Mark S. F. Clarke

Contraction-Induced Cell Wounding and Release of Fibroblast Growth Factor in Heart

The heart hypertrophies in response to certain forms of increased mechanical load, but it is not understood how, at the molecular level, the mechanical stimulus of increased load is transduced into a cell growth response. One possibility is that mechanical stress provokes the release of myocyte-derived autocrine growth factors. Two such candidate growth factors, acidic and basic fibroblast growth factor (aFGF and bFGF, respectively), are released via mechanically induced disruptions of the cell plasma membrane. In the present study, we demonstrate that transient, survivable disruption (wounding) of the cardiac myocyte plasma membrane is a constitutive event in vivo. Frozen sections of normal rat heart were immunostained to reveal the distribution of the wound event marker, serum albumin. Quantitative image analysis of these sections indicated that an average of 25% of the myocytes contained cytosolic serum albumin; ie, this proportion had suffered a plasma membrane wound. Wounding frequency increased approximately threefold after beta-adrenergic stimulation of heart rate and force of contraction. Heparin-Sepharose chromatography, enzyme-linked immunosorbent assay, growth assay coupled with antibody neutralization, and two-dimensional SDS-PAGE followed by immunoblotting were used to demonstrate that both aFGF and bFGF were released from an ex vivo beating rat heart. Importantly, beta-adrenergic stimulation of heart rate and force of contraction increased FGF release. Cell wounding is a fundamental but previously unrecognized aspect of the biology of the cardiac myocyte. We propose that contraction-induced cardiac myocyte wounding releases aFGF and bFGF, which then may act as autocrine growth-promoting stimuli.

A549 Lung Epithelial Cells Grown as Three-Dimensional Aggregates: Alternative Tissue Culture Model for Pseudomonas aeruginosa Pathogenesis

ABSTRACT A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.

Perimembrane Localization of the Estrogen Receptor α Protein in Neuronal Processes of Cultured Hippocampal Neurons

There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) α or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERα were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERα, rabbit polyclonal IgG antibody which does not recognize ERβ was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERα, but abundant labeling for ERα throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 µM antisense oligonucleotide directed against the translation start site of ERα reduced ERα immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERα. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERα in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERα as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.

Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337–2340) previously established a sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis (SDS‐PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (IIa, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS‐PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immuno‐reactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF‐35), and MHCIIa+IIx (mAb SC‐71). Results provide a valuable SDS‐PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

Nicotine stimulation of nerve growth factor receptor expression

Previous studies have suggested that nicotine may have beneficial actions in neurodegenerative disease models. The purpose of the experiments described in this study was to determine whether the long lasting and beneficial effects of nicotine observed previously could be expressed through actions upon nerve growth factor (NGF) receptors. Using a differentiated PC-12 neuronal cell model, we have detected an increase in expression of cell surface NGF receptor protein after acute exposure to nicotine in the micromolar range. In addition, we have also observed a persistent effect upon NGF receptor expression which lasted even after nicotine (nanomolar range) was removed from the tissue culture medium. This increase in cell surface NGF receptor protein was blocked in the presence of mecamylamine, indicating that this effect is likely nicotinic receptor mediated. These results are consistent with the hypothesis that the lasting and beneficial actions of nicotine previously observed in vivo may involve an indirect effect upon the level of neuronal cell surface NGF receptor expression. Our observations offer one possible mechanism for a potential neurotrophic effect of nicotine.

Laser capture microdissection of metachromatically stained skeletal muscle allows quantification of fiber type specific gene expression

Skeletal muscle contains various myofiber types closely associated with satellite stem cells, vasculature, and neurons, thus making it difficult to perform genetic or proteomic expression analysis with sufficient cellular specificity to resolve differences at the individual cell or myofiber type level. Here, we describe the combination of a simple histochemical method capable of simultaneously identifying Type I, IIA, IIB, and IIC myofibers followed by laser capture micro-dissection (LCM) to compare the expression profiles of individual fiber types, myonuclear domains, and satellite cells in frozen muscle sections of control and atrophied muscle. Quantitative RT-PCR (qPCR) was used to verify the integrity of the cell-specific RNAs harvested after histologic staining, while qPCR for specific genes of interest was used to quantify atrophy-associated changes in mRNA. Our data demonstrate that the differential myofiber atrophy previously described by histologic means is related to differential expression of atrophy-related genes, such as MuRF1 and MAFbx (a.k.a. Atrogin-1), within different myofiber type populations. This spatially resolved molecular pathology (SRMP) technique allowed quantitation of atrophy-related gene products within individual fiber types that could not be resolved by expression analysis of the whole muscle. The present study demonstrates the importance of fiber type specific expression profiling in understanding skeletal muscle biology especially during muscle atrophy and provides a practical method of performing such research.

Syringe Loading: A Method for Inserting Macromolecules into Cells in Suspension

Publisher Summary This chapter focuses on the use of syringe loading for inserting macromolecules into cells in suspension. Syringe loading is a simple, efficient, and gentle mechanically based method of loading cells in suspension. The overall loading efficiency of this technique is increased by the presence of the nonionic surfactant Pluronic F-68, which appears to augment the natural tendency of cells to repair plasma membrane wounds. Syringe loading can efficiently introduce into the cell cytoplasm a variety of macromolecules, including dyes, oligonucleotides, and immunoglobulins. Plasmid DNA can also be loaded, although the use of the syringe loading technique for this application remains to be optimized. It is important to watch for excessive pH changes caused by exposure of the bicarbonate-buffered medium to atmospheric air during the procedure as these are potentially harmful to the cells.

The identification of proliferation-related proteins in human endothelial cells as a possible target in tumour therapy.

We report the identification of several proteins which are expressed in proliferating human endothelial cells but are absent in confluent/contact inhibited cells, utilizing a high resolution two dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis system. We also report the detection of a number of proteins specifically induced in the presence of tumour-conditioned media.

Obese mice incur greater myofiber membrane disruption in response to mechanical load compared with lean mice.

Obesity is associated with modified transmembrane signaling events in skeletal muscle, such as insulin signaling and glucose transport. The underlying cause of these obesity‐related effects on transmembrane signaling is still unknown. In general, the function of membrane proteins responsible for transmembrane signaling is modulated by the biochemical makeup of the membrane, such as lipid composition, in which they are embedded. Any obesity‐related alterations in membrane composition would also be predicted to modify membrane biomechanical properties and membrane susceptibility to mechanical load‐induced damage. The primary objective of this study was to investigate whether obesity influences myofiber membrane susceptibility to mechanical damage in skeletal muscle.

An Atherogenic Level of Native LDL Increases Endothelial Cell Vulnerability to Shear-Induced Plasma Membrane Wounding and Consequent Release of Basic Fibroblast Growth Factor

We report here that exposure of large vessel EC to clinically relevant, atherogenic levels of native LDL (240 mg cholesterol/dL) increases the incidence and severity of shear-induced EC plasma membrane wound injury in vitro. The proportion of LDL-treated EC that survived mechanical shearing in suspension was significantly less (∼20%; p < 0.005) than that of control, untreated EC. Moreover, the amount of a fluorescent, cytoplasmic wound marker, detected by flow cytometry, in surviving LDL-treated cells was significantly more (∼2 log units; p < 0.005) than that detected in surviving, control EC. Mechanically sheared LDL-treated EC released significantly more (∼2 fold; p < 0.02) bFGF than sheared, control EC. LDL treatment of EC resulted in an increase of ∼60% in membrane-associated cholesterol, and an increase in the cholesterol/phospholipid ratio from 0.6 to 1.3. Fluorescence anisotropy revealed that the plasma membrane fluidity (PMF) of LDL-treated EC was significantly lower than that of control EC. When ...