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Biomedical subjects

J C Winkelmann

Publications and source records attributed to J C Winkelmann.

At least 19 recordsLinked to original sources
PubMed2001

Colorectal cancer in Russian-speaking Jewish emigrés: community-based screening.

OBJECTIVES: Colorectal cancer (CRC) screening by fecal occult blood testing and flexible sigmoidoscopy is recommended by many authorities for those older than age 50. Ashkenazi Jews have been shown to have a higher level of CRC and polyps than the general population. A subset of Ashkenazi Jews, Russian-speaking Jewish immigrants to the United States (RJIs), have not been studied extensively for CRC and may have additional risk factors not found in other Ashkenazi populations. METHODS: A retrospective chart review was undertaken of fecal occult blood tests, endoscopy reports, and pathology reports of 132 RJIs and 124 non-RJI controls over age 50 between 1987 and 1999 at the Jewish Hospital of Cincinnati Medical Outpatient Clinic. RESULTS: Mean ages at the time of diagnosis or flexible sigmoidoscopy were 68 yr for RJIs and 66 yr for the non-RJI patients. Of the RJI patients, 38.7% had positive findings: 37 (28.0%) with lesions < 2 cm, five (3.8%) with lesions > 2 cm, and nine (6.8%) with CRC. Of the non-RJI control group patients, 16.9% had positive findings: 16 (12.9%) with lesions < 2 cm, three (2.4%) with lesions > 2 cm, and two (1.6%) with CRC. Age- and sex-matched statistical analysis revealed significantly greater CRC and significantly more polyps > 2 cm for the RJI patients (p < 0.003). This is higher than in other studies of Ashkenazis, which show a 2.3% incidence, and in statistics from the National Cancer Institute, which reveal a national CRC incidence rate for those over age 65 to be 0.30%. CONCLUSIONS: RJIs in our study have polyps > 2 cm and CRC at a rate of 10.6%, as compared with 4.0% for in-clinic controls and a national average of 0.30% for patients over age 65. This suggests a need for more aggressive screening of this patient population for CRC.

Aged↗
PubMed1997

Amino-acid substitution in alpha-spectrin commonly coinherited with nondominant hereditary spherocytosis.

Nondominant hereditary spherocytosis (ndHS) is a disorder characterized in some patients by severe hemolytic anemia and marked deficiency of erythrocyte spectrin. This report describes the identification of a variant spectrin chain, alpha-spectrin Bughill or alpha(BH), that is associated with this disorder in a number of patients. Tryptic maps of spectrin from affected individuals revealed an acidic shift in isoelectric point of the alphaII domain peptides at 46 kD and 35 kD. A point mutation at codon 970 of the alpha-spectrin gene (GCT-->GAT), that changes the encoded amino acid from an alanine to an aspartic acid, was identified in genomic DNA of affected patients. The alpha(BH) variant was present in 8 patients with ndHS from five different kindreds but was absent in 4 patients from two other kindreds. The 8 ndHS patients with the alpha(BH) variant appeared to be homozygous for the alpha(BH) variant by analysis of peptide maps of limited tryptic digests of erythrocyte spectrin. However, following genomic DNA analysis, only 2 of these patients were true homozygotes, whereas 6 were found to be doubly heterozygous for the alpha(BH) allele and a second, presumably abnormal, alpha-spectrin gene. These results suggest that, in these 6 patients, the second alpha-spectrin allele is in fact associated with one or more genetic defect(s), causing decreased accumulation of alpha-spectrin. The pattern of transmission of the alpha(BH) allele in certain families suggests that the alpha(BH) amino-acid substitution is not itself responsible for ndHS but is more likely a polymorphic variant that, in some but not all cases, is in linkage disequilibrium with another uncharacterized alpha-spectrin gene defect that itself is a cause of ndHS.

Alleles↗
PubMed1996

Enzyme-linked immunosorbent assay detects a potential soluble form of the erythropoietin receptor in human plasma.

The erythropoietin receptor (EpoR) is a type I transmembrane protein that is a member of the family of hemopoietin receptors. Several members of this family have soluble receptor forms that are secreted by the cells rather than expressed on the cell surface. An alternatively spliced EpoR transcript has been described in human erythroid precursors that, if translated, would produce a truncated, soluble EpoR lacking the transmembrane domain. To determine if the human EpoR is expressed in a soluble form, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for the EpoR, and we analyzed human serum and plasma. Sheep were immunized with a fusion protein (EREx) consisting of glutathione-S-transferase (GST) and the human EpoR extracellular domain. The sheep antiserum was affinity-purified on immobilized EREx, and then used in a two-stage antigen capture ELISA. The plasma from 20 normal subjects was studied with this assay. There was a wide variability in the levels of soluble EpoR in these subjects (range, <10-2,200 ng/ml). An average value of 550 +/- 735 ng/ml for soluble EpoR was obtained in these normals. Protein A adsorption of the test plasma prior to the assay had no effect on the values obtained. Assay of serum from the same normal subjects showed an average decrease of 88% in soluble EpoR levels compared to plasma. There was no correlation between hematocrit and soluble EpoR levels compared to plasma. There was no correlation between hematocrit and soluble EpoR level. This assay may have utility in the further elucidation of erythropoietin physiology.

Adult↗
PubMed1995

A translocated erythropoietin receptor gene in a human erythroleukemia cell line (TF-1) expresses an abnormal transcript and a truncated protein.

We previously identified a translocation breakpoint in exon 8 of the erythropoietin receptor (EpoR) gene in TF-1 cells, a cell line derived from a human erythroleukemia. To investigate the potential pathogenetic significance of this abnormality, we more precisely mapped the breakpoint within exon 8 and studied the expression of the translocated gene by S1 nuclease mapping of EpoR transcripts and chemical crosslinking of labeled erythropoietin (Epo) to TF-1 cell surface receptors. Transcripts from the abnormal gene were found to be highly expressed in relation to normal EpoR transcripts in TF-1 cells. The breakpoint predicted by S1 mapping of abnormal EpoR transcripts agreed closely with that determined by Southern analysis. Chemical cross-linking of 125I-Epo to TF-1 cells showed an abnormal, low-molecular-weight cross-linked species directly recognized by anti-EpoR antibodies and present in considerable excess over the normal EpoR. Karyotype analysis showed that each of 10 TF-1 cell metaphases had, in addition to multiple other alterations, one chromosome 19 with additional chromosomal material translocated onto the short arm at 19p13.3, the location of the EpoR gene. We conclude that the structurally abnormal EpoR gene in TF-1 cells is highly expressed and produces an abnormal protein. We speculate that the chromosomal material brought into the EpoR locus by translocation is responsible for the high level of expression. We hypothesize that this translocation participated in the evolution of the erythroleukemia from which TF-1 cells were derived.

Base Sequence↗
PubMed1995

Localization of an essential ligand binding determinant of the human erythropoietin receptor to a domain N-terminal to the WSXWS motif: implications for soluble receptor function.

The interaction of erythropoietin (Epo) with the erythropoietin receptor (EpoR) supports erythropoiesis. The EpoR is a member of the well-recognized cytokine receptor superfamily characterized by four conserved cysteines and a WSXWS domain in the extracellular portion of the molecule. To localize ligand-binding determinants of the EpoR near the WSXWS domain, we tested the ligand-binding ability of the wild-type human EpoR extracellular domain (EREx), two truncated and three chimeric constructs with the interleukin-2 receptor beta subunit (IL2R beta). Constructs were expressed in E. coli as GST fusion proteins linked to a solid-phase support and assayed for binding to 125I Epo. As previously shown, Epo bound specifically to the expressed extracellular domain, EREx. Epo did not bind to truncated receptors lacking either the entire fifth exon or the WSXWS domain. Epo also did not bind to chimeric receptors that had the amino acids encoded by the fifth exon replaced by IL2R beta or that had the amino acids subsequent to asparagine residue 209 replaced by IL2R beta. Specific binding was demonstrated for a construct in which the WSXWS was replaced by that of IL2R beta. We conclude that the amino acids encoded by this 5' portion of exon 5 of the EpoR are necessary for ligand binding and that the WSXWS domain is necessary for Epo binding but is not involved in ligand-binding specificity. We also speculate that if the putative soluble form of the EpoR is expressed (predicted to lack exon 5), it does not bind Epo and therefore may serve a physiologic purpose other than ligand binding.

Amino Acid Sequence↗
PubMed1995

Polymorphisms of the beta fibrinogen gene and plasma fibrinogen concentration in Caucasian and Japanese population samples.

We reported previously that plasma fibrinogen was significantly higher in U.S. Caucasians than in Japanese, which may contribute to the higher mortality rate of coronary heart disease in the United States than in Japan. To examine the contribution of genetic variations to the race difference in plasma fibrinogen levels, restriction fragment length polymorphisms (RFLPs) of the beta fibrinogen gene were examined in 293 nonsmoking Caucasians and Japanese men and women aged 47-69 years. Three RFLPs were detected by digestion of genomic DNA using the BclI restriction enzyme, polymerase chain reaction (PCR) products using HaeIII and HindIII. The alleles B2 (4.2 kb, BclI digestion), H2 (957 b, HaeIII) and Hd2 (465 b. HindIII) were associated with higher fibrinogen concentrations in previous studies. Because of a strong linkage disequilibrium between HaeIII and HindIII polymorphisms, the data of HindIII was presented. The frequency of the B2 allele was 22% (95% Cl: 17-27%) for Caucasians and 13% (10-17%) for Japanese (the difference: p < 0.01). The respective frequency of the Hd2 allele was 26% (21-31%) and 12% (8-16%) (p < 0.001). After controlling for age, body mass index, alcohol intake, triglycerides, fish intake, and for women, menopausal status and hormone replacement therapy, the adjusted mean fibrinogen level among Caucasians was 289 mg/dl for genotype B1B1 and 301 mg/dl for genotype B1B2 or B2B2 combined (p = 0.18), and 285 mg/dl for Hd1Hd1 and 306 mg/dl for Hd1Hd2 or Hd2Hd2 combined (p = 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

Erythroid-specific processing of human beta spectrin I pre-mRNA.

Erythroid cells express a unique form of beta spectrin I as a result of tissue-specific alternative pre-mRNA processing. Nonerythroid cells that express the beta spectrin I gene include four additional exons at the 3' end of the mature transcript, leading to elongation of the carboxyl terminus of the protein. The nonerythroid beta spectrin I isoform is not present in the red blood cell membrane skeleton; the erythroid isoform is not detected in other cell types. Therefore, developing erythroid cells acquire this tissue-specific pre-mRNA processing activity during differentiation. In the present study, we investigated the developmental timing of erythroid-specific pre-mRNA processing in human erythroid precursors. Partially purified human peripheral blood burst forming uniterythroid (BFU-E) cells were grown in culture for 5 to 12 days. beta Spectrin I mRNA transcripts were analyzed at different time points by S1 nuclease mapping. The processing of beta spectrin I transcripts was found to be exclusively erythroid from day 5 onward, indicating that erythroid-specific processing is not linked temporally to assembly of the mature erythroid membrane skeleton. Human erythroleukemia (HEL) cells had both erythroid and nonerythroid transcripts, indicating that both processing patterns can coexist. Induction of erythroid differentiation in HEL cells using hemin resulted in a partial switch toward the erythroid processing pattern of beta spectrin I transcripts. Using a genomic S1 probe that spans the erythroid polyadenylation signal, we found that a substantial portion of the transcripts detected by the erythroid cDNA S1 probe (in both cultured BFU-E and HEL cells) is incompletely processed pre-mRNA precursors. Poly(A) RNA selection before S1 analysis showed that the unprocessed transcripts are not polyadenylated. We conclude that (1) erythroid-specific pre-mRNA processing activity is present early in erythroid differentiation; (2) beta spectrin I transcripts that are unprocessed at the 3' end accumulate, awaiting either erythroid or nonerythroid processing pathways, from which observation we infer that the regulated alternative pathways are both inefficient; and (3) HEL cells offer a human cell culture model in which to study the balance between the two pre-mRNA processing pathways. We speculate that erythroid cells evolved this tissue-specific pre-mRNA processing machinery for other erythroid genes in addition to beta spectrin I.

Base Sequence↗
PubMed1994

Binding of PH domains of beta-adrenergic receptor kinase and beta-spectrin to WD40/beta-transducin repeat containing regions of the beta-subunit of trimeric G-proteins.

Pleckstrin homology (PH) domains are found in numerous proteins important in signal transduction and cytoskeletal function. Several PH domains are now known to contain a binding site for the beta gamma subunits of trimeric G-proteins (G beta gamma), a finding which naturally raises the question of where on the G beta gamma complex these PH domains bind. Here we demonstrate binding of the PH domains of beta-adrenergic receptor kinase and beta-spectrin to the G beta subunit and not the G gamma subunit in a nitrocellulose gel replica assay. Furthermore, the C-terminal tryptic fragment of G beta containing only 5 WD40/beta-transducin (WD40) repeats also binds these two PH domains. Finally, constructs containing only WD40 repeats of G beta were shown to bind to beta-ARK and beta-spectrin PH domains in solution. These findings suggest that WD40 repeats of G beta are ligands for PH domains and have interesting implications for other proteins containing WD40 sequences.

Amino Acid Sequence↗
PubMed1993

Erythroid and nonerythroid spectrins.

Recent developments have contributed important information to understanding the role of spectrins in the RBC membrane skeleton and nonerythroid cells. Many questions can now be framed, informed by structural knowledge of various spectrin subunit types and alternatively spliced variants, that previously could not have been addressed. Their solution in the coming years will likely lead to further advances with direct relevance to biology and medicine.

Animals↗
PubMed1993

The exon-intron organization of the human erythroid beta-spectrin gene.

The human erythrocyte beta-spectrin gene DNA has been cloned from overlapping human genomic phage and cosmid recombinants. The entire erythroid beta-spectrin mRNA is encoded by 32 exons that range in size from 49 to 871 bases. The exon/intron junctions have been identified and the exons mapped. There is no correlation between intron positions and the repeat units of 106 amino acids within domain II of the beta-spectrin gene. The scatter of the introns over the 17 repeats argues against the 106-amino-acid unit representing a minigene that underwent repeated duplication resulting in the present beta-spectrin gene. In fact, the two largest exons, exon 14 (871 bp) and 16 (757 bp), extend over 4 and 3 repeat units of 106 amino acids, respectively, while repeat beta 10 is encoded by 4 exons. No single position of an intron in the beta-spectrin gene is conserved between any of the 17 beta-spectrin and 22 alpha-spectrin repeat units. The nucleotide sequences of the exon/intron boundaries conform to the consensus splice site sequences except for exon 20, whose 5' donor splice-site sequence begins with GC. The beta-spectrin isoform present in the human brain, the skeletal muscle, and the cardiac muscle is an alternatively spliced product of the erythroid beta-spectrin gene. This splice site is located within the coding sequences of exon 32 and its utilization in nonerythroid tissues leads to the use of 4 additional downstream exons with a size range of 44 to 530 bp.

Amino Acid Sequence↗
PubMed1993

The 270 kDa splice variant of erythrocyte beta-spectrin (beta I sigma 2) segregates in vivo and in vitro to specific domains of cerebellar neurons.

Spectrin isoforms arise from four distinct genes, three of which generate multiple alternative transcripts. With no biochemical restrictions on the assembly of alpha beta heterodimers, more than 25 distinct heterodimeric spectrin species may exist. Whether (and why) this subtle but substantial diversity is realized in any single cell is unknown. To address this question, sequence-specific antibodies to alternatively spliced regions of alpha- and beta-spectrin have been prepared. Reported here is the localization in rat cerebellar neurons at light and electron microscopic levels of an antibody against a unique sequence (beta I sigma 2-A = PGQHKDGQKSTGDERPT) from the 270 kDa transcript of the red cell beta-spectrin gene (spectrin beta I sigma 2). In this version, the 3' sequence of erythroid beta-spectrin (beta I sigma 1) is replaced with an alternative sequence that shares substantial homology with the 3' sequence of non-erythroid beta-spectrin (beta II sigma 1). The antibody to beta I sigma 2-A stains a single protein band at 270 kDa, determined by western blotting, in both rat cerebellum and in cultured cerebellar granule cells, and does not react with beta II sigma 1 spectrin (beta-fodrin). This antibody stains the dendritic spines of Purkinje cells in the molecular layer, and is concentrated at postsynaptic densities (PSDs) adjacent to synapsin I (which is confined to the presynaptic membrane). The soma of Purkinje cells do not stain. In the granular layer, cytoplasmic organelles and the postsynaptic densities of granular cells stain strongly. Astrocytes are also stained. In all cells, plasma membrane staining is confined to postsynaptic densities (PSD). The beta I sigma 2 isoform co-immunoprecipitates with non-erythroid alpha-spectrin (alpha II sigma), even though the distribution of alpha II sigma within neurons only partially overlaps that of beta I sigma 2. No hybrid beta I sigma 2 and beta II sigma 1 (beta-fodrin) spectrin complexes appear to exist. Spectrin beta I sigma 2 is also polarized in cultured rat cerebellar granule cells, where it is abundant in cell bodies but not neurites. The overall distribution of beta I sigma 2 is as a subset of the distribution of spectrins 240/235E previously detected with a generally reactive erythrocyte alpha beta-spectrin antibody. These findings establish the highly precise segregation of a beta-spectrin isoform to distinct cytoplasmic and membrane surface domains, indicate that it is complexed (partially) with non-erythroid alpha-spectrin, and demonstrate that cytoskeletal targeting mechanisms are preserved in cultured granular cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Localization of dystrophin and beta-spectrin in vacuolar myopathies.

We examined the expression of the cytoskeletal proteins dystrophin and beta-spectrin on vacuolar boundaries in vacuolar myopathies. We also localized utrophin, a dystrophin homologue, and laminin, which served as a marker for the basal lamina. Four types of vacuoles were identified. Type 1 vacuoles, found in all diseases, were lined by laminin, dystrophin, and beta-spectrin and arose from infoldings of the basal lamina and sarcolemma into splitting or branching fibers. Type 2 vacuoles were lined by dystrophin and beta-spectrin and were most common in adult acid maltase deficiency, chloroquine myopathy, and periodic paralysis. Traces of utrophin were also noted on the boundaries of some type 2 vacuoles, but only in those fibers that also expressed utrophin on their surface membrane. Type 3 vacuoles were lined by small patches of dystrophin and beta-spectrin and occurred in any vacuolar myopathy. Type 4 vacuoles were unlined by any of the above antigens and were most common in infantile acid maltase deficiency and in the nonlysosomal glycogenoses. Immunoelectron microscopy confirmed the dystrophin label on vacuolar boundaries but revealed no reaction product on any other membranous component within the muscle fiber. We conclude that dystrophin and beta-spectrin provide cytoskeletal support for a species of membrane-bound vacuoles in diverse myopathies.

Dystrophin↗
PubMed1992

Differentiation and erythropoietin receptor gene expression in human erythroid progenitor cells.

Partially purified human burst-forming unit-erythroid (BFU-E) cells from peripheral blood were cultured for 6 to 8 days to obtain colony-forming unit-erythroid (CFU-E) cells. When these BFU-E-derived CFU-E were further purified and recultured in liquid suspension cultures with erythropoietin (EPO), they matured and differentiated into reticulocytes in vitro. A maximum rate of hemoglobin synthesis was observed at day 10 of cumulative culture time by measuring 59Fe incorporation into heme. Withdrawal of EPO from erythroblast cultures at various times during development showed that between day 10 and day 11 (when the majority of the cells are in the polychromatic erythroblast stage), these cells became independent of EPO. The timing of the disappearance of the EPO requirement in these cells coincided with the marked decline in proliferation. Measurement of EPO receptor messenger RNA (mRNA) levels by Northern analysis showed that there is a slight decline during the day 8 to day 10 time period, followed by a rapid decline between days 10 and 14. Binding of 125I-EPO to erythroblasts also showed a steady decline of the cell surface binding during maturation and terminal differentiation. The half-life of the human EPO receptor was 90 minutes in the presence of the transcriptional inhibitor actinomycin D and the half-life measured at two different times during the 8- to 14-day culture period remained constant. These results indicate that human EPO receptor mRNA must be transcribed continuously to maintain the levels seen by Northern analysis. The human cell system described here is well suited for the study of a wide variety of biochemical events during late erythroid differentiation.

Blotting, Northern↗
PubMed1992

Ligand binding properties of the human erythropoietin receptor extracellular domain expressed in Escherichia coli.

We developed an assay to directly measure the ligand binding properties of the cloned human erythropoietin receptor (EpoR). The cDNA encoding the extracellular domain of the human EpoR was amplified by polymerase chain reaction and ligated into the prokaryotic expression vector pGEX3X. Synthesis in Escherichia coli was induced and a soluble glutathione S-transferase fusion protein, EREx, was purified by erythropoietin affinity chromatography. Purified EREx was bound to GSH agarose beads and used in a solid phase ligand binding assay. Specific binding of 125I-erythropoietin to EREx beads was demonstrated. A single affinity class (Kd = 1.5 nM) of the binding site was evident on Scatchard analysis. The Kd of this site is quantitatively equivalent to that of the "low" affinity cellular binding site. Kinetic analysis of ligand binding to EREx revealed both the on and off rates to be rapid, with t1/2 of 60 and 40 s, respectively. EREx ligand binding exhibits no obvious metal ion dependence or cross-competition by other hemopoietins. Antibodies to EREx block the binding of erythropoietin to the cellular EpoR. We conclude that the 66-kDa EpoR protein is capable of specific ligand binding and that no covalent modifications or associated molecules are required for this interaction. We speculate that the "high" affinity cellular binding site (Kd less than 0.2 nM) results from the interaction of the EpoR with another molecule, either additional EpoR or associated subunits, that decreases the ligand off rate.

Base Sequence↗
PubMed1992

Human cardiac and skeletal muscle spectrins: differential expression and localization.

We describe multiple human cardiac and skeletal muscle spectrin isoforms. Cardiac muscle expresses five erythroid alpha,beta spectrin-reactive isoforms with estimated MR's of 280, 274, 270, 255, and 246 kD, respectively. At least one nonerythroid alpha-spectrin of MR 284 kD is expressed in heart. While skeletal muscle shares the 280, 270, and 246 kD erythroid spectrins, it expresses an immunologically distinct 284 kD nonerythroid alpha-spectrin isoform. The 255 kD erythroid beta-spectrin isoform is specific for cardiac tissue. By immunocytochemistry, both erythroid beta- and nonerythroid alpha-spectrins are localized to costameres, the plasma membrane, and the neuromuscular junctional region.

Adult↗
PubMed1992

The human erythropoietin receptor.

Molecular analysis of the human erythropoietin receptor (EpoR) promises to yield a greater mechanistic understanding of erythropoiesis and disease states that affect red cell production. The cloned receptor molecule is a 66 kDa membrane protein that is structurally related to a large superfamily of haemopoietin/growth factor receptors. The 66 kDa EpoR alone is capable of binding to erythropoietin (Epo) with nanomolar affinity. The native EpoR may form dimers before or after binding Epo. EpoR dimers and/or associated molecules are probably necessary for high-affinity Epo binding. The 66 kDa EpoR probably exists as a protein complex with as yet unidentified proteins of 100 and 85 kDa. The molecular mechanism of Epo signal transduction remains largely undefined. The possible role of the EpoR in human diseases has been studied in a variety of clinical conditions. A structurally abnormal EpoR gene has been identified in a human erythroleukemia cell line. In polycythemia vera, red cell progenitors exhibit exaggerated sensitivity to Epo and express only low-affinity EpoR. Some cases of hereditary polycythemia may be due to a mutant EpoR conferring enhanced Epo sensitivity. Other pathologic conditions may also be associated with abnormalities of the EpoR or its associated molecules. Soluble, immunoreactive EpoR is detectable in human serum, but its physiological significance is unknown.

Animals↗