Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.     

Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.     

Apolipoprotein M modulates erythrocyte efflux and tubular reabsorption of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) mediates several cytoprotective functions of HDL. apoM acts as a S1P binding protein in HDL. Erythrocytes are the major source of S1P in plasma. After glomerular filtration, apoM is endocytosed in the proximal renal tubules. Human or murine HDL elicited time- and dose-dependent S1P efflux from erythrocytes. Compared with HDL of wild-type (wt) mice, S1P efflux was enhanced in the presence of HDL from apoM transgenic mice, but not diminished in the presence of HDL from apoM knockout (Apom^−/−) mice. Artificially reconstituted and apoM-free HDL also effectively induced S1P efflux from erythrocytes. S1P and apoM were not measurable in the urine of wt mice. Apom^−/− mice excreted significant amounts of S1P. apoM was detected in the urine of mice with defective tubular endocytosis because of knockout of the LDL receptor-related protein, chloride-proton exchanger ClC-5 (Clcn5^−/−), or the cysteine transporter cystinosin. Urinary levels of S1P were significantly elevated in Clcn5^−/− mice. In contrast to Apom^−/− mice, these mice showed normal plasma concentrations for apoM and S1P. In conclusion, HDL facilitates S1P efflux from erythrocytes by both apoM-dependent and apoM-independent mechanisms. Moreover, apoM facilitates tubular reabsorption of S1P from the urine, however, with no impact on S1P plasma concentrations.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

Antibodies against mouse nerve growth factor interfere in vivo with the development of avian sensory and sympathetic neurones

The monoclonal antibody 27/21 directed against mouse nerve growth factor (NGF) interferes in vivo with the survival of sensory dorsal root ganglion (DRG) neurones during the development of the quail embryo: the number of DRG neurones at embryonic day 11 (E11) was reduced by about 30% in embryos treated with the antibody between E3 and E11. Neurone numbers in the nodose ganglion were not affected. The effect of NGF antibodies on sympathetic neurones was assessed by determining the levels of the adrenergic marker enzyme tyrosine hydroxylase. Both total tyrosine hydroxylase activity and protein levels in sympathetic chains were reduced by about 30% in embryos treated with 27/21 antibody but not in embryos treated with a control antibody. The 27/21 antibody cross-reacts with chick NGF-like activity as shown in vitro by the ability of the antibody to partially block the survival activity of chick-embryo-fibroblast-conditioned medium for E9 chick DRG neurones.     

A consensus linkage map for swine chromosome 7

The First International Workshop on Swine Chromosome 7 (SSC7) was held in Minnesuing, Wisconsin, USA on 21–24 September 1995. The objective was to develop a comprehensive linkage map for porcine chromosome 7 by combining genotypic data from four swine reference populations. Contributions of genotypic data were made from the US Meat Animal Research Center, the University of Minnesota, the PiGMaP consortium and the Scandinavian consortium. Primers for selected sequence tagged site markers, to be genotyped across the reference populations, were exchanged to integrate individual maps of SSC7. Eighty-six loci were genotyped; these loci represented microsatellite, minisatellite, single-strand conformation polymorphism, restriction fragment length polymorphism, erythrocyte antigen and protein polymorphisms. Eighteen genes were mapped, including 12 markers within class I, class II and class III regions (four markers in each class) of the swine major histocompatibility complex. Forty-two markers were either genotyped on more than one population or were included in a haplotype system and used to develop skeletal linkage maps that spanned 147·6, 212·7 and 179·5 cM for the male, female and sex-average maps, respectively. A comprehensive linkage map was developed incorporating those markers with more than 30 informative meioses. The comprehensive map was slightly longer than the skeletal map, at 153·3, 215·3 and 183·8 cM, respectively, with only three intervals greater than 10 cM. These results significantly improve the genetic resolution of the porcine chromosome 7 map and represent an accurate approach for the merging of genetic maps produced in different reference populations.     

Genomic mapping of chemokine and transforming growth factor genes in swine

Five chemokine genes, transforming growth factors alpha, beta 2 and 3 (TGFBA, TGFB-2, and TGFB-3), interleukin 8 (IL-8), and monocyte chemoattractant protein 2 (MCP-2), were mapped to porcine linkage groups on Chromosomes 3q, 10p, 7q, 8, and 12q, respectively. Restriction fragment length polymorphisms (RFLPs) for these genes were developed by Southern blot hybridization after digestion of porcine genomic DNA with BamHI and MspI (TGFBA), BamHI and PvuII (TGFB-2), HindIII (TGFB-3), BglII (IL-8), and PstI (MCP-2) and used to genotype the USDA-MARC Swine Reference Population pigs. Sufficient informative meioses, 61 (TGFBA), 58 (TGFB-2), 28 (TGFB-3), 38 (IL-8), and 156 (MCP-2), were available to pursue two-point pairwise linkage analysis with over 1,000 existing loci in the USDA-MARC genome database to establish initial linkage (LOD > 3). Multi-point analysis with CRIMAP determined the most likely order for each new marker. The assignment of the five chemokine genes in swine concurs with previous porcine/human chromosomal homologies based on results from ZOO-FISH and chromosomal painting experiments. These findings add five new informative Type I markers within a single gene family to the swine genome and may help us understand the genetic basis for disease resistance in livestock. Received: 14 July 1996 / Accepted: 9 November 1996