Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.     

Metabolism of larger high density lipoproteins accumulating in some families of baboons fed a high cholesterol and high saturated fat diet

Progeny of certain baboon sires accumulate lipoproteins in high density lipoprotein-1 (HDL1) when challenged with a high cholesterol, high saturated fat diet. These studies were conducted to determine the apoprotein composition and metabolic fate of HDL1 in the plasma. HDL1 particles containing apoA-I with and without apoE were detected. The majority of particles, however, contained apoA-I without any detectable apoE. To determine the metabolic fate of HDL1 in plasma, HDL1 labeled with iodinated apoA-I from animals with high levels of HDL1 and iodinated apoA-I-labeled autologous HDL were coinjected into both high and low HDL1 animals. The data for the decay of radioactivity in HDL1 and HDL were analyzed by multicompartment modelling. The radioactivity from HDL1 was cleared from the plasma either via direct removal (9.1 +/- 4.7% in low and 21.7 +/- 8.3% in high HDL1 animals) or via its conversion to HDL. A large proportion of radioactivity from HDL1 was rapidly transferred to HDL directly or metabolized via an intermediate compartment. Most of the radioactivity from apoE-poor HDL1, however, was transferred to HDL. Both high and low HDL1 animals catabolized HDL1 and HDL similarly. Low HDL1 animals transferred HDL1 radioactivity to HDL much faster. No detectable radioactivity from HDL was transferred to HDL1. Thus, HDL1 that accumulates in high HDL1 animals is mainly a precursor for HDL. Our hypothesis is that this accumulation of HDL1 is due to the slower cholesteryl ester transfer from HDL to lower density lipoproteins, thus affecting reverse cholesterol transport in high HDL1 baboons.     

Physical and genetic mapping of the protein A gene in the chromosome of Staphylococcus aureus 8325-4

The gene coding for protein A (spa) has been mapped close to nov on the genetic map of the chromosome of Staphylococcus aureus 8325-4. A rapid mapping procedure has been developed which first allowed the region of the chromosome carrying the spa gene to be identified by blot +hybridization of large DNA fragments which had been separated by pulsed-field gel electrophoresis. Restriction endonuclease SmaI fragment G was shown to carry the spa gene. An insertion mutation in spa was constructed by in vitro insertion of a fragment of DNA expressing resistance to kanamycin and neomycin. A spa::Kan(r)Neo(r) mutation was isolated in S. aureus 8325-4 by allele replacement. This provided a selectable marker which allowed the spa gene to be mapped by transformation analysis.     

Two Enzymes, Both of Which Process Recombination Intermediates, Have opposite Effects on Adaptive Mutation in Escherichia Coli

Reversion of a lac(-) frameshift allele carried on an F' episome in Escherichia coli occurs at a high rate when the cells are placed under lactose selection. Unlike Lac(+) mutations that arise during nonselective growth, the production of these adaptive mutations requires the RecA-RecBCD pathway for recombination. In this report, we show that enzymes that process recombination intermediates are involved in the mutagenic process. RuvAB and RecG, E. coli's two enzymes for translocating Holliday junctions, have opposite effects: RuvAB is required for RecA-dependent adaptive mutations, whereas RecG inhibits them.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

A pyrenoid-based carbon-concentrating mechanism is present in terrestrial bryophytes of the class Anthocerotae

It has been widely accepted that carbon assimilation in bryophytes is exclusively based on the conventional C₃ photosynthetic pathway. The occurrence of biochemical CO₂-concentrating mechanisms (C₄ or Crassulacean acid metabolism), which have developed in plants in the last 20–100 million years, has been discounted for bryophytes from studies of the carbon isotope composition (¹³C) of organic material. In contrast cyanobacteria and many algae show active accumulation of dissolved inorganic carbon via biophysical CO₂-concentrating mechanisms which are also found in the photobiont partners in certain lichens. The presence of a pyrenoid, a granular particle within the chloroplast, has been linked with CO₂-concentrating mechanism activity in green algae and lichens and we now show that such a mechanism is categorically associated with the occurrence of a pyrenoid in bryophytes belonging to the class of Anthocerotae. These observations have significant evolutionary implications for the development of terrestrial photosynthesis during the colonisation of the land, raising the intriguing question of why the pyrenoid-based CO₂-concentrating mechanism did not persist in the terrestrial environment.Abbreviations and Symbols CCM carbon-concentrating mechanism - DIG dissolved inorganic carbon (CO₂+HCO ₃ ^- +CO ₂ ^- ) - DW dry weight - K_0.5 external concentration of CO₂ at which half-maximal rates of CO₂ assimilation are reached - Rubisco ribulose-l,5-bisphosphate carboxylase-oxygenase - carbon isotope discrimination (%) - ¹³C carbon isotope ratio (%) This work was supported by the Natural Environment Research Council (GR3/8813) and the Leverhulme Trust. We thank Prof. A. Roy Perry (National Museum of Wales, Cardiff), Dr. B. Coppins and Mr. D. Long (Royal Botanic Garden Edinburgh) for access to herbarium specimens and Mr. M. Fletcher for providing living bryophytes.     

Age-Dependent Impairment of Mitochondrial Function in Primate Brain

Abstract: It has been hypothesized that some of the functional impairments associated with aging are the result of increasing oxidative damage to mitochondrial DNA that produces defects in oxidative phosphorylation. To test this hypothesis, we examined the enzymes that catalyze oxidative phosphorylation in crude mitochondrial preparations from frontoparietal cortex of 20 rhesus monkeys (5-34 years old). Samples were assayed for complex I, complex II-III, complex IV, complex V, and citrate synthase activities. When enzyme activities were corrected for citrate synthase activities (to account for variable degrees of mitochondrial enrichment), linear regression analysis demonstrated a significant negative correlation of the activities of complex I (p < 0.002) and complex IV (p < 0.03) with age but no significant change in complex II-III or complex V activities. Relative to animals 6.9 ± 0.9 years old (n = 7), the citrate synthase-corrected activity of complex I was reduced by 17% in animals 22.5 ± 0.9 years old (n = 6) (p < 0.05) and by 22% in animals 30.7 ± 0.9 years old (n = 7) (p < 0.01). Similar age-related reductions in the activities of complexes I and IV were obtained when enzyme activities were corrected for complex II-III activity. These findings show an age-associated progressive impairment of mitochondrial complex I and complex IV activities in cerebral cortices of primates.     

Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.