Markov random field model for segmenting large populations of lipid vesicles from micrographs

Giant unilamellar lipid vesicles, artificial replacements for cell membranes, are a promising tool for in vitro assessment of interactions between products of nanotechnologies and biological membranes. However, the effect of nanoparticles can not be derived from observations on a single specimen, vesicle populations should be observed instead. We propose an adaptation of the Markov random field image segmentation model which allows detection and segmentation of numerous vesicles in micrographs. The reliability of this model with different lighting, blur, and noise characteristics of micrographs is examined and discussed. Moreover, the automatic segmentation is tested on micrographs with thousands of vesicles and the result is compared to that of manual segmentation. The segmentation step presented is part of a methodology we are developing for bio-nano interaction assessment studies on lipid vesicles.     

Lanosterol metabolism and sterol regulatory element binding protein (SREBP) expression in male germ cell maturation

Expression of genes involved in cholesterol biosynthesis in male germ cells is insensitive to the negative cholesterol feedback regulation, in contrast to cholesterol level-sensitive/sterol regulatory element binding protein (SREBP)-dependent gene regulation in somatic cells. The role of sterol regulatory element binding proteins in spermatogenic cells was an enigma until recently, when a soluble, 55 kDa cholesterol-insensitive form of SREBP2 (SREBP2gc) was discovered [Mol. Cell. Endocrinol. 22 (2002) 8478], being translated from a germ cell-specific SREBP2 mRNA. Our RT-PCR results also show that SREBP2 as well as SREBP1c mRNAs are detectable in prepubertal and postpubertal male germ cells while SREBP1a is not detected. Surprisingly, three SREBP2 immunoreactive proteins (72, 63 and 55 kDa), that are not present in mouse liver nuclei, reside in testis nuclei of prepubertal and adult mice. The 55 kDa protein is likely SREBP2gc, the other two isoforms are novel. HPLC measurements in liver and testes of fasted prepubertal and postpubertal mice showed no significant difference in cholesterol level. However, FF-MAS and lanosterol/testis-meiosis activating sterol (T-MAS) intermediates that are detectable mainly in testes, increase in fasted postpubertal mice which coincides well with the elevated level of 68 kDa SREBP2. Similar to SREBP2gc, the two novel SREBP2 immunoreactive proteins seem to be insensitive to the level of cholesterol.     

Microfacies analysis and palaeoenvironmental interpretation of Lower Oligocene, shallow-water carbonates (Gornji Grad Beds, Slovenia)

Summary The microfacies and palaeoenvironment of Lower Oligocene carbonates of the Gornji Gradbeds from Slovenia are investigated. These beds form part of a transgressive succession overlying both terrigenous sediments (sand-stones and conglomerates) and marine carbonates of Eocene age as well as transgressing directly over Triassic lime-stones. They are followed by foraminiferal rich marls. The carbonates were investigated using multivariate statistical techniques on point counts of thin sections. They are dominated by poorly sorted biogenic rudstones with pack-/wackestone matrix; pack- and grainstones are subordinate. The biogenic components of the carbonates are dominated by coralline red algae (9 genera with 11 species), corals, small benthic, large benthic, and encrusting foraminifera as well as bivalves. Gastropods, bryozoans, brachiopods, echinoderms, serpulids, and green algae are subordinate. The well preserved components allow details pertaining to taxonomy, growth-forms and taphonomic features to be observed. The following carbonate facies are distinguished: 1) nummulitic, 2) bivalve, 3) foraminiferal—coralline algal, 4) grainstone, 5) coralline alga, 6) coralline algal—coral, and 7) coral facies. All the carbonate facies represent fully marine conditions within the photic zone. They are interpreted with respect to substrate composition and stability, water turbulence, terrigenous input and light.     

Nuclear morphometry and AgNOR quantification: computerized image analysis on ovarian mucinous tumor imprints

OBJECTIVE: To determine the morphometric characteristics of nuclei and silver-stained nucleolar organizer regions (AgNORs) on cytologic imprints and their value in differential cytodiagnosis of benign, atypical proliferative (borderline) and malignant ovarian mucinous tumors. STUDY DESIGN: Forty-six mucinous ovarian tumor imprints (16 benign, 15 borderline, 15 malignant), were analyzed. Nuclear area, outline, "shape factor" and "form factor" were measured on Papanicolaou-stained smears. AgNOR quantification included 7 variables related to the number and area of single, cluster, total and relative AgNOR content per nucleus and the size distribution of AgNORs. RESULTS: Nuclear area and shape factor allowed distinguishing borderline and malignant tumors. The nuclear area in benign tumors was larger than that in borderline tumors; malignant tumors had the highest values. Single and cluster AgNORs were statistically significantly different in borderline tumors compared with malignant tumors, except for the cluster AgNOR area. The total AgNOR area, number and relative area increased from benign through malignant tumors, with statistically significant differences among all groups. By AgNOR size distribution, small AgNORs discriminate malignant from borderline and benign tumors. CONCLUSION: Combining nuclear morphometry and AgNOR analysis on cytologic imprints could be a diagnostically useful method in the assessment of mucinous ovarian tumors.     

CREM modulates the circadian expression of CYP51, HMGCR and cholesterogenesis in the liver

We show for the first time that isoforms of the cAMP response element modulator Crem, regulate the circadian expression of Cyp51 and other cholesterogenic genes in the mouse liver. In the wild type mice the expression of Cyp51, Hmgs, Fpps, and Sqs is minimal between CT12 and CT16 and peaks between CT20 and CT24. Cyp51, Fpps, and Sqs lost the circadian behavior in Crem−/− livers while Hmgcr is phase advanced from CT20 to CT12. This coincides with a phase advance of lathosterol/cholesterol ratio, as detected by GC-MS. Overexpression of CREMτ and ICER has little effect on the Hmgcr proximal promoter while they influence expression from the CYP51 promoter. Our data indicate that Crem-dependent regulation of Cyp51 in the liver results in circadian expression of this gene. We propose that cAMP signaling might generally be involved in the circadian regulation of cholesterol synthesis on the periphery.     

Expression of microsomal lanosterol 14alpha-demethylase (CYP51) in an engineered soluble monomeric form

We prepared a soluble monomeric form of bovine cytochrome P450 lanosterol 14α-demethylase (CYP51), which in mammals is a ubiquitously expressed membrane protein of the endoplasmic reticulum. We constructed two variants of bovine CYP51 (bCYP51) with different truncations and modifications in their N-terminal membrane-spanning domains. Both of these were expressed in Escherichia coli at levels of 500 nmol/l. The protein variants were purified and tested for the solubility in the absence of detergent. Variant bCYP51-d1 exhibited ∼10-fold better solubility over variant bCYT51-d2. The bCYP51-d1 eluted as a single peak in size-exclusion chromatography, corresponding to its monomeric form. The activity of bCYP51-d1 is similar to that of recombinant human CYP51 with a non-truncated membrane-spanning region. High solubility and low tendency to non-specific oligomer formation make bCYP51-d1 a promising candidate for successful crystallization, which may finally allow the structural determination of this important mammalian enzyme.     

Anaerobic bacteria in the gut of terrestrial isopod crustaceanPorcellio scaber

Anaerobic bacteria from Porcellio scaber hindgut were identified and, subsequently, isolated using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determined by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts of P. scaber. Sequences from bacteria related to obligate anaerobic bacteria from genera Bacteroides and Enterococcus were retrieved, as well as sequences from 'A1 subcluster' of the wall-less mollicutes. Bacteria from the genus Desulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results indicate the presence of resident anaerobic bacteria in the gut of P. scaber, in spite of apparently unsuitable, i.e. predominantly oxic, conditions.     

Cytochrome P450s in the synthesis of cholesterol and bile acids--from mouse models to human diseases

The present review describes the transgenic mouse models that have been designed to evaluate the functions of the cytochrome P450s involved in cholesterol and bile acid synthesis, as well as their link with disease. The knockout of cholesterogenic Cyp51 is embrionally lethal, with symptoms of Antley-Bixler syndrome occurring in mice, whereas the evidence for this association is conflicting in humans. Disruption of Cyp7a1 from classic bile acid synthesis in mice leads to either increased postnatal death or a milder phenotype with elevated serum cholesterol. The latter is similar to the case in humans, where CYP7A1 mutations associate with high plasma low-density lipoprotein and hepatic cholesterol content, as well as deficient bile acid excretion. Disruption of Cyp8b1 from an alternative bile acid pathway results in the absence of cholic acid and a reduced absorption of dietary lipids; however, the human CYP8B1 polymorphism fails to explain differences in bile acid composition. Unexpectedly, apparently normal Cyp27a1(-/-) mice still synthesize bile acids that originate from the compensatory pathway. In humans, CYP27A1 mutations cause cerebrotendinous xanthomatosis, suggesting that only mice can compensate for the loss of alternative bile acid synthesis. In line with this, Cyp7b1 knockouts are also apparently normal, whereas human CYP7B1 mutations lead to a congenital bile acid synthesis defect in children or spastic paraplegia in adults. Mouse knockouts of the brain-specific Cyp46a1 have reduced brain cholesterol excretion, whereas, in humans, CYP46A1 polymorphisms associate with cognitive impairment. At present, cytochrome P450 family 39 is poorly characterized. Despite important physiological differences between humans and mice, mouse models prove to be an invaluable tool for understanding the multifactorial facets of cholesterol and bile acid-related disorders.     

Prolonged feeding of terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) on TiO (2) nanoparicles. Absence of toxic effect

Nanoparticles of titanium dioxide are one of most widely used nanomaterials in different products in everyday use and in industry, but very little is known about their effects on non- target cells and tissues. Terrestrial isopods were exposed to food dosed with nano-TiO(2) to give final nominal concentration 1000 and 2000 μg TiO(2)/g dry weight of food. The effects of ingested nano-TiO(2) on the model invertebrate Porcellio scaber (Isopoda, Crustacea) after short-term (3 and 7 days) and prolonged (14 and 28 days) dietary exposure was assessed by conventional toxicity measures such as feeding rate, weight change and mortality. Cell membrane destabilization was also investigated. No severe toxicity effects were observed after 3, 7, 14 or 28 days of dietary exposure to nano-TiO(2), but some animals, particularly those exposed to lower concentrations of nanoparticles, had severely destabilized digestive cell membranes. It was concluded that strong destabilization of the cell membrane was sporadic, and neither concentration- nor time-related. Further research is needed to confirm this sporadic toxic effect of nanoparticles.