Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5''-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.     

Complex repetitive arrangements of gene sequence in the candidate region of the spinal muscular atrophy gene in 5q13.

Childhood-onset proximal spinal muscular atrophy (SMA) is a heritable neurological disorder, which has been mapped by genetic linkage analysis to chromosome 5q13, in the interval between markers D5S435 and D5S557. Here, we present gene sequences that have been isolated from this interval, several of which show sequence homologies to exons of beta-glucuronidase. These gene sequences are repeated several times across the candidate region and are also present on chromosome 5p. The arrangement of these repetitive gene motifs is polymorphic between individuals. The high degree of variability observed may have some influence on the expression of the genes in the region. Since SMA is not inherited as a classical autosomal recessive disease, novel genomic rearrangements arising from aberrant recombination events between the complex repeats may be associated with the phenotype observed.     

Immunocytochemical and electrophoretic studies on the localization of the major haemolymph proteins (ceratitins) inCeratitis capitata during development

Summary The developmental profile of the major haemolymph proteins (ceratitins) inCeratitis capitata was studied. Ceratitin concentration in the haemolymph decreases dramatically during the last days of pupal life, while the amounts of ceratitins in whole organism extracts remain unchanged. By electrophoretic, immunological and immunofluorescence techniques it was revealed that ceratitins are reabsorbed by the fat body and a fraction of them is deposited in the cuticle. The possible role of ceratitins is discussed.     

The effect of the elongation of the proximal aorta on the estimation of the aortic wall distensibility

The compliance of the proximal aortic wall is a major determinant of cardiac afterload. Aortic compliance is often estimated based on cross-sectional area changes over the pulse pressure, under the assumption of a negligible longitudinal stretch during the pulse. However, the proximal aorta is subjected to significant axial stretch during cardiac contraction. In the present study, we sought to evaluate the importance of axial stretch on compliance estimation by undertaking both an in silico and an in vivo approach. In the computational analysis, we developed a 3-D finite element model of the proximal aorta and investigated the discrepancy between the actual wall compliance to the value estimated after neglecting the longitudinal stretch of the aorta. A parameter sensitivity analysis was further conducted to show how increased material stiffness and increased aortic root motion might amplify the estimation errors (discrepancies between actual and estimated distensibility ranging from − 20 to − 62%). Axial and circumferential aortic deformation during ventricular contraction was also evaluated in vivo based on MR images of the aorta of 3 healthy young volunteers. The in vivo results were in good qualitative agreement with the computational analysis (underestimation errors ranging from − 26 to − 44%, with increased errors reflecting higher aortic root displacement). Both the in silico and in vivo findings suggest that neglecting the longitudinal strain during contraction might lead to severe underestimation of local aortic compliance, particularly in the case of women who tend to have higher aortic root motion or in subjects with stiff aortas.

     

Evolution of a Complex Locus for Terpene Biosynthesis in Solanum

Functional gene clusters, containing two or more genes encoding different enzymes for the same pathway, are sometimes observed in plant genomes, most often when the genes specify the synthesis of specialized defensive metabolites. Here, we show that a cluster of genes in tomato (Solanum lycopersicum; Solanaceae) contains genes for terpene synthases (TPSs) that specify the synthesis of monoterpenes and diterpenes from cis-prenyl diphosphates, substrates that are synthesized by enzymes encoded by cis-prenyl transferase (CPT) genes also located within the same cluster. The monoterpene synthase genes in the cluster likely evolved from a diterpene synthase gene in the cluster by duplication and divergence. In the orthologous cluster in Solanum habrochaites, a new sesquiterpene synthase gene was created by a duplication event of a monoterpene synthase followed by a localized gene conversion event directed by a diterpene synthase gene. The TPS genes in the Solanum cluster encoding cis-prenyl diphosphate–utilizing enzymes are closely related to a tobacco (Nicotiana tabacum; Solanaceae) diterpene synthase encoding Z-abienol synthase (Nt-ABS). Nt-ABS uses the substrate copal-8-ol diphosphate, which is made from the all-trans geranylgeranyl diphosphate by copal-8-ol diphosphate synthase (Nt-CPS2). The Solanum gene cluster also contains an ortholog of Nt-CPS2, but it appears to encode a nonfunctional protein. Thus, the Solanum functional gene cluster evolved by duplication and divergence of TPS genes, together with alterations in substrate specificity to utilize cis-prenyl diphosphates and through the acquisition of CPT genes.     

Chiral Separation of the Clinically Important Compounds Fucose and Pipecolic Acid Using CE: Determination of the Most Effective Chiral Selector

In this study, simple electrophoretic methods were developed for the chiral separation of the clinically important compounds fucose and pipecolic acid. In recent years, these analytes, and particularly their individual enantiomers, have attracted considerable attention due to their role in biological functions and disorders. The detectability and sensitivity of pipecolic acid and fucose were improved by reacting them with fluorenylmethyloxycarbonyl chloride (FMOC‐Cl) and 5‐amino‐2‐naphthalene‐sulfonic acid (ANSA), respectively. The enantioseparation conditions were optimized by initially investigating the type of the chiral selector. Different chiral selectors, such as polymeric surfactants and cyclodextrins, were used and the most effective ones were determined with regard to resolution and analysis time. A 10‐mM β‐cyclodextrin was able to separate the enantiomers of ANSA‐DL‐fucose and the polymeric surfactant poly(sodium N‐undecanoyl‐LL‐leucine‐valinate) was able to separate the enantiomers of FMOC‐DL‐pipecolic acid, with resolution values of 3.45 and 2.78, respectively. Additional parameters, such as the concentration and the pH of the background electrolyte (BGE), the concentration of the chiral selector, and the addition of modifiers were examined in order to optimize the separations. The addition of the chiral ionic liquid D‐alanine tert‐butyl ester lactate into the BGE was also investigated, for the first time, in order to improve resolution of the enantiomers. Chirality 25:556–560, 2013. © 2013 Wiley Periodicals, Inc.     

Calpain2 protease: A new member of the Wnt/Ca pathway modulating convergent extension movements in Xenopus

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate several physiological processes by limited cleavage of different substrates. The role of Calpain2 in embryogenesis is not clear with conflicting evidence from a number of mouse knockouts. Here we report the temporal and spatial expression of Calpain2 in Xenopus laevis embryos and address its role in Xenopus development. We show that Calpain2 is expressed maternally with elevated expression in neural tissues and that Calpain2 activity is spatially and temporally regulated. Using a Calpain inhibitor, a dominant negative and a morpholino oligonoucleotide we demonstrate that impaired Calpain2 activity results in defective convergent extension both in mesodermal and neural tissues. Specifically, Calpain2 downregulation results in loss of tissue polarity and blockage of mediolateral intercalation in Keller explants without affecting adherens junction turnover. We further show that Calpain2 is activated in response to Wnt5a and that the inhibitory effect of Wnt5a expression on animal cap elongation can be rescued by blocking Calpain2 function. This suggests that Calpain2 activity needs to be tightly regulated during convergent extension. Finally we show that expression of Xdd1 blocks the membrane translocation of Calpain2 suggesting that Calpain2 activation is downstream of Dishevelled. Overall our data show that Calpain2 activation through the Wnt/Ca²⁺ pathway and Dishevelled can modulate convergent extension movements.     

Human TTRV30M localization within podocytes in a transgenic mouse model of transthyretin related amyloidosis: does the environment play a role?

Transthyretin related amyloidosis is a nosological entity that leads to disability, diminished quality of life, all stages of chronic kidney disease and eventually death. Podocytes are polarized, highly differentiated epithelial cells important for proper nephron function. In the present study we investigated whether deposited TTRVal30Met (TTRV30M) molecules could be localized within podocytes in situ under the effect of different housing conditions (i.e. specific pathogen free [SPF] vs. non-SPF). Murine renal glomeruli from human TTRV30M (hTTRV30M) transgenic mice were examined via direct and indirect immunofluorescence techniques for the presence of hTTRV30M, murine serum amyloid P, activated caspase-3 and NPHS1. Association strength and amount of colocalization for NPHS1?ChTTRV30M, NPHS1-activated caspase-3, hTTRV30M-murine serum amyloid P were estimated. Localization of hTTRV30M in podocytes was demonstrated by immuno-electron microscopy. Renal hTTRV30M gene and NPHS1 gene expression levels were estimated. Non-SPF transgenic mice showed increased glomerular hTTRV30M deposition compared to their SPF counterparts. Furthermore increased podocytic localization of hTTRV30M was noticed in non-SPF mice. Glomerular caspase-3 activation was increased only in the non SPF housing conditions. Podocytic caspase-3 activation was increased in SPF and in non-SPF transgenic mice when compared to non transgenic controls. Environmental conditions influence glomerular deposition and podocytic localization of hTTRV30M. In this context increased caspase-3 activation occurred.