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.     

Some Physical and Chemical Properties of Nuclease P1

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP₅₀) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane << ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.     

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.     

Existence of Sixteen 2′→5′ Dinucleotides in Nuclease P1 (Penicillium Nuclease) Digest of Technical Grade Yeast RNA

Sixteen 2′→5′ dinucleotides; (2′–5′)pA-A, pA-G, pA-C, pA-U, pG-A, pG-G, pG-C, pG-U, pC-A, pC-G, pC-C, pC-U, pU-A, pU-G, pU-C, and pU-U were detected in nuclease P₁ digest of a technical grade yeast RNA by means of gel filtration on Sephadex G-10, DEAE-Sephadex A-25 column chromatography in the presence of 7 m urea, paper electrophoresis and paper chromatography. Content of each dinucleotide was about 0.1 to 0.6% of the digest. As the sixteen 2′→5′ dinucleotides were found in all of the digests of technical grade RNA preparations tested, each polynucleotide chain in the preparations may be concluded to contain several per cent of the 2′–5′ minor phosphodiester linkages in addition to the 3′–5′ major phosphodiester linkages.     

Occurrence of Nuclease P1-Malonogalactan Complex

Nuclease P₁ from Penicillium citrinum was found to be produced in a form of complex with malonogalactan (a galactan, 1, 5-β-galactofuranoside polymer esterfied with malonic acid at position 3) in the culture on wheat bran. Neither nuclease P₁-malonogalactan complex nor malonogalactan was produced in a liquid medium. Nuclease P₁-malonogalactan complexes, P₁-MG I, II, and III were purified from an aqueous extract of the culture on wheat bran. The most anionic complex, P₁-MG III, was composed of the protein, carbohydrate and malonic acid in the ratio of 1: 2.6: 0.5 (w/w). The complex was not dissociated by purification procedures including fractionations with acetone and ammonium sulfate, gel filtration and DEAE-cellulose chromatography. A malonogalactan-specific carboxylesterase was found in culture of the same mold on wheat bran. Nuclease P₁-malonogalactan was demalonylated by the esterase to yield nuclease P₁-galactan. The binding of nuclease P₁ to galactan was rather loose so that nuclease P₁-galactan complex was partially dissociated by DEAE-cellulose chromatography. Attempt to reconstitute the complex from nuclease P₁ and malonogalactan upon mixing was unsuccessful. Exogenously supplemented nuclease P₁ did not associate with malonogalactan in the growing culture on wheat bran, either.

Several extracellular enzymes such as RNase, β-galactosidase and protease were also found in a form of complex with malonogalactan in the culture on wheat bran.     

The Photochemical Reaction of Pentachlorophenol

The chemical structure of a yellow C₁₈-compound (IV), isolated from the decomposition products of sodium pentachlorophenoxide (Na-PCP) in an aqueous solution by sunlight, has been determined by chemical and spectroscopic evidences. Some of the chemistry and the absorption spectra of IV and its related compounds containing 3-cyclohexene-1,2-dione and spiroketal structures are also described.     

DNA Methylation Profile Distinguishes Clear Cell Sarcoma of the Kidney from Other Pediatric Renal Tumors

A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms’ tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing’s sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms’ tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.     

Clinical Utility of an Enzyme-Linked Immunosorbent Assay for Detecting Anti-Melanoma Differentiation-Associated Gene 5 Autoantibodies

ObjectiveAutoantibodies to melanoma differentiation-associated gene 5 (MDA5) are specifically expressed in patients with dermatomyositis (DM) and are associated with a subset of DM patients with rapidly progressive interstitial lung disease (RP-ILD). Here, we examined the clinical utility of a newly developed enzyme-linked immunosorbent assay (ELISA) system for detecting these antibodies.MethodsHere we developed an improved ELISA for detecting anti-MDA5 antibodies. We then performed a multicenter clinical study involving 8 medical centers and enrolled 242 adult patients with polymyositis (PM)/DM, 190 with non-PM/DM connective tissue disease (CTD), 154 with idiopathic interstitial pneumonia (IIP), and 123 healthy controls. Anti-MDA5 antibodies in the patients’ serum samples were quantified using our newly developed ELISA, and the results were compared to those obtained using the gold-standard immunoprecipitation (IP) assay. In addition, correlations between the ELISA-quantified anti-MDA5 antibodies and clinical characteristics were evaluated.ResultsIn patients with PM/DM, the anti-MDA5 antibody measurements obtained from the ELISA and IP assay were highly concordant; the ELISA exhibited an analytical sensitivity of 98.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.5% (compared to the IP assay). Anti-MDA5 antibodies were detected in 22.7% of the DM patients, but not in any of the patients with PM, non-PM/DM CTD, or IIP. Clinically amyopathic DM, RP-ILD, arthritis, and fever were more prevalent in DM patients who were anti-MDA5 antibody-positive than in those who were antibody-negative (P ≤ 0.0002 for all comparisons). In addition, anti-MDA5 antibody-positive patients with RP-ILD exhibited higher antibody levels than those without RP-ILD (P = 0.006).ConclusionOur newly developed ELISA can detect anti-MDA5 antibodies as efficiently as the gold standard IP assay and has the potential to facilitate the routine clinical measurement of anti-MDA5 antibodies in patients who suspected to have DM.     

Human Leukocyte Antigen and Systemic Sclerosis in Japanese: The Sign of the Four Independent Protective Alleles,DRB1*13:02, DRB1*14:06, DQB1*03:01, and DPB1*02:01

ObjectiveSeveral studies on associations between human leukocyte antigen (HLA) allele frequencies and susceptibility to systemic sclerosis (SSc) have been reported. Anti-centromere antibodies (ACA) and anti-topoisomerase I antibodies (ATA) are found in SSc patients. Here, we sought to identify HLA alleles associated with SSc in Japanese, and explored their associations with SSc phenotypes including the presence of autoantibodies.MethodsAssociations of HLA-DRB1, DQB1, and DPB1 were analyzed in 463 Japanese SSc patients and 413 controls.ResultsWe found that DRB1*13:02 (P = 0.0011, Pc = 0.0319, odds ratio [OR] 0.46, 95% confidence interval [CI] 0.29–0.73), DRB1*14:06 (P = 6.60X10^-5, Pc = 0.0020, OR 0.05, 95%CI 0.01–0.41), DQB1*03:01 (P = 0.0009, Pc = 0.0150, OR 0.56, 95%CI 0.40–0.79), and DPB1*02:01 (P = 5.16X10^-6, Pc = 8.77X10^-5, OR 0.52, 95%CI 0.39–0.69) were protectively associated with SSc. In addition, these four alleles seemed to be independently associated with the protection against the susceptibility of SSc. On the other hand, we could not find predisposing alleles for overall SSc. With respect to SSc subsets, a tendency for these four alleles to be protectively associated was observed. However, there was a significant association between DRB1*01:01, DRB1*10:01, DQB1*05:01, and DPB1*04:02 and the susceptibility to SSc with ACA. On the other hand, the presence of DRB1*15:02, DQB1*06:01, DPB1*03:01, and DPB1*09:01 was associated with SSc with ATA.ConclusionThus, the present study has identified protective associations of the four HLA class II alleles with overall Japanese SSc and predisposing associations of HLA class II alleles with Japanese SSc subsets.     

A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.