Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans

Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.     

Accumulation of 8-oxo-deoxyguanosine in cardiovascular tissues with the development of hypertension

Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA is associated with mutagenesis and cell death. Little attention has been given to the biological significance of 8-oxo-dG accumulation in cardiovascular tissues during the different stage of hypertension and its prevention. We thus investigated the levels and localization of both 8-oxo-dG accumulation and expression of MTH1, which hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA, in the thoracic aorta prepared from stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wister-Kyoto rats (WKY), aged 5-32 weeks. HPLC-MS/MS analysis revealed that the levels of nuclear 8-oxo-dG in the aorta increased significantly in SHRSP, but not WKY, with aging. Immunohistochemical study revealed that both TUNEL reactivity and 8-oxo-dG immunoreactivity were increased in smooth muscle cells (SMC) and endothelial cells (EC) of the aorta with aging, and they exhibited similar distributions in serial sections. The number of 8-oxo-dG and TUNEL positive cells in EC, but not in SMC, was significantly higher in SHRSP than WKY at 32 weeks of age. In contrast, the expression levels of Mth1mRNA and MTH1 protein in the aorta were similarly decreased both in SHRSP and WKY with aging. However, the number of MTH1 expressing EC was remarkably increased in the older SHRSP compared to the younger ones or age-matched WKY. Hypertension significantly increased not only 8-oxo-dG accumulation but also the expression of MTH1 in EC of the aorta during aging. While accumulation of 8-oxo-dG in SMC of the aorta was slightly increased, the expression of MTH1 protein in SMC was rather decreased by hypertension. We thus suggest that MTH1 may protect EC in the aorta from the oxidative damage increased by hypertension.     

Biosynthetic pathways of the endocannabinoid anandamide

Anandamide (=N-arachidonoylethanolamine) is the first discovered endocannabinoid, and belongs to the class of bioactive, long-chain N-acylethanolamines (NAEs). In animal tissues, anandamide is principally formed together with other NAEs from glycerophospholipid by two successive enzymatic reactions: 1) N-acylation of phosphatidylethanolamine to generate N-acylphosphatidylethanolamine (NAPE) by Ca2+-dependent N-acyltransferase; 2) release of NAE from NAPE by a phosphodiesterase of the phospholipase D type (NAPE-PLD). Although these anandamide-synthesizing enzymes were poorly understood until recently, our cDNA cloning of NAPE-PLD in 2004 enabled molecular-biological approaches to the enzymes. NAPE-PLD is a member of the metallo-beta-lactamase family, which specifically hydrolyzes NAPE among glycerophospholipids, and appears to be constitutively active. Mutagenesis studies suggested that the enzyme functions through a mechanism similar to those of other members of the family. NAPE-PLD is widely expressed in animal tissues, including various regions in rat brain. Its expression level in the brain is very low at birth, and remarkably increases with development. Analysis of NAPE-PLD-deficient mice and other recent studies revealed the presence of NAPE-PLD-independent pathways for the anandamide formation. Furthermore, calcium-independent N-acyltransferase was discovered and characterized. In this article, we will review recent progress in the studies on these enzymes responsible for the biosynthesis of anandamide and other NAEs.     

Altered regulation of phosphatidylinositol 3-kinase signaling in cathepsin D-deficient brain

Cathepsin D (CD) is an essential lysosomal protease and mice lacking this enzyme exhibit neuropathology similar to that observed in brains of patients with neuronal ceroid lipofuscinosces (NCL/Batten disease), a group of autosomal recessive pediatric neurodegenerative diseases. CD-deficient (CD-/-) brains exhibit a dramatic induction of autophagic stress as defined by the aberrant accumulation of autophagosomes, which is concomitant with markers of apoptosis. However, the signaling abnormalities which lead to CD deficiency-induced neurodegeneration are poorly defined. Since phosphatidylinositol-3 kinase (PI3-K) is known to regulate both apoptosis and autophagy, PI3-K-mediated signaling events were assessed in CD-/- brain at P14 and P25-26. Compared to WT littermate controls, CD-/- cortical neurons exhibited a widespread decrease in phosphorylation of Akt (inactivation) and GSK3beta (disinhibition) at P25-26, while levels of total Akt and GSK3beta remained unchanged. This P25-26-specific decrease in phosphorylation of Akt and GSK-3beta in CD-/- brain coincided temporally with markers of apoptosis but followed the induction of autophagic stress observed at both P14 and P25-26. In addition, levels and/or activation of mTOR and Beclin were not affected by CD deficiency, suggesting that the accumulation of autophagosomes is not due to an increased synthesis of autophagosomes but rather from an inhibition of autophagosome recycling, due most likely to a compromise in lysosome function. Together these observations indicate a pronounced decrease in pro-survival PI3-K signaling in CD-/- brain that may contribute to autophagic stress-induced and apoptotic neuropathology.     

Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.     

Circumnutational movement in rice coleoptiles involves the gravitropic response: analysis of an agravitropic mutant and space‐grown seedlings

Plants exhibit helical growth movements known as circumnutation in growing organs. Some studies indicate that circumnutation involves the gravitropic response, but this notion is a matter of debate. Here, using the agravitropic rice mutant lazy1 and space‐grown rice seedlings, we found that circumnutation was reduced or lost during agravitropic growth in coleoptiles. Coleoptiles of wild‐type rice exhibited circumnutation in the dark, with vigorous oscillatory movements during their growth. The gravitropic responses in lazy1 coleoptiles differed depending on the growth stage, with gravitropic responses detected during early growth and agravitropism during later growth. The nutation‐like movements observed in lazy1 coleoptiles at the early stage of growth were no longer detected with the disappearance of the gravitropic response. To verify the relationship between circumnutation and gravitropic responses in rice coleoptiles, we conducted spaceflight experiments in plants under microgravity conditions on the International Space Station. Wild‐type rice seeds were germinated, and the resulting seedlings were grown under microgravity or a centrifuge‐generated 1 g environment in space. We began filming the seedlings 2 days after seed imbibition and obtained images of seedling growth every 15 min. The seed germination rate in space was 92–100% under both microgravity and 1 g conditions. LED‐synchronized flashlight photography induced an attenuation of coleoptile growth and circumnutational movement due to cumulative light exposure. Nevertheless, wild‐type rice coleoptiles still showed circumnutational oscillations under 1 g but not microgravity conditions. These results support the idea that the gravitropic response is involved in plant circumnutation.     

Disruption of phospholipase B gene, PLB1, increases the survival of baker's yeast Torulaspora delbrueckii

Abstract An uracil auxotrophic mutant of baker's yeast Torulaspora delbrueckii , which is resitant to 5-fluoro-orotic acid, was complemented by transformation with YEp24 which harbors 2 μm origin and URA3 derived from Saccharomyces cerevisiae . The phospholipase B in T. delbrueckii cells is active in both acidic and alkaline conditions. However, activity of phospholipase B gene ( PLB1 ) in cells of disruption mutant ( plbI : : URA3 ) was lost in both conditions, which indicates that all phospholipase B activity is encoded by a single gene (or a single polypeptide) in these yeast cells. Over-expression of PLB1 with YEp plasmid vector in T. delbrueckii cells showed ∼ 2.5-fold increase in phospholipase B activity, comparing with that in wild-type cells. Cells of plb1 Δ mutant showed increased survival when cells of plb1 Δ mutant and wild-type strain were incubated in water at 30 °C. Cells of PLB1 -over-expressed strain died rapidly even during the cultivation period, indicating that phospholipase B activity may be a determinant for the survival of this yeast.     

Accumulation of anthranilic acid and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL-serine hydroxamate

During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated. This mutant accumulated five Ehrlich's reagent positive fluorescent substances in the culture medium. Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography-mass spectrometry (LC-MS), and NMR spectrum coincided with that of anthranilic acid. LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299. This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-beta-glucosylamine) by two-dimensional (1)H and (13)C NMR analyses. The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid. N-Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture. In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the fermentation. Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation. The strain TO3002 showed a leaky requirement for L-tryptophan or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.     

Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom

Colominic acid is an 2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A₂ activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity.     

Interaction between a negative regulator (Msa2/Nrd1) and a positive regulator (Cpc2) of sexual differentiation in Schizosaccharomyces pombe

The sexual differentiation of Schizosaccharomyces pombe is controlled by many cellular components which have not been fully characterized. We isolated a gene called msa2 as a multi-copy suppressor of a sporulation abnormal mutant (sam1). Msa2p is identical with Nrd1p which has been characterized as a factor that blocks the onset of sexual differentiation. The yeast two-hybrid system was used to identify Cpc2p, a fission yeast homolog of the RACK1 protein, that interacted with Msa2p/Nrd1p. We confirmed that Msa2p/Nrd1p interacted with Cpc2p in S. pombe cells. An epistatic analysis of msa2/nrd1 and cpc2 suggests that Msa2p/Nrd1p was an upstream regulator for Cpc2p. A localization analysis of Cpc2p and Msa2p/Nrd1p indicates that both proteins were predominantly localized in the cytoplasm. The interaction of negative regulator Msa2p/Nrd1p with positive regulator Cpc2p suggests a new regulatory circuit in the sexual differentiation of S. pombe.