Biomedical subjects

B Alarcón

Publications and source records attributed to B Alarcón.

At least 37 records · Page 2Linked to original sources

PubMed1997

Megalomicin inhibits HIV-1 replication and interferes with gp160 processing.

The inhibitory effects on HIV replication of megalomicin (MGM, an inhibitor of intra-Golgi vesicle transport, have been studied. In experiments at low multiplicity of infection on Jurkat and MT2 cell lines. MGM inhibited the production of p24 antigen, the formation of syncytia, and the induction of apoptosis at concentrations below 5 microM. Furthermore, PCR analysis of genomic DNA showed that, in the presence of MGM, HIV-1 had been eradicated from the culture. MGM also inhibited replication of primary isolates of HIV-1 in blood lymphoblasts and more importantly, at 1 microM, MGM inhibited depletion of CD4+ T cells in cultures of blood lymphocytes from seropositive patients. Finally, MGM inhibited the generation of infectious virions and the processing of the envelope protein precursor gp160 to its mature forms, resulting in the rapid degradation of gp 160. These data suggest that MGM induces a powerful inhibitory effect on HIV-1 replication at nontoxic concentrations by preventing the processing of HIV-1 gp160 envelope protein and the subsequent formation of infectious viral particles.

Anti-Bacterial Agents↗

PubMed1997

Multiple sorting signals determine apical localization of a nonglycosylated integral membrane protein.

In polarized cells, newly synthesized proteins are sorted in the trans-Golgi network and from there delivered to either the apical or basolateral membranes. Madin-Darby canine kidney (MDCK) cells have been widely used as a model system to study sorting determinants to the apical and basolateral surfaces. Whereas sorting signals for basolateral transmembrane proteins seem to reside in their cytoplasmic domains, apical determinants appear to reside in the N-glycans of secretory proteins or in the glycolipid tails of glycosylphosphatidylinositol-linked proteins. We show in this study that a surface-expressed form of CD3-epsilon, a nonglycosylated type I membrane protein, is exclusively targeted to the apical membrane in MDCK cells by a glycolipid-independent transport pathway. Deletion of the cytoplasmic tail does not affect its distribution, whereas deletion of the transmembrane domain results in secretion from both surfaces although still predominantly through the apical membrane. The transmembrane domain of CD3-epsilon appended to rat growth hormone, a secretory protein that lacks apical and basolateral determinants, promotes basolateral localization of the chimeric protein. However, a growth hormone chimera containing both the transmembrane and cytoplasmic domains of CD3-epsilon resulted in localization to the apical and basolateral membranes. These results suggest there are multiple determinants in CD3-epsilon that affect its distribution in polarized MDCK cells. Whereas the transmembrane domain contains a basolateral determinant, the ectodomain and the cytoplasmic domain contain apical determinants.

Animals↗

PubMed1997

1H-NMR analysis of CD3-epsilon reveals the presence of turn-helix structures around the ITAM motif in an otherwise random coil cytoplasmic tail.

The conformation adopted in solution by the cytoplasmic tail of CD3-epsilon has been analyzed by 1H-nmr. The cytoplasmic tail is mostly random coil expect for the amino acids conforming the immunoreceptor tyrosine-based activation motif (ITAM), YxxL/IxxxxxxxY xxL. Although the N-terminal Y xxL sequence of the motif is poorly folded, adopting 6-residue turn-like conformations with the Tyr side chain in two different orientations, the C-terminal Y xxL sequence is placed in a more complex structure involving a set of nonclassical alpha-helix turns and beta-turns that comprises 11 amino acids. This structure is not modified by phosphorylation of the tyrosine residue. The differences in the conformation adopted around the two tyrosines of the ITAM motif suggest that they may play different roles pertaining to either binding signal transducing proteins or, alternatively, proteins involved in other processes such as endoplasmic reticulum location.

Amino Acid Sequence↗

PubMed1997

Differential cytosolic tail dependence and intracellular fate of T-cell receptors internalized upon activation with superantigen or phorbol ester.

Activation of T lymphocytes by T-cell receptor (TCR) ligands such as peptide/MHC complexes, superantigens or anti-TCR mAbs, or by pharmacological activators of protein kinase C such as phorbol esters, results in the internalization and cell surface downregulation of TCRs. We investigated the role of internalization motifs located in the cytosolic region of CD3 gamma in the internalization of TCR complexes induced by enterotoxin superantigens, anti-TCR mAbs or phorbol esters. To this end, a series of CD3 gamma mutants were expressed in a CD3 gamma-deficient variant of the human T-cell line Jurkat. We found that serine126 and the di-leucine motif (Leu131-Leu132) are required for phorbol-ester-induced TCR downregulation, but they are not necessary for enterotoxin superantigen or antibody-induced TCR downregulation. Moreover, the tyrosine-based motifs (residues 138 to 141 and 149 to 152) are not required either for phorbol aster or for superantigen or antibody-induced TCR downregulation. Confocal microscopy analysis reveals that TCR complexes accumulate in an early endocytic/recycling compartment upon activation of cells with phorbol esters, whereas TCRs internalized upon activation with superantigen or anti-TCR mAbs are routed to lysosomes. Consistent with this intracellular localization, TCRs internalized in response to phorbol ester are not degraded and can be reexpressed on the cell surface. In contrast, TCRs internalized upon superantigen activation are degraded.

Antibodies, Monoclonal↗

PubMed1997

Megalomicin disrupts lysosomal functions.

Megalomicin (MGM) has been shown to cause a dilation of the most distal cisternae of the Golgi complex. The effects of MGM on Golgi morphology correlated with an inhibition of protein transport to the trans-Golgi resulting in an accumulation of poorly sialylated glycoproteins. Here we show that the addition of 50 microM MGM caused a rapid swelling of lysosomes in cultured cells and inhibited the degradation of the newly synthesized T cell antigen receptor CD36 subunit. Although MGM did not affect the uptake of fluid phase markers, it prevented their degradation. Interestingly, endocytosed ovalbumin did not colocalize with lysosomes in MGM-treated cells, suggesting an MGM-induced impairment in the delivery to lysosomes. This was confirmed by Percoll density gradients, where the fluid phase marker remained in endosomal fractions, even after long chase times, whereas in control cells the endocytosed marker was located in lysosomes. The effect of MGM was not confined to soluble proteins since it did also inhibit the delivery of the membrane-bound epidermal growth factor receptor to lysosomes. Finally, MGM strongly inhibited the ATP-dependent acidification of lysosomes in vitro, suggesting a possible mechanism for its in vivo activity.

Animals↗

PubMed1996

Apoptosis but not other activation events is inhibited by a mutation in the transmembrane domain of T cell receptor beta that impairs CD3zeta association.

The transmembrane domain of T cell receptor (TCR) beta contains a conserved immunoreceptor tyrosine-based activation-like motif consisting of a duplicated YXXL sequence. The motif is also present in TCRgamma, the equivalent chain to TCRbeta in gammadelta T lymphocytes but is absent in TCRalpha and TCRdelta. To determine the putative role of this sequence in TCR.CD3 complex assembly and signal transduction, a TCRbeta chain cDNA was mutated in the C-terminal tyrosine of the motif, cloned in an expression vector, and transfected into TCRbeta-negative Jurkat cells. Transfectants of the mutated chain (MUT) expressed, on average, much less TCR.CD3 complex on the membrane than wild type TCRbeta transfectants. Radiolabeling experiments suggested that the mutation caused a loose association of the CD3zeta chain resulting in a defective assembly. However, stimulation of high TCR.CD3 expressing wild type and MUT clones with monoclonal antibodies and Staphylococcus aureus enterotoxin B resulted in similar levels of CD25 and CD69 expression, interleukin-2 secretion, and TCR.CD3 complex down-regulation. By contrast, MUT cells were clearly resistant to activation-induced cell death, and they did not express CD95-ligand upon activation. These results suggest a differentiated intracellular signaling pathway leading to apoptosis in which Tyr-TM11 of the immunoreceptor tyrosine-based activation motif-like motif and CD3zeta appear to be involved.

Amino Acid Sequence↗

PubMed1996

Intra-Golgi transport inhibition by megalomicin.

Megalomicin (MGM) is a macrolide antibiotic which has been demonstrated previously to cause an anomalous glycosylation of viral proteins. Here we show that MGM produces profound alterations on Golgi morphology and function. The addition of MGM at 50 microM for 1 h caused a dilation of the Golgi detected by immunofluorescence staining for medial- and trans-Golgi markers. The effect of MGM was clearly more intense on the trans-side of the Golgi, as evidenced in electron microscope preparations. The effect on Golgi morphology was reversible and correlated with an impairment of glycoprotein processing in the trans-Golgi. Thus, although the vesicular stomatitis virus G protein was processed in the presence of MGM to an endoglycosidase H-resistant form, it was poorly sialylated. The sialylation of cellular proteins was also inhibited, resulting in cells with low level of sialylation on the cell surface. However MGM did not inhibit the activities of the galactosyl- or sialyltransferase as measured in vitro. MGM inhibited cis- to medial-, and more strongly, medial- to trans-Golgi transport of vesicular stomatitis virus G protein in an in vitro system, suggesting that the impairment in glycoprotein maturation observed in vivo is the result of intra-Golgi transport inhibition.

Animals↗

PubMed1995

A tyrosine-containing motif mediates ER retention of CD3-epsilon and adopts a helix-turn structure.

The CD3-epsilon endoplasmic reticulum (ER) retention motif has been characterized by mutagenesis and NMR spectroscopy. Tyr177, Leu180 and Arg183 are involved in ER retention. The motif forms an elongated alpha-helix in which the tyrosine and leucine residues are closely apposed, followed by a beta I' turn that places Arg183 in the vicinity of Leu180. The structure formed by Tyr177 and the leucine in position +3 is reminiscent of the beta-turn structure adopted by tyrosine-containing endocytosis signals. Moreover, substitution of the transferrin receptor (TfR) internalization sequence by the CD3-epsilon motif still allowed the rapid internalization of the TfR and, conversely, the chimeric protein resulting from the substitution of the CD3-epsilon motif by the endocytosis signal of the low density lipoprotein receptor was ER located. These data support the idea of a functional homology between the two types of signal.

Amino Acid Sequence↗

PubMed1995

Transferrin receptor induces tyrosine phosphorylation in T cells and is physically associated with the TCR zeta-chain.

In addition to being an iron transporter, the transferrin receptor (TfR) has been shown to play a role in T cell activation. Stimulation of the TfR with specific Abs results in T cell proliferation, IL-2 secretion, and protein kinase C activation. In this paper we have analyzed early events caused by activation of the TfR. We have found several protein substrates to be tyrosine phosphorylated upon TfR stimulation in the human Jurkat T cell line. Interestingly, the TfR induced tyrosine phosphorylation in cell lines expressing TCR but not in TCR-negative mutants. Restoration of the TCR surface expression in these mutants reestablished the ability of the TfR to induce tyrosine phosphorylation. This result suggests that activation through the TfR is functionally dependent upon the expression of the TCR. Moreover, the functional relationship of the TfR with the TCR complex is also supported by data showing that TfR stimulation resulted in the tyrosine phosphorylation of the TCR zeta-chain; conversely, stimulation of the TCR complex resulted in an increased tyrosine phosphorylation of the TfR. More importantly, the TfR is shown to associate physically with the TCR zeta-chain as well as with the zeta-binding ZAP70 tyrosine kinase. The TfR/zeta complex is expressed on the cell surface independent of the expression of the other subunits of the TCR complex. We suggest that the TfR/zeta complex is responsible for transducing the TfR-induced signals, and that it could serve to amplify signals delivered by Ag binding to the TCR.

Animals↗

PubMed1995

T lymphocyte receptor deficiencies.

Signalling through the TCR is mediated by the cytoplasmic tails of the CD3 complex. Deficiencies in the expression of different CD3 components have lead to dramatic, yet dissimilar, effects on T-cell development and to selective deficits in peripheral T-cell subsets. Recent studies of human patients and animal models with CD3 deficiencies are providing insights into the redundant and unique roles of these molecules.

Amino Acid Sequence↗

PubMed1992

An endoplasmic reticulum retention signal in the CD3 epsilon chain of the T-cell receptor.

Isolated polypeptide chains of the T-cell antigen receptor complex are degraded or retained in the endoplasmic reticulum (ER). Assembly of the multisubunit complex allows the individual chains to escape retention in the ER and to be expressed on the cell surface. We engineered a series of deletions in the CD3 epsilon subunit of the human T-cell receptor in order to find the sequences responsible for its retention in the ER. Deletion of amino acids 171 to 180 in the cytosolic tail resulted in the cell-surface expression of the isolated chain. This sequence also promotes retention when it is appended to CD4, a plasma membrane protein. Mutagenesis of the 10-amino-acid CD3 epsilon sequence established that the tyrosine and serine residues are important for ER retention. This and other ER retention signals must be hidden when a complete T-cell receptor complex is assembled in order to allow its expression on the cell surface.

Amino Acid Sequence↗

PubMed1991

A conformational epitope expressed upon association of CD3-epsilon with either CD3-delta or CD3-gamma is the main target for recognition by anti-CD3 monoclonal antibodies.

We have performed immunofluorescence analysis of COS cells transfected with human CD3 genes and detergent permeabilized to define the specificity of several anti-CD3 antibodies. We have found that the mAb OKT3, WT31, UCHT1, and Leu-4 did not stain COS cells singly transfected with the CD3-epsilon chain. However, these antibodies very strongly stained COS cells doubly transfected with a combination of CD3-epsilon plus either CD3-gamma or CD3-delta. By contrast, the antibodies SP34 and APA 1/1, which were raised against isolated SDS-denatured CD3-epsilon protein, gave a strong staining of COS cells singly transfected with CD3-epsilon as well as of the double transfectans. The recognition by this panel of anti-CD3 antibodies of CD3-gamma/epsilon and CD3-delta/epsilon complexes and not of CD3-epsilon alone was assessed by immunoprecipitation. These findings suggest that the most widely used mAb specific for the CD3 complex recognize conformational epitopes on CD3-epsilon, which are expressed when this chain is bound to either CD3-gamma or CD3-delta. It should also be highlighted that antibody WT31 clearly recognizes the CD3 moiety of the TCR/CD3 complex.

Antibodies, Monoclonal↗

PubMed1991

Expression and function of a variant T cell receptor complex lacking CD3-gamma.

A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface-radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3-epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma.

Antibodies, Monoclonal↗

PubMed1991

The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.

Animals↗

PubMed1989

Different functional domains on the transferrin receptor molecule defined by monoclonal antibodies.

Three monoclonal antibodies (mAb) FG 1/5, FG 1/6 and FG 2/12, specific for the human transferrin receptor molecule (TR), have been used to define epitopes on the TR molecule and to block natural killer lysis. FG 2/12 mAb but not FG 1/5 or FG 1/6 blocked [125I-] transferrin binding to the cellular receptor. Furthermore, FG 1/5 and FG 1/6 mAbs competed out the binding of each other to the cells but not significantly that of FG 2/12. As expected, the binding of F2/12 but not of FG 1/5 or FG 1/6 was inhibited by transferrin. In addition, FG 2/12 inhibited in a dose-dependent manner the NK activity of purified T3- large granular lymphocyte effector cells against HeLa or Molt-4 cells but not against K-562 or U937 cells. FG 1/5 preferentially inhibited NK activity against HeLa cells and FG 1/6 mAb was completely uneffective. These inhibitions were stronger at low effector to target cell (E:T) ratios than at high E:T ratios, suggesting that NK cells and anti-TR mAbs compete for the same site in the target cell. It was shown that FG 1/5 and FG 2/12 mAbs blocked cells' conjugate formation by acting at the target cell level. Our results confirm the role of TR as a one of the target structures in NK lysis and suggest that the epitope recognized by NK cells is close to but different from the transferrin binding site.

Antibodies, Monoclonal↗

PubMed1988

Megalomycin C, a macrolide antibiotic that blocks protein glycosylation and shows antiviral activity.

Megalomycin C, a natural macrolide antibiotic showed a potent antiherpetic activity. At concentrations that efficiently prevented HSV-1 multiplication, the compound had no cytotoxic or antiproliferative effects. Viral DNA and protein synthesis took place at normal levels in the presence of the antibiotic, suggesting that neither the translation of viral mRNA, nor the synthesis of viral nucleic acids was affected. The incorporation of mannose and galactosamine into viral proteins was blocked and precursor, but not mature, HSV-1 glycoproteins were detected in the presence of megalomycin C. Non-infectious HSV-1 viral particles were formed when the compound was present, but their glycoproteins were not properly glycosylated.

Anti-Bacterial Agents↗

PubMed1988

Inhibition of natural killer cytotoxicity by extracellular ppp(A2'p5')nA oligonucleotides.

The effect of purified 2'-5' linked polyadenylated oligomers on the human natural killer (NK) activity has been investigated. Permeabilization of NK cells to ppp(A2'p5')2A did not enhance their NK activity at any concentration tested. Furthermore, concentrations higher than 0.1 mM inhibited their NK activity. Similar results were obtained by pre-incubating non-permeabilized intact cells, indicating an extracellular effect of these compounds on cell membranes, which was further investigated. This inhibition by the oligomers was greater (50% inhibition was obtained with 50 micron), when they were added directly to the NK assay, than when the NK cells were pre-incubated. Similar inhibitory effects were observed with ppp(A2'p5')A and ppp(A2'p5')3A oligomers but not with the dephosphorylated "core" molecules or other nucleotides. The inhibition was completely or partially reversed by phorbol esters and by interferon (IFN), respectively. The inhibition was temperature and extracellular Ca2+ dependent. The oligomers did not inhibit the binding of the effector cells to the target cells. The programming for lysis was the step of the lytic mechanism affected, possibly due to the alteration of the Ca2+ influx into the NK cells observed in presence of the ppp(A2'p5')nA.

Adenine Nucleotides↗

PubMed1988

Mechanism of recognition of herpes simplex virus type 1-infected cells by natural killer cells.

Human fibroblast FS-4 cells when infected with herpes simplex virus type 1 (HSV-1) become susceptible to lysis by purified populations of T3- human natural killer (NK) lymphocytes. Blocking of HSV-1 protein synthesis or N-linked glycosylation with pactamycin or tunicamycin, respectively, prevented HSV-1-infected cells from being lysed, suggesting that HSV-1 glycoprotein synthesis is required for recognition by NK cells. However, pactamycin- and tunicamycin-treated cells expressed on their membranes a detectable amount (20 to 40% of the untreated control) of HSV-1 glycoproteins gB, gC and gD, left by the virus during its internalization. Phosphonoformic acid (PFA) blocked HSV-1 DNA replication and inhibited the synthesis and surface expression of newly made gC, gD and gB by 90, 80 and 60% respectively. Despite this reduction, PFA treatment had no effect on NK susceptibility. The target structure recognized seems to be different from those expressed on tumour target cells since there was no competition for the lysis of HSV-1-infected FS-4 by K-562 or HeLa tumour target cells. However, a monoclonal antibody specific for the human transferrin receptor which inhibited NK recognition of tumour cells also blocked NK cytotoxicity of HSV-1-infected cells. In summary our results indicate that although viral glycoprotein synthesis is required, gB, gC and/or gD alone are not the targets for NK recognition of HSV-1-infected cells. In addition, they suggest the involvement of the host cell transferrin receptor in the NK killing process.

Antigens, Viral↗