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Biomedical subjects

A Seth

Publications and source records attributed to A Seth.

At least 109 records · Page 6Linked to original sources
PubMed1998

One step direct detection of recurrent mutations in the breast cancer susceptibility gene, BRCA1.

Germ-line mutations of the BRCA1 gene have been linked to 85% of hereditary breast and ovarian cancers. More than one hundred mutations have been reported, including several which are over represented within the Ashkenazi Jewish (185delAG) and Irish families (1294del40). These recurrent mutations are cost-effective targets for presymptomatic screening of cancer susceptibility. Most current techniques such as single strand conformation polymorphism, heteroduplex analysis, DNA sequencing, and protein translation termination assays are multistep, time consuming methods and require radioactive isotopes for mutation detection. As an alternative, we have developed a single step, non-radioactive allele specific oligonucleotide (ASO) PCR assay designed to target any known mutation. Its application to detection of the BRCA1 185delAG and 1294del40 mutations is described here. The ASO PCR is efficient, highly sensitive, non-radioactive, specific for individual mutations, performed in a single step, and is amenable to large-scale screening for other known mutations.

Alleles↗
PubMed1998

In-line skating: use of protective equipment, falling patterns, and injuries.

OBJECTIVE: To determine the use of protective equipment, reasons for nonuse of protective equipment, and patterns of injury, falls, and stopping techniques of recreational in-line skaters. DESIGN: Survey. PARTICIPANTS: 313 in-line recreational skaters observed skating in the parks, on bicycle trails, and on parkways in Milwaukee, Wisconsin and Columbus, Ohio were asked to participate in a short survey in 1995. MAIN OUTCOME MEASUREMENTS: The frequency of in-line skater use of protective equipment, reasons for nonuse, patterns of injury, and patterns of falls. RESULTS: Protective equipment was worn most of the time in the following proportions of skaters [95% confidence interval in brackets]: wrist guards (51%) [44.7%-58.1%]; knee pads (36%) [29.3%-42.5%]; elbow pads (15%) [8.4%-22.8%]; and helmets (15%) [8.0%-22.0%]. Reasons cited for not using protective equipment included lack of perceived need (47.3%), discomfort (37.5%), cost (15.9%), and undesirable appearance (15.2%). Stopping techniques included skating off into the grass (14.6%) [11.8%-17.4%] and voluntarily falling (3.5%) [2.1%-4.9%]. Causes of involuntary falls included spontaneous loss of balance (32.9%) [28.9%-36.9%], hitting rocks or other small objects (17.3%) [14.4%-20.2%], uneven pavement (17%) [14.2%-19.8%], and failure to stop (11.3%) [8.4%-14.2%]. Site of initial impact after falling included the hands and wrists (44.6%) [38.3%-50.9%], the knees (19.6%) [16.4%-22.8%], and the buttocks region (17.2%) [14.3%-20.1%]. Injuries had occurred in 26% of the skaters; 14% of injuries were fractures. CONCLUSIONS: In-line skaters often do not wear protective equipment because they believe it is not necessary. Recent studies have suggested, however, that protective equipment does prevent injuries. Physicians should counsel their patients who are in-line skaters to use protective equipment.

Accidental Falls↗
PubMed1997

A novel epithelial-expressed ETS gene, ELF3: human and murine cDNA sequences, murine genomic organization, human mapping to 1q32.2 and expression in tissues and cancer.

The ETS family of genes are implicated in cancers such as Ewings sarcoma, acute myeloid leukemia and chronic myelomonocytic leukemia. Further, they have important functions in embryonic development. Hence, identification and characterization of members of this family are important. We identify a novel ETS family member, ELF3, and report its human and murine cDNA sequences. The mouse cDNA has an alternatively spliced transcript with an extra 60 bp inserted. Hence we present the organization of the murine Elf3 gene together with its exon/intron structure. This gene consists of 9 exons and 8 introns spanning 4.8 kb. ELF3 binds and transactivates ETS sequences and interestingly also shows the ability to bind a GGAT-like purine core, a preferential ETS1/ETS2 type binding site. The expression of ELF3, unlike most other ETS family members, is absent in hematopoietic cells and hematopoietic organs in humans and mice. Intriguingly, the gene is specifically expressed in cell lines of epithelial origin and in organs such as lung, stomach, intestine, kidney that have specialized epithelial cells. We localize the human gene to 1q32.2, a region that is amplified in epithelial tumors of the breast, lung and prostate. Finally, we show that ELF3 expression is increased in a lung carcinoma and adenocarcinoma, as compared to normal tissue. ELF3 is also expressed in cell lines derived from lung cancers. These results suggest that this novel ETS gene may be involved in lung tumorigenesis.

Amino Acid Sequence↗
PubMed1997

ETS target genes: identification of egr1 as a target by RNA differential display and whole genome PCR techniques.

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing's sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

3T3 Cells↗
PubMed1997

ETS1, NFkappaB and AP1 synergistically transactivate the human GM-CSF promoter.

Activation of helper T cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony stimulating factor (GM-CSF) is one such cytokine, whose increased expression results mostly from increases in transcription. Cis-acting elements with NFkappaB, AP1 and ETS-like binding motifs have been identified in the promoter region of the GM-CSF gene, and are important or essential for transcriptional activity following T cell activation. ETS1 is a transcription factor of the ETS family that is expressed in T cells. We have previously shown that ETS1 can transactivate GM-CSF in Jurkat T cells, but only after the cells have been stimulated by treatment with PMA and ionomycin, agents that mimic T cell activation. Thus we proposed that ETS1, which is expressed constitutively in Jurkat cells, may act in concert with PMA/ionomycin inducible factors. Here we show that ETS1 can transactivate a GM-CSF reporter construct in unstimulated Jurkat cells, providing that either NFkappaB or AP1 transcription factors are supplied by co-transfection. We confirm that binding of endogenous NFkappaB and AP1 is induced following PMA/ionomycin treatment of T cells. Transactivation by ETS1, NFkappaB and AP1 is synergistic, and mutation of the individual binding sites reveals that the transcriptional activities of these factors are interdependent. Our results suggest that constitutive ETS1, and inducible NFkappaB and AP1, cooperate as part of a higher order transcriptional complex in activated T cells.

Animals↗
PubMed1997

Differential regulation of interstitial collagenase (MMP-1) gene expression by ETS transcription factors.

Expression of interstitial collagenase (MMP-1) has been detected in stromal fibroblasts of various malignant tumors. Here, we have studied the effect of three structurally different ETS transcription factors (ETS-1, ERGB/Fli-1, and PU.1) on MMP-1 promoter activity in NIH3T3 fibroblasts. ETS-1 increased the activity of 3.8 kb MMP-1 promoter construct up to tenfold, while ERGB/Fli-1 or PU.1 alone had no marked effect on basal promoter activity. ETS-1 also markedly potentiated enhancement of MMP-1 promoter by both c-Jun and JunB, whereas ERGB/Fli-1 augmented only the effect of c-Jun. Interestingly, PU.1 abolished induction of MMP-1 promoter by both c-Jun and JunB. Stimulation of MMP-1 promoter by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid was differentially augmented by ETS-1 and ERGB/Fli-1, and abrogated by PU.1. Co-transfection studies with MMP-1 promoter 5'-deletion constructs revealed that AP-1 site was necessary for PU.1-elicited suppression. As compared to control cell lines, PU.1-positive stable cells exhibited clearly weaker binding of c-Jun and JunD containing AP-1 complexes to MMP-1 promoter AP-1 element, as well as marked reduction in basal level and induction of c-jun mRNA by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid, suggesting a novel mechanism for PU.1-mediated inhibition of AP-1 dependent gene expression. These results show that three structurally distinct ETS transcription factors differently modulate AP-1 dependent upregulation of MMP-1 gene expression.

3T3 Cells↗
PubMed1997

FLI1 and EWS-FLI1 function as ternary complex factors and ELK1 and SAP1a function as ternary and quaternary complex factors on the Egr1 promoter serum response elements.

The ETS gene products are a family of transcriptional regulatory proteins that contain a highly conserved and structurally unique DNA binding domain, termed the ETS domain. Several ETS proteins bind to DNA as monomers, however it has been shown that the DNA binding activity is enhanced or modulated in the presence of other factors. By differential display and whole genome PCR techniques, we have recently shown that the Erg1 gene is a target for ETS proteins. The Egr1 promoter contains multiple ETS binding sites, three of which exist as parts of two serum response elements (SREI and SREII). The SRE is a cis-element that regulates the expression of many growth factor responsive genes. ELK1 and SAP1a have been shown to form ternary complexes with SRF on the SRE located in the c-fos promoter. Similarly, we examined whether the ELK1, SAP1a, FLI1, EWS-FLI1, ETS1, ETS2, PEA3 and PU.1 proteins can form ternary complexes with SRF on the Egr1 SREI and II. Our results demonstrate that indeed ELK1, SAPla, FLI1 and EWS-FLI1 are able to form ternary complexes with SRF on Egr1 SREs. In addition, ELK1 and SAP1a can also form quarternary complexes on the Egr1 SREI. However, the proteins ETS1, ETS2, PEA3 and PU.1 were unable to form ternary complexes with SRF on either the Egr1 or c-fos SREs. Our data demonstrate that FLI1 and EWS-FLI1 constitute new members of a subgroup of ETS proteins that can function as ternary complex factors and further implicate a novel function for these ETS transcription factors in the regulation of the Egr1 gene. By amino acid sequence comparison we found that, in fact, 50% of the amino acids present in the B-box of SAP1a and ELK1, which are required for interaction with SRF, are identical to those present in both FLI1 (amino acids 231- 248) and EWS-FLI1 proteins. This B-box is not present in ETS1, ETS2, PEA3 or PU.1 and these proteins were unable to form ternary complexes with SRF and Egrl-SREs or c-fos SRE. Furthermore, deletion of 194 amino terminal amino acids of FLI1 did not interfere with its ability to interact with SRF, in fact, this truncation increased the stability of the ternary complex. The FLI1 protein has a unique R-domain located next to the DNA binding region. This R-domain may modulate the interaction with SRF, providing a mechanism that would be unique to FLI1 and EWS-FLI1, thus implicating a novel function for these ETS transcription factors in the regulation of the Egr1 gene.

Animals↗
PubMed1997

Unmarked gene integration into the chromosome of Mycobacterium smegmatis via precise replacement of the pyrF gene.

After integration into the bacterial chromosome an exogenous gene may be stably expressed without continued selection for the recombinant locus. However, chromosomal integration events occur infrequently, requiring the concomitant integration of a drug resistance marker in order to identify colonies of recombinant cells. The generation of a drug-resistant recombinant strain can both reduce the in vivo applicability of the strain and preclude the use of recombinant vectors which use the same drug resistance marker. We have constructed a plasmid, pINT-delta, which allows recombination of exogenous genes onto the Mycobacterium smegmatis chromosome. The exogenous gene completely replaces the pyrF gene and the resultant strain lacks any exogenous drug resistance marker. The methodologies described herein are general and applicable even to those bacteria for which extrachromosomal plasmids are not available. Using pINT-delta we integrated the lacZ gene into the M. smegmatis chromosome via a precise exchange of lacZ and pyrF. The resultant strain was used to demonstrate that the expression of genes integrated at the pyrF locus is repressed twofold by inclusion of uracil in the growth medium. In addition, we used pINT-delta to construct an M. smegmatis strain with a precise deletion of its pyrF locus. This strain, TSm-627, grows normally in rich medium but does not grow in medium lacking uracil. TSm-627 cells allow the pyrF gene to be used as a selectable marker for growth on medium lacking uracil. In TSm-627 cells, the pyrF gene is also useful as a counterselectable marker on complete medium containing 5'-fluoroorotic acid and uracil. Two pyrF-containing plasmids, designed to exploit the new delta pyrF strain, have been constructed and their possible applications to problems in mycobacteriology are discussed.

Amino Acid Sequence↗
PubMed1997

Facial expression megamix: tests of dimensional and category accounts of emotion recognition.

We report four experiments investigating the perception of photographic quality continua of interpolated ('morphed') facial expressions derived from prototypes of the 6 emotions in the Ekman and Friesen (1976) series (happiness, surprise, fear, sadness, disgust and anger). In Experiment 1, morphed images made from all possible pairwise combinations of expressions were presented in random order; subjects identified these as belonging to distinct expression categories corresponding to the prototypes at each end of the relevant continuum. This result was replicated in Experiment 2, which also included morphs made from a prototype with a neutral expression, and allowed 'neutral' as a response category. These findings are inconsistent with the view that facial expressions are recognised by locating them along two underlying dimensions, since such a view predicts that at least some transitions between categories should involve neutral regions or identification as a different emotion. Instead, they suggest that facial expressions of basic emotions are recognised by their fit to discrete categories. Experiment 3 used continua involving 6 emotions to demonstrate best discrimination of pairs of stimuli falling across category boundaries; this provides further evidence of categorical perception of facial expressions of emotion. However, in both Experiment 1 and Experiment 2, reaction time data showed that increasing distance from the prototype had a definite cost on ability to identify emotion in the resulting morphed face. Moreover, Experiment 4 showed that subjects had some insight into which emotions were blended to create specific morphed images. Hence, categorical perception effects were found even though subjects were sensitive to physical properties of these morphed facial expressions. We suggest that rapid classification of prototypes and better across boundary discriminability reflect the underlying organisation of human categorisation abilities.

Adult↗
PubMed1997

Prevalence of enuresis and other bladder symptoms in patients with active Graves' disease.

OBJECTIVE: To determine the prevalence of bladder symptoms in patients with Graves' disease and assess their abnormalities on urodynamic studies. PATIENTS AND METHODS: The study comprised 30 consecutive patients (13 female, 17 male, mean age 31 years, SD 10) with active Graves' disease. Before and after attaining euthyroidism with carbimazole, their bladder symptoms were assessed using a questionnaire adapted from the American Urologic Association voiding symptom score, the serum levels of total triiodo-L-thyronine (T3), serum thyroxine (T4) and thyroid-stimulating hormone (TSH) were measured by radioimmunoassay, and uroflowmetry, cystometry and perineal muscle electromyography were performed. RESULTS: Twelve of the 30 patients (40%) had the onset of bladder symptoms 1-6 months after the onset of symptoms of Graves' disease; four of the 12 patients had enuresis. Urodynamic studies were possible in five patients and showed reduced flow rates in all, a significant post-void residual volume in four, and enlarged bladder capacity and increased perineal muscle electromyographic activity during the voiding phase in three. Voiding disturbances and urodynamic abnormalities resolved after attaining euthyroidism. CONCLUSION: Significant bladder symptoms can occur in about 40% of patients with active Graves' disease but resolve on euthyroidism; in some patients, urodynamic studies show significant abnormalities which are also reversible.

Adolescent↗
PubMed1996

Assembly, specific binding, and crystallization of a human TCR-alphabeta with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2.

T lymphocytes use TCR-alphabeta to bind and to recognize complexes of antigenic peptides bound to MHC proteins located at the surface of APCs. We have assembled and crystallized this intercellular complex of TCR/peptide/MHC from soluble human TCR-alphabeta and soluble peptide/HLA-A2 complexes. The soluble TCR-alphabeta binds specifically to its in vivo ligand, the complex of HLA-A2, and a peptide from the Tax protein of human T lymphotropic virus type 1. The soluble TCR also binds in vitro to an altered peptide ligand, which appears to be a partial agonist in T cell assays as determined by its ability to elicit different cytolytic and lymphokine secretion responses. Heterodimerization and the antigenic specificity of the TCR do not require its interchain disulfide bond, transmembrane segments, or glycosylations. Crystals of the TCR/peptide/HLA-A2 complex diffract x-rays, providing the means to study in atomic detail the mechanism of Ag-specific cell-cell recognition between T cells and target cells.

Amino Acid Sequence↗
PubMed1996

A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells.

Studies toward the biologic and molecular understanding of programmed cell death have been stimulated by the recent identification of genes and their products that regulate apoptosis. A panel of mAbs has been raised against dying cells in the present study by immunizing mice with apoptotic Jurkat cells. One of these Abs, anti-7A6, was found to react with apoptotic cells. Using ELISA or flow cytometry, little reactivity of anti-7A6 was observed in normal or digitonin-permeabilized human peripheral blood lymphocytes and a number of hemopoietic cell lines tested. The Ab, however, strongly reacted with these cells when they were induced to undergo apoptosis by irradiation or treatment with apoptosis-inducing agents. Cell sorting and DNA fragmentation experiments revealed that 7A6-positive cells, but not 7A6-negative cells, had apparent DNA fragments characteristic of cells undergoing apoptosis. By immunoblot, under reducing conditions, anti-7A6 detected a 38-kDa protein band in the cell lysates prepared from apoptotic cells. Immunoelectron microscopy showed the 7A6 Ag to be localized to the membrane of mitochondria in apoptotic Jurkat cells. These results indicate that anti-7A6 defines a novel epitope on the mitochondrial membrane protein that appears to be exposed on cells undergoing apoptosis, suggesting that the 7A6 molecule may be involved in the molecular cascade of apoptotic cell death.

Animals↗