Genome sequence of Yersinia pestis KIM

We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12. The KIM genome sequence was compared with that of Y. pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and E. coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear "backbone," or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of E. coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.     

Immunodetection of disease-associated mutant PrP, which accelerates disease in GSS transgenic mice

The absence of infectivity-associated, protease-resistant prion protein (PrP^Sc) in the brains of spontaneously sick transgenic (Tg) mice overexpressing PrP linked to Gerstmann–Sträussler Scheinker syndrome, and the failure of gene-targeted mice expressing such PrP to develop disease spontaneously, challenged the concept that mutant PrP expression led to spontaneous prion production. Here, we demonstrate that disease in overexpressor Tg mice is associated with accumulation of protease-sensitive aggregates of mutant PrP that can be immunoprecipitated by the PrP^Sc-specific monoclonal antibody designated 15B3. Whereas Tg mice expressing multiple transgenes exhibited accelerated disease when inoculated with disease-associated mutant PrP, Tg mice expressing mutant PrP at low levels failed to develop disease either spontaneously or following inoculation. These studies indicate that inoculated mutant PrP from diseased mice promotes the aggregation and accumulation of pre-existing pathological forms of mutant PrP produced as a result of transgene overexpression. Thus, while pathological mutant PrP possesses a subset of PrP^Sc characteristics, we now show that the attribute of prion transmission suggested by previous studies is more accurately characterized as disease acceleration.     

The effects of copper on photosynthesis and biomolecules yield in Chlorolobion braunii

Our knowledge of the effects of copper on microalgal physiology is largely based on studies conducted with high copper concentrations; much less is known when environmentally relevant copper levels come into question. Here, we evaluated the physiology of Chlorolobion braunii exposed to free copper ion concentrations between 5.7 × 10^?9 and 5.0 × 10^?6 mol · L^?1, thus including environmentally relevant values. Population growth and maximum photosynthetic quantum yield of PSII were determined daily during the 96 h laboratory controlled experiment. Exponentially‐growing cells (48 h) were analyzed for effective quantum yield and rapid light curves (RLC), and total lipids, proteins, carbohydrates, chlorophyll a and carotenoids were determined. The results showed that growth rates and population density decreased gradually as copper increased in experiment, but the photosynthetic parameters (maximum and effective quantum yields) and photochemical quenching (qP) decreased only at the highest free copper concentration tested (5.0 × 10^?6 mol · L^?1); nonphotochemical quenching (NPQ) increased gradually with copper increase. The RLC parameters Ek and rETRmax were inversely proportional to copper concentration, while α and Im decreased only at 5.0 × 10^?6 mol · L^?1. The effects of copper in biomolecules yield (mg · L^?1) varied depending on the biomolecule. Lipid yield increased at free copper concentration as low as 2.5 × 10^?8 mol · L^?1, but proteins and carbohydrates were constant throughout.     

Evaluation of diuron tolerance and biotransformation by the white-rot fungus Ganoderma lucidum

The white rot basidiomycete Ganoderma lucidum was evaluated for its capability to tolerate and to degrade the herbicide diuron. Diuron at a subtoxic concentration was added at the start of the cultivation in glucose liquid stationary cultures. Under this condition diuron was a laccase inducer. Almost 50% of the initially present diuron was removed after 15 d of cultivation. Two diuron metabolites were found N′-(3,4-dichlorophenyl)-N-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The addition of the cytochrome P450 inhibitors 1-aminobenzotriazole and piperonyl butoxide reduced significantly the capability of the fungus in degrading diuron. The activities of superoxide dismutase and catalase were significantly increased in the mycelial extracts by the presence of diuron. On the other hand, diuron did not cause any significant alteration in the levels of reactive oxygen species. Additionally, laccase could also degrade diuron in vitro and this degradation was increased by the addition of synthetic mediators, 3-ethylbenzthiazoline-6-sulphonic acid and acetylacetone. Significant reduction in the toxicity, as evaluated by the Lactuca sativa bioassay, was observed after G. lucidum treatment. In conclusion, G. lucidum is able to metabolize diuron by intra- and extracellular mechanisms, without the accumulation of toxic products.     

Monophosphoryl Lipid A and Pam3Cys Prevent the Increase in Seizure Susceptibility and Epileptogenesis in Rats Undergoing Traumatic Brain Injury

Five percent of all epilepsy cases are attributed to traumatic brain injury (TBI), which are known as post-traumatic epilepsy (PTE). Finding preventive strategies for PTE is valuable. Remarkable feature of TBI is activation of microglia and subsequent neuroinflammation, which provokes epileptogenesis. The toll-like receptor agonists monophosphoryl lipid A (MPL) and tri-palmitoyl-S-glyceryl-cysteine (Pam3Cys) are safe, well-tolerated and effective adjuvants existing in prophylactic human vaccines. We examined the impact of early injection of MPL and Pam3Cys to rats, on the rate of kindled seizures acquisition following TBI. Rats received a single dose (1 µg/rat) of MPL or Pam3Cys through intracerebroventricular injection. 5 days later, trauma was exerted to temporo-parietal cortex of rats by controlled cortical impact device. After 24 h, traumatic rats underwent amygdala kindling. Brain level of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) was also measured in traumatic rats by immunoblotting. Compared to non-traumatic (sham-operated) rats, traumatic rats showed three times lower seizure threshold (133?±?5 µA vs. 416.3?±?16 µA, p?<?0.001); about three times less number of stimuli to become kindled (5?±?1 vs. 14?±?2, p?<?0.01); longer duration of kindled seizure parameters including entire seizure behavior, generalized seizures, and afterdischarges (p?<?0.001); and a two times increase in the TNF-α level. MPL and Pam3Cys did not change kindling rate and the seizure parameters in sham-operated rats. The MPL- and Pam3Cys-pretreated traumatic rats displayed seizure threshold, speed of kindling, and duration of kindled seizure parameters, similar to the non-traumatic rats. Pretreatment by MPL and Pam3Cys prevented the increase in TNF-α level by trauma. Given that MPL and Pam3Cys currently have clinical use as well-tolerated vaccines with reliable safety, they have the potential to be used in prevention of PTE.     

Lethal and sublethal effects of insecticides on the survival and reproduction of Brevipalpus yothersi (Acari: Tenuipalpidae)

The overuse of insecticides to control vector insects such as Diaphorina citri Kuwayama in citrus groves has altered the population dynamics of pest mites. Among phytophagous mites, population outbreaks of citrus leprosis mite, Brevipalpus yothersi Baker, have been increasingly intense and frequent in Brazilian citrus groves. Despite the great importance of the B. yothersi mite for citrus production, the lethal and sublethal effects of insecticides on this mite have not yet been studied. Therefore, in this study, the effects of insecticides commonly used for D. citri control on B. yothersi mortality, reproduction, and instantaneous growth rate were assessed. For this, two experiments were carried out, one under controlled conditions and another in a greenhouse. The insecticides tested were beta-cyfluthrin, bifenthrin, buprofezin, chlorpyrifos, dimethoate, pyriproxyfen, and thiamethoxam at 0 (control), 0.0625, 0.125, 0.25, 0.5, 1, and twofold the recommended insecticide concentration for D. citri control. The pyriproxyfen insecticide provided high mortality of B. yothersi even at low concentrations. Furthermore, this insecticide negatively interfered with the reproduction of this mite. Beta-cyfluthrin, bifenthrin, buprofezin, chlorpyrifos, dimethoate, and thiamethoxam, in the tested concentrations, showed low impact on citrus leprosis mite. Regarding the reproduction of the mite, no significant increase in fecundity was observed on B. yothersi females exposed to insecticide residues, regardless of the concentration tested. Therefore, the application of these insecticides in the management of pest insects is unlikely to promote an increase in the citrus leprosis mite population.

     

Genotoxicity of diphenyl diselenide in bacteria and yeast

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. This may increase the risk of human exposure to the chemical at the workplace. We have determined its mutagenic potential in the Salmonella/microsome assay and used the yeast Saccharomyces cerevisiae to assay for putative genotoxicity, recombinogenicity and to determine whether DNA damage produced by DPDS is repairable. Only in exponentially growing cultures was DPDS able to induce frameshift mutations in S. typhimurium and haploid yeast and to increase crossing over and gene conversion frequencies in diploid strains of S. cerevisiae. Thus, DPDS presents a behavior similar to that of an intercalating agent. Mutants defective in excision-resynthesis repair (rad3, rad1), in error-prone repair (rad6) and in recombinational repair (rad52) showed higher than WT-sensitivity to DPDS. It appears that this compound is capable of inducing single and/or double strand breaks in DNA. An epistatic interaction was shown between rad3-e5 and rad52-1 mutant alleles, indicating that excision-resynthesis and strand-break repair may possess common steps in the repair of DNA damage induced by DPDS. DPDS was able to enhance the mutagenesis induced by oxidative mutagens in bacteria. N-acetylcysteine, a glutathione biosynthesis precursor, prevented mutagenesis induced by DPDS in yeast. We have shown that DPDS is a weak mutagen which probably generates DNA strand breaks through both its intercalating action and pro-oxidant effect.     

Effect of mouse genotype on interferon production

Upon induction with Newcastle disease virus, peritoneal macrophages derived from C57BL/6 mice produced ten times as much interferon as macrophages derived from BALB/c mice. This suggested that the alleles of theIf-1 locus are expressed in vitro by these cells. Further evidence for this was obtained by studying interferon production by peritoneal macrophages derived from seven recombinant inbred and one congenic line: in each case there was complete correlation between in vivo and in vitro phenotype: macrophages fromIf-1^l mice were low producers in vitro, and macrophages fromIf-1 ^h mice were high producers in vitro.