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The relative coverage, thermal stability, and electronic properties of CH3- and C2H5-functionalized Si(111) surfaces prepared by a two-step chlorination/alkylation procedure have been compared using high-resolution synchrotron photoemission spectroscopy. Whereas the CH3-terminated Si(111) surface showed only one C 2s peak for the occupied orbitals, the C 2s spectra of C2H5-terminated Si(111) surfaces showed a symmetric splitting of the occupied orbitals, as expected for an ethyl moiety bonded to the surface. The C2H5termination resulted in an unpinning of the Si surface Fermi level, with a band bending of ∼0.2 eV, and produced a surface dipole potential step of −0.23(15) eV. The observed close-to-flat-band condition is similar to that of CH3−Si(111) and is consistent with H termination of the non-alkylated Si atop sites in the two-step chlorination/alkylation process. The C2H5-functionalized Si(111) surfaces decomposed at temperatures >300 °C, whereas CH3−Si(111) surfaces were stable up to at least 440 °C. The data clearly highlight the similarities and identify some significant differences between the behavior of the CH3- and C2H5-functionalized Si(111) surfaces.

The process of cell motility involves coordinate signaling events among proteins associated in interactive integrin-linked networks. Mass spectrometric analysis of immunoprecipitation-derived protein mixtures have provided efficient means of identifying proteomes. In this study, we investigate strategies to enhance the detection of interactome proteins for the known signaling module:  PAK1, PIX, GIT1, and paxillin. Our results indicate that near-endogenous expression levels of bait protein enhances the identification of associated proteins, and that phosphatase inhibition augments the detection of specific protein interactions. Following the analysis of a large pool of spectral data, we have identified and mapped clusters of proteins that either share common interactions among the four bait proteins of interest or are exclusive to single bait proteins. Taken together, these data indicate that biochemical manipulations can enhance the ability for LC−MS/MS to identify interactome proteins, and that qualitative screening of multiple samples leads to the compilation of proteins associated with a known plexus.

1-Substituted benzotriazole carboxylic acids have been identified as the first reported examples of selective small-molecule agonists of the human orphan G-protein-coupled receptor GPR109b (HM74), a low-affinity receptor for the HDL-raising drug niacin. No activity was observed at the highly homologous high-affinity niacin receptor GPR109a (HM74A). The high degree of selectivity was attributed to a difference in the amino acid sequence adjacent to a key arginine−ligand interaction allowing somewhat larger ligands to be tolerated by GPR109b.

The heterodimeric splicing factor U2AF plays an important role in 3' splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3' splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AFLG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3' splice site recognition, we sought introns for which the spU2AFLG and spU2AFSM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3' pyrimidine tract. These and other studies performed in fission yeast support a model for 3' splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.

GIT1 is a multidomain protein that is thought to function as an integrator of signaling pathways controlling vesicle trafficking, adhesion and cytoskeletal organization. It regulates ARF GTPases and has binding domains for paxillin and PIX, which is a PAK-binding protein and an exchange factor for Rac. We show that GIT1 cycles between at least three distinct subcellular compartments, including adhesion-like structures, the leading edge and cytoplasmic complexes. The cytoplasmic structures, which also contain paxillin, PAK and PIX, do not detectably co-localize with endosomal Golgi or membrane markers, suggesting that they represent a novel supramolecular complex. The GIT1 cytoplasmic complexes are motile and tended to move toward the cell periphery where they joined existing adhesions. In retracting regions of the cells, the GIT1 complexes moved away from the disassembling adhesions toward the cell body. Using deletion mutants, we have identified domains that target GIT1 to each of the compartments. Localization to adhesions and the leading edge requires the paxillin-binding domain, which comprises the C-terminal 140 residues (cGIT1), whereas targeting to the cytoplasmic complexes requires the central region that contains ankyrin repeats and the PIX-binding domain. Expression of GIT1 or cGIT, but not nGIT1 in which the paxillin-binding domain is deleted, increases the rate of migration and the size and number of protrusions. The latter are inhibited when GIT1 is co-expressed with a kinase-dead PAK, suggesting that the GIT1 interaction with PAK is required for enhanced migration and protrusive activity. Furthermore, GIT1 targets constitutively activated PAK to adhesions and the leading edge via its interaction with paxillin. Since expression of cGIT targets endogenous GIT1 to the leading edge, it appears that the leading edge is the location of GIT1 responsible for these activities. Thus, GIT1 is a component of a motile, multimolecular complex that traffics a set of signaling components to specific locations in the cell where they regulate localized activities.

Urokinase-type plasminogen activator (uPA) binds to the uPA receptor (uPAR) and activates the Ras-extracellular signal-regulated kinase (ERK) signaling pathway in many different cell types. In this study, we demonstrated that endogenously produced uPA functions as a major determinant of the basal level of activated ERK in MDA-MB-231 breast cancer cells. When these cells were cultured in the presence of antibodies that block the binding of uPA to uPAR, the level of phosphorylated ERK decreased substantially. Furthermore, conditioned medium from MDA-MB-231 cells activated ERK in MCF-7 cells and this response was blocked by uPA-specific antibody. The mitogen-activated protein kinase kinase inhibitor, PD098059, decreased expression of uPA and uPAR in MDA-MB-231 cells. Thus, uPA and the uPAR-ERK signaling pathway form a positive feedback loop in these cells. When this feedback loop was disrupted with uPA- or uPAR-specific antibody, uPA mRNA-specific antisense oligodeoxynucleotides or PD098059, cell growth was inhibited and apoptosis was promoted, as determined by the increase in cytoplasmic nucleosomes and caspase-3 activity. Treating the cells simultaneously with PD098059 and uPA- or uPAR-specific antibody did not further promote apoptosis, compared with either reagent added separately, supporting the hypothesis that uPAR and ERK are components of the same cell growth/survival-regulatory pathway. The ability of uPA to signal through uPAR, maintain an elevated basal level of activated ERK and inhibit apoptosis represents a novel mechanism whereby the uPA-uPAR system may affect breast cancer progression in vivo.

Oxidative stress plays an important role in the development of atherosclerosis and contributes to tissue damage that occurs as a consequence, particularly in myocardial infarction and acute stroke. Antioxidant properties of uric acid have long been recognized and, as a result of its comparatively high serum concentrations, it is the most abundant scavenger of free radicals in humans. Elevation of serum uric acid concentration occurs as a physiologic response to increased oxidative stress—for example, during acute exercise—thus providing a counterregulatory increase in antioxidant defenses. In view of its antioxidant properties, uric acid may have potentially important and beneficial effects within the cardiovascular system. We wished to investigate whether administration of uric acid was feasible and if it could have an impact on antioxidant function in vivo. We have, therefore, performed a randomized, placebocontrolled doubleblind study of the effects of systemic administration of uric acid, 1,000 mg, in healthy volunteers, compared with vitamin C, 1,000 mg. We observed a significant increase in serum freeradical scavenging capacity from baseline during uric acid and vitamin C infusion, using two methodologically distinct antioxidant assays. The effect of uric acid was substantially greater than that of vitamin C.

The low density lipoprotein receptor-related protein (LRP) has been reported to regulate cellular migration. In this study, an antisense RNA expression strategy was used to reduce LRP to undetectable levels in HT 1080 fibrosarcoma cells. The LRP-deficient cells demonstrated increased levels of cell-surface uPAR, higher levels of uPA in conditioned medium, increased migration on vitronectin-coated surfaces, and increased invasion of Matrigel. LRP-deficient cells also demonstrated increased levels of phosphorylated extracellular signal-regulated kinase (ERK) in the absence of exogenous stimulants. Antibodies which block binding of endogenously produced uPA to uPAR reduced ERK phosphorylation and migration of LRP-deficient cells to the levels observed with control cells. Inhibitors of ERK activation, including PD098059 and dominant-negative MEK1, also decreased the migration of LRP-deficient but not control cells. By contrast, constitutively active MEK1 stimulated the migration of control but not LRP-deficient cells. Although Matrigel invasion by LRP-deficient cells was inhibited by the proteinase inhibitor, aprotinin, PD098059 in combination with aprotinin was necessary for an optimal effect. Expression of the VLDL receptor in LRP-deficient cells reversed the changes in cellular migration and invasion. These studies demonstrate that binding of endogenously produced uPA to uPAR may serve as a major determinant of basal levels of activated ERK and, by this mechanism, regulate cellular migration and invasion. By regulating the uPA/uPAR system, LRP may also regulate ERK activation, cellular migration, and invasion.

BACKGROUND: Endoscopic ultrasound guided fine needle aspiration biopsy (EUS-FNA) is a recent innovation in the evaluation of gastrointestinal and pulmonary malignancies. AIMS: To review the experience with EUS-FNA of a large single centre. METHODS: 333 consecutive patients underwent EUS-FNA. Follow up data were available on 327 lesions in 317 patients, including 160 lymph nodes, 144 pancreatic lesions, 15 extraintestinal masses, and eight intramural tumours. RESULTS: A primary diagnosis of malignancy was obtained by EUS-FNA in 62% of patients with clinically suspicious lesions. The overall accuracy of EUS-FNA for the diagnosis of malignancy was 86%, with sensitivity of 84% and specificity of 96%. With respect to lesion types, the sensitivity, specificity, and accuracy were 85%, 100%, and 89% for lymph nodes; 82%, 100%, and 85% for pancreatic lesions; 88%, 100%, and 90% for perirectal masses; and 50%, 25%, and 38% for intramural lesions, respectively. Compared with size and sonographic criteria, EUS-FNA in the evaluation of lymph nodes provided superior accuracy and specificity, without compromising sensitivity. Inadequate specimens were obtained from only six patients, including 3/5 with stromal tumors. Only one complication occurred. CONCLUSIONS: EUS-FNA is safe and can readily obtain tissue specimens adequate for cytopathological diagnoses. Compared with size and sonographic criteria, it is a superior modality for the detection of nodal metastases. While providing accurate diagnosis of pancreatic and perirectal malignancies, results suggest the technique is less useful for intramural lesions.

Dehydroepiandrosterone (DHEA) is the only known naturally occurring compound that promotes peroxisome proliferation in rodent liver, and stimulates transcriptional induction of genes involved in lipid metabolism and peroxisomal beta-oxidation. Therefore, we examined mRNA for several such genes in rat liver, specifically acyl-CoA oxidase and the cytochromes P-450 (CYP4A1, CYP4A3, and CYP3A23), after 5 to 6 day treatments with either DHEA, or nafenopin, a known peroxisome proliferator. Acyl-CoA oxidase and CYP4A1 were induced nearly identically by DHEA and nafenopin, with induction being more pronounced in female rats. However, CYP3A23 was induced only by DHEA, suggesting an induction mechanism independent of the peroxisome proliferator activated receptor. Previously, we observed triiodothyronine (T3) suppression of peroxisome proliferator induced CYP4As and we sought to determine whether CYP3A23 might be regulated in a different manner. T3 was found to also suppress DHEA-dependent induction of CYP3A23. CYP4A2 expression in kidney was also negatively regulated by T3. To characterize a putative negative thyroid hormone response element (nTRE) in the 5' flanking region of this gene, a luciferase reporter gene containing a rat CYP4A2 flanking sequence extending to -1865 bp was transfected into HepG2 cells along with human thryroid hormone receptor expression vector. Expression of luciferase activity was unaffected by T3, suggesting the absence of a functional nTRE within this portion of CYP4A2. These data demonstrate gene regulatory activity by DHEA different from that of nafenopin, and a suppressive effect of T3, consistent with indirect regulatory mechanisms not involving an nTRE.

A bis(ethynyl)-substituted porphyrin monomer of Ru(CO) has been prepared and oligomerised by Glaser–Hay coupling to give a cyclic butadiyne-linked trimer. Use of tri(4-pyridyl)triazine as template during the coupling ensured that only a single stereoisomer of the trimer was formed: all three CO groups are forced to be outside the cavity, leaving three potential ruthenium-binding sites facing into the cavity.

Warmer temperatures are expected to increase the incidence of Lyme disease through enhanced tick maturation rates and a longer season of transmission. In addition, there could be an increased risk of disease export because of infected mobile hosts, usually birds. A temperature-driven seasonal model of Borrelia burgdorferi (Lyme disease) transmission among four host types is constructed as a system of nonlinear ordinary differential equations. The model is developed and parametrized based on a collection of lab and field studies. The model is shown to produce biologically reasonable results for both the tick vector (Ixodes scapularis) and the hosts when compared to a different set of studies. The model is used to predict the response of Lyme disease risk to a mean annual temperature increase, based on current temperature cycles in Hanover, NH. Many of the risk measures suggested by the literature are shown to change with increased mean annual temperature. The most straightforward measure of disease risk is the abundance of infected questing ticks, averaged over a year. Compared to this measure, which is difficult and resource-intensive to track in the field, all other risk measures considered underestimate the rise of risk with rise in mean annual temperature. The measure coming closest was “degree days above zero.” Disease prevalence in ticks and hosts showed less increase with rising temperature. Single field measurements at the height of transmission season did not show much change at all with rising temperature.

Osteoarthritis (OA) of the knee is one of the most common joint diseases affecting adults in the United States. For elderly patients with multiple medical comorbidities who do not wish to undergo total knee arthroplasty (TKA), lifestyle modification, pharmacologic management, and injections are the mainstay of therapy. Previously, pain management interventions were limited to intra-articular joint injections and viscosupplementation with hyaluronic acid. Fluoroscopic-guided techniques for radiofrequency ablation (RFA) of the genicular nerves have been previously described and a recent cadaveric study suggests that ultrasound-guided genicular nerve blocks can be performed accurately. We performed an ultrasound-guided radiofrequency ablation of the genicular nerves in 88-year-old woman who had deferred surgical management given her age. Following successful ultrasound guided diagnostic genicular nerve blocks, she proceeded to RFA using the same ultrasound guided technique. The procedure resulted in significant pain relief and improvement in overall function for greater than 6 months. The use of ultrasound provides a relatively rapid and noninvasive method to directly visualize genicular nerves and surrounding vasculature. Our case suggests that, for genicular nerve blockade and RFA, ultrasound may be a useful alternative to fluoroscopy. Not only did the procedure result in significant pain relief that has persisted for greater than 6 months but also more importantly her function status and quality of life were improved.

Mediastinal mass syndrome (MMS) is a complex case that poses many challenges to the anesthesiologist. The cornerstone of management focuses on the potential hemodynamic changes associated with this syndrome. We describe the anesthetic management of a patient with a previously undiagnosed mediastinal mass presenting for emergency neurosurgical surgery.