Objective. Protein kinase C (PKC ), an atypical PKC, has been found to be transcriptionally upregulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKC in interleukin-1 (IL-1)-induced NF-B signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation.Methods. Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKC . Western blot analysis was used to evaluate phosphorylation of PKC and NF-B. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production.Results. Phosphorylation of PKC and NF-B was induced by IL-1 treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKC suppressed IL-1-induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1-induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKC and NO production. Furthermore, small interfering RNA-or short hairpin RNA-mediated knockdown of PKC mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. Conclusion. Our results show that PKC is involved in the regulation of IL-1-induced NF-B signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2.Therefore, inhibition of PKC could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA.Osteoarthritis (OA) is a degenerative joint disorder characterized by destruction and loss of articular cartilage as a result of an imbalance between catabolic and anabolic cartilage matrix metabolism. It is the most common form of arthritis. Chondrocytes are the single cell type present in cartilage, and they play an important role in the pathogenesis of OA. They are the primary source of type II collagen and aggrecan, the most important components of the cartilage matrix. Cartilage loss in OA is characterized by matrix degradation and chondrocyte death.In OA, catabolic factors, including interleukin-1 (IL-1) and tumor necrosis factor (TNF), have been implicated in stimulating chondrocytes to produce matrix-degrading enzymes including aggrecanases (ADAMTS-4 and ADAMTS-5) and other metalloproteinases, resulting in degradation of aggrecan and collagen, respectively (1,2). Catabolic cytokines have also been demonstrated to induce synthesis of type 2 nitric oxide synthase (NOS2), resulting in the formation of nitric oxide (NO), an inducer of chondrocyte apoptosis (3). Studies of inducible NOS-null mice and investiga-
Ectopic expression of recombinant human bone morphogenetic protein 2 (rhBMP2) induces osteogenesis, while ectopic expression of rhBMP12 and rhBMP13 induces the formation of tendon-like tissue. Despite their different in vivo activities, all three ligands bound to the type I bone morphogenic protein receptors (BMPRs), activin receptor-like kinase (ALK)-3 and ALK6, and to the type II BMPRs, activin receptor type-2A, activin receptor type-2B, and BMPR2, with similar affinities. Treatment of C3H10T1/2 cells with rhBMP2 activated SMAD signaling and induced expression of osteoblast markers including osteocalcin mRNA (Ocn). In contrast, treatment with rhBMP12 or rhBMP13 resulted in a dose-dependent induction of a tendon-specific gene (Thbs4) expression with no detectable activation of SMAD 1, 5, and 8. Differential regulation of Thbs4 and Ocn has potential utility as an in vitro biomarker for induction of tenogenic signaling. Such an assay also permits the ability to distinguish between the activities of different BMPs and may prove useful in studies on the molecular mechanisms of BMP tenogenic activity.
ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.
Neratinib (HKI-272) is a small molecule tyrosine kinase inhibitor of the ErbB receptor family currently in Phase III clinical trials. Despite its efficacy, the mechanism of potential cellular resistance to neratinib and genes involved with it remains unknown. We have used a pool-based lentiviral genome-wide functional RNAi screen combined with a lethal dose of neratinib to discover chemoresistant interactions with neratinib. Our screen has identified a collection of genes whose inhibition by RNAi led to neratinib resistance including genes involved in oncogenesis (e.g. RAB33A, RAB6A and BCL2L14), transcription factors (e.g. FOXP4, TFEC, ZNF), cellular ion transport (e.g. CLIC3, TRAPPC2P1, P2RX2), protein ubiquitination (e.g. UBL5), cell cycle (e.g. CCNF), and genes known to interact with breast cancer-associated genes (e.g. CCNF, FOXP4, TFEC, several ZNF factors, GNA13, IGFBP1, PMEPA1, SOX5, RAB33A, RAB6A, FXR1, DDO, TFEC, OLFM2). The identification of novel mediators of cellular resistance to neratinib could lead to the identification of new or neoadjuvant drug targets. Their use as patient or treatment selection biomarkers could make the application of anti-ErbB therapeutics more clinically effective.
Transient and stable expression of foreign genes has been achieved in sweet potato using the particle bombardment system of gene delivery. Callus and root isolates of two genotypes (Jewel and TIS-70357) with positive signs of transformation have been recovered. Tungsten microcarriers coated with plasmid DNA (pBI 221 containing the gusA gene) were accelerated at high velocity using a biolistic device into sweet potato target tissues. Histochemical examination of bombarded leaf and petiole explants revealed that most had cells expressing the gusA gene. When explants were cultured, calli and roots developed in most bombarded tissues. Similar results but with a lower frequency of transformation were observed when the plasmid pBI 121 (with gusA and antibiotic resistance npt II genes) was employed and bombarded explants cultured on an antibiotic selection medium. Subcultured roots and calli were positive for gusA expression when tested even after one year of in vitro culture, and thus the expression of the foreign gene is fairly stable. The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.
This is a rare case report of a patient around 11 years with the complaint of extra mouth who reported to the hospital for removal of that extra mouth. On examination there was accessory oral cavity with small upper and lower lips, seven teeth and saliva was drooling out. Under general anesthesia crevicular incision from 32 to 43 was put and labial gingiva with alveolar mucosa was reflected completely and bone exposed to lower border of mandible. There were seven teeth resembling lower permanent anterior teeth in the accessory mouth, which was excised with the accessory lips. 41 extracted and osteotomy carried out extending the incision from the extracted site and osteotomy carried out. Dermoid cyst both below and above the mylohyoid muscle and rudimentary tongue found and excised and the specimen sent for histopathological examination. The wound was closed and uneventful healing noted to the satisfaction of the patient. This is a rare and interesting case which has been documented.
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