Purification of active calpain by affinity chromatography on an immobilized peptide inhibitor

The 19F-NMR resonance of 5-[19F]fluoropyrimidin-2-one ribonucleoside moves upfield when it is bound by wild-type cytidine deaminase from Escherichia coli, in agreement with UV and X-ray spectroscopic indications that this inhibitor is bound as the rate 3,4-hydrated species 5-fluoro-3,4-dihydrouridine, a transition state analogue inhibitor resembling an intermediate in direct water attack on 5-fluorocytidine. Comparison of pKa values of model compounds indicates that the equilibrium constant for 3,4-hydration of this inhibitor in free solution is 3.5 x 10(-4) M, so that the corrected dissociation constant of 5-fluoro-3,4-dihydrouridine from the wild-type enzyme is 3.9 x 10(-11) M. Very different behavior is observed for a mutant enzyme in which alanine replaces Glu-104 at the active site, and kcat has been reduced by a factor of 10(8). 5-[19F]Fluoropyrimidin-2-one ribonucleoside is strongly fluorescent, making it possible to observe that the mutant enzyme binds this inhibitor even more tightly (Kd = 4.4 x 10(-8) M) than does the native enzyme (Kd = 1.1 x 10(-7) M). 19F-NMR indicates, however, that the E104A mutant enzyme binds the inhibitor without modification, in a form that resembles the substrate in the ground state. These results are consistent with a major role for Glu-104, not only in stabilizing the ES++ complex in the transition state, but also in destabilizing the ES complex in the ground state.     

Survey of the diet of Pima Indians using quantitative food frequency assessment and 24-hour recall. Diabetic Renal Disease Study

OBJECTIVE: A dietary survey was conducted in the Gila River Indian Community in Arizona using two methods of dietary assessment--24-hour recall and quantitative food frequency (QFF) assessment--to determine the usual intake of the population. DESIGN: Interviews were conducted by Pima women who were trained and monitored by a research dietitian. Energy and nutrient intake were calculated using a computerized dietary database that included specific Pima foods. SUBJECTS: An age- and sex-stratified sample of 575 Pima Indians (273 men, 302 women) aged 18 to 74 years participated in the study. STATISTICAL ANALYSES: Spearman correlations were used to compare the results of the two survey methods for energy and each nutrient. Intraclass correlations were used to measure reproducibility. RESULTS: According to the 24-hour recall, mean reported energy intakes within decades of age were 95% to 112% of those in the US population for Pima women, and 76% to 94% of those in the US population for Pima men. Total energy intake assessed using QFF was 30% higher in men and 33% higher in women than the intake assessed using the 24-hour recall method. CONCLUSIONS: A large dietary survey conducted using lay interviewers in a Native-American community was as reproducible as studies conducted in the general US population. The Pima diet was distributed among the major nutrients in a proportion similar to the US diet.     

A phase III, multicenter, open-label, randomized, comparative study evaluating the effect of sevoflurane versus isoflurane on the maintenance of anesthesia in adult ASA class I, II, and III inpatients

STUDY OBJECTIVE: To compare the clinical efficacy and safety of sevoflurane and isoflurane when used for the maintenance of anesthesia in adult ASA I, II, and III inpatients undergoing surgical procedures of at least 1 hour's duration. DESIGN: Phase III, randomized, open-label clinical trial. SETTING: 12 international surgical units. PATIENTS: 555 consenting inpatients undergoing surgeries of intermediate duration. INTERVENTIONS: Subjects received either sevoflurane (n = 272) or isoflurane (n = 283) as their primary anesthetic drug, each administered in nitrous oxide (N2O) (up to 70%) and oxygen (O2) after an intravenous induction using thiopental and low-dose fentanyl. The concentration of volatile drug was kept relatively constant but some titration in response to clinical variable was permitted. Comparison of efficacy was based on observations made of the rapidly and ease of recovery from anesthesia and the frequency of untoward effects for the duration of anesthesia in the return of orientation. Safety was evaluated by monitoring adverse experiences, hematologic and non-laboratory testing, and physical assessments. In 25% of patients (all patients 171 both treatment groups at selected investigational sites), plasma inorganic fluoride concentrations were determined preoperatively, every 2 hours during maintenance, at the end of anesthesia, and at 1, 2, 4, 8, 12, 24, 48, and 72 hours postoperatively. MEASUREMENTS AND MAIN RESULTS: Emergence, response to commands, orientation, and the first request for postoperative analgesia were all more rapid following discontinuation of sevoflurane than following discontinuation of isoflurane (sevoflurane, 11.0 +/- 0.6, 12.8 +/- 0.7, 17.2 +/- 0.9, 46.1 +/- 3.0 minutes, respectively, versus isoflurane, 16.4 +/- 0.6, 18.4 +/- 0.7, 24.7 +/- 0.9, 55.4 +/- 3.2 minutes). The incidence of adverse experiences was similar for sevoflurane and isoflurane patients. Forty-eight percent of patients on the sevoflurane group had no untoward effect versus 39% in the isoflurane group. Three patients who received sevoflurane had serum inorganic fluoride levels 50 microM/I. or greater though standard tests indicated no evidence of associated renal dysfunction. CONCLUSION: Sevoflurane anesthesia, as compared with isoflurane, may be advantageous in providing a smoother clinical course with a more rapid recover.     

Hippocampal 5-HT1A receptor binding site densities, 5-HT1A receptor messenger ribonucleic acid abundance and serotonin levels parallel the activity of the hypothalamo-pituitary-adrenal axis in rats

We have previously demonstrated that susceptibility of the Lewis rat to inflammatory disease, compared to the relatively resistant Fischer F344 rat, is related to a hyporesponsive hypothalamopituitary adrenal axis to inflammatory and other stress mediators. Since 5-HT and the 5HT1A receptor are important stimulators of this axis, we have investigated the levels of 5-HT1A receptor binding sites and encoding mRNA, 5-HT and 5-hydroxyindole acetic acid in various brain regions of Lewis, Harlan Sprague Dawley and Fischer F344 rats. Lewis rats expressed significantly less hippocampal and frontal cortical 5-HT1A receptor binding sites and mRNA than Harlan Sprague-Dawley and Fischer F344 rats. Adrenalectomy increased the number of 5HT1A receptor binding sites and mRNA expression in the hippocampus of all three strains. The levels of hippocampal 5-HT in Fischer F344 rats were significantly greater than levels detected in the same regions for the other two strains. Hypothalamic 5-HT and 5-hydroxyindole acetic acid levels in Harlan Sprague-Dawley rats were higher than the same area from the other two strains. Adrenalectomy increased the levels of 5-hydroxyindole acetic acid in the hypothalamus of all three strains. We conclude that hippocampal 5-HT1A receptor densities and 5-HT levels in the rat parallel the the activity and responsiveness of the hypthalamopituitary-adrenal axis. We have published these data in an earlier report.     

Interleukin-12 restores interferon-gamma production and cytotoxic responses in visceral leishmaniasis

American visceral leishmaniasis (AVL) is associated with the absence of lymphocyte proliferative responses and interleukin (IL)-2 and interferon-gamma (IFN-gamma) production upon stimulation with Leishmania antigen. In contrast, cure of AVL is associated with restoration of these T cell functions. In the present study, the ability of IL-12, a cytokine that acts on NK and T cells to restore cellular immune responses in AVL, was evaluated. Participants of the study included 12 patients with AVL and 7 subjects cured of AVL. The [3H]thymidine uptake and IFN-gamma production in cultures of peripheral blood mononuclear cells (from AVL patients) stimulated with Leishmania chagasi antigen were 882 +/- 1393 cpm and zero, respectively. Addition of IL-12 enhanced the proliferative response to 5097 +/- 6429 cpm (P < .001) and IFN-gamma production to 305 +/- 325 pg/mL (P < .01). IL-12 also restored cytotoxic activity against the K562 cell line. These results indicate that IL-12 has an important role in the regulation of the cellular immune response in human leishmaniasis.     

N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.     

Effects of bronchial vascular engorgement on airway dimensions

Airway vascular engorgement has been suggested to cause luminal narrowing and airflow obstruction. To determine the extent to which changes in bronchial vascular volume could influence airway dimensions, we studied the effects of left atrial pressure elevation on airway morphometry in sheep (n = 17). The bronchial branch of the bronchoesophageal artery was cannulated and perfused with autologous blood (0.6 ml.min-1.kg-1). A balloon-tipped catheter was inserted into the left atrial appendage to elevate left atrial pressure by 10 mmHg, and papaverine was infused into the bronchial artery to eliminate airway smooth muscle tone. Morphological measurements were made from rapidly frozen lungs excised in vivo. Left atrial pressure elevation caused a 79% increase in total vascular area (P = 0.0002). Average airway luminal area was significantly decreased from 86 to 71% of the airway maximal area (P < 0.0001). Noteworthy were the prominent bronchial vessels located within mucosal folds. However, when papaverine was infused during left atrial pressure elevation, despite a comparable total vascular area, luminal narrowing did not occur and remained at 87% of the maximal area (P = 0.6267). In conclusion, we found that engorgement of the bronchial vasculature leads to an increase in the vascular area in regions inside and outside the smooth muscle layer. The associated decrease in luminal area only occurs in the presence of airway smooth muscle tone. This suggests a reflex effect on the airway caused by the vascular engorgement. We conclude that vascular engorgement of the airway wall per se has a negligible effect on airway obstruction.     

Kinetics of cell-wall digestion of orchardgrass and alfalfa silages treated with cellulase and formic acid

The objectives of this study were to determine the effects of cellulase (from Trichoderma longibrachiatum) combined with formic acid, applied before ensiling, on the subsequent concentration and composition of the cell wall and on the extent and rate of in situ cell-wall digestion of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.). Treated and control forages of both plant species were ensiled for at least 60 d before being ruminally digested by two fistulated cows. Analyses of NDF, ADF, and acid detergent lignin were conducted sequentially on original and digested samples. Data were fitted with a first-order, nonlinear model to estimate extents and rates of digestion of potentially digestible NDF, cellulose, and hemicellulose. The concentration of indigestible residue and the discrete lag time before digestion were also determined for the cell-wall components. After ensiling, the mean NDF concentration of treated silages was 19% lower than that of control silages; the effect was greater for orchardgrass than for alfalfa. The extent of digestion of NDF, cellulose, and hemicellulose, respectively, was 33, 37, and 27% lower for treated silages than for control silages. Treatment effects on the extent of digestion varied between plant species. Cellulose from treated orchardgrass was digested 19% more slowly than cellulose from the control silage. Indigestible residue concentrations of NDF, cellulose, and hemicellulose, respectively, were 7, 8, and 7% lower in treated silages than in control silages. Thus, extensive cell-wall degradation by cellulase during ensiling resulted in less digestible cell-wall material for ruminal digestion but greater total cell-wall degradation, including that during ensiling and ruminal incubation, especially during early digestion in the rumen.     

Assessment of cerebral blood flow reserve using functional magnetic resonance imaging

Imaging of activated brain areas based on changes of blood deoxyhemoglobin levels is now possible with MRI. Acetazolamide (ACZ) increases cerebral blood flow (CBF) without changing cerebral oxygen consumption; this results in signal changes observed in gradient echo MR images from the areas with an increase in CBF. We assessed signal changes after ACZ application in seven healthy subjects with a conventional 1.5-T MRI scanner. The susceptibility-sensitized three-dimensional fast low-angle shot (FLASH) sequence was used to visualize signal changes induced by ACZ. We analyzed anatomic localization of different ranges of detected signal changes. ACZ caused significant signal changes in the gray matter and at the edge of the cerebral cortex, the latter corresponding to draining surface veins. No significant differences were seen among different brain areas within the same slice. Using the maximal intensity projection technique, we were able to partially separate signal changes originating in draining veins from signal originating in the gray matter microvasculature. Signal changes from the microvessels reflect cerebrovascular reserve. Blood-oxygen-level-dependent (BOLD) based MRI can evaluate CBF reserve with high spatial and temporal resolution. To assess cerebrovascular reserve, it is necessary to separate signal changes originating in large vessels from signal from brain microvasculature.