Apoptosis in regenerating and denervated, nonregenerating urodele forelimbs.

Denervated limbs of larval salamanders fail to regenerate if amputated and, unlike adult limbs, undergo regression. The cellular basis of the tissue loss is poorly understood. We used TUNEL staining of larval axolotl limbs fixed and sectioned at intervals after bilateral amputation and unilateral denervation to investigate the role of apoptosis during normal limb regeneration and denervated limb regression. In the first week after amputation a small percentage of apoptotic cells was found in both innervated and denervated limbs. During the second week the apoptotic index remained low in the mitotically active mesenchymal cells of the regenerating limbs, but increased twofold in the nondividing, dedifferentiated cells of the regressing limbs. TUNEL-positive cells resembling apoptotic bodies were restricted primarily to the dedifferentiated area beneath the wound epithelium, but were also present within the wound epithelium itself. Macrophages were identified immunohistochemically and were also found in increased numbers in distal areas of the denervated regressing limbs. The results suggest a role for apoptosis in the early phase of normal regeneration and indicate that denervated limb regression involves an increased rate of apoptosis and removal of apoptotic bodies by macrophages and the wound epithelium.

Pharmacological and immunohistochemical characterization of calmodulin-stimulated (Ca(2+)+Mg(2+))-ATPase in cultured porcine aortic endothelial cells.

Plasma membrane (Ca(2+)+Mg(2+))-ATPase and Ca(2+) transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca(2+) signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca(2+)+Mg(2+))-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca(2+)- and/or Mg(2+)-activated ecto-5'-nucleotide phosphohydrolases. Moreover, addition of exogenous calmodulin (10(-9) to 10(-6) mol/L) produced no measurable stimulation of ATPase activities, suggesting a permanently activated state or, alternatively, a complete lack thereof. To establish and verify the presence of a calmodulin-regulated (Ca(2+)+Mg(2+))-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti-(Ca(2+)+Mg(2+))-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca(2+) pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca(2+)+Mg(2+))-ATPase, 10(-5) mol/L calmidazolium (R24571) was added to the isolated plasma membrane preparation, which lowered the (Ca(2+)+Mg(2+))-ATPase activity from 143.0 to 78.15 nmol P(i)/mg protein x min(-1). This calmidazolium-reduced activity could then be stimulated 113.1+/-0.8% in a concentration-dependent manner by the addition of exogenous calmodulin (10(-7) to 2 x 10(-6) mol/L) with an EC(50) of 3.45+/-0.04 x 10(-7) mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by approximately 100 compared with other unopposed calmodulin-stimulated processes. Together, these findings support evidence for the presence of a calmodulin-stimulated plasma membrane (Ca(2+)+Mg(2+))-ATPase activity in cultured porcine aortic endothelial cells.

The use of high-performance size exclusion chromatography (HPSEC) as a molecular weight screening technique for polygalacturonic acid for use in pharmaceutical applications.

Polygalacturonic acid is a linear carbohydrate polymer of monomeric galacturonic acid. It is commercially available as apple and citrus pectins comprised of a mixture of partially methoxylated and/or amidated polygalacturonic acids with molecular weights ranging from 25,000 to > 100,000 Da. Pectin can be chemically or enzymatically hydrolyzed to yield polygalacturonic acid fractions of diverse average molecular weight ranges and polydispersities for a variety of uses. Pectin and polygalacturonic acid are used extensively as gelling agents and stabilizers by the food industry, and have applications as therapeutic, and diagnostic pharmaceutical agents such as the magnetic resonance imaging agent LumenHance. A simple high-performance size exclusion chromatography (HPSEC) method, employing commonly available non-specialized HPLC instrumentation, is described for use as a rapid molecular weight screening technique to determine the average molecular weight range and polydispersity of polygalacturonic acid intended for use in pharmaceutical formulations. A TosoHaas G3000PWXL HPLC column, 50 mM phosphate buffer (pH approximately 6.9) mobile phase, and refractive index detection were used. A molecular weight calibration curve was linear for polysaccharide standards of 180-100,000 Da with a coefficient of correlation of 0.999. The method was employed to screen commercially available polygalacturonic acid raw materials for average molecular weight data (Mn, Mw, and Mp) and polydispersity (Mw/Mn).

Effects of omeprazole paste on healing of spontaneous gastric ulcers in horses and foals: a field trial.

A multicentre, blinded, randomised complete-block, field trial was conducted with 140 horses and foals age 4 weeks-28 years to determine if omeprazole paste is effective and safe in promoting healing of spontaneous gastric ulcers under a variety of field conditions and in different breeds and ages of horses. Horses in the study had gastric ulceration as determined by gastroscopy and were divided into replicates of 4 or 5 animals. One horse in each replicate was assigned randomly to receive an empty omeprazole syringe (sham-dosed control) and the remaining horses received omeprazole paste once daily for 28 days. Gastroscopy was repeated at the end of the study. Horses treated with omeprazole had significantly (P < 0.01) more improvement in ulcer scores at the end of the study compared with controls. Ulcers were improved in 32.4 and 99.0% of the control and omeprazole groups, respectively. Ulcers were completely healed in 8.9 and 86.7% of the control and omeprazole groups, respectively. Under typical field conditions, omeprazole was effective at enhancing healing of spontaneous gastric ulcers in horses of a variety of ages and breeds.

Glucocorticoid-induced apoptosis of dendritic cells in the rat tracheal mucosa.

Dendritic cells are antigen-presenting cells that constitutively express high levels of major histocompatibility complex class II (Ia) antigen on their plasma membrane. Previous studies have shown that the number of dendritic cells in the rat airway mucosa decreases rapidly after glucocorticoid treatment. We sought to determine whether apoptosis contributes to this steroid-induced cell decrease. Dendritic cells in tracheal whole mounts were revealed by immunoperoxidase staining using the OX-6 (anti-Ia) monoclonal antibody. In untreated rats, a dense network of Ia-immunoreactive (Ia+) cells with highly branched cytoplasmic processes was observed just beneath the tracheal epithelium (1,405 +/- 140 cells/mm2 mucosa; mean +/- SEM, n = 6). In rats treated with dexamethasone (10 mg/kg, intraperitoneally), four distinct changes in dendritic cell morphology were evident 4 to 8 h after injection: (1) appearance of large Ia+ granules in cytoplasmic processes, (2) narrowing of cytoplasmic processes, (3) loss of Ia immunoreactivity from the cell surface, and (4) fragmentation of cells into small Ia+ bodies. These changes accompanied a 56% decrease in the number of Ia+ cells over 8 h. The contribution of apoptosis to this decrease in Ia+ cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) of nucleosomal DNA fragments in histologic sections. The number of TUNEL+ bodies increased from a control value of 174 +/- 47 bodies/mm2 mucosa to 2,108 +/- 294 bodies/mm2 mucosa at 4 h and 936 +/- 343 bodies/ mm2 mucosa at 8 h (n = 4 rats per time point). The location of TUNEL+ bodies closely corresponded to that of Ia+ cells stained in adjacent histologic sections. We conclude that apoptosis contributes to the rapid decrease in airway dendritic cells after glucocorticoid treatment.

Spontaneous infectious discitis in adults.

PURPOSE: In adults, discitis most frequently follows spinal surgery. We report 16 adult patients with spontaneously occurring infectious discitis and compare them with an additional 52 patients abstracted from the literature. Infecting organisms, predisposing factors, imaging modalities, and response to therapy are described. PATIENTS AND METHODS: The medical records of adult patients treated for infectious discitis of a community hospital during the past 10 years were reviewed. Postoperative spine patients and patients with primary osteomyelitis were excluded. Sixteen patients were identified with spontaneous primary infection of the disc space. The particulars of comorbid conditions, infection organisms, site of culture, and response to antibiotic therapy were noted and compared to 52 additional cases of spontaneous discitis reported in the literature since 1980. RESULTS: A wide variety of infecting organisms was identified as causing spontaneous discitis, in contrast to previous reports of both postoperative discitis and spontaneous discitis. Nine of 10 patients with positive disc cultures had negative blood cultures. Appropriate antibiotics were curative in all patients but 1, regardless of the duration of symptoms. Nuclear imaging, computed tomography, and magnetic resonance imaging were all useful, although the last appears to be the most sensitive and specific imaging modality for detecting discitis. CONCLUSIONS: Spontaneous infectious discitis is an uncommon cause of low back pain in adults. Nevertheless, it should be considered in any patient with acute or subacute pain. Elevated acute-phase reactants with appropriate imaging modality suggest the diagnosis. given the wide variety of infecting organisms identified, culture of blood and/or disc for the specific causative organism is critical to successful treatment outcome.

Effect of dexamethasone treatment on the regional distribution of neutral endopeptidase activity in the rat trachea.

Treating rats with the glucocorticoid dexamethasone has been shown to reduce the amount of plasma extravasation produced in the trachea by tachykinins released from sensory nerves. We sought to determine whether dexamethasone works by increasing the activity of neutral endopeptidase (NEP), the principal enzyme thought to be responsible for degrading tachykinins in the airways. Rats were treated for 2 days with either saline or 4 mg/kg/day of dexamethasone, a dose we found to be maximally effective in reducing tachykinin-induced plasma extravasation. The tracheas were then removed and processed to reveal NEP-specific histofluorescence. Tissue sections were photographed through a fluorescence microscope, and the relative intensity of fluorescent staining was quantified in five regions of the tracheal wall using computerized image analysis. In the saline-treated rats, the rank order of fluorescent staining was perichondrium > chondrocytes = submucosal >> epithelium > lamina propria. Neither the amount nor distribution of NEP fluorescence was altered in the dexamethasone-treated rats. Biochemical measurements of NEP activity in tracheal homogenates (nmol product/h/mg protein) likewise revealed no significant difference between the two groups (34.1 +/- 3.5 vs 29.0 +/- 3.2; mean +/- SEM, n = 8). These findings suggest that dexamethasone may be working through a mechanism unrelated to NEP activation.

Characterization of ruthenium red as an inhibitor of neurogenic inflammation in the rat trachea.

1. We investigated the ability of ruthenium red, an inorganic dye with capsaicin antagonist properties, to inhibit capsaicin-induced plasma extravasation in the rat trachea. 2. The amount of plasma extravasation produced by intravenous capsaicin was reduced dose-dependently by i.v. ruthenium red. Complete inhibition was achieved with a dose of 5 mumol/kg. 3. The inhibitory effect of ruthenium red persisted for at least 16 hr after its administration, but was not present 24 hr later. 4. Ruthenium red did not reduce the amount of plasma extravasation produced by electrical stimulation of the vagus nerve, nor the amount produced by intravenous substance P or platelet-activating factor. 5. Prior exposure to a high dose of capsaicin reduced the amount of plasma extravasation produced by a second capsaicin exposure 48 hr later. However, giving ruthenium red 30 min before the initial capsaicin exposure largely prevented this loss of sensory nerve function. 6. We conclude that systemic administration of ruthenium red produces long-lasting, selective, and reversible inhibition of capsaicin-induced plasma extravasation in the rat trachea. Moreover, ruthenium red attenuates the long-term, desensitizing effect of capsaicin on sensory nerves.

Home access modifications: effects on community visits by people with physical disabilities.

We examined the effects of access modifications to home entrances of people with physical disabilities on their reported community outings. An interrupted time-series design was used, in which the introduction of ramps was staggered across the homes of 6 people with moderate to severe mobility impairments. Four participants reported increases in weekly outings following installation of ramps at their entrances, and 2 reported a small decrease. These findings suggest that reducing the response requirements of access to and from the residence of people with mobility impairments may increase community visits, but may be insufficient given other environmental barriers in the community.

Differential effects of phosphoramidon and captopril on NK1 receptor-mediated plasma extravasation in the rat trachea.

We sought to confirm the identity of the tachykinin receptor subtype that mediates plasma extravasation in the rat trachea, and assess the respective contributions of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) in regulating this tachykinin-induced response. To achieve these aims, we determined the relative potencies of several natural tachykinins and receptor-selective synthetic agonists, both before and after inhibiting NEP with phosphoramidon and ACE with captopril. We also determined the effects of these peptidase inhibitors, and the NK-1 receptor antagonist L-703,606, on the plasma extravasation produced by capsaicin, which releases tachykinins endogenously from sensory nerve endings. We found that the rank order of potency for producing plasma extravasation in the rat trachea was NK-1 receptor agonist ([Sar9, Met(O2)11] SP) > substance P > neurokinin A > neurokinin B. The NK-2 ([Nle10]NKA (4-10)) and NK-3 ([MePhe7]NKB) receptor agonists were without effect. We observed no change in the relative potencies of these peptides after giving rats phosphoramidon or captopril, which suggests that the different peptide potencies are not simply the consequence of different rates of enzymatic degradation. Nevertheless, the responses to substance P and neurokinin A were clearly potentiated in rats given phosphoramidon, indicating that NEP effectively degrades tachykinins in vivo. No significant potentiation was evident for any peptide in rats given captopril. Similarly, the plasma extravasation produced by capsaicin was potentiated in rats given phosphoramidon, but not in those given captopril. Pretreating rats with L-703,606 abolished the response to capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS)

A contextual-behavioral model of empowerment: case studies involving people with physical disabilities.

When people with disabilities, ethnic minorities, older adults, women, and others lack power, they usually experience adverse conditions disproportionate to other members of society. Empowerment--the process by which people gain some control over valued events, outcomes, and resources--is an important construct for understanding and improving the lives of people of marginal status. This manuscript presents a contextual-behavioral model of empowerment and its application in collaborative research with people with physical disabilities. The eight case studies illustrate 18 tactics for promoting empowerment that flow from the model. The case studies show the use of different combinations of empowerment tactics in a variety of contexts: (a) setting improvement agendas from the perspective of people with disabilities, (b) enforcing ordinances that preserve access to parking spaces designated for people with disabilities, (c) enabling access to homes through housing modifications, (d) enhancing support available through mutual-aid groups, (e) developing skills for recruiting mentors, (f) promoting self-directed behavior change with personal and health concerns, (g) enhancing skills for personal self-advocacy, and (h) building the capacities of groups of people with disabilities for systems advocacy. Finally, we discuss issues that may contribute to research and action related to empowerment.

Influence of repeated treatment with buspirone on central 5-hydroxytryptamine and dopamine synthesis.

The anxiolytic agent buspirone was administered subcutaneously twice a day for 10 days to Sprague-Dawley rats, at a dose of 3 mg kg-1. Controls were given saline. On the eleventh day, the rats were given an injection of NSD-1015, an aromatic L-amino acid decarboxylase inhibitor, 30 min before decapitation. To another group of rats, only one injection of buspirone was given, followed 30 min later by NSD-1015. After a further 30 min the animals were decapitated. The brains were rapidly removed and the raphe nuclei, striatum, hippocampus and cerebellum were dissected out on to dry ice. With the use of HPLC, the four regions of the brain were assayed for 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, reflecting the synthesis of 5-HT and dopamine, respectively. In those rats which had received an acute dose of buspirone, the synthesis of 5-HT was substantially reduced in all four regions of the brain. However, in those rats which had received buspirone for 10 days, no such alterations in the synthesis of 5-HT were observed. The synthesis of dopamine was unchanged in any of the regions of the brain, after the acute dose of buspirone. After 10 days of treatment with buspirone, however, the synthesis of dopamine in the striatum was significantly reduced. These findings suggest that repeated treatment with buspirone reduces the synthesis of dopamine in the striatum but that the synthesis of 5-HT is unaffected.

Mechanism of platelet activating factor-induced vascular leakage in the rat trachea.

Platelet activating factor (PAF) is a phospholipid mediator of inflammation and vascular leakage that may be important in the etiology of asthma. We and others have demonstrated that PAF causes vascular leakage in the rat trachea. In the present study, we attempted to determine how PAF mediates this effect. Vascular leakage was quantitated by measuring the amount of intravascular Evans blue dye extravasated into tracheal tissue. Intravenously administered PAF increased vascular leakage, although Lyso-PAF and Enantio-PAF had no effect. PAF-induced vascular leakage was inhibited in a dose-dependent fashion by the PAF receptor blocker WEB 2086. However, PAF-induced vascular leakage was not inhibited by blockade of cyclooxygenase/lipoxygenase, calmodulin, calcium channels, protein kinase C, histamine receptors, or by destruction of peptidergic sensory nerves. We conclude that PAF causes vascular leakage in the rat trachea by a stereospecific receptor-mediated mechanism that does not depend on arachidonic acid metabolites, calcium, protein kinase C, histamine, or peptidergic sensory nerves.

Calcitonin gene-related peptide potentiates substance P-induced plasma extravasation in the rat trachea.

Antidromic stimulation of vagal sensory nerves is known to produce plasma extravasation in the rat trachea. This neurogenic inflammation is thought to be mediated by substance P or other tachykinins released from sensory nerve endings. We sought to determine whether calcitonin gene-related peptide (CGRP), which is also released from sensory nerve endings, can potentiate substance P-induced plasma extravasation in the rat trachea. To accomplish this, we measured the amounts of Evans blue dye extravasated into the trachea after intravenous injections of substance P alone and combined with CGRP. We found that when substance P and CGRP were injected together, the amount of plasma extravasation produced in the trachea was substantially greater than the amount produced when substance P was injected alone. This potentiation was critically dependent on the dosage of CGRP and was not observed when relatively high dosages were used. We also found that CGRP had a potent hypotensive effect and speculate that reduced blood pressure may account for the lack of potentiation observed at the higher CGRP dosages. Based on these findings, we conclude that CGRP can potentiate substance P-induced plasma extravasation in the rat trachea and may therefore play a role in modulating neurogenic inflammation of the airways.

Effects of platelet-activating factor on vascular permeability and granulocyte recruitment in the rat trachea.

Platelet-activating factor (PAF) is a phospholipid mediator known to produce several features of airway inflammation. We examined the effects of intravenous PAF on vascular permeability and granulocyte recruitment in the rat trachea. To assess vascular permeability, anesthetized rats were given injections of Evans blue dye (30 mg/kg, iv) and PAF (1-10 micrograms/kg, iv), and then their tracheas were removed and assayed spectrophotometrically for dye content. We found that a PAF dosage of 6 micrograms/kg increased the tracheal dye content 7-fold compared to controls. The amount of extravasated dye in the tracheas was significantly increased 1 min after PAF injection, was maximal at 5 min, and had returned to control levels by 10 min. To assess granulocyte recruitment, anesthetized rats were given an injection of PAF (6 micrograms/kg, iv), and then their tracheas were removed and stained to reveal myeloperoxidase-containing neutrophils and eosinophils. We found that the number of neutrophils in the tracheal mucosa was increased 7-fold from controls 5 min after PAF injection, but was not significantly increased 6 h later. The number of eosinophils in the tracheal mucosa was not significantly increased from controls at any time after PAF injection. We conclude that intravenous PAF causes a rapid but transient increase in vascular permeability in the rat trachea, and that intravenous PAF also causes a rapid but transient recruitment of neutrophils into the tracheal mucosa without a similar effect on eosinophils.

Mechanism of tachyphylaxis associated with neurogenic plasma extravasation in the rat trachea.

Electrical stimulation of the vagus nerve of rats is known to produce plasma extravasation in the trachea, presumably by releasing substance P or other tachykinins from sensory nerves. We sought to determine whether the tachyphylaxis that develops after prolonged vagal stimulation results from an inability of sensory nerves to release tachykinins or from an inability of tracheal blood vessels to respond to tachykinins. To induce tachyphylaxis, we electrically stimulated the right vagus nerve of Long-Evans rats for 5 min (5 V, 1 ms, 20 Hz). Then, 10 min later, we gave intravenous injections of capsaicin (0.3 mumol/kg), histamine (18 mumols/kg), or substance P (2.2 nmol/kg), which produce equivalent amounts of plasma extravasation as assessed by the extravasation of Evans blue dye. We found that vagal stimulation reduced the amount of dye extravasation produced by capsaicin but not the amount produced by either histamine or substance P. We also found that pretreating neonatal rats with capsaicin, which destroys tachykinin-containing sensory nerves, reduced the amount of dye extravasation produced by capsaicin but not the amount produced by either histamine or substance P. This finding suggests that capsaicin produces plasma extravasation in the trachea by releasing tachykinins from sensory nerves, whereas histamine and substance P do so by acting directly on tracheal blood vessels. Taken together, our results indicate that prolonged vagal stimulation reduces the ability of sensory nerves to release tachykinins but that tracheal blood vessels remain fully responsive to both histamine and substance P.

Alcohol as a nutrient: interactions between ethanol and carbohydrate.

This study examined metabolic interactions between two nutrients--ethanol and carbohydrate. Both nutrients are metabolized by a common pathway to fatty acids from acetyl-coenzyme A by lipogenic enzymes. The effects of ethanol and carbohydrate on the induction of lipogenic enzymes in livers of rats were examined using two types of base diets differing in carbohydrate and lipid content and using isocaloric substitutions of ethanol, carbohydrate, and fat. Three nonlipogenic enzymes were used for comparison. Isocaloric substitution of both fat and carbohydrate for ethanol was necessary to show the specific effects of alcohol on the activity of lipogenic or nonlipogenic enzymes. Carbohydrate, and not ethanol, induced lipogenic enzymes. Ethanol specifically reduced the activity of lactate dehydrogenase and malic enzyme, but did not affect those of alcohol dehydrogenase or glycerol 3-phosphate dehydrogenase. Ethanol interacted with carbohydrate to increase the activity of ATP citrate lyase. In addition, we studied the effects of ethanol and different kinds of carbohydrates on the growth of rats and on the morphology of their livers and intestines. Ethanol significantly decreased growth characteristics (weight gain, growth rate, and caloric efficiency). Fructose, either as a monosaccharide or in sucrose, decreased this alcohol effect. Sucrose was better than glucose in lowering lipid accumulation in livers of rats. Fragility of intestinal villi was found with an alcohol, low carbohydrate diet, but was not present in alcohol diets with a higher level of carbohydrate. In contrast to carbohydrate, ethanol lacked some characteristics of a nutrient, namely, it did not induce some enzymes involved in its metabolism and did not promote optimum growth.

Reducing risk of pressure sores: effects of watch prompts and alarm avoidance on wheelchair push-ups.

People who use wheelchairs are at risk for developing pressure sores. Regular pressure relief, in the form of a wheelchair push-up, is one way to reduce the likelihood of pressure sores. We examined the effects of antecedent (i.e., instructions, audible prompts) and consequent (i.e., alarm avoidance) events on wheelchair push-ups, using a multiple baseline analysis with 2 participants with spina bifida. Results suggest that the combined procedure was more effective than either antecedent or consequent events alone, and there is some evidence suggesting maintenance of effects over time.

Labelling monoclonal antibodies with yttrium-90.

Using the cyclic DTPA derivatisation procedure developed by Hnatowich, conditions have been optimised for labelling three tumour-associated monoclonal antibodies with 90Y. High labelling efficiencies (greater than 95%) at modest specific activities (1 mCi/mg) could be routinely obtained. Radiochemical stability of the radiolabelled preparations in vitro was good, but radiolysis resulted in early losses of antibody immunoreactivity.