Conformational dynamics of yeast calmodulin in the Ca(2+)-bound state probed using NMR relaxation dispersion

Most calmodulin (CaM) in apo and Ca(2+)-bound states show a dumb-bell-like structure, involving the N- and C-terminal domains, connected with a flexible linker. However, Ca(2+)-bound yeast calmodulin (yCaM) takes on a unique globular structure; the target-binding site of this protein is autoinhibited. We applied NMR relaxation dispersion experiments to yCaM in the Ca(2+)-bound state. The amide (15)N and (1)H(N) relaxation dispersion profiles indicated the presence of conformational dynamics for specific residues at the interface between the N- and C-terminal domains. We conclude that these conformational dynamics were derived from the mobility of the C-terminal domain.     

Unexpected A-form formation of 4'-thioDNA in solution, revealed by NMR, and the implications as to the mechanism of nuclease resistance

Fully modified 4′-thioDNA, an oligonucleotide only comprising 2′-deoxy-4′-thionucleosides, exhibited resistance to an endonuclease, in addition to preferable hybridization with RNA. Therefore, 4′-thioDNA is promising for application as a functional oligonucleotide. Fully modified 4′-thioDNA was found to behave like an RNA molecule, but no details of its structure beyond the results of circular dichroism analysis are available. Here, we have determined the structure of fully modified 4′-thioDNA with the sequence of d(CGCGAATTCGCG) by NMR. Most sugars take on the C3′-endo conformation. The major groove is narrow and deep, while the minor groove is wide and shallow. Thus, fully modified 4′-thioDNA takes on the A-form characteristic of RNA, both locally and globally. The only structure reported for 4′-thioDNA showed that partially modified 4′-thioDNA that contained some 2′-deoxy-4′-thionucleosides took on the B-form in the crystalline form. We have determined the structure of 4′-thioDNA in solution for the first time, and demonstrated unexpected differences between the two structures. The origin of the formation of the A-form is discussed. The remarkable biochemical properties reported for fully modified 4′-thioDNA, including nuclease-resistance, are rationalized in the light of the elucidated structure.     

Postnatal changes in the rheological properties of the aorta in Sprague-Dawley rats.

Postnatal changes in the rheological properties of the aortic wall were investigated in relation to morphological development of the wall in Sprague-Dawley (SD) rats at 3, 8 and 20 weeks old. The mechanical tensile characteristics of the longitudinal wall strip excised from the proximal thoracic aorta were assessed with stress-strain and stress-relaxation tests. Wall tension in the low and medium strain ranges was significantly lower in 3-week-old rats than in 8-week-old rats and in 8-week-old rats than in 20-week-old rats. Wall stress was significantly lower in 3-week-old rats than in 8- and 20-week-old rats mainly in the medium strain range, but was significantly greater in 3-week-old rats than in 8- and 20-week-old rats in the high strain range. The value of incremental elastic modulus at 3 weeks old was significantly smaller than that at 8 and 20 weeks old at a strain of 0.25 and significantly larger than that at 8 and 20 weeks old at a strain of 0.50. The value of relaxation strength at 5 min after the stretching was significantly greater at 3 weeks old than that at 8 and 20 weeks old. The wall was viscoelastic in the low and medium strain ranges at 3 weeks though large wall stress was generated in the high strain range. Histological investigation revealed that the smooth muscle layer, fine elastin fiber connecting thick elastin fibers and wall thickness were thin at 3 weeks old in comparison with those at 8 and 20 weeks old, though there was no significant difference in number of nuclei of the smooth muscle cells among the three age groups. Changes in the tensile characteristics of the wall reflected well those of the microstructure of the wall with growth. The rheological properties and microstructure of the aortic wall were close to maturation at 8 weeks in SD rats.     

Host characteristics and infection level of an intestinal parasite Corynosoma strumosum (Acanthocephala) in the Kuril harbor seal of Erimo Cape,Hokkaido, Japan

The Kuril harbor seal around Hokkaido is presently recovering from a resource crisis while conflicts with local fisheries have become a concern. However, its feeding habits, which are fundamental information for taking proper preventive measures, are still poorly understood. We thus examined the infection status of a trophically-transmitted parasite, Corynosoma strumosum in the seals of Erimo Cape, to assess the host's feeding habits with a practical view of the parasite as a biological indicator. A total of 2802 worms were found from 20 male and 20 female by-caught animals in salmon set nets within local fisheries during August to November 2014. The parasite abundance was explained mainly by the host's developmental stage and intestinal length while weakly affected by gender and body size, through an estimation of generalized linear models combined with hierarchical partitioning. Considering the past records that demersal fishes are the probable main sources of infection, the infection level may owe to individual host differences regarding these sources and/or feeding grounds with relating the host characteristics. This supports that the resource management of Kuril harbor seals requires careful consideration of the individual differences in feeding behavior.     

Structure,interaction and real‐time monitoring of the enzymatic reaction of wild‐type APOBEC3G

Human APOBEC3G exhibits anti‐human immunodeficiency virus‐1 (HIV‐1) activity by deaminating cytidines of the minus strand of HIV‐1. Here, we report a solution structure of the C‐terminal deaminase domain of wild‐type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real‐time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3′ → 5′ order. Virus infectivity factor (Vif) of HIV‐1 counteracts the anti‐HIV‐1 activity of APOBEC3G. The structure of the N‐terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species‐specific sensitivity of APOBEC3G to Vif action.     

Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices.

Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase (AK) and adenine phosphoribosyltransferase (APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase (GART) and 5-aminoimidazole ribonucleotide synthetase (AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5'-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho-d-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8-(14)C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices.