Purification and characterization of maize ribulose-1,5-bisphosphate carboxylase

The ribulose-1,5-bisphosphate carboxylase/oxygenase purified from maize (a C₄ monocot) to homogeneity has a MW of532 000 and sedimentation coeffici     

Blocking of human T lymphocyte functions by anti-Leu-2 and anti-Leu-3 antibodies: differential inhibition of proliferation and suppression

We have shown previously that monoclonal antibodies to the Leu-2 and Leu-3 T cell antigens block the response of their respective subsets in allogeneic MLR. The present study was an effort to explore the mechanism of inhibition and to determine if anti-Leu-2 and anti-Leu-3 antibodies affect the responses to stimuli in addition to alloantigens. Our results indicate that antibodies to Leu-2 and Leu-3 have profound inhibitory effects on proliferation by their respective T cell subsets responding to a variety of stimuli, including specific soluble antigens and alloantigen. This effect was characterized by the following features: a) For optimal inhibition of proliferation, antibody must be present at the onset of antigenic stimulation. b) Inhibition is augmented by increasing the concentration of antibody or decreasing the concentration of antigen. c) Fab fragments of both anti-Leu-2a and anti-Leu-3a antibodies also block proliferation. In addition to their effects on T cell proliferation, anti-Leu-3 antibody blocked T cell-dependent lg synthesis induced in MLR, and anti-Leu-2 antibody prevented the induction, in vitro, of Leu-2+3- suppressor cells of lg synthesis. Taken together, these results suggest that antibodies to antigenic determinants on the Leu-2 and Leu-3 molecules competitively block segments of these structures that bind to alloantigen or nominal antigen. On the other hand, anti-Leu-2a antibody failed to block suppression of the MLR by in vivo activated, antigen-specific Leu-2+3- suppressor cells, which suggests that the Leu-2a epitope does not transmit antigen-specific signals from these differentiated suppressor T cells.     

Analysis of a defect in the H-2 genes of SV40 transformed C3H fibroblasts that do not express H-2Kk

C3H fibroblasts transformed in vitro with SV40 were adapted to in vivo growth. Several clones were isolated from a single, highly oncogenic tumor and those that displayed oncogenic potential also no longer expressed the H-2Kk molecule. Using the technique of Southern blot hybridization, the H-2 genes and integrated SV40 sequences present in the genomic DNA of several of these clones have been examined and compared with both the parent line and normal liver genomic DNA from C3H mice. All H-2Kk negative clones had altered H-2 genes that appeared as a gain and, depending on the restriction endonuclease, loss of hybridizing fragments compared to normal C3H DNA. A 5.5-kb fragment missing from the Sstl digests of the H-2Kk negative variants was mapped to the H-2Kk region of the major histocompatability complex with the use of congenic mice. This provided direct evidence that a mutation had occurred in the H-2Kk region. The integrated SV40 sequences were similar to those already seen in other SV40 transformed cells and not closely linked to any of the H-2 genes. There was no indication that the H-2 mutation was caused by integration of SV40.     

The effect of dexamethasone on pyruvate kinase activity in primary cultures of hepatocytes

Pyruvate kinase activity in primary cultures of hepatocytes isolated from a normal rat was maintained at a constant level similar to that found in vivo (14.0 +/- 2.8 units per mg of DNA) for over 6 days when both dexamethasone and insulin were included in the medium. Yet the pyruvate kinase activity decreased 50% when the cells were cultured for 2 days and 4 days, respectively, in the presence of either dexamethasone or insulin alone. A brief, 10 min incubation of hepatocytes in the presence of dexamethasone was sufficient to maintain the enzyme activity of cells subsequently cultured for 4 days in the presence of insulin. The optimal dexamethasone concentration was 1 microM. Three other glucocorticoids were able to maintain the pyruvate kinase activity in cells cultured in medium containing insulin. The presence of the protein synthesis inhibitors, actinomycin D or cyclohexamide in cells cultured in the presence of dexamethasone and insulin resulted in a 25% decrease in the pyruvate kinase activity. Therefore, it is suggested that the synergistic effect of glucocorticoids and insulin to maintain pyruvate kinase activity in primary cultures of hepatocytes is dependent upon the ability of these cells to maintain protein synthesis.     

Comparative effectiveness of lymphotoxin anticarcinogenic and tumor cell growth-inhibitory activities

Prevention of ultraviolet radiation- or chemical carcinogen-induced morphologic transformation and inhibition of tumor-producing transformed cell growth by lymphotoxin and by normal spleen leukocytes were quantitatively compared to define the antineoplastic activity spectra of these natural immune mediators. When Syrian golden hamster embryo cells seeded for colony formation in culture dishes were treated simultaneously with carcinogen and lymphotoxin, the number of morphologically transformed cell colonies was irreversibly reduced by 50% in the presence of 6 units of lymphotoxin/ml. Lymphotoxin inhibition of tumor cell growth, however, was reversible and 50% reduction in tumor cell growth in three transformed lines required 124, 330, and 477 units/ml. Thus, the anticarcinogenic activity of lymphotoxin can be 20-fold or more greater than its tumor growth-inhibitory activity. Similarly spleen leukocytes also were more effective as an anticarcinogen than as an inhibitor of tumor cell growth, consistent with previous observations that naturally occurring spleen leukocyte antineoplastic activity may result from lymphotoxin secretion.     

The promotion of collagen polymerization by lanthanide and calcium ions.

Ca2+ (1-5 mM) and lanthanide (20-250 microM) ions enhance the rate of polymerization of purified calf skin collagen (1.5 mg/ml) at pH 7.0 in the presence of 30mM-Tris/HCl and 0.2 M-NaCl. Both the nucleation phase and the growth phase of polymerization are accelerated. The activation energy of the growth phase, 239.3 +/- 24.3 kJ/mol (57.2 +/- 5.8 kcal/mol), is decreased to 145.6 +/- 9.6 kJ/mol (34.8 +/- 2.3 kcal/mol) by 5 mM-Ca2+ and to 75.3 +/- 4.6 kJ/mol (18.0 +/- 1.1 kcal/mol) by 25 microM-Sm3+. In contrast, the activation energy of the nucleation phase, 191.6 +/- 23.4 kJ/mol (45.8 +/- 5.6 kcal/mol), is only slightly decreased by Ca2+ or Sm3+. Collagen fibrils formed in the presence of Sm3+ are thinner than control fibrils, and more thermoresistant.     

Degradation of transplanted mitochondrial proteins by hepatocyte monolayers.

Reductively [3H]methylated rat mitochondria and mitochondrial-outer-membrane vesicles and mitochondrial-outer-membrane vesicles where monoamine oxidase is irreversibly labelled by [3H]pargyline have been transplanted into hepatocytes by poly(ethylene glycol)-mediated organelle or organelle-vesicle cell fusion. During subsequent culture of hepatocyte monolayers for 4-5 days, under conditions which mimic endogenous catabolic rates in vivo the transplanted organelle proteins retain their degradation characteristics observed in vivo (e.g. mitochondria: average t 1/2 72.5 h; monoamine oxidase: t1/2 55 h). In all cases protein degradation with first-order kinetics is only observed after an initial lag period (i.e. 24-30 h after fusion). Transplantation of fluorescein-conjugated organelles showed that the fluorescent material is rapidly internalized (average t1/2 1-6 h) and uniformly distributed in the cytoplasm. During a subsequent 18-24 h period (which corresponds to the lag period for intracellular destruction of transplanted mitochondrial material) the transplanted material is translocated to assume a perinuclear distribution. The destruction of transplanted mitochondrial proteins is compared with endogenous mitoribosomally synthesized proteins (average t1/2 52.5 h). Percoll fractionation of cell homogenates containing transplanted mitochondrial outer membranes where the enzyme monoamine oxidase is irreversibly labelled with [3H]pargyline shows a distribution of enzyme similar to lysosomal acid phosphatase. After transplantation of reductively methylated 3H-labelled mitochondrial-outer-membrane vesicles the cells were treated with leupeptin to alter lysosomal density. This treatment leads to the predominant association of acid phosphatase with dense structures, whereas the 3H-labelled transplanted material predominantly does not change density. Therefore transplanted mitochondrial-outer-membrane proteins are found in intracellular vesicular structures from which the proteins are donated for destruction, at least in part, by a lysosomal mechanism.     

Leg chaetotaxy and the classification of the Uropodina (Acari: Mesostigmata)

Details of the segmental chaetotaxy of the legs of 47 species of Uropodina are given. On the basis of the ontogenetic development of the chaetotaxy, the Uropodina may be divided into two groups which coincide with the concepts of the Lower (Polyaspidoidea) and Higher (Uropodoidea) Uropodina of certain authors. Chaetotactic criteria do not support the classification of the Polyaspidini and Trachyuropodini sensu Hirschmann and Z-Nicol within the Oplitinae Hirschmann & Z-Nicol, the Diarthrophallini within the Uroactiniinae Hirschmann & Z-Nicol or the genus Trachytes within the Uropodini.
A critical appraisal is given of the classification of the Uropodidae (based on "Gangmerk-male") by Hirschmann and Z-Nicol.     

Complementary Functioning of Nitrogenase Components from a Blue-Green Alga and a Photosynthetic Bacterium

Nitrogenases from Anabaena cylindrica and Chloropseudomonas ethylicum were partially purified into two components. A. cylindrica fraction I protein complemented fraction II protein from C. ethylicum. However, the reciprocal cross between C. ethylicum fraction I and A. cylindrica fraction II was negative.