Search PubMed⌕ Search

Biomedical subjects

C E Walker

Publications and source records attributed to C E Walker.

At least 19 recordsLinked to original sources
PubMed2001

The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy.

Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline-->leucine mutation at position 50 of human GCAP1 results in cone-rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.

Amino Acid Sequence↗
PubMed2001

Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins.

The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.

3' Untranslated Regions↗
PubMed2000

Functional characterization of missense mutations at codon 838 in retinal guanylate cyclase correlates with disease severity in patients with autosomal dominant cone-rod dystrophy.

Three different mutations in codon 838 of GUCY2D, the gene for retinal guanylate cyclase 1, have been linked to autosomal dominant cone-rod dystrophy at the CORD6 locus. To examine the relationship between enzyme activity and disease severity, the three disease-causing substitutions (R838C, R838H and R838S) and four artificial mutations (R838A, R838E, R838L and R838K) were generated. Assay of GCAP1-stimulated cyclase activity in vitro shows that, compared with wild-type, R838E, R838L and R838K possess only low activity, whereas R838A, R838C, R838H and R838S have activity equal or superior to wild-type at low Ca(2+) concentrations. These four latter mutants showed a higher apparent affinity for GCAP1 than did wild-type. The Ca(2+) sensitivity of the GCAP1 activation was also altered with marked residual activity at high Ca(2+), the effect increasing: wild-type < R838C < R838H << R838A < R838S. Within the photoreceptor, this would result in a failure to inactivate cyclase activity at high physiological Ca(2+ )concentrations. Amongst the three disease-associated mutations, the effect correlates directly with disease severity. The wild-type and R838H mutant displayed a difference in pH sensitivity, with the mutant showing a higher specific activity with pH > 6.0. Site 838 is in the dimerization domain that forms a coiled-coil in the active protein. A computer-aided structure prediction of this region indicates that R838 in the wild-type breaks the structure at four helical turns, and there is an increasing tendency for the structure to continue for further turns in the order R838C < R838H,S,K << R838E < R838A < R838L.

Amino Acid Substitution↗
PubMed1999

A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61beta subunit.

We report here the isolation of a new member of the ADP-ribosylation factor (ARF)-like family (ARL-6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage-restricted expression, ARL-6 mRNA increased with erythropoietin-induced maturation of J2E cells, and decreased with interleukin 6-induced differentiation of M1 monoblastoid cells. In tissues, ARL-6 mRNA was most abundant in brain and kidney. While ARL-6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP-gammaS, like many members of the ARF/ARL family. Using the yeast two-hybrid system, six molecules which interact with ARL-6 were identified including SEC61beta, a subunit of the heterotrimeric protein conducting channel SEC61p. Co-immunoprecipitation of ARL-6 confirmed a stable association between ARL-6 and SEC61beta in COS cells. These results demonstrate that ARL-6, a novel member of the ADP-ribosylation factor-like family, interacts with the SEC61beta subunit.

ADP-Ribosylation Factors↗
PubMed1999

Assessing heparin neutralization following cardiac surgery: sensitivity of thrombin time-based assays versus protamine titration methods.

Adequate assessment of heparin neutralization following cardiac surgery is critical in reducing the patient's exposure to protamine. Both excessive protamine and residual heparin have been associated with postoperative bleeding and poor patient recovery. The activated clotting time (ACT) is the preferred intraoperative heparin monitor, while both protamine titration (i.e. a protamine-containing ACT) and thrombin time methods have been used to detect circulating residual heparin after protamine administration. Following initial protamine dosing using the protamine response test (PRT), postoperative monitoring was employed in the operating room prior to transport of the patient to intensive care. Two point-of-care assays, the thrombin time (TT) and the protamine dose assay (PDA), were evaluated to determine their relative heparin sensitivity and their usefulness to quantitate protamine dose. The PDA, which is based on the ACT, was shown in laboratory and clinical studies to detect residual heparin above 0.25 units/ml and to quantify additional minidoses of protamine (as low as 25 mg) required to obtain complete heparin neutralization. Differential evaluation of the TT and heparin neutralized thrombin time (HNTT) was shown in laboratory studies to be more sensitive to small amounts of residual heparin than the ACT. Clinical evaluations confirmed that additional protamine is required in approximately 12% of cardiac surgical cases managed using the PRT system. Both the PDA and TT/HNTT provided useful postoperative assessment of the adequacy of heparin neutralization. The TT/HNTT had slightly improved heparin sensitivity even in the presence of significant fibrinogen loss. These point-of-care assays provide the opportunity to optimize heparin and protamine management in the cardiac surgery patient.

Anticoagulants↗
PubMed1999

Optimized RNA gel-shift and UV cross-linking assays for characterization of cytoplasmic RNA-protein interactions.

Considerable interest has recently focused on defining the mechanisms involved in the regulation of gene expression at the level of mRNA stability and translational efficiency. However, the assays used to directly investigate interactions between RNA and cytoplasmic proteins have been difficult to establish, and methods are not widely available. Here, we describe a robust method for RNA electrophoretic mobility shift and UV cross-linking assays that allows rapid detection of cytoplasmic RNA-protein interactions. For added convenience to new investigators, these assays use mini-gels with an electrophoresis time of 15-20 min, enabling a high throughput of samples. The method works successfully with many different probes and cytoplasmic extracts from a variety of cell lines. Furthermore, we provide a system to optimize characterization of the RNA-protein complex and troubleshoot most assay difficulties.

Animals↗
PubMed1998

Regulation of EGF-receptor expression by EGF and TGF alpha in epidermoid cancer cells is cell type-specific.

The epidermal growth factor receptor (EGF-R) and its major ligands EGF and transforming growth factor alpha (TGF alpha) play an important role in the development of multiple human tumors. However, little is known of the comparative effects of each ligand on the regulation of EGF-R expression. To investigate this issue we used two similar human epidermoid cancer cell lines that overexpress EGF-Rs (KB and A431). In KB cells, EGF and TGF alpha increased EGF-R mRNA and protein levels by 2-3 fold over 8 h, associated with a greater than 4-fold stabilization of EGF-R mRNA half-life. EGF and TGF alpha also increased transcription of EGF-R mRNA 2-3-fold in KB cells. In contrast, EGF and TGF alpha only minimally increased EGF-R mRNA and protein in A431 cells, without changing EGF-R mRNA half-life. Basal EGF-R mRNA half-life was 2 fold greater in A431 cells than in KB cells (6-7 h versus 2-3 h), whilst the half-life of a mutant 2.6 kb EGF-R mRNA present in A431 cells, which lacks the 3-untranslated region (3'-UTR), was 2 fold greater than the full-length EGF-R mRNA. RNA gel-shift studies demonstrated that KB and A431 cells contain cytoplasmic proteins that bind specifically to an AU-rich sequence from the 3'-UTR of EGF-R mRNA. Taken together, these results demonstrate that in KB cells EGF and TGF alpha upregulate EGF-R expression at both transcriptional and post-transcriptional levels. The identification of AU-rich EGF-R mRNA-specific RNA-binding proteins from epidermoid cancer cells that overexpress EGF-Rs suggests that regulated RNA-protein interactions involving this region may play a central role in modulating EGF-R mRNA stability.

3' Untranslated Regions↗
PubMed1996

Comparison of nucleoside transport binding sites in rabbit iris-ciliary body and cultured rabbit nonpigmented ciliary epithelial cells.

The iris-ciliary body (ICB) is a site of action for topically applied antiglaucoma drugs. Moreover, adenosine has been implicated as a modulator of aqueous humor dynamics. The present study compares the binding of the nucleoside transporter probe, [3H]nitrobenzylthioinosine ([3H]NBMPR), to homogenates prepared from rabbit ICB and a cultured rabbit nonpigmented ciliary epithelial cell line (NPE) to determine whether NPE can be used as an experimental model to study the nucleoside transporter. Linear transformation of the saturation binding data revealed that [3H]NBMPR binds to a homogeneous population of binding sites with similar binding affinities (Kd = 0.3 +/- 0.1 and 0.6 +/- 0.1 nM in NPE and ICB, respectively). However, the maximal binding capacity in NPE (Bmax = 288 +/- 54 fmol/mg protein) was significantly higher than that in ICB (Bmax = 154 +/- 17 fmol/mg protein). Selected inhibitors of the nucleoside transport system and structural analogs of adenosine inhibited the binding in both homogenate preparations with a similar rank order of potency: NBMPR > DPY > CV-1808 > CHA > R-PIA > S-PIA > 2-CADO > NECA. The results suggest that NPE is a useful model which could be used for characterizing the nucleoside transporter in ICB and for the screening of nucleoside transport inhibitors as potential antiglaucoma drugs.

Adenosine↗
PubMed1996

Effects of MaxEPA on salt-induced hypertension: relationship to [3H]nitrobenzylthioinosine binding sites.

We investigated the effects of dietary MaxEPA (a major source of eicosapentaenoic acid in fish oil) supplementation on blood pressure (BP) responses and heart rate (HR) of Dahl salt-sensitive (SS) rats fed low (0.4% NaCl) and high (8.0% NaCl) sodium diets. During a four week treatment period, BP remained normotensive in rats on low salt diet but was significantly elevated in those on high salt diet, causing 50% mortality. MaxEPA diminished the BP elevation and prevented the high salt-induced mortality. HR was not affected by either salt diet alone but was reduced in the presence of MaxEPA. At the end of the treatment period, the distribution of [3H]nitrobenzylthioinosine ([3H]NBMPR) binding, a putative marker of adenosine transport and metabolism, was estimated in selected rat tissues in order to evaluate the role of the purinergic system in the BP lowering effect of MaxEPA. Maximal [3H]NBMPR binding capacity (Bmax) in the kidney and platelets were 39% and 82% lower, respectively, in rats on high salt diet than in those on low salt diet. MaxEPA significantly blunted the decrease in Bmax in the kidney but not in platelets and increased Bmax in heart (48%) of low salt group. There were no changes in dissociation constants (Kd). The results suggest that MaxEPA can attenuate salt-induced hypertension, reduce salt-induced mortality and protect the integrity of kidney NBMPR binding sites in salt-induced hypertension.

Affinity Labels↗
PubMed1992

Behavioral management of toilet training, enuresis, and encopresis.

Toileting problems are a matter of great concern to parents and are a frequent source of family discord, but proper handling of toilet training as well as enuresis and encopresis can ameliorate any untoward effects of these problems. Both the pediatrician and the pediatric psychologist can play a major role in this area.

Behavior Therapy↗
PubMed1992

Effects of exogenous emulsifiers and fat sources on nutrient digestibility, serum lipids, and growth performance in weanling pigs.

Three experiments were conducted to determine whether emulsifiers improve utilization of fat from diets for early-weaned pigs. In Exp. 1, 96 weanling pigs (17 d old) were used in metabolism cages, with main effects of fat source (soybean oil, tallow, lard, and coconut oil) and emulsifier treatment (no emulsifier, lecithin, and lysolecithin as 10% of the added fat). Soybean oil and coconut oil were more digestible than tallow and lard (P < .001). Tallow was more digestible when lecithin and lysolecithin were added (P < .007), and pigs fed lecithin had lower serum triglycerides and cholesterol than pigs fed lysolecithin (P < .03). In Exp. 2, 270 weanling pigs (21 d old) were used in a growth assay. Treatments were 1) control diet; 2) Diet 1 with soybean oil; 3) Diet 1 with tallow; 4, 5, and 6) Diet 3 with lecithin replacing 5, 10, and 30% of the tallow, respectively; and 7, 8, and 9) Diet 3 with lysolecithin replacing 5, 10, and 30% of the tallow, respectively. At d 14 of the experiment, digestibility of tallow was improved more by lecithin than lysolecithin (P < .008). For the total experiment (d 0 to 35), the control pigs had poorer gain:feed ratio than did the pigs fed the fat sources (P < .002). In Exp. 3, 420 weanling pigs (21 d old) were used. Treatments were 1) control diet with soybean oil; 2) Diet 1 with tallow; and 3, 4, and 5) Diet 2 with 10% of the added fat as soybean oil, lecithin, or monoglyceride, respectively. Adding soybean oil, lecithin, and monoglyceride to tallow increased digestibility of total fat (P < .07). From d 0 to 14, pigs fed soybean oil gained weight faster than pigs fed the other treatments (P < .06), and pigs fed tallow without emulsifiers had the lowest ADG. Considering all experiments, addition of emulsifiers increased digestibility of nutrients but had minimal effect on growth performance.

Animal Feed↗
PubMed1980

Sexual attitudes of psychogenic and organic impotence.

Thirty organically impotent men were contrasted with 21 psychogenically impotent men and 17 normal controls. Distinctions among sexual attitudes, sexual behaviors, and sexual interests were evaluated on the Sex Form. Results suggest that impotent men can be reliably distinguished from non-impotent men on the basis of their use of activity rather than fantasy in coping with life's problems and their interest in receiving treatment for their condition. Separate comparisons between the control group and the two impotent groups further suggest that both can be reliably distinguished from normals. It is more difficult, however, to distinguish between organically and psychogenically impotent men on the basis of sexual interests, attitudes, and behavior. The findings suggest, however, that impotent men generally and psychogenically impotent men in particular tend to be individuals who have relatively low sexual drive, diminished sexual knowledge, and who are prone to active ways of coping with stress. The results are discussed in light of previous findings.

Adult↗
PubMed1978

Sexual behavior correlates of female orgasm and marital happiness.

The present study was designed to investigate sexual behavior correlates of marital happiness and female orgasm. Forty-eight female students of Baylor University who were 20 to 35 years old and had been married at least 2 years participated in the survey. A specially compiled 131-item marital relations questionnaire provided data for an intercorrelational analysis. Marital happiness, fidelity, and experience of orgasm correlated with certain specific sexual behaviors. These variables were related also to dissatisfaction with marital sex, which is consistent with previous research. However, when dissatisfaction resulting from performing less desired sexual acts was separated from dissatisfaction resulting from not performing desired sexual acts, the correlations differed, often markedly. Thus, the data suggest that unitary measures of dissatisfaction with marital sex may obscure meaningful differences. Also, information was obtained concerning the orgasmic and multiple orgasmic experience of the women, their extramartial experience, and their relative use of and preference for various intercourse positions and noncoital sexual activities.

Adult↗