Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.     

Lymphoma models for B cell activation and tolerance. VI. Reversal of anti-Ig-mediated negative signaling by T cell-derived lymphokines

We have recently described three "immature" B cell lymphomas which are exquisitely sensitive to growth inhibition by anti-Ig reagents and may serve as models for tolerance induction in normal B cells. These cells are inhibited from cell cycle progression into S after receiving a negative signal in early G1. In this paper, we demonstrate that the growth inhibition by anti-Ig can be prevented and reversed by the addition of supernatants from T cell lines. One such line, called Tova, produces factors which restore normal levels of DNA synthesis in the presence of concentrations of anti-Fab or anti-kappa immunoglobulins which cause up to a 90% inhibition of thymidine incorporation in a 2- to 3-day culture period. This factor is at least partially effective when added up to 24 hr after anti-Ig to unsynchronized lymphoma cells and it does not alter the growth of control cultures. Studies using synchronized lymphoma cells indicated that the T cell factor permitted cycle progression into S when added during the early G1 exposure to anti-kappa and was less effective when added late in G1. Preliminary characterization suggests that both B cell growth factor II (interleukin 5) and B cell stimulatory factor 1 (interleukin 4) have additive activity in this system, although another unidentified lymphokine may also be involved. The relevance of T cell reversal of Ig receptor-mediated negative signaling to neonatal B cell tolerance is emphasized.     

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem, J. 122, 727-740) composed of KNO3, MgCl2, creatine, and ADP. The enzyme is a dimer composed of 2 Mr 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of epsilon 280 nm = 2.22 +/- 0.10 ml X mg-1 X cm-1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.     

Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Km^r callus developing on hypocotyl sections. The totipotency of the Km^r callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Km^r tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Km^r callus and regenerated shoots.Abbreviations npt-II neomycin phosphotransferase II gene - NPT-II neomycin phosphotransferase II - nos nopaline synthase gene promoter - Km^r kanamycin resistant - BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MSD4×2 medium D4×2 based on Murashige & Skoog medium (see Scott & Draper, 1987)     

Chloroplast phosphoproteins: phosphorylation of a 12-kDa stromal protein by the redox-controlled kinase of thylakoid membranes

Activation of the redox-controlled protein kinase of thylakoid membranes is detectable in vivo by measuring radioisotope incorporation into the light-harvesting Chl a/b protein and four photosystem II proteins (8.3, 32, 34, and 44 kDa). In normal barley leaves, the kinase is active under both aerobic and anaerobic (N2) conditions, but in the Chl b-less chlorina f2 mutant it is active only under anaerobic conditions. The responsiveness of this enzyme in the mutant to changes in the gas phase has been exploited to distinguish its protein substrates from those of other leaf protein kinases. Most of the soluble phosphoproteins of normal and mutant leaves (including a conspicuously labeled 67-kDa polypeptide) are labeled equally under both aerobic and anaerobic conditions, indicating that they are not substrates of the redox-controlled protein kinase. The major exception is a 12-kDa phosphoprotein, which is labeled in the mutant only under anaerobic conditions. The 67- and 12-kDa phosphoproteins are located in the chloroplast and are labeled when isolated organelles are incubated with [32P]orthophosphate in the light. When thylakoids and stroma are prepared from chloroplasts and are incubated with [gamma-32P]ATP in vitro, the 12-kDa protein is phosphorylated in the thylakoid preparation and then released from the membranes into the medium. The electron transport inhibitor diuron blocks activation of the redox-controlled kinase and prevents phosphorylation of the 12-kDa protein, which is thus the first example of a soluble protein to be phosphorylated by the thylakoid-bound protein kinase. The 67-kDa protein is phosphorylated by a distinct stromal kinase whose activity is not sensitive to diuron.     

Rat alkaline phosphatase. II. Structural similarities between the osteosarcoma, bone, kidney, and placenta isoenzymes

A mouse monoclonal antibody raised against rat osteosarcoma alkaline phosphatase (AP) was covalently coupled to protein A-Sepharose and used to purify this enzyme from preparations of rat osteosarcoma, calvaria, kidney, and placenta in a single-step procedure. The tissue-specific isoenzymes purified in this manner showed identity in the immunodiffusion reaction with a polyclonal anti-AP antibody, but differed in apparent molecular weight and degree of polydispersity on sodium dodecyl sulfate-polyacrylamide gels. Treatment with N-glycanase abolished these differences, yielding proteins with an apparent molecular weight of 52,000 Da and identical V8 protease digestion patterns. Alkaline phosphatase from these tissues showed no significant difference in amino acid composition and identity in the first 20 N-terminal amino acids. These findings provide structural evidence which supports the hypothesis that the tissue-specific alkaline phosphatase isoenzymes share a common protein sequence subject to different glycosylation pattern.     

The entrapment of [14C]ascorbic acid in human erythrocytes

Radioactively labelled ascorbic acid and dehydroascorbic acid, when incubated with human blood, migrate irreversibly into human red blood cells. Isolation and characterization of the moieties trapped within the cells via infrared spectroscopy established both their identities as L-ascorbic acid. Evidence in the form of the degree of in vitro entrapment of ascorbic acid as a function of the times of incubation and the effect of incubation temperature, anion recognition site inhibitor, and active transport inhibitor on the rate of entrapment support the hypothesis that ascorbic acid is oxidized on or near the surface of the red blood cell to dehydroascorbic acid which migrates through the lipid portion of the cell wall and is reduced back to ascorbic acid within the cell. The resulting L-ascorbic acid can not pass through the cell wall and is therefore entrapped.     

The isolation of a fetal rat liver glutathione S-transferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated from fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Yc (Mr 28,000) and a subunit (Mr 25,500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit 'Yfetus'. The enzyme which we term glutathione S-transferase YcYfetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.     

Gallbladder pressure, compliance, and hysteresis during cyclic volume change

The gallbladder has both storage and contractile properties, but is pressure-volume characteristics have not been fully described. We studied gallbladder pressure, compliance, stress relaxation, and the work performed during infusion-withdrawal of fixed volumes of bile at increments of base-line volume under halothane anesthesia in 13 dogs. Two cannulae were inserted in the gallbladder fundus: one for cyclic infusion-withdrawal of bile, and one for pressure monitoring. Following ligation of the cystic duct, the gallbladder was fully aspirated and then filled to successive predetermined base-line volumes (15, 20, 25, 30, or 35 mL). At each base-line volume, 5 mL of bile was infused and withdrawn at 2.47 mL/min. Studies were performed under basal conditions and with cholecystokinin octapeptide (CCK) infusion (10 or 30 ng.kg-1.h-1iv). Pressure was measured at base-line, after infusion, and after withdrawal. Compliance during infusion (delta V/delta P) was calculated for each cycle. Stress relaxation was defined as the difference between base-line pressure and any reduction in pressure after withdrawal. Hysteresis, the difference between work of infusion and work of withdrawal, was calculated. The results were such that base-line, end-infusion, and end-withdrawal pressures; work of infusion, work of withdrawal, and hysteresis; and stress relaxation all increased significantly with increases in the predetermined base-line volumes (p less than 0.001). Compliance decreased significantly (p less than 0.001) with increasing base-line volume. CCK at 10 ng.kg-1.h-1iv had no effect, but infusion of CCK at 30 ng.kg-1.h-1 significantly (p less than 0.05) increased pressure and work, decreased compliance, and increased stress relaxation compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)