Impact of MCP-1 and CCR-2 gene polymorphisms on coronary artery disease susceptibility

Coronary artery disease (CAD) was the second leading cause of death during the last 3 years in Taiwan. Smooth muscle cells, monocytes/macrophages, and endothelial cells produce monocyte chemoattractant protein-1 (MCP-1) within atherosclerotic plaques following binding to the chemokine receptor-2 (CCR-2). Previous studies have well-documented the association between MCP-1 expression and susceptibility to, or clinicopathological features, of CAD. This study investigated the relationships between MCP-1-2518A/G and CCR-2-V64I genetic polymorphisms and CAD in the Taiwanese population. A total of 608 subjects, including 392 non-CAD controls and 216 patients with CAD, were recruited and subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to evaluate the effects of these two polymorphic variants on CAD. Results indicated a significant association between MCP-1 -2548 gene polymorphism and susceptibility to CAD. GG genotypes (OR = 1.629; 95 % CI = 1.003-2.644), or individuals with at least one G allele (OR = 1.511; 95 % CI = 1.006-2.270), had a higher risk of CAD as compared with AA genotypes. Results also revealed that subjects with at least one A allele of the V64I CCR2 gene polymorphism had significantly increased risk of CAD. G allele in MCP-1-2518 might contribute to higher prevalence of atrial fibrillation in CAD patients (OR = 4.254; p < 0.05). In conclusion, MCP-1-2518G and CCR-2 64I gene polymorphisms represent important factors in determining susceptibility to CAD, and the contribution of MCP-1-2518G could be through effects on atrial fibrillation in CAD patients.     

Insulin detemir enhances proglucagon gene expression in the intestinal L cells via stimulating β-catenin and CREB activities

Insulin therapy using insulin detemir (d-INS) has demonstrated weight-sparing effects compared with other insulin formulations. Mechanisms underlying these effects, however, remain largely unknown. Here we postulate that the intestinal tissues' selective preference allows d-INS to exert enhanced action on proglucagon (Gcg) expression and the production of glucagon-like peptide (GLP)-1, an incretin hormone possessing both glycemia-lowering and weight loss effects. To test this hypothesis, we used obese type 2 diabetic db/db mice and conducted a 14-day intervention with daily injection of a therapeutic dose of d-INS or human insulin (h-INS) in these mice. The body weight of the mice after 14-day daily injection of d-INS (5 IU/kg) was decreased significantly compared with those injected with the same dose of h-INS or saline. The weight-sparing effect of d-INS was associated with significantly elevated circulating levels of total GLP-1 and reduced food intake. Histochemistry analysis demonstrated that d-INS induced rapid phosphorylation of protein kinase B (Akt) in the gut L cells of normal mice. Western blotting showed that d-INS stimulated Akt activation in a more rapid and enhanced fashion in the mouse distal ileum compared with those by h-INS. In vitro investigation in primary fetal rat intestinal cell (FRIC) cultures showed that d-INS increased Gcg mRNA expression as determined by Northern blotting and real-time RT-PCR. Consistent with these in vivo investigations, d-INS significantly increased GLP-1 secretion in FRIC cultures. Consistently, d-INS was also shown to induce rapid phosphorylation of Akt in the clonal gut cell line GLUTag. Furthermore, d-INS increased β-catenin phosphorylation, its nuclear translocation, and enhanced cAMP response element-binding protein (CREB) phosphorylation in a phosphatidylinositol 3-kinase and/or mitogen-activated protein kinase kinase/extracellular signal-regulated kinase-sensitive manner. We suggest that the weight-sparing benefit of d-INS in mice is related to its intestinal tissues preference that leads to profound stimulation of Gcg expression and enhanced GLP-1 secretion in intestinal L cells, potentially involving the activation of insulin/β-catenin/CREB signaling pathways.     

Protein transduction in human cells is enhanced by cell-penetrating peptides fused with an endosomolytic HA2 sequence

Endocytosis has been proposed as one of the primary mechanisms for cellular entry of cell-penetrating peptides (CPPs) and their cargoes. However, a major limitation of endocytic pathway is entrapment of the CPP-cargo in intracellular vesicles from which the cargo must escape into the cytoplasm to exert its biological activity. Here we demonstrate that a CPP tagged with an endosomolytic fusion peptide derived from the influenza virus hemagglutinin-2 (HA2) remarkably enhances the cytosolic delivery of proteins in human A549 cells. To determine the endosome-disruptive effects, recombinant DNA plasmids containing coding sequences of HA2, CPPs and red fluorescent proteins (RFPs) were constructed. The fusion proteins were purified from plasmid-transformed Escherichia coli, and their effects on protein transduction were examined using live cell imaging and flow cytometry. Our data indicate that endocytosis is the major route for cellular internalization of CPP-HA2-tagged RFP. Mechanistic studies revealed that the fusogenic HA2 peptide dramatically facilitates CPP-mediated protein entry through the release of endocytosed RFPs from endosomes into the cytoplasm. Furthermore, incorporating the HA2 fusion peptide of the CPP-HA2 fusion protein improved cytosolic uptake without causing cytotoxicity. These findings strongly suggest that the CPP-HA2 tag could be an efficient and safe carrier that overcomes endosomal entrapment of delivered therapeutic drugs.     

SERS Study of Histamine by Using Silver Film over Nanosphere Structure

Optical properties of histamine and l-histidine have been analyzed by using surface-enhanced Raman scattering (SERS). A silver film over nanosphere (AgFON) structure with 120-nm-thick silver film on polystyrene nanospheres 1,000?nm in diameter is fabricated by nanosphere lithography to enhance the Raman signal excited at the laser wavelength of 532?nm. Normal Raman spectrum and the SERS spectrum of histamine and l-histidine were compared. Further, vibration modes of these molecules were calculated by using density functional method. In the SERS experiment, we were able to measure the Raman spectrum with a histamine concentration as less as 100?pM. This sensitivity is higher than that from high-performance liquid chromatography.     

Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein

Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.     

Clinicodemographic aspect of resectable pancreatic cancer and prognostic factors for resectable cancer

ABSTRACT: BACKGROUND: Pancreatic adenocarcinoma (PCA) is one of the most lethal human malignancies, and radical surgery remains the cornerstone of treatment. After resection, the overall 5-year survival rate is only 10% to 29%. At the time of presentation, however, about 40% of patients generally have distant metastases and another 40% are usually diagnosed with locally advanced cancers. The remaining 20% of patients are indicated for surgery on the basis of the results of preoperative imaging studies; however, about half of these patients are found to be unsuitable for resection during surgical exploration. In the current study, we aimed to determine the clinicopathological characteristics that predict the resectability of PCA and to conduct a prognostic analysis of PCA after resection to identify favorable survival factors. METHODS: We retrospectively reviewed the medical files of 688 patients (422 men and 266 women) who had undergone surgery for histopathologically proven PCA in the Department of Surgery at Chang Gung Memorial Hospital in Taiwan from 1981 to 2006. We compared the clinical characteristics of patients who underwent resection and patients who did not undergo resection in order to identify the predictive factors for successful resectability of PCA, and we conducted prognostic analysis for PCA after resection. RESULTS: A carbohydrate antigen 19-9 (CA 19-9) level of 37 U/ml or greater and a tumor size of 3 cm or more independently predicted resectability of PCA. In terms of survival after resection, PCA patients with better nutritional status (measured as having an albumin level greater than 3.5 g/dl), radical resection, early tumor stage and better-differentiated tumors were associated with favorable survival. CONCLUSIONS: Besides traditional imaging studies, preoperative CA 19-9 levels and tumor size can also be used to determine the resectability of PCA. Better nutritional status, curative resection, early tumor stage and well-differentiated tumors predict the favorable prognosis of PCA patients after resection.     

Extended release of bevacizumab by thermosensitive biodegradable and biocompatible hydrogel

The antibody bevacizumab (Avastin) has been used clinically to treat intraocular neovascular diseases based on its antivascular endothelial growth factor (VEGF) character. The anti-VEGF strategy for retinal neovascular diseases is limited by the short half-life of bevacizumab and thus requires frequent injections. This Article reports the sustained release of bevacizumab from a biocompatible material that is composed of a triblock copolymer of poly(2-ethyl-2-oxazoline)-b-poly(ε-caprolactone)-b-poly(2-ethyl-2-oxazoline) (PEOz-PCL-PEOz). The amphiphilic PEOz-PCL-PEOz triblock copolymer was synthesized in three steps. First, the PEOz was polymerized by methyl p-toluenesulfonate and 2-ethyl-2-oxazoline (EOz), and the living end was terminated by potassium hydroxide methanolic solution. Subsequently, the hydroxyl-PEOz was used as a macroinitiator for the ring-opening polymerization of ε-caprolactone using a Tin(II) octoate catalyst to synthesize the telechelic hydroxylated PEOz-PCL. Finally, the PEOz-PCL-PEOz triblock copolymer was obtained using the 1,6-hexamethylene diisocyanateas a coupling reagent. The PEOz-PCL-PEOz was chemically and molecularly characterized by GPC, (1)H NMR, and FTIR, and its aqueous solution (ECE hydrogel) showed a reversible sol (room temperature)-gel (physiological temperature) phase transition, which serves as an easy antibody-packing system with extended release. The biodegradability of ECE hydrogel was assessed by the porosity formation at different periods by scanning electron microscopy. The ECE hydrogel had no in vitro cytotoxicity on the human retinal pigment epithelial cell line by flow cytometry. The histomorphology and electrophysiology of the rabbit neuroretina were preserved after 2 months of intravitreal injection. In conclusion, the ECE hydrogel has a temperature-sensitive sol-gel phase transition and is effective in vitro. Its intraocular biocompatibility demonstrated its great potential to be widely used in biomedical applications for extended drug release.     

TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death

Self-aggregation of transforming growth factor β (TGF-β)1-induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid β (Aβ) for causing neurodegeneration. Here, we determined that overexpressed TIAF1 exhibits as aggregates together with Smad4 and Aβ in the cancer stroma and peritumor capsules of solid tumors. Also, TIAF1/Aβ aggregates are shown on the interface between brain neural cells and the metastatic cancer cell mass. TIAF1 is upregulated in developing tumors, but may disappear in established metastatic cancer cells. Growing neuroblastoma cells on the extracellular matrices from other cancer cell types induced production of aggregated TIAF1 and Aβ. In vitro induction of TIAF1 self-association upregulated the expression of tumor suppressors Smad4 and WW domain-containing oxidoreductase (WOX1 or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further enhanced Aβ formation. TIAF1 is known to suppress SMAD-regulated promoter activation. Intriguingly, without p53, self-aggregating TIAF1 spontaneously activated the SMAD-regulated promoter. TIAF1 was essential for p53-, WOX1- and dominant-negative JNK1-induced cell death. TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage-independent growth, blocking cell migration and causing apoptosis. Together, TIAF1 shows an aggregation-dependent control of tumor progression and metastasis, and regulation of cell death.