A quantitative assay for insulin-expressing colony-forming progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay. To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 μL of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin βB, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony. In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.     

Effect of oat by-product antioxidants and vitamin E on the oxidative stability of pork from pigs fed diets supplemented with linseed oil

The aim of the experiment was to compare the antioxidative potential of an oat by-product with the effect of vitamin E on the oxidative stability of pork from pigs fed a diet enriched with linseed oil. Thirty-four crossbreed barrows were fed individually from 39 to 109 kg body weight (BW) on one of four diets: a control diet based on barley-triticale-soybean (Diet C), a diet containing an oat byproduct (Diet O), and the same diets supplemented with vitamin E (100 mg/kg diet) (Diets CE and OE, respectively). The oat by-product, comprising oat hulls and bran, was included at 10 and 20% in the grower and finisher diets, respectively. To Diets O and OE, refined rapeseed oil was added to equalise their energy content to Diets C and CE. Compared to Diets C and CE, the inclusion of the oat by-product in Diets O and OE increased the antioxidative capacity of water-soluble and lipid soluble compounds in these diets. Dietary treatment did not influence growth performance, slaughter value, longissimus dorsi (LD) muscle quality measured by nutrient contents, pH, drip loss or colour. Vitamin E supplementation increased the alpha-tocopherol concentration in serum and meat (p < 0.01), and decreased the formation of thiobarbituric acid reactive substances (TBARS) in the fresh and stored LD (p < 0.01). In addition, diets with the oat by-product increased serum alpha-tocopherol concentration (p < 0.01) and decreased the TBARS levels in the fresh and stored LD (p < 0.05), without increasing muscle alpha-tocopherol concentration. The obtained results indicate that the phenolic compounds present in oat by-products have a considerable antioxidant potential and a beneficial effect on the pig organism and oxidative stability of meat. However, dietary inclusion with the oat by-product was not as efficient as supplementation with vitamin E.     

Tracking of wisent–bison–yak mitochondrial evolution

One of the most informative sources which allow the drawing of far-reaching conclusions about the origins and phylogenetics of many species, including domestic animals and humans, is mitochondrial DNA (mtDNA). One of the important research targets should include the identification of similarities between wild and domestic species. The analysis involved the nucleotide sequences of mtDNA of wisent, auroch, bison, yak, bovine reference sequence (BRS) T3, T3a, T3b, T1, T1a, T1'2'3, T2, T3, T4, T5, Q, Q1, P, R, I1, and I2 bovine haplotypes. The non-coding D-loop regions were excluded from the evolutionary analysis and 15,419-bp coding sequences were used in the final dataset. Trees constructed on the basis of whole mitochondrial genomes or on total mtDNA coding sequences alignment were generally in agreement with previous studies on the Bovini tribe. American bison shows stronger maternal relationships to yak than to wisent. It seems that the isolation and divergence of wisent took place early, almost 2 to 1.6 million years ago. This appears to be compatible with the paleontological date, indicating Late Pleistocene speciation of Bison bonasus. The yak/bison mitochondrial transfer model is in agreement with our mutation analysis and phylogenetic tree. The bison/yak mutations were collected in the bison mitochondrial genome before the transfer. After the transfer, the parallel accumulation of unique mutations took place. According to our assessment, the transfer took place at about 700 ky. The characteristic feature of the wisent and bison evolution is the maintenance of mtDNA variability, despite the fact that both species underwent population bottlenecks. Our studies did not reveal any impact of these phenomena populations in the analyzed mitochondrial genomes.     

Specificity of Kirkman-Robbins hepatoma non-histone chromatin proteins: electrophoretic and immunological analyses

The specificity of Kirkman-Robbins hepatoma and hamster liver non-histone chromatin proteins has been studied by comparing polypeptide patterns in polyacrylamide gel electrophoresis and by their immunological activity in the complement fixation test. Non-histone proteins were separated from DNA with a polyethylene glycol-dextran mixture and fractionated by hydroxylapatite chromatography into three classes named NHCP1, NHCP2, and NHCP3. Electrophoretic analysis indicated that among the non-histone proteins of Kirkman-Robbins hepatoma and hamster liver differences mainly of a quantitative nature can be observed. However, the polypeptides with molecular weight 25 000, 31 000, 36 000, 73 000 in NHCP1; 20 000, 40 000 in NHCP2 and 20 000, 23 000, 32 000, 38 000, 44 000, 75 000, 80 000 in NHCP3 were found to be specific for hepatoma chromatin. Application of antibodies against NHCP1, NHCP2 and dehistonized chromatin of Kirkman-Robbins hepatoma revealed that the highest specificity of NHCP2 eluted from hydroxylapatite with 100 mM phosphate buffer at pH 6.8. The NHCP1 of hepatoma shares some common antigenic determinants with analogous proteins of liver. On the other hand non-histone proteins specific for hepatoma dehistonized chromatin can be localized in the NHCP3 and partially in the NHCP1 fractions.     

Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker specific for glandular epithelium and nervous tissue. Further studies should be performed to determine the structure of the tissue epitope recognized.     

Trypanosoma brucei: differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (delta psi) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial delta psi was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.     

Structural analysis of the Lactobacillus rhamnosus strain KL37C exopolysaccharide

The exopolysaccharide from the lactic acid bacterium Lactobacillus rhamnosus strain KL37C isolated from human intestinal flora was prepared by sonication of bacterial cell mass suspended in water followed by centrifugation and cold ethanol precipitation of the supernatant. The polysaccharide material was purified by gel permeation chromatography on an TSK HW-50 column and characterised using chemical and enzymatic methods. On the basis of sugar and methylation analysis and 1H, 13C, 1D and 2D NMR spectroscopy the exopolysaccharide was shown to be composed of the following pentasaccharide repeating unit:-->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->3)-beta-D-Galf-(1-->     

Transgenic rabbit producing human growth hormone in milk

The gene construct WAP(6xHisThr):hGH containing the entire human growth hormone gene (hGH) under the rat whey acidic protein (WAP) promoter regulating the expression in mammary glands of mammals was prepared. The 5' end of the gene was modified by the addition of a sequence encoding six histidine residues and a sequence recognized by thrombin. The gene construct was introduced by microinjection into the male pronucleus of a fertilized oocyte. The founder male rabbit was obtained with the transgene mapping to chromosome 7. The presence of the growth hormone was confirmed in samples of milk collected during the lactation of F1 generation females. The growth hormone can be easily purified by affinity chromatography and cleavage by thrombin to an active form.