Nucleotide sequence analysis of a variant human T-cell leukemia virus (HTLV-Ib) provirus with a deletion in pX-I.

A variant of human T-cell leukemia virus subgroup I (HTLV-I), designated HTLV-Ib, has been isolated from a transformed T-lymphocytic cell line established from a Zairian patient with adult T-cell lymphoma. A recombinant phage clone of the variant provirus, denoted lambda MC-1, hybridizes under high stringency to HTLV-I DNA probes, but 17 of 43 restriction enzyme sites differ from those of HTLV-I, 10 of them clustering within 1.5 kilobases in the env-pX region. Since this variant virus retains its capacity to transform T-cells in vitro, and since a pX product is suspected to be important in transformation, we have determined the nucleotide sequence of the entire pX region of this virus for comparison to the prototype HTLV-I. In addition, the region between the gag and pol genes, parts of the pol and env genes, and a portion of the U3 region of the long terminal repeat sequence were also analyzed. We noted 141 single-base-pair changes among 3,897 base pairs, which were relatively well distributed over those portions of the provirus that were examined. In addition, an 11-base-pair deletion was found which included the potential initiator ATG codon of the first open reading frame of pX (pX-I). The next potential initiator codon predicted by the sequence is followed by 10 codons and then a termination codon. An identical deletion was also demonstrated in the only provirus present in another cell line established from the same patient on a different occasion after transformation in vitro of normal human umbilical cord blood cells. These results indicate that pX-I is not required for transformation.     

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.     

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.     

Screening for mutations in the neurofibromatosis type 2 (NF2) gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. They are usually sporadic but can also occur associated with the neurofibromatosis type 2 (NF2) syndrome. The gene responsible for NF2, recently isolated from chromosome 22, encodes a membrane-organizing protein that shows high sequence homology to a protein family thought to link the cytoskeleton with membrane proteins. Mutations of the NF2 gene have been described in sporadic meningiomas, exclusively in tumors that show loss of heterozygosity (LOH) of 22q. These preliminary results indicate that the NF2 gene is involved in the pathogenesis of at least a subset of meningiomas, where it does indeed behave as a tumor suppressor gene. In order to characterize better the role of the NF2 gene in the genesis of meningiomas we have examined the entire coding sequence of the gene in 125 meningiomas by single-strand conformational polymorphism analysis; furthermore, LOH analysis for markers of 22q has been carried out. Inactivating mutations were identified in 30% of our samples, all of which also showed LOH of 22q. The majority of mutations identified were frameshifts and nonsense mutations, which are predicted to produce a truncated or non-functional protein. We also found two missense and three in-frame deletions that may pinpoint specific regions of the protein critical to its function. Furthermore, the distribution of mutations throughout the gene, suggested that exons 2, 3, 5, 11 and 13 are more frequently involved. Our results reconfirm the importance of the NF2 gene in the pathogenesis of meningiomas and also suggest that there may be a nonrandom clustering of mutations throughout the gene.