Blast-related disinhibition and risk seeking in mice and combat Veterans: Potential role for dysfunctional phasic dopamine release.

Mild traumatic brain injury (mTBI) caused by exposure to high explosives has been called the "signature injury" of the wars in Iraq and Afghanistan. There is a wide array of chronic neurological and behavioral symptoms associated with blast-induced mTBI. However, the underlying mechanisms are not well understood. Here we used a battlefield-relevant mouse model of blast-induced mTBI and in vivo fast-scan cyclic voltammetry (FSCV) to investigate whether the mesolimbic dopamine system contributes to the mechanisms underlying blast-induced behavioral dysfunction. In mice, blast exposure increased novelty seeking, a behavior closely associated with disinhibition and risk for subsequent maladaptive behaviors. In keeping with this, we found that veterans with blast-related mTBI reported greater disinhibition and risk taking on the Frontal Systems Behavior Scale (FrSBe). In addition, in mice we report that blast exposure causes potentiation of evoked phasic dopamine release in the nucleus accumbens. Taken together these findings suggest that blast-induced changes in the dopaminergic system may mediate aspects of the complex array of behavioral dysfunctions reported in blast-exposed veterans.

Tracking the changes in virus taxonomy.

A database and website ( http://www.ictvonline.org/taxonomyReleases.asp ) have been established where the history of changes in virus taxonomy from 1971 to the present day can easily be traced. Each change is linked to a source document confirming the change or, for most changes since 2002, to the taxonomic proposal approved by the International Committee on Taxonomy of Viruses (ICTV).

An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.

T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.

The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein.

H-Y is a transplantation antigen that can lead to rejection of male organ and bone marrow grafts by female recipients, even if the donor and recipient match at the major histocompatibility locus of humans, the HLA (human leukocyte antigen) locus. However, the origin and function of H-Y antigens has eluded researchers for 40 years. One human H-Y antigen presented by HLA-B7 was identified as an 11-residue peptide derived from SMCY, an evolutionarily conserved protein encoded on the Y chromosome. The protein from the homologous gene on the X chromosome, SMCX, differs by two amino acid residues in the same region. The identification of H-Y may aid in transplantation prognosis, prenatal diagnosis, and fertilization strategies.

Multiplicity of N-terminal structures of medium-chain alcohol dehydrogenases. Mass-spectrometric analysis of plant, lower vertebrate and higher vertebrate class I, II, and III forms of the enzyme.

Ten different alcohol dehydrogenases, representing several classes of the enzyme and a wide spread of organisms, were analyzed for patterns of N-terminal structures utilizing a combination of conventional and mass spectrometric peptide analysis. Results show all forms to be N-terminally acetylated and allow comparisons of now 40 such alcohol dehydrogenases covering a large span of forms and origins. Patterns illustrate roles of acetylation in proteins in general, define special importance of the class I N-terminal acetylation, and distinguish separate acetylated structures for all classes, as well as a common alcohol dehydrogenase motif.

Space charge drift from a +/- 400-kV direct current transmission line.

The Minnesota Environmental Quality Board completed a 3-year study of the space charge plume outside the right of way of a bipolar (+/- 400 kV) direct current transmission line in rural west central Minnesota. The purpose of the study was to determine the levels of DC potential gradient and small airs ions in the downwind plume to which humans and animals may be exposed. Potential gradient measurements show that a space charge plume is detectable downwind at least 1600 m from the transmission line. Plume relaxation rates indicate that under certain conditions one could detect the plume at twice that distance or more. Net charge in the plume assumes the polarity of the downwind energized transmission line conductor. The bipolar DC line thus electrically bisects the land through which it passes; on one side of the line there is a net positive space charge, on the other side a net negative space charge. Electric charge in the plume resides on aerosols and small air ions. Polar conductivity data substantiate the fact that small air ions of one polarity in the plume are elevated while those of opposite polarity are suppressed compared to background concentrations found in the rural environment. The resulting charge imbalance persists downwind at least 1600 m, though the median small air ion concentrations of plume polarity have adjusted to within the background range by that time with perpendicular wind speed components between 1 and 6 m/sec.

Analysis of proteins and proteomes by mass spectrometry.

A decade after the discovery of electrospray and matrix-assisted laser desorption ionization (MALDI), methods that finally allowed gentle ionization of large biomolecules, mass spectrometry has become a powerful tool in protein analysis and the key technology in the emerging field of proteomics. The success of mass spectrometry is driven both by innovative instrumentation designs, especially those operating on the time-of-flight or ion-trapping principles, and by large-scale biochemical strategies, which use mass spectrometry to detect the isolated proteins. Any human protein can now be identified directly from genome databases on the basis of minimal data derived by mass spectrometry. As has already happened in genomics, increased automation of sample handling, analysis, and the interpretation of results will generate an avalanche of qualitative and quantitative proteomic data. Protein-protein interactions can be analyzed directly by precipitation of a tagged bait followed by mass spectrometric identification of its binding partners. By these and similar strategies, entire protein complexes, signaling pathways, and whole organelles are being characterized. Posttranslational modifications remain difficult to analyze but are starting to yield to generic strategies.

Substrate specificity of the protease that processes human interleukin-1 beta.

The substrate specificity of the protease which generates mature human interleukin-1 beta (IL-1 beta) from pro-interleukin-1 beta was investigated using synthetic peptide substrates and recombinant pro-IL-1 beta. The requirement of an L-aspartate in the P-1 position was confirmed together with the need for a small hydrophobic residue in the P-1' position (Gly or Ala). It was shown that the enzyme can tolerate conservative substitutions in the P-2 and P-2' positions. We found little difference in the enzyme's ability to cleave denatured and native pro-IL-1 beta, indicating that tertiary structure recognition is not involved in binding. The enzyme did, however, require a peptide of more than six amino acids for cleavage to occur. These results conclusively demonstrate the unusual specificity of this protease.

Modulation of c-Myb-induced transcription activation by a phosphorylation site near the negative regulatory domain.

The c-myb protooncogene encodes a highly conserved transcription factor that functions as both an activator and a repressor of transcription. The v-myb oncogenes of E26 leukemia virus and avian myeloblastosis virus encode proteins that are truncated at both the amino and the carboxyl terminus, deleting portions of the c-Myb DNA-binding and negative regulatory domains. This has led to speculation that the deleted regions contain important regulatory sequences. We previously reported that the 42-kDa mitogen-activated protein kinase (p42mapk) phosphorylates chicken and murine c-Myb at multiple sites in the negative regulatory domain in vitro, suggesting that phosphorylation might provide a mechanism to regulate c-Myb function. We now report that three tryptic phosphopeptides derived from in vitro phosphorylated c-Myb comigrate with three tryptic phosphopeptides derived from metabolically labeled c-Myb immunoprecipitated from murine erythroleukemia cells. At least two of these peptides are phosphorylated on serine-528. Replacement of serine-528 with alanine results in a 2- to 7-fold increase in the ability of c-Myb to transactivate a Myb-responsive promoter/reporter gene construct. These findings suggest that phosphorylation serves to regulate c-Myb activity and that loss of this phosphorylation site from the v-Myb proteins may contribute to their transforming potential.

Mass spectrometric identification of mtb81, a novel serological marker for tuberculosis.

We have used serological proteome analysis in conjunction with tandem mass spectrometry to identify and sequence a novel protein, Mtb81, which may be useful for the diagnosis of tuberculosis (TB), especially for patients coinfected with human immunodeficiency virus (HIV). Recombinant Mtb81 was tested by an enzyme-linked immunosorbent assay to detect antibodies in 25 of 27 TB patients (92%) seropositive for HIV as well as in 38 of 67 individuals (57%) who were TB positive alone. No reactivity was observed in 11 of 11 individuals (100%) who were HIV seropositive alone. In addition, neither sera from purified protein derivative (PPD)-negative (0 of 29) nor sera from healthy (0 of 45) blood donors tested positive with Mtb81. Only 2 of 57 of PPD-positive individuals tested positive with Mtb81. Sera from individuals with smear-positive TB and seropositive for HIV but who had tested negative for TB in the 38-kDa antigen immunodiagnostic assay were also tested for reactivity against Mtb81, as were sera from individuals with lung cancer and pneumonia. Mtb81 reacted with 26 of 37 HIV(+) TB(+) sera (70%) in this group, compared to 2 of 37 (5%) that reacted with the 38-kDa antigen. Together, these results demonstrate that Mtb81 may be a promising complementary antigen for the serodiagnosis of TB.

Mass-spectrometric evaluation of HLA-A*0201-associated peptides identifies dominant naturally processed forms of CTL epitopes from MART-1 and gp100.

Melanoma-reactive human cytotoxic T lymphocytes (CTLs) mediate tumor regression in vivo through specific recognition of MHC-associated peptide epitopes, many of which are encoded by the melanocytic tissue differentiation proteins gp100/Pme117 and MART-1/Melan-A. Vaccines using these peptides may induce protective or therapeutic immunity against melanoma. Rational design of such approaches is aided by a clear understanding of the identity of these antigenic peptides; however, most CTL epitopes described to date were identified indirectly. Especially where these peptides may be used in human clinical trials for the treatment or prevention of cancer, there is substantial need for direct evaluation of HLA-A*0201-associated peptides from MART-1 and gp100 that are naturally processed and presented. To that end, we have isolated peptides directly from HLA-A*0201 molecules of human melanoma cells and have determined that naturally processed epitopes for HLA-A*0201-restricted, melanoma-reactive CTLs include the nonamers MART-1(27-35) (AAGIGILTV), gp100(154-162) (KTWGQYWQV), gp100(209-217) (ITDQVPFSV) and gp100(280-288) (YLEPGPVTA) and the decamer gp100(476-485) (VLYRYGSFSV). Among these, the one that appears to be most abundant at the cell surface is gp100(154-162) (KTWGQYWQV). The others are among the less abundant peptides. HLA-A*0201-restricted CTLs from one melanoma patient who has survived metastatic disease recognized MART-1(27-35) (AAGIGILTV), gp100(280-288) (YLEPGPVTA) and gp100(154-162) (KTWGQYWQV) and were cross-reactive on longer peptides that contained these nonamer sequences. These peptides, identified by both an indirect genetic approach and by a direct peptide approach, can be used for tumor vaccine strategies with confidence that they are identical to the naturally processed peptide epitopes presented at the surface of melanoma cells in association with HLA-A*0201 molecules.

Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel-17/gp100.

Human CD8+ CTL recognize peptides bound to class I MHC molecules on the surface of melanoma cells. Several peptides derived from melanocyte lineage-specific proteins have been identified as epitopes for HLA-A2 restricted melanoma-reactive CTL. Because less than half of melanoma patients express HLA-A2, it is important to identify CTL epitopes restricted by other common MHC molecules including HLA-A1 and -A3. We have generated HLA-A3-restricted human CTL that recognize one or more shared melanoma Ags. All of the melanomas recognized by one of these CTL lines express Pmel-17/gp100, and those that fail to express this Ag are not lysed. This CTL line also specifically recognizes the lymphoblastoid line C1R-A3 following infection with a recombinant vaccinia encoding the melanocyte lineage-specific protein Pmel-17/gp100. Thus, at least one Pmel-17/ gp100 peptide is an epitope for this CTL line. We have identified ALLAVGATK (Pmel-17/gp100 residues 17-25) as an epitope for this CTL line and have shown that it is naturally processed and presented by HLA-A3 on melanoma cells. A second HLA-A3-restricted melanoma-reactive CTL line recognizes at least one additional shared epitope. These findings suggest that cellular immune responses directed against multiple shared melanoma epitopes exist in the 20 to 25% of melanoma patients who express HLA-A3. In addition, immunotherapy directed against Pmel-17/gp100 and other shared melanoma Ags may be useful in a large subset of these patients.

Detection of mammaglobin in the sera of patients with breast cancer.

Current procedures for the diagnosis of breast cancer are cumbersome and invasive, making detection of this disease difficult. A rapid screening test for early detection of breast cancer would allow for better management of this deadly disease. In this report, we show that, with the exception of the skin, mammaglobin mRNA is specifically expressed in mammary tissue and commonly overexpressed in breast cancer. Mammaglobin is not expressed in other types of cancer including colon, lung, ovarian, and prostate cancer. Breast-specific expression of mammaglobin protein was shown using immunohistochemical methods. Mammaglobin is secreted from both established breast cancer cell lines and primary breast carcinoma cells cultured in vitro. Using a monoclonal antibody-based assay for monitoring the presence of mammaglobin in serum, elevated levels of mammaglobin were detected in sera of patients with breast cancer, but not in healthy women. Thus, mammaglobin, which is overexpressed and secreted from breast carcinoma cells, is detectable in sera of patients with breast cancer and may provide a rapid screening test for the diagnosis and management of breast cancer.