Characterization of xenobiotic-metabolizing cytochrome P450 (CYP) forms in ringed and grey seals from the Baltic Sea and reference sites

Earlier studies have shown that members of the cytochrome P4501 (CYP1) enzyme family are constitutively expressed, and are elevated in the livers of ringed seals (Phoca hispida) and grey seals (Halichoerus grypus) living in the heavily polluted Baltic Sea. In this study, we compared the expression profiles of several additional CYP enzymes in the liver and extrahepatic tissues of Baltic ringed and grey seals with the corresponding CYP expression in seals from relatively unpolluted waters. We used marker enzyme activity levels, diagnostic inhibitors and immunoblot analysis to assess members of the CYP2A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A sub-families. Coumarin 7-hydroxylation (COH), a marker of CYP2A activity, was high in the liver and the lungs of all the studied seal populations. The presence of a putative CYP2A form in these seals was further supported by the strong inhibition of COH activity by a chemical inhibitor and by an anti-CYP2A5 antibody. However, antibodies to human and rodent CYP2B, CYP2C and CYP2E forms did not recognize any proteins in these seal species. Dextromethorphan O-demethylation (marker for CYP2D activity) and chlorzoxazone 6-hydroxylation (marker for CYP2E activity) were measurable in the livers of all the seals we studied. Both activities were elevated in the Baltic seal populations, showed a strong positive correlation with CYP1A activity and were at least partly inhibited by a typical CYP1A inhibitor, alpha-naphthoflavone. Further studies are needed to determine the presence and characteristics of CYP2D and CYP2E enzymes in ringed and grey seals. Testosterone 6beta-hydroxylation, a CYP3A marker, showed a relatively high level of activity in the livers of both seal species and was potently inhibited by ketoconazole, a CYP3A-selective inhibitor. The putative CYP3A activity showed an opposing geographical trend to that of CYP2D and CYP2E, since it was elevated in the control area. CYP3A protein levels, revealed by immunoblotting, showed a positive correlation with testosterone 6beta-hydroxylation. We conclude tentatively that CYP2A- and CYP3A-like enzymes are expressed in ringed and grey seals, but that CYP2B- and CYP2C-like ones are not. Further information on the individual contaminant profile is needed before any conclusions can be drawn on a possible connection between the varying CYP expressions and the contaminant load.     

Culturing precision-cut human prostate slices as an in vitro model of prostate pathobiology

Due to the complex morphology of the prostate, it was hypothesized that precision-cut tissue slices from human prostate would provide a unique in vitro model. Precision-cut slices were generated from zones of human prostate and their viability was assessed under conditions of different media for up to 120 h. Slices were also exposed to several concentrations of CdCl₂, which was used as a model toxicant. Maintenance of both stromal and epithelial cells was noted; however, there was a gradual loss of luminal epithelial cells when the medium was not supplemented with dihydrotestosterone (DHT). Minimal leakage of lactate dehydrogenase occurred throughout the incubation. Prostate-specific antigen (PSA) was detected in the medium at all time points, although the rates of secretion fell over time. There was a loss of PSA-positive cells when the medium was not supplemented with DHT, consistent with a loss of luminal cells, whereas PSA-positive cells were maintained in the DHT-supplemented media. A proliferation of basal cells was observed in the presence of media containing 10% fetal bovine serum. Exposure of slices to CdCl₂ demonstrated a dose-response effect ranging from proliferation to complete cellular necrosis. Given the retention of stromal-epithelial interactions and the use of acquired human tissue, prostate slices represent a unique in vitro model for investigating human prostate pathobiology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss

The infection route of Flavobacterium psychrophilum into rainbow trout Oncorhynchus mykiss was studied using bath and cohabitation challenges as well as oral challenge with live feed as a vector. Additionally, the number of bacterial cells shed by infected fish into the surrounding water was determined in the cohabitation experiment and in challenge experiments at 3 different water temperatures. The experiments showed that skin and skin mucus abrasion dramatically enhanced the invasion of F. psychrophilum into the affected fish in bath and cohabitation challenges. Disruption of the skin is discussed as an important invasion route for F. psychrophilum into the fish. The shedding rate of F. psychrophilum by infected fish was associated with water temperature and the mortality of the infected fish. High numbers of F. psychrophilum cells were released into the water by dead rainbow trout during a long time period compared to the numbers of cells shed by live fish. The results emphasise the importance of removing dead and moribund fish from rearing tanks in order to diminish the infection pressure against uninfected fish in commercial fish farms. In immunohistochemical examinations of organs and tissues of orally infected fish, F. psychrophilum cells were detected in only 1 fish out of 31 studied. Mortality of the orally challenged fish was not observed in the experiment.     

Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilic adhesion

Intercellular adhesion molecule-5 (ICAM-5) is a dendritically polarized membrane glycoprotein in telencephalic neurons, which shows heterophilic binding to leukocyte beta(2)-integrins. Here, we show that the human ICAM-5 protein interacts in a homophilic manner through the binding of the immunoglobulin domain 1 to domains 4-5. Surface coated ICAM-5-Fc promoted dendritic outgrowth and arborization of ICAM- 5-expressing hippocampal neurons. During dendritogenesis in developing rat brain, ICAM-5 was in monomer form, whereas in mature neurons it migrated as a high molecular weight complex. The findings indicate that its homophilic binding activity was regulated by nonmonomer/monomer transition. Thus, ICAM-5 displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes.     

2D and Trap‐Assisted 2D Langevin Recombination in Polymer:Fullerene Blends

The impact of trapping on the recombination dynamics in polymer:fullerene blends is clarified using the highly ordered bulk heterojunction (BHJ) blend poly[2,5‐bis(3‐tetradecylthiophen‐2‐yl)thieno[3,2‐b]thiophene] (PBTTT) and [6,6]‐phenyl‐C61‐butyric acid methyl ester (PCBM) at different weight ratios as a model system. The recombination dynamics are determined using both transient charge extraction and steady‐state techniques. The results show that both the decay of photogenerated charge and the light ideality factor at a polymer:fullerene weight ratio of 1:4 are fully consistent with 2D Langevin recombination; in the 1:1 case the recombination is seen to be affected by electron trapping. The theory of 2D Langevin recombination is extended to the case with high trap density in agreement with the observations in the 1:1 case. The recombination capture coefficients are derived both for trap‐assisted and band‐to‐band recombination and it can be seen that anisotropic charge transport reduces the capture coefficients in both cases resulting in a reduced overall recombination.     

Model-Based Quantification of the Systemic Interplay between Glucose and Fatty Acids in the Postprandial State

In metabolic diseases such as Type 2 Diabetes and Non-Alcoholic Fatty Liver Disease, the systemic regulation of postprandial metabolite concentrations is disturbed. To understand this dysregulation, a quantitative and temporal understanding of systemic postprandial metabolite handling is needed. Of particular interest is the intertwined regulation of glucose and non-esterified fatty acids (NEFA), due to the association between disturbed NEFA metabolism and insulin resistance. However, postprandial glucose metabolism is characterized by a dynamic interplay of simultaneously responding regulatory mechanisms, which have proven difficult to measure directly. Therefore, we propose a mathematical modelling approach to untangle the systemic interplay between glucose and NEFA in the postprandial period. The developed model integrates data of both the perturbation of glucose metabolism by NEFA as measured under clamp conditions, and postprandial time-series of glucose, insulin, and NEFA. The model can describe independent data not used for fitting, and perturbations of NEFA metabolism result in an increased insulin, but not glucose, response, demonstrating that glucose homeostasis is maintained. Finally, the model is used to show that NEFA may mediate up to 30–45% of the postprandial increase in insulin-dependent glucose uptake at two hours after a glucose meal. In conclusion, the presented model can quantify the systemic interactions of glucose and NEFA in the postprandial state, and may therefore provide a new method to evaluate the disturbance of this interplay in metabolic disease.     

Structural studies of tri-functional human GART

Human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional GART, contains three enzymatic activities in a single polypeptide chain. We have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. Structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. We also report small angle X-ray scattering models for the full-length protein. These models are consistent with the enzyme forming a dimer through the middle domain. The protein has an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments. This resilient seesaw shape may facilitate internal substrate/product transfer or forwarding to other enzymes in the pathway.     

Decorin in Human Colon Cancer: Localization In Vivo and Effect on Cancer Cell Behavior In Vitro

Decorin is generally recognized as a tumor suppressing molecule. Nevertheless, although decorin has been shown to be differentially expressed in malignant tissues, it has often remained unclear whether, in addition to non-malignant stromal cells, cancer cells also express it. Here, we first used two publicly available databases to analyze the current information about decorin expression and immunoreactivity in normal and malignant human colorectal tissue samples. The analyses demonstrated that decorin expression and immunoreactivity may vary in cancer cells of human colorectal tissues. Therefore, we next examined decorin expression in normal, premalignant and malignant human colorectal tissues in more detail using both in situ hybridization and immunohistochemistry for decorin. Our results invariably demonstrate that malignant cells within human colorectal cancer tissues are devoid of both decorin mRNA and immunoreactivity. Identical results were obtained for cells of neuroendocrine tumors of human colon. Using RT-qPCR, we showed that human colon cancer cell lines are also decorin negative, in accordance with the above in vivo results. Finally, we demonstrate that decorin transduction of human colon cancer cell lines causes a significant reduction in their colony forming capability. Thus, strategies to develop decorin-based adjuvant therapies for human colorectal malignancies are highly rational.     

Quantitative Changes in Gimap3 and Gimap5 Expression Modify Mitochondrial DNA Segregation in Mice

Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.