A mammalian in vitro model to study gangliogenesis from neural crest cells

In spite of considerable advances towards understanding lineages derived from neural crest cells using amphibian and avian embryos, the molecular mechanisms involved in the formation of mammalian peripheral ganglia remain largely unknown, mainly because of the lack of experimental systems that will allow their in vitro manipulation. Here, we present a novel mammalian in vitro model permitting to study gangliogenesis from neural crest cells. This model allowed us to manipulate molecules involved in cell-cell interactions. Our data are in favour of the existence of a hierarchy among adhesion molecules.     

Effect of host sex and litter on the population dynamics of Echinococcus granulosus in dogs.

Twenty-one Beagle dogs consisting of 10 males and 11 females and belonging to 3 litters were infected with 60,000 E. granulosus protoscolices each. They were killed on day 40, the parasites from their intestines recovered, and the number of worms, average number of proglottides per worm, average length per worm, percentage of worms with a uterine cavity, and percentage of egg-bearing worms were determined for each dog and analyzed per sex and litter. On average, the dogs had 1,253 +/- 339 worms (means +/- standard error) with 2.42 +/- 0.1 proglottides, were 1.59 +/- 0.07 mm long, and 25.6 +/- 4.8% of the worms presented a uterine cavity and 1.2 +/- 0.6% bore eggs. The number of worms exhibited a bimodal distribution with 19 dogs having less than or equal to 2,565 worms and 2 greater than or equal to 5,520 worms. Average number of proglottides also showed a bimodal distribution with 7 dogs having less than or equal to 2.1 proglottides per worm and 14 dogs having greater than or equal to 2.4 proglottides per worm. The parasites were significantly more numerous in females than in the males (1,964 +/- 573 vs. 681 +/- 202), had more proglottides (2.67 +/- 0.08 vs. 2.15 +/- 0.16), and were longer (1.72 +/- 0.07 vs. 1.44 +/- 0.11 mm). The percentages of parasites with a uterine cavity (27.8 +/- 5.9 vs. 23.2 +/- 8.1) or bearing eggs (1.0 +/- 0.5 vs. 1.5 +/- 1.8) were comparable in females and males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paired development of hair cells in neuromasts of the teleost lateral line.

Lateral line neuromasts of the bullseye Parapriacanthus ransonetti and the cardinal fish Apogon cyanosoma were examined by scanning electron microscopy. Neuromasts showed large numbers of degenerating hair cells and immature hair cells, suggesting a high degree of hair cell turnover. New hair cells were mainly produced in pairs (fewer than 5% appear singly), the two cells of a pair having opposite but parallel orientations of their mechanosensitive axes. It is suggested that each pair results, directly or indirectly, from a single mitosis. The results further suggest that the axis of mitosis is one of the factors which determine the direction of the hair cell axis of mechanosensitivity.     

BPTI and N-terminal extended analogues generated by factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli

A recombinant gene for BPTI (bovine pancreatic trypsin inhibitor) is expressed in Escherichia coli using a MBP (maltose-binding protein) fusion vector. BPTI is fused through an FXa (blood coagulation factor Xa protease) target sequence (Ile-Glu-Gly-Arg) to the C-terminus of MBP. The MBP moiety of the hybrid protein enables purification in one step utilizing MBP's affinity to cross-linked amylose, and the FXa target sequence allows specific cleavage of the hybrid protein. Effective FXa cleavage is achieved by spacing the FXa target sequence and Arg-1 of the BPTI sequence with four residues (Met-Glu-Ala-Glu). The resulting N-terminal extended BPTI is readily converted to the wild-type sequence by trimming with cathepsin C exopeptidase, for the activity of which the spacing tetrapeptide is optimized. FXa cleavage is prohibited when the target sequence is placed next to Arg-1. In this construction, off-target cleavage at a somewhat homologous sequence (Val-Pro-Gly-Arg) results in five- or six-residue extended BPTI, indicating new details of the FXa specificity. The yield of highly purified recombinant BPTI is 3-6 mg/liter of culture, making the MBP-BPTI expression system convenient for the production of sufficient amounts of protein for NMR studies. 1H NMR is used to analyze the N-extended BPTI analogues.     

Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride

Fluorescence and circular dichroism were used to follow thepH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going frompH 7 to 4, with a midtransitionpH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as thepH was changed from 6 to 2.5, with a midtransitionpH of 4.5. Near UV circular dichroic spectra also showed changes betweenpH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at fivepH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at allpH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable betweenpH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on thepH used. These results are consistent with thepH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.     

A new simplified assay for larval migration inhibition

, , , and 1992. A new simplified assay for larval migration inhibition. International Journal for Parasitology 22: 1183–1185. A simple method is described for the in vitro detection of substances that impair the motility of third-stage larvae of gastro-intestinal nematodes. The test is based on the ability of larvae to freely migrate through selected mesh sizes of nylon sieves and the reduced ability of larvae to migrate after preincubation with, and in the presence of, substances that inhibit or reduce larval motility.     

Bovine babesiosis: failure to induce interferon gamma production in response to Babesia bovis antigens in cattle.

The immune response to Babesia bovis infection or vaccination was evaluated by measuring antibody and interferon gamma (IFN-gamma) production to protective recombinant and crude native B. bovis antigens. Cells from vaccinated or infected cattle failed to produce detectable IFN-gamma when stimulated with B. bovis antigens in vitro. In contrast, antibody was induced by protective recombinant B. bovis antigens. These findings are consistent with the argument that immunity to B. bovis infection is correlated most strongly with humoral rather than cell-mediated immune responses.     

Plasmid transduction using the D3112DeltaH miniphage in Pseudomonas aeruginosa cells]

Plasmid DNA transduction with mini-D3112 delta H, deletion derivative of phage D3112, which lost the genes essential for phage growth but retained the sites required for transposition and packaging was studied. Unlike D3112, mini-D3112 delta H element can transduce plasmids and plasmid markers at frequencies of 10(-5)-10(-8) in rec+ cells of Pseudomonas aeruginosa. Plasmids R1162 and R388 of the size smaller than phage genome were transduced intact. Large plasmids, like RP4 and R151, were deleted under transduction. By this way, we isolated deletion derivatives of RP4. The smallest derivative pN2 contained a 4.5 kb fragment of RP4. Unlike the latter, pN2 plasmid had narrow host range and did not maintain in Escherichia coli cells.     

Fc epsilon receptor mediated Ca2+ influx into mast cells is modulated by the concentration of cytosolic free Ca2+ ions.

The relationship between the Fc epsilon receptor mediated stimulation of mast cells and the Ca2+ signal it induces were studied using thapsigargin (TG), a blocker of the endoplasmic reticulum Ca2+ pump. TG induced, in mucosal mast cells (RBL-2H3 line), a dose-dependent and an InsP3-independent increase in [Ca2+]i (from resting levels of 83-150 nM to 600-680 nM), and a secretory response amounting to 30-50% of that observed upon Fc epsilon RI clustering. The TG induced rise of [Ca2+]i is most probably provided by both arrest of its uptake by the endoplasmic reticulum and influx from the medium. Thus, Ca2+ influx in mast cells may be modulated by the [Ca2+]i level.