BackgroundThe discovery of genetically distinct hantaviruses in shrews (Order Soricomorpha, Family Soricidae) from widely separated geographic regions challenges the hypothesis that rodents (Order Rodentia, Family Muridae and Cricetidae) are the primordial reservoir hosts of hantaviruses and also predicts that other soricomorphs harbor hantaviruses. Recently, novel hantavirus genomes have been detected in moles of the Family Talpidae, including the Japanese shrew mole (Urotrichus talpoides) and American shrew mole (Neurotrichus gibbsii). We present new insights into the evolutionary history of hantaviruses gained from a highly divergent hantavirus, designated Nova virus (NVAV), identified in the European common mole (Talpa europaea) captured in Hungary.Methodology/Principal FindingsPair-wise alignment and comparison of the full-length S- and L-genomic segments indicated moderately low sequence similarity of 54–65% and 46–63% at the nucleotide and amino acid levels, respectively, between NVAV and representative rodent- and soricid-borne hantaviruses. Despite the high degree of sequence divergence, the predicted secondary structure of the NVAV nucleocapsid protein exhibited the characteristic coiled-coil domains at the amino-terminal end, and the L-segment motifs, typically found in hantaviruses, were well conserved. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, showed that NVAV formed a distinct clade that was evolutionarily distant from all other hantaviruses.ConclusionsNewly identified hantaviruses harbored by shrews and moles support long-standing virus-host relationships and suggest that ancestral soricomorphs, rather than rodents, may have been the early or original mammalian hosts.

Recent molecular evidence of genetically distinct hantaviruses in shrews, captured in widely separated geographical regions, corroborates decades-old reports of hantavirus antigens in shrew tissues. Apart from challenging the conventional view that rodents are the principal reservoir hosts, the recently identified soricidborne hantaviruses raise the possibility that other soricomorphs, notably talpids, similarly harbor hantaviruses. In analyzing RNA extracts from lung tissues of the Japanese shrew mole (Urotrichus talpoides), captured in Japan between February and April 2008, a hantavirus genome, designated Asama virus (ASAV), was detected by RT-PCR. Pairwise alignment and comparison of the S-, M-, and L-segment nucleotide and amino acid sequences indicated that ASAV was genetically more similar to hantaviruses harbored by shrews than by rodents. However, the predicted secondary structure of the ASAV nucleocapsid protein was similar to that of rodent-and shrew-borne hantaviruses, exhibiting the same coiledcoil helix at the amino terminus. Phylogenetic analyses, using the maximum-likelihood method and other algorithms, consistently placed ASAV with recently identified soricine shrew-borne hantaviruses, suggesting a possible host-switching event in the distant past. The discovery of a mole-borne hantavirus enlarges our concepts about the complex evolutionary history of hantaviruses.host switching ͉ talpid ͉ evolution ͉ Japan

Axonemal dyneins have two or three globular heads joined by flexible tails to a common base, with each head/tail unit consisting of a single heavy-chain polypeptide of relative molecular mass greater than 400,000. The sizes of the components have been deduced by electron microscopy. The isolated beta heavy chain of sea urchin sperm flagella, which is immunologically identical to that of the embryo cilia, is of particular interest as it retains the capability for microtubule translocation in vitro. Limited proteolysis of the beta heavy chain divides it into two fragments, A and B, which sediment separately at 12S and 6S, and possibly correspond to the head and tail domains of the molecule. Dynein ATPase is the energy-transducing enzyme that generates the sliding movement between tubules that underlies the beating of cilia and flagella of eukaryotes, and possibly also other large intracellular movements. Here we report that the deduced amino-acid sequence of the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla has 4,466 residues and contains the consensus motifs for five nucleotide-binding sites. The probable hydrolytic ATP-binding site can be identified by its location close to or at the V1 site of vanadate-mediated photo-cleavage. The general features of the map of photocleavage and proteolytic peptides reported earlier have been confirmed, except that the map's polarity is reversed. The predicted secondary structure of the beta heavy chain consists of an alpha/beta-type pattern along its whole length. The two longest regions of potential alpha helix, with unbroken heptad hydrophobic repeats 120 and 50 amino acids long, may be of functional importance. But dynein does not seem to contain an extended coiled-coil tail domain.

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The predicted structures are supported by procedures that assess global, regional, and local quality, with the module containing the hydrolytic ATP binding site being supported the most strongly. The structural resemblance of the dynein motor to the hexameric assembly of AAA modules in the hsp100 family of chaperones suggests that the basic mechanism underlying the ATP-dependent translocation of dynein along a microtubule may have aspects in common with the ATP-dependent translocation of polypeptides into the interior compartment of chaperones.

Two related neurodegenerative disorders, Western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD), originally occurred at a high incidence on Guam, in the Kii peninsula of Japan, and in southern West New Guinea more than 50 years ago. These three foci shared a unique mineral environment characterized by the presence of severely low levels of Ca 2؉ and Mg 2؉ , coupled with high levels of bioavailable transition metals in the soil and drinking water. Epidemiological studies suggest that genetic factors also contribute to the etiology of these disorders. Here, we report that a variant of the transient receptor potential melastatin 2 (TRPM2) gene may confer susceptibility to these diseases. TRPM2 encodes a calciumpermeable cation channel highly expressed in the brain that has been implicated in mediating cell death induced by oxidants. We found a heterozygous variant of TRPM2 in a subset of Guamanian ALS (ALS-G) and PD (PD-G) cases. This variant, TRPM2 P1018L , produces a missense change in the channel protein whereby proline 1018 (Pro 1018 ) is replaced by leucine (Leu 1018 ). Functional studies revealed that, unlike WT TRPM2, P1018L channels inactivate. Our results suggest that the ability of TRPM2 to maintain sustained ion influx is a physiologically important function and that its disruption may, under certain conditions, contribute to disease states.calcium ͉ neurodegeneration ͉ oxidative stress ͉ channelopathy ͉ gene environment

The binding of nucleoside triphosphates and related ligands to dynein ATPase from sea urchin sperm flagella has been studied by equilibrium partition analysis in an aqueous biphasic system containing dextran and poly(ethylene glycol). The stoichiometry of binding and the corresponding stepwise binding constants are obtained from direct binding isotherms fitted to the primary data. The results suggest that dynein possesses four different binding sites for nucleoside triphosphates per mole of heavy chain. The stepwise binding constants for MgATP range from approximately 10(4) M-1 to approximately 10(5) M-1. The isolated alpha and beta heavy chains have binding parameters similar to intact dynein. The amount of ADP bound normally is approximately 75% that of ATP, both for the intact dynein and for the separated heavy chains, although full saturation is achieved at high nucleotide concentrations. In the presence of the ATPase inhibitor vanadate, ADP binds with affinities similar to those of ATP, with binding constants close to those of ATP in the absence of vanadate. No appreciable binding of AMP or EDTA/ATP is observed. The substitution of Ca2+ or Fe3+ for Mg2+ does not significantly alter the amount of ATP bound; however, CaATP is bound with a somewhat lower affinity. Scatchard and Hill plots of the binding data and the calculated site-binding constants suggest that ATP and ADP bind in a weakly cooperative manner. These results suggest that the multiple binding of nucleotide to dynein heavy chains occurs at physiological concentrations, putatively at the four binding sites predicted earlier on the basis of their amino acid sequences. The data are consistent with a model in which, in addition to a single catalytic site, nucleotide binding occurs at additional noncatalytic sites that represent an as yet unknown functional aspect of dynein.

The heavy chain of myosin from rabbit skeletal muscle can be cleaved at three sites by irradiation with nearultraviolet light in the presence of 0.1 -1 .O mM vanadate. The sigmoidal dependence upon vanadate concentration, with half-mximal rate occurring at about 0.5 mM vanadate and a sigmoidicity of 2.7, is consistent with the chromophore responsible for cleavage being oligomeric vanadate. Cleavage occurs at two sites located within the head region of the molecule, 23 kDa and 75 kDa from the NH2-terminus; these sites are cleaved equally wcll in heavy meromyosin and subfragment 1. In the presence of 1 mM vanadate, the half-times for cleavage of the 23-kDa and 75-kDa sites are about 15 and 10 min, respectively. The rate of cleavage at both these sites is retarded 2-3-fold by the presence of > 10 pM MgATP. The third photocleavage site is located about 5 -10 kDa from the COOH terminus of the intact heavy chain, and cleaves equally well in the isolated rod and in light meromyosin. Cleavage at this site occurs with a half-time of 138 min, and its rate is unaffected by the presence of MgATP. The vanadate-mediated cleavage of the heavy chains is accompanied by characteristic changes in the myosin ATPase properties, with the Ca2 ', Mg2+ and actin-activated Mg2+ ATPases becoming elevated, whereas the K '/EDTA ATPase becomes inactivated. The sites of photocleavage in the myosin heavy chain might be associated with sites of phosphate binding. ATP inhibits the cleavage at this V2 site and results in cleavage at the V1 site. V2 cleavage is not correlated with any effect on the ATPase activity and the sigmoidal dependence of its rate upon vanadate concentration has been interpreted to indicate that the chromophore is a vanadate oligomer, possibly trivanadate. These photolytic cleavages, in combination with limited tryptic digestion, have proved highly useful in detcrmining a detailed linear map of the dynein heavy chains [ll].As part of a general effort to explore possible photocleavage of other vanadate-binding phosphohydrolases, attention was directed to rabbit skeletal myosin. The inhibition of myosin ATPase by vanadate has been described [12, 131. This paper reports that irradiation of myosin with near-ultraviolet light, in the presence of vanadate concentrations > 0.1 mM, produces photocleavage at three distinct sites on the heavy chains. Cleavage at these sites does not require the presence of ATP and the presence of ATP retards cleavage at two of the sensitive sites. The sites of vanadate-mediated photocleavage may correspond to phosphate-binding sites on the myosin heavy chain. MATERIALS AND METHODSMyosin and actin from rabbit skeletal muscle were prepared by the methods of Sreter [14] and of Spudich and Watt 1151, respectively. Subfragment 1 and heavy meromyosin were prepared by chymotryptic digestion of myosin according to Weeds and Pope [16]. Tryptic light meromyosin and chymotryptic myosin rod were isolated by the procedure of Nyitrai et a]. [17]. Digestion of subfragment 1 by trypsin was carried out at room tempe...