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N Brass

Publications and source records attributed to N Brass.

18 recordsLinked to original sources
PubMed2001

Translation initiation factor eIF-4G is immunogenic, overexpressed, and amplified in patients with squamous cell lung carcinoma.

BACKGROUND: Recently, the authors reported identification and cloning of several novel immunogenic antigens in squamous cell lung carcinoma. Of 14 corresponding genes, 9 mapped in an amplified chromosomal region with the gene for eIF-4G that was amplified most frequently. METHODS: Recombinant eIF-4G was expressed in E. coli and screened with sera from patients with squamous cell carcinoma of the lung and of the head and neck. Protein extracts from squamous cell carcinoma tissues were analyzed for eIF-4G expression by Western blot analysis. Mutation analysis was performed using an automatic DNA sequencer. RESULTS: The authors screened a spectrum of 33 heterologous sera from lung carcinoma patients and detected antibodies against eIF-4G in 5 of these sera (15%). They found no immune response to eIF-4G in 17 sera from squamous cell carcinoma tissues derived from the head and neck. In addition, no antibodies were found to eIF-4G in a group of 17 control sera from individuals without known tumor formation. Sequence analysis of the eIF-4G gave no indication of mutations. To analyze the expression of eIF-4G, a monoclonal antibody was used. Western blot analysis clearly showed overexpression in the tumor tissue compared with the corresponding normal lung tissues. CONCLUSIONS: The translation initiation factor eIF-4G is the first protein in which the gene shows amplification, has increased expression in a human tumor, and induces an immune response in patients. eIF-4G lends itself as a marker for the diagnosis of and possibly as a future therapeutic target in patients with squamous cell lung carcinoma.

Antibody Formation↗
PubMed2000

Gene amplification at chromosome 1pter-p33 including the genes PAX7 and ENO1 in squamous cell lung carcinoma.

Gene amplification is a frequent event in lung cancer, specifically in squamous cell lung carcinoma. Recently, we reported amplifications on chromosomal bands 3q26.1-q26.3 with the genes BCHE and SLC2A2 amplified in 40% of squamous cell lung carcinomas. Here, we identified an amplified domain within chromosomal bands 1pter-p33 in squamous cell lung carcinoma using reverse chromosome painting. A panel of nine genes which have previously been assigned to region 1pter-p33 was tested for amplification using comparative PCR. The ENO1 gene that encodes enolase and the PAX7 gene that encodes a transcription factor were most frequently amplified. Specifically, the gene ENO1 was amplified in six and the gene PAX7 in five out of 37 cases which included both biopsies and paraffin-embedded tissues of squamous cell lung carcinomas. In total, we identified amplifications of at least one gene at bands 1pter-p33 in 10 out of 37 tumors (27%). Together, our data indicate that a novel and frequent amplification unit is present in squamous cell lung carcinoma with the center of the amplified domain in the vicinity of the genes PAX7 and ENO1.

Carcinoma, Squamous Cell↗
PubMed1999

Role of amplified genes in the production of autoantibodies.

A variety of previously published studies have shown the presence of autoantibodies directed against oncogenic proteins in the sera of patients with tumors. Generally the underlying genetic aberration responsible for the induction of an immune response directed against an abnormal protein is unknown. In our studies we analyzed the role of gene amplification in the production of autoantibodies in squamous cell lung carcinoma. We screened a cDNA expression library with autologous patient serum and characterized the isolated cDNA clones encoding tumor expressed antigens termed LCEA (lung carcinoma expressed antigens). As determined by sequence analysis, the 35 identified cDNA clones represent 19 different genes of both known and unknown function. The spectrum of different clones were mapped by polymerase chain reaction (PCR) and fluorescence in-situ hybridization, showing that a majority are located on chromosome 3, which is frequently affected by chromosomal abnormalities in lung cancer. Gene amplification of 14 genes was analyzed by comparative PCR. Nine genes (65% of all analyzed genes) were found to be amplified; furthermore, most of them are also overrepresented in the pool of cDNA clones, suggesting an overexpression in the corresponding tumor. These results strongly suggest that gene amplification is one possible mechanism for the expression of immunoreactive antigens in squamous cell lung carcinoma.

Antigens, Neoplasm↗
PubMed1999

Expression analysis of genes at 3q26-q27 involved in frequent amplification in squamous cell lung carcinoma.

Gene amplifications are known to occur frequently in lung cancer. Recently, we identified gene amplifications at 3q26 in squamous cell lung carcinoma (SCC) using reverse chromosome painting. Here, our aim was to analyse the expression of genes which map within the amplified chromosomal region. The genes which were selected for their known function and their potential involvement in tumour development included the genes for ribosomal protein L22 (RPL22), butyrylcholinesterase (BCHE), glucose transporter 2 (SLC2A2), transferrin receptor (TFRC), thrombopoietin (THPO) and the phosphatidylinositol-3 kinase catalytic alpha polypeptide (PIK3CA). While five genes were expressed in the majority of the 17 samples of SCC, the gene for the glucose transporter 2 (SLC2A2) was expressed in only three cases, excluding SLC2A2 as the target gene of the amplification unit. For a subset of tumours, we determined the amplification status of the six genes. The TFRC, PIK3CA, BCHE, THPO and SLC2A2 genes were amplified in several cases, whereas the RPL22 gene was amplified in only one case. The combined amplification and expression data of this and our previous studies indicate that the amplified region at 3q26 contains several genes that are transcribed in SCC, providing the possibility that several amplified and functionally important genes at 3q26 may be involved in the pathogenesis of SCC.

Blotting, Southern↗
PubMed1999

Five novel immunogenic antigens in meningioma: cloning, expression analysis, and chromosomal mapping.

Tumorigenesis of meningioma has been associated with chromosome 22, most notably the NF2 gene, but additional genes have been implicated in meningioma development. Here, we report the identification of five novel immunogenic antigens expressed in meningioma. An expression library was generated from a meningioma that retained both copies of chromosome 22. Screening with autologous patient serum identified seven cDNA clones that were indicated by antigen-antibody complexes. The clones were sequenced, and sequence comparison revealed that the seven clones represent five different genes, providing evidence that meningiomas express a spectrum of immunoreactive antigens, which were termed meningioma expressed antigens (MGEAs). One gene was identical with the connective tissue growth factor, one gene was in part homologous to an Alzheimer disease-associated gene, and a third gene was in part identical to Homo sapiens molybdenum cofactor biosynthesis proteins A and C mRNA. One gene was partially homologous to previously reported cDNA sequences of unknown function, and the fifth gene showed no significant homologies to sequences deposited in databases. Using somatic hybrid mapping, three genes were localized on chromosome 6, and two genes were localized on chromosomes 3 and 17, respectively. To distinguish the MGEAs from the so-called natural autoantigenes, we also screened the library with 12 sera from individuals without obvious disease. The clones identified by reactivity with normal sera were completely different from the clones identified by screening the same meningioma expression library with serum from the patient bearing the tumor. These data suggest that the newly identified MGEA genes may be useful for diagnosis and possibly therapy of meningioma.

Algorithms↗
PubMed1998

Lymphocyte-specific chemokine receptor CXCR3: regulation, chemokine binding and gene localization.

Expression of CXCR3, the receptor for the CXC chemokines IFN-gamma-inducible 10-kDa protein (IP10) and monokine induced by IFN-gamma (Mig), in human T lymphocytes and their responses to IP10 and Mig were analyzed. About 40 % of resting T lymphocytes (and low numbers of B cells and natural killer cells) stained positive for CXCR3 but these cells did not express CXCR3 transcripts and did not respond to these chemokines. However, treatment with IL-2 with or without addition of phytohemagglutinin for 10 or more days resulted in cultures of fully responsive, CXCR3-positive T lymphocytes. Treatment with anti-CD3 antibodies in the presence or absence of soluble anti-CD28 antibodies was inhibitory. Addition of chondroitin sulfate C to CXCR3-expressing murine pre-B cells allowed the determination of high-affinity binding for Mig and IP10 with Kd of 0.9-1.2 nM and 0.2-0.3 nM, respectively, and 1.3 x 10(4) binding sites per cell. The gene for CXCR3 was localized on human chromosome Xq13 which is in clear contrast to all other chemokine receptor genes, suggesting unique function(s) for this receptor and its ligands that may lie beyond their established role in T cell-dependent immunity.

Chemokine CXCL10↗
PubMed1998

Novel immunogenic antigen homologous to hyaluronidase in meningioma.

By screening a meningioma expression library with autologous serum we identified four cDNA clones representing a novel gene with striking homology to Caenorhabditis elegans hyaluronidase as indicated by BLASTP analysis. In humans hyaluronidase has been implicated in cancer development and three human genes are known to encode proteins with hyaluronidase activity. None of the human genes, however, showed any homology at the nucleotide or amino acid sequence level to the newly isolated antigen we termed meningioma expressed antigen 5 (MGEA5). Somatic cell hybrid mapping and fluorescence in situ hybridization mapped the gene for MGEA5 to chromosomal band 10q24.1-q24.3. Reverse transcription (RT)-PCR and northern blot hybridization revealed expression of the gene encoding MGEA5 in several meningioma and additional human tissues. Expression analysis also indicated an alternative splicing event giving rise to a shorter and altered transcript termed MGEA5s. The expression of MGEA5 and MGEA5s as fusion proteins revealed an approximate molecular weight of 92 and 54 kDa, respectively. Using heterologous sera we found antibodies against MGEA5s in five out of 23 meningioma patients, whereas no immune reaction was detected in 12 control sera from healthy individuals. Confirmation of hyaluronidase activity was independently achieved by turbidometric analysis and a gel matrix assay. A model for involvement of the novel hyaluronidase gene in meningioma development is proposed.

Acetylglucosaminidase↗
PubMed1997

TYMSTR, a putative chemokine receptor selectively expressed in activated T cells, exhibits HIV-1 coreceptor function.

BACKGROUND: Chemokines bind to specific receptors and mediate leukocyte migration to sites of inflammation. Recently, some chemokine receptors, notably CXCR4 and CCR5, have been shown to be essential fusion factors on target cells for infection by human immunodeficiency virus (HIV); the chemokines bound by these receptors have also been shown to act as potent inhibitors of HIV infection. Here, we describe the isolation of a novel, putative chemokine receptor. RESULTS: We have isolated the cDNA for a putative human chemokine receptor, which we have termed TYMSTR (T-lymphocyte-expressed seven-transmembrane domain receptor). The TYMSTR gene is localized to human chromosome 3 and encodes a protein that has a high level of identity with chemokine receptors. TYMSTR mRNA was selectively expressed in interleukin-2-stimulated T lymphocytes but not in freshly isolated lymphocytes and leukocytes or related cell lines. The natural ligand for TYMSTR was not identified among 32 human chemokines and other potential ligands. Cells co-expressing TYMSTR and human CD4 fused with cells expressing envelope glycoproteins of macrophage (M)-tropic HIV-1 as well as T-cell line (T)-tropic HIV-1 isolates. Addition of infectious, T-tropic HIV-1 particles to TYMSTR/CD4-expressing cells resulted in viral entry and proviral DNA formation. CONCLUSIONS: Our findings demonstrate that TYMSTR, in combination with CD4, mediates HIV-1 fusion and entry. The high-level expression of TYMSTR in CD4(+) T lymphocytes and the selectivity of this receptor for T-tropic and M-tropic HIV-1 strains indicates that TYMSTR might function as HIV coreceptor at both early and late stages of infection.

Amino Acid Sequence↗
PubMed1997

Amplification of the genes BCHE and SLC2A2 in 40% of squamous cell carcinoma of the lung.

Gene amplification is a common genetic change in human cancer cells. Previously, we provided the first evidence for gene amplification at chromosome band 3q26 in squamous cell lung carcinoma. In this study, the following analyses were performed: (a) we evaluated biopsies and paraffin-embedded tissues of 16 additional squamous cell lung carcinomas for gene amplification using reverse chromosome painting. Of the 16 tumors, 3 tumors showed an amplification of the entire long arm of chromosome 3, and 3 tumors showed various amplifications on 3q, all of which involved chromosome band 3q26; (b) we tested eight genes encompassing region 3q25-qter in two different tumors to identify amplified genes on chromosome 3q. The genes SI, BCHE, and SLC2A2 were amplified in both tumors; and (c) we analyzed 15 additional paraffin-embedded tissues to determine the amplification frequency of these genes. Of the 15 squamous cell lung carcinomas, 6 showed amplification for at least 1 of the genes, with BCHE and SLC2A2 as the genes most frequently amplified. Together, our reverse chromosome painting data and our PCR analysis indicate gene amplification at 3q26 in 40% of all squamous cell lung carcinomas with BCHE and SLC2A2 as possible target genes of the amplification unit in squamous cell lung carcinoma.

Blotting, Southern↗
PubMed1997

Translation initiation factor eIF-4gamma is encoded by an amplified gene and induces an immune response in squamous cell lung carcinoma.

Amplification of cellular oncogenes is an important mechanism of altered gene expression in human cancers. Using comparative genomic hybridization we recently identified an amplification at 3q26.1-q26.3 in 30% of squamous cell carcinomas of the lung. A variety of methods including microdissection-mediated procedures permit cloning of genes encoded within amplified domains but do not directly lead to the identification of biologically relevant genes. In this study, we have circumvented this problem by combining an immunological and molecular genetic approach to analyze squamous cell lung carcinoma. To identify both amplified and tumor relevant genes, we generated a cDNA expression library from a tumor with the 3q amplification and hybridized the expressed recombinant polypeptides with the autologous serum. Of 400000 cDNA clones we identified 17 antigens which induce an immune response in a patient with squamous cell lung carcinoma. While most clones represent individual genes sequence analysis revealed that four of the 17 cDNAs are nearly identical with the eukaryotic translation initiation factor (eIF)-4gamma recently assigned on 3q. We demonstrated that the gene for eIF-4gamma was amplified within 3q26-q27 in independent squamous cell lung carcinomas. In this study, we report the identification of several antigens which elicit an immune response in a squamous cell lung carcinoma patient including eIF-4gamma. eIF-4gamma is encoded by an amplified gene and possibly plays a crucial part in the development of squamous cell lung carcinoma.

Base Sequence↗
PubMed1997

cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients.

There is increasing evidence that tumor expressed genes induce immune responses in cancer patients. To identify meningioma expressed antigens, we established a meningioma expression library which was screened with autologous serum. Out of 20 positive cDNA clones eight share high sequence homologies as determined by sequence analysis. These eight clones can be grouped into three classes which differ in length and which are characterized by specific sequence variations. The longest open reading frame was found to be 2412 bp encoding an immunoreactive antigen termed meningioma expressed antigen 6 (MEA6). Using five sequence specific primer pairs, somatic hybrid panel mapping revealed locations of the three classes on several human chromosomes including chromosomes 2, 3, 6, 7, 9, 13 and 14. The mapping results were confirmed by fluorescence in situ hybridization. RT-PCR showed consistent expression of all classes in several meningiomas and additional tissues using the same set of primer pairs as for chromosomal mapping. The expression data were confirmed by northern blot analysis. For the predicted amino acid sequence BLASTX revealed a homology to a human C219-reactive peptide which was previously isolated by an antibody directed against p-glycoprotein. Sequence properties of the MEA protein include an acidic activation domain, a proline-rich region and two coiled-coil domains indicating protein binding and activation functions.

Amino Acid Sequence↗
PubMed1996

DNA amplification on chromosome 3q26.1-q26.3 in squamous cell carcinoma of the lung detected by reverse chromosome painting.

Multiple genetic lesions have been reported in small cell lung carcinoma (SCLC), while considerably less information is available on squamous cell carcinoma (SCC). We used reverse chromosome painting to screen a total of nine SCCs for DNA amplifications. In three of the nine SCCs, hybridisation signals were found at chromosome region 3q26.1-q26.3, which does not contain any known oncogene. In one of the three SCCs, there were additional hybridisation signals at 1q, 5p and 6p21.1. The high frequency of a consistent amplification (3q26.1-q26.3) in SCC strongly indicates a novel gene at 3q26.1-q26.3 that is important in the pathology of SCC.

Aged↗
PubMed1995

Assignment of Alu-repetitive sequences to large restriction fragments from human chromosomes 6 and 22.

We have employed a pulsed field gel electrophoresis and Alu hybridization approach for identification of large restriction fragments on chromosome 6 and 22. This technique allows large portions of selected human chromosomes to be visualized as discrete hybridization signals. Somatic cell hybrid DNA which contains chromosome 6 or chromosome 22 was restricted with either Notl or Mlul. The restriction fragments were separated by pulsed field gel electrophoresis (PFGE) and hybridized against an Alu repetitive sequence (Blur 8). The hybridization signals result in a fingerprint-like pattern which is unique for each chromosome and each restriction enzyme. In addition, a continuous pattern of restriction fragments was demonstrated by gradually increasing puls times. This approach will also be suitable to analyze aberrant human chromosomes retained in somatic cell hybrids and can be used to analyze flow sorted human chromosomes. To this end, our method provides a valuable alternative to standard cytogenetic analysis.

Chromosome Mapping↗
PubMed1995

Isolation and localization of transcribed sequences on human chromosome 22.

Recently, we reported the generation of a heteronuclear (hn) cDNA library from a human x rodent somatic cell hybrid retaining human chromosome 22. Here, we report the characterization and localization of 12 cDNA derived clones from this library. Human-specific cDNA sequences have been selectively amplified by inter-Alu PCR. To exclude Alu transcripts, only clones with inserts larger than 500 bp were analyzed. Ten of the 12 clones were localized by PCR on chromosome 22, with four clones mapping on additional chromosomes. One PCR-localized clone and two additional clones were mapped on chromosome 22 by in situ hybridization. Transcripts in human cells were identified for seven of the eight clones analyzed by RT-PCR. None of the clones showed significant sequence similarities within the GenBank and EMBL databases, indicating that these clones represent previously unknown genes. This is the first report on the isolation of chromosome 22-specific transcripts from a human x rodent somatic cell hybrid.

Animals↗
PubMed1994

Strategy for chromosomal assignment of expressed sequences derived from heteronuclear RNA.

The chromosomal localization of transcribed sequences/genes is one of the objectives of the human genome project. Here, we describe a novel strategy for fast and dependable chromosomal assignment of expressed sequences that contain Alu sequences. Alu-PCR was performed on cDNA that was derived from heteronuclear (hn) RNA. hn-cDNA libraries are utilized for the identification of genes from extended human chromosomal regions or entire chromosomes. For chromosomal assignment, Alu-PCR products larger than 500 bp were hybridized against genomic DNA of somatic cell hybrids that was also amplified by Alu-PCR. Hybridization signals obtained within 2-3 h of exposure allow localization of cDNA-derived Alu-PCR products to single chromosomes. This technique is particularly useful for the analysis of cDNA libraries derived from hn-RNA. Using hn-cDNA clones from different chromosomes, we demonstrate the accuracy and reliability of the mapping strategy.

Animals↗
PubMed1993

Identification of chromosome-specific sequence-tagged sites by Alu-PCR.

Recently, methods have been developed for the isolation of expressed sequences from particular human chromosomes. Using Alu consensus sequences as primers, cDNA synthesis has been initiated from interspecies hybrid cell lines that contain single human chromosomes. Alu consensus sequences have also been utilized to amplify human genomic sequences via polymerase chain reaction (PCR). Here, we describe the use of Alu-PCR to isolate expressed sequences from human chromosomes selectively. Heteronuclear (hn) RNA is transcribed into cDNA by using poly-(dT)15 primer sequences. Subsequently, human specific cDNA sequences are amplified by Alu-PCR and cloned into pBluescript. To verify the chromosomal assignment, cloned PCR products are sequenced, converted into STS markers, and tested on a different somatic hybrid that contains human chromosome 22. The method provides a fast, reliable way to identify expressed sequence tagged sites from selected human chromosomes.

Animals↗
PubMed1980

[EEG-changes during haemodialysis in chronic renal insufficiancy (author's transl)].

Longitudinal EEG studies were performed under three different clinical conditions during 15 haemodialysis in chronic renal insufficiency. The relationship of EEG changes to clinical and biochemical parameters and to the subjective state of wellbeing was examined. The relevance of EEG monitoring as a method of assaying the adequacy of haemodialysis was investigated. By means of pre- and post-dialysis examinations it was found in 13 out of 15 measurements that a decrease in Creatinin serum concentration was accompanied by an increase in alpha index and that in 14 out of 15 measurements shift in the power spectrum toward the faster frequencies occurred. In those cases where a reduction in alpha index respectively an increase in theta index was found, the possibility of subacute dialysis encephalopathy must be taken in account. No significant relationship between subjective state of well-being and dialysis was found, with the exception of one patient, who had recently been admitted to the dialysis program. The results demonstrate the diagnostic relevance of EEG monitoring during haemodialysis in chronic renal insufficiency. However, the prognostic relevance remains to be proven with a larger number of patients.

Adult↗