TGFß promotes mesenchymal phenotype of pancreatic cancer cells, in part, through epigenetic activation of VAV1.

The highly homeostasis-resistant nature of cancer cells leads to their escape from treatment and to liver metastasis, which in turn makes pancreatic ductal adenocarcinoma (PDAC) difficult to treat, especially the squamous/epithelial-to-mesenchymal transition (EMT)-like subtype. As the molecular mechanisms underlying tumour heterogeneity remain elusive, we investigated whether epigenetic regulation might explain inter-individual differences in the progression of specific subtypes. DNA methylation profiling performed on cancer tissues prior to chemo/radiotherapy identified one hypermethylated CpG site (CpG6882469) in the VAV1 gene body that was correlated with demethylation of two promoter CpGs (CpG6772370/CpG6772811) in both PDAC and peripheral blood. Transforming growth factor ß treatment induced gene-body hypermethylation, dissociation of DNMT1 from the promoter, and VAV1 expression via SMAD4 and mutant KrasG12D. Pharmacological inhibition of TGFß-VAV1 signalling decreased the squamous/EMT-like cancer cells, promoted nuclear VAV1 localization, and enhanced the efficacy of gemcitabine in prolonging the survival of KPfl/flC mice. Together, the three VAV1 CpGs serve as biomarkers for prognosis and early detection, and the TGFß-VAV1 axis represents a therapeutic target.

Enhanced LPS-induced peritonitis in mice deficiency of cullin 4B in macrophages.

Cullin 4B (CUL4B), a member of the cullin protein family, is a scaffold protein of the CUL4B-RING-E3 ligase complex that ubiquitinates intracellular proteins.CUL4B's targets include cell cycle-regulated proteins and DNA replication-related molecules. In this study, we generated myeloid-specific Cul4b-deficient mice (Cul4b(f/y);LysM-Cre(KI/KI)) to investigate the influence of Cul4b deficiency on innate immunity, especially on the function of macrophages. Our results show that an intraperitoneal injection of lipopolysaccharide (LPS) led to a significant decrease in body weights and increased leukocyte infiltrates with increased chemokines in the peritoneal cavity of Cul4b(f/y);LysM-Cre(KI/KI) mice. However, the proinflammatory cytokines, IL-6 and TNF-α did not increase in LPS-injected Cul4b(f/y);LysM-Cre(KI/KI) mice. Furthermore, bone marrow-derived macrophages from Cul4b(f/y);LysM-Cre(KI/KI) mice secreted higher levels of chemokines but lower levels of TNF-α and IL-6 upon LPS stimulation. Of note, increased proliferation of Cul4b-deficient macrophages was also observed. These results show that myeloid-specific Cul4b deficiency worsens LPS-induced peritonitis. In addition, Cul4b deficiency leads to enhanced DNA replication and proliferation, increased production of chemokines but a decreased production of proinflammatory cytokines of macrophages. Our data highlight a new role of cullin family, CUL4B, in the immune system.

Study on bending behaviour of nickel-titanium rotary endodontic instruments by analytical and numerical analyses.

AIM: To develop analytical models and analyse the stress distribution and flexibility of nickel-titanium (NiTi) instruments subject to bending forces. METHODOLOGY: The analytical method was used to analyse the behaviours of NiTi instruments under bending forces. Two NiTi instruments (RaCe and Mani NRT) with different cross-sections and geometries were considered. Analytical results were derived using Euler-Bernoulli nonlinear differential equations that took into account the screw pitch variation of these NiTi instruments. In addition, the nonlinear deformation analysis based on the analytical model and the finite element nonlinear analysis was carried out. Numerical results are obtained by carrying out a finite element method. RESULTS: According to analytical results, the maximum curvature of the instrument occurs near the instrument tip. Results of the finite element analysis revealed that the position of maximum von Mises stress was near the instrument tip. Therefore, the proposed analytical model can be used to predict the position of maximum curvature in the instrument where fracture may occur. Finally, results of analytical and numerical models were compatible. CONCLUSION: The proposed analytical model was validated by numerical results in analysing bending deformation of NiTi instruments. The analytical model is useful in the design and analysis of instruments. The proposed theoretical model is effective in studying the flexibility of NiTi instruments. Compared with the finite element method, the analytical model can deal conveniently and effectively with the subject of bending behaviour of rotary NiTi endodontic instruments.

Diagnosing primary and metastatic renal cell carcinoma: the use of the monoclonal antibody 'Renal Cell Carcinoma Marker'.

The diagnosis of primary or metastatic renal cell carcinoma (RCC) can be difficult, especially in small biopsies, because of the wide variety of histologic appearances and clinical presentations that RCC can assume. An immunomarker specific for RCC is currently not available. We tested the relevant diagnostic use of the Renal Cell Carcinoma Marker (RCC Ma), a monoclonal antibody, against a normal human proximal tubular brush border antigen. Immunostaining using RCC Ma and the avidin-biotin-peroxidase complex technique was performed on archival tissues from primary and metastatic tumors of renal or nonrenal origin. A total of 122 of 153 primary RCCs (79.7%) were positive [clear cell (84%), papillary (96%), chromophobe (45%), sarcomatoid (25%), and collecting duct (0%)], with > or =10% of tumor cells stained in 93% of cases. None of the 64 primary renal tumors other than RCC, including 15 oncocytomas, was positive. Fifteen of 146 (10.2%) nonrenal primary tumors were positive (5 of 17 breast tumors, 8 of 8 parathyroid adenomas, and 2 of 7 embryonal carcinomas). Forty-two of 63 (67%) metastatic RCCs were positive with > or =10% of cells being stained in 83% of them. Two of 108 (2%) metastases from tumors other than RCCs were positive, both of which were metastatic breast carcinomas; however, only 10% (2 of 19) of metastatic breast carcinomas were positive. RCC Ma is an excellent marker for primary RCC, which should facilitate its diagnosis in a small biopsy. Although RCC Ma remains highly specific (98%) for metastatic RCC, a negative result may not rule out metastatic RCC because of a rather low sensitivity and a focal staining pattern in some of the positive cases. RCC Ma may also facilitate the differential diagnosis between oncocytoma and other types of RCC when they are composed mostly of eosinophilic cells.

Cytochrome P450 CYP2J9, a new mouse arachidonic acid omega-1 hydroxylase predominantly expressed in brain.

A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57-58-kDa 502-amino acid polypeptide that is 70-91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9 cells using a baculovirus system. Microsomes of CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca(2+) channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca(2+) channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.

Molecular cloning, structural analysis, and expression of carp ZP3 gene.

Two types of cDNAs coding for a major component of carp egg membrane were clones from a carp ovarian cDNA library. They encode polypeptides of 422-424 amino acid residues whose sequences are homologous to those of medaka and mammalian ZP3. Similar to the mammalian ZP3 genes, carp ZP3 gene also consists of eight exons and seven introns. Carp ZP3 genes are 2.9 kb in length and present in multiple forms. Carp ZP3 is a glycoprotein of 45 kDa. It was transcribed and translated exclusively in oocytes, in contrast with medaka ZP3, which was synthesized in liver. The transcription of carp ZP3 starts very early in oogenesis, but translation occurs during vitellogenesis, as it is present in vitellogenic but not in previtellogenic oocytes. ZP3 content in oocytes increases as vitellogenesis proceeds.