Assay of oxysterol-binding protein in a mouse fibroblast, cell-free system. Dissociation constant and other properties of the system

Procedures for determining a 7.5 S oxysterol-binding protein in the cytosol fraction of cultured mouse fibroblasts were developed. The methods involved precipitation of cytosolic proteins between 0.3 and 0.4 saturation with (NH4)2SO4, incubation of the proteins with 25-hydroxy[3H]cholesterol at 0 degrees C and analysis by velocity sedimentation of 7.5 S radioactivity or of specific binding using dextran-charcoal to adsorb free sterol. By these means it was shown that binding of the ligand to the protein in a citric acid-phosphate buffer was optimal at pH 5.5 and that the sedimentation rate of the complex was greater at pH 7.4 (7.5 S) than at pH 5.5 (6.9 S). The binding protein was essentially saturated at a diol concentration of about 20 X 10(-9) M. The apparent Kd of the sterol-protein complex was approximately 3.9 X 10(-9) M. Cholesterol did not bind to 25-hydroxycholesterol-binding sites on the 7.5 S protein, whereas several oxysterols that are potent suppressors of 3-hydroxy-3-methylglutaryl CoA reductase also inhibited the binding of 25-hydroxycholesterol. One of these sterols, 5 alpha-cholest-8(14)-en-3 beta-ol-15-one was shown to compete for sites occupied by 25-hydroxycholesterol.     

Peripheral Nerve unmyelinated axons following lumbar sympathectomy

One year after bilateral lumbar sympathectomy for Raynaud's syndrome, a young woman underwent fascicular biopsy of the sural nerve to help elucidate the cause of lower limb pain. The morphological features, density, and diameter spectrum of unmyelinated fibers were normal. The sympathetic nervous system appears to contribute few, if any, axons to the total population of unmyelinated axons in this cutaneous nerve.     

A prospective evaluation of the effect of activated charcoal before oral N-acetylcysteine in acetaminophen overdose

STUDY OBJECTIVE: To evaluate whether activated charcoal (AC) reduces the efficacy of subsequent oral N-acetylcysteine therapy during acute acetaminophen overdose. DESIGN: Prospective observational case series of all acute acetaminophen overdoses reported to three certified regional poison centers. TYPES OF PATIENTS: All patients with acute acetaminophen overdose in whom N-acetylcysteine therapy was initiated within 16 hours after ingestion. INTERVENTIONS: All patients were treated with oral N-acetylcysteine therapy for 72 hours. The decision to use AC was left to the treating physician without input from the investigator. MEASUREMENTS AND RESULTS: One hundred twenty-two patients were evaluated. Maximum recorded SGOT levels of more than 125 U/mL were defined as evidence of hepatotoxicity. AC was used in addition to N-acetylcysteine in 82 of 122 patients. Hepatotoxicity developed in four of 82 patients who received AC versus ten of 40 patients who did not receive AC (P < .005). An increasing dose of N-acetylcysteine provided no additional benefit (P > .05). Spacing the administration of AC and oral N-acetylcysteine less than or more than two hours apart did not affect outcome (P > .05). CONCLUSION: Administration of AC before the administration of oral N-acetylcysteine in acetaminophen overdose does not reduce the efficacy of N-acetylcysteine therapy and may provide some additional hepatoprotective benefit. The practice of increasing the dose of oral N-acetylcysteine therapy after the administration of AC appears unwarranted.     

Evaluation of the skull base by SPECT. A comparison with planar scintigraphy and computed tomography

Computed tomography is currently the standard diagnostic tool for the evaluation of the skull base. The complex anatomy of this area is the primary reason why planar bone scintigraphy is often unsatisfactory; exact localization of abnormalities may be very difficult. These limitations may be overcome by SPECT. Seventeen patients with clinical features of basal skull involvement were assessed by CT, SPECT, and planar scintigraphy. Subsequent clinical diagnoses were malignancy in 15 patients, vasculitis in 1 patient, and osteomyelitis in 1 patient. Computed tomography with IV contrast was performed through the skull base at 5 mm intervals. Planar scintigraphy with Tc-99m MDP was followed by SPECT. Bony involvement compatible with the clinical findings was demonstrated by CT scans in 6 patients, by planar scintigraphy in 7 patients, and by SPECT in 9 patients. The abnormalities that were identified by CT were all identified by SPECT. This study suggests that, in imaging the skull base, SPECT is more sensitive and provides better anatomical localization than planar imaging and appears useful in patients with a negative CT study.     

Variability in mammography use among older women

OBJECTIVE: To determine rates of and explore factors associated with mammography use among older women. DESIGN: Retrospective review of part B (physician) bills submitted to Medicare during 1990. SETTING: Health Care Financing Administration (HCFA) data, including sociodemographic information and part B physician bills for all services delivered to Medicare-eligible women in 1990. PATIENTS/PARTICIPANTS: Women age 65 or older as of January 1, 1990, residing in one of 10 states with part B coverage through December 31, 1990. MEASUREMENTS AND MAIN RESULTS: The outcome was receipt of a mammogram (yes/no). We explored factors associated with mammography use within three age groups: 65 to 74, 75 to 84, and 85+. The factors considered were race, state, median income of ZIP Code of residence (from the 1990 US Census, and used to divide the population into quintiles within each state), and number of primary care visits (0, 1, 2, and 3+). Overall, 15% of women had a mammogram: 20% of women age 65 to 74, 12% of women age 75 to 84, and 4% of women age 85 and older. Mammography use was lowest in Oklahoma and highest in Washington. However, in each state the older the age category, the less the mammography use (e.g., 9% vs 5% vs 2% in Oklahoma and 25% vs 16% vs 5% in Washington for women 65-74, 75-84, and 85+, respectively). Mammography use was lower for black than for white women age 65 to 74 (14% vs 21%, P < .001) and 75 to 84 (9% vs 12%, P < .001). Women in each of these two age groups had lower mammography use if they resided in the lowest income quintile and highest if they resided in the highest income quintile (17% vs 23% 65-74, and 10% vs 13% 75-84, P values < .001). Among the oldest women (those 85+), mammography use was low (4%) and varied minimally by race and income (P = .907 and .003, respectively). In all age groups, mammography use was lowest among women who did not have a primary care visit, was greater among women who had at least one visit, and continued to rise with increasing numbers of visits (all P values < .001). For example, among women age 75 to 84, mammography use increased from 5% to 10%, 14%, and 17% for those with 0, 1, 2, and 3+ visits. CONCLUSIONS: We found that mammography use was less for women who were older, of black race, who did not visit a primary care provider, and who lived in areas with lower median income and certain geographic locations (states). Similar factors influenced mammography use in women age 65 to 74, where there is greater consensus as to who should receive a mammogram, and women age 75 to 84, where there is neither consensus nor data. Surprisingly, neither race nor income had much influence on mammography use among women age 85 or older.     

Functional activity and expression of the myocardial postreceptor adenylyl cyclase system in pressure overload hypertrophy in rat

OBJECTIVE: The aim of the present study was to investigate the functional regulation of the myocardial postreceptor adenylyl cyclase (AC) system in compensated left ventricular hypertrophy (LVH) and the effect of long-term angiotensin converting enzyme (ACE) inhibition. METHODS: Pressure overload LVH was induced in rats by supravalvular aortic banding for 12 weeks. At 12 weeks left ventricular function and inner diameters were analyzed by echocardiography of anesthetized animals, and responsiveness to forskolin (systolic developed pressure) was determined in isolated perfused hearts. Functional activities of AC and the stimulatory G protein Gs were measured as well as mRNA expression (quantitative slot blot analyses) of AC type V, isoforms of Gs alpha and Gi alpha 2. G protein alpha-subunits were also quantified by immunoblotting. Rats were treated with ramipril (Ram, 10 mg/kg per day p.o.) during weeks 7 to 12 to induce regression of LVH or with vehicle (Veh, tap water). RESULTS: Pressure overload induced severe LVH (3.2 +/- 0.09 g/kg in Veh vs. 1.8 +/- 0.03 in sham; P < 0.05) which was significantly reduced by ramipril (2.7 +/- 0.09; P < 0.05 vs. Veh). In-vivo left ventricular function and diameters were unchanged in LVH. In contrast, in hearts with LVH, responsiveness of left ventricles to forskolin was attenuated and basal, GTP gamma S and forskolin as well as manganese chloride-stimulated adenylyl cyclase activity was significantly downregulated by approximately 40% (basal 20.8 +/- 1.9 pmol cAMP/mg per min vs. 34.0 +/- 2.2 in sham; P < 0.01). However, no significant changes of AC type V mRNA were found in hypertrophied left ventricles. Functional activity of the stimulatory G protein Gs was reduced in LVH (48 +/- 7 pmol cAMP/mg per min in Veh vs. 68 +/- 3 in sham), whereas mRNA expression of long and short Gs alpha-isoforms was not altered and that of Gi alpha 2 was only slightly increased in ramipril-treated animals. Western analysis showed no significant differences of Gs alpha or Gi alpha 2 subunits. Long-term blockade of the renin-angiotensin system had no effect on the activity of the adenylyl cyclase system. CONCLUSIONS: Functional desensitization of adenylyl cyclase and stimulatory G protein occurred in rat adaptive LVH prior to the onset of severe left ventricular dysfunction which was not restored by ACE-inhibitor treatment. The desensitization seems not to be mediated by significant changes of mRNA expression of AC type V or abundance of regulatory G proteins.     

Usefulness of multiplane transesophageal echocardiography to improve the assessment of severity of mitral regurgitation

This study was designed to examine the accuracy of multiplane transesophageal echocardiography (TEE) color Doppler measurements in comparison to monoplane or biplane measurements in estimating the severity of mitral regurgitation (MR). Multiplane TEE potentially increases diagnostic accuracy of transesophageal examinations; it is unknown if multiplane is more accurate in assessing the severity of MR than monoplane or biplane TEE. Left ventricular cineangiograms of 91 patients with MR (40 no or mild, 30 moderate, and 21 severe) were compared with systolic pulmonary venous flow reversal and transesophageal color Doppler measurements: jet area and length in the transverse and longitudinal plane, maximal and average of those 2 planes (biplane), and maximal and average of 11 different planes (multiplane). Flow reversal (16 patients) identified severe MR with a specificity of 96% and a sensitivity of 62%; these were 96% and only 10% to 43%, respectively, for color Doppler measurements. In the absence of flow reversal, multiplane maximal jet area predicted severe MR with a sensitivity of 88% and a specificity of 75%, which were 85% and 76%, respectively, for no or mild MR; this did not differ significantly from results obtained by monoplane or biplane measurements. Color Doppler measurements of eccentric jets were not reliable for identification of severe MR. Systolic pulmonary venous flow reversal identifies 2 of 3 patients with severe MR with a high accuracy. In patients without flow reversal, multiplane color Doppler TEE is very capable of assessing MR severity, but biplane and monoplane TEE are equally accurate.     

Design of inhibitors of glycogen phosphorylase: a study of alpha- and beta-C-glucosides and 1-thio-beta-D-glucose compounds

alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase b (Ki = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that, in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several alpha- and beta-anhydroglucoheptonic acid derivatives and 1-deoxy-1-thio-beta-D-glucose analogues have been synthesized and tested in a series of crystallographic and kinetic binding studies with glycogen phosphorylase. The structural results of the bound enzyme-ligand complexes have been analyzed, together with the resulting affinities, in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. This work has shown the following: (i) Similar affinities may be obtained through different sets of interactions. Specifically, in the case of the alpha- and beta-glucose-C-amides, similar Ki's (0.37 and 0.44 mM, respectively) are obtained with the alpha-anomer through interactions from the ligand via water molecules to the protein and with the beta-anomer through direct interaction from the ligand to the protein. Thus, hydrogen bonds through water can contribute binding energy similar to that of hydrogen bonds directly to the protein. (ii) Attempts to improve the inhibition by additional groups did not always lead to the expected result. The addition of nonpolar groups to the alpha-carboxamide resulted in a change in conformation of the pyranose ring from a chair to a skew boat and the consequent loss of favorable hydrogen bonds and increase in the Ki. (iii) The addition of polar groups to the alpha-carboxamide led to compounds with the chair conformation, and in the examples studied, it appears that hydration by a water molecule may provide sufficient stabilization to retain the chair conformation. (iv) The best inhibitor was N-methyl-beta-glucose-C-carboxamide (Ki = 0.16 mM), which showed a 46-fold improvement in Ki from the parent beta-D-glucose. The decrease in Ki may be accounted for by a single hydrogen bond from the amide nitrogen to a main-chain carbonyl oxygen, an increase in entropy through displacement of a water molecule, and favorable van der Waals contacts between the methyl substituent and nonpolar protein residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Case management and preventive services among infants from low-income families

To determine the impact of an experimental approach to case management on use of child health clinic and immunization services, a nonequivalent control group with covariate measures design was employed in a sample of 98 infants from low-income families. The innovative pattern of care featured continuity of care; a single public health nurse (PHN) provided child health care to an infant by integrating case management and preventive services. In contrast, the customary pattern of child health care was characterized by fragmentation of services. Case management was segregated from preventive services, and multiple PHNs delivered care to an infant. As predicted, experimental-group infants (44%) were more likely to achieve adequate child health clinic services than control-group infants (8%) (p < 0.001). Moreover, the cost-effectiveness (C/E) ratio (dollar cost per effective intervention) for adequate child health clinic visits in continuous care ($523) was one-fifth of that in fragmented care ($2,900). The C/E ratio related to adequate immunization was 8% less in continuous care ($359) than in the fragmented approach ($386), although the difference in rates of adequate immunization was nonsignificant (experimental group, 64%; control group, 60%). These findings suggest that continuous PHN care with integrated case management is a more effective, cost-efficient approach to critical child preventive services than the customary, segregated case-management approach.