Murine Sequences Research Articles

A shared MHC supertype motif emerges by convergent evolution in macaques and mice, but is totally absent in human MHC molecules

The SIV-infected rhesus macaque (Macaca mulatta) is the most established model of AIDS disease systems, providing insight into pathogenesis and a model system for testing novel vaccines. The understanding of cellular immune responses based on the identification and study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide-binding motif, provides valuable information to decipher outcomes of infection and vaccine efficacy. Detailed characterization of Mamu-B*039:01, a common allele expressed in Chinese rhesus macaques, revealed a unique MHC:peptide-binding preference consisting of glycine at the second position. Peptides containing a glycine at the second position were shown to be antigenic from animals positive for Mamu-B*039:01. A similar motif was previously described for the Dd mouse MHC allele, but for none of the human HLA molecules for which a motif is known. Further investigation showed that one additional macaque allele, present in Indian rhesus macaques, Mamu-B*052:01, shares this same motif. These “G2” alleles were associated with the presence of specific residues in their B pocket. This pocket structure was found in 6% of macaque sequences but none of 950 human HLA class I alleles. Evolutionary studies using the “G2” alleles points to common ancestry for the macaque sequences, while convergent evolution is suggested when murine and macaque sequences are considered. This is the first detailed characterization of the pocket residues yielding this specific motif in nonhuman primates and mice, revealing a new supertype motif not present in humans.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-011-0598-5) contains supplementary material, which is available to authorized users.

Lack of the Detection of XMRV or Polytropic MLV-Related Sequences in Blood Cells From HIV-1–Infected Patients in Spain

Xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (MLV)-related virus are recently described human gammaretroviruses that have been associated with prostate cancer and chronic fatigue syndrome. These studies have been controversial because a number of laboratories have been unable to find evidence of XMRV in similar groups of patients or controls. Because the existence of XMRV raises many questions, we decided to study its presence in a group of patients infected with HIV-1 with a high proportion of intravenous drug use and coinfection by hepatitis C virus. Forty HIV-1-infected patients under follow-up in our institution were screened for XMRV/MLV by nested polymerase chain reaction using primers targeting the gag and env region. Specific primers for mouse mitochondrial DNA were used to rule out contamination. No evidence of XMRV or polytropic MLV-related sequences was found in any sample from patients or controls. Four samples yielded polymerase chain reaction bands whose sequence corresponded to murine endogenous retroviral sequences, however, contamination with mouse cell DNA was subsequently confirmed. XMRV/MLV viruses do not seem to be associated with HIV-1 infection or intravenous drug use. Contamination of samples or reagents by genomic murine DNA or XMRV vectors could account for the sporadic detection of positive samples for XMRV and related agents.

Evaluation of Residual Promoter Activity in γ-Retroviral Self-inactivating (SIN) Vectors

Therapeutic gene delivery mediated by retroviral vectors has the advantage of stable integration into the host genome. A major safety concern for gene delivery achieved by murine leukemia virus (MLV)-based retroviral vectors is the activation of adjacent cellular genes including oncogenes following integration into the host genome. Self-inactivating (SIN) vectors lacking viral enhancers/promoters in their 3' long terminal repeat (LTR) have been proposed as a means of overcoming this safety concern. However the MLV-based SIN vectors currently used by laboratories to assess insertional mutagenesis, integration site selection, and the potency of transgene expression are not uniform in the composition of their 3' LTRs. We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the messenger RNA (mRNA) level and protein level. We found that viral promoter activity in the 3' LTR is not attenuated in many currently employed SIN vectors. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research.

Phase II study on combination therapy with CHOP-Zenapax for HTLV-I associated adult T-cell leukaemia/lymphoma (ATLL)

Adult T-cell leukaemia lymphoma (ATLL) is an aggressive T-cell malignancy caused by the human T-lymphotropic virus type-1 (HTLV-1) and is associated with a very poor prognosis. Combination chemotherapy has had little impact on the long term survival of these patients. ATLL cells are characterised by the expression of CD25 (IL-2Rα), which is not expressed in normal resting T-cells. Daclizumab (Zenapax®) is a humanised murine anti-CD25 monoclonal antibody, which contains 10% murine CDR sequences. In this prospective trial 15 patients with aggressive ATLL were treated with CHOP-Zenapax (CHOP-Z) to determine the tolerability and feasibility of this novel regimen as well as evaluate its efficacy. Eleven patients had acute ATLL and four had the lymphoma subtype. The main presenting features were elevated LDH (100%), lymphocytosis (73%), lymphadenopathy (67%), skin lesions (40%), hypercalcaemia (53%), and hepato-splenomegaly (27%). Ten (67%) patients received the six scheduled cycles. Complete response (CR) lasting for two months or more was seen in 5 (33%), partial response in 3 (20%), minor response in 1 (7%), and no response in 6 (40%) patients. The median overall survival was 10 months (95% CI: 0.05–20.88) but this was significantly longer among responders (18 months) compared to non responders (3months) (P=0.019). For patients who achieved CR the disease free survival (DFS) was 15 months while the event-free survival (EFS) was 5 months. In conclusion CHOP-Z is safe and in those who achieve a complete response it was associated with prolonged overall survival.

Proline substitutions and threonine pseudophosphorylation of the SH3 ligand of 18.5-kDa myelin basic protein decrease its affinity for the Fyn-SH3 domain and alter process development and protein localization in oligodendrocytes.

The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92-99 (murine sequence -T(92)PRTPPPS(99)-) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP's SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca(2+) influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein-protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP's SH3 ligand domain. These results suggest that MBP's SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function.

DNA Extraction Columns Contaminated with Murine Sequences

Sequences of the novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) have been described in human prostate cancer tissue, although the amounts of DNA are low. Furthermore, XMRV sequences and polytropic (p) murine leukemia viruses (MLVs) have been reported in patients with chronic fatigue syndrome (CFS). In assessing the prevalence of XMRV in prostate cancer tissue samples we discovered that eluates from naïve DNA purification columns, when subjected to PCR with primers designed to detect genomic mouse DNA contamination, occasionally gave rise to amplification products. Further PCR analysis, using primers to detect XMRV, revealed sequences derived from XMRV and pMLVs from mouse and human DNA and DNA of unspecified origin. Thus, DNA purification columns can present problems when used to detect minute amounts of DNA targets by highly sensitive amplification techniques.

Phylogenetic analysis of murine leukemia virus sequences from longitudinally sampled chronic fatigue syndrome patients suggests PCR contamination rather than viral evolution.

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) has been amplified from human prostate cancer and chronic fatigue syndrome (CFS) patient samples. Other studies failed to replicate these findings and suggested PCR contamination with a prostate cancer cell line, 22Rv1, as a likely source. MLV-like sequences have also been detected in CFS patients in longitudinal samples 15 years apart. Here, we tested whether sequence data from these samples are consistent with viral evolution. Our phylogenetic analyses strongly reject a model of within-patient evolution and demonstrate that the sequences from the first and second time points represent distinct endogenous murine retroviruses, suggesting contamination.

A Concerted HIF-1α/MT1-MMP Signalling Axis Regulates the Expression of the 3BP2 Adaptor Protein in Hypoxic Mesenchymal Stromal Cells

Increased plasticity, migratory and immunosuppressive abilities characterize mesenchymal stromal cells (MSC) which enable them to be active participants in the development of hypoxic solid tumours. Our understanding of the oncogenic adaptation of MSC to hypoxia however lacks the identification and characterization of specific biomarkers. In this study, we assessed the hypoxic regulation of 3BP2/SH3BP2 (Abl SH3-binding protein 2), an immune response adaptor/scaffold protein which regulates leukocyte differentiation and motility. Gene silencing of 3BP2 abrogated MSC migration in response to hypoxic cues and generation of MSC stably expressing the transcription factor hypoxia inducible factor 1alpha (HIF-1α) resulted in increased endogenous 3BP2 expression as well as cell migration. Analysis of the 3BP2 promoter sequence revealed only one potential HIF-1α binding site within the human but none in the murine sequence. An alternate early signalling cascade that regulated 3BP2 expression was found to involve membrane type-1 matrix metalloproteinase (MT1-MMP) transcriptional regulation which gene silencing abrogated 3BP2 expression in response to hypoxia. Collectively, we provide evidence for a concerted HIF-1α/MT1-MMP signalling axis that explains the induction of adaptor protein 3BP2 and which may link protein binding partners together and stimulate oncogenic MSC migration. These mechanistic observations support the potential for malignant transformation of MSC within hypoxic tumour stroma and may contribute to evasion of the immune system by a tumour.

PCR Master Mixes Harbour Murine DNA Sequences. Caveat Emptor!

BackgroundXMRV is the most recently described retrovirus to be found in Man, firstly in patients with prostate cancer (PC) and secondly in 67% of patients with chronic fatigue syndrome (CFS) and 3.7% of controls. Both disease associations remain contentious. Indeed, a recent publication has concluded that “XMRV is unlikely to be a human pathogen”. Subsequently related but different polytropic MLV (pMLV) sequences were also reported from the blood of 86.5% of patients with CFS. and 6.8% of controls. Consequently we decided to investigate blood donors for evidence of XMRV/pMLV.Methodology/Principal FindingsTesting of cDNA prepared from the whole blood of 80 random blood donors, generated gag PCR signals from two samples (7C and 9C). These had previously tested negative for XMRV by two other PCR based techniques. To test whether the PCR mix was the source of these sequences 88 replicates of water were amplified using Invitrogen Platinum Taq (IPT) and Applied Biosystems Taq Gold LD (ABTG). Four gag sequences (2D, 3F, 7H, 12C) were generated with the IPT, a further sequence (12D) by ABTG re-amplification of an IPT first round product. Sequence comparisons revealed remarkable similarities between these sequences, endogeous MLVs and the pMLV sequences reported in patients with CFS.Conclusions/SignificanceMethodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent. It is suggested that such contamination is likely to explain the apparent presence of pMLV in CFS.

XMRV: Not a Mousy Virus

The newly discovered mouse-derived human retrovirus, xenotropic murine leukemia virusrelated virus (XMRV), is currently fuelling both the scientific and public debate, where skeptics suggest that results are based on laboratory contamination, while the “believers” have already dressed the virus with highly pathogenic potential. Could the truth lie somewhere in the middle? XMRV was discovered in 2006 in tumor tissue from patients with prostate cancer,1 with a viral genome sequence highly similar to that of mouse xenotropic retroviruses. Sequence analysis suggested that XMRV is a novel recombinant derived from two fragmented endogenous murine viruses integrated in the mouse genome. XMRV was subsequently detected in other prostate cancer tissues and in blood from patients with CFS (chronic fatigue syndrome). However, most other studies failed to replicate these findings, especially outside the USA, suggesting either that the virus has a limited geographical spread, or that positive results were due to contamination of biological reagents or human samples with mouse DNA. Four recent papers indeed show that murine DNA sequences can be detected virtually everywhere,2−5 and that extreme care should be taken when amplifying XMRV sequences. These results certainly put into serious doubt some of the high prevalence results and proposed disease associations that could not be confirmed by others. However, these recent studies do not imply, and in fact did not intend to prove, that all positive results reported thus far are due to contamination, and that XMRV in humans can be dismissed as an artefact. The most important remaining question is: does XMRV infect and replicate in humans, in other words is it a genuine human virus apart from being an easy and frequent contaminant? The relevant subsequent questions would be: “how did XMRV end up in humans” and “is XMRV infection associated with disease”? It is of utmost importance to resolve these questions in a timely manner, as some CFS patients have begun taking antiretroviral drugs that can have side effects. Based on the original XMRV studies, it has also been proposed that CFS patients be banned from donating blood. The best evidence for XMRV replicating in human cells is the detection of proviral integrations flanked by human genome sequences in prostate tissue from eleven patients.6,7 The flanks are human and not mouse sequences, and the insertion sites differ for each clinical sample, thus ruling out a mouse-contamination and

Classical Mus musculus Igκ Enhancers Support Transcription but not High Level Somatic Hypermutation from a V-Lambda Promoter in Chicken DT40 Cells

Somatic hypermutation (SHM) of immunoglobulin genes is initiated by activation-induced cytidine deaminase (AID) in activated B cells. This process is strictly dependent on transcription. Hence, cis-acting transcriptional control elements have been proposed to target SHM to immunoglobulin loci. The Mus musculus Igκ locus is regulated by the intronic enhancer (iE/MAR) and the 3′ enhancer (3′E), and multiple studies using transgenic and knock-out approaches in mice and cell lines have reported somewhat contradictory results about the function of these enhancers in AID-mediated sequence diversification. Here we show that the M. musculus iE/MAR and 3′E elements are active solely as transcriptional enhancer when placed in the context of the IGL locus in Gallus gallus DT40 cells, but they are very inefficient in targeting AID-mediated mutation events to this locus. This suggests that either key components of the cis-regulatory targeting elements reside outside the murine Igκ transcriptional enhancer sequences, or that the targeting of AID activity to Ig loci occurs by largely species-specific mechanisms.

Abstract LB-155: A new syngeneic MYCN-overexpressing neuroblastoma mouse model and MYCN-DNA vaccine

Abstract High-level expression of MycN caused by amplification of the gene characterizes the malignant phenotype of neuroblastoma. Recent studies suggest that MYCN is a suitable target for immunotherapy, but up to now, a syngeneic NB mouse model over expressing MYCN is not available to examine immunotherapeutic strategies in vivo. Here, we report the development of a tetracycline inducible MYCN expressing murine NB cell line syngeneic to A/J mice and a MYCN-DNA vaccine. For this purpose, the murine NB cell line C1300, syngeneic to A/J mice, was stably transfected with a tetracycline inducible MYCN expression vector. Stable transfection was verified by real-time PCR and Western-Blot, revealing high expression levels of MYCN RNA and protein, respectively. Furthermore, a MYCN-DNA vaccine, based on epitopes encoding for three peptides from the murine MYCN protein sequence with high affinity to the A/J mouse MHC class I allele H2-Kk, was designed and tested in vivo for its ability to induce an antigen-specific immune response. Lymphocytes isolated from A/J mice vaccinated with the MYCN vaccine effectively lysed C1300-MYCN +tet cells in cytotoxicity assays in contrast to wild type C1300 cells and lymphocytes from control mice. Lymphocytes from minigene vaccinated mice produced significantly higher amounts of IFN-γ after stimulation with irradiated C1300 cells than lymphocytes from control mice. Interestingly, vaccine induced cytotoxic T lymphocytes also kill parental C1300 tumor cells which show low MYCN expression. We expect that this effect will be enhanced when MYCN-over expressing C1300 cells are employed in a similar cytotoxicity assay which will be reported at the meeting. In summary, we report the development of a new MYCN-DNA vaccine and a murine tetracycline inducible MYCN NB cell line providing an useful syngeneic mouse model for in vivo evaluation of MYCN directed immunotherapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-155. doi:10.1158/1538-7445.AM2011-LB-155

Characterization of a Perilipin‐5 Splice Variant

Perilipin‐5, the newest member of the perilipin family of lipid droplet proteins, has been found to be expressed in oxidative tissues, specifically heart, brown adipose tissue, and fasted liver. This work seeks to characterize a novel splice variant for perilipin‐5 we have termed Perilipin‐5B. Western blots indicate that the protein, of approximately 35 kDa, is present in C2C12 cultured myoblasts and in heart and liver tissue from fed and fasted mice. Highest levels of the protein were present in heart tissue from fed mice. Analysis of murine genomic DNA sequences reveals stop codons found in the introns between exons 7 and 8 and 8 and 9, either of which could give rise to a protein of approximately 35 kDa. Other members of the PAT family, such as Perilipin‐1, have multiple protein products arising from splice variants. The function of perilipin‐5B is yet unknown, but the interesting observation that it is found primarily in fed heart tissue suggests that it plays a role in the oxidation of the lipid droplet. Knowledge of this protein's function will give a more complete picture as to the overall mechanism of lipid storage and metabolism.

Intratumoral IL-12 Gene Therapy Results in the Crosspriming of Tc1 Cells Reactive Against Tumor-associated Stromal Antigens

HLA-A2 transgenic mice bearing established HLA-A2(neg) B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8(+) T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8(+) T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8(+) T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8(+) T cells directly recognized HLA-A2(+) pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8(+) T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8(+) T cells harvested from the blood of HLA-A2(+) normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2(+) cancer patients receiving therapeutic interventions, such as IL-12 gene therapy.

Structural Analysis of a Repetitive Protein Sequence Motif in Strepsirrhine Primate Amelogenin

Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL), the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates.

Overexpression of Glutaminyl Cyclase, the Enzyme Responsible for Pyroglutamate Aβ Formation, Induces Behavioral Deficits, and Glutaminyl Cyclase Knock-out Rescues the Behavioral Phenotype in 5XFAD Mice

Pyroglutamate-modified Aβ (AβpE3-42) peptides are gaining considerable attention as potential key players in the pathology of Alzheimer disease (AD) due to their abundance in AD brain, high aggregation propensity, stability, and cellular toxicity. Overexpressing AβpE3-42 induced a severe neuron loss and neurological phenotype in TBA2 mice. In vitro and in vivo experiments have recently proven that the enzyme glutaminyl cyclase (QC) catalyzes the formation of AβpE3-42. The aim of the present work was to analyze the role of QC in an AD mouse model with abundant AβpE3-42 formation. 5XFAD mice were crossed with transgenic mice expressing human QC (hQC) under the control of the Thy1 promoter. 5XFAD/hQC bigenic mice showed significant elevation in TBS, SDS, and formic acid-soluble AβpE3-42 peptides and aggregation in plaques. In 6-month-old 5XFAD/hQC mice, a significant motor and working memory impairment developed compared with 5XFAD. The contribution of endogenous QC was studied by generating 5XFAD/QC-KO mice (mouse QC knock-out). 5XFAD/QC-KO mice showed a significant rescue of the wild-type mice behavioral phenotype, demonstrating the important contribution of endogenous mouse QC and transgenic overexpressed QC. These data clearly demonstrate that QC is crucial for modulating AβpE3-42 levels in vivo and prove on a genetic base the concept that reduction of QC activity is a promising new therapeutic approach for AD.

Modes of SH3-Domain Interactions of 18.5 kDa Myelin Basic Protein IN Vitro and in Oligodendrocytes

The developmentally-regulated myelin basic proteins (MBPs) play key roles in central nervous system (CNS) myelin formation by oligodendrocytes. They are highly positively-charged, intrinsically disordered, multifunctional proteins having several alternatively-spliced isoforms and combinatorial post-translational modifications. The most common 18.5kDa MBP isoform contains a proline-rich region (murine sequence T92PRTPPPS99) which comprises a minimal SH3-ligand. We have previously shown that 18.5 kDa MBP binds to Fyn, a member of the Src family of tyrosine kinases involved in signaling pathways during CNS development. Here, we have produced an isotopically-labeled fragment of MBP which contains the proline-rich region, and are studying the mode of interaction between MBP and the SH3 domain of Fyn through solution NMR spectroscopy and isothermal titration calorimetry (ITC). We have constructed MBP variants pseudo-phosphorylated at T92 and T95, and with P93G and P96G substitutions to disrupt the conformation of the SH3-ligand. Using ITC, these mutant MBPs are shown to have a decreased affinity towards the SH3-domain of Fyn. Solution NMR spectroscopy shows altered chemical shift patterns for the different variants, confirming that this segment is indeed the SH3-target and that the association can be modulated by phosphorylation. Concomitant over-expression of complementary GFP-tagged MBP forms in cultured oligodendrocytes results in aberrant elongation of membrane processes, increased branching complexity, and in some cases, trafficking of MBP to the nucleus. These data as a whole indicate that MBP's SH3-ligand domain plays a key role in intracellular protein interactions and may be required for proper membrane elaboration in myelin.Acknowledgements: Funded by the Canadian Institutes for Health Research (JMB, GH), NSERC Canada (GH), and by Doctoral Studentships from the Multiple Sclerosis Society of Canada (MDA, GSTS).

Xenotropic and Other Murine Leukemia Virus-Related Viruses in Humans

The recent discovery of a xenotropic murine leukemia virus-related retrovirus (designated as XMRV) in prostate cancer tissues and later in chronic fatigue syndrome (CFS) patients created excitement related to possible association of a virus with these human diseases. However, the failure to reproduce such results in other laboratories raised concerns and much debate among scientists, patient populations, and clinicians over the original findings. This special issue is a collection of recent research reports, that address this controversy. It also includes several expert reviews on various aspects of murine retroviruses, such as virus biology, replication, phylogeny, and pathogenesis, as well as XMRV in prostate cancer. The first paper by A. Rein reviews the murine retrovirus genomic structure, viral structural proteins, and virus replication. The second paper by C. A. Kozak provides a detailed review of murine retrovirus entry into the cell and different receptor usage by different types of murine retroviruses with comparison to XMRV. The third paper by J. Blomberg et al. discusses the phylogenetic analysis of murine retroviruses and other retroviruses. The fourth paper by J. Chakraborty et al. discusses murine retrovirus pathogenesis and a mouse model for transmission of lymphoma by breast milk. The fifth paper by D. E. Kang et al. reviews the discovery, progress, and current status of XMRV findings in prostate cancer patients. The sixth paper by S. Tang and I. K. Hewlett reviews XMRV detection assays and deficiencies in the testing methods. The seventh paper by J. M. Coffin and O. Cingoz provides a detailed review of the controversies related to the XMRV results in human clinical samples and the findings regarding virus origin and discusses the potential sources of contamination that resulted in the misidentification of the virus as a novel human retrovirus. Additionally, the special issue contains various research papers demonstrating the absence of XMRV in various patient populations using sensitive assays for virus detection. The eighth paper by B. Oakes et al. reports the absence of antibodies in CFS patients and healthy controls using two novel sensitive immunoassays. The ninth paper by J. Spindler et al. reports the lack of evidence of XMRV infection in HIV-1 infected men or men at high risk for HIV-1 infection by analyzing PBMCs and plasma samples using sensitive PCR assays and immunoassays. The tenth paper by K. A. Delviks-Frankenberry et al. demonstrates the absence of XMRV in PBMCs and plasma from HIV-1 lymphoma patients using PCR or immunoassays. The eleventh paper by M. J. Robinson et al. indicates the absence of XMRV sequences in prostate cancer samples from diverse populations, B-cell lymphoma patients, as well as UK blood donors. The twelfth paper by M. Kearney et al. reports the use of different methodologies to demonstrate the absence of XMRV in plasma and in some tissue samples from prostate cancer patients. The final, thirteenth paper by P. Sharma et al. describes XMRV infection in the reproductive tissues of rhesus monkeys, proposing the possibility of an animal model for further investigations of virus transmission. This special issue provides the recent thinking and research results of studies on XMRV and other murine leukemia retrovirus-related sequences in humans. Numerous studies have failed to confirm the presence of XMRV in humans, and XMRV has recently been found to be a laboratory-derived rare recombinant, which originated by xenografting a patient's prostate cancer cells in nude mice. A consensus has now emerged that XMRV footprints or infectious XMRV detected in normal human individuals or in some diseased patients represents laboratory contaminations. The information provided in this issue should be of interest to a broad audience including scientists, clinicians, patient populations, and public health agencies. Arifa S. Khan Myra McClure Yoshinao Kubo Paul Jolicoeur

Genomic organization and localization of the NAD-dependent histone deacetylase gene sirtuin 3 (Sirt3) in the mouse

Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which belongs to the Silent information regulator 2 (Sir2) family of histone deacetylases (sirtuin HDACs). The yeast Sir2 protein and its mammalian derivatives play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. We have isolated and characterized the murine Sirt3 genomic sequence, which spans a region of 18,646 bp and which has one single genomic locus. Determination of the exon-intron splice junctions identified murine SIRT3 to be encoded by 7 exons ranging in size from 101 (exon 4) to 420 bp (exon 7). Characterization of the 5' flanking genomic region, which precedes the murine Sirt3 open reading frame, revealed a number of STATx, GATA and SP1 transcription factor binding sites. A CpG island was not detected. The 1,473-bp murine Sirt3 transcript has an open reading frame of 774 bp and encodes a 257-aa protein (cytoplasmic SIRT3) with a predictive molecular weight of 28.8 kDa and an isoelectric point of 5.82. Recently, a 1,406-bp murine SIRT3 splice variant that encodes a 334-aa mitochondrial precursor protein with a molecular weight of 36.6 kDa and an isoelectric point of 7.19 has been described. Fluorescence in situ hybridization analysis identified a single genomic locus for murine Sirt3 gene on chromosome 7F4 and which is neighbored by the Ric8 and PSMD13 genes. Our study brings light and a number of corrections and additions to previous reports on the genomic organization and the genomic sequence of murine Sirt3, which may be of importance in view of studies on potential genetic polymorphisms in relation to cellular respiration, metabolism, aging-related disease and cancer.

Conformational choreography of a molecular switch region in myelin basic protein—Molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments

The 18.5kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5kDa myelin basic protein (residues Glu80–Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80–Val91 and extended poly-proline type II from Thr92–Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.