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Natural elicitors derived from pathogenic microorganisms represent an ecologic strategy to achieve resistance in plants against diseases. Glucosylceramides (GlcCer) are classified as neutral glycosphingolipids. GlcCer were isolated and purified fromFusarium oxysporummycelium.F.oxysporumis a plant pathogenic fungus, abundant in soil and causing severe losses in economically important crops such as corn, tobacco, banana, cotton and passion fruit. In this study we evaluate the capacity of GlcCer in inducing resistance inN.tabacumcv Xanthi plants againstTobacco mosaic virus(TMV). Spraying tobacco plants with GlcCer before virus infection reduced the incidence of necrotic lesions caused by TMV. In addition, plants already infected with the virus showed a reduction in hypersensitive response (HR) lesions after GlcCer treatment, suggesting an antiviral effect of GlcCer. Our investigations showed that GlcCer stimulates the early accumulation of H2O2and superoxide radicals. In addition, the expression of PR-1 (pathogenesis-related 1, with suggested antifungal action), PR-2 (β-1,3-glucanase), PR-3 (Chitinase), PR-5 (Osmotin), PAL (Phenylalanine ammonia-lyase), LOX (Lipoxygenase) and POX (Peroxidase) genes was highly induced after treatment of tobacco plants with GlcCer and induction levels remained high throughout a period of 6 to 120 hours. Our experiments demonstrate that GlcCer induces resistance in tobacco plants against infection by TMV.

A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8+ T cell immunity. While much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as modified vaccinia virus Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required. Here, we compare the form of antigen that supports optimal CD8+ T cell responses for VACV strains WR and MVA using equivalent constructs. We found that for multiple antigens, minimal antigenic constructs (epitope minigenes) that prime CD8+ T cells via the direct presentation pathway elicited optimal responses from both vectors, which was surprising because this finding contradicts the prevailing view in the literature for MVA. We then went on to explore the discrepancy between current and published data for MVA, finding evidence that the expression locus and in some cases the presence of the viral thymidine kinase may influence the ability of this strain to prime optimal responses from antigens that require direct presentation. This extends our knowledge of the design parameters for VACV vectored vaccines, especially those based on MVA.IMPORTANCE Recombinant vaccines based on vaccinia virus and particularly attenuated strains such as MVA are in human clinical trials, but due to the complexity of these large vectors much remains to be understood about the design parameters that alter their immunogenicity. Previous work had found that MVA vectors should be designed to express stable protein in order to induce robust immunity by CD8+ (cytotoxic) T cells. Here, we found that the primacy of stable antigen is not generalizable to all designs of MVA and may depend where a foreign antigen is inserted into the MVA genome. This unexpected finding suggests that there is an interaction between genome location and the best form of antigen for optimal T cell priming in MVA and thus possibly other vaccine vectors. It also highlights that our understanding of antigen presentation by even the best studied of vaccine vectors remains incomplete.

Chemotherapy-induced cognitive impairment, also known as "chemobrain," is a common side effect. The purpose of this study was to examine whether resveratrol, a natural polyphenol that has nootropic effects, could prevent chemobrain and its underlying mechanisms. Mice received three injections of docetaxel, adriamycin, and cyclophosphamide (DAC) in combination, a common chemotherapy regimen, at two-day intervals within one week. Resveratrol (50 and 100 mg/kg per day) was orally administered for three weeks, beginning one week before the DAC treatment. Water maze test and manganese-enhanced magnetic resonance imaging were used to evaluate animals' cognitive performance and brain neuronal activity, respectively. Blood and brain tissues were collected for measurement of cytokines, cytokine regulators, and biomarkers for neuroplasticity. DAC treatment produced a striking cognitive impairment. Cotreatment with 100 mg/kg resveratrol ameliorated DAC-induced cognitive impairment and decreases in prefrontal and hippocampal neuronal activity. Mice co-treated with both doses of resveratrol displayed significantly lower levels of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but markedly higher levels of the anti-inflammatory cytokines IL-4 and IL-10 in several sera and brain tissues than those co-treated with vehicle. Resveratrol modulated the cytokine-regulating pathway peroxisome proliferator activated receptor (PPAR)-γ/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and protected against DAC-induced decreases in the expression of the neuroplasticity biomarkers, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), amino acid neurotransmitter receptors, and calmodulin-dependent protein kinase II (CaMKII). These results demonstrate the efficacy of resveratrol in preventing chemobrain and its association with cytokine modulation and neuroprotection.

Nuclear magnetic resonance (NMR) spectroscopy is an analytical technique that is used routinely for the identification of organic compounds. Chemists apply H and carbon (C)-NMR spectroscopy to determine the structure of synthetic organic molecules and natural products. Liquid chromatography has been interfaced with NMR spectroscopy (LC-NMR), which allows the separation of mixtures into individual anionic surfactants and their subsequent detection by means of proton NMR spectroscopy. The application of LC-NMR requires specialized equipment such as an NMR probe with a flow cell. NMR may be used to quantify the relative ratios of the individual components in mixtures or to measure the average alkyl chain length or the number of ethylene oxide units present in a surfactant. The assignment of fatty acid salts by means of NMR spectroscopy relies mainly on the resonance frequency of the C nucleus of the carbonyl moiety. The signals of the fatty acid chain will provide information on the average chain length of the fatty acid.

We integrate ambipolar quantum dots in silicon fin field-effect transistors using exclusively standard complementary metal-oxide-semiconductor fabrication techniques. We realize ambipolarity by replacing conventional highly doped source and drain electrodes by a metallic nickel silicide with the Fermi level close to the silicon mid-gap position. Such devices operate in a dual mode, as either a classical field-effect or single-electron transistor. We implement a classical logic NOT gate at low temperature by tuning two interconnected transistors into opposite polarities. In the quantum regime, we demonstrate stable quantum dot operation in the few charge carrier Coulomb blockade regime for both electrons and holes.

The role of nongastric H+-K+-ATPase (HKA) in ion homeostasis of macula densa (MD) cells is an open question. To begin to explore this issue, we developed two mathematical models that describe ion fluxes through a nongastric HKA. One model assumes a 1H+:1K+-per-ATP stoichiometry; the other assumes a 2H+:2K+-per-ATP stoichiometry. Both models include Na+and NH4+competitive binding with H+and K+, respectively, a characteristic observed in vitro and in situ. Model rate constants were obtained by minimizing the distance between model and experimental outcomes. Both 1H+(1Na+):1K+(1NH4+)-per-ATP and 2H+(2Na+):2K+(2NH4+)-per-ATP models fit the experimental data well. Using both models, we simulated ion net fluxes as a function of cytosolic or luminal ion concentrations typical for the cortical thick ascending limb and MD region. We observed that 1) K+and NH4+flowed in the lumen-to-cytosol direction, 2) there was competitive behavior between luminal K+and NH4+and between cytosolic Na+and H+, 3) ion fluxes were highly sensitive to changes in cytosolic Na+or H+concentrations, and 4) the transporter does mostly Na+/K+exchange under physiological conditions. These results support the concept that nongastric HKA may contribute to Na+and pH homeostasis in MD cells. Furthermore, in both models, H+flux reversed at a luminal pH that was +/H+exchange for a luminal pH of +homeostasis in the more acidic regions of the renal tubules.

Conventional two-dimensional (2D) transmission electron microscopy of sulfidic hydroprocessing catalysts can be deceiving and give the impression that parts of the support are overloaded with active phase. High-angle annular dark field scanning transmission electron microscopy tomography reveals details on the morphology of MoS2 crystallites and their interaction with the Al2O3 and TiO2 support particles. The three-dimensional (3D) reconstruction shows that the active phase is mainly present as MoS2 single slabs of various shapes aligned with the support. It becomes clear that the surface of the support particles is, in fact, only partly covered by the active phase and the pores remain accessible for reactant molecules.

During the summers of 2002–2013, we measured rates of carbon metabolism in surface waters of six sites across a land-to-lake gradient from the upstream end of drowned river-mouth Muskegon Lake (ML) (freshwater estuary) to 19 km offshore in Lake Michigan (LM) (a Great Lake). Despite considerable inter-year variability, the average rates of gross production (GP), respiration (R) and net production (NP) across ML (604 ± 58, 222 ± 22 and 381 ± 52 µg C L−1 day−1, respectively) decreased steeply in the furthest offshore LM site (22 ± 3, 55 ± 17 and −33 ± 15 µg C L−1day−1, respectively). Along this land-to-lake gradient, GP decreased by 96 ± 1%, whereas R only decreased by 75 ± 9%, variably influencing the carbon balance along this coastal zone. All ML sites were consistently net autotrophic (mean GP:R = 2.7), while the furthest offshore LM site was net heterotrophic (mean GP:R = 0.4). Our study suggests that pelagic waters of this Great Lakes coastal estuary are net carbon sinks that transition into net carbon sources offshore. Reactive and dynamic estuarine coastal zones everywhere may contribute similarly to regional and global carbon cycles.

The thick ascending limb (TAL) is a major NaCl reabsorbing site in the nephron. Efficient reabsorption along that segment is thought to be a consequence of the establishment of a strong transepithelial potential that drives paracellular Na+uptake. We used a multicell mathematical model of the TAL to estimate the efficiency of Na+transport along the TAL and to examine factors that determine transport efficiency, given the condition that TAL outflow must be adequately dilute. The TAL model consists of a series of epithelial cell models that represent all major solutes and transport pathways. Model equations describe luminal flows, based on mass conservation and electroneutrality constraints. Empirical descriptions of cell volume regulation (CVR) and pH control were implemented, together with the tubuloglomerular feedback (TGF) system. Transport efficiency was calculated as the ratio of total net Na+transport (i.e., paracellular and transcellular transport) to transcellular Na+transport. Model predictions suggest that 1) the transepithelial Na+concentration gradient is a major determinant of transport efficiency; 2) CVR in individual cells influences the distribution of net Na+transport along the TAL; 3) CVR responses in conjunction with TGF maintain luminal Na+concentration well above static head levels in the cortical TAL, thereby preventing large decreases in transport efficiency; and 4) under the condition that the distribution of Na+transport along the TAL is quasi-uniform, the tubular fluid axial Cl−concentration gradient near the macula densa is sufficiently steep to yield a TGF gain consistent with experimental data.

The microstructural organization and composition of the corneoscleral shell (CSS) determine the biomechanical behavior of the eye, and are important in diseases such as glaucoma and myopia. However, limited techniques can assess these properties globally, non-invasively and quantitatively. In this study, we hypothesized that multi-modal magnetic resonance imaging (MRI) can reveal the effects of biomechanical or biochemical modulation on CSS. Upon intraocular pressure (IOP) elevation, CSS appeared hyperintense in both freshly prepared ovine eyes and living rat eyes using T2-weighted MRI. Quantitatively, transverse relaxation time (T2) of CSS increased non-linearly with IOP at 0–40 mmHg and remained longer than unloaded tissues after being unpressurized. IOP loading also increased fractional anisotropy of CSS in diffusion tensor MRI without apparent change in magnetization transfer MRI, suggestive of straightening of microstructural fibers without modification of macromolecular contents. Lastly, treatments with increasing glyceraldehyde (mimicking crosslinking conditions) and chondroitinase-ABC concentrations (mimicking glycosaminoglycan depletion) decreased diffusivities and increased magnetization transfer in cornea, whereas glyceraldehyde also increased magnetization transfer in sclera. In summary, we demonstrated the changing profiles of MRI contrast mechanisms resulting from biomechanical or biochemical modulation of the eye non-invasively. Multi-modal MRI may help evaluate the pathophysiological mechanisms in CSS and the efficacy of corneoscleral treatments.

Benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) are two polycyclic aromatic hydrocarbons (PAHs) that exhibit distinctly different mutagenicity and carcinogenicity profiles. Although some studies show that these PAHs produce unstable DNA adducts, conflicting data and arguments have been presented regarding the relative roles of these unstable adducts versus stable adducts, as well as oxidative damage, in the mutagenesis and tumor-mutation spectra of these PAHs. However, no study has determined the mutation spectra along with the stable and unstable DNA adducts in the same system with both PAHs. Thus, we determined the mutagenic potencies and mutation spectra of BP and DBP in strains TA98, TA100 and TA104 of Salmonella, and we also measured the levels of abasic sites (aldehydic-site assay) and characterized the stable DNA adducts (32P-postlabeling/HPLC) induced by these PAHs in TA104. Our results for the mutation spectra and site specificity of stable adducts were consistent with those from other systems, showing that DBP was more mutagenic than BP in TA98 and TA100. The mutation spectra of DBP and BP were significantly different in TA98 and TA104, with 24% of the mutations induced by BP in TA98 being complex frameshifts, whereas DBP produced hardly any of these mutations. In TA104, BP produced primarily GC to TA transversions, whereas DBP produced primarily AT to TA transversions. The majority (96%) of stable adducts induced by BP were at guanine, whereas the majority (80%) induced by DBP were at adenine. Although BP induced abasic sites, DBP did not. Most importantly, the proportion of mutations induced by DBP at adenine and guanine paralleled the proportion of stable DNA adducts induced by DBP at adenine and guanine; however, this was not the case for BP. Our results leave open a possible role for unstable DNA adducts in the mutational specificity of BP but not for DBP.

We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na+ that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ∼330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens’ circulation of Na+ was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium.

Three classes of DNA damage were assessed in human placentas collected (2000–2004) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by 32P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49-312 PAH-DNA adducts/108 nucleotides, were found by IHC/ACIS in 14 immediately fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7–8.6 stable/bulky DNA adducts/108 nucleotides and 0.6–47.2 AB sites/105 nucleotides. For all methods, there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and nonsmokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. Environ. Mol. Mutagen. 52:58–68, 2011. © 2010 Wiley-Liss, Inc.

Detection of the low-frequency (LF; approximately 0.01 Hz) component of renal blood flow, which is theorized to reflect the action of a third renal autoregulatory mechanism, has been difficult due to its slow dynamics. In this work, we used three different experimental approaches to detect the presence of the LF component of renal autoregulation using normotensive and spontaneously hypertensive rats (SHR), both anesthetized and unanesthetized. The first experimental approach utilized a blood pressure forcing in the form of a chirp, an oscillating perturbation with linearly increasing frequency, to elicit responses from the LF autoregulatory component in anesthetized normotensive rats. The second experimental approach involved collection and analysis of spontaneous blood flow fluctuation data from anesthetized normotensive rats and SHR to search for evidence of the LF component in the form of either amplitude or frequency modulation of the myogenic and tubuloglomerular feedback mechanisms. The third experiment used telemetric recordings of arterial pressure and renal blood flow from normotensive rats and SHR for the same purpose. Our transfer function analysis of chirp signal data yielded a resonant peak centered at 0.01 Hz that is greater than 0 dB, with the transfer function gain attenuated to lower than 0 dB at lower frequencies, which is a hallmark of autoregulation. Analysis of the data from the second experiments detected the presence of approximately 0.01-Hz oscillations only with isoflurane, albeit at a weaker strength compared with telemetric recordings. With the third experimental approach, the strength of the LF component was significantly weaker in the SHR than in the normotensive rats. In summary, our detection via the amplitude modulation approach of interactions between the LF component and both tubuloglomerular feedback and the myogenic mechanism, with the LF component having an identical frequency to that of the resonant gain peak, provides evidence that 0.01-Hz oscillations may represent the third autoregulatory mechanism.

Sulfur-containing polycyclic aromatic hydrocarbons (thia-PAHs or thiaarenes) are common constituents of air pollution and cigarette smoke, but only a few have been studied for health effects. We evaluated the mutagenicity in Salmonella TA98, TA100, and TA104 of two sulfur-containing derivatives of benzo[c]phenanthrene, phenanthro[3,4-b]thiophene (P[3,4-b]T), and phenanthro[4,3-b]thiophene (P[4,3-b]T) as well as their dihydrodiol and sulfone derivatives. In addition, we assessed levels of stable DNA adducts (by (32)P-postlabeling) as well as abasic sites (by an aldehydic-site assay) produced by six of these compounds in TA100. P[3,4-b]T and its 6,7- and 8,9-diols, P[3,4-b]T sulfone, P[4,3-b]T, and its 8,9-diol were mutagenic in TA100. P[3,4-b]T sulfone, the most potent mutagen, was approximately twice as potent as benzo[a]pyrene in both TA98 and TA100. Benzo-ring dihydrodiols were much more potent than K-region dihydrodiols, which had little or no mutagenic activity in any strain. P[3,4-b]T sulfone produced abasic sites and not stable DNA adducts; the other five compounds examined, B[c]P, B[c]P 3,4-diol, P[3,4-b]T, P[3,4-b]T 8,9-diol, and P[4,3-b]T 8,9-diol, produced only stable DNA adducts. P[3,4-b]T sulfone was the only compound that produced significant levels of frameshift mutagenicity and induced mutations primarily at GC sites. In contrast, B[c]P, its 3,4-diol, and the 8,9 diols of the phenanthrothiophenes induced mutations primarily at AT sites. P[3,4-b]T was not mutagenic in TA104, whereas P[3,4-b]T sulfone was. The two isomeric forms (P[3,4-b]T and P[4,3-b]T) are apparently activated differently, with the latter, but not the former, involving a diol pathway. This study is the first illustrating the potential importance of abasic sites in the mutagenicity of thia-PAHs.

Two neurohypophysial hormones have been isolated from an avian species, the ostrich, Struthio camelus. Both have been characterized by amino acid analysis and sequence determination. The data obtained suggest that the oxytocin-like hormone is [Ile8-oxytocin] (mesotocin) and the vasopressin-like hormone is [Ile3-vasopressin] (vasotocin). Bioactivity measurements based on urinary conductivity showed vasotocin to be about five times as active as mesotocin.

Although attenuation of anesthetic preconditioning in aged ex vivo heart models has been studied extensively, there are no comparable in vivo studies. To extend previous work and to address a possible mechanism underlying age-related differences, we investigated isoflurane-induced preconditioning and reactive oxygen species (ROS) production in the aged rat heart in vivo.Male Fisher 344 rats were assigned from their respective age groups (young, 3-5 mo; old, 20-24 mo) to either receive 30 min of 1.0 minimum alveolar concentration isoflurane or to a control group. Rats were subjected to coronary artery occlusion for 30 min followed by 2 h of reperfusion. A fluorescent probe for superoxide anion production (dihydroethidium, 1 mg) was administered in the absence of the isoflurane or just before isoflurane exposure in four additional groups. Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and epifluorescence microscopy, respectively.Isoflurane decreased myocardial infarct size of young rats (26.7% +/- 3.0%) compared with young controls (50.9% +/- 1.9%; P0.001), whereas isoflurane did not significantly affect myocardial infarct size of old rats (39.1% +/- 0.9%) compared with old controls (46.5% +/- 2.4%; P0.05). Isoflurane increased ROS levels in young rats (430.5 +/- 95.9 arbitrary units [AU]) compared with young controls (162.7 +/- 25.5 AU; P0.01). In contrast, no significant changes in ROS levels were observed in old animals (316.4 +/- 56.3 AU isoflurane versus 233.8 +/- 59.2 AU control).Reduction in the cardioprotective effects of isoflurane and attenuation of isoflurane-stimulated ROS production were observed in the senescent myocardium in vivo.

We examined the effects of active dehydration by exercise in a hot, humid environment on anaerobic muscular power using a test-retest (euhydrated and dehydrated) design. Seven subjects (age, 27.1 +/- 4.6 years; mass, 86.4 +/- 9.5 kg) performed upper and lower body Wingate anaerobic tests prior to and after a 1.5-hour recovery from a heat stress trial of treadmill exercise in a hot, humid environment (33.1 +/- 3.1C = 55.1 +/- 8.9% relative humidity) until a 3.1 +/- 0.3% body mass loss was achieved. Dehydration was confirmed by a significant body mass loss (P0.001), urine color increase (P = 0.004), and urine specific gravity increase (P = 0.041). Motivation ratings were not significantly different (P = 0.059), and fatigue severity was significantly (P = 0.009) increased 70% in the dehydrated compared to the euhydrated condition. Compared to the euhydrated condition, the dehydrated condition mean power was significantly (P = 0.014) decreased 7.17% in the upper body and 19.20% in the lower body. Compared to the euhydrated condition, the dehydrated condition peak power was significantly (P = 0.013) decreased 14.48% in the upper body and 18.36% in the lower body. No significant differences between the euhydrated and dehydrated conditions were found for decrease in power output (P = 0.219, power = 0.213). Our findings suggest that dehydration of 2.9% body mass decreases the ability to generate upper and lower body anaerobic power. Coaches and athletes must understand that sports performance requiring anaerobic strength and power can be impaired by inadequate hydration and may contribute to increased susceptibility to musculoskeletal injury.

A series of commercial Co−Mo/Al2O3 catalysts, freshly sulfided as well as used in different trickle phase and gas phase reactions, has been characterized by using (scanning) transmission electron m...

Deuteron NMR experiments on dideuterium dissolved in the nematic liquid-crystal solvents 7CB, CCH7, MBBA and PCH7 are reported as a function of temperature. The results are analysed in terms of the average solvent electric field gradient anisotropy (efg) felt by D 2 which is positive with respect to the director in 7CB, CCH7 and PCH7 and negative in MBBA. The order parameter of D 2 is to a large extent rationalized in terms of the interaction between this solvent efg and the solute D 2 molecular quadrupole moment, and its sign is the same as that of the efg.

The environmental pollutant 6-nitrochrysene (6-NC) is a potent carcinogen in several animal models including the rat mammary gland. 6-NC can be activated to intermediates that can damage DNA by simple nitroreduction, ring oxidation, or a combination of ring oxidation and nitroreduction. Only the first pathway (nitroreduction) has been clearly established, and DNA adducts derived from this pathway have been fully characterized in in vitro systems. We also showed previously that the second pathway, ring oxidation leading to the formation of the bay region diol epoxide of 6-NC, is not responsible for the formation of the major DNA adduct in the mammary gland of rats treated with 6-NC. Therefore, in the present study, we explored the validity of the third pathway that involves the combination of both ring oxidation and nitroreduction of 6-NC to form trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C). During the course of this study, we synthesized for the first time 1,2-DHD-6-NHOH-C, N-(deoxyguanosin-8-yl)-6-aminochrysene, and N-(deoxyguanosin-8-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene. Incubation of 1,2-DHD-6-NHOH-C with calf thymus DNA resulted in the formation of three adducts. Upon LC/MS combined with 1H NMR analyses, the first eluting adduct was identified as 5-(deoxyguanosin-N2-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene [5-(dG-N2-yl)-1,2-DHD-6-AC], the second eluting adduct was identified as N-(deoxyguanosin-8-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene, and the last was identified as N-(deoxyinosin-8-yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene. We also report here for the first time that among those adducts identified in vitro, only 5-(dG-N2-yl)-1,2-DHD-6-AC is the major DNA lesion detected in the mammary glands of rats treated with 6-NC.

A mathematical model previously formulated by us predicts that limit-cycle oscillations (LCO) in nephron flow are mediated by tubuloglomerular feedback (TGF) and that the LCO arise from a bifurcation that depends heavily on the feedback gain magnitude, γ, and on its relationship to a theoretically determined critical value of gain, γc. In this study, we used that model to show how sustained perturbations in proximal tubule flow, a common experimental maneuver, can initiate or terminate LCO by changing the values of γ and γc, thus changing the sign of γ - γc. This result may help explain experiments in which intratubular pressure oscillations were initiated by the sustained introduction or removal of fluid from the proximal tubule (Leyssac PP and Baumbach L. Acta Physiol Scand 117: 415–419, 1983). In addition, our model predicts that, for a range of TGF sensitivities, sustained perturbations that initiate or terminate LCO can yield substantial and abrupt changes in both distal NaCl delivery and NaCl delivery compensation, changes that may play an important role in the response to physiological challenge.

Thioarenes, sulfur-containing polycyclic aromatic compounds, are environmental contaminants suspected of posing human health risks. In this study, 5-nitrobenzo[b]naphtho[2,1-d]thiophene (5-nitro-BNT), a nitrated-thioarene, was examined for its mutagenicity, metabolism and subsequent formation of DNA adducts. 5-Nitro-BNT was weakly mutagenic in Salmonella typhimurium strains TA98 and TA100 without Aroclor-1254-induced rat liver S9 (S9), and its activity was increased in the presence of S9. Anaerobic metabolism of 5-nitro-BNT by S9 or xanthine oxidase (XO) produced one major metabolite, identified as 5-amino-BNT by NMR, MS, and UV spectroscopy and by comparison with an authentic standard. Aerobic S9 metabolism of 5-nitro-BNT produced a major metabolite, identified as trans-9,10-dihydroxy-9,10-dihydro-5-nitro-BNT (5-nitro-BNT-9,10-diol). Also present was a minor amount of 5-amino-BNT and trans-9,10-dihydroxy-9,10-dihydro-5-amino-BNT (5-amino-BNT-9,10-diol). DNA adduct analyses were performed using the (32)P-postlabeling assay and reversed-phase HPLC. Three major XO-derived calf thymus DNA adducts were detected. On the basis of their chromatographic mobilities, two adducts were identified as reaction products of 5-nitro-BNT with 2'-deoxyguanosine and one adduct with 2'-deoxyadenosine. Incorporation of allopurinol (a specific XO inhibitor) in the incubation mixture resulted in loss of all three adducts, confirming enzymatic mediation by XO. Aerobic S9 activation of 5-nitro-BNT with calf thymus DNA produced three adducts. On the basis of their chromatographic mobilities, two were identified as reaction products of 5-nitro-BNT with 2'-deoxyguanosine and one with 2'-deoxyadenosine. Incorporation of 1-aminobenzotriazole (a P450 inhibitor) in the incubation mixture resulted in a loss of these adducts, confirming enzymatic mediation by P450. Aerobic S9-catalyzed metabolism of 5-nitro-BNT-9,10-diol produced the same DNA adducts as observed with 5-nitro-BNT. Aerobic S9-catalyzed metabolism of 5-amino-BNT-9,10-diol produced the same deoxyadenosine-derived DNA adducts as observed with 5-nitro-BNT and 5-nitro-BNT-9,10-diol. These results provide additional information that both ring oxidation and nitroreduction are involved in the metabolism, DNA adduct formation and mutagenicity of 5-nitro-BNT.

In order to explain the adduct patterns observed from the human CYP1A1-mediated binding of dibenzo[a, l]pyrene (DB[a, l]P) to DNA, we have investigated the further metabolism and DNA adduct activity of trans-DB[a, l]P-8,9-diol by induced rat liver and human CYP1A1 microsomes. trans-DB[a, l]P-8,9-diol was synthesized and metabolic studies with β-naphthoflavone-induced rat liver microsomes indicated three major metabolites: 2 diastereomers of trans,trans-8,9,11,12-tetrahydro-8,9,11,12-tetrahydroxy-DB[a, l]P and 8,9,13,14-tetrahydro-8,9,13,14-tetrahydroxy-DB[a, l]P. DB[a, l]P when activated by CYP1A1/epoxide hydrase (EH) and calf thymus DNA gave a complex pattern of DNA adducts most of which cochromatograph with syn- and anti-DB[a, l]P fjord region diol epoxide-DNA standards. Two highly polar eluting adducts were also observed, one which cochromatographs with the single major DNA adduct obtained from the CYP1A1/EH activation of trans-DB[a, l]P-8,9-diol. The relative retention time of this adduct sug...

The comparative genotoxic effects of racemic trans-8,9-dihydroxy-8, 9-dihydrodibenzo[a,l]pyrene (trans-DB[a,l]P-8,9-diol), the metabolic K-region dihydrodiol of dibenzo[a,l] pyrene (DB[a,l]P) (dibenzo[def, p]chrysene) and DB[a,l]P in transformable mouse embryo C3H10T(1)/(2)Cl8 (C3H10T(1)/(2)) fibroblasts was investigated. The C3H10T(1)/(2) mouse embryo morphological cell-transforming activities of these polycyclic aromatic hydrocarbons (PAHs) were assayed using concentration-response studies. At concentrations of 33 nM and above both trans-DB[a,l]P-8,9-diol and DB[a,l]P produced significant (and similar) numbers of type II and III foci per dish and numbers of dishes with type II and II foci. Concomitant cytotoxicity studies revealed a reduction in colony survival of approximately 25% up to 198 nM for both PAHs. DNA adducts of trans-DB[a,l]P-8,9-diol and DB[a,l]P in C3H10T(1)/(2) cells were analyzed by a (32)P-post-labeling TLC/HPLC method. No adducts were observed in the DNA of C3H10T(1)/(2) cells treated with trans-DB[a, l]P-8,9-diol at concentrations that induced morphological cell transformation. Under the same exposure and chromatographic conditions, DNA adducts of deoxyadenosine and deoxyguanosine derived from the fjord region anti-DB[a,l]P-11,12-diol-13,14-epoxide and syn-DB[a,l]P-11,12-diol-13,14-epoxide were observed in the DNA of DB[a,l]P-treated cells. These results indicate that trans-DB[a,l]P-8, 9-diol has intrinsic genotoxic activity equal to that of DB[a,l]P, based on morphological cell transformation of mouse embryo fibroblasts. The activity of trans-DB[a,l]P-8,9-diol is apparently not associated with the formation of observable stable covalent DNA adducts. These results suggest that under appropriate conditions, trans-DB[a,l]P-8,9-diol may serve as an intermediate in the genotoxicity of DB[a,l]P.

The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 μM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on l-arginine but notd-arginine, and the responses to CO were inhibited by pretreatment with NG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 μM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord-region dihydrodiol, trans-DB[a,l]P-11, 12-diol, to syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides (syn- and anti-DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using (32)P postlabeling, thin-layer chromatography, and high-pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE-DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a, l]PDE-DNA adducts and unidentified polar adducts at rates threefold and 17-fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti- and syn-DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti-DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn-DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74-82, 1999. Published 1999 Wiley-Liss, Inc.

Metabolic activation studies of dibenzo[a,l]pyrene (DB[a,l]P) (dibenzo[def,p]chrysene), an extremely potent environmental carcinogen, have been focused on metabolism at the fjord region, a region associated with high mutagenic and carcinogenic activities of the corresponding fjord-region DB[a,l]P-11,12-diol-13,14-epoxides. DB[a,l]P is metabolized by beta-naphthoflavone (BNF)- and 3-methylcholanthrene-induced rat liver microsomes and a recombinant human P450 1A1 system to two major dihydrodiols, the K-region dihydrodiol, DB[a,l]P-8,9-dihydrodiol (DB[a,l]P-8,9-diol), and the fjord-region dihydrodiol, DB[a,l]P-11,12-dihydrodiol. We have investigated the further metabolic activation of DB[a,l]P-8,9-diol by BNF-induced rat liver microsomes and a recombinant human P450 1A1 system with epoxide hydrolase to DB[a,l]P-bis-diols and to DNA adducts. (+/-)-trans-DB[a,l]P-8,9-diol was synthesized and resolved into its enantiomers. Racemic trans-DB[a,l]P-8,9-diol was metabolized by BNF-induced rat liver microsomes to six metabolites: two diastereomers of trans,trans-DB[a,l]P-8,9:11,12-bis-diol, two diastereomers of trans,cis-DB[a,l]P-8,9:11,12-bis-diol, and two diastereomers of trans-DB[a,l]P-8,9:13,14-bis-diol as characterized by NMR, MS, and UV spectroscopy. Metabolic studies using both enantiomeric (-)- and (+)-trans-DB[a,l]P-8,9-diol further demonstrated that each diastereomer of trans,trans-DB[a,l]P-8,9:11, 12-bis-diol and trans-DB[a,l]P-8,9:13,14-bis-diol was comprised of two enantiomers. Similarly, incubations of enantiomeric or racemic trans-DB[a,l]P-8,9-diol with a recombinant human P450 1A1 system and epoxide hydrolase also gave the same two enantiomeric mixtures of diastereomers of trans,trans-DB[a,l]P-8,9:11,12-bis-diol and the same two enantiomeric mixtures of diastereomers of trans-DB[a,l]P-8, 9:13,14-bis-diol. This suggested that the microsomal oxidations of (-)- and (+)-trans-DB[a,l]P-8,9-diol were stereospecific. The stereospecific formation of enantiomers of trans-DB[a,l]P-8,9-diol from DB[a,l]P was examined using both BNF-induced rat liver microsomes and a recombinant human P450 1A1 system with epoxide hydrolase. Stereospecificity was observed as both metabolic systems favored the formation of (-)-trans-DB[a,l]P-8,9-diol by 8-9-fold. DNA adduct studies were undertaken using TLC/HPLC 32P-postlabeling techniques. In the presence of a recombinant human P450 1A1 system with epoxide hydrolase, DB[a,l]P gave two groups of calf thymus DNA adducts. The group of later-eluting adducts were identified as arising from syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides, while the more polar early-eluting adducts were derived, in part, from the further activation of trans-DB[a,l]P-8,9-diol. Our data indicate that, in P450 1A1-mediated microsomal incubations, DB[a,l]P is metabolized to trans-DB[a,l]P-8,9-diol which is further metabolized to DB[a,l]P-bis-diols. trans-DB[a,l]P-8,9-diol is metabolically activated to intermediates that can bind to DNA and give DNA adducts similar to those observed with DB[a,l]P. These results indicate that DB[a,l]P can be metabolically activated by both fjord-region and K-region pathways.

Studies have demonstrated differences between the brown bullhead (Ameriurus nebulosus) (BB) and a closely related species of catfish, the channel catfish (Ictalurus punctatus) (CC) in activity of important liver enzymes that may affect their ability to activate or detoxify certain procarcinogens. We have measured the formation and persistence of benzof[a]pyrene-DNA adducts in the liver of these two species using the 32P-postlabeling (PPL) method, along with the effect of prior induction by β-naphthoflavone (βNF). In this time-course study, a total of 80 fish from each species were used. Half were induced with βNF (i.p. injections of 10 mgkg−1 at day 3 and 2) followed by a single benzo[a]pyrene (BaP) i.p. injection (20 mg kg−1). Liver DNA and microsomes were sampled on day 1, 3, 7, 14 and 45 after the BaP dosage—adducts and ethoxyresorufin-O-deethylase (EROD) activity were analyzed. EROD activity in CC was significantly higher at most time points than in BB. In contrast, the BaP-DNA adduct profile revealed a significantly higher level of adducts in the BB than the CC. Adduct levels at most time points were significantly higher in the BB than in the CC at p

We have measured the formation and persistence of benzo[a]pyrene (BaP)-DNA adducts in the liver of two closely related species of fish, the brown bullhead (Ameriurus nebulosus) and the channel catfish (Ictalurus punctatus) using the 32P-postlabeling method. Liver microsomal ethoxyresorufin-O-deethylase (EROD) activity, arylhydrocarbon hydroxylase (AHH) activity, and in vitro microsome-mediated DNA binding were all significantly higher in the channel catfish. In an in vivo time-course experiment, fish were either induced with beta NF followed by a single BaP i.p. injection (20 mg/kg) or treated with corn oil. BaP-DNA adducts and EROD activity in liver were analyzed 1, 3, 7, 14, and 45 days after the BaP dosage. As in the in vitro experiments, EROD activities in channel catfish were significantly higher at most time points than in bullhead liver (p0.05). However, in contrast to the in vitro data, the BaP-DNA adduct profile revealed significantly higher levels of adducts in the bullhead than the channel catfish throughout the time course (p0.05). Prior induction with beta NF did not significantly affect the level or type of adduct binding to DNA in either species. Further characterization of the major adduct by HPLC confirmed it to be the anti-BPDE-dGuo adduct. Analysis of tissue distribution of [14C]BaP in the two species suggested similar absorption and initial distribution, but slower elimination from the liver of bullhead than the catfish. The BaP-adduct profiles were consistent with the relative species susceptibility to polycyclic aromatic hydrocarbon-induced liver neoplasia. EROD activities, however, were negatively associated with adduct levels following in vivo exposure.

Due to its widespread use as a preemergent herbicide, alachlor has been detected as a groundwater contaminant. The procarcinogen, 2,6-dinitrotoluene (DNT), a by-product of the munitions industry and a precursor to polyurethane production, is found in the manufacturing waste stream. This study explores the effect of alachlor treatment on the bioactivation of DNT by examining urine mutagenicity, intestinal enzymes, and hepatic DNA adducts to detect changes in metabolism. Five-week-old male rats were treated daily by gavage with 50 mg/kg of alachlor for up to 5 weeks while control animals received an equal volume of peanut oil. At 1, 3, and 5 weeks following the initial alachlor dose, animals were administered p.o. 75 mg/kg DNT or DMSO. Urine was collected for 24 hr in metabolism cages. Following incubation with sulfatase and beta-glucuronidase, urines were individually concentrated by C-18 solid phase extraction, dried under N2, and prepared for bioassay in Salmonella typhimurium strain TA98 with and without metabolic activation. Urine from peanut oil- and alachlor-treated rots was not mutagenic. Even though calf thymus DNA-alachlor adducts formed in vitro, no hepatic DNA adducts were detected in vivo in these two treatment groups. Interestingly, a significant increase in excretion of mutagenic urine from DNT-treated rats was observed following 3 weeks of alachlor treatment in the absence of S9 (690 +/- 130 vs. 339 +/- 28 revertants/ml) which corresponded to increased DNT-related hepatic DNA adduct formation (5.90 +/- 0.88 adducts/10(8) nucleotides vs. 10.56 x +/- 0.59 adducts/10(8) nucleotides [relative adduct level (RAL)]). Elevation in the production of mutagenic urine from control and treated animals was linked to increases in intestinal nitroreductase and beta-glucuronidase activities; however, the only significant alachlor-related effects were an increase in small intestinal 1-week beta-glucuronidase and 5-week dehydrochlorinase activities. The increased urine mutagenicity and hepatic DNA adduct formation indicates that alachlor has a transient effect on DNT bioactivation that apparently is unrelated to intestinal bioactivation.

To characterize the effects on the rat renal preglomerular microvasculature of insulin-like growth factor I (IGF-I), experiments were performed using the in vitro blood-perfused juxtamedullary nephron preparation. IGF-I induced a reversible vasodilation of pre- but not postglomerular microvessels in a dose-dependent manner (10−9–10−7M). The IGF-I-induced vasodilation was similar in all preglomerular vascular segments: interlobular artery, 11.5 ± 1.2% of control ( n = 16); mid-afferent arterioles, 11.6 ± 1.7% ( n = 24); and juxtaglomerular afferent segments, 16.1 ± 2.8% ( n = 19). Renal autoregulatory capacity was not reduced by IGF-I. Pretreatment with the nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (10−4M) completely inhibited the vasodilatory response to IGF-I. IGF-I induced a rapid increase of NO concentration in intact renal microvessels, monitored by a NO-selective voltametric microelectrode. Pretreatment with the cyclooxygenase inhibitor indomethacin (10−5M) not only abrogated the IGF-I-induced dilation, but, moreover, IGF-I elicited a small but significant (∼10%) vasoconstriction in all preglomerular vessels. These results indicate that the renal vascular effects of IGF-I involve activation of two endogenous vasodilators (NO and vasodilatory prostaglandins). In addition, IGF-I may also release an undefined vasoconstrictor.

C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts were used to study the in vitro carcinogenic activities of dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P). The morphological transforming activities of these rodent carcinogens were compared using replicate concentration-response studies. In concentration ranges where both polycyclic aromatic hydrocarbons (PAHs) were active, DB[a,l]P proved to be four to 12 times as potent as B[a]P based on concentration. At lower concentrations DB[a,l]P was active at 0.10 and 0.20 microM, concentrations where B[a]P was inactive. This makes DB[a,l]P the most potent non-methylated PAH evaluated to date in C3H10T1/2 cells. DNA adducts of DB[a,l]P in C3H10T1/2 cells were analyzed by both TLC and TLC/HPLC 32P-postlabeling methods using mononucleotide 3'-phosphate adduct standards derived from the reactions of anti-DB[a,l]P-11,12-diol-13,14-epoxide (anti-DB[a,l]PDE) and syn-DB[a,l]P-11,12-diol-13,14-epoxide (syn-DB[a,l]PDE) with deoxyadenosine 3'-monophosphate and deoxyguanosine 3'-monophosphate. All of the DNA adducts observed in C3H10T1/2 cells treated with DB[a,l]P were identified as being derived from the metabolism of DB[a,l]P to its fjord region diol epoxides through DB[a,l]P-11,12-diol. The predominant adduct was identified as an anti-DB[a,l]PDE-deoxyadenosine adduct. Other major adducts were anti-DB[a,l]PDE-deoxyguanosine and syn-DB[a,l]PDE-deoxyadenosine adducts with minor amounts of syn-DB[a,l]PDE-deoxyguanosine adducts. These DNA adduct data are consistent with similar findings of DB[a,l]PDE-deoxyadenosine adducts in mouse skin studies and human mammary cells in culture.

The spatial organization of autoregulatory responses (AR) was assessed in couples of afferent arterioles (AA), either grouped as anatomic pairs or branched sequentially along the same arcuate arterial branch (ArcB). With blood-perfused juxtamedullary nephron (JMN) preparations, AR were elicited by raising blood perfusion pressure from 60 to 120 mmHg and quantified by videomicroscopy as pressure-induced constrictions. Paired AA had unequal lengths (long-to-short ratio, 1.9 +/- 0.1; n = 36); however, no statistical difference in AR was found between long and short AA at juxtaglomerular or early AA (EAA) sites. Sequentially branched AA had the same length heterogeneity as paired AA (proximal-to-distal AA length ratio, 2.0 +/- 0.2; n = 30). However, AR exhibited a significant axial gradient, being higher in distal than in proximal AA or ArcB sites. In both AA branching patterns, EAA and nearby sites of the feed arteries had similar AR. Hence, our results are consistent with hemodynamic coupling in paired JMN. Around branching sites, AR are spatially organized in a way consistent with electrotonic vascular coupling.

Immunodominance is a fundamental property of CD8+ T cell responses to viruses and vaccines. It had been observed that route of administration alters immunodominance after vaccinia virus (VACV) infection, but only a few epitopes were examined and no mechanism was provided. We re-visited this issue, examining a panel of 15 VACV epitopes and four routes, namely intradermal (i.d.), subcutaneous (s.c.), intraperitoneal (i.p.) and intravenous (i.v.) injection. We found that immunodominance is sharpened following peripheral routes of infection (i.d. and s.c.) compared with those that allow systemic virus dissemination (i.p. and i.v.). This increased immunodominance was demonstrated with native epitopes of VACV and with herpes simplex virus glycoprotein B when expressed from VACV. Responses to some subdominant epitopes were altered by as much as fourfold. Tracking of virus, examination of priming sites, and experiments restricting virus spread showed that priming of CD8+ T cells in the spleen was necessary, but not sufficient to broaden responses. Further, we directly demonstrated that immunodomination occurs more readily when priming is mainly in lymph nodes. Finally, we were able to reduce immunodominance after i.d., but not i.p. infection, using a VACV expressing the costimulators CD80 (B7-1) and CD86 (B7-2), which is notable because VACV-based vaccines incorporating these molecules are in clinical trials. Taken together, our data indicate that resources for CD8+ T cell priming are limiting in local draining lymph nodes, leading to greater immunodomination. Further, we provide evidence that costimulation can be a limiting factor that contributes to immunodomination. These results shed light on a possible mechanism of immunodomination and highlight the need to consider multiple epitopes across the spectrum of immunogenicities in studies aimed at understanding CD8+ T cell immunity to viruses., Author Summary During an infection, the adaptive immune system responds to many epitopes of the pathogen but the strength of these responses varies widely. This unequal distribution of responses across a range of epitopes is known as immunodominance and understanding why it occurs is a fundamental problem in immunology. It is also relevant to vectored vaccines where the intention is to raise immunity against an antigen of choice, but responses to vector epitopes may dominate. We show that the route of infection changes the extent to which the strongest epitope can dominate CD8+ T cell responses to vaccinia virus (VACV). The cause of this phenomenon is linked to virus spread and therefore the different lymphoid organs that prime T cell responses for each route. We also show that local draining lymph nodes are sites of more robust competition between T cells compared with the spleen, explaining why immunodominance differs according to route. Finally the normally heightened immunodominance after peripheral VACV infection can be reduced by expression the costimulators CD80 (B7-1) and CD86 (B7-2) from the virus. In summary, we have carefully dissected immunodominance using VACV as a model and in doing so exposed general features of CD8+ T cell immunity to pathogens.

This study is a part of an ongoing interdisciplinary project on the health effects of air pollution (Teplice Program) in the highly polluted district of Teplice, Northern Bohemia. In our previous studies we found the relationship between DNA adduct levels detected in white blood cells of selected Teplice population and personal exposure to PAH associated with respirable particles. In this pilot study we used 32P-postlabeling assay for DNA adduct detection in an in vitro model system for evaluation of genotoxic activity of fractionated urban air extractable organic matter (EOM). To identify some of the specific DNA adducts formed we coupled TLC with HPLC analysis of labeled adducts. The urban air particles were collected by high-volume sampler during January-March 1992 in Teplice. EOM was extracted by dichlormethane (DCM) and crude extract was fractionated into five fractions to obtain a gross partition of different chemical classes. Fractions were incubated with calf thymus DNA (dose 100 μg/ml in...

Previous experiments showed that mouse lenses have an intracellular hydrostatic pressure that varied from 335 mm Hg in central fibers to 0 mm Hg in surface cells. Model calculations predicted that in larger lenses, all else equal, pressure should increase as the lens radius squared. To test this prediction, lenses of different radii from different species were studied.All studies were done in intact lenses. Intracellular hydrostatic pressures were measured with a microelectrode-manometer-based system. Membrane conductances were measured by frequency domain impedance analysis. Intracellular Na(+) concentrations were measured by injecting the Na(+)-sensitive dye sodium-binding benzofuran isophthalate.Intracellular hydrostatic pressures were measured in lenses from mice, rats, rabbits, and dogs with radii (cm) 0.11, 0.22, 0.49, and 0.57, respectively. In each species, pressure varied from 335 ± 6 mm Hg in central fiber cells to 0 mm Hg in surface cells. Further characterization of transport in lenses from mice and rats showed that the density of fiber cell gap junction channels was approximately the same, intracellular Na(+) concentrations varied from 17 mM in central fiber cells to 7 mM in surface cells, and intracellular voltages varied from -45 mV in central fiber cells to -60 mV in surface cells. Fiber cell membrane conductance was a factor of 2.7 times larger in mouse than in rat lenses.Intracellular hydrostatic pressure is an important physiological parameter that is regulated in lenses from these different species. The most likely mechanism of regulation is to reduce the density of open Na(+)-leak channels in fiber cells of larger lenses.

Diesel exhaust extracts contain many carcinogenic compounds which have been shown to form polycyclic aromatic hydrocarbon (PAH)- and nitrated PAH-DNA adducts in rodent skin and lung. The aim of this study was to characterize by 32P-postlabeling, TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formed in vitro and in vivo by diesel extracts. The diesel particle extracts had known concentrations of benzo[a]pyrene, benzo[b,j,k]-fluoranthenes (B[b,j,k]F) and chrysene. DNA adducts were analyzed in calf thymus DNA incubated in vitro with PAHs activated by S9 mix and in skin and lung DNA from topically treated mice. The main diesel-derived DNA adduct formed in vitro and in vivo did not co-migrate on HPLC and large TLC plates with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE)-, B[b]F-,B[j]F-,B[k]F-or chrysene-DNA adduct standards. By co-chromatography DNA adducts formed by chrysene from both in vitro and in vivo samples were identified. Nissan diesel extract containing higher PAH concentrations than Volkswagen automobile extract formed skin DNA adducts that co-migrated with chrysene- and anti BPDE- DNA-derived adducts. We conclude that the use of a highly sensitive 32P-postlabeling method combined with HPLC improves the identification of PAH adducts formed by complex mixtures such as diesel exhaust extracts.

Thick ascending limb (TAL) cells are capable of reducing tubular fluid Na+concentration to as low as ∼25 mM, and yet they are thought to transport Na+efficiently owing to passive paracellular Na+absorption. Transport efficiency in the TAL is of particular importance in the outer medulla where O2availability is limited by low blood flow. We used a mathematical model of a TAL cell to estimate the efficiency of Na+transport and to examine how tubular dilution and cell volume regulation influence transport efficiency. The TAL cell model represents 13 major solutes and the associated transporters and channels; model equations are based on mass conservation and electroneutrality constraints. We analyzed TAL transport in cells with conditions relevant to the inner stripe of the outer medulla, the cortico-medullary junction, and the distal cortical TAL. At each location Na+transport efficiency was computed as functions of changes in luminal NaCl concentration ([NaCl]), [K+], [NH4+], junctional Na+permeability, and apical K+permeability. Na+transport efficiency was calculated as the ratio of total net Na+transport to transcellular Na+transport. Transport efficiency is predicted to be highest at the cortico-medullary boundary where the transepithelial Na+gradient is the smallest. Transport efficiency is lowest in the cortex where luminal [NaCl] approaches static head.

Intradermal injection of vaccinia virus in the ear pinnae of mice provides a model of dermal infection and vaccination. The key features of this model are the appearance of a lesion on the surface of the ear that can be measured as a clinical sign of disease and substantial growth of virus in the infected skin in the absence of systemic spread. In addition, infected ears can be easily removed to allow virological, histological, and cellular analyses. Finally, evaluation of the roles of virus (and presumably also host) genes in vaccinia virus pathogenesis in the intradermal model can yield different results than similar experiments using other routes and may reveal otherwise unknown functions.

It has been hypothesized that fluctuations of the ionic composition in the interstitium of juxtaglomerular apparatus (JGA) modulate the function of extraglomerular mesangial cells (MC), thereby participating in tubuloglomerular feedback (TGF) signal transmission. We examined the effects of isosmotic reductions in ambient sodium concentration ([Na+]) and [Cl-] on cytosolic calcium concentration ([Ca2+]i) in cultured rat MC. Rapid reduction of [Na+] or [Cl-] in the bath induced a concentration-dependent rise in [Ca2+]i. MC are much more sensitive to decreases in ambient [Cl-] than to [Na+]; a decrease in [Cl-] as small as 14 mM was sufficient to elicit a detectable [Ca2]i response. These observations suggest that MC can be readily stimulated by modest perturbations of extracellular [Cl-]. Next, we examined whether activation of MC by lowered ambient [Cl-] influences cellular nitric oxide (NO) production. Using an amperometric NO sensor, we found that a 13 mM decrease in ambient [Cl-] caused a rapid, Ca2+/calmodulin-dependent rise in NO release from MC. This response was not inhibitable by dexamethasone, indicating the involvement of the constitutive rather than the inducible type of NO synthase in MC. In addition, the NO release was blunted by indomethacin pretreatment, suggesting that a metabolite(s) of cyclooxygenase regulates the activation of NO synthase in MC. Our findings that small perturbations in external [Cl-] stimulate MC to release NO, a highly diffusible and rapidly acting vasodilator, provide a possible mechanism to explain the transmission of the signal for the TGF response within the JGA.

A mathematical model of the renal vascular and tubular systems was used to examine the possibility that synergistic interactions might occur between the tubuloglomerular feedback (TGF) and myogenic autoregulatory mechanisms in the kidney. To simulate the myogenic mechanism, the renal vasculature was modelled with a resistance network where the total preglomerular resistance varies with intravascular pressure. In addition, a steady-state model of glomerular filtration, proximal and Henle's loop reabsorption, and TGF-modulation of afferent arteriolar resistance was derived. The results show that, if TGF acts on the distal portion of the preglomerular vasculature, then any TGF-induced vasoconstriction should raise upstream intravascular pressure and, thereby, trigger a myogenic response in the more proximal vascular segments, a phenomenon referred to as an ascending myogenic (AMYO) response. The model further predicts that the magnitude of the AMYO response can be similar in magnitude to the TGF-induced increment in afferent resistance. Hence, the effects of TGF excitation on whole kidney hemodynamics may be much greater than pedicted from measurements in single nephrons. Moreover, a significant fraction of the intrinsic myogenic autoregulatory response to increased renal perfusion pressure may result from a synergistic interaction between the TGF and myogenic mechanisms.

This paper describes a multiperiod investment model for processing networks with dedicated and flexible plants. Flexibility can be characterized by feedstocks, products, or any combination, and both batch and continuous plants can be modeled. The model is a multiperiod mixed integer linear programming model which, given a forecast of product demands and pricing, maximizes the net present value of a processing network's operations and expansion decisions over a long time horizon. Application of the model extension is illustrated with a small example. Its impact on an industrial application is also reported.

We fabricated carbon nano-partitions (CNPs) which vertically standing on the metal substrates by radio-frequency magnetron sputtering for dissipating heat applications. Considerable enhancement of the conductive heat transfer has been confirmed and the enhancement depends on the spacing density of nano partitions. The optimum nano-porous density could enhanced the heat transfer when the lower density CNP was made. In our experiments, the enhancement is basically according to the system thermal resistance and it can be reduced by 7.8% to 16.8% which compared with the control samples. Besides, we also make a trial on LEDs. The light output of different colored LEDs responds differently to temperature changes. For the most sensitive color, amber, we can diminish the decrease of light output power by about 15% and the dominant wavelengths shift by about 2nm less than the one is not coated on CNPs.

The anisotropic couplings obtained from the 1H nuclear magnetic resonance (NMR) spectra of partially oriented molecules with internal rotation are difficult to analyse because the order parameters for each conformer cannot be deduced without assumptions. The purpose of this paper is to show how a model for the anisotropic short-range potential can be useful in the interpretation of the high-resolution 1H NMR spectra of the flexible molecules furfural, 2-chlorobenzaldehyde, and 2,2′-dithiophene dissolved in a 55% Merck ZLI 1132-45% N-(p-ethoxybenzylidene)-2,6-dideutero-p'-n-butylaniline (w/w) mixture. In this nematic mixture there is no contribution to the anisotropic potential from the interaction between the solute molecular quadrupole moment and the average electric field gradient of the liquid crystal solvent; the dominant anisotropic intermolecular potential in this mixture is taken to result solely from short-range interactions. We find good agreement between experiment and the order parameters calcu...