Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.     

The Common Ancestor Process Revisited

We consider the Moran model in continuous time with two types, mutation, and selection. We concentrate on the ancestral line and its stationary type distribution. Building on work by Fearnhead (J. Appl. Probab. 39 (2002), 38–54) and Taylor (Electron. J. Probab. 12 (2007), 808–847), we characterise this distribution via the fixation probability of the offspring of all individuals of favourable type (regardless of the offspring’s types). We concentrate on a finite population and stay with the resulting discrete setting all the way through. This way, we extend previous results and gain new insight into the underlying particle picture.     

A real-time PCR assay for DNA-methylation using methylation-specific blockers

DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.     

Variety‐specific Epidemiology of Cercospora beticola Sacc. and Consequences for Threshold‐based Timing of Fungicide Application in Sugar Beet

In Central Europe, fungicides to control leaf spot disease in sugar beet caused by Cercospora beticola are applied based on thresholds of disease incidence (DI, per cent of infected plants). As variety‐specific fungicide application was not analyzed to date, the epidemiology of C. beticola and its effect on white sugar yield (WSY) in varieties with different susceptibility were investigated at seven sites in Germany and Austria in 2004 and 2005. All varieties reached the summary thresholds 5 / 15 / 45% DI in all environments. Fitting a logistic growth curve to DI revealed significant differences among varieties. At high disease pressure, susceptible varieties reached a considerably higher disease severity (DS, per cent of infected leaf area) at harvest and a larger area under disease progress curve (AUDPC) than resistant varieties. Fitting a logistic growth curve to DS showed an increasing differentiation among varieties with time. The growth rate estimated based on the logistic growth curve was the only variable that performed equally well in differentiating varieties under low and high disease pressure. With increasing disease pressure, varieties differed considerably in WSY, but differences between susceptible and resistant varieties were significant only in some environments. The disease‐loss relation between AUDPC and relative WSY was variety‐specific. Resistant varieties had an approximately identical WSY with and without infection and compensated for negative infection effects even at higher AUDPC. Therefore, at high disease pressure, resistant varieties had a higher relative yield compared to susceptible ones. However, our results indicate that there is no need to develop variety‐specific thresholds, but resistant varieties reach the established thresholds later than susceptible ones. Consequently, the time of fungicide application can be delayed in resistant varieties. This will help to reduce the use of fungicides to the bare essentials as requested for the integrated crop protection management.     

Foetal hepatocyte transplantation in a vascularized AV-Loop transplantation model in the rat

The use of foetal liver cells (FLC) in the context of hepatic tissue engineering might permit efficient in vitro expansion and cryopreservation in a cell bank. A prerequisite for successful application of bioartificial liver tissue is sufficient initial vascularization. In this study, we evaluated the transplantation of fibrin gel-immobilized FLC in a vascularized arterio-veno-venous (AV)-loop model. FLC were isolated from embryonic/foetal (ED 16) rat livers and were enriched by using magnetic cell sorting (MACS). After cryopreservation, FLC were labelled by pkh-26. Cells were transplanted in a fibrin matrix into a subcutaneous chamber containing a microsurgically created AV-loop in the femoral region of the recipient rat. The chambers were explanted after 14 days. Subcutaneous implants without an AV-loop and cell-free implants served as controls. Fluorescence microscopy of the constructs was used to identify pkh-26⁺- donor cells. Characterization was performed by RT-PCR and immunhistology (IH) for CK-18 and CD31. Transplantation of FLC using the AV-loop permitted a neo -tissue formation in the fibrin matrix. A high-density vascularization was observed in the AV-loop constructs as shown by CD31 IH. Viable foetal donor cells were detected which expressed CK-18. FLC can be successfully used for heterotopic transplantation. Fibrin matrix permits rapid blood vessel ingrowth from the AV-loop and supports engraftment of FLC. It is therefore an appropriate environment for hepatocyte transplantation in combination with microsurgical vascularization strategies. Transplantation of fibrin gel-immobilized FLC may be a promising approach for the development of highly vascularized in vivo tissue-engineering-based liver support systems.     

Long-distance dispersal of a wolf, <Emphasis Type="Italic">Canis lupus</Emphasis>, in northwestern Europe

Several mammal species have recolonized their historical ranges across Europe during the last decades. In November 2012, a wolf-looking canid was found dead in Thy National Park (56° 56′ N, 8° 25′ E) in Jutland, Denmark. DNA from this individual and nine German wolves were genotyped using a genome-wide panel of 22,163 canine single nucleotide polymorphism (SNP) markers and compared to existing profiles based on the same marker panel obtained from northeastern Polish (n?=?13) wolves, domestic dogs (n?=?13) and known wolf-dog hybrids (n?=?4). The Thy canid was confirmed to be a wolf from the German-western Polish population, approximately 800 km to the southeast. Access to the German reference database on DNA profiles based on 13 autosomal microsatellites of German wolves made it possible to pinpoint the exact pack origin of the Thy wolf in Saxony, Germany. This was the first documented observation of a wolf in Denmark in 200 years and another example of long-distance dispersal of a carnivore.     

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.     

Studies on the intestine section to be sampled in broiler studies on precaecal amino acid digestibility

Measurements of precaecal amino acid digestibility with digesta sampled from slaughtered animals may be affected by the chosen length of the sampled section. The length needs standardization, therefore, when digestibility is understood to be a measure of feedstuff potential. It was our objective to study the change in the net disappearance of amino acids from the lower small intestine of broiler chicken. The section between Meckel's diverticulum and 2 cm anterior of the ileo-caeco-colonic junction was cut into three subsections of equal length: proximal, medial, and terminal. The contents of each subsection were pooled within the birds of each pen (12 in Experiment 1 and 10 in Experiment 2). TiO2 was used as an indigestible marker. Prior to digesta sampling, broilers had been fed the experimental diets for seven days. In Experiment 1, two diets with either soybean meal or a mix of soybean meal and peas as the main protein sources were used. Each diet was allocated to eight pens and feeding commenced on day 14 of age. Net disappearance was significantly affected by diet only in regard to aspartic acid and methionine. No significant interaction between diet and subsection occurred. Net disappearance was significantly affected by subsection for all amino acids. It ranged from 74-92% for individual amino acids without significant differences in the medial and terminal subsections. Net disappearance was, however, between 3% and 9% lower in the proximal subsection. In Experiment 2, diets contained soybean meal as the main protein source and were given to 18 pens from day 22 of age. Again, the effect of subsection on net disappearance was significant for all amino acids. Net disappearance was significantly lower in the proximal than in the middle subsection, and differences ranged from 5-10%. Significant differences in the net disappearance were also found for most of the amino acids between the middle and the terminal subsection ranging from 2-4%. In conclusion, when precaecal amino acid digestibility should be used as a measure for a protein source's potential, digesta sampling should not consider the proximal third of the section between Meckel's diverticulum and the end of the ileum.