Optimization and in vivo evaluation of pyrazolopyridines as a potent and selective PI3Kδ inhibitor

Chemical optimization of pyrazolopyridine 1, focused on cellular potency, isoform selectivity and microsomal stability, led to the discovery of the potent, selective and orally available PI3Kδ inhibitor 5d. On the basis of its desirable potency, selectivity and pharmacokinetic profiles, 5d was tested in the trinitrophenylated aminoethylcarboxymethyl-Ficoll (TNP-Ficoll)-induced antibody production model, and showed higher antibody inhibition than a 4-fold oral dose of the starting compound 1. These excellent results suggest that 5d is a potential candidate for further studies in the treatment of autoimmune diseases and leukocyte malignancies.     

Attempt to simultaneously generate three chiral centers in 4-hydroxyisoleucine with microbial carbonyl reductases

A panel of microorganisms was screened for selective reduction ability towards a racemic mixture of prochiral 2-amino-3-methyl-4-ketopentanoate (rac-AMKP). Several of the microorganisms tested produced greater than 0.5 mM 4-hydroxyisoleucine (HIL) from rac-AMKP, and the stereoselectivity of HIL formation was found to depend on the taxonomic category to which the microorganism belonged. The enzymes responsible for the AMKP-reducing activity, ApAR and FsAR, were identified from two of these microorganisms, Aureobasidium pullulans NBRC 4466 and Fusarium solani TG-2, respectively. Three AMKP reducing enzymes, ApAR, FsAR, and the previously reported BtHILDH, were reacted with rac-AMKP, and each enzyme selectively produced a specific composition of HIL stereoisomers. The enzymes appeared to have different characteristics in recognition of the stereostructure of the substrate AMKP and in control of the 4-hydroxyl group configuration in the HIL product.     

Patchwork-Type Spontaneous Activity in Neonatal Barrel Cortex Layer 4 Transmitted via Thalamocortical Projections

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Metal-catalyzed oxidation of human serum albumin does not alter the interactive binding to the two principal drug binding sites

It is well known that various physiological factors such as pH, endogenous substances or post-translational modifications can affect the conformational state of human serum albumin (HSA). In a previous study, we reported that both pH- and long chain fatty acid-induced conformational changes can alter the interactive binding of ligands to the two principal binding sites of HSA, namely, site I and site II. In the present study, the effect of metal-catalyzed oxidation (MCO) caused by ascorbate/oxygen/trace metals on HSA structure and the interactive binding between dansyl-L-asparagine (DNSA; a site I ligand) and ibuprofen (a site II ligand) at pH 6.5 was investigated. MCO was accompanied by a time-dependent increase in carbonyl content in HSA, suggesting that the HSA was being oxidized. In addition, The MCO of HSA was accompanied by a change in net charge to a more negative charge and a decrease in thermal stability. SDS-PAGE patterns and α-helical contents of the oxidized HSAs were similar to those of native HSA, indicating that the HSA had not been extensively structurally modified by MCO. MCO also caused a selective decrease in ibuprofen binding. In spite of the changes in the HSA structure and ligand that bind to site II, no change in the interactive binding between DNSA and ibuprofen was observed. These data indicated that amino acid residues in site II are preferentially oxidized by MCO, whereas the spatial relationship between sites I and II (e.g. the distance between sites), the flexibility or space of each binding site are not altered. The present findings provide insights into the structural characteristics of oxidized HSA, and drug binding and drug-drug interactions on oxidized HSA.     

The brain neurosecretory cells of the moth Samia cynthia ricini: Immunohistochemical localization and developmental changes of the Samia homologues of the Bombyx prothoracicotropic hormone and bombyxin

We produced mouse antisera against synthetic peptides corresponding to the sequences of the Samia cynthia ricini homologues of the Bombyx mori PTTH and bombyxin. Immunohistochemical analyses of the Samia cephalic neuroendocrine system using these antisera were performed to identify the neurosecretory cells (NSC) containing the PTTH and bombyxin homologues and to examine the developmental changes in their amounts in the NSC. The results show that the PTTH and bombyxin homologues are produced by two pairs of dorsolateral and 16 pairs of dorsomedial NSC of Samia brain, respectively, and both are transported to, and released from, the corpora allata. No clear-cut correlation was found between the fluctuation in the amount of immunoreactive substances in the brain NSC and the endocrinologically anticipated timings of PTTH secretion. From Samia brain extract, two forms of PTTH activity (∼30 kDa and ∼5 kDa) were resolved through Sephadex gel filtration. The ∼30 kDa and ∼5 kDa PTTH seem to represent the PTTH and bombyxin homologues, respectively. We discuss that the ∼30 kDa PTTH homologue is the true PTTH of Samia .     

Purification and characterization of an algal (Dunaliella tertiolecta) protein cross-reacting with an anti-Ga-common antibody

A 26-kDa and a 36-kDa protein that cross-reacted with anti-G_a-common and anti-G_β antibodies, respectively, were detected in Dunaliella cells. The 26-kDa protein was solubilized from a crude membrane fraction with deoxycholate and purified to homogeneity by DE52 and hydroxylapatite chromatography and DEAE-5PW high performance liquid chromatography (HPLC). The hydroxylapatite-purified preparation had GTPγS binding and GTPase activities, but the homogeneous 26-kDa protein had none. The sequence of the 28 N-terminal amino acids of the 26-kDa protein had no homology to any GTP binding protein thus far reported.     

Three-dimensional structure of gurmarin,a sweet taste-suppressing polypeptide

Summary The solution structure of gurmarin was studied by two-dimensional proton NMR spectroscopy at 600 MHz. Gurmarin, a 35-amino acid residue polypeptide recently discovered in an Indian-originated tree Gymnema sylvestre, selectively suppresses the neural responses of rat to sweet taste stimuli. Sequence-specific protons. The three-dimensional solution structure was determined by simulated-annealing calculations on the basis of 135 interproton distance constraints derived from NOEs, six distance constraints for three hydrogen bonds and 16 dihedral angle constraints derived from coupling constants. A total of 10 structures folded into a well-defined structure with a triple-stranded antiparallel -sheet. The average rmsd values between any two structures were 1.65±0.39 Å for the backbone atoms (N, C, C) and 2.95±0.27 Å for all heavy atoms. The positions of the three disulfide bridges, which could not be deterermined chemically, were estimated to be Cys³–Cys¹⁸, Cys¹⁰–Cys²³ and Cys¹⁷–Cys³³ on the basis of the NMR distance constraints. This disulfide bridge pattern in gurmarin turned out to be analogous to that in -conotoxin and Momordica charantia trypsin inhibitor-II, and the topology of folding was the same as that in -conotoxin.Abbreviations DQF-COSY double-quantum-filtered correlated spectroscopy - HOHAHA homonuclear Hartmann-Hahn spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - ppm parts per million; rmsd, root-mean-square deviation - TSP 3-(trimethylsilyl)-2,2,3,3-tetradeutero-propionate     

aproctous,a locus that is necessary for the development of the proctodeum in Drosophila embryos,encodes a homolog of the vertebrate Brachyury gene

The proctodeum of the Drosophila embryo originates from the posterior end of the blastoderm and forms the hindgut. By enhancer-trap mutagenesis, using a P-element-lacZ vector, we identified a mutation that caused degeneration of the proctodeum during shortening of the germ band and named it aproctous (apro). Expression of the lacZ reporter gene, which was assumed to represent expression of the apro gene, began at the cellular blastoderm stage in a ring that encompassed about 10–15% of the egg's length (EL) and included the future proctodeum, anal pads, and posterior-most part of the visceral mesoderm. In later stages, strong expression of lacZ was detected in the developing hindgut and anal pads. Expression continued in the anal pads and epithelium of the hindgut of larvae; the epithelium of the hindgut of the adult fly also expressed lacZ. The spatial patterns of the expression of lacZ in various mutants suggested that the embryonic expression of apro was regulated predominantly by two gap genes, tailless (tll) and huckebein (hkb): tll is necessary for the activation of apro, while hkb suppressed the expression of apro in the region posterior to 10% EL. Cloning and sequencing of the apro cDNA revealed that apro was identical to the T-related gene (Trg) that is a Drosophila homolog of the vertebrate Brachyury gene. apro appears to play a key role in the development of tissues derived from the proctodeum.     

Screening of melanin biosynthesis inhibitors from marine microalgae using Streptomyces bikiniensis bioassay

Summary Inhibitors of melanin biosynthesis from marine microalgae were screened against a melanin-producing microorganism, Streptomyces bikiniensis. From 28 marine microalgal strains, 5 were found showing inhibitory activity. Of these, the extracts (50l, 2g total organic carbon/l) from two marine green algae showed strongly inhibited melanin biosynthesis, but showed less than 30 % inhibitory activity against mushroom tyrosinase.