Biomedical subjects
Publications and source records attributed to R D'Andrea.
Occupational stress may be the cause of psychosomatic and mental disorders. A development of conflicts into a negative direction may trigger a mobbing behaviour at workplace and a mobbing syndrome on the victims. On account of the frequency of mobbing activities at work, it is to assert the need of preventive interventions.
Human interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific alpha chain and a shared subunit (beta(c)). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor alpha chains is the first step in receptor activation, it is the recruitment of beta(c) that allows high-affinity binding and signal transduction to proceed. Thus, beta(c) is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of beta(c). BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of (125)I-IL-5, (125)I-GM-CSF, and (125)I-IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of beta(c). Interestingly, epitope analysis using several beta(c) mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of beta(c), suggesting that ligand contact with beta(c) is a prerequisite for recruitment of beta(c), receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.
BACKGROUND: Since 1993 the 13 VHA Southern New England (VHA-SNE) hospitals have been engaged in a regionally sponsored initiative to analyze and improve selected clinical processes. Nine of these hospitals have chosen to participate in an initiative in which observation units were postulated to offer a tool for improving the care of patients with chest pain-the VHA initiative to Implement Chest Pain Treatment in Observation Units. THE FIVE PHASES: In phase 1 of the initiative, the VHA-SNE's Clinical Benchmarking Work Group reviewed the medical literature, which confirmed longstanding systemic and pervasive problems in the evaluation of chest pain patients. The work group's preferred practice was the outpatient "rule out myocardial infarction [MI] evaluation" program during monitored observation; serial testing can accurately diagnose low- and moderate-probability patients with MI. In Phase 2 the study group surveyed the emergency departments in the nine hospitals, discovering significant variation in admission rates and practice patterns. During phase 3 the work group identified a health care organization demonstrating best-practice performance--one of the few hospitals in the nation with an operational outpatient "rule out MI evaluation" program. A team site-visited that organization and recorded information about its structure and processes. VHA-SNE then published a monograph that identified its current performance, described the best-practice approach, offered strategies to implement the model program, and analyzed the financial implications and return on investment. In phase 4 a pilot hospital implemented the model program, which in phase 5 is being extended to the other hospitals represented in the work group. Information regarding protocols, lessons learned, and barriers to implementation was freely provided.
We have combined retroviral expression cloning with random mutagenesis to identify two activating point mutations in the common signal-transducing subunit (h beta c) of the receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 by virtue of their ability to confer factor independence on the haemopoietic cell line, FDC-P1. One mutation (V449E) is located within the transmembrane domain and, by analogy with a similar mutation in the neu oncogene, may act by inducing dimerization of h beta c. The other mutation (I374N) lies in the extracellular, membrane-proximal portion of h beta c. Neither of these mutants, nor a previously described mutant of h beta c (FI delta, which has a small duplication in the extracellular region), was capable of inducing factor independence in CTLL-2 cells, while only V449E could induce factor independence in BAF-B03 cells. These results imply that the extracellular and transmembrane mutations act by different mechanisms. Furthermore, they imply that the mutants, and hence also wild-type h beta c, interact with cell type-specific signalling molecules. Models are presented which illustrate how these mutations may act and predict some of the characteristics of the putative receptor-associated signalling molecules.
Homeodomain (HD)-containing proteins have been shown to regulate cellular commitment and differentiation in fungal, invertebrate and vertebrate systems. Bone marrow cells synthesizing the CD34 antigen are a complex mix of early, stem and progenitor cells at various stages of commitment to the many haemopoietic lineages. Here, we report the cloning and sequencing of 31 homeobox (HB) sequences, identified using degenerate oligodeoxyribonucleotide primers, in a polymerase chain reaction with cDNA derived from a purified CD34+ population of human haemopoietic cells. Of these sequences, 16 correspond to previously identified genes, and 13 are located within the HOX A, B and C clusters. Ten of the clones most likely represent human homologues of genes identified previously in other species. Five of the clones reported here represent novel HD sequences. The identification of five new genes using a subclass-specific 5' primer, designed from the engrailed and Xanf1 sequences, suggests that there still remain several uncharacterized HB genes in the human genome. Haemopoietic cells purified on the basis of CD34 antigen synthesis are a rich source of regulatory genes consistent with their ability to differentiate into diverse haemopoietic cell types.
The cytokines interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor bind with high affinity to a receptor complex that contains a ligand-specific alpha-chain and a common beta-chain, h beta c. We report here the isolation of a mutant form of h beta c, from growth factor-independent cells, that arose spontaneously after infection of a murine factor-dependent hematopoietic cell line (FDC-P1) with a retroviral h beta c expression construct. Analysis of this h beta c mutation shows that a small (37 amino acid) duplication of extracellular sequence that includes two conserved sequence motifs is sufficient to confer ligand-independent growth on these cells and lead to tumourigenicity. Because this is a conserved region in the cytokine receptor superfamily, our results suggest that the large family of cytokine receptors has the capacity to become oncogenically active.
We have previously reported that the predicted first helix of human interleukin (IL)-3 contains a hydrophilic region encompassing residues Asp21, Glu22, and Thr25 that is crucial for biological activity and IL-3 receptor binding. Using single amino acid substitution mutagenesis, we have now determined that Asp21 and Glu22, but not Thr25, were crucial for full IL-3 activity. Mutant D21R was 30-fold less potent than wild type IL-3 in the stimulation of biological activity. It also exhibited a similar reduction in its ability to bind to the cloned high affinity IL-3 receptor complex (alpha- and beta-chains) or to the receptor alpha-chain alone, indicating that residue 21 is involved in contacts with the alpha-chain. Mutant E22R was approximately 20,000-fold less potent than wild type IL-3 in the stimulation of biological activity and in binding to the IL-3 receptor high affinity complex. However, the binding of E22R to the IL-3 receptor alpha-chain alone was similar to that of wild type IL-3, suggesting that this mutant was defective in interactions with the receptor beta-chain. These results show that two contiguous residues in the N-terminal region of IL-3 mediate binding to the two different chains of the IL-3 receptor and emphasize the functional significance of the conserved Glu in the first helix of the IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 cytokine subfamily.
The Saccharomyces cerevisiae MET17/MET25 gene encoding O-acetyl-L-serine (OAS).O-acetyl-L-homoserine (OAH) sulfhydrylase (EC 4.2.99.10) was overexpressed in Escherichia coli and the gene product was purified to homogeneity, using three steps, with a recovery of 28% from the total cell extract. The gene product has been compared with OAS.OAH sulfhydrylase purified from the yeast cells. These two protein preparations were indistinguishable with respect to their behavior in polyacrylamide gel electrophoresis, both with and without sodium dodecyl sulfate, their specificity for substrate amino acids, Michaelis constant (Km) value for OAH, sensitivity to carbonyl reagents, absorption spectrum, isoelectric point, behavior in HPLC (both ion-exchange chromatography and gel filtration), sensitivity to heat treatment, susceptibility to trypsin digestion, and their N-terminal amino acid sequence. The results obtained imply that the gene product is properly processed in E. coli, and the technique developed in this study to overexpress the gene in bacterial cells provides us with a large amount of the purified preparation of the enzyme. In contrast to a previous report we found that cystathionine gamma-lyase of S.
The Polycomblike gene of Drosophila melanogaster, a member of the Polycomb Group of genes, is required for the correct spatial expression of the homeotic genes of the Antennapaedia and Bithorax Complexes. Mutations in Polycomb Group genes result in ectopic homeotic gene expression, indicating that Polycomb Group proteins maintain the transcriptional repression of specific homeotic genes in specific tissues during development. We report here the isolation and molecular characterisation of the Polycomblike gene. The Polycomblike transcript encodes an 857 amino acid protein with no significant homology to other proteins. Antibodies raised against the product of this open reading frame were used to show that the Polycomblike protein is found in all nuclei during embryonic development. Antibody staining also revealed that the Polycomblike protein is found on larval salivary gland polytene chromosomes at about 100 specific loci, the same loci to which the Polycomb and polyhomeotic proteins, two other Polycomb Group proteins, are found. These data add further support for a model in which Polycomb Group proteins form multimeric protein complexes at specific chromosomal loci to repress transcription at those loci.
We cloned the MET 17 gene of Saccharomyces cerevisiae by functional complementation after transformation of a yeast met 17 mutant. Restriction mapping and nucleotide sequencing of the MET 17 clones revealed that these were from the same genomic region as clones isolated previously and shown to contain the MET 25 gene encoding the enzyme O-acetylhomoserine, O-acetylserine sulphydrylase (OAH-OAS sulphydrylase). Transformation studies with MET 25 clones showed that the MET 17 and MET 25 functions were both endoced in a single transcription unit. We conclude that met 17 and met 25 are both mutations in the structural gene for the OAH-OAS sulphydrylase subunit and that each affects a different functional domain of the enzyme allowing subunit complementation in the met 17 X met 25 diploid. Enzyme assays indicated that the diploid, although not requiring methionine, had a low OAH-OAS sulphydrylase activity (10% of wild type). This is consistent with MET 17 and MET 25 being the same gene. We found that both met 17 and met 25 mutants were devoid of 3' phospho-adenosine 5' phospho-sulphite (PAPS) reductase activity and that this activity was fully restored in the met 17 X met 25 diploid. The possible interactions between OAH-OAS sulphydrylase and PAPS reductase are discussed.
An H5 cDNA clone was used to select H5 genomal recombinants from a chicken Charon 4A library. DNA sequence analysis shows that the H5 gene contains no introns. Putative 5' promoter elements and a 3' polyadenylation site are present within the 1.8 kb of DNA examined. Analysis of 41 kb of DNA surrounding the H5 gene shows that it is not closely linked to either H1 or core histone genes.Images
The DNA sequence of a chicken genomal fragment containing a histone H2A gene has been determined. It contains extensive 5' and 3' flanking regions and encodes a protein identical in sequence to the histone H2A protein isolated from chicken erythrocytes. In the 5' flanking region, a possible "TATA box" and three possible "cap sites" can be recognised upstream from the initiation codon. To the 5' side of the "TATA box" is found an unusual sequence of 21 A's interrupted by a central G residue. It occupies the same relative position as the P. miliaris H2A gene-specific 5' dyad symmetry sequence and the "CCAAT box" seen in other eukaryotic polymerase II genes but is clearly different from both. A significant feature of the 3' non-coding region is the presence of a 23 base-pair sequence that is nearly identical to a conserved region found in sea urchin histone genes. The coding region is extremely GC rich, with strong selection for these bases in the third position of codons. Not a single coding triplet ends in U. No intervening sequences were found in this gene.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The AA. examines different aspects concerning the health of female workers. The following aspects are taken into consideration: sex distribution, general and specific morbidity, work pathology, work accidents, sickness and maternity absences, family life. The AA. recalls the importance of appropriate preventive measures, such as organization of apprenticeship and health education programs. In this connection, the AA. puts forward some proposals which might be useful for a new regulation in the matter and, as a conclusion, stresses the importance of an efficient Public Health Service expecially in this field.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The following aspects are taken into consideration: health, work activity, family life, social life. The A. recalls the importance of preventive measures. Therefore must actued prevention activities to protect women health in accomplishment both Community regulation and Italian legislation.