Calpain regulates actin remodeling during cell spreading.

Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.

Clinical application of ketamine ointment in the treatment of sympathetically maintained pain.

The objective of this study was to determine the clinical efficacy of topical ketamine in relieving sympathectically maintained pain, including complex regional pain syndrome types I and II, involving the upper and/or lower extremities. In an open clinical pilot study of five referral patients diagnosed with sympathetically maintained pain who were unresponsive to conventional modalities, a single dose of topical ketamine was administered. Age, gender, duration or degree of disease and concurrent medication were not controlled. Ketamine was prepared for transdermal delivery in pluronic lethicin organogel (PLO) in calibrated applicators. Concentrations ranged from 10 to `50 mg/mL. Dosage ranged from 10 mg to 700 mg per single application. Each dosage was deternined clinically based on location and suface area of involvement. Pain intensity was measured using a validated mumeric analogue scale (NAS). Ketamine in PLO applied to the uper and/or lower extremities with sympathetically maintained pain resulted in significant pain reduction relative to pretreatment NAS of 65% to 100% . Initial response was within 20 seconds to three minutes, with NAS rating 15 minutes postapplication. No reported side effect occurred on patient follow-up at 24 and 48 hours. Single-dose, topical application of ketamine in PLO (patent pending) appears clinically effective in relieving sympathetically maintained pain of the extremities without apparent side effects. Further controlled studies are warranted to define patient selection, optimize dosage and validate the prominent analgesic effects obtained in this heretofore difficult-to-treat pain syndrome. This was an independent study in joint cooperation with representatives from the Univesity of California at Irvine, Loma Linda University Medical Center and private practice.

Phenylethylamine and piperidine alkaloids in aloe species.

Of the approximately 300 species of ALOE (Liliaceae) native to Africa and Arabia, leaf extracts of 224 species have been examined chromatographically for alkaloids using ninhydrin, Dragendorff's reagent, nitroprusside, and iodoplatinate as revealing agents. From these, 48 (21%) species contained compounds giving a strong colorimetric reaction with at least one of these reagents. Tyramine derivatives were identified in 18 species and piperidine derivatives in a further 6 species. The other coloured zones remain unidentified but often appeared to represent common amino acids. Some taxonomic correlations are suggested. The presence of the toxic hemlock alkaloids in aloes readily available for potential medicinal use by local inhabitants sounds a note of caution against the unthinking use of these otherwise useful plants.