New VMD2 gene mutations identified in patients affected by Best vitelliform macular dystrophy.

PURPOSE: The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin-1 (hBest1) which is a Ca(2+)-sensitive chloride channel. This study was performed to identify disease-specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl(-) channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease. METHODS: Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK-293 cells and the associated Cl(-) current was examined using whole-cell patch clamp analysis. RESULTS: Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non-functional. Furthermore, the Q293H mutant inhibited the function of wild-type bestrophin-1 channels in a dominant negative manner. CONCLUSIONS: This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease-linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane.

Reduced hepatic fatty acid oxidation in fasting PPARalpha null mice is due to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression.

Glucose and fatty acid metabolism (oxidation versus esterification) has been measured in hepatocytes isolated from 24 h starved peroxisome proliferator-activated receptor-alpha (PPARalpha) null and wild-type mice. In PPARalpha null mice, the development of hypoglycemia during starvation was due to a reduced capacity for hepatic gluconeogenesis secondary to a 70% lower rate of fatty acid oxidation. This was not due to inappropriate expression of the hepatic CPT I gene, which was similar in both genotypes, but to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression in the PPARalpha null mouse liver. We also demonstrate that hepatic steatosis of fasting PPARalpha null mice was not due to enhanced triglyceride synthesis.

Comparison of hepatic and renal drug-metabolising enzyme activities in sheep given single or two-fold challenge infections with Fasciola hepatica.

The activity of drug-metabolising enzymes was compared in liver and kidneys of adult sheep given single or two-fold fluke infection. Fascioliasis was induced by oral administration of 200 metacercariae of Fasciola hepatica to female sheep either 10 or 20 weeks (mono-infections) or 10 and 20 weeks (bi-infection) before killing. The parasitic pathology was ascertained at autopsy and by clinical observation of animals. In the liver of both mono- and bi-infected animals, significant decreases (P<0.05) (17-44%) were observed in the microsomal content of cytochrome P450 and in the two measured P450-dependent monooxygenase activities, benzphetamine and ethylmorphine N-demethylations. Moreover, Western blot analysis of microsomes demonstrated a decrease in the expression of cytochrome P4503A subfamily correlative with that of its presumed corresponding activity ethylmorphine N-demethylase. By contrast, the conjugation of chloro-dinitrobenzene to glutathione remained unchanged in liver cytosolic fractions prepared from all these animals. In kidneys, a significant decrease (P<0.05) (30%) in microsomal cytochrome P450 level of 10-week mono-infected sheep was observed whereas there was no change in the other groups of animals. The inflammatory origin and the consequences in terms of pathology and animal productivity of the fascioliasis-induced decreases in tissue-oxidative drug metabolism are discussed, particularly in the case of adult sheep suffering repetitive infections.

The Relative Semi-quantification of mRNA Expression as a Useful Toxicological Endpoint for the Identification of Embryotoxic/Teratogenic Substances.

Embryonic stem cells, which resemble the undifferentiated cells of the epiblast in a blastocyst, are able to differentiate into derivatives of the primary germ layers, including cardiomyocytes. The effects of embryotoxic/teratogenic compounds on the differentiation of cells was examined by semi-quantification of the mRNA expression using the RT-PCR protocol. Alpha and beta myosin heavy chain (alpha-MHC and beta-MHC, respectively) mRNA expression were chosen as tissue-specific markers, characteristic of early cardiac muscle development. Nine chemicals were investigated, chosen according to their in vivo embryotoxic/teratogenic potential in mice. The teratogens all-trans retinoic acid (RA), 5-fluorouracil (5-FU), hydroxyurea (HU), diphenylhydantoin (DPH) and caffeine (Caff) caused a significant reduction in MHC mRNA expression at a dose lower than that required for cytotoxicity. Saccharin (Sacc) had a similar effect, while penicillin G (PenG), isoniazid (Iso) and cytarabine (Ara-C) only showed an effect on MHC mRNA expression when the cells had a significant loss in viability. The inability to identify the strong teratogen Ara-C is related to the inappropriate target tissue.

Establishment of an in vitro reporter gene assay for developmental cardiac toxicity.

This study is based on the unique potential of pluripotent embryonic stem (ES) cells to differentiate in vitro into embryoid bodies containing cell lineages representative of most cell types found in the mammalian fetus. However, the use of wild type ES cells as an in vitro assay for embryotoxicological studies is complicated by the simultaneous development of various cellular phenotypes. This prevents a quantitative assessment of drug effects on one specific cell type. Here we report the effects of 15 chemicals on cardiac differentiation as determined by various specific toxicological endpoints such as morphological inspection (contractile activity), quantitative mRNA analysis and cardiac-specific expression of green fluorescent protein (GFP), used as a quantitative reporter. The data from the different endpoints have been subjected to a statistical analysis, and a preliminary prediction model is proposed. The results demonstrate that genetically-engineered ES cells could provide a valuable tool for estimating the developmental cardiotoxic potential of compounds in vitro and form the basis for automated analysis in a high-throughput system.

Effects of delayed feed intake on body, intestine, and muscle development in neonate broilers.

The short-term effects of delayed feeding (DF) for 2 d posthatching were measured in neonate chicks and compared to early feeding (EF). Chicks from 10 independent families were used in this study to determine whether genetic background control of growth may be influenced by EF and DF. Early feeding maintained significant interfamily body weight variations from hatch to 4 d of age, whereas there were no significant differences from 1 d of age when feeding was delayed to 48 h posthatching. These results suggest that posthatching feeding delay may distort genetic selection by masking the expression of genetic potential and disturbing the estimation of chick breeder value. In DF chicks, overall body growth was delayed until the beginning of feeding and body weight at 6 d of age was 25% lower than EF chicks. Availability of feed after the fasting period was not sufficient to compensate for the retardation of weight gain in either body weight or in intestine and breast muscle weight. However, initiation of intestine growth in DF chicks occurred from 1 d of age despite the lack of feeding, whereas feed intake was essential to enhance muscle growth. The potential for protein synthesis was lower in DF than in EF chicks during the first 2 d posthatching (P < 0.001) and then reached similar values after feed intake. These results confirm that initiation of growth in neonate chicks is improved by earlier feeding after hatching. Awareness of changes in overall body weight caused by posthatching food deprivation, especially in the intestine and muscle might help in the development of new diets which could minimize retardation of body weight gain in chicks.

Is ribosomal capacity a potential metabolic marker of muscle development? Measurement by muscular biopsy.

Metabolic markers of muscle metabolism could help geneticists and nutritionists predict the breast meat development of chickens. The aim of the current study was to test the ribosomal capacity (CS), a potential metabolic marker, and to evaluate a simple biopsy method on the pectoralis major muscle. Ribosomal capacity was measured in three commercial meat chicken genotypes differing in their growth rate. Fast-, medium-, and slow-growing male chickens were fed using three commercial dietary programs of increasing energy and protein concentration (nine treatments). Biopsy was performed at 4 wk of age on the p. major 12 chickens per treatment. Fast-, medium-, and slow-growing chickens were slaughtered at market weight, i.e., 6, 8, and 12 wk of age, respectively, and breast meat (right and left p. major + minor) was dissected. A significant reduction in BW at slaughter (-1 to -3%) and breast meat yield (-4%) occurred only in biopsied medium-growing chickens but not in the other two types. Slow-growing chickens had a significantly lower CS (8.75 microg/mg) than the two other chicken types (9.40 and 9.46 microg/mg for fast- and medium-growing chickens, respectively). No significant dietary effect or interaction of dietary treatment with genotype was measured. The CS was not significantly correlated to breast meat development. Under conditions of the present experiment, CS may not be a relevant marker of subsequent breast meat development at 4 wk of age. The biopsy technique can easily be applied to other markers.

Early-age thermal conditioning reduces uncoupling protein messenger RNA expression in pectoral muscle of broiler chicks at seven days of age.

Early-age thermal conditioning (TC) by exposing young chicks to 40 C for 24 h reduces body temperature (Tb) and has been showed by others to improve long-term resistance of broilers to heat stress. Uncoupling oxidative phosphorylation in pectoral muscle mitochondria might be related to heat production. Fertile eggs were hatched under video control, and 161 pedigree chicks froml2 sires and 22 dams were immediately allocated to two groups (T, a group composed of 81 chicks exposed to TC at 5 d of age, and N, a control group of 80 nonexposed chicks). Body weights and Tb were measured at 2 and 7 d of age. Five pairs (one N and one T) of full sib chicks from families that exhibited the largest difference of Tb variation from 2 to 7 d of age between the two treatments were chosen for pectoral muscle sampling. Avian uncoupling protein (avUCP) messenger RNA expression was measured by reverse transcript-PCR coupled to southern blot in the pectoral muscle of 7-d-old broiler chicks. At 7 d of age, there were no BW differences between treatments and Tb was significantly reduced by TC (-0.13 C on average). Heritability of Tb variation between 2 and 7 d was 0.38 +/- 0.20 (SE) for T chicks and 0.35 +/- 0.17 for N chicks without a significant genetic correlation between the two environments. Expression of avUCP mRNA was significantly (85%) lower in T chicks than in N chicks. Uncoupling protein mRNA expression in pectoral muscle and Tb are quickly adjusted in broiler chicks 24 h after early thermal conditioning.

Fasciola hepatica: influence of gender and liver biotransformations on flukicide treatment efficacy of rats infested and cured with either clorsulon/ivermectin or triclabendazole.

Two fasciolicide preparations have been compared in 130 rats experimentally infected with Fasciola hepatica. Parasitological, immunological, and biochemical parameters have been followed to monitor the efficacy of the treatments. While Fascinex (triclabendazole) efficiently cured both male and female rats when administered as soon as 4 weeks postinfection, treatment with Ivomec-D (clorsulon + ivermectin) displayed a low efficacy on either male or female rats at this time point (54 and 0%, respectively). Moreover, when administered 8 weeks postinfection, the Ivomec-D treatment proved highly efficient on male rats while it displayed little effect on the female population (100 and 53%, respectively). This unexpected result has been related to an overexpression of a P4503A isoform that is observed only in females that have been treated with Ivomec-D. The influence of this P4503A cytochrome on drug metabolism and the need for the incorporation of both genders in clinical trials are discussed.

Paralysie faciale peripherique isolee et serologique positive du VIH : une etude de 6 cas au CNHU de Cotonou (Benin)

Une etude prospective de 6 cas de paralysie faciale peripherique inaugurant un SIDA; observes au CHNU de Cotonou au Benin; est rapportee. La paralysie a constitue une complication initiale de l'infection a VIH dans tous les cas. Elle etait droite dans 4 cas et gauche dans 2 cas; complete dans 5 cas; et de survenue brutale chez tous les patients. Le liquide Cephalo-rachidien (LCR) etait a anormal 2 fois. La recuperation a ete totale dans la plupart des cas. Une redicive a ete pourtant notee

Discrimination de l'infection VIH-1/VIH-2 par l'enzymum test anti HIV-1/HIV-2 et le pepti LAV1; 2

Dans cet article; les auteurs etudient le pouvoir de discrimination entre VIH-1 et VIH-2 de l'Enzymum Test et du PeptiLAV vis-a-vis de serums de seropositifs residents beninois. Les resultats obtenus montrent une concordance parfaite entre l'Enzym Test et le PeptiLAV pour tous les 77 echantillons. On observe aussi une concordance entre le PeptiLAV; l'Enzymum Test et le WB en ce qui concerne les echantillons de seropositifs infectes par le VIH-1 et le VIH-2 seul. Par contre 5 cas de discordance sont observes sur les 28 serums doublement positifs au WB1 et au WB2. Ces 5 serums se comportent comme ceux de seropositifs infectes par le VIH-2 dans le PeptiLAV et l'Enzymum Test. La discordance observee par les auteurs entre le Western Blot et tous les deux autres tests est moindre que celle rapportee dans la litterature a propos des doubles seropositifs. Elle residerait pour une grande part dans les differences des criteres d'interpretation du WB

Séroprévalence de l'antigène HBs du virus de l'hépatite chez les femmes enceintes et leurs enfants

Une etude prospective a determine la seroprevalence de l'antigene HBs (AgHBs) du virus de l'hepatite B (VHB) chez les femmes enceintes et les enfants nes de meres seropositives. La technique utilisee etait l'Elisa; suivie de la confirmation par la methode de neutralisation. Sur 1;017 gestantes testees; 84 (8;26 pour cent) etaient porteuses de l'AgHBs. Parmi celles-ci; 40 avaient ete suivies et etaient restees seropositives apres l'accouchement. Sur les 40 enfants nes de ces dernieres; l'AgHbs etait detecte dans le sang chez 8 d'entre eux (20 pour cent) dans un delai de 1 jour a 11 mois apres la naissance