Endothelin activates the vascular renin-angiotensin system in rat mesenteric arteries

The effects of endothelin on the vascular renin-angiotensin system were examined in isolated perfused rat mesenteric arteries by measuring vascular renin activity and angiotensin II released into the perfusate. Infusion of endothelin (10(-9)M and 10(-11)M) increased the vascular renin activity and angiotensin II release. Pretreatment with nicardipine (10(-6)M), a calcium channel blocker, significantly suppressed these effects of endothelin. These results suggest that endothelin activates the vascular renin-angiotensin system via intracellular calcium metabolism. Vascular angiotensin II produced by endothelin may modulate the local effect of endothelin on the resistance vessels.     

Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A

Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.     

Increased plasma level of endothelin with vasoconstriction after dextran infusion.

Our previous study demonstrated that volume expansion with dextran produced blood pressure elevation due to vasoconstriction 3 hours after the cessation of infusion. To examine whether endogenous endothelin contributes to this vasoconstriction, we measured plasma level of endothelin before, immediately after, and 3 hours after the administration of dextran. Plasma level of endothelin decreased immediately after the administration (from 1.5 +/- 0.3 pg/ml to 1.1 +/- 0.2 pg/ml, P less than 0.05), and increased 3 hours after the administration (2.1 +/- 0.3 pg/ml, P less than 0.05). However, the changes in the plasma level of endothelin did not significantly correlated with those in blood pressure or total peripheral resistance. Thus, vasoconstriction after dextran infusion was accompanied by an increase in the plasma level of endothelin, but further evaluation is needed for the direct role of this peptide in the vasoconstrictive blood pressure elevation.     

Crystal structure of Bacillus sp. GL1 xanthan lyase complexed with a substrate: insights into the enzyme reaction mechanism

Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8 (PL-8), acts exolytically on the side-chains of pentasaccharide-repeating polysaccharide xanthan and cleaves the glycosidic bond between glucuronic acid (GlcUA) and pyruvylated mannose (PyrMan) through a beta-elimination reaction. To clarify the enzyme reaction mechanism, i.e. its substrate recognition and catalytic reaction, we determined crystal structures of a mutant enzyme, N194A, in complexes with the product (PyrMan) and a substrate (pentasacharide) and in a ligand-free form at 1.8, 2.1, and 2.3A resolution. Based on the structures of the mutant in complexes with the product and substrate, we found that xanthan lyase recognized the PyrMan residue at subsite -1 and the GlcUA residue at +1 on the xanthan side-chain and underwent little interaction with the main chain of the polysaccharide. The structure of the mutant-substrate complex also showed that the hydroxyl group of Tyr255 was close to both the C-5 atom of the GlcUA residue and the oxygen atom of the glycosidic bond to be cleaved, suggesting that Tyr255 likely acts as a general base that extracts the proton from C-5 of the GlcUA residue and as a general acid that donates the proton to the glycosidic bond. A structural comparison of catalytic centers of PL-8 lyases indicated that the catalytic reaction mechanism is shared by all members of the family PL-8, while the substrate recognition mechanism differs.     

Visceral Adipose MicroRNA 223 Is Upregulated in Human and Murine Obesity and Modulates the Inflammatory Phenotype of Macrophages

Obesity in humans and mice is typified by an activated macrophage phenotype in the visceral adipose tissue (VAT) leading to increased macrophage-mediated inflammation. microRNAs (miRNAs) play an important role in regulating inflammatory pathways in macrophages, and in this study we compared miRNA expression in the VAT of insulin resistant morbidly obese humans to a non-obese cohort with normal glucose tolerance. miR-223-3p was found to be significantly upregulated in the whole omental tissue RNA of 12 human subjects, as were 8 additional miRNAs. We then confirmed that miR-223 upregulation was specific to the stromal vascular cells of human VAT, and found that miR-223 levels were unchanged in adipocytes and circulating monocytes of the non-obese and obese. miR-223 ablation increased basal / unstimulated TLR4 and STAT3 expression and LPS-stimulated TLR4, STAT3, and NOS2 expression in primary macrophages. Conversely, miR-223 mimics decreased TLR4 expression in primary macrophage, at the same time it negatively regulated FBXW7 expression, a well described suppressor of Toll-like receptor 4 (TLR4) signaling. We concluded that the abundance of miR-223 in macrophages significantly modulates macrophage phenotype / activation state and response to stimuli via effects on the TLR4/FBXW7 axis.     

A toxic substance produced by Nocardia otitidiscaviarum isolated from cutaneous nocardiosis

During our studies on toxic substances from clinically isolated Nocarida, a new isolate identified as Nocardia otitidiscaviarum from cutaneous nocardiosis was found to produce a toxic substance called HS-6 that had strong in vitro as well as in vivo toxicity. The mouse intraperitoneal LD₅₀ value was 1.25 mg/kg and the ED₅₀ value for L1210 cultured cells was 0.3 ng/ml. The structure of HS-6 was determined and found to belong to the 16-membered macrocyclic group with a molecular formula of C₄₃H₆₈O₁₂. HS-6 also showed activity against pathogenic fungi such as Cryptococcus neoformans.     

Fluctuation of the zooplankton community in Lake Biwa during the 20th century: a paleolimnological analysis

Detailed zooplankton records from a 26-cm sediment core with a time resolution of approximately 3–10 years were obtained from Lake Biwa, Japan, to examine the historical variations in the zooplankton community during the 20th century. In the sediments, selected zooplankton remains have fluctuated over the years. Daphnia – large zooplankton herbivores – did not occur from 1900 to 1920, and formed a very minor component of the zooplankton community in the following 30 years, while Bosmina – small zooplankton herbivores – were common during this period. In the mid-1960s, however, when eutrophication was noticeable in this lake, Daphnia numbers increased dramatically and became the dominant zooplankton thereafter. In contrast, Difflugia brevicolla and D. biwae, two amoeboid protozoans that live in connection with the lake bottom environment, occurred abundantly until the late 1950s, but gradually decreased after the mid-1960s. In particular, D. biwae, a species peculiar to this lake, was not found in sediment dated after 1980, suggesting its extinction. These results indicate that the zooplankton community structure changed greatly in the 1960s, and suggest that the eutrophication occurring at this time altered the relative strength of top-down and bottom-up forces on the zooplankton community in Lake Biwa.     

Photosynthesis-dependent Ca2+ influx and functional diversity between phospholipases in the formation of cell polarity in migrating cells of red algae

Unicellular spore cells, designated as monospores (also called archeospores), are well known as migrating plant cells, in which establishment of the anterior-posterior axis directs asymmetrical distribution of F-actin. Since the mechanisms of cell polarity formation are not yet fully elucidated in monospores, we investigated the roles of phosphoinositide signaling systems and Ca²⁺ mobilization in migration. Although we have already found the critical involvement of phosphatidylinositol 3-kinase in the establishment of cell polarity, we recently demonstrated the important roles of extracellular Ca²⁺ influx, phospholipase C (PLC) and phospholipase D (PLD). The remarkable characteristics of these factors are that Ca²⁺ influx depends on photosynthetic activity and that PLC and PLD play roles in the establishment and maintenance of cell polarity, respectively. These findings could provide new insight into the regulation of migration in eukaryotic cells.Key words: Ca²⁺ influx, cell polarity, phospholipase C, phospholipase D, photosynthesis, Porphyra yezoensisMonospores are responsible for asexual and clonal propagation of the marine multicellular red algae Porphyra and have an exceptional characteristic as migrating plant cells.^1–5 Monospores possess a round shape just after release from gametophytic blades (Fig. 1A and B), then undergo morphological change during migration. The establishment of cell polarity leads to the determination of anterior-posterior axis and asymmetrical localization of F-actin (Fig. 1C). After migration, monospores adhere to the substratum in which the apical-basal axis has been established for further development (Fig. 1D). Asymmetrical distribution of F-actin is also found in chemotaxic migration of Dictyostelium cells and leukocytes.^6,7 In these cells, reciprocal local accumulation of phosphoinositides, such as phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P₃] at the leading edge and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] at the trailing side, is critical for the establishment of cell polarity. Phosphatidylinositol 3-kinase (PI3K) and PtdIns(3,4,5)P₃-specific D-3-phosphatase PTEN have been identified as key modulators in the establishment of cell polarity, bringing asymmetrical distribution of these two phosphoinositides in plasma membranes.^6,8 Similarly, we found the involvement of PI3K activity in the establishment of cell polarity in migrating monospores,³ suggesting the evolutional conservation of the function of PI3K in migrating eukaryotic cells. On the other hand, the importance of cell wall synthesis has been found in the maintenance of the cell polarity during monospore migration⁴ as reported in Fucus zygotes.^9,10 Therefore, the establishment and maintenance of cell polarity are thought to be regulated separately in monospores of P. yezoensis. In this addendum, further evidence of differential regulation of cell polarity formation by extracellular Ca²⁺ influx and phospholipases in migrating monospores of red algae is documented according to our recent report.⁵Open in a separate windowFigure 1Establishment and maintenance of cell polarity in monospores from the red alga P. yezoensis. (A) Discharge of unicellular monospores from a multicellular gametophytic blade of P. yezoensis strain TU-1. Scale bar = 20 µm. (B–D) Asymmetrical distribution of F-actin during early development of monospores. F-actin was stained with alex Flour 488 phalloidin. (B) Discharged monospore. (C) Migrating monospore. (D) Adhering monospore. Upper and lower photos in each panel show bright-field and fluorescent images, respectively. Arrow in (C) indicates the direction of migration. Scale bars = 5 µm. (e) Schematic representation of our working hypothesis about the formation of cell polarity required for monospore migration. Photosynthesis-dependent [Ca²⁺]_cyt increase regulates PLC and PI3K for the establishment of cell polarity, while PLD is required for the maintenance of the established cell polarity. DG, diacylgycerol; IP3, inositol-1,4,5-trisphosphate; IP3r, IP3 receptor; PC, phosphatidylcholine.     

Possible role of macrophage-derived soluble mediators in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice.

Pancreatic islets from DBA/2 mice infected with the D variant of encephalomyocarditis (EMC-D) virus revealed lymphocytic infiltration with moderate to severe destruction of pancreatic beta cells. Our previous studies showed that the major population of infiltrating cells at the early stages of infection is macrophages. The inactivation of macrophages prior to viral infection resulted in the prevention of diabetes, whereas activation of macrophages prior to viral infection resulted in the enhancement of beta-cell destruction. This investigation was initiated to determine whether macrophage-produced soluble mediators play a role in the destruction of pancreatic beta cells in mice infected with a low dose of EMC-D virus. When we examined the expression of the soluble mediators interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in the pancreatic islets, we found that these mediators were clearly expressed at an early stage of insulitis and that this expression was evident until the development of diabetes. We confirmed the expression of these mediators by in situ hybridization with digoxigenin-labelled RNA probes or immunohistochemistry in the pancreatic islets. Mice treated with antibody against IL-1beta or TNF-alpha or with the iNOS inhibitor aminoguanidine exhibited a significant decrease in the incidence of diabetes. Mice treated with a combination of anti-IL-1beta antibody, anti-TNF-alpha antibody, and aminoguanidine exhibited a greater decrease in the incidence of disease than did mice treated with one of the antibodies or aminoguanidine. On the basis of these observations, we conclude that macrophage-produced soluble mediators play an important role in the destruction of pancreatic beta cells, resulting in the development of diabetes in mice infected with a low dose of EMC-D virus.     

Crosstalk between oral squamous cell carcinoma cells and cancer-associated fibroblasts via the TGF-β/SOX9 axis in cancer progression

Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial–mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-β1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-β1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-β/SOX9 axis.