Synthesis and biological evaluation of some novel 4'-thio-L-ribonucleosides with modified nucleobase moieties

1,2,3,5-tetra-O-acetyl-4-thio-beta-L-ribofuranose (13) was synthesized by an improved five-step sequence starting from methyl alpha-D-lyxopyranoside. Compound 13 was then converted to the corresponding L-4'-thionucleosides 4-6 and 19 by a modified Vorbrüggen procedure. All of these nucleoside analogues were tested for their antitumour activity in vitro.     

C-H. . .pi interactions in the metal-porphyrin complexes with chelate ring as the H acceptor

Specific C-H. . .pi interactions with the pi-system of porphyrinato chelate ring were found in crystal structures of transition metal complexes from the Cambridge Structural Database and statistical analysis of geometrical parameters for intramolecular and intermolecular interactions was done. By density functional theory calculations on a model system it was evaluated that an interaction energy is above 1.5 kcal/mol and that the strongest interaction occurs when the distance between hydrogen atom and the center of the chelate ring is 2.6 A. This prediction is in good agreement with the distances for intermolecular interactions found in the crystal structures. In many cases the intramolecular interaction distances are much shorter than 2.6 A, and these short distances are caused by geometrical constrains. The C-H. . .pi interactions with chelate ring of porphyrinato ligand can influence the structure, contribute to its stability, and play some role in the function of biomolecules with metalo porphyrins.     

Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid

The fatty acid elongase [often designated FAE or beta-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium beta-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.     

A spin labelling study of immunomodulating peptidoglycan monomer and adamantyltripeptides entrapped into liposomes

The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes. Electron spin resonance (ESR) spectra were studied at 290 and 310 K. The entrapment of the adamantyltripeptides affected the motional properties of all spin labelled lipids, while the entrapment of PGM had no effect. (2). Spin labelled PGM was prepared and the novel compound bearing the spin label attached via the amino group of diaminopimelic acid was chromatographically purified and chemically characterized. The rotational correlation time of the spin labelled molecule dissolved in buffer at pH 7.4 was studied as a function of temperature. The conformational change was observed above 300 K. The same effect was observed with the spin labelled PGM incorporated into liposomes. Such effect was not observed when the spin labelled PGM was studied at alkaline pH, probably due to the hydrolysis of PGM molecule. The study of possible interaction with liposomal membrane is relevant to the use of tested compounds incorporated into liposomes, as adjuvants in vivo.     

Involvement of napsin A in the C- and N-terminal processing of surfactant protein B in type-II pneumocytes of the human lung

Surfactant protein B (SP-B) is a critical component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure. SP-B is synthesized by type-II pneumocytes as a 42-kDa propeptide (proSP-B), which is posttranslationally processed to an 8-kDa surface-active protein. Napsin A is an aspartic protease expressed in type-II pneumocytes. To characterize the role of napsin A in the processing of proSP-B, we colocalized napsin A and precursors of SP-B as well as SP-B in the Golgi complex, multivesicular, composite, and lamellar bodies of type-II pneumocytes in human lungs using immunogold labeling. Furthermore, we measured aspartic protease activity in isolated lamellar bodies as well as isolated human type-II pneumocytes and studied the cleavage of proSP-B by napsin A and isolated lamellar bodies in vitro. Both, napsin A and isolated lamellar bodies cleaved proSP-B and generated three identical processing products. Processing of proSP-B by isolated lamellar bodies was completely inhibited by an aspartic protease inhibitor. Sequence analysis of proSP-B processing products revealed several cleavage sites in the N- and C-terminal propeptides as well as one in the mature peptide. Two of the four processing products generated in vitro were also detected in type-II pneumocytes. In conclusion, our results show that napsin A is involved in the N- and C-terminal processing of proSP-B in type-II pneumocytes.     

7-nitroindazole reduces nitrite concentration in rat brain after intrahippocampal kainate-induced seizure

Kainic acid is an endogenous excitotoxin acting on glutamate receptors, that leads to neurotoxic damage resembling the alterations observed in some neurological disorders. Stimulation of glutamate receptors induces neuronal nitric oxide (NO) release, which in turn modulates glutamate transmission. NO may be a key mediator of excitotoxic neuronal injury in the central nervous system. We investigated the effects of 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor in vivo, on nitrite concentration after kainic acid injection (0.6 mg/ml, pH 7.2) unilaterally into the CA3 region of the rat hippocampus. The accumulation of nitrite, the stable metabolite of NO, was measured by the Griess reaction at different times (5 min, 15 min, 2 h, 48 h and 7 days) following kainate injection in the ipsilateral and contralateral hippocampus, forebrain cortex, striatum and cerebellum homogenates. 7-Nitroindazole (100 microM) can effectively inhibit NO synthesis in rat brain after kainate-induced intrahippocampal neurotoxicity and suppressed nitrite accumulation. The present results suggest that neuronal NO synthase inhibitors may be useful in the treatment of neurological diseases where excitotoxic mechanisms play a role.     

Organic solvents order the dynamic switch II in Ras crystals

Room temperature crystal structures of crosslinked H-Ras bound to GMPPNP were solved in 50% 2,2,2-trifluoroethanol, 60% 1,6-hexanediol, and 50% isopropanol. The disordered switch II region of Ras is ordered in the presence of 2,2,2-trifluoroethanol or 1,6-hexanediol. The overall backbone conformation of switch II in these organic solvents is the same as in the Ras-GMPPNP complexes with RalGDS, PI(3) kinase, and RasGAP, indicating a biologically relevant form. Key polar interactions that stabilize the ordered switch are enhanced in the presence of hydrophobic cosolvents. These results suggest that hydrophobic solvents can be used in general to order short biologically relevant segments of disordered regions in protein crystals by favoring H-bonding interactions between atoms that are highly solvated and mobile in aqueous solution.     

Relationship of the Xeroderma Pigmentosum Group E DNA Repair Defect to the Chromatin and DNA Binding Proteins UV-DDB and Replication Protein A

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB⁻ XP-E cell extracts, but microinjection of the protein into DDB⁻ XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin.The heritable human disorder xeroderma pigmentosum (XP) is chiefly characterized by an increased incidence of benign and malignant skin lesions after exposure to sunlight. Affected individuals fall into one of eight different genetic complementation groups. Cells from the seven complementation groups A through G have reduced nucleotide excision repair (NER) of damaged DNA, while cells from the variant, or V, group are defective in a less-defined process of cellular recovery after DNA damage (11). Genes and proteins representing XP groups A (XP-A) B, C, D, F, and G have all been isolated and found to represent some of the subunits of the core NER recognition and incision machinery. XP-E is the mildest form of the disorder, and cells of this group generally have 40 to 60% of the normal repair level, as shown by autoradiographic measurement of unscheduled DNA synthesis (UDS) after UV irradiation. Cell fusion studies have assigned at least 16 individuals to this form of the disorder (6, 19, 23, 40).There are several indications that a DNA damage binding protein denoted UV-DDB (or DDB) is involved in the primary XP-E defect. The protein has been detected in extracts of vertebrate cells as an activity that preferentially binds damaged oligonucleotides in electrophoretic mobility shift or filter binding assays. The protein has a particular affinity for (6-4) photoproducts in UV-irradiated DNA (10, 15, 16, 34, 41, 43), but UV-DDB also binds to DNA damaged by other agents, including cisplatin and nitrogen mustard (32). The activity has been purified as a single 127-kDa protein (2) and as a complex with two subunits of 127 and 48 kDa (21). Damage-binding activity is missing from some cells in the XP-E group, designated DDB⁻, but is present in other XP-E cell lines, designated DDB⁺ (3, 15, 19, 23). The genes encoding the p127 protein (7, 17, 39) and the p48 protein (7) have been isolated, but DNA sequence features have not yet yielded firm clues about their functions. Microinjection of purified UV-DDB into XP-E cells lacking UV-DDB activity substantially corrects the NER defect, as measured by UDS after UV irradiation, but UV-DDB⁺ cells are not corrected (22). Sequence alterations in the gene for p48 have been reported for several XP-E cell lines (29), and it is possible that these are causative mutations for XP-E.There are also suggestions that the single-stranded DNA binding activity of replication protein A (RPA) is involved in the XP-E defect. RPA is a heterotrimer of three subunits with sizes of 70, 34, and 14 kDa that plays key roles in DNA replication, recombination, and DNA repair (44). It is one of the core components of the eukaryotic nucleotide excision-incision system (1, 12, 28). With regard to XP, it was recently reported that XP-E cell extracts are severely defective in NER in vitro and that RPA can specifically correct the repair defect of these extracts, but not those of extracts of other complementation groups (20). Moreover, it has been found that RPA copurifies to some extent with UV-DDB protein and that the two proteins interact, showing a tighter association with chromatin after UV irradiation of cells (31).The availability of lymphoblastoid cell lines derived from three newly identified XP-E individuals has given us the opportunity to further investigate the possible relationships of UV-DDB and RPA to the molecular defect in XP-E and the influence of these proteins on NER.