vigna radiata (L.) Wilczek var. radiata) cv. K 851. The growing calli were exposed to increasing concentrations of thioproline — an analog of proline, in the medium. A concentration of 3.0 mM thioproline completely inhibited the growth of the cells. However, after 25 days incubation 5 cell clones were obtained which could grow on this concentration of thioproline. Out of them one vigorously growing cell clone was further characterized. This selected clone contained higher endogenous levels of free proline (5 fold) and K⁺ (1.5 fold) and exhibited elevated tolerance, not only to thioproline but also to exogenously applied NaCl in the growth medium, as compared to the normal sensitive callus cells. Higher endogenous levels of free proline and K⁺ appear to impart dual resistance to thioproline and NaCl to the selected cell strain.     

The molecular cloning of the complementary deoxyribonucleic acid for bovine vitamin D-dependent calcium-binding protein: structure of the full-length protein and evidence for homologies with other calcium-binding proteins of the troponin-C superfamily of proteins

We have cloned the cDNA for bovine intestinal vitamin D-dependent calcium-binding protein and, based on the sequence of the DNA, have deduced the structure of the full-length protein. The sequence of the cDNA clone predicts a protein comprised of 78 amino acids with a mol wt of 8788. The mRNA for the protein in bovine duodenum is about 500-600 bases in length. The protein sequence of bovine intestinal calcium-binding protein is 87% homologous with the sequence of porcine intestinal vitamin D-dependent calcium-binding protein and 81% homologous with the sequence of rat intestinal vitamin D-dependent calcium-binding protein. Hydrophilicity plots of the proteins noted above show that despite differences in amino acid sequence the proteins have similar patterns. In addition, the predicted secondary structure of the proteins is similar. Bovine intestinal calcium-binding protein shows 48.6% homology with the alpha-chain and 38.2% homology with the beta-chain of bovine S-100 protein and a similar high degree of homology with the beta-chain of human S-100 protein. The protein also demonstrates 36-43% homology with parvalbumin alpha and beta from various species and with troponin-C. There is some homology with the 28K vitamin D-dependent calcium-binding proteins. Vitamin D-dependent bovine intestinal calcium-binding protein is closely related to other mammalian intestinal calcium-binding proteins and to the S-100 proteins, parvalbumins, and troponin-C.     

Pemphigoid, pemphigus and desmoplakin as antigenic markers of differentiation in normal and tumorigenic mouse keratinocyte lines

Abstract The expression of differentiation stages in a murine epidermal cell transformation model has been investigated as a basis for studies of chemically-induced differentiation. Antibodies in sera of patients with the autoimmune diseases bullous pemphigoid and pemphigus vulgaris exhibit specific reactivity to antigenic determinants of basal and spinous cells, respectively, in sections of mouse and human epidermis. In addition, spinous cells in epidermis are reactive with a mouse monoclonal antibody to desmoplakin, a desmosomal component immunologically distinct from pemphigus. These antibodies were used to identify and attempt to quantify keratinocyte subpopulations in culture based on differentiation stage. Epidermal cell lines were cultured under conditions which favour proliferation (0.02 to 0.04 mm extracellular Ca²⁺, i.e. low Ca²⁺ conditions) or differentiation (0.1 mM to 1.4 mM Ca²⁺), as previously shown using primary cultures of mouse keratinocytes. Two independently-derived normal keratinocyte lines demonstrated Ca²⁺-dependent reactivity with pemphigoid and pemphigus antiserum, like that which has been observed in primary cultures. Furthermore, a Ca²⁺ and time-dependent reactivity with the three antisera was also observed in a papilloma cell line (derived from one of the normal cell lines after treatment in vitro with 7,12-dimethylbenz[α]anthracene). Papilloma cells cultured under conditions of low extracellular Ca²⁺ were comprised of three subpopulations: cells reactive only with pemphigoid anti-serum, cells reactive with pemphigoid and desmoplakin antibody (intracellular location), and cells reactive only with desmoplakin antibody. However, like the normal cell lines, papilloma cells underwent a transition to predominantly a spinous cell population (i.e. reactive with pemphigus and desmoplakin antibody) in response to extracellular Ca²⁺. A slower loss of pemphigoid antibody reactivity was noted in papilloma cells, consistent with an abnormal regulation of differentiation. The attempt to characterize these dynamic transitions from basal to spinous cell subpopulations in culture was considered to be prerequisite for the use of the model to investigate differentiation-inducing agents in carcinoma therapy.     

Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.     

Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria

The malaria parasite, Plasmodium yoelii 17X, causes a self-limited, nonlethal infection characterized, in the blood stage, by preferential invasion of reticulocytes. Previous studies have suggested that immunity to the blood stage infection may be related to enhanced levels of class I MHC Ag on the parasitized reticulocyte surface and can be adoptively transferred to immunodeficient mice by immune CD8+ T cells in the absence of CD4+ T cells. To further examine the mechanisms of CD8+ T cell involvement in immunity to blood stage P. yoelii infection, we performed in vivo CD8 depletion and adoptive transfer experiments. Depletion of CD8+ T cells during primary blood stage infection in BALB/c mice did not diminish the ability of the mice to resolve their infections. Spleen cells from immune BALB/c and C57BL/10 mice were transferred to BALB/c-nu/nu and C57BL/10-nu/nu mice, respectively. The recipient mice were CD4 depleted in vivo to kill any transferred CD4+ T cells. The mice failed to control the infection. Populations of CD4-, CD8+ T cells were transferred from immune CBA/CaJ donors to in vivo CD4-depleted CBA/CaJ recipients. The mice were unable to control the infection. Although immune unfractionated spleen cells transferred rapid protection in all three mouse strains and immune CD4+ T cells transferred immunity in the two mouse strains studied, CD8+ T cells by themselves were neither protective nor did they enhance immunity.