Obba and Sebipora, new polypore genera related to Cinereomyces and Gelatoporia (Polyporales, Basidiomycota)

Cinereomyces clade is a newly proposed monophyletic group of polypores containing currently four genera and five species, including two promising biopulping fungi, Ceriporiopsis rivulosa and C. subvermispora. The Cinereomyces clade is well-delimited in nrDNA-based phylogenetic analysis, but its position in Polyporales remains unclear. Its closest relative may be found in the core polyporoid clade. Only a few morphological characters are common for all the species in the clade, e.g. CB– and CRB+ hyphae, white fruiting bodies, presence of oil, and middle-sized spores. Culturally, the species are unified by producing simple-septate generative hyhpae in the margin, which produce simple-clamped hyphae backwards. The genus Gelatoporia is the correct place for Ceriporiopsis subvermispora. Two new genera are described in the group: Obba to incorporate C. rivulosa and a new austral species, Obba valdiviana, known from southern Argentina and recorded here also from Tasmania, and Sebipora to accommodate a new species from tropical Asia, S. aquosa. ITS sequences imply that Eurasian Gelatoporia subvermispora may belong to a different species from the North American one. G. subvermispora is recorded as new to Indonesia.     

Accumulation of cholesterol precursors and plant sterols in human stenotic aortic valves

The pathogenesis of aortic valve stenosis (AS) is characterized by the accumulation of LDL-derived cholesterol in the diseased valves. Since LDL particles also contain plant sterols, we investigated whether plant sterols accumulate in aortic valve lesions. Serum samples were collected from 82 patients with severe AS and from 12 control subjects. Aortic valves were obtained from a subpopulation of 21 AS patients undergoing valve surgery and from 10 controls. Serum and valvular total cholesterol and noncholesterol sterols were measured by gas-liquid chromatography. Noncholesterol sterols, including both cholesterol precursors and sterols reflecting cholesterol absorption, were detected in serum samples and aortic valves. The higher the ratios to cholesterol of the cholesterol precursors and absorption markers in serum, the higher their ratios in the stenotic aortic valves (r=0.74, P<0.001 for lathosterol and r=0.88, P<0.001 for campesterol). The valvular ratio to cholesterol of lathosterol correlated negatively with the aortic valve area (r= -0.47, P=0.045), suggesting attenuation of cholesterol synthesis with increasing severity of AS. The higher the absorption of cholesterol, the higher the plant sterol contents in stenotic aortic valves. These findings suggest that local accumulation of plant sterols and cholesterol precursors may participate in the pathobiology of aortic valve disease.     

Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) formed a morphologically well-organized epidermis in the absence of feeder cells when grown for 3 weeks on a collagen gel in culture inserts at an air-liquid interface, and developed a permeability barrier resembling that of human skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabasal layers exhibiting the differentiation markers keratin 10, involucrin, and filaggrin. Granular cells with keratohyalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filaments were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the number of keratohyalin granules present, and the quantity and organization of intercellular lipid lamellae in the interstices of the stratum corneum. The morphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rates of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was increased by vitamin C, and western blots confirmed higher levels of profilaggrin and filaggrin, suggesting that vitamin C also influences keratinocyte differentiation in aspects other than the synthesis and organization of barrier lipids. The unique REK cell line in organotypic culture thus provides an easily maintained and reproducible model for studies on epidermal differentiation and transepidermal permeation.     

Cytokeratin immunoreactivity in lobular intraepithelial neoplasia.

Eighteen commercially available antibodies reactive against different cytokeratin proteins were tested on classic examples of lobular intraepithelial neoplasia (LIN) and of ductal intraepithelial neoplasia (DIN) of the breast. About 90% of higher-grade DIN (AIDH and DCIS) show no or substantially diminished reaction with clone 34betaE12 (specified as reactive against keratins 1, 5, 10, and 14 as determined by the manufacturer), while the cells of LIN were found to express the antigen reactive with this antibody. To determine which of these four keratins are present in the cells of LIN, antibodies reactive against these individual four keratins were tested. None of the four antibodies to keratins 1, 5, 10, or 14 reacted with the cells of LIN. To investigate this further, 13 additional monoclonal antibodies to various other keratin proteins were tested on the cells of LIN. Those that successfully reacted with the cells of LIN were further tested on the cells of DIN. All of the individual antibodies reactive with the cells of LIN were also reactive with the cells of DIN to a degree, with clone RCK108 (reactive against keratin 19) coming the closest to demonstrating the reactivity seen with 34betaE12. We conclude that the reactivity seen in the cells of LIN with 34betaE12 is due to either (a) a crossreaction with keratin 19 that is slightly less prominent than the reaction of the individual clone RCK108, (b) a crossreaction with a keratin protein that was not tested (3, 11, 12), (c) a crossreaction with a protein closely resembling keratin in formalin-fixed, paraffin-embedded tissue, or (d) the detection of a mutated or truncated form of keratin 1, 5, 10, or 14 that cannot be detected by the individual monoclonal antibody.     

Cholesterol metabolism during ketoconazole treatment in man

Ketoconazole, an antifungal antibiotic, inhibits cholesterol synthesis by blocking demethylation of lanosterol. Effects of this inhibition were studied on serum cholesterol, lipoproteins and cholesterol precursors, biliary lipid composition, and fecal steroid elimination in five patients with prostate cancer treated with large doses of ketoconazole. The serum level of total cholesterol fell by 27%, that of LDL cholesterol by 41% and that of LDL apoB by 32% with ketoconazole alone; the fall in the total cholesterol level of a patient treated with ketoconazole-cholestyramine was 65%. Serum contents of free lanosterol and dihydrolanosterol increased up to 250 times, yet the total concentrations remained less than 2 mg/dl. Of the other cholesterol precursor sterols only those with delta 8-double bond increased several times, indicating that in addition to 14 alpha-demethylation, ketoconazole also interfered with metabolism of later intermediary sterols to some extent. Compared with serum sterols, lanosterols were enriched in biliary and fecal sterols up to 10-20 times. Fecal lanosterol output increased from 12 to 247 mg/day, and comprised over 20% fecal steroids of endogenous origin. Bile acid synthesis was significantly decreased, the proportion of chenodeoxycholic acid being markedly reduced in both biliary and fecal bile acids. Cholesterol absorption appeared to decrease yet fecal neutral sterol output and cholesterol synthesis were unchanged and the overall sterol synthesis was increased. It thus appears that ketoconazole inhibits cholesterol elimination as bile acids. However, by blocking 14 alpha-demethylation, it results in effective drainage of sterol nucleus as lanosterols into bile and feces, which, in turn, is associated with a marked reduction in low density lipoprotein (LDL) cholesterol level probably through activation of hepatic LDL apoB receptors.     

EGFR Signaling Promotes β-Cell Proliferation and Survivin Expression during Pregnancy

Placental lactogen (PL) induced serotonergic signaling is essential for gestational β-cell mass expansion. We have previously shown that intact Epidermal growth factor –receptor (EGFR) function is a crucial component of this pathway. We now explored more specifically the link between EGFR and pregnancy-induced β-cell mass compensation. Islets were isolated from wild-type and β-cell-specific EGFR-dominant negative mice (E1-DN), stimulated with PL and analyzed for β-cell proliferation and expression of genes involved in gestational β-cell growth. β-cell mass dynamics were analyzed both with traditional morphometrical methods and three-dimensional optical projection tomography (OPT) of whole-mount insulin-stained pancreata. Insulin-positive volume analyzed with OPT increased 1.4-fold at gestational day 18.5 (GD18.5) when compared to non-pregnant mice. Number of islets peaked by GD13.5 (680 vs 1134 islets per pancreas, non-pregnant vs. GD13.5). PL stimulated beta cell proliferation in the wild-type islets, whereas the proliferative response was absent in the E1-DN mouse islets. Serotonin synthesizing enzymes were upregulated similarly in both the wild-type and E1-DN mice. However, while survivin (Birc5) mRNA was upregulated 5.5-fold during pregnancy in the wild-type islets, no change was seen in the E1-DN pregnant islets. PL induced survivin expression also in isolated islets and this was blocked by EGFR inhibitor gefitinib, mTOR inhibitor rapamycin and MEK inhibitor PD0325901. Our 3D-volumetric analysis of β-cell mass expansion during murine pregnancy revealed that islet number increases during pregnancy. In addition, our results suggest that EGFR signaling is required for lactogen-induced survivin expression via MAPK and mTOR pathways.     

Metabolic alteration of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension cultures overexpressing <Emphasis Type="Italic">geraniol synthase</Emphasis> in the plastids or cytosol

Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.     

Female egg dumping and the effect of sex ratio on male egg carrying in a coreid bug

Phyllomorpha laciniata Vill (Heteroptera, Coreidae) is uniqueamong terrestrial insects in that females glue eggs on the backsof other conspecifics. Egg carrying byP. laciniatamales haspreviously been considered as paternal care. We explored femaleoviposition with respect to previous mating experience of femalesand tested whether sex ratio affects male egg-carrying. Thehypothesis that male egg-carrying is a form of paternal carepredicts that a male should always accept eggs after matingwith a female. However, if male egg-carrying is a form of postcopulatorymate guarding rather than paternal care, egg carrying shouldincrease in the presence of other males. When two couples wereplaced together, females laid eggs on the backs of all individualsenclosed, including the backs of other females. However, whena female was accompanied by 2 males, 22 out of 26 females ovipositedon their mating partner. Thus, sexual competition rather thanpaternity alone, affects a male's eagerness to carry eggs. However,even if males sometimes carry their own eggs, females lay eggson the backs of all conspecifics they can easily acquire. Thus,egg carrying in P. laciniata is partially voluntary and partiallythe result of female egg dumping     

Adsorption of dactinomycin to nitrocellulose filters

Dactinomycin is strongly adsorbed to nitrocellulose filters and some of the adsorbed material is slowly released. This affects both the growth and development of embryonic explants and the nucleotide incorporation of HeLa cells cultivated on pretreated filters.