Channel-mediated water movement across enclosed or perfused mouse intrahepatic bile duct units

We previously reported the developmentof reproducible techniques for isolating and perfusing intactintrahepatic bile duct units (IBDUs) from rats. Given the advantages oftransgenic and knockout mice for exploring ductal bile formation, wereport here the adaptation of those techniques to mice and theirinitial application to the study of water transport across mouseintrahepatic biliary epithelia. IBDUs were isolated from livers ofnormal mice by microdissection combined with enzymatic digestion. Afterculture, isolated IBDUs sealed to form intact, polarized compartments,and a microperfusion system employing those isolated IBDUs developed. Aquantitative image analysis technique was used to observe a rapidincrease of luminal area when sealed IBDUs were exposed to a series of inward osmotic gradients reflecting net water secretion; the choleretic agonists secretin and forskolin also induced water secretion into IBDUs. The increase of IBDU luminal area induced by inward osmotic gradients and choleretic agonists was reversibly inhibited by HgCl₂, a water channel inhibitor. With the use of aquantitative epifluorescence technique in perfused mouse IBDUs, a highosmotic water permeability (P_f = 2.5-5.6 × 10² cm/s) was found in response toosmotic gradients, further supporting the presence of water channels.These findings suggest that, as in the rat, water transport acrossintrahepatic biliary epithelia in mice is water channel mediated.

     

A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases

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Skeletal growth of children from the Iron Age site at K2 (South Africa)

Cross-sectional growth data were obtained from the skeletal remains of children from the Iron Age site of K2 near the Limpopo River. Standard measurements of the diaphyseal lengths of the long bones from both limbs were recorded and compared to published skeletal data. For this purpose, data on Eskimo and Aleut skeletons, Libben skeletons, and skeletons from Indian Knoll and Altenerding were used. An attempt to study growth allometrically was made. K2 children were growing as well as children from these other groups. Comparison of data for K2 children with those on living South African “Cape Coloured” rural children, studied during the late 1980s, shows the similarity of growth of both groups. © 1996 Wiley-Liss, Inc.     

Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis

Here we present a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI–TOF/TOF)-based label-free relative protein quantification strategy that involves sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) separation of proteins followed by in-gel trypsin digestion. The main problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high-pressure-assisted in-gel trypsin digestion method that is based on pressure cycling technology (PCT). The PCT approach performed at least as well as the conventional overnight in-gel trypsin digestion approach in parameters such as number of peaks detected, number of peptides identified, and sequence coverage, and the digestion time was reduced to 45 min. The gel/mass spectrometry (MS)-based label-free protein quantification method presented in this work proved the applicability of the signal response factor concept for relative protein quantification previously demonstrated by other groups using the liquid chromatography (LC)/MS platform. By normalizing the average signal intensities of the three most intense peptides of each protein with the average intensities of spiked synthetic catalase tryptic peptides, which we used as an internal standard, we observed spot-to-spot and lane-to-lane coefficients of variation of less than 10 and 20%, respectively. We also demonstrated that the method can be used for determining the relative quantities of proteins comigrating during electrophoretic separation.     

Fungal associations in Asphondylia (Diptera: Cecidomyiidae) galls from Australia and South Africa: implications for biological control of invasive acacias

Gall-forming Asphondylia are well represented on Australian Acacia and have potential for biological control where Australian acacias cause ecological or economic harm, particularly South Africa. Asphondylia in Australia and South Africa are associated with communities of fungi in their galls. In Australia, Botryosphaeria dothidea (as its Dichomera synanamorph) is the most abundant and sometimes the only fungus present and is implicated as the primary species forming a mutualistic relationship with Asphondylia. In the combined analysis of ITS and elongation factor 1-α sequence data, isolates of B. dothidea from Australia and South Africa form distinct sub-clades. Female Asphondylia carry B. dothidea (as Dichomera conidia) in mycangia located posterior to sternite 7. While conidia are always present on field-collected specimens, laboratory-reared females rarely carry conidia. The mechanism and location of spore collection remains unresolved, but needs to be understood if Asphondylia species are to be utilised for biological control of invasive Australian acacias. As B. dothidea is a polyphagous plant pathogen capable of infecting crops of economic importance, including Acacia plantations, the introduction of novel strains of B. dothidea associated with biological control of acacia is undesirable, however endemic forms of the fungus could possibly be exploited by introduced Asphondylia.     

Distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats

To evaluate cell-mediated immunity in influenza-infected cotton rats, we compared the cellular composition of spleen, mediastinal lymph nodes (MLN) and bronchoalveolar lavage (BAL) after primary and secondary infection. There was an increase in cellularity in the MLN after primary infection that was further expanded upon rechallenge. CD4(+) T cells expanded after primary infection, but there was preferential increase in the number of CD4-negative T cells following secondary challenge. After primary infection, a large proportion of the monocytes and NK cells were present in the BAL while a T cell population dominated after secondary infection. CD4(+) T cells were predominant in this population unless the animals had been challenged with heterosubtypic influenza A virus. These studies are the first to show evidence of a memory T cell response to influenza infection in cotton rats and show quantitative and qualitative differences between the recall response to homosubtypic and heterosubtypic viruses.     

Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step, we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells, and then, expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype, expressed myeloperoxidase, and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules, CD1a, CD11c, CD80, CD86, DC-SIGN, and CD40; and were capable of Ag processing, triggering naive T cells in MLR, and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14, and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition, because ES cells can be expanded without limit, they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance.     

Comparative binding of soluble fragments (derCD23, sCD23, and exCD23) of recombinant human CD23 to CD21 (SCR 1-2) and native IgE, and their effect on IgE regulation

IgE, responsible for type I hypersensitivities, is regulated by interactions between its receptor, CD23, and co-receptor CD21. To examine comparative binding of recombinant human CD21 SCR 1-2 and native human IgE to CD23 plus the effect of CD23 on IgE production, we engineered recombinant soluble human CD23 fragments; (1) derCD23, (2) sCD23 and (3) exCD23, formed in vivo by proteolysis. SPR analysis revealed a progressive increment in affinity of soluble fragments for IgE, upon increasing length of CD23 “stalk” domain, exCD23 > sCD23 > derCD23. Soluble CD23 fragments and their oligomeric state are shown to fine-tune the immune response. Oligomers appear more important in enhancing IgE synthesis and monomers lacking the tail residues fail to bind CD21 yet bind membrane IgE and down-regulate IgE synthesis. Co-ligation of membrane IgE and CD21 through soluble CD23 monomers is disturbed. This study supports anti-allergic therapies involving stabilizing membrane CD23, or preventing shedding of soluble CD23.     

Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina)

DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms (ca. 20,000 species) has not been established. We succeeded in generating 167 partial COI sequences (~450 bp) representing ~100 morphospecies from ~650 collections of Agaricomycotina using several sets of new primers. Large introns (~1500 bp) at variable locations were detected in ~5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (~30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms.