Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg(2+)-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.     

Biologic, morphologic, and molecular characterisation of Neospora caninum isolates from littermate dogs

Isolation and biologic and molecular attributes of Neospora caninum from three littermate dogs are described. Tissue cysts were confined to the brain and striated muscles. N. caninum was isolated (isolates NC-6, NC-7, and NC-8) in rodents and cell culture that had been inoculated with brain tissue from the dogs. Schizont-like stages reactive with N. caninum antibodies were seen in cell cultures seeded with bradyzoites released from Percoll-isolated N. caninum tissue cysts from the brain of one dog. Tissue cysts were infective orally to mice and gerbils, but not to cats and dogs. The isolates were also identified as N. caninum by PCR and sequence analysis.     

Isolation and genotyping of Toxoplasma gondii from free-ranging chickens from Argentina

The prevalence of Toxoplasma gondii in free-ranging chickens can be considered a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 29 free-range chickens (Gallus domesticus) from Argentina was investigated. Blood, heart, and brain from each chicken were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT), were found in 19 of 29 (65.5%) chickens. Hearts and brains of seropositive (MAT > or = 1:5) chickens were bioassayed in mice. Toxoplasma gondii was isolated from 9 of 19 seropositive chickens. Genotyping of chicken isolates of T. gondii using the SAG2 locus indicated that 1 was type I, 1 was type II, and 7 were type III. This is the first report of isolation of T. gondii from chickens from Argentina.     

Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells

An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of NT embryos up to 40 days of gestation.     

Experimental infection of ponies with Sarcocystis fayeri and differentiation from Sarcocystis neurona infections in horses

Sarcocystis neurona and Sarcocystis fayeri infections are common in horses in the Americas. Their antemortem diagnosis is important because the former causes a neurological disorder in horses, whereas the latter is considered nonpathogenic. There is a concern that equine antibodies to S. fayeri might react with S. neurona antigens in diagnostic tests. In this study, 4 ponies without demonstrable serum antibodies to S. neurona by Western immunoblot were used. Three ponies were fed 1 x 10(5) to 1 x 10(7) sporocysts of S. fayeri obtained from dogs that were fed naturally infected horse muscles. All ponies remained asymptomatic until the termination of the experiment, day 79 postinoculation (PI). All serum samples collected were negative for antibodies to S. neurona using the Western blot at the initial screening, just before inoculation with S. fayeri (day 2) and weekly until day 79 PI. Cerebrospinal fluid samples from each pony were negative for S. neurona antibodies. Using the S. neurona agglutination test, antibodies to S. neurona were not detected in 1:25 dilution of sera from any samples, except that from pony no. 4 on day 28; this pony had received 1 X 10(7) sporocysts. Using indirect immunofluorescence antibody tests (IFATs), 7 serum samples were found to be positive for S. neurona antibodies from 1:25 to 1:400 dilutions. Sarcocystis fayeri sarcocysts were found in striated muscles of all inoculated ponies, with heaviest infections in the tongue. All sarcocysts examined histologically appeared to contain only microcytes. Ultrastructurally, S. fayeri sarcocysts could be differentiated from S. neurona sarcocysts by the microtubules (mt) in villar protrusions on sarcocyst walls; in S. fayeri the mt extended from the villar tips to the pellicle of zoites, whereas in S. neurona the mt were restricted to the middle of the cyst wall. Results indicate that horses with S. fayeri infections may be misdiagnosed as being S. neurona infected using IFAT, and further research is needed on the serologic diagnosis of S. neurona infections.     

Molecular evolution and genetics of the Saitohin gene and tau haplotype in Alzheimer's disease and argyrophilic grain disease

A single nucleotide polymorphism that results in an amino acid change (Q7R) has been identified in the Saitohin (STH) gene and was initially found to be over-represented in the homozygous state in subjects with late-onset Alzheimer's disease (AD). More extensive studies provide limited support for the association with AD, but confirm an association of the Q allele with progressive supranuclear palsy and argyrophilic grain disease. A homologous sequence was found in the appropriate location of the rat and mouse tau genes, but there was no open reading frame allowing STH expression in these species, suggesting relatively recent evolution of this gene. In some non-human primates, the STH gene was identified, and this was found to differ from the human gene at two of 128 amino acids. All primates in which the STH gene was identified were homozygous for the R allele of STH, suggesting this is the ancestral allele. This observation was surprising, in that the Q allele is more common in human populations, and raises the possibility that natural selection has operated to favor individuals carrying this allele. The STH polymorphism is part of the tau gene haplotype, of which two major variants exist in human populations, the Q being part of the H1 haplotype and the R part of the H2 haplotype. More detailed studies confirm the H2 haplotype to be the ancestral tau gene. This situation is reminiscent of the evolution of the apolipoprotein (ApoE) gene, another locus that is potentially important for the risk of development of AD.     

Isolation, tissue distribution, and molecular characterization of Toxoplasma gondii from chickens in Grenada, West Indies

The prevalence of Toxoplasma gondii in free-range chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 102 free-range chickens (Gallus domesticus) from Grenada was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 53 (52%) chickens with titers of 1:5 in 6, 1:10 in 4, 1:20 in 4, 1:40 in 4, 1:80 in 15, 1:160 in 9, 1: 320 in 5, 1:640 in 4, and 1:1,280 or greater in 2. Hearts, pectoral muscles, and brains of 43 seropositive chickens with MAT titers of 1:20 or greater were bioassayed individually in mice. Tissues of each of 10 chickens with titers of 1:5 and 1:10 were pooled and bioassayed in mice. Tissues from the remaining 49 seronegative chickens were pooled and fed to 4 T. gondii-free cats. Feces of cats were examined for oocysts; they did not shed oocysts. T. gondii was isolated from 35 of 43 chickens with MAT titers of 1:20 or greater; from the hearts, brains, and pectoral muscles of 2, hearts and brains of 20, from the hearts alone of 11, and brains alone of 2. T. gondii was isolated from 1 of 10 chickens with titers of 1:5 or 1:10. All 36 T. gondii isolates were avirulent for mice. Genotyping of these 36 isolates using polymorphisms at the SAG2 locus indicated that 29 were Type III, 5 were Type I, 1 was Type II, and 1 had both Type I and Type III. Genetically, the isolates from Grenada were different from those from the United States; Type II was the predominant type from the United States. Phenotypically, all isolates from Grenada were avirulent for mice, whereas those from Brazil were mouse-virulent. This is the first report of isolation of T. gondii from chickens from Grenada, West Indies.     

First biologic and genetic characterization of Toxoplasma gondii isolates from chickens from Africa (Democratic Republic of Congo, Mali, Burkina Faso, and Kenya)

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. In the present study, prevalence of T. gondii in chickens from Democratic Republic of Congo, Mali, Burkina Faso, and Kenya is reported. The prevalence of T. gondii antibodies in sera of 50 free-range chickens from Congo was 50% based on the modified agglutination test (MAT); antibody titers were 1:5 in 7, 1:10 in 7, 1:20 in 6, 1:40 in 1, and 1:160 or more in 4 chickens. Hearts, pectoral muscles, and brains of 11 chickens with titers of 1:20 or more were bioassayed individually in mice; T. gondii was isolated from 9, from the hearts of 9, brains of 3, and muscles of 3 chickens. Tissues of each of the 14 chickens with titers of 1:5 or 1:10 were pooled and bioassayed in mice; T. gondii was isolated from 1 chicken with a titer of 1:10. Tissues from the remaining 25 seronegative chickens were pooled and fed to 1 T. gondii-free cat. Feces of the cat were examined for oocysts, but none was seen. The results indicate that T. gondii localizes in the hearts more often than in other tissues of naturally infected chickens. Genotyping of these 10 isolates using the SAG2 locus indicated that 8 were isolates were type III, 1 was type II, and 1 was type I. Two isolates (1 type I and 1 type III) were virulent for mice. Toxoplasma gondii was isolated by mouse bioassay from a pool of brains and hearts of 5 of 48 chickens from Mali and 1 of 40 chickens from Burkina Faso; all 6 isolates were avirulent for mice. Genetically, 4 isolates were type III and 2 were type II. Sera were not available from chickens from Mali and Burkina Faso. Toxoplasma gondii antibodies (MAT 100 or more) were found in 4 of 30 chickens from Kenya, and T. gondii was isolated from the brain of 1 of 4 seropositive chickens; this strain was avirulent for mice and was type II. This is the first report on isolation and genotyping of T. gondii from any source from these 4 countries in Africa.     

Pretraining but not Preexposure to the Task Apparatus Prevents the Memory Impairment Induced by Blockade of Protein Synthesis,PKA or MAP Kinase in Rats

Adult male Wistar rats were trained and tested in a step-down inhibitory avoidance task (0.4 mA footshock, 24 h training-test interval). Fifteen minutes before or 0, 1.5 or 3 hours after training, animals received a 0.8 l intrahippocampal infusion of the protein synthesis inhibitor anisomycin (80 g), the PKA inhibitor Rp-cAMP (0.05 g), the MAPK kinase inhibitor PD 098059 (50 M solution) or vehicle (phosphate buffer in saline, pH 7.4). Anisomycin, Rp-cAMP and PD 098059 impaired retention test performance in animals injected at different times, prior and after training. Pretraining with a low footshock intensity (0.2 mA) 24 h before training prevented the amnestic effect of all drugs studied. However, simple preexposure to the inhibitory avoidance apparatus did not alter the amnestic effects of all drugs. The results suggest that memory processing requires hippocampal mechanisms dependent on protein synthesis, PKA and MAPK kinase at different times after training. These findings suggest that weak training must be sufficient to produce some lasting cellular expression of the experience so that the enhancement of consolidation of a previously acquired memory is not dependent on protein synthesis, PKA or MAPK.