Locating the contradiction in our understanding of time.

I offer some clarification concerning the kind of contradiction that Hoerl & McCormack's account could help explain and the scope of the metaphysical intuitions that could be explained by such a theory. I conclude that we need to know more about the sense in which the temporal reasoning system would represent time as a dimension.

Endothelin signaling via guanine exchange factor C3G in renal glomerular mesangial cells.

The guanine nucleotide exchange factor C3G is one of the mediators of endothelin-1 (ET-1) intracellular signaling cascades and is vital for kidney development and homeostasis. The aim of the current study was to analyze the specificity of ET-1-induced signaling via C3G in rat glomerular mesangial cells (GMC) and to investigate the biological significance of C3G during mesangioproliferative glomerulonephritis. In GMC, C3G expression was increased (1) in vivo after induction of the anti-Thy1 model of glomerulonephritis and (2) in cell culture experiments after fetal bovine serum incubation. To examine the consequences of C3G up-regulation, adenovirus-mediated gene transfer of C3G into cultured glomerular cells was done, and the GTP loading of the small G proteins Rap1 and R-Ras was analyzed. Overexpression of C3G in mesangial cells resulted in enhanced activation of Rap1, but failed to affect the GTP-bound status of R-Ras in ET-1-stimulated cells. C3G overexpression led to significant changes in GMC spreading and migration patterns in response to ET-1 stimulation and increased stress fiber formation, which was mimicked by Rap1A overexpression. Together, these findings suggest (1) the existence of regulatory mechanisms resulting in disease-related up-regulation of C3G in GMC and (2) that an increase in the C3G protein level may contribute to the resolution stage of mesangioproliferative glomerulonephritis by reducing GMC sensitivity to ET-1, modulating cellular motility, and actin dynamics.

A novel chromatographic method allows on-line reanalysis of the proteome.

Liquid chromatography combined with electrospray ionization is widely used for direct analysis of polar and labile molecules by LCMS. The on-line coupling in LCMS is a major strength but also causes a principal limitation that each eluting analyte has to be analyzed immediately and is not available for detailed interrogation after the LCMS run. Here we developed a new chromatographic strategy, which removes this limitation. After column separation the flow is split, one portion is analyzed directly, and the other is diverted to a capture capillary. After the direct LCMS run, the flow is switched, and the portion stored in the capillary is analyzed ("replay run"). We describe a setup consisting of an analytical column, a splitting valve, and a focusing column, which performs at full sensitivity and undiminished chromatographic resolution. We demonstrate three principal advantages of this system: nearly continuous MS utilization, duplicate analysis without requirement for additional sample, and targeting of important but undersampled features in the replay run.

Recovery of BK virus large T-antigen-specific cellular immune response correlates with resolution of bk virus nephritis.

BACKGROUND: BK virus (BKV) infection of kidney transplant patients is an increasing problem and is thought to be secondary to potent immunosuppressive therapy. BKV infection progresses to BKV nephritis (BKVN) in approximately 8% of transplants and in half of these cases the graft is lost. METHODS: We used an interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) assay to measure the cellular immune response to peptides encoding BKV large T antigen. Eight kidney transplant patients with BKVN were tested at the time of diagnosis of BKVN and then after resolution of active BKV infection. RESULTS: When total spot counts from all peptide pools were combined, the mean ELISPOT signal per 10,000 cells at the time of BKVN diagnosis was 23.1 (range 3.4-59.7), with a median of 21.8. This increased to 70.2 (range 5.4-189.4) with a median of 37.0 (P=0.1216) after resolution of active BKV infection. To further increase specificity of response, we counted the number of peptide pools with ELISPOT activity of greater than 10 spots per well after subtraction of background. The mean number of pools fitting this criteria at the time of BKVN diagnosis was 2.1 (range 0-8) with a median of 1.5; this increased to 8 (range 1-18) and a median of 6.5 after recovery (P=0.0338). CONCLUSION: This demonstrates that recovery of cellular immune response to large T antigen corresponds with resolution of active BKV infection. This may prove useful in monitoring patients' cellular immunity and recovery from active BKV infection when treated with reduction in immunosuppressive therapy.

CrkIII: a novel and biologically distinct member of the Crk family of adaptor proteins.

As the role for adaptor proteins constantly proliferates, appreciation of their importance has never been higher. The Crk family of adaptor proteins is no exception. Currently comprising four members, v-Crk, CrkI, CrkII and Crk-like protein, we have introduced a fifth member, CrkIII. Cloned by the CORT technique, CrkIII is identical in sequence to CrkII until the second of its two SH3 domains, which is disrupted partway through and results in a nonfunctional domain and a unique C-terminal sequence. We have demonstrated the existence of native CrkIII at the message level using RT-PCR and RNAse protection assays, and at the protein level in mouse fibroblasts. We show that CrkII overexpression is capable of enhancing insulin-stimulated ERK activity, whereas CrkIII is not, thus partially characterizing a novel member of the Crk family and elucidating important effects mediated by the c-terminal SH3 domain.

Biobreeding rat islets exhibit reduced antioxidative defense and N-acetyl cysteine treatment delays type 1 diabetes.

Islet-level oxidative stress has been proposed as a trigger for type 1 diabetes (T1D), and release of cytokines by infiltrating immune cells further elevates reactive oxygen species (ROS), exacerbating ß cell duress. To identify genes/mechanisms involved with diabetogenesis at the ß cell level, gene expression profiling and targeted follow-up studies were used to investigate islet activity in the biobreeding (BB) rat. Forty-day-old spontaneously diabetic lymphopenic BB DRlyp/lyp rats (before T cell insulitis) as well as nondiabetic BB DR+/+ rats, nondiabetic but lymphopenic F344lyp/lyp rats, and healthy Fischer (F344) rats were examined. Gene expression profiles of BB rat islets were highly distinct from F344 islets and under-expressed numerous genes involved in ROS metabolism, including glutathione S-transferase (GST) family members (Gstm2, Gstm4, Gstm7, Gstt1, Gstp1, and Gstk1), superoxide dismutases (Sod2 and Sod3), peroxidases, and peroxiredoxins. This pattern of under-expression was not observed in brain, liver, or muscle. Compared with F344 rats, BB rat pancreata exhibited lower GST protein levels, while plasma GST activity was found significantly lower in BB rats. Systemic administration of the antioxidant N-acetyl cysteine to DRlyp/lyp rats altered abundances of peripheral eosinophils, reduced severity of insulitis, and significantly delayed but did not prevent diabetes onset. We find evidence of ß cell dysfunction in BB rats independent of T1D progression, which includes lower expression of genes related to antioxidative defense mechanisms during the pre-onset period that may contribute to overall T1D susceptibility.

Identification of a serum-induced transcriptional signature associated with type 1 diabetes in the BioBreeding rat.

OBJECTIVE: Inflammatory mediators associated with type 1 diabetes are dilute and difficult to measure in the periphery, necessitating development of more sensitive and informative biomarkers for studying diabetogenic mechanisms, assessing preonset risk, and monitoring therapeutic interventions. RESEARCH DESIGN AND METHODS: We previously utilized a novel bioassay in which human type 1 diabetes sera were used to induce a disease-specific transcriptional signature in unrelated, healthy peripheral blood mononuclear cells (PBMCs). Here, we apply this strategy to investigate the inflammatory state associated with type 1 diabetes in biobreeding (BB) rats. RESULTS: Consistent with their common susceptibility, sera of both spontaneously diabetic BB DRlyp/lyp and diabetes inducible BB DR+/+ rats induced transcription of cytokines, immune receptors, and signaling molecules in PBMCs of healthy donor rats compared with control sera. Like the human type 1 diabetes signature, the DRlyp/lyp signature, which is associated with progression to diabetes, was differentiated from that of the DR+/+ by induction of many interleukin (IL)-1-regulated genes. Supplementing cultures with an IL-1 receptor antagonist (IL-1Ra) modulated the DRlyp/lyp signature (P < 10(-6)), while administration of IL-1Ra to DRlyp/lyp rats delayed onset (P = 0.007), and sera of treated animals did not induce the characteristic signature. Consistent with the presence of immunoregulatory cells in DR+/+ rats was induction of a signature possessing negative regulators of transcription and inflammation. CONCLUSIONS: Paralleling our human studies, serum signatures in BB rats reflect processes associated with progression to type 1 diabetes. Furthermore, these studies support the potential utility of this approach to detect changes in the inflammatory state during therapeutic intervention.