Functional and phenotypic characterization of two HL60 clones resistant to dimethylsulfoxide

Two HL60 clones (C12 and C13) totally insensitive to differentiation induction by dimethylsulfoxide (Me2SO) are described. They have been growing continuously in the presence of the inducer for more than 6 months. The morphological and cytochemical features of the two populations are quite similar to those of the original HL60 cell line, whereas a different karyotype with marked hyperploidy (modal chromosome number of 86 for C12 and 82 for C13) was detected. An antigenic pattern analogous to that of the native HL60 cell line was found in C12 and C13 populations using three monoclonal antibodies differently reactive to myeloid cells. Both clones can be induced to differentiate by retinoic acid (RA) and 12-O-tetradecanoylphorbol 13-acetate (TPA). The pattern of differentiation was assessed by morphological, cytochemical, phenotypical and functional markers. Differentiation of C12 cells by RA and TPA was similar to that observed with native HL60 cells, whereas C13 cells showed lower degrees of sensitivity to RA and TPA. The data presented suggest the existence of different mechanisms for induction of differentiation by Me2SO, RA and TPA. In addition, they are in accordance with previous observations of different degrees of inducibility to differentiation among leukemic cell populations in culture.     

New contributions on extinction tests of essential headache. III. The test with tiapride

The Authors valued effects of the extinction-test with Thiapride (200 mg i.v.) in the primary headaches. They particularly noticed a significant reduction of PGF2-alpha and PRL, together with an increase of 5-HT and beta-ELI in CSF.     

Lipid composition of human serum lipoproteins

1. The lipid compositions of the low-density lipoproteins, the high-density lipoproteins and the ultracentrifugal residue of human serum are presented, with emphasis on certain lipoprotein classes and lipid components not previously described. 2. Except for the lipoproteins with the lowest and highest densities, there is a trend for stepwise successive increase or, respectively, decrease in the relative amounts of the main constituents of lipoproteins. 3. High-density lipoprotein-2 and high-density lipoprotein-3 have different amounts of certain lipids; high-density lipoprotein-2 has relatively more free cholesterol and sphingomyelin; high-density lipoprotein-3 has more free fatty acids, diglycerides and ceramide monohexosides. 4. All the lipoproteins contain hydrocarbons of the alkane series. The greatest amount, which averages 4.4% of total lipid extracted, is in the ultracentrifugal residue; n-alkanes comprise 18-50% of the hydrocarbons. 5. All the lipoproteins contain ceramide monohexosides. The highest relative contents of these glycolipids are in high-density lipoprotein-3 and in the ultracentrifugal residue. 6. The ultracentrifugal residue contains 55% of the total quantity of free fatty acids present in serum. The remaining free fatty acids are distributed among the other lipoprotein classes. 7. The choline-containing phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) comprise about 90% of the phospholipids in all the lipoprotein classes except the low-density lipoprotein-2, which contains about 80% of these phospholipids. 8. The presence of a large amount of lysophosphatidylcholine in the ultracentrifugal residue and the successive decrease of sphingomyelin from the low-density lipoprotein-1 to the ultracentrifugal residue was confirmed. 9. The low-density lipoprotein-2 and the ultracentrifugal residue are characterized by relatively high contents of the lower glycerides.     

A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.     

Functional contribution of cysteine residues to the human immunodeficiency virus type 1 envelope.

Although the envelope gene of human immunodeficiency virus type 1 shows considerable strain variability, cysteine residues of the envelope protein are strongly conserved, suggesting that they are important to the envelope structure. We constructed and analyzed mutants of a biologically active molecular clone of human immunodeficiency virus type 1 in which different cysteines were replaced by other amino acids in order to determine their functional importance. Substitution of cysteines 296 and 331, on either side of a region recognized by type-specific neutralizing antibodies, or on either side (residues 418 and 445) of a region important for CD4 binding, resulted in noninfectious mutants. These mutants were blocked early in the viral life cycle. Their gp160 envelope precursor polypeptides were poorly cleaved, and CD4 binding was also strongly impaired. Similar substitutions in the first variable region (residue 131) or between the first and second variable regions (residue 196) also gave noninfectious mutant virus, but here the block was late in the virus life cycle; these mutants were defective for syncytium formation. Substitution of cys386, between the neutralization and CD4 binding regions, resulted in a virus which retained infectivity but which spread much more slowly than the wild type. As with the cys131 and cys196 mutants, the cys386 mutant appeared to be defective in syncytium formation. These results show that all seven of the tested cysteines are vital for envelope function and suggest that this is likely true for all envelope cysteines. The results further show that regions important for CD4 binding, proteolytic cleavage recognition, and syncytium formation are all multiple and distributed over a relatively large part of the gp120 and therefore are likely dependent on protein tertiary structure.     

Effect of nucleosome distortion on the linking deficiency in relaxed minichromosomes

The wrapping of closed circular DNA on a protein surface, followed by relaxation with a topoisomerase and removal of proteins, produces a characteristic DNA linking deficiency, delta Lk. We show that the magnitude of delta Lk depends upon the surface shape, and we calculate changes in delta Lk caused by particular distortions of the protein wrapping surface. If the DNA remains attached to the surface during distortion, the DNA winding number, phi, is not altered. The change in delta Lk is then equal to the change in the surface linking number, SLk, which is a straightforward measure of the wrapping of the DNA around the surface. For left-handed wrapping, as in a nucleosome, SLk = -n, the number of times that the DNA axis winds around the axis of the protein complex. We calculate values of SLk for the helical wrapping of a constant length of DNA on protein surfaces having the shapes of cylinders and of ellipsoids and hyperboloids of revolution. If the equatorial radius of the protein is fixed, change in shape from a cylinder to a hyperboloid increases SLk, while the corresponding change to an ellipsoid reduces SLk. We apply the general results to the interpretation of experiments in which minichromosomes are relaxed with topoisomerase at various temperatures and delta Lk is determined. The result is that a distortion of the nucleosome core by at most 5% (the change in the radius at the axial extremity relative to the equator) is sufficient to explain the observed delta Lk changes.     

The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase.

The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1 (HSV-1), the product of the UL42 gene, is required for DNA replication both in vitro and in vivo, yet its actual function is unknown. By two independent methods, it was shown that the 65KDBP stimulates the activity of the HSV-1-encoded DNA polymerase (Pol). When Pol, purified from HSV-1-infected cells, was separated from the 65KDBP, much of its activity was lost. However, addition of the 65KDBP, purified from infected cells, stimulated the activity of Pol 4- to 10-fold. The ability of a monoclonal antibody to the 65KDBP to remove the Pol-stimulating activity from preparations of the 65KDBP confirmed that the activity was not due to a trace contaminant. Furthermore, the 65KDBP did not stimulate the activity of other DNA polymerases derived from T4, T7, or Escherichia coli. The 65KDBP gene transcribed in vitro from cloned DNA and translated in vitro in rabbit reticulocyte lysates also was capable of stimulating the product of the pol gene when the RNAs were cotranslated. The product of a mutant 65KDBP gene missing the carboxy-terminal 28 amino acids exhibited wild-type levels of Pol stimulation, while the products of two large deletion mutants of the gene could not stimulate Pol activity. These experiments suggest that the 65KDBP may be an accessory protein for the HSV-1 Pol.     

Some prerequisites for a study of the evolution of cognition in the animal kingdom

A distinction is made between two definitions of animal cognition: the one most frequently employed in cognitive sciences considers cognition as extracting and processing information; a more phenomenologically inspired model considers it as attributing to a form of the outside world a significance, linked to the state of the animal. The respective fields of validity of these two models are discussed along with the limitations they entail, and the questions they pose to evolutionary biologists are emphasized. This is followed by a presentation of a general overview of what might be the study of the evolution of knowledge in animals.     

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.