Multiple analysis of mitochondrial metabolism, autophagy and cell death.

In the perspective to evaluate the toxicity of drug candidates or the exploration of intracellular signaling pathways of cell stress response and pathophysiological conditions, we propose to evaluate cell death, autophagy, mitochondrial network and energetic metabolism by a series of optimized joint protocols for neonatal primary rat cardiomyocytes or H9c2 cardiac cell line in 96 well microtiter plates. We used Digitoxigenin and Digoxin, two cardiac glycosides, and Rapamycin as control drugs, for inhibition of oxidative stress-induced cell death and autophagy induction, respectively.

Localization and (semi-)quantification of fluorescent beads of 2 sizes in chickens over time after simultaneous intratracheal and cloacal administration.

Environmental particles enter the chicken via several routes. Entry via the respiratory and cloacal routes likely activates immune responses. We studied the localization of simultaneous intratracheally and cloacally applied beads of 2 sizes in the chicken body in time, and when possible, semiquantified the amount of beads. Ten broiler hens, 3.5 wk of age, received 1.25 × 10(9) 1.0-µm beads and 1.05 × 10(7) 10-µm fluorescein isothiocyanate (green) labeled cloacally, and simultaneously the same number and same sizes of tetramethylrhodamine isothiocyanate (red) labeled beads intratracheally. The bursa of Fabricius, lung, liver, kidney, gallbladder, spleen, thymus, small intestine (upper ileum), cecum, intestinal luminal contents, aerated bones, feces, and blood, from 2 chickens per moment were sampled at 1 h, 6 h, 24 h, 48 h, and 1 wk after challenge and studied for the presence of beads using fluorescence microscopy. The highest amount of beads was found in organs closest to the application site after 1 h (i.e., the lungs for red beads, and the bursa for green beads). All tissue samples showed all 4 types of beads at all time moments, most of them within 1 h. Lower levels of beads were found in lungs and bursa after 6 h and in all other organs after 24 h, except for the kidneys where levels declined after 48 h. Surprisingly, beads were found in thymus tissue and only relatively few beads were found in the spleen. At 1 h, 1-µm intratracheally applied red beads were also found in the cecal luminal content and cecal tissue, but not in the small intestinal luminal content, suggesting that ceca are capable of excreting small particles entering the body via the respiratory route. The presence of nondegradable and nonimmunogenic beads of different sizes in all sampled organs throughout the whole chicken body for 7 d suggested potentially negative chronic health and welfare risks for the chicken of environmental particles.

Effects of 2 size classes of intratracheally administered airborne dust particles on primary and secondary specific antibody responses and body weight gain of broilers: a pilot study on the effects of naturally occurring dust.

We studied the effects of a concurrent challenge on slow-growing broilers with 1) airborne particles of 2 sizes: fine dust (smaller than 2.5 microns) and coarse dust (between 2.5 and 10 microns) that were directly collected from a broiler house and 2) lipopolysaccharide on intratracheal immunizations with the specific antigen human serum albumin (HuSA) and measured primary and secondary systemic (total) antibody responses and (isotype-specific) IgM, IgG, and IgA responses at 3 and 7 wk of age. All treatments affected immune responses at several ages, heart morphology, and BW gain, albeit the latter only temporarily. Dust particles significantly decreased primary antibody (IgT and IgG) responses to HuSA at 3 wk of age but enhanced IgM responses to HuSA at 7 wk of age. Dust particles decreased secondary antibody responses to HuSA, albeit not significantly. All of the birds that were challenged with dust particles showed decreased BW gain after the primary but not after the secondary challenge. Relative heart weight was significantly decreased in birds challenged with coarse dust, fine dust, lipopolysaccharide, and HuSA at 3 wk of age, but not in birds challenged at 7 wk of age. Morphology (weight, width, and length) of hearts were also affected by the dust challenge at 3 wk of age. The present results indicate that airborne dust particles obtained from a broiler house when intratracheally administered at an early age affect specific humoral immune responsiveness and BW gain of broilers to simultaneously administered antigens differently than when administered at a later age. The hygienic status of broiler houses at a young age may be of importance for growth and immune responsiveness, and consequently, for vaccine efficacy and disease resistance in broilers. The consequences of our findings are discussed.

Effects of repeated intratracheally administered lipopolysaccharide on primary and secondary specific antibody responses and on body weight gain of broilers.

Earlier, we reported that pathogen-associated molecular patterns such as lipopolysaccharide (LPS), when administered intratracheally (i.t.), affected primary and secondary specific antibody responses to antigens administered concurrently, either i.t. or systemically, and also affected BW gain (BWG) of layers and broilers. In the present study, we evaluated the effects of repeated i.t. challenge with LPS concurrently with or before i.t. immunizations with the specific antigens human serum albumin (HuSA) and rabbit gamma globulin (RGG) on primary (HuSA, RGG) and secondary (HuSA) systemic antibody responses and (isotype) IgM and IgG responses at 2 different ages. Broilers were challenged via the trachea at 3 and 7 wk of age with various combinations of LPS, HuSA, and RGG. All treatments affected immune responses at several time points and also affected BWG, albeit temporarily for the latter. Lipopolysaccharide enhanced primary antibody responses to HuSA and to RGG, when challenged concurrently, but birds challenged solely with LPS at 3 wk of age also showed enhanced primary antibody responses to HuSA and RGG given at 7 wk of age. This was true for IgM as well as IgG isotype responses. Lipopolysaccharide challenge negatively affected BWG at 3 wk of age, whereas the negative effects of LPS after a secondary LPS challenge at 7 wk of age were most pronounced in the birds challenged with LPS at 3 wk of age. The present results indicated that LPS, when administered i.t. at a young age, may affect specific humoral immune responsiveness to antigens administered simultaneously and to BWG of broilers, but also when challenged 4 wk later with specific antigens, suggesting an enhanced status of immune reactivity or sensitivity. The hygienic status of broiler houses at a young age may thus influence BWG, immune responsiveness, and, consequently, the vaccine efficacy and disease resistance in broilers at later ages. The consequences of our findings are discussed.

Effects of dust and airborne dust components on antibody responses, body weight gain, and heart morphology of broilers.

Pathogen-associated molecular patterns (PAMP) such as lipopolysaccharide (LPS), lipoteichoic acid, beta-glucans (BGL), and possibly many others are important parts of (fine) dust in animal houses. When intratracheally (i.t.) administered, PAMP affected specific primary and secondary humoral immune responses to concurrently i.t. or systemically administered antigens and BW gain (BWG) of layer chickens. In the present study, we evaluated the effects of i.t. challenge with various PAMP known to be present in dust: LPS, lipoteichoic acid, zymosan-A (containing 1,3 BGL), next to heat-inactivated dust particles as a representative of mechanical stress, a combination of the former components, and NH3 as a chemical component of dust on primary and secondary (total) systemic antibody (Ab) responses and (isotype) IgM and IgG responses to concurrently i.t.-administered human serum albumin (HuSA) in broilers. Birds were challenged via the trachea for 2 consecutive days at 3 and 7 wk of age, respectively. All treatments affected immune responses at several moments, BWG, and heart morphology. beta-Glucans and LPS affected the birds most pronounced and for a prolonged period. Intratracheally administered LPS and BGL significantly enhanced primary and secondary total Ab, IgM Ab, and IgG Ab responses to HuSA. All birds that were challenged with dust, PAMP, or NH3 concurrently with HuSA showed a decreased BWG especially after primary, but also after secondary challenge. Weight, width, and length of hearts were enhanced in dust and PAMP-treated birds as well when these birds were challenged with HuSA. The present results indicated that components of dust such as PAMP when i.t. administered affect humoral immune responsiveness of broilers, which may lead to an enhanced status of immune reactivity. Furthermore, our results suggest that the hygienic status of the environment influences BWG and may affect heart morphology, and as a consequence physiology in broilers. The consequences of our findings with respect to dust, (airborne) PAMP, hygienic conditions in the barn, and immune responsiveness of broilers are discussed.

NADH oxidase activation is involved in arsenite-induced oxidative DNA damage in human vascular smooth muscle cells.

Arsenic is atherogenic, carcinogenic, and genotoxic. Because atherosclerotic plaque has been considered a benign smooth muscle cell tumor, we have studied the effects of arsenite on DNA integrity of human vascular smooth muscle cells. By using single-cell alkaline electrophoresis, apparent DNA strand breaks were detected in a 4-hour treatment with arsenite at a concentration above 1 micromol/L. DNA strand breaks of arsenite-treated cells were increased by Escherichia coli formamidopyrimidine-DNA glycosylase and decreased by diphenylene iodinium, superoxide dismutase, catalase, pyruvate, DMSO, or D-mannitol. Extract from arsenite-treated cells showed increased capacity for producing superoxide when NADH was included in the reaction mixture; however, addition of arsenite to extract from untreated cells did not increase superoxide production. The superoxide-producing ability of arsenite-treated cells was also suppressed by diphenylene iodinium, 4,5-dihydroxy-1, 2-benzenedisulfonic acid disodium salt (Tiron), or superoxide dismutase. Superoxide production and DNA strand breaks in arsenite-treated cells were also suppressed by transfecting antisense oligonucleotides of p22phox, an essential component of NADH oxidase. Treatment with arsenite also increased the mRNA level of p22phox. These results suggest that arsenite activates NADH oxidase to produce superoxide, which then causes oxidative DNA damage. The result that arsenite at low concentrations increases oxidant levels and causes oxidative DNA damage in vascular smooth muscle cells may be important in arsenic-induced atherosclerosis.

Modeling the effects of sodium chloride on degradation of chloramphenicol in aquaculture pond sediment.

Sodium chloride was added to aquaculture pond sediment to determine effects of different salinities on degradation of chloramphenicol (CM). In this experiment, freshwater (0 ppt salinity) eel pond sediment slurries (10% w/v) were amended with sodium chloride to obtain salinities of 12, 24 and 36 ppt. There were no significant differences in sorption rate either between aerobic and anaerobic conditions or among various salinities. Degradation of CM fitted well to the decaying exponential curve. The degradation rates under anaerobic conditions were significantly greater than those under aerobic conditions. As salinity increased, the degradation rates decreased under both aerobic and anaerobic conditions. The differences in degradation rates either between aerobic and anaerobic conditions or among various salinities were attributed to the effects of microbial activities under different environments.

Linkage mapping of Thiel-Behnke corneal dystrophy (CDB2) to chromosome 10q23-q24.

Corneal dystrophy of the anterior basement membrane is a heterogeneous set of diseases characterized by painful, recurrent, bilateral erosions of the cornea, which often result in significant visual impairment. There are several similar but clinically distinct forms of anterior basement membrane/Bowman's membrane disease, including two autosomal dominant forms, Reis-Bücklers and Thiel-Behnke corneal dystrophy. Genes causing autosomal, nonsyndromic corneal dystrophy have been mapped to human chromosomes 1p, 5q, 12q, 16q, 17p, and 20p. Using microsatellite markers closely linked to the known corneal dystrophy loci, we excluded linkage between the known sites and the disease locus in a large, four-generation family with Thiel-Behnke corneal dystrophy. A genome-wide search using a panel of microsatellite markers demonstrated a maximum two-point lod score of 4.0 at 0% recombination between the disease locus in this family and the marker D10S1239, which maps to 10q23-q24. Testing with additional microsatellite markers from 10q places the disease locus between D10S677 and D10S1671, a distance of approximately 12.0 cM, with a maximum multipoint lod score of 5.5. Based on this evidence, we have identified another locus (CDB2) for corneal dystrophy of the anterior basement membrane/Bowman's membrane, Thiel-Behnke type, further demonstrating the exceptional genetic and phenotypic heterogeneity of these diseases.

Arsenite retards DNA break rejoining by inhibiting DNA ligation.

Arsenic has been shown to inhibit methyl methane-sulphonate (MMS)-induced DNA repair but the exact mechanism remains controversial. The purpose of this investigation is to examine which step of DNA repair is most sensitive to arsenite (As) and how As inhibits it. The results from single-cell alkaline electrophoresis, showing post-treatment with As increased DNA strand breaks in MMS-treated cells, suggest that that the excision step seems to be less sensitive to As than later steps. To test this hypothesis, hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC) were used to block DNA polymerization, allowing the DNA strand breaks to accumulate. These experiments indicated that As had weak inhibitory effects on DNA strand break accumulation. However, As inhibited the rejoining of those DNA strand breaks which could be rejoined within 4 h after release from blockage by Hu plus AraC. To further elucidate this mechanism, a cell extract was used to compare the relative sensitivity of the various steps in DNA repair to As. The potency of the As inhibitory effect as deduced from concentration-response curves were: ligation of poly(rA).oligo(dT) > ligation of poly(dA).oligo(dT) approximately DNA polymerization > or = DNA repair synthesis > excision. As is known to inhibit the activity of pyruvate dehydrogenase by interacting with vicinal dithiol groups. Dithiothreitol could effectively remove As inhibition of both the ligation of poly(rA).oligo(dT) and the activity of pyruvate dehydrogenase but had no obvious effect on As inhibition of poly(dA).oligo(dT) ligation. Since DNA ligase III contains vicinal dithiol groups, we postulate that As may inhibit DNA break rejoining by interacting with the vicinal dithiols to inactivate DNA ligation in MMS-treated cells.

Cadmium inhibits DNA strand break rejoining in methyl methanesulfonate-treated CHO-K1 cells.

The cogenotoxicity of Cd has been recognized. This effect may stem from Cd inhibition of DNA repair. We studied the effects of Cd on DNA repair of methyl methanesulfonate (MMS)-damaged Chinese hamster ovary cells (CHO-K1) by single-cell alkaline electrophoresis. The results indicate that in the presence of Cd, DNA strand breaks accumulated in MMS-treated cells. Using hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC) to block DNA polymerization, DNA strand breaks accumulated and Cd had little inhibitory effects on these accumulations. However, Cd inhibited the rejoining of these DNA strand breaks, which could be rejoined 6 hr after release from Hu plus AraC blockage. These results indicate that the potency of Cd inhibition of DNA repair replication and/or ligation may be greater than the inhibition of DNA adduct excision. To further elucidate this mechanism, we used an in vitro cell-free assay system to analyze the Cd effects on DNA repair synthesis, DNA polymerization, and DNA ligation. We have shown a dose-dependent inhibition of these three activities by Cd in CHO-K1 cell extract. The IC50s of Cd were 55, 26, and 10 microM, respectively. Moreover, Cd inhibition of DNA ligation in cell extract could be recovered partially by thiol compounds such as glutathione, beta-mercaptoethanol, dithiothreitol, and metallothionein. Since both in vivo and in vitro studies demonstrated that Cd was more effectively involved in interfering with the DNA ligation step and that thiol agents could partially remove Cd inhibition of DNA ligation, we speculate that part of the Cd inhibition of DNA repair may be through binding of Cd to the proteins participating in DNA ligation.

Adrenal cortical carcinoma with tumor thrombus invasion of inferior vena cava.

A case of adrenal cortical carcinoma with inferior vena cava (IVC) involvement is presented. Ultrasonography, computed tomography, and venacavography all presumptively showed a large mass over the upper pole of the left kidney with tumor thrombus in the IVC. However, aortography demonstrated that this mass was receiving its blood supply from the left inferior phrenic artery, aorta, and left renal artery. Radical surgery, including resection of the tumor and its adjacent organs (kidney, distal pancreas, spleen) and the tumor thrombus in the IVC, with the aid of cardiopulmonary bypass, was performed. We emphasize that adrenal cortical carcinoma can have tumor thrombi invading the IVC, and in such cases we suggest radical surgical removal of the tumor and the thrombus.

Molecular cloning of a novel adenosine receptor gene from rat brain.

We have isolated an adenosine receptor gene (RA2) from a rat brain cDNA library. This novel rat adenosine receptor has 410 amino acids, as deduced from its base sequence, and shows 82% amino acid identity with the dog A2 receptor. Amino acid sequence analysis indicates that RA2 protein contains seven transmembrane domains and belongs to the G protein-coupled receptor family. The variations in amino acid sequences between RA2 protein and the dog A2 receptor are largely confined to the extracellular second loop and the carboxyl terminus.