Synthesis of 1-(β-D-Glycopyranosyl)-3-Deazapyrimidines from 2-Hydroxy and 2-Mercaptopyridines

Abstract

The synthesis of new 4- and 5-substituted-3-cyanopyridine nucleosides has been performed by reacting the silylated pyridines and penta-O-acetyl-α -D-glycopyranose in dichloroethane in the presence of SnCl₄. The free nucleosides were tested for their potential activity against HIV and different types of tumor.     

Identification of oncofetal antigen/immature laminin receptor protein epitopes that activate BALB/c mouse OFA/iLRP-specific effector and regulatory T cell clones

During tumor development in mice and humans, oncofetal Ag/immature laminin receptor (OFA/iLRP)-specific Th1, CTL, and IL-10-secreting T (Ts) cells are induced. The presence of too many Ts or too few effector T cells appears to predict a poor prognosis. We established clones of OFA/iLRP-specific splenic Th1, CTL, and Ts cells from the OFA/iLRP+ MCA1315 fibrosarcoma-bearing BALB/c mice or from BALB/c mice vaccinated with 1 or 10 microg of rOFA/iLRP. The MCA1315 tumor cell-reactive T cell clones were characterized as to surface Ag phenotype, cytokine secretion profile, and specificity for OFA/iLRP presented by syngeneic splenic APC. OFA/iLRP-specific Th1 and Ts clones were established from all mice. OFA/iLRP-specific CTL could be established from all mice except for mice immunized with 10 microg of rOFA/iLRP. Analysis of the proliferation profile of the OFA/iLRP-specific clones to overlapping OFA/iLRP 12-mer peptides that spanned the OFA/iLRP protein sequence defined the epitopes to which the T cell clones responded. There was a similar spatial distribution of the epitopes to which the two types of CD8 T cell clones responded. The nonapeptide epitopes of the Ts clones were located between aa 36 and 147 of OFA/iLRP, while the epitopes of the CTL clones were located between aa 52 and 163. Even though the CTL and Ts epitopes shared part of the protein, all of the CD8 CTL epitopes were distinct and separable from those of CD8 Ts cells.     

Expression, purification, and characterization of His-tagged Staphylococcus xylosus lipase wild-type and its mutant Asp 290 Ala

The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR technique. The sequence corresponding to the mature lipase was subcloned in the pET-14b expression vector, with a strong T7 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. High level expression of the lipase by Escherichia coli BL21 (DE3) cells harbouring the lipase gene containing expression vector was observed upon induction with 0.4 mM IPTG at 37 degrees C. One-step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified His-tagged SXL was 1500 or 850 U/mg using tributyrin or olive oil emulsion as substrate, respectively. It has been proposed that the region near the residue Asp290 could be involved in the selection of the substrate. Therefore, we also mutated the residue Asp 290 by Ala using site-directed mutagenesis. The mutant SXL-D290A was overexpressed in E. coli BL21 (DE3) and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged SXL-D290A mutant was 1000 U/mg using either tributyrin or olive oil emulsion as substrate. A comparative study of the wild type (His(6)-SXL) and the mutant (His(6)-SXL-D290A) proteins was carried out. Our results confirmed that Asp290 is important for the chain length specificity and catalytic efficiency of the enzyme.     

Corneal antifibrotic switch identified in genetic and pharmacological deficiency of vimentin

The type III intermediate filaments (IFs) are essential cytoskeletal elements of mechanosignal transduction and serve critical roles in tissue repair. Mice genetically deficient for the IF protein vimentin (Vim(-/-)) have impaired wound healing from deficits in myofibroblast development. We report a surprising finding made in Vim(-/-) mice that corneas are protected from fibrosis and instead promote regenerative healing after traumatic alkali injury. This reparative phenotype in Vim(-/-) corneas is strikingly recapitulated by the pharmacological agent withaferin A (WFA), a small molecule that binds to vimentin and down-regulates its injury-induced expression. Attenuation of corneal fibrosis by WFA is mediated by down-regulation of ubiquitin-conjugating E3 ligase Skp2 and up-regulation of cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1). In cell culture models, WFA exerts G(2)/M cell cycle arrest in a p27(Kip1)- and Skp2-dependent manner. Finally, by developing a highly sensitive imaging method to measure corneal opacity, we identify a novel role for desmin overexpression in corneal haze. We demonstrate that desmin down-regulation by WFA via targeting the conserved WFA-ligand binding site shared among type III IFs promotes further improvement of corneal transparency without affecting cyclin-dependent kinase inhibitor levels in Vim(-/-) mice. This dissociates a direct role for desmin in corneal cell proliferation. Taken together, our findings illuminate a previously unappreciated pathogenic role for type III IF overexpression in corneal fibrotic conditions and also validate WFA as a powerful drug lead toward anti-fibrosis therapeutic development.     

Conjugation of a 3-(1H-phenanthro[9,10-d]imidazol-2-yl)-1H-indole intercalator to a triplex oligonucleotide and to a three-way junction

A new intercalating nucleic acid monomer M comprising a 4-(1-indole)-butane-1,2-diol moiety was synthesized via a classical alkylation reaction of indole-3-carboxaldehyde followed by a condensation reaction with phenanthrene-9,10-dione in the presence of ammonium acetate to form a phenanthroimidazole moiety linked to the indole ring. Insertion of the new intercalator as a bulge into a Triplex Forming Oligonucleotide resulted in good thermal stability of the corresponding Hoogsteen-type triplexes. Molecular modeling supports the possible intercalating ability of M. Hybridisation properties of DNA/DNA and RNA/DNA three-way junctions (TWJ) with M in the branching point were also evaluated by their thermal stability at pH 7. DNA/DNA TWJ showed increase in thermal stability compared to wild type oligonucleotides whereas this was not the case for RNA/DNA TWJ.     

Nature of non-transferrin-bound iron: studies on iron citrate complexes and thalassemic sera

Despite its importance in iron-overload diseases, little is known about the composition of plasma non-transferrin-bound iron (NTBI). Using 30-kDa ultrafiltration, plasma from thalassemic patients consisted of both filterable and non-filterable NTBI, the filterable fraction representing less than 10% NTBI. Low filterability could result from protein binding or NTBI species exceeding 30 kDa. The properties of iron citrate and its interaction with albumin were therefore investigated, as these represent likely NTBI species. Iron permeated 5- or 12-kDa ultrafiltration units completely when complexes were freshly prepared and citrate exceeded iron by tenfold, whereas with 30-kDa ultrafiltration units, permeation approached 100% at all molar ratios. A g = 4.3 electron paramagnetic resonance signal, characteristic of mononuclear iron, was detectable only with iron-to-citrate ratios above 1:100. The ability of both desferrioxamine and 1,2-dimethyl-3-hydroxypyridin-4-one to chelate iron in iron citrate complexes also increased with increasing ratios of citrate to iron. Incremental molar excesses of citrate thus favour the progressive appearance of chelatable lower molecular weight iron oligomers, dimers and ultimately monomers. Filtration of iron citrate in the presence of albumin showed substantial binding to albumin across a wide range of iron-to-citrate ratios and also increased accessibility of iron to chelators, reflecting a shift towards smaller oligomeric species. However, in vitro experiments using immunodepletion or absorption of albumin to Cibacron blue–Sepharose indicate that iron is only loosely bound in iron citrate–albumin complexes and that NTBI is unlikely to be albumin-bound to any significant extent in thalassemic sera.     

Oritavancin exhibits dual mode of action to inhibit cell-wall biosynthesis in Staphylococcus aureus

Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the d-Ala-d-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan d-Ala-d-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.     

Methionine limitation results in increased hepatic FAS activity, higher liver 18:1 to 18:0 fatty acid ratio and hepatic TAG accumulation in Atlantic salmon, Salmo salar

The current experiment aimed to study whether interactions with lipid metabolism possibly might explain the relative increased liver weight obtained in fish fed sub-optimal methionine levels. A basal diet based on a blend of plant proteins which is low in methionine (1.6 g Met/16 g N) was compared to a methionine adequate diet (2.2 g Met/16 g N) prepared by adding dl-methionine (2.4 g/kg) to the basal diet in the expense of wheat grain. Fish oil was used as the lipid source. The diets were balanced in all nutrients except methionine. The diets were fed to Atlantic salmon (500 g BW) for a period of 3 months. Feed intake did not differ, rendering the intake of all nutrients except methionine equal. Fish fed the low methionine diet had an increased liver size relative to body weight, indicating fat deposition in the liver. Fish given the sub-optimal methionine diet showed about six times higher fatty acid synthase (FAS) activity as compared to the fish fed the adequate methionine diet, indicating a higher de novo lipogenesis. A significant rise in the liver 18:1 to 18:0 fatty acid ratios also supported storage of lipids over fatty acid oxidation. Indeed, methionine limitation resulted in significantly higher TAG concentrations in the liver. Sub-optimal dietary methionine also resulted in lower hepatic taurine concentrations and the total bile acids concentrations were reduced in faeces and tended to be reduced in plasma. Taken together, our data show that salmon fed sub-optimal methionine levels had increased relative liver weight and developed signs commonly described in the early stage of non-alcoholic fatty liver disease in rodent models (increased FAS activity, changed fatty acid ratios and TAG accumulation).     

Increased DNA damage in children caused by passive smoking as assessed by comet assay and oxidative stress

The present study aimed to evaluate the association between the environmental tobacco smoke (ETS) and DNA damage in relation to oxidative stress (OS) in children. Sixty-four children of age 1-8 years, selected from the outpatient clinic of Mansoura University Children Hospital were divided into two groups (23 children/group) based on high (>20 cigarettes/day) or low (<20 cigarettes/day) exposure to ETS at home. Twenty symptom-free children with normal cotinine level and with no exposure to ETS were recruited as controls. The comet assay was used to quantify the level of DNA damage in lymphocytes isolated from all children. Spectrophotometric methods were used to assess the serum level of malondialdehyde (MDA) and activity of glutathione peroxidase (GSH-Px) in erythrocytes. Also, serum level of tocopherol fractions (alpha, gamma, delta) was assessed by high performance liquid chromatography (HPLC). Children exposed to ETS exhibited retarded growth, more chest problems, and gastroenteritis than the control. A significant increase in mean comet tail length indicating DNA damage was observed in ETS-exposed children (P<0.001) compared to controls. ETS-exposed children had significantly (P<0.001) higher MDA level paralleled with significant (P<0.001) decrease in the level of GSH-Px and tocopherol fractions compared with controls. The GSH-Px activity and tocopherol levels were inversely correlated with the increase of ETS exposure. These results show that inhalation of ETS is associated with an increase in the level of oxidants and a simultaneous decrease in the level of antioxidants in the children's blood. This status of oxidant-antioxidant imbalance (OS) may be one of the mechanisms leading to DNA damage detected in lymphocytes of ETS-exposed children. In conclusion, the present study gives an indication of an association between DNA damage and ETS exposure in children.