NMR studies of caldesmon-calmodulin interactions

The binding of the calcium-regulatory protein calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. Two regions of caldesmon have been identified as putative calmodulin-binding domains. We have earlier reported on the binding of one of these domains to calmodulin (Zhang & Vogel (1994) Biochemistry 33, 1163-1171). Here we have studied the binding of CaM to synthetic peptides of CaD which contain: (1) both the first and second CaM-binding domains; (2) the second CaM-binding domain; and (3) the sequence between the first and second CaM-binding domains. Two-dimensional transferred nuclear Overhauser enhancement proton NMR measurements as well as circular dichroism studies of a 22-residue peptide NKETAGLKVGVSSRINEWLTK, which contains the second CaM-binding domain, show that only the C-terminal half of the peptide becomes alpha-helical upon binding to CaM. Somewhat surprisingly, the shorter 9-residue peptide SRINEWLTK was sufficient to form a 1:1 complex with CaM; this peptide appears to bind as a 3(10)-helix. Proton-carbon-13 correlation NMR titration studies with specifically labeled [methyl-13C]methionine CaM were used to study the participation of the hydrophobic regions in both domains of the dumbbell shaped CaM in peptide binding. Binding of a 54-residue CaD peptide containing both CaM-binding domains affects all the 8 Met residues in the two hydrophobic domains of CaM (only Met 76 in the linker region of CaM is not involved), while binding of the second CaM-binding domain of CaD influences principally Met 51, 71, and Met 124, 144. Simultaneous binding to CaM of two peptides comprising the first and the second CaM-binding domains also caused changes to all Met residues except Met 76. Taken together, these data demonstrate that both CaM-binding domains of CaD can bind simultaneously to the two hydrophobic regions of CaM.     

Synthesis and pharmacological evaluation of new flosulide analogues, synthesized from natural safrole

OBJECTIVE: To study the effect of adjuvant chemotherapy after curative hepatic resection in patients with hepatocellular carcinoma. DESIGN: A randomized controlled trial. SETTING: A tertiary referral center. PATIENTS: During a 54-month period, 142 patients with hepatocellular carcinoma underwent hepatic resection at 1 institution. Sixty-six patients who survived the operation and had no demonstrable evidence of residual disease on ultrasonographic examination and hepatic angiographic testing at 1 month after surgery agreed to participate in the study. The median follow-up time was 28.3 months. INTERVENTION: Thirty patients received a combination of intravenous epirubicin hydrochloride (8 doses of 40 mg/m2 each at 6-week intervals) and transarterial chemotherapy using an emulsion of iodized oil and cisplatin (3 courses with a maximum dose of 20 mL each at 2-month intervals). Thirty-six patients had no adjuvant treatment. MAIN OUTCOME MEASURES: Recurrence rate and disease-free survival. RESULTS: A total of 138 courses of intravenous epirubicin was given to the 30 patients. Sixty-one courses of transarterial chemotherapy were given to only 29 of the 30 patients assigned to the treatment group, because the hepatic artery in 1 patient was thrombosed. Six patients (20%) had chemotherapy-related complications with no mortality. Twenty-three of 30 patients in the treatment group and 17 of 36 patients in the control group had recurrences (P=.01). Patients who received adjuvant chemotherapy had a higher incidence of extrahepatic metastases (11 patients vs 5 patients; P=.03). The respective disease-free survival rates at 1, 2, and 3 years were 50%,36%, and 18% for the treatment group and 69%, 53%, and 48% for the control group (P=.04). CONCLUSION: In a group of patients who underwent curative resection of hepatocellular carcinoma, postoperative adjuvant chemotherapy using the present regimen was associated with more frequent extrahepatic recurrences and a worse outcome.     

Effect of alcohol consumption on adult and aged bone: composition, morphology, and hormone levels of a rat animal model

To determine the effect of life-long alcohol consumption on the adult and aged rat model, 4-week-old, female Sprague-Dawley rats were divided into three diet groups. Alcohol-treated animals were fed a modified Lieber-DeCarli diet ad libitum containing 35% ethanol-derived calories, whereas the pair-fed animals (weight-matched to ethanol rats) received an isocaloric liquid diet in which maltose-dextrin substituted calories supplied by ethanol. Chow animals were fed a standard rat chow ad libitum. Proximal tibiae (primarily cancellous bone) and femora (primarily cortical bone) were removed for analysis after 3, 6, 9, 12, or 18 months on the diets. Serum was collected for analysis of calcium levels, the calcium regulating hormones; parathyroid hormone, 25-hydroxyvitamin D, calcitonin, corticosterone, estradiol, testosterone, and IGF-1. Creatinine, SGOT/AST, and SGPT/ALT levels were measured to determine kidney and liver integrity. Previous studies, with young animals, showed that chronic alcohol consumption during the age of bone development reduced bone density and bone mass in both cortical and cancellous bone. The present study demonstrates that these reductions last throughout life, whereas morphological values, such as length and diameter, attain control levels. Calcium regulating hormones and sex hormones are essentially normal and do not appear to be the primary causative agent for adult alcohol-induced osteopenia, but it appears to be due to a more direct effect of alcohol on bone cells.     

The identification of binding constraints: A directional derivative heuristic approach

A heuristic procedure to identify binding constraints in linear programming (LP) problems is based upon an examination of the orientation of the constraining closed half spaces with respect to the orientation of the objective function. The constraints with the minimum difference will be classified as binding. An experiment to test the usefulness of this procedure in solving LP problems is also developed and discussed.