A high-resolution comparative RH map of porcine Chromosome (SSC) 2

A high-resolution comparative map was constructed for porcine Chromosome (SSC) 2, where a QTL for back fat thickness (BFT) is located. A radiation hybrid (RH) map containing 33 genes and 25 microsatellite markers was constructed for this chromosome with a 3000-rad porcine RH panel. In total, 16 genes from human Chromosome (HSA) 11p, HSA19p, and HSA5q were newly assigned to SSC2. One linkage group was observed at LOD 3.0, and five linkage groups at LOD 4.0. Comparison of the porcine RH map with homologous human gene orders identified four conserved segments between SSC2 and HSA11, HSA19, and HSA5. Concerning HSA11, a rearrangement of gene order is observed. The segment HSA11p15.4-q13 is inverted on SSC2 when compared with the distal tip of SSC2p, which is homologous to HSA11p15.5. The boundaries of the conserved segments between human and pig were defined more precisely. This high-resolution comparative map will be a valuable tool for further fine mapping of the QTL area. Received: 10 November 2000 / Accepted: 23 January 2001     

Accessory cell requirements in mitogen-induced rabbitt lymphocyte proliferation in an improved tissue culture system

Summary The impact of an improved culture medium (IMDM⁺), consisting of Iscove’s modified Dulbecco’s medium supplemented with albumin, transferrin, insulin, zinc, 2-mercapthoethanel, and 0.1% fetal bovine serum was investigated in phytohemagglutinin (PHA)-induced rabbit T cell proliferation. At the number of 2×10⁵ cells/well purified T lymphocytes cultured in IMDM⁺ responded 3 to 10 times better than lymphocytes cultured in serum-supplemented RPMI 1640 medium. In these conditions, PHA-induced proliferation seemed not to require the presence of accessory cells. However, at lower cell numbers, T cell proliferation was more efficient when calculated on a per cell basis. At these low cell numbers, optimal proliferation required accessory cells like macrophages or dendritic cells. The appraent absence of this requirement for accessory cells at high T cell concentrations may be explained by the contribution of contaminating macrophages and dendritic cells in the purified T cell fractions.     

alpha-MSH enhances activity-based anorexia

Activity-based anorexia (ABA) is considered an animal model of anorexia nervosa (AN). In ABA, scheduled feeding in combination with voluntary access to running wheels, results in hyperactivity, hypophagia, body weight loss and activation of the HPA axis. Since stimulation of the melanocortin (MC) system has similar effects, this system is a candidate system involved in ABA. Here it is shown that chronic alpha-MSH treatment enhances ABA by increasing running wheel activity (RWA), decreasing food intake and increasing HPA axis activation.     

Chicken Ig-like receptor B2, a member of a multigene family, is mainly expressed on B lymphocytes, recruits both Src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2, and inhibits proliferation

Ig-like inhibitory receptors have been the focus of intensive research particularly in mouse and human. We report the cloning and characterization of three novel inhibitory chicken Ig-like receptors (CHIR) that display a two Ig-domain extracellular structure, a transmembrane region lacking charged residues and a cytoplasmic domain containing two ITIM. The localization of all receptors to a small genomic region and the hybridization pattern indicated that they belong to a multigene family. The genomic structure of the extracellular domain with two exons encoding the signal peptide and single exons for each Ig domain resembled that of all human leukocyte Ig-like receptors and killer cell Ig-like receptors, whereas the exons encoding the C terminus displayed a structure closely resembling killer cell Ig-like receptor genes. A mAb generated against one receptor designated CHIR-B2 reacted with all B cells and a small T cell subset, but not with monocytes, thrombocytes, or various leukocyte-derived cell lines. The mAb immunoprecipitated a 46-kDa protein from bursal cells and transfected cells. The Src homology 2 domain containing protein tyrosine phosphatase (SHP)-2 bound to CHIR-B2 even in unstimulated cells, whereas pervanadate treatment induced the tyrosine phosphorylation and recruitment of several CHIR-B2-associated proteins including SHP-1 and increased levels of SHP-2. Moreover, mAb cross-linking of CHIR-B2 reduced the proliferation of a stable transfected cell line. Together, we have identified a multigene family containing multiple CHIR including one receptor designated CHIR-B2 that is mainly expressed on B lymphocytes and inhibits cellular proliferation by recruitment of SHP-1 and SHP-2.     

The IGF2-intron3-G3072A substitution explains a major imprinted QTL effect on backfat thickness in a Meishan x European white pig intercross

A paternally expressed QTL for muscle growth and backfat thickness (BFT) has previously been identified near the IGF2 locus on the distal tip of pig chromosome 2 (SSC2p) in three experimental F2 populations. Recently, a mutation in a regulatory element of the IGF2 gene was identified as the quantitative trait nucleotide (QTN) underlying the major QTL effect on muscle growth and BFT in crosses between Large White and Wild Boar or Pietrain. This study demonstrates that the IGF2 mutation also controls the paternally expressed QTL for backfat thickness in a cross between Meishan and European Whites. In addition, a comparison of QTL of backfat thickness measured by Hennessy grading probe (HGP) and by ultrasound measurement (USM) was made. In the USM analyses, the IFG2 mutation explains the entire QTL effect on SSC2p, whereas in the HGP analysis the presence of a second minor QTL can not be excluded. Finally, this study shows that this particular IGF2 mutation does not cause the paternally expressed QTL for teat number mapping to the same region of SSC2p as the BFT QTL.     

Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity

Langerhans cells (LC) form a unique subset of dendritic cells (DC) in the epidermis but so far their in vivo functions in skin immunity and tolerance could not be determined, in particular in relation to dermal DC (dDC). Here, we exploit a novel diphtheria toxin (DT) receptor (DTR)/DT-based system to achieve inducible ablation of LC without affecting the skin environment. Within 24 h after intra-peritoneal injection of DT into Langerin-DTR mice LC are completely depleted from the epidermis and only begin to return 4 wk later. LC deletion occurs by apoptosis in the absence of inflammation and, in particular, the dDC compartment is not affected. In LC-depleted mice contact hypersensitivity (CHS) responses are significantly decreased, although ear swelling still occurs indicating that dDC can mediate CHS when necessary. Our results establish Langerin-DTR mice as a unique tool to study LC function in the steady state and to explore their relative importance compared with dDC in orchestrating skin immunity and tolerance.     

Multiparameter Viability Assay for Stress Profiling Applied to the Food Pathogen Listeria monocytogenes F2365

A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This food-borne pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters.     

Manipulation of starch granule size distribution in potato tubers by modulation of plastid division

Starch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery are likely to be similar for all plastids. Essential roles for FtsZ proteins in plastid division in land plants have been revealed. FtsZ forms the so-called Z ring which, together with inner and outer plastid division rings, brings about constriction of the plastid. It has been shown that modulation of the expression level of FtsZ may result in altered chloroplast size and number. To test whether FtsZ is also involved in amyloplast division and whether this, in turn, may affect the starch granule size in crop plants, FtsZ protein levels were either reduced or increased in potato. As shown previously in other plant species, decreased StFtsZ1 protein levels in leaves resulted in a decrease in the number of chloroplasts in guard cells. More interestingly, plants with increased StFtsZ1 protein levels in tubers resulted in less, but larger, starch granules. This suggests that the stoichiometry between StFtsZ1 and other components of the plastid division machinery is important for its function. Starch from these tubers also had altered pasting properties and phosphate content. The importance of our results for the starch industry is discussed.