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Biomedical subjects

E J Wilson

Publications and source records attributed to E J Wilson.

At least 19 recordsLinked to original sources
PubMed1996

The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231S mutant.

The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). At 30 degrees C or 37 degrees C, inactivation rates with 0.3 mM DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate increased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.

Acetylcholinesterase↗
PubMed1996

Epidermal growth factor inhibits carbachol-stimulated canine parietal cell function via protein kinase C.

BACKGROUND & AIMS: Epidermal growth factor (EGF) inhibits secretagogue-stimulated gastric acid secretion via an EGF receptor located on parietal cells. The aim of this study was to examine whether this growth factor inhibited carbachol-stimulated acid secretion through a protein kinase C-dependent mechanism. METHODS: The effect of EGF on carbachol-stimulated aminopyrine uptake, inositol trisphosphate formation, and intracellular Ca2+ ([Ca2+]i) in purified cultured parietal cells was studied. The ability of protein kinase A and C inhibitors to alter the inhibitory action of EGF was assessed. EGF-mediated translocation and activation of protein kinase C in parietal cells were determined. RESULTS: EGF dose dependently inhibited carbachol-stimulated aminopyrine uptake in a pertussis toxin-insensitive, genistein (tyrosine kinase inhibitor)--sensitive manner, with a maximal inhibitory effect (37.5% +/- 6.8%) achieved at 10(-7) mol/L. EGF did not significantly inhibit carbachol-stimulated inositol trisphosphate formation and did not alter the initial transient increase or sustained plateau in [Ca2+]i stimulated by this secretagogue. The protein kinase C inhibitors H-7 and staurosporine dose dependently reversed the inhibitory action of EGF, whereas H-89 (protein kinase A inhibitor) failed to alter the effect of EGF. EGF pretreatment increased the translocation of alpha and beta 1 isoforms of protein kinase C and stimulated kinase activity in parietal cells. EGF did not down-regulate the parietal cell muscarinic receptor. CONCLUSIONS: The inhibitory action of EGF on carbachol-stimulated parietal cell activity seems to involve protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Fasciculin 2 binds to the peripheral site on acetylcholinesterase and inhibits substrate hydrolysis by slowing a step involving proton transfer during enzyme acylation.

The acetylcholinesterase active site consists of a gorge 20 A deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. We report here the association and dissociation rate constants for fasciculin 2 interaction with the human enzyme in the presence of ligands that bind to either the peripheral site or the acylation site. These kinetic data confirmed that propidium is strictly competitive with fasciculin 2 for binding to the peripheral site. In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine bound to the acylation site and formed ternary complexes with the fasciculin 2-bound enzyme in which their affinities were reduced by about an order of magnitude from their affinities in the free enzyme. Steady state analysis of the inhibition of substrate hydrolysis by fasciculin 2 revealed that the ternary complexes had residual activity. For acetylthiocholine and phenyl acetate, saturating amounts of the toxin reduced the first-order rate constant kcat to 0.5-2% and the second-order rate constant kcat/Kapp to 0.2-2% of their values with the uninhibited enzyme. To address whether fasciculin 2 inhibition primarily involved steric blockade of the active site or conformational interaction with the acylation site, deuterium oxide isotope effects on these kinetic parameters were measured. The isotope effect on kcat/Kapp increased for both substrates when fasciculin 2 was bound to the enzyme, indicating that fasciculin 2 acts predominantly by altering the conformation of the active site in the ternary complex so that steps involving proton transfer during enzyme acylation are slowed.

Acetylcholinesterase↗
PubMed1994

Regulatory mechanism of human factor IX gene: protein binding at the Leyden-specific region.

Hemophilia B-Leyden is characterized by the gradual amelioration of bleeding after the onset of puberty. All Leyden phenotype mutations found to date lie within the Leyden-specific region, which spans roughly nt-40 to +20 in the 5' end of the human factor IX gene. With HepG2 cell nuclear extracts, the Leyden-specific region and its immediate neighboring region of the normal factor IX gene showed five DNase I footprints: FP-I (nt +4 to +19), FP-II (nt -16 to -3), FP-III (nt -27 to -19), FP-IV (nt -67 to -49), and FP-V (nt -99 to -77). Protein binding affinities of short oligonucleotides containing sequences of FP-I, FP-II, or FP-III were substantially reduced in the presence of Leyden phenotype mutations in these areas, correlating well with the negative effects of these mutations on factor IX gene expression. A Leyden phenotype mutation at nt -20 (T to A) caused a loss of both footprints FP-III and FP-II but generated a new footprint, FP-III' (nt -34 to -23), partially overlapping with FP-III, indicating mutation-dependent competitive protein binding at these sites. Although the FP-III' area contains an androgen responsive element-like sequence, the nuclear protein that binds at FP-III' is not androgen receptor. The protein was not recognized by anti-androgen receptor antibody and, furthermore, was present not only in liver but also in both androgen receptor-positive and androgen receptor-negative cells in electrophoretic mobility shift assays. The nuclear concentration of this protein increased significantly upon treatment of the HepG2 cells with testosterone. Its binding affinity to an oligonucleotide (-32sub) containing the FP-III' sequence was greatly reduced in the presence of exogenous androgen receptor, suggesting a possible interaction of this protein with androgen receptor. The affinities of both this protein and a protein which binds to FP-III (presumably HNF-4) to -32sub with a mutation at nt -26 were grossly lowered. These findings suggest that the amelioration of hemophilia B-Leyden with a mutation at nt -20 after puberty involves binding of a specific non-androgen receptor nuclear protein at FP-III' and it is able to substitute for the function of a protein bound at FP-III in the normal gene optimally through its elevated interaction with androgen receptor upon a surge of testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Epidermal growth factor and transforming growth factor-alpha directly inhibit parietal cell function through a similar mechanism.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are naturally occurring peptides which are present throughout the gastrointestinal tract and are capable of inhibiting gastric acid secretion. Although previous studies have suggested that TGF-alpha may serve as an autocrine factor regulating parietal cell function, the cellular mechanisms by which it exerts its inhibitory action have not been fully elucidated. In addition, no systematic comparison has been undertaken of the effects of EGF and TGF-alpha on parietal cell function. The aims of the present studies were to compare the actions of EGF and TGF-alpha on basal and stimulated acid secretion by isolated rabbit parietal cells and to elucidate the intracellular mechanisms by which these growth factors inhibit acid secretion stimulated by agents that activate the adenylate cyclase and cyclic AMP second messenger system. Although EGF and TGF-alpha did not alter basal parietal cell function, they both inhibited histamine-stimulated [14C]aminopyrine accumulation in a identical time- and dose-dependent fashion. The maximal effect of approximately 40% inhibition for histamine-stimulated action was achieved with concentrations of 10(-6) M for both EGF and TGF-alpha. The inhibitory effect of EGF and TGF-alpha appeared to be at the postreceptor level as neither growth factor significantly altered binding of histamine to its receptor (H2) on parietal cells. Consistent with this postulated mechanism of action, both EGF and TGF-alpha dose-dependently inhibited forskolin-stimulated aminopyrine uptake with IC50 similar to those required for inhibiting the stimulatory effect of histamine. Of note, neither growth factor inhibited parietal cell activity stimulated by dibutyryl cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗
PubMed1988

Characterization of T lymphocyte responses during primary infection with human immunodeficiency virus.

The present study was undertaken to determine T cell response to primary human immunodeficiency virus (HIV) infection. No significant difference in T cell subsets was found between subjects who later seroconverted and a group of controls. Six subjects had multiple enumerations of T cell subsets done at the time of seroconversion. Initially, total lymphocyte and T cell subset counts were reduced. An inversion of the CD4+:CD8+ ratio due to a rise in the level of CD8+ cells was found later, followed by an appreciable increase in the number of CD8+ cells and further inversion of the CD4+:CD8+ ratio. Finally, the CD8+ cell count returned toward normal but remained higher than the CD4+ cell count; the inverted ratio was maintained. Lymphocyte hyporesponsiveness to mitogens and antigens was found during the seroconversion illness in one subject. In three of five subjects for whom data were available, an increase in the absolute number of CD8+ cells followed a decrease in the serum HIV antigen level.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Should nagarse be used during the isolation of brain mitochondria?

When nagarse is used to isolate brain mitochondria, a proportion of the nagarse stays associated with the mitochondrial fraction. This results in no detrimental affect on the respiratory activities. The nagarse is active in the presence of 2.3% sodium dodecyl sulfate and when samples are prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the nagarse degrades a substantial amount of the mitochondrial proteins.

Animals↗
PubMed1987

HPLC analysis of putative amino acid neurotransmitters released from primary cerebellar cultures.

An HPLC method is described that measures amino acids (putative neurotransmitters and/or neuromodulators) released from primary, dissociated cerebellar cells maintained in monolayer culture. Precolumn derivatization with phenylisothiocyanate, followed by reverse phase chromatography with UV detection was used to quantitate the phenylcarbamyl amino acid derivatives in a chemically defined medium. Quantitation was linear, reproducible and sensitive to one picomole. This method is useful for the measurement of putative neurotransmitters GABA, glutamate, aspartate, taurine and adenosine, and can easily be modified to analyze other amino acids in physiological samples.

Amino Acids↗
PubMed1987

9-(1-3-Dihydroxy-2-propoxymethyl)guanine prevents death but not immunity in murine cytomegalovirus-infected normal and immunosuppressed BALB/c mice.

Immunosuppressed (from treatment with cortisone acetate and anti-thymocyte globulin) and control adult female BALB/c mice, latently infected with murine cytomegalovirus (MCMV) or lethally challenged (10(6) PFU) with MCMV intraperitoneally, were treated with 9-(1-3-dihydroxy-2-propoxymethyl)guanine (DHPG) intraperitoneally. A dose of 3 mg/kg reduced mortality by 50% in lethally challenged normal mice; 10 mg/kg was required in immunosuppressed mice. When 15 mg/kg was given, the onset of treatment could be delayed for 64 h after lethal challenge. DHPG did not prevent the establishment of latent MCMV infection or immunosuppression-induced reactivation. The antibody titer to MCMV in DHPG-treated mice which survived lethal challenge was 41 (reciprocal of geometric mean) 4 to 5 weeks after inoculation; such mice survived a second lethal challenge. When antiserum treatment was begun 64 h and DHPG was begun 72 h after a lethal challenge, most mice survived; most did not when either treatment alone was begun at those times. In summary, DHPG effectively treated lethal MCMV infection even in immunosuppressed mice and even when treatment onset was delayed for 64 h. Treatment did not alter the establishment or reactivation of latent infections or the induction of effective immunity. The administration of DHPG coupled with antiserum treatment may be even more effective than the administration of either alone.

Acyclovir↗
PubMed1987

Effects of oxytocin and vasopressin on the preadipocyte 3T3-F442A cell line.

The 3T3-F442A mouse fibroblast cell line, triggered by factors present in fetal calf serum (FCS), converts either spontaneously or, in the simultaneous presence of FCS and insulin, at an accelerated rate into cells exhibiting the adipocyte phenotype. The effects of the neurohypophysial hormones in differentiated cells on glucose metabolism (glucose oxidation and lipogenesis) were compared with the stimulatory actions of insulin, which had its most pronounced effects in cells differentiated spontaneously with FCS in the absence of insulin. The differentiated 3T3-F442A cells were sensitive to physiological levels of insulin and exhibited manyfold increases in glucose metabolism in response to it. This result demonstrated that these cultured cells respond to insulin, in a manner analogous to freshly isolated adipocytes. In contrast to its insulin-like effects in isolated epididymal adipocytes, oxytocin was not reproducibly able to stimulate glucose metabolism in differentiated 3T3-F442A cells. Vasopressin was similarly inactive. In contrast, both oxytocin and vasopressin blocked adipocyte conversion triggered by FCS, either in the presence or absence of insulin; vasopressin was more potent than oxytocin, indicating that a vasopressin receptor was responsible for the observed inhibition of differentiation. Our work suggests that vasopressin could potentially play a role in the regulation of the adipocyte differentiation process.

1-Methyl-3-isobutylxanthine↗
PubMed1987

The management of late radiation-induced rectal injury after treatment of carcinoma of the uterus.

Sixty-one of 1,418 (4.3 per cent) patients treated with radiation for carcinoma of the uterus from 1963 to 1983 had significant radiation-induced complications of the intestine develop which required a surgical opinion considering further management. Ninety-three per cent of these complications involved the rectum. Florid proctitis resolved within two years of onset in 33 per cent of the patients who were managed conservatively while 22 per cent of the patients died of disseminated disease within the same time period. Surgical treatment was eventually necessary in 39 per cent of the patients who were initially treated conservatively for radiation induced proctitis. Rectal excision with coloanal sleeve anastomosis produced a satisfactory result in eight of 11 patients with severe radiation injury involving the rectum. The incidence of radiation-induced and malignant rectovaginal fistula were similar (1 per cent), but disease-induced symptoms tended to occur earlier after primary treatment (a median of eight months) compared with radiation-induced symptoms (a median of 16 months).

Female↗
PubMed1986

Changes in the phenotype of T-cell subset determinants following murine cytomegalovirus infection.

The murine model provides a particularly apt experimental system in which to evaluate the effects of cytomegalovirus (CMV) infection. CMV exerts a profound "suppression" of the immune response in the mouse and in humans; the infected animal is no longer able to mediate an appropriate response to mitogens or alloantigens. Using fluorocytometry and fluoresceinated monoclonal antibodies directed against the Thy-1.2, Lyt-1, and Lyt-2 cell membrane determinants following a nonlethal intraperitoneal inoculation of weanling BALB/c mice with Smith strain murine cytomegalovirus, significant changes in T-cell subsets were found that are consistent with findings described in the clinical situation. These ratio changes are temporally consistent with the cytotoxic T-lymphocyte population described by others. Finally, novel changes in the antigenic determinant distribution is found which may reflect the appearance of an antigen-committed cytotoxic T-lymphocyte population. This population which peaks at the ninth postinfection day may consist of 20-47% of the T-lymphocyte population and may offer an explanation for the cellular hyporesponsiveness seen following CMV infection.

Animals↗
PubMed1986

The protective effects of hyperimmune anti-murine cytomegalovirus antiserum against lethal viral challenge: the case for passive-active immunization.

The administration of 0.2 ml of hyperimmune anti-mouse cytomegalovirus (CMV) antiserum intraperitoneally (ip) or intravenously provided complete protection against lethal challenge (10(5.8) PFU ip) with murine CMV. Antiserum protection was complete when the antiserum was administered as long as 24 hr after viral challenge. The administration of antiserum had little effect on the titers of virus in the organs of these animals. Ammonium sulfate-treated antiserum provided similar complete protection. Animals rechallenged with 10(6)-10(6.5) PFU of murine CMV 1 month after initial challenge, at a time when the administrated antiserum was no longer detectable, all survived. We conclude that hyperimmune antiserum can provide significant protection against otherwise lethal murine CMV infection, that the protecting material lies within the immunoglobulin fraction, and that long-term immunity results from the combined exposure to virus and antiserum. Such passive-active protection could be useful in protecting against human CMV infection.

Animals↗
PubMed1986

Has the incidence of radiation-induced bowel damage following treatment of uterine carcinoma changed in the last 20 years?

Radiation-induced bowel damage occurred in 4.3% of patients treated primarily by irradiation for uterine carcinoma during the period 1962-1982. There has been a progressive rise in the incidence of radiation damage and radiation-induced rectovaginal fistula during this 20-year period. Radiation from intracavitary sources was a contributory factor in 92% of injured cases. The rising incidence of bowel damage in our patients may be due to an increase in the number of patients receiving a high rectal dose from the intracavitary source. There was a significantly (P less than 0.01) higher incidence of radiation injury in cases of cervical carcinoma compared to endometrial carcinoma. This was because cervical carcinoma tended to present at a more advanced stage than endometrial carcinoma and was more frequently treated with combined external and intracavitary irradiation. There was no significant increase in the incidence of complications among patients undergoing hysterectomy.

Brachytherapy↗
PubMed1986

Myopericarditis and enhanced dystrophic cardiac calcification in murine cytomegalovirus infection.

In mice inoculated with murine cytomegalovirus (MCMV) an acute myopericarditis developed which varied from a focal lymphohistiocytic inflammation to intense inflammation with necrosis and cytomegalic inclusion-bearing cells. Sublethal doses caused focal transient nonspecific chronic inflammation, followed months later by an increased frequency and extent of dystrophic cardiac calcification. When such latently infected hearts were heterotopically transplanted into uninfected animals which were then immunosuppressed (IS), a fatal generalized CMV infection followed. Cytomegalic inclusion-bearing endothelial, fibroblastic, and myocardial cells were seen in the intense inflammation found in hearts taken from mice 4 days after lethal inoculation and transplanted into uninfected mice, which were then IS. These findings may be relevant to human cardiac transplantation because they show that MCMV regularly causes cardiac infection with both acute and chronic consequences; chronic injury may follow a morphologically nonspecific myopericarditis which might not be attributed to CMV infection.

Animals↗