Biomedical subjects

Y Lutz

Publications and source records attributed to Y Lutz.

At least 37 records · Page 2Linked to original sources

PubMed1996

Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene.

The full-length cDNA corresponding to Stra8, a novel gene inducible by retinoic acid (RA) in P19 embryonal carcinoma cells, has been isolated and shown to encode a 45-kD protein. Both Stra8 mRNA and protein were induced in cells treated by all-trans and 9-cis retinoic acids. Two-dimensional gel analysis and dephosphorylation experiments revealed that the two stereoisomers of RA differentially regulate the phosphorylation status of the Stra8 protein, which was shown to exist in differently phosphorylated forms. Subcellular fractionation and immunocytochemistry studies showed that the Stra8 protein is cytoplasmic. During mouse embryogenesis, Stra8 expression was restricted to the male developing gonads, and in adult mice, the expression of Stra8 was restricted to the premeiotic germ cells. Thus, Stra8 protein may play a role in the premeiotic phase of spermatogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Nucleoplasmic and nucleolar distribution of the adenovirus IVa2 gene product.

Sequence elements (DE) located downstream of the adenovirus major late promoter start site have previously been shown to be essential for the activation of this promoter after the onset of viral DNA replication. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner and contribute to this activation. DEF-B corresponds to a dimer of the adenovirus IVa2 gene product (pIVa2, 449 residues), while DEF-A is a heteromeric protein also comprising pIVa2. As revealed by specific immunofluorescence staining of infected cells, pIVa2 is targeted to the nucleus, where it distributes to both nucleoplasmic and nucleolar structures. We have identified the pIVa2 nuclear localization signal (NLS) as a basic peptide element at the C terminus of the protein (residues 432 to 449). An element essential for nucleolar localization (NuLS) has been mapped in the N-terminal part of pIVa2 (between residues 50 and 136). While NuLS activity is dependent upon an intact NLS, we show that both NLS and NuLS functions are independent of specific DNA-binding activity. As visualized by immunoelectron microscopy, pIVa2 is detected in the nucleoplasm at the level of the fibrillogranular network which is active in viral transcription. More surprisingly, pIVa2 accumulates within electron-dense amorphous inclusions found both in the nucleoplasm and in the nucleolus. Altogether, these results suggest that, besides controlling major late promoter transcription, pIVa2 serves additional, as yet unknown functions.

Adenoviridae↗

PubMed1996

Immunohistochemical analysis of retinoic acid receptor-alpha in human breast tumors: retinoic acid receptor-alpha expression correlates with proliferative activity.

Retinoids are known to prevent mammary carcinogenesis in rodents and inhibit the growth of human breast cancer cells in vitro. Previously we demonstrated that retinoid inhibition of proliferation of human breast cancer cell lines is largely mediated by retinoic acid receptor (RAR)-alpha. In this study we describe for the first time the histological distribution of RAR-alpha in 33 breast lesion specimens as determined by immunostaining with RAR-alpha antibody. Nuclear staining was observed in tumor tissue and normal portions of the breast samples. Connective tissue exhibited relative uniform staining, whereas a wide range of RAR-alpha expression was found in the epithelial tumor cells. RAR-alpha protein was expressed at significantly higher levels in tumors with greater proliferative activity as determined by immunostaining with Ki-67 antibody. This suggests that RAR-alpha expression may be altered with tumor progression. Although a positive correlation between RAR-alpha mRNA levels and estrogen receptor status of breast tumors has previously been documented, we did not find such a relationship at the protein level. As RAR-alpha plays a major role in retinoid-mediated growth inhibition of human breast cancer cell in vitro, our findings suggest that patients with highly proliferating tumors could be responsive to retinoid independently of their responsiveness to (anti)-estrogens.

Breast Neoplasms↗

PubMed1995

Polyglutamine expansion as a pathological epitope in Huntington's disease and four dominant cerebellar ataxias.

A polyglutamine expansion (encoded by a CAG repeat) in specific proteins causes neurodegeneration in Huntington's disease (HD) and four other disorders, by an unknown mechanism thought to involve gain of function or toxicity of the mutated protein. The pathological threshold is 37-40 glutamines in three of these diseases, whereas the corresponding normal proteins contain polymorphic repeats of up to about 35 glutamines. The age of onset of clinical manifestations is inversely correlated to the length of the polyglutamine expansion. Here we report the characterization of a monoclonal antibody that selectively recognizes polyglutamine expansion in the proteins implicated in HD and in spinocerebellar ataxia (SCA) 1 and 3. The intensity of signal depends on the length of the polyglutamine expansion, and the antibody also detects specific pathological proteins expected to contain such expansion, in SCA2 and in autosomal dominant cerebellar ataxia with retinal degeneration, whose genes have not yet been identified.

Adult↗

PubMed1995

Characterization of monoclonal antibodies against stromelysin-3 and their use to evaluate stromelysin-3 levels in breast carcinoma by semi-quantitative immunohistochemistry.

Stromelysin-3 (ST3) is a matrix metalloproteinase which is expressed in fibroblastic cells of most human invasive carcinomas and represents a potential new prognostic indicator. Expression of recombinant ST3 forms in Escherichia coli from cDNA constructs indicated that high levels of expression were achieved when the ST3 pro-domain was deleted. The putative mature form of ST3 thus produced and recovered from bacterial inclusion bodies was used to prepare monoclonal antibodies (MAbs) against ST3 by immunization of BALB/C mice. Ten hybridomas producing MAbs against ST3 were obtained and analyzed for their ability to detect endogenous ST3 in breast cancer and in conditioned media from human fibroblasts. One of these MAbs (5ST-4A9) was found to be suitable for the routine detection of ST3 on breast cancer tissue sections, thus opening the possibility to evaluate ST3 prognostic value in breast cancer using semi-quantitative immunohistochemistry.

Animals↗

PubMed1995

The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18.

Nuclear receptors (NRs) bound to response elements mediate the effects of cognate ligands on gene expression. Their ligand-dependent activation function, AF-2, presumably acts on the basal transcription machinery through intermediary proteins/mediators. We have isolated a mouse nuclear protein, TIF1, which enhances RXR and RAR AF-2 in yeast and interacts in a ligand-dependent manner with several NRs in yeast and mammalian cells, as well as in vitro. Remarkably, these interactions require the amino acids constituting the AF-2 activating domain conserved in all active NRs. Moreover, the oestrogen receptor (ER) AF-2 antagonist hydroxytamoxifen cannot promote ER-TIF1 interaction. We propose that TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is a mediator of ligand-dependent AF-2. Interestingly, the TIF1 N-terminal moiety is fused to B-raf in the mouse oncoprotein T18.

Amino Acid Sequence↗

PubMed1995

Two hnRNP-associated proteins share common structural features with the adenoviral 72-kDa protein.

Using our anti-hnRNP monoclonal antibody library Y. Lutz, M. Jacob, and J.-P. Fuchs (1988) Exp. Cell Res., 175, 108-124; P. Mähl, Y. Lutz, E. Puvion, and J.-P. Fuchs, (1989) J. Cell Biol. 109, 1921-1935, we investigated by immunocytofluorescence the fate of a series of speckled-distributed nuclear antigens, after HeLa cells were infected with adenovirus type 2. Although the speckled pattern, which corresponds to the nucleoplasmic fibrillogranular network, including the interchromatin-granule clusters, was still observed during most of the infectious cycle, several antibodies also revealed additional, increasingly fluorescent virus-induced structures. In noninfected cells, two of these antibodies, termed 3F2 and 2A5, recognize two antigens of 33 and 31 kDa, respectively. Western blot analysis showed that this increasing amount of fluorescence observed in infected cells did not reflect an accumulation of the 33- and 31-kDa antigens, but is actually due to the fact that both antibodies also recognize the multifunctional adenovirus 72-kDa single-stranded DNA-binding protein (DBP). Immunoelectron microscopy analyses, including sequential double-labeling, indeed showed that this additional signal precisely colocalizes with the viral 72-kDa DBP, which essentially accumulates over the entire surface of the virus-induced single-stranded DNA accumulation sites. Taken together, our data show that two host-specific hnRNP-associated antigens share common epitopes with the viral 72-kDa DBP.

Adenoviruses, Human↗

PubMed1995

Cellular localization of the Huntington's disease protein and discrimination of the normal and mutated form.

Huntington's disease (HD) results from the expansion of a polyglutamine encoding CAG repeat in a gene of unknown function. The wide expression of this transcript does not correlate with the pattern of neuropathology in HD. To study the HD gene product (huntingtin), we have developed monoclonal antibodies raised against four different regions of the protein. On western blots, these monoclonals detect the approximately 350 kD huntingtin protein in various human cell lines and in neural and non-neural rodent tissues. In cell lines from HD patients, a doublet protein is detected corresponding to the mutated and normal huntingtin. Immunohistochemical studies in the human brain using two of these antibodies detects the huntingtin in perikarya of some neurons, neuropiles, varicosities and as punctate staining likely to be nerve endings.

Amino Acid Sequence↗

PubMed1994

Detection of retinoid X receptors using specific monoclonal and polyclonal antibodies.

Because of the growing importance of the Retinoid X Receptors (RXR alpha, beta and gamma) in the retinoid acid signalling pathway, we have prepared polyclonal and monoclonal antibodies directed against these proteins. For this purpose, either the whole mouse RXR alpha protein expressed in E.Coli, or synthetic peptides corresponding to amino acid sequences common to all RXRs or unique to RXR alpha, beta or gamma, were used as antigens. Antibodies recognizing either all three RXR types (alpha, beta and gamma) or specific for each RXR type were obtained. The antibodies were characterized by immunocytochemistry, immunoblotting, immunoprecipitation and electromobility shift assay (EMSA). These antibodies allowed us to detect the presence of RXR alpha proteins in mouse embryos and in mouse embryonal carcinoma cells (F9 and P19 cell lines) by immunoblotting, immunoprecipitation and EMSA whereas RXR beta could be detected only by EMSA and RXR gamma could not be detected by any of these techniques.

Animals↗

PubMed1994

Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor.

We showed previously that coactivators mediating stimulation by different activators were associated with the TATA-binding protein (TBP) in distinct TFIID complexes. We have characterized a human TBP-associated factor (TAF), hTAFII30, associated with a subset of TFIID complexes. hTAFII30 interacts with the AF-2-containing region E of the human estrogen receptor (ER), but not with ER AF-1 or VP16. An antibody against hTAFII30 inhibited transcriptional stimulation by the ER AF-2 without affecting basal or VP16-activated transcription and allowed the separation of TFIID complex(es) containing hTAFII30 from complexes mediating the activity of VP16. These results directly demonstrate the existence of functionally distinct TFIID populations that share common TAFIIs but differ in specific TAFIIs.

Amino Acid Sequence↗

PubMed1994

p44 and p34 subunits of the BTF2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in DNA repair.

The human BTF2 (TFIIH) transcription factor is a multisubunit protein involved in transcription initiation by RNA polymerase II (B) as well as in DNA repair. In addition to the previously characterized p62 and p89/ERCC3 subunits, we have cloned two other subunits of BTF2, p44 and p34. The gene encoding p44 appeared to be the human counterpart of SSL1, a gene involved in translation and UV resistance in yeast. Interestingly, the p34 subunit also has homology with a domain of SSL1, suggesting that it corresponds to an as yet unidentified protein involved in DNA repair. Both p44 and p34 possess zinc finger domains that may mediate BTF2 binding to nucleic acids.

Amino Acid Sequence↗

PubMed1994

The N-terminal domain of the human TATA-binding protein plays a role in transcription from TATA-containing RNA polymerase II and III promoters.

In eukaryotes, the TATA box binding protein (TBP) is an integral component of the transcription initiation complexes of all three classes of nuclear RNA polymerases. In this study we have investigated the role of the N-terminal region of human TBP in transcription initiation from RNA polymerase (Pol) I, II and III promoters by using three monoclonal antibodies (mAbs). Each antibody recognizes a distinct epitope in the N-terminal domain of human TBP. We demonstrate that these antibodies differentially affect transcription from distinct classes of promoters. One antibody, mAb1C2, and a synthetic peptide comprising its epitope selectively inhibited in vitro transcription from TATA-containing, but not from TATA-less promoters, irrespective of whether they were transcribed by Pol II or Pol III. Transcription by Pol I, on the other hand, was not affected. Two other antibodies and their respective epitope peptides did not affect transcription from any of the promoters tested. Order of addition experiments indicate that mAb1C2 did not prevent binding of TBP to the TATA box or the formation of the TBP-TFIIA-TFIIB complex but rather inhibited a subsequent step of preinitiation complex formation. These data suggest that a defined region within the N-terminal domain of human TBP may be involved in specific protein-protein interactions required for the assembly of functional preinitiation complexes on TATA-containing, but not on TATA-less promoters.

Antibodies, Monoclonal↗

PubMed1994

The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein.

Adrenoleukodystrophy is a severe genetic demyelinating disease associated with an impairment of beta-oxidation of very long chain fatty acids (VLCFA) in peroxisomes. Earlier studies had suggested that a deficiency in VLCFA CoA synthetase was the primary defect. A candidate adrenoleukodystrophy gene has recently been cloned and was found unexpectedly to encode a putative ATP-binding cassette transporter. We have raised monoclonal antibodies against this protein, that detect a 75kDa band. This protein was absent in several patients with adrenoleukodystrophy. Immunofluorescence and immunoelectron microscopy showed that the adrenoleukodystrophy protein (ALDP) is associated with the peroxisomal membrane. Distinct immunofluorescence patterns were observed in cell lines from patients with Zellweger syndrome (a peroxisomal biogenesis disorder) belonging to different complementation groups.

ATP Binding Cassette Transporter, Subfamily D, Mem↗

PubMed1994

Jun and Fos heterodimerize with ATFa, a member of the ATF/CREB family and modulate its transcriptional activity.

Three related clones encoding proteins (ATFa1, 2 and 3) with specific ATF/CRE DNA-binding activities have been isolated from HeLa cell cDNA libraries. All three isoforms have weak effects on the basal activity of the adenovirus E2a promoter. We present evidence suggesting that a C-terminal element of the ATFa molecules negatively interferes with the intrinsic activation function of these proteins. We also show that coexpression of ATFa with c-Jun, Jun-B or Jun-D stimulates ATFa-dependent reporter activity, while coexpression of c-Fos has no effect. Deletion analyses indicate that the metal-binding region of ATFa is dispensible for this effect, but that the domain comprising the leucine-zipper region of ATFa is required. Reciprocal co-immunoprecipitation experiments and electrophoretic band-shift assays with in vitro synthesized proteins reveal direct interactions between ATFa and Jun or Fos. The ATFa/c-Jun heterodimers, but not the ATFa/c-Fos complexes, bind efficiently to ATF, CRE or AP1 sites. The detection of ATFa-Jun complexes in crude extracts from HeLa cells transfected with ATFa and c-Jun expression vectors suggests that such ATFa/c-Jun heterodimers also form in vivo. Altogether these results indicate that the ATFa proteins may contribute to the modulation of the activity of the Jun/Fos complexes by altering their DNA-binding and transcriptional properties.

Activating Transcription Factors↗

PubMed1993

Definition of the DNA-binding site repertoire for the Drosophila transcription factor SNAIL.

The Drosophila gene snail (sna) which encodes a zinc finger protein is essential for dorsal-ventral pattern formation in the developing embryo. We have defined a repertoire of SNAIL (SNA) binding sites using recombinant SNA proteins to select specific binding sequences from a pool of random sequence nucleotides. The bound sequences which were selected by multiple rounds of gel retardation and amplification by the polymerase chain reaction (PCR) were subsequently cloned and sequenced. The consensus sequence, 5'G/A A/t G/A A CAGGTG C/t A C 3', with a highly conserved core of 6 bases, CAGGTG, shares no significant homology with known binding sequences of other Drosophila zinc finger proteins. However, the CAGGTG core is identical to the core motif of aHLH (helix-loop-helix) binding sites. The strongest SNA binding is obtained with sequences containing this core motif whereas reduced binding is seen for sequences with canonical CANNTG HLH motifs. Interestingly, SNA binding is detected in the promoter region of the snail gene. Transient expression in co-transfection experiments using a SNA binding element (SBE) linked to a heterologous promoter indicates that SNA has the ability to function as a transcription activator.

Animals↗

PubMed1993

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

The bone-specific osteocalcin (OC) gene is transcribed only after completion of proliferation in normal diploid calvarial-derived osteoblasts during extracellular matrix mineralization. In contrast, the OC gene is expressed constitutively in both proliferating and nonproliferating ROS 17/2.8 osteosarcoma cells. To address molecular mechanisms associated with these tumor-related modifications in transcriptional control, we examined sequence-specific interactions of transactivation factors at key basal and hormone-responsive elements in the OC gene promoter. In ROS 17/2.8 cells compared to normal diploid osteoblasts, the absence of a stringent requirement for cessation of proliferation to support both induction of OC transcription and steroid hormone-mediated transcriptional modulation is reflected by modifications in transcription factor binding at (i) the two primary basal regulatory elements, the OC box (which contains a CCAAT motif as a central core) and the TATA/glucocorticoid-responsive element domain, and (ii) the vitamin D-responsive element. Particularly striking are two forms of the vitamin D receptor complex that are present in proliferating osteoblasts and osteosarcoma cells. Both forms of the complex are sensitive to vitamin D receptor antibody and retinoic X receptor antibody. After the down-regulation of proliferation, only the lower molecular weight complex is found in normal diploid osteoblasts. Both forms of the complex are present in nonproliferating ROS 17/2.8 cells with increased representation of the complex exhibiting reduced electrophoretic mobility that is phosphorylation-dependent.

Animals↗

PubMed1993

Stromelysin 3 belongs to a subgroup of proteinases expressed in breast carcinoma fibroblastic cells and possibly implicated in tumor progression.

The expression of the stromelysin 3 (ST3) gene, which encodes a putative matrix metalloproteinase, was studied during breast cancer progression. The ST3 gene is expressed in all invasive breast carcinomas, in a number of their metastases, and in some in situ carcinomas where the probability of detecting ST3 transcripts correlates with the known risk of these carcinomas to become invasive. ST3 RNA and protein were specifically detected in fibroblastic cells immediately surrounding the neoplastic cells in both primary and metastatic tumors. This expression pattern distinguishes the ST3 gene from other matrix metalloproteinase genes, most notably from the 72-kDa type IV collagenase gene, which can be expressed in fibroblastic cells distributed throughout the stroma of primary breast carcinomas. Furthermore, high levels of 72-kDa type IV collagenase, but not of ST3 transcripts, are detected in benign breast fibroadenomas. Interestingly, the urokinase and ST3 genes exhibit very similar patterns of expression in breast carcinomas, which suggests that their products may cooperate during cancer progression.

Adenoma↗

PubMed1993

Distinct TFIID complexes mediate the effect of different transcriptional activators.

Multiple chromatographically separable complexes containing the TATA binding protein (TBP), which exhibit different functional properties, exist in HeLa cells. At least three distinct subpopulations of such complexes can be functionally defined as TFIID since they function with RNA polymerase II. Using a partially reconstituted HeLa cell in vitro transcription system and immunoprecipitation with a monoclonal antibody directed against TBP, we show that stimulation of transcription by the chimeric activators GAL-VP16, GAL-TEF-1 and GAL-ER(EF) requires the presence of factors which are tightly associated with these TFIID complexes. Moreover, the activity of GAL-TEF-1 appears to be mediated by at least two chromatographically distinct populations of TFIID. The factor(s) associated with one of these populations is also required for the activity of GAL-ER (EF) and GAL-VP16, while the factor(s) associated with the other population functions selectively with GAL-TEF-1. These two TFIID populations are composed of both common and unique TBP associated factors (TAFs).

Animals↗