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Differential Localization (differential + localization)
Selected AbstractsDifferential Localization of Immunoreactive ,- and ,-subunits of S-100 Protein in Feline TestisANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 2 2000B. C. Cruzana This study investigates the differential localization of the ,- subunit (S100-,) and the ,-subunit (S100-,) of the S-100 protein in the feline testis, using immunohistochemistry with polyclonal antibodies to bovine S-100 protein (S-100) and monoclonal antibodies to bovine S100-, and S100-,. Appreciable differences were observed in the cellular localization of the immunoreactivity of each subunit. S-100 was observed in the Sertoli cells, the epithelial cells of the transitional segment of the seminiferous tubules, Leydig cells and the peritubular cells of the seminiferous tubules, but was not observed in the epithelial cells of straight tubules and the rete testis or in the endothelial cells of blood and lymph vessels. S100-, immunoreactivity was localized in Sertoli cells, peritubular cells and the epithelial cells of the terminal segment of the tubules, whereas S100-, immunoreactivity was localized in Leydig cells. The differential localization of the ,- and ,-subunits of the S-100 protein in the feline testis suggests that this protein is multifunctional and be useful as an investigative tool in studying feline testis function. [source] Differential localization of laminin ,2 and integrin ,4 in primary cultures of the rat gingival epitheliumJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2006Michie Tanno Objectives:, The aim of this study was to investigate the differential immunolocalization of laminin ,2 and integrin ,4 in primary cultures of the rat gingival epithelium. Methods:, The gingival epithelium was obtained from Sprague-Dawley rats and was cultured in serum-free keratinocyte growth medium (DK-SFM). Western blotting analysis, immunofluorescence, confocal laser scanning microscopy (CLSM), and immuno-gold labeling for laminin ,2 and integrin ,4 were employed. CLSM images for laminin and integrin were analyzed in horizontal (x,y axis) and in vertical (x,z axis) sections. Results:, Both laminin ,2 and integrin ,4 were detected by Western blot analysis in the gingival epithelium. Immunolocalization of laminin ,2 was distinct in the cytoplasm to form one or two irregular rings in gingival epithelial cells. By contrast, integrin ,4 was localized diffusely in the cytoplasm. F-actin (indicating actin filaments) was clearly discernible at the periphery of the cytoplasm to form a cellular fringe. In x,z axis images obtained by CLSM, laminin ,2 was recognized as large foci in the most inner portion just above the basal plasma membrane. Integrin ,4 existed in the area where F-actin was labeled surrounding the membrane. Immuno-electron microscopy showed that 10nm colloidal gold particles indicating laminin ,2 were mainly localized at the extracellular portion and in the peripheral cytoplasm, whereas integrin ,4 was distributed in the cytoplasm close to the basal plasma membrane but not in extracellular regions. Conclusions:, In primary cultures of the rat gingival epithelium, both laminin ,2 and integrin ,4 may be produced by the epithelium, and irregular rings of laminin ,2 are formed in areas where gingival cells adhere to the extracellular matrix. [source] Differential localization of carbachol- and bicuculline-sensitive pontine sites for eliciting REM sleep-like effects in anesthetized ratsJOURNAL OF SLEEP RESEARCH, Issue 1 2009VICTOR B. FENIK Summary Carbachol, a cholinergic agonist, and GABAA receptor antagonists injected into the pontine dorsomedial reticular formation can trigger rapid eye movement (REM) sleep-like state. Data suggest that GABAergic and cholinergic effects interact to produce this effect but the sites where this occurs have not been delineated. In urethane-anesthetized rats, in which carbachol effectively elicits REM sleep-like episodes (REMSLE), we tested the ability of 10 nL microinjections of carbachol (10 mm) and bicuculline (0.5 or 2 mm) to elicit REMSLE at 47 sites located within the dorsal pontine reticular formation at the levels -8.00 to -10.80 from bregma (B) (Paxinos and Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1997). At rostral levels, most carbachol and some bicuculline injections elicited REMSLE with latencies that gradually decreased from 242 to 12 s for carbachol and from 908 to 38 s for bicuculline for more caudal injection sites. As the latencies decreased, the durations of bicuculline-elicited REMSLE increased from 104 s to over 38 min, and the effect was dose dependent, whereas the duration of carbachol-elicited REMSLE changed little (104,354 s). Plots of REMSLE latency versus the antero-posterior coordinates revealed that both drugs were maximally effective near B-8.80. At levels caudal to B-8.80, carbachol was effective at few sites, whereas bicuculline-elicited REMSLE to at least B-9.30 level. Thus, the bicuculline-sensitive sites extended further caudally than those for carbachol and antagonism of GABAA receptors both triggered REMSLE and controlled their duration, whereas carbachol effects on REMSLE duration were small or limited by its concurrent REMSLE-opposing actions. [source] Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction,CYTOSKELETON, Issue 7 2008Amy E. Hanlon Newell Abstract A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Participation of protein kinase C , isoform and extracellular signal-regulated kinase in neurite outgrowth of GT1 hypothalamic neuronsJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Youngshik Choe Abstract In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform-selective inhibitors and transfection-based expression of enhanced green fluorescence protein (EGFP)-fused PKC isoforms. 12- O -Tetradecanoylphorbol-13-acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingolTM(a PKC,-selective inhibitor), but not by rottlerinTM (a PKC,-selective inhibitor), indicating that PKC, may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and ,-tubulin. To define the differential localization of PKC isoforms, EGFP-tagged PKC,, PKC,, and PKC, were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKC,-EGFP to growth cones and cell,cell adhesion sites, PKC,-EGFP to the nucleus, and PKC,-EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKC, (PKC,-Cat-EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKC, enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKC,-Cat-EGFP alone increased the neurite-outgrowth and doxycycline treatment further augmented the number of neurite-containing cells. We also examined the involvement of the extracellular signal-regulated kinase (ERK) MAP kinase in TPA-induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKC, with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase-1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKC, isoform-specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway. [source] Differential distribution of voltage-gated potassium channels Kv 1.1,Kv1.6 in the rat retina during developmentJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2007M. Höltje Abstract The discharge behavior of neurons depends on a variable expression and sorting pattern of voltage-dependent potassium (Kv) channels that changes during development. The rodent retina represents a neuronal network whose main functions develop after birth. To obtain information about neuronal maturation we analyzed the expression of subunits of the Kv1 subfamily in the rat retina during postnatal development using immunocytochemistry and immunoelectron microscopy. At postnatal day 5 (P5) all the ,-subunits of Kv1.1,Kv1.6 channels were found to be expressed in the ganglion cell layer (GCL), most of them already at P1 or P3. Their expression upregulates postnatally and the pattern and distribution change in an isoform-specific manner. Additionally Kv1 channels are found in the outer and inner plexiform layer (OPL, IPL) and in the inner nuclear layer (INL) at different postnatal stages. In adult retina the Kv 1.3 channel localizes to the inner and outer segments of cones. In contrast, Kv1.4 is highly expressed in the outer retina at P8. In adult retina Kv1.4 occurs in rod inner segments (RIS) near the connecting cilium where it colocalizes with synapse associated protein SAP 97. By using confocal laser scanning microscopy we showed a differential localization of Kv1.1-1.6 to cholinergic amacrine and rod bipolar cells of the INL of the adult retina. © 2006 Wiley-Liss, Inc. [source] Quantitative proteome analysis of detergent-resistant membranes identifies the differential regulation of protein kinase C isoforms in apoptotic T cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2010Therese Solstad Abstract Several lines of evidence suggest that detergent-resistant membranes (DRMs) (also known as lipid rafts and glycosphingolipid-enriched microdomains) may have a role in signaling pathways of apoptosis. Here, we developed a method that combines DRMs isolation and methanol/chloroform extraction with stable isotope labeling with amino acids in cell culture-based quantitative proteome analysis of DRMs from control and cisplatin-induced apoptotic Jurkat T cells. This approach enabled us to enrich proteins with a pivotal role in cell signaling of which several were found with increased or decreased amounts in DRMs upon induction of apoptosis. Specifically, we show that three isoforms of protein kinase C (PKC) are regulated differently upon apoptosis. Although PKC, which belongs to the group of conventional PKCs is highly up-regulated in DRMs, the levels of two novel PKCs, PKC, and PKC,, are significantly reduced. These alterations/differences in PKC regulation are verified by immunoblotting and confocal microscopy. In addition, a specific enrichment of PKC, in apoptotic blebs and buds is shown. Furthermore, we observe an increased expression of ecto-PKC, as a result of exposure to cisplatin using flow cytometry. Our results demonstrate that in-depth proteomic analysis of DRMs provides a tool to study differential localization and regulation of signaling molecules important in health and disease. [source] Differential Localization of Immunoreactive ,- and ,-subunits of S-100 Protein in Feline TestisANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 2 2000B. C. Cruzana This study investigates the differential localization of the ,- subunit (S100-,) and the ,-subunit (S100-,) of the S-100 protein in the feline testis, using immunohistochemistry with polyclonal antibodies to bovine S-100 protein (S-100) and monoclonal antibodies to bovine S100-, and S100-,. Appreciable differences were observed in the cellular localization of the immunoreactivity of each subunit. S-100 was observed in the Sertoli cells, the epithelial cells of the transitional segment of the seminiferous tubules, Leydig cells and the peritubular cells of the seminiferous tubules, but was not observed in the epithelial cells of straight tubules and the rete testis or in the endothelial cells of blood and lymph vessels. S100-, immunoreactivity was localized in Sertoli cells, peritubular cells and the epithelial cells of the terminal segment of the tubules, whereas S100-, immunoreactivity was localized in Leydig cells. The differential localization of the ,- and ,-subunits of the S-100 protein in the feline testis suggests that this protein is multifunctional and be useful as an investigative tool in studying feline testis function. [source] | |||||||||||||