A practical route to long-chain non-natural α,ω-diamino acids

An efficient method for the synthesis of long-chain α,ω-diamino acids, starting from natural α-amino acids, has been developed. The long-chain skeleton has been generated through condensation between a protected aldehyde, derived from l-aspartic acid, and an ylide obtained from an ω-hydroxy-alkyl phosphonium salt. After conversion of the ω-hydroxy group into an amine, catalytic hydrogenation produced the N,N′-protected α,ω-diamino acid. The present route to α,ω-diamino acids allows the modulation of the chain length depending on the length of the ylide used for the Wittig olefination reaction.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

Cell death in the rat thymus: a minireview

During the last decades, the literature has clearly established the fundamental role of the thymus in the development of an effective immune system. During thymocyte development and maturation, potentially autoreactive thymocytes are eliminated by a process known as apoptosis or programmed cell death responsible for the negative selection occurring within the thymus. This process is in sharp contrast to other types of cell death referred to as necrosis. Actually, three different types of cell death have been recently observed morphologically in the rat thymus, i.e. necrosis, apoptosis and clustered cell death. Moreover, among the numerous factors influencing thymocyte cell death, particular attention has been paid to hormones, chemicals, biological compounds and physical agents that may influence the type and/or the extent of cell death. Finally, a brief overview has been devoted to the contribution of mitochondria, nitric oxide, glutathione and intracellular levels of cations in addition to the activity of genes as cdk2, p53, Fas and members' of the Bcl2 family in modulating rat thymus cell death.     

Relevance of the tumor antigen in the validation of three vaccination strategies for melanoma

Many preclinical studies of cancer immunotherapy are based on the testing of a single vaccination strategy in several tumor models. Moreover, most of those studies used xenogeneic Ags, which, owing to their high immunogenicity, may not represent realistic models for the validation of cancer immunotherapies. To address these issues, we compared the vaccination efficacy of three well established strategies (i.e., naked DNA; peptide-pulsed dendritic cells (DC), or a mixture of peptide and the Escherichia coli toxin LTR72) using the xenogeneic OVA or the naturally expressed tyrosinase-related protein 2 (TRP-2) tumor Ag in the B16 melanoma model. C57BL/6 mice received one to three s.c. injections of peptide-pulsed DC or DNA, or one to four mucosal administrations of peptide-toxin mixture. One to 2 wk later, the animals were challenged s.c. with B16 or B16 cells expressing OVA (B16-OVA). Vaccination of mice with OVA induced in all cases melanoma-specific CTL and protection against B16-OVA. When TRP-2 was used, all three vaccines elicited B16-specific CTL, but only DC pulsed with the immunodominant T cell epitope TRP-2181-188 allowed protection against B16. Even more importantly, a vaccination regimen with TRP-2-pulsed DC, started 24 h after the injection of a lethal number of B16 cells, caused a therapeutic effect in 60% of the challenged animals. Our results strongly emphasize the relevance of the tumor Ag in the definition of immunotherapeutic strategies for cancer, and support the use of peptide-pulsed DC as cancer vaccine in humans.     

Crystal structures of 10alpha-gon-5-enes from the synthetic pathway to desogestrel

X-ray crystallographic studies performed on the product of the ketalization reaction of 13beta-ethyl-11alpha-hydroxy-gon-5-ene-3,17-dione have lead to the unequivocal assignment of the 10alpha stereochemistry to C10, showing that an inversion of configuration occurred during formation of the 3,17-diketal. From the Swern oxidation of this compound, 11alpha-(methylthio)methoxy-10alpha-gonene was obtained as the major product instead of the desired 11-ketone. Modeling studies showed that the configurational instability at C10 is determined by the presence of the 11alpha-hydroxyl group.     

A theoretical study of the conformational behavior of analogues of alpha-L-rhamnose-1-phosphate

The conformational behavior of methyl(2-O-methyl-alpha-L-rhamnopyranosyl)phosphate, together with a group of potentially more stable analogues, was investigated through a DFT approach at the B3LYP/6-31G(d) level; the energy of all the optimized structures was recalculated using a continuum solvent model, C-PCM, choosing water as the solvent. The compounds exhibited several, sometimes tenths of populated conformations so that the overall properties of flexibility and mobility were evaluated. The analogue in which the pyranose oxygen atom is replaced by a methylene group emerges as the best candidate as a mimic of the reference 1-phosphate, in spite of the fact that it lacks the anomeric and exo-anomeric effects. The other analogues result poorer mimics because of a conformational equilibrium at the pyranose ring or of an excessive rigidity of the aglycone moiety.     

Synthesis of novel nitric oxide (NO)-releasing esters of timolol

A novel class of timolol derivatives with nitric oxide (NO)-donating moieties achieved chemical stability yet under physiologically relevant conditions released timolol and NO. Hindered esters A were designed and synthesized, whose ‘triggered’ release relied on enzymatic hydrolysis of the nitrate ester in A to B, that in turn cyclized to liberate timolol.     

Incorporation of 5α-furostan-3β,26-diol into tigogenin by digitalis lanata

2,2′,4,4′-³H₄-dihydrotigogenin was converted by Digitalis lanata plants into tigogenin.     

Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de-adaptation of EUE cells to a hypertonic medium

EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle-related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/1 range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the G0/1 range of DNA contents: this suggests that a G0 block occurs under long-term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.