A Taxonomic Revision of the Wallemia sebi Species Complex

Wallemia sebi is a xerophilic food- and air-borne fungus. The name has been used for strains that prevail in cold, temperate and tropical climates. In this study, multi-locus phylogenetic analyses, using the internal transcribed spacer (ITS) regions, DNA replication licensing factor (MCM7), pre-rRNA processing protein (TSR1), RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2) and a new marker 3´-phosphoadenosine-5´-phosphatase (HAL2), confirmed the previous hypothesis that W. sebi presents a complex of at least four species. Here, we confirm and apply the phylogenetic analyses based species hypotheses from a companion study to guide phenotypic assessment of W. sebi like strains from a wide range of substrates, climates and continents allowed the recognition of W. sebi sensu stricto and three new species described as W. mellicola, W. Canadensis, and W. tropicalis. The species differ in their conidial size, xerotolerance, halotolerance, chaotolerance, growth temperature regimes, extracellular enzyme activity profiles, and secondary metabolite patterns. A key to all currently accepted Wallemia species is provided that allow their identification on the basis of physiological, micromorphological and culture characters.     

Permeation of Calcium through Purified Connexin 26 Hemichannels

Gap junction channels communicate the cytoplasms of two cells and are formed by head to head association of two hemichannels, one from each of the cells. Gap junction channels and hemichannels are permeable to ions and hydrophilic molecules of up to M_r 1,000, including second messengers and metabolites. Intercellular Ca²⁺ signaling can occur by movement of a number of second messengers, including Ca²⁺, through gap junction channels, or by a paracrine pathway that involves activation of purinergic receptors in neighboring cells following ATP release through hemichannels. Understanding Ca²⁺ permeation through Cx26 hemichannels is important to assess the role of gap junction channels and hemichannels in health and disease. In this context, it is possible that increased Ca²⁺ influx through hemichannels under ischemic conditions contributes to cell damage. Previous studies suggest Ca²⁺ permeation through hemichannels, based on indirect arguments. Here, we demonstrate for the first time hemichannel permeability to Ca²⁺ by measuring Ca²⁺ transport through purified Cx26 hemichannels reconstituted in liposomes. We trapped the low affinity Ca²⁺-sensitive fluorescent probe Fluo-5N into the liposomes and followed the increases in intraliposomal [Ca²⁺] in response to an imposed [Ca²⁺] gradient. We show that Ca²⁺ does move through Cx26 hemichannels and that the permeability of the hemichannels to Ca²⁺ is high, similar to that for Na⁺. We suggest that hemichannels can be a significant pathway for Ca²⁺ influx into cells under conditions such as ischemia.     

Studies on the subcommissural organ of Salmo gairdneri

The light microscopic analysis of serial sections of the subcommissural organ (SCO) of the rainbow trout (Salmo gairdneri) shows that the form of the groove-like (in cross section) organ varies over its total length. Its rostral origin is a tunnel-like structure anterior to the orifice of the hollow pineal stalk. The SCO forms the dorsal wall of the brain. Caudally the SCO is increasingly displaced from the surface of the brain by the fibers of the posterior commissure; the organ ends in a tabular area beyond the latter. The orifice of the pineal stalk is surrounded by the ependyma of the SCO that invaginates like a funnel and joins with the ependyma of the pineal stalk after a considerable narrowing. The rudimentary parapineal organ is located on the left side of the brain and is connected with the left habenular ganglion through the parapineal tract. It contacts the third ventricle with a short channel within the ependyma of the SCO. The histological organization of the ependymal and hypendymal cells of the SCO is typical of teleosts. Secretory material is located basally and apically in relation to the nucleus, but there is no indication of a basal secretory release. Basal ependymal processes terminate with broadened endings at the membrana limitans externa. The apical product is discharged into the third ventricle, where it aggregates into the thread-like structure of Reissner's fibre. The SCO cells have no direct contact with cerebral or meningeal blood vessels.     

The telomerase inhibitor Gno1p/PINX1 activates the helicase Prp43p during ribosome biogenesis

We provide evidence that a central player in ribosome synthesis, the ribonucleic acid helicase Prp43p, can be activated by yeast Gno1p and its human ortholog, the telomerase inhibitor PINX1. Gno1p and PINX1 expressed in yeast interact with Prp43p and the integrity of their G-patch domain is required for this interaction. Moreover, PINX1 interacts with human PRP43 (DHX15) in HeLa cells. PINX1 directly binds to yeast Prp43p and stimulates its adenosine triphosphatase activity, while alterations of the G patch abolish formation of the PINX1/Prp43p complex and the stimulation of Prp43p. In yeast, lack of Gno1p leads to a decrease in the levels of pre-40S and intermediate pre-60S pre-ribosomal particles, defects that can be corrected by PINX1 expression. We show that Gno1p associates with 90S and early pre-60S pre-ribosomal particles and is released from intermediate pre-60S particles. G-patch alterations in Gno1p or PINX1 that inhibit their interactions with Prp43p completely abolish their function in yeast ribosome biogenesis. Altogether, our results suggest that activation of Prp43p by Gno1p/PINX1 within early pre-ribosomal particles is crucial for their subsequent maturation.     

Lysophosphatidic Acid Acyltransferase Beta Regulates mTOR Signaling

Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.     

Cytotoxic effect of the immunotoxin constructed of the ribosome-inactivating protein curcin and the monoclonal antibody against Her2 receptor on tumor cells

The toxicity of the curcin on cancer cells allows to consider this protein as the toxic component of an immunotoxin directed to Her2, which is associated with cancer. Reductive amination was proposed to conjugate curcin and an anti-Her2; the binding was tested using Polyacrylamide gel electrophoresis, western blot, and immunocytochemistry. The in vitro cytotoxicity of curcin and the immunotoxin was assessed on breast cancer cell lines SK-BR-3 (Her2⁺) and MDA-MB-231 (Her2^?). IC₅₀ values for curcin were 15.5 ± 8.3 and 18.6 ± 2.4 μg/mL, respectively, statistically equivalent (p < 0.05). While to the immunotoxin was 2.2 ± 0.08 for SK-BR-3 and 147.6 ± 2.5 μg/mL for MDA-MB-231. These values showed that the immunotoxin was seven times more toxic to the SK-BR-3 than curcin and eight times less toxic to the MDA-MB-231. The immunotoxin composed of curcin and an antibody against Her2 and constructed by reductive amination could be a therapeutic candidate against Her2⁺ cancer.