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At least 685 records · Page 38Linked to original sources
PubMed2003

Cutting edge: induced expression of a RhoA-specific guanine nucleotide exchange factor, p190RhoGEF, following CD40 stimulation and WEHI 231 B cell activation.

Stimulation of the B cell surface receptor CD40 induces transcriptional activation and protein expression. To determine which proteins are required for the CD40-mediated B cell activation, we performed a two-dimensional gel electrophoresis of the WEHI 231 B cell lysates. We report in this study the identification of one protein in which the expression was remarkably induced following CD40 stimulation. It was the p190 Rho guanine nucleotide exchange factor (GEF), p190RhoGEF, a recently identified GEF that is specific for RhoA. Overexpression of either p190RhoGEF or RhoA (Q63L), a constitutively active form of RhoA, mimics the effects of CD40 stimulation, such as changes in cellular structure and NF-kappaB activation. These p190RhoGEF overexpression effects are abrogated when coexpressed with a dominant negative form of RhoA (T19N). We also provide evidence for the CD40-mediated cellular changes that are abrogated in cells that are overexpressed with the dominant negative form of either p190RhoGEF (Y1003A) or RhoA (T19N).

Adjuvants, Immunologic↗
PubMedDate not supplied

[Change in the constructive metabolism and ultrastructural organization of Bacillus cereus cells under the influence of a specific autoregulatory factor].

It has been found for the first time that an increase in the concentration of a specific autoregulatory factor in the medium is responsible for the hypometabolic state of vegetative cells; no principal cellular biopolymers are synthesized at this state (DNA, RNA, proteins and phospholipids), and no cellular structures are degraded. When the culture reverses to vegetative growth observed in the same medium, synthesis of the biopolymers is intensified and changes are detected in the ultrastructural organization of the cellular membranous apparatus, the cytoplasm, the nucleoide and the cell wall.

Bacillus cereus↗
PubMed2003

Effect of alteration in the global body plan on the deployment of morphogenesis-related protein epitopes labeled by the monoclonal antibody 12G9 in Tetrahymena thermophila.

We have employed monoclonal antibodies to reinvestigate the janus mutants of the ciliate Tetrahymena thermophila, which cause reversal of circumferential polarity on the dorsal surface of the cell. This reversal brings about frequent ectopic expression of ventral cortical landmarks, such as a "secondary" oral apparatus, on the dorsal surface. The principal antibody employed, FXXXIX-12G9, immunolabels both transient cortical structures not directly associated with basal bodies (the fission line and the postoral meridional filament) and more permanent structures (apical band and oral crescent) that are associated with basal bodies. 12G9-immunolabeling of janus cells has revealed additional phenotypes, including disorder of ciliary rows. Further, this labeling has shown that the postoral meridional filament is often expressed and the apical band is frequently interrupted on the mid-dorsal surface of janus cells irrespective of whether or not these cells express a "secondary" oral apparatus. Of the permanent structures revealed by 12G9 immunofluorescence, modifications of the oral crescent (OC) are associated with prior modifications in the development of basal body-containing structures in the secondary oral apparatus. The formation of the apical band (AB) is also commonly abnormal in janus cells; analysis of specific abnormalities shows that the AB depends both on its initiation at a specific site near the anterior basal body of apical basal body couplets and on the normal location of these couplets just posterior to the fission line. We also have uncovered an intriguing difference in the reactivity of apical-band filaments to the 12G9 antibody in the two non-allelic janus mutants (janA1 and janC2) that we have investigated. Taken together, our observations indicate that the formation of new cellular structures at division depends both upon pre-existing cytoskeletal structures and upon the positional information provided by large-scale cellular polarities.

Animals↗
PubMed1995

Factors limiting adenosine triphosphatase function during high intensity exercise. Thermodynamic and regulatory considerations.

It is widely accepted that a structural organisation favouring interaction between functionally-related enzymes is required for the economy and efficiency of metabolic reactions. Many functionally-related enzymes have been shown to be reversibly bound to cellular structures and to other enzymes at the sites where they are required. Resulting from this binding, close structural proximity and concentration of enzymes, a microenvironment is generated where the product of one enzyme is the substrate of the other. This reduces the diffusion distance for the substrate, saturates binding sites with maximal speed and, as a final outcome, increases the efficiency and economy of function behind these metabolic reactions. Available data indicate that the above-described association between adenosine triphosphatase (ATPase) and enzymes regenerating ATP has an important role in the regulation of ATPase function. A general consensus exists among published studies that the concentration of ATP ([ATP]) is not significantly decreased in fatigued muscle, even in those with severely diminished power output. However, in studies with isolated perfused hearts it has been possible to significantly reduce [ATP] in muscle cells without compromising mechanical activity. An explanation for this discrepancy is connected with local ATP regeneration in the vicinity of ATPase. Furthermore, when ATP regeneration is unable to balance ATP consumption a critical drop in the free energy of ATP hydrolysis is avoided by down-regulation of ATP consumption. The main function of local ATP regeneration is to maintain a low concentration of adenosine diphosphate ([ADP]), and the ADP/ATP ratio in the vicinity of the ATP-binding site of ATPase that is a prerequisite for high thermodynamic efficiency of ATP hydrolysis. Close proximity of creatine kinase and glycolytic enzymes to ATPase and high-affinity binding of substrates generate an ATPase microenvironment, where ADP and ATP are not in free equilibrium with those adenine nucleotides in the surrounding medium. In the physiological range of operation for important cellular ATPases (free energy change of 55 to 60 kJ/mol ATP) only a small fraction of energy, available in ATP, can be utilised, provided that no ATP regeneration takes place. However, ATP regeneration allows utilisation of most of the regenerating capacity, before ATP hydrolysis drops below the critical 55 kJ/mol. The importance of local ATP regeneration increases in parallel with an increase in the rate of ATPase turnover.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed2005

Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci.

The hepatitis C virus (HCV) non-structural protein NS4B induces morphological changes in the endoplasmic reticulum (ER) membrane that may have a direct role in viral RNA replication. A chimeric GFP-NS4B fusion protein located to the ER membrane and to foci that were attached to the ER. These membrane-associated foci (MAFs) could be related to the membrane alterations observed in cells that replicate HCV RNA. The relationship of MAFs to pre-existing cellular structures is not known. Indirect immunofluorescence analysis demonstrated that they did not contain a cellular marker for vesicles, which have been implicated in the replication of other viruses. From photobleaching studies to examine diffusion of NS4B, the GFP-tagged protein had reduced mobility on MAFs compared with on the ER membrane. This slower mobility suggested that NS4B is likely to form different interactions on MAFs and the ER.

Animals↗
PubMed1967

Properties of yeast cell ghosts obtained by ribonuclease action.

Cell ghosts were obtained from Candida utilis and Saccharomyces cerevisiae by the action of some conformational isomers of pancreatic ribonuclease which passed through the cell wall and penetrated the cell membrane. In the interior, the enzyme caused extensive disorganization of the cellular structure as evidenced by the results of vital staining and electron microscopy. Ribonucleic acid was degraded to fragments that leaked out into the suspending medium. Other cytoplasmic constituents, including the amino acid pool, were released, but most of the cellular protein and deoxyribonucleic acid remained in the ghosts. The lesions in the cytoplasmic membrane were too small to be seen by conventional electron microscopy. The membrane clung to the cell wall even after obliteration of most of the intracellular structure.

Amino Acids↗
PubMed1990

Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells.

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.

Albumins↗
PubMedDate not supplied

Some aspects of cataract morphology: a SEM-study.

Two lenses from patients of very advanced age with senile cataracts were processed for SEM, fractured equatorially, sputtered with Au and examined by SEM. In the cross-fracture various areas could be observed. Although the overall structure of the lens-fibres appeared to be intact, higher magnifications showed that the len-fibre material had changed into a brittle structure, with either a granular appearance or a fibrillar character. At other places clearly recrystallization of lens-fibre proteins had taken place, with the formation of finger-like substructures, sometimes organized into plate-like structures or running parallel to each other in a kind of undulating pattern. Between the various areas of chemically changed lens-fibre material certain 'canal-like' areas were found with cellular structures, the so-called 'waterclefts' or 'Wasserspalten'. Structures which, together with the chemical change in the lens proteins, account for the dramatic change in light dispersion.

Aged↗
PubMed2001

Diethylnitrosamine causes pituitary damage, disturbs hormone levels, and reduces sexual dimorphism of certain liver functions in the rat.

The acute toxicity of diethylnitrosamine (DEN) to the liver has been well documented in the literature, but whether DEN also affects the endocrine parameters has been addressed in only a few studies. We thus investigated the effects of DEN on pituitary, serum hormone levels, and certain sex-differentiated liver enzymes in this study. Adult male Wister rats were intraperitoneally injected with DEN at a single dose of 200 mg/kg and were sacrificed at 1, 3, 7, and 35 days after injection; DEN-treated females were included as controls at days 7 and 35. Electron microscopic observation showed that during the first week after injection, all types of granular cells of the anterior pituitary in male animals exhibited cellular damage, including disrupted organelles and cellular structure, as well as pyknotic or lytic nuclei. Many undamaged secretory cells exhibited dilated endoplasmic reticula, hypertrophic Golgi complexes, and peripheral location of secretory granules, which usually are morphologic features of increased cellular activities. In male rats, the serum level of total testosterone decreased and the corticosterone increased 1 day after DEN treatment. The serum level of growth hormone (GH) decreased and the prolactin level increased on day 3. The hepatic expression of the male-specific cytochrome P450 2C11 (CYP2C11) decreased to 1-5% of the normal levels during the first week and was still 50% lower than the normal level on day 35, whereas the female-specific CYP2C12 expression increased only slightly. Activities of the male predominant 16alpha, 16beta, and 6beta hydroxylation of androstenedione by microsome decreased in an in vitro assay, whereas the non-sex-differentiated 7alpha hydroxylation and the female-predominant 5alpha reduction of androstenedione were unaffected. In female rats, decreased serum GH level was observed on day 7. The CYP2C12 expression in females was decreased to about 1% and 80% of the normal levels on day 7 and day 35, respectively, but the CYP2C11 expression was unchanged. These data suggest that in male rats, DEN treatment may cause pituitary damage, disturb serum hormone levels, and induce long-lasting reduction of sexual dimorphism in certain liver functions.

Alkylating Agents↗
PubMed1992

Survival and evolution of vein grafts in otosclerosis surgery: structural and ultrastructural evidence.

The annular ligament of the stapes footplate measures 0.2 mm2 and contains elastic fibers that restrict perilymphatic movement to a molecular level. This resistance of the annular ligament prevents excessive movement of labyrinthine fluids that are potentially hazardous to the membranous and cellular structures of the inner ear. However, if the resistance of the annular ligament is overcome, then inner ear damage can occur. The vein graft, as used in our technique for otosclerosis surgery, occupies a space of 0.2 mm2 between the edge of the stapedotomy hole and the piston and has nearly the same compliance and resistance as the annular ligament of the stapes footplate. The vein graft becomes integrated with the middle-ear mucosa. It develops a rich and active blood supply and the smooth muscle cells and elastic fibers remain, although the latter show some modification of ultrastructural pattern. The graft becomes overlaid by the middle-ear epithelium. Even samples of vein graft that have been present for many years retain surviving elastic fibers, including those that have become fibrotic. In time, the number of elastic fibers increases as a result of the activity of fibroblasts, which build a poorly colored collagen.

Adult↗
PubMed1991

In vivo, real-time confocal imaging.

We have adapted a tandem scanning confocal microscope for real-time, non-invasive imaging of cells under in vivo conditions. This form of in vivo confocal imaging relies on the optical sectioning abilities of the confocal microscope to obtain en face, sequential, reflected light images of cells at various depths, up to 1 mm, within opaque organs in living animals. Of major consideration in the design of an in vivo confocal microscope is maximizing the real-time detection of signals reflected from low contrast structures which can be affected by the microscope design, objective, and image detector systems. Using an in vivo confocal microscope design with a 20 x BioOptics surface contact objective we have obtained live cellular images from selected tissues including cornea, kidney, liver, adrenal, thyroid, epididymis, and muscle and connective tissue of rabbits and rats. Images were captured, digitized, and processed using a DAGE Mti low light level SIT camera coupled to a Gould IP9527 image processor. In vivo images were also compared with conventional bright field light and scanning electron microscopic images of "dead," fixed tissues. Overall, in vivo confocal imaging can provide remarkable detail of living cells comparable to that of conventional microscopic images of "dead," fixed, and stained tissue. A more unique feature of in vivo confocal imaging is the ability to study cellular structure and function sequentially over time in the same organ or tissue and represents a fundamentally new paradigm in microscopy. With continued refinements in the microscope, objective and detection system designs and their consequent improvements in lateral and axial resolution, in vivo confocal microscopy will enable us as observers to see what no one has been able to see before.

Animals↗
PubMed2006

Advances in understanding bacterial outer-membrane biogenesis.

The outer membrane of gram-negative bacteria such as Escherichia coli serves as a protective barrier that controls the influx and efflux of solutes. This allows the bacteria to inhabit several different, and often hostile, environments. The assembly of the E. coli outer membrane has been difficult to study using traditional genetic and biochemical methods, and how all its components reach the outer membrane after being synthesized in the cytoplasm and cytoplasmic membrane, how they are assembled in an environment that is devoid of an obvious energy source, and how assembly proceeds without disrupting the integrity of this essential cellular structure are all fundamental questions that remain unanswered. Here, we review the new approaches that have led to the recent discovery of components of the machinery involved in the biogenesis of this distinctive cellular organelle.

Bacterial Proteins↗
PubMed1997

Chaperonin filaments: the archaeal cytoskeleton?

Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent approximately 4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations >/= 0.5 mg/ml purified chaperonins form filaments in the presence of Mg2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea.

Actin Cytoskeleton↗
PubMed2001

Localization of a developmentally regulated protein in porcine follicles identified by a monoclonal antibody.

OBJECTIVE: In the present study we employed a monoclonal antibody (Mab 3D8) obtained against a rat ovarian antigen and identified a 76 kDa protein in porcine ovarian follicles. METHODS: The localization of this antigen was studied by light and electron-microscopic immunocytochemistry and further characterized by polyacrylamide gel electrophoresis and immunoblotting on nitrocellulose membranes. RESULTS: We found that the antigen recognized by Mab 3D8 is localized in granulosa cells (GCs) and oocytes. The expression of the 76 kDa protein apparently depends on the developmental stage. A particularly strong reaction was observed in cumulus cells and oocytes in early and late antral follicles. In granulosa cells the reaction product was localized in rough endoplasmic reticulum (RER), cis and trans faces of the Golgi stack, the outer nuclear envelope and in numerous transport vesicles budding from the endoplasmic reticulum. In the oocyte the reaction product was localized in structures related to specific endocytosis - small pits at the cell surface, two subsets of endosomes, endocytic carrier vesicles and the prelysosomal compartment (PLC). CONCLUSIONS: The results obtained suggest that porcine oocytes possess the cellular structures, which allow them to bind and internalize this protein, which is most probably produced by granulosa cells. During oocyte development the intracellular site of accumulation of the 76 kDa protein varies, which implies that it is under developmental control. follicle

Animals↗
PubMedDate not supplied

[Ultrastructural changes in the mycelia of Actinomyces hygroscopicus var. var. enhygrus--a proteolytic enzyme producer--during submerged fermentation].

Ultrastructural changes were found to occur in the mycelium of Actinomyces hygroscopicus, strain 33x, which produced an exocellular proteolytic enzyme during submerged fermentation, in both laboratory and semiindustrial conditions (in 100-1 fermenters). In the course of the enzyme accumulation, numerous vacuoles appeared in the hyphae, some hyphae became wider, and the cell walls were more loose. The greatest structural changes were found in the intracytoplasmic membrane systems. The ultrastructural changes of the mycelium are not presumably connected with the accumulation of the enzyme, but are the result of the differentiation of cellular structures during aging of the cells and their transition to the stage of autolysis.

Microscopy, Electron↗
PubMed1996

[Three-dimensional analysis of chromosomes by a computerized fluorescence microscope system].

In order to study spatial arrangement and dynamics of chromosomes and nuclear structures, we have developed a computerized fluorescence microscope workstation. This microscope workstation is capable of rapidly acquiring three-dimensional, multiple-wavelength image data and allows accurate reconstructions of three-dimensional cellular structures stained with multiple fluorescent dyes. Using this microscope system, we have examined dynamics of chromosomes and microtubules in mammalian culture cells by combination of high-resolution analysis in fixed specimens and time-lapse analysis in living cells. A key aspect of such microscopic approaches is a capability of detecting transient events during dynamic biological processes.

Animals↗
PubMed2005

The C-terminal half of TSG101 blocks Rous sarcoma virus budding and sequesters Gag into unique nonendosomal structures.

Retroviral late domains (L domains) are short amino acid sequences in the Gag protein that facilitate the process of budding. L domains act by recruiting the ESCRT complexes, which normally function in the formation of multivesicular bodies. The PTAP late domain of human immunodeficiency virus (HIV) is believed to specifically recruit this machinery by binding the ESCRT protein TSG101. It was recently demonstrated that expression of a C-terminal fragment of TSG101 (TSG-3') blocked the budding of both PTAP-dependent and PPPY-dependent retroviruses. We show here that TSG-3' expression leads to the formation of large spherical entities that we call TICS (TSG-3'-induced cellular structures) in the cytoplasm. Rous sarcoma virus (RSV) and murine leukemia virus (MLV) Gag proteins are selectively recruited to these structures, but HIV type 1 Gag is completely excluded. Experiments with various HIV and RSV vector constructs as well as HIV and RSV chimeras suggest that recruitment to the TICS is late domain independent and does not involve recognition of any single amino acid sequence. TICS appear to have no limiting membrane and do not colocalize with markers for any membranous cellular compartment. Wild-type TSG101 is also recruited to TICS, but most other ESCRT proteins are excluded. These structures are similar in nature to aggresomes, colocalize with the aggresome marker GFP-250, and are highly enriched in ubiquitin but in other ways do not fully meet the description of aggresomes. We conclude that the block to retroviral budding by TSG-3' may be the result of its sequestration of Gag, depletion of free TSG101, or depletion of free ubiquitin.

Animals↗
PubMed1984

Genetics of microtubule systems.

In most eucaryotes the tubulin genes comprise small multigene families with approximately equal numbers of genes for alpha- and beta-tubulin, the structural proteins of microtubules. The recent isolation of tubulin mutations in several species is proving to be a powerful tool for examining the structure and function of specific sets of microtubules. In Drosophila melanogaster, genetic analysis of a testis-specific beta-tubulin gene has shown that a single tubulin gene product may fulfill a number of different microtubule functions. In addition to tubulin mutations, mutations in other genes whose products are involved in the regulation or structure of specific microtubule arrays have also been isolated. The combination of analysis of both classes of mutations is beginning to allow a molecular description of the construction and function of three-dimensional cellular structures. In addition, such studies may also shed light on the evolutionary pressures that gave rise to and serve to maintain small families of genes encoding very similar proteins.

Animals↗