Purification of native myosin filaments from muscle

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.     

Evaluation of bone height in osseous free flap mandible reconstruction: an indirect measure of bone mass

Osseous free flaps have become the preferred method of mandibular reconstruction after oncologic surgical ablation. To elucidate the long-term effects of free flap mandibular reconstruction on bone mass, maintenance or reduction in bone height over time was used as an indirect measure of preservation or loss in bone mass. Factors potentially influencing bone mass preservation were evaluated; these included site of reconstruction (central, body, ramus), patient age, length of follow-up, adjuvant radiotherapy, and the delayed placement of osseointegrated dental implants. A retrospective analysis of patients undergoing osseous free flap mandible reconstruction for oncologic surgical defects between 1987 and 1995 was performed. Postoperative Panorex examinations were used to evaluate bone height and bony union after osteotomy. Fixation hardware was used as a reference to eliminate magnification as a possible source of error in measurement. There were 48 patients who qualified for this study by having at least 24 months of follow-up. There were 27 male and 21 female patients, with a mean age of 45 years (range, 5 to 75 years). Mandibular defects were anterior (24) and lateral (24). Osseous donor sites included the fibula (35), radius (6), scapula (4), and ilium (3). There were between zero and four segmental osteotomies per patient (excluding the ends of the graft). Nineteen percent of all patients had delayed placement of osseointegrated dental implants. Initial Panorex examinations were taken between 1 and 9 months postoperatively (mean, 2 months). Follow-up Panorex examinations were taken 24 to 104 months postoperatively (mean, 47 months). The bony union rate after osteotomy was 97 percent. Bone height measurements were compared by site and type of reconstruction. The mean loss in fibula height by site of reconstruction was 2 percent in central segments, 7 percent in body segments, and 5 percent in ramus segments. The mean loss in bone height after radial free flap mandible reconstruction was 33 percent in central segments and 37 percent in body segments; ramus segments did not lose height. The central and body segments reconstructed with scapular free flaps did not lose height, but one ramus segment lost 20 percent of height. There was no loss in bone height in mandibular body reconstruction with the ilium free flap. Fibula free flaps did not significantly lose bone height when evaluated with respect to age, follow-up, radiation therapy, or dental implant placement. The retention in bone height demonstrated in this study suggests that bone mass is preserved after osseous free flap mandible reconstruction. The greatest amount of bone loss was seen after multiply osteotomized radial free flaps were used for central mandibular reconstruction. The ability of the fibula free flap to maintain mass over time, coupled with its known advantages, further supports its use as the "work horse" donor site for mandible reconstruction.     

Interaction of two phenothiazine derivatives with phospholipid monolayers

This paper addresses the cooperative interaction of two phenothiazine drugs, viz. trifluoperazine (TFP) and chlorpromazine (CPZ), with phospholipid monolayers as the model membrane system. Surface pressure and surface potential isotherms were obtained for mixed Langmuir monolayers of either dipalmitoyl-phosphatidyl-choline (DPPC) or dipalmitoyl-phosphatidyl-glycerol (DPPG) co-spread with TFP or CPZ. The changes in monolayer behavior caused by incorporation of a few molar ratio of drug molecules were practically within the experimental dispersion for the zwitterionic DPPC, and therefore a more refined analysis will be required to probe the interactions in an unequivocal way. For the charged DPPG, on the other hand, the surface pressure and the dipole moment were significantly affected even for TFP or CPZ concentrations as low as 0.002 molar ratio. Overall, the effects from CPZ and TFP are similar, but small differences exist which are probably due to the different protonation properties of the two drugs. For both drugs, changes are more prominent at the liftoff of the surface pressure, i.e. at the gas-condensed phase transition, with the surface pressure and surface potential isotherms becoming more expanded with the drug incorporation. With DPPG/CPZ monolayers, in particular, an additional phase transition appears at higher CPZ concentrations, which resembles the effects from increasing the subphase temperature for a pure DPPG monolayer. The dipole moment for DPPG/CPZ and DPPG/TFP monolayers decreases with the drug concentration, which means that the effects from the charged drugs are not associated with changes in the double-layer potential. Otherwise, the effective dipole moment should increase with the drug concentration. The changes caused in surface pressure and dipole moment by small concentrations of TFP or CPZ can only be explained by some cooperative effect through which the contribution from DPPG molecules changes considerably, i.e. even DPPG molecules that are not neighbor to a CPZ or TFP molecule are also affected. Such changes may occur either through a significant reorientation of the DPPG molecules or to a change in their hydration state. We discuss the cooperativity semi-quantitatively by estimating the number of lipid molecules affected by the drug interaction. CPZ and TFP also affect the morphology of DPPG monolayers, which was confirmed with Brewster angle microscopy. The biological implications from the cooperative, non-specific interaction of CPZ and TFP with membranes are also commented upon.     

Redox regulation of RyR-mediated Ca2+ release in muscle and neurons

Changes in the redox state of the intracellular ryanodine receptor/Ca2+ release channels of skeletal and cardiac muscle or brain cortex neurons affect their activity. In particular, agents that oxidize or alkylate free SH residues of the channel protein strongly enhance Ca(2+)-induced Ca2+ release, whereas reducing agents have the opposite effects. We will discuss here how modifications of highly reactive cysteine residues by endogenous redox agents or cellular redox state influence RyR channel activation by Ca2+ and ATP or inhibition by Mg2+. Possible physiological and pathological implications of these results on cellular Ca2+ signaling will be addressed as well.     

Body size and fecundity of Juxtafabia muliniarum (Brachyura: Pinnotheridae) associated with Saccostrea palmula (Bivalvia: Ostreidae), Costa Rica

Size and fecundity observations of pea crab (Juxtafabia muliniarum) from the paleal cavity of the oyster Saccostrea palmula were made from May 1998 to May 1999. Infestation frequency was 18.52% in a sample of 540 oysters. Of 136 pea crabs, 36% were couples, 60% were single females and 4% were single males. The mean caparace length of J. muliniarum was 5.6 +/- 0.74 mm (range 4.0 to 7.6 mm) for females and 2.71 +/- 0.60 mm (range 1.6 to 4.0 mm) for males. The mean weight was 0.180 +/- 0.084 g (range 0.06 to 0.4 g) for females and 0.011 +/- 0.003 g (range 0.01 to 0.02 g) for males. Ovigerous females (43.75% of all females) were found in all months. The caparace length-fecundity relationship was F = 3904.6 Ln (Lc)--4651.1. The caparace length-weight relationship was P = 6 x 10(-4) Lc3.2122. The mean sex-ratio was 1.0 male: 2.4 females. Saccostrea palmula infected only by females was the dominant group (60.78%). This mollusk is a new host record for the crab.     

Evolutionary maintenance of stigma-height dimorphism in Narcissus papyraceus (Amaryllidaceae)

Stigma-height dimorphism is a sexual polymorphism in which plant populations are composed of two floral morphs that differ significantly in style length but not anther position. The morphs exhibit approach and reverse herkogamy, floral designs that in most species typically occur as monomorphic conditions. We investigated the floral biology of stigma-height dimorphism in the Mediterranean geophyte Narcissus papyraceus (Amaryllidaceae) in an effort to understand the evolutionary forces maintaining stylar polymorphism. Our survey of 66 populations in Spain, Portugal, and Morocco indicated that 56% were dimorphic with the long-styled morph at an average frequency of 0.79. The remaining 44% of populations sampled were monomorphic for the long-styled morph. In dimorphic populations there was a significant positive relation between population size and the frequency of the short-styled morph. Controlled pollinations demonstrated that N. papyraceus is self-sterile with no significant differences in female fertility between intra- and intermorph crosses. Prior self-pollination reduced seed set in flowers that were subsequently cross-pollinated. Estimates of mating patterns using allozyme markers in eight populations indicated that N. papyraceus is largely outcrossing (mean t(m) = 0.81) with no significant differences between monomorphic and dimorphic populations or style morphs. Stigma-height dimorphism in N. papyraceus is maintained in populations by insect-mediated cross-pollination with biased morph ratios and stylar monomorphism likely resulting from the combined influence of the inheritance of the polymorphism, morph-specific differences in assortative mating and founder effects.