Differential expression of PKCα and -ß in the zebrafish retina.

The retina is a complex neural circuit, which processes and transmits visual information from light perceiving photoreceptors to projecting retinal ganglion cells. Much of the computational power of the retina rests on signal integrating interneurons, such as bipolar cells. Commercially available antibodies against bovine and human conventional protein kinase C (PKC) α and -ß are frequently used as markers for retinal ON-bipolar cells in different species, despite the fact that it is not known which bipolar cell subtype(s) they actually label. In zebrafish (Danio rerio) five prkc genes (coding for PKC proteins) have been identified. Their expression has not been systematically determined. While prkcg is not expressed in retinal tissue, the other four prkc (prkcaa, prkcab, prkcba, prkcbb) transcripts were found in different parts of the inner nuclear layer and some as well in the retinal ganglion cell layer. Immunohistochemical analysis in adult zebrafish retina using fluorescent in situ hybridization and PKC antibodies showed an overlapping immunolabeling of ON-bipolar cells that are most likely of the BON s6 and BON s6L or RRod type. However, comparison of transcript expression with immunolabeling, implies that these antibodies are not specific for one single zebrafish conventional PKC, but rather detect a combination of PKC -α and -ß variants.

Protein kinase C α enhances migration of breast cancer cells through FOXC2-mediated repression of p120-catenin.

BACKGROUND: Despite recent advances in the diagnosis and treatment of breast cancer, metastasis remains the main cause of death. Since migration of tumor cells is considered a prerequisite for tumor cell invasion and metastasis, a pressing goal in tumor biology has been to elucidate factors regulating their migratory activity. Protein kinase C alpha (PKCα) is a serine-threonine protein kinase implicated in cancer metastasis and associated with poor prognosis in breast cancer patients. In this study, we set out to define the signaling axis mediated by PKCα to promote breast cancer cell migration. METHODS: Oncomine™ overexpression analysis was used to probe for PRKCA (PKCα) and FOXC2 expression in mRNA datasets. The heat map of PRKCA, FOXC2, and CTNND1 were obtained from the UC Santa Cruz platform. Survival data were obtained by PROGgene and available at http://www.compbio.iupui.edu/proggene . Markers for EMT and adherens junction were assessed by Western blotting and quantitative polymerase chain reaction. Effects of PKCα and FOXC2 on migration and invasion were assessed in vitro by transwell migration and invasion assays respectively. Cellular localization of E-cadherin and p120-catenin was determined by immunofluorescent staining. Promoter activity of p120-catenin was determined by dual luciferase assay using a previously validated p120-catenin reporter construct. Interaction between FOXC2 and p120-catenin promoter was verified by chromatin immunoprecipitation assay. RESULTS: We determined that PKCα expression is necessary to maintain the migratory and invasive phenotype of both endocrine resistant and triple negative breast cancer cell lines. FOXC2 acts as a transcriptional repressor downstream of PKCα, and represses p120-catenin expression. Consequently, loss of p120-catenin leads to destabilization of E-cadherin at the adherens junction. Inhibition of either PKCα or FOXC2 is sufficient to rescue p120-catenin expression and trigger relocalization of p120-catenin and E-cadherin to the cell membrane, resulting in reduced tumor cell migration and invasion. CONCLUSIONS: Taken together, these results suggest that breast cancer metastasis may partially be controlled through PKCα/FOXC2-dependent repression of p120-catenin and highlight the potential for PKCα signal transduction networks to be targeted for the treatment of endocrine resistant and triple negative breast cancer.

An Ultrastructural and Immunohistochemical Analysis of the Outer Plexiform Layer of the Retina of the European Silver Eel (Anguilla anguilla L).

Here we studied the ultrastructural organization of the outer retina of the European silver eel, a highly valued commercial fish species. The retina of the European eel has an organization very similar to most vertebrates. It contains both rod and cone photoreceptors. Rods are abundantly present and immunoreactive for rhodopsin. Cones are sparsely present and only show immunoreactivity for M-opsin and not for L-, S- or UV-cone opsins. As in all other vertebrate retinas, Müller cells span the width of the retina. OFF-bipolar cells express the ionotropic glutamate receptor GluR4 and ON-bipolar cells, as identified by their PKCα immunoreactivity, express the metabotropic receptor mGluR6. Both the ON- and the OFF-bipolar cell dendrites innervate the cone pedicle and rod spherule. Horizontal cells are surrounded by punctate Cx53.8 immunoreactivity indicating that the horizontal cells are strongly electrically coupled by gap-junctions. Connexin-hemichannels were found at the tips of the horizontal cell dendrites invaginating the photoreceptor synapse. Such hemichannels are implicated in the feedback pathway from horizontal cells to cones. Finally, horizontal cells are surrounded by tyrosine hydroxylase immunoreactivity, illustrating a strong dopaminergic input from interplexiform cells.

Expression of PKCα, PKCε, and P-gp in epithelial ovarian carcinoma and the clinical significance.

AIM: To inspect the expression of two protein kinase PKC isozyme hypotype PKCα and PKCε in the epithelial ovarian carcinoma tissue, and investigate their relation with multi-drug resistance with P-glycoprotein (P-gp) medium. MATERIALS AND METHODS: Adopted immunohistochemistry SP method to determine expression of PKCα, PKCε, and P-gp in 64 cases of epithelial ovarian carcinoma, 18 cases of epithelial borderline ovarian carcinoma, 15 cases of epithelial ovarian benign tumor, and 15 cases of normal ovarian tissue. RESULTS: The expression of PKCα, PKCε, and P-gp in the epithelial ovarian carcinoma is obviously higher than expression in the normal, benign. and borderline epithelial ovarian carcinoma; the expression of PKCα, PKCε, and P-gp in the recurrent carcinoma tissue is obviously higher than that in the person with initial treatment; the expression of above-mentioned three indicators in epithelial ovarian carcinoma is unrelated with the pathological type, pathological grade, and clinical stage during initial treatment of the carcinoma; there is a close relation among PKCα, PKCε, and P-gp in epithelial ovarian carcinoma (p < 0.01). It is indicated through research that PKCα, PKCε, and P-gp is related with the survival time and poor prognosis of the patient of epithelial ovarian carcinoma, i.e., the positive expression rate of PKCα, PKCε, and P-gp of the person with recurrent carcinoma is higher than that of the person without recurrent car- cinoma (p < 0.05). However, the survival rate of the patients with positive expression of three indicators is remarkably lower than those with negative expression (p < 0.05). CONCLUSION: There is a consistency between expression of PKCα, PKCε, and P-gp in the epithelial ovarian carcinoma, which indicates that the expression of both plays an important role in generation of drug resistance in chemotherapy of ovarian carcinoma with P-gp medium. Joint detection of three indicators has an active guiding role in judgment of the therapeutic effect of clinical chemotherapy and prognosis estimation of the patient.

The predictive value and potential mechanisms of miRNA-328 and miRNA-378 for brain metastases in operable and advanced non-small-cell lung cancer.

OBJECTIVE: The incidence of brain metastases greatly varies in patients with non-small-cell lung cancer, and molecular markers are considered to predict brain metastases. Therefore, this study sought to identify the predictive value and potential mechanisms of miRNA-328 and miRNA-378 for brain metastases in non-small-cell lung cancer. METHODS: Patients who received a curable surgery for their lung cancer were screened according to our criteria. Formalin-fixed paraffin-embedded samples from the patients were examined for the expression of miRNA-328 and miRNA-378 using real-time polymerase chain reaction and the expression of N-cadherin, E-cadherin, vascular endothelial growth factor, protein kinase Cα and S100B were investigated using immunohistochemical staining. RESULTS: In total, 86 patients were screened for this study and 23 patients were diagnosed with brain metastases during the follow-up period. Comparing patients with and without brain metastases, the expression of miRNA-328 and miRNA-378 in tumor tissues were significantly different (P = 6.2 × 10(-5) and P = 2.8 × 10(-5), respectively). For the patients with brain metastases, the expression of miRNA-328 and miRNA-378 in tumor tissues compared with para-carcinoma tissues were also significantly different (P = 2.2 × 10(-5) and P = 1.6 × 10(-5), respectively). For patients with brain metastases, the association between miRNA-328 and protein kinase Cα was significant (r = 0.591, P = 0.003), but that between miRNA-378 and protein kinase Cα was not significant (r = 0.259, P = 0.232). CONCLUSIONS: The expression of miRNA-328 and miRNA-378 in tumor tissues can be used to predict brain metastases in patients with non-small-cell lung cancer. miRNA-328 might promote brain metastases by regulating the expression of protein kinase Cα. However, the mechanisms of miRNA-378 to promote brain metastases should be studied in the future.

The inhibitory effect of pseudolaric acid B on gastric cancer and multidrug resistance via Cox-2/PKC-α/P-gp pathway.

AIM: To investigate the inhibitory effect of pseudolaric acid B on subcutaneous xenografts of human gastric adenocarcinoma and the underlying molecular mechanisms involved in its multidrug resistance. METHODS: Human gastric adenocarcinoma SGC7901 cells and drug-resistant SGC7901/ADR cells were injected into nude mice to establish a subcutaneous xenograft model. The effects of pseudolaric acid B with or without adriamycin treatment were compared by determining the tumor size and weight. Cyclo-oxygenase-2, protein kinaseC-α and P-glycoprotein expression levels were determined by immunohistochemistry and western blot. RESULTS: Pseudolaric acid B significantly suppressed the tumor growth induced by SGC7901 cells and SGC7901/ADR cells. The combination of pseudolaric acid B and the traditional chemotherapy drug adriamycin exhibited more potent inhibitory effects on the growth of gastric cancer in vivo than treatment with either pseudolaric acid B or adriamycin alone. Protein expression levels of cyclo-oxygenase-2, protein kinaseC-α and P-glycoprotein were inhibited by pseudolaric acid B alone or in combination with adriamycin in SGC7901/ADR cell xenografts. CONCLUSION: Pseudolaric acid B has a significant inhibitory effect and an additive inhibitory effect in combination with adriamycin on the growth of gastric cancer in vivo, which reverses the multidrug resistance of gastric neoplasm to chemotherapy drugs by downregulating the Cox-2/PKC-α/P-gp/mdr1 signaling pathway.

Intracellular TCR-signaling pathway: novel markers for lymphoma diagnosis and potential therapeutic targets.

Despite the immunologic functions of T-cell receptor signaling molecules being extensively investigated, their potential as immunohistochemical markers has been poorly explored. With this background, we evaluated the expression of 5 intracellular proteins-GADS, DOK2, SKAP55, ITK, and PKCα-involved in T-cell receptor signaling in normal and neoplastic hematologic tissue samples, using antibodies raised against fixation-resistant epitopes of the 5 molecules. All 5 antibodies were associated with normal T-cell differentiation. GADS, DOK2, SKAP55, and ITK turned out to be T-cell lineage-specific markers in the setting of lymphoid and myeloid precursor neoplasms but showed differential expression in peripheral T-cell lymphoma (PTCL) subtypes, being detected in PTCL/not otherwise specified (NOS) and angioimmunoblastic T-cell lymphoma but negative in anaplastic large cell lymphoma (ALCL). Peripheral B-cell lymphomas were consistently negative for ITK, with occasional cases showing expression of DOK2 and SKAP55, and a proportion (47%) of hairy cell leukemias were GADS. Notably, PKCα highlighted a defective antigen in both PTCL/NOS (6%) and angioimmunoblastic T-cell lymphoma (10%), mostly negative in ALCL, and was aberrantly expressed in classical Hodgkin lymphoma (65%), Burkitt lymphoma (48%), and plasma cell myeloma (48%). In conclusion, all five molecules evaluated play a role in T-cell differentiation in normal and neoplastic tissues. They can be applied confidently to routine sections contributing primarily to assignment of T-lineage differentiation in the setting of hematopoietic precursor neoplasms (GADS/DOK2/SKAP55/ITK) and for the differential diagnosis between ALCL and PTCL/NOS (GADS/DOK2/SKAP55/ITK) or classical Hodgkin lymphoma (PKCα). Finally, association with specific tumor subtypes may have therapeutic potential.

Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension.

BACKGROUND: Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca(2+) sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. METHODS AND RESULTS: RV samples from PAH patients undergoing heart-lung transplantation were compared to non-failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western-blot analyses using antibodies to protein-kinase-A (PKA), Cα (PKCα) and Ca(2+)/calmoduling-dependent-kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P<0.0001). To test the functional relevance of PKA-, PKCα-, and CamKIIδ-mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca(2+) sensitivity is mediated by sarcomeric troponin I (cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P<0.05) and reduced PKA-mediated cTnI phosphorylation (Ser22/23) (P<0.001). Finally, alterations in Ca(2+)-handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca(2+) clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P<0.05). CONCLUSIONS: Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca(2+) handling proteins contribute to RV diastolic dysfunction in PAH.

Fluorescent polyion complex nanoparticle that incorporates an internal standard for quantitative analysis of protein kinase activity.

We demonstrate a polyion complex (PIC) nanoparticle that contains both a responsive fluorophore and an "internal standard" fluorophore for quantitative measurement of protein kinase (PK) activity. The PK-responsive fluorophore becomes more fluorescent with PK-catalyzed phosphorylation of substrate peptides incorporated in the PIC, while fluorescence from the internal standard remains unchanged during phosphorylation. This new concept will be useful for quantitative PK assays and the discovery of PK inhibitors.

Obesity-associated Gingival Vascular Inflammation and Insulin Resistance.

Obesity is a risk factor for periodontitis, but the pathogenic mechanism involved is unclear. We studied the effects of insulin in periodontal tissues during the state of obesity-induced insulin resistance. Gingival samples were collected from fatty (ZF) and lean (ZL, control) Zucker rats. Endothelial nitric oxide synthase (eNOS) expression was decreased, and activities of protein kinase C (PKC) α, ß2, δ, and ϵ isoforms were significantly increased in the gingiva from ZF rats compared with those from ZL rats. Expression of oxidative stress markers (mRNA) and the p65 subunit of NF-κB was significantly increased in ZF rats. Immunohistochemistry revealed that NF-κB activation was also increased in the gingival endothelial cells from transgenic mice overexpressing NF-κB-dependent enhanced green fluorescent protein (GFP) and on a high-fat vs. normal chow diet. Analysis of the gingiva showed that insulin-induced phosphorylation of IRS-1, Akt, and eNOS was significantly decreased in ZF rats, but Erk1/2 activation was not affected. General PKC inhibitor and an anti-oxidant normalized the action of insulin on Akt and eNOS activation in the gingiva from ZF rats. This provided the first documentation of obesity-induced insulin resistance in the gingiva. Analysis of our data suggested that PKC activation and oxidative stress may selectively inhibit insulin-induced Akt and eNOS activation, causing endothelial dysfunction and inflammation.

Molecular interplays involved in the cellular uptake of octaarginine on cell surfaces and the importance of syndecan-4 cytoplasmic V domain for the activation of protein kinase Cα.

Arginine-rich cell-penetrating peptides (CPPs) are promising carriers for the intracellular delivery of various bioactive molecules. However, many ambiguities remain about the molecular interplays on cell surfaces that ultimately lead to endocytic uptake of CPPs. By treatment of cells with octaarginine (R8), enhanced clustering of syndecan-4 on plasma membranes and binding of protein kinase Cα (PKCα) to the cytoplasmic domain of syndecan-4 were observed; these events potentially lead to the macropinocytic uptake of R8. The cytoplasmic V domain of syndecan-4 made a significant contribution to the cellular uptake of R8, whereas the cytoplasmic C1 and C2 domains were not involved in the process.

Early down-regulation of PKCδ as a pro-survival mechanism in Huntington's disease.

A balance between cell survival and apoptosis is crucial to avoid neurodegeneration. Here, we analyzed whether the pro-apoptotic protein PKCδ, and the pro-survival PKCα and ßII, were dysregulated in the brain of R6/1 mouse model of Huntington's disease (HD). Protein levels of the three PKCs examined were reduced in all the brain regions analyzed being PKCδ the most affected isoform. Interestingly, PKCδ protein levels were also decreased in the striatum and cortex of R6/2 and Hdh(Q111/Q111) mice, and in the putamen of HD patients. Nuclear PKCδ induces apoptosis, but we detected reduced PKCδ in both cytoplasmic and nuclear enriched fractions from R6/1 mouse striatum, cortex and hippocampus. In addition, we show that phosphorylation and ubiquitination of PKCδ are increased in 30-week-old R6/1 mouse brain. All together these results suggest a pro-survival role of reduced PKCδ levels in response to mutant huntingtin-induced toxicity. In fact, we show that over-expression of PKCδ increases mutant huntingtin-induced cell death in vitro, whereas over-expression of a PKCδ dominant negative form or silencing of endogenous PKCδ partially blocks mutant huntingtin-induced cell death. Finally, we show that the analysis of lamin B protein levels could be a good marker of PKCδ activity, but it is not involved in PKCδ-mediated cell death in mutant huntingtin-expressing cells. In conclusion, our results suggest that neurons increase the degradation of PKCδ as a compensatory pro-survival mechanism in response to mutant huntingtin-induced toxicity that can help to understand why cell death appears late in the disease.

Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex.

Here, we report the fluorometric detection of protein kinase Cα (PKCα) activity in a cancerous cell lysate using a polyion complex (PIC) composed of a quencher (BHQ3)-modified chondroitin sulfate [CS(X)] and a dendrimer modified with a cationic peptide substrate (FKKQGSFAKKK-NH(2)) and a near infrared (NIR) fluorophore (Cy5.5) (polymer 1). When polymer 1 formed the PIC with CS(X) through electrostatic interactions, the NIR fluorescence was quenched effectively via Förster resonance energy transfer (FRET) between Cy5.5 and BHQ3. However, this quenched fluorescence was recovered when the pendant peptides in polymer 1 were phosphorylated with PKCα due to the dissociation of the PIC. When PKCα was added to the PIC dispersion, a significant increase in fluorescence intensity was observed, whereas its fluorescence increase was inhibited with a PKCα inhibitor in a concentration-dependent manner. Furthermore, our PIC was robust enough to measure PKCα activity in a cancerous cellular lysate without purification. The PKCα-responsive PIC offers a simple, rapid, sensitive, and robust approach to detect PKCα activity in crude cellular lysates that would be suitable for drug screening formats and cancer diagnosis using crude cellular lysates.

Molecular mechanisms underlying Ca2+-mediated motility of human pancreatic duct cells.

We recently reported that transforming growth factor-ß (TGF-ß) induces an increase in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pancreatic cancer cells, but the mechanisms by which TGF-ß mediates [Ca(2+)](cyt) homeostasis in these cells are currently unknown. Transient receptor potential (TRP) channels and Na(+)/Ca(2+) exchangers (NCX) are plasma membrane proteins that play prominent roles in controlling [Ca(2+)](cyt) homeostasis in normal mammalian cells, but little is known regarding their roles in the regulation of [Ca(2+)](cyt) in pancreatic cancer cells and pancreatic cancer development. Expression and function of NCX1 and TRPC1 proteins were characterized in BxPc3 pancreatic cancer cells. TGF-ß induced both intracellular Ca(2+) release and extracellular Ca(2+) entry in these cells; however, 2-aminoethoxydiphenyl borate [2-APB; a blocker for both inositol 1,4,5-trisphosphate (IP(3)) receptor and TRPC], LaCl(3) (a selective TRPC blocker), or KB-R7943 (a selective inhibitor for the Ca(2+) entry mode of NCX) markedly inhibited the TGF-ß-induced increase in [Ca(2+)](cyt). 2-APB or KB-R7943 treatment was able to dose-dependently reverse membrane translocation of PKCα induced by TGF-ß. Transfection with small interfering RNA (siRNA) against NCX1 almost completely abolished NCX1 expression in BxPc3 cells and also inhibited PKCα serine phosphorylation induced by TGF-ß. Knockdown of NCX1 or TRPC1 by specific siRNA transfection reversed TGF-ß-induced pancreatic cancer cell motility. Therefore, TGF-ß induces Ca(2+) entry likely via TRPC1 and NCX1 and raises [Ca(2+)](cyt) in pancreatic cancer cells, which is essential for PKCα activation and subsequent tumor cell invasion. Our data suggest that TRPC1 and NCX1 may be among the potential therapeutic targets for pancreatic cancer.

Pancreas-specific ablation of beta1 integrin induces tissue degeneration by disrupting acinar cell polarity.

BACKGROUND & AIMS: Integrin contact with basement membrane is a major determinant of epithelial cell polarity. beta1 integrin heterodimers are the primary receptors for basement membrane in pancreatic acinar cells, which function to synthesize and directionally secrete digestive enzymes into a central lumen. Aberrant acinar secretion and exposure of the parenchyma to digestive enzyme activity lead to organ damage and pancreatitis. METHODS: beta1 integrin conditional knockout mice were crossed to Ptf1a-Cre mice to ablate beta1 integrin in the pancreas. Histopathology of aged and cerulein-treated mice were assessed by histology and immunocytochemistry. Directional secretion was determined in vitro by FM1-43 loading with cerulein stimulation. RESULTS: Pancreas-specific ablation of beta1 integrin led to progressive organ degeneration, associated with focal acinar cell necrosis and ductal metaplasia along with widespread inflammation and collagen deposition. beta1 Integrin-null pancreata were highly susceptible to cerulein-induced acute pancreatitis, displaying an enhanced level of damage with no loss in regeneration. Degenerating beta1 integrin-null pancreata were marked by disruption of acinar cell polarity. Protein kinase C epsilon, normally localized apically, was found in the cytoplasm where it can lead to intracellular digestive enzyme activation. beta1 Integrin-null acinar cells displayed indiscriminate secretion to all membrane surfaces, consistent with an observed loss of basolateral membrane localization of Munc18c, which normally prevents basal secretion of digestive enzymes. CONCLUSIONS: Ablation of beta1 integrin induces organ atrophy by disrupting acinar cell polarity and exposing the pancreatic parenchyma to digestive enzymes.

Membrane ERalpha-dependent activation of PKCalpha in endometrial cancer cells by estradiol.

The purpose of this study was to investigate the role of the oestrogen receptor subtypes ERalpha and ERbeta in mediating the non-genomic effects of 17-beta-estradiol (E(2)) in two human endometrial cancer cell lines (RL95-2 and HEC-1A) expressing different levels of these receptor subtypes. Western blotting analysis using phosphorylation site-specific antibodies showed that physiological concentrations of E(2) rapidly (<20 min) activated PKCalpha, but not PKCdelta in the RL95-2 cell line. E(2) had no effect on PKCalpha or PKCdelta activity in the HEC-1A cell line and suppressed basal levels of PKA activity in both cell lines. PKCalpha activation coincided with its membrane translocation. ERalpha was detected in the RL95-2 cell line by Western blotting and RT-PCR but not in the HEC-1A cells, which did express ERbeta. A selective ERalpha agonist PPT had the same effect as E(2) on PKCalpha activation in the RL95-2 cells, but the selective ERbeta agonist DPN had no such effect. A 46kDa variant of ERalpha increased in abundance in the cell membrane within 20 min of E(2) treatment suggesting that ERalpha mediated the E(2) non-genomic effects on PKCalpha through the formation of a membrane associated signalling complex.

Consumption of virgin olive oil influences membrane lipid composition and regulates intracellular signaling in elderly adults with type 2 diabetes mellitus.

We aimed to define changes in membrane fatty acids and signaling proteins induced by virgin olive oil (VOO) consumption in elderly persons with type 2 diabetes (n = 16) compared to a control group (n = 28). The fatty acid composition was determined by gas chromatography and G-protein subunits and protein kinase C alpha (PKCalpha) by immunoblotting. VOO consumption increased the monounsaturated fatty acid content in phospholipids and cholesterol esters in both groups. In contrast, saturated fatty acids were decreased only in phospholipids. The levels of Galphao, Gbeta, and PKCalpha were significantly lower in diabetics than in controls. However, whereas VOO consumption reduced Galphas, Gbeta, and PKCalpha in both groups, reduction in Galphai was observed only in diabetics. These results indicate that long-term VOO consumption modifies the fatty acid composition of plasma membrane, which influences the association of G proteins and PKCalpha with the lipid bilayer. These combined effects probably account for the positive effects of VOO on glycemic homeostasis.

PPARgamma1 attenuates cytosol to membrane translocation of PKCalpha to desensitize monocytes/macrophages.

Recently, we provided evidence that PKCalpha depletion in monocytes/macrophages contributes to cellular desensitization during sepsis. We demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) agonists dose dependently block PKCalpha depletion in response to the diacylglycerol homologue PMA in RAW 264.7 and human monocyte-derived macrophages. In these cells, we observed PPARgamma-dependent inhibition of nuclear factor-kappaB (NF-kappaB) activation and TNF-alpha expression in response to PMA. Elucidating the underlying mechanism, we found PPARgamma1 expression not only in the nucleus but also in the cytoplasm. Activation of PPARgamma1 wild type, but not an agonist-binding mutant of PPARgamma1, attenuated PMA-mediated PKCalpha cytosol to membrane translocation. Coimmunoprecipitation assays pointed to a protein-protein interaction of PKCalpha and PPARgamma1, which was further substantiated using a mammalian two-hybrid system. Applying PPARgamma1 mutation and deletion constructs, we identified the hinge helix 1 domain of PPARgamma1 that is responsible for PKCalpha binding. Therefore, we conclude that PPARgamma1-dependent inhibition of PKCalpha translocation implies a new model of macrophage desensitization.

Adherens junction-associated protein distribution differs in smooth muscle tissue and acutely isolated cells.

This study was designed to examine how smooth muscle (SM) cell (SMC) isolation affects the distribution of some adherens junction (AJ) complex-associated proteins. Immunofluorescence procedures for identifying protein distribution were used on gastrointestinal and tracheal SM tissues and freshly isolated SMCs from dogs and rabbits. As confirmed by force measurements, relaxation, Ca(2+) depletion, and cholinergic activation of SM tissues do not cause significant redistribution of the AJ-associated proteins vinculin, talin, or fibronectin away from the plasma membrane. Unlike SMCs in tissue, freshly isolated SMCs show a variable peripheral/cytoplasmic vinculin and talin distribution that is not altered by activation. Enzymatic treatment of SM tissues (as done for the first step of SMC isolation) results in loss of fibronectin immunoreactivity in SMCs still in the tissue but fails to cause redistribution of vinculin, talin, or caveolin away from the periphery. The loss of fibronectin immunofluorescence with enzymatic digestion correlates significantly with loss of tissue force production. These results confirm that the AJ-associated proteins vinculin and talin do not redistribute throughout SMCs in tissues when relaxed, when generating force, or after enzymatic digestion. In addition, in freshly isolated SMCs, the distribution of these proteins is significantly altered in approximately 50% of the SMCs. The cause of this redistribution is currently unknown, as is the impact on intracellular signaling and mechanics of these cells. Use of these two systems (SMCs in tissues vs. freshly isolated SMCs) provides an ideal situation for studying the role of the AJ in SMC signaling and mechanics.

Distribution of phosphorylated protein kinase C alpha in goldfish retinal bipolar synaptic terminals: control by state of adaptation and pharmacological treatment.

Protein kinase C (PKC) is a signalling enzyme critically involved in many aspects of synaptic plasticity. In cyprinid retinae, the PKC alpha isoform is localized in a subpopulation of depolarizing bipolar cells that show adaptation-related morphological changes of their axon terminals. We have studied the subcellular localization of phosphorylated PKC alpha (pPKC alpha) in retinae under various conditions by immunohistochemistry with a phosphospecific antibody. In dark-adapted retinae, pPKC alpha immunoreactivity is weak in the cytoplasm of synaptic terminals, labelling being predominantly associated with the membrane compartment. In light-adapted cells, immunoreactivity is diffusely distributed throughout the terminal. Western blot analysis has revealed a reduction of pPKC alpha immunoreactivity in cytosolic fractions of homogenized dark-adapted retinae compared with light-adapted retinae. Pharmacological experiments with the isoform-specific PKC blocker Goe6976 have shown that inhibition of the enzyme influences immunolabelling for pPKC alpha, mimicking the effects of light on the subcellular distribution of immunoreactivity. Our findings suggest that the state of adaptation modifies the subcellular localization of a signalling molecule (PKC alpha) at the ribbon-type synaptic complex. We propose that changes in the subcellular distribution of PKC alpha immunoreactivity might be one component regulating the strength of the signal transfer of the bipolar cell terminal.