Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.     

Variation in Storage α-Polyglucans of Red Algae: Amylose and Semi-Amylopectin Types in <Emphasis Type="Italic">Porphyridium</Emphasis> and Glycogen Type in <Emphasis Type="Italic">Cyanidium</Emphasis>

Red algae are widely known to produce floridean starch but it remains unclear whether the molecular structure of this algal polyglucan is distinct from that of the starch synthesized by vascular plants and green algae. The present study shows that the unicellular species Porphyridium purpureum R-1 (order Porphyridiales, class Bangiophyceae) produces both amylopectin-type and amylose-type alpha-polyglucans. In contrast, Cyanidium caldarium (order Porphyridiales, class Bangiophyceae) synthesizes glycogen-type polyglucan, but not amylose. Detailed analysis of alpha-1,4-chain length distribution of P. purpureum polyglucan suggests that the branched polyglucan has a less ordered structure, referred to as semi-amylopectin, as compared with amylopectin of rice endosperm having a tandem-cluster structure. The P. purpureum linear amylose-type polyglucan, which has a lambda(max) of 630 nm typical of amylose-iodine complex and is resistant to Pseudomonas isoamylase digestion, accounts for less than 10% of the total polyglucans. We produced and isolated a cDNA encoding a granule-bound starch synthase (GBSS)-type protein of P. purpureum, which is probably the approximately 60-kDa protein bound tightly to the starch granules, resembling the amylose-synthesizing GBSS protein of green plants. The present investigation indicates that the class Bangiophyceae includes species producing both semi-amylopectin and amylose, and species producing glycogen alone.     

RhoA-mediated Phospholipase D1 signaling is not required for the formation of stress fibers and focal adhesions

The small GTPase RhoA regulates a wide spectrum of cellular functions including transformation and cytoskeletal reorganization. A large number of proteins have been identified as targets of RhoA, but their specific roles in these processes are not clear. Phospholipase D (PLD) was shown to be one such target several years ago; more recent work from our laboratory and others has demonstrated that of the two mammalian PLD isozymes, PLD1 but not PLD2 is activated by RhoA and this activation proceeds through direct binding both in vitro and in vivo. In this study, using a series of RhoA mutants, we have defined a PLD1-specific interacting site on RhoA composed of the residues Asn41, Trp58 and Asp76, using the yeast two-hybrid system, co-immunoprecipitation, and a PLD in vivo assay. The results further substantiate our previous finding that RhoA activates PLD1 through direct interaction. These mutants were then used to investigate the role of PLD1 in the cytoskeletal reorganization stimulated by RhoA signaling. Our results show that PLD1 is not required for the RhoA-mediated stress fiber and focal adhesion formation. The lack of importance of PLD1 signaling in RhoA-mediated cytoskeletal reorganization is further supported by the observation that PLD1 depletion using an shRNA approach and tetracycline-induced overexpression of the wild-type and the catalytically inactive mutant of PLD1 in stable cell lines do not alter stress fiber and focal adhesion formation.     

PVA-gel beads enhance granule formation in a UASB reactor

PVA-gel beads were used as a biocarrier in a lab-scale UASB reactor treating synthetic wastewater composed of corn steep liquor (CSL) with the aim of evaluating its use as a growth nucleus to enhance granule formation. Over 117 days of operation, the organic loading rate was increased to 22.5kgCOD/m(3)/day with an influent COD of about 10.8g/L at an HRT of 12h with COD removal efficiencies greater than 87%. By the end of the study period, the PVA-gel turned black and granule formation was achieved as compared with the formation of much fewer natural granules without the PVA-gel nucleus. No filamentous bacteria were found on the surface or interior of the PVA-gel beads. The PVA-gel granules had an average settling velocity 200m/h (5cm/s), and a biomass attachment of 0.93g VSS/g PVA-gel. The required time for formation of PVA-gel granules was thus demonstrated to be shorter than that of ordinary sludge granules under the experimental conditions used in this study.     

Identification of organoselenium compounds that possess chemopreventive properties in human prostate cancer LNCaP cells

The process of cancer development consists of three sequential stages termed initiation, promotion, and progression. Oxidative stress damages DNA and introduces mutations into oncogenes or tumor suppressor genes, thus contributing to cancer development. Cancer chemoprevention is defined to prevent or delay the development of cancer by the use of natural or synthetic substances. In the present study, we synthesized a series of organoselenium compounds and evaluated their possible chemopreventive properties in human prostate cancer LNCaP cells. Among 42 organoselenium compounds tested, two compounds, 3-selena-1-dethiacephem 13 and 3-selena-1-dethiacephem 14 strongly activated the Nrf2/ARE (antioxidant response element) signaling and thus markedly increased expression of heme oxygenase-1 (HO-1), a phase II antioxidant enzyme. Translocation of Nrf2 to the nucleus preceded HO-1 protein induction by two compounds. The intracellular ROS level was strongly reduced immediately after treatment with these compounds, showing that they are potent antioxidants. Finally, both compounds inhibited cell growth via cell cycle arrest. Our findings suggest that compounds 13 and 14 could not only attenuate oxidative stress through Nrf2/ARE activation and direct ROS scavenging but also inhibit cell growth. Thus, these compounds possess the potential as pharmacological agents for chemoprevention of human prostate cancer.     

Glucose Homeostatic Law: Insulin Clearance Predicts the Progression of Glucose Intolerance in Humans

Homeostatic control of blood glucose is regulated by a complex feedback loop between glucose and insulin, of which failure leads to diabetes mellitus. However, physiological and pathological nature of the feedback loop is not fully understood. We made a mathematical model of the feedback loop between glucose and insulin using time course of blood glucose and insulin during consecutive hyperglycemic and hyperinsulinemic-euglycemic clamps in 113 subjects with variety of glucose tolerance including normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM). We analyzed the correlation of the parameters in the model with the progression of glucose intolerance and the conserved relationship between parameters. The model parameters of insulin sensitivity and insulin secretion significantly declined from NGT to IGT, and from IGT to T2DM, respectively, consistent with previous clinical observations. Importantly, insulin clearance, an insulin degradation rate, significantly declined from NGT, IGT to T2DM along the progression of glucose intolerance in the mathematical model. Insulin clearance was positively correlated with a product of insulin sensitivity and secretion assessed by the clamp analysis or determined with the mathematical model. Insulin clearance was correlated negatively with postprandial glucose at 2h after oral glucose tolerance test. We also inferred a square-law between the rate constant of insulin clearance and a product of rate constants of insulin sensitivity and secretion in the model, which is also conserved among NGT, IGT and T2DM subjects. Insulin clearance shows a conserved relationship with the capacity of glucose disposal among the NGT, IGT and T2DM subjects. The decrease of insulin clearance predicts the progression of glucose intolerance.     

Dynamic patterns in the locomotion and feeding behaviors by the placozoan Trichoplax adhaerence

The placozoan Trichoplax adhaerence is one of the most primitive multi-cellular organisms, and moves about accompanying perpetual changes in its shape. Changes in position, locomotion velocity and the outer shape of the organism were monitored quantitatively with use of a computer image analysis, and their dynamic patterns in free locomotion and upon feeding were analyzed in terms of non-linear dynamics. The organism changed its behavioral patterns discontinuously in response to various concentrations of yeast extracts (food). (1) At low concentrations, the organism moved fast with perpetual random changes in shape. Both locomotion velocity and shape changes exhibited 1/f fluctuations. (2) At high concentrations, the shape of the organism as well as the locomotion exhibited oscillations with periods of about 8 min. These limit cycle oscillations bifurcated into the period 2 at the highest concentration tested. The organism flattened more strongly and the locomotion was more reduced on the whole at higher concentrations. (3) At the intermediate concentrations, two patterns as monitored above appeared: one pattern continued for a while and switched to the other abruptly. (4) The average square displacement of the organism increased linearly with time in all cases, indicating that the locomotion is a Brownian movement. In this way, the feeding behaviors by the placozoan are organized as successive co-operative transitions among non-linear dynamic states.