bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Thrombin Generation Is Associated With Acute Ischemic Stroke but Not With Coronary Heart Disease in the Elderly
Authors: Carcaillon Laure , Alhenc-Gelas Martine , Bejot Yannick , Spaft Christian , Ducimetière Pierre , Ritchie Karen , Dartigues Jean-François , Scarabin Pierre-Yves
Published: April 2011

Objective—

A high thrombin generation level has been associated with the risk of venous thrombosis. Whether changes in this biomarker are relevant to arterial disease remains unknown. We investigated the association of thrombin generation with coronary heart disease (CHD) and acute ischemic stroke (AIS) in the elderly.

Methods and Results—

We used data from the Three-City study, a prospective cohort including 9294 subjects aged >65 years. After 4 years of follow-up, a case-cohort study was established. Using the calibrated automated thrombography method, endogenous thrombin potential and peak height were measured in plasma samples of all CHD and AIS cases and a random sample of 1177 controls. We did not find any significant association between thrombin generation and CHD. In multivariate analyses, high levels of endogenous thrombin potential and peak height were associated with an increased risk of AIS (hazard ratio=1.16 [95% CI, 0.90 to 1.50] and 1.31 [95% CI, 1.01 to 1.69] for a 1 SD increase, respectively). Data also suggested that these associations might be more important in women (hazard ratio=1.55 [95% CI, 1.05 to 2.33] and 1.71 [95% CI, 1.11 to 2.63], respectively) than in men (
P
for interaction=0.04 and 0.08, respectively).

Conclusion—

Thrombin generation emerges as an independent predictor of AIS, particularly in women. Hypercoagulability may have an important role in the pathogenesis of AIS.
Helper-Dependent Adenovirus Is Superior to First-Generation Adenovirus for Expressing Transgenes in Atherosclerosis-Prone Arteries
Authors: Jiang Bo , Qian Kun , Du Liang , Luttrell Ian , Chitaley Kanchan , Dichek David A.
Published: April 2011

Objective—

Vascular gene transfer is a powerful tool for investigating and treating vascular diseases; however, its utility is limited by brevity of transgene expression and vector-associated inflammation. Helper-dependent adenovirus (HDAd), an advanced-generation adenovirus that lacks all viral genes, is superior to first-generation adenovirus (FGAd) in normal rabbit arteries. We compared HDAd to FGAd in arteries of cholesterol-fed rabbits, a model of early atherogenesis in which transgene expression might be decreased, and inflammation increased.

Methods and Results—

Carotid arteries of chow- and cholesterol-fed rabbits were infused with FGAd, HDAd, or medium. HDAd expressed a transgene at least as well in arteries of cholesterol-fed rabbits as in arteries of chow-fed rabbits and expressed more durably than FGAd. In arteries of cholesterol-fed rabbits, HDAd stimulated less intimal growth, lipid deposition, and inflammation than FGAd. Neither vector affected phenylephrine-induced contraction or nitroprusside-mediated relaxation; however, both vectors decreased maximal acetylcholine-stimulated vasorelaxation. The relative absence of intimal growth in HDAd arteries could interfere with the utility of this model for testing atheroprotective genes; however, both coinfusion of FGAd and extension of cholesterol feeding yielded larger intimal lesions, on which atheroprotective genes could be tested.

Conclusion—

HDAd is superior to FGAd for expression of transgenes in atherosclerosis-prone arteries.
Repair of the Injured Adult Heart Involves New Myocytes Potentially Derived From Resident Cardiac Stem Cells
Authors: Angert David , Berretta Remus M. , Kubo Hajime , Zhang Hongyu , Chen Xiongwen , Wang Wei , Ogorek Barbara , Barbe Mary , Houser Steven R.
Published: April 2011

Rationale:

The ability of the adult heart to generate new myocytes after injury is not established.

Objective:

Our purpose was to determine whether the adult heart has the capacity to generate new myocytes after injury, and to gain insight into their source.

Methods and Results:

Cardiac injury was induced in the adult feline heart by infusing isoproterenol (ISO) for 10 days via minipumps, and then animals were allowed to recover for 7 or 28 days. Cardiac function was measured with echocardiography, and proliferative cells were identified by nuclear incorporation of 5-bromodeoxyuridine (BrdU; 7-day minipump infusion). BrdU was infused for 7 days before euthanasia at days 10, 17, and 38 or during injury and animals euthanized at day 38. ISO caused reduction in cardiac function with evidence of myocyte loss from necrosis. During this injury phase there was a significant increase in the number of proliferative cells in the atria and ventricle, but there was no increase in BrdU+ myocytes. cKit+ cardiac progenitor cells were BrdU labeled during injury. During the first 7 days of recovery there was a significant reduction in cellular proliferation (BrdU incorporation) but a significant increase in BrdU+ myocytes. There was modest improvement in cardiac structure and function during recovery. At day 38, overall cell proliferation was not different than control, but increased numbers of BrdU+ myocytes were found when BrdU was infused during injury.

Conclusions:

These studies suggest that ISO injury activates cardiac progenitor cells that can differentiate into new myocytes during cardiac repair.
Matrix Metalloproteinases Are Associated With Increased Blood–Brain Barrier Opening in Vascular Cognitive Impairment
Authors: Candelario-Jalil Eduardo , Thompson Jeffrey , Taheri Saeid , Grossetete Mark , Adair John C. , Edmonds Elaine , Prestopnik Jillian , Wills John , Rosenberg Gary A.
Published: April 2011

Background and Purpose—

Subcortical ischemic vascular disease (SIVD) is a major form of vascular cognitive impairment (VCI) due to small vessel disease. Matrix metalloproteinases (MMPs) are neutral proteases that disrupt the blood–brain barrier and degrade myelin basic protein under conditions of neuroinflammation. Brain tissues and cerebrospinal fluid (CSF) of patients with VCI have increased levels of MMPs. We hypothesized that patients with SIVD have increased MMPs in the CSF, which are associated with increased CSF albumin.

Methods—

We studied 60 patients with suspected VCI. Twenty-five were classified as SIVD, whereas other groups included mixed Alzheimer disease and VCI, multiple strokes, and leukoaraiosis when white matter lesions were present and the diagnosis of VCI was uncertain. MMP-2 and MMP-9 in CSF and plasma were measured by gel zymography and indexed to CSF and plasma albumin. MMP-3 activity was measured by fluorescent assay.

Results—

We found reduced MMP-2 index (
P
<0.001) in the CSF for the full group of patients (SIVD, multiple strokes, mixed Alzheimer disease and VCI, and leukoaraiosis) compared with control subjects, whose CSF was obtained during spinal anesthesia. MMP-3 activity was increased in VCI compared with control subjects (
P
<0.01). In SIVD, MMP-2 index showed a negative correlation with albumin index, which was absent with the MMP-9 index. Combining MMP-2 index and MMP-3 activity separated the patients with SIVD from the control subjects with high specificity (
P
<0.0005).

Conclusions—

Our results support the hypothesis that MMPs are associated with increased CSF albumin and suggest that they may contribute to the pathophysiology of SIVD.
Preexisting Statin Use Is Associated With Greater Reperfusion in Hyperacute Ischemic Stroke
Authors: Ford Andria L. , An Hongyu , D'Angelo Gina , Ponisio Rosana , Bushard Patrick , Vo Katie D. , Powers William J. , Lin Weili , Lee Jin-Moo
Published: April 2011

Background and Purpose—

Statin pretreatment has been associated with improved outcomes in patients with ischemic stroke. Although several mechanisms have been examined in animal models, few have been examined in patients. We hypothesized that patients using statins before stroke onset may have greater reperfusion than patients not using statins.

Methods—

Acute ischemic stroke patients underwent 2 MR scans: within 4.5 (tp1) and at 6 hours (tp2) after stroke onset. Regions of reperfusion were defined by prolonged mean transit time (MTT) at tp1, which normalized at tp2. Four MTT thresholds were assessed to ensure that results were not spuriously based on an arbitrary threshold. Baseline characteristics, relative reperfusion, and change in NIHSS between tp1 and 1-month follow-up (ΔNIHSS) were compared between patients who were using statins at stroke onset and those who were not.

Results—

Thirty-one stroke patients were prospectively enrolled; 12 were using statins and 19 were not. Baseline characteristics did not differ between the 2 groups except the statin group had greater coronary artery disease (
P
=0.03). Patients using statins showed significantly greater reperfusion compared to untreated patients across all MTT thresholds. For MTT of 4 seconds, median relative reperfusion was 50% (interquartile range, 30%–56%) in the preexisting statin group versus 13% (interquartile range, 5%–36%) in the untreated group (
P
=0.014). The statin group had greater ΔNIHSS (8.8±4.0 points) compared to the untreated group (4.4±5.7 points;
P
=0.028).

Conclusions—

Statin use before ischemic stroke onset was associated with greater early reperfusion and NIHSS improvement. Further studies in larger populations are required to confirm our preliminary findings.
Dissociation of Vasospasm and Secondary Effects of Experimental Subarachnoid Hemorrhage by Clazosentan
Authors: Sabri Mohammed , Ai Jinglu , Macdonald R. Loch
Published: April 2011

Background and Purpose—

Endothelin receptor antagonists such as clazosentan decrease large-artery vasospasm after experimental and clinical subarachnoid hemorrhage. We used clazosentan to gain insight into the pathophysiology of subarachnoid hemorrhage by determining if decreasing vasospasm is associated with alleviation of other secondary complications of subarachnoid hemorrhage such as oxidative stress, endothelial nitric oxide synthase dysfunction, microthromboembolism, and neuronal injury.

Methods—

Mice were subjected to subarachnoid hemorrhage by injection of blood into the chiasmatic cistern. They were treated with clazosentan or vehicle by continuous intraperitoneal infusion for 48 hours. Middle cerebral artery vasospasm, superoxide anion radical, peroxynitrite, microthromboemboli, endothelial nitric oxide synthase uncoupling, cerebral blood flow, neuronal injury, and mortality were assessed.

Results—

Clazosentan preserved cerebral blood flow, alleviated vasospasm, and decreased mortality but did not affect superoxide anion radical, peroxynitrite, or microthromboemboli in the brain. Endothelial nitric oxide synthase uncoupling and neuronal injury also were not reduced by clazosentan.

Conclusions—

This study shows large-artery vasospasm is pathophysiologically independent of some other effects of subarachnoid hemorrhage. The findings have implications for development of treatments for this disease.
Sage Philosophy: Revisiting Oruka’s African Ideology
Authors: Binte Masud Sabrina
Published: April 2011

The term African philosophy is considered by Western philosophy either as a subgenre or as an improbability. The findings of Kenyan philosopher H. Odera Oruka provide substantial proof of a major nature that negates this attitude toward African philosophy. This essay demonstrates how Oruka’s Sage philosophy has the consistency and coherence to alter forever any notion of Western superiority in philosophy. Indeed, Oruka, much like Cheikh Anta Diop in the field of anthropology and history, transforms the way we think about African philosophy by taking it away from the Western schools’ perspective to show that oral and proverbial wisdom has a long and noble tradition in human reflection.
Fighting and Voting: Violent Conflict and Electoral Politics
Authors: Dunning Thad
Published: April 2011

Two recent research programs—one on the sources of democratic consolidation and another on the causes and consequences of violent conflict—have tended to evolve in relative isolation. The contributions to this special issue of Journal of Conflict Resolution help to bridge this gap, through explicit theoretical and empirical analysis of the relationship between fighting and voting. Armed conflict and electoral politics may be strategic substitutes, in that political actors may optimally choose to submit to the ballot box or instead attempt to impose their will by force; or they may be strategic complements, in that actors use violence to bolster their electoral aims, or use electoral returns as sources of information on underlying preferences that they exploit in armed campaigns. In either case, the distribution of popular support for contending parties can shape not only the incidence but also the type of armed conflict, and it can also influence the incentives of parties to invest in institutional mechanisms that mitigate commitment problems and help to bring violent conflicts to an end. The contributions to this issue illuminate these themes and demonstrate the value of bringing these separate research programs into closer dialogue.
The Lysosomotropic Agent Monodansylcadaverine Also Acts as a Solvent Polarity Probe
Authors: Niemann Axel , Takatsuki Akira , Elsässer Hans-Peter
Published: April 2011

The autofluorescent substance monodansylcadaverine has recently been reported as a specific in vivo marker for autophagic vacuoles. However, the mechanism for this specific labeling remained unclear. Our results reveal that the common model of ion trapping in acidic compartments cannot completely account for the observed autophagic vacuole staining. Because autophagic vacuoles are characterized by myelin-like membrane inclusions, we tested whether this lipid-rich environment is responsible for the staining properties of monodansylcadaverine. In in vitro experiments using either liposomes or solvents of different polarity, monodansylcadaverine showed an increased relative fluorescence intensity in a hydrophobic environment as well as a Stokes shift dependent on the solvent polarity. To test the effect of autophagic vacuoles or autophagic vacuole lipids on monodansylcadaverine fluorescence, we isolated autophagic vacuoles and purified autophagic vacuole lipids depleted of proteins. Entire autophagic vacuoles and autophagic vacuole lipids had the same effect on monodansylcadaverine fluorescence properties, suggesting lipids as the responsible component. Our results suggest that the in vivo fluorescence properties of monodansylcadaverine do not depend exclusively on accumulation in acidic compartments by ion trapping but also on an effective interaction of this molecule with autophagic vacuole membrane lipids.
The Affinity Binding Sites of Pancreatic Bile Salt-Dependent Lipase in Pancreatic and Intestinal Tissues
Authors: Bruneau Nadine , Lombardo Dominique , Bendayan Moïse
Published: April 2011

In previous studies, we have shown that the bile salt-dependent lipase (BSDL) associates with the Grp94 molecular chaperone, an association that appears to play essential roles in the folding of BSDL. More recently, combined biochemical and immunocytochemical investigations were carried out to show that the transport of BSDL occurs via an association with the Grp94 all along the pancreatic secretory route (ER-Golgi-granules). The Grp94-BSDL complex is secreted with the pancreatic juice into the acinar lumen and reaches the duodenal lumen, where it is internalized by enterocytes. The dissociation of the complex could take place within the endosomal compartment because BSDL continues further on its way to the basolateral membrane of the enterocyte. To localize the affinity binding sites of pancreatic BSDL in pancreatic and duodenal tissues, we have used an affinity-gold ultrastructural technique. BSDL coupled to gold particles appears to interact with specific sites in tissue sections. This was confirmed by another indirect morphological approach using biotin-labeled BSDL and streptavidin-gold complexes on tissue sections. We have shown that BSDL associates with sites in the pancreatic secretory pathway compartments and in the microvilli, the endosomal compartment, and the basolateral membrane of enterocytes. By biochemical approaches, biotin-labeled BSDL displayed affinities with proteins of 180-190 kD in both pancreatic and duodenal tissues. We have also shown that the Grp94-BSDL complexes, which are insensitive to denaturing conditions, are present in pancreatic homogenate but not in duodenal lysate. Thus, BSDL is able to bind protein complexes formed by either BSDL-Grp94 or Grp94 dimers.
Acidic Fibroblast Growth Factor is Present in the Enteric Nervous System of the Large Intestine
Authors: Capetandes Anthony , Salvo Jerry Di , Ronan John J. , Thomas Kenneth A.
Published: April 2011

Acidic fibroblast growth factor (aFGF) is a heparin binding protein that displays pleiotropic activity. The purpose of this study was to document the presence of the translated aFGF product, its mRNA, and its location in the colon. mRNA was extracted from bovine large intestine and reverse transcribed to cDNA. Nested-primer PCR was used to determine the presence of mRNA using primers homologous to the previously published bovine aFGF cDNA. Purification of translated aFGF was performed using an established HPLC protocol. Western blot analysis of the HPLC fractions was performed using two epitopeindependent antibodies against aFGF. Immunohistochemistry employed these antibodies to determine the locus of aFGF expression. The nested-primer PCR product of predicted size was homologous to the published bovine aFGF mRNA sequence, as determined by DNA sequencing. Intestinal aFGF had a mass similar to bovine aFGF isolated from other tissues, and immunocrossreacted with two peptide-based, epitope-independent anti-aFGF antisera on Western blotting. Immunohistochemical analysis of large intestine using these two independent antisera localized aFGF within the myenteric plexus. These data demonstrate that aFGF is present within the myenteric plexus of the enteric nervous system.
Expression of Glutamine Synthetase in Macrophages
Authors: Bode Johannes Georg , Peters-Regehr Thorsten , Kubitz Ralf , Häussinger Dieter
Published: April 2011

We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages.
Direct Eye Visualization of Cy5 Fluorescence for Immunocytochemistry and In Situ Hybridization
Authors: Ferri Gian-Luca , Isola Jorma , Berger Peter , Giro Gabriele
Published: April 2011

Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588-640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650-700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path.
Introduction of a Novel HRP Substrate-Nanogold Probe for Signal Amplification in Immunocytochemistry
Authors: Mayer Gaétan , Leone Robert D. , Hainfeld James F. , Bendayan Moïse
Published: April 2011

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.
Enhanced Labeling Efficiency Using Ultrasmall Immunogold Probes: Immunocytochemistry
Authors: Robinson John M. , Takizawa Toshihiro , Vandré Dale D.
Published: April 2011

Detection of antigen-antibody interactions in immunocytochemistry relies on a reporter system. The most commonly employed reporter systems used are fluorochromes, enzymes, and particulate probes. This article considers the advantages and disadvantages associated with ultrasmall immunogold particles as the reporter system in immunocytochemical applications.
A Practical Technique to Postfix Nanogold-immunolabeled Specimens with Osmium and to Embed Them in Epon for Electron Microscopy
Authors: Sawada Hajime , Esaki Michiyo
Published: April 2011

Nanogold is a tiny gold probe, freely diffusible in cells and tissues, and is suitable for pre-embedding immunohistochemistry. However, it is necessary to develop Nanogold to a larger size so that it can be observed by conventional transmission electron microscopy. Silver enhancement is usually used for visualizing Nanogold, but the silver shell produced is unstable in OsO4 and often becomes invisible after OsO4 postfixation, which is necessary for good visualization of ultrastructure. We used silver enhancement with silver acetate, followed by gold toning with chloroauric acid, to replace the silver shell with a more stable gold in order to observe Nanogold after osmium fixation and Epon embedding. This technique is applicable to various intra- and extracellular antigens. For correlative observation of immunolabled specimens by light and electron microscopy, specimens adhered to slideglasses were embedded in Epon under nonadhesive plastic film. By heating the Epon sheets after polymerization, these supports were removed without difficulty and provided easy correlative observation.
Freeze-drying Allows Double Nonradioactive ISH and Antigenic Labeling
Authors: Louis Huguette , Lavie Julie , Lacolley Patrick , Daret Danièle , Bonnet Jacques , Lamazière Jean-Marie Daniel
Published: April 2011

Because tissue freeze-drying is an excellent way to preserve antigenic conformation, we have tested the feasibility of this technique to reveal nonradioactive in situ hybridization (ISH) of tissue mRNA. We have compared mRNA detection after different methods of tissue preservation, freeze-drying, cryosectioning, and formaldehyde or methanol fixation. Our results show that nonradioactive ISH is more sensitive for tissues preserved by freeze-drying than for other tissue preparations. We have demonstrated that freeze-drying allows combination of ISH and immunohistochemistry for simultaneous detection of mRNA and antigen because with this technique of tissue preservation ISH does not affect the sensitivity or the amount of the detected antigens. This work underscores the fact that tissue freeze-drying is an easy, convenient, and reliable technique for both ISH and immunohistochemistry and achieves excellent structural conditions for nonradioactive detection.
Human Bronchial Epithelial Cells Secrete Laminin 5, Express Hemidesmosomal Proteins, and Assemble Hemidesmosomes
Authors: Michelson Peter H. , Tigue Margaret , Jones Jonathan C.R.
Published: April 2011

Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cell-ECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin α6β4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the α6β4 integrin heterodimer at the site of bronchial epithelial cell-ECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde-fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5-α6β4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung.
Intercellular Adhesion Molecule-1 and Hair Follicle Regression
Authors: Müller-Röver Sven , Bulfone-Paus Silvia , Handjiski Bori , Welker Pia , Sundberg John P. , McKay Ian A. , Botchkarev Vladimir A. , Paus Ralf
Published: April 2011

Although the intercellular adhesion molecule-1 (ICAM-1) is recognized for its pivotal role in inflammation and immune responses, its role in developmental systems, such as the cyclic growth (anagen) and regression (catagen) of the hair follicle, remains to be explored. Here we demonstrate that ICAM-1 expression in murine skin is even more widespread and more developmentally regulated than was previously believed. In addition to endothelial cells, selected epidermal and follicular keratinocyte subpopulations, as well as interfollicular fibroblasts, express ICAM-1. Murine hair follicles express ICAM-1 only late during morphogenesis. Thereafter, morphologically identical follicles markedly differ in their ICAM-1 expression patterns, which become strikingly hair cycle-dependent in both intra- and extrafollicular skin compartments. Minimal ICAM-1 and leukocyte function-associated (LFA-1) protein and mRNA expression is observed during early anagen and maximal expression during late anagen and catagen. Keratinocytes of the distal outer root sheath, fibroblasts of the perifollicular connective tissue sheath, and perifollicular blood vessels exhibit maximal ICAM-1 immunoreactivity during catagen, which corresponds to changes of LFA-1 expression on perifollicular macrophages. Finally, ICAM-1-deficient mice display significant catagen acceleration compared to wild-type controls. Therefore, ICAM-1 upregulation is not limited to pathological situations but is also important for skin and hair follicle remodeling. Collectively, this suggests a new and apparently nonimmunological function for ICAM-1-related signaling in cutaneous biology.
A New Approach to Three-dimensional Reconstructed Imaging of Hormone-secreting Cells and Their Microvessel Environments in Rat Pituitary Glands by Confocal Laser Scanning Microscopy
Authors: Itoh J. , Kawai K. , Serizawa A. , Yasumura K. , Ogawa K. , Osamura R.Y.
Published: April 2011

There has been considerable interest in the relationship between hormone-secreting endocrine cells and their microvessels in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). This study was designed to visualize and to reveal the relationship between hormone-secreting endocrine cells and their microvessel environment in 3D, using rat pituitary glands under various (hyper/hypo) experimental conditions by confocal laser scanning microscopy (CLSM). Female adult Wistar rats were used after bilateral adrenalectomy or ACTH administration for 2 weeks. Clear 3D reconstructed images of ACTH cells, the microvessel network and counterstained nuclei were obtained at a maximal focus depth of 1 mm by CLSM without any background noise. In the hyperfunctional state, slender cytoplasmic processes of hypertrophic stellate ACTH cells frequently extended to the microvessels. In the hypofunctional state, ACTH cells appeared atrophic and round with scanty cytoplasm, and cytoplasmic adhesions to microvessel network patterns were inconspicuous. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways.
Tracking Micrometastasis to Multiple Organs with lacZ-tagged CWR22R Prostate Carcinoma Cells
Authors: Holleran Julianne L. , Miller Carson J. , Culp Lloyd A.
Published: April 2011

SUMMARY Metastasis to organs other than lung is rarely observed in animal model systems of human prostate carcinoma (PCA), with the exception of already metastatic isolates of human PCA cultured for long periods of time. To analyze more directly the evolution of metastatic variants from primary PCA tumor isolates, the lacZ histochemical marker gene was transfected into the CWR22Rv1 cell line isolated from the CWR22R xenograft (primary tumor). Three clones of varying lacZ-expression stability were analyzed for tumorigenicity and progression in athymic nude mice. Clones B and D were highly tumorigenic in the subcutis; however, lacZ expression was highly unstable. In contrast, clone H demonstrated highly stable lacZ expression for >25 passages in culture or in animals. Clone H, injected sc in a PBS vehicle, gave a 15-40% tumorigenic take. All primary tumor-bearing animals exhibited micrometastases in lung and other organs. Clone H injected in a Matrigel vehicle gave 100% tumorigenicity, with all animals displaying micrometastases in lung, liver, and/or bone (lower frequency in brain and kidney). Overall, the relative frequency of micrometastasis to multiple organs was lung>liver=bone>>brain>kidney. Overt metastases were never observed in the lung or bone but were occasionally found in liver. lacZ-transfected clone H CWR22Rv1 cells represent a much more accurate model of metastasis of PCA to the organs normally involved in progression of the human disease. Use of marker gene-tagged cells and other high-resolution molecular techniques will now permit analyses of the earliest events in PCA progression and micrometastasis.
Modified Basement Membrane Composition During Bronchopulmonary Tumor Progression
Authors: Catusse Corinne , Polette Myriam , Coraux Christelle , Burlet Henriette , Birembaut Philippe
Published: April 2011

SUMMARY During tumor progression, the extracellular matrix (ECM) and particularly the basement membrane (BM) appear to be dynamic structures that are not only degraded but also deposited around tumor clusters. In this study we examined by immunohistochemistry the localization of three chains of Type IV collagen (α1, α3 and α5), Type VII collagen, and laminin 5 at different stages of bronchopulmonary cancers. In normal tissues, α1(IV) chain was detected in all BMs (bronchial, vascular, alveolar, and glandular), α5(IV) chain was present only in vascular BM, and laminin 5 and Type VII collagen were co-localized in bronchial and glandular BMs, whereas α3(IV) immunolabeling was totally absent from normal bronchi. In well-differentiated carcinomas, α3(IV) chain staining was found in some neosynthetized BMs interfacing the tumor cell and the stromal compartment, contrasting with the total absence of labeling in normal tissues. α1(IV) chain showed strong reactivity in all BM. Laminin 5 and Type VII collagen were also detected in neosynthetized BM. In poorly differentiated invasive cancers, α3(IV) chain and Type VII collagen were not found, whereas laminin 5 and α1(IV) chain persisted. The most important modifications in BM composition during tumor progression therefore appear to be the appearance of the α3 (IV) chain in well-differentiated carcinomas and its subsequent disappearance in poorly differentiated carcinomas, together with the loss of type VII collagen. α5(IV) chain distribution was restricted in vascular BM of well- and poorly differentiated carcinomas. These results show that the composition of BM is modified during the progression of bronchopulmonary tumor, emphasizing that the BM represents a dynamic element in tumor progression and has an important role in tumor cell invasiveness.
A New Cat Fos Antibody to Localize the Immediate Early Gene c-fos in Mammalian Visual Cortex after Sensory Stimulation
Authors: Gucht Estel Van der , Vandenbussche Erik , Orban Guy A. , Vandesande Frans , Arckens Lutgarde
Published: April 2011

SUMMARY We developed a novel antibody against cat Fos by immunizing rabbits with a 26-amino-acid peptide. Immunocytochemistry on visual cortex of cats undergoing different visual manipulations was applied to test the reliability and the efficacy of this antiserum. One hour of light stimulation after an overnight dark adaptation resulted in strongly induced Fos expression in supra- and infragranular layers of cat primary visual cortex. Short-term monocular deprivation changed the Fos expression profile into a columnar immunostaining related to ocular dominance columns. Fos expression has also been analyzed in cats in which visual input was confined to the right hemisphere by sectioning the left optic tract and the corpus callosum. In the right hemisphere, visual stimulation elicited Fos induction, whereas in the contralateral hemisphere a very low Fos signal was observed. The specificity of this newly synthesized antibody was confirmed by Western blotting. To further establish the applicability of this Fos antiserum, we performed immunostaining on monkey and rat visual cortex. This new cat Fos antibody appears to be excellent for study of Fos expression as a marker for mapping neuronal activity in mammalian brain.
Immunohistochemical Localization of cGMP-binding cGMP-specific Phosphodiesterase (PDE5) in Rat Tissues
Authors: Kotera Jun , Fujishige Kotomi , Omori Kenji
Published: April 2011

SUMMARY We raised a polyclonal antibody against maltose binding protein fusion human cGMP-binding, cGMP-specific phosphodiesterase (PDE5) produced in E. coli. This antibody immunoreacted specifically with recombinant human and rat PDE5 proteins expressed in transfected COS-7 cells and with a native form of PDE5 in extracts of rat platelets, lung, and cerebellum. Immunohistochemical analysis showed that the anti-PDE5 antibody detected immunoactive materials in Purkinje cell layers of the cerebellum, proximal renal tubules, collecting renal ducts, and epithelial cells of pancreatic ducts in rats. Reverse transcriptase-polymerase chain reaction analysis demonstrated that PDE5 transcripts are also present in rat cerebellum, kidney, and pancreas. Here we described a cell-specific localization of PDE5 in various rat tissues, suggesting the possibility of the presence of a cGMP/PDE5 pathway in these tissues.
Morphological Aspects of the Postnatal Development of Submandibular Glands in Male Rats: Involvement of Apoptosis
Authors: Hayashi Hiroyuki , Ozono Satoru , Watanabe Kazuko , Ikuko Nagatsu , Onozuka Minoru
Published: April 2011

SUMMARY We studied the involvement of the apoptotic mechanism(s) in cell differentiation in the developing male rat submandibular gland using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-labeling) assay in combination with light and electron microscopy. Whereas the proacinar cells were completely transformed into acinar cells within 2 weeks after birth, starting on postnatal Day 21, the terminal tubule cells formed vacuoles that disappeared by postnatal Day 35. During this period, positive TUNEL reactivity was seen in the terminal tubule cells, and electron microscopic analysis showed that certain morphological features of apoptosis, including fragmentation of nuclei and the presence of apoptotic bodies in the cytoplasm, were present in and restricted to the terminal tubule cells. These results indicate that, in addition to an autophagocytosis-mediated mechanism, apoptosis may also be involved in reducing the number of terminal tubule cells during postnatal development in the submandibular gland.
AgarCyto: A Novel Cell-processing Method for Multiple Molecular Diagnostic Analyses of the Uterine Cervix
Authors: Kerstens Harold M.J. , Robben Johanna C.M. , Poddighe Pino J. , Melchers Willem J.G. , Boonstra Henk , Wilde Petrus C.M. de , Macville Merryn V.E. , Hanselaar Antonius G.J.M.
Published: April 2011

SUMMARY In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.
Affinity Imaging of Red Blood Cells Using an Atomic Force Microscope
Authors: Grandbois Michel , Dettmann Wolfgang , Benoit Martin , Gaub Hermann E.
Published: April 2011

SUMMARY We used an atomic force microscope (AFM) to produce an image of a mixed layer of group A and O red blood cells with a contrast based only on the measured strength of a specific receptor-ligand pair. The image was obtained by measuring and plotting for each image pixel the adhesion force between the mixed RBC layer and the AFM tip functionalized with Helix pomatia lectin. The high specificity of that lectin for the N-acetylgalactosamine-terminated glycolipids present in the membrane of group A RBCs enabled us to discriminate between the two cell populations and to produce an image based on affinity contrast. The rupture force of the adhesion events leading to the image formation were quantitatively analyzed and compared to rupture forces measured with the same AFM tip on N-acetylgalactosamine tethered to agarose beads. The mean rupture force was found to be 65 pN when measured on the group A RBCs and 35 pN on the agarose beads. These results show that the adhesion, mediated by only a few receptor-ligand pairs, produces sufficient contrast for the affinity image formation.
DNA Fiber-FISH Staining Mechanism
Authors: van de Rijke Frans M. , Florijn Ralph J. , Tanke Hans J. , Raap Anton K.
Published: April 2011

Fluorescence in situ hybridization to DNA fibers (Fiber-FISH) is a high-resolution, wide-ranging physical DNA mapping method that finds increasing application in the study of pathological gene rearrangements. Here we present experiments designed to understand the nature of the discontinuous FISH signal patterns seen after Fiber-FISH. Use of a novel cisplatin-based chemical labeling method enabled us to produce intact biotinlabeled cosmid target DNA molecules. We monitored by immunofluorescence the fate of such cosmid targets during denaturation and hybridization. The same cosmid DNA labeled with digoxigenin by nick-translation was used to analyze the FISH probe signal distribution in a different color. The probe signals proved to be a subset of the target signals remaining after denaturation and hybridization. We argue that the discontinuity of probe signals in Fiber-FISH is mainly caused by loss of target DNA and limited accessibility due to in situ renaturation and attachment. Furthermore, we conclude that FISH sensitivity is determined by hybridization efficiency and not the ability to generate sufficient signal from small probes.
Presence of the Kv1.5 K+ Channel in the Sinoatrial Node
Authors: Dobrzynski Halina , Rothery Stephen M. , Marples David D.R. , Coppen Steven R. , Takagishi Yoshiko , Honjo Haruo , Tamkun Michael M. , Henderson Zaineb , Kodama Itsuo , Severs Nicholas J. , Boyett Mark R.
Published: April 2011

The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K+ channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, ~64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gapjunctions.
Connexin43 Gene Expression and Regulation in the Rodent Seminiferous Epithelium
Authors: Batias Catherine , Siffroi Jean-Pierre , Fénichel Patrick , Pointis Georges , Segretain Dominique
Published: April 2011

Connexin43 (Cx43) is one of the most predominant gap junction proteins found in the testis. We used in situ hybridization and indirect immunofluorescence to study the distribution of Cx43 mRNA and protein in the rodent seminiferous epithelium. During mouse testis maturation, Cx43 mRNA and its corresponding protein were first detected in the adluminal compartment of the growing seminiferous tubules (early postnatal age: Day 12) to become progressively located in the basal compartment at later ages (Days 16, 19, 27). In seminiferous tubules of sexually mature animals, the intensity of the hybridization signal was stage-dependent, with a maximum at Stage VII compared with Stages V and IX of the spermatogenic cycle ( p<0.05). The highest expression of Cx43 mRNA was observed in the supporting Sertoli cells and, to a lesser extent, in the most basally located and less mature germ cells (spermatogonia and spermatocytes). Consistent with these observations, in situ dye coupling was observed between Sertoli cells and basal germ cells. In a mutant mouse deficient for the retinoid X receptor β, which exhibited abnormal spermatogenesis due to altered Sertoli cell function, Cx43 transcripts were markedly decreased in the seminiferous epithelium ( p<0.01). The immunoreactive signal for Cx43 was significantly reduced in seminiferous tubules of the 3-month-old mutant mice ( p<0.05) and undetectable in older animals. These data provide new information about the precise localization of Cx43 mRNA and protein in seminiferous tubules of immature and mature rodent testes. Moreover, they suggest that retinoids, through the RXRβ receptors, could be involved in the control of Cx43 gene expression in Sertoli cells.
Immunocytochemical Evidence Suggesting that Diamine Oxidase Catalyzes Biosynthesis of γ-Aminobutyric Acid in Antropyloric Gastrin Cells
Authors: Hardt Jesper , Larsson Lars-Inge , Hougaard David M.
Published: April 2011

γ-Aminobutyric acid (GABA) is a neurotransmitter that also occurs in a few non-neuronal cell types, where it may serve as a paracrine modulator. GABA is biosynthesized from glutamate by glutamate decarboxylase (GAD) and from putrescine via diamine oxidase (DAO). GAD is demonstrable in several GABA-positive cell types but is undetectable in the GABA-containing gastrin cells and somatostatin cells of the antropyloric mucosa of the stomach. Using two antisera raised against synthetic peptides corresponding to two different regions of rat DAO, we now demonstrate strong reactivity for DAO in gastrinpositive cells of the rat antropyloric mucosa, whereas somatostatin-positive cells as well as other structures of the antrum are unreactive. Western blotting analysis of antrum and colon demonstrate that both antisera react with a single band of 85 kD, consistent with the predicted molecular weight of DAO. Expression of DAO mRNA in the antrum is demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR). Our results strongly indicate that gastrin cells produce GABA via DAO-catalyzed oxidation of putrescine, and experimental data moreover suggest that the biosynthesis of GABA is regulated by the prandial state. Because GABA modulates release of somatostatin, these results point to a new mechanism of paracrine interaction between gastrin cells and somatostatin cells.
Calculation of Maximal Hybridization Capacity (Hmax) for Quantitative In Situ Hybridization: A Case Study for Multiple Calmodulin mRNAs
Authors: Vizi Sandor , Gulya Karoly
Published: April 2011

In estimations of mRNA copy numbers, quantitative in situ hybridization (ISH) is expected to be performed at saturating probe concentration. In practice, however, this condition can rarely be fulfilled when medium to high amounts of transcripts exist and/or in large-scale experiments. To resolve this problem, we developed and tested a double-step procedure involving the use of calmodulin (CaM) I, II, and III [35S]-cRNA probes on adult rat brain sections; the hybridization signals were detected with a phosphorimager. By means of hybridization at increasing probe concentrations for a time sufficient for saturation, saturation curves were created for and maximal hybridization capacity (Hmax) values were assigned to selected brain areas. The Kd values of these various brain areas did not differ significantly, which allowed the creation and use of one calibration graph of Hmax vs hybridized [35S]-cRNA values for all brain areas for a given probe concentration. Large-scale ISH experiments involving a subsaturating probe concentration were performed to estimate Hmax values for multiple CaM mRNAs. A calibration graph corresponding to this probe concentration was created and Hmax values (expressed in ISH copy no/mm2 units) were calculated for several brain regions via the calibration. The value of the method was demonstrated by simultaneous quantification of the total accessible multiple CaM mRNA contents of several brain areas in a precise and economical way.