Metacommunity structure of aquatic gastropods in a river floodplain: the role of niche breadth and drift propensity

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Identification of a novel insulin-like growth factor binding protein gene homologue with tumor suppressor like properties

Here we report the identification of a new insulin-like growth factor binding protein homologue, provisionally designated insulin-like growth factor binding related protein-4 (IGFBP-rP4). IGFBP-rP4 was found to be most closely related to IGFBP-7 with 52% amino acid homology and 43% amino acid identity, and shares a similar domain structure. Semi-quantitative RT-PCR expression analysis demonstrated a pattern of downregulation of this gene in multiple tumor samples including lung and colon cancer, compared to matched adjacent normal tissue. Western blotting revealed a protein of approximately 38kDa expressed in both the cell pellet and secreted into the supernatant of transiently transfected Cos-7 cells. Cos-7 supernatants containing IGFBP-RP4 protein were observed to suppress the growth of HeLa cells in culture compared to vector controls. IGFBP-RP4 directly transiently transfected into HeLa cells also further confirmed the growth suppressive properties of this protein. Together these data suggest that IGFBP-RP4 may be a novel putative tumor suppressor protein.     

Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.     

Quantitative detection of agar-cultivated and rhizotron-grown<Emphasis Type="Italic"> Piloderma croceum</Emphasis> Erikss. &; Hjortst. by ITS1-based fluorescent PCR

A real-time quantitative TaqMan-PCR was established for the absolute quantification of extramatrical hyphal biomass of the ectomycorrhizal fungus Piloderma croceum in pure cultures as well as in rhizotron samples with non-sterile peat substrate. After cloning and sequencing of internal transcribed spacer (ITS) sequences ITS1/ITS2 and the 5.8S rRNA gene from several fungi, including Tomentellopsis submollis, Paxillus involutus, and Cortinarius obtusus, species-specific primers and a dual-labelled fluorogenic probe were designed for Piloderma croceum. The dynamic range of the TaqMan assay spans seven orders of magnitude, producing an online-detectable fluorescence signal during the cycling run that is directly related to the starting number of ITS copies present. To test the confidence of the PCR-based quantification results, the hyphal length of Piloderma croceum was counted under the microscope to determine the recovery from two defined but different amounts of agar-cultivated mycelia. Inspection of the registered Ct values (defined as that cycle number at which a statistically significant increase in the reporter fluorescence can first be detected) in a 10-fold dilution series of template DNA represents a suitable and stringent quality control standard for exclusion of false PCR-based quantification results. The fast real-time PCR approach enables high throughput of samples, making this method well suited for quantitative analysis of ectomycorrhizal fungi in communities of natural and artificial ecosystems, so long as applicable DNA extraction protocols exist for different types of soil.     

Use of a rapid and highly sensitive fluorescamine-based procedure for the assay of plasma lipoproteins

A rapid and sensitive method for determining protein concentrations using fluorescamine has been characterized for use in the analysis of intact lipoproteins. It was shown that there is no interference with the assay due to the presence of lipid-associated turbidity or primary amine content. The assay was shown to be sensitive to as little as 0.3 microgram of lipoprotein and to yield similar results when compared to the Lowry method.     

Tumour Necrosis Factor Alpha,Interferon Gamma and Substance P Are Novel Modulators of Extrapituitary Prolactin Expression in Human Skin

Human scalp skin and hair follicles (HFs) are extra-pituitary sources of prolactin (PRL). However, the intracutaneous regulation of PRL remains poorly understood. Therefore we investigated whether well-recognized regulators of pituitary PRL expression, which also impact on human skin physiology and pathology, regulate expression of PRL and its receptor (PRLR) in situ. This was studied in serum-free organ cultures of microdissected human scalp HFs and skin, i.e. excluding pituitary, neural and vascular inputs. Prolactin expression was confirmed at the gene and protein level in human truncal skin, where its expression significantly increased (p = 0.049) during organ culture. There was, however, no evidence of PRL secretion into the culture medium as measured by ELISA. PRL immunoreactivity (IR) in female human epidermis was decreased by substance P (p = 0.009), while neither the classical pituitary PRL inhibitor, dopamine, nor corticotropin-releasing hormone significantly modulated PRL IR in HFs or skin respectively. Interferon (IFN) γ increased PRL IR in the epithelium of human HFs (p = 0.044) while tumour necrosis factor (TNF) α decreased both PRL and PRLR IR. This study identifies substance P, TNFα and IFNγ as novel modulators of PRL and PRLR expression in human skin, and suggests that intracutaneous PRL expression is not under dopaminergic control. Given the importance of PRL in human hair growth regulation and its possible role in the pathogenesis of several common skin diseases, targeting intracutaneous PRL production via these newly identified regulatory pathways may point towards novel therapeutic options for inflammatory dermatoses.     

Islands within an island: Repeated adaptive divergence in a single population

Physical barriers to gene flow were once viewed as prerequisites for adaptive evolutionary divergence. However, a growing body of theoretical and empirical work suggests that divergence can proceed within a single population. Here we document genetic structure and spatially replicated patterns of phenotypic divergence within a bird species endemic to 250 km² Santa Cruz Island, California, USA. Island scrub‐jays (Aphelocoma insularis) in three separate stands of pine habitat had longer, shallower bills than jays in oak habitat, a pattern that mirrors adaptive differences between allopatric populations of the species’ mainland congener. Variation in both bill measurements was heritable, and island scrub‐jays mated nonrandomly with respect to bill morphology. The population was not panmictic; instead, we found a continuous pattern of isolation by distance across the east–west axis of the island, as well as a subtle genetic discontinuity across the boundary between the largest pine stand and adjacent oak habitat. The ecological factors that appear to have facilitated adaptive differentiation at such a fine scale—environmental heterogeneity and localized dispersal—are ubiquitous in nature. These findings support recent arguments that microgeographic patterns of adaptive divergence may be more common than currently appreciated, even in mobile taxonomic groups like birds.