Molecular characterization of field isolates of lentiviruses of small ruminants.
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Animals↗
Biomedical subjects
C Leroux
Publications and source records attributed to C Leroux.
The expression pattern of alfalfa flavanone 3-hydroxylase promoter-gus fusion in Nicotiana benthamiana correlates with the presence of flavonoids detected in situ.
Flavanone 3-hydroxylase is an enzyme acting in the central part of the flavonoid biosynthesis pathway. It is generally encoded by a single gene and seems to have a key position for the regulation in this pathway. These two features make a single f3h promoter-gus fusion a suitable tool to study both the f3h expression and the regulation of this pathway. We present here the spatial and temporal analysis of the expression of an alfalfa flavanone 3-hydroxylase (f3h) promoter-gus fusion introduced into Nicotiana benthamiana. The Medicago sativa (alfalfa) f3h promoter directed gus expression in flowers, stems, leaves and roots. In flowers, GUS activity was observed in pollen grains, in ovules, in ovary placenta and in the epidermis, medullary parenchyma, trichomes and second cortical cellular layer surrounding the vascular bundles of the peduncle. In stems, GUS activity was detected at the same places as in the peduncle except for the medullary parenchyma. In roots, we found GUS staining in root hairs, epidermis and in the vascular bundles of the elongated zone. Finally, in leaves, the f3h promoter expressed essentially in the stalk cells of the multicellular trichomes. The expression pattern of the f3h-gus fusion was correlated to the presence of flavonoids in situ. These data indicate that this construct can be very useful to study factors controlling the production of flavonoids.
Base Sequence↗
Northern analysis of highly folded goat alpha S1 casein mRNA.
Northern blotting using glyoxal to denature a highly folded mRNA, such as goat alpha S1-Casein E, can lead to the detection of multiple incompletely denatured forms. Formaldehyde appears to be the most suitable agent for Northern blotting due to its effective denaturing capacity and lower toxicity than methylmercuric hydroxide.
Animals↗
Relation of intima-media thickness to atherosclerotic plaques in carotid arteries. The Vascular Aging (EVA) Study.
This study examined the relation between arterial wall thickness and local atherosclerosis in the carotid arteries (CAs) and their specific risk factors. B-mode ultrasonography of the CAs was performed in a cohort of 516 men and 756 women aged 59 to 71 years who had been recruited for the European Vascular Aging Study. Ultrasound examination included measurement of intima-media thickness of the common CA (CCA) and the sites of plaque in the internal CA and bifurcations. Significant associations between increases in CCA intima-media thickness and both the presence and severity of atherosclerotic plaque were found in men and women. Examination of specific risk factors for increases in CCA intima-media thickness in the presence of plaque showed that, after adjustment for sex, both ultrasound measurements were independently related to age, body mass index, hypertension, and ever smoking (versus never smoking). Diabetes and current smoking were associated with intima-media thickness only, whereas hypercholesterolemia was related to plaque only. However, when subjects who were taking lipid-lowering drugs were excluded, lipoproteins and apolipoproteins were more consistently related to intima-media thickness than to plaque. In subjects free from any antihypertensive treatment, both intima-media thickness and plaques were independently associated with systolic blood pressure. After adjustment for sex and other risk factors, the odds ratio for having at least one plaque associated with a 0.10-mm increase in CCA intima-media thickness was 1.18 (95% confidence interval, 1.05 to 1.32). In this relatively aged population, increases in intima-media thickness as measured in the CCAs were clearly related to locally detected atherosclerosis and known risk factors for atherosclerosis. Longitudinal studies are needed to clarify the role of arterial wall thickening in the atherosclerotic process.
Aged↗
[Spiral cholangioscanners and tridimensional reconstructions of the biliary tract in children].
PURPOSE: endoscopic retrograde cholangiography may be difficult or unfeasible in children. It also may be complicated by acute pancreatitis. The purpose of this study was to determine the diagnostic contribution of spiral CT cholangiography in pediatric patients. SUBJECTS AND METHODS: seven patients, age ranging from 10 months to 13 years were explored without general anesthesia. Eight spiral CT cholangiographies were performed. The patients were suspected to have biliary or pancreatic lesions. Spiral CT was performed 60 minutes after slow infusion of iodipamide (Transbilix-Guerbet). The dose was correlated to the body surface. 3D reconstructions were done using the surface rendering or the MIP technique. RESULTS: biliary tract opacification and 3D reconstructions were considered of good quality in 6 out of 8 studies. It was normal in 2 cases. A duplication of the duodenum communicating with the choledocus was found in 1 case. Biliary lithiasis with a stone in the cystic duct was found in 1 case. Two choledocal cysts with bile ducts dilatation were diagnosed. A post operative biliary leak with bile ducts dilatation was found in one case. The spiral CT was not diagnostic in 2 patients: a case of choledocal cyst, the study was of poor quality due to inadequate sedation and a case of Caroli's disease with a high serum bilirubin level. Four patients had an endoscopic or a percutaneous cholangiography: to explore the pancreatic ducts (2 cases), poor CT study due to the inadequate sedation in 1 child, and the case of Caroli's disease. CONCLUSION: spiral CT is feasible even in young children. It should reduce the indications for endoscopic or percutaneous cholangiography. It has to be performed as the first examination in cases of biliary or pancreatic diseases when surgery is contemplated.
Adolescent↗
[Surgical cure of postoperative eventrations with intraperitoneal implantation of dacron mesh. Apropos of 160 operated cases].
A series of 160 patients with large incisional hernia underwent surgical repair involving the intraperitoneal positioning of a Dracon mersh. All patients were reexamined three years after operative procedure. Mortality was 1.2%. Four patients (2.5%) presented deep wound infection with removal of the mesh. Seven recurrences (4.3%) were reported. In conclusion, large incisional abdominal hernia can be treated successfully by an intraperitoneal Dracon mesh.
Adolescent↗
Ovine aortic smooth muscle cells allow the replication of visna-maedi virus in vitro.
Visna-maedi virus induces in sheep an interstitial lung disease characterised by an accumulation of smooth muscle cells (SMC) or myomatosis. Infection by HIV-1 has been recently associated with disorders of the vessel-derived cells: primary pulmonary hypertension, coronary artery disease and smooth muscle tumors in humans. We hypothesized that, besides their regular targets (i.e. macrophages and lymphocytes), lentiviruses could infect smooth muscle cells. Smooth muscle cell cultures derived from ovine aorta were infected with visna-maedi virus strain K1514. The cultured cells were smooth muscle cells as demonstrated by their antigenic expression of alpha-actin and vimentin. The lentiviral infection of the smooth muscle cells was demonstrated by a typical cytopathic effect (syncytia), the expression of virus specific antigens, and the presence of genomic RNA detected by Northern blot analysis and RT PCR. The detection of a reverse transcriptase activity, the presence of viral RNA in supernatants of infected smooth muscle cells detected by RT PCR and their ability to infect ovine permissive fibroblasts demonstrated a productive infection. The ability of smooth muscle cells to be infected by lentiviruses may participate in the pathogenesis of the tissue damage associated with the lentiviruses such as myomatosis in sheep and vascular disease in humans.
Animals↗
Genomic heterogeneity in the pol region of ovine lentiviruses obtained from bronchoalveolar cells of infected sheep from France.
In order to determine the genomic heterogeneity of ovine lentiviruses, we analysed eight isolates from naturally infected sheep from one geographical region of France. A 475 nt fragment in the region of the pol gene coding for reverse transcriptase was amplified by RT-PCR from RNA directly extracted from uncultured bronchoalveolar lavage cells. The resulting PCR fragments were analysed by restriction enzyme digestion, cloned in a TA vector and sequenced. Restriction enzyme analysis showed distinct patterns from the eight isolates, and sequencing showed them to be closely related in both nucleotide (2.3-8.1% variation) and deduced amino acid (0-6.2% variation) sequences. Their amino acid sequences differed from that of visna-maedi virus complete viral genome sequence K1514 by 12.5-15.3%, but from that of caprine arthritis encephalitis virus (CAEV) viral genome sequence Co by only 4.2-6.9%. Phylogenetic analysis showed that the French isolates form a group related to CAEV Co and distant from previously reported ovine lentivirus sequences from different origins.
Amino Acid Sequence↗
Occurrence of a LINE sequence in the 3' UTR of the goat alpha s1-casein E-encoding allele associated with reduced protein synthesis level.
The E allele of the alpha s1-casein (alpha s1-Cas)-encoding gene is associated with a reduced casein content in milk. Structural analysis of mRNA and sequencing of amplified genomic DNA fragments, have revealed that this allele contains a 457-bp insertion within exon 19 (last untranslated exon). This insert is a truncated long interspersed repeated element (LINE) containing part of the ORF-2, the 3' UTR and the poly(A) tail of the original retroposon. This LINE sequence was found to be highly repeated in the goat genome. The threefold reduction in the amount of alpha s1-CasE mRNA in the total, as well as in the polysomal fractions, as compared with its A counterpart, leads one to suppose either a reduced transcriptional rate of allele E or/and a decreased stability of the relevant mRNA. Northern blot and PCR experiments suggested the setting up of highly stable secondary structures involving the 3' UTR of the alpha s1-CasE transcript. The mRNA folding calculations support such an hypothesis, by base-pairing interactions between the E allele messenger poly(A) tail and a long poly(U)-stretch occurring at the 5' end of the insertion. In addition, since A+U-rich motifs are also present in the truncated LINE insertion, we propose that this extra sequence might be responsible for the lower milk alpha s1-Cas content by reducing the allele E mRNA stability.
Alleles↗
A microsatellite within the bovine kappa-casein gene reveals a polymorphism correlating strongly with polymorphisms previously described at the protein as well as the DNA level.
The polymorphism of a (TA)n(CA)n repeat microsatellite present in the third intron of the bovine kappa-casein gene (CASK) has been investigated. The existence of six alleles differing only in the number of dinucleotide repeats has been established. A total of 330 animals belonging to nine different pure bred Bos taurus French breeds or to a cross-bred Bos taurus x Bos indicus population (Créole) were genotyped. The distribution of the microsatellite alleles was examined and clear breed differences were noted. Genotyping of animals by isoelectric focusing (IEF) or restriction fragment length polymorphism (RFLP) (TaqI) was performed, in order to examine the relationship of the microsatellite polymorphism to other previously described CASK polymorphisms, at the protein and DNA levels. Strong correlation was seen, indicating that evolution of the various polymorphisms was not independent, and nine CASK haplotypes were observed.
Alleles↗
Prolactin gene expression in ovine and caprine mammary gland.
The presence of prolactin (PRL) mRNA in the mammary gland of lactating goats and sheep was demonstrated by Northern analysis and RT-PCR. This provides evidence that the PRL gene is transcribed in this tissue. This ectopic expression is not restricted to the lactational period, as PRL transcripts were also found during the last third of pregnancy. Comparison of mammary and pituitary PRL mRNAs showed that they are similar in size but less abundant in mammary gland. In addition, an 847-bp cDNA fragment amplified from mammary retrotranscripts, containing the entire coding region and the major part of the 5' and 3' untranslated regions (UTRs), was found to be identical in sequence to its pituitary counterpart. Primer extension analysis, performed to obtain further information on the structure of the mammary PRL mRNA, has shown that the 5' UTR is 56 nucleotides (nt) long for both species. This is comparable with the size (53 nt) found using the caprine pituitary RNA as template. These results strongly suggest that the PRL gene is not transcribed from a different promoter in mammary gland, as has been demonstrated for placental and lymphocyte cells, but is more likely transcribed from the pituitary-specific promoter. Finally, the presence of PRL mRNA in polysomal fractions suggests that PRL is synthesized in mammary cells.
Amino Acid Sequence↗
From granuloma to fibrosis in interstitial lung diseases: molecular and cellular interactions.
Granuloma is a feature of many chronic interstitial lung diseases, and may serve as a focus for subsequent fibrosis. Granulomas are composed of structured masses of cells of the macrophage lineage, which adopt an epithelioid aspect, interspersed with lymphocytes. They are formed around local centres of irritation. During their resolution, fibroblasts congregate around the structures and may penetrate the interior. In many cases, granulomas can disappear without leaving lasting traces. However, especially when damage has occurred to the surrounding tissue, permanent scarring and fibrosis may occur. Both types of cell present in the granuloma are capable of secreting a number of factors influencing the accumulation and proliferation of fibroblasts, both positively and negatively. The possible roles played by the different factors and, especially, interactions between them are discussed in the light of fibrosis formation. Possible therapeutic interventions are summarized.
Cytokines↗
[Fatigue fractures].
Stress fractures are extremely common microtraumatic lesions which occur in specific areas of healthy bones. Since clinical findings are often misleading and radiological signs are known to be delayed, diagnosis can be uneasy. In some instances, infectious or tumoral diseases have to be ruled out. Such difficult cases are shown to stress on the potential interest of CT scanner and magnetic resonance imaging.
Fractures, Stress↗
Mutations away from splice site recognition sequences might cis-modulate alternative splicing of goat alpha s1-casein transcripts. Structural organization of the relevant gene.
alpha s1-Casein variants F and D, synthesized in goat milk at lower levels than variant A, essentially differ from it by internal deletions of 37 and 11 amino acid residues, respectively. Northern blot analysis of mRNAs encoding alpha s1-casein F and A and sequencing of the relevant cloned cDNAs, as well as sequencing of in vitro amplified genomic fragments, revealed multiple alternatively processed transcripts, from the F allele. Although correctly spliced messengers were identified, most of the FmRNAs lacked three exons. These exons, further identified as exons 9, 10, and 11, together encode the 37 amino acid residues present in alpha s1-casein variant A but missing in variant F. Exon 9 codes for the sequence present in variant A but deleted in variant D. A single nucleotide deletion in exon 9 and two insertions, 11 and 3 base pairs in length, in the downstream intron, were identified as mutations potentially responsible for the alternative skipping of these 3 exons. From a computer-predicted secondary structure it appeared that the 11-base pair insertion might be involved in base-pairing interactions with the intron 5' splice site which might consequently be less accessible to U1 snRNA. We also report here the complete structural organization of the goat alpha s1-casein transcription unit, deduced from polymerase chain reaction experiments. It contains 19 exons scattered within a nucleotide stretch nearly 17-kilobase pairs long.
Amino Acid Sequence↗
Exon-skipping is responsible for the 9 amino acid residue deletion occurring near the N-terminal of human beta-casein.
Interspecies comparison and alignment of the beta-casein N-terminal sequence, taking into account its exon modular splitting derived from the known structural organization of the relevant genes, has revealed that a 9 amino acid residue sequence, corresponding to that encoded by the third exon of the other species genes, is lacking in human beta-casein. Using the polymerase chain reaction technique, we have amplified a human genomic 1-kb fragment, spanning from exon 2 to exon 4, which was subsequently cloned and sequenced. One hundred base pairs (bp) upstream from exon 4 and 737 bp downstream of exon 2, a 27-bp virtual exon 3 sequence, probably skipped during the course of pre-mRNA splicing, was identified. We discuss the possibility that this out-splicing event might be due to the weak strength of the 3' acceptor site and/or to the secondary structure sequestering of the branch site sequence.
Amino Acid Sequence↗
[Epidermoid cyst of the base of the skull].
Epidermoid cysts are rare congenital tumours representing less than 1% of all intracranial tumours. They are derived from the development of abnormally situated ectodermal tissue and are often an incidental finding. We report a case confined to the base of the skull, responsible for an extradural mass.
Adult↗
Restriction fragment length polymorphism identification of goat alpha s1-casein alleles: a potential tool in selection of individuals carrying alleles associated with a high level protein synthesis.
The extensive polymorphism of caprine alpha s1-casein, which is controlled by at least seven autosomal alleles segregating in a Mendelian fashion, was investigated by RFLP analysis. Genomic DNA from 77 lactating goats, whose genotypes had been previously determined by electrophoretic analysis of milk proteins, was digested with 11 restriction endonucleases and Southern blots were probed with a radiolabelled ovine alpha s1-casein cDNA. Three enzymes, PstI, TaqI and Rsa I, allowed the unambiguous identification of known alleles alpha s1-CnA, E and O and of the allelic pairs [alpha s1-CnD and F] and [alpha s1-CnB and C]. Evidence for a second null allele, termed alpha s1-CnO', and for an additional allele, designated alpha s1-CnF', was provided, which leads to the identification of nine alleles at the alpha s1-Cn locus, in this species. Although only 15 out of the 45 expected genotypes could be fully ascertained, this procedure allows the identification at birth of animals carrying the alpha s1-CnA, B or C alleles associated with a high alpha s1- and whole-casein content.
Alleles↗
Integration and germ line transmission of foreign genes microinjected into fertilized trout eggs.
Persistence, integration into host genome, germ line transmission and expression of foreign genes microinjected into cytoplasm of fertilized rainbow trout eggs has been examined. Foreign DNA persisted as large random concatenates in approximately 50% of 6 to 12 month-old trout and exhibited a mosaic pattern between tissues. In some cases, free concatenates were observed indicating that extrachromosomal replication occurred in trout. Approximately 50% of the males had the foreign sequences in sperm DNA and all the examined animals transmitted these sequences to their progeny. The percentage of transgenic offsprings ranged from 10 to 30% and putative junction fragments were identified in Southern blot analysis in some of them. These results strongly support the hypothesis that the injected genes became integrated into the genome host, most likely after the first round of chromosomal replication. We also examined the expression of the microinjected plasmids which contained viral or mammalian promoters linked to human or rat growth hormone gene. In no case could exogenous growth hormone be detected.
Animals↗