Kinetic stability may determine the interaction dynamics of the bifunctional protein DCoH1, the dimerization cofactor of the transcription factor HNF-1α .

The two disparate functions of DCoH1 (dimerization cofactor of HNF-1)/PCD (pterin-4a-carbinolamine dehydratase) are associated with a change in oligomeric state. DCoH dimers enhance the activity of the diabetes-associated transcription factor HNF-1α (hepatocyte nuclear factor-1α), while the PCD activity of DCoH1 homotetramers aids in aromatic amino acid metabolism. These complexes compete for the same interface of the DCoH dimer. Formation of the DCoH1/HNF-1α complex requires cofolding. The homotetramer of the DCoH1 paralogue, DCoH2, interacts with HNF-1α through simple mixing. To further investigate regulation of DCoH/HNF-1α complex formation, we measured the stability of the DCoH1 homotetramer through unfolding studies by intrinsic tryptophan fluorescence. DCoH2 unfolding is reversible. Surprisingly, the DCoH1 homotetramer is resistant to guanidine unfolding but refolds at a much lower guanidine concentration. We show that a point mutation at the DCoH1 tetramer interface, Thr 51 Ser, overcomes the dissociation barrier of the homotetramer and increases the interaction with HNF-1α. The 1.8 Ǻ resolution crystal structure of DCoH1 T51S shows the presence of an ordered water molecule at the tetramer interface, as in DCoH2, which may destabilize the homotetramer. The equilibrium unfolding data were fit to a two-state model with no apparent intermediate. Folding intermediates were detectable by size exclusion chromatography. For wild-type DCoH1 the intermediates changed with time, suggesting a kinetic origin for the unfolding barrier of the homotetramer. We propose an unfolding pathway in which the tetramer unfolds slowly, but the dimer folds reversibly. Implications for regulation of DCoH1/HNF-1α complex formation are discussed.

Learning-set formation by mink, ferrets, skunks, and cats.

The ability of mink, fer rets, skunks, and cats to learn to dis criminate between objects was com pared. Performance of mink and fer rets was similar to that reported for primates. This observation suggests that there is considerable overlap among mammals in ability to form learning sets.

Gabapentin in horses: validation of an analytical method for gabapentin quantitation.

Gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin], is a new anticonvulsant used as adjunctive therapy in the treatment of partial seizures in humans not controlled with standard antiseizure drugs, and it has also been used in veterinary medicine. In performance horses, gabapentin is listed as a class 3 performance-enhancing substance by the Association of Racing Commissioners International, and thus is considered to have the potential to influence the outcome of races. Therefore, we developed and validated a sensitive gas chromatographic-mass spectrometric (GC-MS) method for gabapentin detection. Gamma-aminobutyric acid-d(2) (GABA-d(2)) was used as an internal standard during solid-phase extraction; lacking the cyclohexyl ring of gabapentin, GABA-d(2) formed a lactam structure to only a minor extent. Gabapentin, on the other hand, readily formed a lactam on thermal exposure during trimethylsilyl-derivatization and/or GC analysis; electrospray-ionization MS was employed to verify that the original compound was present as the expected 171 m.w. compound. Extraction efficiency for the assay was about 60%, and a curvilinear standard curve ranging from 50 ng/mL to 3000 ng/mL provided excellent within-run and between-run coefficients of variation and accuracies over a range of low, medium, and high values. The limit of detection, defined as the concentration calculated from the mean response at zero concentration plus two times the standard deviation, was calculated at 7.6 ng/mL; the limit of quantitation, defined as the concentration calculated from the mean of the zero responses plus five times the standard deviation, was calculated at 17 ng/mL. This method will enable accurate quantification of gabapentin in equine biological fluids for use in both pharmacokinetic and forensic studies.

Effects of a quick-release form of bromocriptine (Ergoset) on fasting and postprandial plasma glucose, insulin, lipid, and lipoprotein concentrations in obese nondiabetic hyperinsulinemic women.

OBJECTIVE: To assess the effect on various aspects of carbohydrate and lipid metabolism of administering a quick-release formulation of bromocriptine (Ergoset) to obese, nondiabetic, hyperinsulinemic women. RESEARCH DESIGN AND METHODS: Hourly concentrations of prolactin, glucose, insulin, free fatty acid (FFA), and triglyceride were measured for 24 h before and after approximately 8 weeks of treatment with Ergoset. In addition, fasting lipid and lipoprotein concentrations and the steady-state plasma glucose (SSPG) concentration in response to a continuous infusion of somatostatin, insulin, and glucose were determined before and after Ergoset administration. RESULTS: Circulating prolactin concentrations were dramatically decreased (P < 0.001) following treatment, associated with a significant fall (P < 0.05) in 24-h-long plasma glucose, FFA, and triglyceride concentrations. Neither circulating plasma insulin concentrations nor the ability of insulin to mediate glucose disposal changed with treatment. Finally, fasting total cholesterol fell (P < 0.05) and the ratio of total to HDL cholesterol decreased (P = 0.06) in association with Ergoset treatment. CONCLUSIONS: The fact that significant metabolic improvement was seen in the obese nondiabetic hyperinsulinemic women studied suggests that Ergoset could be of therapeutic benefit in clinical conditions of hyperglycemia and/or dyslipidemia.

Alterations in the glucose-stimulated insulin secretory dose-response curve and in insulin clearance in nondiabetic insulin-resistant individuals.

Plasma glucose and insulin responses to a graded i.v. infusion of glucose were compared in two groups of glucose-tolerant women divided on the basis of their insulin sensitivity. Resistance to insulin-mediated glucose disposal was measured using the insulin suppression test, and the women studied were chosen to represent the highest and lowest quartiles of insulin resistance seen in the normal population. The sensitivity of the pancreatic beta-cell to glucose was assessed by measuring the glucose, insulin, and C peptide concentrations in response to continuous graded i.v. infusions of glucose at rates of 1, 2, 3, 4, 6, and 8 mg/kg x min for 40 min each. In addition, insulin secretion rates in response to the graded glucose infusion, calculated over each sampling period, were derived from deconvolution of peripheral plasma C peptide concentrations, using a two-compartment model of C peptide kinetics and standard parameters for C peptide clearance. Although plasma glucose concentrations were only slightly higher throughout the glucose infusion, the insulin concentrations were approximately doubled in the insulin-resistant subjects. When expressed as a function of the molar increments in plasma glucose achieved during the glucose infusion studies, the insulin-resistant women had a 90% higher (684 +/- 55 vs. 360 +/- 36 pmol/L x mmol/L; P < 0.001) total integrated plasma insulin response as the glucose concentration was increased from 5 to 9 mmol/L. However, the total integrated insulin secretory rate was only increased by 37% (1494 +/- 133 vs. 1093 +/- 125 pmol/mmol/L x min; P < 0.05) in the insulin-resistant group. This discrepancy suggested that insulin clearance was lower in the insulin-resistant subjects, and the calculation of this value, as the ratio of the total secretion of insulin to the area under the plasma insulin curve, was significantly lower in the insulin-resistant group (1.25 +/- 0.05 vs. 1.87 +/- 0.16 L/min x m2; P < 0.005). These results show that the hyperinsulinemia of insulin resistance results from an increase in insulin secretion secondary to a shift to the left of the glucose-stimulated insulin response curve as well as a decrease in insulin clearance.

A comparison of immunocytochemical staining enhancement methods using a rapid microtitre immunocytochemistry assay (MIA).

A method is described which allows rapid and quantitative comparison of immunocytochemical staining procedures. Cells grown and fixed in microtitre plates are probed with increasing dilutions of primary antibody and then stained using the procedures under test; the resulting staining intensities are determined using a microtitre plate reader. The microtitre immunocytochemistry assay (MIA) has been used to compare the sensitivities of enhancement procedures based on immunoperoxidase and immunogold staining. Silver enhancement of DAB staining was found to be the most sensitive technique giving up to 200 fold amplification of the peroxidase staining.

Stimulus properties of thyrotropin-releasing hormone.

Male Sprague-Dawley rats were trained in a two-lever operant discrimination task using 20 mg/kg thyrotropin-releasing hormone (TRH) and saline as cues. Following completion of 40 daily training sessions, 22 of 25 subjects demonstrated a high level of discriminative responding based on the TRH and saline cues. An evaluation of the time course of TRH indicated that the stimulus properties peak between 5 and 15 min and dissipate substantially by 55--65 min. During additional testing, rats showed dose-dependent generalization between the training treatments (20 mg/kg TRH and saline) and novel doses of TRH (1, 5, 10, and 40 MG/KG). However, animals failed to show generalization between the training drug (20 mg/kg TRH) and d-amphetamine sulfate (0.8, 1.6, or 2.4 mg/kg); likewise, animals trained to discriminate d-amphetamine (0.8 or 1.6 mg/kg) from saline failed to show generalization between d-amphetamine and TRH (10, 20, OR 30 MG/KG). Microgram quanitites of TRH (2.5--25 microgram administered into either the lateral or third ventricle elicited dose-dependent generalization to the training drug (TRH 20 mg/kg, i.p.), suggesting a CNS mechanism of action for this effect of TRH.

Stimulus properties of antidepressants in the rat.

Various doses of bupropion HCl (Wellbatrin) (5, 10, and 20 mg/kg), a new phenylaminoketone antidepressant, were employed as cues in a two-lever operant discrimination from saline control injections in rats on an FR10 schedule of food reinforcement. Subjects reached and maintained a high level of discrimination in the O vs 20 mg/kg bupropion stimulus condition but not at the lower doses. In generalization testing, the following compounds produced dose-related responding on the bupropion lever: viloxazine, nomifensine, caffeine, d-amphetamine, cocaine, methylphenidate, and benzylpiperazine. Drugs that failed to show dose-related generalization included phenethylamine, thyrotropin-releasing hormone, imipramine, nortriptyline, amitriptyline, desipramine, mianserin, chlordiazepoxide, diazepam, scopolamine, phenobarbital, and morphine. With the important exception of viloxazine, the generalization profile of bupropion seems to reflect its previously reported locomotor stimulant effects in the rat rather than its antidepressant activity and suggests that species differences exist between man and rat with regard to the pharmacologic activity of this new antidepressant.

Ethnic differences in insulin resistance and its consequences in older Mexican American and non-Hispanic white women.

BACKGROUND: This study was initiated to test the hypothesis that older, healthy, nondiabetic Mexican American women would be relatively resistant to insulin-mediated glucose disposal, hyperinsulinemic, and dyslipidemic as compared to a matched group of non-Hispanic White (NHW) women. METHODS: The study, cross-sectional in nature, involved 14 Mexican American and 19 NHW healthy, normotensive nondiabetic, postmenopausal women of similar age and body mass index. It took place in the General Clinical Research Center at Stanford Medical Center. Measurements were made of fasting plasma glucose, insulin and lipid concentrations, and plasma glucose and insulin concentrations following a 75 gram oral glucose challenge. Resistance to insulin-mediated glucose disposal was estimated by the steady-state plasma glucose (SSPG) concentration achieved at the end of a 3-hour constant infusion of glucose, insulin, and somatostatin. RESULTS: Mexican American women had significantly greater glucose (p < .001) and insulin (p < .001) responses to the oral glucose challenge than did the NHW women. Resistance to insulin-mediated glucose disposal was increased in Mexican American women (SSPG 195 +/- 25 mg/dl compared to 137 +/- 18 mg/dl in NHW; p < .001). While total cholesterol, low density lipoprotein (LDL)-cholesterol, and triglyceride concentrations were not significantly different in the two ethnic groups, high density lipoprotein (HDL)-cholesterol was significantly lower in the Mexican American women (51 mg/dl vs 61 mg/dl; p = .04). CONCLUSION: Older Mexican American women are more insulin resistant, glucose intolerant, and hyperinsulinemic, and have a lower HDL-cholesterol than a matched group of non-Hispanic White peers. These results were observed despite the exclusion of individuals with non-insulin dependent diabetes mellitus (NIDDM).

Feedback training of 36 - 44 HZ EEG activity in the visual cortex and hippocampus of cats: evidence for sensory and motor involvement.

Milk reinforcement was contingent on the occurrence of 36 -44 (40) Hz EEG activity in the left visual cortex (VC) of one group of cats and in the right hippocampus (H) of a second group. Both groups learned to increase 40 Hz activity, and acquisition of reinforcement was associated with immobility. A third group (behavioral controls - BC) was trained by the method of successive approximation to behave in a similar manner to VC and H cats. Training significantly increased 40 Hz activity in all of the following structures, except the hippocampi of VC cats and between the right and left visual cortex of H and BC cats: posterior primary visual cortex (bilateral), anterior primary visual cortex (left), primary motor cortex (bilateral), dorsal hippocampus (bilateral), and midbrain reticular formation (bilateral). Since the behavioral and EEG changes of H and BC animals were similar, immobility appears to be important for increased hippocampal 40 Hz activity produced by feedback training. Testing in darkness enhanced 40 Hz activity in the trained area of VC cats but had no effect on H or BC animals. These results, in conjunction with the observation that VC cats appeared to visually fixate, suggest that VC cats may have learned to increase 40 Hz activity in the visual cortex by altering visual processing.

Laminar flow reduces cases of surgical site infections in vascular patients.

INTRODUCTION: Numerous strategies are employed routinely in an effort to lower rates of surgical site infections (SSIs). A laminar flow theatre environment is generally used during orthopaedic surgery to reduce rates of SSIs. Its role in vascular surgery, especially when arterial bypass grafts are used, is unknown. METHODS: A retrospective review of a prospectively maintained database was undertaken for all vascular procedures performed by a single consultant over a one-year period. Cases were performed, via random allocation, in either a laminar or non-laminar flow theatre environment. Demographic data, operative data and evidence of postoperative SSIs were noted. A separate subgroup analysis was undertaken for patients requiring an arterial bypass graft. Univariate and multivariate logistical regression was undertaken to identify significant factors associated with SSIs. RESULTS: Overall, 170 procedures were analysed. Presence of a groin incision, insertion of an arterial graft and a non-laminar flow theatre were shown to be predictive of SSIs in this cohort. In the subgroup receiving arterial grafts, only a non-laminar flow theatre environment was shown to be predictive of an SSI. CONCLUSIONS: This study suggests that laminar flow may reduce incidences of SSI, especially in the subgroup of patients receiving arterial grafts.

Low volume processing of protein blots in rolling drums.

We have evaluated an improved method for processing protein blots on nitrocellulose or nylon membranes using cylindrical plastic containers. The method, which is directly analogous to the commonly used method of photographic processing in rolling drums, uses small values of reagents which are constantly washed over the blotting membrane by rotating the drum horizontally on a roller mixer. Volumes of reagents used are typically less than one-10th of those required for conventional methods using plastic bags or trays. The efficiency of probing and washing steps are greatly improved, giving an all-round increase in sensitivity, ease of processing, and economy of reagents.

Temporal parameters of d-amphetamine as a discriminative stimulus in the rat.

Three groups of rats were trained on a two-lever operant discrimination using d-amphetamine (0.8, 1.6 or 2.4 mg/kg) and saline as cues. Reinforcement of responding on one lever was associated with the drug and reinforcement on the other lever was associated with saline. Following acquisition, behavioral control was evaluated during 10-min extinction tests at 0, 15, 30, 60, 90, 120, 180 or 240 min after injection of drug or saline. Onset of the stimulus properties of d-amphetamine occurred within 10 min and maximal effects were attained by 15-30 min post-injection. Drug effects were minimal or absent 2-4 h after administration. The time course for all doses of d-amphetamine was identical when each group was tested iwth its respective training dose. When subjects were tested with doses other than their training dose, the time course of the drug effect varied as a function of training dose, test dose and time after injection.

Roles of insulin resistance and obesity in regulation of plasma insulin concentrations.

Plasma glucose, insulin, and C-peptide concentrations were determined in response to graded infusions of glucose, and insulin secretion rates were calculated over each sampling period. Measurements were also made of insulin clearance, resistance to insulin-mediated glucose, uptake, and the plasma glucose, insulin, and C-peptide concentrations at hourly intervals from 8:00 AM to 4:00 PM in response to breakfast and lunch. Plasma glucose, insulin, and C-peptide concentrations were significantly (P < 0.01) higher in obese women in response to the graded intravenous glucose infusion, associated with a 40% (P < 0.005) greater insulin secretory response. Degree of insulin resistance correlated positively (P < 0.05) with the increase in insulin secretion rate in both nonobese (r = 0.52) and obese (r = 0.58) groups and inversely (P < 0.05) with the decrease in insulin clearance in obese (r = -0.46) and nonobese (r = -0.39) individuals. Weight loss was associated with significantly lower plasma glucose, insulin, and C-peptide concentrations in response to graded glucose infusions and in day-long insulin concentrations. Neither insulin resistance nor the insulin secretory response changed after weight loss, whereas there was a significant increase in the rate of insulin clearance during the glucose infusion. It is concluded that 1) obesity is associated with a shift to the left in the glucose-stimulated insulin secretory dose-response curve as well as a decrease in insulin clearance and 2) changes in insulin secretion and insulin clearance in obese women are more a function of insulin resistance than obesity.