Avidin-biotinylated Horseradish Peroxidase Complex Research Articles

91 Expression Pattern of Neuron-Specific Red Fluorescence Protein in Nervous System of Transgenic Dog with Human Synapsin I Promoter

Neurodegenerative disorders, such as Alzheimer’s disease, affect neurons in large areas within the central nervous system. The selective expression of disease-causing and therapeutic genes in susceptible regions and cell types is critical to the generation of animal models. In our previous studies, we reported production of a transgenic dog by nuclear transfer using donor cells that had been stably transfected by vector containing the red fluorescence protein (RFP) and human synapsin I (SynI) promoter, a neuron-specific promoter. Here, we evaluated whether RFP expression in transgenic dog with SynI (SynI dog) had high neuronal specificity and strong transcriptional activity. For confirmation of neuron-specific RFP expression, tissue samples were obtained from a killed 4-year-old SynI dog. Quantification of RFP in heart, kidney, liver, lung, spleen, cerebrum, cerebrum, cerebellum, midbrain, hippocampus, peripheral nerves, skin, and spinal cord of SynI dog was analysed using ELISA (Cell Biolabs, San Diego, CA, USA). Moreover, the distribution of RFP activity in neural system of the SynI dog was determined by immunohistochemistry. The first antibody, rabbit polyclonal RFP antibody (1:200, ab62341, Abcam, Cambridge, MA, USA) was applied, and antibody labelling was visualised by incubation with avidin-biotinylated horseradish peroxidase complex (1:1,000; ABC Elite, Vector Laboratories, Burlingame, CA, USA). In the 4-year-old SynI dog, RFP was observed only in neuronal tissues including brain, spinal cord, and peripheral nerves, but was not detected in non-neuronal tissue such as heart, kidney, and skin. In addition, robust RFP expression was observed in the neurons of the peripheral nerve, spinal cord, and prefrontal cortex. In the hippocampus and cerebellum, the RFP-expressing cells appeared heterogeneous in hippocampus neurons and purkinje cells. In conclusion, we report that human SynI promoter is functional in neural cells of dogs. A neural specific-transgene expressed dog could be applied as a research tool in the study of neurodegenerative disorders. This research was supported by RDA (#PJ010928032017), Korea IPET (#316002-05-2-SB010), NRF (#2016R1D1A1B03932198), and Research Institute for Veterinary Science, the BK21 plus program.

Deficiency of Growth Factor Midkine Exacerbates Necrotizing Glomerular Injuries in Progressive Glomerulonephritis

Inflammatory cell infiltration and fibrin deposition play important roles in the development of crescentic glomerulonephritis (GN). In particular, activation of coagulation is an indispensable factor in crescent formation. However, the mechanisms underlying the pathogenesis of crescent formation have not been completely elucidated. We identified the growth factor midkine (MK) as a novel key molecule in the progression of crescentic GN induced by anti-glomerular basement membrane antibody. Despite the lack of significant differences in autologous and heterologous reactions, MK-deficient (Mdk(-/-)) mice unexpectedly showed a greater number of necrotizing glomerular injuries than wild-type (Mdk(+/+)) mice. Likewise, more tubulointerstitial damage was observed in Mdk(-/-) mice, and this damage positively correlated with glomerular injury. Plasminogen activator inhibitor (PAI)-1 was strongly induced in the injured glomerulus of Mdk(-/-) mice, particularly in crescents and endothelial cells. This enhanced PAI-1 production was associated with an increase in inflammatory cell infiltration and matrix deposition in the glomerulus and the interstitium of Mdk(-/-) mice. In line with these invivo data, primary cultured endothelial cells derived from Mdk(-/-) mice exhibited higher PAI-1 mRNA expression on fibrin challenge and less fibrinolysis than Mdk(+/+) mice. In contrast, the expression of plasminogen activators was not affected. Our combined data suggest that MK leads to a blockade of PAI-1, which is closely associated with the suppression of crescentic GN.

Neurogliaform cells of amygdala: a source of slow phasic inhibition in the basolateral complex

Synaptic inhibition in the amygdala actively participates in processing emotional information. To improve the understanding of interneurons in amygdala networks it is necessary to characterize the GABAergic cell types, their connectivity and physiological roles. We used a mouse line expressing a green fluorescent protein (GFP) under the neuropeptide Y (NPY) promoter. Paired recordings between presynaptic NPY-GFP-expressing (+) cells and postsynaptic principal neurons (PNs) of the basolateral amygdala (BLA) were performed. The NPY-GFP+ neurons displayed small somata and short dendrites embedded in a cloud of highly arborized axon, suggesting a neurogliaform cell (NGFC) type. We discovered that a NPY-GFP+ cell evoked a GABA(A) receptor-mediated slow inhibitory postsynaptic current (IPSC) in a PN and an autaptic IPSC. The slow kinetics of these IPSCs was likely caused by the low concentration and spillover of extracellular GABA. We also report that NGFCs of the BLA fired action potentials phase-locked to hippocampal theta oscillations in anaesthetized rats. When this firing was re-played in NPY+-NGFCs in vitro, it evoked a transient depression of the IPSCs. Presynaptic GABA(B) receptors and functional depletion of synaptic vesicles determined this short-term plasticity. Synaptic contacts made by recorded NGFCs showed close appositions, and rarely identifiable classical synaptic structures. Thus, we report here a novel interneuron type of the amygdala that generates volume transmission of GABA. The peculiar functional mode of NGFCs makes them unique amongst all GABAergic cell types of the amygdala identified so far.

Acidic Fibroblast Growth Factor (FGF) Potentiates Glial-mediated Neurotoxicity by Activating FGFR2 IIIb Protein

Previous studies indicate that astrocytes are the brain cells that express acidic fibroblast growth factor (aFGF) and that the expression is increased upon activation. However, there has been no study investigating the significance of this phenomenon. Here we report that aFGF treatment of IFNγ-stimulated human astrocytes, and LPS/IFNγ-stimulated human microglia, enhances their secretion of inflammatory cytokines and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of aFGF enhancement involves stimulation of the receptor FGFR2 IIIb. We show by RT-PCR that this receptor, but not other FGF receptors, is robustly expressed by astrocytes and microglia. We establish by Western blotting, and immunohistochemistry on postmortem human brain tissue that the FGFR2 IIIb protein is expressed by both of these glial cell types. We blocked the inflammatory stimulant action of aFGF by transfecting microglia and astrocytes with a small inhibitory RNA (siRNA) to FGFR2 IIIb as well as by removal of aFGF using an anti-aFGF antibody. Treatment with bFGF in combination with the stimulants was without effect, but together with aFGF, it partially counteracted the action of aFGF, indicating that it may be a weak antagonist of FGFR2 IIIb. The inflammatory effect was also attenuated by treatment with inhibitors of protein kinase C, Src tyrosine kinase, and MEK-1/2 indicating the involvement of these intracellular pathways. Our data suggest that inhibition of expression or release of aFGF could have therapeutic potential by inhibiting inflammation in neurodegenerative diseases such as Alzheimer disease where many neuroinflammatory molecules are prominently expressed.

Is the positive c-kit immunostaining associated with the presence of cells analogous to the intersticial cells of Cajal in the ciliary muscle?

Interstitial cells of Cajal were identified in the gastrointestinal tract of several species, with close relation to the enteric nervous system. Since it was recognized that interstitial cells of Cajal express the gene product of c-kit, we performed immunohistochemistry for c-kit protein in ciliary muscle specimens of monkeys' eyes. Eight eyes from four adult male new world monkeys (Cebus apella) were studied. After blocking endogenous peroxidase activity and nonspecific protein binding, 1:100 dilution of mouse monoclonal antibody against c-kit human oncoprotein was applied to tissues. Antigen-antibody reaction was visualized using the avidin-biotinylated horseradish peroxidase complex in each slide. We observed some groups of fusiform c-kit expressing cells located amongst muscle bundles of the ciliary muscle. Other pigment cells and mast cells were also observed. C-kit expressing cells observed in the ciliary muscle of Cebus apella, showed no similarity to melanocytes or mast cells and they could be associated with their gastrointestinal interstitial cells of Cajal counterpart.

Immunohistochemical Analysis for the Expression of DR5 TRAIL Receptor and p53 in Non-small Cell Lung Cancer

Background: TRAIL is a promising anticancer agent which induces selective tumor cell death due to a unique receptor system that includes death receptors and decoy receptors. DR5 TRAIL receptor is an originally identified p53-regulated death receptor gene that was induced, by doxorubicine, only in cells with a wild-type p53 status. We investigated that focused on the correlation between the DR5 and p53 expressions in non-small cell lung cancer (NSCLC). Methods: Immunohistochemical analysis, with using avidin-biotinylated horseradish peroxidase complex, was carried out in 89 surgically resected NSCLC formalin-fixed paraffin-embedded tissue sections. As primary antibodies, we used anti-DR5 polyclonal antibody and anti-p53 monoclonal antibody. A negative control was processed with each slide. The positive tumor cells were quantified twice and these values were expressed as percentage of the total number of tumor cells, and the intensity of immunostaining was expressed. The analysis of the DR5 expression was done separately in tumor area and in a nearby region of normal tissue. Results: The DR5 expression was high in the bronchial epithelium (89% of cases) but this was almost absent in type I & II pneumocytes, lymphocytes and smooth muscle cells. High DR5 expression rate in tumor was seen in 28% (15/53) of squamous cell carcinomas, in 47% (15/32) of adenocarcinomas and, in 50% (2/4) of large cell carcinomas. The DR5 expression did not show any statistical significance relationship with the T stage, N stage, or survival. However, the DR5 expression showed significant inverse correlation with the p53 expression. (p

ERK8 Down-regulates Transactivation of the Glucocorticoid Receptor through Hic-5

Extracellular signal-regulated kinase 8 (ERK8) is the most recently identified member of the ERK subfamily of MAPKs. Although other members of the ERK subfamily are established regulators of signaling pathways involved in cell growth and/or differentiation, less is known about ERK8. To understand the cellular function of ERK8, a yeast two-hybrid screen of a human lung library was performed to identify binding partners. One binding partner identified was Hic-5 (also known as ARA55), a multiple LIM domain containing protein implicated in focal adhesion signaling and the regulation of specific nuclear receptors, including the androgen receptor and the glucocorticoid receptor (GR). Co-immunoprecipitation experiments in mammalian cells confirmed the interaction between Hic-5 and both ERK8 and its rodent ortholog ERK7. The C-terminal region of ERK8 was not required for the interaction. Although the LIM3 and LIM4 domains of Hic-5 were sufficient and required for this interaction, the specific zinc finger motifs in these domains were not. Transcriptional activation reporter assays revealed that ERK8 can negatively regulate transcriptional co-activation of androgen receptor and GRalpha by Hic-5 in a kinase-independent manner. Knockdown of endogenous ERK8 in human airway epithelial cells enhanced dexamethasone-stimulated transcriptional activity of endogenous GR. Transcriptional regulation of GRalpha and interaction with its ligand binding domain by ERK8 were dependent on the presence of Hic-5. These results provide the first physiological function for human ERK8 as a negative regulator of human GRalpha, acting through Hic-5, and suggest a broader role for ERK8 in the regulation of nuclear receptors beyond estrogen receptor alpha.

Immunohistochemical Analysis for the Expression of p27, CDK4, and Uteroglobin in Resected Non-small Cell Lung Cancer

Background : It has been reported that the expression of protein which influences on the cell cycle is significantly involved in the development, progress, treatment response, and survival of cancer, and also that the degree of expression of p27 and CDK4 is related to the prognosis. Recent research has revealed that uteroglobin, tumor suppressor gene, is related to cell cycle. This study is focused on the relations between expression of proteins related to cell cycle and clinical index of and survival of NSCLC. Methods : We examined immunohistochemically specimens of 110 surgically resected NSCLCs for expression of p27, CDK, Uteroglobin. Tissue array slide were obtained from 110 surgically resected NSCLCs. Immunohistochemical staining was performed by immuno-peroxidase technique using avidin-biotinylated horseradish peroxidase complex. Results : In 110 patients with resected NSCLCs, the ratio of male to female was 87:13, the median age was yrs. The positive staining of p27 was detected in 75% of the cases. A non-statistically significant trend toward increased p27 expression was observed in smoker and squamous cell cancer. The positive staining of CDK4 was detected in 89%, which was the highest expression of protein among 3 types. The survival ratio of CDK4 negative staining group was higher than that of positive staining group, which was significant diffrernce(P

Distribution and subcellular localization of a growth inhibitory factor in hamster liver and its intracellular partner(s).

The subcellular, intralobular distributions and intracellular partner(s) of a factor which inhibits the proliferation of cell growth (Hashimoto C. et al. (1994) Biochim. Biophys. Acta 1221, 107-117) were determined in hamster livers, using a combination of immunological and biochemical techniques. The IgG fraction from an antiserum raised against the growth inhibitory factor with 37 kDa was shown to be highly specific for the antigen. The nuclear and cytosolic fractions demonstrated inhibitory effects on cell growth and Western blot analysis revealed that both fractions contained the immunoreactive 37 kDa protein with the anti-inhibitory factor IgG but microsomal and mitochondrial fractions did not. The nuclear and cytoplasmic localization of the inhibitory factor were further confirmed by immunochemical staining mediated through the immune IgG and an avidin-biotinylated horseradish peroxidase complex, the parenchymal liver cells were clearly stained, but endothelial and connective tissue cells were not. Although some staining was evident throughout the liver parenchyma, the hepatocytes with most intensively stained nuclei were located in the periportal region. In the liver from hamsters 6 days old or the regenerating hamster livers 3 days after partial hepatectomy, the staining intensity was low and the number of hepatocytes with the inhibitory factor positive nuclei was very few compared with the adult hamster livers. In primary cultures of the isolated hepatocytes from adult hamster the inhibitory factor disappeared from nuclei after incubation for 24-48 h. The extracts of hepatic nuclei from adult hamsters were immunoprecipitated with either the anti-growth inhibitory factor IgG or a monoclonal antibody to the RM protein. The growth inhibitory factor and the RB protein coprecipitated in each case, implying that the proteins were complexed with each other in the nuclei. The RB protein family is composed of two sets of species, an un- or underphosphorylated species and a hyperphosphorylated one. It was suggested that the factor bound preferentially to the un- or underphosphorylated member of the family.

An avidin-biotin-peroxidase assay to detect synthetic peptides bound to polystyrene plates

The simple chemical method described for detecting synthetic peptides bound to polystyrene should facilitate interpretation of studies involving interactions of antibodies or solubilized major histocompatibility complex (MHC) molecules with immobilized peptide antigens. After its application to an ELISA plate, the peptide is biotinylated in situ and avidin-biotinylated horseradish peroxidase complex and substrate are added sequentially. For 36 of 43 peptides tested (11–27 residues long), a colored reaction product confirmed that the peptide was bound. In three of the seven instances of a negative result, peptides were positively detected by binding of antibody. Four instances remained in which it could not be determined whether the peptide did not bind to the plate or whether it was not biotinylated. On the other hand, the biotin-ABC assay positively detected peptide in 18 of 22 instances without evidence of antibody binding, implying seronegativity or a loss of antigenic conformation in the bound peptide. This general method should be applicable to assays of microbial antigens, autoantigens and allergens. Modification of the technique by use of biotin hydrazide should enable monitoring of the binding to polystyrene of carbohydrates, glycolipids or DNA.

Distribution of rabbit mucosal glycoprotein throughout urinary tract

The mucin layer covering the transitional epithelium of the bladder is thought to be an anti-adherence substance for bacteria. We describe the use of an immunoperoxidase staining technique to demonstrate the presence of glyeoprotein lining the urothelium of both the upper and lower urinary tracts of the rabbit Antisera against this glyeoprotein (GPI) were raised in Swiss-Webster mice. The genitourinary tracts of male and female NZW rabbits were removed and sequentially treated with mouse anti-GPI sera, biotin-labelled anti-mouse IgG, and an avidinbiotinylated horseradish peroxidase complex. The results demonstrated that an antigenically similar (or identical) glyeoprotein covers the distal renal tubules and urothelium of the renal pelvis, ureters, bladder, and urethra, suggesting that it may function as an antibacterial defense mechanism throughout the urinary tract.

Primary extranodal non‐Hodgkin's lymphoma of the oral region

Seventy cases (47M, 23F) of primary extranodal non-Hodgkin's lymphoma of the oral region were studied to determine tumor characteristics. The most frequent disease sites were the palate (21 cases), gingiva (17 cases) and parotid gland (13 cases). Each lymphoma was classified according to the criteria of the Working Formulation for Clinical Usage. Only 5.7% of cases were follicular lymphomas while diffuse lymphomas had a high incidence. Histologic subtypes included small lymphocytic (1%), small cleaved cell (7%), mixed small and large cell (20%), large cell (43%), large cell, immunoblastic (17%), lymphoblastic (9%) and small non-cleaved cell lymphoma (3%). Immunologic study utilizing the avidin-biotinylated horseradish peroxidase complex (ABC) technique demonstrated the presence of intracytoplasmic monoclonal immunoglobulin in 24 (34%) of the suggested B-cell lymphoma cases; 20 tumors (28%) were classified as T-cell lymphoma based on a positive reaction for mouse monoclonal antibody (UCHL-1) to T-cell related membrane antigen; 11 tumors (16%) contained intracytoplasmic alpha 1-antitrypsin, suggesting true histiocytic lymphoma; 15 tumors (22%) did not contain immunoglobulin, UCHL-1 or alpha 1-antitrypsin positive cells and showed no definite characteristics.

Substance P-immunoreactive nerves in the rat kidney

An indirect immunohistochemical method in which an avidin-biotinylated horseradish peroxidase complex is bound to the secondary antibody was used to visualize substance P-immunoreactive (SPI) nerves in the rat kidney. Rats were perfused with 2% paraformaldehyde + 0.15% picric acid in 0.1 M phosphate buffer, then transferred to the buffer. After 24–48 h, the kidneys were sectioned with a Vibratome at 200 or 300 μm and incubated in the primary antiserum for 18 h at room temperature. A dense plexus of SPI nerves innervates the rat renal calyx. A small proportion of intrarenal SPI axons innervates interlobular arteries and afferent arterioles, but most perivascular SPI axons terminate on interlobar and arcuate arteries. The densest plexuses are located on segments of interlobar arteries near the hilus of the kidney. Some of these axons probably are nociceptive; others may be chemo- or baroreceptive.

Vasoactive intestinal peptide-immunoreactive nerves in the rat kidney.

An indirect immunohistochemical method in which an avidin-biotinylated horseradish peroxidase complex is bound to the secondary antibody was used to visualize vasoactive intestinal peptide-immunoreactive (VIPI) nerves in the rat kidney. Rats were perfused with 4% paraformaldehyde or 2% paraformaldehyde + 0.15% picric acid in 0.1 M phosphate buffer, then transferred to the buffer. After 24-48 hours, the kidneys were sectioned with a Vibratome at 200 or 300 micron and incubated in the primary antiserum for 18 hours at room temperature. A sparse plexus of VIPI nerves innervates the rat renal calyx. Some VIPI nerves innervate interlobar arteries and each succeeding segment of the arterial tree including afferent arterioles, but most innervate arcuate and interlobular arteries. VIPI axons do not innervate each arcuate artery or each interlobular branch of an arcuate artery with equal density. Although some axons follow each interlobular branch, most form a dense plexus on only one or two branches. Therefore, most VIPI nerves in the rat kidney innervate a restricted segment of the renal arterial tree. These nerves may be efferent and may selectively dilate arcuate and smaller arteries, or they may be afferent and may sense local changes in mechanical or chemical parameters.

Avidin-biotin amplification procedures for the detection of human terminal deoxynucleotidyl transferase in cell smears.

The authors have developed fluorescent avidin and avidin-peroxidase assays to detect human terminal deoxynucleotidyl transferase (TdT) in cell smears. These assays used specifically purified rabbit anti-calf TdT antibody, biotinylated goat anti-rabbit IgG antibody, and either fluorescein isothiocyanate-avidin or avidin-biotinylated horseradish peroxidase complex. The use of phosphate-buffered saline rather than TRIS-buffered saline or borate-buffered saline was essential to obtain optimal results in the fluorescent avidin assay. Fixation of cells with either 0.5% paraformaldehyde or 10% neutral buffered formalin was found to be superior to either no fixation or fixation with cold methanol or cold 0.1% glutaraldehyde in ethanol. The sensitivities of the fluorescent avidin and avidin-peroxidase assays were shown to be identical, based on staining intensities and percentages of positive cells when both assays were performed on cells from 14 patients with acute lymphoblastic leukemia (ALL), and 5 patients with lymphoblastic lymphoma (LL).

Role of immunocytochemistry in diagnosis of prostatic neoplasia by fine needle aspiration biopsy

The efficacy of immunocytochemical staining for prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) was studied in aspiration biopsy specimens from 19 patients. Eighteen patients had prostatic carcinoma and one had hyperplasia of prostate. Specimens were obtained from both the primary tumors and metastatic sites. Immunoperoxidase staining was performed on alcohol-fixed cytology smears (some prepared up to 9 years previously) using appropriate antisera followed by an avidin-biotinylated horseradish peroxidase complex. Results were scored according to the percentage and intensity of positively stained malignant cells. Corresponding histologic specimens were stained and scored in a similar fashion. Correlations were made between the staining characteristics of the tumor markers and grade of tumor, using the University of Texas M.D. Anderson Hospital classification of prostate carcinoma. Overall there was good correlation between cytologic and histologic specimens for the presence of PSA and PSAP, although metastases tended to show fewer positively stained cells than the primary tumor. There was no relationship between tumor grade and percentage of positively stained cells. Ninety-three percent of aspirated primary and secondary prostatic tumors stained positively for PSAP compared with 81% for PSA. In one of 3 patients, negative staining of neoplastic cells by both PSAP and PSA was helpful in confirming the existence of a second primary tumor.

Substance P-immunoreactive fibers in the trigeminal sensory nuclei of snakes with infrared sensitivity

An indirect immunohistochemical method in which an avidin-biotinylated horseradish peroxidase complex is bound to the secondary antibody was used to visualize substance P-immunoreactive (SPI) nerves in the rat kidney. Rats were perfused with 2% paraformaldehyde + 0.15% picric acid in 0.1 M phosphate buffer, then transferred to the buffer. After 24–48 h, the kidneys were sectioned with a Vibratome at 200 or 300 μm and incubated in the primary antiserum for 18 h at room temperature. A dense plexus of SPI nerves innervates the rat renal calyx. A small proportion of intrarenal SPI axons innervates interlobular arteries and afferent arterioles, but most perivascular SPI axons terminate on interlobar and arcuate arteries. The densest plexuses are located on segments of interlobar arteries near the hilus of the kidney. Some of these axons probably are nociceptive; others may be chemo- or baroreceptive.

Distribution and subcellular localization of a hepatic proliferation inhibitor in rat liver.

The subcellular and intralobular distributions of a protein which specifically inhibits the proliferation of normal liver cells were determined in rat liver, using a combination of immunological and biochemical techniques. The IgG fraction from an antiserum raised against the hepatic proliferation inhibitor was isolated by protein A-Sepharose CL-4B chromatography and shown to be highly specific for the antigen using electroimmunodiffusion and affinity chromatography. To determine the intracellular location of the inhibitor, subcellular fractions were prepared from adult rat livers by differential centrifugation. The cytoplasmic fraction contained the biologically active cytostatic inhibitor, whereas the nuclear and mitochondrial fractions were inactive. Cytoplasmic localization of the hepatic proliferation inhibitor was further confirmed by anion exchange high performance liquid chromatography and by double immunodiffusion with the anti-hepatic proliferation inhibitor IgG. When liver sections were subjected to histochemical staining mediated through the immune IgG and an avidin-biotinylated horseradish peroxidase complex, the parenchymal liver cells were stained, but endothelial and connective tissue cells were not. Although some staining was evident throughout the liver parenchyma, the most intensely stained cells were located in the centrilobular region. Moreover, an age-dependent increase in the staining intensity and/or in the number of cells containing the proliferation inhibitor was observed. Preliminary experiments showed that little, if any, staining occurred in hepatocellular carcinoma cells. This highly specific IgG can be used to monitor alterations in the content and location of hepatic proliferation inhibitor in proliferative disorders of the liver.

Use of avidin-biotinylated horseradish peroxidase complex for visualization of spirochetes.

The use of avidin-biotinylated peroxidase as a simple technique for light microscopic visualization of spirochetes is described. The three major genera of spirochetes--Treponema, Borrelia, and Leptospira--were stained with the avidin complex.