Expanding the proteome two-dimensional gel electrophoresis reference map of human renal cortex by peptide mass fingerprinting

Proteomics methodologies hold great promise in basic renal research and clinical nephrology. The classical approach for proteomic analysis couples two-dimensional gel electrophoresis (2-DE) with protein identification by mass spectrometry, to produce more global information regarding normal protein expression and alterations in different physiological and pathological states. In this report we have expanded the identification of proteins in the renal cortex, improving the previously published map to facilitate the study of different diseases affecting the human kidney. About 250 spots were analyzed by peptide mass fingerprinting, 89 proteins and 74 isoforms for some of them were identified and implemented in the normal human renal cortex 2-DE reference map. This more comprehensive view of the proteome of the human renal cortex could be of invaluable help to the differential proteomic display of urological diseases.     

Microarray-based AMASE as a novel approach for mutation detection

Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology.     

Co-localization of susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on human chromosome 1q21

Psoriasis (PS) is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and altered differentiation. Atopic dermatitis (ATOD) is a chronic inflammatory, pruritic and eczematous disease frequently associated with respiratory atopy. These diseases are associated with distinct immunologic abnormalities and represent typical examples of complex diseases triggered by both genetic and environmental factors, as demonstrated by independent twin studies. Genome wide linkage studies have mapped susceptibility loci on several chromosomes (PSORS1-9; ATOD1-5). Four of them overlap on chromosomes 1q21, 3q21, 17q25 and 20p although ATOD is quite distinct from PS and these two diseases rarely occur together in the same patient. An association fine-mapping study has been performed to refine PSORS4 and ATOD2 susceptibility loci on chromosome 1q21 analyzing two independently collected cohorts of 128 PS and 120 ATOD trios. Genotype and haplotype analysis of PSORS4 and ATOD2 led us to detect significant p value for haplotypes defined by MIDDLE and ENDAL16 markers in both PS (p = 0.0000036) and ATOD (p = 0.0276), suggesting a strict co-localization within an interval of 42 kb. This genomic interval contains a single gene, LOR, encoding for loricrin. Polymorphic markers mapping in regulatory and coding regions did not show evidence of association in neither of the two diseases. However, expression profiles of LOR in skin biopsies have shown reduced levels in PS and increased levels in ATOD, suggesting the existence of a specific misregulation in LOR mRNA production.     

Birth of cloned pigs from zona-free nuclear transfer blastocysts developed in vitro before transfer

The objective of this work was to obtain cloned pig offspring by uterine transfer of blastocysts produced by zona-free manipulation. We started by defining the most suitable culture media for growing pig nuclear transfer embryos produced by zona-free micromanipulation comparing NCSU-23aa with Synthetic Oviduct Fluid (SOFaa) and with in vivo culture in the sheep oviduct. We found that parthenogenetic development to day 7 blastocyst in NCSU-23aa and sheep oviduct was significantly superior as compared to SOFaa (61.8%, 64% and 42.4 respectively) although blastocyst cell number was higher in the latter. Interestingly, when we compared the two media for the culture of nuclear transfer (NT) embryos derived from 3 different donor cell lines, we observed lower rates of development with NCSU-23aa (from 24.5% to 32.4%) while with SOFaa the development was significantly higher for two donor cell lines as compared to the third (44.4%, 48.9% and 20.6% respectively). A total of 244 blastocysts grown in SOFaa were transferred in four synchronized sows on day 5 or 6 of development. Two recipients farrowed 6 and 8 piglets corresponding to an efficiency of development to term of 8% and 16% of the transferred embryos respectively. Eleven pigs are now 10 month of age and those that have reached puberty have been proven to be fertile. Finally, this is the first report on the production of cloned pigs derived from the transfer of NT embryos at the blastocyst stage.     

Stringent requirement for HRD1, SEL1L,and OS-9/XTP3-B for disposal of ERAD-LS substrates

Sophisticated quality control mechanisms prolong retention of protein-folding intermediates in the endoplasmic reticulum (ER) until maturation while sorting out terminally misfolded polypeptides for ER-associated degradation (ERAD). The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L_S substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B. These ERAD factors become dispensable for degradation of the same polypeptides when membrane tethered (ERAD-L_M substrates). Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.     

Differentiation and adaptive radiation of amphibious gobies (Gobiidae: Oxudercinae) in semi-terrestrial habitats

During several surveys made in the region of the lower Fly River and delta, Papua New Guinea, nine species of oxudercine gobies (Gobiidae: Oxudercinae) were recorded: Boleophthalmus caeruleomaculatus, Oxuderces wirzi, Periophthalmodon freycineti, Periophthalmus darwini, Periophthalmus novaeguineaensis, Periophthalmus takita, Periophthalmus weberi, Scartelaos histophorus and Zappa confluentus. An exploratory multivariate analysis of their habitat conditions discriminated five guilds, differentially distributed in habitats with different quantities of environmental water and three guilds corresponding to different levels of salinity. A partial correspondence between phylogenetic and ecological categories suggested the presence of parallel adaptive radiations within different genera. In particular, the species found in the most terrestrial habitats (P. weberi) was also found in the widest range of conditions, suggesting that colonization of extreme semi-terrestrial and freshwater habitats by this species was facilitated by eurytypy. It is proposed that these findings provide insight into convergent adaptations for the vertebrate eco-evolutionary transition from sea to land.     

Assessing coral bleaching and recovery with a colour reference card in Watamu Marine Park,Kenya

With this study we estimated the changes in colour, bleaching and mortality of coral colonies from February to December 2007, using the colour reference card method. The study was developed in the Watamu Marine Park lagoon (Kenya), bridging the local summer when seawater temperatures were highest and coral bleaching risk was at its maximum. Seven coral genera were selected, and their colour recorded using a colour reference card (Coral Watch card). Seven different scenarios of bleaching and mortality were observed, varying among the coral genera and between two species in the genus Pocillopora. Twenty percent of the colonies bleached, of which 50% died. Only 15% of the coral that did not bleach died. Branching genera had a higher bleaching incidence than massive and sub-massive genera. Pocillopora showed the highest bleaching susceptibility, followed by Acropora, and the highest level of mortality. Of the two species of Pocillopora considered in this study, P. eydouxi showed higher bleaching and mortality levels, while P. verrucosa bleached less and experienced only partial mortality. Our results evidenced different patterns of coral bleaching and mortality which were easily and clearly detected with the colour card method during both bleaching and a post-bleaching events.