Dopamine release in PC12 cells is mediated by Ca(2+)-dependent production of ceramide via sphingomyelin pathway

A presynaptic membrane disturbance is an essential process for the release of various neurotransmitters. Ceramide, which is a tumor suppressive lipid, has been shown to act as a channel-forming molecule and serve as a precursor of ceramide-1-phosphate, which can disturb the cellular membrane. This study found that while permeable ceramide increases the rate of dopamine release in the presence of a Ca(2+)-ionophore, A23187, permeable ceramide-1-phosphate provoked its release even without the ionophore. The treatment of PC12 cells with the ionophore at concentrations < 2 microM produced ceramide via the sphingomyelin (SM) pathway with a concomitant release of dopamine, and no cell damage was observed. The addition of a Ca(2+) chelator, EGTA, to the medium inhibited the increase in the release of both the ceramide and dopamine. This suggests that ceramide might be produced by Ca(2+) and is implicated in the membrane disturbance associated with the release of dopamine as a result of its conversion to ceramide-1-phosphate. Consistent with these results, this study detected a membrane-associated and neutral pH optimum sphingomyelinase (SMase) whose activity was increased by Ca(2+). Together, these results demonstrate that ceramide can be produced via the activation of a neutral form of SMase through Ca(2+), and is involved in the dopamine release in concert with Ca(2+).     

Time-course of mitochondrial gene expressions in mice brains: implications for mitochondrial dysfunction, oxidative damage, and cytochrome c in aging

The study of aging is critical for a better understanding of many age-related diseases. The free radical theory of aging, one of the prominent aging hypotheses, holds that during aging, increasing reactive oxygen species in mitochondria causes mutations in the mitochondrial DNA and damages mitochondrial components, resulting in senescence. Understanding a mitochondrial gene expression profile and its relationship to mitochondrial function becomes an important step in understanding aging. The objective of the present study was to determine mRNA expression of mitochondrial-encoded genes in brain slices from C57BL6 mice at four ages (2, 12, 18, and 24 months) and to determine how these altered mitochondrial genes influence age-related changes, including oxidative damage and cytochrome c in apoptosis. Using northern blot analysis, in situ hybridization, and immunofluorescence analyses, we analyzed changes in the expression of mitochondrial RNA encoding the mitochondrial genes, oxidative damage marker, 8-hydroxyguanosine (8-OHG), and cytochrome c in brain slices from the cortex of C57BL6 mice at each of the four ages. Our northern blot analysis revealed an increased expression of mitochondrial-encoded genes in complexes I, III, IV, and V of the respiratory chain in 12- and 18-month-old C57BL6 mice compared to 2-month-old mice, suggesting a compensatory mechanism that allows the production of proteins involved in the electron transport chain. In contrast to the up-regulation of mitochondrial genes in 12- and 18-month-old C57BL6 mice, mRNA expression in 24-month-old C57BL6 mice was decreased, suggesting that compensation maintained by the up-regulated genes cannot be sustained and that the down-regulation of expression results in the later stage of aging. Our in situ hybridization analyses of mitochondrial genes from the hippocampus and the cortex revealed that mitochondrial genes were over-expressed, suggesting that these brain areas are critical for mitochondrial functions. Our immunofluorescence analysis of 8-OHG and cytochrome c revealed increased 8-OHG and cytochrome c in 12-month-old C57BL6 mice, suggesting that age-related mitochondrial oxidative damage and apoptosis are associated with mitochondrial dysfunction. Our double-labeling analysis of in situ hybridization of ATPase 6 and our immunofluorescence analysis of 8-OHG suggest that specific neuronal populations undergo oxidative damage. Further, double-labeling analysis of in situ hybridization of ATPase 6 and immunofluorescence analysis of cytochrome c suggest cytochrome c release is related to mitochondrial dysfunction in the aging C57BL6 mouse brain. This study also suggests that these mitochondrial gene expression changes may relate to the role of mitochondrial dysfunction, oxidative damage, and cytochrome c in aging and in age-related diseases such as Alzheimer's disease and Parkinson's disease.     

Magnetic resonance imaging findings in the brains of rabbits infected with Angiostrongylus cantonensis: a long-term investigation

Because magnetic resonance (MRI) imaging is a superior technique in delineating pathological changes in cerebral angiostrongyliasis, it should also be an optimal imaging modality in monitoring long-term changes in the brains of animals infected with Angiostrongylus cantonensis. In this study, MRI and histological techniques were used to observe the changes in the brains of 7 rabbits infected with the third-stage larvae of A. canronensis. Changes were monitored by MRI from day 0 to day 207 postinfection (PI). Hyperintense lesions on T2-weighted MR brain images were first observed on day 22 PI and hallmarks of abnormalities were noted on day 35 PI. Hyperintensities on brain MR images remained up to day 207 PI. Histological examination from days 108 to 207 PI revealed meningeal congestion, choroid plexus inflammation, infarction, granuloma with embedded larva, gliosis, and hemorrhage in the brain tissues. These findings suggest that hosts infected with A. cantonensis may undergo pathological changes in the brain tissues for more than 200 days PI. Moreover, severe abnormalities may occur as early as the fifth week PI.     

A genetic linkage map of Lens sp. based on microsatellite and AFLP markers and the localization of fusarium vascular wilt resistance

Microsatellites have currently become the markers of choice for molecular mapping and marker-assisted selection for key traits such as disease resistance in many crop species. We report here on the mapping of microsatellites which had been identified from a genomic library of lentil (Lens culinaris Medik.). The majority of microsatellite-bearing clones contained imperfect di-nucleotide repeats. A total of 41 microsatellite and 45 amplified fragment length polymorphism (AFLP) markers were mapped on 86 recombinant inbred lines derived from the cross ILL 5588 × L 692-16-1(s), which had been previously used for the construction of a random amplified polymorphic DNA and AFLP linkage map. Since ILL 5588 was resistant to fusarium vascular wilt caused by the fungus Fusarium oxysporum Shlecht. Emend. Snyder & Hansen f.sp. lentis Vasud. & Srini., the recombinant inbreds were segregating for this character. The resulting map contained 283 markers covering about 751 cM, with an average marker distance of 2.6 cM. The fusarium vascular wilt resistance was localized on linkage group 6, and this resistance gene was flanked by microsatellite marker SSR59-2B and AFLP marker p17m30710 at distances of 8.0 cM and 3.5 cM, respectively. These markers are the most closely linked ones known to date for this agronomically important Fw gene. Using the information obtained in this investigation, the development and mapping of microsatellite markers in the existing map of lentil could be substantially increased, thereby providing the possibility for the future localization of various loci of agronomic interest.     

Epizootiologic investigations of parvovirus infections in free-ranging carnivores from Germany

To assess if wild carnivores in Germany play a role in the epizootiology of canine parvovirus (CPV) infection, seroprevalences against CPV in free-ranging carnivores (n=1,496) from selected urban and rural areas were compared. Antibodies against CPV were found in sera from red foxes (Vulpes vulpes; 136 of 1,442; 9%), raccoon dogs (Nyctereutes procyonides; two of 33; 6%), stone martens (Martes foina; four of 13; 31%), and pine martens (Martes martes; one of two) using the hemagglutination-inhibition test and pig erythrocytes. Evidence of CPV infection was detected in all study areas. Antibody titers varied between 10 and 320. In red foxes, the number of reactors did not differ between most urban and rural areas. However, we found significantly more reactors in the most densely populated urban area (Berlin). None of 430 tissue samples (small intestine, spleen, mesenterial lymph nodes) from any species tested for the presence of CPV nucleic acid using polymerase chain reaction yielded an amplification product. Based on our results, we believe that contact between domestic dogs and free-ranging red foxes probably plays a subordinate role in the epizootiology of CPV in Germany.     

Serologic surveillance for selected viral agents in captive and free-ranging populations of Arabian oryx (Oryx leucoryx) from Saudi Arabia and the United Arab Emirates

A total of 294 sera collected between 1999 and 2001 from eight captive and one free-ranging herds of Arabian oryx (Oryx leucoryx) distributed in Saudi Arabia (SA) and the United Arab Emirates (UAE) were assayed for antibodies against 13 selected viral agents. Arabian oryx have been exposed to bluetongue virus (BTV), epizootic hemorrhagic disease virus (EHDV), rinderpest virus (RPV), bovine respiratory syncytial virus (BRSV), bovine adenovirus 3 (BAV-3), cervid herpesvirus-1, foot-and-mouth disease virus, equine herpesvirus 9, and bovine viral diarrhea virus. The high seroprevalence to BTV and EHDV in the UAE and SA indicates that Arabian oryx are likely to be susceptible to infection by these viruses and therefore could act as a source of virus to vectors during the infective stage of infection. Moreover, antibodies were detected against RPV and BRSV in sera from SA and against BAV-3 in sera from the UAE. No antibodies were found against bovine herpesvirus-1, caprine herpesvirus-1, enzootic bovine leucosis virus, and peste des petits ruminants virus. On the basis of these results, caution should be applied when considering translocation of Arabian oryx, and only those proven to be free of infectious agents that might present a risk to other species should be moved.     

TGF-beta control of cell proliferation

This article focuses on recent findings that the type V TGF-beta receptor (TbetaR-V), which co-expresses with other TGF-beta receptors (TbetaR-I, TbetaR-II, and TbetaR-III) in all normal cell types studied, is involved in growth inhibition by IGFBP-3 and TGF-beta and that TGF-beta activity is regulated by two distinct endocytic pathways (clathrin- and caveolar/lipid-raft-mediated). TGF-beta is a potent growth inhibitor for most cell types, including epithelial and endothelial cells. The signaling by which TGF-beta controls cell proliferation is not well understood. Many lines of evidence indicate that other signaling pathways, in addition to the prominent TbetaR-I/TbetaR-II/Smad2/3/4 signaling cascade, are required for mediating TGF-beta-induced growth inhibition. Recent studies revealed that TbetaR-V, which is identical to LRP-1, mediates IGF-independent growth inhibition by IGFBP-3 and mediates TGF-beta-induced growth inhibition in concert with TbetaR-I and TbetaR-II. In addition, IRS proteins and a Ser/Thr-specific protein phosphatase(s) are involved in the TbetaR-V-mediated growth inhibitory signaling cascade. The TbetaR-V signaling cascade appears to cross-talk with the TbetaR-I/TbetaR-II, insulin receptor (IR), IGF-I receptor (IGF-IR), integrin and c-Met signaling cascades. Attenuation or loss of the TbetaR-V signaling cascade may enable carcinoma cells to escape from TGF-beta growth control and may contribute to the aggressiveness and invasiveness of these cells via promoting epithelial-to-mesenchymal transdifferentiation (EMT). Finally, the ratio of TGF-beta binding to TbetaR-II and TbetaR-I is a signal controlling TGF-beta partitioning between two distinct endocytosis pathways and resultant TGF-beta responsiveness. These recent studies have provided new insights into the molecular mechanisms underlying TGF-beta-induced cellular growth inhibition, cross-talk between the TbetaR-V and other signaling cascades, the signal that controls TGF-beta responsiveness and the role of TbetaR-V in tumorigenesis.