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Biomedical subjects

W H Walker

Publications and source records attributed to W H Walker.

At least 19 recordsLinked to original sources
PubMed1992

The v-rel oncogene: insights into the mechanism of transcriptional activation, repression, and transformation.

The v-rel oncogene product from the avian reticuloendotheliosis virus strain T corresponds to a member of the Rel-related family of enhancer-binding proteins that includes both the mammalian 50- and 65-kDa subunits of the NF-kappa B transcription factor complex. However, in contrast to NF-kappa B, v-Rel has been shown to function as a dominant-negative repressor of kappa B-dependent transcription in many mature cell types. We now demonstrate that a highly conserved motif within the Rel homology domain of v-Rel containing a consensus protein kinase A phosphorylation site is required for DNA binding, transcriptional repression, and cellular transformation mediated by this oncoprotein. However, replacement of the serine phosphate acceptor within the protein kinase A site with an alanine did not alter any of these functions of v-Rel, suggesting that phosphorylation at this site is not central to the regulation of this oncogene product. Rather, the inactive mutations appear to identify a functional domain within v-Rel required for these various biological activities. It is notable that these same mutations do not impair the ability of v-Rel to heterodimerize with the 50-kDa subunit of NF-kappa B, suggesting that v-Rel-mediated transcriptional repression likely involves direct nuclear blockade of the kappa B enhancer rather than indirect alterations in the composition of preformed cytoplasmic NF-kappa B complexes. Paradoxically, when introduced into undifferentiated F9 cells, v-Rel functions as a kappa B-specific transcriptional activator rather than as a dominant-negative repressor. These stimulatory effects of v-Rel require both the conserved protein kinase A phosphorylation site and additional unique C-terminal sequences not needed for v-Rel-mediated repression in mature cells. Retinoic acid-induced differentiation of these F9 cells restores the repressor function of v-Rel. These opposing biological actions of v-Rel occurring in cells at distinct stages of differentiation may have important implications for the mechanism of v-Rel-mediated transformation occurring in avian splenocytes.

Amino Acid Sequence↗
PubMed1991

The human placental lactogen genes: structure, function, evolution and transcriptional regulation.

hPL is a member of an evolutionarily related gene family including hGH and hPRL. Expression of hPL is limited to the placenta but its physiological actions are far reaching. hPL has a direct somatotropic effect on fetal tissues, it alters maternal carbohydrate and lipid metabolism to provide for fetal nutrient requirements, and aids in stimulation of mammary cell proliferation. Two hPL genes (hPL3 and hPL4) encoding identical proteins are responsible for the production of up to 1-3 g PL hormone/day. Recent studies have characterized the regulatory controls of hPL expression. At the post transcriptional level, RNA stability may contribute to variable levels of hPL3 vs. hPL4 production. In addition, non-tissue-specific protein-promoter interactions involving the Sp1 transcription factor are necessary for hPL transcription initiation. A transcriptional enhancer located 3' to the hPL3 gene is responsible for the placenta-specific expression of this gene, while an additional enhancer may be located 3' to the hPl4 gene. The hPL enhancer is bound by multiple proteins including at least one placental specific protein that interacts with a TEF-1 motif. Therefore, enhancer-protein interactions most likely play a large part in the high levels of placenta-specific hPL expression.

Amino Acid Sequence↗
PubMed1990

The v-rel oncogene encodes a kappa B enhancer binding protein that inhibits NF-kappa B function.

Studies of NF-kappa B suggest that this enhancer binding activity corresponds to a family of at least four proteins (p50, p55, p75, and p85) differentially induced with biphasic kinetics during T cell activation. While p55 and p50 are closely related to the 50 kd DNA binding subunit of NF-kappa B, p75 and p85 exhibit DNA binding properties that distinguish them from this 50 kd polypeptide and its regulatory subunits I kappa B and p65. All four members of this kappa B-specific protein family are structurally related to the v-Rel oncoprotein and one, p85, appears identical to human c-Rel. v-Rel, but not nontransforming v-Rel mutants, binds to the kappa B enhancer and inhibits NF-kappa B-activated transcription from the IL-2 receptor alpha promoter and HIV-1 LTR. These findings suggest a Rel-related family of kappa B enhancer binding proteins and raise the possibility that the transforming activity of v-Rel is linked to its inhibitory action on cellular genes under NF-kappa B control.

Base Sequence↗
PubMed1990

Human placental lactogen transcriptional enhancer. Tissue specificity and binding with specific proteins.

Human placental lactogen (hPL) and growth hormone (hGH) are thought to be derived from a common ancestral gene and have similar nucleotide and amino acid sequences. Although the genes are similar in structure, they are expressed in different tissues. A transcriptional enhancer has been found 2.2 kilobases 3' of the hPL3 gene at the distal extreme of the hPL/hGH gene cluster. This enhancer is at least 20-fold more active in hPL-producing human choriocarcinoma JEG-3 cells than in non-hPL-producing cells. The enhancer is active when linked to either the hPL3 or SV40 promoter. We have localized the hPL enhancer to a 138-base pair (bp) region that retains tissue specificity in transient transfection assays. Gel mobility shift assays showed that the hPL enhancer interacted specifically with nuclear proteins from JEG-3 cells and placental tissue. Within the 138-bp enhancer, a 22-bp region overlapping a TEF-1 binding site was shown to be protected from DNase I digestion by placental and HeLa nuclear extracts. Placental protein(s) binding this region may be instrumental in tissue-specific activity of the hPL enhancer.

Amino Acid Sequence↗
PubMed1990

NF-kappa B: a family of inducible and differentially expressed enhancer-binding proteins in human T cells.

A sensitive DNA-protein crosslinking approach has been used to characterize four inducible T-cell proteins (50 kDa, 55 kDa, 75 kDa, and 85 kDa) that specifically bind to kappa B enhancer elements. Partial proteolytic mapping revealed a distinct cleavage pattern for three of these proteins. These polypeptides are sequestered as inactive precursors in the cytosol of unstimulated T cells but can be converted into active forms in vivo by phorbol ester stimulation or in vitro by detergent treatment. The induction of these proteins by phorbol ester results in a strikingly biphasic pattern of nuclear expression with the 55-kDa and 75-kDa species appearing within minutes, whereas the 50-kDa and 85-kDa species appear only several hours after cellular stimulation. These data suggest that NF-kappa B-binding activity may not correspond to a single polypeptide but rather a family of at least four inducible and differentially regulated DNA-binding proteins that are expressed with distinct kinetics in human T lymphocytes.

Base Sequence↗
PubMed1990

DNA sequences involved in the transcriptional activation of a human placental lactogen gene.

To identify regulatory elements in the promoter of a human placental lactogen gene (hPL3) that are important for its transcriptional activation, sequences 5' to the start of transcription were linked to the reporter gene chloramphenicol acetyltransferase (CAT) and transiently transfected into JEG-3 cells, a human placental choriocarcinoma cell line. In the presence of the hPL3 enhancer, deletion of the promoter sequence between -142 and -129 basepairs resulted in an 8-fold decrease in CAT activity. Similar results were seen with the SV40 enhancer and the hPL3 promoter in HepG2 liver cells. Nuclear proteins from HepG2, HeLa, and JEG-3 cells formed specific binding complexes with this region of the hPL3 promoter by a gel mobility shift assay, indicating that the DNA-binding protein was not tissue specific. The -142 to -129 basepair region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. An oligonucleotide containing Sp1-binding sites specifically competes for proteins binding the hPL3 promoter, and the methylation interference pattern is similar to that for an Sp1-binding site. This suggests that the hPL3 promoter binds Sp1- or an Sp1-like trans-acting factor, and this binding site is important for transcriptional regulation by the hPL3 enhancer in PL-producing cells.

Base Sequence↗
PubMed1990

[49 day storage of erythrocyte concentrates in blood bags with the PAGGS-mannitol solution].

The red cell preservation solution PAGGS-Mannitol differs from the well documented PAGGS-Sorbitol only by the exchange from Sorbitol in Mannitol. In a clinical investigation with volunteers, up to 21 of the pure PAGGS-Mannitol solution were infused within 4 h. Blood and urine parameters were determined. The solution was well tolerated, no unexpected change of blood and urine parameters was found. The 24 h red cell in vivo survival rate of PAGGS-Mannitol was found to be 74.5 +/- 4.4%, after 49 days storage, a value which was described for PAGGS-Sorbitol before. In vitro data on storage of red cell concentrates with and without buffy coat were determined for hemolysis, 2,3-DPG and ATP using a blood bag system with DEHP and TOTM plastisized PVC. If 1% hemolysis is regarded as acceptable, red cell concentrates can be stored in PAGGS-Mannitol with a TOTM plastisized blood bag system up to 42 days. Under these conditions the amount of plastisizer in the red cell concentrate was found to be only 1% of the amount determined in a standard DEHP plastisized PVC blood bag system.

Adenine↗
PubMed1986

Studies of intestinal permeability in inflammatory diseases using polyethylene glycol 400.

It has been proposed that increased bowel permeability might play a role in the pathogenesis of inflammatory disease. Intestinal permeation was investigated by measuring the 6-hour urinary excretion of polyethylene glycol (PEG) 400 in 40 adult volunteer controls and in patients with inflammatory disease. Of the patients, 15 had Crohn's disease; 7, ulcerative colitis; 2, celiac disease; and 7, rheumatoid arthritis. No significant difference in total urinary excretion over a 6-hour period was found between controls and patients with ulcerative colitis. Patients with Crohn's disease, celiac disease, or with rheumatoid arthritis were found to have significantly decreased urinary excretion of PEG 400. The results of this study indicate that there is no identifiable increase in intestinal permeation as measured by PEG 400 excretion during periods of active inflammatory disease.

Adult↗
PubMed1986

Studies of thyroxine binding to plasma proteins in health and disease.

Thyroxine binding protein characteristics were defined in 6 normal subjects, 4 with thyroxine binding globulin (TBG) deficiency, 3 with TBG increase, one with hyperthyroxinemic dysalbuminemia, 7 with severe non-thyroidal illness, and 3 with chronic renal failure. Free thyroxine was measured by Sephadex partition in plasma to which increasing thyroxine concentrations were added. Deconvolution of the resultant titration data was performed by computer modelling. Abnormalities of thyroxine binding capacities or of binding affinities occur in non-thyroidal illness, chronic renal failure, and sporadically. The patient with hyperthyroxinemic dysalbuminemia had increased thyroxine binding affinity to thyroxine binding prealbumin as well as to albumin. "Free-T4 assays" or estimates of Free-T4 by calculation from total thyroxine and measures of protein binding such as T3-uptake must be expected to be perturbed by these binding protein abnormalities unless such interferences are explicitly demonstrated to be absent.

Adult↗
PubMed1977

A radioimmunoassay for serum ferritin.

We describe the development and evaluation of a serum ferritin radioimmunoassay, in which 125l-labeled ferritin and rabbit anti-ferritin antibody are used. Goat anti-rabbit gamma-globulin antibody, together with polyethylene glycol, is used as the separating reagent. The assay has a working range up to 500 mug of ferritin per litre, and a sample requirement of 75 mul of serum for assay at two dilutions. The assay requires 24 h. it has a sensitivity of 1.5 mug of ferritin per litre and a long-term precision (CV) of 13%. Reference intervals for a population of men were 18-330 mug/litre, with no marked age dependence, while those for a population of women older than 50 years were 18-200 mug/litre. Many apparently healthy women in the 20-50 year age group have much lower concentrations. Serum ferritin concentrations of less than 18 mug/litre are indicative of iron deficiency, defined as the absence of stainable iron in an aspirate of bone marrow.

Adult↗