Molecular evidence of natural hybridization between Fasciola hepatica and F. gigantica.

Nucleotide sequences of two regions, cytochrome c-oxidase subunit 1 (CO1) and NADH dehydrogenase subunit 1 (ND1) of the mitochondrial DNA and two regions, internal-transcribed spacer 2 (ITS2) and the D2 region in the 28S rDNA (28S) of the nuclear DNA were obtained from five Korean worms of the genus Fasciola in order to elucidate their taxonomic status. The CO1 and ND1 regions are all monomorphic in the Korean worms and similar to those of F. gigantica. On the other hand, the ITS2 and D2 regions were found to be polymorphic; that is, out of five worms, two possessed a F. gigantica-type sequence, one, a F. hepatica-type sequence and two possessed sequences of both types indicating an existence of different alleles at the loci. It should be noted that these variations of the ITS2 and D2 regions co-occur at the same individual worms. This was confirmed by sequencing five to six cloned PCR products for each worm. The present study strongly suggests interspecific cross-hybridization between the two species coexisting in Korea.     

Crystal Structure of a Symmetric Football-Shaped GroEL:GroES2-ATP14 Complex Determined at 3.8 Å Reveals Rearrangement between Two GroEL Rings

The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES₂ complex (the “football”-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES₂-ATP₁₄ complex from Escherichia coli at 3.8 Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the “bullet” complex). However, the two GroES-bound GroEL rings form a modified interface by an ~ 7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.     

Rabbit antibody detection with RNA aptamers

Antibody-based detection systems are widely used, but in the cases of immunoprecipitations and enzyme-linked immunoassays, they can be laborious. These techniques require the preparation of at least two kinds of non-cross-reactive immunoglobulin Gs (IgGs), usually made from different species against the single target molecule. Aptamers composed of nucleic acids possess strict recognition ability for the target molecule's three-dimensional structure and, thus, are considered to act like IgG. In this study, experimental trials were designed to combine the advantages of IgG and aptamers. For this purpose, aptamers against rabbit IgG were identified by in vitro selection. One of the obtained aptamers had a dissociation constant lower than 15 pM to the rabbit IgG. It bound specifically to the constant region of the rabbit IgG, and no binding was observed with mouse or goat IgG. Moreover, this aptamer recognized only the native form of rabbit IgG and could not bind the sodium dodecyl sulfate (SDS)-denatured form. These features show the advantage of using the aptamer as a secondary probing agent rather than the usual secondary antibodies.     

Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.     

The gene expression landscape of thermogenic skunk cabbage suggests critical roles for mitochondrial and vacuolar metabolic pathways in the regulation of thermogenesis

Floral thermogenesis has been described in several plant species. Because of the lack of comprehensive gene expression profiles in thermogenic plants, the molecular mechanisms by which floral thermogenesis is regulated remain to be established. We examined the gene expression landscape of skunk cabbage (Symplocarpus renifolius) during thermogenic and post-thermogenic stages and identified expressed sequence tags from different developmental stages of the inflorescences using super serial analysis of gene expression (SuperSAGE). In-depth analysis suggested that cellular respiration and mitochondrial functions are significantly enhanced during the thermogenic stage. In contrast, genes involved in stress responses and protein degradation were significantly up-regulated during post-thermogenic stages. Quantitative comparisons indicated that the expression levels of genes involved in cellular respiration were higher in thermogenic spadices than in Arabidopsis inflorescences. Thermogenesis-associated genes seemed to be expressed abundantly in the peripheral tissues of the spadix. Our results suggest that cellular respiration and mitochondrial metabolism play key roles in heat production during floral thermogenesis. On the other hand, vacuolar cysteine protease and other degradative enzymes seem to accelerate senescence and terminate thermogenesis in the post-thermogenic stage.     

Xylem water-conducting patterns of 34 broadleaved evergreen trees in southern Japan

A dye injection method was used to elucidate the xylem water-conducting pathways of 34 broadleaved evergreen trees growing in southern Japan: two semi-ring-porous, 26 diffuse-porous, five radial-porous and one non-vessel species. The large earlywood vessels in semi-ring-porous species have a water transport function in only the outermost annual ring, as in deciduous ring-porous species. On the other hand, the small vessels in semi-ring-porous species maintain the water transport function in many outer annual rings. For the other xylem-type species, the many vessels in many outer annual rings have a water transport function. In diffuse-porous species, we categorized the water-conducting pattern within the annual rings into two types: d1 type, where water travels through vessels in the whole region; and d2 type, where water travels mainly through the earlywood vessels. The pattern in radial-porous species is similar to that in the d1 type; the pattern in non-vessels species is similar to that in the d2 type. The vessel diameter in radial-porous species is similar to that of the earlywood vessels of semi-ring-porous species. These results suggest that the conduit diameter size is only one of many factors determining the water-conducting pathways of broadleaved evergreen species.     

Nucleotide‐dependent structural fluctuations and regulation of microtubule‐binding affinity of KIF1A

Molecular motors such as kinesin regulate affinity to a rail protein during the ATP hydrolysis cycle. The regulation mechanism, however, is yet to be determined. To understand this mechanism, we investigated the structural fluctuations of the motor head of the single‐headed kinesin called KIF1A in different nucleotide states using molecular dynamics simulations of a Gō‐like model. We found that the helix at the microtubule (MT) binding site intermittently exhibits a large structural fluctuation when MT is absent. Frequency of this fluctuation changes systematically according to the nucleotide states and correlates strongly with the experimentally observed binding affinity to MT. We also showed that thermal fluctuation enhances the correlation and the interaction with the nucleotide suppresses the fluctuation of the helix . These results suggest that KIF1A regulates affinity to MT by changing the flexibility of the helix during the ATP hydrolysis process: the binding site becomes more flexible in the strong binding state than in the weak binding state. Proteins 2015; 83:809–819. © 2015 Wiley Periodicals, Inc.     

A molecular mechanism for copper transportation to tyrosinase that is assisted by a metallochaperone, caddie protein

The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated “caddie,” which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16–1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.     

Degeneration of hrpZ gene in Pseudomonas syringae pv. tabaci to evade tobacco defence: an arms race between tobacco and its bacterial pathogen

The HrpZ harpin of Pseudomonas syringae is known to induce a hypersensitive response (HR) in some plants. In P. syringae pv. tabaci (Pta), the harpin gene hrpZ has been spontaneously disrupted by an internal deletion in its open reading frame and a frame shift. The loss of the ability of the recombinant harpin polypeptide of Pta to induce HR despite the high sensitivity of tobacco plants to harpin led us to investigate the meaning of the disrupted hrpZ gene in the virulence of Pta 6605. The hrpZ gene from P. syringae pv. pisi was introduced into wild-type (WT) Pta. The hrpZ-complemented Pta secreted harpin into the culture medium, but failed to cause disease symptoms by both infiltration and spray inoculation. Inoculation with the hrpZ-complemented Pta induced defence responses in tobacco plants, whereas the defence responses of tobacco plants were not prominent on inoculation with WT Pta. These results indicate that an ancestor of Pta might have disrupted hrpZ by an internal deletion to evade plant defences and confer the ability to cause disease in tobacco plants.     

Long-term treatment with the Na+-glucose cotransporter inhibitor T-1095 causes sustained improvement in hyperglycemia and prevents diabetic neuropathy in Goto-Kakizaki Rats

We examined the effects of T-1095, an orally active inhibitor of Na(+)-glucose cotransporter (SGLT), on the development and severity of diabetes in Goto-Kakizaki (GK) rat, a spontaneous, non-obese model of type 2 diabetes. T-1095 was administered as dietary admixture (0.1% w/w) beginning at 7 weeks of age for 32 weeks. Untreated male GK rats were hyperglycemic compared with Wistar rats. Throughout the study, T-1095 treatment significantly decreased both blood glucose and hemoglobin A(1C) levels in the GK rats. The concomitant increase of urinary glucose excretion indicated that the hypoglycemic action of T-1095 is derived from the enhancement of urinary glucose disposal. Although food intake was not changed in the T-1095-treated rats, the body weight gain was retarded. T-1095 treatment partially ameliorated oral glucose tolerance but not the impaired glucose-induced insulin secretion. Homeostasis model assessment (HOMA) indicated the existence of insulin resistance in GK rats and a significant restoration by T-1095-treatment. There was a reduction of the thermal response in tail-flick testing following long-term hyperglycemia (diabetic neuropathy). Treatment of T-1095 significantly prevented the development of diabetic neuropathy in male GK rats. Sustained improvement of hyperglycemia and prevention of diabetic neuropathy by the T-1095-treatment provide further support the use of SGLT inhibitors for the treatment of diabetes.