Evolutionarily Conserved Sequence Features Regulate the Formation of the FG Network at the Center of the Nuclear Pore Complex.

The nuclear pore complex (NPC) is the portal for bidirectional transportation of cargos between the nucleus and the cytoplasm. While most of the structural elements of the NPC, i.e. nucleoporins (Nups), are well characterized, the exact transport mechanism is still under much debate. Many of the functional Nups are rich in phenylalanine-glycine (FG) repeats and are believed to play the key role in nucleocytoplasmic transport. We present a bioinformatics study conducted on more than a thousand FG Nups across 252 species. Our results reveal the regulatory role of polar residues and specific sequences of charged residues, named 'like charge regions' (LCRs), in the formation of the FG network at the center of the NPC. Positively charged LCRs prepare the environment for negatively charged cargo complexes and regulate the size of the FG network. The low number density of charged residues in these regions prevents FG domains from forming a relaxed coil structure. Our results highlight the significant role of polar interactions in FG network formation at the center of the NPC and demonstrate that the specific localization of LCRs, FG motifs, charged, and polar residues regulate the formation of the FG network at the center of the NPC.

MET in gastric carcinomas: comparison between protein expression and gene copy number and impact on clinical outcome.

BACKGROUND: The aim of this study was to compare gene copy number (GCN) and protein expression of MET and to evaluate their prognostic roles in gastric carcinomas. METHODS: MET protein expression and gene amplification (GA) status were determined by immunohistochemistry (IHC) and silver in-situ hybridisation (SISH), respectively, in a large series of gastric carcinoma. RESULTS: Protein overexpression was observed in 104 of 438 cases, with IHC 2+ in 94 and IHC 3+ in 10, and high polysomy of chromosome 7 and GA were found in 61 and 13 of 381, respectively. Direct comparison revealed a significant correlation between high level of protein expression and increased GCN. All cases with GA showed protein overexpression. Furthermore, all with IHC 3+ showed GA except 1, even which could be categorised as GA according to the ASCO/CAP guideline for human epidermal growth factor receptor 2 assessment. IHC 3+ and GA were significantly associated with poor prognosis. CONCLUSION: MET IHC reflects well on GA, and therefore, it could be a primary screening test for patient selection for anti-MET therapy if GA is a major determinant of drug responsiveness. Also, the prognostic role of MET indicates that anti-MET therapy is a very promising modality in adjuvant treatment for gastric cancer.

A hypoxia-dependent upregulation of hypoxia-inducible factor-1 by nuclear factor-κB promotes gastric tumour growth and angiogenesis.

BACKGROUND: The underlying mechanisms involved in the activation of hypoxia-inducible factor-1 (HIF-1) in gastric cancer remain unclear. As nuclear factor-κB (NF-κB) as well as HIF-1 have been implicated in angiogenesis of various cancers, we investigated their relationship in gastric cancer. METHODS: Nuclear expressions of HIF-1α and NF-κB/RelA were assessed in 251 human gastric carcinoma specimens by immunohistochemical tissue array analysis. Stable human gastric cancer cells, infected with a retroviral vector containing super-suppressive mutant form of IκBα (IκBαM), were used for animal studies as well as cell culture experiments. Xenografted tumours were measured and IκBαM effects on angiogenesis and HIF-1α activation were assessed by immunohistochemistry, western blotting, luciferase reporter assay, and semiquantitative reverse transcription-polymerase chain reaction. In addition, NF-κB effects on the HIF-1α degradation and synthesis were examined. RESULTS: Hypoxia-inducible factor-1α activation positively correlated with RelA activation in clinical gastric cancer samples (P<0.001). The IκBαM overexpression suppressed tumour growth, microvessel density, and HIF-1α activation in xenografted tumours. Cell culture experiments showed that hypoxia-induced HIF-1α expression was reduced by NF-κB inhibition under hypoxic conditions at the translational level. CONCLUSION: The hypoxia-dependent activation of the NF-κB/HIF-1α/VEGF pathway contributes, at least in part, to gastric cancer promotion via enhancement of angiogenesis.

PTEN status switches cell fate between premature senescence and apoptosis in glioma exposed to ionizing radiation.

Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) has frequently been observed in human gliomas, conferring AKT activation and resistance to ionizing radiation (IR) and drug treatments. Recent reports have shown that PTEN loss or AKT activation induces premature senescence, but many details regarding this effect remain obscure. In this study, we tested whether the status of PTEN determined fate of the cell by examining PTEN-deficient U87, U251, and U373, and PTEN-proficient LN18 and LN428 glioma cells after exposure to IR. These cells exhibited different cellular responses, senescence or apoptosis, depending on the PTEN status. We further observed that PTEN-deficient U87 cells with high levels of both AKT activation and intracellular reactive oxygen species (ROS) underwent senescence, whereas PTEN-proficient LN18 cells entered apoptosis. ROS were indispensable for inducing senescence in PTEN-deficient cells, but not for apoptosis in PTEN-proficient cells. Furthermore, transfection with wild-type (wt) PTEN or AKT small interfering RNA induced a change from premature senescence to apoptosis and depletion of p53 or p21 prevented IR-induced premature senescence in U87 cells. Our data indicate that PTEN acts as a pivotal determinant of cell fate, regarding senescence and apoptosis in IR-exposed glioma cells. We conclude that premature senescence could have a compensatory role for apoptosis in the absence of the tumor suppressor PTEN through the AKT/ROS/p53/p21 signaling pathway.

Plasma carotenoids and risk of acute myocardial infarction in the Singapore Chinese Health Study.

BACKGROUND AND AIM: Modification of low-density lipoprotein due to oxidative stress is essential in the development of coronary atherosclerosis. Data of specific carotenoids except ß-carotene on cardioprotective effects in humans are limited. METHODS AND RESULTS: This study examined the associations between plasma concentrations of specific carotenoids and incidence of acute myocardial infarction. The study included 280 incident cases of acute myocardial infarction and 560 matched controls nested within the Singapore Chinese Health Study, a prospective cohort of 63,257 Chinese men and women aged 45-74 years old enrolled in 1993-1998 in Singapore. Retinol and carotenoids in prediagnostic plasma were quantified using high-performance liquid chromatography. High levels of plasma ß-cryptoxanthin and lutein were associated with decreased risk of acute myocardial infarction after adjustment for multiple risk factors for coronary heart disease. For ß-cryptoxanthin, the odds ratio (95% confidence interval) for the highest (Q5) versus the lowest (Q1) quintile was 0.67 (0.37-1.21) (P for trend=0.03). For lutein, the odds ratios (95% confidence intervals) for the combined Q2-Q3 and the combined Q4-Q5 versus Q1 were 0.71 (0.45-1.12) and 0.58 (0.35-0.94) respectively (P for trend=0.03). There was no statistically significant association between other carotenoids or retinol and risk of acute myocardial infarction. CONCLUSIONS: High plasma levels of ß-cryptoxanthin and lutein were associated with decreased risk of acute myocardial infarction. The findings of this study support a cardioprotective role of these two carotenoids in humans.

Prognostic implications of type and density of tumour-infiltrating lymphocytes in gastric cancer.

The study aims to determine whether type and density of tumour-infiltrating lymphocytes (TILs) can predict the clinical course in gastric cancer. Gastric carcinomas (n=220) were immunostained for CD3, CD8, CD20, and CD45RO and evaluated for clinicopathologic characteristics. Number of TILs that immunostained positively for each marker were counted using NIH ImageJ software. Tumours were grouped into low- and high-density groups for each marker (CD3, CD8, CD45RO). The densities of CD3(+), CD8(+), and CD45RO(+) TILs were found to be independent predictors of lymph node metastasis by multivariate analysis with odds ratios (95% CI) of 0.425 (0.204-0.885), 0.325 (0.150-0.707), and 0.402 (0.190-0.850), respectively. Kaplan-Meier survival analysis revealed that patients in the high-density groups for CD3, CD8, and C45RO had a significantly longer survival time than the patients in the corresponding low-density groups, respectively. In multivariate survival analysis, the densities of CD3(+), CD8(+), and CD45RO(+) TILs remained independent prognostic factors with hazard ratios (95% CI) of 0.549 (0.317-0.951), 0.574 (0.347-0.949), and 0.507 (0.298-0.862), respectively. In conclusion, density of TILs was found to be independently predictive of regional lymph node metastasis and patient survival in gastric cancer.

Characteristics of KIT-negative gastrointestinal stromal tumours and diagnostic utility of protein kinase C theta immunostaining.

AIMS: To characterise KIT-negative gastrointestinal stromal tumours (GISTs) clinically, pathologically, immunohistochemically and genetically, and to establish the usefulness of protein kinase C theta (PKC) as a diagnostic marker in KIT-negative GIST. METHODS: 252 consecutive cases of GIST were evaluated for clinicopathological characteristics and immunostained for various antibodies. Mutational analyses of KIT and platelet-derived growth factor receptor alpha (PDGFRA) were also performed in 62 cases. RESULTS: 20 (7.9%) GISTs showed negative immunostaining for KIT. KIT-negative GISTs were more likely to originate from omentum or peritoneum, have an epithelioid histology, and be classified as high risk. The overall survival rate of patients with KIT-negative GISTs (5-year survival rate 68.7% (SD 10.7%)) was lower than that of patients with KIT-positive GISTs (5-year survival rate, 79.9% (3.0%)) (p = 0.042, log-rank test). Negative KIT expression was an independent prognostic factor in multivariate Cox regression analysis when the risk of aggressive behaviour and the status of imatinib treatment were adopted as covariates. KIT-negative GISTs also showed lower expression rates of CD34, Bcl-2, and PKC than KIT-positive GISTs; mutational analysis revealed that 30% of KIT-negative GISTs harboured a PDGFRA exon 18 mutation. Immunostaining on PKC showed that 93.9% of all GISTs expressed PKC protein. However, 21.9% of 64 mesenchymal tumours other than GIST also showed positivity on PKC. CONCLUSIONS: KIT-negative GISTs had characteristics that differ from those of KIT-positive GISTs, and negative KIT expression was an independent prognostic indicator for overall survival of patients. Although PKC is a sensitive diagnostic marker for GIST, its usefulness is limited because of low sensitivity and low specificity in KIT-negative GISTs.

Susceptibility of various oral bacteria to antimicrobial peptides and to phagocytosis by neutrophils.

BACKGROUND AND OBJECTIVE: The aim of this study was to compare the susceptibility of nonperiodontopathic and periodontopathic bacteria to major defense mechanisms for bacterial clearance in gingival sulcus. MATERIAL AND METHODS: Twenty strains of 13 oral bacterial species were studied for their susceptibility to phagocytosis by human neutrophils and to the antimicrobial peptides LL-37 and human beta defensin-3. The minimum inhibitory concentrations of LL-37 and human beta defensin-3 were determined by a liquid dilution assay, and susceptibility to phagocytosis was examined by a flow cytometric phagocytosis assay. RESULTS: The minimum inhibitory concentrations of LL-37 and human beta defensin-3 varied greatly, depending on the strain and species. Although a significant difference between the non- and periodontopathic groups was not observed, the red-complex bacteria were more resistant to LL-37 than the others (p=0.004). The susceptibility of oral bacteria to phagocytosis was quite variable, depending on the species but not on the strains. The periodontopathic bacteria, especially Actinobacillus actinomycetemcomitans and the red-complex triad, were more resistant to phagocytosis than were the nonperiodontopathic bacteria (p=0.0003). In addition, bacteria resistant both to antimicrobial peptides and to phagocytosis were more common in the periodontopathic group. CONCLUSION: Our results indicate that immune evasion may contribute to the pathogenicity of some periodontopathic bacteria.

Oxidative burden in prediabetic and diabetic individuals: evidence from plasma coenzyme Q(10).

AIM: Individuals with diabetes and prediabetes are at risk of vascular injury. However, the exact mechanisms are unclear. The mitochondria mobile electron carrier coenzyme Q(10) (CoQ(10)) is a potent lipophilic antioxidant. We hypothesize that oxidative stress, detectable as changes in plasma CoQ(10) concentrations and composition, plays an important role in vascular disease in diabetes. METHODS: We measured plasma CoQ(10) concentrations (including reduced ubiquinol and oxidized ubiquinone subfractions) in 60 subjects with normal glucose tolerance [NGT; fasting plasma glucose (FPG) < 5.5 mmol/l], 63 with impaired fasting glucose (IFG; FPG 5.6-6.9 mmol/l) and 69 with Type 2 diabetes (DM; FPG > 6.9 mmol/l). RESULTS: In men and women, the total CoQ(10)/total cholesterol ratio was reduced in DM (mean +/-sd) [male (M) 0.09 +/- 0.04; female (F) 0.07 +/- 0.04] compared with NGT (0.29 +/- 0.08; 0.21 +/- 0.07) and IFG (0.27 +/- 0.07; 0.23 +/- 0.07) (DM vs. NGT and IFG P = 0.001). A stepwise reduction in the plasma ubiquinol fraction (ubiquinol/total CoQ10) was observed from NGT (M 0.93 +/- 0.06; F 0.95 +/- 0.06) compared with IFG (0.43 +/- 0.25; 0.41 +/- 0.15) and DM (0.24 +/- 0.11; F 0.29 +/- 0.16) (DM vs. IFG vs. NGT P = 0.001). In contrast, the plasma ubiquinone/ubiquinol ratio increased from NGT (M 0.08 +/- 0.07, F 0.06 +/- 0.08) to IFG (2.14 +/- 1.84, 1.75 +/- 1.04) to DM (4.77 +/- 4.88, 3.81 +/- 3.71) (DM vs. IFG vs. NGT P = 0.001). These differences remained after adjusting for age, body mass index and FPG. CONCLUSIONS: The change in CoQ(10) with increasing FPG concentration suggests an increase in oxidative burden, already evident in the prediabetic IFG individuals. This increase in oxidative stress might contribute to the increased risk of vascular disease.

The afterglow and elliptical host galaxy of the short gamma-ray burst GRB 050724.

Despite a rich phenomenology, gamma-ray bursts (GRBs) are divided into two classes based on their duration and spectral hardness--the long-soft and the short-hard bursts. The discovery of afterglow emission from long GRBs was a watershed event, pinpointing their origin to star-forming galaxies, and hence the death of massive stars, and indicating an energy release of about 10(51) erg. While theoretical arguments suggest that short GRBs are produced in the coalescence of binary compact objects (neutron stars or black holes), the progenitors, energetics and environments of these events remain elusive despite recent localizations. Here we report the discovery of the first radio afterglow from the short burst GRB 050724, which unambiguously associates it with an elliptical galaxy at a redshift z = 0.257. We show that the burst is powered by the same relativistic fireball mechanism as long GRBs, with the ejecta possibly collimated in jets, but that the total energy release is 10-1,000 times smaller. More importantly, the nature of the host galaxy demonstrates that short GRBs arise from an old (> 1 Gyr) stellar population, strengthening earlier suggestions and providing support for coalescing compact object binaries as the progenitors.