MHC antigen expression by melanomas recovered from mice treated with allogeneic mouse fibroblasts genetically modified for interleukin-2 secretion and the expression of melanoma-associated antigens

Recent approaches toward the immunotherapy of neoplastic disease involve the introduction of expression-competent genes for interleukin-2 (IL-2) into autologous malignant cells. Treatment of tumor-bearing experimental animals with the IL-2-secreting cells successfully induces partial and at times complete remissions. In most instances, however, although delayed, progressive tumor growth continues. Here, certain of the characteristic of B16 melanomas (H-2^b) persisting in C57BL/6 mice (H-2^b) treated with an IL-2-secreting, melanoma-antigen-positive cellular immunogen (RLBA-IL-2 cells) are described. Unlike the melanoma cells first injected, B16 cells recovered from mice treated with RLBA-IL-2 cells were deficient in the experssion of MHC class I, but not class II determinants. Deficient MHC class I expression correlated with the cells' resistance to cytotoxic T lymphocytes (CTL) from the spleens of mice immunized with RLBA-IL-2 cells. Melanomas persisting in mice treated with non-IL-2-secreting, melanoma-antigen-positive cell constructs (RLBA-ZipNeo cells) were also deficient in the expression of MHC class I determinants, and the melanoma cells were resistant to CTL from mice immunized with RLBA-ZipNeo cells. Thus, the expression of melanoma-associated antigens rather than IL-2-secretion correlated with deficient MHC class I expression by the persistent melanomas. This point was substantiated by the expression of MHC class I antigens by melanomas persisting in mice treated with IL-2-secreting, melanoma-antigen-negative LM cells (LM-IL-2); it was equivalent to that of melanomas in untreated mice. The involvement of MHC class I antigens in the immune resistance of persistent melanoma cells from mice treated with the melanoma-autigen-positive immunogens was indicated by the effect of interferon (IFN) orN-methyl-N-nitro-N-nitrosoguanidine (MNNG) on the susceptibility of the cells to anti-melanoma CTL. Treatment of the resistant melanomas with IFN or MNNG stimulated MHC class I antigen expression and restored the cells' sensitivity to CTL from mice immunized with IL-2-secreting or nonsecreting, melanoma-antigen-positive cellular immunogens. Prior treatment of the treated cells with antibodies to MHC class I determinants inhibited the cells' susceptibility to CTL from mice immunized with RLBA-IL-2 cells.     

How does the G protein,Gi2, transduce mitogenic signals?

Serpentine receptors coupled to the heterotrimeric G protein, G_i2, are capable of stimulating DNA synthesis in a variety of cell types. A common feature of the G_i2-coupled stimulation of DNA synthesis is the activation of the mitogen-activated protein kinases (MAPKs). The regulation of MAPK activation by the G_i2-coupled thrombin and acetylcholine muscarinic M₂ receptors occurs by a sequential activation of a network of protein kinases. The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation. MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK). Thus, Raf and MEKK converge at MEK to regulate MAPK. G_i2-coupled receptors are capable of activating MEK and MAPK by Raf-dependent and Raf-independent mechanisms. Pertussis toxin catalyzed ADP-ribosylation of α_i2 inhibits both the Raf-dependent and-independent pathways activated by G_i2-coupled receptors. The Raf-dependent pathway involves Ras activation, while the Raf-independent activation of MEK and MAPK does not involve Ras. The Raf-independent activation of MEK and MAPK most likely involves the activation of MEKK. The vertebrate MEKK is homologous to the Ste11 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The yeast Ste11 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins. MEKK appears to be conserved in the regulation of G protein-coupled signal pathways in yeast and vertebrates. Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system. Defining MEKK and Raf as a divergence in the MAPK regulatory network provides a mechanism for differential regulation of this system by G_i2-coupled receptors as well as other receptor systems, including the tyrosine kinases.     

Enhanced expression of group II phospholipase A2 in human hepatocellular carcinoma

Enzyme activity, protein contents, and mRNA contents of group II phospholipase A₂ (PLA₂) in hepatocellular carcinoma (HCC) surgically obtained from 8 patients were compared with those in either its neighboring liver tissues or control liver tissues. The PLA₂ specific activity towards the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was significantly greater in the tumor tissues (6.62 ± 1.46 nmol/min/mg) than those in the surrounding liver tissues (1.33 ± 0.22 nmol/min/mg) and controls (0.43 ± 0.04 nmol/min/mg). The results of immunoblot analysis using a specific anti-human group II PLA₂ antibody and of Northern blot analysis using a human group II PLA₂ cDNA as a probe demonstrated that group II PLA₂ was responsible for the increased enzyme activity. The contents of immunoreactive group II PLA₂ in the tumor tissues (8.81 ± 1.24 ng/mg) were significantly higher than those in the surrounding liver tissues (1.77 ± 0.27 ng/mg); those in the control tissues were below the analytical range of the method used. The group II PLA₂ mRNA was also significantly increased in the tumor tissues, compared with that in the surrounding liver tissues, whereas it was not detectable in th controls. This indicates that group II PLA₂ in HCC is induced at the pretranslational level.     

Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice

Summary We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family.     

Effect of exchangeable aluminium on the reduction of potato scab

The severity of the incidence of the fungal disease, potato scab, varies with different soil groups at the same soil pH. At a soil pH of 5.3, potato scab is easily controlled in soils of western Hokkaido (soil group A) by simply decreasing soil pH, but in soils from eastern Hokkaido (soil group B) it is not so easily controlled. The difference appears to be due to higher levels and exchangeable aluminium in Group A.Addition of sufficient aluminium or ferrous sulfate to a group B soil decreased the incidence of potato scab in a field experiment. Higher levels of aluminium sulfate depressed crop production. It is concluded that aluminium ions control the incidence of potato scab in acid soils. It is suggested that, in soils with low exchange acidity Y1, potato scab can be controlled by adding sufficient aluminium to increase their exchange acidity Y1 to above 7–8.     

15N2 measurement of nitrogen fixation by legumes and actinorhizals: theory and practice

A gas-tight chamber has been constructed to calibrate the ¹⁵N isotope dilution method against direct ¹⁵N₂ measurements. The theoretical basis for such estimates is given, and the practical problems associated with the experiments are discussed.     

Ca2+ transport and chemoreception in Paramecium

Intracellular Ca²⁺ levels in Paramecium must be tightly controlled, yet little is understood about the mechanisms of control. We describe here indirect evidence that a phosphoenzyme intermediate is the calmodulin-regulated plasma membrane Ca²⁺ pump and that a Ca²⁺-ATPase activity in pellicles (the complex of cell body surface membranes) is the enzyme correlate of the plasma membrane pump protein. A change in Ca²⁺ pump activity has been implicated in the chemoresponse of paramecia to some attractant stimuli. Indirect support for this is demonstrated using mutants with different modifications of calmodulin to correlate defects in chemoresponse with altered Ca²⁺ homeostasis and pump activity.Abbreviations EGTA ethyleneglycol tetra-acetate - ER endoplasmic reticulum - IBMX isobutyl methylxanthine - I _che index of chemokinesis - Mops 3-[N-morpholino] propanesulfonic acid - PEI phosphoenzyme intermediate - STEN sucrose, TRIS, EDTA, sodium chloride - TCA trichloroacetic acid - TRIS tris[hydroxymethyl] aminomethane     

Evaluation of the role of State transitions in determining the efficiency of light utilisation for CO2 assimilation in leaves

Wheat leaves were exposed to light treatments that excite preferentially Photosystem I (PS I) or Photosystem II (PS II) and induce State 1 or State 2, respectively. Simultaneous measurements of CO₂ assimilation, chlorophyll fluorescence and absorbance at 820 nm were used to estimate the quantum efficiencies of CO₂ assimilation and PS II and PS I photochemistry during State transitions. State transitions were found to be associated with changes in the efficiency with which an absorbed photon is transferred to an open PS II reaction centre, but did not correlate with changes in the quantum efficiencies of PS II photochemistry or CO₂ assimilation. Studies of the phosphorylation status of the light harvesting chlorophyll protein complex associated with PS II (LHC II) in wheat leaves and using chlorina mutants of barley which are deficient in this complex demonstrate that the changes in the effective antennae size of Photosystem II occurring during State transitions require LHC II and correlate with the phosphorylation status of LHC II. However, such correlations were not found in maize leaves. It is concluded that State transitions in C₃ leaves are associated with phosphorylation-induced modifications of the PS II antennae, but these changes do not serve to optimise the use of light absorbed by the leaf for CO₂ assimilation.Abbreviations Fm, Fo, Fv maximal, minimal and variable fluorescence yields - Fm, Fv maximal and variable fluorescence yields in a light adapted state - LHC II light harvesting chlorophyll a/b protein complex associated with PS II - q_P photochemical quenching - A₈₂₀ light-induced absorbance change at 820 nm - _{PS I}, _{PS II} relative quantum efficiencies of PS I and PS II photochemistry - _{CO 2} quantum yield of CO₂ assimilation     

Biochemical characterization of the boar sperm 42 kilodalton protein tyrosine kinase: Its potential for tyrosine as well as serine phosphorylation towards microtubule-associated protein 2 and histone H 2B

The majority of cellular responses to changing environmental conditions is regulated by protein kinases. Spermatozoa have many special properties, including motility with demonstrated chemotaxis, the ability to undergo capacitation, and the acrosome reaction, which are in part controlled by extracellular signals and in which sperm kinases are considered to be involved. We have previously reported that there is a protein kinase activity, which phosphorylates the synthetic substrate poly-(Glu, Tyr) with a Km value of 2.3 μM, and is inhibited by the tyrosine kinase inhibitor tyrphostin, in the protein extract from boar spermatozoa (Berruti and Porzio, 1992: Biochim Biophys Acta 1118:149–154). Now we have demonstrated that the enzyme is cytosolic, is active as a monomer of M_r 42,000, is stimulated by Mg²⁺ > Mn²⁺ but not by Ca²⁺, is renaturable, and can phosphorylate native protein substrates such as microtubule-associated protein 2 (MAP2) and histone H2B both on the tyrosine and serine residues. N-terminal sequence analysis suggests that it is a novel protein. These new findings imply that the boar sperm 42 kD kinase may be a novel member of the emerging class of dual-specificity protein kinases, and they raise the intriguing question of its function in the protein kinase network mediating signal transduction in mammalian spermatozoa. © 1994 Wiley-Liss, Inc.