2,3-Dideoxy-3-Phthalimidopentoses in the Synthesis of 3′-Amino-2′,3′-Dideoxynucleosides

Abstract

3′-Amino-3′deoxythymidine is a very effective drug in vivo against L 1210 leukemia. It mives 1441 increase in lifespan with very little drug-induced toricitylil. Therefore, it was attractive to synthesize a large series of cuialogues, but unfortunately, such compounds are only achievable through a 1inear synthesis via the corresponding nucleoside which typically is transformed into the 3′-azido derivative and finally reduced.     

Synthesis and Evaluation of Oligodeoxynucleotides Containing 3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine: Introduction of a Novel Class of Phosphodiester Internucleoside Linkages

Abstract

3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine (5) was synthesized and converted into the phosphoramidite building block 8. Novel oligodeoxynucleotide analogues containing 4′-C-hydroxymethyl phosphodiester internucleoside linkages were synthesized on an automated DNA-synthesizer. The hybridization properties and enzymatic stability were studied on oligomers with one to four modifications. The 3′-end modified oligodeoxynucleotides were resistent towards 3′-exonuclease degradation and showed only moderate lowered affinity towards complementary DNA compared with oligodeoxynucleotides bearing modifications in the middle.     

Convenient Synthesis of 4-C-Branched Lactones and 3′-C-Branched 2′,3′-Dideoxynucleosides

Abstract

The regio- and stereoselective photocatalysed addition of 2-propanol and cyclopentanol to (5S)-hydroxymethylfuran-2(5H)-one (1) gave 4-C-branched lactones 2 and 3 after selective silylations. The lactones 2 and 3 were radically deoxygenated affording lactones 4 and 5, respectively. As an example, compound 2 was transformed without purification of the intermediates into an anomeric mixtures of deprotected 3′-C-branched 2′, 3′-dideoxynucleosides 6 by the following reaction sequence: silylation, reduction, acetylation, coupling with silylated thymine and desilylation.     

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: Interactions with human β2 integrins and erythrocytes

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α_Lβ₂ (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express β₂ integrin. Cells expressing α_Mβ₂ (CD11b/CD18) or α_Xβ₂ (CD11c/CD18) were killed as efficiently as cells expressing α_Lβ₂. Erythrocytes, which do not express β₂ integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β₂ chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α_Mβ₂ and α_Xβ₂ as novel receptors for LtxA.     

A postmortem study of the corpus callosum in the common minke whale (Balaenoptera acutorostrata)

Cetaceans diverged from terrestrial mammals approximately 53 mya and have evolved independently since then. During this time period, they have developed a complex nervous system with many adaptations to the marine environment. This study used stereological methods to estimate the total number and diameter of the myelinated fibers in the corpus callosum of the common minke whale (Balaenoptera acutorostrata) (n= 4). The total number of callosal fibers was estimated to 55.3 × 10⁶ (range: 49.0 × 10⁶–59.1 × 10⁶). Despite large variations of the callosal area (350–950 mm²), there was little variation in total fiber number. The fibers with diameters ranging from 0.822 to 1.14 μm were the most frequent, which is similar to results obtained in the human brain using the same method. There was no systematic distribution of large‐, middle‐, or small‐sized fibers along the rostrocaudal axis of the corpus callosum. This study indicated that the corpus callosum of the common minke whale is small and has few fibers compared to terrestrial mammals.     

Modulation of i-motif thermodynamic stability by the introduction of UNA (unlocked nucleic acid) monomers

The influence of acyclic RNA derivatives, UNA (unlocked nucleic acid) monomers, on i-DNA thermodynamic stability has been investigated. The 22 nt human telomeric fragment was chosen as the model sequence for stability studies. UNA monomers modulate i-motif stability in a position-depending manner. The largest destabilization is observed for position C14, while UNA placed in position A12 causes significant increase of i-DNA thermodynamic stability. CD curves of UNA-modified variants imply no structural changes relative to the native i-motif.     

Postglacial migration supplements climate in determining plant species ranges in Europe

The influence of dispersal limitation on species ranges remains controversial. Considering the dramatic impacts of the last glaciation in Europe, species might not have tracked climate changes through time and, as a consequence, their present-day ranges might be in disequilibrium with current climate. For 1016 European plant species, we assessed the relative importance of current climate and limited postglacial migration in determining species ranges using regression modelling and explanatory variables representing climate, and a novel species-specific hind-casting-based measure of accessibility to postglacial colonization. Climate was important for all species, while postglacial colonization also constrained the ranges of more than 50 per cent of the species. On average, climate explained five times more variation in species ranges than accessibility, but accessibility was the strongest determinant for one-sixth of the species. Accessibility was particularly important for species with limited long-distance dispersal ability, with southern glacial ranges, seed plants compared with ferns, and small-range species in southern Europe. In addition, accessibility explained one-third of the variation in species' disequilibrium with climate as measured by the realized/potential range size ratio computed with niche modelling. In conclusion, we show that although climate is the dominant broad-scale determinant of European plant species ranges, constrained dispersal plays an important supplementary role.     

Photoligation of self-assembled DNA constructs containing anthracene-functionalized 2'-amino-LNA monomers

Efficient synthesis of a novel anthracene-functionalized 2′-amino-LNA phosphoramidite derivative is described together with its incorporation into oligodeoxynucleotides. Two DNA strands with the novel 2′-N-anthracenylmethyl-2′-amino-LNA monomers can be effectively cross-linked by photoligation at 366 nm in various types of DNA constructs. Successful application of three differently functionalized 2′-amino-LNA monomers in self-assembled higher ordered structures for simultaneous cross-linking and monitoring of assembly formation is furthermore demonstrated.     

The sterol-binding protein Kes1/Osh4p is a regulator of polarized exocytosis

Oxysterol-binding protein (OSBP)-related protein Kes1/ Osh4p is implicated in nonvesicular sterol transfer between membranes in Saccharomyces cerevisiae. However, we found that Osh4p associated with exocytic vesicles that move from the mother cell into the bud, where Osh4p facilitated vesicle docking by the exocyst tethering complex at sites of polarized growth on the plasma membrane. Osh4p formed complexes with the small GTPases Cdc42p, Rho1p and Sec4p, and the exocyst complex subunit Sec6p, which was also required for Osh4p association with vesicles. Although Osh4p directly affected polarized exocytosis, its role in sterol trafficking was less clear. Contrary to what is predicted for a sterol-transfer protein, inhibition of sterol binding by the Osh4p Y97F mutation did not cause its inactivation. Rather, OSH4(Y97F) is a gain-of-function mutation that causes dominant lethality. We propose that in response to sterol binding and release Osh4p promotes efficient exocytosis through the co-ordinate regulation of Sac1p, a phosphoinositide 4-phosphate (PI4P) phosphatase, and the exocyst complex. These results support a model in which Osh4p acts as a sterol-dependent regulator of polarized vesicle transport, as opposed to being a sterol-transfer protein.     

ErbB2-associated changes in the lysosomal proteome

Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal membrane proteome was significantly altered by the ectopic expression of an active form of the ErbB2 oncogene, which renders the cells highly metastatic. The furthermost ErbB2-associated changes included increased levels of CD63, S100A11 and ferritin heavy chain. Overall, our data introduce the antibody-based purification of lysosomes as a suitable method for the characterization of lysosomes from a variety of pathological conditions with altered lysosomal density and stability.