bluuebunny/crossref_metadata_2025 huggingface dataset:
Peroxisome Subpopulations of the Rat Liver: Isolation by Immune Free Flow Electrophoresis
Authors: Völkl Alfred , Mohr Heribert , Fahimi H. Dariush
Published: April 2011

Peroxisomes (POs) are a heterogenous population of cell organelles which, in mammals, are most abundant in liver and kidney. Although they are usually isolated by differential and density gradient centrifugation, isolation is hampered by their high fragility, sensitivity to mechanical stress, and their sedimentation characteristics, which are close to those of other major organelles, particularly microsomes. Consequently, until now only the so-called “heavy” POs with a buoyant density of 1.22-1.24 g/cm3 have been highly purified from rat liver, whereas the other subpopulations also present in that tissue have escaped adequate characterization. The purification of these subpopulations has become an essential task in view of the functional significance of POs in humans, and the putative importance of peroxisomal subpopulations in the biogenesis of this organelle. Here we used an alternative novel approach to density gradient centrifugation, called immune free flow electrophoresis (IFFE). IFFE combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle complexed to an antibody against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided that the pH of the electrophoresis buffer is adjusted to pH ∼8.0, the pI of IgG molecules. Because of this reduced electrophoretic mobility, IgG-coupled particles can be separated in an electric field from those that are noncoupled and hence more mobile. The IFFE technique has been recently applied for isolation of regular POs (ρ = 1.22-1.24 g/cm3) from a light mitochondrial fraction of rat liver. We succeeded in isolating different subpopulations of POs by applying IFFE to heavy, light, and postmitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The PO subfractions obtained differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic POs.
Induction of Tubular Peroxisomes by UV Irradiation and Reactive Oxygen Species in HepG2 Cells
Authors: Schrader Michael , Wodopia Ralf , Fahimi H. Dariush
Published: April 2011

Peroxisomes in the human hepatoblastoma cell line HepG2 exhibit a high degree of plasticity. Whereas in confluent cultures they appear as small (0.1-0.3 μm) spherical particles, they undergo dramatic changes, forming elongated tubules measuring up to 5 μm on separation of cells and cultivation at low density. We recently showed that several growth factors, including nerve growth factor (NGF), induce the formation of tubular peroxisomes and that this induction is sensitive to K 252b, a specific tyrosine kinase inhibitor, suggesting the involvement of this signal transduction pathway. Because tyrosine kinase is also involved in signal transduction via the reactive oxygen species (ROS), we have analyzed in this study the effects of UV irradiation, H2O2, and oxygen on tubulation of peroxisomes. UVC irradiation induced a significant increase in formation of tubular peroxisomes (40-50% of cells) and this effect was dose-dependently inhibited by pretreatment with N-acetyl cysteine, confirming the involvement of ROS in the UV effect. Furthermore, H2O2 also directly induced the tubulation of peroxisomes, although to a lesser extent. Finally, cultivation under hypoxic conditions (1.5% O2) drastically reduced the inducing effect of fetal calf serum on tubulation of peroxisomes, suggesting the involvement of oxygen-mediated signaling. Taken together, our observations indicate that ROS induce the tubulation of peroxisomes in HepG2 cells. Because peroxisomes harbor most of the enzymes for catabolism of ROS, the tubulation and expansion of the peroxisome compartment could have a cell rescue function against the destructive effects of ROS.
Alexa Dyes, a Series of New Fluorescent Dyes that Yield Exceptionally Bright, Photostable Conjugates
Authors: Panchuk-Voloshina Nataliya , Haugland Rosaria P. , Bishop-Stewart Janell , Bhalgat Mahesh K. , Millard Paul J. , Mao Fei , Leung Wai-Yee , Haugland Richard P.
Published: April 2011

Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges.
Cerium-based Histochemical Demonstration of Oxidative Stress in Taurocholate-induced Acute Pancreatitis in Rats: A Confocal Laser Scanning Microscopic Study
Authors: Telek Géza , Scoazec Jean-Yves , Chariot Jacques , Ducroc Robert , Feldmann Gerard , Rozé Claude
Published: April 2011

Direct in vivo histological detection of oxygen-derived free radicals (OFRs) in inflammatory conditions is not fully resolved. We report an application of cerium histochemistry (in which capture of OFRs by Ce atoms results in laser-reflectant cerium-perhydroxide precipitates) combined with reflectance confocal laser scanning microscopy (CLSM) to demonstrate the evolution of oxidative stress in taurocholate-induced acute pancreatitis (AP) in rats. Animals were perfused with CeCl3 in vivo and cryostat sections of pancreata were studied by CLSM. Vascular endothelium was immunolabeled for PECAM-1. OFR production by isolated polymorphonuclear leukocytes (PMNs) incubated in vitro with CeCl3 was quantified by image analysis. In the pancreas, strong OFR-derived cerium reflectance signals were seen in acinar cells at 1-2 hr, capillaries and small venules were frequently engorged by cerium precipitates, and adherent PMNs presented weak intracellular reflectance signals. At 8-24 hr, acinar cell OFR production decreased, whereas adherent/transmigrated PMNs displayed abundant intra- and pericellular reflectance. PECAM-1 expression was unchanged. PMNs from ascites or blood showed significant ( p<0.01) time-dependent OFR production, plateauing from 2 hr. The modified cerium capture/CLSM method allows the co-demonstration of in vivo oxidative stress and cellular structures labeled with fluorescent markers. In vivo oxidative stress was shown histologically for the first time in experimental AP.
Current Cytochemical Techniques for the Investigation of Peroxisomes: A Review
Authors: Fahimi H. Dariush , Baumgart Eveline
Published: April 2011

The past decade has witnessed unprecedented progress in elucidation of the complex problems of the biogenesis of peroxisomes and related human disorders, with further deepening of our understanding of the metabolic role of this ubiquitous cell organelle. There have been many recent reviews on biochemical and molecular biological aspects of peroxisomes, with the morphology and cytochemistry receiving little attention. This review focuses on the state-of-the-art cytochemical techniques available for investigation of peroxisomes. After a brief introduction into the use of the 3,3′-diaminobenzidine method for localization of catalase, which is still most commonly used for identification of peroxisomes, the cerium technique for detection of peroxisomal oxidases is discussed. The influence of the buffer used in the incubation medium on the ultrastructural pattern obtained in rat liver peroxisomes in conjunction with the localization of urate oxidase in their crystalline cores is discussed, particularly since Tris-maleate buffer inhibits the enzyme activity. In immunocytochemistry, quantitation of immunogold labeling by automatic image analysis enables quantitative assessment of alterations of proteins in the matrix of peroxisomes. This provides a highly sensitive approach for analysis of peroxisomal responses to metabolic alterations or to xenobiotics. The recent evidence suggesting the involvement of ER in the biogenesis of “preperoxisomes” is mentioned and the potential role of preembedding immunocytochemistry for identification of ER-derived early peroxisomes is emphasized. The use of GFP expressed with a peroxisomal targeting signal for the investigation of peroxisomes in living cells is briefly discussed. Finally, the application of in situ hybridization for detection of peroxisomal mRNAs is reviewed, with emphasis on a recent protocol using perfusion-fixation, paraffin embedding, and digoxigenin-labeled cRNA probes, which provides a highly sensitive method for detection of both high- and low-abundance mRNAs encoding peroxisomal proteins.
Localization of the Organic Anion Transporting Polypeptide 2 (Oatp2) in Capillary Endothelium and Choroid Plexus Epithelium of Rat Brain
Authors: Gao Bo , Stieger Bruno , Noé Birgitta , Fritschy Jean-Marc , Meier Peter J.
Published: April 2011

In this study we investigated the distribution of a recently cloned polyspecific organic anion transporting polypeptide (Oatp2) in rat brain by nonradioactive in situ hybridization histochemistry and immunofluorescence microscopy. The results demonstrate that Oatp 2 is expressed in brain capillary and in plexus epithelial cells. At the blood-brain barrier (BBB), Oatp 2 expression could be co-localized with the endothelial marker vWF (von Willebrand factor) but not with the astrocyte marker GFAP (glial fibrillary acidic protein). In choroid plexus epithelial cells, Oatp 2 could be localized to the basolateral cell pole, whereas the first member of the Oatp gene family of membrane transporters to be cloned (Oatp 1) co-localized with the α1subunit of Na,K-ATPase at the apical plasma membrane domain. Because Oatp 1 and Oatp 2 have been previously shown to mediate transmembrane transport of a wide variety of amphipathic organic compounds, including many drugs and other xenobiotics, the histochemical localization of Oatp 2 at the BBB and of Oatp 1 and Oatp 2 in the choroid plexus imply a role for these transporters in the active exchange of amphipathic solutes between the blood, brain, and cerebrospinal fluid compartments.
Developmental Expression of Dynamin in the Chick Retinotectal System
Authors: Bergmann Mathias , Grabs Detlev , Rager Günter
Published: April 2011

Dynamin I, a GTPase involved in the endocytic cycle of synaptic vesicle membranes, is believed to support axonal outgrowth and/or synaptogenesis. To explore the temporal and spatial patterns of dynamin I distribution in neuronal morphogenesis, we compared the developmental expression of dynamin with the expression of presynaptic membrane proteins such as SV2, synaptotagmin, and syntaxin in the chick primary visual pathway. Western blots of retina and tectum revealed a steady increase of synaptotagmin and syntaxin from embryonic Day 7 (E7) to E11, whereas for the same time frame no detectable increase of dynamin was found. Later stages showed increasing amounts of all tested proteins until the first postnatal week. Immunofluorescence revealed that SV2, synaptotagmin, and syntaxin are present in retinal ganglion cell axons from E4 on. In later stages, the staining pattern in the retina and along the visual pathway paralleled the formation and maturation of axons. In contrast, dynamin is not detectable by immunofluorescence in the developing retina and optic tectum before synapse formation. Our data indicate that, in contrast to the early expression of synaptotagmin, SV2, and syntaxin during axonal growth, dynamin is upregulated after synapse formation, suggesting its function predominantly during and after synaptogenesis but not in axonogenesis.
Murine Progesterone Receptor Expression in Proliferating Mammary Epithelial Cells During Normal Pubertal Development and Adult Estrous Cycle: Association with ERα and ERβ Status
Authors: Zeps Nikolajs , Bentel Jacqueline M. , Papadimitriou John M. , Dawkins Hugh J.S.
Published: April 2011

The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-α (ERα). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [3 H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [3 H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERβ suggesting that the role of ERβ in mediating normal estrogen-induced responses should be further studied.
Intracellular Localization of Hyaluronan in Proliferating Cells
Authors: Evanko Stephen P. , Wight Thomas N.
Published: April 2011

Hyaluronan is a high molecular weight glycosaminoglycan found in the extracellular matrix of many tissues, where it is believed to promote cell migration and proliferation. It was recently shown that hyaluronan-dependent pericellular matrix formation is a rapid process that occurs as cells detach during mitosis. Growing evidence for intracellular hyaluronan in tissues in vivo, together with evidence of intracellular hyaluronan binding molecules, prompted us to examine hyaluronan distribution and uptake as well as hyaluronan binding sites in cells and their relationship to cell proliferation in vitro, using a biotinylated hyaluronan binding protein and fluorescein-labeled hyaluronan. In permeabilized smooth muscle cells and fibroblasts, hyaluronan staining was seen in the cytoplasm in a diffuse, network-like pattern and in vesicles. Nuclear hyaluronan staining was observed and confirmed by confocal microscopy and was often associated with nucleoli and nuclear clefts. After serum stimulation of 3T3 cells, there was a dramatic increase in cytoplasmic hyaluronan staining, especially during late prophase/early prometaphase of mitosis. In contrast, unstimulated cells were negative. There was a pronounced alteration in the amount and distribution of hyaluronan binding sites, from a mostly nucleolar distribution in unstimulated cells to one throughout the cytoplasm and nucleus after stimulation. Exogenous fluorescein-labeled hyaluronan was taken up avidly into vesicles in growing cells but was localized distinctly compared to endogenous hyaluronan, suggesting that hyaluronan in cells may be derived from an intracellular source. These data indicate that intracellular hyaluronan may be involved in nucleolar function, chromosomal rearrangement, or other events in proliferating cells.
Quantification of Protein A-Gold Staining for Peroxisomal Enzymes by Confocal Laser Scanning Microscopy
Authors: Ogiwara Naoko , Usuda Nobuteru , Yamada Masashi , Johkura Kohei , Kametani Kiyokazu , Nakazawa Ayami
Published: April 2011

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific β-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.
Satellite Cells on Isolated Myofibers from Normal and Denervated Adult Rat Muscle
Authors: Kuschel Roland , Yablonka-Reuveni Zipora , Bornemann Antje
Published: April 2011

Satellite cells (SCs) in normal adult muscle are quiescent. They can enter the mitotic program when stimulated with growth factors such as basic FGF. Short-term denervation stimulates SC to enter the mitotic cycle in vivo, whereas long-term denervation depletes the SC pool. The molecular basis for the neural influence on SCs has not been established. We studied the phenotype and the proliferative capacity of SCs from muscle that had been denervated before being cultured in vitro. The expression of PCNA, myogenin, and muscle (M)-cadherin in SCs of normal and denervated muscle fibers was examined at the single-cell level by immunolabeling in a culture system of isolated rat muscle fibers with attached SCs. Immediately after plating (Day 0), neither PCNA nor myogenin was present on normal muscle fibers, but we detected an average of 0.5 M-cadherin+ SCs per muscle fiber. The number of these M-cadherin+ cells (which are negative for PCNA and myogenin) increased over the time course examined. A larger fraction of cells negative for M-cadherin underwent mitosis and expressed PCNA, followed by myogenin. The kinetics of SCs from muscle fibers denervated for 4 days before culturing were similar to those of normal controls. Denervation from 1 to 32 weeks before plating, however, suppressed PCNA and myogenin expression almost completely. The fraction of M-cadherin+ (PCNA−/myogenin−) SCs was decreased after 1 week of denervation, increased above normal after denervation for 4 or 8 weeks, and decreased again after denervation for 16 or 32 weeks. We suggest that the M-cadherin+ cells are nondividing SCs because they co-express neither PCNA or myogenin, whereas the cells positive for PCNA or myogenin (and negative for M-cadherin) have entered the mitotic cycle. SCs from denervated muscle were different from normal controls when denervated for 1 week or longer. The effect of denervation on the phenotypic modulation of SCs includes resistance to recruitment into the mitotic cycle under the conditions studied here and a robust extension of the nonproliferative compartment. These characteristics of SCs deprived of neural influence may account for the failure of denervated muscle to fully regenerate.
Regulation of Cell Surface Tissue Transglutaminase: Effects on Matrix Storage of Latent Transforming Growth Factor-β Binding Protein-1
Authors: Verderio Elisabetta , Gaudry Claire , Gross Stephane , Smith Colin , Downes Sandra , Griffin Martin
Published: April 2011

Using a cytochemical approach, we examined the role of tissue transglutaminase (tTgase, Type II) in the incorporation of latent TGF-β binding protein-1 (LTBP-1) in the extracellular matrix of Swiss 3T3 fibroblasts in which tTgase expression can be modulated through a tetracycline-controlled promoter. Increased tTgase expression led to an increased rate of LTBP-1 deposition in the matrix, which was accompanied by an increased pool of deoxycholate-insoluble fibronectin. Matrix deposition of LTBP-1 could also be reduced by the competitive amine substrate putrescine. Immunolocalization at the fluorescence and electron microscopic level showed that extracellular tTgase is located at the basal and apical surfaces of cells and at cell-cell contacts, and that LTBP-1 is co-distributed with cell surface tTgase, suggesting an early contribution of tTgase to the binding of LTBP-1 to matrix proteins. LTPB-1 was also found to co-localize with both intracellular and extracellular fibronectin, and increased immunoreactivity for LTBP-1 and fibronectin was found in large molecular weight polymers in the deoxycholate-insoluble matrix of fibroblasts overexpressing tTgase. We conclude that regulation of tTgase expression is important for controlling matrix storage of latent TGF-β1 complexes and that fibronectin may be one extracellular component to which LTBP-1 is crosslinked when LTBP-1 and tTgase interact at the cell surface.
A High-resolution, Fluorescence-based Method for Localization of Endogenous Alkaline Phosphatase Activity
Authors: Cox William G. , Singer Victoria L.
Published: April 2011

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins.
Immunohistochemical Localization of Cytochrome P450 CYP1B1 in Breast Cancer with Monoclonal Antibodies Specific for CYP1B1
Authors: McFadyen Morag C.E. , Breeman Suzanne , Payne Simon , Stirk Chris , Miller Iain D. , Melvin William T. , Murray Graeme I.
Published: April 2011

Cytochrome P450 CYP1B1 is a recently identified member of the CYP1 P450 family. We have shown that this P450 displays increased expression in several types of human cancer, indicating that CYP1B1 is a potential tumor biomarker. In this study we developed monoclonal antibodies (MAbs) to CYP1B1 that are effective on formalin-fixed, paraffin-embedded tissue sections and investigated the presence of CYP1B1 in a series of primary breast cancers. The MAbs were generated using a synthetic peptide coupled to carrier protein as the immunogen. The MAbs specifically recognized CYP1B1 and did not recognize either CYP1A1 or CYP1A2, related CYP1 forms. The MAbs were tested by immunohistochemistry and were found to be effective on formalin-fixed, paraffin-embedded tissue sections. The majority of breast cancers showed positive immunoreactivity for CYP1B1, and in each case CYP1B1 was specifically localized to tumor cells. The presence of CYP1B1 in breast cancer cells is likely to contribute to their metabolism of estradiol because CYP1B1 is a specific estradiol hydroxylase.
Antigen Retrieval in Prion Protein Immunohistochemistry
Authors: Everbroeck Bart Van , Pals Philippe , Martin Jean-Jacques , Cras Patrick
Published: April 2011

Transmissible spongiform encephalopathies are a group of neurodegenerative diseases occurring in both humans and animals and are most likely caused by prions. Neuropathological confirmation of the clinical diagnosis has been a problem because of the difficulty in epitope retrieval from formalin-fixed, paraffin-embedded brain specimens. Many different protocols for the detection of prions in brain tissue have been used. Thus far, picric and/or formic acid, steam autoclaving at 121C of sections, microwave treatment, and 4 M guanidine thiocyanate treatment have been suggested. The objective of our experiment was to obtain the standard pretreatment(s) resulting in optimal immunostaining. In the experiment, successive tissue slides of brain specimens of several Creutzfeldt-Jakob disease and control patients were stained using different combinations of pretreatments. Using densitometric analysis, several well-defined locations per section were examined and prion immunostaining was quantified. The results showed that autoclaving is necessary for antigen retrieval and cannot be substituted by microwave treatment. The best results were obtained when the following combination was used in the specified order: 15 min saturated picric acid, 10 min steam autoclaving at 121C, 5 min 88% formic acid, and 2 hr 4 M guanidine thiocyanate at 4C.
Observation of Fibronectin Distribution on the Cell Undersurface Using Immunogold Scanning Electron Microscopy
Authors: Goto Tetsuya , Wong Kung S. , Brunette Donald M.
Published: April 2011

Immunogold staining followed by observation with scanning electron microscopy (SEM) has been quite effective in showing the distribution of proteins on dorsal cell surfaces. However, observation of proteins on the ventral cell surface using SEM has not been developed to the same extent. In this study, human gingival fibroblasts cultured on titanium-coated wafers were embedded in resin. After fracturing the wafers off the embedded cells, the undersurface of the cell was exposed by argon gas glow discharge etching. After 15 min of glow discharge etching, the resin covering the cell undersurface was completely removed. The distribution of fibronectin (FN) on the cell undersurface was demonstrated using an anti-FN antibody and colloidal gold (30 nm) conjugated with IgG. The undersurface was then coated with carbon or gold-palladium and observed by SEM. Using back-scattered electron detection, gold beads could be identified in high contrast. On cells cultured for 5 hr, gold beads were distributed randomly on the entire cell undersurface. However, a line of gold beads was sometimes observed close to the edge of the cell. These results indicated that this immunogold/SEM etching method provides a powerful means for studying cell adhesion molecules on the cell undersurface.
SPARC, a Matricellular Glycoprotein with Important Biological Functions
Authors: Yan Qi , Sage E. Helene
Published: April 2011

SPARC (secreted protein, acidic and rich in cysteine) is a unique matricellular glycoprotein that is expressed by many different types of cells and is associated with development, remodeling, cell turnover, and tissue repair. Its principal functions in vitro are counteradhesion and antiproliferation, which proceed via different signaling pathways. SPARC consists of three domains, each of which has independent activity and unique properties. The extracellular calcium binding module and the follistatin-like module have been recently crystallized. Specific interactions between SPARC and growth factors, extracellular matrix proteins, and cell surface proteins contribute to the diverse activities described for SPARC in vivo and in vitro. The location of SPARC in the nuclear matrix of certain proliferating cells, but only in the cytosol of postmitotic neurons, indicates potential functions of SPARC as a nuclear protein, which might be involved in the regulation of cell cycle progression and mitosis. High levels of SPARC have been found in adult eye, and SPARC-null mice exhibit cataracts at 1–2 months of age. This animal model provides an excellent opportunity to confirm and explore some of the properties of SPARC, to investigate cataractogenesis, and to study SPARC-related family proteins, e.g., SC1/hevin, a counteradhesive matricellular protein that might functionally compensate for SPARC in certain tissues.
Expression Pattern of Glutamine Synthetase Marks Transition from Collecting into Conducting Hepatic Veins
Authors: Lamers Wouter H. , Vermeulen L.M. Vermeulen , Hakvoort B.M. Hakvoort , Moorman F.M. Moorman
Published: April 2011

The expression of glutamine synthetase (GS) is confined to a rim of hepatocytes surrounding the efferent hepatic veins in all mammalian species investigated. In rat liver, a two- to three-cell thick layer of GS-positive (GS+) hepatocytes uniformly surrounds the two to four terminal branching generations of the hepatic vein that collect blood from sinusoids (central veins). With increasing diameter of the efferent vessel, this multilayered rim of GS+ hepatocytes becomes confined to patches surrounding the decreasing number of central vein outlets. The remaining part of the wall of these sublobar hepatic veins is bordered by a onecell thick layer of GS+ hepatocytes. Around still larger veins, this single-cell layer of GS+ hepatocytes gradually disappears. The expression pattern of GS is therefore a convenient biological parameter to delimit sinusoidal draining (“collecting”) from nondraining (“conducting”) surfaces in the wall of the efferent hepatic vessels. The hepatocytes surrounding a single tree of central veins together form a compound liver lobule.
Localization of Carbonic Anhydrase in Guinea Pig Bowman's Glands
Authors: Okamura Hiro-oki , Sugai Naonori , Suzuki Kazunori
Published: April 2011

We examined the histochemical localization of carbonic anhydrase (CA) in Bowman's glands by light and electron microscopy. Neither CAI nor CAII was detected immunohistochemically in the duct cells. However, by enzyme histochemistry the duct cells revealed electron-dense precipitates demonstrative of CA in the microvilli and intercellular digitations. The reaction product was also noted in small vesicles in the cytoplasm of duct cells. In cells of the acini, the well-developed short microvilli, basolateral cell membrane, and mitochondria along the basolateral membrane showed strong deposits indicating CA activity. Dense reaction product of CA was also detected in a small core within the electron-lucent granules of the secretory cells, although CAI and CAII were not detected by immunostaining in the secretory granules. Although the functional significance of CA in Bowman's glands is obscure, the enzyme may play a role in regulation of pH and ion balance in the mucous layer covering the olfactory epithelium. The presence of CA activity in the ducts suggests that these structures are not simple tubes serving as a conduit for secretory substances but participate in modifying the luminal content by secreting CA.
Human Ocular Drusen Possess Novel Core Domains with a Distinct Carbohydrate Composition
Authors: Mullins Robert F. , Hageman Gregory S.
Published: April 2011

Ocular drusen are extracellular deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane. Although the presence of large and/or numerous drusen in the macula is a significant risk factor for development of age-related macular degeneration (AMD), a major cause of irreversible blindness, little is known about their origin or composition. We have expanded on our previous investigations related to drusen-associated glycoconjugates by examining lectin binding patterns after removal of terminal sialic acid residues. Strikingly, intense and distinct labeling of drusen subdomains is revealed by Arachea hypogea agglutinin (PNA) after neuraminidase treatment. PNA binding is confined to discrete domains within both hard and soft drusen. These “cores” are positioned centrally within drusen and are typically juxtaposed to Bruch's membrane. Only one core per druse is observed. PNA labeling of drusen cores does not co-localize with associated lipids and is abrogated by digestion with O-glycosidase but not N-glycosidase. The association of cores with small drusen suggests that they may participate in drusen biogenesis.
Expression of Menkes Copper-transporting ATPase, MNK, in the Lactating Human Breast: Possible Role in Copper Transport into Milk
Authors: Ackland M. Leigh , Anikijenko Peter , Michalczyk Agnes , Mercer F.B. Mercer
Published: April 2011

The Menkes copper ATPase (MNK) is a copper efflux ATPase that is involved in copper homeostasis. Little is known about the intracellular localization and cell-specific function of the MNK in human tissues. To investigate a possible role for this protein in lactation, we measured its expression in sections of tissue from nonlactating and lactating human breast. Western blot analysis showed that MNK expression was greater in lactating tissue than in nonlactating tissue. By confocal immunofluorescence, the MNK was detected in luminal epithelial cells of the alveoli and ducts but not in myoepithelial cells. In the nonlactating breast epithelial cells, the MNK had a predominantly perinuclear distribution. In lactating breast tissue, the distribution of the MNK was markedly altered. Lactating epithelial cells showed a granular, diffuse pattern, which extended beyond the perinuclear region of the cell. This pattern was similar to that observed in a previous study in which cultured CHO cells were exposed to high copper concentrations. Our results suggest that relocalization of the MNK is a physiological process, which may be mediated by copper levels in the breast or by hormones and other events taking place during lactation. A vesicular pathway for copper from the Golgi into milk, similar to that of calcium, is proposed.
CD44 Expression Marks the Onset of Keratinocyte Stratification and Mesenchymal Maturation into Fibrous Dermis in Fetal Human Skin
Authors: Tuhkanen Anna-Liisa , Ågren Ulla M. , Tammi Markku I. , Tammi Raija H.
Published: April 2011

The cell surface glycoprotein CD44 is involved in active cell movement, cancer metastasis, and morphogenesis. We studied its expression in fetal human skin using an antibody specific for CD44v3 and another that recognizes all CD44 forms. In embryonic and early fetal skin, only cells with dendritic morphology expressed CD44. The first keratinocyte expression of CD44 occurred in the basal cells on the eleventh week. Later, the suprabasal cells also turned positive, whereas periderm and the terminally differentiated cells remained negative at all stages. Maturation of the early mesenchyme towards dermis at the eleventh week was associated with an increase in the number of CD44-positive cells, and later the fibrous extracellular matrix also became CD44-positive. During hair induction, the epithelium showed a transient downregulation of CD44. Later, the follicular cells regained CD44. Cells in the primordial dermal papilla displayed a continuously strong signal. The sweat gland anlagen showed faint CD44 positivity. Exon 3 was present in the CD44 of keratinocytes and their derivatives but was absent in dermis. CD44 expression in human fetal skin is a relatively late event, associated with maturation and adult-type differentiation both in epidermal keratinocytes and in dermal fibroblastic cells.
Binding of a Putative and a Known Chaperone Protein Revealed by Immunogold Labeling Transmission Electron Microscopy: A Suggested Use of Chaperones as Probes for the Distribution of Their Target Proteins
Authors: Thorpe Julian R. , Rulten Stuart L. , Kay John E.
Published: April 2011

Parvulins are a distinct family within the peptidyl-prolyl cis-trans isomerase group of proteins that catalyse the cis-trans isomerization of proline-containing peptides. The intracellular distribution of a novel human parvulin homologue (hEPVH) has been investigated in a human kidney cell line (HEK 293) by immunogold labeling transmission electron microscopy (TEM). This showed hEPVH to be distributed throughout HEK 293 cells but in highest concentration within mitochondria. Unexpectedly, preabsorption of anti-hEPVH antiserum with recombinant hEPVH exaggerated the observed immunolabel density in a pattern that mirrored that of the endogenous hEPVH. The hEPVH protein itself was then used to label sections, and the specificity of its binding was confirmed with the use of polyclonal and monoclonal antibodies in conjunction with homologous and irrelevant protein controls. The pattern of hEPVH binding also mirrored that of endogenous hEPVH. A known chaperone protein, Pin1, was also found to bind to cells in a pattern mirroring that of the endogenous protein. This lends considerable weight to our hypothesis that hEPVH is binding to its target protein(s) within the cell, reflecting its postulated chaperone function. Finally, we suggest that chaperone proteins in general might be used as TEM probes for their target (or substrate) proteins.
Expanding the scope of post-conflict justice: Individual, state and societal responsibility for mass atrocity
Authors: Subotic Jelena
Published: April 2011

Over the past two decades, a new international regime of individual criminal accountability has emerged as a dominant regulatory mechanism to address gross human rights violations. At the same time, states are still pursuing claims against each other for human rights abuses in international courts. These two concepts of responsibility — individual and state — are not only fundamentally at odds with one another; they also exclude the third, critical aspect of political accountability — societal responsibility for past violence. This triple accountability — of individual perpetrators who committed the crimes, of the state that hired them to implement the practices, and of society that supported or tacitly approved repressive state policies — is a complex political condition that the current transitional justice framework is ill equipped to deal with. Individualization of accountability serves the retributive purpose of justice, but it is woefully inadequate to address the collective political ideologies that made such heinous crimes possible in the first place. Domestic elites can be enthusiastic supporters of individual human rights trials — not because they want to bring about justice, but because they want to shield the state and society from complicity in past crimes. To address this paradox, this article presents a new framework of post-conflict accountability that includes individual, state, and societal responsibility for human rights violations. The framework is then applied to the case of Serbian responsibility for war crimes committed in Bosnia.
Origins of the Palestinian refugee problem: Changes in the historical memory of Israelis/Jews 1949-2004
Authors: Nets-Zehngut Rafi
Published: April 2011

The major historical issue in the Israeli—Arab/Palestinian conflict is the causes for the 1948 Palestinian exodus. Among the Israelis/Jews there are two main narratives regarding this issue: the Zionist one — the refugees fled, for various reasons; and the critical one — some fled while others were expelled by the Jewish/Israeli security forces. This article explores the way the Israeli/Jewish historical memory (i.e. the Israeli/Jewish research community) related to this historical issue from 1949 until 2004. According to the findings, until 1957 this memory exclusively presented the Zionist narrative. However, from 1958 to 1976 this Zionist trend largely continued but was accompanied by considerable critical studies. Later, from 1977 to 2004, this memory was characterized by the almost exclusive adoption of the critical narrative (with major increase in its significance since 1988). These findings contradict the way the literature relates to this memory as almost exclusively Zionist until the late-1980s. Other aspects of this memory are also discussed, such as the explanations for its characteristics, the significance of non-academic scholars, the contribution of scholars who reside externally to the given country, state—research community relations, the influence of present interests on the portrayal of the past, and gender issues. The findings have theoretical implications for collective and historical memories.
Third-party trade, political similarity, and dyadic conflict
Authors: Peterson Timothy M
Published: April 2011

A growing literature maintains that trade is pacifying to interstate relations, and recent work on trade networks suggests that this pacifying effect extends to indirect trade ties. In this article, it is argued that third-party trade can also have an aggravating effect within dyads, by threatening to alter the dyadic balance of capabilities. For the state trading outside the dyad, trade gains from third parties provide incentives to demand change in the dyadic status quo and finance violent conflict when dyadic disputes arise. For the state whose dyadic partner trades with third parties, potential for the trading state to grow increasingly more powerful encourages action to prevent erosion of the non-trading state’s relative power. These aggravating effects are conditional, however, on dyadic political similarity, as concerns for relative capability shifts decrease when states do not view their dyadic partners as threats. Hypotheses derived from this argument are tested on data spanning 1885 to 2000. Results support these hypotheses, suggesting important implications for the literature on trade and conflict. When extending the relative gains arguments associated with realism beyond the dyad, a clear, yet conditional, aggravating effect of third-party trade emerges.
Sleeping hegemons: Third-party intervention following territorial integrity transgressions
Authors: van der Maat Eelco
Published: April 2011

It is not clear why some territorial transgressions lead to intervention by the international community and others do not. As most territorial transgressions lead to a reaction from the international community but a few, however, do not, this study examines the main determinants for non-intervention by capable members of the international community, following violation of the territorial integrity norm by a deviant state. This article focuses on which characteristics of the norm-transgressing state, the conflict, and the state capable of enforcing the norm affect the occurrence of non-intervention. This comparative study examines six major alleged transgressions of the territorial integrity norm — the occupation of Tibet, the Suez crisis, the 1967 six-day war, the Indonesian occupation of East Timor, the Moroccan occupation of the Western Sahara, and the 1989 Gulf crisis — using a multi-methodological approach based on qualitative methods, most notably fuzzy-set Qualitative Comparative Analysis. Analysis reveals that the absence of high security costs to the hegemon in combination with (a) strategically important security relations between the transgressor and the hegemon or (b) absence of military and economic vulnerability are generally necessary and sufficient for non-intervention.
Lessons from the Volunteering Legacy of the 2002 Commonwealth Games
Authors: Nichols Geoff , Ralston Rita
Published: April 2011

Potential exists for a more multifaceted and interrelated volunteering legacy from mega sporting events than has been recognised by previous research, including not only the continuation and development of volunteering activity, but also the contribution that activity makes to the social inclusion of volunteers, the economic contribution to the development of events in the region, the development of a skilled volunteer workforce and raising the standard of event volunteer management. This paper provides evidence for that claim via a case study of Manchester Event Volunteers—a volunteer development organisation established after the 2002 Commonwealth Games, which is still operating seven years later and provides a role model for volunteer broker organisations. The case study shows that local government played a key role in generating a volunteer legacy, but that legacy planning was limited by the imperative of running the event. Implications for similar events, such as the 2012 Olympics, are discussed.
The Inaugural Kenneth N. Waltz Annual Lecture A World Order Without Superpowers
Authors: Buzan Barry
Published: April 2011

The category of superpower, as distinct from great power, has become naturalized in the discourses about international relations. But ‘superpower’ has only become common usage since the end of the Second World War and in modern history cannot meaningfully be applied much further than the 19th century. This article argues that superpowers are a historically contingent phenomenon whose emergence rested on the huge inequality of power between the West and the rest of the world that developed during the 19th century. As this inequality diminishes, the most likely scenario for world politics is decentred globalism, in which there will be no superpowers, only great powers. The largest section of the article uses a framework of material and social factors to show why the US is unlikely to remain a superpower, and why China and the EU are unlikely to become superpowers. The following three sections use the same framework to look more briefly at why a world with only great powers is likely to take a more regionalized form; why this might produce a quite workable, decentralized, coexistence international society with some elements of cooperation; and what the possible downsides of a more regionalized international order might be, focusing particularly on the problem of regional hegemony. The conclusions offer five policy prescriptions for living in a decentred globalist world.
Catholic and Non-Catholic NGOs Fighting HIV/AIDS in Sub-Saharan Africa
Authors: Ferrari Lisa L.
Published: April 2011

While governments in sub-Saharan Africa work to provide in-country relief for the HIV/AIDS crisis, much health care and infrastructure comes from local or international non-governmental organizations (NGOs). The literature on NGOs suggests that collaboration increases their efficacy. Many non-Catholic NGOs do not work collaboratively with Catholic NGOs on HIV/AIDS, though the Catholic Church has rich and varied resources at its disposal for relief work. Observers often characterize the incompatibility of Catholic and non-Catholic NGOs as tactical, especially with regard to condom use. However, divergent issue framing is a critical and more fundamental distinction between the two groups. Contrasting the Catholic Church’s unique spiritual frame with the scientific frame of many non-Catholic NGOs highlights the epistemological and teleological differences between the two. Reconciling these differing approaches, or finding ways to cooperate despite them, is a key element of promoting broader NGO collaboration on HIV/AIDS relief work. This theoretical analysis suggests directions for future empirical research.