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Abstract: Noxa is a Bcl-2 homology domain-containing pro-apoptotic mitochondrial protein. Noxa mRNA and protein expression are upregulated by dsRNA or virus, and ectopic Noxa expression enhances cellular sensitivity to virus or dsRNA-induced apoptosis. Here we demonstrate that Noxa null baby mouse kidney (BMK) cells are deficient in normal cytopathic response to lytic viruses, and that reconstitution of the knockout cells with wild-type Noxa restored normal cytopathic responses. Noxa regulation by virus mirrored its regulation by proteasome inhibitors or ER stress inducers and the ER stress response inhibitor salubrinal protected cells against viral cytopathic effects. Noxa mRNA and protein were synergistically upregulated by IFN or dsRNA when combined with ER stress inducers, leading to Noxa/Mcl-1 interaction, activation of Bax and pro-apoptotic caspases, degradation of Mcl-1, loss of mitochondrial membrane potential and initiation of apoptosis. These data highlight the importance of ER stress in augmenting the expression of Noxa following viral infection. [Copyright &y& Elsevier]

Abstract: Objective: To determine the incidence of subsequent abnormal cervical or vaginal cytology and confirmatory histology in women completing five-years of surveillance for cervical cancer without recurrence. Methods: Following IRB approval, a tumor registry database identified women managed for all stages of cervical cancer from 1990 to 2003 who after completion of 60months of active surveillance following primary therapy underwent continued vaginal or cervical cytologic surveillance. Retrospective review was performed to determine demographics, clinicopathologic variables, vaginal or cervical cytology and outcomes. Results: Sixty-one women were identified with a median age at diagnosis of 41 (range 23–81). 72% of women were Caucasian, 16% were African–American with the remainder primarily Asian. Squamous cell carcinoma was the most common histology and present in 47 women (77%) with an equal proportion of women having G1 and G2 tumors. 80% of patients had early stage disease (Stages IA1–IIA). Median follow-up after completing five-years of active surveillance for all patients was 143months and a total of 303 Pap tests were performed with the mean/median number of five cytologic evaluations per patient. A total of 17 (5.6%) [95% CI, 3.5–8.8%] abnormal Pap tests were reported, which led to the performance of three diagnostic procedures. One case of moderate vaginal dysplasia was diagnosed and treated. Conclusions: Continued annual cytologic screening is of low yield in women completing five-years of surveillance that have remained free of recurrence. The incorporation of newer testing modalities including HPV testing may allow increases in the screening interval in this group of patients at relatively low risk for recurrence. [Copyright &y& Elsevier]

Abstract: Human urine contains a large number of proteins and peptides (the urinary proteome). Global analysis of the human urinary proteome is important for understanding urinary tract diseases. Bladder cancer is the most common urological cancer with higher incidence rates in endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to use the proteomic approach to establish urinary protein biomarkers of bladder cancer. ADAM28, identified by proteomic approaches and confirmed by Western blotting, showed significant differences compared with normal individuals, so it may be a biomarker of bladder cancer. [Copyright &y& Elsevier]

Abstract: Objective: Obesity is characterized by low levels of adiponectin, an adipocytes derived hormone, and by an inflammatory component. Endothelial dysfunction is often found in overweight/obesity, diabetes, and atherosclerosis. Advanced glycation end products (AGEs) induce endothelial dysfunction and are linked to diabetes and increased atherogenicity and inflammation. The aim of the study was to investigate the possible link between adiponectin and N(epsilon)-(carboxymethyl) lysine (CML), the predominant adduct of circulating AGEs in overweight patients, and, in an in vitro model, to test the hypothesis that adiponectin acts as modulator of endothelial dysfunction, induced by AGEs. Results: In 108 overweight patients, plasma levels of CML correlated inversely with adiponectin levels. Pre-incubation of human vein endothelial cells (HUVECs) with physiological concentrations of adiponectin, followed by stimulation with AGEs, reduced vascular adhesion molecule-1 (VCAM-1) and E-selectin expression, as assessed by surface enzyme immunoassay. Conclusions: Taken together, these findings demonstrate an inverse correlation between CML and adiponectin levels in overweight patients and a protective role of adiponectin on endothelial dysfunction induced by AGEs, suggesting its key role in the treatment of the vascular complications of obesity/metabolic syndrome. [Copyright &y& Elsevier]

Abstract: An important consideration in the manufacture of products derived from animal or human sources is the virus reduction capacity of the manufacturing process as estimated using validated bench-scale models of relevant manufacturing steps. In these studies, manufacturing process intermediates are spiked with virus and processed using the bench-scale model and the resulting viral titres of input and output samples are typically determined using cell-based infectivity assays. In these assays, the Spearman-Kärber (SK) method is commonly used to estimate titres when there is one or more positive observation (i.e., the presence of any viral cytopathic effect). The SK method is most accurate when the proportion of positive observations ranges from <0.1 to >0.9 across dilutions but can be biased otherwise. Maximum likelihood (ML) based on a single-hit Poisson model is an alternative widely used estimation method. We compared SK with ML and found the methods to have similar properties except for situations in which the concentration of virus is low but measurable. In this case, the SK method produces upwardly biased estimates of titres. Based on our results, we recommend the use of either ML or SK at most virus concentrations; however, at low virus concentrations ML is preferred. [Copyright &y& Elsevier]

Abstract: Objective: Heredity is a major cause of ovarian cancer and during recent years the contribution from germline mismatch repair (MMR) gene mutations linked to Lynch syndrome has gradually been recognized. Methods: We characterized clinical features, tumor morphology and mismatch repair defects in all ovarian cancers identified in Swedish and Danish Lynch syndrome families. Results: In total, 63 epithelial ovarian cancers developed at mean 48 (range 30–79) years of age with 47% being early stage (FIGO stage I). Histologically, endometrioid (35%) and clear cell (17%) tumors were overrepresented. The underlying MMR gene mutations in these families affected MSH2 in 49%, MSH6 in 33% and MLH1 in 17%. Immunohistochemical loss of the corresponding MMR protein was demonstrated in 33/36 (92%) tumors analyzed. Conclusion: The combined data from our cohorts demonstrate that ovarian cancer associated with Lynch syndrome typically presents at young age as early-stage, non-serous tumors, which implicates that a family history of colorectal and endometrial cancer should be specifically considered in such cases. [Copyright &y& Elsevier]

Abstract: Dystonia is a motor sign characterized by involuntary muscle contractions which produce abnormal postures. Genetic factors contribute significantly to primary dystonia. In comparison, secondary dystonia can be caused by a wide variety of metabolic, structural, infectious, toxic and inflammatory insults to the nervous system. Although classically ascribed to dysfunction of the basal ganglia, studies of diverse animal models have pointed out that dystonia is a network disorder with important contributions from abnormal olivocerebellar signaling. In particular, work with the dystonic (dt) rat has engendered dramatic paradigm shifts in dystonia research. The dt rat manifests generalized dystonia caused by deficiency of the neuronally restricted protein caytaxin. Electrophysiological and biochemical studies have shown that defects at the climbing fiber-Purkinje cell synapse in the dt rat lead to abnormal bursting firing patterns in the cerebellar nuclei, which increases linearly with postnatal age. In a general sense, the dt rat has shown the scientific and clinical communities that dystonia can arise from dysfunctional cerebellar cortex. Furthermore, work with the dt rat has provided evidence that dystonia (1) is a neurodevelopmental network disorder and (2) can be driven by abnormal cerebellar output. In large part, work with other animal models has expanded upon studies in the dt rat and shown that primary dystonia is a multi-nodal network disorder associated with defective sensorimotor integration. In addition, experiments in genetically engineered models have been used to examine the underlying cellular pathologies that drive primary dystonia. This article is part of a Special Issue entitled “Advances in dystonia”. [Copyright &y& Elsevier]

Abstract: The flowers and leaves buds of Buddleja officinalis MAXIM (Buddlejaceae) are used to treat eye troubles, hernia, gonorrhea and liver troubles in Asia. To elucidate the protective effects of linarin isolated from B. officinalis on the response of osteoblast to oxidative stress, osteoblastic MC3T3-E1 cells were pre-incubated with linarin for 1h before treatment with 0.3mM H2O2 for 48h, and markers of osteoblast function and oxidative damage were examined. Linarin significantly (P <0.05) increased cell survival, alkaline phosphatase (ALP) activity, collagen content, calcium deposition, and osteocalcin secretion and decreased the production of receptor activator of nuclear factor-kB ligand (RANKL), protein carbonyl (PCO), and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells in the presence of hydrogen peroxide. These results demonstrate that linarin can protect osteoblasts against hydrogen peroxide-induced osteoblastic dysfunction and may exert anti-resorptive actions, at least in part, via the reduction of RANKL and oxidative damage. [Copyright &y& Elsevier]

Abstract: Enteroviruses (EV) frequently infect the central nervous system (CNS) and induce neurological diseases. Although the CNS is composed of many different cell types, the spectrum of tropism for each EV is considerable. These viruses have the ability to completely shut down host translational machinery and are considered highly cytolytic, thereby causing cytopathic effects. Hence, CNS dysfunction following EV infection of neuronal or glial cells might be expected. Perhaps unexpectedly given their cytolytic nature, EVs may establish a persistent infection within the CNS, and the lasting effects on the host might be significant with unanticipated consequences. This review will describe the clinical aspects of EV-mediated disease, mechanisms of disease, determinants of tropism, immune activation within the CNS, and potential treatment regimes. [Copyright &y& Elsevier]

Abstract: The endoplasmic reticulum (ER) is an organelle in which most membrane and secretory proteins are synthesized. If these proteins are not folded correctly, unfolded proteins accumulate in the ER lumen, causing a cellular situation known as ER stress. Recently, many studies on the relationship between ER stress and diseases have been reported. Thus, studies of ER stress in vivo should yield information that is useful in pathology. Model mice have been developed as a powerful tool to visualize ER stress in vivo, but this approach depends on transgenic technology. Here, we report on a method of detecting ER stress in vivo by Raman spectroscopy. Our experiments revealed that two specific Raman bands were reduced in both cultured cells and animal tissues in an ER stress dependent manner. This suggests that Raman spectroscopy could be a useful tool to detect ER stress in vivo without transgenic technology. [Copyright &y& Elsevier]

Abstract: MM5 was a bacterial strain isolated from moribund clam (Meretrix meretrix) collected from a farm with mass mortality outbreak. Primary genotypic and phenotypic identification including 16S rDNA sequence analysis, multilocus sequence analysis (MLSA) of four housekeeping genes (gapA, ftsZ, mreB and topA) and biochemical tests suggested that strain MM5 was a Vibrio species closest to but different from Vibrio furnissii. Our previous study indicated that MM5 could induce a high mortality of M. meretrix (). Quantitative challenge test was performed in this study to further evaluate the pathogenic potential of MM5, which showed that at 84h post-inoculation, the cumulative mortalities of the MM5-injected group were significantly higher than those of control groups (P <0.05). Cytopathological and histopathological features of the clam infected by MM5 were carried out by transmission electron microscopy (TEM) and Hematoxylin and Eosin (H&E) staining, respectively. Cytopathologically, foci of MM5 were found in hepatocytes of the clam infected by MM5. In addition, cytopathological lesion was detected in foot of infected clam. Histopathologically, MM5 was detected in different tissues of infected clam, including hepatopancreas, mantle and gill. Challenge test combined with pathological features indicated that MM5 was virulent to M. meretrix. [ABSTRACT FROM AUTHOR]

Abstract: Lamin A is a component of the nuclear lamina that plays a major role in the structural organization and function of the nucleus. Lamin A is synthesized as a prelamin A precursor which undergoes four sequential post-translational modifications to generate mature lamin A. Significantly, a large number of point mutations in the LMNA gene cause a range of distinct human disorders collectively known as laminopathies. The mechanisms by which mutations in lamin A affect cell function and cause disease are unclear. Interestingly, recent studies have suggested that alterations in the normal lamin A pathway can contribute to cellular dysfunction. Specifically, we and others have shown, at the cellular level, that in the absence of mutations or altered splicing events, increased expression of wild-type prelamin A results in a growth defective phenotype that resembles that of cells expressing the mutant form of lamin A, termed progerin, associated with Hutchinson–Gilford Progeria syndrome (HGPS). Remarkably, the phenotypes of cells expressing elevated levels of wild-type prelamin A can be reversed by either treatment with farnesyltransferase inhibitors or overexpression of ZMPSTE24, a critical prelamin A processing enzyme, suggesting that minor increases in the steady-state levels of one or more prelamin A intermediates is sufficient to induce cellular toxicity. Here, to investigate the molecular basis of the lamin A pathway toxicity, we characterized the phenotypic changes occurring in cells expressing distinct prelamin A variants mimicking specific prelamin A processing intermediates. This analysis demonstrates that distinct prelamin A variants differentially affect cell growth, nuclear membrane morphology, nuclear distribution of lamin A and the fundamental process of transcription. Expression of prelamin A variants that are constitutively farnesylated induced the formation of lamin A aggregates and dramatic changes in nuclear membrane morphology, which led to reduced levels of the basal transcription factor TATA-binding protein (TBP) and global transcription, and severely limited cell growth. Expression of a prelamin A variant that cannot be farnesylated, although did not appreciably influence cell growth, resulted in the formation of lamin A nucleoplasmic foci and caused, in a minor subpopulation of cells, changes in nuclear morphology that were accompanied by reduced levels of TBP and transcription. In contrast, expression of mature lamin A did not affect any of these parameters. These data demonstrate that accumulation of any partially processed prelamin A protein alters cellular homeostasis to some degree, even though the most dramatic effects are caused by variants with a permanently farnesylated carboxyl-terminal tail. [Copyright &y& Elsevier]

Abstract: To investigate hierarchy in human prostate epithelial cells, we generated recombinant lentiviruses, infected primary cultures and cell lines, and followed their fate in vitro. The lentiviruses combined constitutive promoters including CMV and β-actin, or late-stage differentiation promoters including PSCA (prostate stem cell antigen) and PSAPb (prostate specific antigen/probasin) driving expression of monomeric, dimeric and tetrameric fluorescent proteins. Significantly, rare CD133+ cells from primary prostate epithelial cultures were successfully infected and activation of late-stage promoters was observed in basal epithelial cultures following induction of differentiation. Lentiviruses also infected CD133+ cells within the P4E6 cell line. However, promoter silencing was observed in several cell lines (P4E6, BPH-1, PC3). We examined the promoter methylation status of the lentiviral insertions in heterogeneously fluorescent cultures from PC3 clones and found that DNA methylation was not the primary mechanism of silencing of the CMV promoter. We also describe limitations to the lentivirus system including technical challenges due to low titers and low infection efficiency in primary cultures. However, we have identified a functional late-stage promoter that indicates differentiation from a basal to a luminal phenotype and demonstrate that this strategy for lineage tracking of prostate epithelial cells is valid with further optimisation. [ABSTRACT FROM AUTHOR]

Abstract: Although transmission of pandemic influenza A virus H1N1 2009 is still occurring globally, little has been reported about how this outbreak has affected the safety of plasma derivatives. To evaluate the safety of plasma derivatives, dedicated virus clearance processes used during their production were investigated for their effectiveness in eliminating this virus of recent concern. In this study, influenza A virus H1N1 strain A/NWS/33 (H1N1) was chosen as a surrogate. H1N1 was completely inactivated by fraction IV fractionation as well as pasteurization during the manufacture of albumin. H1N1 was also effectively removed into the precipitate by fraction III fractionation and completely inactivated by low pH incubation as well as pasteurization during the manufacture of intravenous immunoglobulin. H1N1 was completely inactivated within 1 min of solvent/detergent treatment using 0.3% tri (n-butyl) phosphate and 1.0% Triton X-100 and also completely inactivated within 10 min of dry-heat treatment at 98 °C during the manufacture of factor VIII. H1N1 was completely removed by virus filtration process using Viresolve NFP filter and also completely inactivated by pasteurization during the manufacture of anti-thrombin III. These results indicate that all the virus clearance processes commonly used have sufficient H1N1 reducing capacity to achieve a high margin of safety. [Copyright &y& Elsevier]

Abstract: HCV-JFH1 yields subclones that develop cytopathic plaques (Sekine-Osajima Y, et al., Virology 2008; 371:71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection. [ABSTRACT FROM AUTHOR]

Abstract: Three different cell lines (murine macrophages, HeLa and osteosarcoma cells) were assayed in order to check for the manifestation of the cytopathic effects of three strains of Acanthamoeba recently isolated in our laboratory from contact lens cases: CLC-16, CLC-41.r and CLC-51-l. Adhesion and cytotoxicity assays were carried out with these strains and the type strain Acanthamoeba castellanii Neff as a control. Briefly, the ability of these amoebae to bind to the three cell lines was calculated and supernatants were examined for cytotoxicity by measuring lactate dehydrogenase released as an estimate of cytotoxicity using a commercial detection kit. The three strains showed high adhesion and cytotoxicity levels when tested in the three cell lines. This study demonstrates the ability of these amoebae to degrade any of the tested cell lines. To the best of our knowledge, this is the first report of the in vitro effects of acanthamoebae on osteosarcoma cells. [Copyright &y& Elsevier]

Abstract: Protective effects of recombinant human granulocyte macrophage colony stimulating factor (rHuGM-CSF) on H1N1 influenza virus infection was studied in vivo and in vitro. Mice were infected with H1N1 influenza A viruses and rHuGM-CSF at doses of 0.34, 0.67, and 1.34mgkg−1 d−1 was administrated for 7days before the mice were infected with influenza virus and continued for a further 3days. Compared with control mice, rHuGM-CSF was demonstrated to increase the survival rate of the infected mice by 50.0%, 55.6%, and 80.0% and increased the mean survival days by 25.7%, 30.0%, and 46.8%, respectively. Histopathological study of the lungs in pneumonia mice found that pre-treatment with rHuGM-CSF significantly ameliorated lung injury induced by influenza virus infection. In vitro study demonstrated that when rHuGM-CSF were co-incubated with peripheral blood mononuclear cells (PBMCs), the PBMCs culture supernatant induced a dose-dependent reduction of virus-induced cytopathic effect (CPE) in Madin-Darby canine kidney (MDCK) cells in vitro. These results suggested that rHuGM-CSF might be an effective and potential protection for H1N1 influenza virus-induced pneumonia. [Copyright &y& Elsevier]

Abstract: Influenza virus leads to acute respiratory disease resulting in seasonal epidemics and periodic pandemics. Little is known about the signaling events that regulate host defense to influenza. One particular pathway, the c-Jun amino-terminal kinase (JNK) cascade is activated following influenza infection and blocking JNK leads to enhanced viral replication. We hypothesize that Mixed Lineage Kinase 3 (MLK3), an upstream regulator of JNK, is involved in the host response to influenza. To test this, wild-type and MLK3−/− mice were infected with pathogenic strain of influenza A virus, A/PR/8/34 (PR8). Although, cellular and humoral immune responses were similar between wild-type and MLK3−/− hosts, the viral load in the lungs was comparatively higher in MLK3−/− mice at day 8 post-infection. Consistent with this, MLK3−/− murine lung fibroblast and epithelial cells had prolonged survival and increased virion production following infection compared to wild-type. These findings support a role for MLK3 in viral production during influenza infection. [Copyright &y& Elsevier]

Abstract: A promising approach for preventing allograft rejection involves shifting the balance between cytopathic and regulatory T cells to dominance of the latter cell type. Nonspecific lymphodepletion was conducted by administration of depleting anti-CD4 and anti-CD8 antibodies to reduce effector T cells and adoptive transfer of ex vivo-expanded host Treg cells by stimulation with donor dendritic cells to augment the Treg cell compartment. Evaluation of an MHC-mismatched skin allograft model revealed that combined therapy with these two protocols consistently induced modest prolongation of allograft survival, although all skin grafts were eventually rejected. The administration of IL-2/anti-IL-2 complexes significantly improved the efficacy of combination therapy via promoting the expansion of adoptively transferred Treg cells as well as endogenous recipient Treg cells. We conclude that Treg cell therapy combined with lymphodepletion is of practical benefit for the control of allograft rejection, but requires supplementary measures to promote immune tolerance. [Copyright &y& Elsevier]

Abstract: High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5′ end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectious progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo. [Copyright &y& Elsevier]

Abstract: Small molecule amplifiers of heat shock response have shown promising results in rescuing stress related injury through chaperone amplification. Herein, we report the results of a high content target-based primary screening of several known bioactive libraries. Screening resulted in the identification of three potent gedunin derivatives and a sappanone A derivative. Western blot results confirmed compound-induced activation of HSF1 and increased expression level of HSP70. These compounds rescued cells from cell death caused by proteasome inhibitor MG-132 and RNAi knockdown of HSF1 significantly reversed the cytoprotective effects, confirming an HSF1-dependent mechanism of action. These HSF1 amplifiers were tested in two mammalian cell based models of Huntington’s disease (HD) and found to improve survival. Therefore, these screening hits may have therapeutic potential for HD and possibly other protein conformational disorders. [Copyright &y& Elsevier]

Abstract: In diabetes, hyperglycemia and the associated formation of advanced glycation end-products (AGE) and AGE-modified low density lipoproteins (AGE-LDL) can directly affect the cells of the vascular wall. We hypothesize that AGE-LDL may act directly and induce oxidant and inflammatory alterations in human endothelial cells (HEC), this effect being amplified by high glucose. To test this assumption, the activity of NADPH oxidase (NADPHox) was evaluated and the expression of its subunits (p22phox, NOX4, and p67phox), of the AGE receptor (RAGE), and of the monocyte chemoattractant protein-1 (MCP-1) were assessed by real-time PCR and Western blot in confluent EA.hy926 cells incubated with AGE-LDL for 24 and 48h, in normal and high glucose conditions. Exposure of HEC for 48h to AGE-LDL in 5mM glucose induced an increase of RAGE expression (50%), NADPHox activity (107%), p22phox and NOX4 mRNA (50% and 188%, respectively) and MCP-1 expression (80%). AGE-LDL-stimulated p22phox expression by activating p38 MAP kinase and NF-kB, and MCP-1 expression by activating NF-kB, as demonstrated by the use of specific inhibitors (SB203580 and Bay11-7085). The addition of 25mM glucose in the culture medium enhanced the effect of AGE-LDL, but also of nLDL, on RAGE, p22phox, NOX4, p67phox, and MCP-1 gene expression. In conclusion, AGE-LDL induce an oxidative stress and a pro-inflammatory state in human endothelial cells. Both AGE-LDL and nLDL in the presence of high glucose amplify their effect, revealing a link between hyperlipidemia, diabetes, and endothelial cell dysfunction. [Copyright &y& Elsevier]

Abstract: Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation. [Copyright &y& Elsevier]

Abstract: The dynamics of astral and midzone microtubules (MTs) must be separately regulated during cell division, but the mechanism of selective stabilization of midzone MTs is poorly understood. Here we show that, in HT1080 cells, activation of Rho is required to stabilize midzone MTs, and to maintain the midzone structures after anaphase onset or during cytokinesis. Ect2-depleted cells undergoing conventional cytokinesis (cytokinesis A) or contractile ring-independent cytokinesis (cytokinesis B) formed abnormally thin bundles of midzone MTs. C3-loaded mitotic cells with inactivated Rho showed similar but more severe disorganization of midzone MTs. In addition, the bundles of astral MTs were abnormally abundant along the cell periphery in both Ect2-depleted and C3-loaded mitotic cells. Mitotic kinesin-like protein 1 (MKLP1), a component of the spindle midzone required for bundling of MTs, was localized only in the narrower equatorial regions in Ect2-depleted cells, and disappeared from the midzone accompanying the progression of the mitotic phase in C3-loaded cells. Stabilization of MTs by taxol was sufficient to maintain the midzone structures in C3-loaded mitotic cells. These results, when combined with a preceding analysis on another, microtubule-associated Rho GEF (C.J. Bakal, D. Finan, J. LaRose, C.D. Wells, G. Gish, S. Kulkarni, P. DeSepulveda, A. Wilde, R. Rottapel, The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9529–9534), suggest that mammalian cells have two potential steps that require active Rho for the stabilization of midzone MTs during mitosis and cytokinesis. [Copyright &y& Elsevier]

Abstract: Objective: Many ovarian carcinomas are presumed to arise within ovarian cortical inclusion cysts (CICs). This study examined the frequency of ovarian CICs in relation to epidemiologic risk factors in women with BRCA1 and BRCA2 (BRCA+) mutations. Methods: BRCA+ women who underwent risk-reducing bilateral salpingo-oophorectomy were studied (n =74). Fifteen demographic variables (e.g., age at time of surgery, age at first birth, age at menopause, body mass index (BMI), gravidity) from a review of the medical records and three pathologic variables (cystic and atretic follicles, corpora lutea) were recorded. Statistical associations were made using T-test or Chi Square analysis and logistic regression analysis for p-trend. Results: Women whose ovaries contained 7 for more CICs were older at first birth (p =0.034), surgery (p =0.059), menopause (p =0.046) and had a higher BMI (p =0.034) than those with <7 CICs. Regression analysis revealed a significant association between CICs and increasing BMI (p =0.01). Conclusions: CICs correlate with greater body mass index, similar to low-grade serous and endometrioid tumors and in contrast to high-grade serous carcinoma and its putative precursor in the fallopian tube. A model is presented for ovarian and tubal pathways to pelvic cancer that are linked to different microscopic precursors with distinct epidemiologic correlates. [Copyright &y& Elsevier]

Abstract: Objective: The aim of this study was to validate the role of p16INK4a immunocytochemistry as a marker of cervical epithelial neoplasia in cytology and biopsy samples. Methods: During the period of 4 years a total of 501 immunocytochemistry stains in cytology and biopsy samples were performed. The cytology smears were obtained from two independent laboratories of women with low-grade squamous intraepithelial lesion interpretation of Pap test or ThinPrep. Additionally, a selection of paraffin blocks from cases diagnosed previously with squamous intraepithelial lesion and cervical cancer were immunostained. Results: Among 312 cytology samples analyzed by immunocytochemistry, 36.2% were positive for p16INK4a. Negative cases were followed-up with conventional Pap smear and 86.9% showed regression. Among positive patients, 14 underwent surgery and 100% exhibited squamous intraepithelial lesion. In additional immunohistochemical analysis of selected biopsies from our archive, we found that the ratio of positivity for p16INK4a increased from 40% in L-SIL/CIN1 to 69.8% in H-SIL/CIN2 to 100% in H-SIL/CIN3 and squamous cell carcinoma. A relatively low number (24%) of cases with squamous metaplasia revealed focal and/or sporadic staining in the superficial layers of the epithelium. All the cases of cervical adenocarcinoma and mesonephric carcinoma were positive for p16INK4a. Conclusion: p16INK4a is a useful additional marker for interpretation of problematic Pap smears and reduces the variability during evaluation of suspicious biopsies of the uterine cervix. [Copyright &y& Elsevier]

Abstract: Objective: This prospective study was performed to evaluate the clearance of high-risk human papillomavirus (HPV) infection and incidence of cytologic abnormality and cervical intraepithelial neoplasia (CIN) during the follow-up of persistent infection in Korean women. Methods: A total of 4170 women who were screened for cervical cancer, were aged 30 years or older, had no abnormal last Pap smear results for 3 years, had no history of treatment for cervical neoplastic disease, and were not pregnant were analyzed for high-risk HPV prevalence using the Hybrid capture (HC) II assay. The 224 women with normal cytology but positive for high-risk HPV DNA using the HC-II assay were analyzed for their clearance of HPV infection. Results: The median time to clearance in women with initially normal cytology was 7.5 months from initial detection (95% CI, 5.2–9.8 months). There were significant differences in the median time to clearance (4.5 vs. 14.5 months, p <0.001) of high-risk HPV infection between women with the initial relative light unit/cutoff (RLU/CO) ratio values <10.0 and ≥10.0, as determined by the HC-II assay. In Kaplan–Meier analysis, probability of development of cytologic abnormalities, CIN and high-grade CIN was 38.2%, 21.7% and 8.5% respectively at 24 months of persistent high-risk HPV infection. Conclusion: The prevalence and clearance of high-risk infection in Korean women was similar to that in Western countries. Persistent high-risk HPV infection was associated with high viral load. [Copyright &y& Elsevier]

Abstract: Objective: Randomized controlled trial evaluating a topical treatment for cervical intraepithelial neoplasia 2 and 3 (CIN 2+) using cidofovir. Methods: Fifty-three women with a biopsy-proven CIN 2+ were randomly assigned, 6 weeks before their planned conisation, either 3 applications of 3 ml 2% cidofovir in Intrasite® gel in a cervical cap or a placebo (the same volume of Intrasite® alone). A cervical sample for high-risk types of human papillomaviruses (HPV) (Hybrid Capture 2 or HC2) was taken before treatment and before conisation. The cone was submitted for pathological examination, and subsequently, along with the initial biopsy, to in situ hybridization (ISH) for high-risk HPV. Results: Forty-eight patients were treated and followed according to the protocol, (23 cidofovir, and 25 placebo). Fourteen of the 23 cones were free of any CIN (60.8%) in the cidofovir group. Only 5 of 25 cones were free of any CIN (20%) in the placebo group (p <0.01). The difference remained significant in the ITT group (p <0.05). In the per-protocol and ITT populations, we observed more frequent viral clearance in the cidofovir group, but the difference was significant only when evaluated by ISH and not by HC2. No systemic toxicity was observed. Cervico-vaginal side effects of cidofovir were limited, and not statistically different from placebo. Conclusion: The medical topical treatment with cidofovir, at this point, cannot replace conisation, but it is a promising candidate for topical chemotherapy of CIN 2+ lesions; a larger prospective randomized study is needed to confirm our results. [Copyright &y& Elsevier]

Background: Tumor derived pancreatic stellate cells (TDPS) cells are key cellular components of the pancreatic tumor microenvironment. These stellate cells can release growth factors, proteases, and extracellular matrix proteins that may stimulate the spread of pancreatic cancer. We sought to determine whether TDPS cells promote the local invasion of pancreatic cancer cells and mechanisms involved. Methods: TDPS and panc-1 cells were grown in coculture to determine directional migration and panc-1 invasiveness was quantified using Matrigel invasion chambers, comparing TDPS cells to human foreskin fibroblasts (HFFs). ELISA was used to determine the secretion of growth factors, proteases, and extracellular matrix proteins from TDPS cells and HFFs, and then siRNAs used to knockdown expression of factors. Results: In coculture panc-1 cells migrate toward TDPS cells, creating nests of cancer cells within the stromal cells. TDPS cells promote the invasion of panc-1 cells and release thrombospondin 2 (TSP-2), whereas HFFs did not. When TSP-2 expression is reduced in TDPS cells using selective siRNAs, pancreatic cancer cell invasion was inhibited. Conclusion: Tumor-derived pancreatic stellate cells stimulate pancreatic cancer cell invasion, likely through release of TSP-2. Targeting pro-invasive elements, such as TSP-2, within the tumor microenvironment may inhibit local invasion, thus permitting more patients to undergo curative resection of pancreatic cancer. [Copyright &y& Elsevier]

Abstract: Recent studies indicate that the biophysical properties of the cellular microenvironment strongly influence a variety of fundamental cell behaviors. The extracellular matrix’s (ECM) response to mechanical force, described mathematically as the elastic modulus, is believed to play a particularly critical role in regulatory and pathological cell behaviors. The basement membrane (BM) is a specialization of the ECM that serves as the immediate interface for many cell types (e.g. all epithelial cells) and through which cells are connected to the underlying stroma. Matrigel is a commercially available BM-like complex and serves as an easily accessible experimental simulant of native BMs. However, the local elastic modulus of Matrigel has not been defined under physiological conditions. Here we present the procedures and results of indentation tests performed on Matrigel with atomic force microscopy (AFM) in an aqueous, temperature controlled environment. The average modulus value was found to be approximately 450Pa. However, this result is considerably higher than macroscopic shear storage moduli reported in the scientific literature. The reason for this discrepancy is believed to result from differences in test methods and the tendency of Matrigel to soften at temperatures below 37°C. [Copyright &y& Elsevier]

Abstract: The mesangial cell of the renal glomerulus is exposed to circulating toxic substances and is a target involved in the glomerular component of chronic occupational and environmental exposure to cadmium. We review evidence for the involvement of cadmium in mesangial cell pathology, including effects on cell signaling, oncogene expression, and cell death. Previously we have shown that cadmium can inhibit apoptosis initiated through both the extrinsic (death ligand receptor) and intrinsic (mitochondrial) pathways, whereas exposure of mesangial cells to 10 μM CdCl2 for 6 h initiates caspase-independent cell death through both apoptotic and apoptotic-like (annexin V positive, propidium iodide staining) mechanisms. Apoptotic death is dependent upon activation of Ca2+/calmodulin-dependent protein kinase II (CaMK-II). In the present study we show that low level exposure of mesangial cells to Cd2+ (0.5 μM) initiates cell survival signals including PI3 kinase/Akt signaling, also dependent on CaMK-II, that are eventually overcome resulting in caspase-dependent cell death. These studies underscore the roles of cell signaling in various modes of cell death, and in particular the central role of CaMK-II in cadmium toxicology of the mesangial cell. [Copyright &y& Elsevier]

Abstract: We previously reported that, in mice, B cells are a reservoir for bacteria during Salmonella infection. Here, we show that, within the bone marrow, B cells and their precursors are targeted for infection by Salmonella enterica serovar typhimurium. Our data suggest that B cells within the bone marrow may be a bacterial niche during chronic Salmonella infection. [Copyright &y& Elsevier]

Abstract: Objective: To assess the compliance of HSIL patients to the national Dutch routine follow-up protocol in the first 2 years after LLETZ and to determine if based on the status of excision margins, follow-up intervals could be modified. Methods: A prospective cohort study was performed in patients, referred because of an abnormal Pap smear between 1996 and 2004 and treated for HSIL with LLETZ. The Dutch national routine follow-up protocol orders a Pap smear after 6, 12 and 24 months, respectively. Follow-up results were completed by using PALGA, the nationwide network and registry of histo- and cytopathology in the Netherlands. To assess compliance to the follow-up protocol, adequate follow-up was defined as three cervical smears taken after 6 (+/−3), 12 (+/−3) and 24 (+/−3) months, respectively. Results: Compliance to the first 2 years follow-up protocol declined from 86.2% to 64.8% to 51.2% for first, second and third follow-up cervical smears, respectively. Patients with involved excision margins had a three times higher overall risk of developing a subsequent HSIL after LLETZ as compared to patients with free excision margins (HR: 3.2, 95% CI=1.3–7.9, p =0.01). Risk for diagnosing HSIL during the first 12 months of follow-up for patients with free excision margins was only 1%. Conclusions: Compliance to the Dutch national routine follow-up protocol in HSIL patients after LLETZ is only moderate. For HSIL patients with free excision margins after LLETZ the first cytological follow-up interval can safely be increased to 12 months. [Copyright &y& Elsevier]

Abstract: Lettuce infectious yellows virus (LIYV) encodes a 26 kDa protein (P26) previously shown to associate with plasmalemma deposits (PLDs), unique LIYV-induced cytopathologies located at the plasmalemma over plasmodesmata pit fields in companion cells and phloem parenchyma. To further characterize the relationship of P26 and PLDs, we assessed localization and cytopathology induction of P26 expressed from either LIYV or a heterologous Tobacco mosaic virus (TMV) vector using green fluorescent protein (GFP) fusions, immunofluorescence microscopy, biochemical fractionation, and transmission electron microscopy (TEM). TEM analyses demonstrated that P26 not only associated with, but induced formation of PLDs in the absence of other LIYV proteins. Interestingly, PLDs induced by P26-expressing TMV were no longer confined to phloem cells. Putative P26 orthologs from two other members of the genus Crinivirus which do not induce conspicuous PLDs exhibited fractionation properties similar to LIYV P26 but were not associated with any PLD-like cytopathology. [Copyright &y& Elsevier]

Summary: Lectin AAL (Agrocybe aegerita lectin) from the edible mushroom A. aegerita is an antitumor protein that exerts its tumor-suppressing function via apoptosis-inducing activity in cancer cells. The crystal structures of ligand-free AAL and its complex with lactose have been determined. The AAL structure shows a dimeric organization, and each protomer adopts a prototype galectin fold. To identify the structural determinants for antitumor effects arising from the apoptosis-inducing activity of AAL, 11 mutants were prepared and subjected to comprehensive investigations covering oligomerization detection, carbohydrate binding test, apoptosis-inducing activity assay, and X-ray crystallographic analysis. The results show that dimerization of AAL is a prerequisite for its tumor cell apoptosis-inducing activity, and both galactose and glucose are basic moieties of functional carbohydrate ligands for lectin bioactivity. Furthermore, we have identified a hydrophobic pocket that is essential for the protein''s apoptosis-inducing activity but independent of its carbohydrate binding and dimer formation. This hydrophobic pocket comprises a hydrophobic cluster including residues Leu33, Leu35, Phe93, and Ile144, and is involved in AAL''s function mechanism as an integrated structural motif. Single mutants such as F93G or I144G do not disrupt carbohydrate binding and homodimerization capabilities, but abolish the bioactivity of the protein. These findings reveal the structural basis for the antitumor property of AAL, which may lead to de novo designs of antitumor drugs based on AAL as a prototype model. [Copyright &y& Elsevier]

Abstract: Goniothalamin oxide (1) is a styryl lactone which was isolated from bark and leaves of several Goniothalamus species. This natural product has some interesting biological properties such as larvicidal and tripanocidal activities. However, no studies on the antiproliferative profile of goniothalamin oxide (1) and its stereoisomers have been reported yet. Here, goniothalamin epoxide (1), isogoniothalamin epoxide (2) and their enantiomers were prepared via epoxidation of (R)-and (S)-goniothalamin (4). A 3:2 molar ratio in favor of goniothalamin oxide (1) and ent-1 was observed from (R)- and (S)-4, respectively, when 3-chloroperbenzoic acid (mCPBA) was employed while an increase to 6:1 molar ratio was achieved with (S,S)-Jacobsen’s catalyst. Antiproliferative activity of these epoxides revealed that ent-isogoniothalamin oxide (ent-2) was the most active against the eight cancer cell lines studied. These results indicate that 6S, 7R and 8R absolute configurations are beneficial for the activity of these epoxides. [Copyright &y& Elsevier]

Abstract: Mitochondrial cytopathies are characterized by a large variability of clinical phenotypes and severity. The 14487T>C mutation in mtDNA has been recently described to be associated with Leigh syndrome. The 12297T>C mutation has been described in isolated dilated cardiomyopathy patients. Here, we report a family with multiple members who harbor both mutations, with only a few individuals who are affected with Leigh syndrome. Mitochondrial whole genome sequencing analysis in the proband’s muscle specimen detected two nearly homoplasmic mutations: 14487T>C (M63V in ND6) and 12297T>C in the tRNA Leu (CUN) gene. These two mutations were also detected in the blood, urine sediments, hair follicles, and buccal swab samples of all matrilineal relatives tested. All individuals tested were nearly homoplasmic for the 12297T>C mutation, but had variable degrees of heteroplasmy for 14487T>C. We also screened for the frequency of these two mutations. Of 268 patients with Leigh or Leigh-like disease, one case was found to harbor the 14487T>C mutation (0.3%), and one had the 12297T>C mutation (0.3%). Neither mutation was detected in the 88 patients meeting MELAS syndrome criteria nor in the 56 patients with respiratory chain complex I or I+III deficiency. In conclusion, the 14487T>C mutation appears as the primary etiology of Leigh syndrome in this family, demonstrating the high level of heteroplasmy needed for a clinically significant phenotype with this mutation. The 12297T>C mutation was not associated with dilated cardiomyopathy for the family members who were clinically evaluated and who were shown by testing to be nearly homoplasmic for that mutation. [Copyright &y& Elsevier]

Abstract: Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of β1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects. [Copyright &y& Elsevier]

Abstract: Elevated levels of oxidatively induced DNA lesions have been reported in malignant pancreatic tissues relative to normal pancreatic tissues. However, the ability of the pancreatic cancer cells to remove these lesions has not previously been addressed. This study analyzed the effectiveness of the pancreatic cancer cell line, BxPC-3 to repair 8-hydroxyguanine (8-OH-Gua) relative to a nonmalignant cell line. We show that BxPC-3 cells repair 8-OH-Gua less effectively than the nonmalignant cells. This repair deficiency correlated with significant downregulation of the hOGG1 protein and the corresponding mRNA (30-fold lower than GAPDH) in BxPC-3 cell line. The repair defect was complemented in vivo by transient transfection of the hOGG1 gene and in vivo by recombinant hOGG1. These results are the first to show a deficiency of 8-OH-Gua repair in BxPC-3 cells, implicating this defect in the risk factor of pancreatic cancer. [Copyright &y& Elsevier]

This review explores the developments leading up to the establishment of the cell theory and cellular pathology and their subsequent refinements and applications while focusing on the individuals who have made seminal advances in the field. The links between cell biology, cell pathology and cell injury research are emphasized. Recognition also is given to the importance of technological advances in microscopy, histology, biochemical and molecular methods for discovery in cell biology and cell pathology. Particular attention is focused on the work of Rudolph Virchow and his former students in the formulation of the cell theory in biology and pathology and John F. R. Kerr and colleagues who identified and developed a comprehensive characterization of apoptosis, thereby giving impetus to the contemporary field of cell injury research. Cell injury research remains an important and fruitful field of ongoing inquiry and discovery. [ABSTRACT FROM AUTHOR]

Abstract: Stat3 was initially recognized as a transcription factor and mediates the nuclear action of many different cytokines and growth factors. In addition to its roles in normal cell function, the inappropriate activation of Stat3 in tumor cells has attracted the attention of tumor biologists and has led to the consideration of Stat3 as a drug target. The induction of Stat3 activity under physiological circumstances is transient and many different levels of activation and deactivation have been defined. In addition to kinases and phosphatases, the SOCS proteins and the PIAS proteins have been recognized as negatively regulating components, which fine-tune the extent and the duration of Stat3 function. Its nuclear cytoplasmic shuttling is exquisitely regulated and adds to the complexity of Stat3 action. Newly discovered associations with cytoplasmic molecules suggest functions outside the conventional transcriptional regulation context. High molecular weight transcription complexes suggest that Stat3 might assume roles in transcriptional induction as well as in transcriptional suppression. The aberrant activation in tumor cells and the central function of Stat3 in the communication between cells of the immune system and tumor cells are of great interest for translational research projects and innovative drug development. [Copyright &y& Elsevier]

Abstract: Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the β-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms. [Copyright &y& Elsevier]

Abstract: LYRIC/AEG-1 is a unique protein that has been shown to promote tumor cell migration and invasion through activation of the transcription factor NF-κB. We performed yeast two-hybrid screening to detect LYRIC/AEG-1 associated proteins, and identified BCCIP, a CDKN1A and BRCA2-associated protein involved in cell cycle regulation and DNA repair. Here, we demonstrate association between LYRIC/AEG-1 and BCCIP in mammalian cells, and define the region of interaction. Co-expression of the two proteins resulted in decreased levels of BCCIPα, an effect partially abrogated by proteasome inhibition. A truncated LYRIC/AEG-1 construct lacking the interaction region did not alter BCCIPα protein levels. Coincidentally, it was observed that overexpression of BCCIPα in DU145 prostate tumor cells induced an apparent neuroendocrine differentiation. In summary, our data suggest LYRIC/AEG-1 is a negative regulator of BCCIPα, promoting proteasomal degradation either through direct interaction, or potentially through an indirect mechanism involving downstream effects of the NF-κB signaling pathway. [Copyright &y& Elsevier]

Abstract: Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of cellular sequestration. The contributions of organelles and metabolic processes that may mimic drug transport were defined in human vascular smooth muscle cells. We hypothesized that procainamide-induced vacuolar cytopathology is driven by intense pseudotransport mediated by the vacuolar (V)-ATPase and pursued the characterization of vesicular trafficking alterations in this model. Large amounts of procainamide were taken up by intact cells (maximal in 2 h, reversible upon washout, apparent KM 4.69 mM; fluorometric determination of cell-associated drug). Procainamide uptake was extensively prevented or reversed by pharmacological inhibition of the V-ATPase with bafilomycin A1 or FR 167356, decreased at low extracellular pH and preceded vacuolar cell morphology. However, the uptake of procainamide was unaffected by mitochondrial poisons that reduced the uptake of rhodamine 6G. Large vacuoles induced by millimolar procainamide were labeled with the late endosome/lysosome markers Rab7 and CD63 and the autophagy effector LC3; their osmotic formation (but not procainamide uptake) was reduced by extracellular mannitol and parallel to LC3 II formation. Procainamide-induced vacuolization is associated with defective endocytosis of fluorophore-labeled bovine serum albumin, but not with induction of the unfolded protein response. The contents of a vacuole subset slowly (≥24 h) become positive for Nile red staining (phospholipidosis-like response). V-ATPase-driven ion trapping is a form of intense cation pseudotransport that concerns the uncharged form of the drugs, and is associated with a vacuolar, autophagic and evolutive cytopathology and profound effects on vesicular trafficking. [Copyright &y& Elsevier]

Abstract: Mammalian stanniocalcin-2 (STC2) is a secreted glycoprotein hormone with a putative role in unfolded protein response and apoptosis. Here we reported that STC2 expression was sporadically abrogated in human cancer cells by transcriptional silencing associated with CpG island promoter hypermethylation. Direct sequencing of bisulfite-modified DNA from a panel of seven human cancer cell lines revealed that CpG dinucleotides in STC2 promoter was methylated in human ovarian epithelial cancer (SKOV3, OVCAR3 and CaOV3), pancreatic cancer (BxP3), colon adenoma (HT29), and leukemia (Jurkat cells). STC2 CpG island hypermethylation was accompanied with a low basal STC2 expression level. Treatment of these cancer cells with 5-aza-2′-deoxycytidine (5-aza-CdR), an inhibitor of DNA methylation significantly induced STC2 expression. Using SKOV3 cells as a model, the link between DNA demethylation and STC2 expression was consistently demonstrated with hydralazine treatment, which was shown to reduce the protein level of DNA methyltransferase 1 (DNMT1) but stimulated STC2 expression. Two human normal surface ovarian cell-lines (i.e. IOSE 29 and 398) showed no methylation at CpG dinucleotides in the examined promoter region and were accompanied with high basal STC2 levels. Hypoxia stimulated STC2 expression in SKOV3 cells was markedly increased in 5-aza-CdR pretreated cells, showing that DNA methylation may hinder the HIF-1 mediated activation. To elucidate this possibility, RNA interference studies confirmed that endogenous HIF-1α was a key factor for STC2 gene activation as well as in the synergistic induction of STC2 expression in 5-aza-CdR pretreated cells. Chromatin immunoprecipitation (ChIP) assay demonstrated the binding of HIF-1α to STC2 promoter. The binding was increased in 5-aza-CdR pretreated cells. Collectively, this is the first report to show that STC2 was aberrantly hypermethylated in human cancer cells. The findings demonstrated that STC2 epigenetic inactivation may interfere with HIF-1 mediated activation of STC2 expression. [Copyright &y& Elsevier]

Abstract: Although altered gene expression clearly causes failure of the neocortex to form properly, many causes of neocortical dysplasia arise from environmental or unknown factors. Our lab studies a model of cortical dysplasia induced by injection of methylazoxymethanol (MAM) into pregnant ferrets on embryonic day 33 (E33), which shares many features of neocortical dysplasia in humans. E33 MAM treatment results in characteristic deficits that include dramatic reduction of layer 4 in somatosensory cortex, widespread termination of thalamic afferents, and altered distribution of GABAergic elements. We determined the ability of immature cells to migrate into MAM-treated cortex using ferret neural progenitor cells obtained at E27 and E33 and mouse neural progenitor cells obtained at E14. When these cells were transplanted into organotypic cultures obtained from normal and E33 MAM-treated ferret cortex prepared on postnatal day 0 (P0), all progenitor cells migrated similarly in both hosts, preferentially residing in the upper cortical plate. The site of transplantation was significant, however, so that injections into the ventricular zone were more likely to reach the cortical plate than transplants into the intermediate zone. When similar cells were transplanted into ferret kits, ∼P7–P9, and allowed to survive for 2–4 weeks, the donor cells migrated differently and also reached distinct destinations in normal and MAM-treated hosts. MAM-treated cortex was more permissive to invasion by donor cells as they migrated to widespread aspects of the cortex, whereas transplants in normal host cortex were more restricted. E27 neural progenitor cells populated more cortical layers than later born E33 neural progenitor cells, suggesting that the fate of transplanted cells is governed by a combination of extrinsic and intrinsic factors. [Copyright &y& Elsevier]

Abstract: Toll-like receptors (TLRs) are involved in the production of inflammatory mediators upon specific ligands stimuli. Chemokines are important inflammatory mediators capable of chemoattracting diverse immune cells. In addition to normal immune cells, the expression of TLRs and chemokines has been detected in various tumor cells. However, the roles of TLRs and chemokines expressed by tumor cells in the processes of tumor progression and immune escape have not been fully elucidated. Here we report that TLR4 ligation by lipopolysaccharide (LPS) significantly promotes CT-26 colon cancer cells to produce chemokine CCL20 via activation of TLR4 signaling pathways. We find that LPS treatment of CT-26 cells can significantly increase the chemoattraction of immature dendritic cells (DC) by the autocrine CCL20. Our studies suggest that TLR4 expressed by tumor cells may be involved in the induction of chemokines like CCL20, providing a potential linkage between chronic inflammation and tumor immune escape. [Copyright &y& Elsevier]

Introduction: Pancreatic cancer is an extremely virulent form of cancer with few effective treatments. Catechin and inositol hexaphosphate (IP6), two naturally occurring molecules found in green tea and high-fiber foods, respectively, are compounds that have been shown to demonstrate anti-proliferative effects when administered as single therapeutic agents against a number of cancers. We hypothesized that, alone and in combination, IP6 and catechin would be effective against pancreatic cancer. Materials and methods: Pancreatic (PANC-1 and MIAPACA) cancer cell lines were cultured and treated with IP6 (0.8 mm/well), catechin (100uM/well), and the combination of the two. Cell viability was measured by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) at 24, 48, and 72 h. Vascular endothelial growth factor (VEGF) was measured in the cell supernatants by ELISA. Apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC). Results: The combination of catechin and IP6 significantly inhibited proliferation in the PANC-1 cell line at 24, 48, and 72 h compared to single agents (P < 0.001). Growth of the MIAPACA cell line was inhibited (P < 0.01) by each agent alone, but additive inhibitory effects were not seen. An increase in early apoptosis was attributed to catechin therapy in both cell lines (P < 0.01). The combination of these agents also increased early apoptotic activity when compared to the control (P < 0.001). IP6 reduced VEGF in both cell lines (P < 0.01). In combination, catechin and IP6 amplified VEGF reduction compared to each agent in MIAPACA and control (P < 0.002). Conclusions: These results, combined with the prevalence of these compounds in safe, naturally occurring foods, make catechin and IP6 attractive therapies for treatment, and possibly in preventative trials, of pancreatic cancer. [Copyright &y& Elsevier]

Abstract: A cryptopleurine analogue, 7-methoxycryptopleurine, a phenanthroquinolizidine, was first found to exert potent anti-inflammatory activity in vitro and in vivo as well as have remarkable cytotoxic activity against cancer cells. The non-planar structure between the two major moieties, phenanthrene and indolizidine/quinolizidine, played a crucial role in the activity of phenanthroindolizidines or phenanthroquinolizidines in terms of cytotoxic effects on cancer cells and anti-inflammatory activity. We also showed that increase in planarity and rigidity of the indolizidine/quinolizidine moiety and change of the amine group into an amide by introducing a keto group to phenanthroindolizidines or phenanthroquinolizidines at the equivalent position 9 of tylophorine significantly reduced their activities. Moreover, in general, phenanthroquinolizidines are more potent than their respective phenanthroindolizines. [Copyright &y& Elsevier]

Abstract: Bladder cancer is the fourth most common cancer in men and ninth most common in women. It has a protracted course of progression and is thus an ideal candidate for chemoprevention strategies and trials. This study was conducted to evaluate the chemopreventive/antiproliferative potential of (−)-epigallocatechin gallate (EGCG, the major phytochemical in green tea) against bladder cancer and its mechanism of action. Using the T24 human bladder cancer cell line, we found that EGCG treatment caused dose- and time-dependent inhibition of cellular proliferation and cell viability, and induced apoptosis. Mechanistically, EGCG inhibits phosphatidylinositol 3′-kinase/Akt activation that, in turn, results in modulation of Bcl-2 family proteins, leading to enhanced apoptosis of T24 cells. These findings suggest that EGCG may be an important chemoprevention agent for the management of bladder cancer. [Copyright &y& Elsevier]