Phylogenetic relationships inferred from ribosomal its sequences and biogeographic patterns in representatives of the genus Calopteryx (Insecta: Odonata) of the West Mediterranean and adjacent West European zone.

Western Europe is a reinvasion zone for the riverine dragonfly genus Calopteryx (Insecta: Odonata). Reinvasion may have been from central West Asia or from the West Mediterranean refugium. Phylogenetic relationships of West Mediterranean and West European taxa of the genus Calopteryx from different localities were inferred from sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal RNA genes. Twenty-six taxa belonging to the species groups C. splendens, C. meridionalis, C. haemorrhoidalis, C. virgo, C. xanthostoma, and C. exul were analyzed, with two North American species, C. amata and C. aequabilis, as outgroup. Sequence data and phylogenetic analyses were used to infer biogeographical patterns. The ribosomal spacers (ITS1 and ITS2) and the intervening 5.8S rDNA gene were amplified by PCR and sequenced. The ITS2 sequences of the West Mediterranean and West European calopterygids show no length variation but the ITS1 region was slightly variable in length. The sequence variation for ITS1 and ITS2 regions between different West Mediterranean and West European calopterygids was 14.5 and 6.1%, respectively. Phylogenetic relationships inferred from ITS sequences only partly confirm morphological data. A monophyletic origin of all West Mediterranean and West European species emerged. They are separated into two main clades; the splendens-like forms and the virgo/meridionalis/haemorrhoidalis group. Intraspecific variability, indicating different stages of speciation, was detected only in West Mediterranean representatives (e.g., C. xanthostoma) but not in invasive representatives in West Europe. The North African endemic C. exul is more closely related to the Italian C. s. caprai than to C. splendens sensu strictu. Based on the present information, Cretan populations are the only splendens-like taxa in addition to C. s. caprai that deserve subspecies status.     

Improved liquid chromatographic determination of serum cortisol with double internal standardization compared to radioimmunoassay and fluorometry, and evaluated by isotope dilution/mass spectrometry

A sensitive and specific high-performance liquid chromatographic (HPLC) method for the determination of cortisol in only 200 microliters of serum is described. Cortisol and two internal standards, 19-nortestosterone (IS1) and 6 alpha-methylprednisolone (IS2) are extracted with dichloromethane and analyzed on a C18 reversed-phase column eluted with a mobile phase of methanol:water at a flow rate of 0.75 ml/min. Ultraviolet absorption at 254 nm is used for detection and quantitation is performed by peak height ratio measurement. Using 200 microliters of serum, the lower limit of detection for cortisol is 10 ng/ml, the analytical recovery is 104 +/- 3.6% (n = 8), and the day-to-day precision was 1.69% at a level of 90 ng/ml (n = 16). Cortisol values obtained by this method were generally lower than those obtained by radioimmunoassay or by fluorometry. A serum pool was analyzed both by HPLC and by isotope dilution/mass spectrometry (ID/MS). A mean value of 90.1 ng/ml was obtained by HPLC (n = 16, CV = 1.7%), whereas ID/MS yielded a mean of 90.8 ng/ml (n = 28, CV = 0.4%). These results clearly demonstrate the high specificity and the accuracy of the HPLC procedure. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of medication by either of these two products.     

Restoration of complex V deficiency caused by a novel deletion in the human TMEM70 gene normalizes mitochondrial morphology

We report a fragmented mitochondrial network and swollen and irregularly shaped mitochondria with partial to complete loss of the cristae in fibroblasts of a patient with a novel TMEM70 gene deletion, which could be completely restored by complementation of the TMEM70 genetic defect. Comparative genomics analysis predicted the topology of TMEM70 in the inner mitochondrial membrane, which could be confirmed by immunogold labeling experiments, and showed that the TMEM70 gene is not restricted to higher multi-cellular eukaryotes. This study demonstrates that the role of complex V in mitochondrial cristae morphology applies to human mitochondrial disease pathology.     

Isolation and identification of pathogenic Naegleria australiensis (Amoebida, Vahlkampfiidae) from a spa in northern Italy.

Samples from therapeutic swimming pools and mud basins were cultured for free-living amoebae. Seven strains of pathogenic Naegleria species were isolated. Although some of the strains were as virulent as Naegleria fowleri, the etiological agent of primary amoebic meningoencephalitis, they were identified as Naegleria australiensis with the indirect fluorescent-antibody technique. The virulence of the isolates for mice corresponded with the cytopathic effect for Vero cells. The N. australiensis strains were isolated from swimming pools with water temperatures ranging from 32 to 35 degrees C and from mud with temperatures from 25 to 43 degrees C. The presence of pathogenic N. australiensis in the swimming pools did not correlate with bacterial indicators.     

Genetic Variation in the Free-Living Amoeba Naegleria fowleri

In this study, 30 strains of the pathogenic free-living amoeba Naegleria fowleri were investigated by using the randomly amplified polymorphic DNA (RAPD) method. The present study confirmed our previous finding that RAPD variation is not correlated with geographical origin. In particular, Mexican strains belong to the variant previously detected in Asia, Europe, and the United States. In France, surprisingly, strains from Cattenom gave RAPD patterns identical to those of the Japanese strains. In addition, all of these strains, together with an additional French strain from Chooz, exhibited similarities to South Pacific strains. The results also confirmed the presence of numerous variants in Europe, whereas only two variants were detected in the United States. The two variants found in the United States were different from the South Pacific variants. These findings do not support the previous hypothesis concerning the origin and modes of dispersal of N. fowleri.     

Analysis of mPygo2 mutant mice suggests a requirement for mesenchymal Wnt signaling in pancreatic growth and differentiation

Pygopus has recently been identified in Drosophila as an essential component of the nuclear complex required for canonical Wnt signaling. Here, we have investigated the role of the mammalian pygopus ortholog, mPygo2, in pancreas development. We show that a null mutation of mPygo2 in mice causes pancreas hypoplasia due to decreased progenitor cell proliferation after embryonic day (e) 12.5. During the same time window, mPygo2-deficient embryos begin to display a reduction in endocrine progenitors and consequently a decrease in islet endocrine cell mass. Consistent with its function after e12.5, late-developing endocrine cell types, such as beta, delta and PP cells, are specifically reduced, while the earlier-forming alpha cells develop normally. We find canonical Wnt signaling to be predominantly active in the mesenchyme at the time when mPygo2 is required and demonstrate the dependence of Wnt signal transduction on mPygo2. Furthermore, conditional deletion of mPygo2^flox allele in the pancreatic epithelium does not phenocopy the defects in mPygo2-null mutants. Since mPygo2 is expressed in the pancreatic mesenchyme and the role of the mesenchyme in epithelial progenitor cell expansion is well documented, our findings suggest an indirect role for mPygo2 in epithelial growth and differentiation through regulation of mesenchymal signals. Together, our data suggest a previously unappreciated role for mesenchymal Wnt signaling in regulating pancreatic organ growth and cell differentiation.     

The identification of vahlkampfiid amoebae by ITS sequencing

We have determined the internal transcribed spacer (ITS) sequences (including the 5.8S ribosomal DNA) of 30 strains of 14 species belonging to eight vahlkampfiid genera. Each previously described species has a specific ITS sequence, except for Tetramitus aberdonicus, Tetramitus thorntoni, and Tetramitus jugosus, which have identical ITS sequences. The latter three may therefore constitute a single species despite their apparent phenotypic differences. The ITS sequence appears to be conserved within a species. The species Willaertia magna appears to be ubiquitous. The 5.8S rDNA sequences of Singhamoeba horticola and Learamoeba waccamwensis indicate that they do not represent different genera, but both belong to the genus Tetramitus. The ITS sequences of 16 undescribed vahlkampfiid isolates were determined. Based on these sequences, seven isolates were identified as belonging to described species, while nine probably represent seven new species. Five of these presumed new species belong to the genus Tetramitus, and one each to the genera Vahlkampfia and Paravahlkampfia.