Evaluation of Francisella tularensis ΔpdpC as a candidate live attenuated vaccine against respiratory challenge by a virulent SCHU P9 strain of Francisella tularensis in a C57BL/6J mouse model

Francisella tularensis, which causes tularemia, is an intracellular gram‐negative bacterium. F. tularensis has received significant attention in recent decades because of its history as a biological weapon. Thus, development of novel vaccines against tularemia has been an important goal. The attenuated F. tularensis strain ΔpdpC, in which the pathogenicity determinant protein C gene (pdpC) has been disrupted by TargeTron mutagenesis, was investigated as a potential vaccine candidate for tularemia in the present study. C57BL/6J mice immunized s.c. with 1 × 10⁶ CFUs of ΔpdpC were challenged intranasally with 100× the median lethal dose (LD₅₀) of a virulent SCHU P9 strain 21 days post immunization. Protection against this challenge was achieved in 38% of immunized C57BL/6J mice administered 100 LD₅₀ of this strain. Conversely, all unimmunized mice succumbed to death 6 days post challenge. Survival rates were significantly higher in vaccinated than in unimmunized mice. In addition, ΔpdpC was passaged serially in mice to confirm its stable attenuation. Low bacterial loads persisted in mouse spleens during the first to tenth passages. No statistically significant changes in the number of CFUs were observed during in vivo passage of ΔpdpC. The inserted intron sequences for disrupting pdpC were completely maintained even after the tenth passage in mice. Considering the stable attenuation and intron sequences, it is suggested that ΔpdpC is a promising tularemia vaccine candidate.     

Discrimination of macaques by macaques: the case of Sulawesi species

A series of work by the first author have demonstrated that many macaque species show a visual preference for the pictures of their own species when the monkeys actively press a lever to see the pictures. We expanded this study to Sulawesi macaques kept as a pet by local people with slight modification. All seven species of Sulawesi macaques were passively exposed to a variety of colored slides of Sulawesi macaques. The experimenter recorded the duration of visual fixation onto the pictures. Male monkeys of all the seven species clearly watched the pictures of their own species for longer duration than those of the other species. Such visual preference suggested that the seven Sulawesi macaques discriminate each other species and, thus, they may not be integrated into fewer number of species. This visual preference may work to prevent overall intergradation of the Sulawesi macaques who sometimes have hybrid zones only in limited areas. This preference was in general weaker in female monkeys. In one species,Macaca ochreata, females actively avoided to see the pictures of conspecifics. These results may be related to how female monkeys interact with other individuals.     

Efficient folding of the insect neuropeptide eclosion hormone by protein disulfide isomerase.

Eclosion hormone is an insect neuropeptide that consists of 62 amino acid residues including three disulfide bonds. We have previously reported its hypothetical 3D structure consisting mainly of three alpha-helices. In this paper, we report the effects of chaperone proteins on the refolding of denatured eclosion hormone in a redox buffer containing reduced and oxidized glutathione. Urea-denatured eclosion hormone was spontaneously reactivated within 1 min with a yield of more than 90%, while beta-mercaptoethanol-denatured eclosion hormone was reactivated in a few minutes with a yield of 75%. Under the same experimental conditions, eclosion hormone treated with beta-mercaptoethanol and urea was reactivated slowly with a yield of 47% over a period of 2 h. Protein disulfide isomerase, a eucaryotic chaperone protein, markedly increased the reactivation yield and rate of the totally denatured hormone. GroE oligomers slightly improved the reactivation yield but peptidyl prolyl isomerase had no influence on yield or rate. We propose that the folding pathway of eclosion hormone involves at least two rate-limiting steps, and that protein disulfide isomerase is likely to be involved in the folding in insect neuronal cells.     

Antiparallel pleated beta-sheets observed in crystal structures of N,N-bis(trichloroacetyl) and N,N-bis(m-bromobenzoyl) gramicidin S.

Despite intensive efforts, the structures of gramicidin S (GS) [cyclo(-Val-Orn-Leu-d-Phe-Pro-)(2)] and its analogues have not been elucidated by the X-ray diffraction method, except for the GS-urea complex (Hull et al., Nature 275, 206-207, 1978; Tishchenko et al., Acta Cryst. D53, 151-159, 1997). We focused on the acetylation of GS to obtain suitable crystals for X-ray diffraction. The amino groups of Orn residues were capped with trichloroacetic and m-bromobenzoic acids. Both trichloroacetyl and m-bromobenzoyl GSs (TcGS and BzGS, respectively) are hydrophobic and their properties are similar to those of acetyl-GS (AcGS). Although it is well known that AcGS yields hexagonal crystals, TcGS and BzGS yield monoclinic and orthorhombic crystals in aqueous dimethylformamide solution, respectively. Their cell volumes were approximately one-fourth or one-eighth of the hexagonal cell volume. The crystal structures of TcGS and BzGS were determined as the first examples of acetylated GS analogues: TcGS, C(64)H(90)N(12)O(12)Cl(6). 3(C(3)H(7)NO), M(r) = 1651.47, monoclinic, P2(1), a = 15.4366(6) A, b = 18.5312(4) A, c = 16.4774(6) A, beta = 14.160(2) degrees, V = 4300.6(2) A(3), Z = 2; and BzGS, C(64)H(98)N(12)O(12)Br(2). 1.54(H(2)O), M(r) = 1535.21, orthorhombic, P2(1)2(1)2(1), a = 16.748(10) A, b = 18.834(5) A, c = 28.558(10) A, V = 9008(7) A(3), Z = 4. Both these peptide molecules formed an antiparallel pleated beta-sheet, and pseudo twofold symmetries existed in the repeated sequence. beta-Turns formed at the fragments of d-Phe-Pro were classified into type II' based on their characteristics. The peptide conformations of TcGS and BzGS were similar to each other, and these structural features agreed with those of structures proposed by the previous studies.     

Identification of intracellular target proteins of the calcium-signaling protein S100A12.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.     

18O-Labelling pattern of okadaic acid from H218O in dinoflagellate Prorocentrum lima elucidated by tandem mass spectrometry.

Okadaic acid is a metabolite of the unicellular algae dinoflagellate. Its biosynthesis has attracted considerable attention since the skeletal structure was shown to be synthesized via an unprecedented route. However, its relevant intermediates or enzymes are unknown. In the course of our previous investigations on the oxygen source of okadaic acid by tandem mass spectrometry (CID MS/MS), we determined the level of 18O incorporation for each oxygen site from 18O2 and [18O2]acetate. In the present study, we examined H218O-labelling patterns of okadaic acid from dinoflagellates in comparison with salinomycin from actinomycetes and has provided intriguing information regarding biosynthesis. Unexpectedly, oxygen atoms originating from acetate were not labelled from H218O; this can not be accounted for by the usual metabolic route where acetyl-CoA is biosynthesized via pyruvate. Similar experiments for salinomycin revealed that all of its oxygen atoms derived from acetate or propionate were labelled by H218O. Another interesting feature is that two oxygen sites were derived from both O2 and H2O while the others were labelled only from O2. These results imply that an oxidation mechanism other than those in actinomycetes polyethers may be involved in the biosynthesis of okadaic acid.     

The sugary-type isoamylase gene from rice and Aegilops tauschii: characterization and comparison with maize and arabidopsis.

Genes for an isoamylase-like debranching enzyme have been isolated from rice and Aegilops tauschii, the donor of the D genome to wheat. The structures of the genes are very similar to each other and to the maize SU1 isoamylase gene and consist of 18 exons spread over approximately 7.5 kb. Southern analysis and fluorescent in situ hybridization showed the Ae. tauschii gene to be located in the proximal region of the short arm of chromosome 7D, thus showing synteny with the localization of the rice isoamylase gene on rice chromosome 8. Analysis of the expression pattern of wheat sugary isoamylase genes indicates that they are strongly expressed in the developing endosperm 6 days after flowering. Three distinct Sugary-type cDNA sequences were isolated from the wheat endosperm that are likely to correspond to the products of the three genomes. The deduced amino acid sequence of rice and wheat Sugary-type isoamylase is compared with other sequences available in the database and the results demonstrate that there are three types of isoamylase sequences in plants: those containing 18 exons (the Sugary-type isoamylase gene), those containing 21 exons, and those containing only 1 exon. It is possible that different combinations of isoamylase genes are expressed in different tissues.     

Effect of insulin-like growth factor-I (IGF-I) on femoral bone modeling in growing mice.

The time-dependent effects of daily dosing of IGF-I (1.21 mg/g) on the linear growth of the femur were investigated in mice. The femoral length and volume and the number of osteoclasts were significantly greater after IGF-I injection as compared to the non-injected control, suggesting that the IGF-I imbalance might cause a quick turnover cycle of the bone resulting in the altered femoral modeling.