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Biomedical subjects

A Israël

Publications and source records attributed to A Israël.

At least 37 records · Page 2Linked to original sources
PubMed2000

In vivo identification of lymphocyte subsets exhibiting transcriptionally active NF-kappaB/Rel complexes.

To analyze the NF-kappaB/Rel activity pattern in a living organism, we previously generated transgenic mice carrying a kappaB-dependent lacZ gene. In situ analysis of both primary and secondary lymphoid organs revealed a strong NF-kappaB transcriptional activity in antigen-presenting cells, some endothelial cells and sinus lining cells of the lymph node capsula with very little activity in lymphocytes and thymocytes. Using fluorescein-di-beta-D-galactopyranoside (FDG) as a vital substrate for the beta-galactosidase, we re-examined by flow cytometry the NF-kappaB/Rel transcriptional activity in our mouse model. We report here that such constitutive NF-kappaB/Rel activity was significantly detected in thymocytes at the CD44+CD25(-) stage. This constitutive activity extended with CD25 expression to the majority of the CD44(-)CD25(+) thymocytes and was then restricted to a few mature T cells. In the spleen, constitutive NF-kappaB/Rel activity was found in most B cells, unlike T cells which were largely negative. Virgin IgD(+) B cells expressed higher levels of NF-kappaB transcriptional activity than other B cell types. Altogether, these results suggest that NF-kappaB/Rel complexes are key players in the in vivo differentiation of IgD(+) B lymphocytes and possibly CD25(+) thymocytes.

Animals↗
PubMed1999

IkappaBepsilon-deficient mice: reduction of one T cell precursor subspecies and enhanced Ig isotype switching and cytokine synthesis.

Three major inhibitors of the NF-kappaB/Rel family of transcription factors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, have been described. To examine the in vivo role of the most recently discovered member of the IkappaB family, IkappaBepsilon, we generated a null allele of the murine IkappaBepsilon gene by replacement of all coding sequences with nlslacZ. Unlike IkappaBalpha nullizygous mice, mice lacking IkappaBepsilon are viable, fertile, and indistinguishable from wild-type animals in appearance and histology. Analysis of beta-galactosidase expression pattern revealed that IkappaBepsilon is mainly expressed in T cells in the thymus, spleen, and lymph nodes. Flow cytometric analysis of immune cell populations from the bone marrow, thymus, spleen, and lymph nodes did not show any specific differences between the wild-type and the mutant mice, with the exception of a reproducible 50% reduction of the CD44-CD25+ T cell subspecies. The IkappaBepsilon-null mice present constitutive up-regulation of IgM and IgG1 Ig isotypes together with a further increased synthesis of these two isotypes after immunization against T cell-dependent or independent Ags. The failure of observable augmentation of constitutive nuclear NF-kappaB/Rel-binding activity is probably due to compensatory mechanisms involving IkappaBalpha and IkappaBbeta, which are up-regulated in several organs. RNase-mapping analysis indicated that IL-1alpha, IL-1beta, IL-1Ra, and IL-6 mRNA levels are constitutively elevated in thioglycolate-elicited IkappaBepsilon-null macrophages in contrast to GM-CSF, G-CSF, and IFN-gamma, which remain undetectable.

Animals↗
PubMed1999

Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines.

During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells.

Down-Regulation↗
PubMed1999

Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain.

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-kappaB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.

Amino Acid Sequence↗
PubMed1999

Direct association and nuclear import of the hepatitis B virus X protein with the NF-kappaB inhibitor IkappaBalpha.

The X protein of hepatitis B virus (HBV) is a transcriptional activator which is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. It has been suggested that X acts as a nuclear coactivator or stimulates several signal transduction pathways by acting in the cytoplasm. One of these pathways leads to the nuclear translocation of NF-kappaB. A recent report indicates that X activates NF-kappaB by acting on two cytoplasmic inhibitors of this family of transcription factors: IkappaBalpha and the precursor/inhibitor p105. We demonstrate here that X directly interacts with IkappaBalpha, which is able to transport it to the nucleus by a piggyback mechanism. This transport requires a region of IkappaBalpha (the second ankyrin repeat) which has been demonstrated to be involved in its nuclear import following NF-kappaB activation. Using deletion mutants, we showed that amino acids 249 to 253 of IkappaBalpha (located in the C-terminal part of the sixth ankyrin repeat) play a critical role in the interaction with X. This small region overlaps one of the domains of IkappaBalpha mediating the interaction with the p50 and p65 subunits of NF-kappaB and is also close to the nuclear export sequence of IkappaBalpha, therefore providing a potential explanation for the nuclear accumulation of IkappaBalpha with X. This association can also be observed upon the induction of endogenous IkappaBalpha by tumor necrosis factor alpha (TNF-alpha) treatment of Chang cells expressing X. In accordance with this observation, band shift analysis indicates that X induces a sustained NF-kappaB activation following TNF-alpha treatment, probably by preventing the reassociation of newly synthesized nuclear IkappaBalpha with DNA-bound NF-kappaB complexes.

Animals↗
PubMed1998

The Notch1 receptor is cleaved constitutively by a furin-like convertase.

The Notch receptor, which is involved in numerous cell fate decisions in invertebrates and vertebrates, is synthesized as a 300-kDa precursor molecule (p300). We show here that proteolytic processing of p300 is an essential step in the formation of the biologically active receptor because only the cleaved fragments are present at the cell surface. Our results confirm and extend recent reports indicating that the Notch receptor exists at the plasma membrane as a heterodimeric molecule, but disagree as to the nature of the protease that is responsible for the cleavage that takes place in the extracellular region. We report here that constitutive processing of murine Notch1 involves a furin-like convertase. We show that the calcium ionophore A23187 and the alpha1-antitrypsin variant, alpha 1-PDX, a known inhibitor of furin-like convertases, inhibit p300 processing. When expressed in the furin-deficient Lovo cell line, p300 is not processed. In vitro digestion of a recombinant Notch-derived substrate with purified furin allowed mapping of the processing site to the carboxyl side of the sequence RQRR (amino acids 1651-1654). Mutation of these four amino acids (and of two secondary dibasic furin sites located nearby) completely abolished processing of the Notch1 receptor.

Calcimycin↗
PubMed1998

Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation.

We have characterized a flat cellular variant of HTLV-1 Tax-transformed rat fibroblasts, 5R, which is unresponsive to all tested NF-kappaB activating stimuli, and we report here its genetic complementation. The recovered full-length cDNA encodes a 48 kDa protein, NEMO (NF-kappaB Essential MOdulator), which contains a putative leucine zipper motif. This protein is absent from 5R cells, is part of the high molecular weight IkappaB kinase complex, and is required for its formation. In vitro, NEMO can homodimerize and directly interacts with IKK-2. The NEMO cDNA was also able to complement another NF-kappaB-unresponsive cell line, 1.3E2, in which the protein is also absent, allowing us to demonstrate that this factor is required not only for Tax but also for LPS, PMA, and IL-1 stimulation of NF-kappaB activity.

Amino Acid Sequence↗
PubMed1998

Cytosol is the prime compartment of hepatitis B virus X protein where it colocalizes with the proteasome.

The hepatitis B virus X protein plays an important role in the regulation of viral genome expression and has also been implicated in the development of liver cancer associated with chronic viral infection. Several effects have been attributed to X but their biological relevance remains elusive. One of the confusing issues has been so far the uncertainty concerning its cellular location. To gain insight into the mechanism(s) how X exerts its effects, we have analysed its subcellular distribution and its dependency on the cell cycle. We used two complementary approaches namely, immunolocalization using a cell line stably expressing X, and characterization of the dynamics of X location in living cells by means of the reporter gene GFP. Our data clearly define the cytosol as the prime location of X, irrespectively of the cell cycle and show in addition the close attachment of a fraction of X to the nuclear membrane. However, X does not associate with any cytoplasmic vesicles and organelles so far tested. In contrast, our study provides strong evidence for the codistribution of X with the cytosolic fraction of proteasomes. In pulse-chase experiments, X decayed with a half-life of less than 30 min and proteasome-inhibitors did not modify its turnover, suggesting that X colocalization with the proteasome does not simply point to its degradation pathway. The proteolytic processing of the p105 precursor of the p50 subunit of the NF-kappaB transcription factor, which has been shown to be proteasome-dependent, is markedly slow down in the presence of X. These findings suggest that X modulates the processing rate of p105 by acting presumably at the level of the proteasome. Thus, targeting of proteasomes by X might be one of the pathways employed by this viral protein to subvert cellular functions.

Animals↗
PubMed1998

[Contraception by subcutaneous Norplant implants. 300 cases].

It is a prospective study for the evaluation of the efficiency and the acceptability of Norplant contraceptive subdermal implants within the Senegalese female population. Three hundred women were selected on the basis of precise criteria between December 1986 and July 1991. Every patient included in the study signed a form of voluntary consent with the agreement of the consort, and was bound to come to control examinations until the removal of the implants. The insertion was made under local anaesthesia between the 1st and the 7th day of the cycle, or starting from the 6th week of post-partum in the other cases. The average age of patients was 32, and the average parity four children. 50 percent of women did not want any other children. The average insertion time is 4.8 minutes. The analysis of side-effects on 248 regularly followed women mostly reveals menstrual troubles like amenorrhea, spottings, menorrhagia. The fitful local prurigo was noted in 35 cases, and accidental pregnancy in 4 cases. The removal of implants within the first six months was made on 11 women (4.4%). As such, the Norplant implants constitutes a highly efficient method, well tolerated by Senegalese women.

Adolescent↗
PubMed1997

Reconstitution of the NF-kappa B system in Saccharomyces cerevisiae for isolation of effectors by phenotype modulation.

NF-kappa B is a ubiquitous transcription factor that contributes to the induction of many genes playing a central role in immune and inflammatory responses. The NF-kappa B proteins are subject to multiple regulatory influences including post-translational modifications such as phosphorylation and proteolytic processing. A very important component of this regulation is the control of their subcellular localization: cytoplasmic retention of NF-kappa B is achieved through interaction with I kappa B molecules. In response to extracellular signals, these molecules undergo degradation, NF-kappa B translocates to the nucleus and activates its target genes. To investigate novel proteins involved in this dynamic response, we have reconstituted the NF-kappa B/I kappa Beta system in the yeast Saccharomyces cerevisiae. We have successively introduced p65, the main transcriptional activator of the NF-kappa B family, which leads to the activation of two reporter genes controlled by kappa B sites, and the I kappa B alpha inhibitory protein, which abolishes this activation. By transforming such a yeast strain with a cDNA library we have performed a genetic screen for cDNAs encoding proteins capable of either dissociating the p65/I kappa B alpha complex or directly transactivating the expression of the reporter genes. The efficiency of our screen was demonstrated by the isolation of a cDNA encoding the p105 precursor of the p50 subunit of NF-kappa B. We also used this system to test stimuli known to activate signalling pathways in yeast, in order to investigate whether the related mammalian cascades might be involved in NF-kappa B activation. We showed that yeast endogenous kinase cascades activated by pheromone, hypo- or hyperosmotic shock cannot modulate NF-kappa B activity in our system, and that the p38 human MAP kinase does not act directly on the p65/I kappa B alpha complex.

DNA, Complementary↗
PubMed1997

IkappaB alpha is a target for the mitogen-activated 90 kDa ribosomal S6 kinase.

The activity of transcription factor NFkappaB is regulated by its subcellular localization. In most cell types, NFkappaB is sequestered in the cytoplasm due to binding of the inhibitory protein IkappaB alpha. Stimulation of cells with a wide variety of agents results in degradation of IkappaB alpha which allows translocation of NFkappaB to the nucleus. Degradation of IkappaB alpha is triggered by phosphorylation of two serine residues, i.e. Ser32 and Ser36, by as yet unknown kinases. Here we report that the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1) is an IkappaB alpha kinase. p90rsk1 phosphorylates IkappaB alpha at Ser32 and it physically associates with IkappaB alpha in vivo. Moreover, when the function of p90rsk1 is impaired by expression of a dominant-negative mutant, IkappaB alpha degradation in response to mitogenic stimuli, e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA), is inhibited. Finally, NFkappaB cannot be activated by TPA in cell lines that have low levels of p90rsk1. We conclude that p90rsk1 is an essential kinase required for phosphorylation and subsequent degradation of IkappaB alpha in response to mitogens.

Animals↗
PubMed1997

Regulation of IkappaBbeta degradation. Similarities to and differences from IkappaBalpha.

The transcription factor NF-kappaB (nuclear factor-kappaB) is neutralized in nonstimulated cells through cytoplasmic retention by IkappaB inhibitors. In mammalian cells, two major forms of IkappaB proteins, IkappaBalpha and IkappaBbeta, have been identified. Upon treatment with a large variety of inducers, IkappaBalpha and IkappaBbeta are proteolytically degraded, resulting in NF-kappaB translocation into the nucleus. Recent observations suggest that phosphorylation of serines 32 and 36 and subsequent ubiquitination of lysines 21 and 22 of IkappaBalpha control its signal-induced degradation. In this study we provide evidence that critical residues in the NH2-terminal region of IkappaBbeta (serines 19 and 23) as well as its COOH-terminal PEST region control IkappaBbeta proteolysis. However Lys-9, the unique lysine residue in the NH2-terminal region of IkappaBbeta, is not absolutely required for its degradation. We also demonstrate that following stimulation, an underphosphorylated nondegradable form of IkappaBbeta accumulates. Surprisingly, our data suggest that unlike IkappaBalpha, IkappaBbeta is constitutively phosphorylated on one or two of the critical NH2-terminal serine residues. Thus, phosphorylation of these sites is necessary for degradation but does not necessarily constitute the signal-induced event that targets the molecule for proteolysis.

Animals↗
PubMed1997

I kappa B epsilon, a novel member of the I kappa B family, controls RelA and cRel NF-kappa B activity.

We have isolated a human cDNA which encodes a novel I kappa B family member using a yeast two-hybrid screen for proteins able to interact with the p52 subunit of the transcription factor NF-kappa B. The protein is found in many cell types and its expression is up-regulated following NF-kappa B activation and during myelopoiesis. Consistent with its proposed role as an I kappa B molecule, I kappa B-epsilon is able to inhibit NF-kappa B-directed transactivation via cytoplasmic retention of rel proteins. I kappa B-epsilon translation initiates from an internal ATG codon to give rise to a protein of 45 kDa, which exists as multiple phosphorylated isoforms in resting cells. Unlike the other inhibitors, it is found almost exclusively in complexes containing RelA and/or cRel. Upon activation, I kappa B-epsilon protein is degraded with slow kinetics by a proteasome-dependent mechanism. Similarly to I kappa B-alpha and I kappa B, I kappa B-epsilon contains multiple ankyrin repeats and two conserved serines which are necessary for signal-induced degradation of the molecule. A unique lysine residue located N-terminal of the serines appears to be not strictly required for degradation. Unlike I kappa B- alpha and I kappa B-beta, I kappa B-epsilon does not contain a C-terminal PEST-like sequence. I kappa B-epsilon would, therefore, appear to regulate a late, transient activation of a subset of genes, regulated by RelA/cRel NF-kappa B complexes, distinct from those regulated by other I kappa B proteins.

Amino Acid Sequence↗
PubMed1997

I kappa B proteins: structure, function and regulation.

The Rel/NF-kappa B transcription factors represent the paradigm of regulation of transcriptional activation through sub-cellular localization. In unstimulated cells, NK-kappa B exists in an inactive state in the cytoplasm complexed to the inhibitory I kappa B molecules. Upon stimulation, I kappa B is rapidly degraded, freeing NF-kappa B to translocate to the nucleus and to activate the expression of its target genes. In this chapter, we will summarize what is known about the structure of I kappa B molecules, their functions, the mechanisms of I kappa B degradation, and the most common upstream signaling pathway (that is, serine phosphorylation) that leads to I kappa B degradation. Finally, we will discuss alternative mechanisms for induction of NF-kappa B through regulation of I kappa B activity.

Animals↗
PubMed1997

Control of NF-kappa B activity by the I kappa B beta inhibitor.

The transcription factor NF-kappa B is maintained in an inactive cytoplasmic state by I kappa B inhibitors. In mammalian cells, I kappa B alpha and I kappa B beta proteins have been purified and shown to be the inhibitors of NF-kappa B through their association with the p65 or c-Rel subunits. In addition, we have isolated a third NF-kappa B inhibitor, I kappa B epsilon (1). Upon treatment with a large variety of inducers, I kappa B alpha, I kappa B beta are proteolytically degraded, resulting in NF-kappa B translocation into the nucleus. Here we show that in E29.1 T cell hybridoma I kappa B alpha and I kappa B beta are equally associated with p65 and that I kappa B beta is degraded in response to TNF alpha in contrast to what has been originally published. Our data also suggest that, unlike I kappa B alpha, I kappa B beta is constitutively phosphorylated and resynthesized as a hypophosphorylated form. The absence of slow migrating forms of I kappa B beta following stimulation suggests that the phosphorylation does not necessarily constitute the signal-induced event which targets the molecule for proteolysis.

Animals↗
PubMed1996

Phosphorylation of p105 PEST sequence via a redox-insensitive pathway up-regulates processing of p50 NF-kappaB.

The p105 Rel protein has dual functions; it is the precursor of the p5O subunit of NF-kappaB, and it acts as an IkappaB-like inhibitor to retain other Rel subunits in the cytoplasm. We have investigated the posttranslational regulation of p105 following activation of Jurkat T cells and find that a rapid and sustained phosphorylation of p105 is induced. The inducible phosphorylation occurs on multiple serines in the C-terminal-most 150 amino acids of the molecule, a region rich in Pro, Glu, Ser, and Thr residues. Phosphorylation of p105 in Jurkat cells treated with phorbol 12-myristate 13-acetate/ionomycin or with okadaic acid, another activator of NF-kappaB, is correlated with an increase in proteolytic processing to p5O. Intact PEST sequences are required for the phorbol 12-myristate 13-acetate/ionomycin-induced p105 processing, as a 68-amino acid C-terminal deletion abolishes the response to stimulation. When compounds that block Ikappa B alpha phosphorylation and degradation were tested, the serine protease inhibitors L-1-tosylamido-2-phenylethyl chloromethyl ketone and 1-chloro-3-tosyl-amido-7-amino-2-heptanone blocked inducible p105 phosphorylation, but the antioxidants pyrrolidine dithiocarbamate and butylated hydroxyanisol did not. Thus, while regulation of the p105 IkappaB resembles that of lkappaBa, involving inducible serine phosphorylation and proteolysis of the inhibitory ankyrin repeat domain, it depends on a different, redox-insensitive, signaling pathway.

Amino Acid Sequence↗