Chemical toxicity testing in vitro using cytochrome P450-expressing cell lines, such as human CYP1B1

This protocol describes how to use cytochrome P450-dependent monooxygenase (CYP)-expressing cell lines in toxicity testing of chemicals in vitro. Selected cells amenable to permanently grow in culture are genetically manipulated to stably express single CYP enzymes originating from any species of interest. This expression can be characterized by, for example, determining CYP mRNA content, CYP protein level (western blotting or in situ immunofluorescence) and CYP-mediated enzyme activity (substrate conversion assays). These cells can be used to determine substrate specificities and species differences, e.g., in the bioactivation of drugs. Once constructed, CYP-expressing cells can serve as a straightforward and reliable tool in toxicity testing and the corresponding assays could be adapted for high-throughput analysis. Using these cells, enzyme assays can be performed in a matter of hours. This protocol is exemplified with V79 fibroblasts from Chinese hamster (Cricetulus griseus), modified to express human cytochrome P450 1B1 (CYP1B1). These cells are characterized for their CYP1B1-linked properties by in situ immunofluorescence and their activity in the 7-ethoxyresorufin-O-deethylase enzyme assay. This is followed by an assay showing metabolic activation of the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene by CYP1B1, along with the toxicological endpoints of cytotoxicity and micronucleus formation.     

Transcriptomic analysis of the sulfate starvation response of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes a number of infections in humans, but is best known for its association with cystic fibrosis. It is able to use a wide range of sulfur compounds as sources of sulfur for growth. Gene expression in response to changes in sulfur supply was studied in P. aeruginosa E601, a cystic fibrosis isolate that displays mucin sulfatase activity, and in P. aeruginosa PAO1. A large family of genes was found to be upregulated by sulfate limitation in both isolates, encoding sulfatases and sulfonatases, transport systems, oxidative stress proteins, and a sulfate-regulated TonB/ExbBD complex. These genes were localized in five distinct islands on the genome and encoded proteins with a significantly reduced content of cysteine and methionine. Growth of P. aeruginosa E601 with mucin as the sulfur source led not only to a sulfate starvation response but also to induction of genes involved with type III secretion systems.     

Long-term cultures of barley synthesize and correctly deposit seed storage proteins

Summary Long-term cultures of four different cultivars of barley (Hordeum vulgare L.) have been established. Both callus and suspension cultures formed embryogenic structures at high frequency even after more than 18 months of culture. These compact proembryogenic cell clusters synthesize seed storage globulins whereas loose cell aggregates in callus culture and suspension cultures of fine dispersed consistency were free of globulins. Globulin synthesis was especially intense in compact structures of callus cultures established from suspension culture-derived protoplasts. Within the cells storage globulins are deposited in the vacuolar compartment as in zygotic embryos. The molecular data provided recommend the system for studies on factors determining seed protein gene expression and intracellular protein transport.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Why two are not enough: degradation of p-toluenesulfonate by a bacterial community from a pristine site in Moorea, French Polynesia

It has been demonstrated previously that Enterococcus faecalis produces secreted endoglycosidases that enable the bacteria to remove N-linked glycans from glycoproteins. One enzyme potentially responsible for this activity is EF0114, comprising a typical GH18 endoglycosidase domain and a GH20 domain. We have analyzed the other candidate, EF2863, and show that this predicted single domain GH18 protein is an endo-β-N-acetylglucosaminidase. EF2863 hydrolyzes the glycosidic bond between two N-acetylglucosamines (GlcNAc) in N-linked glycans of the high-mannose and hybrid type, releasing the glycan and leaving one GlcNAc attached to the protein. The activity of EF2863 is similar to that of the well known deglycosylating enzyme EndoH from Streptomyces plicatus. According to the CAZy nomenclature, the enzyme is designated EfEndo18A.     

Co‐occurrence of bobcats,coyotes, and ocelots in Texas

Interspecific competition among carnivores has been linked to differences in behavior, morphology, and resource use. Insights into these interactions can enhance understanding of local ecological processes that can have impacts on the recovery of endangered species, such as the ocelot (Leopardus pardalis). Ocelots, bobcats (Lynx rufus), and coyotes (Canis latrans) share a small geographic range overlap from South Texas to south‐central Mexico but relationships among the three are poorly understood. From May 2011 to March 2018, we conducted a camera trap study to examine co‐occurrence patterns among ocelots, bobcats, and coyotes on the East Foundation's El Sauz Ranch in South Texas. We used a novel multiseason extension to multispecies occupancy models with ≥2 interacting species to conduct an exploratory analysis to examine interspecific interactions and examine the potential effects of patch‐level and landscape‐level metrics relative to the occurrence of these carnivores. We found strong evidence of seasonal mutual coexistence among all three species and observed a species‐specific seasonal trend in detection. Seasonal coexistence patterns were also explained by increasing distance from a high‐speed roadway. However, these results have important ecological implications for planning ocelot recovery in the rangelands of South Texas. This study suggests a coexistence among ocelots, bobcats, and coyotes under the environmental conditions on the El Sauz Ranch. Further research would provide a better understanding of the ecological mechanisms that facilitate coexistence within this community. As road networks in the region expand over the next few decades, large private working ranches will be needed to provide important habitat for ocelots and other carnivore species.     

Modified pressure distribution patterns in walking following reduction of plantar sensation

The aim of the present study was to investigate the influence of reduced plantar sensation on pressure distribution patterns during gait of 40 healthy subjects (25.3+/-3.3 yr, 70.8+/-10.6 kg and 176.5+/-7.8 cm) with no history of sensory disorders. Plantar sensation in the subjects was reduced by using an ice immersion approach, and reduced sensitivity was tested with Semmes-Weinstein monofilaments. All subjects performed six trials of barefoot walking over a pressure distribution platform under normal as well as iced conditions. Plantar cutaneous sensation was significantly reduced after the cooling procedure (p<0.0001). Pressure distribution analysis showed substantially modified plantar pressure distribution patterns during the roll-over process (ROP) under iced conditions. Analysis of peak pressures revealed significant reductions under the toes and under the heel (p<0.001). The contact time and the relative impulse for the whole foot did not change significantly between the two conditions. For the different areas, a significant load shift from the heel and toes towards the central and lateral forefoot and the lateral midfoot was observed. The results indicate the strong influence of reduced afferent information of the sole of the foot on the ROP in walking.     

Comparative immobilization of wild felids in Thailand

We immobilized individuals of four free-ranging felid species, leopard cat (Prionailurus bengalensis), clouded leopard (Neofelis nebulosa), Asiatic golden cat (Catopuma temminckii), and marbled cat (Pardofelis marmorata) with ketamine hydrochloride and xylazine hydrochloride (KH-XH) and with tiletamine hydrochloride and zolazepam hydrochloride (TH-ZH) between March 1998 and July 2002. Mean (+/-SD) dose of KH and XH was 26.51+/-5.71 mg/kg and 1.89+/-0.43 mg/kg, respectively (n=25), and mean dose of TH-ZH was 11.61+/-3.39 mg/kg (n=28). Dose was significantly correlated with induction time (P<0.001) and duration of anesthesia (P<0.05), but not with recovery time. There were significant differences between the drug combinations in time to induction (P<0.03) and time to anesthesia (P<0.01); recovery times were not significantly different. We conclude that immobilization of these felids with TH-ZH and KH-XH is effective and safe, but TH-ZH is preferred because of the smaller volume of drug necessary for sedation, faster time to induction, and absence of prolonged muscle rigidity during anesthesia.