Rapid recovery of nuclear and mitochondrial genes by genome skimming from Northern Hemisphere freshwater crayfish

Molecular phylogenetics has benefited tremendously from the advent of next‐generation sequencing, enabling quick and cost‐effective recovery of whole mitogenomes via an approach referred to as ‘genome skimming’. Recently, genome skimming has been utilised to recover highly repetitive nuclear genes such as 18S and 28S ribosomal RNA genes that are useful for inferring deeper evolutionary relationships. To address some outstanding issues in the relationships among Northern Hemisphere freshwater crayfish (Astacoidea), we sequenced the partial genome of crayfish species from Asian, North American and European genera and report the successful recovery of whole mitogenome sequences in addition to three highly repetitive nuclear genes, namely histone H3, 18S and 28S ribosomal RNA. Consistent with some previous studies using short mtDNA and nuclear gene fragments, phylogenetic analyses based on the concatenation of recovered mitochondrial and/or nuclear sequences recovered the Asian cambarid lineage as basal to all astacids and North American cambarids, which conflicts with the current taxonomic classification based on morphological and reproduction‐related characters. Lastly, we show that complete H3, 18S and 28S ribosomal RNA genes can also be consistently recovered from a diverse range of animal taxa, demonstrating the potential wide utility of genome skimming for nuclear markers.     

Transmission cycles of Borrelia burgdorferi and B. bissettii in relation to habitat type in northwestern California

This study was undertaken to determine which rodent species serve as primary reservoirs for the Lyme disease spirochete Borrelia burgdorferi in commonly occurring woodland types in inland areas of northwestern California, and to examine whether chaparral or grassland serve as source habitats for dispersal of B. burgdorferi‐ or B. bissettii‐infected rodents into adjacent woodlands. The western gray squirrel (Sciurus griseus) was commonly infected with B. burgdorferi in oak woodlands, whereas examination of 30 dusky‐footed woodrats (Neotoma fuscipes) and 280 Peromyscus spp. mice from 13 widely‐spaced Mendocino County woodlands during 2002 and 2003 yielded only one infected woodrat and one infected deer mouse (P. maniculatus). These data suggest that western gray squirrels account for the majority of production by rodents of fed Ixodes pacificus larvae infected with B. burgdorferi in the woodlands sampled. Infections with B. burgdorferi also were rare in woodrats (0/47, 0/3) and mice (3/66, 1/6) captured in chaparral and grassland, respectively, and therefore these habitats are unlikely sources for dispersal of this spirochete into adjacent woodlands. On the other hand, B. bissettii was commonly detected in both woodrats (22/47) and mice (15/66) in chaparral. We conclude that the data from this and previous studies in northwestern California are suggestive of a pattern where inland oak‐woodland habitats harbor a B. burgdorferi transmission cycle driven primarily by I. pacificus and western gray squirrels, whereas chaparral habitats contain a B. bissettii transmission cycle perpetuated largely by I. spinipalpis, woodrats, and Peromyscus mice. The dominant role of western gray squirrels as reservoirs of B. burgdorferi in certain woodlands offers intriguing opportunities for preventing Lyme disease by targeting these animals by means of either host‐targeted acaricides or oral vaccination against B. burgdorferi.     

Catalysis by Glomerella cingulata cutinase requires conformational cycling between the active and inactive states of its catalytic triad

Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 Å. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 Å away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.     

fMRI mapping of cortical centers following visual stimulation in postnatal pigs of different ages

Classical studies have demonstrated that the visual centers in primates consist of cortical areas V1, V2 and V4 and their branches. However, nothing is known about how these visual areas change in postnatal development. In the present studies, therefore, pigs aged 2, 4, and 6 months old, were stimulated visually with a colored checker board and the active sites in the cortex, cerebellum and brainstem recorded using functional magnetic resonance imaging (fMRI). In pigs aged 2 months old, visual stimulation induced an increase in activation of sites in the V2 and V4 cortical areas, as well as in the areas of the inferior aspect of the parietal and middle aspect of the temporal cortices, but not in the medial and caudal occipital cortex (V1 area). At 4 months old, the V1 area was also activated, and by 6 months old, an inferior sector in the prefrontal cortex was also activated. As the pigs aged, functional active sites were further demonstrated in the cerebellum and the brainstem, which probably had to do with action memory, and the control of the ocular muscles, respectively. It is concluded that the visual pathway of the pig mainly involves cortical areas that mature at 6 months of age.     

Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms

Background

Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform.

Methodology/Principal Findings

In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples.

Conclusion/Significance

Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.     

Tropomyosin position on F-actin revealed by EM reconstruction and computational chemistry

Electron microscopy and fiber diffraction studies of reconstituted F-actin-tropomyosin filaments reveal the azimuthal position of end-to-end linked tropomyosin molecules on the surface of actin. However, the longitudinal z-position of tropomyosin along F-actin is still uncertain. Without this information, atomic models of F-actin-tropomyosin filaments, free of constraints imposed by troponin or other actin-binding proteins, cannot be formulated, and thus optimal interfacial contacts between actin and tropomyosin remain unknown. Here, a computational search assessing electrostatic interactions for multiple azimuthal locations, z-positions, and pseudo-rotations of tropomyosin on F-actin was performed. The information gleaned was used to localize tropomyosin on F-actin, yielding an atomic model characterized by protein-protein contacts that primarily involve clusters of basic amino acids on actin subdomains 1 and 3 juxtaposed against acidic residues on the successive quasi-repeating units of tropomyosin. A virtually identical model generated by docking F-actin and tropomyosin atomic structures into electron microscopy reconstructions of F-actin-tropomyosin validated the above solution. Here, the z-position of tropomyosin alongside F-actin was defined by matching the seven broad and narrow motifs that typify tropomyosin's twisting superhelical coiled-coil to the wide and tapering tropomyosin densities seen in surface views of F-actin-tropomyosin reconstructions. The functional implications of the F-actin-tropomyosin models determined in this work are discussed.     

A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome

Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase®, Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase®, Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes. The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4?±?0.9 vs. 68.1?±?2.2 %, P?<?0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6?±?1.1 vs. 27.8?±?0.9 nmol/min/μg protein, P?<?0.001). The levels of M6P and sialic acid were not significantly different (2.4?±?0.1 vs 2.4?±?0.3 mol/mol protein for M6P and 12.3?±?0.7 vs. 12.4?±?0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (K_uptake, 5.09?±?0.96 vs. 6.50?±?1.28 nM protein, P?=?0.017). In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.