Functional expression of pig renal organic anion transporter 3 (pOAT3)

With the cloning of pig renal organic anion transporter 1 (pOAT1) (Biochimie 84 (2002) 1219) we set up a model system for comparative studies of cloned and natively isolated membrane located transport proteins. Meanwhile, another transport protein involved in p-aminohippurate (PAH) uptake on the basolateral side of the proximal tubule cells was identified, designated organic anion transporter 3 (OAT3). To explore the contribution of pOAT1 to the PAH clearance in comparison to OAT3, it was the aim of this study to extend our model by cloning of the pig ortholog of OAT3. Sequence comparisons of human organic anion transporter 3 (hOAT3) with the expressed sequence tag (EST) database revealed a clone and partial sequence of the pig renal organic anion transporter 3 (pOAT3) ortholog. Sequencing of the entire open reading frame resulted in a protein of 543 amino acid residues encoded by 1632 base pairs (EMBL Acc. No. AJ587003). It showed high homologies of 81%, 80%, 76%, and 77% to the human, rabbit, rat, and mouse OAT3, respectively. A functional characterization of pOAT3 in Xenopus laevis oocytes yielded an apparent Km (Kt) for [3H]estrone sulfate of 7.8 +/- 1.3 microM. Moreover, pOAT3 mediated [3H]estrone sulfate uptake was almost abolished by 0.5 mM of glutarate, dehydroepiandosterone sulfate, or probenecid consistent with the hallmarks of OAT3 function.     

Multiple forms of secretory phospholipase A2 in plants

Multiple secretory phospholipase A2 (sPLA2) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of this genetic and biochemical heterogeneity has provided important clues to the regulation and function of the individual enzymes. An increasing body of evidence shows that their lipid products, lysophospholipids and free fatty acids, mediate a variety of cellular responses, including plant growth, development, and responses to stress and defense. This review discusses the newly-acquired information on plant sPLA2s including the molecular and biochemical characteristics, and signaling functions of each isoform.     

Polymorphisms of the prion protein gene (PRNP) in Hanwoo (Bos taurus coreanae) and Holstein cattle

Polymorphisms in the prion protein gene (PRNP) in humans and sheep correlate with susceptibility to transmissible spongiform encephalopathies (TSEs). Bovine spongiform encephalopathy (BSE) has been reported in British and Japanese cattle; it has occurred thus far in Holstein cattle. BSE in Hanwoo (Bos taurus coreanae) cattle has not been diagnosed up to now. To characterize the bovine PRNP polymorphisms in Korean cattle, we analyzed the open reading frame (ORF) of PRNP in 120 Hanwoo (beef) cattle and 53 Holstein (dairy) cattle. Three polymorphisms were found, the third position of codon 78 (G-->A), the third position of codon 192 (C-->T), and the deletion of a single octa-repeat. An analysis of codon 78 revealed no difference in the genotype (P = 0.2026) or allele (P = 0.7180) frequencies between Hanwoo and Holstein animals. However, there were significant differences in the genotype (P < 0.0001) and allele (P < 0.0001) frequencies at PRNP codon 192 between Hanwoo and Holstein animals. The rate of Holstein animals with deletion of a single octa-repeat was 91.5% undeleted homozygotes, 8.5% heterozygotes (with R3 deletion), and 0% deleted homozygotes. However, none of the 120 Hanwoo animals had any octa-repeat deletions. The genotype (P < 0.0001) and allele (P < 0.0001) frequencies of a single octa-repeat-deletion were also significantly different between Hanwoo and Holstein animals.     

Expression of transglutaminase substrate activity on Candida albicans germ tubes through a coiled, disulfide-bonded N-terminal domain of Hwp1 requires C-terminal glycosylphosphatidylinositol modification

By serving as a microbial substrate for epithelial cell transglutaminase, Hwp1 (Hyphal wall protein 1) of Candida albicans participates in cross-links with proteins on the mammalian mucosa. Biophysical properties of the transglutaminase substrate domain were explored using a recombinant protein representative of the N-terminal domain of Hwp1 and were similar to other transglutaminase substrates, the small proline-rich proteins of cornified envelopes found in stratified squamous epithelia. Recombinant Hwp1 lacks alpha and beta structures by circular dichroism and likely exists as a disulfide-cross-linked coiled-coil. The transglutaminase substrate property prompted a unique approach for investigating the features of surface Hwp1 on germ tubes. A lysine analog, 5-(biotinamido)pentylamine, was cross-linked to germ tubes catalyzed by transglutaminase 2 prior to cell fractionation, immunoprecipitation, and detection with streptavidin conjugates. The majority of the transglutaminase-modifiable Hwp1 was covalently attached to the beta-glucan of hyphae by the C terminus of Hwp1 via a glycosylphosphatidylinositol remnant anchor. A putative precursor of cell wall forms of Hwp1 was identified in the cell extract and in the culture medium. Hwp1 was modified by relatively short N-linked glycans, and the molecular size of the protein was reduced by hypomannosylation when expressed in O-glycosylation mutant strains. Hwp1 combines features of mammalian transglutaminase substrate proteins with characteristics of fungal cell wall proteins to form an unconventional adhesin at the hyphal wall of C. albicans.     

Metabolic profiling of plasma from discordant schizophrenia twins: correlation between lipid signals and global functioning in female schizophrenia patients

(1)H NMR spectroscopy-based metabonomic analysis was employed to investigate plasma samples from 21 pairs of monozygotic twins discordant for schizophrenia and 8 pairs of age-matched healthy twins in an effort to disentangle genetic and epigenetic components of schizophrenia. We identified alterations in the lipid profile of both affected and unaffected schizophrenia twins. Additionally, there is a close association of VLDL/LDL signals and Global Functioning Scores in female twins suffering from schizophrenia. Our results further support a link between metabolic disturbances and the etiopathology of schizophrenia.     

CO2 sensing in fungi and beyond

Carbon dioxide is not only an important gaseous molecule for maintenance of the biosphere homeostasis, but is also a crucial signalling cue in living cells. Fungal pathogens, including Candida albicans and Cryptococcus neoformans, must adapt to dramatic changes in CO2 levels during colonization and subsequent infection of their human host. Recent reports provide insight into how pathogenic fungi sense environmental CO2 and the role of carbonic anhydrase and fungal adenylyl cyclase in CO2 sensing.     

Small GTPases Rap1 and RhoA regulate superoxide formation by Rac1 GTPases activation during the phagocytosis of IgG-opsonized zymosans in macrophages

Phagocytic NADPH oxidase plays a critical role in superoxide generation in macrophage cells. Small GTPases, including Rac1 and Rac2, have been implicated in the regulation of NADPH oxidase activity. Rap1, which has no effect in a cell-free system of oxidase activation, recently has been proven to colocalize with cytochrome b(558). In addition, neutrophils from rap1A(-/-) mice reduce fMLP-stimulated superoxide production. Here, we tried to determine whether Rap1 also plays a role in the production of superoxide. IgG-opsonized zymosan (IOZ) particles treatment induced Rap1 activation and superoxide generation. Knock-down of Rap1 by si-Rap1 suppressed IOZ-induced superoxide formation. Sh-RhoA also reduced superoxide levels, but 8CPT-2Me-cAMP, an activator of Epac1 (a guanine nucleotide exchange factor (GEF) of Rap1), could recover the levels to the control value. When cells were stimulated by IOZ, Rap1 and Rac1 were translocated to the membrane, and then interacted with p22(phox). 8CPT-2Me-cAMP rescued sh-RhoA-induced reduction of the interaction between Rac1 and p22(phox), and enhanced lysophosphatidic acid (LPA)-induced increase of their interaction. Moreover, Rac1 activity was increased by both LPA and 8CPT-2Me-cAMP when treated with IOZ particles. Si-Vav2 impaired GTP-Rac1 levels in response to 8CPT-2Me-cAMP/IOZ. Phosphorylation of RhoA activates Rac1 in response to IOZ by the enhanced binding of phospho-RhoA to RhoGDI, leading to the release of Rac1 from the Rac1-RhoGDI complex. In conclusion, IOZ treatment induces Rap1 activation and phosphorylation of RhoA, which in turn cause Rac1 activation and promote Rac1 translocation to the membrane leading to binding with p22(phox) that activates NADPH oxidase and produces superoxide.     

The interaction between propagule pressure,habitat suitability and density‐dependent reproduction in species invasion

Seedling recruitment limitations create a demographic bottleneck that largely determines the viability and structure of plant populations and communities, and pose a core restriction on the colonization of novel habitat. We use a shade‐tolerant, invasive grass, Microstegium vimineum, to examine the interplay between seed and establishment limitations – phenomena that together determine recruitment success but usually are investigated individually. We add increasing amounts of seed to microhabitats containing variable levels of leaf litter thickness – with reduced leaf litter simulating disturbance – to investigate whether reduced seed limitation overcomes the establishment limitation posed by litter cover. We do this across gradients in understory light, moisture and temperature, and quantify germination, survival, and then per capita adult biomass and reproduction in order to understand the implications for invasion across the landscape. We find that the combined effects of seed and establishment limitation influence recruitment; however, propagule pressure overwhelms the inhibitory effects of leaf litter thickness. Leaf litter reduces germination by 22–57% and seedling survival by 13–15% from that observed on bare soil. However, density‐dependent reproduction compensates as 1–3 plants can produce far more seeds (approx. 525) than are required for persistence. As such, just a few plants may establish in understory forest habitat and subsequently overwhelm establishment barriers with copious propagule production. These results, for a widespread, invasive plant, are consistent with the emerging perspective for native plants that seed and establishment limitation jointly influence recruitment. The ability for an exotic plant species to compensate for low population densities with high per capita seed production, that then overrides establishment limitations, makes its invasive potential daunting. Further work is required to test if this is a common mechanism underlying plant invasions.