Variations in the DNA methylation and polypeptide patterns of adult hazel (Corylus avellana L.) associated with sequential in vitro subcultures

Summary The polypeptide and DNA methylation patterns of leaves from adult hazel trees maintained by sequential in vitro subcultures were analyzed. Qualitative and quantitative variations were found in the in vitro tissues as compared to both adult and juvenile forms. From the comparisons between different tree sources it may be concluded that hazel trees under in vitro conditions show specific biochemical and molecular patterns. The specificity of the induced changes could be a prerequisite related to the higher morphogenic potential of adult plants when they are subjected to sequential in vitro subcultures.Abbreviations BAP 6-benzylaminopurine - BSA bovine seroalbumin - EDTA ethylenediaminetetraacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - kbp Kilobases - kD kilodaltons - LSU Rubisco Large Subunit - PAGE polyacrylamide gel electrophoresis - Rf relative mobility of polypeptides - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)aminomethane     

Cells with High Cyclophilin A Content Support Replication of Human Immunodeficiency Virus Type 1 Gag Mutants with Decreased Ability To Incorporate Cyclophilin A

Gag polyprotein-mediated incorporation of cellular cyclophilin A (CyPA) into virions is essential for the formation of infectious human immunodeficiency virus type 1 (HIV-1) virions. Either a point mutation in Gag (P222A) or drugs which bind CyPA decrease virion incorporation of CyPA and interfere with HIV-1 replication. We have found that lymphoid cells varied greatly in their CyPA content and that cells with high CyPA content supported the replication of P222A HIV-1 Gag mutants. These experiments demonstrated that a higher cellular CyPA content of some cells was able to compensate for the decreased binding affinity of P222A mutant Gag for CyPA, allowing virus replication to occur.     

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.     

Effects of low temperature, high salinity and exogenous ABA on the synthesis of two chloroplastic drought-induced proteins in Solanum tuberosum

Two chloroplastic proteins of 32 and 34 kDa were previously shown to be substantially synthesized in response to a progressive water deficit in whole Solanum tuberosum plants (G. Pruvot, S. Cuiné, N. Gault, G. Peltier and P. Rey, unpublished data; G. Pruvot, S. Cuiné, G. Peltier and P. Rey. 1996. Planta 198: 471–479). These chloroplastic drought-induced stress proteins, named CDSP 32 and CDSP 34, accumulated in the stroma and in the thylakoids, respectively. In this study, we investigated the effects of low temperature and high salinity on the synthesis of the CDSP proteins. Whereas the CDSP 32 synthesis was not modified in response to a cold treatment, an increased synthesis of CDSP 32 was observed in salt-stressed plants, resulting in accumulation of the protein. The thylakoid CDSP 34 protein exhibited enhanced synthesis and substantial accumulation in response to cold and high salinity. A significant increase in the leaf abscisic acid content (at least 2.5-fold) was measured in plants subjected to water deficit, high salinity or low temperature. The contribution of ABA to the synthesis of the two proteins was investigated by spraying well-watered plants with a 100 μ M / ABA solution for 15 days. This treatment resulted in a 15-fold increase in the leaf ABA content. Whereas synthesis of the CDSP 32 protein was not affected by exogenous ABA, synthesis of the CDSP 34 protein was substantially enhanced. Based on these results, we conclude that ABA likely mediates the increased synthesis of CDSP 34 upon drought, low temperature and high salinity and suggest that another signal, likely related to high osmolarity, is involved in the induction of CDSP 32 synthesis.     

Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme.

The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr.     

Effects of adrenal medullectomy and passive immunization with an anti-CRF antiserum on the corticotropin secretion in response to stress in Long-Evans rats

Ether stress-induced ACTH and corticosterone secretion is similar in 60 days adrenal medullectomized and intact Long-Evans Rats. Passive immunisation with a 41-CRF antiserum impairs corticotropic function after stress. This decrease is more pronounced in adrenal medullectomized than in intact animals. These results suggest that epinephrine plays a minor role on the stress induced ACTH secretion. This role can be demonstrated only after immunoneutralisation of endogenous 41-CRF.     

The role of interleukin 1 and interleukin 2 in human T colony formation

We investigated the roles of interleukin 1 (IL1) and interleukin 2 (IL2) on T colony formation by PHA-stimulated peripheral blood lymphocytes (PBL). Purified T cells stimulated by PHA could not generate T colonies as did PBL. Media conditioned by PHA-stimulated PBL (PHA-LCM) contained IL2 and a T colony-promoting activity (TCPA) which induced T colony formation in PHA-stimulated purified T cells. IL2 and TCPA are coeluted in the same peak of 18,000 molecular weight after gel filtration chromatography. Moreover, TCPA present in the PHA-LCM could be absorbed on IL2-sensitive cells which possessed specific receptors for IL2. These results suggest that TCPA and IL2 are related entities. Monocytes or IL1 (a monokine released by activated monocytes) also induced T colony formation in purified T cells. Phorbol myristate acetate (PMA) could replace monocytes in the induction of T colony. Monocytes, IL1, or PMA are known to be crucial requirements for IL2 production by PHA-stimulated T cells. This combined with the fact that IL2 participates in T colony formation suggests that monocytes induce T colony formation through IL2 production.