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Bladder cancer (BC) is the 10th most common malignancy worldwide, with an estimated 573,000 new cases and 213,000 deaths in 2020. Available therapeutic approaches are still unable to reduce the incidence of BC metastasis and the high mortality rates of BC patients. Therefore, there is a need to deepen our understanding of the molecular mechanisms underlying BC progression to develop new diagnostic and therapeutic tools. One such mechanism is protein glycosylation. Numerous studies reported changes in glycan biosynthesis during neoplastic transformation, resulting in the appearance of the so-called tumor-associated carbohydrate antigens (TACAs) on the cell surface. TACAs affect a wide range of key biological processes, including tumor cell survival and proliferation, invasion and metastasis, induction of chronic inflammation, angiogenesis, immune evasion, and insensitivity to apoptosis. The purpose of this review is to summarize the current information on how altered glycosylation of bladder cancer cells promotes disease progression and to present the potential use of glycans for diagnostic and therapeutic purposes. [ABSTRACT FROM AUTHOR]

Simple Summary: By studying the cancer-inducing ability of polyomaviruses, several milestones in cancer research crucially contributing to our current understanding of, e.g., the tumor suppressor proteins p53 and RB1 have been achieved. However, only with the discovery of Merkel cell polyomavirus (MCPyV) and its linkage to the highly aggressive Merkel cell carcinoma (MCC) in 2008 has a human polyomavirus-induced cancer been identified. Since then, intensive research has uncovered many details of the interaction of the virus with its human host, as well as many molecular mechanisms by which the MCPyV-encoded oncoproteins the so-called T antigens mediate oncogenic transformation. Surprisingly, many differences to the previously known polyomaviruses have been observed. In this review, we summarize the current knowledge on MCPyV and MCC and discuss some of the open questions. The best characterized polyomavirus family member, i.e., simian virus 40 (SV40), can cause different tumors in hamsters and can transform murine and human cells in vitro. Hence, the SV40 contamination of millions of polio vaccine doses administered from 1955–1963 raised fears that this may cause increased tumor incidence in the vaccinated population. This is, however, not the case. Indeed, up to now, the only polyomavirus family member known to be the most important cause of a specific human tumor entity is Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma (MCC). MCC is a highly deadly form of skin cancer for which the cellular origin is still uncertain, and which appears as two clinically very similar but molecularly highly different variants. While approximately 80% of cases are found to be associated with MCPyV the remaining MCCs carry a high mutational load. Here, we present an overview of the multitude of molecular functions described for the MCPyV encoded oncoproteins and non-coding RNAs, present the available MCC mouse models and discuss the increasing evidence that both, virus-negative and -positive MCC constitute epithelial tumors. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (ESCs) can differentiate into a range of cell types during development, and this pluripotency is regulated by various extrinsic and intrinsic factors. Mucin-type O-glycosylation has been suggested to be a potential factor in the control of ESC pluripotency, and is characterized by the addition of N-acetylgalactosamine (GalNAc) to serine or threonine residues of membrane-anchored proteins and secreted proteins. To date, the relationship between mucin-type O-glycosylation and signaling in ESCs remains undefined. Here, we identify the elongation pathway via C1GalT1 that synthesizes T antigen (Galβ1-3GalNAc) as the most prominent among mucin-type O-glycosylation modifications in ESCs. Moreover, we show that mucin-type O-glycosylation on the Wnt signaling receptor frizzled-5 (Fzd5) regulates its endocytosis via galectin-3 binding to T antigen, and that reduction of T antigen results in the exit of the ESCs from pluripotency via canonical Wnt signaling activation. Our findings reveal a novel regulatory mechanism that modulates Wnt signaling and, consequently, ESC pluripotency. [ABSTRACT FROM AUTHOR]

Cells are covered with glycans. The expression and distribution of specific glycans on the surface of a cell are important for various cellular functions. Imaging these glycans is essential to aid elucidation of their biological roles. Here, utilizing methods of direct fluorescent glycan imaging, in which fluorescent sialic acids are directly incorporated into substrate glycans via recombinant sialyltranferases, we report the differential distribution of N- and O-glycans and variable expression of sialyl-T antigen on HeLa cells. While the expression of N-glycans tends to be more peripheral at positions where cell–cell interaction occurs, O-glycan expression is more granular but relatively evenly distributed on positive cells. While N-glycans are expressed on all cells, sialyl-T antigen expression exhibits a wide spectrum of variation with some cells being strongly positive and some cells being almost completely negative. The differential distribution of N- and O-glycans on cell surface reflects their distinctive roles in cell biology. [ABSTRACT FROM AUTHOR]

JC polyomavirus (JCPyV) infects 50 to 80% of the population and is the causative agent of a fatal demyelinating disease of the central nervous system (CNS). JCPyV presents initially as a persistent infection in the kidneys of healthy people, but during immunosuppression, the virus can reactivate and cause progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types, oligodendrocytes and astrocytes. Until recently, the role of astrocytes has been masked by the pathology in the myelin-producing oligodendrocytes, which are lytically destroyed by the virus. To better understand how astrocytes are impacted during JCPyV infection, the temporal regulation and infectious cycle of JCPyV were analyzed in primary normal human astrocytes (NHAs). Previous research to define the molecular mechanisms underlying JCPyV infection has mostly relied on the use of cell culture models, such as SVG-A cells (SVGAs), an immortalized, mixed population of glial cells transformed with simian virus 40 (SV40) T antigen. However, SVGAs present several limitations due to their immortalized characteristics, and NHAs represent an innovative approach to study JCPyV infection in vitro. Using infectivity assays, quantitative PCR, and immunofluorescence assay approaches, we have further characterized JCPyV infectivity in NHAs. The JCPyV infectious cycle is significantly delayed in NHAs, and the expression of SV40 T antigen alters the cellular environment, which impacts viral infection in immortalized cells. This research establishes a foundation for the use of primary NHAs in future studies and will help unravel the role of astrocytes in PML pathogenesis. [ABSTRACT FROM AUTHOR]

Often glycosidase assays are based on small-molecule compounds where a glycan of interest is linked to a chromophore allowing for easy detection of cleavage of the glycoside bond. However, such compounds only resemble part of the more complex substrate molecule for enzymes acting on glycoconjugates of glycopeptides or glycoproteins. Nonetheless, the advantage is obvious as enzyme activity is readily recorded and kinetic parameters easily obtained. This is not often the case with glycopeptides or glycoproteins as these may reveal increased complexity in terms of heterogeneity in protein-glycan stoichiometry and restricted enzyme accessibility. However, a kinetic analysis of glycan release from glycopeptides could provide information complementary to that of small-molecule substrates, especially if providing kinetic parameters that are immediately comparable. We have characterized the steady state kinetics of wild type and mutant variants of Bifidobacterium longum endo-α- N -acetylgalactosaminidase, by recording the enzymatic release of Galβ(1–3)GalNAc from bovine glycomacropeptide pre-treated with sialidase to remove sialic acid units. Differences between previously reported kinetic constants obtained with synthetic substrates and those obtained in the present work demonstrate an influence of the peptide moiety on the kinetic properties of endo-α- N -acetylgalactosaminidase. The devised assay and data handling method determines the accessible substrate concentration as well as the steady state kinetic parameters, K M and k cat , for glycoconjugates of glycopeptides described by the same units as obtained from using small-molecule substrates and thus allows for a direct comparison. Image 1 • Assay for enzymatic O-linked 2- N -Acetyl hexosamine glycan release. • Analysis tools for quantitative data of enzymatic O-linked glycan release. • Preparation of glycopeptides for enzymes acting on O-linked hexosamine glycans. [ABSTRACT FROM AUTHOR]

Intense study of three families of small tumour viruses with double-stranded DNA genomes, carried out over 50 years, has had a profound impact on biology. The polyomaviruses and papillomaviruses have circular DNA genomes of approximately 5000 and approximately 8000 base-pairs, respectively, and thus encode only a handful of proteins. Adenoviruses have a 32 000-base-pair linear DNA genome, still far smaller than the three billion-base-pair human genome. Members of all three virus families can transform cultured cells to tumorigenicity and cause tumours in experimental animals. Several human papillomaviruses (HPV) and at least one polyomavirus are oncogenic in humans. Early analysis of these viruses, particularly the polyomavirus SV40, led to the development of many powerful experimental tools, including restriction mapping, site-directed mutagenesis, gene transfer, genome-wide sequencing and recombinant DNA. These tools have since been refined and used to study cellular genes, revolutionizing our understanding of biology. These tools were also applied to the viruses themselves. Analysis of the virus life cycle and the effect of these viruses on cells yielded important new insights into many aspects of gene expression, DNA replication, cell biology and carcinogenesis. These studies have also led to vaccination strategies to prevent infection and cancer in humans. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'. [ABSTRACT FROM AUTHOR]

The human JC polyomavirus (JCPyV) infects the majority of the population worldwide and presents as an asymptomatic, persistent infection in the kidneys. In individuals who are immunocompromised, JCPyV can become reactivated and cause a lytic infection in the central nervous system resulting in the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection is initiated by interactions between the capsid protein viral protein 1 (VP1) and the α2,6-linked sialic acid on lactoseries tetrasaccharide c (LSTc), while JCPyV internalization is facilitated by 5-hydroxytryptamine 2 receptors (5-HT2Rs). The mechanisms by which the serotonin receptors mediate virus entry and the signaling cascades required to drive viral infection remain poorly understood. JCPyV was previously shown to induce phosphorylation of extracellular signal-regulated kinase (ERK), a downstream target of the mitogen-activated protein kinase (MAPK) pathway, upon virus entry. However, it remained unclear whether ERK activation was required for JCPyV infection. Both ERK-specific small interfering RNA (siRNA) and ERK inhibitor treatments resulted in significantly diminished JCPyV infection in both kidney and glial cells yet had no effect on the infectivity of the polyomavirus simian virus 40 (SV40). Experiments characterizing the role of ERK during steps in the viral life cycle indicate that ERK activation is required for viral transcription, as demonstrated by a significant reduction in production of large T antigen (TAg), a key viral protein associated with the initiation of viral transcription and viral replication. These findings delineate the role of the MAPK-ERK signaling pathway in JCPyV infection, elucidating how the virus reprograms the host cell to promote viral pathogenesis. IMPORTANCE Viral infection is dependent upon host cell factors, including the activation of cellular signaling pathways. These interactions between viruses and host cells are necessary for infection and play an important role in viral disease outcomes. The focus of this study was to determine how the human JC polyomavirus (JCPyV), a virus that resides in the kidney of the majority of the population and can cause the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML) in the brains of immunosuppressed individuals, usurps a cellular signaling pathway to promote its own infectious life cycle. We demonstrated that the activation of extracellular signal-regulated kinase (ERK), a component of the mitogenactivated protein kinase (MAPK) pathway, promotes JCPyV transcription, which is required for viral infection. Our findings demonstrate that the MAPK-ERK signaling pathway is a key determinant of JCPyV infection, elucidating new information regarding the signal reprogramming of host cells by a pathogenic virus. [ABSTRACT FROM AUTHOR]

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's r, 0.625; P < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTagdependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific. IMPORTANCE HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) is a globally important human pathogen that causes a broad spectrum of disease ranging from mild superficial infections to severe invasive diseases with high morbidity and mortality. Currently, there is no vaccine available for human use. GAS produces a vast array of virulence factors including multiple adhesin molecules. These mediate binding of the bacteria to host tissues and are essential in the initial phases of infection. Prophylactic vaccination with adhesins is a promising vaccine strategy and many GAS adhesins are currently in development as vaccine candidates. The most advanced candidates, having entered clinical trials, are based on the M protein, while components of the pilus and a number of fibronectin-binding proteins are in pre-clinical development. Adhesin-based vaccines aim to induce protective immunity via two main mechanisms: neutralisation where adhesin-specific antibodies block the ability of the adhesin to bind to host tissue and opsonisation in which adhesin-specific antibodies tag the GAS bacteria for phagocytosis. This review summarises our current knowledge of GAS adhesins and their structural features in the context of vaccine development. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans. [ABSTRACT FROM AUTHOR]

A new immunological investigation was carried out to study the association between non-Hodgkin lymphoma and Simian virus 40 (SV40). To this end, a new indirect ELISA was employed with two mimotopes from SV40 large T antigen (Tag), the viral oncoprotein, to analyse for specific reactions to antibodies in sera from non-Hodgkin lymphoma patients and controls, represented by healthy subjects (HS) and breast carcinoma (BC) patients. This study allowed us to assay a new sera collection from non-Hodgkin lymphoma patients (NHL, n = 254). To verify the association between NHL and SV40 Tag, two totally independent cohorts were analysed: NHL1 n = 150 and NHL2 n = 104. The epidemiological survey included sera from HS1, n = 150; HS2, n = 104 and BC, n = 78. This new indirect ELISA revealed that antibodies against SV40 Tag mimotopes are detectable in NHL1 and NHL2 sera with a prevalence of 37 and 36%, respectively. The prevalence of SV40-antibodies detected in both NHL1 and NHL2 cohorts differs statistically from controls, at 19% for HS1 ( p < 0.01), HS2 ( p < 0.05) and BC patients ( p < 0.05). This study, carried out with an immunological assay with specific Tag oncoprotein mimotopes of Simian virus 40, reports the presence of IgG antibodies against the large Tumour antigen in non-Hodgkin lymphomas for the first time. Our immunological data with two independent NHL cohorts show a statistically significant association between Simian virus 40 Tag and non-Hodgkin lymphoma. These results suggest that SV40-positive non-Hodgkin lymphomas could be treated differently from those tested SV40-negative. [ABSTRACT FROM AUTHOR]

Human milk is a source of glycoconjugates, sialylated forms of which enrich the newborn immature immune system and are crucial for their proper development and well-being. Here, we analyzed the expression of α2,3-/α2,6-sialylated glycotopes on skim milk glycoproteins over lactation. Milk samples were analyzed by lectin-blotting using α2,3- and α2,6- sialic acid specific Maackia amurensis (MAA) and Sambucus nigra (SNA) lectins and sialyl- and asialyl-T antigen specific Artocarpus integrifolia (Jacalin) and Arachis hypogaea (PNA) lectins. The reactivities of MAA, SNA, Jacalin and PNA with milk glycoproteins showed that they are heavily decorated with α2,3-/α2,6-linked sialic acid and sialyl-T antigen and to a lesser degree with asialyl-T antigen. Despite individual differences of particular glycoproteins, a sharp and significant decline of α2,6-sialylated glycotopes and sialyl-T antigens and a weaker but significant decrease of α2,3-sialylated glycotopes and asialyl-T antigens on milk glycoproteins during milk maturation was observed. The expression of α2,3-/α2,6-sialylated glycotopes, sialyl- and asialyl-T antigens corresponds to milk maturation but differs in relation to the analyzed glycoprotein. Sialylated milk glycoproteins are considered as a part of innate immunity provided to neonates. Further investigations are needed to understand if they may be useful in milk banking to control the biochemical quality of milk. [ABSTRACT FROM AUTHOR]

Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors. [ABSTRACT FROM AUTHOR]

Background: Merkel cell carcinoma (MCC) is an aggressive skin cancer with a recurrence rate of >40%. Of the 2000 MCC cases per year in the United States, most are caused by the Merkel cell polyomavirus (MCPyV). Antibodies to MCPyV oncoprotein (T-antigens) have been correlated with MCC tumor burden. The present study assesses the clinical utility of MCPyV-oncoprotein antibody titers for MCC prognostication and surveillance.Methods: MCPyV-oncoprotein antibody detection was optimized in a clinical laboratory. A cohort of 219 patients with newly diagnosed MCC were followed prospectively (median follow-up, 1.9 years). Among the seropositive patients, antibody titer and disease status were serially tracked.Results: Antibodies to MCPyV oncoproteins were rare among healthy individuals (1%) but were present in most patients with MCC (114 of 219 patients [52%]; P < .01). Seropositivity at diagnosis independently predicted decreased recurrence risk (hazard ratio, 0.58; P = .04) in multivariate analyses adjusted for age, sex, stage, and immunosuppression. After initial treatment, seropositive patients whose disease did not recur had rapidly falling titers that became negative by a median of 8.4 months. Among seropositive patients who underwent serial evaluation (71 patients; 282 time points), an increasing oncoprotein titer had a positive predictive value of 66% for clinically evident recurrence, whereas a decreasing titer had a negative predictive value of 97%.Conclusions: Determination of oncoprotein antibody titer assists in the clinical management of patients with newly diagnosed MCC by stratifying them into a higher risk seronegative cohort, in which radiologic imaging may play a more prominent role, and into a lower risk seropositive cohort, in which disease status can be tracked in part by oncoprotein antibody titer. Cancer 2017;123:1464-1474. © 2016 American Cancer Society. [ABSTRACT FROM AUTHOR]

Electrochemical glycoprotein sensors have proven to be a great alternative for the detection and characterization of cancer, therapeutic design, and monitoring. A combination of the glycoproteins lubricin (LUB) and peanut agglutinin (PNA) has been successfully implemented for the detection of tumour-associated short glycans directly in bodily fluids, e.g. whole blood. However, such an approach has so far only been studied using a surface-bound redox mediator engineered with the recognition element, PNA. In this paper, we extend the lubricin-peanut agglutinin (LUB-PNA) interface for monitoring glycan binding interactions using redox species in solution. Cyclic voltammetry and electrochemical impedance spectroscopy measurements using the LUB-PNA interface showed that faradaic electron transfer of ferrocyanide redox species in solution is altered in presence of the glycoprotein containing cancer-associated T-antigen glycoprotein (asialofetuin, ASF), while it does not alter in presence of the sialylated glycoform of ASF, fetuin (FET), bovine serum albumin (BSA) and Tn-antigen. The limit of detection (LoD) of ASF is 39 nM in PBS. In addition, this highly specific LUB-PNA sensor allows for the detection of ASF in human blood. [Display omitted] [ABSTRACT FROM AUTHOR]

Glycosylation is one of the major posttranslational modifications of proteins. N-glycosylation (Asn-linked) and O-glycosylation (Ser/Thr-linked) are the two main forms. Abnormal O-glycosylation is frequently observed on the surface of tumor cells, and is associated with an adverse outcome and poor prognosis in patients with cancer. O-glycans (Tn, sTn, and T antigen) can be synthesized in the Golgi apparatus with the aid of several glycosyltransferases (such as T-synthase and ST6GalNAc-I) in a suitable environment. The unique molecular chaperone of T-synthase is Cosmc, which helps T-synthase to fold correctly in the endoplasmic reticulum. Dysregulation of these glycosyltransferases, molecular chaperones, or the environment is involved in the dysregulation of O-glycans. Tn, sTn, and T antigen neo- or over-expression occurs in many types of cancer including gastric, colon, breast, lung, esophageal, prostate, and endometrial cancer. This review discusses the major synthetic pathway of O-glycans and the mechanism by which Tn, sTn, and T antigens promote tumor metastasis. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer is the most lethal gynecological malignancy due to its frequent recurrence and drug resistance even after successful initial treatment. Accumulating scientific evidence indicates that subpopulations of cancer cells with stem cell-like properties, such as so-called side population (SP) cells, are primarily responsible for these recurrences. A better understanding of SP cells may provide new clues for detecting and targeting these cancer-initiating cells and ultimately help to eradicate cancer. Changes in glycosylation patterns are remarkable features of SP cells. Here, we isolated SP cells from ovarian cancer cell lines and analyzed their glycosylation patterns using multiple glycomic strategies. Methods: Six high-grade serous ovarian cancer cell lines were used for SP cell isolation. Among them, HO8910 pm, which contained the highest proportion of SP cells, was used for glycomic analysis of SP cells. Cell lysate of SP cells and main population cells was applied to lectin microarray and mass spectrometry for glycan profiling. Differently expressed glycan structures were further verified by lectin blot, flow cytometry, and real-time PCR analysis of their relevant enzymes. Results: Expression of core fucosylated N-glycan and tumor-associated Tn, T and sT antigens were increased in SP cells. By contrast, SP cells exhibited decreased hybrid glycan, α2,3-linked sialic glycan and multivalent sialyl-glycan. Conclusions: Glycan structures, such as Tn, T, sT antigens, and core fucosylation may serve as biomarkers of ovarian cancer stem cells. [ABSTRACT FROM AUTHOR]

Human polyomaviruses are generally latent but can be reactivated in patients whose immune systems are suppressed. Unfortunately, current therapeutics for diseases associated with polyomaviruses are non-specific, have undefined mechanisms of action, or exacerbate the disease. We previously reported on a class of dihydropyrimidinones that specifically target a polyomavirus-encoded protein, T antigen, and/or inhibit a cellular chaperone, Hsp70, that is required for virus replication. To improve the antiviral activity of the existing class of compounds, we performed Biginelli and modified multi-component reactions to obtain new 3,4-dihydropyrimidin-2(1 H )-ones and -thiones for biological evaluation. We also compared how substituents at the N-1 versus N-3 position in the pyrimidine affect activity. We discovered that AMT580-043, a N-3 alkylated dihydropyrimidin-2(1 H )-thione, inhibits the replication of a disease-causing polyomavirus in cell culture more potently than an existing drug, cidofovir. [ABSTRACT FROM AUTHOR]

T antigen (Galβ1-3GalNAcα1-Ser/Thr), a core 1 mucin-type O -glycan structure, is synthesized by Drosophila core 1 β1,3-galactosyltrasferase 1 (dC1GalT1) and is expressed in various tissues. We previously reported that dC1GalT1 synthesizes T antigen expressed in hemocytes, lymph glands, and the central nervous system (CNS) and that dC1GalT1 mutant larvae display decreased numbers of circulating hemocytes and excessive differentiation of hematopoietic stem cells in lymph glands. dC1GalT1 mutant larvae have also been shown to have morphological defects in the CNS. However, the functions of T antigen in other tissues remain largely unknown. In this study, we found that glycans contributed to the localization of neuromuscular junction (NMJ) boutons. In dC1GalT1 mutant larvae, NMJs were ectopically formed in the cleft between muscles 6 and 7 and connected with these two muscles. dC1GalT1 synthesized T antigen, which was expressed at NMJs. In addition, we determined the function of mucin-type O -glycans in muscle cells. In dC1GalT1 mutant muscles, myofibers and basement membranes were disorganized. Moreover, ultrastructural defects in NMJs and accumulation of large endosome-like structures within both NMJ boutons and muscle cells were observed in dC1GalT1 mutants. Taken together, these results demonstrated that mucin-type O -glycans synthesized by dC1GalT1 were involved in the localization of NMJ boutons, synaptogenesis of NMJs, establishment of muscle cell architecture, and endocytosis. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSCs) are present in hematopoietic organs and differentiate into mature blood cells as required. Defective HSCs have been implicated in the human autoimmune disease Tn syndrome, which results from the failure of the core 1 β 1,3-galactosyltransferase 1 enzyme (C1β3GalT1) to synthesize T antigen. In both mice and humans, a reduced level of T antigen is associated with a reduction in blood cell numbers. However, the precise roles of T antigen in hematopoiesis are unknown. Here, we show that the Drosophila T antigen, supplied by plasmatocytes, is essential for the regulation of HSCs. T antigen appears to be an essential factor in maintaining the extracellular environment to support filopodial extensions from niches that are responsible for transmitting signaling molecules to maintain the HSCs. In addition, our results revealed that the clotting factor, hemolectin, disrupted the hemolymph environment of C1β3GalT1 mutants. This study identified a novel mucin function for the regulation of HSCs that may be conserved in other species. [ABSTRACT FROM AUTHOR]

Abstract: A series of STn-MUC1 and ST-MUC1 glycopeptides containing naturally occurring and non-natural sialic acids have been chemoenzymatically synthesized from Tn-MUC1 glycopeptide using one-pot multienzyme (OPME) approaches. In situ generation of the sialyltransferase donor cytidine 5′-monophosphate-sialic acid (CMP-Sia) using a CMP-sialic acid synthetase in the presence of an extra amount of cytidine 5′-triphosphate (CTP) and removal of CMP from the reaction mixture by flash C18 cartridge purification allow the complete consumption of Tn-MUC1 glycopeptide for quantitative synthesis of STn-MUC1. A Campylobacter jejuni β1–3GalT (CjCgtBΔ30-His6) mutant has been found to catalyze the transfer of one or more galactose residues to Tn-MUC1 for the synthesis of T-MUC1 and galactosylated T-MUC1. Sialylation of T-MUC1 using Pasteurella multocida α2–3-sialyltransferase 3 (PmST3) with Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) and Escherichia coli sialic acid aldolase in one pot produced ST-MUC1 efficiently. These glycopeptides are potential cancer vaccine candidates. [Copyright &y& Elsevier]

The Merkel cell polyomavirus (MCPyV), identified in humans in 2008, is associated with a relatively rare but aggressive neuroendocrine skin cancer, the Merkel cell carcinoma (MCC). MCC incidence is increasing due to the advancing age of the population, the increase in damaging sun exposure and in the number of immunocompromised individuals. MCPyV must be considered as the etiological agent of MCC and thus is the first example of a human oncogenic polyomavirus. MCPyV infection is common, and seroprevalence studies indicate that widespread exposure begins early in life. The majority of adults have anti-MCPyV antibodies and there is a growing body of evidence that healthy human skin harbors resident or transient MCPyV suggesting that MCPyV infection persists throughout life. However, the mode of transmission, the host cells, and the latency characteristics of this virus remain to be elucidated. In addition, it is still not clear whether MCPyV is associated with diseases or lesions other than Merkel cell carcinoma. The etiologic role of MCPyV in MCC opens up opportunities to improve the understanding of this cancer and to potentially improve its treatment. [ABSTRACT FROM AUTHOR]

Abstract: Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high mortality rate. The majority of MCC (70–80%) harbor clonally integrated Merkel cell polyomavirus (MCV) in the tumor genome and express viral T antigen oncoproteins. The characterization of an early passage MCV-positive MCC cell line MS-1 is described, and its cellular, immunohistochemical, and virological features to MCV-negative (UISO, MCC13, and MCC26) and MCV-positive cell lines (MKL-1 and MKL-2) were compared. The MS-1 cellular genome harbors integrated MCV, which preserves an identical viral sequence from its parental tumor. Neither VP2 gene transcripts nor VP1 protein are detectable in MS-1 or other MCV-positive MCC cell lines tested. Mapping of viral and cellular integration sites in MS-1 and MCC tumor samples demonstrates no consistent viral or cellular gene integration locus. All MCV-positive cell lines show cytokeratin 20 positivity and grow in suspension. When injected subcutaneously into NOD scid gamma (NSG) mice, MS-1 forms a discrete macroscopic tumor. Immunophenotypic analysis of the MS-1 cell line and xenografts in mice show identical profiles to the parental tumor biopsy. Hence, MS-1 is an early passage cell line that provides a useful in vitro model to characterize MCV-positive MCC. [Copyright &y& Elsevier]

The mechanism of SV40 DNA replication is certainly not completely understood. The proteins that are necessary for replication have been known for quite some time, but how they work together to form a nanomachine capable of faithfully replicating the virus DNA is only partially understood. Some of the proteins involved have been crystallized and their 3D structures determined, and several EM reconstructions of SV40 T antigen have been generated. In addition, there is a fair amount of biochemical data that pinpoints the sites of interaction between various proteins. With this information, various models were assembled that show how the SV40 DNA replication nanomachine could be structured in three dimensional space. This process was aided by the use of a 3D docking program as well as fitting of structures. The advantage of the availability of these models is that they are experimentally testable and they provide an insight into how the replication machine could work. Another advantage is that it is possible to quickly compare newly published structures to the models in order to come up with improved models. [ABSTRACT FROM AUTHOR]

Abstract: New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg’s ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection. [Copyright &y& Elsevier]

Abstract: Simian virus 40 (SV40) large T antigen (TAg) is a multifunctional oncoprotein essential for productive viral infection and for cellular transformation. We have used microarray analysis to examine the global changes in cellular gene expression induced by wild-type T antigen (TAgwt) and TAg-mutants in mouse embryo fibroblasts (MEFs). The expression profile of approximately 800 cellular genes was altered by TAgwt and a truncated TAg (TAgN136), including many genes that influence cell cycle, DNA-replication, transcription, chromatin structure and DNA repair. Unexpectedly, we found a significant number of immune response genes upregulated by TAgwt including many interferon-stimulated genes (ISGs) such as ISG56, OAS, Rsad2, Ifi27 and Mx1. Additionally, we also observed activation of STAT1 by TAgwt. Our genetic studies using several TAg-mutants reveal an unexplored function of TAg and indicate that the LXCXE motif and p53 binding are required for the upregulation of ISGs. [ABSTRACT FROM AUTHOR]

Abstract: BK virus (BKV) is a ubiquitous human pathogen that establishes a lifelong persistent infection in kidney epithelial cells. BKV reactivation within these cells results in a lytic infection in immunocompromised patients. Little is known about the specific interactions of BKV and the host cell during persistence and reactivation. We performed global cellular gene expression analyses using microarrays to characterize the global effect of BKV on primary kidney epithelial cells during the viral life cycle. Our results demonstrate that BKV primarily activates genes involved in cell cycle regulation and apoptosis (58% and 44% of upregulated genes at 48 and 72 h post-infection, respectively). Surprisingly, we observed that only four genes were downregulated during infection and that only two genes directly involved in the inflammatory response were differentially expressed. These results provide information about how BKV interacts with a cell type in which it both establishes persistence and undergoes lytic reactivation. [Copyright &y& Elsevier]

Abstract: BK virus (BKV) is a polyomavirus that ubiquitously infects the human population. Following a typically subclinical primary infection, BKV establishes a life-long persistent infection in the kidney and urinary tract. BKV is known to reactivate and cause severe disease in immunosuppressed patients, particularly renal and bone marrow transplant patients. Infection of BKV in rodent animal models or cells in culture often results in tumor formation or transformation, respectively. When co-expressed with activated oncogenes, BKV large tumor antigen drives the transformation of primary human cells. An etiological role of BKV in human cancer, however, remains controversial. Multiple reports have demonstrated conflicting results in regards to the presence of BKV sequences and/or proteins in various tumor types. This review compiles the most recent findings of BKV detection in a number of human cancers. Due to the lack of conclusive causality data from these studies, there does not appear to be a definitive association between BKV and human cancers. [Copyright &y& Elsevier]

Abstract: Simian Virus 40 (SV40) and Mouse Polyoma Virus (PY) are small DNA tumor viruses that have been used extensively to study cellular transformation. The SV40 early region encodes three tumor antigens, large T (LT), small T (ST) and 17KT that contribute to cellular transformation. While PY also encodes LT and ST, the unique middle T (MT) generates most of the transforming activity. SV40 LT mediated transformation requires binding to the tumor suppressor proteins Rb and p53 in the nucleus and ST binding to the protein phosphatase PP2A in the cytoplasm. SV40 LT also binds to several additional cellular proteins including p300, CBP, Cul7, IRS1, Bub1, Nbs1 and Fbxw7 that contribute to viral transformation. PY MT transformation is dependent on binding to PP2A and the Src family protein tyrosine kinases (PTK) and assembly of a signaling complex on cell membranes that leads to transformation in a manner similar to Her2/neu. Phosphorylation of MT tyrosine residues activates key signaling molecules including Shc/Grb2, PI3K and PLCγ1. The unique contributions of SV40 LT and ST and PY MT to cellular transformation have provided significant insights into our understanding of tumor suppressors, oncogenes and the process of oncogenesis. [Copyright &y& Elsevier]

Abstract: Duplication of the simian virus 40 (SV40) genome is the best understood eukaryotic DNA replication process to date. Like most prokaryotic genomes, the SV40 genome is a circular duplex DNA organized in a single replicon. This small viral genome, its association with host histones in nucleosomes, and its dependence on the host cell milieu for replication factors and precursors led to its adoption as a simple and powerful model. The steps in replication, the viral initiator, the host proteins, and their mechanisms of action were initially defined using a cell-free SV40 replication reaction. Although our understanding of the vastly more complex host replication fork is advancing, no eukaryotic replisome has yet been reconstituted and the SV40 paradigm remains a point of reference. This article reviews some of the milestones in the development of this paradigm and speculates on its potential utility to address unsolved questions in eukaryotic genome maintenance. [Copyright &y& Elsevier]

Abstract: microRNAs (miRNAs) are post-transcriptional regulators of gene expression that play a role in viral infection. We have developed a method to identify viral-encoded miRNAs from viruses in which abundant amounts of infected material is limiting. We show that Merkel Cell Polyomavirus (MCV), a recently identified human virus associated with cancer, encodes a miRNA. This miRNA is expressed from the late strand, lies antisense to the early transcripts and negatively regulates expression of chimeric reporters containing a portion of the early transcripts. Interestingly, different viral isolates have sequence polymorphisms in the pre-miRNA region that result in amino acids substitutions but fully preserve the processing and activity of the miRNAs. [Copyright &y& Elsevier]

Abstract: The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak but is stimulated by J domain-containing proteins. We identified pyrimidinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators. In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70. [Copyright &y& Elsevier]

Nephropathy associated with BK virus has emerged as an important cause of allograft failure in renal transplant recipients. Here we exploited a recently developed novel monocyte based solid phase T cell selection system, in which monocytes are immobilized on solid support, for antigen-specific T cell purification. The underlying hypothesis of this new method is that antigen-specific T cells recognize, bind their cognate antigens faster than non-specific T cells and are concentrated on the surface after removing the non-adherent cells by washing. Moreover, activated antigen-specific T cells proliferate more rapidly than non-specific T cells, further increasing the frequency and purity of antigen-specific T cells. Optimal selection times for BK virus-specific T cells are studied. Our data demonstrated that T cell selection can usually increase the frequency of antigen-specific T cells by >10 fold, whereas T cell expansion following the selection boost the frequency of antigen-specific T cells by another ~10 fold. This new T cell selection system is superior to traditional stimulation method (i.e. simply mixing antigen presenting cells and lymphocytes together) in generating antigen-specific T cells. This inexpensive and simple T cell selection system can produce large quantity of highly purified BK virus-specific T cells within 1-2 weeks after initial T cell activation. [ABSTRACT FROM AUTHOR]

JC virus-specifi c CD8+ cytotoxic T lymphocytes are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy. However, very few JC virus T cell epitopes restricted to MHC class I have been defined. Of the two HLA-A*0201-restricted JCV epitopes, VP1p36 and VP1p100, studies have shown that they are conserved T cell epitopes of polyomaviruses. The cross-recognition associated to these epitopes has complicated the efforts of understanding the dynamics of immune response to JC virus. Based on the previously identified HLA-A*0201 binding T cell epitope of Simian virus 40 T antigen P281-289 (KCDDVLLLL) and BK virus T antigen P558-566 (SLQNSEFLL), T cell epitopes of JC Virus T antigen P282-290 (KCEDVFLLM) and P557-565 (SLSCSEYLL) were identified. In this report, we demonstrated that JC Virus P282-290 and P557-565 were able to stimulate T cell responses in healthy donors' PBMCs and CD8+ cytotoxic T lymphocytes raised with both peptides could recognize and lyse their targets. Most importantly, there were no T cell cross-recognitions between JC Virus, BK Virus and SV40 virus. Therefore, JCV T-ag epitopes P282-290 and P557-565 could be better antigen epitopes compared to VP1p36 and VP1p100 to study the dynamics of cellular immune response to JCV in PML patients. In addition, as a HLA-A*0201 binding T cell epitope, both peptides could be a valuable component of immunotherapies aiming at increasing the cellular immune response against JCV for the treatment of progressive multifocal leukoencephalopathy. [ABSTRACT FROM AUTHOR]

Simian virus 40 large T antigen is a J-domain-containing protein with multiple functions. Among its numerous activities, T antigen can bind heat shock cognate 70 (hsc70) but the biological significance of this interaction has not been fully understood. Here, we show that T antigen can act as an hsc70 co-chaperone enhancing the protein-folding ability of the hsc70 chaperone machine. We also show that T antigen exerts its function in collaboration with the mammalian homologue of DnaJ. Moreover, we show that the participation of T antigen in the hsc70 chaperone machine has cell-type-specific characteristics. [ABSTRACT FROM AUTHOR]

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. The use of DNA-based plasmid vectors to achieve transient and stable expression of siRNA has been developed to avoid the problems of double-stranded oligonucleotides transfection. These vectors direct the transcription of small hairpin RNAs (shRNAs) from a polymerase-III (H1 or U6)-RNA gene promoter. However, numerous disadvantages remain, including low transfection efficiency and difficulty in transfecting primary cells. To overcome some of these problems, the use of viral vectors for siRNA delivery has been described. Retroviral, adenoviral, adeno-associated and herpes viral shRNAs delivery systems have been successfully used to silence genes, in vitro and in vivo. The use of a herpes simplex virus type 1 (HSV-1)-based amplicon vector for siRNA delivery into mammalian cells, using human polyomavirus BK (BKV)-transformed cells as a model system is described. The results demonstrate the ability of amplicon vectors to inhibit the expression of BKV T-Ag and tumorigenicity of BKV-transformed cells. We show that the use of the amplicon vector is highly efficient for the delivery of siRNA molecules. The unique ability of these vectors to deliver multiple copies of siRNA may provide a useful tool in the development of novel anticancer therapy.Gene Therapy (2007) 14, 459–464. doi:10.1038/sj.gt.3302878; published online 19 October 2006 [ABSTRACT FROM AUTHOR]

We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA. [ABSTRACT FROM AUTHOR]

Previous work has shown that the Simian Virus 40 T antigen (T antigen) cannot transform mouse embryo fibroblasts (MEFs) that do not express the type 1 insulin-like growth factor receptor (IGF-IR). We have now investigated the mechanism(s) by which the transforming activity of T antigen is affected by IGF-IR signaling. We demonstrate that transformation by T antigen of MEFs and several other cell lines requires an insulin receptor substrate-1 (IRS-1) phosphorylated on tyrosines. If IRS-1 is not expressed, or is serine phosphorylated or otherwise inactive, T antigen fails to transform cells in culture. For instance, while T antigen cannot transform 32D myeloid cells (that do not express IRS-1), its transforming activity is restored by the expression of a wild-type IRS-1, but not of an IRS-1 mutated at the PI3K binding sites. The importance of IRS-1 activation of PI3K in T-antigen transformation is supported by the finding that a constitutively activated p110 subunit of PI3K, a target of IRS-1, overcomes the inability of T antigen to transform MEFs with a serine phosphorylated IRS-1. Taken together, these results indicate that the IRS-1/PI3K signaling is one of the mechanisms regulating transformation by the SV40 T antigen. We propose that the requirement for a tyrosyl-phosphorylated IRS-1 provides a mechanism to explain the failure of T antigen to transform MEFs with deleted IGF-IR genes. [ABSTRACT FROM AUTHOR]

Natural O ‐glycoproteins such as the Thomsen‐Friedenreich antigen or gangliosides contain the motif Galβ1‐3GalNAc as an important disaccharide with significant biologic activity. The arrangement of spatial functionalities in this structure are of particular interest with regard to the development of potential leads en route to pharmaceuticals. Therefore, it was desired to obtain access to a range of modified derivatives of the aforementioned motif paying particular attention to introducing specific deoxy functions instead of hydroxyl groups. [ABSTRACT FROM AUTHOR]

Primary human renal proximal tubule epithelial cells (RPTECs) are of limited use for basic research and for clinical applications due to their limited lifespan in culture. Here we used two lentivirus vectors carrying the human telomerase (hTERT) and the SV40T antigen (Tag) flanked by loxP sites to reversibly immortalize RPTECs. Transduced RPTEC clones continued to proliferate while retaining biochemical and functional characteristics of primary cells. The clones exhibited contact-inhibited, anchorage- and growth factor-dependent growth and did not form tumors in nude mice, suggesting that the cells were not transformed. Transient Cre expression in these cells led to efficient proviral deletion, upregulation of some renal specific activities, and decreased growth rates. Ultimately, the cells underwent replicative senescence, indicating intact cell cycle control. Thus, reversible immortalization allows the expansion of human RPTECs, leading to large production of RPTECs that retain most tissue-specific properties.Oncogene (2004) 23, 5950-5957. doi:10.1038/sj.onc.1207801 Published online 21 June 2004 [ABSTRACT FROM AUTHOR]

Targeted tumorigenesis in transgenic mice has been a powerful tool for the study of gene expression and oncogenesis, as well as for the production of differentiated immortal cell lines from rare cell types. Follicle-stimulating hormone (FSH) is secreted by the gonadotrope cells of the anterior pituitary gland and plays a pivotal role in mammalian reproduction. Here we have used the regulatory region of the ovine FSHβ gene to direct expression of the SV40 T antigen oncogene to gonadotrope cells in the pituitary of transgenic mice. Two of five transgenic mouse lines bearing this fusion gene rapidly developed pituitary tumors, with appearance of adenomatous foci as early as 6 weeks of age, resulting in death by 12 weeks of age in both genders. Histologic examination of tumor development over time revealed that increases in cell proliferation and dysplasia were accompanied by decreases in synthesis of pituitary hormones, indicating dedifferentiation of the pituitary cells. Histological features observed in these tumors were in agreement with this rapid transformation of cell phenotype. Tumors were multifocal in origin, and the most highly transformed cell types observed consisted of giant pale basophilic cells with enormous hyperploid nuclei associated with infiltrating neuronal-like cells, which were very abundant at later stages of tumor development. Mitotic indices were much higher in transgenic than wild-type pituitaries, as expected. Morphologic analysis of the gonads of these transgenic mice showed no major developmental differences, as compared to wild-type littermates, however the length of the seminiferous tubules in transgenic males was greater than age-matched wild-type animals. Despite this phenotype difference, both genders were fertile, with normal sperm development observed in males and normal estrous cycle stages in females. Moreover, while 8–10-week-old transgenic males had much lower blood levels of FSH than littermates, transgenic female FSH levels were the same as those of wild-type females. These animals offer a unique and potentially useful model of organ-specific tumorigenesis, where a multistage pathway of tumor development is evident, both histologically and temporally. Study of such models will advance our knowledge on the physiological and molecular mechanisms involved in gene expression as well as tumor formation. [Copyright &y& Elsevier]

Medulloblastomas represent about 25% of all pediatric intracranial neoplasms. These highly malignant tumors arise from the cerebellum, affecting mainly children between ages 5 and 15. Although the etiology of medulloblastomas has not yet been elucidated, several reports suggest that both the cellular protein insulin-like growth factor I (IGF-I) and the early protein of the human polyomavirus JC (JCV T antigen) may contribute to the development of these tumors. The results of this study show a potential functional cooperation between these two proteins in the process of malignant transformation. Both medulloblastoma cell lines and medulloblastoma biopsies are characterized by the abundant presence of the IGF-I receptor (IGF-IR) and its major signaling molecule, insulin receptor substrate 1 (IRS-1). Importantly, IRS-1 is translocated to the nucleus in the presence of the JCV T antigen. Nuclear IRS-1 was detected in T antigen–positive cell lines and in T antigen–positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JCV T antigen binding was localized within the N-terminal portion of IRS-1 molecule and the competition for IRS-1 T antigen binding by a dominant-negative mutant of IRS-1 inhibited growth and survival of JCV T antigen-transformed cells in anchorage-independent culture condition. [ABSTRACT FROM AUTHOR]

Sialyl-Tn, Tn and T antigens are caused by aberrant or incomplete glycosylation of apomucins and are related to the aggressiveness of malignant neoplasms. Using 41 liver samples from patients with cholangiocarcinoma (including four with cirrhosis), 21 with combined hepatocellular– cholangiocellular carcinoma and 17 with hepatocellular carcinoma, the expression of sialyl-Tn, Tn and T antigens were characterized immunohistochemically and the correlation with apomucin profiles was evaluated. The prevalence of sialyl-Tn, Tn and T antigens expression was 89, 95 and 51% in cholangiocarcinoma without cirrhosis; 25, 75, and 0% in cholangiocarcinoma with cirrho- sis; 29, 90, and 48% in combined hepatocellular– cholangiocellular carcinoma; and 0, 12 and 6% in hepatocellular carcinoma, respectively. Sialyl-Tn antigen was frequently expressed in cholangiocarcinoma without cirrhosis compared with cholangiocarcinoma with cirrhosis and combined hepatocellular–cholangiocellular carcinoma (P < 0.01). Although sialyl-Tn expression was associated with MUC1, MUC6 and MUC7 expression, the expression sites among them were not identical in the individual cases. These data suggest that the different expressions of sialyl-Tn antigen among cholangiocarcinoma without cirrhosis, cholangiocarcinoma with cirrhosis and combined hepatocellular–cholangiocellular carcinoma may reflect the biological features inherent to these tumors, such as the ability of invasion. [ABSTRACT FROM AUTHOR]

The mechanism of IgA deposition in the kidneys in IgA nephropathy is unknown, Mesangial IgA is of the IgA 1 subclass, and since no consistent antigenic target for the IgA 1 has been described, we have investigated the glycosylation of the molecule, as a potential non-immunological abnormality which may contribute to its deposition. IgA 1 is rich in carbohydrate, carrying N-linked moieties in common with IgG, but also O-linked sugars, which are rare in serum proteins, and not expressed by IgG or IgA2. Lectin binding assays were designed to examine the expression of terminal galactose on the N-linked carbohydrate chains of purified serum IgG and IgA1, and the O-linked sugars of IgAI and Cl inhibitor (one of the very few other serum proteins with O-linked glycosylation). No evidence was found for abnormalities of N-linked glycosylation of either isotype in IgA nephropathy compared with matched controls. However, in IgA nephropathy, reduced terminal galactosylation of the hinge region O-linked moieties was demonstrated; this was not seen in Cl inhibitor, which showed normal or increased galactosylation of the O-linked sugars. This abnormality of IgA1 has considerable implications for the pathogenesis of IgA nephropathy, since the O-linked sugars lie in an important functional location within the IgA1 molecule, close to the ligand of Fc receptors. Changes in the carbohydrates in this site may therefore affect interactions with receptors and extracellular proteins, leading to anomalous handling of the IgA1 protein in this condition, including failure of normal clearance mechanisms, and mesangial deposition. [ABSTRACT FROM AUTHOR]

The article presents a case study of 144 hospitalized patients with cellulitis cases in Tokyo, Japan. It mentions that two of them got the disease from impetigo and ecthyma, while 14 of them had cellulitis on the lower extremities. It states that they undergone several medical examinations including T serotyping of group A streptococcus (GAS) and T-antigen serological typing and found that majority of patients with pharyngitis have cellulitis and nonbullous impetigo.

During polyomavirus (PyV) infection, host proteins localize to subnuclear domains, termed viral replication centers (VRCs), to mediate viral genome replication. Although the protein composition and spatial organization of VRCs have been described using high-resolution immunofluorescence microscopy, little is known about the temporal dynamics of VRC formation over the course of infection. We used live cell fluorescence microscopy to analyze VRC formation during murine PyV (MuPyV) infection of a mouse fibroblast cell line that constitutively expresses a GFP-tagged replication protein A complex subunit (GFP-RPA32). The RPA complex forms a heterotrimer (RPA70/32/14) that regulates cellular DNA replication and repair and is a known VRC component. We validated previous observations that GFP-RPA32 relocalized to sites of cellular DNA damage in uninfected cells and to VRCs in MuPyV-infected cells. We then used GFP-RPA32 as a marker of VRC formation and expansion during live cell microscopy of infected cells. VRC formation occurred at variable times post-infection, but the rate of VRC expansion was similar between cells. Additionally, we found that the early viral protein, small TAg (ST), was required for VRC expansion but not VRC formation, consistent with the role of ST in promoting efficient vDNA replication. These results demonstrate the dynamic nature of VRCs over the course of infection and establish an approach for analyzing viral replication in live cells. [ABSTRACT FROM AUTHOR]

• Inflammatory factors could affect the O-glycosylation of mucin. • NE promoted O-glycosylation of MUC5AC by the help of Cosmc. • Cosmc acted as a signal conducting factor in MUC5AC aberrant O-glycosylation stimulated by NE. • PI3K played an important role in the signaling pathway between NE and Cosmc, and subsequent O-glycosylation modification. Mucin 5AC (MUC5AC) is a highly O-glycosylated mucin secreted by human bronchial epithelial cells during pulmonary inflammatory diseases. T antigen, a component of the MUC5AC glycans, is the product of the O-glycosylation transferase T-synthase and its chaperone Cosmc. Since the expression of Cosmc is mediated by signaling pathways and inflammatory factors affecting mucin O-glycosylation, we analyzed the impact of neutrophil elastase (NE)-mediated Cosmc and T antigen expression in BEAS-2B cells derived from human bronchial epithelial cells. The expression of Cosmc and T antigen in human lung tissue was analyzed by immunohistochemistry. Cellular immunohistochemistry and western blot analysis demonstrated elevated expression of T antigen in BEAS-2B cells after NE stimulation. Altered Cosmc expression in BEAS-2B cells after NE stimulation was analyzed by confocal microscopy, western blot analysis and quantitative RT-PCR. To assess the biological implications of Cosmc function for T-synthase activity and T antigen synthesis after NE stimulation, BEAS-2B cells were transfected with shRNA to silence the expression of Cosmc. The changes in signaling pathways were analyzed by western blotting. The expression of Cosmc and T antigen increased in lung tissue exposed to chronic inflammation. The expression of Cosmc and T antigen increased in NE-stimulated BEAS-2B cells. NE induced increases in T antigen expression and T-synthase transferase activity in BEAS-2B cells expressing Cosmc, highlighting the importance of Cosmc in the relationship between NE and T antigen. Cosmc and phosphatidylinositol-3-kinase (PI3K) played important roles in the signaling pathway that stimulated hyperexpression of T antigen. [ABSTRACT FROM AUTHOR]

Merkel cell carcinoma is a rare, lethal cancer histopathologically composed of cells showing similarity with mechanoreceptor Merkel cells. Merkel cell tumors manifest in two distinct forms. While a virus called Merkel cell polyomavirus is involved in the pathogenesis of one form of Merkel tumors, the other is driven by ultraviolet (UV)-linked mutations. In this study we investigated 18 cases, from the Indian population, of Merkel cell carcinoma for immunohistochemical (IHC) expression of Merkel cell polyomavirus (MCV) T antigen, including 12 cases tested by PCR, to identify viral etiopathology. We tested the tumors with two sensitive antibodies (CM2B4 and Ab3), targeting the viral large T antigen protein and with PCR primers targeting the N terminus of T antigen. Overall, we observed 38.8% (7/18) tumors displaying positive IHC expression of Merkel cell polyomavirus T antigen and 25% (3/12) tumors showing positive results, by both, immunohistochemistry and PCR. This constitutes the first report from India showing implication of MCV in Merkel cell carcinomas. Moreover, this is one of the larger series of Merkel cell carcinomas, tested for MCV, by both immunohistochemistry and PCR, in this part of the world. These results further indicate that a slightly more number of such cases in India are likely to be caused by UV-linked damage, as opposed to Merkel cell polyomavirus mediated tumorigenesis, which is definitely implicated in a subset of cases. • We investigated 18 Merkel cell carcinoma cases, from the Indian population to identify viral etiopathology. • Overall, 38.8% (7/18) tumors displayed positive IHC expression for MCV T antigen using antibodies CM2B4 and Ab3. • Three of the twelve tumors, (25%) showed positive MCV presence, by both, immunohistochemistry and PCR. • Our study is the first report from India showing implication of Merkel cell polyomavirus (MCV) in Merkel cell carcinomas. [ABSTRACT FROM AUTHOR]

As the phylogenetic organization of mammalian polyomaviruses is complex and currently incompletely resolved, we aimed at a deeper insight into their evolution by identifying polyomaviruses in host orders and families that have either rarely or not been studied. Sixteen unknown and two known polyomaviruses were identified in animals that belong to 5 orders, 16 genera, and 16 species. From 11 novel polyomaviruses, full genomes could be determined. Splice sites were predicted for large and small T antigen (LTAg, STAg) coding sequences (CDS) and examined experimentally in transfected cell culture. In addition, splice sites of seven published polyomaviruses were analyzed. Based on these data, LTAg and STAg annotations were corrected for 10/86 and 74/86 published polyomaviruses, respectively. For 25 polyomaviruses, a spliced middle T CDS was observed or predicted. Splice sites that likely indicate expression of additional, alternative T antigens, were experimentally detected for six polyomaviruses. In contrast to all other mammalian polyomaviruses, three closely related cetartiodactyl polyomaviruses display two introns within their LTAg CDS. In addition, the VP2 of Glis glis (edible dormouse) polyomavirus 1 was observed to be encoded by a spliced transcript, a unique experimental finding within the Polyomaviridae family. Co-phylogenetic analyses based on LTAg CDS revealed a measurable signal of codivergence when considering all mammalian polyomaviruses, most likely driven by relatively recent codivergence events. Lineage duplication was the only other process whose influence on polyomavirus evolution was unambiguous. Finally, our analyses suggest that an update of the taxonomy of the family is required, including the creation of novel genera of mammalian and non-mammalian polyomaviruses. [ABSTRACT FROM AUTHOR]