Genetic depletion of Soat2 diminishes hepatic steatosis via genes regulating de novo lipogenesis and by GLUT2 protein in female mice.

Depletion of the cholesterol esterifying enzyme acyl-Coenzyme A: cholesterol acyltransferase 2 (ACAT2, encoded by Soat2) protects mice from atherosclerosis, diet-induced hypercholesterolemia, and hepatic steatosis when fed high-cholesterol diet. The glucose transporter 2 (GLUT2) represents the main gate of glucose uptake by the liver. Lipid synthesis from glucose (de novo lipogenesis; DNL) plays a pivotal role in the development of hepatic steatosis. Inhibition of DNL is a successful approach to reverse hepatic steatosis, as shown by different studies in mice and humans. Here we aimed to investigate whether depletion of Soat2 per se can reduce hepatic steatosis, also in the presence of very low levels of cholesterol in the diet, and the underlying mechanisms. Female Soat2-/- and wild type mice were either fed high-fat or high-carbohydrate diet and both contained <0.05% (w/w) cholesterol. Analysis in serum, liver, muscles and adipose tissues were performed. We found Soat2-/- mice fed high-fat, low-cholesterol diet to have less hepatic steatosis, decreased expression of genes involved in DNL and lower hepatic GLUT2. Similar findings were found in Soat2-/- mice fed high-carbohydrate, low-cholesterol diet. CONCLUSION: Depletion of Soat2 reduces hepatic steatosis independently of the presence of high levels of cholesterol in the diet. Our study provides a link between hepatic cholesterol esterification, DNL, and GLUT2.

Feeding efficiency of planktivores under disturbance, the effect of water colour, predation threat and shoal composition.

The consumption of phantom midge Chaoborus flavicans larvae by Perca fluviatilis showed clear response to water colour, predation threat and shoal composition with the most significant negative effect for water colour. In the case of Rutilus rutilus, no similar combined response was observed and the total prey consumption was significantly negatively affected by predation threat of Esox lucius. The results suggest that differences in life-history traits may result in disparity in species-specific responses to disturbance.

Bioanalysis in clinical development of tasquinimod using liquid chromatography/tandem mass spectrometry.

Tasquinimod (ABR-215050) is an oral drug in clinical development for treatment of patients with castrate resistant prostate cancer. This paper describes a method for the determination of tasquinimod in human plasma. The method is based on liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) using stable isotope labeled tasquinimod as internal standard (IS). The plasma samples were processed by protein precipitation using acidic acetonitrile containing the IS. The precipitated samples were centrifuged and the supernatant was injected directly into the LC-MS/MS system. Chromatographic separation was performed on a reversed phase column using fast gradient elution, with a total run cycle time of 4 min. The method was validated with respect to accuracy, precision, dynamic range, lower limit of quantification, selectivity and robustness. Furthermore, the stability of tasquinimod in spiked plasma, in processed extracts and in incurred samples was thoroughly studied. The method was validated in the range of 1.0-2400 nmol/L, defining the lower and upper limits of quantification. The repeatability, reproducibility and overall bias were 1.5-7.1%, 3.5-7.4%, and 1.3-4.7%, respectively, in the range of 1-2000 nmol/L. Excellent selectivity was demonstrated in the validation, as well as in study samples from both healthy volunteers and cancer patients. Robustness was demonstrated by the calculated carry-over as low as 0.06%, and by an incurred sample reproducibility (ISR) experiment where 97% of the reanalyzed samples fulfilled the acceptance criteria of 20% deviation from initial analysis result. Also, tasquinimod was found to be stable in all investigated matrices, including in incurred samples. In an incurred sample stability (ISS) investigation, tasquinimod was demonstrated to be stable for 24 months, and 97% of the reanalyzed samples were within 20% from the initial analysis result. In conclusion, the method was demonstrated to be accurate, precise, robust and reliable for the determination of tasquinimod. The method was successfully used in several clinical studies for the support of pharmacokinetic and pharmacodynamic evaluations.

Enhanced production of 24S-hydroxycholesterol is not sufficient to drive liver X receptor target genes in vivo.

BACKGROUND: Oxysterols such as 24S-hydroxycholesterol (OHC) and 27-OHC are intermediates of cholesterol excretion pathways. In addition, they are putative endogenous agonists of the liver X receptor (LXR) class of nuclear hormone receptors and are thought to be important mediators of cholesterol-dependent gene regulation. 24S-OHC is one of the most efficient endogenous LXR agonists known and is present in the brain and in the circulation at relatively high levels. OBJECTIVES: To explore the regulatory importance of 24S-OHC in vivo. DESIGN: We developed a transgenic mouse model in which human cholesterol 24-hydroxylase, the enzyme responsible for the formation of 24S-OHC, was expressed under the control of a promoter derived from the ß-actin gene. RESULTS: Both male and female transgenic mice had elevated levels of cerebral, plasma, biliary and faecal 24S-OHC. According to the faecal excretion results, production of 24S-OHC was increased four- to sevenfold. Gene expression profiling revealed that the elevated production of 24S-OHC did not result in the anticipated activation of LXR target genes in the brain or liver. CONCLUSION: In spite of the fact that 24S-OHC is a highly effective agonist of LXRs in vitro, it is not a critical activator of target genes to this nuclear receptor in vivo, either in the brain or in the liver.

Diet shifts and food selection of perch Perca fluviatilis and roach Rutilus rutilus in humic lakes of varying water colour.

The field data from four humic lakes suggested that water colour may have both direct and indirect effects on inter- and intra-specific interactions of perch Perca fluviatilis and roach Rutilus rutilus. The results agree with suggestions that, compared with R. rutilus, P. fluviatilis may be an inferior forager on zooplankton in highly coloured water. As an indirect effect, water colour decreases the coverage of macrophytes and limits suitable littoral habitats, benefiting R. rutilus over P. fluviatilis. Perca fluviatilis benefiting from complex habitats does not have the advantage in macrophyte-poor highly coloured water.

Environmental factors regulate the effects of roach Rutilus rutilus and pike Esox lucius on perch Perca fluviatilis populations in small boreal forest lakes.

In this study of 18 small boreal forest lakes, the effects of abiotic and biotic factors (roach Rutilus rutilus and pike Esox lucius) on various population variables of perch Perca fluviatilis were examined. As a single variable, the gillnet catch per unit effort (CPUE) of R. rutilus was negatively related to the mean mass of small (< 200 mm) and the growth rate of young (1-2 years) P. fluviatilis. The mean mass of large (> or = 200 mm) P. fluviatilis was the highest at intermediate CPUE of R. rutilus. Redundancy analysis including environmental factors and P. fluviatilis population variables suggested that 'predation-productivity-humus' gradient affected P. fluviatilis populations by decreasing the CPUE and mean mass of small individuals but increasing these variables of large individuals. The CPUE of R. rutilus and lake area had a negative effect on small and a positive effect on large P. fluviatilis growth rate. In small boreal forest lakes, P. fluviatilis populations are affected by the partially opposite forces of competition by R. rutilus and predation by E. lucius, and the intensity of these interactions is regulated by several environmental factors.

Use of a fluorescently labeled poly-caspase inhibitor for in vivo detection of apoptosis related to vascular-targeting agent arsenic trioxide for cancer therapy.

Arsenic trioxide (ATO, Trisenox) is a potent anti-vascular agent and significantly enhances hyperthermia and radiation response. To understand the mechanism of the anti-tumor effect in vivo we imaged the binding of a fluorescently-labeled poly-caspase inhibitor (FLIVO) in real time before and 3 h or 24 h after injection of 8 mg/kg ATO. FSaII tumors were grown in dorsal skin-fold window chambers or on the rear limb and we observed substantial poly-caspase binding associated with vascular damage induced by ATO treatment at 3 and 24 h after ATO injection. Flow cytometric analysis of cells dissociated from the imaged tumor confirmed cellular uptake and binding of the FLIVO probe. Apoptosis appears to be a major mode of cell death induced by ATO in the tumor and the use of fluorescently tagged caspase inhibitors to assess cell death in live animals appears feasible to monitor and/or confirm anti-tumor effects of therapy.

Cholestatic liver disease in adults may be due to an inherited defect in bile acid biosynthesis.

BACKGROUND: An increasing number of treatable inborn errors of bile acid synthesis have been described, primarily in infants with severe cholestatic liver disease. RESULTS: The present patient, whose two older siblings had died from progressive cholestatic liver disease, developed neonatal cholestasis and rickets but recovered during the childhood years and follow-up was terminated at 12 years of age. The patient presented again at 26 years of age with jaundice and pathological liver function tests. This was normalized upon treatment with ursodeoxycholic acid. Electrospray mass spectrometry of urine showed predominance of unsaturated bile acids, characteristic of 3beta-hydroxy-Delta5-C27-steroid dehydrogenase/isomerase (HSD3B7) deficiency. The activity of HSD3B7 in cultured fibroblasts was less than 5% of normal. A single homozygous mutation was found in exon 4 leading to an amino acid exchange (S162R) and loss of enzyme activity. CONCLUSION: This case illustrates that infants with an inherited absence of HSD3B7 may survive the neonatal period of life and childhood without treatment with bile acids. A low level of sulphation of the abnormal trihydroxy bile acid formed as a result of enzyme deficiency may be of importance for survival. The possibility that liver disease presenting in the adult may be due to a mutation in the HSD3B7 gene should be considered, especially in cases with familial occurrence of liver disease and earlier periods of liver dysfunction.

Unique patient with cerebrotendinous xanthomatosis. Evidence for presence of a defect in a gene that is not identical to sterol 27-hydroxylase.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder believed to be exclusively caused by mutations in the CYP27A1 gene coding for the enzyme sterol 27-hydroxylase. Common findings in CTX are tendon xanthomas, cataracts and progressive neurological dysfunction. Here, we characterize an adult female patient with tendon xanthomas and classic biochemical findings of CTX (i.e. high levels of bile alcohols and cholestanol and extremely low levels of 27-hydroxycholesterol in plasma). Additionally, sterol 27-hydroxylase activity in cultured monocyte-derived macrophages from this patient was <5% of normal. Sequencing the CYP27A1 gene uncovered that the patient is heterozygous for two previously undescribed base substitutions in exon 8, C478A and C479A, which are expected to affect the haeme-binding domain of the enzyme. When expressed in HEK293 cells, the corresponding protein had only 8% of normal enzymatic activity. No other mutation was found in the open reading frame of the CYP27A1 gene, intron-exon boundaries or in the 5'-untranslated region up to 5000 bp distal to the translational start site. Sequencing mRNA isolated from leucocytes from the patient revealed a 1 : 1 ratio of mutated and nonmutated species, with total mRNA levels that were not significantly different from the controls. It is concluded that the patient is heterozygous for two mutations affecting one allele of the CYP27A1 gene and with at least one additional yet undefined gene that is of critical importance for the activity of sterol 27-hydroxylase.

Leptin increases T-cell immune response in birds.

Leptin is a 16 kDa protein hormone that besides being a satiety factor also functions as a pleiotropic molecule regulating endocrine and immune functions. The aim of this study was to investigate the role of leptin on mitogen stimulated T-lymphocyte proliferation in birds. In the first experiment (in vitro), peripheral blood was collected from turkeys and lymphocytes were isolated from samples. Recombinant chicken leptin was added at concentrations of 0, 10(-9), 10(-8), 10(-7), and 10(-6) M prior to mitogen stimulation with Concavalin A. BrdU incorporation allowed us to detect T-cell proliferation using intracellular labeling of nucleic acids. Mitogen activation induced with Concavalin A caused a proliferation of T-cells that was positively correlated with the concentration of leptin. In the second experiment (in vivo), asian blue quail were fitted with osmotic pumps releasing leptin and injected with phytohemagglutinin (PHA) in their wing-webs before, during, and after leptin administration. The response to mitogen was greater in leptin treated birds during the leptin administration, but not before or after. These findings demonstrate that leptin enhances mitogen stimulated T-cell proliferation in birds. The results correspond with previous reports on mammals.

Cerebrotendinous xanthomatosis: molecular characterization of two Scandinavian sisters.

Cerebrotendinous xanthomatosis (CTX) is a hereditary disorder, which is inherited as an autosomally recessive disease, causing production of cholesterol and cholestanol xanthomas and mental retardation. The disease is caused by mutations in the gene for sterol 27-hydroxylase (CYP27A1). The only CTX patients diagnosed in Scandinavia are two Norwegian sisters from a consanguineous marriage. Here we have characterized the mutation and its functional consequences for the enzyme. Analysis of genomic DNA from cultured fibroblasts identified a base exchange C > T in position 1441, causing arginine at amino acid position 441 to be replaced by tryptophan. The same mutation was introduced by mutagenesis in the complimentary DNA (cDNA) for CYP27, ligated into the expression vector pcDNA4/HisMax and transfected into HEK293 cells. The mutated enzyme had less than 5% of the enzyme activity compared with the native enzyme. No abnormal catalytic products could be identified in the cell culture medium. Probably the mutation affects the haem binding within the holoenzyme. The mutation has also previously been reported in a Japanese family. This is the second example of a CTX-causing mutation that has been recognized in more than one population.

Structure and chromosomal assignment of the sterol 12alpha-hydroxylase gene (CYP8B1) in human and mouse: eukaryotic cytochrome P-450 gene devoid of introns.

Sterol 12alpha-hydroxylase (CYP8B1) is a hepatic cytochrome P-450 that controls the ratio of cholic acid over chenodeoxycholic acid in bile and thus controls the solubility of cholesterol. Both the human and the mouse CYP8B1 complementary DNA and gene were cloned and structurally characterized. Surprisingly, the genomic DNA from both species was found to lack introns. The major transcript of the human gene was estimated to be 3950 bp, and the putative promoter region was estimated to be at least 1360 bp. The murine structural gene was found to span approximately 3 kb. By using FISH and radiation hybrid mapping techniques, the human CYP8B1 gene was located to chromosome 3p21.3-p22, whereas FISH mapped the murine counterpart to chromosome 9qF4, a region that is homologous to the third human chromosome. The results from the chromosome mapping and Southern blotting indicated that the gene is present in a single copy. Transcription of the mouse and human CYP8B1 genes was initiated from a position situated 51 and 35 bases, respectively, downstream of a consensus TATA box. A homology of 21% for the promoter regions of mouse and human may indicate differences in transcriptional regulation. Although a potent induction of CYP8B1 mRNA was observed upon starvation of mice, the mechanism behind this effect was not revealed by analysis of the promoter for potential cis-acting elements. In the human promoter, several possible cis-acting regions were identified but none of them could be directly related to bile acid metabolism. After transfection of COS cells with the human coding region, mRNA and enzymatic activity for the 12alpha-hydroxylase were identified. This is the first mammalian cytochrome P-450 gene reported to lack introns. The importance of this structural feature for evolution and gene regulation is discussed.

Simple, hybrid deep venous thrombosis/pulmonary embolus prophylaxis after total hip arthroplasty.

A 7.1% deep venous thrombosis rate followed total hip arthroplasty in 56 patients using a hybrid prophylactic regimen against deep venous thrombosis and pulmonary embolus. There were no bleeding complications, no symptomatic pulmonary emboli, and no unexplained deaths. The regimen consisted of an initial loading dose of warfarin, usually 10 mg, the night of surgery followed by 2.5 mg/day for 3 weeks, with dosage adjustments only in cases of over-anticoagulation. This regimen was combined with elevated sling suspension of the operative leg, bilateral pedal intermittent pneumatic compression devices, and early mobilization. This prophylactic regimen is simple, inexpensive, efficacious, and compatible with an early hospital discharge.

Molecular cloning and expression of rabbit sterol 12alpha-hydroxylase.

Sterol 12alpha-hydroxylase is an important enzyme in bile acid biosynthesis, responsible for the balance between formation of cholic acid and chenodeoxycholic acid. The enzyme has been purified to apparent homogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, K., and Coon, M. J. (1992) J. Biol. Chem. 267, 21319-21323), and we here describe the cloning and sequencing of a cDNA coding for this enzyme. After tryptic digestion of purified protein in a polyacrylamide gel, eight different peptides were isolated and sequenced. Using oligonucleotides deduced from the amino acid sequences, clones were isolated from a rabbit liver cDNA library. In addition to several overlapping clones, one full-length clone was obtained that coded for a polypeptide of 500 amino acids, corresponding to a molecular mass of 57 kDa. All of the eight peptides and the reported NH2-terminal amino acid sequence were matched against the sequence. The peptide sequence showed a 39% similarity with human prostacyclin synthase (CYP8) and 31% similarity with the rate-limiting enzyme in over-all synthesis of bile acids, the cholesterol 7alpha-hydroxylase (CYP7) of the rabbit. The similarity with most other sterol cytochrome P-450 hydroxylases was less. Thus, this species of cytochrome P-450 should belong to a group of its own, here denoted CYP12. Transfection of COS cells with the coding part of the cDNA resulted in a significant expression of sterol 12alpha-hydroxylase activity toward 7alpha-hydroxy-4-cholesten-3-one. Northern blotting showed that the enzyme was exclusively expressed in the liver. The major mRNA fraction in rabbit liver had a size of approximately 2.9 kilobases, and those found in rat and human liver were about 2.5 and 4.5 kilobases, respectively. Fasting of rats and mice led to a severalfold increase in both enzyme activity and mRNA levels. In contrast, starvation of rabbits had little or no stimulatory effect on enzyme activity and mRNA levels.