1,2,3,4,6-Penta-O-galloyl-ß-D-glucose, a bioactive compound in Elaeocarpus sylvestris extract, inhibits varicella-zoster virus replication.

The aim of this study was to establish the effect of a 70% ethanol extract of Elaeocarpus sylvestris (ESE) on varicella-zoster virus (VZV) replication and identify the specific bioactive component(s) underlying its activity. ESE induced a significant reduction in replication of the clinical strain of VZV. Activity-guided fractionation indicated that the ethyl acetate (EtOAc) fraction of ESE contains the active compound(s) inhibiting VZV replication. High-Performance Liquid Chromatography coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (HPLC-Q-TOF-MS/MS) analysis of the EtOAc fraction of ESE facilitated the identification of 13 chemical components. Among these, 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG) markedly suppressed VZV-induced c-Jun N-terminal kinase (JNK) activation, expression of viral immediate-early 62 (IE62) protein and VZV replication. Our results collectively support the utility of PGG as a potential candidate anti-viral drug to treat VZV-associated diseases.

Localizing PRL-2 expression and determining the effects of dietary Mg(2+) on expression levels.

The phosphatase of regenerating liver (PRL) is a group of protein tyrosine phosphatases that play a key role in cancer progression and metastasis. We previously showed that PRL-2 modulates intracellular Mg(2+) levels and sustains cancer phenotypes by binding to the Mg(2+) transporter CNNM3. However, the physiological functions of PRL-2 in animals remain largely unknown. To better understand which cell types are associated with PRL-2 function, we characterized its expression in mouse tissues using a PRL-2 ß-galactosidase reporter mouse model. Our results demonstrated that PRL-2 was ubiquitously expressed, with the highest expression levels observed in the hippocampal pyramidal neurons, ependymal cells, cone and rod photoreceptor cells, endocardium, vascular and bronchial smooth muscle, and collecting ducts in the kidney. On the other hand, PRL-2 expression was undetectable or very low in the parenchymal cells of the liver and pancreas. Our results also indicated that PRL-2 is involved in cell-type-specific Mg(2+) homeostasis and that PRL-2 expression is potentially inversely regulated by dietary Mg(2+) levels.

[Research of nasopharyngeal carcinoma and the applicationof epstein-barr virus immediate early protein].

Epstein-barr virus is closely related to the occurrence and development of nasopharyngeal carcinoma;EB virus immediate early protein Zta and Rta is the control factor for the virus to enter the replication and cleavage stage.At present,ELISA method is commonly used to detect serum Zta and Rta related antibodies in the clinical as one of the serological indicators for early nasopharyngeal cancer screening and the auxiliary diagnosis.

Tumor Necrosis Factor-α Underlies Loss of Cortical Dendritic Spine Density in a Mouse Model of Congestive Heart Failure.

BACKGROUND: Heart failure (HF) is a progressive disorder characterized by reduced cardiac output and increased peripheral resistance, ultimately leading to tissue perfusion deficits and devastating consequences for several organs including the brain. We previously described a tumor necrosis factor-α (TNF-α)-dependent enhancement of posterior cerebral artery tone and concomitant reduced cerebral blood flow in a mouse model of early HF in which blood pressure remains minimally affected. HF is often associated with cognitive impairments such as memory deficits, even before any overt changes in brain structure and function occur. The pathophysiology underlying the development of cognitive impairments in HF is unknown, and appropriate treatment strategies are lacking. METHODS AND RESULTS: We used a well-established mouse model in which HF was induced by experimental myocardial infarction produced by permanent surgical ligation of the left anterior descending coronary artery (infarct size ≈25% of the left ventricular wall). Ligated mice developed enlarged hearts, congested lungs, and reduced cardiac output and blood pressure, with elevated peripheral resistance within 6 to 8 weeks after ligation. In this study, we demonstrated the significance of the proinflammatory cytokine TNF-α during HF-mediated neuroinflammation and associated impaired hippocampus-independent nonspatial episodic memory function. Augmented cerebral TNF-α expression and microglial activation in HF mice, indicative of brain inflammation, were accompanied by morphological changes and significant reduction of cortical dendritic spines (61.39±8.61% for basal and 61.04±9.18% for apical spines [P<0.001]). The significance of TNF-α signaling during the observed HF-mediated neurodegenerative processes is supported by evidence showing that sequestration or genetic deletion of TNF-α ameliorates the observed reduction of cortical dendritic spines (33.51±7.63% for basal and 30.13±6.98% for apical spines in wild-type mice treated with etanercept; 17.09±6.81% for basal and 17.21±7.29% for apical spines in TNF-α(-/-)). Moreover, our data suggest that alterations in cerebral serum and glucocorticoid-inducible kinase 1 (SgK1) expression and phosphorylation during HF may be TNF-α dependent and that an increase of SgK1 phosphorylation potentially plays a role in the HF-associated reduction of dendritic spine density. CONCLUSIONS: Our findings demonstrate that TNF-α plays a pivotal role in HF-mediated neuroinflammation and associated alterations of cortical dendritic spine density and has the potential to reveal novel treatment strategies for HF-associated memory deficits.

Preparation and characterization of polyclonal antibody against Kaposi's sarcoma-associated herpesvirus lytic gene encoding RTA.

Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293 T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzyme-linked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene.

Human cytomegalovirus and primary intracranial tumours: frequency of tumour infection and lack of correlation with systemic immune anti-viral responses.

AIMS: Human cytomegalovirus (HCMV) is a ubiquitous beta human herpesvirus able to influence infected cell survival and proliferation and to modulate the host immune response. As there is accumulating evidence that HCMV is detected in primary intracranial astrocytic tumours, in this study we looked for the presence of HCMV in intracranial tumours and tried to correlate this eventual presence with the anti-HCMV systemic immunoreactivity and with the detection of HCMV in peripheral blood. METHODS: In this study, we analysed 43 glioblastomas (GBM), 14 oligodendrogliomas (OL) and 20 meningiomas (MG) by immunofluorescence (IF) targeting HCMV immediate early antigen (IE1) and by nested PCR (nPCR) amplifying HCMV glycoprotein B (gB). RESULTS: Detection of IE1 by IF showed the presence of HCMV in 70% of GBM, 57% of OL and 85% of MG, in contrast to gB nPCR, which detected HCMV in only 50% of GBM, 38% of OL and 46% of MG. Unexpectedly, HCMV DNA and antigens were detected within GBM, OL and MG of patients that exhibit negative viral serology. More surprisingly, PCR on the peripheral blood did not detect HCMV in patients with a HCMV-positive tumour. CONCLUSIONS: Our results are in agreement with previous observations demonstrating HCMV in glial tumours and highlight the presence of HCMV in meningiomas. We also showed that anti-HCMV specific systemic immunoreactivity and detection of HCMV in peripheral blood are not predictive of HCMV presence in primary intracranial tumours.

Unique expression pattern of viral proteins in human herpesvirus 8-positive plasmablastic lymphoma: a case report.

BACKGROUND: Human herpesvirus 8 (HHV8)-positive plasmablastic lymphoma is a disease which correlates with acquired immunodeficiency syndrome (AIDS). Little is known about the pathogenesis of the disease due to its rarity. We report an autopsy case about AIDS related HHV-8-positive plasmablastic lymphoma and presents an examination about HHV8 related proteins for the disease by using immunohistochemical techniques. CASE PRESENTATION: Two kinds of tumors complicated the male AIDS patient: one was HHV-8-positive plasmablastic lymphoma and the other was Kaposi's sarcoma (KS). Immunohistochemically, the lymphoma cells were positive for HHV8-associated lytic early proteins as well as HHV8 latency-associated nuclear antigen 1 (LANA-1), and, on the other hand, the lymphoma cells were negative for lytic immediately early proteins. KS was positive for only LANA-1. CONCLUSION: These findings indicate that the lymphoma cells acquired an ability to proliferate without de novo HHV8 replication. Moreover, the onset mechanisms of HHV-8-positive plasmablastic lymphoma may be different from those of KS.

Fluorescent tagging and cellular distribution of the Kaposi's sarcoma-associated herpesvirus ORF45 tegument protein.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the Gammaherpesvirinae subfamily. We report here the construction of a dual fluorescent-tagged KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses green fluorescent protein (GFP) and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confirmed that this virus is properly expressed and correctly replicates and that the mCherry-ORF45 protein is incorporated into the virions. Using this labeled virus, we describe the dynamics of mCherry-ORF45 expression and localization in newly infected cells as well as in latently infected cells undergoing lytic induction and show that mCherry can be used to monitor cells undergoing the lytic viral cycle. This virus is likely to enable future studies monitoring the dynamics of viral trafficking and tegumentation during viral ingress and egress. IMPORTANCE: The present study describes the construction and characterization of a new recombinant KSHV genome BAC16 clone which expresses mCherry-tagged ORF45. This virus enables the tracking of cells undergoing lytic infection and can be used to address issues related to the trafficking and maturation pathways of KSHV virions.

Genetic inactivation of Nupr1 acts as a dominant suppressor event in a two-hit model of pancreatic carcinogenesis.

BACKGROUND: Nuclear protein 1 (Nupr1) is a major factor in the cell stress response required for Kras(G12D)-driven formation of pancreatic intraepithelial neoplastic lesions (PanINs). We evaluated the relevance of Nupr1 in the development of pancreatic cancer. METHODS: We investigated the role of Nupr1 in pancreatic ductal adenocarcinoma (PDAC) progression beyond PanINs in Pdx1-cre;LSL-Kras(G12D);Ink4a/Arf(fl/fl)(KIC) mice. RESULTS: Even in the context of the second tumorigenic hit of Ink4a/Arf deletion, Nupr1 deficiency led to suppression of malignant transformation involving caspase 3 activation in premalignant cells of KIC pancreas. Only half of Nupr1-deficient;KIC mice achieved PDAC development, and incident cases survived longer than Nupr1(wt);KIC mice. This was associated with the development of well-differentiated PDACs in Nupr1-deficient;KIC mice, which displayed enrichment of genes characteristic of the recently identified human classical PDAC subtype. Nupr1-deficient;KIC PDACs also shared with human classical PDACs the overexpression of the Kras-activation gene signature. In contrast, Nupr1(wt);KIC mice developed invasive PDACs with enriched gene signature of human quasi-mesenchymal (QM) PDACs. Cells derived from Nupr1-deficient;KIC PDACs growth in an anchorage-independent manner in vitro had higher aldehyde dehydrogenase activity and overexpressed nanog, Oct-4 and Sox2 transcripts compared with Nupr1(wt);KIC cells. Moreover, Nupr1-deficient and Nurpr1(wt);KIC cells differed in their sensitivity to the nucleoside analogues Ly101-4b and WJQ63. Together, these findings show the pivotal role of Nupr1 in both the initiation and late stages of PDAC in vivo, with a potential impact on PDAC cell stemness. CONCLUSIONS: According to Nupr1 status, KIC mice develop tumours that phenocopy human classical or QM-PDAC, respectively, and present differential drug sensitivity, thus becoming attractive models for preclinical drug trials.

The prevalence of human cytomegalovirus DNA in gliomas of Brazilian patients.

Members of the Herpesviridae family have been implicated in a number of tumours in humans. At least 75% of the human population has had contact with cytomegalovirus (HCMV). In this work, we screened 75 Brazilian glioma biopsies for the presence of HCMV DNA sequences. HCMV DNA was detected in 36% (27/75) of the biopsies. It is possible that HCMV could be a co-factor in the evolution of brain tumours.

The prevalence of human cytomegalovirus DNA in gliomas of Brazilian patients

Members of the Herpesviridae family have been implicated in a number of tumours in humans. At least 75% of the human population has had contact with cytomegalovirus (HCMV). In this work, we screened 75 Brazilian glioma biopsies for the presence of HCMV DNA sequences. HCMV DNA was detected in 36% (27/75) of the biopsies. It is possible that HCMV could be a co-factor in the evolution of brain tumours.

Immunohistochemical detection of intra-neuronal VZV proteins in snap-frozen human ganglia is confounded by antibodies directed against blood group A1-associated antigens.

Varicella-zoster virus (VZV) causes chickenpox, establishes latency in trigeminal (TG) and dorsal root ganglia (DRG), and can lead to herpes zoster upon reactivation. The VZV proteome expressed during latency remains ill-defined, and previous studies have shown discordant data on the spectrum and expression pattern of VZV proteins and transcripts in latently infected human ganglia. Recently, Zerboni and colleagues have provided new insight into this discrepancy (Zerboni et al. in J Virol 86:578-583, 2012). They showed that VZV-specific ascites-derived monoclonal antibody (mAb) preparations contain endogenous antibodies directed against blood group A1 proteins, resulting in false-positive intra-neuronal VZV staining in formalin-fixed human DRG. The aim of the present study was to confirm and extend this phenomenon to snap-frozen TG (n=30) and DRG (n=9) specimens of blood group genotyped donors (n=30). The number of immunohistochemically stained neurons was higher with mAb directed to immediate early protein 62 (IE62) compared with IE63. The IE63 mAb-positive neurons always co-stained for IE62 but not vice versa. The mAb staining was confined to distinct large intra-neuronal vacuoles and restricted to A1(POS) donors. Anti-VZV mAb staining in neurons, but not in VZV-infected cell monolayers, was obliterated after mAb adsorption against blood group A1 erythrocytes. The data presented demonstrate that neuronal VZV protein expression detected by ascites-derived mAb in snap-frozen TG and DRG of blood group A1(POS) donors can be misinterpreted due to the presence of endogenous antibodies directed against blood group A1-associated antigens present in ascites-derived VZV-specific mAb preparations.

Human cytomegalovirus and mucoepidermoid carcinoma of salivary glands: cell-specific localization of active viral and oncogenic signaling proteins is confirmatory of a causal relationship.

Human cytomegalovirus (hCMV) infection is common. Although still controversial, there is growing evidence that active hCMV infection is associated with a variety of malignancies, including brain, breast, lung, colon, and prostate. Given that hCMV is frequently resident in salivary gland (SG) ductal epithelium, we hypothesized that hCMV would be important to the pathogenesis of SG mucoepidermoid carcinoma (MEC). This was initially supported by our finding that purified CMV induces malignant transformation in SG cells in an in vitro mouse model, and utilizes a pathogenic pathway previously reported for human MEC. Here we present the histologic and molecular characterizations of 39 human SG MECs selected randomly from a repository of cases spanning 2004-2011. Serial sections were obtained from formalin-fixed, paraffin embedded, tissue blocks from previous incisional or excisional biopsies. Immunohistochemical assays were performed for active hCMV proteins (IE1 and pp65) and the activated COX/AREG/EGFR/ERK signaling pathway. All four prospective causal criteria for viruses and cancer are fully satisfied: (1) protein markers for active hCMV are present in 97% of MECs; (2) markers of active hCMV are absent in non-neoplastic SG tissues; (3) hCMV-specific proteins (IE1, pp65) are in specific cell types and expression is positively correlated with severity; (4) hCMV correlates and colocalizes with an upregulation and activation of an established oncogenic signaling pathway (COX/AREG/EGFR/ERK). Thus, the evidential support reported here and previously in a mouse model is strongly confirmatory of a causal relationship between hCMV and SG mucoepidermoid carcinoma. To our knowledge, this is the first demonstration of hCMV's role in human oncogenesis that fully responds to all of Koch's Postulates as revised for viruses and cancer. In the absence of any contrary evidence, hCMV can reasonably be designated an "oncovirus."

The immediate early gene Ier2 promotes tumor cell motility and metastasis, and predicts poor survival of colorectal cancer patients.

Here, we report unbiased screens for genes expressed in metastatic tumor cells that are associated with cell motility. These screens identified Ier2, an immediate early gene of unknown function, as potentially having a role in tumor cell motility and metastasis. Knockdown of Ier2 in 3T3 fibroblasts inhibited their motility upon relief of contact inhibition in monolayer wounding assays. Furthermore, ectopic Ier2 expression promoted the motility and invasiveness of poorly metastatic 1AS pancreatic tumor cells in vitro. Relief of contact inhibition was associated with translocation of the Ier2 protein from the cytoplasm to the nucleus in both 3T3 fibroblasts and 1AS tumor cells. Importantly, ectopic Ier2 expression in 1AS cells stimulated metastasis formation when cells were implanted into experimental animals. Furthermore, we found elevated Ier2 expression in a wide variety of human tumor types. This correlated with poor metastasis-free and overall survival in patients with colorectal adenocarcinomas. Together, these data reveal Ier2 as a new player in the regulation of tumor progression and metastasis, and suggest that Ier2 may be useful prognostically and therapeutically in the management of cancer.

Lack of TNF-alpha promotes caspase-3-independent apoptosis during murine cytomegalovirus retinitis.

PURPOSE: Both caspase-dependent and caspase-independent apoptosis contribute to retinal damage during murine cytomegalovirus (MCMV) retinitis, and TNF-α is among the inducers of apoptosis. The aim of this study was to determine the contribution of TNF-α by studying virus replication and apoptosis in immunosuppressed (IS) TNF-α(-/-) mice. METHODS: IS TNF-α(-/-) mice or wild-type mice were inoculated with MCMV by the supraciliary route. Injected eyes were examined by plaque assay, electron microscopy, Western blot analysis (caspase-3, caspase-8, caspase-12, Bid, NF-κB, cFlip, XIAP), staining for MCMV early antigen, and TUNEL assay. RESULTS: Although the titer of MCMV was similar in both groups, significantly more apoptotic cells were observed in the retinas of IS TNF-α(-/-) mice than in those of wild-type mice. The level of active caspase-3 was similar in both groups; however, more activated proteins for genes involved in the mitochondrial pathway (cleaved caspase-8, tBid) and endoplasmic reticulum (ER) stress (cleaved caspase-12) and, though less active, NF-κB subunits and antiapoptotic proteins (XIAP and cFlip) were detected in the TNF-α(-/-) eyes compared with wild-type mice. CONCLUSIONS: Although TNF-α is an inducer of apoptosis, the results of this study suggest that TNF-α is also antiapoptotic by the following mechanism: TNF-α activation of NF-κB promotes the production of the antiapoptosis genes, c-flip or XIAP, which, in turn, inhibit the activation of caspase-8 and the mitochondrial pathway or the activation of caspase-12 and ER stress.

The detection of CMV pp65 and IE1 in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor affecting children and adults, with the majority of affected individuals dying from their disease by 2 years following diagnosis. Other groups have reported the association of cytomegalovirus (CMV) with GBM, and we sought to confirm these findings in a large series of patients with primary GBM from our institution. Immunohistochemical analysis of paraffin embedded tissue sections was performed on 49 newly diagnosed GBM tumors, the largest series reported to date. We confirmed the presence of CMV pp65 on 25/49 (51%) and of IE1 on 8/49 (16%) of these tumors. While pp65 and IE1 are generally found in the nucleus of cells that are permissibly infected by CMV, GBM in this series had mostly cytoplasmic staining, with only 16% having nuclear staining for one or both of these antigens. We infected GBM cell lines with a laboratory strain of CMV, and found that most of the staining was cytoplasmic, with some perinuclear localization of IE1. To test the potential for CMV infected GBM cells to be recognized by CMV pp65 and IE1 specific cytotoxic T lymphocytes (CTL), we used CMV infected GBM cell lines in cytotoxicity assays with human leukocyte antigen partially matched CMV CTL. Lysis of CMV infected GBM tumor cells was accentuated by pre-treating these cell lines with either the demethylating agent decitabine or interferon-γ, both of which were shown to increase MHC Class I and II expression on tumor cells in vitro. These studies confirm the presence of CMV pp65 or IE1 on approximately half of GBM, with the possibility that CMV positive tumor cells can be recognized by CMV pp65/IE1 specific T cells.

Up-regulation of cell cycle arrest protein BTG2 correlates with increased overall survival in breast cancer, as detected by immunohistochemistry using tissue microarray.

BACKGROUND: Previous studies have shown that the ADIPOR1, ADORA1, BTG2 and CD46 genes differ significantly between long-term survivors of breast cancer and deceased patients, both in levels of gene expression and DNA copy numbers. The aim of this study was to characterize the expression of the corresponding proteins in breast carcinoma and to determine their correlation with clinical outcome. METHODS: Protein expression was evaluated using immunohistochemistry in an independent breast cancer cohort of 144 samples represented on tissue microarrays. Fisher's exact test was used to analyze the differences in protein expression between dead and alive patients. We used Cox-regression multivariate analysis to assess whether the new markers predict the survival status of the patients better than the currently used markers. RESULTS: BTG2 expression was demonstrated in a significantly lower proportion of samples from dead patients compared to alive patients, both in overall expression (P = 0.026) and cell membrane specific expression (P = 0.013), whereas neither ADIPOR1, ADORA1 nor CD46 showed differential expression in the two survival groups. Furthermore, a multivariate analysis showed that a model containing BTG2 expression in combination with HER2 and Ki67 expression along with patient age performed better than a model containing the currently used prognostic markers (tumour size, nodal status, HER2 expression, hormone receptor status, histological grade, and patient age). Interestingly, BTG2 has previously been described as a tumour suppressor gene involved in cell cycle arrest and p53 signalling. CONCLUSIONS: We conclude that high-level BTG2 protein expression correlates with prolonged survival in patients with breast carcinoma.

MCAF1 and synergistic activation of the transcription of Epstein-Barr virus lytic genes by Rta and Zta.

Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle. The two proteins often collaborate to activate the transcription of EBV lytic genes synergistically. This study demonstrates that Rta and Zta form a complex via an intermediary protein, MCAF1, on Zta response element (ZRE) in vitro. The interaction among these three proteins in P3HR1 cells is also verified via coimmunoprecipitation, CHIP analysis and confocal microscopy. The interaction between Rta and Zta in vitro depends on the region between amino acid 562 and 816 in MCAF1. In addition, overexpressing MCAF1 enhances and introducing MCAF1 siRNA into the cells markedly reduces the level of the synergistic activation in 293T cells. Moreover, the fact that the synergistic activation depends on ZRE but not on Rta response element (RRE) originates from the fact that Rta and Zta are capable of activating the BMRF1 promoter synergistically after an RRE but not ZREs in the promoter are mutated. The binding of Rta-MCAF1-Zta complex to ZRE but not RRE also explains why Rta and Zta do not use RRE to activate transcription synergistically. Importantly, this study elucidates the mechanism underlying synergistic activation, which is important to the lytic development of EBV.

Expression of varicella-zoster virus immediate-early regulatory protein IE63 in neurons of latently infected human sensory ganglia.

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediate-early 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained.

Comparative proteomic analysis of mouse melanoma cell line B16, a metastatic descendant B16F10, and B16 overexpressing the metastasis-associated tyrosine phosphatase PRL-3.

Metastasis is a complex, multistep process by which a cancer cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary cancer. A deeper understanding of the molecular events underlying metastasis will provide information that will be useful for the development of new diagnostic and therapeutic strategies. The B16 and B16F10 mouse melanoma cell lines are widely used as model system for studying many aspects of cancer biology including metastasis. Compared with B16, which has a low metastatic potential, the highly metastatic cell line B16F10 displayed a higher metastatic ability along with higher expression levels of the metastasis-associated phosphatase of regenerating liver-3 (PRL-3). B16 cells transfected with PRL-3 cDNA (B16-PRL3) had metastatic abilities comparable to those of Bl16F10 cells. To study the molecular mechanisms that underlie metastasis, the proteomes of the B16, B16F10, and B16-PRL3 cell lines were compared using two-dimensional differential in-gel electrophoresis. Proteins that varied significantly in levels between these cell lines were selected and identified using mass spectrometry. Interestingly, many proteins, especially those present in membrane fractions, were similarly up- or downregulated in both the Bl16F10 and B16-PRL3 cells lines compared to B16 cell lines. The list of similarly regulated proteins included heat shock protein 70, fascin-1, septin-6, ATP synthase beta subunit, and bone morphogenic protein receptor type IB. These proteins may play a causal role in PRL-3-mediated metastasis. These investigations open an avenue for the further characterization of the molecular mechanisms that underlie metastasis.