Interplay between PI3K/Akt and MAPK signaling pathways in DNA-damaging drug-induced apoptosis

In order to elucidate the role of the mitogen-activated protein kinases, including JNK, p38 MAPK and ERK, as well as the survival-associated PI3K/Akt signaling pathway, in the response to chemotherapy, we have conducted a comparative study regarding the effects of doxorubicin on these pathways. Doxorubicin was determined to elicit the apoptosis of NIH3T3 cells in a dose-dependent manner. Prior to cell death, both Akt and p38 MAPK were transiently activated, and subsequently inactivated almost wholly, whereas ERK and JNK evidenced sustained activations in response to the drug treatment. The inhibition of PI3K/Akt and p38 MAPK both accelerated and enhanced doxorubicin-induced apoptosis and ERK inhibition apparently exerted negative effect on apoptosis. The modulation of PI3K/Akt activation by treatment of LY294002 or expression of Akt mutants such as Akt-DN or Myr-Akt exerted a significant effect on the activation of ERK1/2. We also observed that PI3K/Akt and sustained ERK activation were associated intimately with the etoposide-induced apoptosis. Taken together, our results clearly suggest that the differential regulation of the PI3K/Akt, ERK1/2, and p38 MAPK signaling pathways are crucial in the context of DNA-damaging drug-induced apoptosis, and this has compelled us to propose that the sustained activation of ERK1/2 pathway may be generally involved in the apoptosis induced by anticancer DNA-damaging drugs, including doxorubicin and etoposide.     

Binding modes of V(=O)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various synthetic DNAs studied by polarized spectroscopy

The interactions between water-soluble cationic oxovanadyl[meso-tetrakis(4-N-methylpyridiumyl)]porphyrin (VOTMPyP) and various synthetic polynucleotide including poly[d(A-T)2], poly[d(G-C)2], and poly[d(I-C)2] were studied using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy. When VOTMPyP formed a complex with poly[d(A-T)2] and poly[d(I-C)2], a positive CD signal at low [VOTMPyP]/[DNA] ratios (R ratios) and strong excitonic CD signals at above R > or = 0.15 were induced. The appearance of the CD spectra of the VOTMPyP-poly[d(G-C)2] complex were very different: a small negative CD at low R ratios and very small excitonic CD at high R ratios were observed. Considering the facts that the minor grooves of the former two polynucleotides resemble and the major groove of poly[d(I-C)2] is similar with that of poly[d(G-C)2], it is conclusive that VOTMPyP binds to the minor groove of all DNA at lower R ratios while they stack at the outside of DNA at higher R ratios. The binding geometry of VOTMPyP to all polynucleotides studied by LD seemed to be homogenous, irrespective of the R ratio. It has been found that VOTMPyP can have five- and six-fluxional coordination states. Comparing the absorption spectra of VOTMPyP complexed with poly[d(A-T)2] and poly[d(G-C)2], the distinctive absorptions of the five- and six-coordinated species were observed at lower R ratios which centered at 420-430 nm and 442 nm, respectively. While the six-coordinated VOTMPyP favored the poly[d(A-T)2], the five-coordinated species favored the poly[d(G-C)2] at the low R ratios. As the stacked species increased with an increasing R ratio, the six-coordinated species became the major bound species. These observations lead us to conclude that the guanine base' amino group plays a crucial role not only in determining the binding mode of VOTMPyP but also in the conversion of the six-coordinated species to the five-coordinated species.     

Cortical bone remodeling rates in a sample of African American and European American descent groups from the American Midwest: comparisons of age and sex in ribs

This study employs regression analysis to explore population and sex differences in the pattern of age-associated bone loss, as reflected by histomorphometric variables that are measures of intracortical and endocortical bone remodeling. A comparison of an African American sample from the Washington Park Cemetery in St. Louis, Missouri, and a European American rib sample composed of cadavers, autopsies, and forensic cases from Missouri reveals the existence of complex age-associated patterns for differences in measures of intracortical remodeling and cortical area. Females from the two samples express similar bone dimensions and dynamics. The African American females appear to lose more bone than their male counterparts, but this difference is absent in the European American sample. When age-associated patterns are considered, it is in the younger cohorts that African Americans exhibit greater relative cortical area than European Americans, but this is reversed in the older ages, when the latter group manifests greater bone mass. The European American males consistently differ in the slopes and intercepts for the variables compared to the other groups, and differences are highly significant with African American females, with the former group maintaining bone mass while the latter exhibit a more rapid bone loss. Achieving larger relative cortical area due to smaller endosteal area, coupled with better bone quality due to lower intracortical porosity early in life, may be a mechanism by which African Americans, especially females, maintain adequate bone mass in older ages, which buffers them from bone loss and related fragility fractures despite higher rates of intracortical remodeling and endosteal expansion later in life. These results suggest that both genetic and environmental factors are responsible for the differences in bone remodeling and bone mass observed between these samples.     

Tick-Borne Rickettsial Pathogens in Ticks and Small Mammals in Korea

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.     

Stimulation of cyclic AMP production by the Caenorhabditis elegans muscarinic acetylcholine receptor GAR-3 in Chinese hamster ovary cells

Among the three G-protein-linked acetylcholine receptors (GARs) in Caenorhabditis elegans (C. elegans), GAR-3 is structurally and pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). Using Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the major alternatively spliced isoform of GAR-3, we observed that carbachol stimulated cyclic AMP (cAMP) production in a dose- and time-dependent manner. The stimulating effect of carbachol was abolished by atropine, a muscarinic antagonist, indicating that the cAMP production is specifically mediated by GAR-3b. When the cells were treated with BAPTA-AM and EGTA, which reduce the cytosolic Ca(2+) level, carbachol-stimulated cAMP accumulation was inhibited by approximately 56%. Inhibition of protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate (PMA) or by GF109203X decreased carbachol-stimulated cAMP production by as much as 68%. It thus appears that Ca(2+) and PKC are critically involved in GAR-3b-mediated cAMP formation. We also observed that carbachol-stimulated cAMP production was further enhanced by pertussis toxin (PTX) treatment. This observation indicates that GAR-3b couples to a PTX-sensitive G protein, presumably Gi, to attenuate the cAMP accumulation. Taken together, our data show that GAR-3b stimulates cAMP production in CHO cells and suggest that GAR-3b couples to both stimulatory and inhibitory pathways to modulate the intracellular cAMP level.     

Integrated electromyography and performance outcomes to inertial resistance exercise

Integrated electromyography (IEMG) and performance outcomes from resistance exercise may be influenced by gender, repetition order, and contractile mode. A novel strength training apparatus employing inertial resistance operates unlike standard exercise equipment and may therefore evoke different IEMG and performance outcomes. Subjects performed 3-set, 10-repetition calf press workouts on an inertial device while IEMG and flywheel velocity were recorded. Dependent variables were examined with 2 (men, women) x 2 (first vs. 10th repetition) x 2 (concentric, eccentric) analyses of variance. Performance outcomes showed gender-by-repetition and gender-by-contractile-mode interactions, with men's 10th-repetition and men's concentric data, respectively, causing these effects. Medial gastrocnemius (MG) IEMG showed a gender-by-repetition interaction resulting from men's first-repetition data. Greater 10th-repetition performance despite higher first-repetition MG IEMG may result from heightened triceps surae elastic energy utilization and/or maintained cross-bridges during transitions from lengthening-to-shortening actions. Inertial strength training may improve performance outcomes without additional motor unit recruitment.     

E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis

The von Hippel-Lindau tumor suppressor, pVHL, forms part of an E3 ubiquitin ligase complex that targets specific substrates for degradation, including hypoxia-inducible factor-1alpha (HIF-1alpha), which is involved in tumor progression and angiogenesis. It remains unclear, however, how pVHL is destabilized. Here we show that E2-EPF ubiquitin carrier protein (UCP) associates with and targets pVHL for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1alpha. UCP is detected coincidently with HIF-1alpha in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma and colon cancer cells. UCP level correlates inversely with pVHL level in most tumor cell lines. In vitro and in vivo, forced expression of UCP boosts tumor-cell proliferation, invasion and metastasis through effects on the pVHL-HIF pathway. Our results suggest that UCP helps stabilize HIF-1alpha and may be a new molecular target for therapeutic intervention in human cancers.     

Quantitative analysis of representative proteome components and clustering of Helicobacter pylori clinical strains

BACKGROUND: Several Helicobacter pylori proteins have been reported to be associated with severe symptoms of gastric disease. However, expression levels of most of these disease-associated proteins require further evaluation in order to clarify their relationships with gastric disease patterns. Representative proteome components of 71 clinical isolates of H. pylori were analyzed quantitatively to determine whether the protein expression levels were associated with gastric diseases and to cluster clinical isolates. METHODS: After two-dimensional electrophoresis (2-DE) of H. pylori isolates, spot intensities were analyzed using pdquest 2-D Gel Analysis Software. The intensities of 10 representative protein spots, identified by peptide fingerprinting using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) or peptide sequencing using quadrupole TOF MS, were subjected to the nonparametric Mann-Whitney test and hierarchical agglomerative cluster analysis. The relationship between clusters and gastric diseases was analyzed by the chi-squared test. RESULTS: Although the spot intensities of the 10 representative proteins were highly variable within each gastric disease group, the expression levels of CagA, UreB, GroEL, EF-Tu, EF-P, TagD, and FldA showed some significant differences among the gastric disease patterns. On the basis of the 10 target protein intensities, hierarchical agglomerative cluster analysis generated a dendrogram with clusters indicative of chronic gastritis/gastric cancers and gastric/duodenal ulcers. CONCLUSION: These results indicated that quantitative analysis of proteome components is a feasible method for examining disease-associated proteins and clustering clinical strains of H. pylori.     

Crystal structure of a beta-catenin/BCL9/Tcf4 complex

The canonical Wnt pathway plays critical roles in embryonic development, stem cell growth, and tumorigenesis. Stimulation of the Wnt pathway leads to the association of beta-catenin with Tcf and BCL9 in the nucleus, resulting in the transactivation of Wnt target genes. We have determined the crystal structure of a beta-catenin/BCL9/Tcf-4 triple complex at 2.6 A resolution. Our studies reveal that the beta-catenin binding site of BCL9 is distinct from that of most other beta-catenin partners and forms a good target for developing drugs that block canonical Wnt/beta-catenin signaling. The BCL9 beta-catenin binding domain (CBD) forms an alpha helix that binds to the first armadillo repeat of beta-catenin, which can be mutated to prevent beta-catenin binding to BCL9 without affecting cadherin or alpha-catenin binding. We also demonstrate that beta-catenin Y142 phosphorylation, which has been proposed to regulate BCL9-2 binding, does not directly affect the interaction of beta-catenin with either BCL9 or BCL9-2.     

SUMO-specific protease SUSP4 positively regulates p53 by promoting Mdm2 self-ubiquitination

The p53 tumour suppressor has a key role in the control of cell growth and differentiation, and in the maintenance of genome integrity. p53 is kept labile under normal conditions, but in response to stresses, such as DNA damage, it accumulates in the nucleus for induction of cell-cycle arrest, DNA repair or apoptosis. Mdm2 is an ubiquitin ligase that promotes p53 ubiquitination and degradation. Mdm2 is also self-ubiquitinated and degraded. Here, we identified a novel cascade for the increase in p53 level in response to DNA damage. A new SUMO-specific protease, SUSP4, removed SUMO-1 from Mdm2 and this desumoylation led to promotion of Mdm2 self-ubiquitination, resulting in p53 stabilization. Moreover, SUSP4 competed with p53 for binding to Mdm2, also resulting in p53 stabilization. Overexpression of SUSP4 inhibited cell growth, whereas knockdown of susp4 by RNA interference (RNAi) promoted of cell growth. UV damage induced SUSP4 expression, leading to an increase in p53 levels in parallel with a decrease in Mdm2 levels. These findings establish a new mechanism for the elevation of cellular p53 levels in response to UV damage.