bluuebunny/crossref_metadata_2025_split huggingface dataset:
Binding and distribution of fluorescently labeled filamin in permeabilized and living cells
Authors: Mittal Balraj , Sanger Jean M. , Sanger Joseph W. 
Published: February 2005

AbstractThis study report the first development of a fluorescently labeled filamin. Smooth muscle was labeled with fluorscent dyes in order to study its interaction with stress fibers and myofibrils, both in living cells and in permeabilized cells. The labeled filamin bounds to the Z bands of isolated cross‐striated myofibrils and to the Z bands and intercalated discs in both permeabilized embryonic cardiac myocytes and in frozen sections of adult rat venticle. In permeabilized embryonic chick myotubes, filamin bound to early myotubes but was absent at later stages. In living embryonic chick myotubes, the fluorescently labeled filamin was incorporated into the Z bands of myofibirls during early and late stages of develoment but was absent during an intermediate stages. In living cardiac myocytes, filamin‐IAR was incorporated into nascent as well as fully formed sarcomeres throughout develoment. In permeabilized nonmuslce cells, labeled filamin bound to attachment plaques and foci of polygonal networks and to the dense bodies in stress fibers. The periodic bands of filamin in stress fibers had a longer spacing in fibroblasts than in epithelial cells. When injected into living cells, filamin was readily incorporated into stress fibers in a striated pattern. The fluorescent filamin bands were broader in injected cells, however, than they were in permeabilized cells. We have interpreted these results from living and permeabilized cells to mean that native filamin is distributed along the full lengh of the actin filaments in the stress fibers, with a higher concentration present in the dense bodies. A sarcomeric model is presented indicating the position of filamin with respect to other proteins in the stress fibers.
Structure and mass analysis of 12S and 19S dynein obtained from bull sperm flagella
Authors: Marchese‐Ragona Silvio P. , Gagnon Claude , White Daniel , Isles Marthe Belles , Johnson Kenneth A. 
Published: February 2005

AbstractThe Brookhaven scanning transmission electron microscpe (STEM) was used to elucidate the structures and masses of 12S and 19S dynein extracted from bull sperm flagella. The 12S particle was a single globular particle with an average mass of 311 ± 10 kdaltons. The 19S dynein particles consisted of two globular heads joined to a common base. The average mass of the 19S particle was 1.6 ± 0.04 × 106 daltons. Thus, with the exception of the larger mass, the bull sperm 19S dynein molecule resembles the two‐headed 21S dynein obtained from sea urchin sperm flagella and the 18S dynein obtained from Chlamydomonas with the possibility of a third head giving rise to the 12S particle. The structure, mass and polypeptide composition of bull sperm flagella dynein is compared with outer arm dyneins previously obtained from Chlamydomonas, Tetrahymena, and sea urchin sperm flagella.
The position of the microtubule‐organizing center relative to the nucleus is independent of the direction of cell migration in dictyostelium discoideum
Authors: Sameshima Masazumi , Imai Yoshihisa , Hashimoto Yohichi 
Published: February 2005

AbstractDictyostelium amoebae can migrate in several different modes. We tested for correlations of the direction of cell locomotion with the relative positions of the nucleus and microtubule‐organizing center (MTOC). Five cases were analyzed on electron micrographs with a microcomputer. Each mode of movement showed characteristic locations of the MTOC relative to the nucleus; however, they differed in the various cases. In randomly migrating interphase amoebae, the number of cells with the MTOC located behind the nucleus was twice as great as those with the MTOC located ahead of the nucleus. During chemotactic migration toward folic acid, cells with the MTOC behind the nucleus were more numerous, with a concomitant reduction of anterior MTOCs.When amoebae aggregated on agar plates, a posterior location of the MTOC was most strikingly favored, whereas in cells aggregating under submerged conditions, the MTOC was indifferently anterior or posterior to the nucleus. (It may be significant that EDTA‐resistant cell‐cell adhesion was fully expressed in the former cells, but weaker in the latter.) Finally, in the case of chemotactically migrating cells from dissociated pseudoplasmodia, which adhere by means of other molecules, the MTOC was consistently ahead of the nucleus. Thus the MTOC shows no necessary preferential position anterior or posterior to the nucleus; its position, rather, correlates with the type of migration and perhaps with the nature of cell‐cell adhesion.
A monoclonal antibody to the Ca2+ ‐ATPase of cardiac sarcoplasmic reticulum cross‐reacts with slow type I but not with fast type II canine skeletal muscle fibers: An immunocytochemical and immunochemical study
Authors: Jorgensen Annelise O. , Arnold Wayne , Pepper David R. , Kahl Steven D. , Mandel Frederick , Campbell Kevin P. 
Published: February 2005

AbstractCa2+ ‐ATPase of the sarcoplasmic reticulum was localized in cryostat sections from three different adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with monoclonal antibodies to the Ca2+ ‐ATPase Type I (slow) myofibers were strongly labeled for the Ca2+ ‐ATPase with a monoclonal antibody (II D8) to the CA2+ ‐ATPase of canine cardiac sarcoplasmic reticulum; the type II (fast) myofibers were labeled at the level of the background with monoclonal antibody II D8. By contrast, type II (fast) myofibers were strongly labeled for Ca2+ ‐ATPase of rabbit skeletal sarcoplasmic reticulum. The subcellular distribution of the immunolabeling in type I (slow) myofibers with monoclonal antibody II D8 corresponded to that of the sarcoplasmic reticulum as previously determined by electron microscopy. The structural similarity between the canine cardiac Ca2+ ‐ATPase present in the sarcoplasmic reticulum of the canine slow skeletal muscle fibers was demonstrated by immunoblotting. Monoclonal antibody (II D8) to the cardiac Ca2+ ‐ATPase binds to only one protein band present in the extract from either cardiac or type I (slow) skeletal muscle tissue. By contrast, monoclonal antibody (II H11) to the skeletal type II (fast) Ca2+ ‐ATPase binds only one protein band in the extract from type II (fast) skeletal muscle tissue. These immunopositive proteins coelectrophoresed with the Ca2+ ‐ATPase of the canine cardiac sarcoplasmic reticulum and showed an apparent Mr of 115,000. It is concluded that the Ca2+ ‐ATPase of cardiac and type I (slow) skeletal sarcoplasmic reticulum have at least one epitope in common, which is not present on the Ca2+ ‐ATPase of sarcoplasmic reticulum in type II (fast) skeletal myofibers. It is possible that this site is related to the assumed necessity of the Ca2+ ‐ATPase of the sarcoplasmic reticulum in cardiac and type I (slow) skeletal myofibers to interact with phosphorylated phospholamban and thereby enhance the accumulation of Ca2+ in the lumen of the sarcoplasmic reticulum following β–adrenergic stimulation.
Analysis of bacterial flagellar rotation
Authors: Khan Shahid 
Published: February 2005

AbstractBacterial flagella have rotary motors at their base; embedded in the cytoplasmic membrane and powered by transmembrane ion gradients instead of ATP. Assays have been developed to measure the torque output of individual motors over a wide regime of load, to correlate the energizing proton flux with rotation speed and relate through genetic analysis motor structure to function. These assays promise substantial advances in understanding mechanochemical coupling in these motors. Here, I summarize the present status of our understanding of energy transduction in bacterial flagella and compare this with the case for muscle.
High‐performance optical filters for fluorescence analysis
Authors: Marcus David A. 
Published: February 2005

AbstractRecent advances in thin film optical coating technology significantly improve the filters available for fluorescence spectroscopy. Bandpass and long‐ and shortpass filters with very sharply defined edges can provide from 10−5 to 10−6 blocking within 10–15 nm of the transmission region and are ideal for use as excitation and emission filters. A variety of nonpolarizing dichroic beamsplitters for use in epi‐illumination configurations or in multiple emission configurations provides optimum longpass, shortpass, band reflection, or bandpass spectral control. These dichroics, used with high‐performance bandpass, longpass, or shortpass filters, form matched sets that optimize the signal‐to‐noise ratio and system efficiency for fluorescence spectroscopic systems in single or multiple dye applications. Specially designed dichroic beamsplitters are used to reduce excitation filter overheating. Other dichroic beamsplitters efficiently separate two planes of polarization in a narrow wavelength band. Rejection band filters can be used to measure the fluorescent dye Indo 1 with very low emission signals.
In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg
Authors: Rebhun Lionel I. , Palazzo Robert E. 
Published: February 2005

AbstractSpindles may be isolated from sea urchin eggs so that some mitotic processes can be reactivated in vitro. The isolation media allow spindles to remain stable for days. Transfer of the spindles to reactivation media results in loss of birefringence and breakdown of the matrix within which the microtubules function. If, however, tubulin and either guanosine triphosphate or adenosine triphosphate are present in these media so that tubulin can cycle, the spindles do not break down but grow in size and birefringence and show some of the movements of in vivo spindles. The most prominent is that of anaphase B if the mitotic apparatuses (MAs) have been isolated at a time when anaphase was initiated. When isolated during metaphase, MAs either do not show chromosome movement or, if they do, it is a random movement which causes redistribution of the chromosomes on the spindle surface. In either case, such metaphase spindles grow in size and birefringence. Thus under the proper conditions, cycling microtubules can interact with the spindle matrix to induce chromosome movements which resemble those seen in in vivo cells in the case of anaphase B and show some aspects of anaphase A in at least half the spindles isolated at metaphase, although such movements are not coordinated to show a true anaphase movement.
Isolation and reactivation of highly‐coupled newt lung cilia
Authors: Hard Robert , Cypher Christopher , Schabtach Eric 
Published: February 2005

AbstractThe paired lungs of the newt, Taricha granulosa, are simple, unbranched sacs, 3.5–5.0 cm in length. The inner epithelium overlying the pulmonary vein is differentiated into a mucociliary tract that extends the entire length of the lung. Populations of single, demembranated ciliary axonemes, 12–13 μm in length, can be isolated by extracting whole lungs or primary cultures of the ciliated epithelium with Triton X‐100. The motile capabilities of the isolated axonemes are the highest yet obtained for any ciliary model. When exposed to a suitable reactivating medium containing Mg2+ and ATP, nearly 100% of the axonemes become motile. Uniform reactivation of high quality requires short extraction times, minimization of mechanical damage, and strict adherence to optimal conditions throughout the extraction, storage, and reactivation procedures. Significant deviations from either pH 7.0 or 0.12 M salt can lead to a rapid, irreversible decrease in the beat frequency of reactivated axonemes. Both DTT and EDTA serve to stabilize their motility. The isolated axonemes beat at 29.5 Hz in the presence of 1.75 mM ATP at 21°C, matching the beat frequencies measured for cultured cells at the same temperature. With 5 mM ATP, beat frequencies over 40 Hz are measured. Our results show that neither the plasma membrane, accessory structures, nor hydrodynamic coupling of cilia are required for this activity and imply that the lack of these factors is not responsible for the low motile capabilities of ciliary models isolated previously.
Fast axonal transport alterations in amyotrophic lateral sclerosis (ALS) and in parathyroid hormone (PTH)‐treated axons
Authors: Breuer Anthony C. , Atkinson Mark B. 
Published: February 2005

AbstractVideo‐enhanced contrast techniques have been used to study fast axonal transport of organelles in diseased and normal human axons. A broad perspective on the importance of axonal transport in the pathogenesis of human neurological disorders is presented and problems in dealing with human nerve summarized. Results from analysis of organelle traffic in axons from motor nerve in patients with amyotrophic lateral sclerosis (ALS) show: (1) higher mean speed of anterograde organelles, (2) lower mean speed of retrograde organelles, and (3) lower retrograde organelle traffic density. Hyperparathyroidism, another human clinical syndrome, can mimic ALS. The effect of parathyroid hormone (PTH) on axons in vitro is to increase the mean speed of both anterograde and retrograde organelle traffic. The dose response curve and time course of the PTH effect are delineated. Dihydropyridine calcium channel antagonists block the PTH effect, implicating extracellular calcium in the alteration of organelle traffic speed. The results are discussed in relation to neuronal function and the regulation of fast axonal transport.
Involvement of myxamoebal fragmin in the Ca2+‐induced reorganization of the microfilamentous cytoskeleton in flagellates of Physarum polycephalum
Authors: Uyeda Taro Q. P. , Hatano Sadashi , Furuya Masaki 
Published: February 2005

AbstractWhen flagellates of Physarum polycephalum were treated with Triton X‐100 and more than 10−5 M Ca2+, the microfilamentous cytoskeleton disintegrated, as seen by staining with rhodamine‐phalloidin, and myxamoebal fragmin became associated with the Triton‐insoluble cytoskeleton as demonstrated by immunofluorescence microscopy and immunoblotting. The association of myxamoebal fragmin with the cytoskeleton was reversed by the subsequent addition of excess EGTA. When flagellates were permeabilized in the absence of Ca2+, myxamoebal fragmin did not associate with the cytoskeleton and diffused out of the cells. Subsequent treatment of these cells with Ca2+ was ineffective in inducing either the association of myxamoebal fragmin with the cytoskeleton or the disintegration of the microfilamentous cytoskeleton. However, treatment of these permeabilized flagellates with 10 μg/ml purified myxamoebal fragmin and 1 mM Ca2+ caused the disintegration of the microfilaments. Therefore, we conclude that myxamoebal fragmin participates in the Ca2+‐induced disintegration of the microfilamentous cytoskeleton in these permeabilized cells. Rapid cooling of flagellates caused the reversible association of myxamoebal fragmin with the Triton‐insoluble cytoskeleton in vivo. Thus myxamoebal fragmin may also participate in the reorganization of the microfilamentous cytoskeleton induced in vivo by the cold treatment.
Cell‐cycle modulation of MPM‐2‐specific spindle pole body phosphorylation in Aspergillus nidulans
Authors: Engle Dorothy B. , Doonan John H. , Morris N. Ronald 
Published: February 2005

AbstractMPM‐2 is a monoclonal antibody that interacts with mitosis‐specific phosphorylated proteins in many different organisms. Immunocytochemistry of tissue culture cells has shown that MPM‐2 stains centrosomes, chromosomes, kinetochores, and spindles. In this paper, we demonstrate that MPM‐2 staining colocalizes with the spindle pole body (SPB) of Aspergillus nidulans and that SPB staining varies during the mitotic cycle. In an unsynchronized population, about one‐fourth to one‐third of the cells stain with MPM‐2 at the spindle plaques or SPBs. Nuclei in mitosis have two SPBs localized at the ends of the spindle, both of which stain with MPM‐2. To determine when MPM‐2 staining appears, we have examined the effects of temperature‐sensitive cell‐cycle mutations that block nuclear division in S or G2. Only a very small fraction of cells blocked in S‐phase stain with MPM‐2. In contrast, a large fraction of cells blocked in G2 stain brightly at the SPB. These data suggest that MPM‐2 reactivity of SPBs appears in G2. Moreover, the fact that cells blocked in G2 showed MPM‐2 staining but no spindles suggests that reactivity of SPBs occurs prior to mitosis but is not sufficient to trigger spindle formation. When G2‐blocked cells were downshifted to permissive temperature, they generated a mitotic spindle with an SPB at each end. Both SPBs stained with MPM‐2 in all of the mitotic cells.
Protease inhibitor and substrates block motility and microtubule sliding of sea urchin and carp spermatozoa
Authors: Cosson Marie‐Paule , Gagnon Claude 
Published: February 2005

AbstractThe effects of protease substrates and inhibitor, which have been previously shown to inhibit mammalian sperm motility (de Lamirande, E., and Gagnon, C. [1986] J. Cell Biol. 102:1378–1383), were investigated using reactivated sea urchin and carp spermatozoa as models of “9 + 2” flagella. Aprotinin in the 2 to 20 μM range interfered with sperm motility by reducing both the beat frequency and the percentage of motile spermatozoa. These inhibitory effects of aprotinin were reversible either by dilution or by the addition of high concentrations of MgATP to the incubation medium. Protease substrates with a lys–ester bond, such as N‐α‐benzyloxycarbonyl‐lys thiobenzyl ester (BLT), also affected motility, but in the 0.1 to 0.5 mM range. As with aprotinin, both the flagellar beat frequency and the percentage of motile spermatozoa were partially and completely decreased, respectively. Analysis of the beat frequencies as a function of MgATP concentration in the presence and absence of 6 μM aprotinin indicated that this protease inhibitor affects sperm motility by decreasing the maximal flagellar beat frequency rather than by altering the axoneme's apparent Km for MgATP. Furthermore, aprotinin concentrations that blocked flagellar reactivation completely inhibited the sliding of microtubules from trypsinized axonemes. Basic proteins or polypeptides of pI close to that of aprotinin (10.3) were also potent inhibitors of the reactivation of motility. However, the characteristics of their inhibition of flagellar beat frequencies and reversibility of their effects suggested that they might be acting on sites different from those sensitive to aprotinin. The inhibitory effects of protease inhibitor and substrates, as well as results of experiments showing the absolute requirement of an intact ester bond for the inhibitory action of protease substrates, suggest that the involvement of a protease in the reactivation of 9 + 2 flagellar beating might be considered as a possible mechanism to explain aprotinin and BLT actions.
Acrylamide treatment of PtKl cells causes dephosphorylation of keratin polypeptides
Authors: Eckert Barry S. , Yeagle Philip L. 
Published: February 2005

AbstractTreatment of PtKl cells with 5 mM acrylamide for 4 hr results in alterations in the distribution of keratin filaments within the cells. This effect is reversible within 18 hr. Labeling of PtKl cells with 32P demonstrates that there are four phosphorylated keratins, having Mr of 56 k, 53 k, 45 k, and 40 k. Phosphate associated with these polypeptides appears to turn over with a t1/2 of 12 hr. Incubation of labeled cells in 5 mM acrylamide results in approximately 50% dephosphorylation of keratins within 2 hr, which is 3 times faster than normal turnover. Recovery of cells from acrylamide is accompanied by rephosphorylation of keratins within 18 hr. Analysis by 31P NMR spectroscopy shows that acrylamide treatments are accompanied by a transient decrease in soluble inorganic phosphate. This is followed by a rapid increase in Pi which gradually returns to normal levels. These studies show a strong correlation between phosphorylation of PtKl cell keratins and morphological response of keratin filaments to acrylamide. These observations suggest that normal distribution of keratin filaments may be, in part, mediated by protein phosphorylation.
Adhesion of cells to protein carpets: Do cells' feet have to be black?
Authors: Opas Michal 
Published: February 2005

AbstractIn most physiological situations, cell contact with a substratum is mediated by proteins of extracellular matrix. Therefore, an increasing number of cellsubstratum adhesion studies employ substrata covered with one or more proteins of extracellular matrix. To visualize the most adhesive cell structures, focal contacts and focal adhesions, the interference reflection microscopy has been widely used. It has been generally accepted that these strongly adhesive structures can be seen as black streaks in interference reflection microscopy. Calculations are presented herein, which although simplified, suggest that when cells are plated on protein‐covered substrata, their focal contacts may not always appear black in interference reflection microscopy.
Centripetal transport of cytoplasm, actin, and the cell surface in lamellipodia of fibroblasts
Authors: Fisher Gregory W. , Conrad Patricia A. , Debiasio Robbin L. , Taylor D. Lansing 
Published: February 2005

AbstractWound healing in Swiss 3T3 cultures was investigated with video‐enhanced contrast (VEC) microscopy. The formation of protrusions at the leading edge of cells along wounds was investigated in detail during the spreading stage, which usually lasted from 1 to 4 hr postwounding. Lamellipodia exhibited a continuous rearward, or centripetal, transport of a variety of cellular constituents at rates of ∼0.26 μm/sec from the leading edge. The lamellipodia were also the sites of lateral migration as well as extension and retraction of actin microspikes. Actin fibers oriented transversely to the direction of movement were also observed to transport centripetally at similar rates. These fibers may in part give rise to large actin fibers forming at the interface between the base of the lamellipodia and the lamellae. Beads 0.5 μm in diameter attached to the dorsal surfaces of lamellipodia also transported centripetally at rates of ∼0.21 μm/sec. Thus there is an apparent correlation between transport of a variety of structures within lamellipodia and with surface movements of lamellipodia.