Comprehensive analysis of lymph node stroma-expressed Ig superfamily members reveals redundant and nonredundant roles for ICAM-1, ICAM-2, and VCAM-1 in lymphocyte homing

Although it is well established that stromal intercellular adhesion molecule-1 (ICAM-1), ICAM-2, and vascular cell adhesion molecule-1 (VCAM-1) mediate lymphocyte recruitment into peripheral lymph nodes (PLNs), their precise contributions to the individual steps of the lymphocyte homing cascade are not known. Here, we provide in vivo evidence for a selective function for ICAM-1 > ICAM-2 > VCAM-1 in lymphocyte arrest within noninflamed PLN microvessels. Blocking all 3 CAMs completely inhibited lymphocyte adhesion within PLN high endothelial venules (HEVs). Post-arrest extravasation of T cells was a 3-step process, with optional ICAM-1-dependent intraluminal crawling followed by rapid ICAM-1- or ICAM-2-independent diapedesis and perivascular trapping. Parenchymal motility of lymphocytes was modestly reduced in the absence of ICAM-1, while ICAM-2 and alpha4-integrin ligands were not required for B-cell motility within follicles. Our findings highlight nonredundant functions for stromal Ig family CAMs in shear-resistant lymphocyte adhesion in steady-state HEVs, a unique role for ICAM-1 in intraluminal lymphocyte crawling but redundant roles for ICAM-1 and ICAM-2 in lymphocyte diapedesis and interstitial motility.

MET inhibition in tumor cells by PHA665752 impairs homologous recombination repair of DNA double strand breaks

Abnormal activation of cellular DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems has broad implications for both cancer biology and treatment. Recent studies suggest a potential link between DNA repair and aberrant activation of the hepatocyte growth factor receptor Mesenchymal-Epithelial Transition (MET), an oncogene that is overexpressed in numerous types of human tumors and considered a prime target in clinical oncology. Using the homologous recombination (HR) direct-repeat direct-repeat green fluorescent protein ((DR)-GFP) system, we show that MET inhibition in tumor cells with deregulated MET activity by the small molecule PHA665752 significantly impairs in a dose-dependent manner HR. Using cells that express MET-mutated variants that respond differentially to PHA665752, we confirm that the observed HR inhibition is indeed MET-dependent. Furthermore, our data also suggest that decline in HR-dependent DNA repair activity is not a secondary effect due to cell cycle alterations caused by PHA665752. Mechanistically, we show that MET inhibition affects the formation of the RAD51-BRCA2 complex, which is crucial for error-free HR repair of double strand DNA lesions, presumably via downregulation and impaired translocation of RAD51 into the nucleus. Taken together, these findings assist to further support the role of MET in the cellular DNA damage response and highlight the potential future benefit of MET inhibitors for the sensitization of tumor cells to DNA damaging agents.

Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes

The cytidine deaminase AID hypermutates immunoglobulin genes but can also target oncogenes, leading to tumorigenesis. The extent of AID's promiscuity and its predilection for immunoglobulin genes are unknown. We report here that AID interacted broadly with promoter-proximal sequences associated with stalled polymerases and chromatin-activating marks. In contrast, genomic occupancy of replication protein A (RPA), an AID cofactor, was restricted to immunoglobulin genes. The recruitment of RPA to the immunoglobulin loci was facilitated by phosphorylation of AID at Ser38 and Thr140. We propose that stalled polymerases recruit AID, thereby resulting in low frequencies of hypermutation across the B cell genome. Efficient hypermutation and switch recombination required AID phosphorylation and correlated with recruitment of RPA. Our findings provide a rationale for the oncogenic role of AID in B cell malignancy.

Placing Identity: Town, Land, and Authenticity in Nunavut, Canada

Recent demographic changes have made settlement patterns in the Canadian Arctic increasingly urban. Iqaluit, capital of Canada’s newest territory, Nunavut, is home to the largest concentration of Inuit and non-Inuit populations in the Canadian North. Despite these trends, Inuit cultural identity continues to rest heavily on the perception that to learn how to be authentically Inuit (or to be a better person), a person needs to spend time out on the land (and sea) hunting, fishing, trapping, and camping. Many Inuit also maintain a rather negative view of urban spaces in the Arctic, identifying them as places where Inuit values and practices have been eclipsed by Qallunaat (‘‘white people’’) ones. Some Inuit have even gone so far as to claim that a person is no longer able to be Inuit while living in towns like Iqaluit. This article examines those aspects of Canadian Inuit identity, culture, and tradition that disfavor the acceptance of an urban cultural identity. Based on ethnographic research conducted on Baffin Island in the mid 1990s and early 2000s, the many ways Iqaluit and outpost camp Inuit express the differences and similarities between living on the land and living in town are described. Then follows an examination of how the contrast of land and town is used in the rhetoric of Inuit politicians and leaders. Finally, a series of counterexamples are presented that favor the creation of an authentic urban Inuit identity in the Arctic, including recent attempts on the part of the Nunavut Territorial Government to make education and wage employment in the region more reliant on Inuit Qaujimajatuqangit, or Inuit traditional knowledge.1

Molecular Charge-Transfer Cross Sections and Their Correlation with Reactant Ion Structures

Charge-transfer cross sections have been obtained by using time-of-flight techniques, and results correlated with reaction energetics and theoretical structures computed by self-consistent field-molecular orbital methods. Ion recombination energies, structures, heats of formation, reaction energy defects, and 3.0-keV charge-transfer cross sections are presented for reactions of molecular and fragment ions produced by electron bombardment ionization of CH30CH, and CH$l molecules. Relationships between experimental cross sections and reaction energetics involving different ion structures are discussed.

Charge transfer reactions of organic ions containing oxygen: Correlation between reaction energetics and cross sections

Cross sections for charge transfer reactions of organic ions containing oxygen have been obtained using time-of-flight techniques. Charge transfer cross sections have been determined for reactions of 2.0 to 3.4 keV ions produced by electron impact ionization of oxygen containing molecules such as methanol, ethanal and ethanol. Experimental cross section magnitudes have been correlated with reaction energy defects computed from ion recombination energies and target ionization energies. Large cross sections are observed for reacting systems with small energy defects.

Insight into the mechanism of transient trapping in micellar electrokinetic chromatography

Transient trapping is a new mechanism of on-line sample concentration and separation that has recently been presented. It involves the injection of a short length of micellar solution in front of the sample, making it similar to sweeping in partial-filling MEKC. Here, we examine the mechanism of transient trapping by the use of computer simulations and compare it to sweeping in MEKC for the two analytes, sulforhodamine B and 101. The simulation results confirm the mechanism for concentration and separation originally proposed. The mechanism for concentration is similar to sweeping since the analytes are picked and accumulated by the micelles that penetrate the sample zone. The mechanism for separation is however quite unique since the concentrated analytes are trapped for a few seconds on the sample/micelle boundary before they are released as the concentration of micelle is reduced as it undergoes electromigration dispersion and the analytes separate down a micelle gradient. Simulation results suggested that a significant contribution of band broadening arises from the micelle gradient, with shallower gradients resulting in broader peaks. However, this is offset by an increase in selectivity, such that resolution was enhanced even though the peaks are broader. Transient trapping analysis with similar resolution to those obtained by sweeping MEKC could be achieved in 1/10 of the time and 1/4 of the capillary length, which results in a 2-3 times increase in sensitivity.

Annulation of Tetrathiafulvalene to the Bay Region of Perylenediimide: Fast Electron-Transfer Processes in Polar and Nonpolar Solvents

A tetrathiafulvalene donor has been annulated to the bay region of perylenediimide through a 1H-benzo-[d]pyrrolo[1,2-a]imidazol-1-one spacer affording an extended pi-conjugated molecular dyad (TTF-PDI). To gain insight into its ground- and excited-state electronic properties, the reference compound Ph-PDI has been prepared via a direct Schiff-base condensation of N,N'-bis(1-octylnonyl) benzoperylene-1',2':3,4:9,10-hexacarboxylic-1',2'-anhydride-3,4:9,10-bis (imide) with benzene-1,2-diamine. Both the experimental and the computational (DFT) results indicate that TTF-PDI exhibits significant intramolecular electronic interactions giving rise to an efficient photoinduced charge-separation process. Free-energy calculations verify that the process from TTF to the singlet-excited state of PDI is exothermic in both polar and nonpolar solvents. Fast adiabatic electron-transfer processes of a compactly fused, pi-conjugated TTF-PDI dyad in benzonitrile, 2-methyltetrahydrofuran, anisole and toluene were observed by femtosecond transient absorption spectral measurements. The lifetimes of radical-ion pairs slightly increase with decreasing the solvent polarities, suggesting that the charge-recombination occurs in the Marcus inverted region. By utilizing the nanosecond transient absorption technique, the intermolecular electron-transfer process in a mixture of has been observed via the triplet excited PDI for the first time.

Synthesis and Surface Modification of CdSe and CdS Quantum Dots Exhibiting High Quantum Yield

The thesis investigates the effect of surface treatment with various reducing and oxidizing agents on the quantum yield (QY) of CdSe and CdS quantum dots (QDs). The QDs, as synthesized by the organometallic method, contained defect sites on their surface that trapped photons and prevented their radiative recombination, therefore resulting in adecreased QY. To passivate these defect sites and enhance the QY, the QDs were treated with various reducing and oxidizing agents, including: sodium borohydride (NaBH4), calcium hydride (CaH2), hydrazine (N2H4), benzoyl peroxide (C14H10O4), and tert-butylhydroperoxide (C4H10O2). It was hypothesized that the reducing/oxidizing agents reduced the ligands on the QD surface, causing them to detach, thereby allowing oxygen from atmospheric air to bind to the exposed cadmium. This cadmium oxdide (CdO) layeraround the QD surface satisfied the defect sites and resulted in an increased QY. To correlate what effect the reducing and oxidizing agents were having on the optical properties of the QDs, we investigated these treatments on the following factors:chalcogenide (Se vs. S), ligand (oleylamine vs. OA), coordinating solvent (ODE vs.TOA), and dispersant solvent (chloroform vs. toluene) on the overall optical properties of the QDs. The QY of each sample was calculated before and after the various surface treatments from ultra-violet visible spectroscopy (UV-Vis) and fluorescence spectroscopy data to determine if the treatment was successful.From our results, we found that sodium borohydride was the most effective surface treatment, with 10 of the 12 treatments resulting in an increased QY. Hydrazine, on the other hand, was the least effective treatments, as it quenched the QD fluorescence in every case. From these observations, we hypothesize that the effectiveness of the QD surface treatments was dependent on reaction rate. More specifically, when the surface treatment reaction happened too quickly, we hypothesize that the QDs began to aggregate, resulting in a quenched fluorescence. Furthermore, we believe that the reactionrate is dependent on concentration of the reducing/oxidizing agents, solubility of the agents in each solvent, and reactivity of the agents with water. The quantum yield of the QDs can therefore be maximized by slowing the reaction rate of each surface treatment toa rate that allows for the proper passivation of defect sites.

The habitat, double life, citizenship, and forgetfulness of IgA

Immunoglobulin A (IgA) is the main secretory immunoglobulin of mucous membranes and is powerfully induced by the presence of commensal microbes in the intestine. B cells undergo class switch recombination to IgA in the mucosa-associated lymphoid tissues, particularly mesenteric lymph nodes (MLNs) and Peyer's patches, through both T-dependent and T-independent pathways. IgA B cells primed in the mucosa traffic from the intestinal lymphoid structures, initially through the lymphatics and then join the bloodstream, to home back to the intestinal mucosa as IgA-secreting plasma cells. Once induced, anti-bacterial IgA can be extremely long-lived but is replaced if there is induction of additional IgA specificities by other microbes. The mucosal immune system is anatomically separated from the systemic immune system by the MLNs, which act as a firewall to prevent penetration of live intestinal bacteria to systemic sites. Dendritic cells sample intestinal bacteria and induce B cells to switch to IgA. In contrast, intestinal macrophages are adept at killing extracellular bacteria and are able to clear bacteria that have crossed the mucus and epithelial barriers. There is both a continuum between innate and adaptive immune mechanisms and compartmentalization of the mucosal immune system from systemic immunity that function to preserve host microbial mutualism.

Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine.

Genetic and pharmacologic manipulation of oxidative stress after neonatal hypoxia-ischemia

Oxidative stress is a critical component of the injury response to hypoxia-ischemia (HI) in the neonatal brain, and this response is unique and at times paradoxical to that seen in the mature brain. Previously, we showed that copper-zinc superoxide-dismutase (SOD1) over-expression is not beneficial to the neonatal mouse brain with HI injury, unlike the adult brain with ischemic injury. However, glutathione peroxidase 1 (GPx1) over-expression is protective to the neonatal mouse brain with HI injury. To further test the hypothesis that an adequate supply of GPx is critical to protection from HI injury, we crossed SOD1 over-expressing mice (hSOD-tg) with GPx1 over-expressing mice (hGPx-tg). Resulting litters contained wild-type (wt), hGPx-tg, hSOD-tg and hybrid hGPx-tg/hSOD-tg pups, which were subjected to HI at P7. Confirming previous results, the hGPx-tg mice had reduced injury compared to both Wt and hSOD-tg littermates. Neonatal mice over-expressing both GPx1 and SOD1 also had less injury compared to wt or hSOD-tg alone. A result of oxidative stress after neonatal HI is a decrease in the concentration of reduced (i.e. antioxidant-active) glutathione (GSH). In this study, we tested the effect of systemic administration of alpha-lipoic acid on levels of GSH in the cortex after HI. Although GSH levels were restored by 24h after HI, injury was not reduced compared to vehicle-treated mice. We also tested two other pharmacological approaches to reducing oxidative stress in hSOD-tg and wild-type littermates. Both the specific inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7NI), and the spin-trapping agent alpha-phenyl-tert-butyl-nitrone (PBN) did not reduce HI injury, however. Taken together, these results imply that H2O2 is a critical component of neonatal HI injury, and GPx1 plays an important role in the defense against this H2O2 and is thereby neuroprotective.

Gamma-tocopherol supplementation inhibits protein nitration and ascorbate oxidation in rats with inflammation

Gamma-tocopherol (gammaT) complements alpha-tocopherol (alphaT) by trapping reactive nitrogen oxides to form a stable adduct, 5-nitro-gammaT [Christen et al., PNAS 94:3217-3222; 1997]. This observation led to the current investigation in which we studied the effects of gammaT supplementation on plasma and tissue vitamin C, vitamin E, and protein nitration before and after zymosan-induced acute peritonitis. Male Fischer 344 rats were fed for 4 weeks with either a normal chow diet with basal 32 mg alphaT/kg, or the same diet supplemented with approximately 90 mg d-gammaT/kg. Supplementation resulted in significantly higher levels of gammaT in plasma, liver, and kidney of control animals without affecting alphaT, total alphaT+gammaT or vitamin C. Intraperitoneal injection of zymosan caused a marked increase in 3-nitrotyrosine and a profound decline in vitamin C in all tissues examined. Supplementation with gammaT significantly inhibited protein nitration and ascorbate oxidation in the kidney, as indicated by the 29% and 56% reduction of kidney 3-nitrotyrosine and dehydroascorbate, respectively. Supplementation significantly attenuated inflammation-induced loss of vitamin C in the plasma (38%) and kidney (20%). Zymosan-treated animals had significantly higher plasma and tissue gammaT than nontreated pair-fed controls, and the elevation of gammaT was strongly accentuated by the supplementation. In contrast, alphaT did not significantly change in response to zymosan treatment. In untreated control animals, gammaT supplementation lowered basal levels of 3-nitrotyrosine in the kidney and buffered the starvation-induced changes in vitamin C in all tissues examined. Our study provides the first in vivo evidence that in rats with high basal amounts of alphaT, a moderate gammaT supplementation attenuates inflammation-mediated damage, and spares vitamin C during starvation-induced stress without affecting alphaT.