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Biomedical subjects

E Lazarides

Publications and source records attributed to E Lazarides.

At least 19 recordsLinked to original sources
PubMed1999

Soluble vascular cell adhesion molecule (VCAM)-Fc fusion protein induces leukotriene C4 secretion in platelet-activating factor-stimulated eosinophils.

Eosinophil adhesion to vascular cell adhesion molecule-1 (VCAM-1) is important for cellular recruitment into allergic inflammatory sites. To determine whether eosinophil adhesion to VCAM-1 affects cell function, leukotriene C4 (LTC4) was measured. Human eosinophils were incubated with platelet-activating factor (PAF) in the presence or absence of soluble VCAM-Fc fusion protein (sVCAM-Fc) or immobilized VCAM-Fc. sVCAM-Fc induced a concentration-dependent increase in LTC4 secretion, which was dependent on the presence of PAF and not blocked by cyclic peptides shown to inhibit alpha4beta1-dependent adhesion. Likewise, soluble ICAM-Fc induced a concentration-dependent LTC4 secretion. LTC4 secretion was induced by the calcium ionophore, A23187, and the combination of sVCAM-Fc and A23187 had synergistic properties. It is interesting to note that Mn2+ or anti-beta1 monoclonal antibody, TS2/16, inhibited LTC4 secretion induced by sVCAM-Fc and PAF. Eosinophil adhesion to VCAM-Fc or interleukin-1 beta-stimulated endothelial cells did not induce LTC4 secretion. These data suggest that sVCAM-Fc-induced LTC4 secretion depends on distinct signals from those of eosinophil adhesion.

Amino Acid Sequence↗
PubMed1998

Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13-35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1-10), second loop (amino acids 37-51), and carboxy-terminus (amino acids 56-71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.

Amino Acid Sequence↗
PubMed1995

Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action.

Tubulin polymerization into microtubules is a dynamic process, with the equilibrium between growth and shrinkage being essential for many cellular processes. The antineoplastic agent taxol hyperstabilizes polymerized microtubules, leading to mitotic arrest and cytotoxicity in proliferating cells. Using a sensitive filtration-calorimetric assay to detect microtubule nucleating activity, we have identified epothilones A and B as compounds that possess all the biological effects of taxol both in vitro and in cultured cells. The epothilones are equipotent and exhibit kinetics similar to taxol in inducing tubulin polymerization into microtubules in vitro (filtration, light scattering, sedimentation, and electron microscopy) and in producing enhanced microtubule stability and bundling in cultured cells. Furthermore, these 16-membered macrolides are competitive inhibitors of [3H]taxol binding, exhibiting a 50% inhibitory concentration almost identical to that of taxol in displacement competition assays. Epothilones also cause cell cycle arrest at the G2-M transition leading to cytotoxicity, similar to taxol. In contrast to taxol, epothilones retain a much greater toxicity against P-glycoprotein-expressing multiple drug resistant cells. Epothilones, therefore, represent a novel structural class of compounds, the first to be described since the original discovery of taxol, which not only mimic the biological effects of taxol but also appear to bind to the same microtubule-binding site as taxol.

Animals↗
PubMed1995

Antagonistic role of vitamin D3 and retinoic acid on the differentiation of chicken hematopoietic macrophages into osteoclast precursor cells.

An in vitro culture model of osteoclast differentiation is described which is derived from homogeneous populations of chick yolk sac and peripheral blood macrophages. In primary cultures, both types of macrophages undergo a proliferative phase, become quiescent after reaching high cell densities, then aggregate and eventually form large multinucleated giant cells (MNGCs), presumably by fusion. These MNGCs can be characterized as premature osteoclasts on the basis of several morphological and biochemical criteria, although they do not undergo the final differentiation step rendering them competent to resorb bone in vitro. Clonal analysis of single cell-derived colonies indicates that all macrophages have the potential to differentiate into these osteoclast-like cells under these culture conditions. Both retinoic acid and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] modulate macrophage growth, but in an antagonistic manner. Although retinoic acid strongly promotes macrophage proliferation and impedes MNGC formation, 1,25-(OH)2D3 inhibits proliferation and changes the kinetics of MNGC formation. Combination experiments reveal that the proliferative signals induced by retinoic acid can override the signal to differentiate induced by 1,25-(OH)2D3. Our results indicate that even though retinoic acid and vitamin D3 act through homologous receptors, they have dramatically opposing effects on macrophage differentiation toward osteoclast progenitors.

Animals↗
PubMed1995

Multinucleated cells can continuously generate mononucleated cells in the absence of mitosis: a study of cells of the avian osteoclast lineage.

The multinucleated bone-resorbing osteoclast has a hematopoietic origin. We have demonstrated previously that osteoclasts are derived from the monocytic lineage by fusion of mononuclear macrophage precursors. Using an in vitro-osteoclast differentiation model derived from pure populations of chick macrophage cultures, osteoclast-like multinucleated giant cells (MNGCs) can be formed by fusion following an active proliferation phase. However, after reaching a peak with 70% of the culture being MNGCs, a new round of expansion of the mononuclear cells is observed. The following experiments suggest that these mononuclear cells were derived directly from the MNGCs by a budding process, selectively from the central zone of the apical surface. After microinjection of the membrane-impermeable probe, Lucifer Yellow, into single MNGCs, initially only diffuse fluorescence, limited to the whole MNGC injected, was observed. However, after 24-48 hours fluorescent mononuclear cells were observed adjacent but distinct from the injected MNGC. To confirm that these mononuclear cells were indeed derived from a parent MNGC, single MNGCs were cloned into single wells. Within a week, the MNGC was surrounded by mononuclear cells, which eventually populated the entire well. These mononuclear cells could then give rise to a second generation of MNGCs following a three-week period of culture. To determine whether this process required mitosis, MNGCs were cultured for three days in the presence of the mitotic inhibitor, Ara-C, prior to microinjection with Lucifer Yellow. Fluorescent mononuclear cells were still seen to arise from a single injected MNGC under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Identification of a novel Ca(2+)-regulated protein that is associated with the marginal band and centrosomes of chicken erythrocytes.

We have identified a novel Ca(2+)-regulated protein, p23, that is expressed specifically in avian erythrocyte and thrombocyte lineages. Sequence analysis of this 23 kDa protein reveals that it bears no homology to any known sequence. In mature definitive erythrocytes p23 exists in equilibrium between a soluble and a cytoskeletal bound pool. The cytoskeletal fraction is associated with the marginal band of microtubules, centrosomes and nuclear membrane under conditions of low free [Ca2+]. An increase in free [Ca2+] to 10(-6) M is sufficient to induce dissociation of > 95% of bound p23 from its target cytoskeletal binding sites, yet this [Ca2+] has little effect on calmodulin-mediated MB depolymerization. Analysis of p23 expression and localization during erythropoiesis together with results from heterologous p23 expression in tissue cultured cells demonstrated that this protein does not behave as a bone fide microtubule-associated protein. In addition, the developmental analysis revealed that although p23 is expressed early in definitive erythropoeisis, its association with the MB, centrosome and nuclear membrane occurs only in the final stages of differentiation. This cytoskeletal association correlates with marked p23 stabilization and accumulation at a time p23 expression is being markedly downregulated. We hypothesize that the mechanism of p23 association to the MB and centrosomes may be induced in part by a decrease in intracellular [Ca2+] during the terminal stages of definitive erythropoiesis.

Amino Acid Sequence↗
PubMed1995

Novel centrosomal protein reveals the presence of multiple centrosomes in turkey (Meleagris gallopavo) bnbn binucleated erythrocytes.

The phenotype of the bnbn hemolytic anemia mutation in the domestic turkey is manifested as binucleation specifically in the definitive erythrocyte lineage, most likely as the consequence of anomolous centrosomal activity (Bloom et al., 1970; Searle and Bloom, 1979). Here we have identified in turkey two variants of the novel, centrosomally-associated erythroid-specific protein p23. One variant is Ca(2+)-sensitive and is highly homologous to its chick counterpart (Zhu et al., 1995, accompanying paper). The other, p21 is a truncated form resulting from a 62 amino acid deletion from the 3' end and a 40 amino acid insertion at the 5' end, and appears to lack Ca(2+)-sensitivity. These proteins are localized at the marginal band, centrosomes and nuclear membrane of differentiated erythrocytes. Anti-p23/p21 immunofluorescence revealed the presence of multiple centrosomes in bnbn erythrocytes. We therefore undertook a detailed genetic analysis to determine whether the p21 variant represented the bn mutation. Initial tests of normal BnBn and mutant bnbn individuals suggested that the p23/p21 proteins might be encoded by the Bn/bn genes. However, further genetic tests demonstrated independent segregation for these two genetic loci. Thus, these proteins are encoded by the heretofore undescribed genes, p23/p21, mapping to an autosomal locus in the turkey genome.

Amino Acid Sequence↗
PubMed1995

Taxol-induced mitotic block triggers rapid onset of a p53-independent apoptotic pathway.

BACKGROUND: At therapeutic concentrations, the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis, similar to the mechanism of cell death induced by other antineoplastic agents. However, taxol has shown efficacy against drug-refractory cancers, raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block, aberrant mitosis, and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or, for those cells progressing through aberrant mitosis, arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast, G1-arrested, multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase, both wt p53-expressing and p53-null MEFs responded similarly to taxol, showing rapid onset of DNA nicking and apoptosis. However, p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive, and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest, which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent.

Animals↗
PubMed1994

K+ currents expressed from the guinea pig cardiac IsK protein are enhanced by activators of protein kinase C.

We have isolated cardiac cDNA and genomic clones encoding the guinea pig IsK protein. The deduced amino acid sequence is approximately 78% identical to the rat, mouse, and human variants of this channel, and the structure of the gene encoding the protein is also similar to that in other species. For example, the gene is present only once in the haploid genome, the protein-coding sequence is present on a single uninterrupted exon, an intron exists in the 5' untranslated domain, and multiple alternative polyadenylation sites are used in processing the transcript. Expression of the guinea pig protein in Xenopus oocytes results in a slowly activating, voltage-dependent K+ current, IsK, similar to those expressed previously from the rat, mouse, and human genes. However, in sharp contrast to the rat and mouse currents, activation of protein kinase C with phorbol esters increases the amplitude of the guinea pig IsK current, analogous to its effects on the endogenous IKs current in guinea pig cardiac myocytes. Mutagenesis of the guinea pig cDNA to alter four cytoplasmic amino acid residues alters the phenotype of the current response to protein kinase C from enhancement to inhibition, mimicking that of rat and mouse IsK currents. This mutation is consistent with reports that phosphorylation of Ser-102 by protein kinase C decreases the current amplitude. These data explain previously reported differences in the regulatory properties between recombinant rat or mouse IsK channels and native guinea pig IKs channels and provide further evidence that the IsK protein forms the channels that underlie the IKs current in the heart.

Amino Acid Sequence↗
PubMed1992

Methylations of 70,000-Da heat shock proteins in 3T3 cells: alterations by arsenite treatment, by different stages of growth and by virus transformation.

We have characterized the basic amino acid methylation of three members of the 70,000-Da heat shock protein superfamily, hsp68, hsc70, and BiP, in Balb/c 3T3 cells. It appears that a lysyl residue is the only methylation site in BiP and that both lysyl and arginyl residues are methylated in hsp68 and hsc70. In all cases, epsilon-N-trimethyllysine is the predominant methyllysine species. Both NG-monomethylarginine and NG,NG-dimethylarginine are identified as the methylarginine species. The stoichiometry of the methylation is indirectly determined by using the amount of actin methylation as a reference. Three, four, and four methyl groups are incorporated into lysyl residues of hsp68, hsc70, and BiP, respectively. The level of lysyl methylation in hsc70 remains unchanged under different growth conditions. On the other hand, the arginyl methylation in hsc70 varies considerably. In confluent Balb/c 3T3 cells, there are 1.8 and 1.3 methyl groups in dimethylarginine and monomethyl-arginine, respectively. In nonconfluent cells, the amount of monomethylarginine is similar to that in confluent cells, but dimethylarginine is not detectable. Furthermore, in both confluent and nonconfluent cells, the level of monomethylarginine is reduced 5- to 10-fold after arsenite treatment. However, in 3T3 cells transformed by Rous sarcoma virus (SR-RSV 3T3 cells), the level of arginine methylation is constitutively lower and cannot be reduced further by arsenite.

3T3 Cells↗
PubMed1992

Continuous growth of vimentin filaments in mouse fibroblasts.

We have investigated the dynamics of intermediate filament assembly in vivo by following the fate of heterologous chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. Using RNase protection, metabolic protein pulse-chase and immunofluorescence microscopy, we have examined the fate of newly assembled subunits under physiological conditions in situ. Following induction and subsequent removal of inducer, chicken vimentin mRNA had a half-life of approximately 6 h while both chicken and mouse vimentin protein polymer had long half-lives--roughly equivalent to the cell generation time. Moreover, following deinduction, chicken vimentin immunolocalization progressed from a continuous (8-10 h chase) to a discontinuous (> or = 20 h chase) pattern. The continuous chicken vimentin staining reflects the uniform incorporation of chicken vimentin throughout the endogenous mouse vimentin network while the discontinuous or punctate chicken vimentin staining represents short interspersed segments of assembled chicken vimentin superimposed on the endogenous polymer. This punctate staining pattern of chicken vimentin was present throughout the entire array of intermediate filaments, with no bias toward the perinuclear region. These results are consistent with a continuous growth model of intermediate filament assembly, wherein subunit addition occurs at discrete sites located throughout the cytoskeleton.

3T3 Cells↗
PubMed1991

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1.

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.

Actins↗
PubMed1990

Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly.

We have assessed the mechanism of intermediate filament assembly by assaying the sites of incorporation of chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. The localization of newly synthesized vimentin was determined by immunofluorescence and immunoelectron microscopy at short time periods of induced synthesis, using antibodies specific for chicken vimentin. Under conditions where neither the soluble subunit pools nor the steady-state distribution of endogenous filaments are affected, newly synthesized vimentin incorporates into the vimentin filament network at numerous and discrete sites throughout the cell. Over time, the pattern of newly assembled vimentin converts to a continuous array coincident with preexisting vimentin filaments. These results are consistent with a novel mechanism of intermediate filament assembly, whereby growth of intermediate filaments occurs by topographically restricted and localized subunit addition, necessitating a transient disruption of filament integrity.

Animals↗
PubMed1990

Vimentin downregulation is an inherent feature of murine erythropoiesis and occurs independently of lineage.

In mammalian erythropoiesis, the mature cells of the primitive lineage remain nucleated while those of the definitive lineage are anuclear. One of the molecular and structural changes that precedes enucleation in cells of the definitive lineage is the cessation in the expression of the gene for the intermediate filament (IF) protein vimentin and the removal of all vimentin filaments from the cytoplasm. We show here that in immature primitive cells vimentin is synthesized and forms a cytoplasmic network of IFs. As differentiation proceeds in vivo, vimentin gene expression is downregulated in these cells; this is accompanied by the loss of vimentin filaments from the cytoplasm. This loss temporally coincides with the nucleus becoming freely mobile within the cytoplasm, suggesting that, while IF removal is not directly linked to the physical process of enucleation, it may be a prerequisite for the initiation of nuclear mobility in both lineages. These changes are also observed in early primitive cells cultured in vitro, suggesting that they constitute an intrinsic part of the murine erythroid differentiation program independent of lineage and hematopoietic microenvironment.

Animals↗
PubMed1988

Alternative primary structures in the transmembrane domain of the chicken erythroid anion transporter.

Isolation and characterization of the chicken erythroid anion transporter (band 3) cDNA clone, pCHB3-1, revealed that the chicken erythroid band 3 polypeptide is 844 amino acids in length with a predicted mass of 109,000 daltons. This polypeptide is composed of a hydrophilic N-terminal cytoplasmic domain and a hydrophobic C-terminal transmembrane domain. The approximately 90 N-terminal amino acids of the human and murine erythroid band 3 polypeptides are absent in the predicted sequence of the chicken erythroid band 3 polypeptide. The absence of this very acidic N-terminal region is consistent with the lack of binding of glyceraldehyde-3-phosphate dehydrogenase to chicken erythroid band 3, as well as the relatively basic isoelectric point observed for this molecule. The remainder of the cytoplasmic domain shows little similarity to the cytoplasmic domain of the murine and human erythroid band 3, with the exception of the putative ankyrin-binding site, which is highly conserved. In contrast, the transmembrane domain of the chicken band 3 polypeptide is very similar to that of the murine erythroid and human nonerythroid band 3 polypeptides. The transmembrane domain contains 10 hydrophobic regions that could potentially traverse the membrane 12 to 14 times. In addition, a variant of chicken erythroid band 3, pCHB3-2, was cloned in which one of the hydrophobic regions of pCHB3-1 is lacking. The transcript complementary to pCHB3-2 accumulated in chicken erythroid cells in a similar manner as the transcript complementary to pCHB3-1 during embryonic development. This is the first example of a transporter protein or ion channel with alternative primary structures in its membrane-spanning segments.

Amino Acid Sequence↗
PubMed1988

The erythroid membrane skeleton: expression and assembly during erythropoiesis.

One aspect of erythropoiesis entails the biogenesis of a membrane skeleton. Analysis of the expression and assembly of the various components of this multisubunit structure illustrates the problem of spatial and temporal regulation of cytoskeletal morphogenesis, departures from simple self-assembly, and the various levels of regulation that might be operative in this aspect of cellular morphogenesis in development. Additionally, it reveals a new insight into the mechanisms underlying the molecular defects in certain hereditary hemolytic anemias.

Animals↗
PubMed1988

Gelsolin sensitivity of microfilaments as a marker for muscle differentiation.

The ability of porcine smooth muscle gelsolin to sever actin filaments was used to study alterations in the organization of F-actin containing structures during skeletal myogenesis. In permeabilized fibroblasts and unfused myoblasts, gelsolin induced complete degradation of the actin cytoskeleton. After fusion of myoblasts to multinucleated myotubes, gelsolin removed a substantial amount of actin, revealing fibers with a sarcomere-like arrangement of gelsolin-insensitive actin. These fibrils were much thinner and had shorter sarcomeres than fully differentiated myofibrils. The proportion of gelsolin-resistant fibrils increased during differentiation, resulting in almost complete inertness of mature myofibrils. Fibrils isolated from adult muscle were also found nearly resistant to gelsolin. Extraction of tropomyosin and myosin in buffer of high ionic strength prior to gelsolin treatment reestablished the susceptibility to the severing protein, both in myotubes and isolated myofibrils. Only small remnants of phalloidin-stainable material were retained. We therefore conclude that during myotube differentiation either an increased interaction of actin with actin-binding proteins (e.g., myosin and tropomyosin), or the assembly of muscle-specific isoforms of these proteins protect the filaments against degradation by actin severing proteins.

Actin Cytoskeleton↗