Diagnostic Tests for Primary Renal Hypouricemia

Primary renal hypouricemia is a genetic disorder characterized by defective renal uric acid (UA) reabsorption with complications such as nephrolithiasis and exercise-induced acute renal failure. The known causes are: defects in the SLC22A12 gene, encoding the human urate transporter 1 (hURAT1), and also impairment of voltage urate transporter (URATv1), encoded by SLC2A9 (GLUT9) gene. Diagnosis is based on hypouricemia (<119 μmol/L) and increased fractional excretion of UA (>10%). To date, the cases with mutations in hURAT1 gene have been reported in East Asia only. More than 100 Japanese patients have been described. Hypouricemia is sometimes overlooked; therefore, we have set up the flowchart for this disorder. The patients were selected for molecular analysis from 620 Czech hypouricemic patients. Secondary causes of hyperuricosuric hypouricemia were excluded. The estimations of (1) serum UA, (2) excretion fraction of UA, and (3) analysis of hURAT1 and URATv1 genes follow. Three transitions and one deletion (four times) in SLC22A12 gene and one nucleotide insertion in SLC2A9 gene in seven Czech patients were found. Three patients had acute renal failure and urate nephrolithiasis. In addition, five nonsynonymous sequence variants and three nonsynonymous sequence variants in SLC2A9 gene were found in two UK patients suffering from acute renal failure. Our finding of the defects in SLC22A12 and SLC2A9 genes gives further evidence of the causative genes of primary renal hypouricemia and supports their important role in regulation of serum urate levels in humans.     

Oligonucleotides Containing Acyclic Nucleoside Analogues with Carbamate Internucleoside Linkages

Abstract

Synthesis of 2′-deoxy-2′,3′-secothymidine t and its dimer t?t, where the two 2′-deoxy-2′,3′-secothymidine t units are connected via a carbamate, ?=3′-NH-CO-O-5′, internucleoside linkage has been achieved. These building blocks were protected in the 5′-position, converted into their phosphoramidites, or attached onto CPG, and then used for “chimeric oligonucleotide” synthesis.     

Efficient Synthesis of 2′-Deoxynucleoside 3′-C-Phosphonates: Reactivity of Geminal Hydroxyphosphonate Moiety

Abstract

In this report we present a novel, simple way for the synthesis of 3′-C-phosphonate derivatives of all four basic 2′-deoxynucleosides in both fully protected and deprotected forms. The reactivity of the geminal hydroxy phosphonate moiety located at the 3′-carbon atom of the nucleoside was studied with respect to the use of this type of nucleoside phosphonic acid for the preparation of short oligonucleotides, namely, dinucleoside monophosphate analogues.     

Synthesis and Evaluation of Acyclic Nucleotide Analogs

Abstract

Acyclic nucleotide analogs derived from antiviral 9-(2-phosphonylmethoxyethyl)adenine by modification at the side chain or by alternation of the heterocyclic base were synthesized and investigated for their antiviral activity.     

Synthesis and Some Properties of Modified Oligonucleotides. II. Oligonucleotides Containing 2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl Pyrimidine Nucleosides

Abstract

In order to find the effects of unnatural nucleosides on the stability of duplex, several oligonucleotides containing 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-uracil(FAU),-cytosine (FAC) and -thymine (FMAU) were synthesized by two alternative approaches: phosphoramidite method on an ABI 392 synthesizer and H-phosphonate procedure on our GeneSyn I universal module synthesizer. It was shown from the melting profiles that the presence of FMAU has a large stabilizing effect on the duplex. Replacement of thymidine with FAU, or deoxycytidine with FAC resulted in the formation of less stable duplexes. Temperature-dependent CD spectroscopy demonstrated that the structures of the fluorine containing oligomers are very similar to those of unmodified oligomers.     

3′-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors

3′-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3′-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3′-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem–loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation—CPSF, CstF, and CF I_m—are present in Drosophila nuclear extracts in a stable supercomplex.     

Alien parasite hitchhikes to Patagonia on invasive bumblebee

The worldwide trade in bumblebees can lead to the spread of diseases, which in turn has been claimed as a factor in bumblebee decline. Populations of the introduced Bombus terrestris, which invaded NW Patagonia, Argentina, in 2006, harbor the highly pathogenic protozoan Apicystis bombi. We asked whether A. bombi had been co-introduced with B. terrestris, and if so, whether spillover occurred to the two resident bumblebee species in the region: the introduced European Bombus ruderatus and the native Bombus dahlbomii. We searched for A. bombi by means of PCR in samples of B. ruderatus and B. dahlbomii collected before and after the invasion of B. terrestris and in samples of the latter. We found no A. bombi in samples of B. ruderatus and B. dahlbomii collected before B. terrestris invasion, whereas post invasion, A. bombi was present in all 3 species. The identity of the parasite was established by sequencing the 18S region, which was identical for the three bumblebee species and also matched the European sequence, confirming it to be A. bombi. This is the first report of A. bombi in B. ruderatus and B. dahlbomii. Moreover, our results suggest that Patagonia had been free of A. bombi until this parasite was co-introduced with B. terrestris, and spilled over in situ to these two previously resident species. Finally, our findings provide indirect circumstantial evidence of a potential link between the population collapse and geographic retraction of B. dahlbomii and the introduction of this novel parasite.     

Eye trematode infection in small passerines in Peru caused by Philophthalmus lucipetus,an agent with a zoonotic potential spread by an invasive freshwater snail

Until now, four species of eye trematodes have been found in South America. Of them, Philophthalmus lucipetus (synonymized with Philophthalmus gralli) displays a broad host spectrum, with at least 30 bird species (prevalently large water birds), five mammal species and humans serving as definitive hosts, and with snails Fagotia (Microcolpia) acicularis, Amphimelania holandri, Melanopsis praemorsa and Melanoides tuberculata serving as intermediate hosts. When examining a total of 50 birds of ten species in the wetland of Pantanos de Villa, Lima, Peru in July 2011, eye trematodes were identified visually in the edematous conjunctival sac of 11 (48%) out of 23 resident many-colored rush tyrants Tachuris rubrigastra. Based on morphometric characteristics, the trematodes were identified as P. lucipetus. ITS2 and CO1 gene of the examined specimens combined showed a 99% similarity to an Iranian isolate of Philophthalmus sp. from the intermediate host Melanoides tuberculata, an invasive freshwater snail, suggesting that these two isolates represent the same species with a wide geographical range. Moreover, the prevalence of infection with the philophthalmid cercariae was 31% in 744 Melanoides tuberculata examined in Pantanos de Villa in 2010. It is evident that P. lucipetus occurs throughout the world as well as locally, including Eurasia and South America. Here we report this trematode for the first time in Peru, and we were the first to sequence any of the South American eye trematodes. Low host specificity of P. lucipetus and the invasive character of Melanoides tuberculata as a competent intermediate host suggest that eye trematodosis caused by P. lucipetus may emerge frequently in various parts of the world, especially in the tropics. Increase of the zoonotic potential of the P. lucipetus associated with this invasive snail spreading across the world is predictable and should be of interest for further research.