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PubMedDate not supplied

HeLa cell adhesion on various collagen-grafted surfaces.

Cell adhesion is important to develop cell microarrays and biocompatible materials. Collagen has been reported to be able to improve cell adhesion. In this paper, two collagen coating methods (collagen grafted directly on the substrate and chitosan-modified substrate) were carried out, on which the adhesive behaviors of HeLa cells were studied. An atomic force microscope and a surface potential meter were used to characterize morphologies and electric polarization of these surfaces. It was found that surface electric polarization and the its durability and surface topography were key factors to cell adhesion. Collagen (1 mg/mL) grafted on 1% chitosan-modified surface showed the best adhesion of HeLa cell. This work might be helpful to the practical application of cell microarray chips.

Cell Adhesion↗

PubMedDate not supplied

Proteome annotations and identifications of the human pulmonary fibroblast.

We hereby report on a three year project initiative undertaken by our research team encompassing large-scale protein expression profiling and annotations of human primary lung fibroblast cells. An overview is given of proteomic studies of the fibroblast target cell involved in several diseases such as asthma, idiopatic pulmonary disease, and COPD. It has been the objective within our research team to map and identify the protein expressions occurring in both activated-, as well as resting cell states. The JGGL database www.2DDB.org has been built around these data, allowing advanced hypothesis building using the interactive query bioinformatic tools developed. Gene ontology has been applied to these annotations, classifying and correlating protein expressions to function. The localization as well as the biological processes involved for the annotations are being presented including an annotation-, and sequence-identification strategy, resulting in close to 2000 protein identities. Both gel based, high resolution 2D-gels, and liquid-phase separation (three-dimensional HPLC), as well as the combination of gel- and LC-based approaches (1D-gels and nano-capillary LC, reversed-phase) were utilized. Protein sequencing and structure identities were acquired by a combination of MALDI-, and electrospray-mass spectrometry techniques. Phenotypical and morphological characterizations were also made for this human disease target cell in both stimulated- and resting-cell states. The use of functional assays that demonstrate the key regulating role of growth factors and cytokine stimuli such as PDGF, TGF-beta, and EGF and the effect of ECM molecules such as Biglycan, are also presented and discussed.

Amino Acid Sequence↗

PubMed1998

Isolation of a petite mutant from a histidine auxotroph of Candida albicans and its characterization.

Respiration-deficient (petite) mutations have been induced in various yeasts, which are categorized as petite-positive. Candida albicans was classified among the petite-negative yeasts. Since then, a few reports have appeared, describing the isolation of petite mutants in C. albicans. We report in the present study on the isolation of a petite mutant of C. albicans-SAR1. This mutant was isolated from a histidine auxotroph of C. albicans after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, thus our petite mutant carries a double mutation. SAR1 was characterized morphologically, biochemically and ultrastructurally. The results revealed differences from the wild type in respect to morphological, physiological and biochemical characteristics. Electron microscopy showed that the cells of the petite mutant contain only very few mitochondria that looked 'thread like' without any cristae. The significance of the mutation in the virulence of the mutant vs. that of the wild-type is being assessed.

Candida albicans↗

PubMed1989

Cultured buccal epithelium: an in vitro model derived from the hamster pouch for studying drug transport and metabolism.

Hamster pouch buccal epithelium (HPBE) was isolated and grown in primary cultures on rat-tail collagen-coated growth surfaces. The cultured pouch buccal epithelium (CPBE) was characterized morphologically with electron microscopy as stratified multilayers of epithelial cells with well-developed tonofibrillar-desmosomal complexes. Only the superficial layer of the cultured cells exhibited evidence of terminal differentiation. Alkaline phosphatase, alcohol dehydrogenase, and aminopeptidase activities in the primary cultured cells were determined by biochemical assays and found to be similar to those of homogenates of freshly excised hamster pouch epithelium. In addition, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), keratins and total proteins associated with the cultured cells were similar to those of freshly excised HPBE. The permeability characteristics of the cultured cells was determined by placing cultured cells grown on permeable polycarbonate disks in a Side-Bi-Side diffusion apparatus and quantitating the transcellular flux of tritium-labeled water, fluorescein, and fluorescein isothiocyanate dextrans (MW 3800 to 150,000). The cultured cells were least permeable on the third day of culture and were not permeable to substances with a MW greater than about 18,000. Our results indicate that primary cultures of hamster pouch epithelium exhibit biochemical properties similar to those of the excised hamster pouch epithelium from which they were derived. The morphological and permeability characteristics of cultured hamster epithelium were similar to those of nonkeratinized stratified oral epithelia typical of buccal mucosa in man, rabbit, and other species. CPBE, as described here, represents a potentially useful tool for in vitro drug transport, metabolism, pharmacology, and toxicology studies.

Animals↗

PubMed1997

Isotropic solutions of phospholipid bicelles: a new membrane mimetic for high-resolution NMR studies of polypeptides.

In order to illustrate the utility of phospholipid bicelles [Sanders, C.R. and Schwonek, J.P. (1992) Biochemistry, 31, 8898-8905] as a membrane mimetic for high-resolution NMR studies, we have recorded two-dimensional 1H NMR spectra of the tetradecameric peptide mastoparan Vespula lewisii in an isotropic aqueous solution of dimyristoyl and dihexanoyl phosphatidylcholine. Mastoparan is largely unstructured in water, but assumes a well-defined helical conformation in association with the bilayers. A pronounced periodicity of the sequential NH chemical shifts provides strong evidence that the helix axis of this short peptide is parallel, rather than perpendicular, to the bilayer plane. The bicellar solutions still require in-depth morphological characterization, but they appear to be ideal media for NMR determination of the mode of binding and the structure of membrane-associated peptides and proteins.

Amino Acid Sequence↗

PubMed1998

Adaptive cytoprotection against deoxycholate-induced injury in human gastric cells in vitro: is there a role for endogenous prostaglandins?

The majority of previous work investigating adaptive cytoprotection has involved in vivo studies, which have suggested that this protective response is in large part mediated by endogenous prostaglandins (PGs). The aim of this study was to investigate adaptive cytoprotection under in vitro conditions in human gastric cells and to better delineate the role of endogenous PGs in this protective response. AGS cells (a human gastric carcinoma cell line) were characterized morphologically and subsequently used for all experiments. Sodium deoxycholate was used as both the mild irritant and the damaging agent, and cell injury was quantified using both a commercial viability/cytotoxicity kit as well as transepithelial permeability studies. Finally, endogenous PG synthesis in response to varying concentrations of deoxycholate was determined. AGS cells were determined to be morphologically similar to gastric mucous cells. Pretreatment of cells with low-dose deoxycholate significantly attenuated injury upon subsequent exposure to damaging concentrations of deoxycholate, and this protection was determined to be dependent upon both concentration and duration of mild irritant exposure. Preincubation of AGS cells with indomethacin reversed protection induced by mild irritant pretreatment and also significantly increased cellular susceptibility to injury. Results of the permeability studies closely paralleled those assessing cell mortality. While deoxycholate exposure increased PG synthesis, the concentrations required were much higher than those needed to initiate protection. Adaptive cytoprotection exists in AGS cells under in vitro conditions independent of intact blood flow, neural innervation, or circulating humoral mediators. While this protection is reversed by indomethacin, it appears that this reversal results from increased cellular injury secondary to diminished basal PGs, rather than inhibition of endogenous PG synthesis.

Cell Membrane Permeability↗

PubMedDate not supplied

Development of electrical membrane properties in cultured avian neural crest.

In previous studies of the development of membrane excitability in vertebrate neurones, a calcium current has commonly been observed first, later replaced by a sodium current. We have now examined the development of membrane currents in explant cultures of mesencephalic neural crest cells from the quail embryo. Some of these cells constitute the precursors for the ciliary and trigeminal ganglia and in certain conditions can be characterized morphologically as neurones after only a few hours in culture. We report here that two membrane currents are present in neurones after 1 day in culture, a voltage-and time-dependent potassium current and a leakage current. On the second day in culture, voltage-dependent sodium and calcium currents can be detected. With time the sodium and calcium currents increase in magnitude and all four currents are present for at least 7 days in culture. This onset of electrical excitability differs from that described in other vertebrate neurones both in vitro and in vivo, but resembles the sequence observed in neurones of the developing grasshopper.

Action Potentials↗

PubMed1988

Acetylcholine inhibits identified interneurons in the cat lateral geniculate nucleus.

The transmission of visual information from retina to cortex through the dorsal lateral geniculate nucleus (LGNd) is controlled by non-retinal inputs. Enhanced visually evoked responses in cat LGNd relay cells during periods of increased alertness have been attributed in large part to increased rate of acetylcholine (ACh) release by fibres ascending from the brainstem reticular formation. ACh can modulate geniculate visual responses in vivo, but comparatively little is known about the underlying ionic mechanisms of these cholinergic actions. Although direct excitation of LGNd relay neurons has been shown in vitro, the situation is complicated because cholinergic axons form numerous and complex synapses not only with relay cells, but also with inhibitory interneurons, and electrical activation of the brainstem cholinergic neurons reduces inhibitory postsynaptic potentials in the LGNd. We report here that morphologically characterized interneurons in the cat LGNd possess distinctive electrophysiological properties in comparison with those of relay cells and are inhibited by ACh through a muscarinic receptor-mediated increase in potassium conductance. Together the direct excitation of relay cells and inhibition of intrageniculate interneurons allow the ascending cholinergic system to exert a powerful facilitatory influence over the transfer of visual information to the cerebral cortex.

Acetylcholine↗

PubMed1998

Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis.

Various molecules such as cytokines and anticancer drugs, as well as factor deprivation, rapidly induce apoptosis (programmed cell death), which is morphologically characterized by cell shrinkage and the blebbing of plasma membranes and by nuclear condensation. Caspases, particularly caspase 3, are proteases that are activated during apoptosis and which cleave substrates such as poly(ADP-ribose) polymerase, actin, fodrin, and lamin. Apoptosis is also accompanied by the internucleosomal degradation of chromosomal DNA. In the accompanying Article, we have identified and molecularly cloned a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD). Here we show that caspase 3 cleaves ICAD and inactivates its CAD-inhibitory effect. We identified two caspase-3 cleavage sites in ICAD by site-directed mutagenesis. When human Jurkat cells were transformed with ICAD-expressing plasmid, occupation of the receptor Fas, which normally triggers apoptosis, did not result in DNA degradation. The ICAD transformants were also resistant to staurosporine-induced DNA degradation, although staurosporine still killed the cells by activating caspase. Our results indicate that activation of CAD downstream of the caspase cascade is responsible for internucleosomal DNA degradation during apoptosis, and that ICAD works as an inhibitor of this process.

Animals↗

PubMed1980

Kininogenase activity in plasma membranes and cell organelles from rabbit kidney cortex: subcellular localization of renal kallikrein by free-flow electrophoresis and density-gradient fractionation.

Subcellular fractions were prepared from a rabbit kidney cortex homogenate by density gradient and free-flow electrophoresis techniques. After enzymatic and morphologic characterization, we determined the kininogenase activity in the different fractions. This activity was present in those plasma membranes that also contained high specific activities Na-K-ATPase and in lysosomal-like particles. No activity was found in the lumen, that is, the microvillous part of the proximal tubule cell. The kallikrein-like nature of this kininogenase activity was established by several methods.

Animals↗

PubMed2001

The pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (Macaca fascicularis).

Cynomolgus monkeys (Macaca fascicularis) were exposed by fine-particle aerosol to lethal doses of monkeypox virus, Zaire strain. Death, attributable to fibrinonecrotic bronchopneumonia, occurred 9 to 17 days postexposure. Lower airway epithelium served as the principal target for primary infection. The relative degree of involvement among lymphoid tissues suggested that tonsil, mediastinal, and mandibular lymph nodes were also infected early in the course of the disease, and may have served as additional, although subordinate, sites of primary replication. The distribution of lesions was consistent with lymphatogenous spread to the mediastinal lymph nodes and systemic dissemination of the virus through a monocytic cell-associated viremia. This resulted in lesions affecting other lymph nodes, the thymus, spleen, skin, oral mucosa, gastrointestinal tract, and reproductive system. The mononuclear phagocyte/dendritic cell system was the principal target within lymphoid tissues and may also have provided the means of entry into other systemic sites. Hepatic involvement was uncommon. Lesions at all affected sites were characterized morphologically as necrotizing. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining of select lesions suggested that cell death within lymphoid and epithelial tissues was due in large part to apoptosis. Skin and mucosal surfaces of the respiratory and gastrointestinal tracts also exhibited variable proliferation of epithelial cells and subjacent fibroblasts. Epithelial intracytoplasmic inclusion bodies, consistent with Guarnieri bodies, were usually inconspicuous by light microscopy, but when present, were most readily apparent in the stratified squamous epithelium of the oral mucosa and epidermis. Multinucleated syncytial cells were also occasionally observed in the stratified squamous epithelium of the tongue, tonsil, and skin, and in the intestinal mucosa. Monkeypox virus antigen was readily demonstrated by immunohistochemistry using anti-vaccinia mouse polyclonal antibodies as well as anti-monkeypox rabbit polyclonal antibodies. Detectable poxviral antigen was limited to sites exhibiting obvious morphologic involvement and was most prominent within epithelial cells, macrophages, dendritic cells, and fibroblasts of affected tissues. The presence of poxviral antigen, as determined by immunohistochemistry, correlated with ultrastructural identification of replicating virus. Concurrent bacterial septicemia, present in one monkey, was associated with increased dissemination of the virus to the liver, spleen, and bone marrow and resulted in a more rapidly fatal clinical course.

Aerosols↗

PubMed2021

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Animals↗

PubMed2003

Staphylococcus aureus alpha-toxin-induced cell death: predominant necrosis despite apoptotic caspase activation.

Recent data suggest that alpha-toxin, the major hemolysin of Staphylococcus aureus, induces cell death via the classical apoptotic pathway. Here we demonstrate, however, that although zVAD-fmk or overexpression of Bcl-2 completely abrogated caspase activation and internucleosomal DNA fragmentation, they did not significantly affect alpha-toxin-induced death of Jurkat T or MCF-7 breast carcinoma cells. Caspase inhibition had also no effect on alpha-toxin-induced lactate dehydrogenase release and ATP depletion. Furthermore, whereas early assessment of apoptosis induction by CD95 resulted solely in the generation of cells positive for active caspases that were, however, not yet permeable for propidium iodide, a substantial proportion of alpha-toxin-treated cells were positive for both active caspases and PI. Finally, electron microscopy demonstrated that even in the presence of active caspases, alpha-toxin-treated cells displayed a necrotic morphology characterized by cell swelling and cytoplasmic vacuolation. Together, our data suggest that alpha-toxin-induced cell death proceeds even in the presence of activated caspases, at least partially, in a caspase-independent, necrotic-like manner.

Amino Acid Chloromethyl Ketones↗

PubMed1999

Granulocytic differentiation of myeloid progenitor cells by p130, the retinoblastoma tumor suppressor homologue.

The retinoblastoma protein (pRB) and the related pocket proteins, p107 and p130, play crucial roles in mammalian cell cycle control. Recent studies indicate that these pocket proteins are also involved in cellular differentiation processes. We demonstrate in this work that the pRB-related p130 selectively accumulates during the in vitro differentiation of the myeloid progenitor cell, 32Dcl3, into granulocyte in response to granulocyte-colony stimulating factor (G-CSF). This G-CSF-dependent granulocytic differentiation is blocked by the adenovirus E1A oncoprotein, which binds to and inactivates the pRB family of pocket proteins including p130. Furthermore, enforced overexpression of p130 but not pRB inhibits the myeloid cell proliferation that is concomitantly associated with granulocytic differentiation morphologically characterized by nuclear segmentation. However, simple G1-cell cycle arrest induced by cytokine deprivation or ectopic overexpression of the p27 cyclin-dependent kinase inhibitor, or inhibition of E2F activities by dominant negative DP-1 is not sufficient to trigger granulocytic differentiation. The differentiation-promoting activity of p130 in myeloid cells requires both the pocket domain and the spacer domain. Our results indicate that the pRB-related p130 plays a critical role in myeloid cell differentiation and suggest that coupling of cell cycle exit with the cellular differentiation program may be specifically achieved by p130.

Adenovirus E1A Proteins↗

PubMed2001

The Brn-3b POU family transcription factor regulates the cellular growth, proliferation, and anchorage dependence of MCF7 human breast cancer cells.

The Brn-3b POU domain containing transcription factor is expressed in the developing sensory nervous system as well as in epithelial cells of the breast, cervix, and testes. Brn-3b functionally interacts with the estrogen receptor (ER) and in association with the ER, regulates transcription from estrogen responsive genes. In addition, Brn-3b expression is elevated in breast tumours compared to levels in normal mammary cells. To explore the role of Brn-3b in breast cancer, we established stable cell lines derived from the MCF7 human breast cancer cell line which had been transfected with Brn-3b sense or anti-sense constructs. The Brn-3b over-expressing cell lines exhibited increased growth rate, reached confluence at a higher saturation density, had higher proliferative activity, and an enhanced ability to form colonies in soft agar when compared to the control empty vector transfected cells. Likewise, the Brn-3b anti-sense cell lines showed reduced cellular growth and proliferation, reached confluence at a lower density, and exhibited a decreased ability to form colonies in soft agar when compared to the vector controls. Five to ten per cent of the Brn-3b over-expressing cells exhibited a severely altered morphology characterized by reduced adherence to tissue culture plastic, increased cell size, and a vacuolar cell shape. These results thus further indicate a role for the Brn-3b transcription factor in regulating mammary cell growth and suggest that its elevation in breast cancer is of functional significance.

Adenocarcinoma↗

PubMed2004

Electrochemical fabrication of single-walled carbon nanotubes-DNA complexes by poly(ethylenedioxythiophene) and photocurrent generation by excitation of an intercalated chromophore.

It was found that single-walled carbon nanotubes (SWNTs) solubilized into water by complexation with DNA (salmon testes) can be readily deposited on the ITO electrode by electrochemical oxidative polymerization of ethylenedioxythiophene (EDOT). The driving force for this novel deposition is an electrostatic interaction between the anionic charges of wrapping DNA and the cationic charges of poly(EDOT) formed in the oxidative polymerization process. The presence of poly(EDOT), SWNTs and DNA in the composite was confirmed by measurements of UV-Vis, IR, resonance Raman spectra, cyclic voltammetry (CV) and confocal laser scanning microscopy (CLSM). The composite adsorbed a DNA intercalator (ethidium bromide: EB) very efficiently, which is regarded to be further evidence for inclusion of DNA. The surface morphology, characterized by CLSM, SEM and AFM, featured the network structure consisting of 0.5-ca. 10 microm nanorods. Very interestingly, we found that photoexcitation of EB bound to the DNA generates the photocurrent, indicating that the excited energy of EB is injected into SWNTs, which is collected by the electroconductive poly(EDOT) film on the ITO electrode. We believe, therefore, that the present system is a very convenient method to explore new materials related to redox and photochemical functions.

Biosensing Techniques↗

PubMed2005

Oligofunctional amphiphiles featuring geometric core group preorganization: synthesis and study of Langmuir and Langmuir-Blodgett films.

Based on the principle of supramolecular preorganization, a new type of oligofunctional amphiphile, of which compounds 1-4 are representative structures, has been designed and synthesized. The typical feature of their structure is a highly rigid and geometrically well-defined central unit composed of ethynylene substituted aromatic spacers with different numbers of amphiphilic segment groups (also of a rigid geometric design) attached to it. The molecules form well-defined Langmuir films when spread from a solution at the air/water interface or when a 10(-4) M aqueous CaCl2 solution was used as the subphase. By analysis of the surface pressure-surface area (pi-A) isotherms, information on the packing behavior and orientation of the amphiphilic molecules depending on the molecular structure could be obtained. Morphological characterization of the dynamic process of monolayer compression at the air/water interface was carried out by Brewster angle microscopy, illustrating several phase states visualized as snap shots. Thin monolayer films produced on a 10(-4) M aqueous CaCl2 subphase can be transferred to a mica solid support by the Langmuir-Blodgett technique. Tapping mode atomic force microscopy reveals a surface topography of the monofilms composed of 1 and 3 that differ in roughness and also in the properties of elasticity, hardness and adhesive strength. X-Ray crystal structure analysis of three relevant intermediate compounds of the synthesis were successfully determined giving an indication of the potential structural features inherent in the new amphiphiles.

Crystallography, X-Ray↗

PubMed2003

Attenuation of glucocorticoid functions in an Anx-A1-/- cell line.

The Ca(2+)- and phospholipid-binding protein Anx-A1 (annexin 1; lipocortin 1) has been described both as an inhibitor of phospholipase A(2) (PLA(2)) activity and as a mediator of glucocorticoid-regulated cell growth and eicosanoid generation. Here we show that, when compared with Anx-A1(+/+) cells, lung fibroblast cell lines derived from the Anx-A1(-/-) mouse exhibit an altered morphology characterized by a spindle-shaped appearance and an accumulation of intracellular organelles. Unlike their wild-type counterparts, Anx-A1(-/-) cells also overexpress cyclo-oxygenase 2 (COX 2), cytosolic PLA(2) and secretory PLA(2) and in response to fetal calf serum, exhibit an exaggerated release of eicosanoids, which is insensitive to dexamethasone (10(-8)- 10(-6) M) inhibition. Proliferation and serum-induced progression of Anx-A1(+/+) cells from G(0)/G(1) into S phase, and the associated expression of extracellular signal-regulated kinase 2 (ERK2), cyclin-dependent kinase 4 (cdk4) and COX 2, is strongly inhibited by dexamethasone, whereas Anx-A1(-/-) cells are refractory to the drug. Loss of the response to dexamethasone in Anx-A1(-/-) cells occurs against a background of no apparent change in glucocorticoid receptor expression or sensitivity to non-steroidal anti-inflammatory drugs. Taken together, these observations suggest strongly that Anx-A1 functions as an inhibitor of signal-transduction pathways that lead to cell proliferation and may help to explain how glucocorticoids regulate these processes.

Animals↗