Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.     

Immunohistochemical localization of D-aspartate oxidase in porcine peripheral tissues

d-Aspartate (d-Asp) is an endogenous substance in mammals. Degradation of d-Asp is carried out only by d-aspartate oxidase (DDO). We measured DDO activity in porcine tissues, and produced an anti-porcine DDO antibody to examine the cellular localization of DDO. All the tissues examined showed DDO activities, whereas the substrate d-Asp was not detected in kidney cortex, liver, heart, and gastric mucosa. In the kidney, intensive immunohistochemical staining for DDO was found in the epithelial cells of the proximal tubules. In the liver, the epithelial cells of interlobular bile ducts, liver sinusoid-lining cells with cytoplasmic processes, and the smooth muscle cells of arterioles were strongly stained for DDO. In the heart, cardiomyocytes and the smooth muscle cells of arterioles showed DDO-immunoreactivity. In the gastric mucosa, only the chief cells were DDO-positive. These newly identified DDO-positive cells seem to actively degrade d-Asp to prevent an excess of d-Asp from exerting harmful effects on the respective functions of porcine tissues.     

Ecological differentiation and habitat unsuitability maintaining a ground beetle hybrid zone

Exogenous selection via interactions between organisms and environments may influence the dynamics of hybrid zones between species in multiple ways. Two major models of a hybrid zone allowed us to hypothesize that environmental conditions influence hybrid zone dynamics in two ways. In the first model, an environmental gradient determines the mosaic distribution at the boundary between ecologically differentiated species (mosaic hybrid zone model). In the second model, a patch of unsuitable habitat traps a hybrid zone between species whose hybrids are unfit (tension zone model). To test these, we examined the environmental factors influencing the spatial structure of a hybrid zone between the ground beetles Carabus maiyasanus and C. iwawakianus using GIS‐based quantification of environmental factors and a statistical comparison of species distribution models (SDMs). We determined that both of the hypothetical processes can be important in the hybrid zone. We detected interspecific differences in the environmental factors in presence localities and their relative contribution in SDMs. SDMs were not identical between species even within contact areas, but tended to be similar within the range of each species. These results suggest an association between environments and species, and provide evidence that ecological differentiation between species plays a role in the maintenance of the hybrid zone. Contact areas were characterized by a relatively high temperature, low precipitation, and high topological wetness. Thus, the contact areas were regarded as being located in an unsuitable habitat with a drier climate, where those populations are likely to occur in patches with limited precipitation concentrated. A comparison of spatial scales suggests that exogenous selection via environmental factors may be weaker than endogenous selection via genitalic incompatibility.     

A New Fungal Isolate,Penidiella sp. Strain T9, Accumulates the Rare Earth Element Dysprosium

With an aim to develop a highly efficient method for the recovery of rare earth elements (REEs) by using microorganisms, we attempted to isolate dysprosium (Dy)-accumulating microorganisms that grow under acidic conditions from environmental samples containing high concentrations of heavy metals. One acidophilic strain, T9, which was isolated from an abandoned mine, decreased the concentration of Dy in medium that contained 100 mg/liter Dy to 53 mg/liter Dy after 3 days of cultivation at pH 2.5. The Dy content in the cell pellet of the T9 strain was 910 μg/mg of dry cells. The T9 strain also accumulated other REEs. Based on the results of 28S-D1/D2 rRNA gene sequencing and morphological characterization, we designated this fungal strain Penidiella sp. T9. Bioaccumulation of Dy was observed on the cell surface of the T9 strain by elemental mapping using scanning electron microscopy-energy dispersive X-ray spectroscopy. Our results indicate that Penidiella sp. T9 has the potential to recover REEs such as Dy from mine drainage and industrial liquid waste under acidic conditions.     

A simple purification method for a glucosyltransferase complex from Streptococcus mutans OMZ 176 with a high yield

1. Glucosyltransferase was purified from Streptococcus mutans OMZ 176 by ammonium sulfate fractionation and gel filtration. 2. The overall purification was about 15,000-fold with a yield of 53%. 3. The purified enzyme appeared to be a multienzyme complex consisting of three different components with molecular weights of 158,000, 153,000 and 149,000, respectively.     

Location of the catalytic nucleophile of phospholipase D of Streptomyces antibioticus in the C-terminal half domain.

Phospholipase D (PLD) of Streptomyces antibioticus was labelled with fluorescent-labelled substrate, 1-hexanoyl-2-{6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)-amino]hexanoyl}-sn-glycero-3-phosphocholine, when it was incubated with the substrate and the reaction followed by SDS/PAGE. Mutant enzymes lacking the catalytic activity were not labelled under the same conditions, indicating that labelling of the PLD occurred as the result of its catalytic action. This confirmed that the labelled protein was the phosphatidyl PLD intermediate. PLDs contain two copies of the highly conserved catalytic HxKxxxxD (HKD) motif. Therefore, two protein fragments were separately prepared with recombinant strains of Escherichia coli. One of the fragments was the N-terminal half of the intact PLD containing one HKD motif, and the other was the C-terminal half with the other motif. An active enzyme was reconstructed from these two fragments, and therefore designated fragmentary PLD (fPLD). When fPLD was subjected to the labelling experiment, only the C-terminal half was labelled. Therefore, it was concluded that the catalytic nucleophile that bound directly to the phosphatidyl group of the substrate was located on the C-terminal half of PLD, and that the N-terminal half did not contain such a nucleophile.     

Gene for a protein capable of enhancing lateral root formation.

Analysis of genes preferentially expressed in hairy roots caused by infection with Agrobacterium rhizogenes has provided insights into the regulation of lateral root formation. A hairy root preferential cDNA, HR7, has been cloned from hairy roots of Hyoscyamus niger. HR7 encodes a novel protein partially homologous to a metallocarboxypeptidase inhibitor and is expressed exclusively in the primordium and base of lateral roots in hairy roots. Overexpression of HR7 in transgenic roots of H. niger dramatically enhances the frequency of lateral root formation. The results of this study indicate that expression of HR7 plays a critical role in initiating lateral root formation.     

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. II. Stoichiometry of holoenzyme association and energetics of subunit association

The self-association pattern of D-amino acid oxidase holoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was examined by the low-angle laser light-scattering method. As to the results of nonlinear least-squares analysis of the apparent weight-average molecular weight (Mwapp) versus protein concentration (c) data, the following three models fitted equally well the data over the concentration range of 0.03-11.4 mg/ml: 1) the model of isodesmic indefinite self-association of the monomer where the dimerization constant differs from the isodesmic association constant, 2) the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, and 3) the model which involves the trimerization of the monomer and isodesmic indefinite self-association of the trimer. In a more limited concentration range (0.3-11.4 mg/ml), a model of isodesmic indefinite self-association of the stable dimer where the dimer does not dissociate into the monomers cannot be excluded from the above three models. Measurements with the concentration range lowered to 0.03 mg/ml enabled us to exclude unequivocally the model involving such a stable dimer and to extrapolate the Mwapp data to the Mr of the monomer at infinite dilution as in the case of the apoenzyme. The observed sedimentation boundary profiles were qualitatively consistent with the idealized boundary profiles calculated with the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, so this model is the most probable of the models examined. These results provide the first evidence that the association mode of the holoenzyme is different from that of the apoenzyme, i.e. isodesmic indefinite self-association of the monomer (Tojo, H., Horiike, K., Shiga, K., Nishina, Y., Watari, H., and Yamano, T. (1985) J. Biol. Chem. 260, 12607-12614). The overall linkage scheme, between binding of coenzyme FAD and subunit association, was considered, and the overall free energy change in each process in the scheme was calculated. The total stabilization energies of the intersubunit interaction in the holoenzyme relative to the apoenzyme were found to be -2.2 kcal/mol at the dimerization step and -0.5 kcal/mol at the step of the addition of the dimer to any 2i-mer (i = 1,2, ...).     

Thermodynamic characterization of hog kidney D-amino acid oxidase apoenzyme in concentrated guanidine hydrochloride solution. Preferential interaction with the solvent components and the molecular weight of the monomeric unit

This paper describes the physical characterization of the monomeric unit of hog kidney D-amino acid oxidase apoenzyme in 6 M guanidine hydrochloride (GuHCl) solution by means of differential refractometry, densimetry, light scattering, equilibrium sedimentation, and high-speed gel filtration chromatography. In 6 M GuHCl solution, the oxidase interacts preferentially with GuHCl: the values of the preferential interaction parameter are 0.11 +/- 0.03 (S.D.) g/g of protein by densimetry and 0.14 +/- 0.04 g/g of protein by refractometry. The volume change, delta V, of the oxidase on transfer from the native to the denatured state is -350 ml/mol. The molecular weight of the monomeric apoenzyme is 39,600 +/- 1,700 by light scattering and 38,000 +/- 1,200 by high-speed equilibrium sedimentation. The values of the molecular weight estimated by the empirical methods, i.e., sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and high-speed gel filtration chromatography in 6 M GuHCl, agree well with those obtained by the thermodynamic methods mentioned above. These results confirm definitely that the complex of the apoenzyme with SDS normally behaves in the same manner as those of standard proteins in SDS-gel electrophoresis. This is also supported in this study by the analysis of the electrophoretic data at several gel concentrations by Ferguson plots. The molecular weight of quasi-D-amino acid oxidase apoenzyme was also examined by the empirical methods.     

Rejoining between 9q+ and Philadelphia chromosomes results in normal-looking chromosomes 9 and 22 in Ph1-negative chronic myelocytic leukemia

Summary Rearrangement of the breakpoint cluster region (bcr) and the chromosomal location of c-abl and 3-bcr were studied in two patients with Philadelphia chromosome (Ph¹)-negative chronic myelocytic leukemia (CML). One patient (patient 1) had a normal karyotype and the other (patient 2), 46,XY,inv(3)(q21q26). Both patients showed the bcr rearrangement by Southern blot analysis with a 1.2 kb 3-bcr probe. In situ hybridization studies demonstrated the location of the homologous sequences of bcr on chromosome 22 in patient 1, and on chromosomes 9 and 22 in patient 2. These findings indicate that the morphologically normal-looking chromosomes 9 and 22 in patient 2 are the result of a retranslocation between chromosomes 9q+ and 22q-, abnormalities which were first formed by a standard Ph¹ translocation.