Endarterectomy is not indicated for moderate symptomatic carotid stenosis.
Explore the source record for details and available documents.
Journal Article↗
Biomedical subjects
M D Walker
Publications and source records attributed to M D Walker.
A role of Hsp60 in autoimmune diabetes: analysis in a transgenic model.
A pathogenic role for self-reactive cells against the stress protein Hsp60 has been proposed as one of the events leading to autoimmune destruction of pancreatic beta cells in the diabetes of nonobese diabetic (NOD) mice. To examine this hypothesis, we generated transgenic NOD mice carrying a murine Hsp60 transgene driven by the H-2E alpha class II promoter. This would be expected to direct expression of the transgene to antigen-presenting cells including those in the thymus and so induce immunological tolerance by deletion. Detailed analysis of Hsp60 expression revealed that the endogenous gene is itself expressed strongly in thymic medullary epithelium (and weakly in cortex) yet fails to induce tolerance. Transgenic mice with retargeted Hsp60 showed overexpression of the gene in thymic cortical epithelium and in bone marrow-derived cells. Analysis of spontaneous T-cell responses to a panel of self and heterologous Hsp60 antigens showed that tolerance to the protein had not been induced, although responses to an immunodominant 437-460 epitope implicated in disease were suppressed, probably indicating an epitope shift. This correlated with changes in disease susceptibility: insulitis in transgenic mice was substantially reduced so that pathology rarely progressed beyond periislet infiltration. This was reflected in a substantial reduction in hyperglycemia and disease. These data indicate that T cells specific for some epitopes of murine Hsp60 are likely to be involved in the islet-cell destruction that occurs in NOD mice.
Animals↗
NOD mouse diabetes: the ubiquitous mouse hsp60 is a beta-cell target antigen of autoimmune T cells.
In the NOD mouse, the onset of beta-cell destruction is associated with spontaneous development of T-lymphocytes reactive to members of the 60 kDa heat shock protein (hsp60) family, including the Mycobacterial (MT) and the human (H) hsp60 molecules. Diabetes in the NOD mouse is a spontaneous tissue-specific autoimmune disease occurring without prior immunization. Therefore, it has been suggested that the anti-hsp60 T cells involved in the autoimmune diabetes of NOD mice might reflect molecular mimicry between MT-hsp60 and a beta-cell tissue specific molecule sharing similar T cell epitopes, the p277 peptide of hsp60 in particular. We cloned and expressed the mouse hsp60 cDNA from a beta-cell tumour. This mouse beta-cell hsp60 cDNA was found to be identical in sequence to the hsp60 of mouse fibroblasts. We further report that NOD spleen cells and an NOD diabetogenic T cell clone C9 responded to the recombinant mouse hsp60 and to its peptide M-p277 to the same extent as to H-hsp60 and H-p277. Splenocytes of mice of other strains did not respond to p277. Moreover, treatment of 3 month old NOD mice with the non-modified self M-p277 peptide was as efficient as H-p277, from which it differs in one amino acid, in halting progression of the disease. Thus, anti-H-p277 T cells modulating diabetes in the NOD mouse are autoreactive, and are targeted at the mouse beta-cell hsp60, which is not tissue specific. These findings raise the question of how a non-tissue specific molecule may be a target of a tissue-specific autoimmune disease.
Amino Acid Sequence↗
An activation domain of the helix-loop-helix transcription factor E2A shows cell type preference in vivo in microinjected zebra fish embryos.
The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is implicated in cell-specific gene expression in several cell lineages. Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells. To analyze this preferential activity in an in vivo setting, we adapted a system involving transient gene expression in microinjected zebra fish embryos. Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid. The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16. The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site. Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three activation domains. To determine the cell types in which the activation domains were functioning, a reporter plasmid encoding beta-galactosidase and then in situ staining of whole embryos were used. Expression of ADI led to activation in all major groups of cell types of the embryo (skin, sclerotome, myotome, notochord, and nervous system). On the other hand, ADII led to negligible expression in the sclerotome, notochord, and nervous system and much more frequent expression in the myotome. Parallel experiments conducted with transfected mammalian cells have confirmed that ADII shows significant activity in myoblast cells but little or no activity in neuronal precursor cells, consistent with our observations in zebra fish. This transient-expression approach permits rapid in vivo analysis of the properties of transcription activation domains: the data show that ADII functions preferentially in cells of muscle lineage, consistent with the notion that certain activation domains contribute to selective gene activation in vivo.
Animals↗
Noninvasive high-voltage measurement in mammography by crystal diffraction spectrometry.
Wavelength dispersive crystal diffraction spectrometry has been applied to the measurement of the accelerating voltage on an x-ray source in a prototype experiment in the mammographic source. The results indicate that this noninvasive approach can yield determinations of such voltages within 0.1 kV, a level of imprecision that appears adequate for high-level standardization of such potentials.
Biophysical Phenomena↗
Early treatment of stroke with monosialoganglioside GM-1. Efficacy and safety results of the Early Stroke Trial.
BACKGROUND AND PURPOSE: The Early Stroke Trial (EST) is a randomized, double-blind, placebo-controlled trial to assess the effect of monosialoganglioside GM-1 in improving recovery in patients who experienced an ischemic supratentorial stroke. METHODS: Sixteen clinical centers recruited 805 patients, of whom 792 were confirmed to be eligible. Treatment, consisting of a first dose of either 200 mg GM-1 or placebo, was initiated within 5 hours of the onset of stroke; a second dose of either 100 mg GM-1 or placebo was administered 12 hours later. Thereafter, patients received a daily injection of 100 mg GM-1 or placebo intravenously from day 2 through 10 and intramuscularly from day 11 through 21. Patients were followed up for a total of 4 months. RESULTS: Survival was similar in the two treatment groups. Improvement in neurological status, as measured by the change in Canadian Neurological Scale score between baseline and 4-month assessments, was greater in the group receiving GM-1; the observed difference between treatment groups was 0.22 (P = .06). A post hoc analysis in the subgroup of patients treated within 4 hours showed a statistically significant difference, with Canadian Neurological Scale mean improvement of 0.41 (P = .016). GM-1 use was not associated with differences in frequency, nature, or severity of adverse experiences. CONCLUSIONS: These findings suggest that GM-1 is safe in the dose and treatment schedule used and that its efficacy in ischemic stroke is greater when given soon after onset of stroke.
Adult↗
The E2A gene product contains two separable and functionally distinct transcription activation domains.
The E2A gene encodes transcription factors of the helix-loop-helix (HLH) family which are implicated in cell-specific transcriptional control in several cell lineages, including pancreatic beta cells. In the present work, we show by deletion mapping of both the E2A protein itself and the Gal4-E2A fusion protein that the protein contains at least two distinct activation domains. One domain (located between amino acids 1 and 153) functions efficiently in a variety of mammalian cell lines. The second domain (located between amino acids 369 and 485) functions preferentially in pancreatic beta cell lines. The latter domain shows a pattern of heptad repeats of leucine residues characteristics of "leucine zipper" transcription factors; site-directed mutagenesis of leucines within this repeat led to substantial reductions in activity. The selective properties of this activation domain may contribute to cell-specific transcription directed by the E2A gene.
Adenoviridae↗
Cell-specific expression of helix-loop-helix transcription factors encoded by the E2A gene.
The E2A gene encodes transcription factors of the helix-loop-helix family that are implicated in cell-specific gene expression as part of dimeric complexes that interact with E box enhancer elements. It has previously been shown that transcripts of the E2A gene can be detected in a wide range of cell types. We have now examined expression of the mouse E2A gene at the protein level using polyclonal antisera directed against distinct portions of the E2A protein to probe blots of cellular extracts. A 73 kDa protein was identified by this analysis: this protein is highly enriched in cell lines of B lymphoid origin as compared to pancreatic beta-cells and fibroblast cells. The detection of this protein selectively in extracts of lymphoid cells correlates with the presence of the E box-binding activity LEF1/BCF1 in these cells; this binding activity was previously shown to be efficiently recognized by antiserum directed against E2A gene products. Transfection of cells with full length E2A cDNA leads to appearance of protein co-migrating with the 73 kDa protein on SDS gel electrophoresis and co-migrating with LEF1/BCF1 on mobility shift analysis. Our results are consistent with the view that the DNA-binding activity LEF1/BCF1 is a homodimer of E2A proteins; the selective appearance of this putative cell-specific transcription factor in B lymphoid cells seems to be attributable, at least in part, to the elevated E2A protein concentrations in these cells.
Animals↗
NF-kappa B activates the HIV promoter in neurons.
Human immunodeficiency virus (HIV) infection of the brain leads to massive neuronal damage, resulting in the AIDS (acquired immunodeficiency syndrome) dementia complex (ADC). A recent study using transgenic mice indicates that neurons possess transcription factors capable of activating the HIV promoter. To identify these, we transfected two types of primary cultures of rat neurons with HIV promoter-reporter gene constructs. The two kappa B regulatory sites in the HIV long terminal repeat (LTR) are shown to be essential for strong promoter activity. Two proteins present in neurons, BETA and an NF-kappa B-like protein, can bind the kappa B sites. These proteins are shown to belong to distinct families of transcription factors. Mutation analysis and transfection of a dominant negative NF-kappa B mutant, indicate that the neuronal NF-kappa B-like activity mediates HIV promoter activation. cDNA cloning, biochemical and immunological analyses indicate that neuronal NF-kappa B is similar to NF-kappa B of other tissues. Transfections of primary neuron cultures with an HIV promoter-beta-galactosidase construct show that within these cultures, neurons are indeed the cells that highly activate the HIV promoter. Thus, analogous to the situation in T-lymphocytes and macrophages, NF-kappa B is an activator of HIV transcription in neurons.
AIDS Dementia Complex↗
Positive and negative regulation of insulin gene transcription.
Explore the source record for details and available documents.
Animals↗
Subunit structure of cell-specific E box-binding proteins analyzed by quantitation of electrophoretic mobility shift.
Expression of insulin and immunoglobulin genes is dependent on the presence of E boxes (consensus sequence CAXXTG) within the enhancer regions. These sequences are recognized by cell-specific nuclear factors IEF1 (insulin enhancer factor 1) and LEF1 (lymphoid enhancer factor 1). Although IEF1 and LEF1 are distinct by several parameters, they are both recognized by antisera to the mouse helix-loop-helix (HLH) protein A1 (a homolog of the human protein E47, product of the E2A gene). This suggests that A1/E47 or a close relative is a component of both complexes. In order to further characterize the complexes, we have used in vitro translated DNA-binding proteins of known size to verify that electrophoretic mobility shift analysis can be used to estimate the molecular weight of DNA-binding proteins from both the HLH family and the leucine zipper family. Under the conditions used, migration is relatively insensitive to changes in protein charge. This analysis, in combination with mixing experiments between nuclear extracts and in vitro translated HLH proteins, indicates that IEF1 and LEF1 are dimeric complexes. IEF1 behaves as a complex of two proteins, one of which is 67 kDa and is recognized by antibodies to A1, and the second of which is 25 kDa. LEF1 on the other hand, appears to be a complex of two proteins of 67 kDa. The size of the 67-kDa subunits is consistent with that reported for the full-length E2A gene products. The 25-kDa subunit of IEF1 forms DNA-binding heterodimers with A1 but not MyoD and is present in a limited range of cell types, features characteristic of class B HLH proteins such as MyoD and achaete-scute. Taken together, the data support the idea that the E2A gene products are involved directly in regulation of insulin and immunoglobulin gene expression; regulation of the insulin gene apparently requires, in addition, the 25-kDa HLH protein (designated IESF1 for insulin enhancer-specific factor 1).
Animals↗
Carotid endarterectomy: a little more light at the end of the tunnel.
Explore the source record for details and available documents.
Carotid Stenosis↗
Insulin-producing cells contain a cell-specific repressor activity that functions through multiple E-box sequences.
The cis-acting DNA element known as the E box (consensus sequence CAxxTG) plays an important role in the transcription of a number of cell-specifically expressed genes. The rat insulin I gene, for example, contains two such sequences (IEB1 and IEB2) that are recognized specifically by a characteristic beta cell nuclear factor insulin enhancer factor 1 (IEF1). To define the role of these elements better, we tested for cooperative interactions between the IEB sequences. Transfection experiments were performed with a series of plasmids containing the elements separated by different distances. Transcriptional activity in vivo is only modestly affected (less than two-fold) when the distances between the IEB elements are changed by a half-integral number of double-helical turns. Surprisingly, plasmids bearing four and six copies of the IEB motif showed sharply reduced activity as compared to those with two copies. In vitro DNA-binding studies revealed that this effect was not due to inability of IEF1 to bind to multiple copies of IEB. Moreover, multiple copies of the IEB sequence were able to inhibit activity of a cis-linked Moloney sarcoma virus (MSV) or insulin enhancer upon transfection to beta cells but not to other cell types. The above data are consistent with the view that beta cells contain a cell-specific repressor molecule capable of binding to multiple copies of IEB and thereby inhibiting transcription. This interpretation was further strengthened by in vivo competition and trans-activation experiments. The beta-cell-specific repressor activity identified by these studies may play an important role in mediating gene expression in insulin-producing cells, perhaps by regulating the access of helix-loop-helix transcription factors to E-box sequence elements.
3T3 Cells↗
B-cell factor 1 is required for optimal expression of the DRA promoter in B cells.
The X box in the DRA promoter of the human histocompatibility complex is required for expression of the DRA gene in B cells. We show that a B-cell factor binds to a sequence that is clearly distinguishable from binding sites for the previously described X box binding nuclear proteins RF-X, NF-X, NF-Xc, NF-S, hXBP, and AP-1. Mutations in the DRA X box that disrupt the binding of this factor result in a lower level of gene expression, as does the presence of Id (a trans-dominant regulatory protein that negatively regulates helix-loop-helix proteins). Furthermore, this factor is recognized by antibodies directed against the helix-loop-helix protein A1, a mouse homolog of the immunoglobulin enhancer binding proteins E12/E47, and it binds to sequences in other genes that were previously shown to bind these proteins. By these criteria, this factor is BCF-1.
B-Lymphocytes↗
Design and baseline results of the monosialoganglioside early stroke trial. The EST Study Group.
BACKGROUND AND PURPOSE: The Early Stroke Trial is a randomized, placebo-controlled, double-masked, multicenter study to assess the safety and efficacy of monosialoganglioside in patients who have suffered an ischemic stroke of the cerebral hemispheres. METHODS: Only patients who could be evaluated and treated within 5 hours after the onset of stroke were considered; within each center, subjects were stratified by age, sex, and clinical severity. Patients were randomly allocated to receive a specified sequence of intravenous and intramuscular doses of either monosialoganglioside or identical-appearing placebo for 21 days. Patients were followed up for 4 months after randomization. Neurological status was measured primarily by using the Canadian Neurological Scale. After assessing the effect of treatment on survival, the principal measure of efficacy will be the change in neurological status between baseline and the 4-month follow-up among survivors. RESULTS: Sixteen clinical centers, 15 in Europe and one in North America, entered a total of 792 eligible patients during a 36-month recruitment period (from May 1987 to April 1990). In our series there were more men than women, and the relative frequency of patients increased with advancing age. The most frequently associated cardiovascular conditions were hypertension, atrial fibrillation, and peripheral vascular disease. Approximately 46% of the patients were admitted to a hospital within 1 hour and 81%, within 2 hours after the onset of stroke. About 22% first received the study treatment within 3 hours and 57%, within 4 hours. CONCLUSIONS: This study demonstrates the feasibility of large-scale trials with the onset of treatment within 5 hours after an ischemic stroke.
Adult↗
Effect of short-term fasting/refeeding on epidermal growth factor content in the gastrointestinal tract of suckling rats.
Epidermal growth factor (EGF) is trophic for varying regions of the developing gastrointestinal tract (GIT) of suckling rats. The presence of large amounts of EGF in milk from various species, combined with low production of EGF by suckling animals, led to speculation that milk is a major source of EGF for suckling rats. We report that short-term fasting (8 hr) of 12-day-old suckling rats resulted in a significant decrease in the levels of immunoreactive EGF (irEGF) in the GIT. Pups refed by lactating mothers for 1 to 4 hr exhibited an increase in irEGF to original levels, whereas pups fed a rat milk substitute by gastric gavage did not have an increase in irEGF content. The irEGF levels in the GIT of pups that were manually fed normal rat milk, or rat milk substitute supplemented with EGF, returned to the prefasted levels. Fasted suckling rats refed 2 ml of rat milk in 2 h exhibited significantly higher level of irEGF in the GIT than did those refed with 0.5 ml in 45 min. Since rat milk irEGF exists in three distinct forms (A, B, and C; C is equal to authentic submandibular gland EGF, the irEGF forms in the GIT were characterized by native polyacrylamide gel electrophoresis. In the stomach luminal contents of the fed suckling rats, only the larger form, Peak B, was observed. Both the luminal content and the mucosa scrapings of all other segments of all groups contained only Form D (comigrating with desarginyl EGF), a metabolic derivative of EGF. All forms were immunoreactive, exhibited receptor binding, and stimulated DNA synthesis in growth-arrested fibroblasts. The rapid changes in EGF within the GIT of suckling rats suggest the EGF can acutely modify some GIT functions of suckling rats.
Animals↗
Distribution and characterization of helix-loop-helix enhancer-binding proteins from pancreatic beta cells and lymphocytes.
Transcription of a number of mammalian genes is controlled in part by closely-related DNA elements sharing a CAxxTG consensus sequence (E boxes). In this report, we survey cell extracts from a variety of mammalian cell lineages for ability to bind to the E box denoted IEB1/kappa E1, which plays an important role in expression of both insulin and immunoglobulin kappa genes. Insulin enhancer factor 1 (IEF1), a binding activity previously identified in beta cells, was also present in pituitary endocrine cells but absent in 7 other mammalian cell lines tested. A distinct binding activity, lymphoid enhancer factor 1 (LEF1), was observed in several lymphoid cell lines, but was absent from all nonlymphoid cells tested. IEF1 and LEF1 were distinct according to electrophoretic mobility, and DNA binding specificity. As previously reported, both beta cell and lymphoid cell factors are recognized by antibodies to helix-loop-helix (HLH) proteins, indicating that they may contain functional helix-loop-helix dimerization domains. To directly demonstrate this, we showed that the binding factors are able to interact in vitro with the HLH domain of a characterized HLH protein. These results support the notion that HLH proteins play a key role in cell-specific transcriptional regulation in cells from endocrine and lymphocyte lineages.
Animals↗
Acute spinal-cord injury.
Explore the source record for details and available documents.
Cost-Benefit Analysis↗