Electrically excitable normal rat kidney fibroblasts: A new model system for cell-semiconductor hybrids.
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In testing various designs of cell-semiconductor hybrids, the choice of a suitable type of electrically excitable cell is crucial. Here normal rat kidney (NRK) fibroblasts are presented as a cell line, easily maintained in culture, that may substitute for heart or nerve cells in many experiments. Like heart muscle cells, NRK fibroblasts form electrically coupled confluent cell layers, in which propagating action potentials are spontaneously generated. These, however, are not associated with mechanical disturbances. Here we compare heart muscle cells and NRK fibroblasts with respect to action potential waveform, morphology, and substrate adhesion profile, using the whole-cell variant of the patch-clamp technique, atomic force microscopy (AFM), and reflection interference contrast microscopy (RICM), respectively. Our results clearly demonstrate that NRK fibroblasts should provide a highly suitable test system for investigating the signal transfer between electrically excitable cells and extracellular detectors, available at a minimum cost and effort for the experimenters. (+info)
Helicobacter pylori can be induced to assume the morphology of Helicobacter heilmannii.
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Cultures of Helicobacter pylori obtained from the American Type Culture Collection (strain 43504) were grown as isolated colonies or lawns on blood agar plates and in broth culture with constant shaking. Examination of bacterial growth with Gram-stained fixed preparation and differential interference contrast microscopy on wet preparations revealed that bacteria grown on blood agar plates had a morphology consistent with that normally reported for H. pylori whereas bacteria from broth cultures had the morphologic appearance of Helicobacter heilmannii. Bacteria harvested from blood agar plates assumed an H. heilmannii-like morphology when transferred to broth cultures, and bacteria from broth cultures grew with morphology typical of H. pylori when grown on blood agar plates. Analysis by PCR of bacteria isolated from blood agar plates and broth cultures indicated that a single strain of bacteria (H. pylori) was responsible for both morphologies. (+info)
Evidence for multiple promoter elements orchestrating male-specific regulation of the her-1 gene in Caenorhabditis elegans.
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The sex-determining gene her-1 is required for male development in Caenorhabditis elegans. In XO males, two her-1 mRNAs, her-1a and her-1b, are transcribed from two separate promoters: P1, located in the 5'-flanking region, and P2, located in the large second intron. In XX hermaphrodites, accumulation of both her-1 transcripts is repressed by the sdc genes, which in turn are negatively regulated by the xol-1 gene. When introduced into a xol-1(y9) background, transgenic arrays, including 3.4 kb of her-1 intron 2 sequence (P2), result in phenotypes that mimic those of sdc(lf) mutants, including suppression of XO lethality and masculinization of both XX and XO animals. The masculinization, but not the suppression of XO lethality, is dependent on endogenous her-1 activity. These effects could therefore result from sequestration (titration) of sdc gene products by sequences in the arrays, causing derepression of her-1 (masculinizing effect) and disruption of the dosage compensation machinery (allowing survival of XO animals). We used these effects as an assay in a deletion analysis of the two her-1 promoter regions to define potential cis-regulatory sites required for the putative titration. Several regions in P2 contributed to these effects. P1 was effective only in combination with certain P2 sequences and only if a particular P1 site previously implicated in her-1 repression was intact. These results suggest that normal repression of transcription from P1 in XX animals may involve cooperative interaction with sequences in the P2 region. In experiments to test for a possible role of the her-1b transcript in regulation of sdc genes, no significant effects could be demonstrated. (+info)
The immunological synapse: a molecular machine controlling T cell activation.
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The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor-ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of the synapse was a determinative event for T cell proliferation. (+info)
Transformations in flagellar structure of Rhodobacter sphaeroides and possible relationship to changes in swimming speed.
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Rhodobacter sphaeroides is a photosynthetic bacterium which swims by rotating a single flagellum in one direction, periodically stopping, and reorienting during these stops. Free-swimming R. sphaeroides was examined by both differential interference contrast (DIC) microscopy, which allows the flagella of swimming cells to be seen in vivo, and tracking microscopy, which tracks swimming patterns in three dimensions. DIC microscopy showed that when rotation stopped, the helical flagellum relaxed into a high-amplitude, short-wavelength coiled form, confirming previous observations. However, DIC microscopy also revealed that the coiled filament could rotate slowly, reorienting the cell before a transition back to the functional helix. The time taken to reform a functional helix depended on the rate of rotation of the helix and the length of the filament. In addition to these coiled and helical forms, a third conformation was observed: a rapidly rotating, apparently straight form. This form took shape from the cell body out and was seen to form directly from flagella that were initially in either the coiled or the helical conformation. This form was always significantly longer than the coiled or helical form from which it was derived. The resolution of DIC microscopy made it impossible to identify whether this form was genuinely in a straight conformation or was a low-amplitude, long-wavelength helix. Examination of the three-dimensional swimming pattern showed that R. sphaeroides changed speed while swimming, sometimes doubling the swimming speed between stops. The rate of acceleration out of stops was also variable. The transformations in waveform are assumed to be torsionally driven and may be related to the changes in speed measured in free-swimming cells. The roles of and mechanisms that may be involved in the transformations of filament conformations and changes in swimming speed are discussed. (+info)
Doublecortin, a stabilizer of microtubules.
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X-linked lissencephaly is a severe brain malformation affecting males. Recently it has been demonstrated that the doublecortin gene is implicated in this disorder. In order to study the function of Doublecortin, we analyzed the protein upon transfection of COS cells. Doublecortin was found to bind to the microtubule cytoskeleton. In vitro assays (using biochemical methods, DIC microscopy and electron microscopy) demonstrate that Doublecortin binds microtubules directly, stabilizes them and causes bundling. In vivo assays also show that Doublecortin stabilizes microtubules and causes bundling. Doublecortin is a basic protein with an iso-electric point of 10, typical of microtubule-binding proteins. However, its sequence contains no known microtubule-binding domain(s). The results obtained in this study with Doublecortin and our previous work on another lissencephaly gene ( LIS1 ) emphasize the central role of regulation of microtubule dynamics and stability during neuronal morphogenesis. (+info)
Pearling in cells: a clue to understanding cell shape.
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Gradual disruption of the actin cytoskeleton induces a series of structural shape changes in cells leading to a transformation of cylindrical cell extensions into a periodic chain of "pearls." Quantitative measurements of the pearling instability give a square-root behavior for the wavelength as a function of drug concentration. We present a theory that explains these observations in terms of the interplay between rigidity of the submembranous actin shell and tension that is induced by boundary conditions set by adhesion points. The theory allows estimation of the rigidity and thickness of this supporting shell. The same theoretical considerations explain the shape of nonadherent edges in the general case of untreated cells. (+info)
The Caulobacter crescentus smc gene is required for cell cycle progression and chromosome segregation.
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The highly conserved SMC (Structural Maintenance of Chromosomes) proteins function in chromosome condensation, segregation, and other aspects of chromosome dynamics in both eukaryotes and prokaryotes. A null mutation in the Caulobacter crescentus smc gene is conditionally lethal and causes a cell cycle arrest at the predivisional cell stage. Chromosome segregation in wild-type and smc null mutant cells was examined by monitoring the intracellular localization of the replication origin and terminus by using fluorescence in situ hybridization. In wild-type cells, the origin is located at the flagellated pole of swarmer cells and, immediately after the initiation of DNA replication in stalked cells, one of the origins moves to the opposite pole, giving a bipolar localization of the origins. The terminus moves from the end of the swarmer cell opposite the origin to midcell. A subpopulation of the smc null mutant cells had mislocalized origins or termini, showing that the smc null mutation gives DNA segregation defects. Nucleoid morphology was also abnormal. Thus, we propose that the Caulobacter chromosomal origins have specific cellular addresses and that the SMC protein plays important roles in maintaining chromosome structure and in partitioning. The specific cell cycle arrest in the smc null mutant indicates the presence of a cell cycle checkpoint that senses perturbations in chromosome organization or segregation. (+info)