Interleukin-8 secretion by cultured oral epidermoid carcinoma cells induced with nicotine and/or arecoline treatments.

Interleukin-8 (IL-8) is one of the multifunctional cytokines that can play a role on immune and inflammatory activities. Other in vitro observations indicated that IL-8 is a growth factor for keratinocytes. However, as the role of IL-8 in oral cancer cells is unclear, this study is thus designed to examine IL-8 secretion in cultured oral epidermoid carcinoma KB CCL17 cells treated with nicotine and/or arecoline. The cultures were treated with nicotine (1 or 100 microM) and arecoline (1 or 100 microM), alone or both, for 72 hrs. Enzyme-linked immunosorbent assay (ELISA) was used to examine IL-8 concentrations in culture supernatants. A repeated measure analysis of variance was used to identify differences among the treatments. Nicotine and arecoline, single or combined treatment, increased IL-8 secretion in KB CCL17 cells. When monoclonal 1 microgram/ml of antibody was added against IL-1 alpha or IL-1 beta in the treatment, IL-8 concentration significantly decreased compared with the non-added one. Exposure of cells to antibody against IL-1 alpha or IL-1 beta showed no significant increase in cell growth as compared with the control (medium alone). However, incubation of cells for 72 hrs in the presence of nicotine and/or arecoline treatments and antibody against IL-1 alpha or IL-1 beta significantly increased cell growth as compared with the antibody free one. It was concluded that IL-8 secretion by KB CCL17 cells may be partially mediated by IL-1 which could inhibit the KB CCL17 cell growth. Thus, IL-8 may be a vital participant in the cascade of interacting cytokines during smoking and areca quid chewing, inducing inflammation in oral cancer.

Evidence for an improvement in cognitive function following treatment of Schistosoma japonicum infection in Chinese primary schoolchildren.

A double-blind, placebo-controlled, treatment trial was conducted in Sichuan, China to investigate the unique and combined effects on the cognitive function (working memory) of children after treating geohelminth infections with albendazole and treating Schistosoma japonicum infection with praziquantel. One hundred eighty-one children 5-16 years of age participated. At baseline, the praziquantel and placebo groups were similar in all background characteristics. Three months after praziquantel treatment, there was a significant reduction in the prevalence and intensity of S. japonicum infection. There were significant age group by praziquantel treatment interaction effects in three of the five cognitive tests, Fluency, Picture Search, and Free Recall, with effects being strongest in the youngest children (5-7 years old). Exploratory analysis within the youngest children showed a significant positive main effect of treatment on Fluency (P < 0.001), after controlling for sex, anthropometric, and parasitic and iron status. There was also a treatment by height-for-age interaction (P = 0.03) and a treatment by iron status interaction (P = 0.024) on Fluency. There was a treatment by S. japonicum intensity interaction (P < 0.001) on Free Recall, but the main effect of treatment on Picture Search was not significant (P = 0.058). Younger children and those who are physically the most vulnerable are likely to benefit the most from the treatment of S. japonicum infection in terms of improved performance on tests of working memory.

Syk is activated by phosphotyrosine-containing peptides representing the tyrosine-based activation motifs of the high affinity receptor for IgE.

Engagement of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on the surface of mast cells induces tyrosine phosphorylation of numerous cellular proteins. Syk, one of several non-receptor protein tyrosine kinases implicated in Fc epsilon RI signaling, is activated following receptor cross-linking and associates with phosphorylated gamma subunits of Fc epsilon RI. We previously showed that the Src homology 2 (SH2) domains of Syk bind with high affinity to the conserved tyrosine-based activation motif (TAM) of the gamma subunit in vitro. In this report, we show that a tyrosine-phosphorylated gamma TAM peptide induced tyrosine phosphorylation of Syk in RBL-2H3 cell lysates and stimulated Syk kinase activity 10-fold in vitro, with half-maximal activation at 1-2 microM. A similar beta subunit TAM peptide showed much lower stimulation of Syk tyrosine phosphorylation and kinase activity. Phosphopeptide-induced activation was inhibited by an antiserum to the carboxyl-terminal tail of Syk, suggesting that those amino acids are also involved in Syk activation. These results indicate that the catalytic domain of Syk may be regulated by intramolecular interactions with adjacent domains and suggest that Syk binding to phosphorylated gamma subunits following Fc epsilon RI engagement in vivo stimulates Syk kinase activity.

Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon tyrosine activation motifs in RBL-2H3 cells.

Two wnt genes in Caenorhabditis elegans.

wnt genes encode secretory glycoproteins that have been implicated in growth control and development in mice, frogs and insects. In this report we examine properties of two wnt genes recently identified in the nematode Caenorhabditis elegans. The first gene, Ce-wnt-1, was previously identified by a polymerase chain reaction-based screen of genomic DNA, and the second, Ce-wnt-2, was fortuitously encountered in a survey of clones in a cDNA library by the Caenorhabditis Genome Project. Full-length or nearly full-length cDNAs representing both mRNAs encode proteins that are similar in length, sequence and functional domains to other Wnt proteins. Primary products of 372 and 362 amino acids begin with a hydrophobic signal peptide, include two potential N-linked glycosylation sites and contain the 22 cysteine residues conserved throughout the wnt family. In contrast to mammalian and insect wnt genes with four or five exons and conserved intron-exon boundaries, Ce-wnt-1 has nine coding exons; only one of the eight identified introns interrupts the coding sequence at a position homologous to an intron position in other wnt genes. The major transcript derived from Ce-wnt-1 is 1.4 kb in length, and the 22 nucleotides at its 5' end are added by a trans-splicing mechanism. Ce-wnt-2 is also expressed via a single major transcript, 1.5 kb in length. Both RNAs are detectable in all larval forms and adults, but they are most abundant at the embryonic stage. Ce-wnt-1 is localized to the left arm of chromosome II and Ce-wnt-2 maps to a cluster of genes on chromosome IV.

Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro.

The retroviral integrase (IN) protein is essential for integration of retroviral DNA into the host cell genome. To identify functional domains within the protein and to assess the importance of conserved residues, we performed site-directed mutagenesis of HIV-1 IN and analyzed the mutants in vitro for IN-mediated activities: 3' processing (att site-specific nuclease activity), strand transfer (the joining of att site oligonucleotides to target DNA), disintegration (the reverse of strand transfer), and integration site selection. Changing the conserved residue His-16 either to Cys or to Val in a proposed zinc-finger region had minimal effect on IN activities. Alteration of two highly conserved amino acid residues, Asp-116-->Ile and Glu-152-->Gly, each resulted in complete or nearly complete loss of 3' processing, strand transfer, and disintegration, whereas alteration of another conserved residue, Trp-235-->Glu, had no demonstrable effect on any of the activities in vitro. Two mutants, Asp-64-->Val and Arg-199-->Cys delta, each demonstrated differential effects on IN activities. Asp-64-->Val has no demonstrable strand transfer or disintegration activity yet maintains 3' processing activity at a diminished level. Arg-199-->Cys delta, which lacks part of the carboxyl terminus of IN, has impaired strand transfer activity without loss of disintegration activity. Use of a target site selection assay showed that all of our mutants with strand transfer activity maintain the same integration pattern as wild type IN. We conclude that not all highly conserved IN residues are essential for IN activities in vitro, zinc coordination by the proposed zinc-finger domain may not be required for the activities assayed, alteration of single residues can yield differential effects on IN activities, and target site selection into naked DNA is not necessarily altered by changes in strand transfer activity.

A mammalian factor that binds telomeric TTAGGG repeats in vitro.

We have identified a DNA-binding activity with specificity for the TTAGGG repeat arrays found at mammalian telomeres. This factor, called TTAGGG repeat factor (TRF), is present in nuclear extracts of human, mouse, and monkey cells. TRF from HeLa cells was characterized in detail by electrophoretic mobility shift assays. It binds double-stranded TTAGGG repeats in linear and circular DNAs. Single-stranded repeats are not recognized. The optimal site for TRF appears to contain more than six contiguous TTAGGG repeats. Tandem arrays of TAGGG, TTTAGGG, TTTTAGGG, TTGGGG, and TTAGGC repeats do not bind TRF well, indicating that TRF preferentially recognizes the telomeric repeat sequence present at mammalian chromosome ends. The apparent molecular mass of this factor, based on recovery of TRF from sodium dodecyl sulfate-polyacrylamide gels, is approximately 50 kDa. We suggest that TRF binds along the length of mammalian telomeres.

Structure and variability of human chromosome ends.

Mammalian telomeres are thought to be composed of a tandem array of TTAGGG repeats. To further define the type and arrangement of sequences at the ends of human chromosomes, we developed a direct cloning strategy for telomere-associated DNA. The method involves a telomere enrichment procedure based on the relative lack of restriction endonuclease cutting sites near the ends of human chromosomes. Nineteen (TTAGGG)n-bearing plasmids were isolated, two of which contain additional human sequences proximal to the telomeric repeats. These telomere-flanking sequences detect BAL 31-sensitive loci and thus are located close to chromosome ends. One of the flanking regions is part of a subtelomeric repeat that is present at 10 to 25% of the chromosome ends in the human genome. This sequence is not conserved in rodent DNA and therefore should be a helpful tool for physical characterization of human chromosomes in human-rodent hybrid cell lines; some of the chromosomes that may be analyzed in this manner have been identified, i.e., 7, 16, 17, and 21. The minimal size of the subtelomeric repeat is 4 kilobases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14 kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14 kb long, telomeres in somatic cells are several kilobase pairs shorter and very heterogeneous in length. Additional telomere reduction occurs in primary tumors, indicating that somatic telomeres are unstable and may continuously lose sequences from their termini.

A second chain of human CD8 is expressed on peripheral blood lymphocytes.

Human CD8 has been thought to consist of disulfide-linked homodimers and homomultimers of a single polypeptide chain homologous to mouse and rat CD8 alpha. In contrast, mouse and rat CD8 are composed of disulfide-linked heterodimers of alpha and beta chains. We have now isolated and sequenced cDNA clones encoding a human homologue of mouse and rat CD8 beta. One such clone was inserted into an expression vector and its encoded product was shown to be expressed on the cell surface after cotransfection into L cells with the human CD8 alpha gene. A second form of human CD8 beta cDNA encoding a protein with an altered cytoplasmic tail was similarly transfected, but its product could not be demonstrated on the cell surface. CD8 beta was further shown to be expressed on the surface of almost all CD8+ human peripheral blood T cells. These data provide the first evidence that human CD8 is a heterodimeric protein.

A computational approach to the spectrophotometric determination of stability constants-II Application to metalloporphyrin-axial ligand interactions in non-aqueous solvents.

The ability of the computer program SQUAD to deduce a plausible equilibrium model, associated stability constants and spectra of individual species is described. The original version of SQUAD has been extensively modified and these changes are detailed. In particular a "user-friendly" method of data input has been implemented that simplifies familiarization with the program. Brevity of program code has been sacrificed in favour of the new data input and error-checking features of SQUAD, with beneficial results. The application of SQUAD to five non-aqueous metalloporphyrin-axial ligand interactions exemplifies the program's ability to handle widely different types of equilibrium systems.