In vivo mutagenicity of the carcinogenic 1-nitropyrene. A model of a potential asbestos exposure

The environmental carcinogen 1-nitropyrene was orally and intraperitoneally administered to rats in a single dose of 30 mmol/kg. Mutagenicity of excreted urine was tested in Salmonella typhimurium TA 98 and 100 strains. The mutagenic pattern of urine in case of oral exposure proved to be completely different as compared to the intraperitoneal administration. Frame-shift mutagen(s) was/were detected only after enzymatic deconjugation of sulphate or glucuronide metabolites within the first 24 h. Base-pair substitution-type mutagenicity was only detected in the urine samples collected after intraperitoneal treatment. Since environmental asbestos exposure involves carcinogenic effects of adsorbed polycyclic aromatic hydrocarbons, this animal model provides a useful tool for testing fiber-associated nitroarenes, in both mechanistic and risk assessment studies.     

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm - with or without anchoring them in the plasma membrane -, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised either by adding a C-terminal stabilisation signal or by anchoring the protein on the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.     

Expression of CD44 and L-selectin in the innate immune system is required for severe joint inflammation in the proteoglycan-induced murine model of rheumatoid arthritis

Proteoglycan (PG)-induced arthritis, a murine model of rheumatoid arthritis, is characterized by autoimmunity against mouse cartilage PG and chronic joint inflammation. L-selectin (CD62L) and CD44 are major adhesion molecules on leukocytes that regulate their homing to lymph nodes and entry into inflamed tissues. In the present study, we studied the requirement for CD44 and CD62L expression for mediating lymphocyte homing, thus permitting the development of autoimmunity vs mediating the entry of leukocytes into the joints, thus allowing inflammation in PG-induced arthritis. We immunized wild-type, CD44 knockout (KO), CD62L KO, and double (CD44/CD62L) KO BALB/c mice with PG and monitored the effects of gene deficiencies on PG-specific immunity, arthritis severity, leukocyte trafficking, and the ability of lymphocytes to adoptively transfer disease to syngeneic SCID mice. Single and double KO mice demonstrated reduced PG-specific spleen cell proliferation, but the production of Th cytokines and autoantibodies was comparable in KO and wild-type mice. KO leukocytes had reduced ability to adhere tightly to the synovial endothelium in arthritic joints. This diminished leukocyte adhesion correlated with the magnitude of granulocyte (neutrophil) influx and the severity of inflammation, which were both reduced in the joints of KO mice. However, transfer of spleen cells from mildly arthritic KO donors to SCID hosts resulted in development of severe arthritis. Our results indicate that CD44 and CD62L expression in the cells of the innate immune system (granulocytes) is important for their efficient influx into the joints and also suggest that granulocytes play a crucial role in arthritis progression.     

Azidomorphine is an agonist of high-affinity opioid receptor binding sites

Azidomorphine at low concentration (10^–9 M) inhibits the high-affinity binding site of labeled naloxone in rat brain membrane preparations. In the presence of Na⁺ and guanine nucleotides the displacement curves of azidomorphine are increased toward high concentrations, whereas Mg²⁺ ions decrease the IC₅₀ values; This demonstrates the agonist behavior of azidomorphine in binding experiments. When compared with morphine, azidomorphine displayed five-fold lower IC₅₀ values. Based on the presented results, azidomorphine appears to be a good candidate for photoaffinity labeling of opiate receptors.     

Fluorimetric determination of 5-hydroxytryptamine and catecholamines in the central nervous system and heart of Locusta migratoria migratorioides

5-Hydroxytryptamine (5HT) was measured in the nervous and heart tissues of Locusta migratoria migratorioides in concentrations of 2·34 and 2·69 μg/g wet weight respectively. Among catecholamines, dopamine was found in the nervous tissue (1·31 μg/g wet weight) as well as in the heart (2·42 μg/g wet weight). Noradrenaline was present only in the brain, but in smaller amounts (0·24 μg/g wet weight) than dopamine. Adrenaline was found neither in the ganglia nor in the heart.On the basis of their occurrence in the CNS, 5HT, dopamine, and noradrenaline, and in the heart 5HT and dopamine can be regarded as transmitter or modulator substances.     

β-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

β-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [³H]dihydro-l-alprenolol ([³H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [³H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, β-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of B_max = 12.01 fmol/10⁶ cells (7200 sites/cell), K_d = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with l-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k₊ = 8.31·10⁷ M^?1 · min^?1 and k_? = 0.28 min^?1 from the association results, and k_? = 0.24 min^?1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; K_d, as $\text{(k}{}_{\mathrm{?}}\text{/k}{}_{\mathrm{+}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>n</mtext>}}$ was found to be 101 pM. Analysis of the equilibrium binding of [³H]DHA to rat and mouse living brain cells resulted in values of B_max = 13.04 fmol/10⁶ cells (7800 sites/cell), K_d = 43.85 pM and n = 2.52, and B_max = 8.08 fmol/10⁶ cells (4800 sites/cell), K_d = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the β-receptor in mammalian objects, too. The [³H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, K_d in the range 1–2 nM, and a B_max of 10³–10⁴ sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the β-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.     

In situ bioremediation of hydrocarbon in soil

Laboratory and field experiments were carried out for bioremediation of soils contaminated by fuel oil and motor oil. Bioventing was combined with the application of selected bacteria and dissolved nutrients. In the field experiments, soil gas was evacuated by air pumps from the permeable boreholes. The process was followed by both soil and gas analysis. Biodegradation of oil contamination and the microbial activity was measured by the oil and cell concentration in the soil. In 2 months, the oil content decreased considerably, and the cell number increased by one order of magnitude or more. The evacuated gas was tested for CO₂, O₂ and volatilized hydrocarbon content. The CO₂ level proves the presence of biodegradation: a permanent high value about ten times higher than normal, could be measured for 2 months, followed by a slow decrease in the third month. Volatilized hydrocarbon content was the highest in the first 2 d. After a continuous decrease, it dropped under the threshold of measurability for the third month. Selective biodegradation of hydrocarbon mixtures (oily wastes) was investigated as well: gas Chromatographic oil analysis showed the changes in the oil composition. The appropriate microflora was working in an ideal commensalism, and as a result, all of the hydrocarbon components were degraded nearly to the same extent.     

Construction of a basic genetic map for alfalfa using RFLP, RAPD, isozyme and morphological markers

The genetic map for alfalfa presented here has eight linkage groups representing the haploid chromosome set of the Medicago species. The genetic map was constructed by ordering the linkage values of 89 RFLP, RAPD, isozyme and morphological markers collected from a segregating population of 138 individuals. The segregating population is self-mated progeny of an F1 hybrid plant deriving from a cross between the diploid (2n=2x=16) yellow-flowered Medicago sativa ssp. quasifalcata and the diploid (2n=2x=16) blue-flowered M. sativa ssp. coerulea. The inheritance of many traits displayed distorted segregation, indicating the presence of lethal loci in the heterozygotic parent plants. In spite of the lack of uniform segregation, linkage groups could be assigned and the order of the markers spanning > 659 centimorgans could be unambiguously determined. This value and the calculated haploid genome size for Medicago (1n=1x=1.0 x 10⁹ bp) gives a ratio of < 1500 kb per centimorgan.     

Microbial food web in a large shallow lake (Lake Balaton, Hungary)

Seasonal variations of phyto-, bacterio- and colourless flagellate plankton were followed across a year in the large shallow Lake Balaton (Hungary). Yearly average chlorophyll-a concentration was 11 µg 1^–1, while the corresponding values of bacterioplankton and heterotrophic nanoflagellate (HNF) plankton biomass (fresh weight) were 0.24 mg 1^–1 and 0.35 mg 1^–1, respectively. About half of planktonic primary production was channelled through bacterioplankton on the yearly basis. However, there was no significant correlation between phytoplankton biomass and bacterial abundance. Bacterial specific growth rates were in the range of 0.009 and 0.09 h^–1, and ended to follow the seasonal changes in water temperature. In some periods of the year, predator-prey relationships between the HNF and bacterial abundance were obvious. The estimated HNF grazing on bacteria varied between 3% and 227% of the daily bacterial production. On an annual basis, 87% of bacterial cell production was grazed by HNF plankton.