Search PubMed⌕ Search

Biomedical subjects

G Damiani

Publications and source records attributed to G Damiani.

At least 73 records · Page 4Linked to original sources
PubMed1988

Characterization, localization, and biosynthesis of acetylcholinesterase in K 562 cells.

K 562 cell acetylcholinesterase (AChE), identifiable by active site labeling with radioactive diisopropylfluorophosphate (DFP), showed a Mr around 55,000 in both a crude lysate and a purified sample. The K 562 AChE was reactive with one polyclonal and two monoclonal antibodies produced against human erythrocyte AChE. Subcellular localization, investigated by assay on cell fractions, showed that AChE is membrane bound and that it is located on the cell surface as well as on microsomal and Golgi membranes. Biosynthesis of new enzyme molecules, after inactivation of the constitutive AChE with the irreversible inhibitor DFP, allowed us to follow the kinetics of reappearance in the intracellular compartment and at the cell surface (4 and 8 h, respectively).

Acetylcholinesterase↗
PubMed1988

Effects of a monoclonal anti-calpain antibody on responses of stimulated human neutrophils. Evidence for a role for proteolytically modified protein kinase C.

A monoclonal antibody directed against the Ca2+-requiring proteinase (calpain) of human neutrophils was employed to assess the role of this proteinase in mediating the responses to stimuli such as phorbol 12-myristate 13-acetate or fMet-Leu-Phe. In the presence of either phorbol 12-myristate 13-acetate or fMet-Leu-Phe the antibody is taken up by the neutrophils, and a marked inhibition of intracellular calpain is observed. The decreased calpain activity is accompanied by (a) a significant decrease in the proteolytic conversion of native protein kinase C (Ca2+/phospholipid-dependent enzyme) to the soluble form that does not require Ca2+ or phospholipids for activity; (b) a marked increase in the production of superoxide anion; and (c) a decrease in the exocytosis of granule contents. The increase in superoxide production can be attributed to a more prolonged association of native protein kinase C with the plasma membrane, thus enhancing the phosphorylation of membrane proteins that precedes O(2-) production (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Sacco, O., and Horecker, B. L. (1986), Biochem. Biophys. Res. Commun. 140, 1121-1126). The decreased exocytosis can be attributed to a decreased phosphorylation of certain cytoskeletal proteins, catalyzed by the soluble form of protein kinase C (Pontremoli, S., Melloni, E., Michetti, M., Sparatore, B., Salamino, F., Sacco, O., and Horecker, B. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3604-3608); the subsequent reorganization of the cytoskeleton appears to be related to degranulation. These effects of the monoclonal anti-calpain provide direct evidence for an essential role for calpain in the activation of human neutrophils.

Animals↗
PubMed1988

The pyrazine-binding protein and olfaction.

1. The present results provide circumstantial evidence, but not a proof, that the Pyrazine-binding Protein is an odorant carrier molecule of fundamental importance. 2. At first sight a role for a secretory protein in olfaction is not obvious. 3. Odorants freely diffuse in air, in water and in lipids, and the use of carrier proteins, would seem superfluous unless a very special combination with the odorant occurs [Gaupp E. (1902) In Anatomie des Frosches, 2nd Edn, pp. 673. Vieweg-Verlag, Braunschweig]. 4. The possibility should be considered that the Pyrazine-binding Protein and the urinary proteins belong to a large family of species-specific secretory molecules which, with the odorant bound, directly stimulate the receptor cell.

Animals↗
PubMed1988

Vincristine-resistant erythroleukemia cell line has marked increased sensitivity to hexamethylenebisacetamide-induced differentiation.

Hexamethylenebisacetamide (HMBA)-induced murine erythroleukemia (MEL) differentiation is a multistep process. Commitment is the capacity to express terminal cell division and characteristics of the differentiated phenotype even after the cells are removed from culture with inducer. Culture of MEL cell line 745A.DS19 (DS19) with HMBA causes commitment to terminal differentiation after a latent period of about 10-12 hr. Previous studies have shown that during this latent period, HMBA causes a number of metabolic changes, including modulation in expression of certain protooncogenes. We now report the development of a MEL cell line (designated V3.17) derived from DS19 that is resistant to vincristine and is (i) markedly more sensitive to HMBA, (ii) induced to commitment without a detectable latent period, and (iii) resistant to the effects of phorbol ester and dexamethasone, which are potent inhibitors of HMBA-mediated DS19 differentiation. We suggest that this V3.17 MEL cell line may express a factor that circumvents HMBA-mediated early events, which prepare the cells for commitment to terminal differentiation.

Acetamides↗
PubMed1988

Generation and characterization of monoclonal antibodies to 28-, 35-, and 65-kilodalton proteins of Mycobacterium tuberculosis.

Three monoclonal antibodies (H60.15, H61.3, and H105.10) directed to protein antigens of Mycobacterium tuberculosis were obtained and characterized. H60.15 recognizes a protein with a molecular mass of 28 kilodaltons (kDa) with broad cross-reactivity on a panel of 12 species and strains of mycobacteria. H61.3 reacts with a 35-kDa protein present in M. tuberculosis, Mycobacterium bovis BCG, and M. africanum. On the basis of the antigen molecular masses and competition experiments with other monoclonal antibodies, H60.15 and H61.3 seem to be the first described monoclonal antibodies to these M. tuberculosis proteins. H105.10 binds to the cross-reactive 65-kDa protein present in mycobacteria. Epitope mapping of H105.10 was performed by using the M. leprae DNA sublibrary available in bacteriophage lambda gt11 for this antigen and revealed that its epitope resides in the region from amino acids 20 to 54. The 28-, 35-, and 65-kDa antigens isolated by immunoblotting and presented on nitrocellulose to pleural effusion T cells from tuberculosis patients induced a proliferative response, indicating the presence of T-cell epitopes. These observations indicate that two protein antigens should be added to the list of antigens detectable in M. tuberculosis by monoclonal antibodies. The common feature of such proteins, the elicitation of an immune response of limited or broad cross-reactivity for mycobacteria, encourages the search for their role in the pathogenesis of mycobacterioses.

Animals↗
PubMed1988

Human peripheral blood and pleural fluid eosinophils can be induced by immune complexes to release IgG immune complexes and aggregated IgE.

The ability of IgG and IgE immune complexes and of phorbol myristate acetate (PMA), a soluble membrane activator, to stimulate hydrogen peroxide (H2O2) release and to induce oxygen radical-mediated cytotoxic activity by human peripheral blood (PBL) eosinophils and by PBL neutrophils was evaluated in normal volunteers and patients with hypereosinophilic malignant pleural effusions due to lung cancer. PMA stimulated a significant respiratory burst. Similar results were obtained with IgG IC stimulation, although the levels of H2O2 were lower. Agg IgE induced H2O2 release only by PBL and PE eosinophils and not by neutrophils. PMA stimulation resulted in detectable cytotoxic activity. IgG IC generated both PBL and PE eosinophil and PBL neutrophil cytotoxicity. Agg IgE induced significant cellular cytotoxicity in both PBL and PE eosinophils. This study suggests that eosinophil oxidative metabolic burst and cytotoxic activity stimulated by IgG and IgE immune complexes could represent a possible mechanism of parenchymal injury in eosinophilic disorders.

Adult↗
PubMed1988

Monoclonal antibodies: modulation of cellular activities and identification of heterogeneity of functional response in human neutrophils.

We have studied the effect of the two antibodies, MF 25.1 and GF 26.7.3, on leucocyte function: these antibodies did not mimic the effect of cellular stimuli, but the pre-treatment of neutrophils with MF 25.1 and GF 26.7.3 modulated several cell activities. Both antibodies significantly reduced superoxide anion production in response to various stimuli. Despite similar cellular response MF 25.1 and GF 26.7.3 bound to different antigenic determinants, as shown both by immunoblotting studies and by treating neutrophils first with GF 26.7.3 and subsequently with MF 25.1, and vice-versa; the inhibition of superoxide production corresponded, in fact, to the total of that seen for each antibody. Moreover, in the absence of attractant, measures of locomotion of neutrophils treated with monoclonal antibody showed that 43% of the subjects responded to antibody binding by twofold activation of spontaneous movement. Chemotaxis and lysosomal enzyme release were not affected by antibody treatment.

Antibodies, Monoclonal↗
PubMed1987

Sequence and functional analysis of a divergent promoter from a cryptic plasmid of Lactobacillus acidophilus 168 S.

We have characterized three of at least five plasmids borne by Lactobacillus acidophilus 168 S. Restriction mapping indicates extensive sequence homology between at least two of them (p1 and p3). We have cloned them in Escherichia coli, and for the smallest (p1) we present the sequence of a region with two divergently arranged promoters which probably share a symmetrical (TTTAAA)-35 box and function efficiently in E. coli cells; an open reading frame contiguous to the promoter, which codes for a 120 amino acid protein of unknown function, and is transcribed in E. coli; and a transcription termination sequence next to this open reading frame. The promoter region contains an AT cluster which is similar to that of the ori2 region of the E. coli F plasmid, and is probably involved in the control of the replication of p1.

Base Sequence↗
PubMed1987

Lactobacillus protoplast transformation.

A method for the transformation of Lactobacillus protoplasts by plasmid DNA is reported. The procedure involves polyethylene glycol treatment of protoplasts to induce DNA uptake. A transformation efficiency ranging from 5 to 1000 transformants per microgram of DNA is achieved; the efficiency of protoplast regeneration ranged from 10 to 20%.

DNA, Bacterial↗
PubMed1986

Deficiency of neutrophil membrane antigen detected by monoclonal antibody in rheumatoid arthritis.

Monoclonal antibodies (mAbs) against cell surface antigens and receptors are instrumental in defining specific membrane markers. mAbs GF 26.7.3 and MF 25.1 against human neutrophils modulated the activation mechanism of superoxide anion production induced by formyl-peptide and PMA in all subject. However, treatment with mAb MF 25.1 of neutrophils from patients with rheumatoid arthritis did not have any effect. This may suggest that the antigen which MF 25.1 binds is absent in rheumatoid conditions. This confirms our previous data showing that defective expression of membrane components is associated with neutrophil dysfunction.

Antibodies, Monoclonal↗
PubMed1986

Heterologous expression in Bacillus subtilis. II. In vitro removal of the attenuator sequence of the Escherichia coli his operon allows expression of the cloned hisG gene in B. subtilis.

The promoter-proximal region of the Escherichia coli histidine (his) operon, including the promoter, the attenuator and the hisG gene, as well as the first of the nine structural genes of the his operon, have been cloned in Bacillus subtilis. In this host, the hisG gene could not be expressed because its transcription appeared to be irreversibly terminated at the attenuator (Ferretti et al., 1984). When the attenuator plus various lengths of the two bordering regions were removed, one of the attenuatorless sequences cloned in B. subtilis allowed the progression of transcription and complementation of the corresponding hisA mutation in this Gram-positive host. The deletion removed a 349-bp segment which contained the his attenuator and promoter. In B. subtilis, the productive transcription of the hisG gene started at a site in pAT153 and terminated in pC194. Sequence analysis of the deletion indicates that the E. coli ribosome-binding site of the his operon was used for the translation of the E. coli hisG gene mRNA in B. subtilis cells, which can thus grow in the absence of histidine.

Bacillus subtilis↗
PubMed1986

Cytolytic effects of neutrophils: role for a membrane-bound neutral proteinase.

A neutral serine proteinase, purified 250-fold from the plasma membrane fraction of human neutrophils, differs in its catalytic and molecular properties from the well-known neutral proteinases present in azurophil (primary) granules. Stimulation of neutrophils with low concentrations of phorbol 12-myristate 13-acetate (PMA) results in the release into the medium of the membrane-bound proteinase and the concomitant production of oxygen radicals. These concentrations of PMA also induce full cytolytic activity measured with 51Cr-labeled ox erythrocytes. A role for the neutral serine proteinase in the cytolytic activity of PMA-stimulated neutrophils is supported by the following observations: (i) the lytic activity of the stimulated neutrophils is correlated with the quantity of neutral proteinase present in the membranes; (ii) the extracellular medium from PMA-stimulated neutrophils causes the cytolysis of 51Cr-labeled erythrocytes that have been exposed to nonlytic concentrations of H2O2; (iii) cytolysis of H2O2-treated erythrocytes is also observed with the crude proteinase solubilized from neutrophil membranes or with the purified proteinase from the same source; and (iv) in each case the cytolytic activity is proportional to the proteinase activity present and is prevented by the addition of serine proteinase inhibitors. We conclude that cytolysis of target cells by PMA-activated neutrophils can result from the cooperative effects of oxygen radicals and the membrane-bound neutral serine proteinase. The participation of enzymes from specific (secondary) granules is excluded because, with the low concentrations of PMA employed, very little release of secondary granule constituents is observed.

Cytotoxicity, Immunologic↗
PubMed1986

Alveolar macrophage stimulation of T-cell proliferation in autologous mixed lymphocyte reactions. Role of HLA-DR antigens.

Alveolar macrophages act as accessory cells in lymphocyte response to mitogens or alloantigens. Because the autologous mixed lymphocyte reaction (MLR), in which HLA-DR-positive non-T cells stimulate the proliferation of autologous T lymphocytes, represents a good model to study macrophage-T cell interaction, we examined and compared the ability of human alveolar macrophages and peripheral blood-derived monocytes to induce T-cell proliferation in autologous MLR. Maximal T lymphocyte proliferation was observed in both alveolar-macrophage- and blood-monocyte-stimulated autologous MLR at a T cell to alveolar macrophage or blood monocyte ratio of 4:1, but the ability to stimulate T-cell proliferation was lower for alveolar macrophages than for blood monocytes (p less than 0.01). Because HLA-DR antigens modulate monocyte-T cell interaction, we quantified the proportions of HLA-DR-positive cells in alveolar macrophage and blood monocyte suspensions and determined the inhibitory effects on T-cell proliferation of masking HLA-DR antigens on stimulator cells with monoclonal antibodies. The proportions of HLA-DR-positive cells were higher in alveolar macrophage than in blood suspensions (p less than 0.01); interestingly, however, the preincubation of the stimulator cells with anti-HLA-DR monoclonal antibodies inhibited to a similar extent both alveolar-macrophage- and blood-monocyte-stimulated autologous MLR (p greater than 0.2). These studies indicate that alveolar macrophages are less effective than blood monocytes are as stimulator cells in autologous MLR and that, although the masking of HLA-DR molecules results in inhibition of autologous MLR, T-cell proliferation is not dependent on the numbers of stimulator cells bearing HLA-DR antigens. The autologous MLR may represent a good model to study the functions of alveolar macrophages during their interaction with autologous T cells in health and disease.

Adult↗
PubMed1986

Effect of a monoclonal antibody against GPs IIb-IIIa on platelet aggregation and ATP secretion.

A murine monoclonal antibody against glycoproteins IIb-IIIa of platelet membrane completely abolished platelet aggregation induced by epinephrine, arachidonic acid, and low concentration of collagen and thrombin, but it had only minor inhibitory effects on aggregation induced by ADP, higher amounts of collagen and thrombin, and these agents combined in pairs. The simultaneous studies of aggregation and ATP secretion demonstrated that aggregation plays an essential role in stimulating the platelet-release reaction, except for the thrombin-induced ATP secretion that seems to be largely dependent on fibrinogen binding to platelet surface.

Adenosine Triphosphate↗
PubMed1985

Role of class II histocompatibility antigens in Staphylococcus aureus protein A-induced activation of human T lymphocytes.

The capacity of peripheral blood monocytes and B lymphocytes to support staphylococcal protein A (SpA)-induced proliferation of autologous and allogeneic T cells, as well as the role of major histocompatibility complex (MHC) class I and II molecules in this activation process, were investigated. Highly purified peripheral T lymphocytes did not proliferate in response to SpA, but their response was reconstituted by both irradiated (or mitomycin C-treated) monocytes and B lymphocytes. The effect of B cells on the SpA-induced T-cell response could not be explained by a contamination of residual accessory cells because long-term continuous B-cell lines restored SpA-induced T-cell DNA synthesis as effectively as did monocytes. Support of SpA responsiveness by B cells could not be accounted for by polyclonal binding of SpA to cell surface immunoglobulins, since the ability of SpA-unreactive and SpA-reactive B cells was comparable. The cells from two human leukemic lines--K562 and Raji--showed the same ability in supporting the pokeweed mitogen-induced T-cell response, but the class II-positive Raji cells were much more effective than class II-negative K562 cells in restoring the T-cell responsiveness to SpA. Monoclonal antibodies specific for monomorphic determinants of MHC class II antigens, as well as their F(ab')2 fragments, consistently inhibited the SpA-induced proliferative response, whereas antibodies specific for MHC class I antigens were without effect. The antibodies specific for class II antigens appeared to act at the level of accessory cell, since pretreatment with these antibodies inhibited the ability of SpA-pulsed monocytes or Raji cells to present SpA to autologous or allogeneic T lymphocytes, respectively. These data indicate that either monocytes or normal and lymphoblastoid B cells can act as accessory cells for the proliferative response of human T cells to soluble SpA and that monomorphic determinants of MHC class II molecules play an important role in this activation process.

Histocompatibility Antigens↗
PubMed1985

Establishment and characterization of a new human cloned myelomonocytic cell line (ZC-1.6).

A human cell line, ZC, derived from the blood of a patient with acute myelomonocytic leukemia, was established and cloned. One of the clones, ZC-1.6, was subsequently characterized. As for its morphology and cytochemistry, ZC-1.6 clone shares a number of features with immature cells of the monocytic or myelocytic lineages. Surface marker analysis shows positivity for 4F2 (100% of the cells), and OKM1 (38%) monoclonal antibodies, and presence of surface HLA-D/DR antigens (100%) and Fc (11%) and complement (C3b) receptors (100%). Functional capabilities of ZC-1.6 cells include adherence, spreading, and phagocytosis of latex and opsonized zymosan particles. Despite its morphological immaturity, the ZC-1.6 clone produces relevant amounts of O2- in the presence of different stimuli (phorbol myristate acetate, opsonized zymosan, or latex particles). The production of O2- by ZC-1.6 cells is the first evidence that reactive oxygen intermediate production may represent an early feature of cells of the myelomonocytic lineage.

Adult↗