Duplication detection in Japanese Duchenne muscular dystrophy patients and identification of carriers with partial gene deletions using pulsed-field gel electrophoresis

DNA samples from 21 unrelated Japanese patients with Duchenne muscular dystrophy (DMD) with nondeletion-type abnormality in the dystrophin gene and three samples from possible deletion carriers were analyzed using pulsed-field gel electrophoresis (PFGE). Among the 21 patients, 7 were found to carry partial duplications of the dystrophin gene spanning 50–400 kb. Of these 7 patients, 4 carried duplications corresponding to the major hot-spot regions for deletions (7.5–8.5 kb from the 5 end of cDNA), whereas two cases contained duplications in a region about 10 kb from the 5 end of cDNA, where causative mutations are reported to be rare. Only 1 case was found to contain a duplication of a region about 1 kb from the 5 end of cDNA, which is the reported duplication prone region. A combination of Southern blot analyses of conventional agarose gel electrophoresis and PFGE was confirmed to be useful, not only for detecting duplications and deletions, per se, but also for identifying carriers in the affected family.     

A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis

The epidermis of higher plants generates the cuticle layer that covers the outer surface of each plant. The cuticle plays a crucial role in plant development, and some mutants with defective cuticle exhibit morphological abnormalities, such as the fusion of organs. The way in which the cuticle forms and its contribution to morphogenesis are poorly understood. Conventional detection of the cuticle by transmission electron microscopy (TEM) requires laborious procedures, which include fixation, staining with osmium, and preparation of ultra-thin sections. It is also difficult to survey entire surfaces of expanded leaves because of the limited size of specimens that can be examined. Thus, TEM is unsuitable for large-scale screening for mutants with defective cuticle. We describe here a rapid and inexpensive method, designated the toluidine-blue (TB) test, for detection of cuticular defects in whole leaves. We demonstrated the validity of the TB test using mutants of Arabidopsis thaliana, including abnormal leaf shape1 (ale1), fiddlehead (fdh), and five eceriferum (cer) mutants, in which the structure and/or function of the cuticle is abnormal. Genetic screening for mutants using the TB test allowed us to identify seven loci. The cuticle-defective regions of leaves of the mutants revealed five intrinsic patterns of surface defects (classes I through V), suggesting that formation of functional cuticle on leaves involves various spatially regulated factors.     

Regulation of replication at the R/G chromosomal band boundary and pericentromeric heterochromatin of mammalian cells

Mammalian chromosomes consist of multiple replicons; however, in contrast to yeast, the details of this replication process (origin firing, fork progression and termination) relative to specific chromosomal domains remain unclear. Using direct visualization of DNA fibers, here we show that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. To support this, fluorescence in situ hybridization (FISH)-based replication profiles at the human 1q31.1 (R-band)-32.1 (G-band) regions revealed that replication timing switched around at the putative R/G chromosomal band boundary predicted by marked changes in GC content at the sequence level. Thus, the slowdown of replication fork movement is thought to be the general property of the band boundaries separating the functionally different chromosomal domains. By simultaneous visualization of replication fork movement and pericentromeric heterochromatin sequences on DNA fibers, we observed that this region is duplicated by many replication forks, some of which proceed unidirectionally, that originate from clustered replication origins. We showed that histone hyperacetylation is tightly associated with changes in the replication timing of pericentromeric heterochromatin induced by 5-aza-2'-deoxycytidine treatment. These results suggest that, similar to the yeast system, histone modification is involved in controlling the timing of origin firing in mammals.     

Novel nuclear signaling pathway mediates activation of fibroblast growth factor-2 gene by type 1 and type 2 angiotensin II receptors

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1',3'-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.     

Molecular and immunological significance of chimpanzee major histocompatibility complex haplotypes for hepatitis C virus immune response and vaccination studies

The chimpanzee is a critical animal model for studying cellular immune responses to infectious pathogens such as hepatitis B and C viruses, human immunodeficiency virus, and malaria. Several candidate vaccines and immunotherapies for these infections aim at the induction or enhancement of cellular immune responses against viral epitopes presented by common human major histocompatibility complex (MHC) alleles. To identify and characterize chimpanzee MHC class I molecules that are functionally related to human alleles, we sequenced 18 different Pan troglodytes (Patr) alleles of 14 chimpanzees, 2 of them previously unknown and 3 with only partially reported sequences. Comparative analysis of Patr binding pockets and binding assays with biotinylated peptides demonstrated a molecular homology between the binding grooves of individual Patr alleles and the common human alleles HLA-A1, -A2, -A3, and -B7. Using cytotoxic T cells isolated from the blood of hepatitis C virus (HCV)-infected chimpanzees, we then mapped the Patr restriction of these HCV peptides and demonstrated functional homology between the Patr-HLA orthologues in cytotoxicity and gamma interferon (IFN-gamma) release assays. Based on these results, 21 HCV epitopes were selected to characterize the chimpanzees' cellular immune response to HCV. In each case, IFN-gamma-producing T cells were detectable in the blood after but not prior to HCV infection and were specifically targeted against those HCV peptides predicted by Patr-HLA homology. This study demonstrates a close functional homology between individual Patr and HLA alleles and shows that HCV infection generates HCV peptides that are recognized by both chimpanzees and humans with Patr and HLA orthologues. These results are relevant for the design and evaluation of vaccines in chimpanzees that can now be selected according to the most frequent human MHC haplotypes.     

Phosphatidylinositol and PI-4-monophosphate recover amyloid beta protein-induced inhibition of Cl- -ATPase activity

The neuronal Cl- -ATPase/pump is a candidate for an outwardly directed active Cl- transport system, which requires phosphatidylinositol-4-monophosphate (PI4P) for its optimal activity. We previously reported that low concentrations (1-10 nM) of amyloid beta proteins (Abetas, Abeta1-42, Abeta25-35), the neurotoxic peptides in Alzheimer's disease, reduced Cl- -ATPase activity in cultured rat hippocampal neurons without any changes in the activities of Na+/K+-ATPase or anion-insensitive Mg(2+)-ATPase, and decreased PI, PIP, and PIP2 levels in neuronal plasma membranes (Journal of Neurochemistry 2001, 78, 569-579). In this study, we examined the effects of exogenously applied PI and PI4P on the Abeta25-35-induced changes in Cl- -ATPase activity, the intracellular concentration of Cl- ([Cl- ]i), and glutamate neurotoxicity using primary cultured rat hippocampal neurons. The Abeta decreased Cl- -ATPase activity to 47% of control and increased [Cl- ]i in hippocampal pyramidal cell-like neurons to a level 3 times higher than the control. The addition of PI (50-750 nM) or PI4P (50-150 nM) dose-dependently blocked the inhibitory effects of Abeta on Cl- -ATPase activity. High doses of PI (750 nM) and PI4P (100-150 nM) reduced Na+/K+-ATPase activity to 41% and 35% of control, respectively, but this inhibition was attenuated by the co-application of phosphatidylserine (PS, 1 micro M). PI or PI4P (75 nM each) reversed the Abeta-induced increase in [Cl-]i. In the Abeta-exposed culture, stimulation by glutamate (10 micro M, 10 min) resulted in an increase in DNA fragmentation and decreases in cell viability. Addition of PI or PI4P prevented the Abeta-induced aggravation of glutamate neurotoxicity. Thus, PI and PI4P were demonstrated to prevent Abeta-induced decreases in Cl- -ATPase activity and increases in neuronal [Cl- ]i in parallel with the attenuation of Abeta-induced aggravation of glutamate neurotoxicity.     

Expression of swelling- and/or pH-regulated chloride channels (ClC-2, 3, 4 and 5) in human leukemic and normal immune cells

Chloride channels on immune cells reportedly play important roles in cell volume regulation, cell proliferation and immune functions, but they are not well characterized at the molecular level. We examined the expression of swelling-and/or pH-regulated chloride channels (ClC-2, 3, 4 and 5) in human leukemic cell lines [Jurkat and Hut-78 (T cells), Raji and Daudi (B cells), K-562 and HL-60 (myeloid cells)] and T cells, B cells and neutrophils from 8 normal subjects to clarify the difference of their expression among different cell types and maturity. Semi-quantitative RT-PCR and Northern blot analysis showed that ClC-3 was most abundantly expressed in all cells regardless of the cell types and maturity, while expression of ClC-2 was weak in these cells. Expression of ClC-4 was observed mainly in leukemic B cell lines, and in B cells and neutrophils from normal subjects. ClC-5 was expressed in all cell lines, while it was observed in only T and B cells but not in neutrophils from normal subjects. Thus, these chloride channels (ClC-2, 3, 4 and 5) showed distinct distribution among human immune cells, suggesting that they have specific roles in these cells. Molecular identification of chloride channels in leukocytes of different types and maturity may provide a new approach for the treatment of leukemia.     

Increase in CD14+HLA-DR−/low myeloid-derived suppressor cells in hepatocellular carcinoma patients and its impact on prognosis

Myeloid-derived suppressor cells (MDSCs) are known as key immune regulators in various human malignancies, and it is reported that CD14⁺HLA-DR^?/low MDSCs are increased in hepatocellular carcinoma (HCC) patients. However, the host factors that regulate the frequency and the effect on the prognosis of HCC patients are still unclear. We investigated these issues and clarified the relationships between a feature of MDSCs and host factors in HCC patients. We examined the frequency of MDSCs in 123 HCC patients, 30 chronic liver disease patients without HCC, and 13 healthy controls by flow cytometric analysis. The relationships between the clinical features and the frequency of MDSCs were analyzed. In 33 patients who received curative radiofrequency ablation (RFA) therapy, we examined the impact of MDSCs on HCC recurrence. The frequency of MDSCs in HCC patients was significantly increased. It was correlated with tumor progression, but not with the degree of liver fibrosis and inflammation. In terms of serum cytokines, the concentrations of IL-10, IL-13, and vascular endothelial growth factor were significantly correlated with the frequency of MDSCs. In HCC patients who received curative RFA therapy, the frequency of MDSCs after treatment showed various changes and was inversely correlated with recurrence-free survival time. The frequency of MDSCs is correlated with tumor progression, and this frequency after RFA is inversely correlated with the prognosis of HCC patients. Patients with a high frequency of MDSCs after RFA should be closely followed and the inhibition of MDSCs may improve the prognosis of patients.     

Escherichia coli PriA protein, two modes of DNA binding and activation of ATP hydrolysis

Escherichia coli PriA protein plays crucial roles in processing of arrested replication forks. PriA serves as a sensor/stabilizer for an arrested replication fork and eventually promotes restart of DNA replication through assembly of a primosome. PriA carries a 3' terminus binding pocket required for its high affinity binding to a specific arrested fork as well as for its biological functions. We show here that PriA binds to DNA in a manner either dependent on or independent of 3' terminus recognition. The former mode of binding requires the 3' terminus binding pocket present at the N-terminal half of the 181-residue DNA binding domain and exhibits specific bipartite interaction on the template DNA. The latter mode is independent of the pocket function, but requires the C-terminal half of the same domain. ATP hydrolysis activity of PriA can be stimulated in vitro by either of the two binding modes. We propose architecture of PriA bound to various arrested replication fork structures and discuss its implication in helicase activation and ATP hydrolysis.