Intermediates in the oxidative pathway from torulene to torularhodin in the red yeasts Cystofilobasidium infirmominiatum and C. capitatum (Heterobasidiomycetes, Fungi)

Two red Cystofilobasidium spp. isolated from spring sap-flows of Betula pendula were analysed for their carotenoid content. In Cystofilobasidium infirmominiatum, three unusual pigments were detected and identified by structure elucidation as oxidised torulene derivatives. These included 16'-hydroxytorulene and torularhodinaldehyde, two carotenoids known so far only from chemical synthesis or as postulated biosynthetic intermediates en route to torularhodin. Unprecedented formation of beta-apo-2'-carotenal was also observed. The production of these pigments in pure culture was dependent on enhanced oxidative stress caused by cultivation in well-aerated (indented) flasks with or without 2% ethanol (16'-hydroxytorulene), or with 100 microM duroquinone (torularhodinaldehyde and beta-apo-2'-carotenal). Among these three pigments, only 16'-hydroxytorulene was detected in C. capitatum. Torularhodin, a common end product of carotenoid oxidation in red yeasts, was not produced by either species under any incubation conditions. Biosynthetic aspects of incomplete oxidation of torulene by these Cystofilobasidium spp. are discussed.     

Independence of in vitro iron absorption from mucosal transferrin content in rat jejunal and ileal segments

Isolated non blood-perfused intestinal segments from normal and iron-deficient rats were used in vitro. A modification of the luminal perfusion method according to Fisher and Parsons allowed the comparison of iron and transferrin quantities in the serosal fluid at 15 min intervals. Iron transfer in jejunal and ileal segments was directly proportional to the luminal iron concentration within a dose range of 1 to 100 mumol/l, did not show saturation characteristics and was linear over time. Jejunal segments from iron-deficient rats transferred about twice as much iron as the jejunal controls. In ileal segments there was no difference in iron transfer between iron-deficient and control rats; in both cases transfer amounted to approx. 10% of jejunal controls. An exponential correlation was found, when the decreasing transferrin content of the tissue was plotted against the cumulative water transport. Transferrin and albumin release from jejunal and ileal segments into the absorbate cumulated asymptotically, which is typical for wash-out phenomena. As iron transfer cumulated linearly while transferrin release cumulated in an asymptotic manner, the capacity of transferrin to bind iron ions is exceeded roughly 100 times by molar equivalents of iron in the last absorbate fractions. Independence of iron transfer from mucosal transferrin quantities is concluded. As the molar transferrin/albumin ratios do not show significant differences between plasma and the sequence of absorbate samples, a wash-out from the gut's interstitial space is assumed, which makes plasma the most likely origin of transferrin in the mucosa.     

Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.

Methods

Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.

Results

COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.

Conclusions

In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.     

Is there a typical germinal center? A large-scale immunohistological study on the cellular composition of germinal centers during the hapten-carrier-driven primary immune response in mice

Germinal centers (GCs) are complex, multicell-type, transient structures that form in secondary lymphatic tissues in response to T cell-dependent stimulation. This process is crucial to the adaptive immune response because it is the source of affinity maturation and long-lived B cell memory. Our previous studies showed that the growth of murine splenic GCs is nonsynchronized, involving broad-volume distributions of individual GCs at any time. This raises the question whether such a thing as a typical GC exists. To address this matter, we acquired large-scale confocal data on GCs throughout the course of the 2-phenyl-5-oxazolone chicken serum albumin-driven primary immune response in BALB/c mice. Semiautomated image analysis of 3457 GC sections revealed that, although there is no typical GC in terms of size, GCs have a typical cellular composition in that the cell ratios of resident T cells, macrophages, proliferating cells, and apoptotic nuclei are maintained during the established phase of the response. Moreover, our data provide evidence that the dark zone (DZ) and light zone (LZ) compartments of GCs are about the same size and led us to estimate that the minimal cell loss rate in GCs is 3% per hour. Furthermore, we found that the population of GC macrophages is larger and more heterogeneous than previously thought, and that despite enrichment of T cells in the LZ, the DZ of murine splenic GCs is not poor in T cells. DZ and LZ differ in the T cell-to-macrophage ratio rather than in the density of T cells.     

Generalist Life Cycle Aids Persistence of Alexandrium ostenfeldii (Dinophyceae) in Seasonal Coastal Habitats of the Baltic Sea

In seasonal environments, strong gradients of environmental parameters can shape life cycles of phytoplankton. Depending on the rate of environmental fluctuation, specialist or generalist strategies may be favored, potentially affecting life cycle transitions. The present study examined life cycle transitions of the toxin producing Baltic dinoflagellate Alexandrium ostenfeldii and their regulation by environmental factors (temperature and nutrients). This investigation aimed to determine whether genetic recombination of different strains is required for resting cyst formation and whether newly formed cysts are dormant. Field data (temperature and salinity) and sediment surface samples were collected from a site with recurrent blooms and germination and encystment experiments were conducted under controlled laboratory conditions. Results indicate a lack of seasonal germination pattern, set by an endogenous rhythm, as commonly found with other dinoflagellates from the Baltic Sea. Germination of quiescent cysts was triggered by temperatures exceeding 10°C and combined nutrient limitation of nitrogen and phosphorus or a drop in temperature from 16 to 10°C triggered encystment most efficiently. Genetic recombination was not mandatory for the formation of resting cysts, but supported higher numbers of resistant cysts and enhanced germination capacity after a resting period. Findings from this study confirm that A. ostenfeldii follows a generalist germination and cyst formation strategy, driven by strong seasonality, which may support its persistence and possibly expansion in marginal environments in the future, if higher temperatures facilitate a longer growth season.     

Female mice carrying a defective Alox15 gene are protected from experimental colitis via sustained maintenance of the intestinal epithelial barrier function

Lipoxygenases (ALOXs) are involved in the regulation of cellular redox homeostasis. They also have been implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators and play a role in the pathogenesis of inflammatory diseases, which constitute a major health challenge owing to increasing incidence and prevalence in all industrialized countries around the world. To explore the pathophysiological role of Alox15 (leukocyte-type 12-LOX) in mouse experimental colitis we tested the impact of systemic inactivation of the Alox15 gene on the extent of dextrane sulfate sodium (DSS) colitis. We found that in wildtype mice expression of the Alox15 gene was augmented during DSS-colitis while expression of other Alox genes (Alox5, Alox15b) was hardly altered. Systemic Alox15 (leukocyte-type 12-LOX) deficiency induced less severe colitis symptoms and suppressed in vivo formation of 12-hydroxyeicosatetraenoic acid (12-HETE), the major Alox15 (leukocyte-type 12-LOX) product in mice. These alterations were paralleled by reduced expression of pro-inflammatory gene products, by sustained expression of the zonula occludens protein 1 (ZO-1) and by a less impaired intestinal epithelial barrier function. These results are consistent with in vitro incubations of colon epithelial cells, in which addition of 12S-HETE compromised enantioselectively transepithelial electric resistance. Consistent with these data transgenic overexpression of human ALOX15 intensified the inflammatory symptoms. In summary, our results indicate that systemic Alox15 (leukocyte-type 12-LOX) deficiency protects mice from DSS-colitis. Since exogenous 12-HETE compromises the expression of the tight junction protein ZO-1 the protective effect has been related to a less pronounced impairment of the intestinal epithelial barrier function.