Genetic interactions between BLM and DNA ligase IV in human cells

BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure. To explore this, we generated BLM(-/-) and BLM(-/-)LIG4(-/-) cells from the human pre-B cell line Nalm-6. BLM(-/-) cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression. Interestingly, these phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. We propose that the absence of BLM leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by NHEJ. In addition, the NHEJ pathway per se was marginally affected by BLM deficiency, as evidenced by x-ray sensitivity and I-SceI-based DSB repair assays. More intriguingly, however, these experiments revealed the presence of an alternative, DNA ligase IV-independent end-joining pathway, which was significantly affected by the loss of BLM. Collectively, our results provide the first evidence for genetic interactions between BLM and NHEJ in human cells.     

A novel HLA-A*3303-restricted minor histocompatibility antigen encoded by an unconventional open reading frame of human TMSB4Y gene

Female-to-male hemopoietic stem cell transplantation (HSCT) elicits T cell responses against male-specific minor histocompatibility (H-Y) Ags encoded by the Y chromosome. All previously identified H-Y Ags are encoded by conventional open reading frames, but we report in this study the identification of a novel H-Y Ag encoded in the 5'-untranslated region of the TMSB4Y gene. An HLA-A*3303-restricted CD8(+) CTL clone was isolated from a male patient after an HSCT from his HLA-identical sister. Using a panel of cell lines carrying Y chromosome terminal deletions, a narrow region controlling the susceptibility of these target cells to CTL recognition was localized. Minigene transfection and epitope reconstitution assays identified an 11-mer peptide, EVLLRPGLHFR, designated TMSB4Y/A33, whose first amino acid was located 405 bp upstream of the TMSB4Y initiation codon. Analysis of the precursor frequency of CTL specific for recipient minor histocompatibility Ags in post-HSCT peripheral blood T cells revealed that a significant fraction of the total donor CTL response in this patient was directed against the TMSB4Y epitope. Tetramer analysis continued to detect TMSB4Y/A33-specific CD8(+) T cells at least up to 700 days post-HSCT. This finding underscores the in vivo immunological relevance of minor histocompatibility Ags derived from unconventional open reading frame products.     

Focal adhesion kinase mediates endothelin-induced cyclin D3 expression in rat cultured astrocytes

Focal adhesion kinase (FAK), a non-receptor type tyrosine kinase, is involved in the G1/S phase cell cycle transition of astrocytes induced by endothelin-1 (ET-1). In this study, the roles of FAK in the expression of cyclin D1 or D3, which are pivotal in G1/S phase transition, were examined in cultured astrocytes. Accompanied with increases in bromodeoxyuridine (BrdU) incorporation, ET-1 (100 nm) increased the numbers of cyclin D1- and D3-positive astrocytes. PD98059 (a MEK inhibitor) and PP-2 (a Src kinase inhibitor) inhibited ET-induced cyclin D1 expression and BrdU incorporation without affecting cyclin D3 expression. In contrast, cytochalasin D, lovastatin (a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor) and Y-27632 (a rho-kinase inhibitor) prevented both cyclin D3 expression and BrdU incorporation. FAK phosphorylation by ET-1 was inhibited by cytochalasin D, lovastatin and Y-27632, but not by PD98059 or PP-2. Transfection with wild-type FAK increased expression of cyclin D3 in astrocytes, while that of cyclin D1 was not affected. Dominant-negative FAK mutants prevented an ET-induced increase in cyclin D3 expression, but not D1. These results suggest that activation of FAK causes cyclin D3 expression in cultured astrocytes, which would underlie the FAK-mediated astrocytic G1/S phase transition by ET-1.     

A comparison of the emission efficiency of four common green fluorescence dyes after internalization into cancer cells

In vivo optical imaging to enhance the detection of cancer during endoscopy or surgery requires a targeted fluorescent probe with high emission efficiency and high signal-to-background ratio. One strategy to accurately detect cancers is to have the fluorophore internalize within the cancer cells permitting nonbound fluorophores to be washed away or absorbed. The choice of fluorophores for this task must be carefully considered. For depth of penetration, near-infrared probes are ordinarily preferred but suffer from relatively low quantum efficiency. Although green fluorescent protein has been widely used to image tumors on internal organs in mice, green fluorescent probes are better suited for imaging the superficial tissues because of the short penetration distance of green light in tissue and the highly efficient production of signal. While the fluorescence properties of green fluorophores are well-known in vitro, less attention has been paid to their fluorescence once they are internalized within cells. In this study, the emission efficiency after cellular internalization of four common green fluorophores conjugated to avidin (Av-fluorescein, Av-Oregon green, Av-BODIPY-FL, and Av-rhodamine green) were compared after each conjugate was incubated with SHIN3 ovarian cancer cells. Using the lectin binding receptor system, the avidin-fluorophore conjugates were endocytosed, and their fluorescence was evaluated with fluorescence microscopy and flow cytometry. While fluorescein demonstrated the highest signal outside the cell, among the four fluorophores, internalized Av-rhodamine green emitted the most light from SHIN3 ovarian cancer cells both in vitro and in vivo. The internalized Av-rhodamine green complex appeared to localize to the endoplasmic vesicles. Thus, among the four common green fluorescent dyes, rhodamine green is the brightest green fluorescence probe after cellular internalization. This information could have implications for the design of tumor-targeted fluorescent probes that rely on cellular internalization for cancer detection.     

Larval development and settlement of a whale barnacle

Larval development and settlement of whale barnacles have not previously been described, unlike intertidal barnacles. Indeed, the mechanisms of the association between barnacles and whales have not been studied. Here we describe the larval development and settlement of the whale barnacle, Coronula diadema, and possible involvement of a cue from the host in inducing larval settlement. Eight-cell stage embryos were collected from C. diadema on a stranded humpback whale, incubated in filtered seawater for 7 days, and nauplius larvae hatched out. When fed with Chaetoceros gracilis, the nauplii developed to stage VI, and finally metamorphosed to the cypris stage. The larval development looked similar to that of intertidal barnacles with planktotrophic larval stages. The cyprids did not settle in normal seawater, but did settle in polystyrene Petri dishes when incubated in seawater with a small piece of skin tissue from the host whale. This strongly suggests the involvement of a chemical cue from the host whale tissue to induce larval settlement.     

Alpneumines A–H,new anti-melanogenic indole alkaloids from Alstonia pneumatophora

Eight new indole alkaloids, alpneumines A–H (1–8) were isolated from the Malaysian Alstonia pneumatophora (Apocynaceae) and their structures were determined by MS and 2D NMR spectroscopic methods. Alpneumines E and G (5 and 7), vincamine, and apovincamine showed anti-melanogenesis in B16 mouse melanoma cells.     

Transcardiac adiponectin gradient is independently related to endothelial vasomotor function in large and resistance coronary arteries in humans

Adiponectin, an adipocyte-derived protein, has been shown to have vasculoprotective effects. This study examined the possible relationship between coronary vasomotor function and the transcardiac gradient of adiponectin, reflecting adiponectin utilization and/or accumulation in the coronary vascular bed. The epicardial diameter and blood flow response of the left anterior descending coronary artery to intracoronary infusions of ACh was analyzed in 108 consecutive subjects who had a normal coronary angiogram and left ventriculogram. Adiponectin levels were measured by ELISA in plasma obtained from the aortic root (Ao) and the anterior interventricular vein (AIV). Adiponectin levels in the AIV were lower than levels in the Ao. In multivariate linear regression analysis, the transcardiac gradient of adiponectin (Ao - AIV levels) showed a positive correlation with increases in epicardial coronary diameter and coronary blood flow in response to ACh that was independent of traditional coronary risk factors. The transcardiac gradient of adiponectin was not significantly associated with the coronary dilator response to isosorbide dinitrate and the coronary flow response to sodium nitroprusside. In other groups of patients with coronary spastic angina (n = 41) or microvascular angina (n = 32) who had impaired coronary vasomotor responses, there was no significant gradient of adiponectin between the Ao and AIV. The transcardiac gradient of adiponectin may modulate endothelial vasomotor function in large and resistance coronary arteries and may play a role in the pathogenesis of diseases presenting with coronary vasomotor dysfunction.     

Identification and analysis of Ku70 and Ku80 homologs in the koji molds Aspergillus sojae and Aspergillus oryzae

Ku genes play a key role in the non-homologous end-joining pathway. We have identified Ku70 and Ku80 homologs in the koji molds Aspergillus sojae and Aspergillus oryzae, and have constructed the disruption mutants of Ku70, Ku80, and Ku70-80 to characterize the phenotypic change in these mutants. Neither Ku70- nor Ku80-disrupted strains show hypersensitivity to the DNA damaging agents methylmethane sulfonate (MMS) and phleomycin. Moreover, undesirable phenotypes, such as poor growth or repressed conidiospore formation, were not observed in the Ku-disrupted A. sojae and A. oryzae.     

cDNA cloning and phylogenetic and expression analyses of actin in symbiotic dinoflagellates (Symbiodinium spp.)

Three cDNAs encoding actins were identified in two culturable strains (clades A and F) of the symbiotic dinoflagellates Symbiodinium spp. In a molecular phylogenetic analysis these actin sequences formed a monophyletic group with known dinoflagellate actins, remote from Syact-p that had been isolated from a clade A Symbiodinium strain (HG39). One of the newly identified actin sequences (SyAct-F1) was the most closely related to partial actin cDNA sequences (named AGfact-p and AFcact-p) isolated from adult colonies of two reef corals (Galaxea fascicularis and Favites chinensis) that were inhabited by Symbiodinium spp., suggesting the possibility that the latter two were from the symbionts. Partial AFcact-p sequences could be amplified by PCR using genomic DNA prepared from a symbiotic adult colony of F. chinensis as the template, but not from planula larvae in which zooxanthellae could not be detected, also arguing for the origin of AFcact-p in the symbiont. An expression analysis showed that the levels of the SyAct-A1 mRNA were comparable in symbiotic and non-symbiotic states, and also in motile and non-motile phases in a cultured condition, suggesting its usefulness as a constitutively expressed control gene in expression analysis of Symbiodinium mRNAs.