Tick-Borne Rickettsial Pathogens in Ticks and Small Mammals in Korea

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.     

The adenosine dimethyltransferase KsgA recognizes a specific conformational state of the 30S ribosomal subunit

The methyltransferase KsgA modifies two adjacent adenosines in 16S rRNA by adding two methyl groups to the N(6) position of each nucleotide. Unlike nearly all other rRNA modifications, these modifications and the responsible enzyme are highly conserved phylogenetically, suggesting that the modification system has an important role in ribosome biogenesis. It has been known for some time that KsgA recognizes a complex pre-30S substrate in vitro, but there is disagreement in the literature as to what that substrate can be. That disagreement is resolved in this report; KsgA is unable to methylate 30S subunits in the translationally active conformation, but rather can modify 30S when in an experimentally well established translationally inactive conformation. Recent 30S crystal structures provide some basis for explaining why it is impossible for KsgA to methylate 30S in the translationally active conformation. Previous work identified one set of ribosomal proteins important for efficient methylation by KsgA and another set refractory methylation. With the exception of S21 the recent crystal structures of 30S also instructs that the proteins important for KsgA activity all exert their influence indirectly. Unfortunately, S21, which is inhibitory to KsgA activity, has not had its position determined by X-ray crystallography. A reevaluation of published biophysical data on the location also suggests that the refractory nature of S21 is also indirect. Therefore, it appears that KsgA solely senses the conformation 16S rRNA when carrying out its enzymatic activity.     

Induction of lung glutathione and glutamylcysteine ligase by 1,4-phenylenebis(methylene)selenocyanate and its glutathione conjugate: role of nuclear factor-erythroid 2-related factor 2

The synthetic organoselenium agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione (GSH) conjugate (p-XSeSG) are potent chemopreventive agents in several preclinical models. p-XSC is also an effective inducer of GSH in mouse lung. Our objectives were to test the hypothesis that GSH induction by p-XSC occurs through upregulation of the rate-limiting GSH biosynthetic enzyme glutamylcysteine ligase (GCL), through activation of antioxidant response elements (AREs) in GCL genes via activation of nuclear factor-erythroid 2-related factor 2 (Nrf2). p-XSC feeding (10 ppm Se) increased GSH (230%) and upregulated the catalytic subunit of GCL (GCLc) (55%), extracellular-related kinase (220%), and nuclear Nrf2 (610%) in lung but not liver after 14 days in the rat (P<0.05). Similarly, p-XSeSG feeding (10 ppm) induced lung GCLc (88%) and GSH (200%) (P<0.05), whereas the naturally occurring selenomethionine had no effect. Both p-XSC and p-XSeSG activated a luciferase reporter in HepG2 ARE-reporter cells up to threefold for p-XSC and greater than or equal to fivefold for p-XSeSG. Luciferase activation by p-XSeSG was associated with enhanced levels of GSH, GCLc, and nuclear Nrf2, which were significantly reduced by co-incubation with short interfering RNA targeting Nrf2. The dependence of GCL induction on Nrf2 was confirmed in Nrf2-deficient mouse embryonic fibroblasts, in which p-XSeSG induced GCL subunits in wild-type but not in Nrf2-deficient cells (P<0.05). These results indicate that p-XSC may act through the Nrf2 pathway in vivo and that p-XSeSG is the putative metabolite responsible for such activation, thus offering p-XSeSG as a less toxic, yet highly efficacious, inducer of GSH.     

Cytochrome c oxidase dysfunction in oxidative stress

Cytochrome c oxidase (CcO) is the terminal oxidase of the mitochondrial electron transport chain. This bigenomic enzyme in mammals contains 13 subunits of which the 3 catalytic subunits are encoded by the mitochondrial genes. The remaining 10 subunits with suspected roles in the regulation, and/or assembly, are coded by the nuclear genome. The enzyme contains two heme groups (heme a and a3) and two Cu(2+) centers (Cu(2+) A and Cu(2+) B) as catalytic centers and handles more than 90% of molecular O(2) respired by the mammalian cells and tissues. CcO is a highly regulated enzyme which is believed to be the pacesetter for mitochondrial oxidative metabolism and ATP synthesis. The structure and function of the enzyme are affected in a wide variety of diseases including cancer, neurodegenerative diseases, myocardial ischemia/reperfusion, bone and skeletal diseases, and diabetes. Despite handling a high O(2) load the role of CcO in the production of reactive oxygen species still remains a subject of debate. However, a volume of evidence suggests that CcO dysfunction is invariably associated with increased mitochondrial reactive oxygen species production and cellular toxicity. In this paper we review the literature on mechanisms of multimodal regulation of CcO activity by a wide spectrum of physiological and pathological factors. We also review an array of literature on the direct or indirect roles of CcO in reactive oxygen species production.     

Modular and scalable load-wall sled buck for pure-lateral and oblique side impact tests

A considerable majority of side impact sled tests using different types of human surrogates has used a load-wall design not specific to subject anthropometry. The use of one load-wall configuration cannot accurately isolate and evaluate regional responses for the same load-wall geometry. As the anatomy and biomechanical responses of the human torso depends on the region, and anthropomorphic test devices continue to advance and accommodate regional differences, it is important to obtain specific data from sled tests. To achieve this goal, the present study designed a scalable modular load-wall consisting of the shoulder, thorax, abdomen, and superior and inferior pelvis, and lower limb plates. The first five plates were connected to a vertical fixture and the limb plate was connected to another fixture. The width, height, and thickness, and the gap between plates were modular. Independent adjustments in the coronal and sagittal planes allowed region-specific positioning depending on surrogate anthropometry, example pelvis width and seated height. Two tri-axial load cells were fixed on the contralateral face of each plate of the load-wall to record impact force-time histories. The load-wall and vertical fixture design can be used to conduct side impact tests with varying vectors, pure-lateral to anterior and posterior oblique, by appropriately orienting the load-wall with respect to the surrogate. The feasibility of the design to extract region-specific biomechanical data was demonstrated by conducting pure-lateral and anterior oblique sled tests using two different surrogates at a velocity of 6.7m/s. Uses of this design are discussed for different applications.     

Sulfated, low molecular weight lignins inhibit a select group of heparin-binding serine proteases

Sulfated low molecular weight lignins (LMWLs), designed as oligomeric mimetics of low molecular weight heparins (LMWHs), have been found to bind in exosite II of thrombin. To assess whether sulfated LMWLs recognize other heparin-binding proteins, we studied their effect on serine proteases of the coagulation, inflammatory and digestive systems. Using chromogenic substrate hydrolysis assay, sulfated LMWLs were found to potently inhibit coagulation factor XIa and human leukocyte elastase, moderately inhibit cathepsin G and not inhibit coagulation factors VIIa, IXa, and XIIa, plasma kallikrein, activated protein C, trypsin, and chymotrypsin. Competition studies show that UFH competes with sulfated LMWLs for binding to factors Xa and XIa. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold.     

Dynamic mutation-selection balance as an evolutionary attractor

The vast majority of mutations are deleterious and are eliminated by purifying selection. Yet in finite asexual populations, purifying selection cannot completely prevent the accumulation of deleterious mutations due to Muller's ratchet: once lost by stochastic drift, the most-fit class of genotypes is lost forever. If deleterious mutations are weakly selected, Muller's ratchet can lead to a rapid degradation of population fitness. Evidently, the long-term stability of an asexual population requires an influx of beneficial mutations that continuously compensate for the accumulation of the weakly deleterious ones. Hence any stable evolutionary state of a population in a static environment must involve a dynamic mutation-selection balance, where accumulation of deleterious mutations is on average offset by the influx of beneficial mutations. We argue that such a state can exist for any population size N and mutation rate U and calculate the fraction of beneficial mutations, ε, that maintains the balanced state. We find that a surprisingly low ε suffices to achieve stability, even in small populations in the face of high mutation rates and weak selection, maintaining a well-adapted population in spite of Muller's ratchet. This may explain the maintenance of mitochondria and other asexual genomes.     

Characterization of temperature inducible promoters from a novel rolling circle replicating plasmid of Enterococcus faecium DJ1

Limited studies have been performed on the characterization of small size plasmids of Enterococcus faecium with the intention of evaluating the strength of their promoters in Escherichia coli. The complete nucleotide sequence (3.825 Kb) and structural organization of E. faecium DJ1 cryptic plasmid pNJAKD is presented. Seven promoter sequences from the pNJAKD plasmid of E. faecium have been identified. The regions coding for the putative promoters were either amplified using PCR based techniques or chemically synthesized as oligonucleotides of different sizes. These were subsequently cloned in the pEGFP vector at the Pvu II site. The efficiency of putative promoter fragments were measured using the intensity of eGFP fluorescence in E. coli JM101, DH5α and BL21(DE3), among which AKD3 exhibited moderate to strongest promoter activity at temperatures of 30, 37, and 42°C.