Optical Resolution and Determination of Absolute Configurations of α-Isopropyl-4-substituted Phenylacetic Acids and Insecticidal Activities of Their 5-Benzyl-3-furylmethyl Esters

In connection with disclosure of a new class of insecticides, the modified phenylacetates, six new optically active α-isopropy-4-substituted phenylacetic acids whose substituents are respectively 4-methyl,4-methoxy, 4-fluoro, 4-chloro, 4-bromo and 3,4-methylenedioxy group were prepared by optical resolution and their absolute configurations were determined by comparative ORD with α-isopropylphenylacetic acid derivatives whose absolute configuration is known as (S)-(²). Esters of the (S)-(²)-acids with 5-benzyl-3-furylmethanol were nearly twice toxic to Musca domestica than those of the racemic esters. Optical purities of the resolved acids were determined by GLC and NMR (with Eu-FOD) as (?)-methyl esters.     

Production of Acidic Actinomycin Congeners by a Mutant Strain of Streptomyces antibioticus,No. B-1625

Streptomyces sp. No. B-1625, which was identified as a strain of Streptomyces antibioticus, is a typical producer of actinomycin, but also produces minor acidic antibiotic components (FA), besides actinomycins X₂, D and X_0β. The FA-components, which were obtained with a high-producing mutant, 11M-21, showed antibacterial and antitumor activities, and also similar visible and UV absorption spectra to those characteristic of actinomycin. The FA-components were separated into five components, FA₁ FA_2α, FA_2β, FA_3α and FA_3β, on TLC. Among them, one component, FA_3β, isolated in a purified state as an orange powder, has a composition of C, 52.97: H, 6.34: N, 10.48%, and is active against B. subtilis at a MIC of 5mcg/ml. The FA_3β component showed pK_a′ of 5.4 and 12.0 and λ_max at 443, 427 and 233 nm. From these properties, FA_3β is considered to be an acidic actinomycin congener.     

Bio-functional pickles that reduce blood pressure of rats

Addition of γ-aminobutyric acid (GABA) and angiotensin converting enzyme (ACE)-inhibitory peptides to the pickles was studied in order to develop a new type of pickles that reduce blood pressure. Based on the outcome of these studies, a new type of fermentation bed composed of rice bran and white miso has been successfully developed. The advantage of such pickles is that they not only contain both GABA and ACE-inhibitory peptides, but also that their taste and flavor are excellent, with colors close to the original ones. The new type of pickles could temporarily reduce blood pressure in two types of rats, spontaneously hypertensive rats and NaCl-sensitive model rats. Thus, the newly developed pickles appear to be beneficial for pickle business.     

Effect of PLCβ4 in the thalamus on the EEG in REM sleep

Sleep and Biological Rhythms -     

The clinicopathological significance of angiogenesis in hindgut neuroendocrine tumors obtained via an endoscopic procedure

Background

As the World Health Organization grading system for gastroenteropancreatic-neuroendocrine tumors (GEP-NETs) may not always correlate with tumor progression, it is imperative that other independent predictors of tumor progression be established. To identify such predictors, we conducted a retrospective histopathological study of hindgut NETs, obtained from endoscopic procedures, and used statistical analyses to evaluate predictive factors.

Methods

We first obtained clinicopathological data of cases of hindgut NETs. Tissue sections from tumor samples were prepared and subjected to pathological examination. In particular, we calculated the microvessel density (MVD) and lymphatic microvessel density (LMVD) values, and performed appropriate statistical analyses.

Results

A total of 42 cases of hindgut NETs were selected for the study, 41 from the rectum and 1 from the sigmoid colon. Based on the Ki-67 labeling index, 34 cases were classified as NET G1 tumors and 8 as NET G2 tumors. MVD values ranged from 1.4/mm² to 73.9/mm² and LMVD values from 0/mm² to 22.9/mm². MVD and LMVD were identified as risk factors for venous and lymphatic invasion of hindgut NETs. Moreover, MVD positively correlated with the maximum diameter of the tumor.

Conclusions

Tumor progression of NETs may cause angiogenesis and lymphangiogenesis, via an unknown mechanism, as well as lymphovascular invasion. Angiogenesis likely plays an important role in occurrence and progression in the initial phase of hindgut NETs.

     

Lead optimization and efficacy evaluation of quinazoline-based BET family inhibitors for potential treatment of cancer and inflammatory diseases

Extensive optimization of quinazoline-based lead 8 is described. The structure-activity relationship studies indicate the S-configuration is preferred for the phenylmorpholine substitution. Together with incorporation of a (2-hydroxyl-2-methylpropyl)pyrazole moiety at the 2-position leads to analogs with comparable potency and marked improvement in the pharmacokinetic profile over our previously reported lead compounds. Further in vivo efficacy studies in Kasumi-1 xenograft mouse model demonstrates that the selected inhibitors are well tolerated and highly efficacious in the inhibition of tumor growth. Additionally, the representative analog 19 also demonstrated significant improvement of arthritis severity in a collagen-induced arthritis (CIA) mouse model. These results indicate potential use of these quinazoline-based BET inhibitors for treatment of cancer and inflammatory diseases. A brief discussion of the co-crystallized structure of 19 with BRD4 (BD1) is also highlighted.     

Structure-activity relationship study of estrogen receptor down-regulators with a diphenylmethane skeleton

Selective estrogen receptor (ER) down-regulators (SERDs) are pure ER antagonists that also induce ER degradation upon binding to the receptor. Although SERDs have been developed for the treatment of ER-positive breast cancers for nearly a decade, their precise mechanism(s) of action and structure-activity relationship are still unclear. Generally, Western blotting is used to examine the effects of SERDs on ER protein levels, but the methodology is low-throughput and not quantitative. Here, we describe a quantitative, high-throughput, luciferase-based assay for the evaluation of SERDs activity. For this purpose, we established stable recombinant HEK-293 cell lines expressing ERα fused with emerald luciferase. We also designed and synthesized new diphenylmethane derivatives as candidate SERDs, and evaluated their SERDs activity using the developed system in order to examine their structure-activity relationship, taking EC₅₀ as a measure of potency, and E_max as a measure of efficacy.     

Visualization of RecA filaments and DNA by fluorescence microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.     

Structure of the antitumour enzyme L-methionine gamma-lyase from Pseudomonas putida at 1.8 A resolution

l-Methionine gamma-lyase (EC 4.4.1.11, MGL_Pp) from Pseudomonas putida is a multifunctional enzyme, which belongs to the gamma-family of pyridoxal-5'-phosphate (PLP) dependent enzymes. In this report, we demonstrate that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 A resolution. Detailed information of the overall structure of MGL_Pp supplies a clear picture of the substrate- and PLP-binding pockets. Tyr59 and Arg61 of neighbouring subunits, which are strongly conserved in other gamma-family enzymes, contact the phosphate group of PLP. These residues are important as the main anchor within the active site. Lys240, Asp241 and Arg61 of one partner monomer and Tyr114 and Cys116 of the other partner monomer form a hydrogen-bond network in the MGL active site which is specific for MGLs. It is also suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug.