The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC₅₀ = 8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED₅₀ = 4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment     

Anti-inflammatory roles of mesenchymal stromal cells during acute Streptococcus pneumoniae pulmonary infection in mice

Background

Pneumonia is the fourth leading cause of death worldwide, and Streptococcus pneumoniae is the most commonly associated pathogen. Increasing evidence suggests that mesenchymal stromal cells (MSCs) have anti-inflammatory roles during innate immune responses such as sepsis. However, little is known about the effect of MSCs on pneumococcal pneumonia.

Identification of mono-, oligo-, and polysaccharides secreted from soybean roots

The mist culture system was conducted to study secreted polysaccharides from soybean ( Glycine max) roots grown for 15 days. Roots were rinsed with distilled water (DW) for 15 min, then with 30 mM oxalic acid (OXA) for 15 min to remove ionically bound sugar. Released sugars were further fractionated into low (L) and high (H) molecular weight fractions with Sephadex G-10. DW rinsing released 190 microg neutral sugar (NS) and 62 microg uronic acid (UA) per plant, while 374 microg NS and 70 microg UA per plant were released by OXA rinsing. Acetylation analysis revealed that the L fraction by DW and OXA mainly consisted of glucose (Glc), pinitol, and UA, whereas the H fraction mainly consisted of arabinose (Ara), galactose (Gal), Glc, and UA. The presence of rhamnose (2%-6%) in both fractions suggests secretion of rhamnogalacturonans. Methylation analysis revealed that the H fraction by DW and OXA contained T-Ara, 3-, 6-, and 3,6-Gal, suggesting the presence of type II arabinogalactan and arabinogalactan proteins. HPLC analysis detected mono-, di-, and tri-GalA in the L fraction by DW and OXA. Substances corresponding to sucrose, kojibiose, cello- and laminari-oligosaccharides were also found in root exudates.     

Involvement of digalactosyldiacylglycerol in cellular thermotolerance in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The effects of digalactosyldiacylglycerol (DGDG) deficiency on photosynthesis at high temperatures were examined using a dgdA mutant of Synechocystis sp. PCC 6803 incapable of DGDG biosynthesis. The dgdA mutant cells showed significant growth retardation when the temperature was increased from 30 to 38°C, although wild-type cells grew normally. The degree of growth retardation was enhanced by increasing light intensity. In addition, dgdA mutant cells showed increased sensitivity to the photoinhibition of photosynthesis when illuminated at 38°C. Analysis of photosynthesis in intact cells suggested that the inhibition of repair processes and accelerated photodamage resulted in growth retardation in dgdA mutant cells at high temperatures.     

Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis.     

Crystal Structures of Ethanolamine Ammonia-lyase Complexed with Coenzyme B12 Analogs and Substrates

N-terminal truncation of the Escherichia coli ethanolamine ammonia-lyase β-subunit does not affect the catalytic properties of the enzyme (Akita, K., Hieda, N., Baba, N., Kawaguchi, S., Sakamoto, H., Nakanishi, Y., Yamanishi, M., Mori, K., and Toraya, T. (2010) J. Biochem. 147, 83–93). The binary complex of the truncated enzyme with cyanocobalamin and the ternary complex with cyanocobalamin or adeninylpentylcobalamin and substrates were crystallized, and their x-ray structures were analyzed. The enzyme exists as a trimer of the (αβ)₂ dimer. The active site is in the (β/α)₈ barrel of the α-subunit; the β-subunit covers the lower part of the cobalamin that is bound in the interface of the α- and β-subunits. The structure complexed with adeninylpentylcobalamin revealed the presence of an adenine ring-binding pocket in the enzyme that accommodates the adenine moiety through a hydrogen bond network. The substrate is bound by six hydrogen bonds with active-site residues. Argα¹⁶⁰ contributes to substrate binding most likely by hydrogen bonding with the O1 atom. The modeling study implies that marked angular strains and tensile forces induced by tight enzyme-coenzyme interactions are responsible for breaking the coenzyme Co–C bond. The coenzyme adenosyl radical in the productive conformation was modeled by superimposing its adenine ring on the adenine ring-binding site followed by ribosyl rotation around the N-glycosidic bond. A major structural change upon substrate binding was not observed with this particular enzyme. Gluα²⁸⁷, one of the substrate-binding residues, has a direct contact with the ribose group of the modeled adenosylcobalamin, which may contribute to the substrate-induced additional labilization of the Co–C bond.     

Nutrients distribution in the coastal water of East Asia relative to the Kuroshio

Nutrients in the coastal water of East Asia were surveyed from the point of contribution of the diluted water to the open water relative to the Kuroshio, the western boundary current. The Kuroshio runs along eastern Asia, sensing changes in environment in the coastal region. The nutrients-depleted water from the Kuroshio, close to the coastal water, provides heat energy and high saline water. The fresh water from the river contributes to the coastal water as diluted water and exhibits the entrainments of nutrient water into the Kuroshio. The nutrients are important parameters to monitor the interactions among the Kuroshio and inland waters. Nutrients exhibited a wide range of ratio between nitrate to phosphate, nitrate to silicate, and silicate to phosphate in East Asia as divergences from the Redfield ratios. The distribution of nutrients could also be monitored as the distribution of chlorophyll-a concentration using the ocean color sensors. Contrasts in chlorophyll-a concentration between the oligotrophic water of the Kuroshio and the trophic water of the coastal region exhibit the mixing process of two water masses. Although the chlorophyll-a concentrations estimated by the current ocean color algorithm over the coastal water are of insufficient accuracy, the distribution of chlorophyll-a concentration exhibits a variation of seasonal consumption of nutrients and response to the Kuroshio and river runoff.     

Transient hyper-17-OHPnemia unrelated to cross-reactions with residual fetal adrenal cortex products

OBJECTIVE: To clarify the pathogenesis of transient hyper-17alpha-hydroxyprogesteronemia, we initiated a laboratory investigation in a pre-term infant with persistently high serum 17alpha-hydroxyprogesterone (17-OHP) until 2 months of age. METHODS: Serum 17-OHP level was measured by high-performance liquid chromatography and radioimmunoassay, and gene analysis of CYP21A2 (21-hydroxylase) was performed. RESULT: Serum 17-OHP level on the 29th day of life was 25.4 ng/ml, and the urinary steroid profile showed low pregnanetriolone. Gene analysis of 21-hydroxylase disclosed no mutation, and 17-OHP normalized by 3 months of age without specific treatment. CONCLUSION: Transient elevations in 17-OHP, which do not appear related to cross-reactions with products of a residual fetal adrenal cortex, may occur in the first few months of life.     

Characterization of acatalasemic erythrocytes treated with low and high dose hydrogen peroxide. Hemolysis and aggregation

The effects of hydrogen peroxide on normal and acatalasemic erythrocytes were examined. Severe hemolysis of acatalasemic erythrocytes and a small tyrosine radical signal (g = 2.005) associated with the formation of ferryl hemoglobin were observed upon the addition of less than 0.25 mM hydrogen peroxide. However, when the concentration of hydrogen peroxide was increased to 0.5 mM, acatalasemic erythrocytes became insoluble in water and increased the tyrosine radical signal. Polymerization of hemoglobin and aggregation of the erythrocytes were observed. On the other hand, normal erythrocytes exhibited only mild hemolysis by the addition of hydrogen peroxide under similar conditions. From these results, the scavenging of hydrogen peroxide by hemoglobin generates the ferryl hemoglobin species (H-Hb-Fe(IV)=O) plus protein-based radicals (*Hb-Fe(IV)=O). These species induce hemolysis of erythrocytes, polymerization of hemoglobin, and aggregation of the acatalasemic erythrocytes. A mechanism for the onset of Takarara disease is proposed.     

mDia2 induces the actin scaffold for the contractile ring and stabilizes its position during cytokinesis in NIH 3T3 cells

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.