Residues Specifically Involved in Down-Regulation but Not Internalization of the m1 Muscarinic Acetylcholine Receptor

Abstract: Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down-regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down-regulation alone. Four mutations were identified that specifically impaired down-regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down-regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [³⁵S]GTPγS binding. However, the level of second messenger stimulation correlated poorly with the extent of down-regulation. In summary, several mutations of the m1 receptor selectively affect down-regulation, demonstrating that internalization and down-regulation represent distinct events driven by different cellular mechanisms.     

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+ Cell Populations

Human cytomegalovirus (HCMV) is a significant human pathogen that achieves lifelong persistence by establishing latent infections in undifferentiated cells of the myeloid lineage, such as CD34⁺ hematopoietic progenitor cells. When latency is established, viral lytic gene expression is silenced in part by a cellular intrinsic defense consisting of Daxx and histone deacetylases (HDACs) because pp71, the tegument transactivator that travels to the nucleus and inactivates this defense at the start of a lytic infection in differentiated cells, remains in the cytoplasm. Because the current in vitro and ex vivo latency models have physiological and practical limitations, we evaluated two CD34⁺ myeloblastic cell lines, KG-1 and Kasumi-3, for their ability to establish, maintain, and reactivate HCMV experimental latent infections. Tegument protein pp71 was cytoplasmic, and immediate-early (IE) genes were silenced as in primary CD34⁺ cells. However, in contrast to what occurs in primary CD34⁺ cells ex vivo or in NT2 and THP-1 in vitro model systems, viral IE gene expression from the laboratory-adapted AD169 genome was not induced in the presence of HDAC inhibitors in either KG-1 or Kasumi-3 cells. Furthermore, while the clinical strain FIX was able to reactivate from Kasumi-3 cells, AD169 was not, and neither strain reactivated from KG-1 cells. Thus, KG-1 and Kasumi-3 experimental latent infections differ in important parameters from those in primary CD34⁺ cell populations. Aspects of latency illuminated through the use of these myeloblastoid cell lines should not be considered independently but integrated with results obtained in primary cell systems when paradigms for HCMV latency are proposed.     

The Receptor Complex Associated with Human T-Cell Lymphotropic Virus Type 3 (HTLV-3) Env-Mediated Binding and Entry Is Distinct from,but Overlaps with,the Receptor Complexes of HTLV-1 and HTLV-2

Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4⁺ and CD8⁺ T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4⁺ T cells, and HTLV-2 SU, which primarily binds to activated CD8⁺ T cells. Binding studies with heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), two molecules important for HTLV-1 entry, revealed that these molecules also enhance HTLV-3 SU binding. However, unlike HTLV-1 SU, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, for HTLV-1, glucose transporter 1 (GLUT-1) functions at a postbinding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4⁺ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1, and GLUT-1. These results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.The primate T-cell lymphotropic virus (PTLV) group of deltaretroviruses consists of three types of human T-cell lymphotropic viruses (HTLVs) (HTLV-1, HTLV-2, HTLV-3), their closely related simian T-cell lymphotropic viruses (STLVs) (STLV-1, STLV-2, STLV-3), an HTLV (HTLV-4) for which a simian counterpart has not been yet identified, and an STLV (STLV-5) originally described as a divergent STLV-1 (5-7, 30, 35, 37, 38, 45, 51, 53). HTLV-1 and HTLV-2, which have a 70% nucleotide homology, differ in both their pathobiology and tropism (reviewed in reference 13). While HTLV-1 causes a neurological disorder (tropical spastic paraparesis/HTLV-1-associated myelopathy) and a hematological disease (adult T-cell leukemia/lymphoma) (15, 42, 55), HTLV-2 is only rarely associated with tropical spastic paraparesis/HTLV-1-associated myelopathy-like disease and is not definitively linked to any lymphoproliferative disease (12, 20). In vivo, both HTLV-1 and HTLV-2 infect T cells. Although HTLV-1 is primarily found in CD4⁺ T cells, other cell types in the peripheral blood of infected individuals have been found to contain HTLV-1, including CD8⁺ T cells, dendritic cells, and B cells (19, 29, 33, 36, 46).Binding and entry of retroviruses requires specific interactions between the Env glycoproteins on the virus and cell surface receptor complexes on target cells. For HTLV-1, three molecules have been identified as important for entry, as follows: heparan sulfate proteoglycans (HSPGs), neuropilin-1 (NRP-1), and glucose transporter 1 (GLUT-1) (16, 22, 26, 28, 29, 34, 39, 44). Recent studies support a model in which HSPG and NRP-1 function during the initial binding of HTLV-1 to target cells, and GLUT-1 functions at a postattachment stage, most likely to facilitate fusion (29, 34, 49). Efficient HTLV-2 binding and entry requires NRP-1 and GLUT-1 but not HSPGs (16, 26, 39, 49).This difference in the molecules required for binding to target cells reflects differences in the T-cell tropisms of these two viruses. Activated CD4⁺ T cells express much higher levels of HSPGs than CD8⁺ T cells (26). In infected individuals, HTLV-1 is primarily found in CD4⁺ T cells, while HTLV-2 is primarily found in CD8⁺ T cells (21, 43, 46). In vitro, HTLV-1 preferentially transforms CD4⁺ T cells while HTLV-2 preferentially transforms CD8⁺ T cells, and this difference has been mapped to the Env proteins (54).We and others have reported the discovery of HTLV-3 in two Cameroonese inhabitants (6, 7, 53). We recently uncovered the presence of a third HTLV-3 strain in a different population living several hundred kilometers away from the previously identified groups (5), suggesting that this virus may be common in central Africa. Since the HTLV-3 sequences were obtained by PCR amplification of DNA isolated from peripheral blood mononuclear cells (PBMCs) of infected individuals, little is known about its tropism and pathobiology in vivo. Based on the correlation between HSPG expression levels and viral tropisms of HTLV-1 and HTLV-2, we reasoned that knowledge about the HTLV-3 receptors might provide insight into the tropism of this virus. We therefore generated vectors expressing HTLV-3 Env proteins and used them to begin to characterize the receptor complex used by HTLV-3 to bind and enter cells.     

Genetic Variations in the Androgen Receptor Are Associated with Steroid Concentrations and Anthropometrics but Not with Muscle Mass in Healthy Young Men

Objective

The relationship between serum testosterone (T) levels, muscle mass and muscle force in eugonadal men is incompletely understood. As polymorphisms in the androgen receptor (AR) gene cause differences in androgen sensitivity, no straightforward correlation can be observed between the interindividual variation in T levels and different phenotypes. Therefore, we aim to investigate the relationship between genetic variations in the AR, circulating androgens and muscle mass and function in young healthy male siblings.

Design

677 men (25–45 years) were recruited in a cross-sectional, population-based sibling pair study.

Methods

Relations between genetic variation in the AR gene (CAGn, GGNn, SNPs), sex steroid levels (by LC-MS/MS), body composition (by DXA), muscle cross-sectional area (CSA) (by pQCT), muscle force (isokinetic peak torque, grip strength) and anthropometrics were studied using linear mixed-effect modelling.

Results

Muscle mass and force were highly heritable and related to age, physical activity, body composition and anthropometrics. Total T (TT) and free T (FT) levels were positively related to muscle CSA, whereas estradiol (E₂) and free E₂ (FE₂) concentrations were negatively associated with muscle force. Subjects with longer CAG repeat length had higher circulating TT, FT, and higher E₂ and FE₂ concentrations. Weak associations with TT and FT were found for the rs5965433 and rs5919392 SNP in the AR, whereas no association between GGN repeat polymorphism and T concentrations were found. Arm span and 2D:4D finger length ratio were inversely associated, whereas muscle mass and force were not associated with the number of CAG repeats.

Conclusions

Age, physical activity, body composition, sex steroid levels and anthropometrics are determinants of muscle mass and function in young men. Although the number of CAG repeats of the AR are related to sex steroid levels and anthropometrics, we have no evidence that these variations in the AR gene also affect muscle mass or function.     

A Protein Kinase Activity Is Associated with and Specifically Phosphorylates the Neural Cell Adhesion Molecule LI

The neural cell adhesion molecule L1 is a phosphorylated integral membrane glycoprotein that is recovered from adult mouse brain by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, 80, and 50 kilodaltons (L1-200, L1-180, L1-140, L1-80, and L1-50, respectively). In the present study, we show that two kinase activities are associated with immunopurified L1: One specifically phosphorylates L1-200 and L1-80 but not L1-180, L1-140, or L1-50. This pattern of phosphorylation corresponds to the one described for L1 after metabolic phosphate incorporation into cultures of cerebellar cells. In both cases, serine is the main amino acid that is labeled by radioactive phosphate. The kinase activity is not activated by Ca2+, calmodulin, phosphatidylserine, diolein, cyclic AMP, or cyclic GMP, a result suggesting that the enzyme is distinct from Ca2+/calmodulin-dependent kinases, from protein kinase C, or from cyclic AMP/cyclic GMP-dependent kinases and may belong to the independent kinase group. The other kinase phosphorylates only casein but not L1, utilizes GTP as well as ATP, and is strongly inhibited by heparin. Because the primary structure of the L1 protein does not contain consensus sequences characteristic for known kinases, we believe that the catalytic activities detectable in immunopurified L1 are due to kinases that are strongly enough associated with L1 to withstand the stringent purification procedures.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

The Bacterium Endosymbiont of Crithidia deanei Undergoes Coordinated Division with the Host Cell Nucleus

In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three-dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells.     

Genetic Variation in the TNF Gene Is Associated with Susceptibility to Severe Sepsis,but Not with Mortality

Background

Tumor necrosis factor (TNF) and TNF receptor superfamily (TNFR)-mediated immune response play an essential role in the pathogenesis of severe sepsis. Studies examining associations of TNF and lymphotoxin-α (LTA) single nucleotide polymorphisms (SNPs) with severe sepsis have produced conflicting results. The objective of this study was to investigate whether genetic variation in TNF, LTA, TNFRSF1A and TNFRSF1B was associated with susceptibility to or death from severe sepsis in Chinese Han population.

Methodology/Principal Findings

Ten SNPs in TNF, LTA, TNFRSF1A and TNFRSF1B were genotyped in samples of patients with severe sepsis (n = 432), sepsis (n = 384) and healthy controls (n = 624). Our results showed that rs1800629, a SNP in the promoter region of TNF, was significantly associated with risk for severe sepsis. The minor allele frequency of rs1800629 was significantly higher in severe sepsis patients than that in both healthy controls (P_adj = 0.00046, odds ratio (OR)_adj = 1.92) and sepsis patients (P_adj = 0.002, OR_adj = 1.56). Further, we investigated the correlation between rs1800629 genotypes and TNF-α concentrations in peripheral blood mononuclear cells (PBMCs) of healthy volunteers exposed to lipopolysaccharides (LPS) ex vivo, and the association between rs1800629 and TNF-α serum levels in severe sepsis patients. After exposure to LPS, the TNF-α concentration in culture supernatants of PBMCs was significantly higher in the subjects with AA+AG genotypes than that with GG genotype (P = 0.007). Moreover, in patients with severe sepsis, individuals with AA+AG genotypes had significantly higher TNF-α serum concentrations than those with GG genotype (P_adj = 0.02). However, there were no significant associations between SNPs in the four candidate genes and 30 day mortality for patients with severe sepsis.

Conclusions/Significance

Our findings suggested that the functional TNF gene SNP rs1800629 was strongly associated with susceptibility to severe sepsis, but not with lethality in Chinese Han population.     

Birth of Cone Bipolar Cells,but Not Rod Bipolar Cells,Is Associated with Existing RGCs

Retinal ganglion cells (RGCs) play important roles in retinogenesis. They are required for normal retinal histogenesis and retinal cell number balance. Developmental RGC loss is typically characterized by initial retinal neuronal number imbalance and subsequent loss of retinal neurons. However, it is not clear whether loss of a specific non-RGC cell type in the RGC-depleted retina is due to reduced cell production or subsequent degeneration. Taking advantage of three knockout mice with varying degrees of RGC depletion, we re-examined bipolar cell production in these retinas from various aspects. Results show that generation of the cone bipolar cells is correlated with the existing number of RGCs. However, generation of the rod bipolar cells is unaffected by RGC shortage. Results report the first observation that RGCs selectively influence the genesis of subsequent retinal cell types.     

Childhood Adversity Is Associated with Adult Theory of Mind and Social Affiliation,but Not Face Processing

People vary substantially in their ability to acquire and maintain social ties. Here, we use a combined epidemiological and individual differences approach to understand the childhood roots of adult social cognitive functioning. We assessed exposure to 25 forms of traumatic childhood experiences in over 5000 adults, along with measures of face discrimination, face memory, theory of mind, social motivation, and social support. Retrospectively-reported experiences of parental maltreatment in childhood (particularly physical abuse) were the most broadly and robustly associated with adult variations in theory of mind, social motivation, and social support. Adult variations in face discrimination and face memory, on the other hand, were not significantly associated with exposure to childhood adversity. Our findings indicate domains of social cognition that may be particularly vulnerable to the effects of adverse childhood environments, and suggest mechanisms whereby environmental factors might influence the development of social abilities.     

The Feline Calicivirus Leader of the Capsid Protein Is Associated with Cytopathic Effect

Open reading frame 2 (ORF2) of the feline calicivirus (FCV) genome encodes a capsid precursor that is posttranslationally processed to release the mature capsid protein (VP1) and a small protein of 124 amino acids, designated the leader of the capsid (LC). To investigate the role of the LC protein in the virus life cycle, mutations and deletions were introduced into the LC coding region of an infectious FCV cDNA clone. Three cysteine residues that are conserved among all vesivirus LC sequences were found to be critical for the recovery of FCV with a characteristic cytopathic effect in feline kidney cells. A cell-rounding phenotype associated with the transient expression of wild-type and mutagenized forms of the LC correlated with the cytopathic and growth properties of the corresponding engineered viruses. The host cellular protein annexin A2 was identified as a binding partner of the LC protein, consistent with a role for the LC in mediating host cell interactions that alter the integrity of the cell and enable virus spread.     

Angiopoietin-2 and Biliary Diseases: Elevated Serum,but Not Bile Levels Are Associated with Cholangiocarcinoma

Background

The diagnosis of cholangiocarcinoma (CC) is challenging especially in patients with primary sclerosing cholangitis (PSC) and often delayed due to the lack of reliable markers. Angiopoietin-2 (Angpt-2) has been employed as a biomarker of angiogenesis and might be involved in tumor neoangiogenesis.

Aim

To evaluate the diagnostic potential of Angpt-2 as a biomarker to detect patients with CC.

Methods

Bile and serum Angpt-2 levels were measured in patients with CC (n = 45), PSC (n = 74), CC complicating PSC (CC/PSC) (n = 11) and patients with bile duct stones (n = 37) in a cross sectional study. Diagnostic accuracy of Angpt-2 was compared to carbohydrate antigen 19-9 (CA19-9). Fluorescent immunohistochemistry from human CC liver tissue samples was performed to localize the origin of Angpt-2.

Results

Serum Angpt-2 concentration was significantly elevated in patients with CC compared to control patients (p<0.05). Diagnostic accuracy of Angpt-2 as determined by receiver operating characteristic (ROC) analysis resulted in a higher area under the curve (AUC) value compared to CA19-9 (AUC: 0.85 versus 0.77; 95% confidence interval (CI): 0.74–0.93 versus 0.65–0.87, respectively). Angpt-2 was also detectable in bile, but was not associated with the presence of CC. Immunohistochemistry revealed a strong induction of Angpt-2 expression in the tumor vasculature.

Conclusions

Circulating Angpt-2 in serum might be a promising protein candidate locally derived from the tumor vasculature in patients with CC. Measurement of Angpt-2 in serum may be useful for diagnosis and further clinical management of patients with CC.     

Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.     

Interaction of Poliovirus with Its Purified Receptor and Conformational Alteration in the Virion

Polypeptides of amino acids 1 to 241 (PVR241) and 1 to 330 (PVR330) of the human poliovirus receptor (hPVR) were produced in a baculovirus expression system. PVR241 contained extracellular domains 1 and 2 of hPVR, and PVR330 contained extracellular domains 1, 2, and 3. These peptides were purified by immunoaffinity column chromatography with an anti-hPVR monoclonal antibody (MAb). After the purification, PVR241 and PVR330 appeared to retain their native conformation as judged by reactivity with an anti-PVR MAb that recognized domain 1 of hPVR in a conformation-dependent manner. The virulent Mahoney strain of poliovirus type 1 was mixed with the purified PVRs in various concentrations. An average of at least 43 PVR330 molecules were able to bind to one virion particle under the conditions used. The equilibrium dissociation constant between the PVR330 molecule and the PVR binding site (canyon) on the virion was determined to be 4.50 ± (0.86) × 10⁻⁸ M at 4°C. Higher rates of conformational change of the virus (160S) to 135S and 80S particles were observed as the concentration of PVR330 was increased. In this in vitro system, the ratio of the amount of the 135S particle to that of the 80S particle seemed to be always constant. After the disappearance of the 160S particle, the amount of the 80S particle was not increased by further incubation at 37°C. These results suggested that the 80S particle was not derived from the 135S particle under the conditions used in this study.     

Demographic and Lifestyle Characteristics,but Not Apolipoprotein E Genotype,Are Associated with Intelligence among Young Chinese College Students

Background

Intelligence is an important human feature that strongly affects many life outcomes, including health, life-span, income, educational and occupational attainments. People at all ages differ in their intelligence but the origins of these differences are much debated. A variety of environmental and genetic factors have been reported to be associated with individual intelligence, yet their nature and contribution to intelligence differences have been controversial.

Objective

To investigate the contribution of apolipoprotein E (APOE) genotype, which is associated with the risk for Alzheimer’s disease, as well as demographic and lifestyle characteristics, to the variation in intelligence.

Methods

A total of 607 Chinese college students aged 18 to 25 years old were included in this prospective observational study. The Chinese revision of Wechsler Adult Intelligence Scale (the fourth edition, short version) was used to determine the intelligence level of participants. Demographic and lifestyle characteristics data were obtained from self-administered questionnaires.

Results

No significant association was found between APOE polymorphic alleles and different intelligence quotient (IQ) measures. Interestingly, a portion of demographic and lifestyle characteristics, including age, smoking and sleep quality were significantly associated with different IQ measures.

Conclusions

Our findings indicate that demographic features and lifestyle characteristics, but not APOE genotype, are associated with intelligence measures among young Chinese college students. Thus, although APOE ε4 allele is a strong genetic risk factor for Alzheimer’s disease, it does not seem to impact intelligence at young ages.     

High Expression of HuR in Cytoplasm,but Not Nuclei,Is Associated with Malignant Aggressiveness and Prognosis in Bladder Cancer

Introduction

Human antigen R (HuR) regulates the stability of mRNA and is associated with cell proliferation, angiogenesis, and lymphangiogenesis. However, the clinical significance and pathological role of HuR in bladder cancer remains unclear. The main objective of this investigation was to clarify the relationships between HuR expression and clinical significance and cancer cell proliferation, angiogenesis, lymphangiogenesis, and expressions of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF)-A, -C, and -D.

Methods

All expressions were examined by immunohistochemical techniques in 122 formalin-fixed specimens of bladder cancer patients. HuR expression was evaluated separately with cytoplasmic and nuclear staining. Cell proliferation, angiogenesis and lymphangiogenesis were measured as the percentage of Ki-67-positive cell (proliferation index, PI), CD34-stained vessels (microvessel density, MVD), and D2-40-stained vessels (lymph vessel density, LVD). Relationships between each HuR expression and clinicopathological features, prognosis, and expressions of COX-2 and VEGFs were analyzed by multi-variate analyses. HuR expression was also investigated in 10 mice of N-Butyl-N-[4-hydroxybutil] nitrosamine (BBN) induced bladder cancer model.

Results

In human tissues, high cytoplasmic expression was seen in 5% and 25.4% of normal and cancer cells, respectively. Nuclear HuR expression bore no significant relationship to any pathological features. However, cytoplasmic HuR expression appeared positively associated with pT stage and grade (P<0.001). In mouse tissues, similar trends were confirmed. Cytoplasmic expression correlated with PI, MVD, and LVD, as well as expression of VEGF-A and -C, but not VEGF-D. High cytoplasmic expression of HuR was a significant predictor of metastasis and cause-specific survival, and was identified as a prognostic correlative factor for metastasis (hazard ratio, 4.75; P = 0.028) in a multivariate analysis model that included pathological features.

Conclusions

Cytoplasmic HuR appears to play important roles in cell proliferation, progression, and survival of bladder cancer patients. Its expression was associated with angiogenesis, lymphangiogenesis, and expressions of VEGF-A and –C.     

Consumption of Green Tea,but Not Black Tea or Coffee,Is Associated with Reduced Risk of Cognitive Decline

Our objective was to determine whether the consumption of green tea, coffee, or black tea influences the incidence of dementia and mild cognitive impairment (MCI) in older people. We conducted a population-based prospective study with Japanese residents aged >60 years from Nakajima, Japan (the Nakajima Project). Participants received an evaluation of cognitive function and blood tests. The consumption of green tea, coffee, and black tea was also evaluated at baseline. Of 723 participants with normal cognitive function at a baseline survey (2007–2008), 490 completed the follow up survey in 2011–2013. The incidence of dementia during the follow-up period (mean ± SD: 4.9±0.9 years) was 5.3%, and that of MCI was 13.1%. The multiple-adjusted odds ratio for the incidence of overall cognitive decline (dementia or MCI) was 0.32 (95% CI: 0.16–0.64) among individuals who consumed green tea every day and 0.47 (95% CI: 0.25–0.86) among those who consumed green tea 1–6 days per week compared with individuals who did not consume green tea at all. The multiple-adjusted odds ratio for the incidence of dementia was 0.26 (95% CI: 0.06–1.06) among individuals who consumed green tea every day compared with those who did not consume green tea at all. No association was found between coffee or black tea consumption and the incidence of dementia or MCI. Our results indicate that green tea consumption is significantly associated with reduced risk of cognitive decline, even after adjustment for possible confounding factors.     

ERCC1 Single Nucleotide Polymorphism C8092A,but Not Its Expression Is Associated with Survival of Esophageal Squamous Cell Carcinoma Patients from Fujian Province,China

Esophageal carcinoma is one of the world''s deadliest cancers. Esophageal squamous cell carcinoma (ESCC) is more frequent than adenocarcenoma (AC) in China. Platinum-based chemotherapy with surgical resection is a common treatment approach for ESCC; however, the treatment response is uncertain. Evidence suggests polymorphisms in genes encoding excision repair cross-complementing group 1 (ERCC1), a protein involved in nuclear excision repair (NER), may help predict response to cisplatin and other platinum-based chemotherapeutics. Multiple ERCC1 single nucleotide polymorphisms (SNPs) have been associated with platinum chemotherapy response. Two common SNPs occur at the C8092A and C118T loci. Our study aimed to determine if 1) an association exists between ERCC1 tumor expression and patient survival, 2) whether adjuvant therapy influence on survival is related to histological ERCC1 presence in tumor cell nuclei, and 3) whether other clinicopathological characteristics in a cohort of patients following surgery for various stages of ESCC are associated with tumor ERCC1 expression. One hundred eight patients were included in the study, and tumor biopsy was collected for genotyping and immunohistochemical analysis of ERCC1. Sixty-seven patients (62%) received no adjuvant therapy, and the rest had either platinum-based chemotherapy (28.5%), radiotherapy (6.5%) or both treatments (2.8%). Log-rank analysis revealed no significant connection between tumor ERCC1 expression (P = 0.12) or adjuvant therapy (P = 0.56) on patient survival. Also, non-parametric Mann-Whitney analysis showed no significant link between tumor size or nodus tumor formation and ERCC1 presence in patients in the study. Interestingly, C8092A SNP showed significant association with patient survival (P = 0.01), with patients homozygous for the mutant allele showing the most significantly reduced survival (P = 0.04) compared to those homozygous for the dominant allele (CC). Our results provide novel insight into the genotypic variation of patients from Quanzhou, Fujian province China.