De novo insertion of an<Emphasis Type="Italic"> Alu</Emphasis> sequence in the coding region of the<Emphasis Type="Italic"> CLCN5</Emphasis> gene results in Dent's disease

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and eventual renal failure. Various types of mutations in the renal chloride channel gene, CLCN5, have been identified in patients with this disease. We studied a Spanish patient with Dent's disease and found, by polymerase chain reaction amplification of the CLCN5 exons, an abnormally large exon 11. Sequencing analysis revealed that this was attributable to the insertion in codon 650 of an Alu element of the "young" Ya5 subfamily. The Alu element was inserted with the same orientation as the CLCN5 gene and arose de novo on the maternal chromosome. Polymorphism analysis indicated that the insertion occurred in the germline of the maternal grandfather. The presence of a long poly(A) tract and evidence for a 16-bp target-site duplication implied that the Alu element was integrated by retrotransposition. This mutation predicts a truncated ClC-5 protein that lacks part of the carboxy-terminus and is likely to result in loss of function of the chloride channel. Insertions of Alu sequences, which are rarely found in coding regions, have occasionally been reported to cause other genetic diseases. However, this is the first report of a retrotransposon insertion in the CLCN5 gene associated with Dent's disease.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Catabolism of volatile sulfur compounds precursors by Brevibacterium linens and Geotrichum candidum, two microorganisms of the cheese ecosystem

Two Brevibacterium linens strains and the cheese-ripening yeast Geotrichum candidum were compared with regard to their ability to produce volatile sulfur compounds (VSCs) from three different precursors namely L-methionine, 4-methylthio-2-oxobutyric acid (KMBA) and 4-methylthio-2-hydroxybutyric acid (HMBA). All microorganisms were able to convert these precursors to VSCs. However, although all were able to produce VSCs from L-methionine, only G. candidum accumulated KMBA when cultivated on this amino acid, contrary to B. linens suggesting that the transamination pathway is not active in this microorganism. Conversely, a L-methionine gamma-lyase activity--which catalyses the one step L-methionine to methanethiol (MTL) degradation route--was only found in B. linens strains. Several other enzymatic activities involved in the catabolism of the precursors tested were investigated. KMBA transiently accumulated in G. candidum cultures, and was then reduced to HMBA by a KMBA dehydrogenase (KDH) activity. This activity was not detected in B. linens. Despite no HMBA dehydrogenase (HDH) was found in G. candidum, a strong HMBA oxidase (HOX) activity was measured in this microorganism. This latter activity was weakly active in B. linens. KMBA and HMBA demethiolating activities were found in all the microorganisms. Our results illustrate the metabolic diversity between cheese-ripening microorganisms of the cheese ecosystem.     

Artificial chaperone-assisted refolding of bovine carbonic anhydrase using molecular assemblies of stimuli-responsive polymers

An artificial chaperone, which can decrease the protein aggregation and increase the reactivation yield of denatured protein in a fashion similar to natural chaperone, was newly developed using stimuli-responsive polymers. It has previously been reported that the addition of poly(propylene oxide)-phenyl-poly(ethylene glycol) (PPOn-Ph-PEG) with the unit number of PPO (n) 33 could enhance the refolding of bovine carbonic anhydrase (Kuboi et al. J. Chromatogr. B 2000, 243, 213). PPO-Ph-PEG with a large PPO chain (n = 50) was synthesized and the surface properties were characterized by both the relative fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) and the fluidity determined by diphenylhexatriene (DPH). The variation of ANS intensity and DPH fluidity is shown in a diagram as functions of temperature and polymer concentration. The high values of ANS intensity and fluidity of PPO50-Ph-PEG were obtained in a relatively wide conditional range (more than 0.08 mM and more than 15 degrees C) although the conditions showing the high values of PPO33-Ph-PEG were restricted (more than 0.1 mM and more than 40 degrees C). It was also found that molecular assemblies of PPOn-Ph-PEG with diameters of 7-18 nm were formed in the above conditions. On the basis of the surface properties of their polymer self-assemblies, the possibility of using them as an artificial chaperone was investigated. The effect of the addition of PPOn-Ph-PEG on the reactivation yield of a model protein, carbonic anhydrase from bovine (CAB), and the optical density of the solution was examined at various temperatures and concentrations. The reactivation yield of CAB was strongly enhanced and the aggregate formation (the optical density) was suppressed by adding PPOn-Ph-PEG in the above conditions, which show high ANS intensity and DPH fluidity. Especially in the presence of 0.1 mM PPO50-Ph-PEG, the reactivation yield of CAB reached approximately 100% at 40-55 degrees C. It was thus found that self-assemblies of the present polymer could be utilized as an artificial chaperone by selecting suitable stimuli conditions.     

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type-specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.     

Cytokine regulation of periportal fibrosis in humans infected with Schistosoma mansoni: IFN-gamma is associated with protection against fibrosis and TNF-alpha with aggravation of disease

Hepatic periportal fibrosis, which affects 5-10% of subjects infected by Schistosoma mansoni, is caused by the T cell-dependent granuloma that develop around schistosome eggs. Experimental models of infection have shown that granuloma and fibrosis are tightly regulated by cytokines. However, it is unknown why advanced periportal fibrosis occurs only in certain subjects. The goal of the present study was to evaluate the cytokine response of S. mansoni-infected subjects with advanced liver disease in an attempt to relate susceptibility to periportal fibrosis with an abnormal production of cytokines that regulate granuloma and fibrosis. Fibrosis was evaluated by ultrasound on 795 inhabitants of a Sudanese village in which S. mansoni is endemic: advanced periportal fibrosis was observed in 12% of the population; 35% of the affected subjects exhibited signs of portal hypertension. Age (odds ratio (OR), 11.5), gender (OR, 4.2), and infection levels (OR, 2.2) were significantly (p < or = 0.01) associated with hepatic fibrosis. Cytokines produced by egg-stimulated blood mononuclear cells from 99 subjects were measured (75 with no or mild fibrosis; 24 subjects with advanced fibrosis). Multivariate analysis of cytokine levels showed that high IFN-gamma levels were associated with a marked reduction of the risk of fibrosis (p = 0.01; OR, 0.1); in contrast, high TNF-alpha levels were associated with an increased risk (p = 0.05; OR, 4.6) of periportal fibrosis. Moreover, infection levels were negatively associated with IFN-gamma production. These results with observations in experimental models strongly suggest that IFN-gamma plays a key role in the protection of S. mansoni-infected patients against periportal fibrosis, whereas TNF-alpha may aggravate the disease.     

A tomato enzyme catalyzing the phosphorylation of 3,4-dihydroxy-2-butanone

A riboflavin biosynthesis ribB mutant of Escherichia coli deficient of 3,4-dihydroxy-2-butanone 4-phosphate synthase was complemented with a cDNA library from Lycopersicon esculentum. The complementing gene was isolated and expressed in E. coli. The resulting protein was shown to specify a 62 kDa protein which phosphorylates dihydroxyacetone, both enantiomers of 3,4-dihydroxy-2-butanone, and several other aldoses and ketoses. Sequence analysis revealed homology to dihydroacetone kinases (dak) genes from plants, animals, fungi and some eubacteria. Genes with similarity to the 5' part of the dak gene from tomato were found in many other eubacteria. The physiological role of the dak gene is still incompletely known.     

Quantitative high-performance liquid chromatography/electrospray ionization tandem mass spectrometric analysis of 2- and 3-series prostaglandins in cultured tumor cells

This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE(2), PGE(3), and other closely related prostaglandins from cultured cells using liquid chromatography/electrospray ionization tandem mass spectrometry. This method permits quantification of selected individual prostaglandins derived either from arachidonic acid (AA) or eicosapentaenoic acid (EPA) from cell extracts without tedious derivatization, lengthy sample preparation, and separation required by GC-MS- or HPLC-UV-based methods. The validation assessment showed that the quantitative determination is linear (r(2)>0.999) for both PGE(2) and PGE(3) in the range tested (1-500 ng/ml, 0.0028-1.4 microM) and a coefficient of variation lower than 10% was obtained for samples analyzed on 3 separate days. The detection limit was 2.5 pg for both PGE(2) and PGE(3). Extraction efficiency of PGE(2) and PGE(3) from cell suspensions ranged from 89.4 to 98.2%. As an application of the method, prostaglandins formed by EPA in human lung cancer A549 cells were determined. A 62% reduction of PGE(2) formation was noted when A549 cells were treated with 10 microM of EPA. Concomitantly, EPA increased formation of PGE(3) by 10-fold in A549 cells. This is the first report that unequivocally demonstrates that EPA can be converted to PGE(3) by cyclooxygenase in human cancer cells.     

Method of non-destructive mechanical testing of new surface coatings for prostheses]

Research into new surface coatings and surface processing methods for prostheses is subject to numerous studies. The aim of this study was to test an innovative biomechanical measuring method for the examination of the ingrowth of bone implants. Using a transcortical model, coated (n=14) or uncoated (n=14) titanic cylinders were implanted into the lateral condyle of 28 New Zealand White Rabbits. After 6 weeks or 6 months the animals were sacrificed and the osseointegration of the implants was evaluated biomechanically and histologically. Up to traction of 50 N the load dependent movement between bone and testing cylinder did not lead to a destruction of the bone-implant-interface. Therefore, biomechanical and histological investigations could be performed in the same specimen. The results of both evaluations showed a significant correlation (correlation coefficient -0.79; p < 0.01) and were absolutely reproducible. With the method of non-destructive mechanical testing, it is possible to halve the number of required animals. Additionally, the results of the biomechanical and histological analysis can be compared and thus serve as an internal control. In summary, the method of non-destructive mechanical testing represents an ideal tool to study new surface coatings and surface processing methods for prostheses.     

MITOCHONDRIAL DNA SEQUENCE VARIATION AMONG NATURAL POPULATIONS OF THE TRINIDAD GUPPY,POECILIA RETICULATA

Guppies were sampled from eight populations representing four river drainage basins in northern Trinidad, and from one population on the nearby island of Tobago. For each individual, a 465 base pair (bp) segment of the control region of the mitochondrial genome was sequenced. The resulting DNA sequences were subjected to sequence divergence calculations and the populations were linked by maximum parsimony analysis to determine their phylogenetic relationships. Mitochondrial DNA (mtDNA) sequence variation was found both within and between river drainages, correlated with the geographic features of northern Trinidad. The variation observed exists primarily between drainages, particularly between the Oropuche drainage and all other Trinidad drainages examined. Estimates of time of divergence between guppy populations of different drainages, based on mtDNA sequence variation, ranged from 100,000 to 200,000 for the most recently separated populations and from 600,000 to 1.2 million years between the Oropuche populations and all others examined. Examination of fish from northeastern South America will be required to determine whether these populations differentiated in their present locations or were the result of separate invasions of Trinidad from different Venezuelan sources. However, genetic isolation of these populations appears to predate the current physical separation of the island of Trinidad from the Venezuelan mainland.