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M D Walker

Publications and source records attributed to M D Walker.

At least 19 recordsLinked to original sources
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65-kDa heat shock protein.

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells resident in the pancreatic islets. We recently discovered that the pathogenesis of diabetes in NOD strain mice was associated with T-cell reactivity to an antigen cross-reactive with a mycobacterial 65-kDa heat shock protein. To identify peptide epitopes critical to the insulin-dependent diabetes mellitus of NOD mice, we studied the specificities of helper T-cell clones capable of causing hyperglycemia and diabetes. We now report the identification of a functionally important peptide within the sequence of the human variant of the 65-kDa heat shock protein molecule. T-cell clones recognizing this peptide mediate insulitis and hyperglycemia. Alternatively, the T cells can be attenuated and used as therapeutic T-cell vaccines to abort the diabetogenic process. Moreover, administration of the peptide itself to NOD mice can also down-regulate immunity to the 65-kDa heat shock protein and prevent the development of diabetes. Thus, T-cell vaccination and specific peptide therapy are feasible in spontaneous autoimmune diabetes.

Animals↗
PubMed1991

The community hospital-based stroke programs in North Carolina, Oregon and New York--V. Stroke diagnosis: factors influencing the diagnostic evaluation of patients following acute stroke.

Among the 4129 patients of the Community Hospital-based Stroke Program, 30% had an unspecified stroke diagnosis. Since specific diagnosis and, perhaps, eventual treatment, derives in part from diagnostic testing, we examined the effect of clinical condition, geographic and demographic factors on the incidence of certain diagnostic tests after acute stroke. In this multivariable analysis, race, sex, history of hypertension and history of diabetes did not influence the chance of having any test, but older age strongly reduced the chances of receiving extensive evaluation. When CT scanning was available, the utilization of a CT as well as other diagnostic studies including cerebral angiography, radionuclide brain scan, EEG and EKG was increased. The odds of receiving a CT scan increased if the patient was married, and decreased with a history of previous stroke. A history of previous TIA increased the chance of having a cerebral angiogram while a history of cardiac disease decreased the chance. There were striking regional geographic differences in the use of CT, radionuclide brain scanning and cerebral angiography which may, in part, reflect differences between the availability of these technologies in urban and rural hospitals. These results indicate that evaluation of stroke patients remains heterogenous.

Aged↗
PubMed1991

Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1.

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

Animals↗
PubMed1990

A cDNA from a mouse pancreatic beta cell encoding a putative transcription factor of the insulin gene.

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5' flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5-10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine beta cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5' flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression.

Amino Acid Sequence↗
PubMed1989

Individual protein-binding domains of the insulin gene enhancer positively activate beta-cell-specific transcription.

A beta-cell-specific enhancer is located in the 5'-flanking DNA of the rat insulin 1 gene. Two homologous 8-base-pair sequences in the enhancer (IEB1 and IEB2) significantly stimulated transcription from a heterologous promoter (two- to fourfold) in a cell-specific fashion. When the elements were combined or duplicated, more than 50% of the activity of the intact enhancer was obtained. These two cis-acting elements appear to play a dominant role in the positive control of beta-cell-specific transcription of the insulin gene.

Animals↗
PubMed1988

Identification and characterization of transcriptional regulatory regions associated with expression of the human apolipoprotein E gene.

Multiple cis-acting regulatory elements have been mapped within a 1-kilobase fragment spanning nucleotides -651 through +356 of the human apolipoprotein E gene using a transient expression system based on the chloramphenicol acetyltransferase gene as well as DNase I footprinting techniques. A 651-base pair 5'-flanking region of the human apolipoprotein E gene was capable of directing chloramphenicol acetyltransferase gene expression over a 48-fold range among the various cultured cell lines tested. Deletion analysis of this 651-base pair upstream region linked to either the chloramphenicol acetyltransferase gene or the intact apolipoprotein E structural sequences revealed at least three regulatory domains within the proximal 383 nucleotides. One of these domains contained a GC box transcriptional control element. Further analysis demonstrated that the other two domains contained enhancer-like activity. These enhancer-like elements were located within the nucleotides spanning -366 to -246 and -193 to -124. A third enhancer element was identified in the first intron, within nucleotides +44 to +262. Changing the distance of the three enhancer elements from the transcription start site and reversing their orientation did not significantly alter their effects on transcription rates. However, enhancer activity was influenced by the promoter and cell line that were used. DNase I footprinting assays showed that specific sequences within two of these elements (-193 to -124 and +44 to +262) bind proteins in nuclear extracts from HepG2 and Chinese hamster ovary cells. A protein footprint also was identified for a GC box element at nucleotides -59 to -45. Thus, control of apolipoprotein E gene expression is the result of a complex interaction of several different regulatory elements.

Acetyltransferases↗
PubMed1988

Biosynthetic regulation of endogenous hamster insulin and exogenous rat insulin II in transfected HIT cells.

To investigate mechanisms underlying biosynthetic regulation of an insulin gene, the rat insulin II gene was introduced into hamster beta-cells (HIT) by cotransfection with the neomycin phosphotransferase-selectable marker. The insulin gene fragment was 2.2 kilobases (kb) in length and contained all exons, introns, and approximately 700 base pairs (bp) of 5'-flanking DNA and 300 bp of 3'-flanking DNA. The HIT cell was known to have endogenous hamster insulin production under regulation by glucose and dexamethasone. In a pool of stably transfected cells (HIT M62pR2), rat insulin II and hamster insulin were produced at comparable rates. Glucose (20 mM) stimulated cellular [3H]leucine labeling of both hamster insulin and rat insulin II by approximately twofold. Addition of 10(-6) M dexamethasone to media containing 11.1 mM glucose inhibited biosynthesis of both hamster insulin and rat insulin II by greater than 90%. Thus, with both positive and negative biosynthetic regulation, changes in the cellular labeling of exogenous rat insulin II were qualitatively and quantitatively similar to those of the endogenous hamster insulin. These data suggest that the 2.2-kb rat insulin II gene fragment contained sufficient information for both expression and apparently "normal" biosynthetic regulation of exogenous rat insulin II (when compared with endogenous hamster insulin) in response to glucose and dexamethasone.

Animals↗
PubMed1988

Dose-escalation study of intravenous nicardipine in patients with aneurysmal subarachnoid hemorrhage.

A dose-escalation study of the calcium ion entry blocking drug nicardipine was performed using large dose infusions in 67 patients with recent aneurysmal subarachnoid hemorrhage (SAH). A safe, potentially therapeutic dose of the drug was determined. Patients admitted within 7 days of SAH from a documented cerebral aneurysm were entered into the study if no spasm was present on the initial angiogram. Nicardipine was administered as a continuous intravenous infusion throughout the 14-day period after SAH, regardless of the timing of surgery. To determine the safest possible dose, nicardipine was administered at seven dose levels from 0.01 to 0.15 mg/kg/hr. The total daily doses ranged from 27.7 mg to 375.0 mg. A follow-up angiogram was carried out on all 67 patients 7 to 10 days after SAH. Computerized tomography and neurological examinations were used to determine the presence of cerebral infarction. No major adverse effects, unexpected reactions, or permanent sequelae could be attributed to nicardipine. A decline in blood pressure was noted following administration of the drug. This occurred more frequently among patients given the largest dose but did not produce clinical problems or require discontinuation of the drug. Favorable outcomes were noted in 52 patients (78%). Vasospasm was found by arteriography in 31 patients (46%). A dose-related trend was noted: only eight (24%) of 33 patients treated at the highest dose level (approximately 10 mg/hr) developed arteriographic evidence of vasospasm. Symptomatic vasospasm was diagnosed in only two (6%) of 33 patients treated with this dose. Of the 34 patients receiving the lower dose levels, angiographic spasm was observed in 68% and symptomatic vasospasm in 27%. No deaths due to vasospasm occurred. Nicardipine appears to prevent both vasospasm and cerebral ischemia after SAH. A multicenter randomized double-blind trial to test this hypothesis is planned.

Adult↗