Interaction between the polyol pathway and non-enzymatic glycation on aortic smooth muscle cell migration and monocyte adhesion

We investigated for the interaction between the polyol pathway and enhanced non-enzymatic glycation, both implicated in the pathogenesis of diabetic atherosclerosis, in the activation of aortic smooth muscle cell (SMC) function. Mouse aortas and primary cultures of SMCs from wildtype (WT) mice and transgenic (TG) mice expressing human aldose reductase (AR) were studied regarding changes in AR activity, and SMC gene activation, migration and monocyte adhesion, in response to advanced glycation end-product modified BSA (AGE-BSA). Results showed that AGE-BSA increased AR activity in both WT and TG aortas, with greater increments (p < 0.05) in TG aortas which, basally, had elevated AR activity (2.8 fold of WT). These increments were attenuated by zopolrestat, an AR inhibitor. Similar AGE-induced increments in AR activity were observed in primary cultures of aortic SMCs from WT and TG mice (60% and 100%, respectively, P < 0.01). Such increments were accompanied by increases in intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) mRNA levels (both P < 0.05), activation of membrane-associated PKC-beta1 (P < 0.05) as well as increased SMC migration and Tamm-Horsfall protein (THP)-1 monocyte adhesion to SMCs (both p < 0.01), with all changes being significantly greater in TG SMCs (P < 0.05) and suppressible by either zopolrestat or transfection with an AR antisense oligonucleotide. Our findings suggest that the effects of AGEs on SMC activation, migration and monocyte adhesion are mediated partly through the polyol pathway and, possibly, PKC activation. The greater AGE-induced changes in the TG SMCs have provided further support for the dependency of such changes on polyol pathway hyperactivity.     

Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values

To facilitate the identification of candidate molecular biomarkers that are linked to the pathogenesis of hepatocellular carcinoma (HCC), we investigated protein-expression profiles of 146 tissue specimens including 67 pairs of tumors and adjacent non-tumors resected from HCC patients as well as 12 normal livers by 2-DE. Among the 1800 spots displayed in the liver proteome, a total of 90 protein species were found to be significantly different between the three groups (P < 0.05). Three of the top candidate markers up-regulated in HCC, with high receiver operating characteristic (ROC) curves, were identified by MS/MS analysis and belonged to the chaperone members: heat-shock protein (Hsp)27, Hsp70 and glucose-regulated protein (GRP)78. Over-expression of these chaperone proteins in HCC tissues was confirmed by Western blotting and immunohistochemistry. In correlation with clinico-pathological parameters, expression of Hsp27 was linked to alpha-fetoprotein level (P = 0.007) whereas up-regulation of GRP78 was associated with tumor venous infiltration (P = 0.035). No significant association of Hsp70 with any pathologic features was observed. The present HCC proteome analysis revealed that in response to the stressful cancerous microenvironment, tumor cells strived to increase the expression of chaperone proteins for cyto-protective function and to enhance tumor growth and metastasis.     

Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer

A glycomic approach is developed to identify oligosaccharide markers for ovarian cancer by rapidly profiling globally released oligosaccharides. Glycoproteins shed by cancer cells are found in the supernatant (or conditioned media) of cultured cells. In this approach, shed glycoproteins are profiled for their oligosaccharide content using beta-elimination conditions. Changes in glycosylation are monitored by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Because shed glycoproteins would also be found in serum, similar glycan profiling was performed to observe potential oligosaccharide markers. Oligosaccharide profiling data on a limited set of patient and normal serum samples were studied to determine potential glycan markers in ovarian cancer. We were able to demonstrate the presence of at least 15 unique serum glycan markers in all patients but absent in normal individuals. To determine the structure of the glycan biomarkers, a number of the ions were isolated and further analyzed using infrared multiphoton dissociation (IRMPD). One major advantage of this approach is that glycans are examined directly from patient sera without the need to obtain cancer biopsy specimens or to purify glycosylated proteins from these specimens.     

Methylation analysis by DNA immunoprecipitation

DNA methylation regulates gene expression primarily through modification of chromatin structure. Global methylation studies have revealed biologically relevant patterns of DNA methylation in the human genome affecting sequences such as gene promoters, gene bodies, and repetitive elements. Disruption of normal methylation patterns and subsequent gene expression changes have been observed in several diseases especially in human cancers. Immunoprecipitation (IP)‐based methods to evaluate methylation status of DNA have been instrumental in such genome‐wide methylation studies. This review describes techniques commonly used to identify and quantify methylated DNA with emphasis on IP based platforms. In an effort to consolidate the wealth of information and highlight critical aspects of methylated DNA analysis, sample considerations, experimental and bioinformatic approaches for analyzing genome‐wide methylation profiles, and the benefit of integrating DNA methylation data with complementary dimensions of genomic data are discussed. J. Cell. Physiol. 222: 522–531, 2010. © 2009 Wiley‐Liss, Inc.     

Soluble and surface-bound aminopeptidase in eosinophilic blood cells from Mytilus edulis

This study focused on soluble and surface-bound aminopeptidase (AP) in hemocytes from Mytilus edulis and on the identification of the enzyme-producing blood cells. The cell extract hydrolyzed alanine p-nitroanilide (Ala-pNA) with an optimum between pH 6.4 and 7.0. Following native gradient PAGE of extract, alanyl methoxy-naphthylamide (AMNA) was converted by one band with an estimated molecular weight of 375 kDa; it included at least ten putative AP-isozymes with isoelectric points between pH 4.5 and 5.8. In addition to this soluble form, electron microscopy revealed simultaneous conversion of AMNA on the surface of blood cells. Individual mussels expressed AP-molecules in 23-39% of their hemocytes. These cells were shown to represent eosinophilic granulocytes.     

Involvement of calcium mobilization from caffeine-sensitive stores in mechanically induced cell cycle arrest in the dinoflagellate Crypthecodinium cohnii

Mechanical loads can profoundly alter cell growth and cell proliferation. The dinoflagellates are especially sensitive to mechanical stimulation. Many species will be arrested in cell cycle in response to turbulence or shear stress. We demonstrate here that mechanical shaking and caffeine, the ryanodine-receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate Crypthecodinium cohnii. Dantrolene, a ryanodine-receptor antagonist, dose-dependently inhibited both shaking-induced and caffeine-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently and reversibly induced cell cycle arrest in dinoflagellates. Prolonged shaking substantially abolished the magnitude of caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores through ryanodine receptors. Fluorescence-conjugated ryanodine gave positive labeling, which could be blocked by ryanodine, in the cortice of C. cohnii cells. In addition, caffeine or shaking mobilized intracellular chlortetracycline (CTC)-positive membrane-bound calcium, which could be similarly depleted by t-BuBHQ, a SERCA pump inhibitor. Prior treatment with shaking or caffeine also inhibited the ability of the other agent in mobilizing CTC-positive calcium. CTC-positive microsomal fractions could also be induced to release calcium by caffeine and cADPR, the ryanodinee receptor modulator. t-BuBHQ, but not calcium ionophores, induced cell cycle arrest, and the calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. These data culminate to suggest that mobilization or depletion of caffeine-sensitive calcium stores, but not calcium elevation per se, is involved in the induction of cell cycle arrest by mechanical stimulation. The present study establishes the role of caffeine-sensitive calcium stores in the regulation of cell cycle progression.