PubMed Health⌕ Search

Biomedical subjects

D Milan

Publications and source records attributed to D Milan.

At least 55 records · Page 3Linked to original sources
PubMed1996

Porcine linkage and cytogenetic maps integrated by regional mapping of 100 microsatellites on somatic cell hybrid panel.

Recently two main genetic maps [Rohrer et al. Genetics 136, 231 (1994); Archibald et al. Mamm. Genome 6, 157 (1995)] and a cytogenetic map [Yerle et al. Mamm. Genome 6, 175 (1995)] for the porcine genome were reported. As only a very few micro-satellites are located on the cytogenetic map, it appears to be important to increase the relationships between the genetic and cytogenetic maps. This document describes the regional mapping of 100 genetic markers with a somatic cell hybrid panel. Among the markers, 91 correspond to new localizations. Our study enabled the localization of 14 new markers found on both maps, of 54 found on the USDA map, and of 23 found on the PiGMaP map. Now 21% and 43% of the markers on the USDA and PiGMaP linkage maps respectively are physically mapped. This new cytogenetic information was then integrated within the framework of each genetic map. The cytogenetic orientation of the USDA linkage maps for Chromosomes (Chrs) 3, 8, 9, and 16 and of PiGMaP for Chr 8 was determined. USDA and PiGMaP linkage maps are now oriented for all chromosomes, except for Chrs 17 and 18. Moreover, the linkage group "R" from the USDA linkage map was assigned to Chr 6.

Animals↗
PubMed1996

Assignment of markers by using polymerase chain reaction on pools of swine flow-sorted chromosomes.

Gene chromosomal assignment can be realized not only by somatic hybrid panels but also by spot-blot hybridization or polymerase chain reaction (PCR) of flow-sorted chromosomes. We propose a swine chromosome assignment strategy by PCR amplification on pooled chromosomal DNA, which allows assignment despite possible chromosomal contamination during sorting. Each pool contains three different chromosomes, each chromosome being present in one or two pools. We present concordant results obtained for eight markers already mapped to different swine chromosomes and we assign the somatostatin gene to chromosome 13, a new marker in the pig genome.

Animals↗
PubMed1996

A somatic cell hybrid panel for pig regional gene mapping characterized by molecular cytogenetics.

A panel of 27 pig x rodent somatic cell hybrids was produced and characterized cytogenetically. The first step of this study consisted of hybridizing a SINE probe to GTG-banded metaphases of each hybrid clone in order to count and identify the normal pig chromosomes and to detect rearranged ones. The second step consisted of using the DNA of each clone as a probe after pIRS-PCR (porcine interspersed repetitive sequence-polymerase chain reaction) amplification to highly enrich it in pig sequences. These probes, hybridized to normal pig metaphase chromosomes, enabled the identification of the complete porcine complement in the hybrid lines. Whole chromosomes and fragments were characterized quickly and precisely, and results were compared. In addition to this cytogenetic characterization, molecular verification was also carried out by using primers specific to six microsatellites and to one gene previously mapped to pig chromosomes. The results obtained allow us to conclude that we have produced a panel that is informative for all porcine chromosomes. This panel constitutes a highly efficient tool to establish not only assignments of genes and markers but also regional localizations on pig chromosomes.

Animals↗
PubMed1996

Sequence analysis and genetic mapping of porcine chromosome 11 centromeric S0048 marker.

We report the existence of a new family of swine centromeric satellite DNA composed of a 51-bp repeat unit, most specifically found on pig chromosome 11 centromere and with less specificity at the centromeric region of other meta- and submetacentric chromosomes. This satellite DNA family, which has no homologies with the Mc1 and Ac2 families published previously, was named Mc2. We designed a specific primer set for PCR amplification of this centromeric satellite DNA. Specificity of amplification was checked by using a porcine somatic cell hybrid panel and by FISH. Furthermore, the development of a PCR-RFLP marker of Mc2 repetition allowed its genetic mapping on the PiGMaP reference families panel. The centromere of chromosome 11 was thus integrated to the genetic map previously published.

Animals↗
PubMed1996

The hemodynamic abnormalities in short-term insulin deficiency: the role of prostaglandin inhibition.

It has been suggested that the hemodynamic derangements present in diabetic ketoacidosis are the results not only of profound volume depletion but also of the effects of increased production of vasodilating prostaglandins (PGs), principally PGI2, released by adipose tissue. In animal and in vitro models, prostaglandin synthesis is increased during insulin deficiency. We assessed the effects of short-term ketosis on the metabolic and hemodynamic variables of 10 IDDM patients free from long-term complications and of 9 normal control subjects after a 7-day randomized double-blind indomethacin (INDO) (50 mg q.i.d.) or placebo treatment period. Calf blood flow (CBF), postocclusive reactive hyperemia (PORH), and recovery half-time (an index of overall perfusion) after PORH were measured by plethysmography. Left ventricular and myocardial functions were also studied in each different condition during placebo and INDO treatment in IDDM patients. During placebo treatment, the increase in CBF during ketosis was higher (1.75 +/- 0.29 ml / min / 100 ml muscle) than during INDO (0.85 +/- 0.17 ml / min) / 100 ml muscle; P = 0.007). PORH was similar in baseline conditions, during ketosis, and in recovery in both the placebo and INDO arms. Recovery half-time significantly increased during placebo (10 +/- 2; 200%; P < 0.01) but not during INDO (1 +/- 1; 106%; NS) treatment. In normal control subjects, insulin deficiency did not induce any significant effect on hemodynamic variables. In IDDM patients, during placebo treatment, ketosis increased both the cardiac index (from 3.4 +/- 0.7 to 4.1 +/- 0.81 / min / m; P < 0.01) and the stroke index (from 42 +/- 8 to 49 +/- 7 ml/m2; P < 0.01) without changes in left ventricular ejection fraction but with a significant increase in both left and right ventricular end-diastolic volumes. Metabolic recovery induced a normalization of these parameters. INDO treatment significantly blunted these alterations. In summary, we showed that during acute insulin deficiency, INDO-sensitive mechanisms mediate vascular disturbances. Moreover, INDO treatment was capable of completely preventing the cardiac venous return and the left ventricular alterations. INDO does not interfere with the overall ketogenetic process or with insulin-induced metabolic recovery.

3-Hydroxybutyric Acid↗
PubMed1995

A linkage map with microsatellites isolated from swine flow-sorted chromosome 11.

We have developed a simple and efficient method to construct partial libraries of swine Chromosome (Chr) 11, starting with only 300 flow-sorted copies. DNA is amplified by PARM-PCR with primer containing at the 5'-end the sequence AGCU-. After amplification, digestion of PCR products with uracil DNA glycosylase generates cohesive ends corresponding to the SstI site. The amplified fragments can then be ligated in vector linearized with the SstI enzyme. Using five different primers, we PARM-PCR amplified and cloned swine Chr 11 DNA. These chromosome-specific libraries have been used to develop 14 different (TG)n microsatellites. Ten of these markers were assigned to Chr 11 by PCR analysis of a panel of Pig-Rodent somatic hybrids and by linkage analysis of the 171 individuals of the PiGMaP reference families. A complete linkage map of 147 cM of this chromosome was then realized by integrating existing markers.

Animals↗
PubMed1995

Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie.

Scrapie is a transmissible degenerative disease of the central nervous system occurring naturally in sheep. It belongs to the group of prion diseases also affecting man in which an abnormal isoform of the host-encoded prion protein (PrP) accumulating in the brain is responsible for neuronal death. Three main polymorphisms have been described in the sheep PrP gene, at positions 136, 154 and 171. A strong association between susceptibility/resistance to natural scrapie and a dimorphism at codon 136 of the ovine PrP gene has been reported in several breeds, including Romanov. This dimorphism, however, is not found in all scrapie-affected breeds. We have compared the PrP genotypes of Lacaune sheep obtained from enzootically affected flocks with those of apparently healthy sheep. A third variant at codon 171 was also evidenced. The results were compared with those obtained in a single experimental Romanov flock orally challenged with nematode parasites in which scrapie suddenly appeared and killed 80% of the sheep. We present evidence that, even in different epizootological circumstances, the major genetic factor controlling the susceptibility/resistance to natural scrapie in sheep, is represented by codon 171 genotype of the PrP gene. We also suggest that a modification of the allelic effects of codon 136 can occur in heavily infected animals.

Animals↗
PubMed1995

Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes.

One of the most widely studied simple sequences in the mammalian genome is the (TG)n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)10 probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.

Animals↗
PubMed1994

The latch region of calcineurin B is involved in both immunosuppressant-immunophilin complex docking and phosphatase activation.

The immunosuppressants cyclosporin A and FK506, when complexed with their intracellular receptors, prevent T cell activation by directly binding to the phosphatase calcineurin. We have used molecular modeling and mutagenesis to identify sites on calcineurin important for this interaction. We have created calcineurins that are resistant to both cyclosporin A and FK506 by mutating specific residues in CnB, a calcium-binding protein that regulates the catalytic subunit, CnA. Significantly, on a model of CnB, these mutations map to the latch region, an element of tertiary structure that forms when CnB binds CnA. In addition, we show that this latch region plays an important role in activating the catalytic subunit CnA. These results suggest a molecular mechanism for suppression of calcineurin by cyclosporin A and FK506 involving their binding to the same region of CnB used for allosterically activating CnA.

Amino Acid Isomerases↗
PubMed1993

Accurate characterization of porcine bivariate flow karyotype by PCR and fluorescence in situ hybridization.

The 19 chromosomal pairs of the swine karyotype are resolved into 18 peaks denoted A to Q and Y by dual-beam flow cytometry. The chromosomal content of six peaks has previously been determined by analyzing male/female differences, karyotypes of animals carrying translocations, and PCR studies of genes with known assignments. For the remaining chromosomes, putative assignments to flow peaks were deduced from comparison of DNA contents, determined by flow cytometry, and chromosomal size. We present here the complete characterization of the pig bivariate flow karyotype using the PARM-PCR technique combined with fluorescence in situ hybridization. Chromosome-specific probes were generated by PCR amplification of 300 sorted chromosomes with primers under nonspecific conditions and used to paint chromosomes by FISH. The chromosomal content of each peak was identified: peaks A (chromosome 1), B (13), C (6), D (2), E (14), F (3), G (7), H (9,4,X), H1 (9), I (15), J (8), K (5), L (10), M (12), N (16), O (11), P (17), Q (18), Y (Y). We were able to characterize perfectly the pig bivariate flow karyotype. Such techniques could be applied to any other species.

Animals↗
PubMed1993

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor.

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.

Amino Acid Sequence↗
PubMed1993

A PCR-based method to amplify DNA with random primers: determining the chromosomal content of porcine flow-karyotype peaks by chromosome painting.

We present here a new PCR-based technique that allows the production of several micrograms of DNA from only 300 flow-sorted chromosomes. During the first two PCR cycles, the annealing temperature is decreased to 30 degrees C, and numerous random loci are amplified under nonspecific conditions. As demonstrated here for pig chromosomes 1 and 18, the PCR products may be used to identify the chromosomal content of the flow-karyotype peaks of any species by fluorescence in situ hybridization.

Animals↗
PubMed1993

Clinical assessment of low molecular weight heparin effects in peripheral vascular disease.

This study was carried out, using the double-blind method vs placebo, on 36 patients suffering from stage II peripheral vascular disease (PVD) according to Leriche, to check the clinical and hemorrheologic effects of the administration of a new low molecular weight heparin (LMWH). After a one-month washout period, the patients were randomly assigned either to the group treated with the LMWH (15,000 aXaU/day sc) or to the group treated with indistinguishable placebo. At the start of the study and after three and six months of treatment, clinical, instrumental, and laboratory tests were performed to assess local and systemic efficacy and tolerance. The LMWH under study caused a statistically significant increase in claudication time, with a parallel increase in the absolute claudication distance and in the interval free of pain. The drug also led to a significant increase in activated partial thromboplastin time, the values of which, however, remained within the normal limits, and to a marked reduction in blood viscosity. No significant variation was observed in the tolerability parameters in the two treatment groups, and no local or systemic adverse reactions occurred.

Aged↗
PubMed1992

The gene map of the pig (Sus scrofa domestica L.): a review.

A review of the present status of the porcine gene map is given with references. A total of 84 loci have now been studied, and genes have been assigned to 17 chromosomes. Among them, six chromosomes are defined by only one marker. No loci have been attributed yet to three chromosomes.

Animals↗
PubMed1991

Activator-dependent and activator-independent defective recombinant retroviruses from bovine leukemia virus.

The replication-competent bovine leukemia virus (BLV) has been modified for use as a vector for foreign genes. The gag, pol, env, and pX regions of the virus were replaced by an exogenous nuclear location signal LacZ (nlsLacZ) or SVnlsLacZ gene. Transfection of the ovine cell line FLK-BLV, which expresses all BLV proteins from a wild-type provirus, with this viral DNA resulted in a viral titer of 10(4) CFU/ml. The inclusion of a large portion of the gag region did not significantly increase the titer. Both activator-dependent and activator-independent retroviruses were constructed. In activator-dependent vectors, the expression of the insert was dependent on the presence of the Tax protein, which activated the BLV long terminal repeat. In activator-independent vectors, the expression of the insert was constitutive because of the presence of an internal promoter. Infections with the recombinant retrovirus were inhibited by specific neutralizing antibodies. The structure of the transduced genetic material was not rearranged. BLV vectors encoding a reporter nlsLacZ gene, the product of which can be detected in single cells, greatly simplified studies of their biological properties. Determination of the host range of BLV vectors established that BLV-based recombinant retroviruses are effective in the transduction of genes in a variety of species and cell types.

Animals↗
PubMed1990

Xenotropic and amphotropic pseudotyped recombinant retrovirus to transfer genes into cells from various species.

The ability to transfer genes into cells from different species with murine recombinant retroviruses was evaluated with the SVnls LacZ reporter gene. Mouse and cat packaging cell lines can be used to transfer amphotropic pseudotype, in human, mouse, cat, rabbit, sheep, horse and beef cells and with a very low efficiency in pig and avian cells. Xenotropic pseudotype recombinant retroviruses, produced in cat and rabbit packaging cell lines, transferred genes with the same efficiency as amphotropic retroviruses in human, cat, rabbit and sheep cells. In contrast to amphotropic retroviruses, xenotropic retroviruses infect beef, pig and horse cells with a high efficiency. These results emphasize the need to determine carefully the producer cell line (the type of helper virus and the species origin of the cell) for efficient transfer of genes in cells and embryos.

Animals↗