Identity of common phosphoprotein substrates stimulated by interleukin 2 and diacylglycerol suggests a role of protein kinase C for IL 2 signal transduction

Interleukin 2 (IL 2) is a polypeptide growth factor essential for the proliferation and differentiation of T lymphocytes, large granulocytic lymphocytes, and, potentially, cells of the antibody-producing lineage, B lymphocytes. Many of the biological properties of IL 2 may be mimicked or potentiated by a potent class of tumor promoters, phorbol esters. Phorbol esters have recently been shown to associate with and activate a unique phospholipid/Ca2+-dependent phosphotransferase, protein kinase C (PK-C). Utilizing two-dimensional gel electrophoresis, we have compared the IL 2 and diacylglycerol-induced protein phosphorylation patterns of several IL 2-dependent murine cell lines. Both IL 2 and synthetic diacylglycerol, 1-oleyl-2-acetylglycerol (OAG), stimulated phosphorylation of a number of protein substrates in intact cells compared to unstimulated controls. Three groups of substrates were identified; the first showed increased phosphorylation following stimulation with either IL 2 or OAG, while the second and third groups showed increased phosphorylation following stimulation with IL 2 but not OAG, and with OAG but not IL 2, respectively. Here, we characterize the kinetics of phosphorylation of one cellular substrate, p68, which appears to be phosphorylated in response to direct activators of PK-C or lymphoid or myeloid growth factors in their respective lineage cell lines. The observation that IL 2 also stimulates a unique series of phosphoproteins in addition to those induced by direct PK-C activators suggests that IL 2 may initiate additional protein kinase activities, unrelated to PK-C, which may also be critical for the ligand-receptor signal transduction process regulating growth and gene expression.     

Clustering of null mutations in the EcoRI endonuclease

EcoRI endonuclease mutants were isolated in a methylase-deficient background following in vitro hydroxylamine mutagenesis of plasmid pKG2 (Kuhn et al.: Gene 44:253-263, 1986). Mutants which survived high-level endonuclease expression (IPTG induction) were termed null mutants. Sixty-two of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein. The complete endonuclease gene was sequenced for 27 null mutants. This group was found to consist of 20 single base-change missense mutations, 6 double mutations, and 1 triple mutation. Ten of the 20 single mutations were clustered between residues 139 and 144. When examined with respect to the structure of the EcoRI-DNA complex (McClarin et al.: Science 234:1526-1541, 1986), these alterations were found to fall predominantly into two classes: substitutions at the protein-DNA interface or substitutions at the protein-protein (dimer) interface. Protein from several of the mutants was purified and sized by using HPLC. Wild-type EcoRI endonuclease and protein from three of the DNA interface mutations (Ala139----Thr, Gly140----Ser, Arg203----Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144----Lys, Glu152----Lys, Gly210----Arg) migrated as monomers.     

Estimation of uncertainties in X-ray refinement results by use of perturbed structures

The uncertainties in the refined parameters for a 1.5-A X-ray structure of carbon-monoxy (FeII) myoglobin are estimated by combining energy minimization with least-squares refinement against the X-ray data. The energy minimizations, done without reference to the X-ray data, provide perturbed structures which are used to restart conventional X-ray refinement. The resulting refined structures have the same, or better, R-factor and stereochemical parameters as the original X-ray structure, but deviate from it by 0.13 A rms for the backbone atoms and 0.31 A rms for the sidechain atoms. Atoms interacting with a disordered sidechain, Arg 45 CD3, are observed to have larger positional uncertainties. The uncertainty in the B-factors, within the isotropic harmonic motion approximation, is estimated to be 15%. The resulting X-ray structures are more consistent with the energy parameters used in simulations.     

Molecular biology of Philadelphia chromosome in chronic granulocytic leukaemia and acute lymphoblastic leukaemia

In classical t(9;22) translocation, as observed in chronic granulocytic leukemia (CGL), a hybrid DNA unit is produced, including a rearranged PHL gene, previously known as bcr (breakpoint cluster region) plus the translocated c-abl gene from chromosome 9: a hybrid bcr-abl protein, p210 is formed, with increased tyrosine kinase activity. Such DNA rearrangement, with a p210 protein synthesis, is also found in cases of Philadelphia-positive acute lymphoblastic leukemia (ALL), but in apparently similar cases the bcr gene is not rearranged, and a novel p190 abl-related protein can be found; c-abl rearrangement has also been observed.It is thus established that correlations between cytogenetic and molecular events can be found in CGL and ALL, as in other haemopoietic malignancies: translocation and possible rearrangement of the c-abl oncogene seem of particular importance in this case.     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

Acquisition of a brief behavioral experience in the presence of neuron-specific and D2-CAM/N-CAM-specific antisera

The effect of intraventricular infusion of D2-CAM/N-CAM directed antibodies prior to the acquisition of a passive-avoidance paradigm is described. The antisera used in this study were the neuron specific anti-BPM and a D2-CAM/N-CAM specific serum, anti-D2. Anti-BPM reliably inhibited paradigm acquisition when recall was ascertained at 24 and 48 hours and no effect was noted with absorbed anti-BPM or in sham-operated animals. This effect was time-dependent and no inhibition of memory formation was noted when the antiserum was administered at 6 and 10 hours after training. In contrast, infusion of anti-D2 had no effect on paradigm acquisition. These findings are discussed in relation to the potential synaptogenic events associated with memory formation.     

Stimulation of phospholipase D activity and indication of acetylcholine synthesis by oleate in rat brain synaptosomal preparations

It had been previously demonstrated that the oleate activation of synaptosomal membrane phospholipase D liberated choline which was available for acetylcholine formation. The present investigations were undertaken to determine if oleate might have an effect on choline uptake by synaptosomes. It was observed that oleate interfered with choline uptake when incubations were carried out at 37°C but uptake was stimulated at 3°C. Oleate was the most effective fatty acid of several tested. Preliminary observations suggest the presence of a membranous form of choline acetyltransferase.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.