New developments for the design,synthesis and biological evaluation of potent SARS-CoV 3CLpro inhibitors

A series of trifluoromethyl, benzothiazolyl or thiazolyl ketone-containing peptidic compounds as SARS-CoV 3CL protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. Three candidates had encouraging results for the development of new anti-SARS compounds.     

IFN-alpha beta promote priming of antigen-specific CD8+ and CD4+ T lymphocytes by immunostimulatory DNA-based vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-alphabeta and -gamma, TNF-alpha, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8(+) and CD4(+) lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-alphabeta pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8(+) CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4(+) Th cells by ISS-based vaccines.     

The Effect of Cork Pieces on Pseudohypericin Production in Cells ofHypericum perforatumShoots

The stimulating effect of cork pieces on hypericin and pseudohypericin biosyntheses was studied in cells of shoots regenerated from the callus cultures of St. John's wort (Hypericum perforatumL.). The addition of the cork matrix slightly stimulated shoot growth and enhanced pseudohypericin biosynthesis about threefold (to 0.4 mg/g dry wt). Pseudohypericin production increased proportionally with the amount of cork material added (from 1 to 4 mg/ml of growth medium). Further increase in the amount of cork pieces inhibited both pseudohypericin production and shoot growth. Organic and aqueous extracts of cork pieces did not affect the production of these substances.     

Combinatorial evaluation of the chiral discrimination of permethylated carbohydrates using fast-atom bombardment mass spectrometry

The chiral discrimination abilities of several variously permethylated carbohydrates toward various amino acid 2-propyl esters were combinatorially evaluated from the relative peak intensity of the 1:1 diastereomeric complex ions with the deuterium-labeled L-amino acid 2-propyl ester protonated ion and with the unlabeled D-amino acid 2-propyl ester protonated ions in FAB mass spectrometry. The chiral discrimination abilities evaluated using FAB mass spectrometry approximately corresponded to the ratio of the association constants (K(R)/K(S)) toward each enantiomer in the solution. Therefore, this evaluation method is very useful for the screening of the chiral discrimination abilities of carbohydrates and their derivatives.     

Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase

Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a beta-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each k(cat)/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6A resolution revealed that KLrP is a (beta/alpha)8 TIM barrel, in which Glu(165) and Glu(373) at the carboxyl termini of beta-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer.     

Ultrastructure of cementum formation on partially formed teeth in dogs

Cementum crystals and matrix vesicles on the root surface of partially formed teeth in dogs were examined with a transmission electron microscope. Fine filamentous crystals were observed in the cementum calcifying fronts. The running pattern was mainly parallel to the root surface in the apical region and perpendicular to the root surface in lateral and coronal regions. Matrix vesicles were observed at the apical half of the periodontium, but not observed at the coronal region. These findings suggest that the parallel-arranged cementum would become the light-microscopic lamellar type and the perpendicular one the light-microscopic dense-line structure when fully developed. Moreover, cementum formation occurs due to two kinds of mechanisms: participation of matrix vesicles and secondary calcification (= additional cementogenesis).     

Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7. We found that TGF-beta stimulates Smurf2 expression. TGF-beta activated the Smurf2 promoter in a TGF-beta responsive cell lines, whereas IL-1alpha, PDGF and epidermal growth factor did not. TGF-beta-mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor-like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad-binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF-beta-mediated Smurf2 induction. These results suggest that TGF-beta stimulates Smurf2 expression by Smad-independent pathway such as PI3K/Akt pathway via TGF-beta receptor.     

Arterial receptors for adrenal steroids and transport of electrolytes in vascular smooth muscle

The role of arterial receptors to mineralocorticoids (MC) and glucocorticoids (GC) in the induction by MC and GC of changes in transmembrane transport of sodium (Na+) and water was investigated. Implantation of Silastic rubber strips impregnated with 11-desoxycorticosterone acetate (DOCA) in rabbits was followed by a marked increase in vascular smooth muscle cell membrane permeability to Na+ and hypertension. Both of these effects were preventable with progesterone, an anti-MC at the steroid-receptor level, implanted in relative excess simultaneously with DOCA, in approximately 50% of the implanted animals. The other 50% were hydroxylating in vivo progesterone to 11-desoxycorticosterone (DOC) efficiently enough not to yield the necessary ratio of progesterone to DOC for the sufficient MC receptor blockage. In vascular smooth muscle cell culture, grown in the presence of steroids, GC but not MC increased intracellular water space. This increase was preventable by a potent synthetic anti-GC,RU 38486, a steroid with high affinity for GC receptors, added to culture medium along with GC. These results provide evidence that both the in vivo effect of MC on Na+ permeability and the induction of hypertension, and the in vitro effect of GC on water transport in cultured vascular smooth muscle cells are elicited through the receptor-mediated molecular mechanism(s) for action of these steroids in the arterial wall.