Angiotensin IV protects cardiac reperfusion injury by inhibiting apoptosis and inflammation via AT4R in rats

Angiotensin IV (Ang IV) is formed by aminopeptidase N from Ang III by removing the first N-terminal amino acid. Previously, we reported that Ang III has some cardioprotective effects against global ischemia in Langendorff heart. However, it is not clear whether Ang IV has cardioprotective effects. The aim of the present study was to evaluate the effect of Ang IV on myocardial ischemia-reperfusion (I/R) injury in rats. Before ischemia, male Sprague-Dawley rats received Ang IV (1 mg/kg/day) for 3 days. Anesthetized rats were subjected to 45 min of ischemia by ligation of left anterior descending coronary artery followed by reperfusion and then, sacrificed 1 day or 1 week after reperfusion. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations, and infarct size were measured. Quantitative analysis of apoptotic and inflammatory proteins in ventricles were performed using Western blotting. Pretreatment with Ang IV attenuated I/R-induced increases in plasma CK and LDH levels, and infarct size, which were blunted by Ang IV receptor (AT₄R) antagonist and but not by antagonist for AT₁R, AT₂R, or Mas receptor. I/R increased Bax, caspase-3 and caspase-9 protein levels, and decreased Bcl-2 protein level in ventricles, which were blunted by Ang IV. I/R-induced increases in TNF-α, MMP-9, and VCAM-1 protein levels in ventricles were also blunted by Ang IV. Ang IV increased the phosphorylation of Akt and mTOR. These effects were attenuated by co-treatment with AT₄R antagonist or inhibitors of downstream signaling pathway. Myocardial dysfunction after reperfusion was improved by Ang IV. These results suggest that Ang IV has cardioprotective effect against I/R injury by inhibiting apoptosis via AT₄R and PI3K-Akt-mTOR pathway.     

1H, 13C and 15N resonance assignment of phage T4 endoribonuclease RegB

RegB is involved in the control of the phage T4 life cycle. It inactivates the phage early mRNAs when their translation is no more required. We determined its structure and identified residues involved in substrate binding. For this, all backbone and 90% of side-chain resonance frequencies were assigned.     

Structural biology of human cannabinoid receptor-2 helix 6 in membrane-mimetic environments

We detail the structure and dynamics of a synthetic peptide corresponding to transmembrane helix 6 (TMH6) of human cannabinoid receptor-2 (hCB2) in biomembrane-mimetic environments. The peptide’s NMR structural biology is characterized by two α-helical domains bridged by a flexible, nonhelical hinge region containing a highly-conserved CWFP motif with an environmentally sensitive, Pro-based conformational switch. Buried within the peptide’s flexible region, W²⁵⁸ may hydrogen-bond with L²⁵⁵ to help stabilize the Pro-kinked hCB2 TMH6 structure and position C²⁵⁷ advantageously for interaction with agonist ligands. These characteristics of hCB2 TMH6 are potential structural features of ligand-induced hCB2 activation in vivo.     

Discovery of new low-molecular-weight p53–Mdmx disruptors and their anti-cancer activities

Although several p53–Mdm2-binding disruptors have been identified to date, few studies have been published on p53–Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200–300 selectively inhibited the p53–Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53–Mdmx interaction over the p53–Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53–Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.     

Development of new Malt1 inhibitors and probes

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) is a promising therapeutic target for the treatment of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL). Several research groups have reported on the development of Malt1 inhibitors and activity-based probes for in vitro and in situ monitoring and modulating Malt1 activity. In this paper, we report on two activity-based Malt1 probes (6 and 7) and a focused library of 19 new Malt1 inhibitors. Our peptide-based probe 6 labels Malt1 in an activity-based manner. In contrast, probe 7, derived from the known covalent inhibitor MI-2, labels both wild type and catalytically inactive Cys to Ala mutant Malt1, suggesting that MI-2 inhibits Malt1 by reacting with a nucleophilic residue other than the active site cysteine. Furthermore, two of our inhibitors (9, apparent IC₅₀ 3.0 μM, and 13, apparent IC₅₀ 2.1 μM) show good inhibitory activity against Malt1 and outperform MI-2 (apparent IC₅₀ 7.8 μM) in our competitive activity-based protein profiling assay.     

Structural-Thermodynamic Relationships of Interactions in the N-Terminal ATP-Binding Domain of Hsp90

Despite its importance as a target in anti-cancer therapeutics and the numerous rational-based inhibitor design efforts aimed at it, there are only limited data available on structural-thermodynamic relationships of interactions of the N-terminal ATP-binding domain of Hsp90 (N-Hsp90). Here, we redress this by presenting an investigation of binding of nucleotides and ansamycin compounds to this domain. Interactions of nucleotides with N-Hsp90 are relatively weak (> 10 μM) and are strongly enthalpy driven over the temperature range 10-25 °C. Geldanamycin (GA) and its analogues 17-AAG [17-(allylamino)-17-demethoxy-GA] and 17-DMAG (17-N,N-dimethylaminoethylamino-17-demethoxy-GA) bind more strongly and have a dominant favourable enthalpic contribution over the temperature range investigated. We investigated the temperature dependence of the enthalpic contribution to binding. We found that while the ansamycin compounds have the commonly observed negative value, the nucleotides show a negligible or even a positive ΔC_p of binding. These data represent the first observation of a single binding site for which interactions with different ligands result in both negative and positive ΔC_p values. By addressing the likely impact of the potential contributions from protein-ligand interactions, we are able to attribute the anomalous ΔC_p for the nucleotides largely to a change in the conformation of the domain structure and local motion in the lid region of N-Hsp90 with the concomitant exposure of hydrophobic amino acid side chains.     

Immortalization up‐regulated protein promotes tumorigenesis and inhibits apoptosis of papillary thyroid cancer

The incidence of thyroid cancer is increasing in recent years worldwide, but the underlying mechanisms await further exploration. We utilized the bioinformatic analysis to discover that Immortalization up‐regulated protein (IMUP) could be a potential oncogene in the papillary thyroid cancer (PTC). We verified this finding in several databases and locally validated cohorts. Clinicopathological features analyses showed that high expression of IMUP is positively related to malignant clinicopathological features in PTC. Braf‐like PTC patients with higher IMUP expression had shorter disease‐free survival. The biological function of IMUP in PTC cell lines (KTC‐1 and TPC‐1) was investigated using small interfering RNA. Our results showed that silencing IMUP suppresses proliferation, migration and invasion while inducing apoptosis in PTC cell lines. Changes of the expression of apoptosis‐related molecules were identified by real‐time quantitative polymerase chain reaction and Western blotting. We also found that YAP1 and TAZ, the critical effectors in the Hippo pathway, were down‐regulated when the IMUP is silenced. Rescue experiments showed that overexpression of YAP1 reverses the tumour inhibitory effect caused by IMUP knockdown. Our study demonstrated that IMUP has an oncogenic function in PTC and might be a new target gene in the treatment of PTC.     

Nonhistone chromatin proteins of B-lymphocytes stimulated by lipopolysaccharide Two-dimensional gel electrophoresis analysis of proteins synthesized and phoshorylated during the initiation of lymphocyte differentiation

Synthesis and phosphorylation of nonhistone chromatin and nucleoplasmic proteins during the first 24 h of activation of mouse B-lymphocytes by the B-cell mitogen lipopolysaccharide have been studied by two-dimensional gel electrophoretic analysis. Although little change occurs in the nucleoplasmic proteins, it has been shown that the incorporation of [³⁵S]methionine into nonhistone chromatin proteins is selectively stimulated. The degree of stimulation and the kinetics of synthesis are characteristic for each individual protein; some proteins exhibit increased incorporation only 4 h after addition of mitogen, while others are synthesized de novo between 8 and 24 h. After 72 h stimulation, the majority of nonhistone chromatin protein synthesis occurs in the highly differentiated lymphoblasts and plasma cells actively secreting IgM, very little synthesis taking place in the small lymphocytes. Analysis of nuclear proteins from lymphocytes stimulated for 2 h showed no selective stimulation of phosphorylation. These observations suggest that nonhistone chromatin proteins play an important role in the regulation of gene expression in B-lymphocytes.     

Expression of the vesicular stomatitis virus membrane glycoprotein gene in Bacillus subtilis

Abstract The molecularly cloned gene encoding the vesicular stomatitis virus (VSV) membrane glycoprotein G was modified and joined to a Bacillus subtilis secretion vector constructed from the plasmid pUB110 and containing the promoter and signal sequence regions of the α-amylase (a secretory protein) gene from Bacillus amyloliquefaciens . The regions encoding the NH₂-terminal signal peptide and the COOH-terminal hydrophobic transmembrane domains of the VSV gene were deleted to facilitate the secretion of the G protein in soluble form. The truncated G protein was found to be expressed in B. subtilis . The expression level was low, probably due to rapid proteolytic degradation of the protein and, contrary to what was expected, almost all of the protein remained cell-associated.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.