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At least 1,063 records · Page 59Linked to original sources
PubMed2006

Characterisation of rat corneal cells that take up soluble antigen: an in vivo and in vitro study.

The aim of the present study was to determine the capacity of resident corneal and limbal dendritic cells (DC) and macrophages to capture antigen (Ag) in vivo and compare this to their capacity in vitro to take up Ag during organ culture conditions. To investigate Ag uptake in vivo 3 microl (30 microg) of fluorescently labelled Dextran, bovine serum albumin (BSA) or ovalbumin (OVA) were either placed on the intact ocular surface or injected into the anterior chamber (AC) or subconjunctival space of the Lewis rat eye. The presence of Ag+ cells in the cornea was assessed using intravital fluorescence video microscopy. Animals were sacrificed 24h after Ag injections or topical application and the distribution and phenotype of Ag+ cells were analysed ex vivo by fluorescence and confocal microscopic analysis of immunostained and unstained corneal tissue wholemounts or frozen sections. Corneal buttons and corneoscleral rims from naive Lewis rats were placed in organ culture conditions in the presence of LPS with or without FITC-Dextran for 48 h and 72 h. The explants were examined by epi-fluorescence microscopy and the phenotype of Ag+ cells in the supernatant from the organ cultures was analyzed by flow cytometry using a range of macrophage and DC markers. In vivo observations and microscopic examination of corneas 24h following Ag topical application failed to reveal evidence of Ag retention by ocular cells. Those in which Ag had been placed in the AC or subconjunctival space revealed Ag+ cells within the corneal stroma. The distribution of Ag+ cells displayed a centripetal gradient, the most marked uptake of Ag being by cells in the circumferential limbal zone. Immunophenotypic studies revealed that Ag uptake was predominantly performed by cells that were CD68+, CD172+ but rarely MHC class II+, a profile characteristic of macrophages. Occasional Ag+ keratocytes were noted. In vitro studies of corneal buttons placed in culture conditions revealed that cells from the limbal zone, but not the central cornea, were able to take up Ag from the supernatant. Significant numbers of the cells that had migrated from the corneal buttons and captured fluorescent labelled Ag in the presence of LPS were revealed by flow cytometry to consist of CD163+ and CD11b+ macrophages, but none expressed the DC markers CD11c or OX62 and they were also generally MHC class II(-). In conclusion the present study revealed that macrophages and keratocytes in the corneal stroma and limbal episcleral tissue have the capacity to internalise fluorescent mock Ag injected into the AC or subconjunctival space or in culture conditions. The failure to demonstrate significant Ag trapping ability by corneal or limbal stromal or epithelial DC may either be due to the rarity of such cells or their lack of Ag trapping ability.

Administration, Topical↗
PubMed2005

Potassium homeostasis influences the locomotion and encystment of zoospores of plant pathogenic oomycetes.

Zoospores of plant pathogenic oomycetes exhibit distinct swimming speeds and patterns under natural conditions. Zoospore swimming is influenced by ion homeostasis and changes in the ionic composition of media. Therefore, we used video microscopy to investigate swimming patterns of five oomycete species in response to changes in potassium homeostasis. In general, zoospore speed tended to be negatively correlated with zoospore size. Three Phytophthora species (Phytophthora palmivora, Phytophthora megakarya, and Phytophthora infestans) swam in straight patterns with speeds ranging from 50 to 250 microm/s whereas two Pythium species (Pythium aphanidermatum and Pythium dissotocum) swam at similar speeds ranging from 180 to 225 microm/s with a pronounced helical trajectory and varying amplitudes. High external concentrations of potassium salts reduced the swimming speed of Ph. palmivora and induced encystment. This was not observed for Py. aphanidermatum. Application of the potassium ionophores gramicidin, nigericin and valinomycin resulted in reduced swimming speeds and changes in the swimming patterns of the Phytophthora species. Therefore, potassium ions play a key role in regulating zoospore behavior.

Anti-Infective Agents↗
PubMed2006

Ontogeny of the Spitzenkörper in germlings of Neurospora crassa.

The Spitzenkörper (Spk) is a highly dynamic and pleomorphic complex located at the hyphal apex of filamentous fungi. Most studies revealing the structure and behavior of the Spk have been conducted on mature vegetative hyphae of filamentous fungi, including both main leading hyphae and branches. However, these reports do not address whether the observations can be extended to germ tubes. By enhanced phase-contrast video-microscopy and laser scanning confocal microscopy we have analyzed the intracellular changes prior to the appearance of a Spk in germlings of Neurospora crassa. Observations began at the early stages of spore germination and were carried out until a conspicuous Spk could be observed at the apex of germ tubes. Before a Spk could be observed, young germ tubes (<150 microm) displayed a uniform distribution of organelles such as nuclei, mitochondria, and cytoplasmic granules along the length of the cells. Once the germlings started reaching lengths of more than approximately 150 microm, visible organelles experienced a displacement towards the subapical region of the cell and a small exclusion zone free of organelles (0.6+/-0.3 microm) formed at the apex. The position of this exclusion zone within the apex seemed to determine the germling growth direction, which was highly erratic. Few minutes after it first appeared, upon growth of the germling, the exclusion zone started to become occupied by an accumulation of material that gradually concentrated into a light gray body that we describe as an immature Spk. During this phase the presence of a Spk in the apical dome was not constant. Approximately 30 min later, the immature Spk became more robust and gradually acquired its typical phase-dark appearance, while the growth direction of the germ tube became less wavering. The formation of a mature phase-dark Spk coincided with the stabilization of the growth direction of the germling, therefore suggesting that it is at this stage when the transition from germling to vegetative hypha occurs.

Fluorescent Dyes↗
PubMed2005

Regeneration of human auditory nerve. In vitro/in video demonstration of neural progenitor cells in adult human and guinea pig spiral ganglion.

Time lapse video recordings of cultured adult human and guinea pig spiral ganglion (hSG and gpSG) show that mitogen responsive progenitor/stem cells develop in the form of spheres that proliferate and differentiate into mature neurons and glia cells. Neurospheres, cultured with EGF and bFGF showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). Newly formed BrdU labelled cells were positive for beta-tubulin, and also for GFAP demonstrating that neuronal cells were derived from a dividing population of progenitor cells. Dissociated spheres cultured either with glia cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), induced differentiation of the progenitor cells. Video microscopy showed that neurons develop from subcultured spheres maintained for up to four weeks. Neurons showed fasciculation and migration with a speed of 10-30 microm/h, and some cells had up to 6 mm long neurites coexpressing TrkB and TrkC receptors. Precise dissection suggests that the neurons formed are cochlea-specific. The results suggest that the mammalian auditory nerve has the capability for self-renewal and replacement. Transplantation of progenitor cells together with established means to induce neural differentiation and fiber growth may facilitate strategies for better repair and treatment of auditory neuronal damage.

Adult↗
PubMed2006

Intracellular osmotic gradient generation in rat ventricle myocyte by using a micro-perfusion system.

This experimental study describes the fabrication and analysis of a micro-perfusion system that can be used in many bioengineering experiments to create rapid, large regional intracellular changes within single ventricular myocytes. The myocyte was a kind of osmometer since the cell volume was found to be strongly dependent on the perfusion solution osmolarity. This volume change was measured, indirectly, by measuring the cell width change using video-microscopy and image analysis software. Jacob's equation was used to model these results successfully. Some dual perfusion experiments to see the effects of the localized perfusion of different osmotic solutions to generate an osmotic gradient inside myocytes were also investigated. This device can be useful for studying the effects of localized pH or osmotic gradients inside myocytes, estimating intracellular ion diffusion rates, and inducing regional changes in other important intracellular ions.

Animals↗
PubMed2005

Spatial and temporal development of chondrocyte-seeded agarose constructs in free-swelling and dynamically loaded cultures.

Dynamic deformational loading has been shown to significantly increase the development of material properties of chondrocyte-seeded agarose hydrogels, however little is known about the spatial development of the material properties within these constructs. In this study, a technique that combines video microscopy and optimized digital image correlation, was applied to assess the spatial development of material properties in tissue-engineered cartilage constructs cultured in free-swelling and dynamically-loaded conditions (3h/day, 5 days/week, and maintained in free-swelling conditions when not being loaded) over a 6-week period. Although homogeneous at day 0, both free-swelling and dynamically loaded samples progressively developed stiffer outer edges and a softer central region. The distribution of GAGs and collagens were shown to mimic this profile. These results indicate that although dynamic loading augments the development of bulk properties in these samples, possibly by overcoming some of the diffusion limitation and nutrient transport issues, the overall profile of construct properties in the axial direction remains qualitatively the same as in free-swelling culture conditions. Poisson's ratio of these constructs increased over time in culture with increased fixed charged density contributed by the GAGs, but this increase was significantly less in dynamically loaded samples by day 42. Polarized light microscopy of Picrosirius Red labeled samples, at an angle perpendicular to the direction of loading, suggests that these differences in Poisson's ratio may be due to improved organization of collagen network in the dynamically loaded samples.

Animals↗
PubMed2005

Extended time range modeling of myelin growth.

When some poorly water-soluble solid surfactants are contacted with water, several microstructures are observed as part of the dissolution process of the surfactants in water. One such microstructure called "myelin," which is observed when a surfactant like phosphatidylcholine is contacted with water, is the subject of this paper. In this study we have used video microscopy to investigate myelin growth over a wide time range, namely 0.25-700 s, and found that existing models do not correctly express myelin growth over extended time ranges. When studied over a wide time range, the myelin growth was found to evolve over three distinct regimes, namely ballistic, diffusional, and subdiffusional regimes. The underlying growth models are physically explained and mathematically expressed. A relationship is derived between the width of myelin and the growth rate at long times. The estimated width of myelin is consistent with experiments.

Journal Article↗
PubMed2006

Transport of ions through the oil phase of W(1)/O/W(2) double emulsions.

Using a capillary video microscopy technique, the ion transport at liquid-liquid interfaces and through a surfactant-containing emulsion liquid membrane was visually studied by preparing a double emulsion globule within the confined space of a thin-walled, transparent, cylindrical microtube. NaCl and AgNO(3) were selected as the model reactants and were prepared to form a NaCl/AgNO(3) pair across the oil film. By observing and measuring the formed AgCl deposition, it was found that both Cl(-) and Ag(+) could transport through a thick oil film and Ag(+) was transported faster than Cl(-). Interestingly, the ion transport was significantly retarded when the oil film became extremely thin (<1 microm). The results suggested that the transport of ions mainly depends on the "reverse micelle transport" mechanism, in which reverse micelles with entrapped ions and water molecules can be formed in a thick oil film and their construction will get impeded if the oil film becomes extremely thin, leading to different ion transport rates in these two cases. The direction of ion transport depends on the direction of the osmotic pressure gradient across the oil film and the ion transport is independent of the oil film thickness in the investigated thick range. Ions with smaller Pauling radii are more easily entrapped into the formed reverse micelles and therefore will be transported faster through the oil film than bigger ions. Oil-soluble surfactants facilitate ion transport; however, too much surfactant in the oil film will slow down the ion migration. In addition, this study showed no support for the "molecular diffusion" mechanism of ion transport through oils.

Journal Article↗
PubMed2006

DNA/cationic polymer complex attachment on a human vascular endothelial cell monolayer exposed to a steady laminar flow.

This study evaluated for the first time the binding of pDNA/polymer complexes (polyplexes) on a human lung microvascular endothelial cell (HLMEC) monolayer under flow conditions. A slide of a HLMEC monolayer was mounted on a parallel flow chamber connected to an open flow system from a reservoir containing fluorescent polyplexes to a syringe. A precise pump allowed their passage through the chamber under a range of shear stresses. The binding of polyethyleneimine (PEI)- and histidylated polylysine (His)-polyplexes was carried out over 30 min by time-lapse video microscopy. At 10 microg pDNA/ml in 10% serum, we found that 360+/-80 PEI- and 250+/-50 His-polyplexes were bound per 1000 cells at a shear stress of 0.3-1 dyn/cm(2). This number dropped to approximately 100 at 2 dyn/cm(2). These polyplexes exhibited differences in their interactions with the cell membrane. Concerning PEI-polyplexes, there was a shear threshold effect allowing a maximum binding at 0.06 dyn/cm(2) and a higher binding reduction (77%) at 5 microg/ml pDNA in 100% serum. The polyplex binding was augmented by 300% with PEI bearing tetraglucose moiety. This set-up is potentially helpful to screen a wide array of endothelial cells ligands prior in vivo experiments.

Cells, Cultured↗
PubMed2006

A deletion variant study of the functional role of the Salmonella flagellin hypervariable domain region in motility.

The eubacterial flagellum is a complex structure with an elongated extracellular filament that is composed primarily of many subunits of a flagellin protein. The highly conserved N and C termini of flagellin are important in its export and self-assembly, whereas the middle sequence region varies greatly in size and composition in different species and is known to be deletion-tolerant. In Salmonella typhimurium phase 1 flagellin, this "hypervariable" region encodes two solvent-exposed domains, D2 and D3, that form a knob-like feature on flagella fibers. The functional role of this structural feature in motility remains unclear. We investigated the structural and physiological role of the hypervariable region in flagella assembly, stability and cellular motility. A library of random internal deletion variants of S. typhimurium flagellin was constructed and screened for functional variants using a swarming agar motility assay. The relative cellular motility and propulsive force of ten representative variants were determined in semi-solid and liquid medium using colony swarming motility assays, video microscopy and optical trapping of single cells. All ten variants exhibited diminished motility, with varying extents of motility observed for internal deletions less than 75 residues and nearly complete loss of motility for deletions greater than 100 residues. The mechanical stability of the variant flagella fibers also decreased with increasing size of deletion. Comparison of the variant sequences with the wild-type sequence and structure indicated that all deletions involved loss of hydrophobic core residues, and removal of both partial and complete segments of secondary structure in the D2 and D3 domains. Homology modeling predicted disruptions of secondary structures in each variant. The hypervariable region D2 and D3 domains appear to stabilize the folded conformation of the flagellin protein and contribute to the mechanical stability and propulsive force of the flagella fibers.

Agar↗
PubMed2005

Insulin enhances mitochondrial inner membrane potential and increases ATP levels through phosphoinositide 3-kinase in adult sensory neurons.

We tested the hypothesis that neurotrophic factors control neuronal metabolism by directly regulating mitochondrial function in the absence of effects on survival. Real-time whole cell fluorescence video microscopy was utilized to analyze mitochondrial inner membrane potential (Delta Psi(m)), which drives ATP synthesis, in cultured adult sensory neurons. These adult neurons do not require neurotrophic factors for survival. Insulin and other neurotrophic factors increased Delta Psi(m) 2-fold compared with control over a 6- to 24-h period (P < 0.05). Insulin modulated Delta Psi(m) by activation of the phosphoinositide 3-kinase (PI 3-K) pathway. Insulin also induced rapid and long-term (30 h) PI 3-K-dependent phosphorylation of Akt and cAMP response element binding protein (CREB). Additionally, insulin elevated the redox state of the mitochondrial NAD(P)H pool, increased hexokinase activity (first committed step of glycolysis), and raised ATP levels. This study demonstrates that insulin utilizes the PI 3-K/Akt pathway to augment ATP synthesis that we propose contributes to the energy requirement for neurotrophic factor-driven axon regeneration.

Activating Transcription Factor 1↗
PubMed2005

Mutation of retS, encoding a putative hybrid two-component regulatory protein in Pseudomonas aeruginosa, attenuates multiple virulence mechanisms.

Two-component regulatory systems play an important role in bacterial virulence. We report that mutation of a Pseudomonas aeruginosa gene designated retS (previously designated fimK; accession number PA4856) encoding a putative hybrid two-component regulator, attenuates multiple virulence mechanisms. The retS mutant was selected from a Tn5 transposon library of the cytotoxic P. aeruginosa strain PA103 based upon expression of a small-colony phenotype suggestive of reduced surface-associated "twitching" motility, a property dependent upon type IV pili. Subsequent analysis revealed that the mutant expressed pilin, albeit at lower levels than wild-type PA103. In a murine model of corneal infection, retS mutation was associated with delayed disease development and altered pathology. In vitro, retS mutants demonstrated loss of acute cytotoxic activity towards corneal epithelia as determined by trypan blue exclusion and by LDH release assays (P<0.0001). This coincided with loss of ExsA-regulated type III secretion. Mutation of retS also impaired ExsA-independent pathogenic mechanisms. When compared to the exsA mutant of PA103, retS mutants exhibited reduced epithelial adherence and invasion and reduced intracellular survival within the cells after invasion. Time-lapse video microscopy revealed that retS mutants, compared to exsA mutants, had a reduced capacity to access, and move along, the basal cell surfaces of corneal epithelial cell monolayers. Taken together, these data suggest that the protein encoded by retS regulates various properties of P. aeruginosa including both ExsA-dependent and ExsA-independent virulence mechanisms.

Animals↗
PubMed2005

Increased substance P content in nerve fibers associated with mesenteric veins from deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

This study examined sensory nerves associated with mesenteric arteries and veins in sham and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Reactivity of arteries and veins to substances released from sensory nerves was also studied in vitro using computer-assisted video microscopy. Co-localization of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity (ir) was used to evaluate perivascular sensory nerves. Radioimmunoassay was used to quantify SP- and CGRP-ir content. Immunohistochemical studies revealed a plexus of SP/CGRP-ir nerves associated with arteries and veins. The intensity of SP-ir, but not CGRP-ir labeling was greater in arteries and veins from DOCA-salt compared to sham rats. RIA measurements revealed that the CGRP-ir content of arteries and veins was higher than the SP-ir content but there was a significant increase in SP-ir, but not CGRP-ir, content in arteries and veins from DOCA-salt rats. SP (0.03-1 microM) contracted veins and the NK-3 receptor agonist, senktide, mimicked this effect. There were no differences in SP or senktide reactivity of veins from sham or DOCA-salt rats. SP, but not senktide, relaxed KCl (40 mM) preconstricted arteries. CGRP (0.3 microM), acetylcholine (10 microM) and capsaicin (1 microM) relaxed KCl-preconstricted arteries and veins. The NK-1 receptor agonist, substance P methyl ester relaxed arteries but not veins. These data indicate that DOCA-salt hypertension is associated with upregulation of SP content in perivascular nerves. NK-3 receptors mediate venoconstriction which is unchanged in DOCA-salt hypertension. Increased release of SP from perivenous nerves might contribute to the increased venomotor tone in DOCA-salt hypertension.

Animals↗
PubMed2004

Perillyl alcohol-mediated inhibition of lung cancer cell line proliferation: potential mechanisms for its chemotherapeutic effects.

Perillyl alcohol (POH) is currently being tested in clinical trials as an anticancer agent, though its mechanism of action has not been definitively established. We treated two human lung cancer cell lines, H322 and H838, with POH to determine its antitumor properties. A sulforhodamine B (SRB) cell proliferation assay was used to determine the effects of POH after 1 and 5 days of treatment with 0.25, 0.5, 0.75, 1.0, and 1.5 mM POH. After 1 day of treatment, little difference could be seen between the lowest and highest concentrations of POH. However, after 5 days, both cell lines showed a dose-dependent decrease in cell proliferation that ranged from 15% to 83%. A clonogenic assay confirmed these results-while there was no significant effect of POH after 1 day of exposure, a dose-dependent decrease in colony formation, ranging from 15% to 100%, was seen after 5 days of treatment. Time-lapse video microscopy revealed that apoptotic cells were evident within 24-48 h of treatment with 1.5 mM POH. The appearance of apoptotic cells was preceded by increased caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP) as POH activated caspase-3 activity 3-6-fold. Nuclear staining with 4',6-diamidino-2-phenylindole (DAPI) confirmed the classical characteristics of apoptosis in POH-treated cells. DNA microarray expression analysis was performed following 8 and 24 (H322) or 8 and 48 (H838) h of treatment with 1.5 mM POH. While a large number of genes were up- or downregulated in the two cell lines at various times after POH treatment, the levels of expression of only eight genes were up- or down-related in both cell lines at both of the time points examined. The significance of these genes as potential mediators of POH action is still uncertain, but the limited number of commonly up- or downregulated genes detected by microarray expression analysis suggests that POH may mediate its effects via posttranscriptional mechanisms. Our results suggest that POH may have potential use as an anticancer drug that stimulates or sensitizes lung tumor cells to apoptosis, and this effect may depend on genetic lesions present in tumor cells.

Antineoplastic Agents↗
PubMed2005

Vascular reactivity of mesenteric arteries and veins to endothelin-1 in a murine model of high blood pressure.

We characterized vascular reactivity to endothelin-1 (ET-1) in mesenteric vessels from DOCA-salt hypertensive and SHAM control mice and assessed the effect that endothelial-derived vasodilators have on ET-1-induced vasoconstriction. Changes in the diameter of unpressurized small mesenteric arteries and veins (100- to 300-microm outside diameter) were measured in vitro using computer-assisted video microscopy. Veins were more sensitive than arteries to the contractile effects of ET-1. There was a decrease in arterial maximal responses (E(max)) compared to veins, this effect was larger in DOCA-salt arteries. The selective ET(B) receptor agonist, sarafotoxin 6c (S6c), contracted DOCA-salt and SHAM veins but did not contract arteries. The ET(B) receptor antagonist, BQ-788 (100 nM), but not the ET(A) receptor antagonist, BQ-610 (100 nM), blocked S6c responses. BQ-610 partially inhibited responses to ET-1 in mesenteric veins from DOCA-salt and SHAM mice while BQ-788 did not affect responses to ET-1. Co-administration of both antagonists inhibited responses to ET-1 to a greater extent than BQ-610 alone suggesting a possible functional interaction between ET(A) and ET(B) receptors. Responses to ET-1 in mesenteric arteries were completely inhibited by BQ-610 while BQ-788 did not affect arterial responses. Nitric oxide synthase inhibition potentiated ET-1 responses in veins from SHAM but not DOCA-salt mice. There was a prominent role for ET-mediated nitric oxide release in DOCA-salt but not SHAM arteries. In summary, these studies showed a differential regulation of ET-1 contractile mechanisms between murine mesenteric arteries and veins.

Algorithms↗
PubMed2004

The human small glutamine-rich TPR-containing protein is required for progress through cell division.

Eukaryotic organisms from yeast to human harbor genes encoding the small glutamine-rich tetratricopeptide repeat-containing (SGT) protein. Work presented here demonstrated the presence of human SGT (hSGT) protein in a panel of human cell lines and throughout the cell cycle. To identify cellular processes in which hSGT is involved, knock down populations were analyzed which were generated through transfection of hsgt-specific small interfering RNA. Most strikingly, depletion of hSGT led to reduced proliferation of the affected cell populations while the mitotic index was increased. Time-lapse video microscopy revealed that cells from hSGT-depleted populations were unable to complete cell division due to mitotic arrest which was frequently followed by cell death. Further evidence for a role in cell division was given by the accumulation of hSGT in the midzone and the midbody, and by a mitosis-specific migration pattern of hSGT as detected by Western blotting after SDS-PAGE or two-dimensional gel electrophoresis. In conclusion, results obtained in this study demonstrate that hSGT protein is a constitutive component of all human cell lines tested and that this protein is essential for successful completion of cell division.

Amino Acid Motifs↗
PubMed2004

A dynamic podosome-like structure of epithelial cells.

Focal contacts and hemidesmosomes are cell-matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin, and zyxin. We also show that the maintenance of the actin core and hemidesmosomes is dependent on actin polymerization, src-family kinases, and Grb2, but not on microtubules. Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of beta4 integrin subunit to the actin core before its positioning at hemidesmosomes. When 804G cells are induced to migrate, actin cores as well as hemidesmosomes disappear and beta4 integrin subunit becomes co-localized with dynamic actin at leading edges. We show that podosome-like structures are not unique to cells of mesenchymal origin, but also appear in epithelial cells, where they seem to be related to basement membrane adhesion.

Actins↗
PubMed2005

Single cell analysis of G1 check points-the relationship between the restriction point and phosphorylation of pRb.

Single cell analysis allows high resolution investigation of temporal relationships between transition events in G1. It has been suggested that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is the molecular mechanism behind passage through the restriction point (R). We performed a detailed single cell study of the temporal relationship between R and pRb phosphorylation in human fibroblasts using time lapse video-microscopy combined with immunocytochemistry. Four principally different criteria for pRb phosphorylation were used, namely (i) phosphorylation of residues Ser795 and Ser780, (ii) degree of pRb-association with the nuclear structure, a property that is closely related with pRb phosphorylation status, (iii) release of the transcription factor E2F-1 from pRb, and (iv) accumulation of cyclin E, which is dependent on phosphorylation of pRb. The analyses of individual cells revealed that passage through R preceded phosphorylation of pRb, which occurs in a gradually increasing proportion of cells in late G1. Our data clearly suggest that pRb phosphorylation is not the molecular mechanism behind the passage through R. The restriction point and phosphorylation of pRb thus seem to represent two separate check point in G1.

Cell Cycle Proteins↗