Diagnostic evaluation of 2', 5'-oligoadenylate synthetase activities and antibodies against Epstein-Barr virus and Coxiella burnetii in patients with chronic fatigue syndrome in Japan

To investigate the association of viral infections with chronic fatigue syndrome (CFS), we assayed 2', 5'-oligoadenylate synthetase (2-5AS) activities in peripheral blood mononuclear cells from CFS patients in Japan. These patients were diagnosed in two hospitals, H1 and H2, located in different areas of the country. The activities were detected in 19 (86%) and 7 (32%) of each of the 22 patients in H1 and H2, respectively, while they were detected in only four (11%) out of the 38 healthy controls. IFN-alpha was similarly detected in a few CFS patients and healthy controls. We also assayed the antibody titers against Epstein-Barr virus (EBV) and Coxiella burnetii in these patients. The EBV anti-EA-IgG antibodies were detected in two (9%) and seven (32%) of each of the 22 patients in H1 and H2, respectively. Anti-C. burnetii IgG antibodies were detected in six (27%) out of 22 patients in H1 but not in 22 patients in H2, while they were detected in one (11%) of the nine healthy controls. Some CFS patients may be associated with EBV or C. burnetii infection. There were some statistical correlations between the 2-5AS activities and antibody titers of EA-IgG (P < 0.05, Student's t-test) but not to the antibody titers of C. burnetii. The up-regulation of 2-5AS activities suggests immunological dysfunctions with some virus infections in the CFS patients. Our results indicate that 2-5AS activities are useful for a diagnostic marker of CFS and for exploring the complicated pathogenesis of CFS.     

Strict paternal transmission of mitochondrial DNA of Chlamydomonas species is explained by selection against maternal nucleoids

Summary.  The non-Mendelian inheritance of organelle DNA is common in most plants and animals. Here we examined inheritance mechanisms involved in the transfer of mitochondrial DNA. We successively backcrossed (to F₅) two interfertile strains of the unicellular isogamous haploid algae Chlamydomonas reinhardtii and Chlamydomonas smithii to match nuclear backgrounds and examine transmission patterns of mitochondrial DNA by PCR analysis of cob gene sequences. Mitochondrial DNA was strictly transmitted paternally. To investigate the behavior of parental mitochondrial DNA, we used F₅ progeny to form zygotes and isolated single zygotes. The results showed selective disappearance of maternal mitochondrial nucleoids occurred between 3 and 6 h after zygote formation. Received July 11, 2002; accepted September 28, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan.     

Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome

Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries approximately 500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed. --The nucleotide sequence data were uploaded to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AG020727 to AG025611 and AG205093 to AG215049.     

Transblot and cytochemical identification of avidin-interacting proteins in mitochondria of cultured cells

Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.     

Spy1, a histidine-containing phosphotransfer signaling protein, regulates the fission yeast cell cycle through the Mcs4 response regulator

Common histidine-to-aspartate (His-to-Asp) phosphorelay signaling systems involve three types of signaling components: a sensor His kinase, a response regulator, and a histidine-containing phosphotransfer (HPt) protein. In the fission yeast Schizosaccharomyces pombe, two response regulators, Mcs4 and Prr1, have been identified recently, and it was shown that they are involved in the signal transduction implicated in stress responses. Furthermore, Mcs4 appears to be involved in mitotic cell-cycle control. However, neither the HPt phosphotransmitter nor His kinase has been characterized in S. pombe. In this study, we identified a gene encoding an HPt phosphotransmitter, named Spy1 (S. pombe YPD1-like protein). The spy1(+) gene showed an ability to complement a mutational lesion of the Saccharomyces cerevisiae YPD1 gene, which is involved in an osmosensing signal transduction. The result from yeast two-hybrid analysis indicated that Spy1 interacts with Mcs4. To gain insight into the function of Spy1, a series of genetic analyses were conducted. The results provided evidence that Spy1, together with Mcs4, plays a role in regulation of the G(2)/M cell cycle progression. Spy1-deficient cells appear to be precocious in the entry to M phase. In the proposed model, Spy1 modulates Mcs4 in a negative manner, presumably through a direct His-to-Asp phosphorelay, operating upstream of the Sty1 mitogen-activated protein kinase cascade.     

Glucocorticoid‐inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death

Pathogenic strains of Pseudomonas syringae pv. tomato carrying the avrRpt2 avirulence gene specifically induce a hypersensitive cell death response in Arabidopsis plants that contain the complementary RPS2 disease resistance gene. Transient expression of avrRpt2 in Arabidopsis plants having the RPS2 gene has been shown to induce hypersensitive cell death. In order to analyze the effects of conditional expression of avrRpt2 in Arabidopsis plants, transgenic lines were constructed that contained the avrRpt2 gene under the control of a tightly regulated, glucocorticoid-inducible promoter. Dexamethasone-induced expression of avrRpt2 in transgenic lines having the RPS2 gene resulted in a specific hypersensitive cell death response that resembled a Pseudomonas syringae-induced hypersensitive response and also induced the expression of a pathogenesis-related gene (PR1). Interestingly, high level expression of avrRpt2 in a mutant rps2–101C background resulted in plant stress and ultimately cell death, suggesting a possible role for avrRpt2 in Pseudomonas syringae virulence. Transgenic RPS2 and rps2 plants that contain the glucocorticoid-inducible avrRpt2 gene will provide a powerful new tool for the genetic, physiological, biochemical, and molecular dissection of an avirulence gene-specified cell death response in both resistant and susceptible plants.     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.     

Spatial Change of Cruciate Ligaments in Rat Embryo Knee Joint by Three-Dimensional Reconstruction

This study aimed to analyze the spatial developmental changes of rat cruciate ligaments by three-dimensional (3D) reconstruction using episcopic fluorescence image capture (EFIC). Cruciate ligaments of Wister rat embryos between embryonic day (E) 16 and E20 were analyzed. Samples were sectioned and visualized using EFIC. 3D reconstructions were generated using Amira software. The length of the cruciate ligaments, distances between attachment points to femur and tibia, angles of the cruciate ligaments and the cross angle of the cruciate ligaments were measured. The shape of cruciate ligaments was clearly visible at E17. The lengths of the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) increased gradually from E17 to E19 and drastically at E20. Distances between attachment points to the femur and tibia gradually increased. The ACL angle and PCL angle gradually decreased. The cross angle of the cruciate ligaments changed in three planes. The primordium of the 3D structure of rat cruciate ligaments was constructed from the early stage, with the completion of the development of the structures occurring just before birth.