Biomedical subjects

C W Stevens

Publications and source records attributed to C W Stevens.

At least 19 recordsLinked to original sources

PubMed2000

Selective opioid receptor agonist and antagonist displacement of [3H]naloxone binding in amphibian brain.

Opioid receptor ligands have been shown to elicit antinociception in mammals through three distinct types of receptors designated as mu, delta and kappa. These opioid receptors have been characterized and cloned in several mammalian species. Radioligand binding techniques were employed to characterize the sites of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid receptor antagonist which has not been characterized in R. pipiens. Kinetic analyses of [3H]naloxone in the amphibian yielded a K(D) of 6.84 nM while the experimentally derived K(D) value from saturation experiments was found to be 7.11 nM. Density data were also determined from saturation analyses which yielded a B(max) of 2170 fmol/mg. Additionally, K(i) values were calculated in competition studies for various unlabelled mu-, delta- and kappa-opioid receptor ligands to isolate their site of action. Highly selective antagonists for mu-, delta- and kappa-opioid receptors yielded nearly identical K(i) values against [3H]naloxone.

Animals↗

PubMed2000

Novel approaches to locally advanced unresectable non-small cell lung cancer.

The management of advanced non-small cell lung cancer (NSCLC) is rapidly evolving. Advances in combined chemo-radiation therapy have led to improvements in patient survival which are statistically significant, but most patients still succumb to their disease. New chemotherapeutic agents, such as taxanes (paclitaxel, docetaxel), topoisomerase inhibitors (topotecan, irinotecan), and novel analogs (gemcitabine, vinorelbine), may offer the promise of improved outcome, but have not yet been tested in phase III trials. Molecular therapeutics, such as gene therapy, drugs that target specific oncogene activation (such as Ki-ras inactivation by farnesyl transferase inhibitors), and hypoxic cell toxins (such as tirapazamine), are in clinical trials. The optimum use of these agents awaits more rapid and widespread molecular diagnostics. Finally, technological advances in radiotherapy will allow higher tumor doses, while minimizing doses to dose-limiting normal structures, such as the esophagus, normal lung and heart. We describe a move towards molecular strategies, both for therapy and diagnostics, that may result in more effective treatment. While the outcome for patients with advanced non-small cell lung carcinoma is still poor, new agents are being developed rapidly and offer the hope of improved survival.

Antineoplastic Agents↗

PubMed1999

Spinal administration of selective opioid antagonists in amphibians: evidence for an opioid unireceptor.

In mammals, opioids act by interactions with three distinct types of receptors: mu, delta, or kappa opioid receptors. Using a novel assay of antinociception in the Northern grass frog, Rana pipiens, previous work demonstrated that selective mu, delta, or kappa opioids produced a potent antinociception when administered by the spinal route. The relative potency of this effect was highly correlated to that found in mammals. Present studies employing selective opioid antagonists, beta-FNA, NTI, or nor-BNI demonstrated that, in general, these antagonists were not selective in the amphibian model. These data have implications for the functional evolution of opioid receptors in vertebrates and suggest that the tested mu, delta, and kappa opioids mediate antinociception via a single type of opioid receptor in amphibians, termed the unireceptor.

Acetic Acid↗

PubMed1999

Characterization of [3H]-diprenorphine binding in Rana pipiens: observations of filter binding enhanced by naltrexone.

Initial studies were undertaken to examine the properties of [3H]-diprenorphine binding to Rana pipiens whole brain tissue using naltrexone for the definition of nonspecific binding. Saturation analysis demonstrated the binding of [3H]-diprenorphine to be saturable with a K(D) value of 0.65 nM and a Bmax value of 287.7 fmol/mg protein. Unlabeled diprenorphine dose-dependently displaced [3H]-diprenorphine from a single noninteractive site in competition studies which yielded a Ki of 0.22 nM. However, control studies in the absence of tissue revealed significant binding of [3H]-diprenorphine to the filter alone. Interestingly, [3H]-diprenorphine in the presence of unlabeled naltrexone as well as with unlabeled naloxone showed significantly greater binding to the filter than did [3H]-diprenorphine alone. Given this observation of increased nonspecific binding, an artificially low Bmax value would be expected. It is our hypothesis that the unlabeled nonspecific drug forms a complex with [3H]-diprenorphine preventing it from being effectively washed through the filter or the unlabeled drug itself is blocking the flow of [3H]-diprenorphine through the filter. The latter is unlikely however as other binding studies done in our lab using the radioligand [3H]-naloxone with unlabeled naltrexone do not show significant binding to the filter.

Animals↗

PubMed1999

Radiation-induced recombination is dependent on Ku80.

We have recently shown that irradiating cells prior to transfection induces recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors. We hypothesized that Ku proteins, which have previously been shown to be involved in both recombination and the repair of DNA damage after irradiation, would likely be important mediators of radiation-induced recombination. The present work demonstrates that Ku80 is essential for radiation-induced recombination. While human and hamster Ku80 are equally effective at restoring the transfection efficiency and radiation resistance of xrs-5 cells, human Ku80 is much more effective at radiation-induced recombination than hamster Ku80. This difference is not due to differences in Ku80 expression or DNA end-binding activity, but it may be due to structural differences between human and hamster Ku80.

Animals↗

PubMed1998

DNA damaging agents improve stable gene transfer efficiency in mammalian cells.

Gene therapy is an evolving discipline which today relies primarily on viral systems for gene transfer. The primary reason that plasmid vectors have not been widely used for gene therapy trials is their relatively low rate of stable gene transfer. We show here that both ionizing irradiation and hydrogen peroxide can each increase the gene transfer efficiency of plasmids. Hydrogen peroxide improves gene transfer in a linear dose-dependent manner. At equitoxic doses, hydrogen peroxide improves gene transfer by 20-fold over untreated cells and approximately 5 times above that seen for radiation, and this improvement correlates with both the total amount of DNA damage induced and the amount of residual damage after 4 hr of repair. These data suggest that DNA damaging agents may be useful to improve human gene therapy.

Animals↗

PubMed1997

Supraspinal administration of opioids with selectivity for mu-, delta- and kappa-opioid receptors produces analgesia in amphibians.

Previous results using an amphibian model showed that systemic and spinal administration of opioids selective for mu, delta and kappa-opioid receptors produce analgesia. It is not known whether non-mammalian vertebrates also contain supraspinal sites mediating opioid analgesia. Thus, opioid agonists selective for mu (morphine; fentanyl), delta (DADLE, [D-Ala2, D-Leu5]-enkephalin; DPDPE, [D-Pen2, D-Pen5]-enkephalin) and kappa (U50488, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulfonate; CI977, (5R)-(544alpha,744alpha,845beta)-N-methyl-N-[7-(1-p yrr olidinyl)-1-oxaspiro[4,5]dec-8yl]-4-benzofuranaceta mide++ + monohydrochloride) opioid receptors were tested for analgesia following i.c.v. administration in the Northern grass frog, Rana pipiens. Morphine, administered at 0.3, 1, 3 and 10 nmol/frog, produced a dose-dependent and long-lasting analgesic effect. Concurrent naltrexone (10 nmol) significantly blocked analgesia produced by i.c.v. morphine (10 nmol). ED50 values for the six opioids ranged from 2.0 for morphine to 63.9 nmol for U50488. The rank order of analgesic potency was morphine > DADLE > DPDPE > CI977 > fentanyl > U50488. These results show that supraspinal sites mediate opioid analgesia in amphibians and suggest that mechanisms of supraspinal opioid analgesia may be common to all vertebrates.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1997

High-efficiency stable gene transfer of adenovirus into mammalian cells using ionizing radiation.

We report a novel method for targeting adenovirus-mediated gene delivery. By irradiating mammalian cells prior to adenoviral transduction, adenoviral gene transfer is greatly improved and the adenoviral genome integrates into cellular DNA. In this work, human and rodent cell lines were irradiated and subsequently transduced with the adenovirus vector Ad5CMVlacZ. Initial levels of transduction were as much as 40-fold higher in irradiated cells, and this improvement in transduction was radiation dose dependent. The duration of lacZ expression in irradiated cells was also much longer than in nonirradiated cells and reached a plateau after 21 days. At doses of 7 Gy, long-term (< 50 day) expression of lacZ could be detected in 15% of cells by flow cytometry. This long-lasting expression of lacZ was due to viral DNA integration into the host genome. Thus, pretreatment of cells with ionizing radiation improves both immediate transduction efficiency and duration of transgene expression. This may lead to the development of new protocols combining radiation and gene therapy in treating human malignancy.

3T3 Cells↗

PubMed1997

Spinal mu, delta and kappa opioids alter chemical, mechanical and thermal sensitivities in amphibians.

Previously we demonstrated the use of chemical (topical acetic acid), thermal (radiant heat) and mechanical (von Frey filament) stimuli as quantifiable behavioral response assays in the northern grass frog, Rana pipiens. Furthermore, response thresholds in all of these sensory modalities are significantly elevated by systemic morphine injections, which can be antagonized by naltrexone. The present study employed these three sensory assays to assess changes in chemical, mechanical and thermal sensitivities following spinal administration of mu, delta and kappa opioids. Significant elevations were observed across all three sensory modalities in each subtype category and these effects were abolished by prior systemic administration of naltrexone. However, naltrexone antagonism of morphine produced hyperalgesia in both the mechanical and thermal modalities. The results support other recent work demonstrating that the spinal site for opioid analgesia is present in amphibians and that the thermal, mechanical and acetic acid assays are measures of true nociceptive activity in the amphibian.

Analgesics, Opioid↗

PubMed1997

The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide.

Instability of hydrogen peroxide solutions was noted during the experimental exposure of human cells in culture to hydrogen peroxide in experiments designed to study the production and repair of DNA single-strand breaks. A hydrogen peroxide concentrate was diluted into culture medium, which was then added to experimental cell cultures at various times, with all cultures assessed for DNA damage at 2 h. Only cells treated by the first addition had observable DNA damage. This result was unexpected since these cells had had the maximum repair time. It was determined that the hydrogen peroxide had been eliminated by the culture medium. To determine the mechanism of this elimination, 200 microM hydrogen peroxide was added to various cell culture components, and the solutions were assayed for hydrogen peroxide after 1 h at 37 degrees C. Although most components (except the balanced salts) showed some hydrogen peroxide degradation, it was found that sodium pyruvate was most effective, by a wide margin, in eliminating hydrogen peroxide and its toxic effects. This was confirmed by addition of pyruvate to balanced salt solutions or buffers, and observing the same elimination of hydrogen peroxide. We subsequently found a few earlier reports describing the decarboxylation reaction between hydrogen peroxide and pyruvate, but no kinetic measurements have been published and there seems to be no general appreciation for the very high efficiency of this reaction. The present work presents a preliminary assessment of the importance of pyruvate in the study of hydrogen peroxide and other reactive oxygen species in mammalian cell culture.

Azides↗

PubMed1996

Spinal administration of adrenergic agents produces analgesia in amphibians.

Direct intraspinal injection of the catecholamines epinephrine and norepinephrine, and the alpha-adrenergic agents dexmedetomidine and clonidine, produced a dose-dependent elevation of pain thresholds in the Northern grass frog, Rana pipiens. Significant analgesic effects were noted for at least 4 h. The analgesic effect of intraspinal dexmedetomidine or epinephrine was blocked by systemic pretreatment with the alpha 2-adrenoceptor antagonists, yohimbine and atipamezole, but not with the alpha 1-adrenoceptor antagonist, prazosin. Dose-response analyses showed that dexmedetomidine, epinephrine, norepinephrine had similar analgesic potencies, but clonidine was significantly less potent. Analgesia was observed without accompanying motor or sedative effects. These results suggest that alpha 2-adrenoceptor mechanisms which mediate analgesia may have evolved early in vertebrate evolution and that descending epinephrine-containing fibers in the amphibian nervous system may be the source of endogenous catecholamines regulating nociceptive sensitivity in the amphibian spinal cord.

Analgesics, Non-Narcotic↗

PubMed1996

Ionizing radiation greatly improves gene transfer efficiency in mammalian cells.

The vast majority of clinical protocols involving gene therapy today rely on viral vectors for gene transduction. The primary reason that plasmid vectors have not been widely used for gene therapy trials is their relatively low rate of stable gene transfer. We show here that ionizing radiation can improve plasmid transfection efficiency in both normal and neoplastic human and mouse cells. As high as 1,400-fold improvement in transfection efficiency can be seen in primary human fibroblasts treated with 9 Gy. Radiation improves transfection efficiency in a dose-dependent manner of only linearized plasmid DNA in transformed or immortalized cells, but of both linearized and supercoiled plasmid in normal human fibroblasts. The gene transfer dose-response curves are linear for neoplastic cell lines and exponential for primary cell lines. This suggests that radiation can improve gene integration by at least two mechanisms, one that may require free DNA ends and one that does not. The 2-hr delay described here, from the time of irradiation to the beginning of enhanced gene integration, suggests an inducible process that becomes active after the bulk of the radiation damage has been repaired. Our data further suggest that radiation may be useful to target human gene therapy using plasmid vectors.

Animals↗

PubMed1996

Thermal, mechanical and chemical peripheral sensation in amphibians: opioid and adrenergic effects.

The acetic acid test (AAT) is a quantifiable assay of the response to noxious chemical stimuli on the hindlimb of the northern grass frog, Rana pipiens. AAT is sensitive to both opioid and adrenergic agonist modulation. The present study introduces the novel use of mechanical and thermal stimulus behavioral assays in comparison with the established acetic acid test in studying nociception in frogs. We evaluated mechanical and thermal responses and their sensitivity to systemic morphine (MOR) or dexmedetomidine (DEX) administrations with comparison to AAT. MOR produced dose-related elevations of response thresholds in all three sensory tests, whereas DEX produced elevations in the thermal and AAT assays but had no effect on sensitivity to non-noxious mechanical stimuli. The results suggest a distinct separation of sensory modalities in the frog similar to that observed in mammals and indicate the usefulness of this amphibian model in further studies of nociception.

Acetates↗

PubMed1996

Relative analgesic potency of mu, delta and kappa opioids after spinal administration in amphibians.

The analgesic effects of 12 opioid agonists in amphibians were measured using the acetic acid test. Spinal administration of dermorphin, [D-Ala2,NMePhe4,Gly-ol]-enkephalin, fentanyl and morphine (mu opioids); [D-Ser2,Leu5-Thr6]-enkephalin, [D-Ala2,D-Leu5]-enkephalin, [D-Pen2,D-Pen5]-enkephalin and deltorphin (delta opioids); and Cl977 [(5R)-(544 alpha,744 alpha,845 beta)-N-methyl-N-[7-(1-pyrrolidinyl)-1- oxaspiro[4,5]dec-8yl]-4-benzofuranacetamide monohydrochloride, bremazocine), U50488 (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide methanesulfonate) and nalorphine (kappa opioids) produced a dose-dependent and long-lasting analgesia in the Northern grass frog, Rana pipiens. With all opioids, time course experiments showed that this analgesic effect lasted for at least 4 hr, with no untoward effects observed within each dosage range used. The analgesic effects of the 12 opioids were blocked by systemic naltrexone pretreatment. Comparison of dose-response curves demonstrated that the rank order of potency was such that, in general, mu opioids > delta opioids > kappa opioids. Dose-response curves obtained in the presence of a fixed dose of naltrexone showed the greatest shift for [D-Ser2,Leu5-Thr6]-enkephalin, less so for [D-Ala2,NMePhe4,Gly-ol]-enkephalin and the least shift for bremazocine. ED50 values for mu and delta opioids in the amphibian acetic acid test were significantly correlated to ED50 values of the same opioids reported in the literature for the rodent TF test. These results show that a spinal site of opioid analgesia is present in amphibians and supports the utility of this alternative, nonmammalian model for studies of opioid analgesia and pain research.

Analgesics, Opioid↗

PubMed1995

Changes of opioid binding density in the rat spinal cord following unilateral dorsal rhizotomy.

Mu, delta and kappa opioid receptors in the vertebrate spinal cord mediate the potent antinociceptive effects of opioid agonists administered onto the spinal cord. The present experiments were conducted to determine the effect of unilateral dorsal rhizotomy on mu, delta and kappa spinal opioid binding sites. Measurements of opioid binding were made at 1, 2, 4 or 8 days after rhizotomy and comparisons were made to intact animals. The changes in mu, delta and kappa opioid binding sites were determined by receptor autoradiography using the highly selective radioligands [3H]sufentanil, [3H]DPDPE and [3H]U69593, respectively. Within autoradiograms of each spinal cord, three regions on each side of the spinal cord were targeted for densitometric analysis: laminae I-II (medial), V (lateral) and X. When effects of unilateral rhizotomy within animals were assessed by comparison of the density of binding on the side ipsilateral to the rhizotomy to the contralateral side, decreases in the binding of all three radioligands were observed in laminae I-II on the side of the spinal cord ipsilateral to the rhizotomy at 2-8 days postlesion. A significant reduction in binding was also noted for mu and delta sites in lamina V after 8 days and for delta binding in lamina X at 2 and 4 days on the side ipsilateral to the rhizotomy. However, when densities of binding sites were compared with the corresponding regions in control, it was clear that dorsal rhizotomy resulted in significant changes in opioid binding on both sides of the spinal cord; changes differed for each type of opioid binding site. On the contralateral side of the spinal cord, rhizotomy caused a significant decrease of mu opioid sites 1 day after the lesion and showed partial recovery by day 8. Delta opioid sites were also significantly decreased as early as 1 day postlesion, but did not recover. Kappa opioid sites did not change at 1 day after the rhizotomy but increased on day 2, decreased on day 4 and fully recovered 8 days after rhizotomy. The present results support the hypothesis that a significant proportion of spinal mu, delta and kappa opioid binding sites are present on the central terminations of primary afferents. Finally the present data are the first to report a contralateral effect of the unilateral rhizotomy on spinal opioid binding sites. The contralateral changes in binding were specific to the type of opioid site examined, time after the surgery and region of the spinal cord examined.

Animals↗

PubMed1995

Analgesia produced by immobilization stress and an enkephalinase inhibitor in amphibians.

The role of endogenous opioids in modulating pain transmission in amphibians was examined by two methods known to activate endogenous opioids in mammals. Analgesia was assessed using the acetic acid test in the Northern grass frog, Rana pipiens. One or 2 h of immobilization produced a significant analgesia lasting for at least 90 min. Systemic, but not spinal, administration of naloxone before immobilization prevented the analgesic effects seen in saline-pretreated controls. Spinal administration of the enkephalinase inhibitor, thiorphan, but not bestatin (both at 100 nmol/frog), produced significant analgesia. The analgesic effect of thiorphan was blocked by coadministration of intraspinal naloxone. These data are the first to suggest a role for endogenous opioid modulation of noxious stimuli in lower vertebrates by examination of stress-induced analgesia and the action of agents that inhibit enkephalin degradation.

Acetates↗

PubMed1994

Analgesic potency of alpha adrenergic agents after systemic administration in amphibians.

The analgesic and behavioral effects produced by systemic adrenergic agonists dexmedetomidine (0.1-3 nmol/g), clonidine (100-1000 nmol/g), epinephrine (1-30 nmol/g) and norepinephrine (10-300 nmol/g) were determined in Rana pipiens using the acetic acid test. Each agonist produced a dose-dependent analgesic effect that was sustained for at least 4 hr with all agonists. The analgesic effect of epinephrine and dexmedetomidine was observed 15 min after agonist administration and continued for more than 8 hr. Dexmedetomidine was the most potent agonist followed by epinephrine, norepinephrine and clonidine, and the relative potencies compared to epinephrine = 1.0 were 0.01 (clonidine), 0.02 (norepinephrine) and 4.83 (dexmedetomidine). Pretreatment with selective alpha-2 receptor antagonists, yohimbine and atipamezole, significantly decreased the analgesic effect of dexmedetomidine (80 and 87%) and clonidine (66 and 60%), whereas the selective alpha-1 receptor antagonist, prazosin, had no effect on dexmedetomidine but augmented clonidine analgesia. All animals treated with alpha adrenergic agonists retained corneal, righting and hind limb withdrawal reflexes and exhibited normal behavior. These studies demonstrate that systemic adrenergic agonists produce analgesia in amphibians, with a similar order of potency as reported in mammalian studies, and suggest that this analgesia is mediated by adrenergic alpha-2 receptors.

Adrenergic alpha-Agonists↗

PubMed1994

Analgesic potency of mu and kappa opioids after systemic administration in amphibians.

The relative analgesic potency of 11 opioid agents was assessed by using the acetic acid test in amphibians. Systemic administration of the mu agonists, fentanyl, levorphanol, methadone, morphine, meperidine and codeine; the partial mu agonist, buprenorphine; and the kappa agonists nalorphine, bremazocine, U50488 and CI-977 was made by s.c. injection into the dorsal lymph sac of the Northern grass frog, Rana pipiens. All agents produced a dose-dependent and long-lasting analgesia which persisted for at least 4 hr. The analgesic effects of single doses of each agent were significantly blocked or reduced by pretreatment with naltrexone. Systemic opioids produced log dose-response curves which yielded ED50 values ranging from 1.4 nmol/g for fentanyl to 320.9 nmol/g for nalorphine. Comparison of ED50 values gave a rank order of analgesic potency = fentanyl > CI-977 > levorphanol > U50488 > methadone > bremazocine > morphine > buprenorphine > meperidine > codeine > nalorphine. The relative analgesic potency of mu opioids in amphibians was significantly correlated with relative analgesic potency of these same agents obtained on the mouse writhing and hot plate tests. These data suggest that the amphibian model may serve as an adjunct or alternative model for the testing of opioid agents. Furthermore, given the inactivity of kappa opioids on rodent hot plate and tail-flick tests, the acetic acid test in amphibians may be especially well-suited for the assessment of opioid analgesia after administration of kappa-selective opioids.

Analgesics↗