Diastereoselective annulation of 4-hydroxypyran-2H-ones with enantiopure 2,3-dideoxy-alpha,beta-unsaturated sugar aldehydes derived from respective glycals

One-pot condensations of 4-hydroxypyran-2H-ones 1 and 2, respectively, with various enantiopure 2,3-dideoxy-alpha,beta-unsaturated carbohydrate enals in the presence of l-proline in EtOAc at room temperature generated pyrano-pyrones. It was observed that, while benzyl-protected carbohydrate enals on condensation with 1 or 2 under the above conditions produced an inseparable diastereomeric mixture in a ratio of 1:1, the acyl-protected carbohydrate enals on treatment with 1 or 2 under identical conditions yielded products with moderate to very high diastereoselectivity. A remarkable asymmetric induction was noticed from the C-4 stereogenic center of the acyl-protected carbohydrate enals. An almost complete diastereoselectivity was observed in those reactions that involved condensation of 1 with acetyl-protected enals 5 and 7. The reaction of 2 with 5 also proceeded diastereoselectively to furnish the corresponding annulated product. The reaction presumably took place by C-1,2-addition of the pyrone onto the iminium salt of the alpha,beta-unsaturated carbohydrate enal generated in situ, followed by beta-elimination and cyclization of the 1-oxatriene involving a 6pi-electron electrocyclic process to yield a 2H,5H-pyrano[3,2-c]pyran-5-one derivative.     

Mild one-pot preparation of glycosyl bromides

Mild one-pot protocols for the preparation of glycosyl bromides and alkyl bromides via in situ generation of HBr is reported here.     

Carbohydrate analysis by a phenol-sulfuric acid method in microplate format

Among many colorimetric methods for carbohydrate analysis, the phenol-sulfuric acid method is the easiest and most reliable method. It has been used for measuring neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. This method is used widely because of its sensitivity and simplicity. In its original form, it required 50-450 nmol of monosaccharides or equivalent for analysis and thus is inadequate for precious samples. A scaled-down version requiring only 10-80 nmol of sugars was reported previously. We have now modified and optimized this method to use 96-well microplates for high throughput, to gain greater sensitivity, and to economize the reagents. This modified and optimized method allows longer linear range (1-150 nmol for Man) and excellent sensitivity. Moreover, our method is more convenient, requiring neither shaking nor covering, and takes less than 15 min to complete. The speed and simplicity of this method would make it most suitable for analyses of large numbers of samples such as chromatographic fractions.     

Structural determination of novel lacto-N-decaose and its monofucosylated analogue from human milk by electrospray tandem mass spectrometry and 1H NMR spectroscopy

We have isolated and characterised two neutral oligosaccharides, one nonfucosylated and the other monofucosylated, from human milk that are based on the doubly branched lacto-N-decaose core. Their structures have been determined by a combined use of electrospray tandem mass spectrometry (ES-MS/MS) and NMR spectroscopy. The sequences of the three branches resulted from the double-branching, including the identity and location of the blood-group-related Lewis determinant and partial linkages, were elucidated by the unique method of high sensitivity negative-ion ES-MS/MS analysis. Their full structure assignment was completed by methylation analysis and 1H NMR. The monofucosylated lacto-N-decaose, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc is a novel sequence, whereas the nonfucosylated lacto-N-decaose, Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc, has not been isolated and identified as an individual oligosaccharide.     

Leishmanin skin test in guinea pig with a single purified protein of Leishmania major

A series of hybridomas was produced by fusion of SP2/0 myeloma cells with spleen cells of mice immunized with Leishmania major (L. major). The reactivity of secreted monoclonal antibodies (mAbs) was evaluated against available leishmanin antigen by enzyme linked immunosorbent assay. Only one hybridoma designated as 7F9 secreted IgG1 mAb which was shown to be reactive with leishmanin. This mAb was further tested against four species of Leishmania (L. donovani, L. tropica, L. infantum, L. major) and a recombinant gp63. Among the four species tested it was shown to be only reactive with promastigotes of L. major. The antigen recognized by this mAb was purified and analyzed from both sonicated and supernatant cultures of L. major by immunoaffinity chromatography and reverse phase high performance liquid chromatography. The purified antigen, which gave a single band of 56kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis elicited a strong delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized with L. major. It was almost of the same degree as that produced by leishmanin. These results suggest that an L. major-specific antigen is an alternative as a specific diagnostic skin test reagent, which could lead to a better understanding of the mechanism of DTH in L. major.     

Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats

Recent evidence indicates that cell-cycle regulating proteins are involved in apoptotic process in post-mitotic neurons. In this study, we examined cell-cycle regulators for G1/S cell-cycle progression after a transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion. In the cerebral frontoparietal cortex, we observed a marked induction of Cyclin D1 (a coactivator of Cdks), and proliferating cell nuclear antigen (PCNA), together with upregulated Cdk kinase activities. This process is accompanied with multiple phosphorylation of retinoblastoma (Rb) protein at Cdk phosphorylation sites in neurons from the ischemic cortex. We further examined DNA synthesis by the incorporation of BrdU, a nucleotide analog that incorporates into newly synthesized DNA. Within 24-h of reperfusion after 60-min occlusion, substantial BrdU-positive neurons were observed in the ischemic cortex. Inhibition of Cdk4 activity during this ischemia/reperfusion is highly neuroprotective. These results suggest that ischemia/reperfusion cerebral damage induces signalings at the G1/S cell-cycle transition, and may constitute a critical step in the neuronal apoptotic pathway in ischemia/reperfusion induced neuronal damage.     

Polyomavirus EGFP-pseudocapsids: analysis of model particles for introduction of proteins and peptides into mammalian cells

A vector for preparation of mouse polyomavirus capsid-like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C-terminal part of the VP3 minor protein (EGFP-VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP-VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co-stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion.     

Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a rare clinically heterogeneous genodermatosis due to genetic defects in type VII collagen gene (COL7A1). Identification of COL7A1 mutations is a challenge since this gene comprises 118 exons and more than 300 mutations scattered over the gene have been reported. Here, we describe for the first time the use of denaturing high performance liquid chromatography (DHPLC) for COL7A1 mutation detection. To validate the method, exon-specific DHPLC conditions were applied to screen DNA samples from patients carrying known COL7A1 mutations. Abnormal DHPLC profiles were obtained for all known mutations. Subsequent DHPLC analysis of 17 DEB families of unknown genotype allowed the identification of 21 distinct mutations, 9 of which were novel. The DHPLC mutation detection rate was significantly higher compared with our mutation scanning rate with conventional techniques (97% vs 86%), indicating DHPLC as the method of choice for COL7A1 molecular characterization in DEB patients.     

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

Highly specific expression of luciferase gene in lungs of naïve nude mice directed by prostate-specific antigen promoter

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10(9)PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.