bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in the effect of Ca2+ on isolated microtubules from cod and cow brain
Authors: Strömberg Elisabeth , Wallin Margareta
Published: February 2005

Isolated microtubules from cod and cow brains were compared with respect to their response to calcium ions. The effect of Ca2+ on cod microtubules was found to be temperature dependent. In contrast to cow microtubules, cod microtubules assembled at 18°C. At this temperature the assembly was inhibited by Ca2+ concentrations of 2 mM and higher. This was also found for cow microtubules at 37°C. However, at 30°C there was no effect of 2 mM Ca2+ of the amount of assembly or disassembly of cod microtubules consisting of only tubulin or of tubulin and microtubule‐associated proteins (MAPs). The morphology was affected though, since some coiled ribbons formed from tubulin and MAPs. The calcium‐binding calmodulin did not alter the effect of calcium on cod microtubules markedly. At higher Ca2+ concentrations (>4 mM), coiled ribbons were formed from cod tubulin and MAPs, but mainly amorphous aggregates and very few coiled ribbons were formed from cod tubulin alone, indicating that the Ca2+ effect is modulated by cod MAPs. The modulatory effect of cod MAPs was however not species specific, since both cod and cow MAPs had the same effect on cod microtubules, in spite of a different protein composition. A MAP‐dependent effect of Ca2+ was also found for cow microtubule proteins. The assembly of pure cow tubulin, as well as that of cow tubulin and MAPs, was inhibited by 2 mM Ca2+. In the presence of 10 and 20 mM Ca2+, pure cow tubulin formed amorphous aggregates, rings, and even paracrystals, while the assembly of cow tubulin and MAPs was inhibited. Our results suggest therefore that the effect of Ca2+ can be moderated by MAPs, but depends on intrinsic properties of the different tubulins. © 1994 Wiley‐Liss, Inc.
Intact alpha‐actinin molecules are needed for both the assembly of actin into the tails and the locomotion of Listeria monocytogenes inside infected cells
Authors: Dold Frederick G. , Sanger Jean M. , Sanger Joseph W.
Published: February 2005

After the infectious bacterium, Listeria monocytogenes, is phagocytosed by a host cell, it leaves the lysosome and recruits the host cell's cytoskeletal proteins to assemble a stationary tail composed primarily of actin filaments cross‐linked with alpha‐actinin. The continual recruitment of contractile proteins to the interface between the bacterium and the tail accompanies the propulsion of the bacterium ahead of the elongating tail. When a bacterium contacts the host cell membrane, it pushes out the membrane into an undulating tubular structure or filopodium that envelops the bacterium at the tip with the tail of cytoskeletal proteins behind it. Previous work has demonstrated that alpha‐actinin can be cleaved into two proteolytic fragments whose microinjection into cells interferes with stress fiber integrity. Microinjection of the 53 kD alpha‐actinin fragment into cells infected with Listeria monocytogenes, induces the loss of tails from bacteria and causes the bacteria to become stationary. Infected cells that possess filopodia when injected with the 53 kD fragment lose their filopodia. These results indicate that intact alpha‐actinin molecules play an important role in the intracellular motility of Listeria, presumably by stabilizing the actin fibers in the stationary tails that are required for the bacteria to move forward. Fluorescently labeled vinculin associated with the tails when it was injected into infected cells. Talin antibody staining indicated that this protein, also, is present in the tails. These observations suggest that the tails share properties of attachment plaques normally present in the host cells. This model would explain the ability of the bacterium (1) to move within the cytoplasm and (2) to push out the surface of the cell to form a filopodium. The attachment plaque proteins, alpha‐actinin, talin, and vinculin, may bind and stabilize the actin filaments as they polymerize behind the bacteria and additionally could also enable the tails to bind to the cell membrane in the filopodia. © 1994 Wiley‐Liss, Inc.
Movement of Myzostomum spermatozoa: Calcium ion regulation of swimming direction
Authors: Ishijima Sumio , Ishijima Sanae A. , Afzelius Björn A.
Published: February 2005

Spermatozoa of the small myzostomid worm Myzostomum cirriferum usually swim with the flagellum foremost but occasionally stop and then swim with the head foremost. The spermatozoa have axoneme of the 9+0 type; thus each lacks the central pair microtubules. The flagellum emerges in the anterior end of the cell body and attaches to it with junctions. To understand the mechanism regulating the swimming direction of the spermatozoa, we recorded the sperm and their flagellar movements using a video camera with a high‐speed shutter. The effects of calcium and viscosity on these movements were also examined.

The cell body with the flagellum attached to it formed a curved plate during beating, while the free portion of the flagellum beats with small helical bends. Motive force to propel a spermatozoon was mainly due to the bends in the cell body. The spermatozoa reversed the direction of their swimming as a result of a change in the direction of bend propagation. The direction of bend propagation was regulated by calcium; the bends in the cell body propagated from the end of the head toward the free portion of the flagellum at low concentrations of Ca2+, whereas the direction of bend propagation was reversed at high concentrations of this ion. High viscosity of the medium stimulated a change in the direction of bend propagation. © 1994 Wiley‐Liss, Inc.
Assembly and bundling of marginal band microtubule protein: Role of tau
Authors: Sanchez Ivelisse , Cohen William D.
Published: February 2005

Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22°C. The bundles, which were readily visible by video‐enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6–11 μm in length and 2–5 MTs in thickness. No inter‐MT cross‐bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS‐PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP‐2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt‐extractable and heat‐stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were “unbundled” by subtilisin or by high salt (0.5–1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt‐induced unbundling was reversible with desalting. However, reversibility was observed only after salt‐induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor. © 1994 Wiley‐Liss, Inc.
Effects of 6‐dimethylaminopurine on the length of the cell cycle and on the state of phosphorylation of putative intermediate filament proteins in sea urchin embryos
Authors: St‐Pierre Johanne , Vincent Michel , Dufresne Louise
Published: February 2005

The effects of 6‐dimethylaminopurine (6‐DMAP) on the length of the cell cycle and on the state of phosphorylation of a putative intermediate filament protein, p117, have been studied in sea urchin embryos. Embryos were transferred into sea water containing 600 μM 6‐DMAP at 0.5, 2 or 5 min after insemination, and incubated for 30 or 90 min. The effects of 6‐DMAP on cell cycle length were studied by determining the time required for completion of mitosis upon return of the embryos in normal sea water. In all instances, except for the embryos transferred 0.5 min after insemination (AI) and incubated for 30 min, the duration of the M phase was shortened compared to controls, being faster in the embryos incubated for 90 minutes compared to the 30 min incubation period. However, embryos transferred 0.5 min AI have a longer M‐phase than those transferred 2 minutes or later after fertilization, suggesting that between 0.5 and 2 min after fertilization, critical phosphorylating events occur which affect the commitment of the cells to enter M‐phase.

To study the pattern of p117 phosphorylation during the cell cycle, the eggs were transferred 2 minutes after fertilization in presence of 600 μM 6‐DMAP and with 200 μCi/ml of 32P‐orthophosphate. Analyses of 32P‐labelled proteins after exposure of SDS‐PAGE gels and their corresponding blots suggested that phosphorylation of p117 greatly increases at the time of pronuclear fusion, and then declines slightly at prophase‐metaphase. This decrease is markedly enhanced when the cells are treated with 6‐DMAP during metaphase in order to induce a premature breakdown of the mitotic apparatus. A causal link is suggested between the level of phosphorylation of p117 and its state of assembly. © 1994 Wiley‐Liss, Inc.
Intermittent swimming in the spermatozoa of the lugworm Arenicola marina (L.) (Annelida: Polychaeta)
Authors: Pacey A. A. , Cosson J. C. , Bentley M. G.
Published: February 2005

Motile spermatozoa of the polychaete Arenicola marina were observed to swim intermittently. On the basis of the behaviour of the flagellum, the quiescent periods can be classified into two main types. The first are those in which, although the generation of the flagellar wave appears to be initiated, its passage down the axoneme appears blocked. This results in the formation of an acute bend (of approximately 2.65 rad) in the proximal region of the flagellum with the remainder of the axoneme remaining straight. These have been termed Type I quiescent periods and are very similar to the “cane‐shaped” configuration which has been described in the spermatozoa of some sea urchins. Sperm may also enter a Type II quiescent period, in which both the propagation and the generation of flagellar waves appears blocked. The flagellum of such sperm appears straight or slightly curved and they can remain in this configuration for several minutes. With increased intensity and duration of irradiation, the length of time spent in Type II quiescent period was increased significantly. Both types of quiescent period were (1) reduced in duration and frequency by deletion of calcium from artificial sea water (ASW); (2) either abolished or reduced in duration by the addition of 1 mM cadmium chloride to ASW. In addition, flagellar waveforms very similar to those displayed by spermatozoa in Type I quiescent periods could be induced (if only for a short time) by the addition of the divalent cation ionophore A23187 to ASW. It is suggested that this type of behaviour may be induced following an influx of calcium into the intraflagellar compartment of spermatozoa and that this may be mediated by certain intensities and wavelengths of light. © 1994 Wiley‐Liss, Inc.
Subtraction scanning acoustic microscopy reveals motility domains in cells in vitro
Authors: Veselý P. , Lüers H. , Riehle M. , Bereiter‐Hahn J.
Published: February 2005

Scanning acoustic microscopy (SAM) observes all mechanical properties of living cells. Subtraction of the SAM images (SubSAM) of live cells was developed as a method for investigating minimal changes in cellular topography and elasticity. The image formation in the SubSAM takes into account the motion of cell mass as well as the changes of tension. High spatial and temporal resolution of the SubSAM revealed the structure of motile processes that develops at increasing time intervals, thus allowing the arising complexity of motion to be registered and investigated. Independent spots of activity emerge on a quiescent background as motility domains; they may change position, divide, merge, or disappear after a long time interval. In addition, zones of quiescence were identified over central parts of cytoplasmic lamellae. Nonmalignant (Ep: tadpole epidermal cells, XTH2: endothelial cells from tadpole hearts, 3T3 cells) and neoplastic cells (K2 cells of rat fibrosarcoma, A870N cells selected from K2) were investigated with the SubSAM. Three types of domains of subcellular cytoplasmic motility were identified in time series of two‐dimensional SubSAM images in normal and neoplastic cells. Of them only the wave‐like domain is self‐evident, being derived from ruffling and protruding activity at the cell margin. Two other domains wait for detailed analysis. The oscillating domain is a visualization of tension within the cell(s), and the nucleating domain indicates intracellular processes possibly preceding locomotion. Differences in motile domains were found between low K2 and high A870N metastatic cells. The dynamics of motility domains of the A870N cells resembled that of the highly motile Ep cells. Cell morphotype and motile activity of the A870N cells are significantly influenced by the pH of the medium. It became evident that identification of the otherwise invisible motile domains in living cells by SubSAM opens a new approach to a characterization of cell motility in vitro and to an understanding of early cellular reactions to various stimuli. © 1994 Wiley‐Liss, Inc.
Characteristics of pronuclear migration in Beroe ovata
Authors: Rouvière Christian , Houliston Evelyn , Carré Danièle , Chang Patrick , Sardet Christian
Published: February 2005

In the large eggs (∼1 mm) of the ctenophore Beroe ovata, female pronuclei migrate long distances to join stationary male pronuclei in the peripheral cytoplasm that surrounds the yolky interior. We have investigated the mechanism of nuclear migration using time lapse video recording, automated image analysis, visualization of microtubules by immunofluorescence and rhodamine‐tubulin injection, and electron microscopy. Female pronuclei migrated at average speeds of 0.2 μm/sec, and were found to show periodic oscillations in velocity. Alternating phases of acceleration and deceleration occurred with an average periodicity of 235 seconds covering distances of 47 μm (about 3 times the nuclear diameter). Migration velocities and velocity oscillations were similar in fertilized and unfertilized eggs; however, changes in migration direction were much more frequent in unfertilized eggs. Characteristic deformations of the pronuclear membrane and occasional rotation of the nuclear contents were observed during migration. Inhibitor studies indicated that microtubules are required for nuclear migration. In fertilized eggs the top of the nucleus was found to move through the dense layer of aligned sperm aster microtubules. The frequent changes in direction of pronuclear migration in unfertilized eggs reflect the random organization of the microtubule layer in the absence of sperm derived centrosomes. Densely packed endoplasmic reticulum was found intermeshed with sperm aster microtubules and connected extensively with the nuclear membrane during migration. Most nuclear pores were grouped in an infolding of the nuclear membrane. We suggest that in fertilized eggs the female pronucleus is transported to the minus ends of sperm aster microtubules using motor molecules attached either to the outer nuclear membrane and/or to the network of connecting ER. © 1994 Wiley‐Liss, Inc.
Concentration of cofilin, a small actin‐binding protein, at the cleavage furrow during cytokinesis
Authors: Nagaoka Rie , Abe Hiroshi , Kusano Ken‐Ichi , Obinata Takashi
Published: February 2005

Cofilin is a small actin‐binding protein which reguiates actin polymerization in a pH‐dependent manner. Immunofluorescence microscopy with a monoclonal antibody for cofilin revealed that this protein is temporarily concentrated at the contractile ring during cytokinesis. Cofilin appeared to accumulate rapidly at the contractile ring during late stages of furrowing, and was finally enriched at the midbody. The concentration of cofilin at the contractile ring was observed in several kinds of cultured cells. Furthermore, cofilin introduced into living cells by a microinjection method was also concentrated at the contractile ring. These results suggest that cofilin is involved in actin reorganization during cytokinesis. © 1995 Wiley‐Liss, Inc.
Dynamics of triton‐insoluble and triton‐soluble F‐actin pools in calcium‐activated human polymorphonuclear leukocytes: Evidence for regulation by gelsolin
Authors: Watts Raymond G. , Deaton Jane D. , Howard Thomas H.
Published: February 2005

Gelsolin, a Ca++ activated, 90 kd actin binding protein, can regulate actin polymerization in polymorphonuclear leukocytes (PMNs) via severing of filaments to dissolve gels or by capping of filament ends to limit polymerization. In Triton‐lysed PMNs, 30% of gelsolin is bound to the Triton‐soluble F‐actin (TSF) pool and none is bound to the Triton‐insoluble F‐actin (TIF) pool. Calcium‐activated PMNs exhibit concurrent temporal and quantitative TIF growth and TSF and total F‐actin loss. To determine if gelsolin plays a role in regulating TSF pool size, we monitored gelsolin‐actin interactions and TIF, TSF and G‐actin content at 5 second intervals in PMNs activated with the calcium ionophore, ionomycin. Actin pools were measured by NBDphallacidin binding and by gel scans and expressed relative to basal; gelsolin‐actin interactions were measured as change in the amount of EGTA‐resistant gelsolin:actin (G:A) complexes and by immunoblot quantification of gelsolin in actin pools. In basal PMNs, 33% of PMN gelsolin is bound in 1:1 EGTA‐resistant G:A complexes and TSF and TIF retain 30% and 0% of PMN gelsolin, respectively. By 20 seconds after ionomycin addition, TSF decreases, TIF increases and a fraction of gelsolin repartitions from the TSF to the TIF pool. At maximum change (60 seconds), total F‐actin (TIF + TSF) and TSF decrease and TIF increases by 25%; gelsolin is bound to both TSF and TIF (35% of total gelsolin in each pool), and 1:1 EGTA‐resistant G:A complexes increase from 33% to 70%. No changes occur in cells activated by ionomycin in the absence of Ca++. The data show Ca++ activated TIF growth and TSF loss are temporally and quantitatively associated with an increase in the percent of gelsolin bound to actin and the translocation of gelsolin from TSF to TIF. This is unique, since no other PMN activator is known to repartition gelsolin into TIF actin. Further, the Ca++ activated initial increase in TIF concurrent with a fall in TSF without a change in total F‐actin or G‐actin content suggest that TIF grows initially only by TSF annealing/cross‐linking to TIF. Gelsolin may regulate these events. © 1995 Wiley‐Liss, Inc.
Comparative study of the colchicine binding site and the assembly of fish and mammalian microtubule proteins
Authors: de Pereda J. M. , Wallin M. , Billger M. , Andreu J. M.
Published: February 2005

Isolated microtubules from cod (Gadus morhua) are apparently more stable to colchicine than bovine microtubules. In order to further characterize this difference, the effect of the colchicine analogue 2‐methoxy‐5‐(2,3,4‐trimethoxyphenyl)‐2,4,6‐cyclo heptatrien‐1‐one (MTC) was studied on assembly, as measured by turbidity and sedimentation analysis, and on polymer morphology. MTC has the advantage to bind fast and reversible to the colchicine binding site of tubulin even at low temperatures. It was found to bind to one site in cod brain tubulin, with affinity (6.5 ± 1.5) × 105M 1at both low or high temperature, similarly to bovine brain tubulin. However, the effect of the binding differed. At substoichiometric concentrations of MTC bovine brain microtubule assembly was almost completely inhibited, while less effect was seen on the mass of polymerized cod microtubule proteins. A preformed bovine tubulin‐colchicine complex inhibited the assembly of both cod and bovine microtubules at substoichiometric concentrations, but the effect on the assembly of cod microtubules was less. At higher concentrations (5 × 10−5 to 1 × 10−3M), MTC induced a large amount of cold‐stable spirals of cod proteins, whereas abnormal polymers without any defined structure were formed from bovine proteins. Spirals of cod microtubule proteins were only formed in the presence of microtubule associated proteins (MAPs), indicating that the morphological effect of MTC can be modulated by MAPs. The effects of colchicine and MTC differed. At 10−5M colchicine no spirals were formed, while at 10−4M and 10−3M, a mixture of spirals and aggregates was found. The morphology of the spirals differed both from vinblastine spirals and from the spirals previously found when cod microtubule proteins polymerize in the presence of high Ca2concentrations. The present data show that even if the colchicine binding site is conserved between many different species, the bindings have different effects which seem to depend on intrinsic properties of the different tubulins. © 1995 Wiley‐Liss, Inc.
Growth cone enrichment and cytoskeletal association of non‐receptor tyrosine kinases
Authors: Helmke Steve , Pfenninger Karl H.
Published: February 2005

Fetal rat brain (E18) expresses at least three c‐src‐like, membrane‐associated non‐receptor tyrosine kinases: c‐src, fyn, and lyn. c‐src and fyn are the most abundant and are highly enriched in a subcellular fraction of nerve growth cones (GCPs). To study the cytoskeletal association of these tyrosine kinases, Triton X‐100‐resistant fractions were prepared from GCPs. All three non‐receptor tyrosine kinases are associated with the cytoskeleton to a significant degree with the relative affinities: fyn > c‐src > lyn. The binding is sensitive to ionic strength and to phosphotyrosine, but not to phosphoserine or phosphothereonine. To investigate the regulation of this association we used phosphatese inhibitors to increase phosphotyrosine levels in GCPs. This resulted in the release of c‐src from the cytoskeleton. Under these conditions tyrosine phosphorylation was increased selectively in released c‐src and primarily on tyrosine 527. Cytoskeletally bound c‐src had a higher specific kinase activity than Triton X‐100‐soluble c‐src. These findings indicate that src family members interact in a regulated manner with the cytoskeleton in non‐transformed cells. This regulation is explained by a model in which c‐src binds to the cytoskeleton via its SH2 domain and is released when phosphorylated tyrosine‐527 binds to this domain intramolecularly, inhibiting kinase activity. © 1995 Wiley‐Liss, Inc.
A 62‐kDA mitotic apparatus protein required for mitotic progression is sequestered to the interphase nucleus by associating with the chromosomes during anaphase
Authors: Ye Xiaojian , Sloboda Roger D.
Published: February 2005

A protein component of 62‐kDa (p62) in the mitotic apparatus of the sea urchin embryo has been shown to be important for the proper progression of mitosis [Dinsmore and Sloboda, 1989: Cell 57:127–134]. To study the subcellular distribution of p62 during the cell cycle of sea urchin embryos, indirect immunofluorescence microscopy was used coupled to a modified detergent extraction procedure. The improved fluorescent images obtained by this procedure provide new information concerning the subcellular localization of p62 during the cell cycle that could not be obtained with previous conventional staining procedures [Johnston and Sloboda, 1992: J. Cell Biol. 119:843–854]. Using affinity purified antibodies to p62, we observed a cell cycle‐dependent localization of p62 to the chromosomes/chromatin. Prior to nuclear envelope breakdown of the first or second cell cycle, p62 localizes to chromatin in the nucleus. During mitosis, p62 associates with the region of the spindle occupied by the microtubules of the mitotic apparatus. As anaphase proceeds, but before the nuclear envelope reforms, p62 becomes progressively associated with the chromosomes. Thus, p62 is incorporated into the forming interphase nucleus due to its association with chromosomes during late anaphase, rather than by active translocation into the newly formed daughter nuclei through the nuclear pores. The protein is not unique to marine embryos, as demonstrated by immunofluorescence of Y‐1 cells, a mouse adrenal tumor cell line In these cells, the localization of p62 is similar to the localization of the protein in echinoderm embryos, suggesting its possible function in mitotic progression in mammalian somatic cells as well. © 1995 Wiley‐Liss, Inc.
Topographic compensation: Guidance and directed locomotion of fibroblasts on grooved micromachined substrata in the absence of microtubules
Authors: Oakley C. , Brunette D. M.
Published: February 2005

Fibroblats cultured on grooved substrata align themselves and migrate in the direction of the grooves, a phenomenon called contact guidance. Microtubules have been deemed important for cell polarization, directed locomotion, and contact guidance. Because microtubules were the first cytoskeletal element to align with the grooves when fibroblasts spread on grooved substrata, we investigated the consequences of eliminating the influence of microtubules by seeding fibro‐blasts onto smooth and grooved micromachined substrata in the presence of colcemid. Fibroblasts were examined by time‐lapse cinematography and epifluorescence or confocal microscopy to determine cell shape and orientation and the distribution of cytoskeletal or associated elements including actin filaments, vinculin, intermediate filaments, microtubules, and kinesin.

As expected, cells spreading on smooth surfaces in the presence of colcemid did not polarize or locomote. Surprisingly however, by 24 hours, cells spread on grooves in the presence of colcemid were morphologically indistinguishable from controls spread on grooves. Both groups were aligned and polarized with the direction of the grooves and demonstrated directional locomotion along the grooves. In the absence of microtubules, kinesin localized to some of the aligned stress fibers and to leading edges of cells spreading on grooves. The grooved substratum compensated for the microtubule deficiency by organizing and maintaining an aligned actin filament framework. Thus, microtubules are not required to establish or maintain stable, polarized cell shapes or directed locomotion, provided an alternate oriented cytoskeletal component is available.
Differential localization of cytoplasmic myosin ii isoforms a and b in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro
Authors: Conrad Abigail H. , Conrad Gary W. , Jaffredo Thierry
Published: February 2005

Two principal isoforms of cytoplasmic myosin II, A and B (CMIIA and CMIIB), are present in different proportions in different tissues. Isoform‐specific monoclonal and polyclonal antibodies to avian CMIIA and CMIIB reveal the cellular distributions of these isoforms in interphase and dividing embryonic avian cardiac, intestinal epithellal, spleen, and dorsal root ganglia cells in primary cell culture. Embryonic cardiomyocytes react with antibodies to CMIIB but not to CMIIA, localize CMIIB in stress‐fiber‐like ‐structures during interphase, and markedly concentrate CMIIB in networks in the cleavage furrow during cytokinesis. In contrast, cardiac fibroblasts localize both CMIIA and CMIIB in stress fibers and networks during interphase, and demonstrate slight and independently regulated concentration of CMIIA and CMIIB in networks in their cleavage furrows. V‐myc‐immortalized cardiomyocytes, an established cell line, have regained the ability to express CMIIA, as well as CMIIB, and localize both CMIIA and CMIIB in stress fibers and networks in interphase cells and in cleavage furrows in dividing cells. Conversely, some intestinal epithelial, spleen, and dorsal root ganglia interphase cells express only CMIIA, organized primarily in networks. Of these, intestinal epithelial cells express both CMIIA and CMIIB when they divide, whereas some dividing cells from both spleen and dorsal root ganglia express only CMIIA and concentrate it in their cleavage furrows. These results suggest that within a given tissue, different cell types express different isoforms of CMII, and that cells expressing either CMIIA or CMIIB alone, or simultaneously, can form a cleavage furrow and divide.
Manipulation of bovine sperm metabolism and motility using anoxia and phosphodiesterase inhibitors
Authors: Schoff Patrick K. , First Neal L.
Published: February 2005

Bovine sperm that were subjected to extended anoxia (2.5 h) in the absence of glycolytic substrates then diluted into oxygenated medium were immotile but metabolically active, producing ATP from lactate via oxidative phosphorylation. In response to anoxia sperm ATP titers dropped from 15–20 μmoles/108 cells to 1–2 μmoles/108 cells in the first 5 min then remained extremely low until reoxygenation. Cyclic AMP titers declined slowly over the anoxic period, but did not show the same scale of depression as ATP. After dilution and re‐oxygenation ATP recovered to pre‐anoxia levels within 1 min, and cAMP rose to about the pre‐anoxia levels. However, motility, which varied quantitatively and qualitatively between ejaculates prior to anoxic treatment, was substantially depressed after extended anoxia in all cases; progressive motility was almost non‐existent in post‐anoxic sperm. Addition of isobutylmethylxanthine or Cibacron Blue F3GA, both putative phosphodiesterase inhibitors, stimulated a transient peak of cAMP, which was accompanied by motility stimulation. These techniques provide a protocol to manipulate and dissect the biochemical pathways of motility initiation in mammalian sperm.
Role of the cytoskeleton in the reaction of fibroblasts to multiple grooved substrata
Authors: Wójciak‐Stothard B. , Curtis A. S. G. , McGrath M. , Sommer I. , Wilkinson C. D. W. , Monaghan W.
Published: February 2005

The role of the cytoskeleton and cell attachments in the alignment of baby hamster kidney fibroblasts to ridge and groove substratum topography was investigated using confocal scanning microscopy. This was carried out with normal cells and cells treated with the cytoskeleton modifiers cytochalasin D, colcemid, and taxol. Actin was localised with fluorescent phalloidin. Tubulin, Vinculin, and intracellular adhesion molecule‐1 were visualised by indirect immunofluoresence. The spreading, elongation, and orientation of the cells after 24 h of culture in these conditions were measured on grooves of 5, 10, and 25 μm width and 0.5, 1, 2, and 5 μm depth. We have also observed events over the first 30 min of cell attachment. Five minutes after cell attachment, F‐actin condensations were seen close to the intersection of groove wall and ridge top, that is, at a topographic discontinuity. The condensations were often at right angles to the groove edge and showed a periodicity of 0.6 μm. Vinculin arrangement at the early stages of cell spreading was similar to that of actin. Organisation of the microtubule system followed later, becoming obvious at about 30 min after cell plating. The Curtis and Clark theory (that cell react to topography primarily at lines of discontinuity in the substratum by actin nucleation) is supported by these results. The use of cytoskeletal poisons did not entirely abolish cell reaction to grooves. Colocemid increased cell spreading and reduced cell orientation and elongation. Cytochalasin D reduced cell spreading, orientation, and elongation. Taxol reduced cell elongation but did not affect cell spreading and orientation. We conclude that the aggregation of actin along groove/ridge boundaries is a primary driving event in determining fibroblast orientation on microgrooved substrata.
cAMP/ATP relationship in the activation of trout sperm motility: Their interaction in membrane‐deprived models and in live spermatozoa
Authors: Cosson Marie‐Paule , Cosson Jacky , Andrè Françoise , Billard Roland
Published: February 2005

Live trout spermatozoa initiate flagellar motility for only a short period (30 sec at 18°C) during which their mean beat frequency decreases steadily from 60 to 20 Hz. Motility then stops abruptly. Investigations of the activation of movement in demembranated sperm points to cyclic‐AMP being necessary for reactivation (half effect at 0.5μm) in some conditions. cAMP acts mainly by increasing the percentage of motile cells and not the beat frequency (BF) of the flagellar axoneme. Dibutyryl cAMP does not initiate movement or prolong motility of live sperm.

The initiation of movement of demembranted trout sperm was investigated in various incubation conditions relative to previous phases of in vivo movement and to ATP concentration. In the absence of cAMP and in the presence of ATP lower than 25 μM, all sperm celi models were active with BF up to 15‐20 Hz whatever their previous physiological condition. In contrast, at ATP concentrations above 100 μM, the fraction of active spermatozoa decreased proportionally but the BF of the active ones increased so that, at 1 mM ATP up to 20 μM restored activity to 100% sperm models with a similar BF of 65 Hz.

At ATP concentrations higher than 25 μM, cAMP was necessary in a concentration dependent manner in the reactivation, but not in the demembranation meduim. This dependence was found to be unrelated to a previous in vivo phase of movement. The antagonistic effects of ATP vs. cAMP were tested at various concentrations of both nucleotides: the apparent affinity for cAMP, measured as the concentration restoring movement of 50% cell models, was decreased from 15 nM at 0.1 mM ATP to 0.5 μM at 1 mM ATP; conversely, the affinity for ATP, measured as the concentration giving rise to the half maximal beat frequency, was not significautly affected when the concentration of cAMP was raised to 0.5 mM. Preincubation with phosphodiesterase (PDE) resulted in motility of 100% of sperm models even at low ATP concentration. This tends to show that cAMP must be constantly present to sustain motility.
Suppression of syntheses of high molecular weight nonmuscle tropomyosins in macrophages
Authors: Nakamura Yohko , Sakiyama Shigeru , Takenaga Keizo
Published: February 2005

In mouse fibroblasts, at least five TM isoforms are identified and they can be grouped into the high (TM1, TM2, and TM3) and low molecular weight TM isoforms (TM4 and TM5). Suppression of one of the high molecular weight tropomyosin (TM) isoforms in nonmuscle cells is implicated to be one of the causes for disorganization of actin microfilament bundles and subsequent changes in cell motility and cell shape. In this study, we studied the expression of tropomyosin isoforms in macrophages that exhibit high motility and ability to change cell shape. Two‐dimensional gel electrophoresis followed by Western blot analysis using polyclonal anti‐TM antiserum revealed that the high molecular weight TM isoforms were lacking in both resident and activated mouse peritoneal macrophages. Analyses of newly synthesized TM isoforms, Northern blot analyses using isoform‐specific cDNA probes, and immunostaining with monoclonal anti‐TM antibody that recognizes only the high molecular weight TM isoforms also demonstrated that the syntheses of the high molecular weight TM isoforms (TM1, TM2, and TM3) were completely suppressed, whereas the low molecular weight TM isoforms (TM4 and TM5) were expressed in macrophages. These results indicate that macrophages intrinsically lack the high molecular weight TM isoforms. In order to obtain information about cellular localization of the low molecular weight TM isoforms in macrophages, they were immunostained with polyclonal anti‐TM antiserum that recognizes both the high and low molecular weight TM isoforms. The results showed that the low molecular weight TM isoforms were co‐localized with F‐actin in punctate and short fibrous structures. In addition, we performed in situ hybridization analysis to examine localizations of the TM mRNAs in fibroblasts and macrophages. The results showed that TM mRNAs were localized throughout the cytoplasm. © 1995 Wiley‐Liss, Inc.
Actin filaments in honeybee‐flight muscle move collectively
Authors: Trombitéas K. , Pollack G. H.
Published: February 2005

To investigate the pattern of actin‐filament translation in the intact myofibrillar matrix, we carried out electron micrographic experiments on the “rigor‐stretch” model of insect‐flight muscle. In this model, thin filaments are mechanically severed from their connections to the Z‐line and may then slide freely over the myosin filament when activated. The model is similar to the in vitro motility assay in that untethered actin filaments slide over myosin, but here the natural filament lattice is retained: sliding takes place through the lattice of thick filaments. We find, in this model, that while the extent of thin filament translation is variable from sarcomere to sarcomere, filaments never translate far enough to enter the opposite I‐band. Unlike the in vitro motility assay, where the actin filament translates over the entire thick filament even with “incorrectly” polarized crossbridges as the sole driver, in this intact filament‐lattice model, cross‐bridges are apparently unable to move filaments in both directions. We also find that the pattern of filament translation is collective. Although the extent of translation may vary among sarcomeres, in any given half‐sarcomere all actin filaments translate by the same degree. Further, the extent of translation is is the same in both halves of a given sarcomere. In rare instances where the extent of translation exhibited a transverse gradient across the myofibrillar half‐sarcomere, the gradient was similar on both sides of the sarcomere. Filament translation within the sarcomere is thus collective. Some mechanism ensures that nearby but distinctly separated actin filaments move together and that cooperative‐like behavior therefore extends to the supramolecular level.
Nebulette: A 107 kD nebulin‐like protein in cardiac muscle
Authors: Moncman Carole L. , Wang Kuan
Published: February 2005

A 107‐kD protein has been identified in primary cultures of chicken embryonic cardiomyocytes by immunoprecipitations with certain anti‐nebulin monoclonal antibodies (mAbs). These mAbs, prepared against a fragment of human skeletal muscle nebulin located near the carboxyl terminus, detect a 107‐kD protein in extracts of adult chicken heart, adult mouse heart, and adult rabbit heart by immunoblot analysis. A partial cDNA corresponding to this protein has been isolated by immunological screening of a chicken heart cDNA expression vector library. The partial cDNA encodes a 380‐amino acid open reading frame composed entirely of nebulin‐like 35‐residue modules marked by the highly conserved sequence motifs: SXXXYK and TPD. The open reading frame exhibits 60–85% homology with skeletal muscle nebulins from a variety of species. This cDNA recognizes an ˜8‐kb transcript in cardiac RNA and does not hybridize to skeletal muscle RNAs by northern analysis. Immunofluorescence localization of this nebulin‐like protein in primary cultures of chicken cardiomyocytes and embryonic chicken cardiac myofibrils indicates that the protein is localized to the I‐Z‐I complex of the myofibrils, extending approximately 25% of the thin filament length. Comparisons of the distribution of this protein relative to actin, myosin, and titin in spreading cardiomyocytes suggest that the cardiac nebulin‐like protein becomes aligned with the nascent myofibrils early during myofibrillogenesis. To distinguish this petite nebulin‐like protein from the 600–900 kD skeletal muscle nebulin, we have named it nebulette. © 1995 Wiley‐Liss, Inc.
Polarity and nucleation of microtubules in polarized epithelial cells
Authors: Meads Tim , Schroer Trina A.
Published: February 2005

Microtubules oriented in the apicobasal axis of columnar epithelial cells arranged with a uniform polarity with minus ends toward the apical surface, suggesting that these cytoskeletal filaments might serve as a substrate for polarized movement of membrane vesicles within the cell. It is not known whether hepatocytes, a cuboidal epithelium in which transcellular transport is a requisite step in normal apical membrane biogenesis, contain microtubules arranged with a similar polarity. In the present study, we explore the question of microtubule polarity and possible mechanisms for nucleation in the epithelial cell lines WIF‐B (hepatocyte), Caco‐2 (intestine), and Madin‐Darby canine kidney (MDCK). Caco‐2 microtubules in the apicobasal axis had uniform polarity with minus ends nearest the apical surface. After cold and nocodazole‐induced depolymerization, microtubule regrowth initiated in the apical region in all three cell types. The apex of WIF‐B and Caco‐2 cells contained two pools of γ‐tubulin: one associated with centrosomes and the other delocalized under the apical membrane. Non‐centrosomal γ‐tubulin was present in complexes that sedimented between 10S and 29S; both forms could bind microtubules. The presence of both centrosomal and noncentrosomal γ‐tubulin in apical cytoplasm suggests multiple mechanisms by which microtubule nucleation might occur in epithelial cells. © 1995 Wiley‐Liss, Inc.
Silicon crystal growth from the melt: Analysis from atomic and macro scales
Authors: Kakimoto K. , Liu L. , Kitashima T. , Murakawa A. , Hashimoto Y.
Published: February 2005

The effect of impurity concentration on thermal conductivity of natural and isotope silicon by using equilibrium molecular dynamics simulation is investigated. It was found that the concentrations of the impurities such as boron, phosphor and arsene play an important role in the propagation of phonons in silicon crystals. It was also clarified that a mass difference of impurities and host crystals results in degradation of thermal conductivity of silicon. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Study of microindentation hardness of different planes of gadolinium calcium oxyborate single crystals
Authors: Sangwal K. , Kłos A.
Published: February 2005

The microhardnessHVof gadolinium calcium oxyborate single crystals has been investigated on the planes of different orientations as a function of applied load and indenter orientation. It was found that: (1) on the (010) plane microhardness is practically independent of indenter orientation and indenter diagonald, and is constant, (2) on the (100) and (001) planes both normal and reverse indentation size effects occur in the indentation diagonal intervals below and above a critical valuedc, and (3) at loads exceeding about 30 g all indentation impressions are accompanied by cracks, the formation of which does not depend on the orientations of indentations. The observations of indentation size effect were analysed by using Hays‐Kendall's approach, the proportional specimen resistance model of Li and Bradt, and the strain gradient plasticity theory. The analysis of the data revealed that: (1) indentation size effect on the different faces of gadmium calcium oxyborate may be explained satisfactorily by the proportional specimen resistance model and the strain gradient plasticity theory, (2) the models explain the dependence of microhardnessHVon indentation diagonaldin the rangeddcthe excess indentation pressure, and the energy associated with it, is expended in creating and developing radial and lateral cracks around indentations, and (3) in the rangedH0(001) >H0(100), and may be explained in terms of the process of rupturing of successive planes perpendicular to the direction of penetration of indenter. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Instability of Al‐Cu‐Co single quasicrystals at low temperature
Authors: Bogdanowicz W.
Published: February 2005

Decomposition process of the Al‐Cu‐Co single quasicrystals (falling apart into small pieces) after annealing at 100 °C for 20 h was studied by X‐ray topography, high‐resolution electron microscopy, X‐ray diffractometry and optical microscopy. It was found that the decomposition of the single quasicrystals is preceded by the creation of a large number of antiphase microdomains and the deformation of the clusters occurs in the two‐fold directions of [01$\bar 1$00] type. The decomposition begins with the propagation of cracks in the directions of [01$\bar 1$00] type. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
X‐ray characterization of thick GaN layers grown by HVPE
Authors: Korbutowicz R. , Kozłowski J. , Dumiszewska E. , Serafińczuk J.
Published: February 2005

The crystallographic structures of the various Metalorganic Vapour Phase Epitaxy (MOVPE) thin GaN epitaxial layers deposited on (00.1) sapphire substrates are described and compared with the structural properties of the thick gallium nitride layers deposited by Hydride Vapour Phase Epitaxy (HVPE) on the top of them. The crystallographic structure and the quality of epitaxial GaN layers obtained in this way are determined. Additionally thick HVPE GaN layers deposited on these composite substrates (MOVPE‐GaN/sapphire) are compared with the thick HVPE GaN layers grown directly on sapphire substrates. It was found that HVPE thick GaN epilayers on sapphire are comparable with the HVPE thick GaN layers deposited on composite substrates. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Evolution of stress and structure in Cu thin films
Authors: Chocyk D. , Zientarski T. , Proszynski A. , Pienkos T. , Gladyszewski L. , Gladyszewski G.
Published: February 2005

Evolution of stresses developing in Cu thin films during and after deposition by thermal evaporation in UHV system is studied. Thin films were deposited on 100 µm thick Si substrate at room temperature. Deposition rates for the films were changed between 0.2 Å/s and 2.0 Å/s, while the total thickness was changed from 7.7 nm to 155 nm. Deformation analysis of crystalline lattice and microstructure was performed by x‐ray diffraction measurements (θ‐2θ scans, “sin2ψ” method). The surface morphology of film was studied by atomic force microscopy. The average stress in the films was determined by measuring the radius of curvature of samples. For thin films three stages of stress evolution (compressive, tensile and compressive) were we distinguished. This behavior is characteristic for materials with a Volmer‐Weber mode. A three‐dimensional molecular dynamics technique was applied for simulating the stress calculation during thin film growth. The results obtained from the simulation are consistent with the experimental results. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Ontogeny of urine preference and its relationship to NH4Cl preference and sodium hunger in suckling rat pups
Authors: Leshem Micah , del Canho Sonia
Published: February 2005

We chart the postnatal ontogeny of urine preference in the suckling rat. Twelve‐day‐old sucklings, when offered urine, NH4Cl, or NaCl, ingest more urine and NH4Cl than NaCl. When rendered sodium hungry by ivc renin or by sodium depletion, these sucklings prefer urine and NH4Cl to NaCl, dilute urine, or an NaCl and KCl mineral mix equimolar to urine; however, by 18 days of age, urine and NH4Cl are no longer preferred to NaCl. Hence, urine preference in the suckling may be specific and preparatory for the variety of purposes urine preference serves in the adult rat, and it might guide the pup to urinary sodium in the nest. Since preference for urine and NH4Cl covary during postnatal development, the high preference for NH4Cl in midterm sucklings might be because its ammonium flavor is similar to urine. © 2005 Wiley Periodicals, Inc. Dev Psychobiol 46: 111–117, 2005.