Adiponectin mediates cardioprotection in oxidative stress-induced cardiac myocyte remodeling

Reactive oxygen species (ROS) induce matrix metalloproteinase (MMP) activity that mediates hypertrophy and cardiac remodeling. Adiponectin (APN), an adipokine, modulates cardiac hypertrophy, but it is unknown if APN inhibits ROS-induced cardiomyocyte remodeling. We tested the hypothesis that APN ameliorates ROS-induced cardiomyocyte remodeling and investigated the mechanisms involved. Cultured adult rat ventricular myocytes (ARVM) were pretreated with recombinant APN (30 μg/ml, 18 h) followed by exposure to physiologic concentrations of H(2)O(2) (1-200 μM). ARVM hypertrophy was measured by [(3)H]leucine incorporation and atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) gene expression by RT-PCR. MMP activity was assessed by in-gel zymography. ROS was induced with angiotensin (ANG)-II (3.2 mg·kg(-1)·day(-1) for 14 days) in wild-type (WT) and APN-deficient (APN-KO) mice. Myocardial MMPs, tissue inhibitors of MMPs (TIMPs), p-AMPK, and p-ERK protein expression were determined. APN significantly decreased H(2)O(2)-induced cardiomyocyte hypertrophy by decreasing total protein, protein synthesis, ANF, and BNP expression. H(2)O(2)-induced MMP-9 and MMP-2 activities were also significantly diminished by APN. APN significantly increased p-AMPK in both nonstimulated and H(2)O(2)-treated ARVM. H(2)O(2)-induced p-ERK activity and NF-κB activity were both abrogated by APN pretreatment. ANG II significantly decreased myocardial p-AMPK and increased p-ERK expression in vivo in APN-KO vs. WT mice. ANG II infusion enhanced cardiac fibrosis and MMP-2-to-TIMP-2 and MMP-9-to-TIMP-1 ratios in APN-KO vs. WT mice. Thus APN inhibits ROS-induced cardiomyocyte remodeling by activating AMPK and inhibiting ERK signaling and NF-κB activity. Its effects on ROS and ultimately on MMP expression define the protective role of APN against ROS-induced cardiac remodeling.     

Self-Replication Reactions Dependent on Tertiary Interaction Motifs in an RNA Ligase Ribozyme

RNA can function both as an informational molecule and as a catalyst in living organisms. This duality is the premise of the RNA world hypothesis. However, one flaw in the hypothesis that RNA was the most essential molecule in primitive life is that no RNA self-replicating system has been found in nature. To verify whether RNA has the potential for self-replication, we constructed a new RNA self-assembling ribozyme that could have conducted an evolvable RNA self-replication reaction. The artificially designed, in vitro selected ligase ribozyme was employed as a prototype for a self-assembling ribozyme. The ribozyme is composed of two RNA fragments (form R1·Z1) that recognize another R1·Z1 molecule as their substrate and perform the high turnover ligation reaction via two RNA tertiary interaction motifs. Furthermore, the substrate recognition of R1·Z1 is tolerant of mutations, generating diversity in the corresponding RNA self-replicating network. Thus, we propose that our system implies the significance of RNA tertiary motifs in the early RNA molecular evolution of the RNA world.     

Vildagliptin Stimulates Endothelial Cell Network Formation and Ischemia-induced Revascularization via an Endothelial Nitric-oxide Synthase-dependent Mechanism

Dipeptidyl peptidase-4 inhibitors are known to lower glucose levels and are also beneficial in the management of cardiovascular disease. Here, we investigated whether a dipeptidyl peptidase-4 inhibitor, vildagliptin, modulates endothelial cell network formation and revascularization processes in vitro and in vivo. Treatment with vildagliptin enhanced blood flow recovery and capillary density in the ischemic limbs of wild-type mice, with accompanying increases in phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS). In contrast to wild-type mice, treatment with vildagliptin did not improve blood flow in ischemic muscles of eNOS-deficient mice. Treatment with vildagliptin increased the levels of glucagon-like peptide-1 (GLP-1) and adiponectin, which have protective effects on the vasculature. Both vildagliptin and GLP-1 increased the differentiation of cultured human umbilical vein endothelial cells (HUVECs) into vascular-like structures, although vildagliptin was less effective than GLP-1. GLP-1 and vildagliptin also stimulated the phosphorylation of Akt and eNOS in HUVECs. Pretreatment with a PI3 kinase or NOS inhibitor blocked the stimulatory effects of both vildagliptin and GLP-1 on HUVEC differentiation. Furthermore, treatment with vildagliptin only partially increased the limb flow of ischemic muscle in adiponectin-deficient mice in vivo. GLP-1, but not vildagliptin, significantly increased adiponectin expression in differentiated 3T3-L1 adipocytes in vitro. These data indicate that vildagliptin promotes endothelial cell function via eNOS signaling, an effect that may be mediated by both GLP-1-dependent and GLP-1-independent mechanisms. The beneficial activity of GLP-1 for revascularization may also be partially mediated by its ability to increase adiponectin production.     

Prolamellar bodies formed by cyanobacterial protochlorophyllide oxidoreductase in Arabidopsis

In angiosperms, chlorophyll biosynthesis is light dependent. A key factor in this process is protochlorophyllide oxidoreductase (POR), which requires light to catalyze the reduction of protochlorophyllide to chlorophyllide. It is believed that this protein originated from an ancient cyanobacterial enzyme that was introduced into proto‐plant cells during the primary symbiosis. Here we report that PORs from the cyanobacteria Gloeobacter violaceus PCC7421 and Synechocystis sp. PCC6803 function in plastids. First, we found that the G. violaceus POR shows a higher affinity to its substrate protochlorophyllide than the Synechocystis POR but a similar affinity to plant PORs. Secondly, the reduced size of prolamellar bodies caused by a knockdown mutation of one of the POR genes, PORA, in Arabidopsis could be complemented by heterologous expression of the cyanobacterial PORs. Photoactive protochlorophyllide in the etioplasts of the complementing lines, however, was retained at a low level as in the parent PORA knockdown mutant, indicating that the observed formation of prolamellar bodies was irrelevant to the assembly of photoactive protochlorophyllide. This work reveals a new view on the formation of prolamellar bodies and provides new clues about the function of POR in the etioplast–chloroplast transition.     

Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach

Summary A method involving rapid freezing followed by substitution fixation was developed, using acrolein as a fixative. This was then applied to several cytochemical stainings, and showed well preserved and clear cell structures. Membranes were apparently negatively stained and the ultrastructure of mitochondria, rough endoplasmic reticulum and Golgi apparatus was clearly discernible. The mitochondrial and cytoplasmic matrices were stained rather densely compared with routine chemically fixed preparations, implying a good preservation of matrix substrances. Periodic acid-thiocarbohydrazide-silver proteinate staining was applied to the present method. The mucous granules of surface covering epithelial cells indicated fine staining of bipartite structure and the Golgi apparatus of mucous cells showed clear staining differences based on polarity. Postembedding lectin-ferritin and immunocytochemical stainings were applicable to the present preparations and stable stainings of secretory granules were obtained. A low temperature embedding material, Lowicryl K4M, was also examined. The cell preservation of these samples was not as good as those embedded in Epon, but the rough endoplasmic reticulum and Golgi apparatus of chief cells were stained with anti-pepsinogen antibody as were the secretory granules. The present method was also applicable to light microscopy.     

Microbial production of cis,cis-muconic acid from benzoic acid

Summary The authors isolated numerous microorganisms with the capacity to assimilate large amounts of benzoate from many soil samples. Several of them were selected and subjected to mutation mainly by ultraviolet irradiation. One mutant lacking active muconate-lactonizing enzyme, the parent strain of which was identified as belonging to the genus Arthrobacter, was isolated and found to be capable of producing cis, cis-muconic acid with a quantitative yield of 44.1 g/l over 48 h in a 30 1 jar fermentor by successive feeding of small amounts of benzoate. This mutant, however, was more sensitive to high concentrations of the substrate than the parent strain. As few intermediates and isomers other than cis, cis-muconic acid were accumulated in the large fermentor, a large amount of pure cis, cis-muconic acid was easily obtained from the broth by salting out and recrystallization at a high recovery rate.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.