Biomedical subjects

R L Evans

Publications and source records attributed to R L Evans.

At least 37 records · Page 2Linked to original sources

PubMed1999

Radiation-induced progressive decrease in fluid secretion in rat submandibular glands is related to decreased acinar volume and not impaired calcium signaling.

The mechanism(s) of radiation-induced salivary gland dysfunction is poorly understood. In the present study, we have assessed the secretory function (muscarinic agonist-stimulated saliva flow, intracellular calcium mobilization, Na+/K+/2Cl- cotransport activity) in rat submandibular glands 12 months postirradiation (single dose, 10 Gy). The morphological status of glands from control and irradiated rats was also determined. Pilocarpine-stimulated salivary flow was decreased by 67% at 12 months (but not at 3 months) after irradiation. This was associated with a 47% decrease in the wet weight of the irradiated glands. Histological and morphometric analysis demonstrated that acinar cells were smaller and occupied relatively less volume and convoluted granular tubules were smaller but occupied the same relative volume, while intercalated and striated ducts maintained their size but occupied a greater relative volume in submandibular glands from irradiated compared to control animals. In addition, no inflammation or fibrosis was observed in the irradiated tissues. Carbachol- or thapsigargin-stimulated mobilization of Ca2+ was similar in dispersed submandibular gland cells from control and irradiated animals. Further, [Ca2+]i imaging of individual ducts and acini from control and irradiated groups showed, for the first time, that mobilization of Ca2+ in either cell type was not altered by the radiation treatment. The carbachol-stimulated, bumetanide-sensitive component of the Na+/K+/ 2Cl- cotransport activity was also similar in submandibular gland cells from control and irradiated animals. These data demonstrate that a single dose of gamma radiation induces a progressive loss of submandibular gland tissue and function. This loss of salivary flow is not due to chronic inflammation or fibrosis of the gland or an alteration in the neurotransmitter signaling mechanism in the acinar or ductal cells. The radiation-induced decrease in fluid secretion appears to be related to a change in either the water-handling capacity of the acini or the number of acinar cells in the gland.

Animals↗

PubMed1998

Evidence for a physiological role of NH4+ transport on the secretory Na(+)-K(+)-2Cl- cotransporter.

The secretory Na(+)-K(+)-2Cl- cotransporter in salivary acinar cells is responsible for driving the transepithelial Cl- fluxes that give rise to fluid secretion. We demonstrate that the application of the muscarinic agonist carbachol to rat parotid acini results in an intracellular acid load that can be blocked by bumetanide, a specific inhibitor of the cotransporter. One component of this bumetanide-sensitive acid load is ouabain-sensitive while a second is dependent on the presence of sub-millimolar concentrations of NH4+ in our media. Our data indicate that this latter effect arises from NH4+ entry on the cotransporter operating in a Na(+)-NH4(+)-2Cl- cotransport mode and that at physiological NH4+ levels in the rat (approximately 0.1 mM), 10-15% of the acinar Cl- entry occurs via this route. We suggest that Na(+)-NH4(+)-2Cl- cotransport may also play a significant physiological role in other cell types and that this mode of operation of the secretory Na(+)-K(+)-2Cl- cotransporter could account for the currently unexplained presence of this protein in a number of tissues.

Ammonia↗

PubMed1998

p53 Mutations in hairless SKH-hr1 mouse skin tumors induced by a solar simulator.

In this study, we investigated whether the spectrum of p53 mutations in skin tumors induced in hairless SKH-hr1 mice by a solar simulator (290-400 nm) are similar to those found in skin tumors induced in C3H mice by UV radiation from unfiltered (250-400 nm) and Kodacel-filtered (290-400 nm) FS40 sunlamps. Analysis of tumor DNA for p53 mutations revealed that 14 of 16 (87.5%) SkH-hr1 skin tumors induced by the solar simulator contained mutations. Single C-->T transitions at dipyrimidine sequences located on the nontranscribed DNA strand were the most predominant type of p53 mutation. Remarkably, 52% of all p53 mutations in solar simulator-induced SKH-hr1 skin tumors occurred at codon 270, which is also a hotspot in C3H skin tumors induced by unfiltered and Kodacel-filtered FS40 sunlamps. However, T-->G transversions, which are hallmarks of UVA-induced mutations, were not detected in any of the solar simulator-induced skin tumors analyzed. These results demonstrate that the p53 mutation spectra seen in solar simulator-induced SKH-hr1 skin tumors are similar to those present in -unfiltered and Kodacel-filtered FS40 sunlamp-induced C3H skin tumors. In addition, our data indicate that the UVA present in solar simulator radiation does not play a role in the induction of p53 mutations that contribute to skin cancer development.

Animals↗

PubMed1998

Hospital-based rehabilitative care versus outpatient services: effect on functioning and health status.

PURPOSE: The goal of this clinical trial was to examine the long-term impact of rehabilitative care on the health status of patients diagnosed with a disabling disorder. METHOD: Study patients consisted of first-time hospitalizations from diagnostic groups commonly admitted for inpatient rehabilitation, including nervous, circulatory, and musculoskeletal disorders or injury. Patients were randomly assigned to inpatient rehabilitation (n = 43) or to outpatient follow-up (n = 42) in which the usual medical services were provided but no scheduled rehabilitative therapies were offered. Specific objectives of the study were to determine the effects of impatient rehabilitation on: (1) functional ability, (2) health and mental health status, (3) personal adjustment, and (4) family function. Cost and use of health-care resources were descriptively assessed. RESULTS: Analysis of covariance found no significant treatment effects, either at 6 months or at 1 year, for any of the variables under study. In addition, there were no differences between groups in their use of nursing homes, length of hospital stay, mortality, or in the number of hospital readmissions or clinic visits during the first year after hospital discharge. Use of rehabilitation services and cost of care was significantly higher than outpatient services. The findings were consistent with previous studies for most outcomes, with the major exception being functional improvements. Contrary to earlier studies, rehabilitation was not found to effectively produce lasting functional outcomes. However, study conditions may not have fully corresponded to those of previous studies, and further research is needed. The patient sample was representative of a full inpatient service and therefore more heterogeneous than samples reported in prior studies, but the small sample size (due to reductions in the number of admitted patients to the rehabilitation unit during the course of the study) precluded subgroup analysis of diagnostic groupings. CONCLUSION: The findings suggest that hospital-based rehabilitative care does not have lasting benefits, and that alternative care or supportive follow-up by a subacute-care facility may be needed to assist patients in maintaining functional gains and health benefits.

Adult↗

PubMed1998

New insights into the upregulation and function of the salivary Na+-K+-2Cl- cotransporter.

In many exocrine epithelia, the Na+-K+-2Cl- cotransporter is the main provider of cellular chloride entry during transepithelial salt and water secretion. Because of its accessibility and hormonal responsiveness, the salivary gland has recently emerged as a convenient preparation in which to study the regulation and characteristics of this transport protein. In this review, we summarize recent findings from our laboratory which demonstrate that muscarinic, alpha1-adrenergic and peptidergic stimulation of rat parotid acinar cells induce a dramatic (up to twenty-fold) upregulation of Na+-K+-2Cl- cotransporter activity. Our results indicate that this effect is dependent on the rise in intracellular calcium concentration ([Ca2+]i) that accompanies stimulation, and is not a consequence of the KCl loss and the concomitant cell shrinkage associated with fluid secretion. In addition, we show that the effect of muscarinic stimulation on the cotransporter can be blocked by inhibitors of phospholipase A2, by a general inhibitor of arachidonic acid metabolism, and by specific inhibitors of the cytochrome P450 pathway. These data argue strongly for the involvement of a product of the cytochrome P450 pathway of arachidonic acid metabolism in upregulation of the salivary Na+-K+-2Cl- cotransporter.

Animals↗

PubMed1998

In vitro studies of the regulation of mucous gland secretion.

Our laboratory is concerned with elucidation of mechanisms regulating the exocrine secretion of mucin glycoproteins by mucous cells of salivary mucous glands. As a model system for our studies we use short-term cultures of acinar structures isolated from rat sublingual glands. Recent results are discussed from an on-going study of cross-talk between the two primary signaling pathways regulating exocrine secretion from isolated sublingual acini: the muscarinic cholinergic and vasoactive intestinal peptide (VIP) pathways. The combination of muscarinic agonist (carbachol) and VIP elicits a secretion equivalent to about 150% of the additive sum of the secretory responses to each agonist alone. This synergistic secretory response is only observed at submaximal concentrations of carbachol. VIP thus serves to decrease the EC50 for carbachol nearly three-fold. Results are thus far consistent with the hypothesis that a sustained rise in the intracellular concentration of calcium ions induced by VIP accounts for synergistic secretion and the heightened sensitivity to carbachol.

Animals↗

PubMed1997

Upregulation of Na(+)-K(+)-2Cl- cotransporter activity in rat parotid acinar cells by muscarinic stimulation.

1. The effects of fluid secretory stimuli on the Na(+)-K(+)-2Cl- cotransporter in rat parotid acini were investigated. Cotransporter activity was measured using NH4+ as a K+ surrogate and following cotransporter-mediated NH4+ fluxes by monitoring intracellular pH. 2. A dramatic upregulation (15- to 20-fold) of acinar Na(+)-K(+)-2Cl- cotransporter activity was induced by muscarinic, alpha 1-adrenergic and peptidergic stimuli. A half-maximal effect of the muscarinic agonist carbachol was observed at approximately 0.5 microM. 3. Our results indicate that the rise in intracellular calcium concentration ([Ca2+]i) which accompanies these stimuli is both a necessary and a sufficient condition for this effect; but it is not a consequence of the KCl loss and concomitant isotonic shrinkage caused by increased [Ca2+]i as it persists when these effects are prevented. 4. The effect of muscarinic stimulation on the cotransporter can, however, be blocked by inhibitors of phospholipase A2 (4-bromophenacylbromide and manoalide), by a general inhibitor of arachidonic acid metabolism (5,8,11,14-eicosatetraynoic acid) and by specific inhibitors of the cytochrome P450 pathway (methoxsalen and ketoconazole). 5. These latter results argue strongly for the involvement of a product of the cytochrome P450 pathway of arachidonic acid metabolism in upregulation of the salivary Na(+)-K(+)-2Cl- cotransporter. 6. Owing to the complexity of the arachidonic acid cascade a wide variety of agents could potentially interfere with this upregulation of the cotransporter, and thereby result in decreased salivary fluid production. We suggest that such an effect could underlie the dry mouth (xerostomia) that occurs as an unexplained side-effect of many commonly prescribed medications.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1997

Evidence that up-regulation of the parotid Na(+)-K(+)-2Cl- cotransporter by hypertonic shrinkage is not duplicated by isotonic shrinkage.

The application of Ca2+ mobilizing secretagogues to rat parotid acini results in a significant decrease in cell volume (15-30%) due to isotonic salt loss. It is often assumed that the effects of such an isotonic volume decrease can be mimicked by anisotonic cell shrinkage. We demonstrate that the Na(+)-K(+)-2Cl- cotransporter in these cells is up-regulated by Ca2+ mobilizing secretagogues as well as by cell shrinkage in hypertonic media. However, we find that although the protein kinase inhibitors staurosporine (0.3 microM) and K252a (0.6 microM) significantly blunt the latter up-regulation, they are without effect on the former. These observations suggest that hypertonic and isotonic shrinkage do not result in the activation of the same intracellular signalling pathways, and indicate that anisotonic volume perturbations may not provide good experimental models of physiologic isotonic volume changes.

Ammonium Chloride↗

PubMed1997

Sunlight and skin cancer: inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens.

UV-induced mutations in the p53 tumor suppressor gene play an essential role in skin cancer development. We report here that such mutations can be detected in UV-irradiated mouse skin months before the gross appearance of skin tumors. Application of SPF-15 sunscreens to mouse skin before each UV irradiation nearly abolished the frequency of p53 mutations. These results indicate that p53 mutation is an early event in UV skin carcinogenesis and that inhibition of this event may serve as an early end point for assessing protective measures against skin cancer development.

Animals↗

PubMed1997

Molecular analysis of the human vitamin D binding protein (group specific component, Gc) in tuberous sclerosis complex (TSC).

Group specific component (Gc) is an abundant plasma protein whose functional role is not clearly established. Gc protein is synthesised in the liver and is known to bind vitamin D, vitamin D metabolites, and G actin; Gc protein is also implicated in macrophage activation. Several polymorphic electrophoretic variants of Gc protein are found in all human populations; the most common alleles are Gc-1f, Gc-1s, and Gc-2. In previous studies, Gc allele frequencies, determined using isoelectric focusing or immunofixation or both, were significantly different in patients with tuberous sclerosis complex (TSC) from matched controls, with an excess of Gc-2 in patients. Linkage association between Gc and TSC is unlikely since the Gc locus maps to chromosome 4q12, whereas the two common forms of TSC map to 9q34 and 16p13.1, respectively. However, a direct cause and effect relationship between Gc protein and TSC symptoms is possible. To investigate further the relationship between the Gc locus and TSC, two Gc restriction site polymorphisms, HaeIII and StyI, were typed in 43 unrelated white subjects with TSC. The frequencies of the restriction site polymorphisms in the TSC patients did not differ from those in control populations. Therefore a direct association between Gc type and TSC is unlikely. The previously reported association was either spurious or the result of typing errors in plasma from subjects with underlying abnormalities in plasma proteins.

Base Sequence↗

PubMed1997

Evaluating rehabilitation medicine: effects on survival, function, and home care.

The goal of this study was to measure the clinical impact of rehabilitation on adults diagnosed with a disabling disorder in four major diagnostic groups (nervous, circulatory, musculoskeletal, and injury). To summarize the current knowledge in this area, a meta-analysis of rehabilitation studies was also completed. Specific objectives of the clinical trial were to determine the effects of inpatient rehabilitation on: (1) survival, (2) function, (3) home care, and related variables such as family function and use of health care resources. Patients hospitalized for the first time with a disabling condition (n = 85) were randomly assigned to inpatient rehabilitation (n = 43) or to outpatient follow-up (n = 42) in which the usual medical services were provided but no scheduled rehabilitative therapies were offered. To compare the two groups, analyses of covariance were conducted for functional ability, health care use, survival, health status, personal adjustment and family function. The between subjects factor was inpatient rehabilitation versus the control group. The within subjects factor was time of assessment (index, six months, and 1 year). No significant treatment effect was found at six months or one year for any of the variables under study using analyses of covariance. There were also no differences between groups in their use of nursing homes, length of hospital stay, survival, or in the number of hospital readmissions or clinic visits during the first year after hospital discharge. Rehabilitation did cost significantly more than medical care, primarily due to the cost of inpatient services. Some clinical trials have noted a treatment effect on functional ability but not on mortality, need for skilled care, or mental health status. The current study is consistent with these previous findings except for the lack of impact on physical function. This exception may be due to the fact that prior studies looked only at homogeneous groups, whereas the current study utilized heterogeneous grouping across four major diagnostic categories. Any apparent benefit may not be detectable across disability groups and may require more specialized scrutiny, or even tailored rehabilitative care, to detect a difference. It is recommended that health care systems evaluate the benefits of subacute rehabilitative care and consider outpatient programs that can be provided at home for implementation.

Activities of Daily Living↗

PubMed1996

Interactions between secretin and acetylcholine in the regulation of fluid secretion by isolated rat pancreatic ducts.

1. Interlobular ducts were isolated from the rat pancreas and maintained in short-term tissue culture. Fluid secretion from these isolated ducts was measured using micropuncture techniques, intracellular calcium concentration ([Ca2+]i) by fura-2 microspectrofluorimetry, and cyclic AMP by radioimmunoassay. 2. Applying secretin and ACh simultaneously to ducts caused either a stimulation or an inhibition of fluid secretion depending on the doses employed. 3. The inhibitory effect of secretin and ACh could be relieved by atropine, and by the protein kinase C (PKC) inhibitors staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7). 4. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12, 13-dibutyrate (PDBu) inhibited secretin-evoked fluid secretion. 5. ACh and TPA also inhibited fluid secretion stimulated by the adenylate cyclase activator, forskolin. 6. Neither secretin nor the PKC activators and inhibitors had any effect on either the increase in [Ca2+]i evoked by ACh or the increase in intracellular cyclic AMP evoked by secretin and forskolin. 7. We conclude that the inhibitory effect of combined doses of secretin and ACh on ductal fluid secretion is probably mediated by PKC at a point in the secretory mechanism distal to the generation of intracellular messengers.

Acetylcholine↗

PubMed1996

Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells.

Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.

Animals↗

PubMed1996

Elevation of intracellular cAMP by noradrenaline and vasoactive intestinal peptide in striated ducts isolated from the rabbit mandibular salivary gland.

Salivary gland intralobular ducts are responsible for the modification of the electrolyte composition of the primary fluid secreted by the acini. However, the intracellular messengers that regulate this and other intralobular duct cell processes have not been fully characterized. To investigate the possibility that cAMP-mobilizing agonists may be involved, intralobular (striated) ducts were isolated from the rabbit mandibular salivary gland by tissue dissociation and microdissection and maintained in tissue culture overnight. Individual duct fragments were stimulated with the secretory agonists noradrenaline, vasoactive intestinal peptide (VIP) and substance P and their cAMP content measured by acetylated radioimmunoassay. Both noradrenaline and VIP elevated intracellular cAMP content concentration dependently, but substance P did not. The response to noradrenaline was blocked by the beta-adrenoceptor antagonist propranolol, but not by the alpha-adrenoceptor antagonist prazosin. Application of the VIP analogue [D-p-Cl-Phe6, Leu17]-VIP decreased the VIP-induced cAMP response. These results demonstrate that striated intralobular duct cells possess beta-adrenoceptors and peptidergic receptors that are coupled to adenylate cyclase and activated by noradrenaline and VIP, respectively. By elevating ductal cAMP content, these agonists may regulate both the electrolyte content of the primary saliva and the secretion of protein(s) from the ducts.

Adenylyl Cyclases↗

PubMed1996

Ion transport systems in human labial acinar cells.

Human labial acini were assayed for the presence of ion transport systems associated with salivary fluid secretion using microfluorometric methods. Na(+)-K(+)-Cl- contransport and Na+/H+ exchange activities (determined by their bumetanide and amiloride sensitivities, respectively) were found at levels approximately 50% of those seen in similarly assayed rat parotid acini, but little, if any, C1-/HCO3-exchange activity was observed. Also, when human labial acini were stimulated with the muscarinic agonist carbachol, little evidence of the intracellular acidification associated with HCO3- secretion by other salivary glands was found. Na+/H+ exchange activity in human labial acini was downregulated (approximately 40%) by beta-adrenergic stimulation and upregulated (approximately threefold) by muscarinic stimulation. In contrast, beta-adrenergic stimulation produced only a marginally significant increase in Na(+)-K(+)-Cl- cotransport activity, and muscarinic stimulation was without effect. We include that basolateral Na(+)-K(+)-Cl- cotransport appears to be the dominant mechanism driving Cl- secretion and thereby fluid secretion in this tissue.

Adrenergic beta-Agonists↗

PubMed1996

Risk screening for adverse outcomes in subacute care.

Purpose of this study was to identify variables near hospital admission that could identify patients at risk for placement, death, or readmission. The goals were to assess sensitivity and specificity of screening strategies in predicting adverse outcomes that deter or affect home health care. We evaluated whether generic screens might efficiently identify type of outcome. Criteria reported in the literature were used to predict outcomes for 1,332 admissions. Factors that discriminated type of outcome included comorbidity, mental status, living arrangement, transfer to special care, prior admission, iatrogenic trauma, and pending litigation. We conclude risk scores can accurately predict outcome of hospital treatment, which may be useful in targeting patients for intervention. Using billing data, although rather insensitive, was the most cost-effective strategy.

Confidence Intervals↗

PubMed1996

Meta-analysis with longitudinal studies: controlling for analytical bias.

A recent study published in an international medical journal presented an opportunity to demonstrate the difficulties of interpreting meta-analysis results with longitudinal data. We conducted a new meta-analysis using identical data from the published study and showed contradictory results.

Bias↗

PubMed1995

Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus.

Whereas baculovirus expression systems have been extensively used for high-level expression of steroid receptors and receptors coupled to adenylate cyclase, there are few studies on peptide receptors coupled to phospholipase C (PLC). In the present study we have expressed the murine gastrin-releasing peptide receptor (mGRP-R) in Sf9 cells using a recombinant baculovirus and characterized it structurally and functionally. mGRP-R was detectible 12 h post infection with recombinant baculovirus carrying mGRP-R cDNA and became maximal at 60 h post infection (Bmax = 6 pmol/mg protein), which is a 4-60-fold greater density than is found in native tissues. The mGRP-R in Sf9 cells assessed by affinity labeling or immunoblotting was smaller than that in native tissues (M(r) = 51 kD vs 82 kD), and the difference was due to the extent of glycosylation. In Sf9 cells the mGRP-R had at least two of the four potential extracellular glycosylation sites glycosylated, whereas in the native receptor all four were approximately equally glycosylated. In Sf9 cells the glycosylation was entirely biantennary complex, in contrast to the native mGRP-R, where it was entirely tri- and tetraantennary complex N-linked oligosaccharides. Affinity labeling studies revealed a band with an apparent molecular mass about 40 kDa higher than the 51-kDa mGRP-R band. The intensity of this band correlated with the extent of functional G protein coupling, suggesting that it may represent an mGRP-R-G protein complex. In binding studies the affinity of the mGRP-r in Sf9 cells for the agonists bombesin (Bn), GRP, and neuromedin B (NMB) varied differently with infection time: with Bn the affinity decreased 3-fold with longer infection times, with GRP it remained unchanged, and with NMB it decreased 10-fold. GPP(NH)p inhibited binding of either [125I]Tyr4Bn or [125I]GRP at 24 h post infection, but not at 96 h post infection. Agonists activated PLC, increasing both [3H]IP and [Ca2+]i; however, the efficacy of each agonist decreased with infection time. These results demonstrate that by the use of recombinant baculovirus infected Sf9 cells the PLC-linked receptor mGRP-R can be expressed in amounts significantly greater than those in native tissues. The mGRP-R expressed in these Sf9 cells is incompletely glycosylated and has less complex N-linked oligosaccharide chains, yet it is fully coupled to G proteins and activates phospholipase C, similar to the native receptor, if short infection times are used.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗