Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans.

Engagement of the T cell antigen receptor (TCR)1 results in the sequential activation of the Src (p56 lck /p59 fyn ) and Syk (Syk/ZAP-70) families of protein-tyrosine kinases (PTKs) (1-3). Both families of PTKs are required for normal T cell development and function (4 -9). In resting T cells, the TCR chain is constitutively phosphorylated and associated with 11). Tyrosine phosphorylation of the receptor-associated ZAP-70 by p56 lck is a requisite modification resulting in the up-regulation of ZAP-70 catalytic activity (12, 13). Therefore, the recruitment of the CD4 and CD8 co-receptors into the TCR complex positions p56 lck to trans-phosphorylate ZAP-70. Phosphorylation and activation of ZAP-70, in turn, is required for both an increase in phosphoinositide metabolism and activation of the ras pathway.2 The integration of these downstream signals gives rise to transcriptional activation of cytokine genes and a resultant elevation in cytokine synthesis and secretion (15). While the up-regulation of ZAP-70 catalytic activity is required for TCR function, little is known about the cellular proteins which serve as substrates for this PTK.SLP-76 is a recently identified molecule which undergoes tyrosine phosphorylation upon TCR cross-linking (16 -19). This protein associates with both the SH3 domain of Grb2 and an SH2 domain of PLC␥. SLP-76 is structurally characterized by a C-terminal SH2 domain, a region enriched in proline residues which probably serves as the site for Grb2 binding, and an N-terminal motif which contains three tandemly repeated DYE(S/P)P sequences. We demonstrate here that SLP-76 is phosphorylated by ZAP-70 and that phosphorylation of these repeated tyrosine motifs is required for optimal generation of IL-2 in response to TCR ligation. In addition, overexpression of SLP-76 augments TCR-mediated transcriptional activation of the IL-2 gene, while mutation of the SH2 domain attenuates this response. Together, these studies identify SLP-76 as a physiologic substrate for ZAP-70 and suggest a mechanism by which TCR-induced activation of ZAP-70 regulates both the calcium and ras pathways. EXPERIMENTAL PROCEDURESCells and Antibodies-Jurkat and Sf9 cells (Pharmingen) were maintained as described previously (12). The mouse monoclonal antibody (mAb) H3 was generated against the SLP-76 SH2 domain. SLP-76 polyclonal antisera (22652) was generated against a peptide spanning amino acids 301-318 of human SLP-76. C305 is an anti-Jurkat Ti␣/␤-mAb (20); 4G10 (UBI) and PY20 (Santa Cruz) are anti-phosphotyrosine mAbs; 2F3.2 is an anti-ZAP-70 mAb (UBI), and 9E10 is an anti-myc mAb. Anti-GST mAb was purchased from Santa Cruz.Construction of Plasmids-A full-length SLP-76 cDNA was generated by reverse transcription PCR from murine T-cell mRNA. This cDNA was appended with a myc-epitope at the 3Ј end of the coding cDNA. Mutations were generated by PCR-directed mutagenesis and confirmed using standard dideoxy sequencing methods. These constructs were then subcloned into the pApuro vector (21) and the baculoviral vector pVL13...

The threshold at which antigen triggers lymphocyte activation is set by the enzymes that regulate tyrosine phosphorylation. Upon T cell activation, the protein tyrosine phosphatase SHP-1 was found to bind to the protein tyrosine kinase ZAP-70. This interaction resulted in an increase in SHP-1 phosphatase activity and a decrease in ZAP-70 kinase activity. Expression of a dominant negative mutant of SHP-1 in T cells increased the sensitivity of the antigen receptor. Thus, SHP-1 functions as a negative regulator of the T cell antigen receptor and in setting the threshold of activation.

A great deal of research over the last century has focused on drowsiness/alertness detection, as fatigue-related physical and cognitive impairments pose a serious risk to public health and safety. Available drowsiness/alertness detection solutions are unsatisfactory for a number of reasons: 1) lack of generalizability, 2) failure to address individual variability in generalized models, and/or 3) they lack a portable, un-tethered application. The current study aimed to address these issues, and determine if an individualized electroencephalography (EEG) based algorithm could be defined to track performance decrements associated with sleep loss, as this is the first step in developing a field deployable drowsiness/alertness detection system. The results indicated that an EEG-based algorithm, individualized using a series of brief "identification" tasks, was able to effectively track performance decrements associated with sleep deprivation. Future development will address the need for the algorithm to predict performance decrements due to sleep loss, and provide field applicability.

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Understanding how amyloid-β peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric Aβ interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high Aβ concentrations required for most biochemical analyses, the metastable nature of Aβ aggregates, and the complex variety of Aβ species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small Aβ(1-40) oligomers bind to living neuroblastoma cells at physiological Aβ concentrations. Single particle fluorescence intensity measurements indicate that cell-bound Aβ species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound Aβ oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary Aβ species that interact with neurons at physiological concentrations.

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Prior exposure to social disruption stress (SDR) exacerbates both the acute and chronic phase of Theiler's murine encephalomyelitis virus infection (TMEV; [Johnson, R.R., Storts, R., Welsh, T.H., Jr., Welsh, C.J., Meagher, M.W., 2004. Social stress alters the severity of acute Theiler's virus infection. J. Neuroimmunol. 148, 74--85; Johnson, R.R., Prentice, T.W., Bridegam, P., Young, C.R., Steelman, A.J., Welsh, T.H., Welsh, C.J.R., Meagher, M.W., 2006. Social stress alters the severity and onset of the chronic phase of Theiler's virus infection. J. Neuroimmunol. 175, 39--51]). However, the neuroimmune mechanism(s) mediating this effect have not been determined. The present study examined whether stress-induced increases in the proinflammatory cytokine interleukin-6 (IL-6) contributes to the adverse effects of SDR on acute TMEV infection. Experiment 1 demonstrated that SDR increases central and peripheral levels of IL-6 and that this effect is reversed by intracerebral ventricular infusion of neutralizing antibody to IL-6 prior to each of six SDR sessions. Although SDR reduced the sensitivity of spleen cells to the anti-inflammatory effects of corticosterone, the neutralizing antibody to IL-6 did not alter this effect. To investigate whether stress-induced increases in IL-6 contribute to the exacerbation of acute TMEV infection, Experiment 2 examined whether intracerebral administration of neutralizing antibody to IL-6 during SDR would prevent the subsequent exacerbation of acute TMEV infection. Experiment 3 then replaced the social stress with intracerebral infusion of IL-6 to assess sufficiency. As expected, prior exposure to SDR subsequently increased infection-related sickness behaviors, motor impairment, CNS viral titers, and CNS inflammation. These deleterious effects of SDR were either prevented or significantly attenuated by intracerebral infusion of neutralizing antibody to IL-6 during the stress exposure period. However, infusion of IL-6 alone did not mimic the adverse effects of SDR. We conclude that IL-6 is necessary but not sufficient to exacerbate acute TMEV infection.