bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuregulin Receptor ErbB2 Localization at T-tubule in Cardiac and Skeletal Muscle
Authors: Hideho Ueda , Oikawa Akihiro , Nakamura Akinori , Terasawa Fumiko , Kawagishi Kyutaro , Moriizumi Tetsuji
Published: April 2011

Previous studies have indicated that ErbB receptors for neuregulins play an important role in cardiac development and muscle spindle formation during embryogenesis; however, little is known about their functions in adulthood. Recent reports indicate that breast cancer therapy with humanized monoclonal ErbB2 antibody induces cardiomyopathy, suggesting that ErbB2 serves as a crucial signaling receptor, even in the adult heart. Here, we examine ErbB2 expression and localization in both cardiac and skeletal muscle of adult mice via immunoblot and immunohistochemistry. ErbB2 was detected as a band ~185 kD molecular mass in each cardiac and skeletal muscle extraction. Confocal images of double labeling showed that ErbB2 was colocalized with caveolin-3 in cardiac muscle and with dihydropyridine receptor in skeletal muscle, suggesting that ErbB2 was localized at the T-tubule. In addition, immunoelectron micrographs clearly demonstrated that ErbB2 was located at the T-tubule in both types of muscle. Taken together, the results of the present study suggest that neuregulin-ErbB2 signaling plays a role in the physiological function of cardiac and skeletal muscle, even in adulthood.
Evaluation of Pancreatic Amylase mRNA upon Cholinergic Stimulation of Secretion
Authors: Gingras Diane , Bendayan Moïse
Published: April 2011

The primary function of the exocrine pancreas consists of the synthesis and secretion of several digestive enzymes. It is well established that amylase secretion by rat pancreatic tissue or by isolated acinar cells in culture can be stimulated by the cholinergic agonist carbachol. However, the effect of this secretagogue on enzyme synthesis remains unclear. Some studies demonstrated increases in rates of synthesis, whereas others reported increases in secretion with or without decreases in synthesis. We have evaluated changes in pancreatic amylase mRNA and total RNA after a single injection of carbachol and under fasting conditions. Two approaches in molecular morphology were applied on rat pancreatic tissue: in situ hybridization and RNase A-gold. Both revealed decreases in RNA labeling at the level of the rough endoplasmic reticulum (RER) 5 min after stimulation of secretion and after fasting. Gradual recovery was registered 15 and 30 min after stimulation of secretion. Northern blotting confirmed drastic decreases in amylase mRNA 5 min after stimulation and after fasting. The combination of such different approaches has demonstrated drastic decreases in RNA at the RER level, reflecting declines in rates of synthesis at the translational level under all conditions tested.
Localization of CD44 (Hyaluronan Receptor) and Hyaluronan in Rat Mandibular Condyle
Authors: Nakamura Hiroaki , Kato Ryoko , Hirata Azumi , Inoue Miho , Yamamoto Toshio
Published: April 2011

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.
Raman Spectroscopic Evaluation of Efficacy of Current Paraffin Wax Section Dewaxing Agents
Authors: Faoláin Eoghan Ó , Hunter Mary B. , Byrne Joe M. , Kelehan Peter , Lambkin Helen A. , Byrne Hugh J. , Lyng Fiona M.
Published: April 2011

During a spectroscopic study to identify biochemical changes in cervical tissue with the onset of carcinogenesis, residual paraffin wax contributions were observed on almost all dewaxed formalin-fixed paraffin-processed (FFPP) tissue sections examined. Subsequently, the present study was formulated to evaluate the efficacy of current dewaxing agents using Raman spectroscopy. Three cervical FFPP sections were subjected to each of the protocols. Sections were dewaxed using four common dewaxing protocols, namely, xylene, Histoclear, heat-mediated antigen retrieval (HMAR) using xylene and citrate buffer, and Trilogy (combined deparaffinization and unmasking of antigens). The potential for hexane as a dewaxing agent was also evaluated. Sections were dewaxed in multiple dewaxing cycles using xylene, Histoclear, and hexane. Residual paraffin wax contributions remained at 1062 cm−1, 1296 cm−1, and 1441 cm−1. HMAR using xylene and citrate buffer, and HMAR using Trilogy, showed a similar efficacy, resulting in incomplete removal of wax. Hexane was shown to be the most effective dewaxing agent, resulting in almost complete removal of wax. Immunohistochemistry was carried out on dewaxed slides, and those dewaxed with hexane displayed a stronger positivity (≍28%). Implications for histopathology and immunohistochemistry are considered, as well as problems that residual wax poses for spectroscopic evaluation of dewaxed FFPP sections with a view to disease diagnosis.
Tissue Distribution and Subcellular Localization of Rabbit Liver Metalloendopeptidase
Authors: Nakagawa Kazunori , Kawabata Shun-ichiro , Nakashima Yutaka , Iwanaga Sadaaki , Sueishi Katsuo
Published: April 2011

We have previously isolated rabbit liver microsomal metalloendopeptidase (MEP) as a candidate for the processing enzyme of vitamin K-dependent plasma proteins. A cDNA coding for MEP has revealed that it is structurally related to metalloendopeptidase-24.15, which catalyzes the proteolytic processing of several bioactive peptides. In this study we examined the tissue distribution and subcellular localization of MEP by light and electron microscopic immunohistochemical methods, in addition to Northern blot analysis. Chicken polyclonal antibodies were raised by using synthetic peptides AG1 (Met31-Asn46) and AG3 (Asp537-Gly551) derived from the sequence of MEP. Both anti-AG1 and anti-AG3 antibodies reacted specifically with MEP, as judged by Western blotting and immunohistochemical methods. Both antibodies gave an identical staining distribution, which was localized on the luminal cell surfaces and in the cytoplasm of the following organs: liver, brain, lungs, kidneys, esophagus, stomach, duodenum, pancreas, placenta, epididymis, uterus, ovary, and oviduct. Northern blot analysis revealed that the expression of MEP mRNA is similar to its immunohistochemical distribution except in the heart. These results suggest that MEP may participate more closely in a degradation role in peptide metabolism in various tissues than in a processing role of the proprotein, like metalloendopeptidase-24.15.
Detection of Mitochondrial DNA Deletions in Human Skin Fibroblasts of Patients with Pearson's Syndrome by Two-color Fluorescence In Situ Hybridization
Authors: Corput Mariëtte P.C. van de , Ouweland Johannes M.W. van den , Dirks Roeland W. , Hart Leen M. ‘t , Bruining G. Jan. , Maassen J. Antonie , Raap Anton K.
Published: April 2011

Pearson's marrow/pancreas syndrome is a disease associated with a large mitochondrial DNA (mtDNA) deletion. The various tissues of a patient contain heteroplasmic populations of wild-type (WT) and deleted mtDNA molecules. The clinical phenotype of Pearson's syndrome is variable and is not correlated with the size and position of the deletion. The histo- and cytological distribution of WT and deleted mtDNA molecules may be factors that correlate with the phenotypical expression of the disease. Here we introduce a new application of two-color FISH to visualize WT and deleted mtDNA simultaneously in a cell population of in vitro cultured skin fibroblasts of two patients with Pearson's syndrome. At the third passage of culturing, fibroblasts showed a remarkable heterogeneity of WT and deleted mtDNA: about 90% of the cells contained almost 100% WT mtDNA, and 10% of the cells contained predominantly deleted mtDNA. At the tenth passage of culturing, fibroblasts showed a reduction of intercellular heteroplasmy from 10% to 1%, while intracellular heteroplasmy was maintained. This new approach enables detailed analysis of distribution patterns of WT and deleted mtDNA molecules at the inter- and intracellular levels in clinical samples, and may contribute to a better understanding of genotype-phenotype relationships in patients with mitochondrial diseases.
Analysis of Localization of Adult T-cell Leukemia-derived Factor in the Transient Ischemic Rat Retina After Treatment with OP-1206 α-CD, a Prostaglandin E1 Analogue
Authors: Yamamoto Miho , Ohira Akihiro , Honda Osamu , Sato Norihito , Furuke Keizo , Yodoi Junji , Honda Yoshihito
Published: April 2011

Prostaglandin E1 (PGE1) is commonly used in therapy for obstructive diseases, including ischemic retinopathy, in which pathogenetic reactive oxygen intermediates are responsible. However, the mechanism(s) of PGE1 in reducing tissue damage is still unclear. Adult T-cell leukemia-derived factor/human thioredoxin (ADF) is induced by oxidative stresses and has protective activity against oxidative cellular injury. To evaluate the possible involvement of ADF in the tissue-protective effect of PGE1, we analyzed ADF expression immunohistochemically using a rat transient retinal ischemia model. Rats were treated orally with 300 μg/kg/day OP-1206 α-cyclodextrin clathrate (OP-1206), a stable PGE1 analogue, for 14 days after photodynamic retinal vascular thrombosis by rose Bengal. Rats without any OP-1206 treatment were used as controls. In the OP-1206-treated rats, minimal retinal atrophy due to ischemia/reperfusion was observed histologically up to 14 days, whereas in the non-treated rats the inner layer of the retina became markedly atrophic. In parallel with the histological change, after 14 days following thrombosis ADF immunoreactivity was preserved on retinal pigment epithelial cells in the OP-1206-treated rats, whereas it was diminished in the non-treated rats. These findings suggest an important role for ADF in the OP-1206-dependent suppression of retinal tissue damage caused by oxidative insult.
Expression of HGF, Its Receptor c-met, c-myc, and Albumin in Cirrhotic and Neoplastic Human Liver Tissue
Authors: Ljubimova Julia Y. , Petrovic Lidija M. , Wilson Steven E. , Geller Stephen A. , Demetriou Achilles A.
Published: April 2011

Hepatocellular carcinoma (HCC) is a common type of cancer, with approximately 260,000 new cases each year, and liver cirrhosis is generally considered a major predisposing factor for HCC. However, specific changes of gene expression in liver cirrhosis and HCC remain obscure. The expression of genes for hepatocyte growth factor (HGF), its receptor c-met proto-oncogene, c-myc proto-oncogene, and albumin was analyzed. Gene expression was studied by PCR in seven normal human livers, nine cases of hepatitis C cirrhosis, 12 cases of alcoholic cirrhosis, two cases of liver adenoma, and 12 cases of HCC. HGF and c-met protein were revealed by immunofluorescent staining. HGF mRNA was not expressed in normal livers but was detected in adenomas, in 80% of HCC, and in some cirrhoses. Paraffin-embedded and fresh-frozen tissue samples yielded similar results. Immunohistochemical data correlated with PCR results regarding the overexpression of the HGF/ c-met system in HCC. Albumin gene expression was decreased in HCC vs normal livers, consistent with altered function of tumor hepatocytes. The elevated expression of the HGF/ c-met system in HCC may play a role in tumor development and/or progression. Tissue localization studies of HGF and its receptor c-met protein support the existence of both autocrine and paracrine mechanisms of action of HGF in HCC vs only a paracrine mechanism in normal liver.
Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA
Authors: Sailer Brian L. , Nastasi Anthony J. , Valdez Joseph G. , Steinkamp John A. , Crissman Harry A.
Published: April 2011

Deuterium oxide (D2O) increases both the fluorescence lifetime and the fluorescence intensity of the intercalating dyes propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis coupled with conventional and phase-sensitive flow cytometry to compare the alterations in intensity and lifetime of various DNA-binding fluorochromes bound to DNA and Chinese hamster ovary (CHO) cells in the presence of D2O vs phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a differential enhancement of intensity and lifetime based on the mode of fluorochrome-DNA interaction. The fluorescence properties of intercalating probes, such as 7-aminoactinomycin D (7-AAD) and ethidium homodimer II (EthD II) were enhanced to the greatest degree, followed by the probes TOTO and YOYO, and the non-intercalating probes Hoechst 33342 (HO) and 4',6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D2O, indicating that when staining is performed in PBS, much of the MI fluorescence is quenched by the solvent environment. Apoptotic subpopulations of HL-60 cells had a shorter lifetime compared to nonapoptotic subpopulations when stained with EthD II. These results indicate that accessibility of the dye molecules to the solvent environment, once bound to DNA, leads to the differential enhancement effects of D2O on fluorescence intensity and lifetime of these probes.
Biochemical Properties and Cytochemical Localization of Ouabain-insensitive, Potassium-dependent p-Nitrophenylphosphatase Activity in Rat Atrial Myocytes
Authors: Zinchuk Vadim S. , Kobayashi Toshihiro , Saz Eva Garcia del , Seguchi Harumichi
Published: April 2011

Enzyme activity that represents ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase (p-NPPase) was assessed in rat atrial myocytes by biochemical and cytochemical procedures. No activity was detected in parallel experiments with ventricular myocytes. Fixed tissues were incubated in a reaction medium containing Tricine buffer, p-nitrophenylphosphate (p-NPP), KCl, MgCl2, CaCl2, CeCl3, Triton X-100, levamisole, and ouabain. Final pH was adjusted to 7.5. Biochemical studies showed that accumulation of p-nitrophenol in the medium was increased proportionally in accordance with the amount of incubated tissue. This activity was optimal with incubation at pH 7.5 and in the presence of KCl. Approximately 70% of the enzyme was inhibited by 2 mM CeCl3. Electron microscopic observations revealed reaction product (RP) at sites of ouabain-insensitive, potassium-dependent p-NPPase activity as electron-dense precipitate localized at the inner surface of the plasma membrane and at the T-tubules of atrial myocytes. Control experiments indicated that the activity was strongly inhibited by sodium orthovanadate and was repressed by omeprazole and 1,3-dicyclohexylcarbodiimide. X-ray microanalysis confirmed the presence of cerium within the cytochemical RP. The ouabain-insensitive, K-dependent p-NPPase activity detected in the present study is considered to be an isoform of a P-type, H-transporting, K-dependent adenosine triphosphatase (H,K-ATPase).
Pathway and Speed of Calcium Movement from Blood to Mineralizing Enamel
Authors: Kawamoto Tadafumi , Shimizu Masaharu
Published: April 2011

We studied by autoradiography the distribution of 45Ca in the enamel organ of frozen rats 4.3, 6.1, 7.8, 10.6 and 13.7 sec after an IV injection. The intercellular junctions of the proximal side of the smooth-ended ameloblast (SA) and the distal side of the ruffle-ended ameloblast (RA) were closed to calcium. The junctions of the distal side of SA, the proximal side of RA, and both sides of the secretory stage ameloblasts were not. The time required for calcium to pass through the ameloblast layer was less than 1.8 sec in the secretory stage and SA region. The time in the RA region was 3.5–6.3 sec. In the transitional region from RA to SA, a band of strong radioactivity appeared from the papillary layer of RA region towards the enamel of the SA region. The radioactivity in the secretory stage enamel increased almost linearly with time. The diffusion speed of calcium in the enamel was more than 50 μm for 1.8 sec in the maturation stage and less than 15 μm for 9.4 sec in the secretory stage. These results indicate that in the secretory and SA regions calcium moves to the enamel surface through the intercellular spaces of ameloblasts and in the RA region via RA cells.
Intracellular Distribution of Oligonucleotides Delivered by Cationic Liposomes: Light and Electron Microscopic Study1
Authors: Lappalainen Katriina , Miettinen Riitta , Kellokoski Jari , Jääskeläinen Ilpo , Syrjänen Stina
Published: April 2011

Synthesized oligonucleotides are used in anti-sense and anti-gene technology to control gene expression. Because cells do not easily take up oligonucleotides, cationic liposomes have been employed to facilitate their transport into cells. Athough cationic liposomes have been used in this way for several years, the precise mechanisms of the delivery of oligonucleotides into cells are not known. Because no earlier reports have been published on the liposomal delivery of oligonucleotides at the ultrastructural level, we performed a study, using electron microscopy, on the cellular uptake and intracellular distribution of liposomal digoxigenin-labeled oligodeoxynucleotides (ODNs) at several concentrations (0.1, 0.2, and 1.0 μM) in CaSki cells. Two cationic lipids (10 μM) were compared for transport efficiency: polycationic 2,3-dioleoyloxy- N-[2(sperminecarboxamido)ethyl]- N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and monocationic dimethyl-dioctadecylammonium bromide (DDAB). Both liposomes contained dioleoyl-phosphatidylethanolamine (DOPE) as a helper lipid. Endocytosis was found to be the main pathway of cellular uptake of liposomal ODNs. After release from intracellular vesicles, ODNs were carried into the perinuclear area. The nuclear membrane was found to be a barrier against the penetration of ODNs delivered by liposomes into the nucleus. Release from vesicles and transport into the nuclear area was faster when the oligo-DDAB/DOPE complex had a positive net charge (0.1 and 0.2 μM ODN concentrations), and only under this condition were some ODNs found in nucleoplasm. Although DOSPA/DOPE could also efficiently deliver ODNs into the cytosol, no ODNs were found in nucleoplasm. These findings suggest that both the type of liposome and the charge of the oligo-liposome complex are important for determination of the intracellular distribution of ODNs.
Comparison of Immunologic Amplification vs Enzymatic Deposition of Fluorochrome-conjugated Tyramide as Detection Systems for FISH
Authors: Macechko P. Timothy , Krueger Lisa , Hirsch Betsy , Erlandsen Stanley L.
Published: April 2011

Using various sizes and dilutions of hapten-conjugated DNA probes, we compared catalyzed reporter deposition (CARD) to fluorochrome-conjugated antibody layering (immunological method) for amplifying FISH signals. Cosmid and phage probes that contained human DNA inserts of 40 kb and 15 kb, respectively, and were mapped to chromosome 15q11.2 were used to evaluate these amplification methods. The probes were used either at standard concentrations (10 ng/μl) or at dilutions up to 1:40 (0.25 ng/μl). Detection of FISH signals using either immunological (three antibody layers) or CARD methods were comparable when the undiluted (10 ng/μl) or 1:4 dilution (2.5 ng/μl) of the cosmid probe was used. Use of a single fluorochrome-conjugated antibody layer produced very weak FISH signals. However, addition of an unlabeled secondary antibody followed by a third antibody conjugated to the same fluorochrome (i.e., two rounds of amplification) produced a strong signal that was detected at a 1:4 probe dilution but was not successfully detected at probe dilutions of 1:10 or greater. In contrast, intense probe signals were produced with the CARD method at all probe dilutions, particularly when coupled to extended hapten-antibody incubation times. The 15-kb phage probe was difficult to detect at a 1:4 dilution with the standard immunologic amplification methods but was readily detected with the CARD method. These data suggest that CARD may be useful for FISH in that (a) less probe may be needed and therefore valuable probe reagents may be conserved, and (b) smaller targets may be detected, thus extending the range of this technique.

(J Histochem Cytochem 45:359–363, 1997)
Fluorochrome-labeled Tyramides: Use in Immunocytochemistry and Fluorescence In Situ Hybridization
Authors: Gijlswijk Rob P. M. van , Zijlmans Henry J. M. A. A. , Wiegant Joop , Bobrow Mark N. , Erickson Thomas J. , Adler Karl E. , Tanke Hans J. , Raap Anton K.
Published: April 2011

The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluoresence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods. (J Histochem Cytochem 45:375–382, 1997)
Immunocytochemical Localization of Na+,K+-ATPase in the Calcium-transporting Sternal Epithelium of the Terrestrial Isopod Porcellio scaber L. (Crustacea)
Authors: Ziegler Andreas
Published: April 2011

Terrestrial isopods store large amounts of calcium carbonate between the epithelium and the old cuticle of the first four anterior sternites before molt. During the formation of these sternal CaCO3 deposits, large amounts of calcium are transported across the anterior sternal epithelium from the base to the apical side of the integument, and in the reverse direction during resorption of the deposit. A monoclonal antibody against the avian α-subunit of Na+,K+-ATPase was used to localize Na+,K+-ATPase in the anterior and the posterior sternal epithelium of Porcellio scaber. Semithin cryosections 0.5 μm thick were used for immunofluorescence microscopy and ultrathin cryosections for immunogold electron microscopy. The Na+,K+-ATPase was localized in the basolateral plasma membrane of the posterior and anterior sternal epithelium. The apical plasma membrane, including cytoplasmic extensions into the newly secreted cuticle, was virtually devoid of the enzyme. This pattern of immunolocalization was not affected by the direction of transepithelial calcium transport associated with the deposition and resorption phases of the molt cycle.

(J Histochem Cytochem 45:437–446, 1997)
Ultrastructural Localization of Binding Sites for the Lectins RCA I, WGA, and LTA in the Preimplantation Mouse Embryo
Authors: Miosge Nicolai , Dresp Werner , Herken Rainer
Published: April 2011

In biological tissues, specific carbohydrate moieties of the oligosaccharide chains of glycoproteins can be localized by lectin binding. Such carbohydrate moieties are among the factors that mediate cell-cell or cell-matrix interactions during pre- and postimplantation embryonic development. Binding sites for the lectins RCA I, WGA, and LTA were localized in preimplantation mouse embryos at the ultrastructural level with the help of postembedding lectin gold cytochemistry. WGA and RCA I binding sites, but no LTA binding sites, were present in the zona pellucida. WGA and RCA I binding sites were found at cell surfaces of morulae and in cells of the inner cell mass of blastocysts, suggesting that N-acetylglucosamine-, terminal β-galactosyl-, and N-acetylgalactosamine-rich glycoproteins might be involved in cell-cell and cell-matrix interactions. WGA binding sites were found predominantly in electron lucid vesicles of the blastomeres, whereas RCA I was detected in electron dense vesicles of the compacted morula and later in the polar trophoblast cells. This allows early identification of blastomere cells that later differentiate into the polar trophoblast. (J Histochem Cytochem 45:447–453, 1997)
A Procedure for In Situ Hybridization Combined with Retrograde Labeling of Neurons: Application to the Study of Cell Adhesion Molecule Expression in DiI-labeled Rat Pyramidal Neurons
Authors: Fujimori Kazuhiro E. , Takauji Rumiko , Yoshihara Yoshihiro , Tamada Atsushi , Mori Kensaku , Tamamaki Nobuaki
Published: April 2011

We have devised a simple method that combines retrograde labeling of projecting neurons and in situ hybridization histochemistry to examine mRNA expression in the retrogradely labeled neurons. First, projecting neurons were retrogradely labeled in vivo by injection of the lipophilic neuronal tracer DiI. The fluorescence of the labeled neurons in the brain slices was photoconverted into stable DAB precipitate by green light illumination. The slices were cut into thinner sections and processed for detection of specific mRNA by in situ hybridization. Using this highly sensitive method, we demonstrate here that the corticospinal tract neurons in newborn rats express mRNA for the cell adhesion molecule L1. TAG-1 mRNA was not detected in these neurons. Therefore, the present method provides an important tool to study the molecular expression of projection neurons during the development of neuronal circuitry. (J Histochem Cytochem 45:455–459, 1997)
Widefield Microscopy Images of Tissue Sections by Computer Imaging Techniques
Authors: Ikeda Isamu , Urushihara Kazunobu , Ono Tomomichi
Published: April 2011

A fine photomicrograph covering one whole specimen is very useful in the study of skin histopathology. However, because it is almost impossible to take such a picture with a conventional photomicroscope, we attempted to make one with the aid of a computer. The large field was divided into small fields, which were individually recorded through a photomicroscope. The images were then digitized and processed with a computer to reconstruct the largefield image. A fine seamless image was reconstructed with this method. We can thus extend the field of a photomicroscope with the aid of computer imaging techniques, without impairing the quality. (J Histochem Cytochem 45:461–466, 1997)
Biotin and Digoxigenin as Labels for Light and Electron Microscopy in Situ Hybridization Probes: Where Do We Stand?
Authors: Chevalier Jacques , Yi Jing , Michel Odile , Tang Xue-Ming
Published: April 2011

Biotin was recently applied to detect cellular DNA or RNA. In combination with avidin, streptavidin or antibody, it can be conjugated with fluorescent dye, enzyme, ferritin, or gold. However, emphasis has recently been placed on the false-positive results that are obtained when this probe is used, because endogenous biotin may sometimes interfere with specific signals. Digoxigenin appears to be an interesting alternative because it is present exclusively in Digitalis plants as a secondary metabolite. We discuss in this review the efficiency and the respective advantages and disavantages of these two probes for in situ hybridization, mainly at the electron microscopic level.
Sequential Expression of Bone Matrix Proteins During Rat Calvaria Osteoblast Differentiation and Bone Nodule Formation In Vitro
Authors: Nefussi Jean R. , Brami Gabriel , Modrowski Dominique , Obcuf Martine , Forest Nadine
Published: April 2011

We investigated the expression of osteocalcin (OC), bone sialoprotein (BSP), osteonectin (ON), and alkaline phosphatase (ALP) during cell differentiation and bone nodule formation by fetal rat calvaria cells, using immunofluorescent and immunogold techniques at light and electron microscopic levels. Six hours after plating all proteins were expressed in calvaria cells. However, expression was not detected during the proliferation phase after plating. Cell morphological modifications were observed in osteoblastic cells expressing ALP, OC, and BSP, but not ON. During the matrix formation phase, all proteins were expressed with various intensities and OC was limited to differentiated osteoblastic cells. EM observations demonstrated that BSP was selectively associated with clusters of needle-like crystals, but not with collagen fibers, in mineralization foci and in the mineralized matrix. OC was localized intracellularly and in all the extracellular compartments, and was concentrated at the mineralization front. ON was distributed uniformly throughout the osteoid and mineralized matrix, which was intensely labeled. The results show that the expression of bone matrix proteins during differentiation of calvaria cells and nodule formation in vitro duplicate what is observed during osteogenesis in vivo.
Immunohistochemical Localization of Lipocortin 1 in Rat Brain Is Sensitive to pH, Freezing, and Dehydration
Authors: McKanna James A. , Zhang Ming-Zhi
Published: April 2011

Lipocortin 1 (LC1, annexin 1) has received considerable attention as a substrate for protein kinases, as a Ca++- and phosphatidylserine-binding protein, and as a mediator of glucocorticoid anti-inflammatory effects. However, there has been confusion over localization of LC1 immunoreactivity (LC1-ir), which reportedly localizes to neurons and/or to astrocytes or microglia in rat brain. To test whether these contradictory data arise from unusual properties of the antigen, we developed a novel brain slice model to determine fixation and staining variables. The specificity of anti-LC1 sera was ensured by pre-absorption and affinity purification with immobilized recombinant LC1. Specific LC1-ir was detected in ramified microglia of brains perfused with acidified aldehydes and embedded in paraffin. However, commonly used immunohistochemical procedures have unexpected profound effects. LC1-ir was eliminated by fixation with neutral/alkaline aldehydes, by freezing before strong acid-aldehyde fixation, or by staining without partial de/rehydration before the primary serum. The sensitivity of LC1 epitopes to proton and water activities may reflect molecular properties important to LC1's roles in vivo. True LC1-ir was not detected in normal neurons or astrocytes.
Phosphatidylinositol Transfer Protein in Lung: Cellular and Subcellular Localization
Authors: Ibrahim Abdul-Manaf A. , Funkhouser Jane D.
Published: April 2011

Determination of the cellular distribution of phosphatidylinositol transfer protein in rat lung by immunocytochemistry revealed that the protein is more readily observed in the nonciliated bronchial epithelial cells (Clara cells) than in other lung cells. By light microscopy, the phosphatidylinositol transfer protein (PtdIns-TP) was localized to the dome-shaped apical region of Clara cells that were identified by staining with an antibody to Clara cell protein. Further investigation by electron microscopy revealed that the PtdIns-TP accumulated at the limiting membrane surrounding secretory granules and at the apical plasma membrane. This localization is compatible with the proposed roles for PtdIns-TP in formation of vesicles and exocytosis of secretory granules and, when considered in the context of the proposed role of PtdIns-TP in phosphatidylinositide metabolism, suggests that phosphatidylinositides may be involved in the mechanisms regulating Clara cell secretion.
The Distribution of Laminins and Fibronectins Is Modulated During Extravillous Trophoblastic Cell Differentiation and Decidual Cell Response to Invasion in the Human Placenta
Authors: Korhonen Matti , Virtanen Ismo
Published: April 2011

We studied the distribution of laminin (Ln) α1–α3, β1–β3, and γ1 chains, and of the extradomain-A (EDA) and EDB and the oncofetal epitope of fibronectin (Onc-Fn) in extravillous trophoblastic cells and decidua in the human placenta by immunohistochemistry. We found that the transition from villous to extravillous trophoblast was accompanied by emergence of immunoreactivity for EDA-, EDB-, and Onc-Fn among the cells. Furthermore, whereas the villous trophoblastic basement membrane (BM) contains Ln α1, α2, β1, β2, and γ1 chains, immunoreactivity for Ln α1, β1, and γ1, but not for Ln α2 and β2 chains, was detected in association with extravillous trophoblastic cells. Interestingly, although immunoreactivity for the Ln α1, α2, β1, β2, and γ1 chains was detected in all decidual cell BMs, EDB-Fn and Onc-Fn were detected only in decidua that had been invaded by the trophoblast. In summary, our results describe distinct changes in the distribution of Ln and Fn isoforms during the differentiation of villous trophoblast into extravillous trophoblastic cells. Furthermore, EDB- and Onc-Fn are preferentially found in decidua that has been invaded by the trophoblast, indicating that the deposition of these Fn isoforms reflects a decidual cell response to invasion.
Distribution of Casein-like Proteins in Various Organs of Rat
Authors: Onoda Makoto , Inano Hiroshi
Published: April 2011

Casein-like proteins were detected in various organs of rat by use of a specific antiserum raised against rat milk caseins. The antiserum specifically recognized α1-, α2-, β-, and γ-caseins in rat milk by Western blot analysis, whereas no immunoreactive band was observed in sera of rat and fetal bovine and in bovine caseins. Immunohistochemical studies of this antiserum on formalin-fixed mammary glands showed that immunoreactive caseins were localized to the apical portion of the cytoplasm in lactating mammary epithelial cells and in the luminal secretion, which indicates a directional secretion of caseins to the lumen by the mammary epithelial cells. With this antiserum, immunoreactive substances were detected in various organs, including the pancreatic ducts and islets of Langerhans, the secretory ducts of salivary glands, zona fasciculata cells and ganglion cells of adrenal gland, distal tubules and convoluted collecting tubules of kidney, epithelial cells of bronchioles and large pneumocytes of the lung, hair follicles, sebaceous glands, and the prickle cell layer of skin, uterine glands and epithelium of the endometrium, hepatic bile ducts, and brain. In Western blot analysis, major immunoreactive substances in the above organ extracts showed a similarity in molecular weight to α2-casein of rat milk. Skin was the only tissue that expressed both α2- and β-caseins. There were no other immunoreactive bands with similarity to β- and γ-caseins in the other organ extracts, but higher molecular weight immunoreactive bands (> 100 kD) were detected in some organ extracts, such as salivary gland, kidney, liver, lung, and uterus. These findings suggest that the α2-casein-like substance is localized not only in the mammary gland but also in a variety of organs and may play an important role as a functional molecule in those organs.
Differential Staining of Apoptotic Nuclei in Living Cells: Application to Macronuclear Elimination in Tetrahymena
Authors: Mpoke Solomon S. , Wolfe Jason
Published: April 2011

Acridine orange (AO) has been used as a vital fluorescent stain to identify apoptotic cells in Drosophila, but little is known about what structures are stained. We explored the specificity of AO staining while studying nuclear apoptosis in Tetrahymena. Using AO alone or together with the vital nuclear stain Hoechst 33342 (HO), we find that lysosomes are generally clustered around the degenerating nucleus and that such nuclei are stained an orange-red color, like lysosomes. Significantly, the combined dyes, more so than with AO alone, distinguish between apoptotic and normal (or necrotic) nuclei by a clear color difference. Moreover, these dyes differentially stain apoptotic and normal nuclei in avian chondrocytes. The differential staining results are nullified in fixed cells or in cytoskeletal preparations treated with RNAse. Similarly, lysosomotrophic agents eliminate the differential staining. Our results are consistent with acidification of the apoptotic nucleus, possibly by fusion with lysosomes. However, even under basic conditions, the macronucleus condenses and is eliminated, suggesting that, if the nucleus is becoming acidified, acidification by itself is not essential for nuclear elimination. The differential staining procedure may provide a useful method for specifically identifying apoptotic cells and separating them for further analysis. (J Histochem Cytochem 45:675–683, 1997)
Cellular Distribution of Branched-chain Amino Acid Aminotransferase Isoenzymes Among Rat Brain Glial Cells in Culture
Authors: Bixel M. Gabriele , Hutson Susan M. , Hamprecht Bernd
Published: April 2011

The first step in the catabolism of branched-chain amino acids (BCAA), reversible transamination, is catalyzed by one of the two isoforms of branched-chain amino acid aminotransferase (BCAT). The mitochondrial isoenzyme (BCATm) is widely distributed among tissues, whereas the cytosolic isoenzyme (BCATc) is restricted to only a few organs. Remarkably, BCATc is the prominent isoenzyme found in brain. The physiological significance of the subcellular compartmentation of BCAT is still not understood. To contribute to the elucidation of the cellular distribution of the two isoenzymes in brain, we used cultured rat glial cells in an immunocytochemical study to determine the pattern of BCAT isoenzyme expression by glial cells. Antiserum against BCATm generated a punctate staining pattern of astroglial cells, confirming the mitochondrial location of this isoenzyme. In contrast, the cytosol of galactocerebroside-expressing oligodendroglial cells and O2A progenitor cells displayed intense staining only for BCATc. In addition, subpopulations of astroglial cells exhibited BCATc immunoreactivity. The presence of BCATm in astrocytes is consistent with the known ability of these cells to oxidize BCAA. Furthermore, our results on BCATc provide support for the hypothesis that BCATs are also involved in nitrogen transfer from astrocytes to neurons.
Improved In Situ Hybridization to HIV with RNA Probes Derived from PCR Products
Authors: Cone Richard W. , Schlaepfer Erika
Published: April 2011

These experiments tested the hypothesis that a pool of PCR-derived RNA probes with defined length and even representation of the target sequences could produce more specific and intense in situ hybridization signals than randomly size-reduced, plasmid-derived RNA probes. In situ hybridization was performed with sense and anti-sense HIV-1 RNA probes that were derived from PCR products tailed with the T7 RNA polymerase promoter or from plasmid DNA. In situ hybridization using a pool of seven anti-sense or sense PCR-derived RNA probes (1805 nucleotides of HIV sequence, 257 nucleotides average probe length) was compared with hybridization using anti-sense or sense RNA probes made from a plasmid representing the HIV-1 env gene (3151 nucleotides of HIV-1 target). The pooled PCR-derived probes resulted in stronger in situ hybridization signals and less background than those produced with plasmid-derived RNA probes. This method for creating PCR-derived RNA probes improves the feasibility of synthesizing multiple, discrete RNA probes for studies of specific mRNA expression because it does not require the subcloning steps used to construct plasmids. PCR-derived RNA probes may provide a viable alternative to the use of plasmid-derived RNA probes for in situ hybridization.
Use of Enzyme Overlay Membranes to Survey Proteinase Activity in Frozen Sections: Cathepsin-like and Plasmin-like Activity in Regenerating Newt Limbs
Authors: Day Frances A. , Neufeld Daniel A.
Published: April 2011

We present a method that permits extremely simple and rapid screening of proteolytic enzyme activity in sectioned tissues. Enzyme overlay membranes (EOMs) are custom-made membranes designed to fluoresce at sites of specific proteolytic enzyme activity after separation of proteins by gel electrophoresis. EOMs, selected to detect either plasmin-like or cathepsin B-like activity, have been used in a novel way to document the distribution of enzyme activity in frozen sectioned tissues. When moistened membranes were placed in contact with sectioned regenerating newt limbs, a fluorescent pattern of enzyme activity was generated. In limbs at 3 hr post amputation, cathepsin B-like activity was prominent across the amputation site but plasmin-like activity was distributed in dermal and deeper proximal tissues, suggesting different roles for these two classes of enzymes. EOM enzymology in situ (EEI) on frozen sectioned tissues may be a widely useful technique to display distribution and level of activity of proteolytic enzymes in various systems.
Differential Expression of c-fos In Vitro by All Anterior Pituitary Cell Types During the Estrous Cycle: Enhanced Expression by Luteinizing Hormone but Not by Follicle-stimulating Hormone Cells
Authors: Armstrong Jennifer , Childs Gwen V.
Published: April 2011

C-fos expression appears in some activated cell types. Because of dynamic changes in gonadotropes during the estrous cycle, this study was initiated to determine if fos might be expressed in gonadotropes before any period of activation. We detected c-fos and pituitary antigens in dissociated anterior pituitary cells by dual-labeling immunocy-tochemistry. The highest percentages of cells with fos protein were found in proestrous rat populations. In diestrous and proestrous populations, dual labeling showed that 6–9% of pituitary cells contained fos with adrenocorticotropin, thyroid-stimulating hormone, prolactin, or growth hormone antigens. In contrast, only 0.8–3% contained fos with luteinizing hormone (LH) or follicle-stimulating hormone (FSH) antigens. We then tested the hypothesis that gonadotropes might increase fos expression earlier in the cycle. In populations from metestrous rats, c-fos labeling was found in 45% of LH cells compared to only 23% of LH cells in the proestrous group. This suggests that proportionately more LH cells are being activated to produce fos early in the cycle. Perhaps fos is used in translation of LHβ antigens or gonadotropin-releasing hormone (GnRH) receptor mRNAs. In contrast, less than 1% of all pituitary cells expressed fos with FSH at all stages of the cycle (only 6–12% of FSH cells). This differential expression suggests one mechanism behind the regulation of non-parallel storage and release of gonadotropin antigens.
Prohormone Convertases in Mouse Submandibular Gland: Co-localization of Furin and Nerve Growth Factor
Authors: Farhadi Hooman , Pareek Sangeeta , Day Robert , Dong Weijia , Chrétien Michel , Bergeron John J. M. , Seidah Nabil G. , Murphy Richard A.
Published: April 2011

Nerve growth factor (NGF) in mouse submandibular glands (SGs) is generated from a 35-kD precursor by proteolytic enzymes that have yet to be identified. Prohormone convertases (PCs) cleave the NGF precursor in vitro, and in this study we questioned whether PCs could process salivary NGF in vivo. mRNA coding for PC2 (but not PC1) was detected on Northern blots of SG mRNA and also by in situ hybridization within parasympathetic neurons of intralobular ganglia. Northern blot and in situ hybridization analyses also detect mRNA coding for furin. In SGs of male mice, furin mRNA levels are high at birth and remain high throughout development. In glands from female mice, levels decline during postnatal development and are lower in adults than in newborns. Immunocytochemistry detects furin immunoreactivity in pro-acinar and ductal cells of glands from newborn and pubescent mice. In glands of adults, furin immunoreactivity is detectable in acinar cells but highest levels are present in NGF-containing granular convoluted tubule cells. These data, taken together with those from previous studies, suggest that furin is a candidate processing enzyme for NGF in mouse submandibular glands.
Modulation of Protein Kinases and Microtubule-associated Proteins and Changes in Ultrastructure in Female Rat Pituitary Cells: Effects of Estrogen and Bromocriptine
Authors: Matsuno Akira , Takekoshi Susumu , Sanno Naoko , Utsunomiya Hirotoshi , Ohsugi Yoshitaka , Saito Nobuhito , Kanemitsu Hideaki , Tamura Akira , Nagashima Tadashi , Osamura R. Yoshiyuki , Watanabe Keiichi
Published: April 2011

This study focused on the intracellular signal transduction system and microtubule-associated proteins (MAPs), such as MAP-2 and Tau protein. The modulation of these proteins and their correlation with ultrastructural changes were investigated in rat pituitary prolactin (PRL) cells. Adult female Wistar rats were treated with estrogen and bromocriptine and their pituitary glands were removed for analysis of the expression of tubulin, MAP-2, Tau protein, protein kinase C (PKC), and calcium calmodulin (CaM) kinase. Western blot analysis showed that estrogen increased and bromocriptine decreased the expression of PKCα, β1, β2, CaM kinase α, β, MAP-2, and Tau protein. MAP-2 and Tau protein, which are cytosolic proteins, being translated on free ribosomes, were associated with the membrane of whirling rough endoplasmic reticulum (RER) in estrogen-treated cells and dissociated with vesiculated RER induced by bromocriptine. These results suggested that the modulation of MAP-2 and Tau protein may reflect changes of PKC and CaM kinase, and that the quantitative changes and intracellular modulation of MAPs induced by estrogen and bromocriptine, i.e., estrogen-induced association and bromocriptine-induced dissociation of MAP-2 and Tau protein with membrane of RER, may reflect the dynamics of microtubules and are associated with structural changes in the RER and changes in the synthesis and intracellular transport of PRL.
The Epidermal Stem Cell Compartment: Variation in Expression Levels of E–Cadherin and Catenins Within the Basal Layer of Human Epidermis
Authors: Molès Jean-Pierre , Watt Fiona M.
Published: April 2011

The basal layer of the epidermis contains two types of proliferating keratinocyte: stem cells, with high proliferative potential, and transit amplifying cells, which are destined to undergo terminal differentiation after a few rounds of division. It has been shown previously that two- to three-fold differences in the average staining intensity of fluorescein-conjugated antibodies to β1 integrin subunits reflect profound differences in the proliferative potential of keratinocytes, with integrin-bright populations being enriched for stem cells. In the search for additional stem cell markers, we have stained sections of normal human epidermis with antibodies to proteins involved in intercellular adhesion and quantitated the fluorescence of individual cell-cell borders. In the basal layer, patches of brightly labeled cells were detected with antibodies to E-cadherin, β-catenin, and γ-catenin, but not with antibodies to P-cadherin, α-catenin, or with pan-desmocollin and pan-desmoglein antibodies. In the body sites examined, palm and foreskin, integrinbright regions were strongly labeled for γ-catenin and weakly labeled for E-cadherin and β-catenin. Our data suggest that there are gradients of both cell-cell and cell-extracellular matrix adhesiveness within the epidermal basal layer and that the levels of E-cadherin and of β-and γ-catenin may provide markers for the stem cell compartment, stem cells expressing relatively higher levels of γ-catenin and lower levels of E-cadherin and β-catenin than other basal keratinocytes.