In Vitro Transformation of Chlorinated Parabens by the Liver S9 Fraction: Kinetics, Metabolite Identification, and Aryl Hydrocarbon Receptor Agonist Activity.

We investigated the kinetics of in vitro transformation of a dichlorinated propyl paraben (2-propyl 3,5-dichloro-4-hydroxybenzoate; Cl2PP) by the rat liver S9 fraction and assessed the aryl hydrocarbon receptor (AhR) agonist activity of the metabolite products identified in HPLC and GC/MS analysis and by metabolite syntheses. The results indicated that the chlorination of Cl2PP reduced its degradation rate by approximately 40-fold. Two hydroxylated metabolite products showed AhR agonist activity of up to 39% of that of the parent Cl2PP when assessed in a yeast (YCM3) reporter gene assay. The determination of the metabolic properties of paraben bioaccumulation presented here provides further information on the value of risk assessments of chlorinated parabens as a means to ensure human health and environmental safety.

Silent information regulator 2 proteins encoded by Cryptosporidium parasites.

Screening in a database has revealed that Cryptosporidium hominis encodes a silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase. Cellular localization of the protein, ChSir2, was analyzed by the use of the social amoeba Dictyostelium discoideum as a model system. Fluorescent microscopic analysis showed that ChSir2 fused with green fluorescent protein was localized in the D. discoideum nucleus. D. discoideum expressing ChSir2 grew faster and reached higher cell density than did D. discoideum harboring a control vector. These results suggest that ChSir2 is a nucleus-localizing protein that plays an important role in the growth of C. hominis. We cloned and sequenced the genes for Sir2 orthologs encoded by three isolates of C. hominis, two isolates of Cryptosporidium parvum and one isolate of Cryptosporidium meleagridis. The orthologs conserve critical catalytic or NAD-binding residues but do not have similarity with human Sir2 proteins (SIRTs). Cryptosporidium Sir2 orthologs would therefore be attractive therapeutic targets. The Cryptosporidium orthologs were classified into four variants based on their nucleotide sequences. Each of the four variants produces its own unique restriction fragment length polymorphism pattern by digestion with TfiI.

Occurrence and AhR activity of brominated parabens in the Kitakami River, North Japan.

Parabens are used as preservatives in pharmaceuticals and personal care products (PPCPs). Parabens react with aqueous chlorine, which is used in disinfection processes, leading to the formation of halogenated parabens. In the presence of Br-, parabens and HOBr (formed via oxidation of Br-) can react to form brominated parabens. Brominated parabens may result in pollution of river water through effluent discharge from sewage treatment plants. The present study involved measuring brominated paraben concentrations in the Kitakami River, northern Japan, which flows through urban and agricultural areas. Aryl hydrocarbon receptor (AhR) agonist activity was also assessed using a yeast (YCM3) reporter gene and HepG2 ethoxyresorufin O-deethylase (EROD) assays. Dibrominated methylparaben (Br2MP), ethylparaben (Br2EP), propylparaben (Br2PP), butylparaben (Br2BP), and benzylparaben (Br2BnP), and monobrominated benzylparaben (Br1BnP) were detected in 25-100% of river samples during the sampling period from 2017 to 2018 at median concentrations of 8.1-28 ng/L; the highest concentrations were measured during the low flow season (November) in urban areas (P < 0.01). In the yeast assay, 12 compounds exhibited AhR activity (activity relative to ß-naphthoflavone; 4.4 × 10-4-7.1 × 10-1). All monobrominated parabens exhibited higher activity than their parent parabens, however, further bromination reduced or eliminated their activity. In the EROD assay, five compounds caused significant induction of CYP1A-dependent activity at 100 µM (P < 0.05). Monobrominated i-butylparaben (Br1iBP) and s-butylparaben (Br1sBP), Br1BnP, and Br2BP exhibited activity in both yeast and EROD assays. We found novel aspects of brominated parabens originating from PPCPs.

Analysis of Sir2E in the cellular slime mold Dictyostelium discoideum: cellular localization, spatial expression and overexpression.

It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A-D) showing sequence similarity to human homologues of Sir2 (SIRT1-3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E-green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum.

A homolog of Escherichia coli RecA in mitochondria of the cellular slime mold Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum expresses a gene encoding a 452-amino-acid polypeptide that is 47% identical to Escherichia coli RecA. A recA-deficient E. coli, JE6651, was transformed by pYSN1, which was designed to express the truncated form of the D. discoideum gene, and used in suppression assays. The viability of the transformant, JE6651(pYSN1), increased following UV irradiation or mitomycin C treatment. Phage lambda (red(-) gam(-)), which required RecA activity for DNA packaging, formed plaques on a lawn of JE6651(pYSN1). These results indicate that the gene product has a DNA recombination activity. Fluorescence of D. discoideum protein fused with GFP was detected in mitochondria. The gene disruption mutant was hypersensitive to UV-light (254nm), mitomycin C and H(2)O(2), indicating that D. discoideum recA is important for survival following exposure to DNA damaging agents.

Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

Photomanipulation of antibiotic susceptibility and biofilm formation of Escherichia coli heterologously expressing photoactivated adenylyl cyclase.

A cyaA-deficient Escherichia coli strain was transformed by a plasmid carrying the gene for BsPAC, a photoactivated adenylyl cyclase identified from a Beggiatoa sp., and was subjected to an antibiotic susceptibility assay and biofilm formation assay under a light or dark condition. Cells expressing BsPAC that were incubated under blue light (470 nm) were more susceptible to fosfomycin, nalidixic acid and streptomycin than were cells incubated in the dark. Cells expressing BsPAC formed more biofilms when incubated under the light than did cells cultured in the dark. We concluded from these observations that it is possible to determine the importance of cAMP in antibiotic susceptibility and biofilm formation of E. coli by photomanipulating the cellular cAMP level by the use of BsPAC. A site-directed mutant of BsPAC in which Tyr7 was replaced by Phe functioned even in the dark, indicating that Tyr7 plays an important role in photoactivation of BsPAC. Results of mutational analysis of BsPAC should contribute to an understanding of the molecular basis for photoactivation of the protein.

Developmental and Spatial Expression of sir2 Genes in the Cellular Slime Mold Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum grows as unicellular free-living amoebae in the presence of nutrients. Upon starvation, the amoebae aggregate and form multicellular structures that each consist of a stalk and spores. D. discoideum encodes at least four proteins (Sir2A, Sir2B, Sir2C, and Sir2D) homologous to human SIRT. RT-PCR and WISH analyses showed that the genes for Sir2A, Sir2C, and Sir2D were expressed at high levels in growing cells but at decreased levels in developing cells, whereas the gene encoding Sir2B was expressed in the prestalk-cell region in the developmental phase.

Identification of a penicillin-sensitive carboxypeptidase in the cellular slime mold Dictyostelium discoideum.

Penicillin binding proteins (PBPs) are penicillin-sensitive DD-peptidases catalyzing the terminal stages of bacterial cell wall assembly. We identified a Dictyostelium discoideum gene that encodes a protein of 522 amino acids showing similarity to Escherichia coli PBP4. The D. discoideum protein conserves three consensus sequences (SXXK, SXN and KTG) that are responsible for the catalytic activities of PBPs. The gene product prepared in the cell-free translation system showed carboxypeptidase activity but the activity was not detected in the presence of penicillin G. These results demonstrate that the D. discoideum gene encodes a eukaryotic form of penicillin-sensitive carboxypeptidase.

Multinucleation of the sodC-deficient Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum expresses three genes (sodA, sodB and sodC) encoding the extracellular Cu/Zn superoxide dismutases. Following H(2)O(2) treatment, the expression of sodA and sodB increased while that of sodC decreased. The sodC null strain formed multinucleate cells in a shaking culture. These results suggest that sodC plays a unique role in Dictyostelium discoideum.