黑蕊虎耳草中岩白菜素没食子酸酯类及其对丙型肝炎丝氨酸蛋白酶的抑制作用

研究了黑蕊虎耳草(Saxifraga melanocentra)中岩白菜素衍生物的化学和生物活性,从其地上部分分离纯化得到一个新的岩白菜素没食子酸酯,主要通过1维和2维核磁共振波谱鉴定结构为11-氧-(4-氧甲基没食子酰)岩白菜素[11-O-(4-O-methylgalloyl) bergenin(1)] ,该化合物对丙型肝炎丝氨酸蛋白酶(HCVNS3serine protease)具有抑制活性,其IC50为0.32 mg/mL。     

CoMFA and CoMSIA 3D-QSAR analysis of diaryloxy-methano-phenanthrene derivatives as anti-tubercular agents

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) based on three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were conducted on a series (44 compounds) of diaryloxy-methano-phenanthrene derivatives as potent antitubercular agents. The best predictions were obtained with a CoMFA standard model (q (2)=0.625, r (2)=0.994) and with CoMSIA combined steric, electrostatic, and hydrophobic fields (q (2)=0.486, r (2)=0.986). Both models were validated by a test set of seven compounds and gave satisfactory predictive r (2) values of 0.999 and 0.745, respectively. CoMFA and CoMSIA contour maps were used to analyze the structural features of the ligands to account for the activity in terms of positively contributing physicochemical properties: steric, electrostatic, and hydrophobic fields. The information obtained from CoMFA and CoMSIA 3-D contour maps can be used for further design of phenanthrene-based analogs as anti-TB agents. The resulting contour maps, produced by the best CoMFA and CoMSIA models, were used to identify the structural features relevant to the biological activity in this series of analogs. Further analysis of these interaction-field contour maps also showed a high level of internal consistency. This study suggests that introduction of bulky and highly electronegative groups on the basic amino side chain along with decreasing steric bulk and electronegativity on the phenanthrene ring might be suitable for designing better antitubercular agents.     

Isolation, purification and characterization of a DPP-III homologue from goat brain

A dipeptidyl peptidase (DPP) from goat brain has been purified. The purified enzyme showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). It is a monomer with molecular weight of 69kDa with a pI of 4.5. The K(m) was estimated to be 39microM for Arg-Arg-4-methoxy-beta-naphthylamide (Arg-Arg-4mbetaNA). This enzyme is strongly inhibited by commonly used metallochelators and sulfhydryl reagents. Among various beta-naphthylamides examined, Arg-Arg-4mbetaNA was the most rapidly hydrolyzed substrate. Although, initially it was thought to be the DPP-III but on the basis of its molecular weight and inhibition studies, it was concluded that this enzyme is a functional homologue of DPP-III.     

Glycogen metabolism in tissues from a mouse model of Lafora disease

Laforin, encoded by the EPM2A gene, by sequence is a member of the dual specificity protein phosphatase family. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons, muscle and other tissues. Glycogen metabolizing enzymes were analyzed in a transgenic mouse over-expressing a dominant negative form of laforin that accumulates Lafora bodies in several tissues. Skeletal muscle glycogen was increased 2-fold as was the total glycogen synthase protein. However, the -/+glucose-6-P activity of glycogen synthase was decreased from 0.29 to 0.16. Branching enzyme activity was increased by 30%. Glycogen phosphorylase activity was unchanged. In whole brain, no differences in glycogen synthase or branching enzyme activities were found. Although there were significant differences in enzyme activities in muscle, the results do not support the hypothesis that Lafora body formation is caused by a major change in the balance between glycogen elongation and branching activities.     

Anti-inflammatory effects of 7-methoxycryptopleurine and structure-activity relations of phenanthroindolizidines and phenanthroquinolizidines

A cryptopleurine analogue, 7-methoxycryptopleurine, a phenanthroquinolizidine, was first found to exert potent anti-inflammatory activity in vitro and in vivo as well as have remarkable cytotoxic activity against cancer cells. The non-planar structure between the two major moieties, phenanthrene and indolizidine/quinolizidine, played a crucial role in the activity of phenanthroindolizidines or phenanthroquinolizidines in terms of cytotoxic effects on cancer cells and anti-inflammatory activity. We also showed that increase in planarity and rigidity of the indolizidine/quinolizidine moiety and change of the amine group into an amide by introducing a keto group to phenanthroindolizidines or phenanthroquinolizidines at the equivalent position 9 of tylophorine significantly reduced their activities. Moreover, in general, phenanthroquinolizidines are more potent than their respective phenanthroindolizines.     

Expression of the Longin domain of TI-VAMP impairs lysosomal secretion and epithelial cell migration

BACKGROUND INFORMATION: TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein; also called VAMP7) belongs to the Longin subfamily of v-SNAREs (vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors). The regulatory N-terminal extension, called the Longin domain, of TI-VAMP has been shown previously to have a dual biochemical function: it inhibits the capacity of TI-VAMP to form SNARE complexes and it binds to the delta subunit of the AP-3 (adaptor protein 3) complex in early endosomes, thereby targeting TI-VAMP to late endosomes. RESULTS: We have generated MDCK (Madin-Darby canine kidney) cell lines expressing the Longin domain of TI-VAMP coupled to GFP (green fluorescent protein) in a doxycycline-dependent manner. As expected, AP-3delta (AP-3 delta subunit) is not properly localized in Longin-expressing cells. We have shown that the expression of the Longin domain impairs lysosomal secretion, as determined by the release of a pre-internalized fluorescent fluid-phase marker and by electron microscopy of the membrane-associated released particles. Membrane repair following mechanical wounding, a process requiring lysosomal secretion, is also impaired in cells expressing the Longin domain. Furthermore, cell migration, assessed by wound healing of MDCK monolayers, is also inhibited. CONCLUSIONS: The results of the present study suggest that the expression of the Longin domain of TI-VAMP regulates lysosomal secretion of epithelial cells and provide molecular evidence for a role of the late endocytic system in cell migration.     

Complementary selectivity to (S)-1-phenyl-1,2-ethanediol-forming Candida parapsilosis by expressing its carbonyl reductase in Escherichia coli for (R)-specific reduction of 2-hydroxyacetophenone

An (R)-specific carbonyl reductase from Candida parapsilosis CCTCCM203011 (CprCR) was shown to catalyze the asymmetric reduction of 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol (PED), which is a critical chiral building block in organic synthesis. The gene (rcr) encoding CprCR was cloned based on the amino acid sequences of tryptic fragments of the enzyme. Sequence analysis revealed that rcr is comprised of 1008 nucleotides encoding a 35 977 Da polypeptide, and shares similarity to proteins of the medium-chain dehydrogenase/reductase (MDR) superfamily. Recombinant rcr expressed in Escherichia coli showed a specific 2-hydroxyacetophenone-reducing activity. Using rcr expressing cells, (R)-PED was obtained by asymmetric reduction, which is complementary in enantiomeric configuration to (S)-PED obtained by using whole cells of C. parapsilosis. After optimization of reaction conditions, (R)-PED was produced at 95.5% enantiomeric excess with a yield of 92.6% when isopropanol was used for cofactor regeneration.     

Mechanotransduction of keratinocytes in culture and in the epidermis

The epidermis, like many other tissues, reacts to mechanical stress by increasing cell proliferation. Mechanically stressed skin regions often develop thicker skin and hyperkeratosis. Interestingly, a large number of skin diseases are accompanied by epidermal proliferation and hyperkeratosis even under normal mechanical stress conditions. Although, some of the molecular pathways of mechanical signaling involving integrins, the epidermal growth factor receptor and mitogen-activated protein kinases are known it is still unclear, how mechanical force is sensed and transformed into the molecular signals that induce cell proliferation. This review focuses on the molecules and pathways known to play a role in mechanotransduction in epidermal keratinocytes and discusses the pathways identified in other well-studied cell types.     

Degradation of 3-O-methylgallate in Sphingomonas paucimobilis SYK-6 by pathways involving protocatechuate 4,5-dioxygenase

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate and 3-O-methylgallate (3MGA), respectively. 3MGA is metabolized via multiple pathways involving 3MGA 3,4-dioxygenase, protocatechuate 4,5-dioxygenase (LigAB), and gallate dioxygenase whereas protocatechuate is degraded via the protocatechuate 4,5-cleavage pathway. Here the secondary role of LigAB in syringate metabolism is investigated. The reaction product of 3MGA catalyzed by His-tagged LigAB was identified as 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD) and 2-pyrone-4,6-dicarboxylate (PDC), indicating that 3MGA is transformed to CHMOD and PDC by both reactions catalyzed by DesZ and LigAB. Mutant analysis revealed that the 3MGA catabolic pathways involving LigAB are functional in SYK-6.     

A comparative study of different glycosylation methods for the synthesis of D-mannopyranosides of Nalpha-fluorenylmethoxycarbonyl-trans-4-hydroxy-L-proline allyl ester

The synthesis of Nalpha-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-O-acetyl)-alpha-d-mannopyranosyl]-l-proline allyl ester and Nalpha-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-O-benzoyl)-alpha-d-mannopyranosyl]-l-proline allyl ester is described. Glycosylation using K?nigs-Knorr conditions with a benzoyl protected glycosyl donor provided the optimum method. Removal of the allyl ester gave two mannosylated building blocks suitable for solid phase glycopeptide synthesis.