Loss of α1β1 Soluble Guanylate Cyclase,the Major Nitric Oxide Receptor,Leads to Moyamoya and Achalasia

Moyamoya is a cerebrovascular condition characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and the compensatory development of abnormal “moyamoya” vessels. The pathophysiological mechanisms of this condition, which leads to ischemic and hemorrhagic stroke, remain unknown. It can occur as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes). Here, we describe an autosomal-recessive disease leading to severe moyamoya and early-onset achalasia in three unrelated families. This syndrome is associated in all three families with homozygous mutations in GUCY1A3, which encodes the α1 subunit of soluble guanylate cyclase (sGC), the major receptor for nitric oxide (NO). Platelet analysis showed a complete loss of the soluble α1β1 guanylate cyclase and showed an unexpected stimulatory role of sGC within platelets. The NO-sGC-cGMP pathway is a major pathway controlling vascular smooth-muscle relaxation, vascular tone, and vascular remodeling. Our data suggest that alterations of this pathway might lead to an abnormal vascular-remodeling process in sensitive vascular areas such as ICA bifurcations. These data provide treatment options for affected individuals and strongly suggest that investigation of GUCY1A3 and other members of the NO-sGC-cGMP pathway is warranted in both isolated early-onset achalasia and nonsyndromic moyamoya.     

Influenza A viruses limit NLRP3‐NEK7‐complex formation and pyroptosis in human macrophages

Pyroptosis is a fulminant form of macrophage cell death, contributing to release of pro‐inflammatory cytokines. In humans, it depends on caspase 1/4‐activation of gasdermin D and is characterized by the release of cytoplasmic content. Pathogens apply strategies to avoid or antagonize this host response. We demonstrate here that a small accessory protein (PB1‐F2) of contemporary H5N1 and H3N2 influenza A viruses (IAV) curtails fulminant cell death of infected human macrophages. Infection of macrophages with a PB1‐F2‐deficient mutant of a contemporary IAV resulted in higher levels of caspase‐1 activation, cleavage of gasdermin D, and release of LDH and IL‐1β. Mechanistically, PB1‐F2 limits transition of NLRP3 from its auto‐repressed and closed confirmation into its active state. Consequently, interaction of a recently identified licensing kinase NEK7 with NLRP3 is diminished, which is required to initiate inflammasome assembly.     

Scar myofibroblasts of the infarcted rat heart express natriuretic peptides

The present study examined whether natriuretic peptide expression in the scar of post-myocardial infarcted (MI) rats was derived at least in part by residing myofibroblasts. ANP and BNP mRNA levels were significantly increased in the non-infarcted left ventricle and scar of 1-week post-MI male rats, as compared to the left ventricle of normal rats. The infarct region contained myofibroblasts and contracted cardiac myocytes residing predominantly in the epicardial border zone. In primary passage scar-derived myofibroblasts, alpha-myosin heavy chain mRNA was undetectable, whereas ANP, BNP, as well as adrenomedullin and corin mRNA expression persisted. In 1-3 day cultured primary passage myofibroblasts, prepro-ANP, mature ANP, and BNP staining was observed in the cytoplasm/perinuclear region co-incident with unorganized alpha-smooth muscle actin. Following 4-7 days in culture, myofibroblasts expressed organized alpha-smooth muscle actin filaments. However, natriuretic peptides were predominantly detected in the nucleus and cytoplasm, and thin filaments occupying the perinuclear region were positive for prepro-ANP and BNP. Isoproterenol treatment of first passage scar myofibroblasts increased protein synthesis and induced BNP mRNA expression, whereas ANP mRNA levels remained unchanged. By contrast, neither ANP nor BNP mRNAs were induced following exposure to AII despite increased protein synthesis. These data highlight the novel observation that scar myofibroblasts synthesized ANP, BNP, adrenomedullin, and expressed the pro-convertase corin. Constitutive and sympathetic-driven natriuretic peptide synthesis by myofibroblasts may in part influence reparative fibrosis.     

Morphological and biochemical evidence for partial nuclear localization of annexin 1 in endothelial cells.

Using immunofluorescence, an affinity-purified anti-annexin-1 polyclonal antibody showed both cytoplasmic and nuclear staining, whereas antibodies against annexins 2, 5 and 6 labelled almost exclusively the cytoplasm of cultured endothelial cells. This was further confirmed by immunogold labelling and electron microscopy using a monoclonal antibody, annexin 1 being detected close to the plasma membrane, in the cytoplasm, as well as inside the nucleus. Finally, using immunoblotting, purified nuclei were shown to contain annexin 1, which was not removed by EDTA treatment. These data open some new perspectives in the understanding of annexin function, including possible involvement in nucleoskeleton dynamics and regulation of proliferation through cell signalling.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway

In acute promyelocytic leukaemia (APL), arsenic trioxide induces degradation of the fusion protein encoded by the PML-RARA oncogene, differentiation of leukaemic cells and produces clinical remissions. SUMOylation of its PML moiety was previously implicated, but the nature of the degradation pathway involved and the role of PML-RARalpha catabolism in the response to therapy have both remained elusive. Here, we demonstrate that arsenic-induced PML SUMOylation triggers its Lys 48-linked polyubiquitination and proteasome-dependent degradation. When exposed to arsenic, SUMOylated PML recruits RNF4, the human orthologue of the yeast SUMO-dependent E3 ubiquitin-ligase, as well as ubiquitin and proteasomes onto PML nuclear bodies. Arsenic-induced differentiation is impaired in cells transformed by a non-degradable PML-RARalpha SUMOylation mutant or in APL cells transduced with a dominant-negative RNF4, directly implicating PML-RARalpha catabolism in the therapeutic response. We thus identify PML as the first protein degraded by SUMO-dependent polyubiquitination. As PML SUMOylation recruits not only RNF4, ubiquitin and proteasomes, but also many SUMOylated proteins onto PML nuclear bodies, these domains could physically integrate the SUMOylation, ubiquitination and degradation pathways.     

Chemoenzymatic synthesis of 6omega -modified maltooligosaccharides from cyclodextrin derivatives

Regioselectively substituted maltooligosaccharides were prepared by enzymatic transformation of modified cyclodextrins by using simultaneously two different enzymes: cyclodextrin glucanotransferase (CGTase) and amyloglucosidase. Oligosaccharides were obtained in very good yields and their structures were identified by 1D and 2D NMR spectroscopy. These results provided new information about the specificity of the catalytic sites of CGTase and amyloglucosidase. They also offered new ways for the synthesis of regioselectively modified maltooligosaccharides.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

Early effects of temperate agroforestry practices on soil organic matter and microbial enzyme activity

Plant and Soil - A field experiment was conducted to evaluate the effects of alley cropping systems on microbial activity and soil organic matter (SOM) pools. We hypothesized that enzyme activity...