The NAD(+) salvage pathway modulates cancer cell viability via p73.

The involvement of the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD(+) salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD(+) salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.

Oncolytic reovirus induces intracellular redistribution of Ras to promote apoptosis and progeny virus release.

Reovirus is a naturally oncolytic virus that preferentially replicates in Ras-transformed cells and is currently undergoing clinical trials as a cancer therapeutic. Ras transformation promotes reovirus oncolysis by enhancing virion disassembly during entry, viral progeny production, and virus release through apoptosis; however, the mechanism behind the latter is not well understood. Here, we show that reovirus alters the intracellular location of oncogenic Ras to induce apoptosis of H-RasV12-transformed fibroblasts. Reovirus infection decreases Ras palmitoylation levels and causes accumulation of Ras in the Golgi through Golgi fragmentation. With the Golgi being the site of Ras palmitoylation, treatment of target cells with the palmitoylation inhibitor, 2-bromopalmitate (2BP), prompts a greater accumulation of H-RasV12 in the Golgi, and a dose-dependent increase in progeny virus release and subsequent spread. Conversely, tethering H-RasV12 to the plasma membrane (thereby preventing its movement to the Golgi) allows for efficient virus production, but results in basal levels of reovirus-induced cell death. Analysis of Ras downstream signaling reveals that cells expressing cycling H-RasV12 have elevated levels of phosphorylated JNK (c-Jun N-terminal kinase), and that Ras retained at the Golgi body by 2BP increases activation of the MEKK1/MKK4/JNK signaling pathway to promote cell death. Collectively, our data suggest that reovirus induces Golgi fragmentation of target cells, and the subsequent accumulation of oncogenic Ras in the Golgi body initiates apoptotic signaling events required for virus release and spread.

Gigacycle fatigue behavior by ultrasonic nanocrystalline surface modification.

Nanocrystalline surface layer up to 84 microm in thick is produced on a specimen made of Al6061-T6 alloy by means of surface treatment called ultrasonic nanocrystalline surface modification (UNSM) technique. The refined grain size is produced in the top-layer and it is increased with increasing depth from the top surface. Vickers microhardness measurement for each nanocrystalline surface layer is performed and measurement results showed that the microhardness is increased from 116 HV up to 150 HV, respectively. In this study, fatigue behavior of Al6061-T6 alloy was studied up to 10(7)-10(9) cycles by using a newly developed ultrasonic fatigue testing (UFT) rig. The fatigue results of the UNSM-treated Al6061-T6 alloy specimens were compared with those of the untreated specimens. The microstructure of the untreated and UNSM-treated specimens was characterized by means of scanning electron microscopey (SEM) and transmission electron microscopey (TEM).

Interference of hepatitis C virus replication in cell culture by antisense peptide nucleic acids targeting the X-RNA.

The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is the essential catalytic enzyme for viral genome replication. It initiates minus-strand RNA synthesis from a highly conserved 98-nt sequence, called the X-RNA, at the 3'-end of the plus-strand viral genome. In this study, we evaluated the antiviral effects of peptide nucleic acids (PNAs) targeting the X-RNA. Our in vitro RdRp assay results showed that PNAs targeting the three major stem-loop (SL) domains of X-RNA can inhibit RNA synthesis initiation. Delivery of X-RNA-targeted PNAs by fusing the PNAs to cell-penetrating peptides (CPPs) into HCV-replicating cells effectively suppressed HCV replication. Electrophoretic mobility shift assays revealed that the PNA targeting the SL3 region at the 5'-end of X-RNA dissociated the viral RdRp from the X-RNA. Furthermore, delivery of the SL3-targeted PNA into HCV-infected cells resulted in the suppression of HCV RNA replication without activation of interferon ß expression. Collectively, our results indicate that the HCV X-RNA can be effectively targeted by CPP-fused PNAs to block RNA-protein and/or RNA-RNA interactions essential for viral RNA replication and identify X-RNA SL3 as an RdRp binding site crucial for HCV replication. In addition, the ability to inhibit RNA synthesis initiation by targeting HCV X-RNA using antisense PNAs suggests their promising therapeutic potential against HCV infection.

tbx20, a new vertebrate T-box gene expressed in the cranial motor neurons and developing cardiovascular structures in zebrafish.

The T-box genes constitute a family of transcriptional regulator genes that have been implicated in a variety of developmental processes ranging from the formation of germ layers to the regionalization of the central nervous system. In this report we describe the cloning and expression pattern of a new T-box gene from zebrafish, which we named tbx20. tbx20 is an ortholog of two other T-box genes isolated from animals of different phyla - H15 of Drosophila melanogaster and tbx-12 of Caenorhabditis elegans, suggesting that the evolutionary origin of this gene predates the divergence between the protostomes and deuterostomes. During development, tbx20 is expressed in embryonic structures of both mesodermal and ectodermal origins, including the heart, cranial motor neurons, and the roof of the dorsal aorta.

Axial variation in the threespine stickleback: relationship to Hox gene expression.

Despite mounting evidence that key developmental regulator genes are involved in significant macroevolutionary changes, there have been few studies demonstrating the functional significance of variation in such genes for the generation of population-level variation. In this study we examined and compared the expression domains of three Hox gene homeobox sequences in embryos derived from two morphologically distinct populations of the threespine stickleback, Gasterosteus aculeatus. We found within-population variation in the location of anterior limits, particularly in more 5' Hox genes whose anterior expression domains showed graded distributions of transcripts over several somites. However, despite considerable and statistically significant differences in the anteroposterior pattern of the axial and median skeletons between the two stickleback populations, this phenotypic variation was not found to be correlated with any of the variation in Hox gene expression. The possible functional significance of the combinatorial Hox code in fish species is discussed with respect to the buffering of development in fluctuating environments, and it is argued that population and quantitative genetic perspectives should also be taken into account in considering the function and evolution of Hox genes.

Expression patterns of threespine stickleback hox genes and insights into the evolution of the vertebrate body axis.

Understanding the patterning mechanisms that operate to promote differentiation of individual segments along the main body axis is an important goal of both developmental and evolutionary biology. In order to gain a better insight into the role of Hox genes in generating diversity of axial plans seen in vertebrates, we have cloned 11 homeobox sequences from an acanthopterygian teleost, the threespine stickleback, and analyzed the expression of 7 of these during embryogenesis. Transcripts are observed in a variety of tissues, including the neural tube, paraxial mesoderm, lateral plate mesoderm, pectoral fins, pronephric ducts, as well as some neural crest-derived structures. Anterior limits of expression in the central nervous system and paraxial mesoderm exhibited both similarities and differences to those of mouse and zebrafish homologs. In both stickleback and zebrafish embryos expression limits within the paraxial mesoderm were detected only within the trunk region in which ribs are attached to all vertebrae. The finding of this pattern in two divergent teleosts as well as in various tetrapod species supports the hypothesis that a Hox precode was present prior to the divergence of ray-finned and lobe-finned fishes and was subsequently used to generate different types of vertebrae in tetrapods. We also describe a dynamic pattern of expression of several stickleback Hox genes associated with the development of the caudal paraxial mesoderm, which suggests uncoupling of the process of segmentation from segmental identity determination. We propose that in fishes the patterning of the tail region is under the control of a separate mechanism from the trunk, which utilizes Hox genes in a different manner.

Axial variation in the three-spine stickleback: genetic and environmental factors.

Subtle differences in the pattern of arrangement of types of vertebrae and associated median skeletal structures between a benthic and limnetic species pair of three-spine stickleback from Paxton Lake, British Columbia, are typical of those found throughout the range of the Gasterosteus aculeatus species complex. We established laboratory colonies from just three individuals of each species, and studied the effect of three generations of inbreeding on axial morphology. There was sufficient divergence in the location of individual elements between families to regenerate close to the entire range of axial diversity seen in threespine sticklebacks worldwide. Analysis of the patterns of variance and covariance between the axial locations of elements provides evidence for the action of both meristic and homeotic processes in the generation of morphological divergence within each species. Hybrid sticklebacks produced by the cross of limnetic and benthic parents tend to have intermediate morphologies, with dominance of either parental type evident for some elements. Effects of temperature and salinity were found to be small in direct comparison with between-family effects, and varied according to genetic background. These results demonstrate that considerable genetic variation for axial morphology is maintained in natural populations of three-spine stickleback, and that differences between populations may be brought about rapidly by changes in frequency of alleles that have coordinated effects along the body axis.

Midterm follow-up of necrotic bleb excision and advancement of the fornical conjunctiva.

Mitomycin C has improved the success rate of glaucoma filtering surgery in patients at high risk for surgical failure. However chronic hypotony is marked by decreased vision and a late-onset leaking bleb after filtration surgery using mitomycin C. Bleb excision and conjunctival advancement is the method of choice to repair bleb leakage and chronic hypotony. Five eyes from five patients were received glaucoma filtration surgery with topical mitomycin C. All of the patients' blebs were avascular and transparent. The reasons for bleb excision were two spontaneous bleb leaks, two traumatic bleb leaks and one case of severe irritation. The mean follow-up period was 18.4 +/- 8.3 months (ten to 29 months). Cataract surgery was combined in one eye. Postoperative intraocular pressure (IOP) increased from 2.3 +/- 1.5 mmHg to 9.5 +/- 3.7 mmHg at nine months postoperatively in four eyes. It went from 28 mmHg to 40 mmHg in one patient with uveitis, for whom a second trabeculectomy with mitomycin C; 0.4 mg/ml for 3 minutes, was performed. After surgery, IOP decreased to 4 mmHg in three months. Postoperative visual acuity improved four snellen lines in three eyes. A partially avascular bleb recurred in three eyes, a corneal bleb in one eye and blepharoptosis, which disappeared spontaneously at four months postoperatively, in one eye. Necrotic bleb excision and advancement of fornical conjunctiva were useful methods to increase IOP and to improve visual acuity for the patient experiencing irritation symptoms, and for leaking blebs, and hypotonic maculopathy.