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At least 883 records · Page 49Linked to original sources
PubMed2006

Simultaneous quantification of oxidative stress and cell spreading using 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein.

BACKGROUND: Mitochondrial dysfunction may lead to increased oxidative stress and consequent changes in cell spreading. Here, we describe and validate a novel method for simultaneous quantification of these two parameters. METHODS: Human skin fibroblasts were loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-H(2)DCF), and its oxidative conversion into CM-DCF was monitored as a function of time by video-rate confocal microscopy and real-time image averaging. Cell size was determined after binarization of the acquired images. RESULTS: At the lowest practical laser output, CM-DCF formation occurred with zero order kinetics, indicating that [CM-H(2)DCF] was not rate-limiting and that the rate of [CM-DCF] formation (V(CM-DCF)) was a function of the cellular oxidant level. Analysis of fibroblasts of a healthy control subject and a patient with a deficiency of NADH:ubiquinone oxidoreductase, the first complex of the oxidative phosphorylation system, revealed a significant increase in cellular oxidant level in the latter cells that was, however, not accompanied by a change in cell spreading. Conversely, chronic treatment with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a derivative of vitamin E, markedly decreased the oxidant level and cell spreading in both control and patient fibroblasts. CONCLUSIONS: We present a reliable method for simultaneous quantification of oxidant levels and cell spreading in living cells.

Antioxidants↗
PubMed1989

Maize streak virus genes essential for systemic spread and symptom development.

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The ;+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary ;-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.

Journal Article↗
PubMed2002

Attachment, spreading, and matrix formation by human gingival fibroblasts on porous-structured titanium alloy and calcium polyphosphate substrates.

Porous calcium polyphosphate (CPP) structures represent promising resorbable implant systems that can promote anchorage to connective tissues. Previous studies focused on chondrocyte interactions with CPP, but there are limited data on interactions of soft connective tissue cells with these materials. We studied attachment, spreading, and matrix formation by human gingival fibroblasts when cultured on amorphous and crystalline CPP. Comparison with porous Ti6Al4V substrates of similar volume percent, porosity, and pore size distribution provided evaluations of fibroblast interactions with rapid, moderate, and nonbiodegradable systems, respectively. Cells were incubated on substrates in medium containing ascorbic acid and evaluated at 3, 24, 48, 72, and 96 h after plating. Attached cell counts, cytoplasmic actin filament area, and immunostained extracellular type 1 collagen or fibronectin were quantified by morphometric analyses using epifluorescence microscopy. Cell morphology and substrate interactions were evaluated by scanning electron microscopy. Spreading, attachment, and matrix production were similar for both CPP substrates. In contrast, titanium alloy substrates exhibited threefold more attachment and twofold more spreading than CPP substrates. The area per cell of immunostained extracellular collagen and fibronectin was similar for the three different substrates. The results indicate that the crystallinity and, hence, degradation rate of CPP substrates does not substantially affect the interactions of fibroblasts with CPP materials but that compared with titanium alloy substrates, spreading and attachment are inhibited.

Alloys↗
PubMed1988

Attachment and spreading of human gingival fibroblasts on potentially bioactive glasses in vitro.

The attachment and spreading of cultured fibroblasts on potentially bioactive glasses (bioglasses) of ten different compositions were studied. Human gingival fibroblasts were allowed to attach and spread on bio-glasses for 1-72 h. Unreactive silica glass and cell culture polystyrene served as controls. The attachment and spreading of cells were examined by 3H-thymidine labeling of cells, planimetric analysis, cytological staining, immunocytochemistry, and scanning electron microscopy. The cell attachment to bioglasses and silica glass and the cell spreading on bioglasses were slower and cell morphology more elongated compared to control plastic. In spite of great differences in bioglass compositions no great differences in cell behavior on these surfaces were detected. Thus the initial events in the tissue-implant interface might be independent on the bioglass composition, and furthermore the differences in the organization of the tissue-implant interface in vivo might depend on the nature of the surrounding tissues and subsequent changes of the implant surface and the extracellular environment.

Adult↗
PubMed2004

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel.

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.

Animals↗
PubMed2006

The effects of short-term stimulation on fibroblast spreading in an in vitro 3D system.

Although the short-term effects of mechanical and biochemical stimulation on cell protein and DNA production have recently begun to be evaluated using 3D models, the effects that such stimulation have on cell morphology and adaptation remains unclear. Using a custom-built bioreactor, we were able to create a systematic model to examine the short-term effects of stimulation on cell morphology in a 3D model, specifically by evaluating cell spreading as the short-term indicator of cell adaptation. Fibroblasts were seeded on a porous poly(L-lactic acid) scaffold and cultured in a computerized bioreactor for 24 h under various uniaxial strains (0, 0.6, 3, 6%) and frequencies (0.0125, 0.125 Hz). Also, the effects of transforming growth factor (TGF-beta1) (1, 10, 100 ng/mL) were examined on static, nonstimulated cells-scaffold constructs after 24 h. Fibroblasts that had been subjected to mechanical stretching were found to exhibit significantly more spreading than the static control group. Conversely, TGF-beta1 between 1 and 100 ng/mL did not produce any significant difference in fibroblast spreading from the control groups after 24 h. Collectively, the findings suggest that cell morphology and adaptation may be affected by short-term mechanical stimulation, as seen by increased cell spreading by the fibroblasts under these experimental conditions.

Animals↗
PubMed2006

Integrin signaling and cell spreading mediated by phorbol 12-myristate 13-acetate treatment.

Spreading of SNU16mAd gastric carcinoma cells was previously shown to be regulated via a signaling network from transforming growth factor beta1 (TGFbeta1) to integrins signaling, through a mediation of protein kinase C delta (PKCdelta). However, in the previous study, the roles of PKCdelta appeared complicated. In this study to clarify the roles of PKCdelta in the spreading of the gastric carcinoma cells, we questioned if PKC activation via phorbol 12-myristate 13-acetate (PMA) treatment could mimic the TGFbeta1 effects. An acute PMA treatment increased phosphorylations of focal adhesion (FA) kinase, paxillin, c-Src, and cofilin, just as TGFbeta1 did. Furthermore, cell spreading mediated by TGFbeta1- or acute PMA treatment correlated with activation of RhoA, which regulates actin reorganization and FA formation. However, stress fiber formation was prominent in TGFbeta1-treated cells, compared to cortical actin organization in PMA-treated cells. Altogether, these observations indicate that acute PMA treatment could mimic the TGFbeta1 mechanisms for cell spreading through subtly different effects on actin reorganization.

CSK Tyrosine-Protein Kinase↗
PubMed1985

Human spreading factor: synthesis and response by HepG2 hepatoma cells in culture.

Human serum spreading factor (SF) is a cell adhesion and spreading-promoting glycoprotein purified from serum or plasma that mediates effects in a wide variety of animal cell culture systems. HepG2 human hepatoma cells were found to synthesize and secrete SF into culture medium. Quantitative immunoassay of the protein indicated a concentration of about 1 microgram/ml in 48 hr-conditioned medium from confluent cultures. Although fibronectin also was synthesized and secreted into the culture medium, HepG2 cell spreading was observed in response to human serum SF, but not in response to human plasma fibronectin. Immunoprecipitation of SF from culture medium of cells metabolically-labeled with leucine, fucose or glucosamine identified a single form of the molecule of approximately 70,000 daltons. Treatment of cultures with tunicamycin inhibited incorporation of fucose and glucosamine into immunoprecipitated SF, but did not prevent synthesis and secretion of the protein. Electrophoretic analysis and cell spreading assays showed that SF secreted by tunicamycin-treated HepG2 cells was of molecular weight (mw) approximately 60,000, and was biologically active.

Carcinoma, Hepatocellular↗
PubMed1995

Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading.

Guinea pig bone marrow megakaryocytes were cultured on a type I rat tail collagen gel which stimulated proplatelet formation. Proplatelet formation was inhibited by monoclonal antibody LM609 to the alpha v beta 3 integrin (VnR), but not by monoclonal antibodies to the alpha 5, alpha 6, beta 1, or IIb beta 3(GPIIb-IIIa) integrin proteins. Megakaryocytes cultured on a plastic surface and stimulated with thrombin undergo a spreading and an adhesion reaction. This reaction is blocked in a dose-dependent manner by the tetrapeptide RGDS and by the monoclonal antibody PG2 to the GPIIb-IIIa integrin, but not by the monoclonal antibody LM609 to the VnR. Immunoprecipitation and affinity chromatography experiments demonstrate that guinea pig megakaryocytes have distinct GPIIb-IIIa and VnR integrins with similar electrophoretic mobility. Spreading was significantly inhibited in a dose-dependent fashion by drugs which elevate cellular cyclic AMP, including forskolin, dibutyryl cAMP, and isobutylmethylxanthine. In contrast to spreading, megakaryocyte proplatelet formation was stimulated by these agents in a dose-dependent manner. Megakaryocyte spreading was stimulated by the protein kinase C (PKC) activator phorbol myristate acetate (PMA) and inhibited by the PKC inhibitors Calphostin C and K5720 in a dose-dependent manner. PKC inhibitors did not inhibit megakaryocyte proplatelet formation. These results demonstrate that the closely related VnR and GPIIb-IIIa integrins regulate different aspects of megakaryocyte morphological change and appear to be associated with different second messenger systems.

Animals↗
PubMed1985

An inhibitory factor against monocyte spreading in the sera of patients with systemic lupus erythematosus.

The effect of the sera of patients with systemic lupus erythematosus (SLE) on monocyte function was studied using cell spreading as an indicator. Monocyte spreading induced by exogenous stimuli was shown to be inhibited by SLE sera. Gel filtration of SLE sera on Sephadex G-200 revealed that the factor responsible for this inhibition had a molecular weight of about 50,000. Pretreatment of monocytes with the inhibitory factor led to suppression of cell spreading induced by subsequent stimulation, but this hyporeactivity was reversible. Spreading of monocytes was rapidly aborted by the addition of this inhibitory factor. Thus, the inhibitory factor appeared to affect monocyte itself, but its effect seemed to be transient.

Cell Adhesion↗
PubMed1981

The spread and persistence of influenza viruses in normal and cyclophosphamide-treated mice.

The persistence and extrapulmonary spread of three strains of influenza virus, the mouse neuro-adapted A/NWS virus, the wild-type strain A/Victoria/75, and a recombinant virus RIT4050, bearing surface antigens derived from A/Victoria/75, were studied in both normal and cyclophosphamide-treated CBA mice following either intranasal or intracerebral inoculation. All three viruses showed increased lethality in mice in the presence of cyclophosphamide but exhibited distinctive patterns of replication and spread. The recombinant virus RIT4050 showed a reduced ability to replicate, persist, and spread in CBA mice compared to either A/NWS or A/Victoria/75 viruses, and in general, the A/NWS virus persisted to a greater extent than the A/Victoria/75 virus in both normal and treated mice. However, in the presence of cyclophosphamide, no extrapulmonary spread of A/NWS virus was observed. The reasons for the differences are discussed.

Administration, Intranasal↗
PubMed1992

Capsaicin-sensitive peptidergic neurons are involved in the zosteriform spread of herpes simplex virus infection.

The intraneuronal transport of herpes simplex virus (HSV) is an essential component in disease pathogenesis. Capsaicin, a neuropharmacologic agent lacking direct antiviral activity, has been shown to protect animals against HSV-induced disease. It has been hypothesized that capsaicin acts by interfering with the intraneuronal transport of virus. Since animal models have been useful in studying the spread of virus, we used two guinea pig models of zosteriform herpes to examine the effect of capsaicin on HSV spread. Capsaicin was subcutaneously administered to Hartley guinea pigs prior to intravaginal or cutaneous HSV-2 inoculation. Treatment did not prevent the development of herpetic vesicles at the site of inoculation but significantly reduced the zosteriform spread of lesions in male and female animals. Further, after recovery from primary infection, capsaicin-treated male guinea pigs were observed to have fewer days with recurrent herpetic lesions. These results suggest that capsaicin-sensitive nerve fibers play a role in the pathogenesis of primary and recurrent HSV infections. Capsaicin appears to reduce the severity of cutaneous HSV infections by interfering with the spread of virus.

Animals↗
PubMed1984

Neurons cultured from developing rat brain attach and spread preferentially to laminin.

Neurons, mechanically dissociated from newborn rat brain and identified by immunostaining for neurofilaments, attached preferentially to laminin-coated coverslips without need of an underlying glial monolayer. The most extensive neurite outgrowth was seen when 20-30 micrograms/ml of laminin was used to coat the coverslips. Higher concentrations of laminin (greater than 30 micrograms/ml) supported single neurons to spread on the coverslips. Fibronectin coating of the coverslips allowed glial cells to attach more rapidly than on uncoated surfaces, but it did not support neuronal spreading or neurite outgrowth. Spreading of neurons and neurite outgrowth were completely inhibited by preincubation of laminin-coated coverslips with laminin antibodies but were unaffected by fibronectin antibodies. These results indicate that laminin is an attachment and spreading factor for central neurons in culture and suggest the presence of a laminin receptor on the neuronal cell surfaces.

Animals↗
PubMed1995

Spread of symptoms in idiopathic torsion dystonia.

We reviewed the histories of 115 patients who had idiopathic dystonia and who were 21 years of age and younger at onset of symptoms and 472 patients with onset older than age 21. Patients with onset in the lower extremities tended to be younger at onset (mean age 8.4 years), have rapid spread of symptoms to other body parts, and to develop generalized dystonia. Patients with onset in the upper extremities tended to be older at onset (mean age 11.2 years) and were less likely to develop generalized dystonia, but were more likely to experience spread of symptoms many years after the disease began. Almost 20% of patients younger than age 22 began with torticollis, and 67% of these remained focal after a mean 14.9 years. In adults, dystonic symptoms remained focal in the majority, but approximately 15-30% of patients presenting with blepharospasm, torticollis, laryngeal or brachial dystonia eventually experienced dystonia outside the initially involved segment. Almost 50% of children and adolescents with dystonia reported a family history of dystonia, compared with approximately 10-13% of patients with torticollis, laryngeal or brachial dystonia. Less than 3% of patients with blepharospasm reported a family history of dystonia. There were only minor differences in the pattern of spread of dystonic symptoms between Jewish and non-Jewish patients. Although younger patients were more likely to report a family history of dystonia, patterns of spread were the same for familial and sporadic patients in the same age range.

Adolescent↗
PubMed2006

Neoplastic lumbosacral radiculoplexopathy in prostate cancer by direct perineural spread: an unusual entity.

Neoplastic lumbosacral plexopathy occurs with some abdominal and pelvic malignancies. Patients present with severe pain radiating from the low back down to the lower extremities, and this progresses to weakness. Neoplastic lumbosacral plexopathy is virtually always associated with known malignancy or obvious pelvic metastatic disease. Uncommonly, prostate cancer can present as a lumbosacral plexopathy occurring through direct pelvic spread. We describe two cases of lumbosacral radiculoplexopathy from infiltrative prostate cancer without evidence of other pelvic or extraprostatic spread. The probable etiology of tumor spreading along prostatic nerves into the lumbosacral plexus (i.e., perineural spread) is discussed as are the potential mechanisms for this unusual mode of cancer dissemination.

Aged↗
PubMed2001

Patterns of spread of adenocarcinoma in the prostate as related to cancer volume.

BACKGROUND: Peripheral zone (PZ) and transition zone (TZ) cancers of the prostate remain confined to their zone of origin under 4 cc volume, with progressive molding to TZ boundary. In PZ cancer, growth in perineural spaces over 4 cc volume directs cancer toward the base, around subcapsular nerve trunks, and often transcapsular. This tendency to stereotyped patterns of cancer spread in the prostate is investigated systematically here for the first time. METHODS: Cancers in 571 radical prostatectomy specimens were sorted by zone of origin and tumor volume. A traced map of each cancer at 3 mm transverse intervals was assessed for location, contour, selected linear measures and the "transverse (largest) reference plane". RESULTS: Spread along prostate capsule characterized all but the smallest PZ cancers and was most extensive transversely. By 4 cc volume, most PZ cancers' transverse reference plane filled one side of PZ. Above 4 cc, bilateral spread, TZ invasion, and nodularity progressively increased, but dominant growth was toward the base along nerves to the superior pedicle; here capsule penetration was most common. TZ cancers arose mainly in anterior-mid TZ, invading anterior fibromuscular stroma (AFM) while small. AFM was massively invaded in many large tumors. Larger TZ cancers (> 4 cc) invaded anterolateral PZ but seldom penetrated posterior PZ. CONCLUSIONS: Patterns and extent of spread of carcinoma in the prostate are stereotyped following a few principles regarding stromal interactions. Using these, sequential maps were presented of evolving prostate cancer contours at consecutive increasing volumes.

Adenocarcinoma↗
PubMedDate not supplied

Comparison in spatial spreads of secondary electron information between scanning ion and scanning electron microscopy.

Monte Carlo simulations have been carried out to compare the spatial spreads of secondary electron (SE) information in scanning ion microscopy (SIM) with scanning electron microscopy (SEM). Under Ga ion impacts, the SEs are excited by three kinds of collision-partners, that is, projectile ion, recoiled target atom, and target electron. The latter two partners dominantly contribute to the total SE yield gamma for the materials of low atomic number Z2. For the materials of high Z2, on the other hand, the projectile ions dominantly contribute to gamma. These Z2 dependencies generally cause the gamma yield to decrease with an increasing Z2, in contrast with the SE yield delta under electron impacts. Most of the SEs are produced in the surface layer of about 5lambda in depth (lambda: the mean free path of SEs), as they are independent of the incident probe. Under 30 keV Ga ion impacts, the spatial spread of SE information is roughly as small as 10 nm, decreasing with an increasing Z2. Under 10 keV electron impacts, the SEI excited by the primary electrons has a small spatial spread of about 5lambda, but the SEII excited by the backscattered electrons has a large one of several 10 to several 100 nanometers, decreasing with an increasing Z2. The main cause of a small spread of SE information at ion impact is the short ranges of the projectile ions returning to the surface to escape as backscattered ions, the recoiled target atoms, and the target electrons in collision cascade. The 30 keV Ga-SIM imaging is better than the 10 keV SEM imaging in spatial resolution for the structure/material measurements. Here, zero-size probes are assumed.

Journal Article↗
PubMed1996

Evidence for a K+ channel requirement in spreading of rat basophilic leukemia cells on fibronectin-coated surfaces.

We investigated the ionic requirements for the early events of cell-extracellular matrix interactions leading to cell spreading. We found that potassium ions were required specifically in several cell types. Adhesion to fibronectin- (FN) coated surfaces was independent of K+ in the medium. In contrast, cells that adhered to FN in the absence of K+ failed to spread. This requirement for K+ occurred only during a discrete time frame: in the first 15 minutes following adhesion. Moreover, we identified a specific trans-membrane flux of the radioactive K+ analog 86Rb+, the kinetics of which correlated with this requirement. Both this ion flux and cell spreading were blocked by the K+ -channel inhibitors tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Our results suggest that this K+ ion flux and the channels that regulate it are important in regulating the initial responses to adhesion that lead to spreading.

Animals↗