A new helminth parasite of fish: Spirocamallanus freitasi sp. n. (Nematoda - Camallanidae)

A new species of nematode is described, Spirocamallanus freitasi sp. n. The worms were collected from fishes (Bergiaria westermanni, Pimelodus maculatus and Pimelodus sp.) living in Três Marias Dam (São Francisco River) in the State of Minas Gerais, Brazil. Spirocamallanus freitasi sp. n. differs from Procamallanus iheringi, P. amarali, P. macaensis, P. (Spirocamallanus) pimelodus, P. (S.) solani and P. (S.) pereirai by having digitated larger spicule and from P. cruzi by having not digitated terminal parts of both spicules. It differs also from P. (S.) intermedius by having not larger spicule divided in two undigitated branches and six to nine sclerotized bands in the buccal capsule; P. rarus has three to four sclerotized bands and tridigitated larger branch of longer speicule, differing from S. feitasi sp. n.

A new species of Limnoderetrema (Trematoda, Digenea) from the freshwater Atherinid fish Basilichthys australis Eigenmann, 1927 from the south of Chile

Limnoderetrema tolosai sp. n. (Trematoda, Digenea) within an autochthonous freshwater fish, the silverside Basilichthys autralis Eigenmann, 1927, from Lakes Riñihue and Ranco in Southern Chile is described. The species is distinguished from Limnoderetrema minutum (Manter, 1954) by the presence of one spine in the cirrus and cecal bifurcation nearer to ventral sucker than to pharinx. It is proposed Limnoderetrema macrophallus (Szidat & Nani, 1951) n. comb. (originally Steganoderma). Limnoderetrema tolosai differs from L. macrophallus since it cirrus has a distal spine and by its vitelline follicles distribution. It seems that Limnoderetrema spp. of South America are highly specific unlike L. minutum of New Zealand.

On some family related parasites (Nematoda, Cucullanidae) from the marine fish Paralonchurus brasiliensis (Steindachner, 1875) (Pisces, Ostraciidae)

Dichelyne (Cucullanellus) elongatus (Tornquist, 1931) Petter, 1974 and Cucullanus pulcherrimus Barreto, 1918, from Paralonchurus brasiliensis (Steind., 1875) are redescribed and two specimens of Dichelyne (Cucullanellus) sp. are also reported in this host, despite lack of previously, was again identified in Brazil since its original description and posterior illustration. The present findings represent also a new host record for the referred genera: Dichelyne Jagerskiold, 1902 and Cucullanus Mueller, 1777. D. (C.) elongatus is also referred in Brazil for the first time.

Comparative allometry growth of some marine fish digenetic trematodes

Allometric growth variation was compared for Plagioporus idoneus, Lepocreadium pegorchis, Opecoeloides furcatus, Bacciger israelensis, Aphanurus stossichi and Parahurleytrema trachinoti collected from East Mediterranean fishes. The pharynx, the oral and the ventral sucker diameters always showed a negative allometry. The other parameters tested were variable with the species. We study the effects of some environmental factors: the influence of the host species is analysed in Plagioporus idoneous, wich parasitizes Oblada melanura, Diplodus sargus and D. vulgaris and in Lepocreadium pegorchis, wich parasitizes Pagellus erythrinus, Lithognathus mormyrus and Spicara smaris; the influence of the microhabitat and the intensity of infection is analysed in Bacciger israelensis and Aphanurus stossichi, both parasites of Boops boops. We report significant differences with the host species, for the allometric growth of the testes; the effect of the microhabitat was revealed by the hindbody allometric value; no significant difference was detected in relation with the intensity of infection.

Desciption of Cribomazocraes travassosi n. sp. (Monogenea: Mazocraeidae), a fish parasite from the Atlantic Ocean

Cribomazocraes travassosi n. sp. is described from Harengula clupeola (Cuvier, 1829) from Rio de Janeiro, coast, Brazil. It differs from C. nagibinae, Mamaev, 1981 in the size and shape of opisthohaptor and lappet and in the smaller size of anchors. From C. bychowskyi it also differs in the extension of vittelaria. A key for the species of the genus is presented.

Characterization of pip5k3 fleck corneal dystrophy-linked gene in zebrafish.

PIKfyve is a kinase encoded by pip5k3 involved in phosphatidylinositols (PdtIns) pathways. These lipids building cell membranes have structural functions and are involved in complex intracellular regulations. Mutations in human PIP5K3 are associated with François-Neetens mouchetée fleck corneal dystrophy [Li, S., Tiab, L., Jiao, X., Munier, F.L., Zografos, L., Frueh, B.E., Sergeev, Y., Smith, J., Rubin, B., Meallet, M.A., Forster, R.K., Hejtmancik, J.F., Schorderet, D.F., 2005. Mutations in PIP5K3 are associated with François-Neetens mouchetee fleck corneal dystrophy. Am. J. Hum. Genet. 77, 54-63]. We cloned the zebrafish pip5k3 and report its molecular characterization and expression pattern in adult fish as well as during development. The zebrafish PIKfyve was 70% similar to the human homologue. The gene encompassed 42 exons and presented four alternatively spliced variants. It had a widespread expression in the adult organs and was localized in specific cell types in the eye as the cornea, lens, ganglion cell layer, inner nuclear layer and outer limiting membrane. Pip5k3 transcripts were detected in early cleavage stage embryos. Then it was uniformly expressed at 10 somites, 18 somites and 24 hpf. Its expression was then restricted to the head region at 48 hpf, 72 hpf and 5 dpf and partial expression was found in somites at 72 hpf and 5 dpf. In situ on eye sections at 3 dpf showed a staining mainly in lens, outer limiting membrane, inner nuclear layer and ganglion cell layer. A similar expression pattern was found in the eye at 5 dpf. A temporal regulation of the spliced variants was observed at 1, 3 and 5 dpf and they were also found in the adult eye.

Two new genera of Paramphistomidae (Trematoda, Digenea) from freshwater fish of Rondônia State, Brazil

Two new genera and species of Paramphistomidae (Trematoda, Digenea) are described from freshwater fish of Rondônia State, Brazil. Pronamphistoma cichlasomae gen. et sp. nov. from Cichlasoma severum (Heckel) is characterized as having a small, robust body with an anterior collar-like expansion, diverticula within the wall of the oral sucker and vitellaria in lateral fanshaped configurations. Micramphistoma ministoma gen. et sp. nov. from Hypopomus sp lacks oral diverticula, has anteriorly situated parallel tests and a very small postero-ventral acebtabulum. Neither of the new forms closely resembles any known genus.

Manipulating keratinocyte stem cell proliferation and differentiation using RNA interference

ABSTRACT : The epidermis, the outermost compartment of the skin, is a stratified and squamous epithelium that constantly self-renews. Keratinocytes, which represent the main epidermal population, are responsible for its cohesion and barrier function. Epidermal renewal necessitates a fine equilibrium between keratinocyte proliferation and differentiation. The keratinocyte stem cell, located in the basal cell layer, is responsible for epidermal homeostasis and regeneration during the wound healing process. The transcription factor p63 structurally belongs to the p53 superfamily. It is expressed in the basal and supra-basal cell layers of stratified epithelia and is thought to be important for the renewal or the differentiation of keratinocyte stem cells (Yang et al., 1999; Mills et al., 1999). In order to better understand its function, we established an in vitro model of p63 deficient human keratinocyte stem cells using a shp63 mediated RNA interference. Knockdown of endogenous p63 induces downregulation of cell-adhesion genes as previously described (Carroll et al., 2006). Interestingly, the replating of attached p63-knockdown keratinocytes on a feeder layer results in a loss of attachment and proliferation. They are no longer clonogenic. However, if the same population are replated in a fibrin matrix, extended fibrinolysis is reported, a common process in wound healing, suggesting that p63 regulates the fibrinolytic pathway. This result was confirmed by Q-PCR and shows that the urokinase pathway, which mediates fibrinolysis, is upregulated. Altogether, these findings suggest a mechanism in which the fine tuning of p63 expression promotes attachment or release of the keratinocyte stem cell from the basement membrane by inducing genes of adhesion and/or of fibrinolysis. This mechanism may be important for epidermal self-renewal, differentiation as well as wound healing. Its misregulation may be partly responsible for the p63 knockout phenotype. The downregulation of p63 also induces a decrease in LEKTI expression. LEKTI (lymphoepithelial Kazal-type serine protease inhibitor) is a serine protease inhibitor encoded by the Spink5 gene. It is expressed and secreted in the uppermost differentiated layers of stratified epithelia and plays a role in the desquamation process. When this gene is disrupted, humans develop the Netherton syndrome (Chavanas et al., 2000b). It is a dermatosis characterized by hair dysplasias, ichtyosiform erythroderma and impairment in epidermal barrier function promoting inflammation similarly as in psoriasis with inflammatory infiltrate in excess. TNFα (tumor necrosis factor alpha) and EDA1 (ectodysplasin A1) are two transmembraneprecursors that belong to the TNF superfamily, which is involved in immune and inflammation regulation (Smahi et al., 2002). We suggest that the secreted serine protease inhibitor LEKTI plays a role in the regulation of TNFα and EDA1 precursor cleavage and absence of LEKTI induces excess of inflammation. To investigate this hypothesis, we induced downregulation of Spink5 expression in rat keratinocyte stem cells by using a shSpink5 mediated RNA interference approach. Interestingly, expression of TNFα and EDA1 is modified after knockdown of Spink5 by Q-PCR. Moreover, downregulation of Spink5 induces loss of cohesiveness between keratinocytes and colonies adopt a scattered phenotype. Altogether, these preliminary data suggest that downregulation of LEKTI may play a role in the inflammatory response in Netherton syndrome patients, by regulating TNFα expression.

RNA-driven cyclin-dependent kinase regulation: when CDK9/cyclin T subunits of P-TEFb meet their ribonucleoprotein partners.

The positive transcription elongation factor (P-TEFb) consists of CDK9, a cyclin-dependent kinase and its cyclin T partner. It is required for transcription of most class II genes. Its activity is regulated by non-coding RNAs. The 7SK cellular RNA turns the HEXIM cellular protein into a P-TEFb inhibitor that binds its cyclin T subunit. Thus, P-TEFb activity responds to variations in global cellular transcriptional activity and to physiological conditions linked to cell differentiation, proliferation or cardiac hypertrophy. In contrast, the Tat activation region RNA plays an activating role. This feature at the 5' end of the human immunodeficiency (HIV) viral transcript associates with the viral protein Tat that in turn binds cyclin T1 and recruits active P-TEFb to the HIV promoter. This results in enhanced P-TEFb activity, which is critical for an efficient production of viral transcripts. Although discovered recently, the regulation of P-TEFb becomes a paradigm for non-coding RNAs that regulate transcription factors. It is also a unique example of RNA-driven regulation of a cyclindependent kinase.

A new, automated, four-colour interphase FISH approach for the simultaneous detection of specific aneuploidies of diagnosis and prognosis significance in high hyperdiploid ALL

In hyperdiploid acute lymphoblastic leukaemia (ALL), the simultaneous occurrence of specific aneuploidies confers a more favourable outcome than hyperdiploidy alone. Interphase (I) FISH complements conventional cytogenetics (CC) through its sensitivity and ability to detect chromosome aberrations in non-dividing cells. To overcome the limits of manual I-FISH, we developed an automated four-colour I-FISH approach and assessed its ability to detect concurrent aneuploidies in ALL. I-FISH was performed using centromeric probes for chromosomes 4, 6, 10 and 17. Parameters established for automatic nucleus selection and signal detection were evaluated (3 controls). Cut-off values were determined (10 controls, 1000 nuclei/case). Combinations of aneuploidies were considered relevant when each aneuploidy was individually significant. Results obtained in 10 ALL patients (1500 nuclei/patient) were compared with those by CC. Various combinations of aneuploidies were identified. All clones detected by CC were observed by I-FISH. I-FISH revealed numerous additional abnormal clones, ranging between 0.1 % and 31.6%, based on the large number of nuclei evaluated. Four-colour automated I-FISH permits the identification of concurrent aneuploidies of prognostic significance in hyperdiploid ALL. Large numbers of cells can be analysed rapidly by this method. Owing to its high sensitivity, the method provides a powerful tool for the detection of small abnormal clones at diagnosis and during follow up. Compared to CC, it generates a more detailed cytogenetic picture, the biological and clinical significance of which merits further evaluation. Once optimised for a given set of probes, the system can be easily adapted for other probe combinations.

Rhipidocotyle gibsoni n. sp. from a Brazilian Freshwater Fish and Rhipidocotyle froesi n.sp. for r. baculum (linton, 1905) of Eckmann (1932) (Bucephalidae; Digenea)

Rhipidocotyle gibsoni n.sp. is described from Acestrorhynchus lacustris from Paraná River, brazil. It is most closely related to r. froesi n. sp. and to R. eckmanni in the shape of the cephalic hood, differing in the extent of the uterus, in the position of the vitelline follicles and in that the host is a freshwater fish. Rhipidocotyle froesi n. sp. is proposed for the marine specimens described by Eckmann (1932) as R. baculum (Linton, 1905).

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Monophyletic origin of multiple clonal lineages in an asexual fish (Poecilia formosa).

Despite the advantage of avoiding the costs of sexual reproduction, asexual vertebrates are very rare and often considered evolutionarily disadvantaged when compared to sexual species. Asexual species, however, may have advantages when colonizing (new) habitats or competing with sexual counterparts. They are also evolutionary older than expected, leaving the question whether asexual vertebrates are not only rare because of their 'inferior' mode of reproduction but also because of other reasons. A paradigmatic model system is the unisexual Amazon molly, Poecilia formosa, that arose by hybridization of the Atlantic molly, Poecilia mexicana, as the maternal ancestor, and the sailfin molly, Poecilia latipinna, as the paternal ancestor. Our extensive crossing experiments failed to resynthesize asexually reproducing (gynogenetic) hybrids confirming results of previous studies. However, by producing diploid eggs, female F(1) -hybrids showed apparent preadaptation to gynogenesis. In a range-wide analysis of mitochondrial sequences, we examined the origin of P. formosa. Our analyses point to very few or even a single origin(s) of its lineage, which is estimated to be approximately 120,000 years old. A monophyletic origin was supported from nuclear microsatellite data. Furthermore, a considerable degree of genetic variation, apparent by high levels of clonal microsatellite diversity, was found. Our molecular phylogenetic evidence and the failure to resynthesize the gynogenetic P. formosa together with the old age of the species indicate that some unisexual vertebrates might be rare not because they suffer the long-term consequences of clonal reproduction but because they are only very rarely formed as a result of complex genetic preconditions necessary to produce viable and fertile clonal genomes and phenotypes ('rare formation hypothesis').