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Biomedical subjects

J W Carpenter

Publications and source records attributed to J W Carpenter.

At least 19 recordsLinked to original sources
PubMed2004

Simultaneous extraction and quantitation of fentanyl and norfentanyl from primate plasma with LC/MS detection.

The quantitation of both fentanyl and its desalkyl metabolite, norfentanyl, in plasma using LC/MS has not been previously described. The detection and quantitation of fentanyl and norfentanyl was achieved using LC/MS detection. The liquid-liquid extraction used toluene as the organic phase. Chromatography was carried out using a Zirchrom-PBD (50 mm x 2.1 mm, 3 microm) column with a mobile phase of acetonitrile-ammonium acetate (10 mM), citrate (0.1 mM, pH 4.4) (45:55, v/v) with a flow rate of 0.3 ml/min. Mass spectroscopy detection was performed using ESI in the positive mode. The LOQ for fentanyl was 25 pg/ml and norfentanyl was 50 pg/ml. For the concentrations of 75, 250, and 750 pg/ml, respectively, fentanyl had inter-day precisions of 6.6, 7.2, and 6.6% with accuracies of 4.0, 5.1, and 5.1% and intra-day precisions of 1.6, 1.9, and 1.9% with accuracies of 11.6, 9.4, and 8.4%, and norfentanyl had inter-day precisions of 7.4, 0.3, and 0.7% with accuracies of 9.1, 8.8, and 12.3% and intra-day precisions of 5.3, 1.4, and 0.1% with accuracies of 10.9, 8.9, and 12.8%. The recoveries of fentanyl were 85, 92, and 75% and of norfentanyl were 40, 49, and 46% at the 75, 250, and 750 pg/ml concentrations, respectively.

Animals↗
PubMed2001

Persistent right aortic arch and aberrant left subclavian artery in a white Bengal tiger (Panthera tigris).

A 3-mo-old male white Bengal tiger (Panthera tigris) presented with the chief complaint of regurgitation of solid food since weaning at 2 mo of age. Compared with its littermates, the tiger was in poor body condition and weighed only 10.3 kg when its littermates were estimated at 20-25 kg. Thoracic radiographs showed a megaesophagus cranial to the heart base. A contrast esophagram more clearly outlined the megaesophagus, and fluoroscopy demonstrated normal motility of the caudal esophagus. Endoscopic examination revealed a structure coursing dorsally from right to left over the esophagus and a constrictive band on the left of the esophagus at the heart base. Nonselective angiography confirmed the presence of a persistent right aortic arch, as well as an aberrant left subclavian artery. A left fourth intercostal thoracotomy was performed, and the ligamentum arteriosum was double ligated and divided. The left subclavian artery did not cause significant compromise of the esophagus and was not manipulated at surgery. The tiger recovered well from anesthesia and surgery. Solid food was slowly introduced over a 2-mo period without any regurgitation. The cub gained weight rapidly after surgery.

Animals↗
PubMed1997

14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain.

Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.

14-3-3 Proteins↗
PubMed1997

Structural and functional analysis of a mutant Ras protein that is insensitive to nitric oxide activation.

Ras proteins cycle between active, guanosine triphosphate (GTP)-bound and inactive, guanosine diphospate (GDP)-bound states to mediate signal transduction pathways that promote cell growth and differentiation. It is believed that the major physiological mechanism for Ras activation is via interaction with guanine-nucleotide exchange factors (GEFs). This interaction is highly regulated and results in elevated levels of Ras-GTP by facilitating GDP dissociation. Recently, a novel mechanism of Ras activation has been proposed, whereby nitric oxide (NO) modification of Cys-118, like GEF interaction, populates Ras in its biologically active form by stimulating GDP release. Here, we describe characterization of a variant of Ras, C118S, that is insensitive to NO modification. We have measured the GTPase activity and the GDP dissociation rate of the C118S mutant and found them to be similar to wild-type Ras. We have also analyzed the structure of this mutant using multidimensional heteronuclear NMR methods. Analysis of chemical shifts and distance restraints demonstrates that this mutation has not disrupted the structure of the protein. These results suggest that NO modification of Cys-118 may not alter Ras structure and that the basis of Ras activation by NO is destabilization of a crucial interaction between residues in the GDP-binding pocket and the nucleotide. We have also found that this mutant is a more stable form of Ras at concentrations required for NMR studies, probably due to the removal of a surface-accessible cysteine residue. This stable variant may facilitate structural and biochemical investigations of Ras and other guanine-nucleotide-binding proteins containing a cysteine at this position.

GTP Phosphohydrolases↗
PubMed1996

The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.

Amino Acid Sequence↗
PubMed1995

Complications with the use of carfentanil citrate and xylazine hydrochloride to immobilize domestic horses.

Carfentanil citrate, the only opioid approved in the United States for immobilizing large exotic animals, increasingly has been used to chemically restrain exotic horses, such as Prezwalski's horses (Equus przewalskii) and wild horses (E caballus). Because carfentanil's duration of action is long and renarcotization may develop 2 to 24 hours after administration of antagonists, a study was designed to compare the physiologic effects of opioid antagonists, using domestic horses chemically restrained with xylazine hydrochloride and carfentanil. The study was terminated after the initial 3 horses developed severe tachycardia and hypertension, which resulted in the death of 1 horse from pulmonary edema. Although it was possible that the clinical findings in these horses may have resulted from use of an inadequate dosage of carfentanil or xylazine, or both, analysis of the results more likely indicated that domestic and exotic horses may respond differently to carfentanil, and domestic horses may not be a good model for use in studies of carfentanil.

Analgesics, Opioid↗
PubMed1995

Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily.

Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.

3T3 Cells↗
PubMed1994

Ultrasonographic detection of adrenal gland tumors in two ferrets.

Hyperadrenocorticism is recognized as a common medical condition in middle- to old-aged ferrets. Because diagnosis of adrenocortical tumors in ferrets, using results of adrenocorticotropic hormone stimulation or dexamethasone suppression tests, is unreliable, ultrasonography was used as an alternative to laparotomy for the detection of tumors in 2 ferrets. The diagnosis was confirmed by surgical excision and histologic examination of the adrenal gland. In a typical manifestation of the condition, clinical signs in 1 ferret included vulvar swelling and serous to mucopurulent vaginal discharge, which resolved after the adrenalectomy.

Adenocarcinoma↗
PubMed1994

Development of an experimental model of hypothyroidism in cockatiels (Nymphicus hollandicus).

Hypothyroidism is a possible predisposing factor in a number of disorders of companion psittacine birds. We developed and validated a thyroid-stimulating hormone (TSH) response testing protocol for cockatiels (Nymphicus hollandicus), using 0.1 IU of TSH/bird given IM, with blood sample collection at 0 and 6 hours after TSH, and a commercial radioimmunoassay for thyroxine (T4). This protocol was used to document a seasonal sex difference in stimulated T4 values--females responded with higher T4 values than those in males in summer--and a stress-induced depression of baseline T4 values was detected in a group of cockatiels with normal TSH response. An experimental model for mature-onset hypothyroidism in cockatiels was created by radiothyroidectomizing cockatiels with 3.7 MBq (100 microCi) of 131I/bird given IV. Induction of the hypothyroid state was confirmed by baseline T4 concentration, TSH response test results, thyroid pertechnetate scintigraphy, and gross and microscopic examinations. Classical signs of hypothyroidism (eg, hypercholesterolemia, obesity, poor feathering) were lacking or mild at 48 days after thyroid ablation.

Animals↗
PubMed1993

Effects of exercise cessation on lipids and lipoproteins in distance runners and power athletes.

The purpose of this study was to determine the effects of short-term exercise cessation on lipid and lipoprotein profile and insulin sensitivity in highly trained runners (n = 12; mean age 19.9 years) and power athletes (n = 12; mean age 24.4 years). Following 14 days of exercise cessation, running time to exhaustion and maximal oxygen uptake decreased by 9.2% and 4.8% (P < 0.05) in the runners, while in the power athletes one repetition maximum squat and bench press did not change (P > 0.05). No changes occurred in body composition. Data from a 2-h oral glucose tolerance test revealed an impairment of the glycemic state in all athletes (P < 0.05). In contrast, exercise cessation did not significantly (P > 0.05) alter plasma levels of cholesterol, triglycerides, and low density (LDL) and high density lipoprotein (HDL). No changes were observed in HDL2, HDL2b, and HDL3 subfractions, LDL diameter, and qualitative LDL pattern (P > 0.05). These data thus suggest that despite a decrease in insulin sensitivity, short-term exercise cessation, independent of exercise mode, was insufficient to alter plasma lipid and lipoprotein profiles in well-trained athletes.

Adolescent↗
PubMed1992

Influence of morbid obesity and non-insulin-dependent diabetes mellitus on high-density lipoprotein composition and subpopulation distribution.

The chemical composition, subpopulation distribution and peak hydrated density of high-density lipoprotein (HDL) were examined in 18 morbidly obese women with either normal glucose tolerance or with non-insulin-dependent diabetes mellitus (NIDDM), and in 15 age-matched lean control subjects. Similar measurements were made in the morbidly obese subjects after gastric bypass surgery for weight loss. In the diabetic group, HDL was relatively protein-enriched and cholesterol- and cholesterol ester-poor compared with the lean controls. The same trend was seen in the nondiabetic group, although the cholesterol ester difference was not significant. In both cases, both electrophoretic and density gradient analysis showed that plasma HDL contained more of the HDL3 and less of the HDL2 subfractions than that for the lean controls. Following surgery in the diabetic group, the protein percentage of HDL decreased, and that of cholesterol ester increased; for the nondiabetic group, the protein decreased and phospholipid increased. In the diabetic group, a shift of the electrophoretic HDL subpopulation distribution toward more HDL2 accompanied these changes. Average hydrated peak density of HDL shifted from the HDL3 to the HDL2 range for the diabetic group following surgery; for the nondiabetic group the peak density also decreased but still remained within the HDL3 range. In all cases, the differences seen were more pronounced among the diabetic patients. These changes in the properties of HDL after gastric bypass surgery may favorably influence the risk for coronary heart disease usually associated with diabetes.

Adult↗
PubMed1991

Lipogenic potential of liver from morbidly obese patients with and without non-insulin-dependent diabetes.

Intra-abdominal liver biopsies were obtained during surgery from fasted obese patients with non-insulin-dependent diabetes mellitus (NIDDM), obese normoglycemic controls, and lean controls. Lipid synthesis was studied in freshly isolated hepatocytes and liver homogenates from the three groups of subjects. Incorporation of 3H2O into the lipids of hepatocytes was determined in the absence and presence of insulin (0.1 mumol/L). The activities of five enzymes involved in fatty acid synthesis, and the incorporation of 14C-glycerol-3-phosphate into lipids were determined in liver homogenates. Basal lipid synthesis by hepatocytes was not different in the three groups of patients. Insulin stimulated lipogenesis by 8% +/- 30% in the lean controls, 33% +/- 8% in the obese controls and 17% +/- 6% in the NIDDM patients. No significant differences in the activities of the five enzymes that are involved in de novo fatty acid synthesis among the three groups of patients were observed. Similarly, incorporation of 14C-glycerol-3-phosphate by liver homogenates, in the presence of saturating or submaximal concentrations of fatty acids, did not differ among the three groups. These results show that under the experimental conditions of this study, including the fasted state of the patients, the basal capacity of liver of NIDDM patients to synthesize fatty acids or glycerides is the same as that of liver from obese and lean controls. Thus, it is likely that an increase in fatty acid flux into a liver with normal lipogenic potential may contribute to the increased synthesis of triglycerides by the liver of these patients in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Influence of obesity, impaired glucose tolerance, and NIDDM on LDL structure and composition. Possible link between hyperinsulinemia and atherosclerosis.

The possible causes of the enhanced risk for coronary heart disease (CHD) were examined in morbidly obese women with normoglycemia, impaired glucose tolerance (IGT), and non-insulin-dependent diabetes mellitus (NIDDM) before and after gastric bypass surgery. Compared with age-matched lean women, plasma lipid and apolipoprotein concentrations of the obese women before surgery favored atherogenesis. The risk for CHD may further be exacerbated in the IGT and NIDDM groups by the prevalence of smaller and denser low-density-lipoprotein (LDL) particles. LDL size correlated negatively with plasma insulin levels independent of triglyceride levels, age, or body mass index (BMI). After surgery, BMI, plasma insulin, and triglyceride levels decreased, but LDL size increased, and LDL density decreased. Neither cholesterol nor LDL cholesterol levels were affected after surgery, but high-density-lipoprotein cholesterol was increased in all patients after surgery. Although the mechanisms underlying the changes in the properties of LDL could not be determined from this study, these changes appear to be of benefit in reducing CHD risk in these patients.

Adult↗
PubMed1989

Alterations in low-density lipoproteins in subjects with abdominal adiposity.

Abdominal adiposity, as indexed by the waist to hip girth ratio (WHR), has been associated with increased risk and incidence of coronary heart disease (CHD). The purpose of this study was to determine if this enhanced risk is related to alterations in the structure of low density lipoproteins (LDL). LDL were isolated from nine nonobese men with an average WHR of 1.046 and nine nonobese men with a WHR of 0.94, who were matched on age (45.6 +/- 2.7 v 47.7 +/- 2.3 mean +/- SEM) percent body fat (26.5 +/- 0.5 v 26.1 +/- 0.9), and body mass index (27.3 +/- 0.6 v 26.3 +/- 0.6). The average molecular weight of LDL from the subjects with a high WHR was lower than that of subjects with low WHR (2.70 v 3.02 x 10(6) d), the average hydrated density higher (1.050 v 1.040 g/mL), and the mobility (Rf) on 2% to 16% polyacrylamide gradient gel electrophoresis higher. Subfractionation by equilibrium density ultracentrifugation showed that the LDL of subjects with a high WHR was predominantly in the heavy density range (1.038 to 1.048 g/mL) compared with the LDL of subjects with low WHR, which was in the lighter density range (1.030 to 1.040 g/mL). Chemical analysis of the subfractions showed that the peak density fractions of LDL of subjects with a high WHR had a lower cholesterol to protein ratio than the peak density fractions of LDL of subjects with low WHR. Electron microscopy of these peak density fractions showed that LDL of subjects with high WHR was smaller than that of subjects with low WHR. These characteristics of LDL of subjects with abdominal adiposity closely resemble the properties of LDL of patients with documented CHD. It is concluded that the increased risk of CHD associated with abdominal adiposity may be due in part to the alterations in LDL characteristics, and that these alterations in LDL are independent of the degree of obesity.

Abdomen↗