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B Webb

Publications and source records attributed to B Webb.

At least 37 records · Page 2Linked to original sources
PubMed1997

Ethanol effects on cultured embryonic hippocampal neuronal calcium homeostasis are altered by nerve growth factor.

The neurotoxic effect of acute ethanol treatment (AET) may lead to an alteration in the regulation of calcium (Ca2+) homeostasis in hippocampal neurons. Ca2+ homeostasis could be affected by AET when neurons are at rest or after depolarizing activity during synaptic transmission. It has been shown that nerve growth factor (NGF) can ameloriate some types of neurotoxicity by stabilizing Ca2+ homeostasis. Previously, we observed that ethanol (EtOH) changed unstimulated (basal) and potassium (K+)-stimulated intracellular calcium ([Ca2+]i) in embryonic septohippocampal neurons (Webb et al., Brain Res. 729:176-189, 1996). The purpose of the present study is to determine the effects of NGF and EtOH on neuronal Ca2+ homeostasis in cultured embryonic hippocampal neurons. The hypotheses tested were the following: EtOH alters Ca2+ homeostasis in hippocampal neurons; NGF modulates Ca2+ homeostasis in hippocampal neurons; and NGF treatment alters the effect of EtOH on [Ca2+]i in hippocampal neurons. Our results indicated that hippocampal neuronal cultures treated with EtOH had lower basal [Ca2+]i than untreated neurons. EtOH decreased K+-stimulated (30 mM KCI) changes in [Ca2+]i in a dose-dependent manner. During K+ stimulation, 20 ng/ml of NGF slowed and reduced the increase in [Ca2+]i. Hippocampal neurons treated with NGF increased or did not change basal [Ca2+]i and did not change or increase K+-stimulated [Ca2+]i in response to EtOH. These responses were dose-related and indicated that NGF could alter the response of hippocampal neurons to EtOH. In conclusion, AET results in the alteration of Ca2+ homeostasis in unstimulated and depolarized cultured embryonic hippocampal neurons. NGF and EtOH independently and collectively affected the regulation of Ca2+ homeostasis in this neuronal population. Changes in [Ca2+]i can disrupt normal cellular function and contribute to cell death. Therefore, alteration of Ca2+ homeostasis may be an underlying mechanism involved in EtOH toxicity. NGF may ameliorate the toxic effects of EtOH by regulating Ca2+ homeostasis.

Animals↗
PubMed1996

Calcium homeostasis in cultured embryonic rat septohippocampal neurons is altered by ethanol and nerve growth factor before and during depolarization.

Ethanol and nerve growth factor (NGF) affect the survival of septohippocampal (SH) neurons. The effect of ethanol and NGF on calcium (Ca2+) homeostasis in these neurons was investigated in this study. Changes in intracellular-free Ca2+ concentration ([Ca2+]i) were measured using indo-1 in cultured embryonic (E21) SH neurons before stimulation (basal) and during stimulation with 30 mM potassium cloride (KCl+). SH neurons were treated with 0, 100, 200, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (-NGF). NGF treatment decreased, while ethanol did not affect basal [Ca2+]i. The combination of ethanol and NGF treatment led to increases in basal [Ca2+]i. While [Ca2+]i was lower during stimulation with KCl+ following ethanol or NGF treatment, ethanol and NGF treatment together led to significantly greater increases or decreases in [Ca2+]i compared to similarly treated NGF neurons. Responses of SH neurons were compared to those of medial septal (MS) neurons. Changes in [Ca2+]i during treatment with ethanol and/or NGF were reduced in SH neurons compared with MS neurons. We conclude that changes in Ca2+ homeostasis can occur in SH neurons in the presence of ethanol and/or NGF. The changes following ethanol treatment are enhanced by NGF. By altering Ca2+ homeostasis, NGF may enhance the survival of SH neurons during ethanol-induced neurotoxicity.

Animals↗
PubMed1996

Contribution of vagal afferents to the volume-timing response to expiratory loads in neonatal lambs.

Expiratory loads elicit volume-dependent reflex increases in expiratory duration (TE) in adult animals. This reflex is mediated by pulmonary vagal afferents. It was hypothesized that this same reflex in newborn lambs is mediated by vagal afferents. Single-breath graded expiratory loads were presented to 4 day old, anesthetized neonatal lambs through a facemask (FM), via tracheostomy (TR) and after the cervical vagi were severed bilaterally in the TR animals (VAG). In FM and TR studies, expired volume (VE) decreased and TE increased with increasing load magnitude. There was no significant difference in the VE-TE curves for FM and TR. These results demonstrate that pulmonary vagal afferents are mediating the volume-related reflex modulation of TE during expiratory loading in the anesthetized neonatal lamb which is unaffected by bypass (TR) of upper airway afferents.

Animals↗
PubMed1996

Cultured postnatal rat medial septal neurons respond to acute ethanol treatment and nerve growth factor by changing intracellular calcium levels.

Ethanol neurotoxicity results in the loss of neurons during the development of the nervous system. Nerve growth factor (NGF) can ameliorate the neurotoxic effects of ethanol (EtOH) in rat medial septal (MS) neurons. These experiments study the effects of EtOH and NGF on neuronal calcium (Ca2+) homeostasis in cultured postnatal day of birth (PO) rat MS neurons. Previously, we observed that EtOH and NGF modulate intracellular Ca2+ levels [Ca2+]i) in unstimulated and high potassium stimulated (30 mM KCl) cultured rat embryonic day 21 (E21) MS neurons (Webb et al., Brain Res 701:61-74, 1995). The purpose of the present study was to explore whether the effects of EtOH and NGF on Ca2+ homeostasis were altered by developmental stage. The hypotheses tested were the following: treatment with EtOH affects Ca2+ homeostasis in postnatal day of birth (PO) rat MS neurons by causing transient and persistent changes in [Ca2+]i; NGF modulates Ca2+ homeostasis in MS neurons by regulating [Ca2+]i; the action of NGF changes the response of MS neurons to EtOH, thus altering Ca2+ homeostasis; and that EtOH and/or NGF effects on Ca2+ homeostasis are developmentally regulated. Our results indicated that behaviorally relevant levels of EtOH caused a rapid transient increase in basal [Ca2+]i, whereas there was no effect of NGF on basal [Ca2+]i. Ethanol and NGF interacted, resulting in the lowering of [Ca2+]i. During stimulation with high K+, EtOH inhibited the change in [Ca2+]i. NGF partially ameliorated this effect of higher levels of EtOH, allowing [Ca2+]i to increase. NGF and the lowest level of EtOH potentiated the high K+ stimulated increase in [Ca2+]i. Ethanol and NGF effects on [Ca2+]i were different in the PO neurons compared with our previously published observations in E21 neurons. Therefore, these data suggest that EtOH neurotoxicity and NGF protection involve mechanisms that regulate neuronal Ca2+ homeostasis, and the magnitude of these effects depend on developmental stage.

Animals↗
PubMed1995

Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization.

Ethanol and nerve growth factor (NGF) affect the survival of cholinergic neurons in the rat medial septum. To investigate whether calcium (Ca2+) homeostasis in these neurons is affected by ethanol or NGF treatment, changes in intracellular free Ca2+ concentration ([Ca2+]i) were studied in embryonic (E21) cultured medial septal neurons before stimulation (basal) and during stimulation with high potassium (K+). Changes in [Ca2+]i across time were measured in cultures of neurons treated without ethanol or with 100, 200, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (-NGF). Changes in [Ca2+]i were analyzed from fluorescence images, using indo-1. The effect of ethanol or NGF treatment was to reduce the rise in basal [Ca2+]i. The combination of ethanol and NGF treatment in +NGF neurons led to increases in basal [Ca2+]i with the greatest increase in basal [Ca2+]i occurring with 200 mg% ethanol. The effect of ethanol or NGF was to increase [Ca2+]i during stimulation with high K+. The greatest increases in [Ca2+]i occurred with 100 and 800 mg% ethanol. Together, ethanol and NGF treatment in +NGF-treated neurons led to significantly greater increases or decreases in K+ stimulated changes in [Ca2+]i compared to similarly treated -NGF neurons. We conclude that in medial septal neurons (before and during depolarization) changes in Ca2+ homeostasis occur in the presence of ethanol or NGF. The changes in [Ca2+]i following ethanol treatment are greater when NGF is present.

Animals↗
PubMed1995

An improved method for the quantification and recovery of rat uterine nuclear type II [3H]estradiol binding sites immobilized on a glass fiber matrix.

An improved assay for measuring ligand binding to extracted nuclear type II estrogen binding sites which involves preimmobilization on glass fiber filters is described. At least two classes of specific estrogen binding sites have been demonstrated in rat uterus as well as in a variety of other tissues and species and have been designated as type I and type II. Although the endogenous ligand to the type II binding site has recently been identified as methyl p-hydroxyphenyllactate (MeHPLA), tritiated estrogens are generally used for radiolabeling this site due to the susceptibility of MeHPLA to enzymatic hydrolysis in in vitro assays. After extracting the type II site from the nuclear matrix, ligand binding and protein stability appear to be significantly enhanced by first immobilizing the site on an artificial matrix, such as hydroxylapatite, before incubating with radiolabeled ligand. Immobilization of the extracted site on glass fiber filters results in higher specific binding and lower nonspecific binding when compared to hydroxylapatite and a number of other immobilization matrices. The glass fiber ligand exchange procedure for measuring type II binding can also be performed on smaller samples and requires less time than other methods. Type II sites are significantly stabilized when immobilized on glass and exhibit sigmoidal binding curves when incubated with increasing concentrations of [3H]estradiol and [3H]estrone and display inhibition data characteristic of that observed using more traditional assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Metal ion binding to apo, holo, and reconstituted horse spleen ferritin.

The binding of Cd2+, Zn2+, Cu2+, Ni2+, Co2+, Mn2+, and Mg2+ to apo, holo, reconstituted horse spleen ferritin (HoSF), and native holo HoSF with phosphate removed was measured by gel-exclusion chromatography. Three classes of strong binding interactions (Kd < 10(-7) M) with apo HoSF at pH 7.5 were found for the various M2+ studied: high stoichiometric binding (30-54 M2+/HoSF) for Cd2+, Zn2+, Cu2+, with two protons released per metal bound; intermediate binding (16 M2+/HoSF) for Ni2+ and Co2+, with one proton released per metal bound; and low levels of binding (2-12 M2+/HoSF) for Mn2+, Mg2+, and Fe2+, with < 0.5 protons released per metal bound. M2+ binding to apo HoSF was nearly abolished at pH 5.5, except for Fe2+ and Cu2+, which remained unaffected by pH alteration. Holo HoSF bound much higher levels of M2+, a result directly attributable to the presence of phosphate binding sites. This conclusion was confirmed by decreased binding of M2+ to HoSF reconstituted in the absence of phosphate and by native holo HoSF with phosphate chemically removed. The binding of Cd2+ to apo HoSF was 54 per HoSF, but in the presence of developing core, the amount bound decreased to about 30 Cd2+/HoSF. This result indicated that Cd2+ and developing core were competing for the same sites on the HoSF interior, suggesting that 24 of the Cd2+ were bound to the inside surface. No other M2+ studied bound to the interior of HoSF by this criterion. Several of the M2+ appeared to bind strongly to the phosphate-free mineral core surface in reconstituted HoSF.

Animals↗
PubMed1995

A method for labeling embryonic rat medial septal region projection neurons, in vitro, using fluorescent tracers.

A retrograde labeling method is described in which rat embryonic (E18, E21) and postnatal (P1) medial septal neurons were labeled with succinyl wheat germ agglutin-fluorescein, fluorescent green microspheres, or 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil) following in vitro hippocampal injections. The brains were removed and immediately immersed in oxygenated Tyrode solution. Dye was pressure injected into the hippocampus bilaterally. After incubating the brain in oxygenated Tyrode, the medial septal region was removed. The neurons were dissociated and cultured at medium density in 35 mm dishes with a hole in the bottom covered by a coverslip with a grid. The neurons were observed with a low light system, and cell counts were made at 5, 24, and 48 h. Labeled and unlabeled neurons showed considerable neurite outgrowth and acetylcholinesterase activity in culture. Highly reproducible labeling was obtained, with Dil giving the best results. Dil labeled the neurons in vitro, was retained during culture for 1 week, and was compatible with cell survival.

Acetylcholinesterase↗
PubMed1995

Effects of coumestrol on estrogen receptor function and uterine growth in ovariectomized rats.

Isoflavonoids and related compounds such as coumestrol have classically been categorized as phytoestrogens because these environmentally derived substances bind to the estrogen receptor (ER) and increase uterine wet weight in immature rats and mice. Assessment of the binding affinities of isoflavonoids for ER and subsequent effects on uterine growth suggest these compounds are less active estrogens than estradiol and therefore may reduce the risk of developing breast or prostate cancer in humans by preventing estradiol binding to ER. With the renewed interest in the relationships between environmental estrogens and cancer cause and prevention, we assessed the effects of the phytoestrogen coumestrol on uterotropic response in the immature, ovariectomized rat. Our studies demonstrated that in this animal model, coumestrol is an atypical estrogen that does not stimulate uterine cellular hyperplasia. Although acute (subcutaneous injection) or chronic (multiple injection or orally via drinking water) administration of coumestrol significantly increased uterine wet and dry weights, the phytoestrogen failed to increase uterine DNA content. The lack of true estrogenic activity was characterized by the inability of this phytoestrogen to cause cytosolic ER depletion, nuclear ER accumulation, or the stimulation of nuclear type II sites which characteristically precede estrogenic stimulation of cellular DNA synthesis and proliferation. In fact, subcutaneous or oral coumestrol treatment caused an atypical threefold induction of cytosolic ER without corresponding cytosolic depletion and nuclear accumulation of this receptor, and this increased the sensitivity of the uterus to subsequent stimulation by estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Iron core formation in horse spleen ferritin: magnetic susceptibility, pH, and compositional studies.

Horse spleen ferritin (HoSF) reconstituted with small iron cores ranging in size from 8 to 500 iron atoms was studied by magnetic susceptibility and pH measurements to determine when the added Fe3+ begins to aggregate and form antiferromagnetically coupled clusters and also to determine the hydrolytic state of the iron at low iron loading. The Evans NMR magnetic susceptibility measurements showed that at iron loadings as low as 8 Fe3+/HoSF, at least half of the added iron atoms were involved in antiferromagnetic exchange interactions and the other half were present as isolated iron atoms with S = 5/2. As the core size increased to about 24 iron atoms, the antiferromagnetic exchange interactions among the iron atoms increased until reaching the limiting value of 3.8 Bohr magnetons per iron atom, the value present in holo HoSF. HoSF containing eight or more Fe3+ to which eight Fe2+ were added showed that the Fe2+ ions were at sites remote from the Fe3+ and that the resulting HoSF consisted of individual, noninteracting Fe2+ and the partially aggregated Fe3+. pH measurements for core reduction showed that Fe(OH)3 was initially present at all iron loadings but that in the absence of iron chelators the reduced iron core is partially hydrolyzed. Proton induced x-ray emission spectroscopy showed that Cl- is transported into the iron core during reduction, forming a stable chlorohydroxy Fe(II) mineral phase.

Animals↗
PubMed1994

Molecular entrapment of small molecules within the interior of horse spleen ferritin.

A procedure for trapping small molecules inside the interior of horse spleen ferritin (HoSF) and methods for characterizing HoSF and its small entrapped molecules are described. HoSF is first dissociated into subunits by adjustment to pH 2 in the presence of the small molecules to be trapped. The pH of the dissociated HoSF is then increased to 7 at which time the dissociated subunits reassemble reforming the 24-mer HoSF, thereby trapping solvent within its interior. HoSF is then separated from unbound molecules by dialysis, ultrafiltration, and/or ammonium sulfate precipitation. Sephadex G-25 and DEAE chromatographic methods were also used to separate HoSF from unbound small molecules. Capillary electrophoresis (CE) was used to demonstrate the association of small molecules with HoSF after the pH-induced unfolding-refolding process. The pH indicator neutral red was clearly associated with HoSF and presumed trapped within the ferritin interior. Acid/base titrations suggested that the trapped indicator had a different pKa than the free indicator, a result which indicates that the ferritin interior is different than the external solution. The utility of using trapped molecules for gaining information on ferritin function is proposed and discussed.

Ammonium Sulfate↗
PubMed1994

Chromatographic resolution of the type II estrogen binding site and a tyrosinase-like enzymatic activity from rat uterine nuclei.

Nuclear extracts from estradiol-treated rat uteri which contain type II estrogen binding sites have recently been found to also contain a tyrosinase-like estradiol metabolizing activity. A recent study suggested that both the binding and enzymatic activities are significantly increased in the presence of micromolar concentrations of copper and ascorbate, display a number of common biochemical sensitivities, and share similar ligand/substrate binding affinities. Levels of both activities are significantly increased in uterus in response to hormone (estrogen) stimulation. These and other similarities indicate a possible relationship between the enzymatic and binding activities. A detailed chromatographic examination of these two activities in the present study revealed that while the type II sites and estradiol metabolizing activity exhibited virtually identical chromatographic properties on DEAE-high-performance liquid chromatography they are readily resolved on other chromatographic matrices, including phosphocellulose, DNA-cellulose, and S-Sepharose. These results demonstrate that type II binding sites are distinct from the tyrosinase-like enzyme activity previously described in rat uterine nuclear extracts.

Animals↗
PubMed1994

Vagally mediated volume-dependent modulation of inspiratory duration in the neonatal lamb.

Inspiratory mechanical loads elicit a reflex volume-timing response in human infants and experimental animals. In adult animals, this reflex has been shown to be mediated by vagal afferents. It was hypothesized that the volume-related regulation of inspiratory duration would also be vagally mediated in the newborn. Single-breath graded inspiratory loads were presented to 4-day-old anesthetized neonatal lambs breathing through a facemask, after tracheostomy, and after bilateral cervical vagotomy. Inspired volume decreased and inspiratory duration (TI) increased with increasing load magnitude. The increase in TI was greater with facemask loads than with loads presented after tracheostomy for equivalent changes in inspired volume. The volume-related prolongation of TI after tracheostomy was abolished after cervical bilateral vagotomy. The results demonstrate that the volume-timing reflex in neonatal lambs after tracheostomy is mediated by vagal afferents. However, in the intact lamb, there is also a significant contribution to this reflex by upper airway afferents.

Airway Resistance↗
PubMed1993

Leiomyosarcoma of the rectum and anal canal.

Leiomyosarcoma of the rectum and anal canal is rare and the optimal treatment is not clear. Eight patients with isolated anorectal leiomyosarcoma treated surgically were reviewed. The age ranged from 44 to 76 years (median 63 years) and the follow up ranged from 6 months to 4.5 years (median 2 years). All patients were symptomatic at presentation. All tumours involved the muscularis propria of the low and/or mid-rectum with three tumours also involving the anal sphincters. The tumour size ranged from 1.2 to 10 cm (median 4 cm). Mucosal involvement occurred in only three patients and there was no lymph node involvement. All showed microscopic infiltration at the advancing border, despite macroscopic circumscription. Only one patient was thought to have a tumour sufficiently small (3 cm) and localized on clinical and intrarectal ultrasound examinations (UST2N0) to be suitable for wide local excision. That patient remained tumour-free after 2 years. The remaining patients (88%) were treated by abdomino-perineal resection. The disease free interval in this latter group ranged from 3 months to 4.5 years. All recurrences were detected within 15 months of surgery and the mean interval from detection of recurrence to death was 9 months. Using a histological grading system that included tumour differentiation, mitotic count and amount of necrosis, high grade sarcomas were associated with a worse prognosis. Other factors associated with a poor outcome included large tumour size (> 6-7 cm) and prior incomplete local excision.

Adult↗
PubMed1991

Serum estradiol concentration required to maintain body weight, attractivity, proceptivity, and receptivity in the ovariectomized female rat.

Female hooded rats (230 to 260 g) were ovariectomized and given a subcutaneous implant of an estradiol-filled Silastic tube. The length of the tube was varied in order to produce a variety of serum estradiol levels. In the first experiment, animals were weighed over a 6-week period following surgery and then tested for sexual responsiveness to a male. The results demonstrated that ovariectomized females with an implant maintaining a serum estradiol concentration at about 15 pg/ml maintained body weight at the same level as that of intact females. A smaller implant gave rise to a higher weight gain and a larger implant to a lower weight gain. All implants resulted in a continuous state of receptivity. In a second experiment, ovariectomized females were implanted with smaller estradiol-filled implants in order to determine the threshold for maintaining proceptivity and receptivity. The results indicated that with a serum estradiol concentration below 15 pg/ml, the frequency of lordosis and of ear wiggling and darting decreased. Progesterone injections facilitated both proceptive and receptive behavior. In addition, following progesterone injections, the time required for a male to mount a female 10 times was decreased in females with low or no estradiol replacement. These results indicate that a constant concentration of estradiol at about the mean level present throughout the estrous cycle will result in normal body weight regulation and will maintain sexual behaviors that normally occur only during estrus. These results emphasize that Silastic implants of estradiol do not mimic normal endocrine function since, even at low levels, estradiol implants produce continuous receptivity.

Animals↗
PubMed1991

Inspiratory resistive load detection in conscious dogs.

The physiological mechanisms mediating the detection of mechanical loads are unknown. This is, in part, due to the lack of an animal model of load detection that could be used to investigate specific sensory systems. We used American Foxhounds with tracheal stomata to behaviorally condition the detection of inspiratory occlusion and graded resistive loads. The resistive loads were presented with a loading manifold connected to the inspiratory port of a non-rebreathing valve. The dogs signaled detection of the load by lifting their front paw off a lever. Inspiratory occlusion was used as the initial training stimulus, and the dogs could reliably respond within the first or second inspiratory effort to 100% of the occlusion presentations after 13 trials. Graded resistances that spanned the 50% detection threshold were then presented. The detection threshold resistances (delta R50) were 0.96 and 1.70 cmH2O.l-1.s. Ratios of delta R50 to background resistance were 0.15 and 0.30. The near-threshold resistive loads did not significantly change expired PCO2 or breathing patterns. These results demonstrate that dogs can be conditioned to reliably and specifically signal the detection of graded inspiratory mechanical loads. Inspiration through the tracheal stoma excludes afferents in the upper extrathoracic trachea, larynx, pharynx, nasal passages, and mouth from mediating load detection in these dogs. It is unknown which remaining afferents (vagal or respiratory muscle) are responsible for load detection.

Afferent Pathways↗