Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.      

The physiological basis of seed dormancy in Avena fatua. II. On the involvement of alternative respiration in the stimulation of germination by sodium azide

Chromatography on DEAE-cellulose of an extract from etiolated leaves of sorghum ( Sorghum vulgare Pers. cv. INRA 450), a C₄ plant, gave only one form of phosphoenol pyruvate carboxylase with functional and regulatory properties of a C₃ type plant enzyme. Greening of the leaves resulted in a significant increase in activity. This increase was due to the appearance of a new form of the enzyme, which eluted at lower ionic strength and exhibited new properties. This form was glucose-6-P activated and showed a sigmoidal curve response to the concentration of the substrate phosphoerralpyruvate. These kinetic properties are typical of a C₄ plant enzyme.     

WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 μM). This effect was not detected for tolaasin I at the concentrations tested (< 28 μM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.     

Kaolin-based particle film technology affects tomato physiology,yield and quality

Environmental stress can affect development and yield of tomato plants. This study was undertaken to investigate the underlying mechanism asserted by kaolin on tomato physiology by evaluating its effect on leaf, canopy and inner fruit temperatures, gas exchange at the leaf and canopy scales, above ground biomass, yield and fruit quality.The study was carried out under field conditions in Southern Italy. Treatments were plants treated with kaolin-based particle film (Surround^® WP) suspension and untreated plants (control).Kaolin application slightly increased leaf and canopy scale temperatures by 1.0 and 0.4 °C, respectively, transpiration rate decreased at both scales. On calm days (wind speed <0.5 m s^?1) with a prevalently clear sky at midday, inner fruit temperature (t_f) of kaolin-treated plants was 4.4 °C lower than the t_f of control plants, while in days with clear sky-windy, and cloudy-calm, the t_f did not differ.At leaf scale, net assimilation was reduced by 26% in kaolin-coated treatments. Stomatal conductance decreased by 53%, resulting in reductions of 34 and 15% in transpiration and internal CO₂ concentration, respectively. Gas exchange parameters measured at canopy scale were similarly affected. In kaolin-treated plants, assimilation and evapotranspiration rates were reduced by 17 and 20%, respectively, while dark respiration was not affected. Above ground dry biomass decreased by 6.4%.Marketable yield in kaolin-treated plants was 21% higher than those measured in control plants; this is possibly related to the 96 and 79% reduction in sunburned fruit and those damaged by insects, respectively, and to the 9% increase in mean fruit weight. Kaolin treatment increased lycopene fruit content by 16%, but did not affect total soluble solids content, fruit dry matter, juice pH, titratable acidity or tomato fruit firmness. The use of kaolin-based particle film technology would be an effective tool to alleviate heat stress and to reduce water stress in tomato production under arid and semi-arid conditions.     

Sedentary life style of Neotropical sedge wrens promotes song imitation

To what extent has the style of song development among songbirds coevolved with other life history strategies? Among Cistothorus wrens in North America, it seems that sedentary or site-faithful habits of marsh wrens, C. palustris, favour song imitation, but seminomadic habits of sedge wrens, C. platensis, favour song improvisation, whereby each male generates a large but unique song repertoire. In this study, we tested whether more sedentary populations of sedge wrens in the Neotropics would imitate songs. At our primary study site near Cartago, Costa Rica, breeding birds were colour-banded during 1995 and 1996, and follow-up surveys revealed that the birds remained at this site the year round. Extensive tape recording and analysis of songs showed that males had large song repertoires (200-300+ songs), and that many songs were shared among neighbouring males. In addition, males only 27 km distant, at La Pastora, used different songs. Furthermore, matched countersinging, in which two males answer each other with identical song types, was recorded near Brasilia, in Brazil. The sharing of songs among permanent neighbours, microgeographical variation in song, and matched countersinging can be achieved only through song imitation, thus revealing a striking difference in the style of song development among different populations of the sedge wren. In the Neotropics, having predictable neighbours throughout life appears to have favoured song imitation, so that individuals can interact using a common, learned code typical of the local population; among more mobile populations in North America, however, individuals improvise large repertoires of species-typical songs, thereby enabling singing males to communicate with any individual, no matter what the population of origin. Strategies of song development must correlate with life history features, and further surveys are needed to make sense of the great diversity of singing behaviours among songbirds. Copyright 1999 The Association for the Study of Animal Behaviour.     

Pollination ecology and breeding system of the very narrow coastal endemic Seseli farrenyi (Apiaceae). Effects of population fragmentation

The reproductive biology of Seseli farrenyi (Apiaceae), a very narrow endemic to Cape Creus (Catalonia, Spain), including flowering timing patterns, quantity and quality of pollination services (type and frequency of pollinators, pollen carryover, pollen deposition on stigmas and reproductive success measured as fruit set), and breeding system was studied. Given the decline of population size detected in the last twenty years, we also analyzed the effects of fragmentation on pollination mechanisms. Protandry along with strong synchrony of floral development within umbels and sequential inflorescence emission within individual stalks, produces sexual phase alternation that promotes a strong outcrossing despite its non-specific pollination system and its (at least partial) self-compatibility. This pronounced xenogamy is supported by results of the insect exclusion test, hand-pollination experiments, and high P/O ratio. S. farrenyi flowers received visits from at least 28 species of insects, including wasps, small bees, ants, flies, syrphid flies, beetles and stink bugs, with different pollen carry-overs. Heterospecific pollen on stigmas decreased notably during the season (50% to 2.5%), averaging 12%. In the small population the stigmatic pollen loads and seed set decreased, but there was no effect of pollinator visitation rates. It was more affected by the composition of pollinators and their efficiency. The wind had a considerable effect on the plant. Some conservation measures are proposed.     

Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.     

Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.