Initial Transformations in the Biodegradation of Benzothiazoles by Rhodococcus Isolates

Benzothiazole-2-sulfonate (BTSO₃) is one of the side products occurring in 2-mercaptobenzothiazole (MBT) production wastewater. We are the first to isolate an axenic culture capable of BTSO₃ degradation. The isolate was identified as a Rhodococcus erythropolis strain and also degraded 2-hydroxybenzothiazole (OBT) and benzothiazole (BT), but not MBT, which was found to inhibit the biodegradation of OBT, BT, and BTSO₃. In anaerobic resting cell assays, BTSO₃ was transformed into OBT in stoichiometric amounts. Under aerobic conditions, OBT was observed as an intermediate in BT breakdown and an unknown compound transiently accumulated in several assays. This product was identified as a dihydroxybenzothiazole. Benzothiazole degradation pathways seem to converge into OBT, which is then transformed further into the dihydroxy derivative.     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Isolation and characteristics of 2-hydroxybenzothiazole-degrading bacteria

Several strains capable of growth on 2-oxybenzothiazole (OBT) were isolated. They were all Gram-positive, spore-forming rods, tentatively identified as Bacillus species. Their mean generation time on OBT was 12 h with a cell yield coefficient of 0.63. The strains could also degrade benzothiazole but not 2-mercaptobenzothiazole (MBT) or its sulphonate form. MBT was inhibitory. The biodegradation was inhibited by a 5% NaCl concentration.     

2-Mercaptobenzothiazole degradation in laboratory fed-batch systems

Rubber additives manufacture yields waste-waters with recalcitrant and/or toxic benzothiazole compounds. Biodegradation of such compounds was investigated in fed-batch systems. 2-Mercaptobenzothiazole (MBT) was best degraded by a mixture of MBT-history and non-MBT-history sludge. Concentrations up to 200 mg·l^–1 were removed. From 100 mg·l^–1 onwards, high percentages of the recalcitrant disulphide were accumulated in the sludge. MBT slowed down the biodegradation of benzothiazole-2-sulphonate. MBT and benzothiazole did not mutually influence their degradation. Under some experimental conditions high levels of unidentified so-called polar compounds were formed.     

Parameters affecting the degradation of benzothiazoles and benzimidazoles in activated sludge systems

It was found that benzothiazole, 2-oxybenzothiazole and 2-benzothiazolesulphonate were degraded in activated sludge systems. 2-Mercaptobenzothiazole (MBT) was more resistant, although the first step in MBT degradation seemed to be transformation to the sulphonate form. At higher MBT concentrations, it was transformed into a disulphide, which accumulated in the sludge. MBT was also found to be mainly responsible for the toxicity of rubber chemical waste-water towards activated sludges. It inhibited the degradation of the other hetrocycles. Only at concentrations of around 20 ppm was MBT degraded. Mercaptobenzimidazole ranked second in resistance to degradation. Correspondence to: H. Verachtert     

Synthesis of 6-N-(benzothiazol-2-yl)deoxyadenosine and its exciton-coupled circular dichroism

6-N-(Benzothiazol-2-yl)deoxyadenosine (A^BT) was synthesized and incorporated into DNAs. Although, the multipoint benzothiazole (BT) modification of oligodeoxynucleotides reduced the stability of duplexes with their complementary strands, it induced the strong exciton coupling between BT moieties. The circular dichroism (CD) spectra of this exciton coupling interaction were observed at wavelengths above 300 nm and overlapping with the UV absorption bands of nucleotides could be avoided. The shapes of the CD spectra due to this interaction were strongly influenced by the helicity of two BT groups.     

Benzothiazole degradation by Rhodococcus pyridinovorans strain PA: evidence of a catechol 1,2-dioxygenase activity

The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by Rhodococcus pyridinovorans strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ (1)H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional (1)H-(13)C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by (1)H and (19)F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this Rhodococcus strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.     

Genome‐wide DNA methylation analysis using next‐generation sequencing to reveal candidate genes responsible for boar taint in pigs

Boar taint (BT) is an offensive flavor observed in non‐castrated male pigs that reduces the carcass price. Surgical castration effectively avoids the taint but is associated with animal welfare concerns. The functional annotation of farm animal genomes for understanding the biology of complex traits can be used in the selection of breeding animals to achieve favorable phenotypic outcomes. The characterization of pig epigenomes/methylation changes between animals with high and low BT and genome‐wide epigenetic markers that can predict BT are lacking. Reduced representation bisulfite sequencing of DNA methylation patterns based on next‐generation sequencing is an efficient technology to identify candidate epigenetic biomarkers associated with BT. Three different BT levels were analyzed using reduced representation bisulfite sequencing data to calculate the methylation levels of cytosine and guanine dinucleotide (CpG) sites. The co‐analysis of differentially methylated CpG sites identified by this study and differentially expressed genes identified by a previous study found 32 significant co‐located genes. The joint analysis of GO terms and pathways revealed that methylation and gene expression of seven candidate genes were associated with BT; in particular, FASN plays a key role in fatty acid biosynthesis, and PEMT might be involved in estrogen regulation and the development of BT. This study is the first to report the genome‐wide DNA methylation profiles of BT in pigs using next‐generation sequencing and summarize candidate genes associated with epigenetic markers of BT, which could contribute to the understanding of the functional biology of BT traits and selective breeding of pigs against BT based on epigenetic biomarkers.     

Some biochemical properties of higher plant tubulins

Tubulin was isolated by a combination of affinity (ethyl N-phenylcarbamate-Sepharose) and ion exchange (DEAE-Sephacel) chromatography from mung bean and cultured carrot suspension cells. SDS-PAGE (Blose 1981) of mung bean tubulin has shown it to consist of two major subunits (MBT1 and MBT2) and a minor subunit (MBT3). Tubulin isolated from carrot cells was resolved into only two bands on SDS-PAGE (slow moving subunit was named CT1). However, the faster moving subunit on SDS-PAGE was resolved into two bands (CT2 and CT3) on SDS-4M urea-PAGE. On SDS-4M urea-PAGE, CT1 migrated faster than CT2, CT3. By contrast in SDS-4M urea-PAGE, mung bean tubulin remains unresolved. Mammalian tubulin could be resolved into alpha and beta-subunits in both electrophoretic systems. Monoclonal antibodies to mammalian alpha and beta-tubulin subunits (MCA-T alpha and MCA-T beta, respectively) and Western blot analysis clearly demonstrated a cross-reactivity of MCA-T alpha with MBT2, MBT3, CT2 and CT3, while MCA-T beta showed cross-reactivity with MBT1 and CT1. Although MBT2, MBT3, CT2 and CT3 are immunologically related to the alpha-subunit of mammalian tubulin, their migration on SDS-PAGE was reversed with respect to MBT1 or CT1, which were immunologically related to the beta-subunit of mammalian tubulin. Peptide mapping patterns also supported above the results.     

Distribution and bioaccumulation of butyltins in the edible gastropod Odontocymbiola magellanica

Butyltins (BTs) were found in sediments and body tissues of the edible gastropod Odontocymbiola magellanica, in which imposex has been recorded since 2000. BTs in sediments ranged from < MDL to 174.8?ng (Sn) g^?1 for TBT, < MDL to 19.2?ng (Sn) g^?1 for DBT, and < MDL to 71.8?ng (Sn) g^?1 for MBT. In body tissues BTs varied from??1 for TBT, DBT and MBT, respectively. BT concentrations were higher in gonads and digestive glands than in the albumen gland and foot (edible). The highest concentrations of BTs in both sediments and gastropods were found in the harbour area, decreasing with distance to the harbour and areas with less maritime traffic. The Biota-Sediment Accumulation Factor (BSAF) in the different organs was between 0.02–0.42, 0.09–0.35 and 0.08–5.25 for TBT, DBT and MBT, respectively. There were positive correlations between concentrations of BTs in sediments and gastropod body tissues, suggesting that xenobiotic accumulation in O. magellanica occurs mainly through contaminated sediments, rather than water or the food chain. Considering current sediment quality guidelines, our results indicate that acute toxic effects would be expected from TBT exposure, which represents a serious environmental threat for the benthic community. Although the levels of BTs found in the foot of this edible gastropod did not exceed the recommended Tolerable Daily Intake in polluted areas, they should be monitored to ensure the safety of seafood consumers. The alternative antifouling biocides Irgarol and Diuron were not detected in sediments.     

Photobleaching of pheomelanin increases its phototoxic potential: Physicochemical studies of synthetic pheomelanin subjected to aerobic photolysis

Although melanin is a photoprotective pigment, its elevated photochemical reactivity could lead to various phototoxic processes. Photoreactivity of synthetic pheomelanin, derived from 5‐S‐cysteinyldopa (5SCD‐M) and its photodegradation products obtained by subjecting the melanin to aerobic irradiation with UV‐visible light, was examined employing an array of advanced physicochemical methods. Extensive photolysis of 5SCD‐M was accompanied by partial bleaching of the melanin, modification of its paramagnetic properties, and significant increase in the ability to photogenerate singlet oxygen. The changes correlated with a substantial decrease in the melanin content of benzothiazine (BT) units and increase of modified benzothiazole (BZ) units. Synthetically prepared BZ exhibited higher efficiency to photogenerate singlet oxygen than the synthetic BT, and the free radical form of BZ, unlike that of BT, did not show measurable spin density on nitrogen atom, which was confirmed by quantum chemical calculations. Formation of modified BZ units in the photobleached 5SCD‐M is responsible for the paramagnetic and photochemical changes of the melanin and its elevated phototoxic potential. Given a relatively constant pheomelanin–eumelanin ratio, such undesirable changes could occur in individual of all skin types.     

Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation

Epidermal growth factor (EGF) stimulates the homodimerization of EGF receptor (EGFR) and the heterodimerization of EGFR and ErbB2. The EGFR homodimers are quickly endocytosed after EGF stimulation as a means of down-regulation. However, the results from experiments on the ability of ErbB2 to undergo ligand-induced endocytosis are very controversial. It is unclear how the EGFR-ErbB2 heterodimers might behave. In this research, we showed by subcellular fractionation, immunoprecipitation, Western blotting, indirect immunofluorescence, and microinjection that, in the four breast cancer cell lines MDA453, SKBR3, BT474, and BT20, the EGFR-ErbB2 heterodimerization levels were positively correlated with the ratio of ErbB2/EGFR expression levels. ErbB2 was not endocytosed in response to EGF stimulation. Moreover, in MDA453, SKBR3, and BT474 cells, which have very high levels of EGFR-ErbB2 heterodimerization, EGF-induced EGFR endocytosis was greatly inhibited compared with that in BT20 cells, which have a very low level of EGFR-ErbB2 heterodimerization. Microinjection of an ErbB2 expression plasmid into BT20 cells significantly inhibited EGF-stimulated EGFR endocytosis. Coexpression of ErbB2 with EGFR in 293T cells also significantly inhibited EGF-stimulated EGFR endocytosis. EGF did not stimulate the endocytosis of ectopically expressed ErbB2 in BT20 and 293T cells. These results indicate that ErbB2 and the EGFR-ErbB2 heterodimers are impaired in EGF-induced endocytosis. Moreover, when expressed in BT20 cells by microinjection, a chimeric receptor composed of the ErbB2 extracellular domain and the EGFR intracellular domain underwent normal endocytosis in response to EGF, and this chimera did not block EGF-induced EGFR endocytosis. Thus, the endocytosis deficiency of ErbB2 is due to the sequence of its intracellular domain.     

5-Benzothiazole substituted pyrimidine derivatives as HCV replication (replicase) inhibitors

Based on a previously identified HCV replication (replicase) inhibitor 1, SAR efforts were conducted around the pyrimidine core to improve the potency and pharmacokinetic profile of the inhibitors. A benzothiazole moiety was found to be the optimal substituent at the pyrimidine 5-position. Due to potential reactivity concern, the 4-chloro residue was replaced by a methyl group with some loss in potency and enhanced rat in vivo profile. Extensive investigations at the C-2 position resulted in identification of compound 16 that demonstrated very good replicon potency, selectivity and rodent plasma/target organ concentration. Inhibitor 16 also demonstrated good plasma levels and oral bioavailability in dogs, while monkey exposure was rather low. Chemistry optimization towards a practical route to install the benzothiazole moiety resulted in an efficient direct C-H arylation protocol.     

Discrepancies in the determination of sperm concentration using Bürker-Türk, Thoma and Makler counting chambers

Determination of sperm concentration by use of a haemocytometer or counting chamber is an important step in semen evaluation and is also used for calibration or validation of instruments. Three experiments were carried out to determine the precision and accuracy of the Makler chamber, the Thoma haemocytometers (50 and 100 microm deep, TH-50, TH-100) and the Bürker-Türk (BT) haemocytometer. The first experiment confirmed that precision in sperm count by use of the haemocytometers (TH-50 and BT) can be increased if a higher number of sperm are counted. In contrast, the precision of the Makler chamber was relatively unaffected by the number of sperm counted and the coefficient of variation for this chamber was significantly (P < 0.05) higher than for the two haemocytometers. Experiment 2 confirmed the low precision of the Makler chamber and also showed that the TH-50 haemocytometer underestimated sperm concentration by approximately 25% in comparison to the Makler chamber and the BT haemocytometer. Experiment 3 demonstrated a slight underestimation of sperm count by the TH-100 haemocytometer in comparison to the BT haemocytometer, but both haemocytometers yielded acceptable precision (coefficients of variation were 10.4% and 9.4%, respectively). In comparison, the precision of the Makler chamber was significantly poorer (coefficient of variation 18.6%). When used for validation of a flow cytometric method which determines sperm concentration, the Makler chamber caused a higher degree of scattering of the points around the regression line than when the flow cytometric method was validated against the BT haemocytometer. It thus appears that the poor precision of the Makler chamber also affects the accuracy. It is concluded that duplicate counts by at least two technicians is recommended to achieve high precision but, that particular caution should be exerted with regard to the precision and accuracy of the used counting device.