The preparation and properties of isolated chicken hepatocytes

Chicken hepatic parenchymal cell suspensions, isolated by an optimised collagenase digestion, were used for a study of hepatic glucose metabolism and its control in the chicken. Characterisation of this iri vitro preparation showed the parenchymal cells immediately after isolation to be similar to those of whole liver, both morphologically and metabolically. This similarity suggested that metabolic studies with isolated hepatocytes might confidently be extrapolated to the situation in the intact animal. However the preparation quality was dependent on collagenase contaminants and all preparations exhibited decreased viability throughout subsequent incubations.Glycogen metabolism in isolated hepatocyte suspensions favoured glycogenolysis and under no conditions was net glycogen synthesis observed. Gluconeogenesis from added precursors was difficult to discern with fed chicken hepatocytes due to the high basal glucose production but was readily demonstrated at a constant rate over a two hour incubation with starved chicken hepatocytes.The gluconeogenic effectiveness of precursors was generally similar in isolated hepatocytes and in chickens in vivo. The greater effectiveness of lactate compared with pyruvate, observed with both systems (unlike the rat), is probably a consequence of impaired hydrogen ion transfer during pyruvate gluconeogenesis due to the mitochondrial location of phosphoenolpyruvate carbcxykinase in the chicken. Synergistic interactions between substrates were shown to occur and be important for interpretation of results from isolated hepatocytes for extrapolation to the situation in vivo. Glycerol wasPhysiological concentrations of glucagon stimulated glycogenolysis and gluconeogenesis from precursors entering the glycolytic pathway above and below the triose phosphate dehydrogenase step. Although it was possible to assign a glucagon control point between triose phosphate and glucose in chicken liver, that between pyruvate and phosphoenol pyruvate (postulated for the rat) was not observed

Liver-enriched transcription factors are critical for the expression of hepatocyte marker genes in mES-derived hepatocyte-lineage cells

<p>Abstract</p> <p>Background</p> <p>Induction of stem cell differentiation toward functional hepatocytes is hampered by lack of knowledge of the hepatocyte differentiation processes. The overall objective of this project is to characterize key stages in the hepatocyte differentiation process.</p> <p>Results</p> <p>We established a mouse embryonic stem (mES) cell culture system which exhibited changes in gene expression profiles similar to those observed in the development of endodermal and hepatocyte-lineage cells previously described in the normal mouse embryo. Transgenic mES cells were established that permitted isolation of enriched hepatocyte-lineage populations. This approach has isolated mES-derived hepatocyte-lineage cells that express several markers of mature hepatocytes including albumin, glucose-6-phosphatase, tyrosine aminotransferase, cytochrome P450-3a, phosphoenolpyruvate carboxykinase and tryptophan 2,3-dioxygenase. In addition, our results show that the up-regulation of the expression levels of hepatocyte nuclear factor-3α, -4α, -6, and CCAAT-enhancer binding protein-β might be critical for passage into late-stage differentiation towards functional hepatocytes. These data present important steps for definition of regulatory phenomena that direct specific cell fate determination.</p> <p>Conclusion</p> <p>The mES cell culture system generated in this study provides a model for studying transition between stages of the hepatocyte development and has significant potential value for studying the molecular basis of hepatocyte differentiation <it>in vitro</it>.</p

Engineering Chinese Hamster Ovary cells for enhanced protein secretion

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The Legal Background to Community Based Fisheries Management in Bangladesh

This booklet, produced as an output from the Community Based Fisheries Management Project -- 2nd Phase(CBFM-2), aims to summarise the legal knowledge and experiences built up and challenges faced during the five years of CBFM-2 implementation. It also suggests a set of legal and policy interventions to ensure future sustainability. The project has established community control over 116 water bodies, spread over 48 Upazilas (sub-district) in 22 districts in Bangladesh. With 130 Community Based Organisations (CBOs), formed under this project, the communities were given the responsibility for management of 116 water bodies -- government owned fisheries (jalmohals) and privately owned seasonal water bodies -- closed beels, open beels, river sections and floodplains. The CBFM-2 project has been managed by the Department of Fisheries in partnership with the WorldFish Center and 11 implementing NGOs -- Banchte Shekha, BRAC, Caritas, CNRS, CRED, GHARONI, Proshika, SDC, SHISUK, and the specialist NGOs FemCom for media communications and BELA for legal support and assistance

All smiles are positive, but some smiles are more positive than others.

Disagreement as to whether all smiling or specific types of smiling index positive emotion early in life was addressed by examining when infants produced different types of smiling and other facial expres-sions. Thirteen infants were observed weekly from 1 to 6 months of age. Smiling alone—without cheek raising or mouth opening—was relatively more likely than periods without smiling both when mothers were smiling and when infants were gazing at their mothers &apos; faces. Cheek-raise (Duchenne) smiling was relatively more likely than smiling alone only when mothers were smiling. Open-mouth (play) smiling was relatively more likely than smiling alone only when infants were gazing directly at mothers &apos; faces. Smiling involving both cheek raising and mouth opening was relatively likely both when mothers were smiling and when infants were gazing at mothers &apos; faces and became increasingly likely with age when both conditions co-occurred. The cheek-raise and open-mouth dimensions of smiling appear to be associated with, respectively, the amplification of processes of sharing positive affect and of visual engagement that are present to a lesser degree in smiling alone. In infancy, positive emotions such as joy are hypothesized to motivate and organize desired actions (Blehar, Lieberman, &amp; Ainsworth, 1977; Cohn, Campbell, &amp; Ross, 1991; Malatesta, Cul

Biomineralisation in the Palaeozoic oceans: evidence for simultaneous crystallisation of high and low magnesium calcite by phacopine trilobites

The chemical composition and microstructure of the calcite cuticles of eleven species of phacopine trilobites have been investigated by electron beam imaging, diffraction, and microanalysis, and results reveal that the lenses of their schizochroal eyes differed significantly in chemical composition from the rest of the cuticle in vivo. Apart from the eye lenses, most cuticles are inferred to have escaped extensive recrystallisation because their constituent crystals are sub-micrometre in size and have a preferred orientation that is consistent between species. Their current compositions of ~1.4 to 2.4 mol% MgCO3 are likely to be close to original values, although as they commonly luminesce and contain detectable manganese and iron, some diagenetic alteration has taken place. The associated lenses have a microstructure that is suitable for focusing light, yet are optically turbid owing to the presence within calcite of micropores and crystals of microdolomite, apatite, celestite and pyrite. The microdolomite indicates that lenses recrystallised from an original high-Mg calcite composition and this is supported by the presence of nanometre-scale modulated microstructures in both the calcite and dolomite. These lenses currently contain ~1 to 6 mol% MgCO3, and by comparison with the proportion of magnesium lost from echinoderm stereom in the same thin sections, may have contained ~7.5 mol% MgCO3 in vivo. In some samples, more extensive diagenetic alteration is evidenced by recrystallisation of the cuticle including lenses to coarse equant calcite or enrichment of the cuticle, but not necessarily the lenses, in magnesium accompanying replacement by a Mg–Fe phyllosilicate. The phacopine trilobites had to modify partition coefficients for magnesium considerably in order to grow lenses with contrasting compositions to the rest of their cuticles, and such a strong vital effect on biomineralisation suggests that incorporation of magnesium was essential for functioning of their calcite optical s

Compuchtational prediction of expression and solubility of recombinant biopharmaceuticals

Protein based therapeutics have emerged as a successful class of pharmaceutical. However, it is well known that much of the current therapeutic protein discovery and development processes is based around existing molecular frameworks and that novel formats offer significant challenges for expression. Efficient production of protein is required to meet the growing demands and increasing expectation of the patients and healthcare providers. Major obstacles during biopharmaceutical production are linked to the efficiency of the protein expression system and the biophysical properties of proteinbased products that can lead to aggregation and subsequent problems for purification, quality and effectiveness. Computational tools have been developed to aid prediction of protein solubility and aggregation propensity to support enhanced certainty of optimal generation of product with desirable properties. In this study, we have used an in-house computational tool for prediction of soluble protein expression (developed around protein structure and surface electrostatic properties of human erythropoietin, HuEPO) was developed in E. coli and has been validated experimentally with several bacteriallyexpressed model protein variants. The application of the computational approach has been extended to mammalian expression platforms. A significant inverse correlation was observed between positive surface patches and the expressability of HuEPO in transient mammalian cells (HEK and CHO cell lines). Mechanistically the differential expression operates at a level post-transcriptionally, associated with ribosome-secretory complex engagement, protein stability or secretory processes. The results demonstrate the potential of application of a predictive computational algorithm as a design tool in rational protein engineering to improve expression of novel protein formats in mammalian systems as well as E.coli. In summary, optimization of molecular patches on the surface of proteins may be a viable strategy to enhance protein soluble expression and therefore a potential solution for development of novel proteins that might otherwise fit into the category of “difficult-toexpress” proteins

Identifying opportunities in cell engineering for the production of ‘difficult to express’ recombinant proteins

There is a growing demand for production of recombinant proteins of many structural varieties in mammalian expression systems, either as therapeutics or for protein characterisation. However, certain recombinant proteins are “difficult to express” in mammalian expression systems requiring extensive cell line and process optimisation which, as a result, can have significant consequences for drug development processes. The Tissue Inhibitors of Metalloproteinase (TIMP) protein family, TIMP-2, -3 and -4, are naturally secreted proteins that share significant structural homology (~50% identity and ~70% similarity in amino acid sequence), but show profound differences in secretion in mammalian expression systems. Computational sequence analysis of the TIMPs shows areas of significant amino acid difference mainly locating to flexible loop regions. This study has investigated the molecular mechanisms that selectively restrict expression of recombinant proteins of extensive sequence similarity. The loci of the molecular steps that limit successful expression have been defined by quantitative real-time polymerase chain reaction, proteomic analyses, cellular fractionation and immunofluorescence microscopy. All three TIMPs were readily detectable at mRNA and protein level within the cell but only TIMP-2 was secreted effectively into the culture medium. Analysis of protein localisation showed intracellular protein for all three TIMPs, mainly co-localised in the organellar and cytoskeleton fractions. In addition, immunofluorescence microscopy showed all three TIMPs to be detectable within the endoplasmic reticulum. TIMP-3, which was not secreted, was detected within the cell in both expected glycosylated and non-glycosylated forms. Treatment of intracellular TIMP-3 with glycosidases suggests the presence of an immature high mannose glycoform. Knockout of the TIMP-3 glycan site did not result in secretion. These data suggest that the post-translational processing of poorly expressed TIMPs limits transit through the secretory pathway. To overcome this challenge, cell engineering of limiting secretory pathway components could enhance production of these “difficult to express” recombinant proteins

Variation in the Response of Three Different Pinus Radiata Kraft Pulps to Xylanase Treatments

Two xylanase preparations (Pulpzyme HC and Xylanasc E) were assessed for their ability to enhance the refining properties of three different Pinus radiata kraft pulps. Both preparations selectively solubilized a significant proportion of the available xylan; however, xylanase E proved to be more aggressive, regardless of the pulp type. The selective removal of pulp xylan improved pulp beatability by increasing the apparent densities of the resultant handsheets over their corresponding controls. There were, however, variations in the response of the different pulp types, with an unbleached kappa 70 pulp showing the greatest improvement in sheet densification, as compared to an isothermal-cooked (kappa 33) and a fully bleached pulp. In general, xylanase treatments improved tear strength at a given density without significant loss in tensile strength and intrinsic fiber strength. These results suggest that xylanase treatments may be a means of enhancing the collapsibility/flexibility of certain kraft fibers while maintaining intrinsic strength