Biomedical subjects

D Stafford

Publications and source records attributed to D Stafford.

At least 19 recordsLinked to original sources

PubMed2004

A conserved role for retinoid signaling in vertebrate pancreas development.

Retinoic acid (RA) signaling plays critical roles in the regionalization of the central nervous system and mesoderm of all vertebrates that have been examined. However, to date, a role for RA in pancreas and liver development has only been demonstrated for the teleost zebrafish. Here, we demonstrate that RA signaling is required for development of the pancreas but not the liver in the amphibian Xenopus laevis and the avian quail. We disrupted RA signaling in Xenopus tadpoles, using both a pharmacological and a dominant-negative strategy. RA-deficient quail embryos were obtained from hens with a dietary deficiency in vitamin A. In both species we found that pancreas development was dependent on RA signaling. Furthermore, treatment of Xenopus tadpoles with exogenous RA led to an expansion of the pancreatic field. By contrast, liver development was not perturbed by manipulation of RA signaling. Taken together with our previous finding that RA signaling is necessary and sufficient for zebrafish pancreas development, these data support the hypothesis that a critical role for RA signaling in pancreas development is a conserved feature of the vertebrates.

Animals↗

PubMed2001

Four loops of the catalytic domain of factor viia mediate the effect of the first EGF-like domain substitution on factor viia catalytic activity.

The presence of tissue factor is essential for factor VIIa (FVIIa) to reach its full catalytic potential. The previous work in this laboratory demonstrated that substitution of the EGF1 domain of factor VIIa with that of factor IX (FVII((IXegf1))a) results in a substantial decrease in TF-binding affinity and catalytic activity. Supporting simulations of the solution structures of Ca(2+)-bound factor VIIa and FVII((IXegf1))a with tissue factor are provided. Mutants are generated, based on the simulation model, to study the effect of EGF1 substitution on catalytic activity. The simulations show larger Gla-EGF1 and EGF1-EGF2 inter-domain motions for FVII((IXegf1))a than for factor VIIa. The catalytic domain of the chimeric factor VIIa has been disturbed and several surface loops in the catalytic domain of FVII((IXegf1))a (Loop 170s (170-182), Loop 1 (185-188) and Loop 2 (221A-225)) manifest larger position fluctuations than wild-type. The position of Loop 140s (142-152) of FVII((IXegf1))a, near the N terminus insertion site of the catalytic domain, shifts relative to factor VIIa, resulting in a slight alteration of the active site. The results suggest that these four loops mediate the effect of the EGF1 domain substitution on the S1 site and catalytic residues. To test the model, we prepared mutations of these surface loops, including four FVII mutants, D186A, K188A, L144A and R147A, a FVII mutant with multiple mutations (MM3: L144A+R147A+D186A) and a FVII mutant with Loop 170s partially deleted, Loop 170s(del). The catalytic activities towards a small peptidyl substrate decreased 2.4, 4.5 and 9-fold for Loop 170s(del)a (a, activated), L144Aa and D186Aa, respectively, while MM3a lost almost all catalytic activity. The combined results of the simulations and mutants provide insight into the mechanism by which tissue factor enhances factor VIIa catalytic activity.

Amino Acid Substitution↗

PubMed2001

Repeated exposure to inhaled toluene induces behavioral and neurochemical cross-sensitization to cocaine in rats.

RATIONALE: Toluene is a solvent found in many commercial products and is frequently abused by inhalation. Whether previous exposure to toluene alters subsequent responses to other drugs of abuse is not known. OBJECTIVES: This study determined the effects of repeated toluene exposure on the acute motor-stimulant response to cocaine and on cocaine-induced dopamine (DA) concentrations in the nucleus accumbens (NAc). METHODS: One week following bilateral cannulae implantation over the NAc, 27 adult, male Wistar rats began a daily 30-min exposure regimen to either toluene (8,000 ppm) or air for ten sessions. Approximately 24 h or 96 h after their last exposure, animals were injected with either saline or cocaine (15 mg/kg, i.p.) and locomotor activity and DA concentrations in the NAc were measured. RESULTS: Exposure to toluene rendered the rats immobile, and the time required for recovery of normal posture decreased across the ten sessions. In all animals tested, systemic cocaine administration enhanced both locomotor activity and DA concentrations in the NAc. These increases, however, were significantly greater in rats previously exposed to toluene. CONCLUSIONS: Overall, these findings show that repeated toluene exposure enhances behavioral and neurochemical responses to subsequent cocaine administration.

Administration, Inhalation↗

PubMed2001

A comparison of cocaine, GBR 12909, and phentermine self-administration by rhesus monkeys on a progressive-ratio schedule.

The dopamine reuptake inhibitor GBR 12909 and the dopamine releaser phentermine may have potential for the treatment of cocaine abuse in humans. Pre-session treatment with either drug can decrease cocaine-maintained responding in rhesus monkeys while not affecting food-maintained responding. Both drugs are self-administered, but in some reports the patterns of responding they maintain differ from typical cocaine-reinforced responding. This study compared self-administration of cocaine (1--100 microg/kg/inj), GBR 12909 (3--100 microg/kg/inj), and phentermine (10--170 microg/kg/inj) in rhesus monkeys on a progressive-ratio schedule. Individual unit doses of each drug were available across several consecutive sessions. Cocaine self-administration was typical: the average number of ratios completed per session was a bitonic (increasing/decreasing) function of unit dose. Phentermine self-administration was variable across subjects (two of four monkeys self-administered reliably); one subject exhibited clear signs of behavioral toxicity. Self-administration of GBR 12909 was similarly variable across subjects. In the two subjects that self-administered GBR 12909 reliably, self-administration of small to mid-sized unit doses was enhanced following exposure to large unit doses. These data indicate that differences in self-administration of these drugs can be observed under progressive ratio procedures. Further, the data add to existing evidence suggesting that phentermine and GBR 12909 have at least moderate potential to be abused by humans.

Adrenergic Agents↗

PubMed2001

Dysregulation of insulin secretion in children with congenital hyperinsulinism due to sulfonylurea receptor mutations.

Mutations in the high-affinity sulfonylurea receptor (SUR)-1 cause one of the severe recessively inherited diffuse forms of congenital hyperinsulinism or, when associated with loss of heterozygosity, focal adenomatosis. We hypothesized that SUR1 mutations would render the beta-cell insensitive to sulfonylureas and to glucose. Stimulated insulin responses were compared among eight patients with diffuse hyperinsulinism (two mutations), six carrier parents, and ten normal adults. In the patients with diffuse hyperinsulinism, the acute insulin response to intravenous tolbutamide was absent and did not overlap with the responses seen in either adult group. There was positive, albeit significantly blunted, acute insulin response to intravenous dextrose in the patients with diffuse hyperinsulinism. Graded infusions of glucose, to raise and then lower plasma glucose concentrations over 4 h, caused similar rises in blood glucose but lower peak insulin levels in the hyperinsulinemic patients. Loss of acute insulin response to tolbutamide can identify children with diffuse SUR1 defects. The greater response to glucose than to tolbutamide indicates that ATP-sensitive potassium (KATP) channel-independent pathways are involved in glucose-mediated insulin release in patients with diffuse SUR1 defects. The diminished glucose responsiveness suggests that SUR1 mutations and lack of KATP channel activity may contribute to the late development of diabetes in patients with hyperinsulinism independently of subtotal pancreatectomy.

ATP-Binding Cassette Transporters↗

PubMed2000

Abuse liability of the anesthetic propofol: self-administration of propofol in rats under fixed-ratio schedules of drug delivery.

RATIONALE: Previous reports suggest that propofol (PPF) may have abuse potential in humans. Hence, we hypothesized that PPF could reinforce self-administration behavior in other species. Positive reinforcing effects of PPF could be interpreted as an index of abuse liability. OBJECTIVE: Acquisition and maintenance of i.v. PPF self-administration were examined in 12 rats. METHODS: Six rats were initially given access to methohexital (MHX, 2.0 mg/kg per infusion) under a fixed ratio (FR) 1 schedule, while the other six were initially given access to PPF (1.7 mg/kg per infusion). Once stable responding was established, various doses of PPF (0.56, 1.0, and 1.7 mg/kg per infusion) and vehicle (Intralipid 20%) were made available. RESULTS: The number of PPF infusions per session was an inverse function of dose, with 0.56 mg/kg and 1.0 mg/kg per infusion maintaining significantly more infusions per session than vehicle in most rats under the FR 1 schedule. For some rats, the number of vehicle infusions per session was equal to or greater than the number of PPF infusions. Increasing the response requirement to FR 5 decreased the number of vehicle infusions per session in these rats, while PPF maintained a higher number of infusions than vehicle under this FR value in six of seven rats. CONCLUSION: PPF served as a reinforcer in rats under FR schedules of i.v. drug delivery, adding to the extant evidence that it has abuse potential.

Anesthetics, Intravenous↗

PubMed2000

Modeling zymogen protein C.

A solution structure for the complete zymogen form of human coagulation protein C is modeled. The initial core structure is based on the x-ray crystallographic structure of the gamma-carboxyglutamic acid (Gla)-domainless activated form. The Gla domain (residues 1-48) is modeled from the x-ray crystal coordinates of the factor VII(a)/tissue factor complex and oriented with the epidermal growth factor-1 domain to yield an initial orientation consistent with the x-ray crystal structure of porcine factor IX(a). The missing C-terminal residues in the light chain (residues 147-157) and the activation peptide residues 158-169 were introduced using homology modeling so that the activation peptide residues directly interact with the residues in the calcium binding loop. Molecular dynamics simulations (Amber-particle-mesh-Ewald) are used to obtain the complete calcium-complexed solution structure. The individual domain structures of protein C in solution are largely unaffected by solvation, whereas the Gla-epidermal growth factor-1 orientation evolves to a form different from both factors VII(a) and IX(a). The solution structure of the zymogen protein C is compared with the crystal structures of the existing zymogen serine proteases: chymotrypsinogen, proproteinase, and prethrombin-2. Calculated electrostatic potential surfaces support the involvement of the serine protease calcium ion binding loop in providing a suitable electrostatic environment around the scissile bond for II(a)/thrombomodulin interaction.

1-Carboxyglutamic Acid↗

PubMed2000

Response requirements and unit dose modify the effects of GBR 12909 on cocaine-maintained behavior.

Previous studies found that GBR 12909 can decrease cocaine-maintained responding at doses that do not affect food-maintained responding. In this study, the effects of GBR 12909 (0.3-3.0 mg/kg) were further examined by varying the response requirement and unit dose of cocaine. Rhesus monkeys earned food or cocaine under a multiple fixed-ratio (FR) schedule. The FR for food was always 30, but the FR for cocaine was varied from 10-130 and the unit dose was varied from 5.6-56.0 microg/kg per injection. Doses of GBR 12909 were tested in an ascending order, for 5 consecutive sessions each. GBR 12909 selectively decreased cocaine-maintained responding in all monkeys in at least 1 condition. These effects were enhanced with large response requirements and/or small unit doses. The results demonstrate that environmental variables can influence the selectivity of GBR 12909's effects and contribute to a growing debate concerning the evaluation of potential pharmacotherapies for drug abuse.

Animals↗

PubMed1999

Oxygenated analogues of 1-[2-(Diphenylmethoxy)ethyl]- and 1-[2-[Bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR 12909) as potential extended-action cocaine-abuse therapeutic agents.

An investigation into the preparation of potential extended-release cocaine-abuse therapeutic agents afforded a series of compounds related to 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (1a) and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (1b) (GBR 12935 and GBR 12909, respectively), which were designed, synthesized, and evaluated for their ability to bind to the dopamine transporter (DAT) and to inhibit the uptake of [(3)H]-labeled dopamine (DA). The addition of hydroxy and methoxy substituents to the benzene ring on the phenylpropyl moiety of 1a-1d resulted in a series of potent and selective ligands for the DAT (analogues 5-28). The hydroxyl groups were included to incorporate a medium-chain carboxylic acid ester into the molecules, to form oil-soluble prodrugs, amenable to "depot" injection techniques. The introduction of an oxygen-containing functionality to the propyl side chain provided ketones 29 and 30, which demonstrated greatly reduced affinity for the DAT and decreased potency in inhibiting the uptake of [(3)H]DA, and benzylic alcohols 31-36, which were highly potent and selective at binding to the DAT and inhibiting [(3)H]DA uptake. The enantiomers of 32 (34 and 36) were practically identical in biological testing. Compounds 1b, 32, 34, and 36 all demonstrated the ability to decrease cocaine-maintained responding in monkeys without affecting behaviors maintained by food, with 34 and 36 equipotent to each other and both more potent in behavioral tests than the parent compound 1b. Intramuscular injections of compound 41 (the decanoate ester of racemate 32) eliminated cocaine-maintained behavior for about a month following one single injection, without affecting food-maintained behavior. The identification of analogues 32, 34, and 36, thus, provides three potential candidates for esterification and formulation as extended-release cocaine-abuse therapeutic agents.

Animals↗

PubMed1999

Preclinical research on cocaine self-administration: environmental determinants and their interaction with pharmacological treatment.

It has been asserted that any comprehensive understanding of cocaine abuse and its treatment will require attention to both behavioral and pharmacological variables. Although the preclinical literature evaluating the effects of pharmacological variables on cocaine self-administration has been extensively reviewed, no comprehensive review of the effects of environmental variables on cocaine self-administration has been published. The present review summarizes and critiques the preclinical findings on environmental determinants of cocaine self-administration. The influence of environmental variables on the effects of pharmacological interventions on cocaine self-administration are also described. Several environmental variables have been shown to affect cocaine self-administration, including unit dose, schedule of cocaine delivery, schedules of nondrug stimuli, behavioral history, conditioned stimuli, food deprivation, exposure to stress, and rearing environment. Among these variables, unit dose, schedule of cocaine delivery, availability of alternative nondrug reinforcers, food deprivation, and rearing environment have also been shown to alter pharmacological treatment effects on cocaine self-administration. Thus, drug effects on cocaine self-administration are malleable and dependent upon the environmental context within which they occur. Suggestions for future research on the effects of these and other environmental variables on cocaine self-administration and its pharmacological treatment are presented.

Animals↗

PubMed1999

Effects of phentermine on responding maintained by progressive-ratio schedules of cocaine and food delivery in rhesus monkeys.

Previous reports indicate that intravenous pretreatment with phentermine can decrease cocaine-maintained responding without affecting food-reinforced responding under fixed-ratio schedules. The present experiments were designed to explore the generality of this effect using progressive-ratio schedules of reinforcement and different routes of phentermine administration. Unit doses of cocaine and food-pellet magnitudes were identified that maintained similar breaking points, and the effects of acute exposure to phentermine were assessed. In Experiment 1, a 'conventional' (one-trial) progressive-ratio schedule was used, in which response requirements increased after each reinforcer delivery; in Experiment 2, a 'modified' (five-trial) progressive-ratio schedule was used, in which response requirements increased after every five reinforcer deliveries. In one group of monkeys, responding was maintained by food; in another, cocaine infusions maintained responding. Phentermine (0.1-5.6mg/kg, intramuscularly (i.m.)) dose-dependently decreased breakpoints on both progressive-ratio schedules. There were no differences in phentermine's effects on cocaine- and food-maintained behavior. In Experiment 3, intravenous administration of phentermine had largely similar effects. Taken together with results from previous reports, these data suggest that the effects of phentermine pretreatment are influenced by the behavioral procedure used to maintain responding and/or by the efficacy of the food and cocaine reinforcers.

Animals↗

PubMed1998

Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells.

Cystic fibrosis (CF) is a lethal inherited disease that results from abnormal chloride conduction in epithelial tissues. ClC-2 chloride channels are expressed in epithelia affected by CF and may provide a key "alternative" target for pharmacotherapy of this disease. To explore this possibility, the expression level of ClC-2 channels was genetically manipulated in airway epithelial cells derived from a cystic fibrosis patient (IB3-1). Whole-cell patch-clamp analysis of cells overexpressing ClC-2 identified hyperpolarization-activated Cl- currents (HACCs) that displayed time- and voltage-dependent activation, and an inwardly rectifying steady-state current-voltage relationship. Reduction of extracellular pH to 5.0 caused significant increases in HACCs in overexpressing cells, and the appearance of robust currents in parental IB3-1 cells. IB3-1 cells stably transfected with the antisense ClC-2 cDNA showed reduced expression of ClC-2 compared with parental cells by Western blotting, and a significant reduction in the magnitude of pH-dependent HACCs. To determine whether changes in extracellular pH alone could initiate chloride transport via ClC-2 channels, we performed 36Cl- efflux studies on overexpressing cells and cells with endogenous expression of ClC-2. Acidic extracellular pH increased 36Cl- efflux rates in both cell types, although the ClC-2 overexpressing cells had significantly greater chloride conduction and a longer duration of efflux than the parental cells. Compounds that exploit the pH mechanism of activating endogenous ClC-2 channels may provide a pharmacologic option for increasing chloride conductance in the airways of CF patients.

Cell Line↗

PubMed1998

2-amino-4H-3,1-benzoxazin-4-ones as inhibitors of C1r serine protease.

A series of 2-amino-4H-3,1-benzoxazin-4-ones have been synthesized and evaluated as inhibitors of the complement enzyme C1r. C1r is a serine protease at the beginning of the complement cascade, and complement activation by beta-amyloid may represent a major contributing pathway to the neuropathology of Alzheimer's disease. Compounds such as 7-chloro-2-[(2-iodophenyl)-amino]benz[d][1,3]oxazin-4-one (32) and 7-methyl-2-[(2-iodophenyl)amino]benz[d][1,3]oxazin-4-one (37) show improved potency compared to the reference compound FUT-175. Many of these active compounds also possess increased selectivity for C1r compared to trypsin and enhanced hydrolytic stability relative to 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1).

Complement C1r↗

PubMed1998

Progressive-ratio schedules of drug delivery in the analysis of drug self-administration: a review.

Drugs, like other reinforcers, can vary in their relative abilities to support operant responding. Considerable research has been designed to obtain useful measures of a given drug's or dose's "reinforcing efficacy" and to identify the ways in which a variety of behavioral and pharmacological variables impact these measures. Progressive-ratio schedules of drug delivery generate an index of a drug's or dose's reinforcing efficacy (the breaking point) and are being used increasingly as tools in the analysis of drug self-administration. Progressive-ratio schedules of drug delivery have been used to characterize the effects of pretreatment drugs, lesions, drug deprivation, physical dependence, and repeated non-contingent drug exposure on breaking points. Behavioral factors, including food restriction and electric shock, and organismic factors, including gender and strain, have also been investigated using progressive-ratio schedules of drug delivery. To the extent that breaking points provide an index of reinforcing efficacy, these studies demonstrate that a wide range of variables can influence the reinforcing efficacy of self-administered drugs. The objectives of this review are to critique existing research themes, outline potential limitations of progressive-ratio procedures, and to suggest potentially fruitful uses of these procedures in future research.

Algorithms↗

PubMed1998

How breast cancer treatment decisions are made by women in North Dakota.

BACKGROUND: Although equally effective, women in rural midwestern states choose modified radical mastectomy (MRM) over breast conservation surgery for early stage breast cancer. This study assessed treatment decisions by the women of North Dakota. METHODS: Surveys were sent to women treated for early stage breast cancer from 1990 through 1992. Separate surveys were sent out to surgeons in the state. The questions assessed physician and patient perceptions of treatment and the decision making process. RESULTS: A majority of surgeons believed that the long-term and disease-free survival was equal and that the preference for choosing MRM was due mostly to inconvenience of radiotherapy. The women reported that the surgeon was the most influential in the treatment decision and that concerns over radiation, duration of treatment, and travel restrictions all were factors in the decision. CONCLUSIONS: Education of surgeons and patients plus the early involvement of the radiation oncologist in discussing options is essential in the treatment of early stage breast cancer.

Aged↗

PubMed1998

Effects of step size and break-point criterion on progressive-ratio performance.

Key pecking by pigeons was maintained by arithmetic progressive-ratio schedules of food delivery. Successive conditions arranged different step sizes, and each condition remained in effect until behavior appeared stable. Each session continued until a period of time passed in which no key pecks were recorded (the break-point criterion); both a 5-min and a 15-min criterion were tested across a range of step sizes. Average breaking points (i.e, the largest ratio completed) were relatively unaffected by step-size magnitude, whereas the average number of ratios completed and average response rates generally declined across increasing step sizes. Within sessions, preratio pauses were relatively short and fairly constant in duration as the ratio increased; pause durations increased rapidly near the end of a session. The relation between the average number of completed ratios and step size was described well by a power function [y = b(xa), in which y represents the average number of completed ratios, x represents the step size, and a and b are fitted parameters]. Increasing the break-point criterion from 5 to 15 min resulted in increased values of b, whereas parameter a was relatively unaffected and was close to -1 (consistent with the lack of effect of step size on breaking point). This function also provided an excellent description of data drawn from previous reports.

Animals↗

PubMed1996

Effects of ICE-like protease and calpain inhibitors on neuronal apoptosis.

Both ice-like protease and calpain have been shown to be involved in apoptosis in non-neuronal cells. Cultured rat cerebellar granule neurons undergo apoptosis when exposed to low potassium-containing medium. Calpain inhibitors 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) and N-acetyl-Leu-Leu-Met-CHO (calpain inhibitor II) as well as interleukin-beta 1 converting enzyme (ICE)-like protease inhibitor Z-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) protect against such apoptotic death. They also reduce DNA laddering and the number of apoptotic nuclei. Staurosporine treatment also evokes apoptosis in human neuroblastoma SH-SY5Y. While Z-D-DCB is again anti-apoptotic, calpain inhibitors only provide modest effects in this model. Our results suggest that ICE-like protease plays a critical role in neuronal apoptosis whereas the contributions of calpain are more cell-type dependent.

Animals↗

PubMed1996

Synaptic inputs to ON parasol ganglion cells in the primate retina.

In primates, the retinal ganglion cells that project to the magnocellular layers of the lateral geniculate nucleus have distinctive responses to light, and one of these has been identified morphologically as the parasol ganglion cell. To investigate their synaptic connections, we injected parasol cells with Neurobiotin in lightly fixed baboon retinas. The five ON-center cells we analyzed by electron microscopy received approximately 20% of their input from bipolar cells. The major synaptic input to parasol cells was from amacrine cells via conventional synapses and, in this respect, they resembled alpha ganglion cells of the cat retina. We also found the gap junctions between amacrine cells and parasol ganglion cells that had been predicted from tracer-coupling experiments. To identify the presynaptic amacrine cells, ON-center parasol cells were injected with Neurobiotin and Lucifer yellow in living macaque retinas, which were then fixed and labeled by immunofluorescence. Two kinds of amacrine cells were filled with Neurobiotin via gap junctions: a large, polyaxonal cell containing cholecystokinin and a smaller one without cholecystokinin. There were also appositions between cholecystokinin-containing amacrine cell processes and parasol cell dendrites. Cholinergic amacrine cell processes often followed parasol cell dendrites and made extensive contacts. In other mammals, the light responses of polyaxonal amacrine cells like these and cholinergic amacrine cells have been recorded, and the effects of acetylcholine and cholecystokinin on ganglion cells are known. Using this information, we developed a model of parasol cells that accounts for some properties of their light responses.

Afferent Pathways↗