Structural centrosome aberrations promote non‐cell‐autonomous invasiveness

Centrosomes are the main microtubule‐organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein‐like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP‐induced structural centrosome aberrations trigger the escape (“budding”) of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP‐induced centrosome aberrations trigger a re‐organization of the cytoskeleton, which stabilizes microtubules and weakens E‐cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non‐cell‐autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes.     

Tumor necrosis factor-alpha supports the survival of osteoclasts through the activation of Akt and ERK.

Differentiated osteoclasts have a short life span. We tested various cytokines and growth factors for the effects on the survival of purified mature osteoclasts. In the absence of any added factors, osteoclasts exhibited the survival rate of less than 25% after a 24-h incubation. Among the tested factors, tumor necrosis factor-alpha (TNF-alpha) was found to increase the survival rate to approximately 80%. The TNF-alpha-enhanced survival of osteoclasts appeared to be associated with reduction in apoptosis and suppression of caspase activation. The antiapoptotic signaling pathways involved in the TNF-alpha-induced osteoclast survival were investigated. TNF-alpha treatment increased the phosphorylation of Akt in osteoclasts, which was suppressed by a phosphatidylinositol 3-kinase inhibitor LY294002 and an Src family kinase-selective inhibitor PP1. These inhibitors also attenuated the TNF-alpha stimulation of osteoclast survival. In addition an increase in the phosphorylation of ERK was observed upon TNF-alpha stimulation. PD98059, a specific inhibitor of the ERK-activating kinase MEK-1, abolished the TNF-alpha-induced ERK phosphorylation and osteoclast survival, and in these responses the involvement of Grb2 and ceramide was observed. These results suggest that TNF-alpha promotes the survival of osteoclasts by engaging the phosphatidylinositol 3-kinase Akt and MEK/ERK signaling pathways.     

Gene conversion and natural selection in the evolution of X-linked color vision genes in higher primates

During higher primate evolution, gene conversion seems to have occurred often between the red and green photo-pigment genes, which are tandemly linked on the X chromosome. To understand this phenomenon better, intron 4 sequences of the red and green pigment genes of a male human (an Asian Indian), a male chimpanzee, and a male baboon were amplified by PCR and sequenced. The data show that the intron 4 sequences between the two genes have been strongly or completely homogenized in the three species studied. Apparently recent gene conversion events have occurred in introns 4 of the red and green pigment genes in humans and chimpanzees. Two or more conversion events may have occurred at different times in introns 4 of the two pigment genes in baboons. The divergence between the two genes is significantly lower in intron 4 than in exons 4 and 5 in each species, contrary to the usual situation that introns evolve faster than exons. It is most likely that strong natural selection for maintaining the distinct functions of exons 4 and 5 of the red and green pigment genes has acted against sequence homogenization of these exons.      

Biochemical characterization of deblocking aminopeptidases from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1

Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis. In this study, the TkDAP1 and TkDAP2 genes were cloned and expressed in Escherichia coli. The two proteins were purified homogeneity and analyzed by gel filtration chromatography and electron microscopy. TkDAP1 showed two oligomers, which were identified as an octodecimer and a dodecamer. TkDAP2 produced three native forms: octodecimer, dodecamer, and trimer. Dodecamer assembly was the main form in the two proteins. Finally, TkDAP1 was found to have higher deblocking aminopeptidase activity on the substrates of Ac-Leu-pNA and Ac-Ala-Ala-Ala, while TkDAP2 had higher aminopeptidase activity on the substrates of Leu-pNA and Ala-Ala-Ala-pNA.     

Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae

Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer.     

Variation of telomerase activity and morphology in porcine mesenchymal stem cells and fibroblasts during prolonged in vitro culture

The purpose of this study was to examine the telomerase activity, population doubling time (PDT), morphological alterations, and the cell cycle status with activity of senescence-associated-?-galactosidase in porcine mesenchymal stem cells (MSCs) and fibroblasts during an extended in vitro culture. MSCs and fibroblasts were isolated from bone marrow and ear skin of a miniature pig, respectively, and cultured up to 20 passages. The analysis was carried out in MSCs and fibroblasts at 1, 5, 10, 15, and 20 passages. Relative telomerase activity (RTA) levels were significantly (P?相似文献   

The role of survivin in angiogenesis during zebrafish embryonic development

Background  

Survivin is the smallest member of the inhibitor of apoptosis (IAP) gene family. Recently, the zebrafish survivin-1 gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse survivin gene. Here we investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5' untranslated region (UTR) (Sur_UTR) and sequences flanking the initiation codon (Sur_ATG) of zebrafish survivin-1 gene were injected into embryos at 1–4 cell stage. Vasculature was examined by microangiography and GFP expression in Tg(fli1:EGFP) ^y1 embryos. Results: In embryos co-injected with Sur_UTR and Sur_ATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) and dorsal longitudinal anastomotic vessels (DLAV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of Sur_UTR and Sur_ATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression. Conclusion: Survivin is involved in angiogenesis during zebrafish development and may be under VEGF regulation.     

Specificity and enzymatic mechanism of the editing exonuclease of Escherichia coli DNA polymerase III.

Exonucleolytic editing is a major contributor to the fidelity of DNA replication by the multisubunit DNA polymerase (pol) III holoenzyme. To investigate the source of editing specificity, we have studied the isolated exonuclease subunit, epsilon, and the pol III core subassembly, which carries the epsilon, theta, and alpha (polymerase) subunits. Using oligonucleotides with specific terminal mismatches, we have found that both epsilon and pol III core preferentially excise a mispaired 3' terminus and therefore have intrinsic editing specificity. For both epsilon and pol III core, exonuclease activity is much more effective with single-strand DNA; with a double-strand DNA, the exonuclease is strongly temperature-dependent. We conclude that the epsilon subunit of pol III holoenzyme is itself a specific editing exonuclease and that the source of specificity is the greater melting capacity of a mispaired 3' terminus.     

Single nucleotide polymorphisms and the linkage disequilibrium at the LDL receptor gene in Koreans

The low-density lipoprotein (LDL) receptor gene is one of the important genes in cardiovascular biology, but gives difficulty in single nucleotide polymorphism (SNP) marker choice. Twelve SNPs at the LDL receptor locus were genotyped in 117 Korean individuals. Six SNPs (rs8111982, rs12983082, rs1003723, rs5925, rs6413504, and rs2738464) were polymorphic in the Korean sample. And 8 SNPs (rs5931, rs811 1982, rs12983082, rs11669576, rs1003723, rs5925, rs641 3504, and rs2738464) were polymorphic in at least one population among the five ethnic groups (European: CEU; Chinese: CHB; Japanese: JPT; African:YRI; and Korean: KOR). Phylogenetic trees constructed based on the twelve SNPs showed a pattern clustering KOR with CHB and JPT as more closely related than either YRI or CEU. The plot of the pairwise linkage disequilibrium (LD) values for the exon 13 SNP (rs5925) against the physical distances between pairs of SNPs showed a tendency of LD decrease in the upstream of rs5925, while the low r² values are present in the downstream of rs5925 (≥0.20). The three SNPs (rs12983082, rs1003723, and rs5925) which are ethnically polymorphic are likely to be useful SNP markers for the LDL receptor locus. The interblock region between exon 13 and exon 18 delimited by the less stringent block definition (four gamete rule) contains a feasible recombination hotspot in the LDL receptor gene in Koreans.