Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.     

Mutagenesis of human granulocyte colony stimulating factor

To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis. The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity. N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture. Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo.     

Peritraumatic distress, watching television, and posttraumatic stress symptoms among rescue workers after the Great East Japan earthquake

Background

The Great East Japan Earthquake of March 11, 2001 left around 20,000 dead or missing. Previous studies showed that rescue workers, as well as survivors, of disasters are at high risk for posttraumatic stress disorder (PTSD). This study examined the predictive usefulness of the Peritraumatic Distress Inventory (PDI) among rescue workers of Disaster Medical Assistance Teams (DMATs) deployed during the acute disaster phase of the Great East Japan Earthquake.

Methodology/Principal Findings

In this prospective observational study, the DMAT members recruited were assessed 1 month after the earthquake on the PDI and 4 months after the earthquake on the Impact of Event Scale-Revised to determine PTSD symptoms. The predictive value of the PDI at initial assessment for PTSD symptoms at the follow-up assessment was examined by univariate and multiple linear regression analysis. Of the 254 rescue workers who participated in the initial assessment, 173 completed the follow-up assessment. Univariate regression analysis revealed that PDI total score and most individual item scores predicted PTSD symptoms. In particular, high predictive values were seen for peritraumatic emotional distress such as losing control of emotions and being ashamed of emotional reactions. In multiple linear regression analysis, PDI total score was an independent predictor for PTSD symptoms after adjusting for covariates. As for covariates specifically, watching earthquake television news reports for more than 4 hours per day predicted PTSD symptoms.

Conclusions/Significance

The PDI predicted PTSD symptoms in rescue workers after the Great East Japan Earthquake. Peritraumatic emotional distress appears to be an important factor to screen for individuals at risk for developing PTSD among medical rescue workers. In addition, watching television for extended period of time might require attention at a time of crisis.     

Differential expression of ribosomal proteins in human normal and neoplastic colorectum.

Ribosomal proteins are a major component of ribosomes and play critical roles in protein biosynthesis. Recently it has been shown that the ribosomal proteins also function during various cellular processes that are independent of protein biosynthesis therefore called extraribosomal functions. In this study we have, for the first time, determined the expression profile of 12 ribosomal proteins (Sa, S8, S11, S12, S18, S24, L7, L13a, L18, L28, L32, and L35a) in normal epithelia of human colorectal mucosa using immunohistochemistry (IHC) and then compared their expression patterns with those of colorectal cancer. In the normal mucosa, ribosomal proteins were largely associated with the ribosomes of mucosal epithelia, and the expression level of ribosomal proteins, except for S11 and L7 proteins, was markedly increased in associated with maturation of the mucosal cells. On the other hand, these ribosomal proteins were markedly decreased in colorectal cancer compared with the normal mucosa. By contrast, S11 and L7 ribosomal proteins were rarely associated with the ribosomes of colorectal epithilia except immature mucosal cells, whereas their expression levels were significantly enhanced in colorectal cancer cells. In addition, L7 ribosomal protein was detected in the secretory granules of the enterochromaffin cells in the colorectal mucosa and in carcinoma cells expressing chromogranin A. These results indicate that the expression of ribosomal proteins is differentially regulated not only in normal mucosa but also in carcinoma of human colorectum, and suggest an extraribosomal function of L7 ribosomal protein in neuroendocrine function.     

Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase

We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney > testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.     

Differential effects of ret and TRKB on axonal branching and survival of parasympathetic neurons

Interactions between neurons and their targets of innervation influence many aspects of neural development. To examine how synaptic activity interacts with neurotrophic signaling, we determined the effects of blocking neuromuscular transmission on survival and axonal outgrowth of ciliary neurons from the embryonic chicken ciliary ganglion. Ciliary neurons undergo a period of cell loss due to programmed cell death between embryonic Days (E) 8 and 14 and they innervate the striated muscle of the iris. The nicotinic antagonist d‐tubocurarine (dTC) induces an increase in branching measured by counting neurofilament‐positive voxels (NF‐VU) in the iris between E14‐17 while reducing ciliary neuron survival. Blocking ganglionic transmission with dihyro‐β‐erythroidin and α‐methyllycacontine does not mimic dTC. At E8, many trophic factors stimulate neurite outgrowth and branching of neurons placed in cell culture; however, at E13, only GDNF stimulates branching selectively in cultured ciliary neurons. The GDNF‐induced branching at E13 could be inhibited by BDNF. Blocking ret signaling in vivo with a dominant negative (dn)ret decreases survival of ciliary and choroid neurons at E14 and prevents dTC induced increases in NF‐VU in the iris at E17. Blocking TRKB signaling with dn TRKB increases NF‐VU in the iris at E17 and decreases neuronal survival at E17, but not at E14. Thus, RET promotes survival during programmed cell death in the ciliary ganglion and contributes to promoting branching when synaptic transmission is blocked while TRKB inhibits branching and promotes maintenance of neuronal survival. These studies highlight the multifunctional nature of trophic molecule function during neuronal development. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Atorvastatin-induced cardioprotection is mediated by increasing inducible nitric oxide synthase and consequent S-nitrosylation of cyclooxygenase-2

We determined the effects of cyclooxygenase-1 (COX-1; SC-560), COX-2 (SC-58125), and inducible nitric oxide synthase (iNOS; 1400W) inhibitors on atorvastatin (ATV)-induced myocardial protection and whether iNOS mediates the ATV-induced increases in COX-2. Sprague-Dawley rats received 10 mg ATV.kg(-1).day(-1) added to drinking water or water alone for 3 days and received intravenous SC-58125, SC-560, 1400W, or vehicle alone. Anesthesia was induced with ketamine and xylazine and maintained with isoflurane. Fifteen minutes after intravenous injection rats underwent 30-min myocardial ischemia followed by 4-h reperfusion [infarct size (IS) protocol], or the hearts were explanted for biochemical analysis and immunoblotting. Left ventricular weight and area at risk (AR) were comparable among groups. ATV reduced IS to 12.7% (SD 3.1) of AR, a reduction of 64% vs. 35.1% (SD 7.6) in the sham-treated group (P < 0.001). SC-58125 and 1400W attenuated the protective effect without affecting IS in the non-ATV-treated rats. ATV increased calcium-independent NOS (iNOS) [11.9 (SD 0.8) vs. 3.9 (SD 0.1) x 1,000 counts/min; P < 0.001] and COX-2 [46.7 (SD 1.1) vs. 6.5 (SD 1.4) pg/ml of 6-keto-PGF(1alpha); P < 0.001] activity. Both SC-58125 and 1400W attenuated this increase. SC-58125 did not affect iNOS activity, whereas 1400W blocked iNOS activity. COX-2 was S-nitrosylated in ATV-treated but not sham-treated rats or rats pretreated with 1400W. COX-2 immunoprecipitated with iNOS but not with endothelial nitric oxide synthase. We conclude that ATV reduced IS by increasing the activity of iNOS and COX-2, iNOS is upstream to COX-2, and iNOS activates COX-2 by S-nitrosylation. These results are consistent with the hypothesis that preconditioning effects are mediated via PG.     

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.     

pKJ1, a naturally occurring conjugative plasmid coding for toluene degradation and resistance to streptomycin and sulfonamides.

Pseudomonas sp. TA8 isolated by m-toluate enrichment from an aqueous sample metabolized toluene and m- and p-xylenes via the meta cleavage pathway, and manifested specific resistance to streptomycin and sulfonamides. A variety of experiments revealed that the pKJ1 plasmid of about 150 megadaltons carried by TA8 specified both the toluene and xylene degradative function (the Tol function) and streptomycin/sulfonamide resistance. The deletion of a segment of pKJ1 (about 22 megadaltons) resulted in the loss of the Tol function. pKJ1 was not assigned to Pseudomonas incompatibility group P-1, P-2, P-3, or P-9.