Protein Kinase C Alpha Expression Research Articles

Salivary protein kinase C alpha and novel microRNAs as diagnostic and therapeutic resistance markers for oral squamous cell carcinoma in Indian cohorts.

Oral squamous cell carcinoma (OSCC) is the second leading cause of cancer-related morbidity and mortality in India. Tobacco, alcohol, poor oral hygiene, and socio-economic factors remain causative for this high prevalence. Identification of non-invasive diagnostic markers tailored for Indian population can facilitate mass screening to reduce overall disease burden. Saliva offers non-invasive sampling and hosts a plethora of markers for OSCC diagnosis. Here, to capture the OSCC-specific salivary RNA markers suitable for Indian population, we performed RNA-sequencing of saliva from OSCC patients (n = 9) and normal controls (n = 5). Differential gene expression analysis detected an array of salivary RNAs including mRNAs, long non-coding RNAs, transfer-RNAs, and microRNAs specific to OSCC. Computational analysis and functional predictions identified protein kinase c alpha (PRKCA), miR-6087, miR-449b-5p, miR-3656, miR-326, miR-146b-5p, and miR-497-5p as potential salivary indicators of OSCC. Notably, higher expression of PRKCA, miR-6087 and miR-449b-5p were found to be associated with therapeutic resistance and poor survival, indicating their prognostic potential. In addition, sequencing reads that did not map to the human genome, showed alignments with microbial reference genomes. Metagenomic and statistical analysis of these microbial reads revealed a remarkable microbial dysbiosis between OSCC patients and normal controls. Moreover, the differentially abundant microbial taxa showed a significant association with tumor promoting pathways including inflammation and oxidative stress. Summarily, we provide an integrated landscape of OSCC-specific salivary RNAs relevant to Indian population which can be instrumental in devising non-invasive diagnostics for OSCC.

EGCG promotes PRKCA expression to alleviate LPS-induced acute lung injury and inflammatory response

Acute lung injury (ALI), which could be induced by multiple factors such as lipopolysaccharide (LPS), refer to clinical symptoms of acute respiratory failure, commonly with high morbidity and mortality. Reportedly, active ingredients from green tea have anti-inflammatory and anticancer properties, including epigallocatechin-3-gallate (EGCG). In the present study, protein kinase C alpha (PRKCA) is involved in EGCG protection against LPS-induced inflammation and ALI. EGCG treatment attenuated LPS-stimulated ALI in mice as manifested as improved lung injury scores, decreased total cell amounts, neutrophil amounts and macrophage amounts, inhibited the activity of MPO, decreased wet-to-dry weight ratio of lung tissues, and inhibited release of inflammatory cytokines TNF-α, IL-1β, and IL-6. PRKCA mRNA and protein expression showed to be dramatically decreased by LPS treatment while reversed by EGCG treatment. Within LPS-stimulated ALI mice, PRKCA silencing further aggravated, while PRKCA overexpression attenuated LPS-stimulated inflammation and ALI through MAPK signaling pathway. PRKCA silencing attenuated EGCG protection. Within LPS-induced RAW 264.7 macrophages, EGCG could induce PRKCA expression. Single EGCG treatment or Lv-PRKCA infection attenuated LPS-induced increases in inflammatory factors; PRKCA silencing could reverse the suppressive effects of EGCG upon LPS-stimulated inflammatory factor release. In conclusion, EGCG pretreatment inhibits LPS-induced ALI in mice. The protective mechanism might be associated with the inhibitory effects of PRKCA on proinflammatory cytokine release via macrophages and MAPK signaling pathway.

PRKCA Overexpression Is Frequent in Young Oral Tongue Squamous Cell Carcinoma Patients and Is Associated with Poor Prognosis.

Simple SummaryOver the last decades, the incidence of tongue cancer has risen among young patients who often show an aggressive course of disease. At the same time, exposure to the major risk factors, alcohol and tobacco, has decreased, indicating that a novel risk factor may be involved. In this study, we found that high expression of protein kinase C alpha (PRKCA) is frequent among young patients without alcohol and smoking history and associated with a poor prognosis. Our results suggest that PRKCA levels may serve as a molecular marker of an emerging high-risk subgroup of young tongue cancer patients. Elucidation of the underlying mechanisms may clarify whether PRKCA expression itself promotes disease progression and which genetic or environmental factors trigger its upregulation. Oral tongue squamous cell carcinomas (OTSCCs) have an increasing incidence in young patients, and many have an aggressive course of disease. The objective of this study was to identify candidate prognostic protein markers associated with early-onset OTSCC. We performed an exploratory screening for differential protein expression in younger (≤45 years) versus older (>45 years) OTSCC patients in The Cancer Genome Atlas (TCGA) cohort (n = 97). Expression of candidate markers was then validated in an independent Austrian OTSCC patient group (n = 34) by immunohistochemistry. Kaplan–Meier survival estimates were computed, and genomic and mRNA enrichment in silico analyses were performed. Overexpression of protein kinase C alpha (PRKCA) was significantly more frequent among young patients of both the TCGA (p = 0.0001) and the Austrian cohort (p = 0.02), associated with a negative anamnesis for alcohol consumption (p = 0.009) and tobacco smoking (p = 0.02) and poorer overall survival (univariate p = 0.02, multivariate p< 0.01). Within the young subgroup, both overall and disease-free survival were significantly decreased in patients with PRKCA overexpression (both p < 0.001). TCGA mRNA enrichment analysis revealed 332 mRNAs with significant differential expression in PRKCA-upregulated versus PRKCA-downregulated OTSCC (all FDR ≤ 0.01). Our findings suggest that PRKCA overexpression may be a hallmark of a novel molecular subtype of early-onset alcohol- and tobacco-negative high-risk OTSCC. Further analysis of the molecular PRKCA interactome may decipher the underlying mechanisms of carcinogenesis and clinicopathological behavior of PRKCA-overexpressing OTSCC.

Association of PRKCA expression and polymorphisms with layer duck eggshell quality

ABSTRACT 1. Eggshell quality is important for the poultry industry. Calcium is deposited during eggshell formation, and protein kinase C alpha (PRKCA) is involved in transmembrane transport of calcium ions in cells. However, the biological function of PRKCA in poultry is still not understood. Therefore, the aim of this study was to explore the association of mRNA expression and single nucleotidepolymorphisms (SNPs) of the PRKCA gene with eggshell quality in laying ducks. 2. The mRNA expression and SNPs of the PRKCA gene were detected by real-time fluorescence quantitative PCR (qRT-PCR) and sequencing of PCR products in 45-week-old female Sansui ducks, which is a high production layer duck breed in China. The association of mRNA expression and SNPs in the PRKCA gene with layer duck eggshell traits was analysed using SPSS (v18.0) software. 3. The results demonstrated that PRKCA mRNA was widely expressed in all examined tissues, and expression was highest in kidney and lowest in the gizzard. Furthermore, the PRKCA mRNA level in uterus was significantly positively correlated with eggshell strength and eggshell weight (P < 0.05). Three novel SNPs, the synonymous mutations of g.9571770 T > C in exon 5, g.9583222 C > T and g.9583227 G > A in exon 7, were found in the PRKCA gene, giving four haplotypes and 10 diplotypes, which affected the mRNA secondary structure and free energy. The g.9583222 C > T and g.9583227 G > A mutations were significantly associated with eggshell strength (P < 0.05). Diplotype H1H1 was advantageous for increasing the strength and thickness of an eggshell. 4. In conclusion, the study showed that the mRNA transcription and genetic variation in the PRKCA gene could significantly affect the strength of duck eggshell and that the PRKCA gene is an important candidate gene for improving eggshell quality in poultry.

Upregulation of miR-92a-2-5p potentially contribute to anorectal malformations by inhibiting proliferation and enhancing apoptosis via PRKCA/β-catenin

Anorectal malformations (ARMs) is one of the most common gastrointestinal anomalies. Previous research revealed that miR-92a-2-5p was upregulated in ARMs. However, the underlying roles remains unknown. The current study was to further investigate the spatiotemporal expression patterns of miR-92a-2-5p and its target gene protein kinase C alpha (PRKCA) predicted by bioinformatic method, and to explore their potential functions in anorectal malformations (ARMs). Rat models with ethylenethiourea-induced ARMs were made for subsequent experiments. Direct target relationship between miR-92a-2-5p and PRKCA was validated using a luciferase reporter assay. The spatiotemporal expression pattern of miR-92a-2-5p was evaluated using fluorescence in situ hybridization (FISH), while the expression of PRKCA was revealed by immunohistochemical staining and western blotting. IEC-6 cells were transfected with mimics/mimics NC (Negative control)/inhibitor/inhibitor NC of miR-92a-2-5p or si-PRKCA/si-PRKCA NC, respectively. Then the downstream molecules of miR-92a-2-5p, PRKCA and β-catenin, were subsequently detected. Meanwhile, apoptosis and viability assays were measured. Dual luciferase assay confirmed the direct regulatory relationship between miR-92a-2-5p and PRKCA. FISH revealed that miR-92a-2-5p was expressed with a higher level in ARMs fetuses. Further analyses of PRKCA showed lower protein expression level in ARMs group, which was opposite to miR-92a-2-5p. In vitro experiments revealed that overexpression of miR-92a-2-5p or knockdown of PRKCA can down-regulate PRKCA, up-regulate and facilitate nuclear localization of β-catenin, increase apoptosis and decrease proliferation of IEC-6. Taken together, these findings suggest that aberrantly high expression of miR-92a-2-5p potentially contribute to ARMs by inhibiting proliferation and enhancing apoptosis of intestinal cells via negatively regulating PRKCA/β-catenin.

Jowiseungki decoction affects diabetic nephropathy in mice through renal injury inhibition as evidenced by network pharmacology and gut microbiota analyses

BackgroundJowiseungki decoction (JSD) is a prescription commonly used for the treatment of diabetic complications or diabetic nephropathy (DN) in traditional medicine clinics. However, the underlying therapeutic mechanisms of JSD are still unclear.MethodsStreptozotocin (STZ)-induced DN mice were administered 100 and 500 mg/kg JSD for 4 weeks, and the therapeutic mechanisms and targets of JSD were analyzed by network pharmacology and gut microbiota analyses.ResultsJSD significantly decreased the increase in food and water intake, urine volume, fasting blood glucose, serum glucose and triglyceride levels, and urinary albumin excretion. JSD administration significantly increased the decrease in insulin secretion and creatinine clearance and reduced the structural damage to the kidney tissues. Moreover, JSD administration significantly inhibited the expression of protein kinase C-alpha (PKC-α), transforming growth factor beta-1 (TGF-β1), α-smooth muscle actin (α-SMA), nuclear factor-κB (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in the kidney tissues of DN mice, while it significantly increased the phosphorylation of insulin receptor substrate 1 (IRS-1), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (Akt). In the network pharmacological analysis, JSD obviously influenced phosphatase binding, protein serine/threonine kinase, and mitogen-activated protein kinase (MAPK)-related signaling pathways. Our data suggest that JSD can improve symptoms in STZ-induced DN mice through the inhibition of kidney dysfunction, in particular, by regulating the PKCα/PI3K/Akt and NF-κB/α-SMA signaling pathways. Gut microbiota analysis can help to discover the pharmaco-mechanisms of the influence of JSD on bacterial diversity and flora structures in DN.ConclusionJSD can improve the symptoms of DN, and the underlying mechanism of this effect is renal protection through the inhibition of fibrosis and inflammation. JSD can also change bacterial diversity and community structures in DN.

Heroin-based crack induces hyperalgesia through β-arrestin 2 redistribution and phosphorylation of Erk1/2 and JNK in the periaqueductal gray area

Continuous use of crack induces hyperalgesia which is related to drug tolerance. Despite cumulative evidence based on the growth rate of crack abuse, no serious study has been focused on the mechanisms of crack-induced hyperalgesia. This study aimed to elucidate whether extracellular signal-regulated kinases (Erk1/2)/β-arrestin pathways are involved in the crack-induced hyperalgesia. Fifty adult male Wistar rats were randomly divided into five groups: normal saline (NS), crack (0.9 mg/kg/day), heroin (1 mg/kg/day), crack + barbadin (100 μM), and heroin + barbadin groups, which received their intraperitoneal (i.p) treatments for four weeks. The thermal sensitivity was assessed using the hot-plate test. Moreover, phosphorylation of the Erk1/2 and JNK, as well as expression of protein kinase C-alpha (PKC-α), Mu-receptor (MOR), and β-arrestin 2 were determined in the whole lysate and membrane fraction using immunoblotting assay in the periaqueductal gray (PAG) area. The results demonstrated that chronic administration of crack and heroin significantly decreased hind-paw withdrawal latency compared to the NS group. Furthermore, crack as well as heroin administration increased phosphorylated Erk1/2 and JNK in the PAG. In addition, membrane β-arrestin 2 and PKC-α were significantly increased in the crack and heroin-received groups, while membrane MOR expression was decreased in the PAG. Nevertheless, co-administration of barbadin, an inhibitor of β-arrestin, and crack or heroin reversed all these changes. Our findings may partially confirm the role of β-arrestin 2 and PKC rearrangements, Erk1/2 and JNK phosphorylation in crack-induced hyperalgesia and provide potential therapeutic targets to attenuate crack-induced hyperalgesia.

MiR-634 is a Pol III-dependent intronic microRNA regulating alternative-polyadenylated isoforms of its host gene PRKCA

BackgroundThe protein kinase C alpha (PRKCA) gene, coding for a Th17-cell-selective kinase, shows a complex splicing pattern, with at least 2 stable alternative transcripts characterized by an alternative upstream polyadenylation site. Polymorphisms in this gene were associated with several conditions, including multiple sclerosis, asthma, schizophrenia, and cancer. The presence of a microRNA (miRNA), i.e. miR-634, within intron 15 of the PRKCA gene, suggests the intriguing possibility that this miRNA might play a role in the susceptibility to these pathologies. MethodsHere, we characterized miR-634 expression profile and searched for its putative targets using a combination of RT-PCR and gene reporter assays. ResultsThe quantitative analysis of PRKCA and miR-634 transcripts in a panel of human tissues and cell lines revealed discordant expression profiles, suggesting the presence of an independent miR-634 promoter and/or a possible direct role of miR-634 in modulating PRKCA expression. Functional studies demonstrated the existence of a miRNA-specific promoter, which was shown to be Pol-III-dependent. Furthermore, transfection experiments showed that miR-634 is able to target its host gene by specifically down-regulating the shorter alternative-polyadenylated isoforms. ConclusionsMiR-634 is a Pol III-dependent intronic miRNA, which could target its host gene through a “first-order” negative feedback. General significanceMiR-634 is one of the few characterized examples of Pol-III-dependent intronic miRNAs. Its independent transcription from the host gene suggests caution in using expression profiles of host genes as proxies for the expression of the corresponding intronic miRNAs.

Functional variations modulating PRKCA expression and alternative splicing predispose to multiple sclerosis.

The protein kinase C alpha (PRKCA) gene, encoding a Th17-cell-selective kinase, was repeatedly associated with multiple sclerosis (MS), but the underlying pathogenic mechanism remains unknown. We replicated the association in Italians (409 cases, 723 controls), identifying a protective signal in the PRKCA promoter (P = 0.033), and a risk haplotype in intron 3 (P = 7.7 × 10(-4); meta-analysis with previously published data: P = 4.01 × 10(-8)). Expression experiments demonstrated that the protective signal is associated with alleles conferring higher PRKCA expression levels, well fitting our observation that MS patients have significantly lower PRKCA mRNA levels in blood. The risk haplotype was shown to be driven by a GGTG ins/del polymorphism influencing the heterogeneous nuclear ribonucleoprotein H-dependent inclusion/skipping of a PRKCA alternative exon 3*. Indeed, exon 3* can be present in two different versions in PRKCA mRNAs (out-of-frame 61 bp or in-frame 66 bp long), and is preferentially included in transcripts generated through a premature polyadenylation event. The GGTG insertion downregulates 3* inclusion and shifts splicing towards the 66 bp isoform. Both events reduce the nonsense-mediated mRNA-decay-induced degradation of exon 3*-containing mRNAs. Since we demonstrated that the protein isoform produced through premature polyadenylation aberrantly localizes to the plasma membrane and/or in cytoplasmic clusters, dysregulated PRKCA 3* inclusion may represent an additional mechanism relevant to MS susceptibility.

Recent Patents Concerning Modulators of Protein Kinase C

Protein kinase C (PKC) comprises a family of 10 serine/threonine kinases divided into 3 subfamilies: classical, novel and atypical. These isoenzymes represent one of the major mediators of signal transduction, and most may be associated with several pathogenic processes including malignant transformation or cancer and metastasis. Moreover, some activated isoenzymes are also involved in other diseases such as infarct, rejection due to incomplete histocompatibility in organ transplantation, pain, diabetic macular edema, etc. Here, we review several patents related to inhibitors of PKC that represent a new and promising strategy for the prevention and treatment of these illnesses. Among these inhibitors, we included antisense oligonucleotides as another useful strategy to treat infectious and autoimmune diseases associated with misregulated expression of PKC and tumour necrosis factor alpha (TNF-α). On the other hand, two different activators of PKC and their applications related to neurodegenerative diseases have also been reviewed in this work.

Polyphenol-Rich Extracts from Solanum nigrum Attenuated PKC α-Mediated Migration and Invasion of Hepatocellular Carcinoma Cells

Solanum nigrum L. (SN) has exhibited multiple biological effects such as anti-inflammation and antiproliferation. Protein kinase C (PKC) regulates cellular functions including proliferation, migration, and invasion. In the present investigation, we demonstrated that 12-o-tetradecanoylphobor-13-acetate (TPA) and constitutively activated PKC alpha significantly increased migration and invasion of HepG2 cells, while treatment with water or polyphenol extracts of SN (SNWE or SNPE) attenuated TPA-induced migration and invasion. SNWE and SNPE reduced TPA-elicited PKC alpha expression in a dose-dependent manner and obviously inhibited TPA-induced phosphorylation of p38 and ERK, respectively. Constitutively activated PKC alpha (caPKC alpha) significantly reversed the inhibitory effects of SNWE and SNPE on ERK and p38 activation. However, the antimigration effect of SNWE and SNPE could not be abrogated by caPKC alpha. Our results revealed the antimigration and anti-invasion effects of both extracts derived from SN, which may act as a promising therapeutic agent for the treatment of hepatocellular carcinoma.

Dose-related changes in retinal function and PKC-alpha expression in rabbits on vigabatrin medication

To investigate, in a rabbit model, the effect of two different doses of vigabatrin (VGB) on retinal function and morphology. Twenty-nine rabbits of mixed strain were divided into two groups, receiving either high-dose (n = 15) or low-dose (n = 14) oral VGB treatment (cumulative dose 29.8 +/- 2.9 g and 14.2 +/- 0.6 g respectively). Ten rabbits receiving water served as control animals. The rabbits underwent three baseline ff-ERG measurements before initiation of VGB medication and two ff-ERG registrations during treatment, after 8 and 12-14 weeks respectively. At the end of the study, the expression of protein kinase C-alpha (PKC-alpha), gamma amino butyric acid (GABA) A receptors, vimentin, glial fibrillary acidic protein (GFAP) and peanut agglutinin (PNA) was examined in retinal sections from all rabbits. In animals of the high-dose group, the ff-ERG measurements revealed a significant decrease of isolated rod b-wave amplitudes, combined rod-cone b-wave amplitudes and amplitudes of oscillatory potentials (OPs); OP1, OP2 and OP3. In the low-dose group, the b-wave amplitudes of combined rod-cone responses as well as OP2 and OP3 were significantly reduced. PKC-alpha labeling demonstrated a dose-related translocation of the enzyme in rod bipolar cells, also revealing a significant decline of the number of PKC-alpha labeled rod bipolar cells in treated animals. Vimentin labeling showed a dose-related deviant labeling pattern of Müller cells, with strikingly low labeling intensity in the outer parts of the cells; in the outer limiting membrane (OLM) as well as the outer nuclear layer (ONL). Labeling with antibodies against GABA A receptors and GFAP, as well as PNA staining, revealed no differences between treated animals and controls. In this study, VGB medication was associated, in a dose-related manner, with a decrease of ff-ERG amplitudes as well as with altered protein expression in rod bipolar cells and Müller cells, suggesting alterations of inner retinal function. The dose-related morphological and electrophysiological changes indicate a retinal pathology that may explain the constricted visual fields seen in some patients treated with VGB.

The variation of intracellular distribution and translocation of the protein kinase C alpha among human lung cancer cell line with different metastasis potential.

Protein Kinase C (PKC) is one of the key kinases in the cell signal transduction passway. There are more reports about it's ability on cell proliferation, but fewer on invasion and metastasis in the past; and it's mechanisms are unclear. The aim of this study is to analyze the variation of intracellular distribution and translocation of the protein kinase C alpha among human high-metastatic large cell lung cancer cell line with different metastasis potential,in order to investigate the correlation between the lung carcinoma invasion and metastasis and the PKC isoforms. Using Western blot and laser scanning confocal microscope (LSCM) method. The distribution of PKC alpha in cytosol and plasma membrane and translocation were detected among different metastatic potential human pulmonary carcinoma cells L9981, L9981-pLXSN and L9981-nm23-H1 before and after treatment with PKC inhibitor Calphostin C, by Western blot and LSCM. PKC alpha in L9981 and L9981- pLXSN was mainly expressed on membrane, which was remarkably higher than those in L9981-nm23-H1 cell line (P =0.001); while expression of PKC alpha in cytoslol in L9981 and L9981-pLXSN cell lines, was lower than those in L9981-nm23-H1 cell line (P <0.001). The expression of PKC alpha in cytosol in L9981-nm23-H1 cell line was remarkably higher than those in L9981 and L9981-pLXSN cell lines (P <0.001), while expression of PKC alpha in plasma membrane in L9981-nm23-H1 cell line, was significantly lower than those in L9981 and L9981- pLXSN cell lines (P =0.001). PKC alpha is mainly located in nucleus and perinucleus in L9981 and L9981-pLXSN cells, which was in active status, In L9981-nm23-H1 cell line, PKC alpha is mainly located in soluble cytosolic section, which was in inactive status. After treated with PKC inhibitor Calphostin C, the expression of PKC alpha in membrane in L9981, L9981-pLXSN and L9981-nm23-H1 was downregulated, and PKC alpha were observed mainly located in cytosolic site in all the three cell lines, which was mainly in inactive status. The study suggests that PKC isoforms is closely correlated with human lung cancer invasion and metastasis.

Overexpression of protein kinase cα mRNA may be an independent prognostic marker for gastric carcinoma

The variability of the prognosis of gastric carcinoma drives extensive researches for novel prognostic markers. The aims of this study were to correlate the expression of protein kinase Calpha (PKCalpha) mRNA with clinicopathological parameters and to evaluate the significant value of PKCalpha in gastric carcinoma prognosis. PKCalpha mRNA levels were analyzed in tumor/non-tumor pairs of gastric tissues from surgical specimens of 41 patients with gastric carcinoma employing quantitative real-time polymerase chain reaction. Expression of PKCalpha in gastric carcinoma was also examined using immunohistochemistry. PKCalpha mRNA expression was significantly upregulated in gastric carcinoma (P = 0.007). Overexpression of PKCalpha mRNA was correlated with distant metastasis (P = 0.040). Patients with high PKCalpha mRNA expression had a significantly poorer overall survival compared with patients with low PKCalpha mRNA expression (P = 0.0113). The uni-variate Cox regression analysis showed that high PKCalpha mRNA expression (P = 0.0363) and depth of invasion (P = 0.0443) were two significant prognostic markers for gastric carcinoma. In backward stepwise multi-variate analysis, PKCalpha mRNA overexpression was also proved to be an independent prognostic marker for gastric carcinoma (P = 0.0275). Our results suggest that overexpression of PKCalpha mRNA has correlation with distant metastasis and may be an independent prognostic marker for gastric carcinoma.

Retinal function and PKC alpha expression after focal laser photocoagulation

To examine the effects of focal laser photocoagulation on general and local retinal function and to relate electrophysiological findings with changes in protein kinase C (PKC) alpha expression. Twelve rabbits were treated with 70 spots of laser photocoagulation in the central cone-rich retina. The operated eyes were investigated with electroretinography (full-field ERG and multifocal electroretinography, mfERG) preoperatively and at 1, 3, and 5 weeks after surgery. The expression of PKC alpha was examined at all three time points using immunohistochemistry, and PKC alpha mRNA levels were quantified using real-time polymerase chain reaction (PCR). Immunohistochemistry for glial fibrillary acidic protein (GFAP) and hematoxylin and eosin staining was employed to monitor the extent and dynamics of the morphological response. The full-field ERG revealed a significant increase in b-wave amplitudes derived from the isolated rod response (blue light) at all three time points after surgery (p < 0.05). Supernormal b-wave amplitudes were also found for the combined rod-cone response at 3 weeks (white light), and for the isolated cone response (light-adapted 30-Hz flicker) at 5 weeks after treatment. In the mfERG, amplitudes derived from the central retina did not change postoperatively, while the implicit time was significantly increased at all time points. Immunohistochemistry for PKC alpha revealed a reduced expression of the enzyme in rod bipolar cells 1 and 3 weeks after laser treatment compared with untreated controls. Five weeks postoperatively, no PKC alpha labeling in rod bipolar cells was found in any part of the retina. Real-time PCR 1 and 3 weeks after treatment displayed a decreased level of PKC alpha mRNA compared to the controls. Immunolabeled tissue sections from laser-treated eyes displayed GFAP expression in Müller cells in the treated as well as untreated retina 1 week postoperatively. At 3 and 5 weeks, GFAP labeling was less pronounced and was concentrated around the laser-treated spots. Focal laser treatment in the rabbit eye induces local and wide-spread alterations in both rod- and cone-mediated retinal function in the form of supernormal b-wave amplitudes in the full-field ERG and increased latency in the mfERG. The electrophysiological abnormalities are accompanied by a progressive down-regulation of the PKC alpha isoenzyme in rod bipolar cells, reaching far beyond the treated area. PKC alpha is down-regulated directly by impaired protein synthesis, and also possibly indirectly by protein consumption related to GFAP up-regulation. The results indicate that focal laser photocoagulation interferes with PKC-alpha-mediated inhibitory regulation of inner retinal signal transmission.

On the role of methacrylic acid copolymers in the intracellular delivery of antisense oligonucleotides

The delivery of active biomacromolecules to the cytoplasm is a major challenge as it is generally hindered by the endosomal/lysosomal barrier. Synthetic titratable polyanions can overcome this barrier by destabilizing membrane bilayers at pH values typically found in endosomes. This study investigates how anionic polyelectrolytes can enhance the cytoplasmic delivery of an antisense oligonucleotide (ODN). Novel methacrylic acid (MAA) copolymers were examined for their pH-sensitive properties and ability to destabilize cell membranes in a pH-dependent manner. Ternary complex formulations prepared with the ODN, a cationic lipid and a MAA copolymer were systematically characterized with respect to their size, zeta potential, antisense activity, cytotoxicity and cellular uptake using the A549 human lung carcinoma cell line. The MAA copolymer substantially increased the activity of the antisense ODN in inhibiting the expression of protein kinase C-alpha. Uptake, cytotoxicity and antisense activity were strongly dependent on copolymer concentration. Metabolic inhibitors demonstrated that endocytosis was the major internalization pathway of the complexes, and that endosomal acidification was essential for ODN activity. Confocal microscopy analysis of cells incubated with fluorescently-labeled complexes revealed selective delivery of the ODN, but not of the copolymer, to the cytoplasm/nucleus. This study provides new insight into the mechanisms of intracellular delivery of macromolecular drugs, using synthetic anionic polyelectrolytes.

Effect of cigarette smoke extract on the role of protein kinase C in the proliferation of passively sensitized human airway smooth muscle cells.

To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A1, A2 and A3 and the group B was sub-divided into the group of B1, B2, B3, B4 and B5. No other agents were added to the group A1 and B1. The cells of group A2 and B2 were stimulated with 5% CSE for 24 h. HASMCs from group A3 and B3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5%) for 24 h. PKC inhibitor Ro-31-8220 (5 micromol/L) was added to the HASMCs of group B4 for 24 h. The cells from group B5 were stimulated with Ro-31-8220 (5 micromol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-a in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-alpha mRNA and the A value of PKC-alpha protein in HASMCs from group B1, B2 and B3 were significantly increased compared to those of group A1, A2 and A3 correspondingly and respectively (P< 0.01). The proliferation of HASMCs of group A2 and B2 stimulated with CSE and group A3 and B3 stimulated with CSE and PMA were also significantly enhanced when group A1, A2 and A3 and group B1, B2 and B3 compared to each other (P<0.05, P<0.01, respectively). The percentage of S phase, absorbency (A) value, 3H-TdR incorporation rate, the ratios of A value of PKC-alpha mRNA and the A value of PKC-alpha protein in HASMCs from group B4 treated with Ro-31-8220 and group B5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B1 and B2 correspondingly and respectively (P<0.05, P<0.01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.

Expression of protein kinase C isoenzymes in benign hyperplasia and carcinoma of prostate

Protein kinase C family consists of 11 isoforms, classified into 3 categories according to their structure and mechanisms of activation. These isoenzymes are involved in processes, which maintain intracellular homeostasis. Alterations in activity, amount or distribution of protein kinase C (PKC) isoenzymes may cause cellular proliferation or induce apoptosis. We have studied and compared the expression levels of several PKC isoforms in benign prostatic hyperplasia (BPH) and prostate cancer (PCa). These are PKCs alpha (alpha), beta (beta), delta (delta), epsilon (epsilon), zeta (zeta), eta (eta), which have been detected as major isoforms in prostate tissue. Paraffin sections of 25 benign prostatic hyperplasia (BPH) and 25 of prostatic carcinoma (PCa) were examined for expression of PKC alpha, beta, delta, epsilon, zeta, and eta. Expression of PKC beta was examined in additional 3 BPH and 3 PCa using Western blot analysis. We found a significant high level of expression of PKC isoforms alpha, beta, epsilon and eta in PCa compared to BPH (p<0.01). Using backward logistic regression, we found changes in PKC epsilon expression to be most significant between malignant compared to benign tumor tissue specimens. Immunostaining for PKCs alpha, beta and eta in addition to PKC epsilon may aid in distinguishing between benign and malignant prostatic disease.

Differential influence of increased polyol pathway on protein kinase C expressions between endoneurial and epineurial tissues in diabetic mice.

To explore the relationship between polyol pathway and protein kinase C (PKC), we examined PKC activities and expressions of PKC isoforms separately in endoneurial and vessel-rich epineurial tissues in diabetic mice transgenic for human aldose reductase (Tg). Tg and littermate control mice (Lm) were made diabetic by streptozotocin at 8 weeks of age and treated orally with aldose reductase inhibitor (ARI) (fidarestat 3-5 mg/kg/day) or placebo for 12 weeks. At the end, compared with non-diabetic state, sorbitol contents were increased 6.4-fold in endoneurium and 5.1-fold in epineurium in diabetic Tg, whereas the increase was detected only in endoneurium in diabetic Lm. Endoneurial PKC activity was significantly reduced in diabetic Tg. By contrast, epineurial PKC activity was increased in both diabetic Lm and diabetic Tg and there was no significant difference between the two groups. These changes were all corrected by ARI treatment. Consistent with the changes of PKC activities, diabetic Tg showed decreased expression of PKC alpha in endoneurium, whereas there was an increased expression of PKC beta II in epineurium in both diabetic Tg and diabetic Lm. These findings suggest the presence of dichotomous metabolic pathway between neural and vascular tissues in the polyol-PKC-related pathogenesis of diabetic neuropathy.

Emodin ameliorates glucose-induced matrix synthesis in human peritoneal mesothelial cells

Emodin ameliorates glucose-induced matrix synthesis in human peritoneal mesothelial cells. Prolonged exposure of human peritoneal mesothelial cells (HPMC) to high glucose concentrations in peritoneal dialysate is the principal factor leading to matrix accumulation and thickening of the peritoneal membrane, accompanied by progressive deterioration of transport functions. These changes are mediated in part through protein kinase C (PKC) activation and the induction of transforming growth factor-beta 1 (TGF-beta 1). Emodin (3-methyl-1,6,8 trihydroxyanthraquinone) has previously been demonstrated to reduce cell proliferation and fibronectin synthesis in cultured mesangial cells. How emodin modulates glucose-induced abnormalities in HPMC has not been elucidated and thus constitutes the theme of this study. We investigated the effects of emodin on the expression of PKC alpha, TGF-beta 1, fibronectin, and collagen type I in HPMC, and its effects on HPMC proliferation under physiologic (5 mmol) or high (30 mmol) glucose concentrations. Exposure of HPMC cultured with 5 mmol or 30 mmol D-glucose to emodin (20 microg/mL) resulted in an initial lag of proliferation by 2.3 to 2.7 days, but did not affect cell viability or morphology at confluence. D-glucose (30 mmol) induced TGF-beta 1 secretion in a time-dependent manner (3.72 +/- 0.29 and 4.30 +/- 0.50 pg/microg cellular protein at 24 hours and 48 hours respectively, compared to 2.13 +/- 0.23 and 2.65 +/- 0.32 pg/microg cellular protein at 24 hours and 48 hours, respectively for 5 mmol glucose; P < 0.001 at both time points). Such induction was ameliorated by emodin (20 microg/mL) (TGF-beta 1 concentration 2.25 +/- 0.15 and 2.96 +/- 0.33 pg/microg cellular protein at 24 hours and 48 hours, respectively, in the presence of emodin and 30 mmol D-glucose; P < 0.001 compared to 30 mmol D-glucose alone at both time points). Induction of TGF-beta 1 synthesis by 30 mmol D-glucose was associated with induction of PKC alpha, phosphorylation of cAMP-responsive element binding protein (CREB) and activating transcription factor-1 (ATF-1), and increased fibronectin and type I collagen translation. Emodin abrogated all these effects of concentrated glucose. Immunohistochemical staining showed that 30 mmol D-glucose induced cytoplasmic, perinuclear, and extracellular fibronectin and type I collagen expression by HPMC. Emodin reduced 30 mmol D-glucose-induced cytoplasmic and extracellular matrix synthesis to near basal levels. Our findings demonstrate that emodin ameliorates the undesirable effects of concentrated glucose on HPMC via suppression of PKC activation and CREB phosphorylation, and suggest that emodin may have a therapeutic potential in the prevention or treatment of glucose-induced structural and functional abnormalities in the peritoneal membrane.