Isolation and characterization of three Dictyostelium myosin-I isozymes

Using ion exchange chromatography and an ATP-dependent actin precipitation step, we have isolated three myosin-I isozymes that, together, account for most of the K+EDTA-ATPase activity recovered from extracts of Dictyostelium. The two major myosin-I isozymes, present in approximately equal amounts, had apparent molecular masses of 125 kDa on SDS gels and have been identified by amino acid sequence analysis as the products of the Dictyostelium myosin-IB (DMIB) and myosin-ID (DMID) genes. DMIB, with a specific K+EDTA-ATPase activity 10-fold higher than DMID, was responsible for most of the activity in cell extracts. The third isozyme, present in low amounts, had an apparent molecular mass of 137 kDa on SDS gels and is too large to be the product of any of the known myosin-I genes. DMIB eluted from DE53 cellulose columns as two distinct peaks (II and III). Addition of the phosphatase inhibitor okadaic acid to the extraction buffer increased the fraction of DMIB recovered from growth phase cells in peak III from 35 to 70%. DMIB isolated from peak III, but not from peak II, displayed a significant level of actin-activated MgATPase activity. These results indicate that peak III represents a phosphorylated, actin-activatable form of DMIB.     

Methotrexate in the management of severe steroid dependent asthma

AIMS: This study was designed to assess the efficacy of low dose methotrexate, 15 mg weekly, as a steroid-sparing agent in asthmatic patients requiring long-term oral prednisone treatment. METHODS: The study was a randomised, double blind, placebo controlled, cross over study of 48 weeks duration. Eleven patients with severe steroid-dependent asthma were included. A successful outcome was defined as a reduction in mean prednisone requirements of 7 mg daily compared to baseline requirements, during active treatment. RESULTS: Two patients were required to be withdrawn owing to methotrexate-related adverse effects. The mean prednisone dose for patients who completed the study was 14.4 mg per day (95% CI; 13.6, 15.1) during active treatment, and 12.9 mg per day (95% CI: 12.2, 13.6) during placebo treatment (NS). Only one patient reduced his individual dose requirements by more than 7 mg per day, whereas in three patients prednisone requirements actually increased during active treatment. There were no significant differences in symptom scores, pulmonary function data, and exacerbations between active and placebo treatments. CONCLUSION: No significant steroid-sparing effect was obtained using low dose methotrexate in this study. This negative outcome may be attributable to the small population of patients studied, low baseline FEV1, and the omission of a steroid minimisation run-in period. Our results highlight the importance of careful patient selection and a painstaking approach in the management of patients with steroid-dependent asthma.     

Serum lipids and incidence of coronary heart disease. Findings from the Systolic Hypertension in the Elderly Program (SHEP)

BACKGROUND: The association of serum lipids with coronary heart disease has been studied extensively in middle-aged men and, to a lesser extent, in similar women. Less well defined are lipid variables predictive of CHD in individuals of age > or = 60 years. METHODS AND RESULTS: The Systolic Hypertension in the Elderly Program recruited 4736 persons (mean age, 72 years; 14% were black; and 43% were men). Mean systolic and diastolic blood pressures were 170 and 77 mm Hg, respectively. Baseline mean total cholesterol was 6.11 mmol/L (236 mg/dL); HDL cholesterol, 1.39 mmol/L (54 mg/dL); and non-HDL cholesterol, 4.72 mmol/L (182 mg/dL). Triglyceride levels were 1.62 mmol/L (144 mg/dL) for fasting participants and 1.78 mmol/L for the total group. LDL cholesterol, estimated in fasting samples with triglycerides of < 4.52 mmol/L, averaged 3.98 mmol/L (154 mg/dL). Mean follow-up was 4.5 years. In multivariate Cox regression analyses, baseline total, non-HDL, and LDL cholesterol levels and the ratios of total, non-HDL, and LDL to HDL cholesterol were significantly related to CHD incidence. HDL cholesterol and triglycerides were not significant in these analyses. In fasting participants with triglyceride levels of < 4.52 mmol/L, a 1.03 mmol/L (40 mg/dL) higher baseline total, non-HDL, or LDL cholesterol was associated with a 30% to 35% higher CHD event rate. CONCLUSIONS: The results of this study support the concept that serum lipids are CHD risk factors in older Americans.