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Biomedical subjects

A Rubartelli

Publications and source records attributed to A Rubartelli.

At least 37 records · Page 2Linked to original sources
PubMed1996

Nerve growth factor is an autocrine survival factor for memory B lymphocytes.

Production of nerve growth factor (NGF) was assessed in cultures of human T and B lymphocytes and macrophages. NGF was constitutively produced by B cells only, which also expressed surface p140trk-A and p75NGFR molecules and hence efficiently bound and internalized the cytokine. Neutralization of endogenous NGF caused disappearance of Bcl-2 protein and apoptotic death of resting lymphocytes bearing surface IgG or IgA, a population comprising memory cells, while surface IgM/IgD "virgin" B lymphocytes were not affected. In vivo administration of neutralizing anti-NGF antibodies caused strong reduction in the titer of specific IgG in mice immunized with tetanus toxoid, nitrophenol, or arsonate and reduced numbers of surface IgG or IgA B lymphocytes. Thus, NGF is an autocrine survival factor for memory B lymphocytes.

Animals↗
PubMed1996

HIV-tat protein is a heparin-binding angiogenic growth factor.

Transgenic animal studies have linked the expression of the HIV-1 tat gene to the appearance of Kaposi's sarcoma (KS)-like lesions. We have recently shown that recombinant tat is angiogenic in vivo, and that tat angiogenic response is enhanced by heparin. Also in the rabbit cornea model, recombinant HIV-1 tat alone is poorly angiogenic, but gives a good response when combined with heparin. Like many angiogenic growth factors, tat has a basic domain similar to that of several heparin binding angiogenic factors, including FGF, VEGF and HGF, suggesting that this region is important in endothelial cell activation. We show that tat binds heparin sepharose with a high affinity, similar to bFGF. Binding of tat to the cell surface is also modulated by heparin. Biological activities of tat, such as induction of endothelial cell growth, migration and invasion in vitro are all enhanced by low concentrations and inhibited by high concentrations of heparin, as has been shown for other heparin-binding angiogenic factors. Heparan sulfate is also effective, whereas the unsulfated polysaccharide K5 does not enhance tat activity. Furthermore, a peptide encompassing the tat basic domain is able to induce growth and migration of endothelial cells, while an adjacent peptide is not. Our data indicate that the tat basic domain plays a key role in its vascular cell activation properties, and strongly suggest that extracellular HIV-tat is essentially a 'new' heparin-binding angiogenic factor.

Amino Acid Sequence↗
PubMed1995

High rates of thioredoxin secretion correlate with growth arrest in hepatoma cells.

Thioredoxin (TRX), a disulfide-reducing intracellular dithiol enzyme, is synthesized by both normal liver cells and the hepatocarcinoma cell line HepG2. Only the former, however, secrete abundant TRX extracellularly. When cultured in mild reducing conditions, HepG2 cells but not normal hepatocytes increase the rate of TRX secretion and undergo growth inhibition accompanied by morphological changes. Also, recombinant TRX inhibits proliferation of HepG2 cells. In contrast, exogenous thiols and TRX stimulate proliferation of a B-cell lymphoma line, indicating that different cell types respond differently to variations in the extracellular redox potential.

Acetylcysteine↗
PubMed1995

Synthesis and secretion of interleukin-1 alpha and interleukin-1 receptor antagonist during differentiation of cultured keratinocytes.

Keratinocytes produce interleukin-1 alpha (IL-1 alpha) and the epithelial variant of its inhibitor, interleukin-1 receptor antagonist (icIL-1ra). Both IL-1 alpha and icIL-1ra lack a secretory signal peptide; however, some icIL-1ra is found in the supernatants of cultured keratinocytes. The lack of correlation with the release of the cytosolic enzyme lactate dehydrogenase suggests that icIL-1ra can be actively secreted. Brefeldin A fails to block icIL-1ra release, suggesting that this protein may be externalized by keratinocytes through a leaderless pathway of secretion. Only minute amounts of soluble extracellular IL-1 alpha are detected: however, both IL-1 alpha and icIL-1ra can be released from the external face of the keratinocyte plasma membrane by mild acidic treatment, suggesting that IL-1 alpha can also be secreted by keratinocytes. The observation of membrane-associated IL-1 alpha and icIL-1ra might reflect an autocrine loop of regulation. Support for this hypothesis comes from the finding that keratinocytes, when exposed to exogenous recombinant IL-1 alpha, increase their content in both IL-1 alpha and IL-1ra mRNA. When keratinocytes are subjected to counterflow centrifugal elutriation, three major cell populations are obtained, representing three different degrees of keratinocyte differentiation. Cells from all populations synthesize IL-1 alpha and IL-1ra: however, while IL-1 alpha is uniformly distributed in cells from all maturational stages, IL-1ra accumulates in large, more differentiated keratinocytes. Changes in the ratio of IL-1ra to IL-1 alpha production and secretion by keratinocytes at different degrees of maturation might contribute to the control of growth and differentiation of human skin.

Cell Differentiation↗
PubMed1995

Entry of exogenous polypeptides into the nucleus of living cells: facts and speculations.

Although the plasma membrane acts as an impermeable barrier to most macromolecules, some exogenous proteins (for example fibroblast growth factor, HIV-1 Tat and lactoferrin) can gain access into the cytosol and reach the nucleus of living cells. How are these exogenous polypeptides selected over and above other extracellular proteins? How and where do they cross the cell membrane? Why do cells need to take up exogenous transcription factors when sophisticated signal-transduction pathways are available? Here, we review the current knowledge on these issues and discuss some mechanistic and physiological implications of this unconventional and direct way of taking messages to the nucleus.

Journal Article↗
PubMed1995

Nuclear translocation of an exogenous fusion protein containing HIV Tat requires unfolding.

OBJECTIVE: To characterize the transcellular transport of HIV-1 Tat. HIV-1 Tat contains a putative localization signal and no leader peptide; however, it can be released from virus-infected cells and taken up by uninfected cells. DESIGN AND METHODS: We constructed a chimeric protein between Tat and dihydrofolate reductase (DHFR), a cytosolic enzyme that binds tightly to the folate analogue methotrexate (MTX). As confirmed by protease sensitivity assays, binding to MTX results in stabilization of the three-dimensional structure of the DHFR domain. The nuclear translocation of recombinant proteins was monitored by both functional [transcellular transactivation of a long terminal repeat-chloramphenicol acetyl transferase (LTR-CAT) reporter gene] and biochemical (subcellular localization in HeLa cells of exogenous radiolabelled proteins) assays and the effects of MTX-induced stabilization were evaluated. RESULTS: When in vitro translated proteins are added to HeLa cells in culture, both wild-type Tat and the chimeric protein Tat-DHFR are taken up by target cells and accumulate in the nucleus, unlike wild-type DHFR. Cells transfected with Tat-DHFR, when co-cultured with cells harbouring a LTR-CAT gene, induce transactivation of the reporter gene to the same extent as cells expressing wild-type Tat. These findings indicate that Tat can mediate the internalization of unrelated polypeptides. Pre-treatment of Tat-DHFR with MTX blocks the nuclear translocation of the chimeric protein. MTX has no effect on wild-type Tat. CONCLUSION: HIV-1 Tat can act as a vector to drive polypeptides into the nucleoplasm of living cells. The inhibitor effects of MTX on the nuclear localization of Tat-DHFR suggest that an unfolding step is required for the internalization of exogenous Tat.

Cell Nucleus↗
PubMed1993

Post-translational regulation of interleukin 1 beta secretion.

In view of the key role played by interleukin 1 (IL-1) beta in inflammation, its production is likely to be precisely regulated. Previous studies have shown that IL-1 beta biosynthesis is controlled at the transcriptional and translational levels. We have investigated whether post-translational events also play a role in regulating the production of bioactive IL-1 beta. IL-1 beta, which lacks a signal sequence, is released by activated monocytes through a novel pathway of secretion, alternative to the classical endoplasmic reticulum-Golgi route. Secretion of mature 17 kDA IL-1 beta is increased when pulse-labelled activated monocytes are chased in the presence of heat-aggregated immunoglobulins or of various drugs. Febrile temperatures inhibit secretion of mature IL-1 beta, but only reduce its synthesis: treatment with cycloheximide restores secretion. Processing of the 33 kDa precursor to the 17 kDa mature molecule is inhibited when the external pH is 8 or higher: under these conditions, release of unprocessed, biologically inactive 33 kDa IL-1 beta is observed. Thus, secretion of IL-1 beta is regulated by post-translational mechanisms which operate at the level of both proteolytic processing and extracellular export.

Biological Transport↗
PubMed1993

Production and secretion of interleukin 1 receptor antagonist in monocytes and keratinocytes.

IL-1 receptor antagonist (IL-1ra) is a newly described member of the IL-1 family, isolated from supernatants of Ig stimulated monocytes, that binds competitively to IL-1 receptors without stimulating target cells. Also epithelial cells produce IL-1ra in a form which lacks a secretory signal sequence. Here we have compared the biosynthesis and secretion of IL-1ra in monocytes and keratinocytes. Our data show that monocytes produce two molecular forms of IL-1ra, of 18 Kd and 23 Kd respectively, which differ in the degree of glycosylation. Both forms are secreted via the "classical" endoplasmic reticulum (ER)-Golgi secretory pathway. By contrast keratinocytes produce IL-1ra in a molecular form of 20 Kd, which is not N-glycosylated: 20 Kd IL-1ra is detectable in supernatants of keratinocytes, although in small amounts. The presence of IL-1ra in keratinocytes cultures fluids is not inhibited by Brefeldin A (BFA), suggesting a possible secretion through the leaderless secretory pathway.

Biotechnology↗
PubMed1992

Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway.

Thioredoxin, despite its function as an intracellular disulfide reducing enzyme and its lack of a signal sequence, has been found to play some roles extracellularly. Here we show that thioredoxin is actively secreted by a variety of normal and transformed cells, including fibroblasts, airway epithelial cells, and activated B and T lymphocytes. Neither brefeldin A nor dinitrophenol, two drugs that block transport through the exocytic pathway, inhibit secretion of thioredoxin, indicating that the latter does not follow the classical ER-Golgi route. The secretory mechanism for thioredoxin shares several features with the alternative pathway described for interleukin-1 beta, such as the potentiating effect on secretion of several unrelated drugs and the sensitivity to methylamine. However, unlike interleukin-1 beta, thioredoxin is not detected in membrane-bound compartments of secreting cells. In addition, when COS7 are transfected with plasmids encoding pro-interleukin-1 beta or thioredoxin, only the latter is detectable extracellularly.

Animals↗
PubMed1990

Interleukin-1 and interleukin-2 control granulocyte- and granulocyte-macrophage colony-stimulating factor gene expression and cell proliferation in cultured acute myeloblastic leukemia.

In vitro proliferation of leukemic cells purified from 10 cases of acute myeloblastic leukemia (AML) was analyzed in basal conditions or in the presence of exogenous recombinant (r) Interleukin (IL) 1. In parallel, blasts from 5 of these patients were studied for granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte-CSF (G-CSF) mRNA. IL-1 augmented the spontaneous AML cell proliferation in all cases and induced de novo expression or increased amounts of GM-CSF and/or G-CSF transcripts in 4 of the 5 cases evaluated. IL-1-induced AML cell proliferation was modulated by neutralizing anti-GM-CSF or anti-G-CSF antibodies in those cases in which CSF mRNAs were induced or increased by exogenous cytokine. In the same cases, biosynthetic labelling and immunoprecipitation studies using monospecific anti-GM-CSF antibodies showed that IL-1 also increased the levels of GM-CSF protein synthesis. Addition of neutralizing anti-IL-1 antibodies to AML cell cultures completely abolished ongoing GM-CSF synthesis, suggesting that endogenous IL-1 is needed to maintain autocrine production of CSFs. The effects of rIL-2 were investigated in a larger series of 21 patients. The cytokine reduced spontaneous AML cell proliferation in 8 cases. It caused complete disappearance of GM-CSF mRNA in 1 case, and marked reduction of G-CSF mRNA in 2 cases. Increased AML cell proliferation was observed in 2 of 21 cases. These findings suggest that expression of CSF genes and cell proliferation in AML are under the control of different cytokines acting in autocrine or paracrine fashion.

Antibodies↗
PubMed1990

A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence.

Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.

Brefeldin A↗
PubMed1990

A novel pathway for secretory proteins?

In eukaryotes, most proteins which are transported to the extracellular space, into mitochondria or into chloroplasts are synthesized as precursor polypeptides containing cleavable N-terminal signal or targeting sequences. We have searched the literature for proteins that are exported from the cytosol without being proteolytically processed. Some of these proteins contain uncleaved signal or targeting sequences. However, among secretory proteins there is a class that does not possess hydrophobic signal sequences and appears to leave the cell by a secretory pathway clearly distinct from the classical route through the endoplasmic reticulum and Golgi apparatus.

Acylation↗
PubMed1989

MLR3 molecule is an activation antigen shared by human B, T lymphocytes and T cell precursors.

MLR3 molecule is a membrane glycoprotein (mol. mass range 28-34 kDa) present on activated, but not resting human peripheral T cells, B cells and thymocytes. Its kinetics of appearance on the cell surface (3 h after the addition of the inductive signal to the cells) suggests that it is an early activation antigen. The proliferative response of cultured T and B lymphocytes and thymocytes to different activation signals is inhibited by the addition of MLR3 monoclonal antibody. Moreover the antibody in combination with non-mitogenic doses of phorbol myristate acetate leads to proliferation of thymocytes and resting B and T lymphocytes. In the latter, synthesis of interleukin 2 is also induced. Biochemical analysis of MLR3 antigen indicates that it is a phosphorylated protein with N-linked sugar moieties. Together these data suggest a role for MLR3 antigen in the signal transduction process during activation, both for mature lymphocytes and for T cell precursors.

Animals↗
PubMed1989

Regulation of IgM biosynthesis in human chronic lymphocytic leukemia. Normal and neoplastic B cells respond differently to TPA.

According to the pattern of IgM biosynthesis (membrane expression and secretion), human B cell chronic lymphocytic leukemias (B-CLLs) were subgrouped into four classes, namely: class I: membrane- secretion-; class II: membrane+ secretion-; class III: membrane+ secretion+; class IV: membrane- secretion+. Abundant membrane mu chain mRNA was present in cells from all cases, indicating that translational and/or post translational events were responsible for the absence of surface IgM in classes I and IV. Similarly, post translational events blocked IgM secretion in non secreting B-CLL cells. In B-CLLs from classes I, II and III, TPA induced IgM secretion by up-regulating secretory mu chain mRNA. By contrast, in normal B cells, TPA induced down-regulation of the secretory form of Ig mRNA, irrespective of the maturational stage of the cell. These observations indicate that IgM biosynthesis is modulated differently by TPA in normal and malignant B cells.

B-Lymphocytes↗
PubMed1989

Interleukin 1 as an autocrine growth factor for acute myeloid leukemia cells.

Production of interleukin 1 (IL-1) by leukemic cells was studied in 13 cases of acute myeloid leukemia. Intracytoplasmic immunofluorescence studies showed that the cells invariably contained the cytokine. Endogenous labeling studies demonstrated that acute myeloid leukemia cells produced either only the 33-kDa propeptide or both the propeptide and the 17-kDa mature form of IL-1 beta. The 33-kDa propeptide IL-1 alpha was always produced but was less frequently released. Involvement of IL-1 in leukemic cell growth was investigated using two antibodies specific for IL-1 subtypes, which inhibited spontaneous cell proliferation in the six cases studied. After acid treatment of the cells, a surface receptor for IL-1 could be demonstrated, which mediated 125I-labeled IL-1-specific uptake by leukemic cells. Furthermore, recombinant IL-1 alpha or IL-1 beta induced significant cell proliferation in 10 of 12 cases. The above findings were uncorrelated with the cytologic type (French-American-British classification) of leukemia. Our studies suggest that IL-1 may act as an autocrine growth factor in most cases of acute myeloid leukemia.

Cell Division↗
PubMed1989

Production of interleukin-1 by bone marrow myeloma cells.

Plasma cells isolated from bone marrow (BM) aspirates of 12 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of undetermined significance (MGUS) were analyzed for production of cytokines with bone-resorbing activity, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and lymphotoxin (LT). Culture supernatants of plasma cells from MM, but not from MGUS or normal donor, invariably contained high amounts of IL-1-beta and lower amounts of IL-1-alpha. With a single exception, TNF/LT biologic activity was not detected in the same supernatants. IL-6 was present in two of five supernatants tested. Normal B lymphocytes released both IL-1 and TNF/LT activities for four days after activation in vitro; however, production of these cytokines ceased at the final stage of plasma cell. Unexpectedly, the mRNA extracted from MM plasma cell hybridized with TNF- and LT-specific, as well as IL-1-specific probes, although the culture supernatants did not contain detectable TNF/LT biologic activity. When tested in the fetal rat long bone assay, MM plasma cell supernatants displayed a strong osteoclast-activating factor (OAF) activity, which was greatly reduced but not completely abolished by neutralizing anti-IL-1 antibodies. Anti-TNF or anti-LT antibodies were ineffective in the same test. We conclude that the IL-1 released in vivo by malignant plasma cells has a major role in pathogenesis of lytic bone lesions of human MM.

Biological Factors↗