Cardiopathic effects of dichloroacetate in the fetal Long-Evans rat.

Dichloroacetic acid (DCA) is a by-product of the chlorine disinfection of water and may occur in treated water at levels exceeding 100 micrograms/L. Previous studies revealed teratogenic effects, particularly heart malformations, at high doses (900-2,400 mg/kg given on days 6-15 of pregnancy). In a series of three studies, groups of 7-10 Long-Evans rats were dosed with 1,900 mg/kg of DCA on days 6-8, 9-11, or 12-15; with 2,400 mg/kg on days 10, 11, 12, or 13; and with 3,500 mg/kg on days 9, 10, 11, 12, or 13, in an attempt to determine the most sensitive period and further characterize the heart defect. In a fourth study, six dams were treated with 1,900 mg/kg of DCA days 6-15 of pregnancy, and 56 fetuses were harvested for light microscopy of the heart. Eight control fetuses from four litters were also examined. No heart malformations were seen in the groups treated with 1,900 mg/kg DCA days 6-8 but were present in the group treated on days 9-11 and 12-15, with the higher incidence occurring on days 12-15. Single doses of 2,400 mg/kg DCA given on days 10, 11, 12, or 13 resulted in a much lower incidence of cardiac malformations, which occurred only on days 10 and 12. The high dose of DCA (3,500 mg/kg) did not increase the incidence of heart defects but showed that dosing on day 9 as well as on days 10 and 12 would produce the defect. The defects seen were characterized as high interventricular septal defects (H-IVSD). Light microscopy showed that the defect was caudal to the semilunar valves, with the anterior right wall of the aorta communicating with the right ventricle. Another aspect of the defect is at the level of the semilunar valves, with the right cusp or sinus of Valsalva in communication with the right ventricle. The defects are discussed more fully and methods for further study suggested.     

Performance of the Columbia,Missouri, treatment wetland

Constructed treatment wetlands have served the City of Columbia, MO, for fourteen years. Four free water surface wetland units in series, comprised of 23 cells, are an addition to the activated sludge wastewater treatment plant, for the purpose of added biochemical oxygen demand (BOD) and total suspended solids (TSS) control. The system operates year-round, and supplies water to the Eagle Bluffs Conservation Area for wetland maintenance. The cattail wetlands processed an average of 57,000 m³/d, at a water depth of 20 cm. The resulting detention time was approximately 2 days, and the hydraulic loading was 13 cm/d. Water temperatures were warm leaving the treatment plant and in the wetlands in winter, because of the short detention. The period of record average carbonaceous biochemical oxygen demand (CBOD) leaving the wetlands was 5.0 mg/L, and the TSS was 14.7 mg/L. Dissolved oxygen was depressed in summer, likely because of the high sediment demand. Nutrient concentrations were only minimally reduced, total nitrogen (TN) by 22% and total phosphorus (TP) by 6%. However, load reductions were maximal, 98 t/yr for nitrogen, and 3.6 t/yr for phosphorus. Fecal coliforms were reduced by 98%, and E. coli by 95%. First order rate coefficients were high for CBOD (64 m/yr), nitrate (61 m/yr) and organic nitrogen (42 m/yr), but relatively low for ammonia (8 m/yr) and phosphorus (5.7 m/yr). Nitrogen removal was strongly affected by vegetative uptake. Sediment accretion in the wetland inlets was substantial, at 1.6 cm/yr in the inlets to the upstream wetland units. Muskrats caused vegetation damage, and waterfowl use was high in winter, causing TSS excursions.     

Cytokine Responses to Novel Antigens in an Indian Population Living in an Area Endemic for Visceral Leishmaniasis

Background

There are no effective vaccines for visceral leishmaniasis (VL), a neglected parasitic disease second only to malaria in global mortality. We previously identified 14 protective candidates in a screen of 100 Leishmania antigens as DNA vaccines in mice. Here we employ whole blood assays to evaluate human cytokine responses to 11 of these antigens, in comparison to known defined and crude antigen preparations.

Methods

Whole blood assays were employed to measure IFN-γ, TNF-α and IL-10 responses to peptide pools of the novel antigens R71, Q51, L37, N52, L302.06, J89, M18, J41, M22, M63, M57, as well as to recombinant proteins of tryparedoxin peroxidase (TRYP), Leishmania homolog of the receptor for activated C kinase (LACK) and to crude soluble Leishmania antigen (SLA), in Indian patients with active (n = 8) or cured (n = 16) VL, and in modified Quantiferon positive (EHC^+ve, n = 20) or modified Quantiferon negative (EHC^−ve, n = 9) endemic healthy controls (EHC).

Results

Active VL, cured VL and EHC^+ve groups showed elevated SLA-specific IFN-γ, but only active VL patients produced IL-10 and EHC^+ve did not make TNF-α. IFN-γ to IL-10 and TNF-α to IL-10 ratios in response to TRYP and LACK antigens were higher in cured VL and EHC^+ve exposed individuals compared to active VL. Five of the eleven novel candidates (R71, L37, N52, J41, and M22) elicited IFN-γ and TNF-α, but not IL-10, responses in cured VL (55–87.5% responders) and EHC^+ve (40–65% responders) subjects.

Conclusions

Our results are consistent with an important balance between pro-inflammatory IFNγ and TNFγ cytokine responses and anti-inflammatory IL-10 in determining outcome of VL in India, as highlighted by response to both crude and defined protein antigens. Importantly, cured VL patients and endemic Quantiferon positive individuals recognise 5 novel vaccine candidate antigens, confirming our recent data for L. chagasi in Brazil, and their potential as cross-species vaccine candidates.     

Developmental toxicity of dichloroacetate in the rat.

Dichloroacetic acid (DCA) is a principal by-product of the chlorine disinfection of water containing humic and fulvic acids, and is also a drug of interest in the therapeutic management of metabolic disorders. The developmental effects of DCA were evaluated in the pregnant Long-Evans rat. In two separate studies, animals were dosed by oral intubation on gestation days 6-15 (plug = 0) with 0, 900, 1,400, 1,900 or 2,400 mg/kg/day and 0, 14, 140, or 400 mg/kg/day. The vehicle control was distilled water. Maternal observations included clinical signs, weight change, and gross evaluation of organ weights and uterine contents at necropsy (day 20). Corpora lutea were counted and uteri stained for implantation sites. Live fetuses were examined for external, skeletal, and soft tissue malformations. Seven dams died during treatment (1,400 mg 1/19, 1,900 mg 2/19, 2,400 mg 4/21), and maternal weight gain was reduced at all except the lowest treatment levels. Liver, spleen, and kidney weights increased in a dose-related manner. The mean percentage of resorbed implants per litter was significantly elevated at greater than or equal to 900 mg/kg/day. Live fetuses showed dose-dependent reductions in weight and length at doses above 140 mg/kg. Statistically significant frequencies of soft tissue malformations ranged from 2.6% (140 mg/kg) to 73% (2,400 mg/kg). These were principally in the cardiovascular system and predominantly comprised defects between the ascending aorta and the right ventricle. Skeletal malformations were not observed in significant numbers in any dose group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence emission spectra of plant leaves and plant constituents

Summary The UV-B radiation (e.g. 337 nm) induced blue fluorescence (BF) and red chlorophyll fluorescence spectra (RF) of green leaves from plants with different leaf structure were determined and the possible nature and candidates of the blue fluorescence emission investigated. The blue fluorescence BF is characterized by a main maximum in the 450 nm region and in most cases by a second maximum/shoulder in the 530 nm region. The latter has been termed green fluorescence GF. The red chlorophyll fluorescence RF, in turn, exhibits two maxima in the 690 and 730 nm region. In general, the intensity of BF, GF and RF emission is significantly higher in the lower than the upper leaf side. The ratio of BF to RF emission (F450/F690) seems to vary from plant species to plant species. BF and GF emission spectra appear to be a mixed signal composed of the fluorescence emission of several substances of the plant vacuole and cell wall, which may primarily arise in the epidermis. Leaves with removed epidermis and chlorophyll-free leaves, however, still exhibit a BF and GF emission. Candidates for the blue fluorescence emission ( max near 450 nm) are phenolic substances such as chlorogenic acid, caffeic acid, coumarins (aesculetin, scopoletin), stilbenes (t-stilbene, rhaponticin), the spectra of which are shown. GF emission ( max near 530 nm) seems to be caused by substances like the alkaloid berberine and quercetin. Riboflavine, NADPH and phyllohydroquinoneK ₁ seem to contribute little to the BF and GF emission as compared to the other plant compounds. Purified natural-carotene does not exhibit any blue fluorescence.     

Enhanced meta-analysis of acetylcholine binding protein structures reveals conformational signatures of agonism in nicotinic receptors

The soluble acetylcholine binding protein (AChBP) is the default structural proxy for pentameric ligand‐gated ion channels (LGICs). Unfortunately, it is difficult to recognize conformational signatures of LGIC agonism and antagonism within the large set of AChBP crystal structures in both apo and ligand‐bound states, primarily because AChBP conformations in this set are nearly superimposable (root mean square deviation < 1.5 Å). We have undertaken a systematic, alignment‐free approach to elucidate conformational differences displayed by AChBP that cleanly differentiate apo/antagonist‐bound from agonist‐bound states. Our approach uses statistical inference based on both crystallographic states and conformations sampled during long molecular dynamics simulations to select important inter‐C_α distances and map their collective values onto functional states. We observe that binding of (nAChR) agonists to AChBP elicits clockwise rotation of the inner β‐sheet with respect to the outer β‐sheet, causing tilting of the cys‐loop away from the five‐fold axis, in a manner quite similar to that speculated for α‐subunits of the heteromeric nAChR structure (Unwin, J Mol Biol 2005;346:967), making this motion potentially important in transmission of the gating signal to the transmembrane domain of a LGIC. The method is also successful at discriminating partial from full agonists and supports the hypothesis that a particularly controversial ligand, lobeline, is in fact an LGIC antagonist.     

Inter-��-trypsin Inhibitor Promotes Bronchial Epithelial Repair after Injury through Vitronectin Binding

Pulmonary epithelial injury is central to the pathogenesis of many lung diseases, such as asthma, pulmonary fibrosis, and the acute respiratory distress syndrome. Regulated epithelial repair is crucial for lung homeostasis and prevents scar formation and inflammation that accompany dysregulated healing. The extracellular matrix (ECM) plays an important role in epithelial repair after injury. Vitronectin is a major ECM component that promotes epithelial repair. However, the factors that modify cell-vitronectin interactions after injury and help promote epithelial repair are not well studied. Inter-α-trypsin inhibitor (IaI) is an abundant serum protein. IaI heavy chains contain von Willebrand A domains that can bind the arginine-glycine-aspartate domain of vitronectin. We therefore hypothesized that IaI can bind vitronectin and promote vitronectin-induced epithelial repair after injury. We show that IaI binds vitronectin at the arginine-glycine-aspartate site, thereby promoting epithelial adhesion and migration in vitro. Furthermore, we show that IaI-deficient mice have a dysregulated response to epithelial injury in vivo, consisting of decreased proliferation and epithelial metaplasia. We conclude that IaI interacts not only with hyaluronan, as previously reported, but also other ECM components like vitronectin and is an important regulator of cellular repair after injury.Epithelial injury is a crucial component in the pathogenesis of many lung diseases. Bronchial epithelial injury occurs chronically in asthmatic patients (1, 2). Furthermore, alveolar and bronchiolar epithelial injury are early triggers in idiopathic pulmonary fibrosis (3) and in lung transplant rejection (4), respectively. In acute respiratory distress syndrome, diffuse alveolar epithelial injury initiates the inflammatory and fibrotic response that leads to lung dysfunction (5). It is now believed that a dysregulated response to epithelial injury ultimately causes fibroproliferation, scar formation, and respiratory failure in acute as well as chronic lung injury (3). It is therefore important to understand the epithelial repair process after pulmonary epithelial injury, if we are to develop causal treatments for these diseases.The mechanisms governing epithelial repair are incompletely understood. Epithelial repair encompasses cell proliferation, migration, and differentiation. All of these processes require cell interactions with the extracellular matrix (ECM).² ECM components like tenascin C, fibronectin, and vitronectin promote epithelial regeneration through integrin binding. Vitronectin (Vn) is a pluripotent 75-kDa plasma and ECM glycoprotein that regulates a number of biological processes such as coagulation, complement activation, and wound healing. Vn promotes cell adhesion and migration via binding primarily to integrins α_vβ₁, α_vβ₃, α_vβ₅, and α_vβ₆. After cell binding, Vn can protect bronchial epithelial cells from apoptosis (6) by inducing Akt phosphorylation and preventing caspase and Fas-associated with death domain (FADD) activation (7). Vn also binds to non-integrin cell receptors such as urokinase-type plasminogen activator receptor to promote changes in cell morphology, migration, and signal transduction (8). Consequently, Vn deficiency impairs bronchial (6) and alveolar (9) epithelial repair.Cell-Vn interactions are modulated by other extracellular factors. For example, plasminogen activator inhibitor 1 (PAI-1) is bound to circulating Vn and forms multimers with Vn upon extravasation to the extracellular space (10), thus possibly activating Vn into an adhesive form (11). Furthermore, Vn possesses several domains that can function as possible ligands, such as a somatomedin B domain, an arginine-glycine-aspartate (RGD) domain, and a heparin-binding domain. However, the extent to which other serum or ECM factors may interact with Vn and influence cell-Vn interactions is unclear.In this report, we investigated possible interactions between Vn and the serum and ECM protein inter-α-trypsin inhibitor. Inter-α-trypsin inhibitor (IaI) is a complex protein found in relatively high concentrations in mammalian plasma. It is made up of a light chain (called bikunin for its two Kunitz domains), which confers the protease inhibitory activity, as well as two heavy chains (12). The precise functions of the heavy chains are unknown. Heavy chains contain a von Willebrand Type A (vWA) domain, and they have been shown to bind to hyaluronan and thereby stabilize the extracellular matrix. However, vWA domains are fairly promiscuous and can bind to a large array of proteins, including RGD domains. Furthermore, IaI is expressed by epithelial cells under stress conditions and is incorporated into de novo ECM structures produced by stressed epithelia (13). We therefore hypothesized that IaI may interact with Vn and thus promote epithelial survival after injury.     

Activating immunity in the liver. II. IFN-beta attenuates NK cell-dependent liver injury triggered by liver NKT cell activation

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10-100 ng/mouse) of alpha-galactosyl ceramide (alphaGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following alphaGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to alphaGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-gamma response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG(-/-) or HBs-tg/RAG(-/-)) hosts via the portal vein elicited IFN-gamma responses of liver NK cells in situ. IFN-beta down-regulates the pathogenic IL-12/IFN-gamma cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-beta suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (alphaGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-gamma response of the specifically activated liver NKT cells. In vivo, IFN-beta attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.