Eight-residue Abeta peptides inhibit the aggregation and enzymatic activity of Abeta42

Insoluble Aβ1–42 is the main component of the amyloid plaque. We have previously demonstrated that exposure to low pH can confer the molten globule state on soluble Aβ1–42 in vitro [Biochem. J. 361 (2000) 547] and unfolding experiments with guadinine hydrochloride (GdnHCl) have now confirmed this observation. The molten globule state of the protein has many biological properties and understanding the mechanisms of its formation is an important step in devising a therapeutic strategy for Alzheimer's disease (AD). We therefore investigated the ability of a series of synthetic eight-residue peptides derived from Aβ1–42 to inhibit the acid-induced aggregation of Aβ1–42 and identified the potent peptides to be Aβ15–22, Aβ16–23 and Aβ17–24. A1-antichymotrypsin, a member of the serine proteinase inhibitor (serpin) family is another major component of the amyloid plaque. In the present study, we investigated the proteolytic activity of Aβ1–42 against casein at different pHs. Chemical modification of amino acid residues in Aβ1–42 indicated that serine and histidine residues, but not aspartic acid, are necessary for enzymatic activity, suggesting that it is a serine proteinase. Amino acid substitution studies indicate that glutamic acids at positions 11 and 22 participate indirectly in proteolysis and we surmise that amino acid residues 29–42 are required to stabilize the conformer. A study of metal ions suggested that Cu²⁺ affected the enzymatic activity, but Zn²⁺ and Fe²⁺ did not. Interestingly, Aβ14–21 and Aβ15–22 were the only peptides that inhibited the proteolytic activity of Aβ42. Therefore, Aβ15–22 may control both aggregation of Aβ1–42 at acidic pH and its proteolytic activity at neutral pH. Consequently, we suggest that it may be of use in the therapy of Alzheimer's disease.     

Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense

The crystal structure of a microbial transglutaminase from Streptoverticillium mobaraense has been determined at 2.4 A resolution. The protein folds into a plate-like shape, and has one deep cleft at the edge of the molecule. Its overall structure is completely different from that of the factor XIII-like transglutaminase, which possesses a cysteine protease-like catalytic triad. The catalytic residue, Cys(64), exists at the bottom of the cleft. Asp(255) resides at the position nearest to Cys(64) and is also adjacent to His(274). Interestingly, Cys(64), Asp(255), and His(274) superimpose well on the catalytic triad "Cys-His-Asp" of the factor XIII-like transglutaminase, in this order. The secondary structure frameworks around these residues are also similar to each other. These results imply that both transglutaminases are related by convergent evolution; however, the microbial transglutaminase has developed a novel catalytic mechanism specialized for the cross-linking reaction. The structure accounts well for the catalytic mechanism, in which Asp(255) is considered to be enzymatically essential, as well as for the causes of the higher reaction rate, the broader substrate specificity, and the lower deamidation activity of this enzyme.     

Efficient uptake and rapid degradation of plasmid DNA by murine dendritic cells via a specific mechanism

In spite of the important roles of dendritic cells in DNA-based therapies, the cellular uptake mechanism of plasmid DNA (pDNA) in dendritic cells is poorly understood. The present study was undertaken to investigate the binding and uptake of pDNA in vitro using a murine dendritic cell line, DC2.4 cells. A significant and time-dependent cellular association of [32P]pDNA with DC2.4 cells was observed at 37 degrees C and this fell markedly at 4 degrees C. The binding and uptake of [32P]pDNA were significantly inhibited by cold pDNA, polyinosinic acid (poly[I]), dextran sulfate, or heparin, but not by polycytidylic acid (poly[C]), dextran, or EDTA, suggesting that a specific mechanism mediated by a receptor like the macrophage scavenger receptor may be involved. The TCA precipitation experiments showed that DC2.4 cells rapidly endocytosed and degraded a significant amount of [32P]pDNA at 37 degrees C and released the degradation products into the medium. The pDNA degradation was also significantly inhibited by poly[I], but not poly[C]. The rate of pDNA degradation by DC2.4 cells was significantly higher than that by macrophages. A confocal microscopic study using fluorescein-labeled pDNA confirmed the rapid internalization and degradation of pDNA by the dendritic cells. Taken together, these results indicate that pDNA is efficiently taken up and rapidly digested by the dendritic cells via a specific mechanism. These findings may suggest the important role of the dendritic cells in the innate immune system for host defense.     

Comparison of bacteriological, genetic and pathological characters between Escherichia coli O115a,c:K(B) and Citrobacter rodentium.

Murine pathogenic Escherichia coli O115a,c:K(B) (MPEC) is the causative agent of mouse megaenteron, the pathology of which resembles that of transmissible murine colonic hyperplasia caused by Citrobacter rodentium. We compared their genetic and pathological features to reveal the relationship between these two bacteria. To evaluate the genetic distances, 16S rDNA genes were sequenced and biochemical reactions were tested. Mouse strain susceptibility tests, using CF1 MPEC-susceptible germfree mice and BALB/cA(Jic) resistant mice were performed. MPEC strains and C. rodentium showed more than 99.6% identity by comparison of 16S rDNA gene sequences. All results from biochemical reactions and the mouse strain susceptibility tests were identical. It is proposed that MPEC should be reclassified as C. rodentium.     

Elimination of Pasteurella pneumotropica from a contaminated mouse colony by oral administration of Enrofloxacin.

Enrofloxacin, a fluoroquinolone bactericidal antibiotic, was administered in an attempt to eradicate Pasteurella pneumotropica (P. pneumotropica) from a contaminated mouse colony. Contaminated mice, maintained within 4 animal rooms, were administered Enrofloxacin in drinking water at a daily dosage of 25.5 mg/kg for 2 weeks. Following one week of Enrofloxacin treatment, mice were selected randomly from each room and examined for P. pneumotropica. This procedure was repeated two or three times until all mice examined tested negative for the Pasteurella strain. With the exception of one room, treated mice consistently tested negative for P. pneumotropica for up to 45 weeks following completion of Enrofloxacin treatment. Thus, oral administration of Enrofloxacin significantly eliminated P. pneumotropica from a contaminated mouse colony.     

Studies on the development of an ELISA kit for microbiological monitoring. 2. Improvement of the prototype ELISA kit with special references to mouse hepatitis virus antigen

Improvement of the mouse hepatitis virus (MHV) antigen in a prototype ELISA kit was performed. Equivalent divalent antigens of MHV Nu-67 and S strains with a protein concentration of 10 micrograms/ml showed the best sensitivity and specificity for the detection of MHV and sialodacryoadenitis/Parker's rat coronavirus antibodies in mice and rats, respectively. An increase in the reliability of macroscopic evaluation of both antibody tests is expected by using the newly manufactured kit with the improved antigen.     

Determination of an antigen suitable for enzyme-linked immunosorbent assay of the antibody to Bordetella bronchiseptica in guinea pigs

To establish an enzyme-linked immunosorbent assay (ELISA) technique for the serological diagnosis of infections caused by Bordetella bronchiseptica (B. bronchiseptica) in guinea pigs, the authors recently assessed the usefulness of three antigen preparations derived from the bacterial cell components: sonication antigen (S-Ag), cell surface antigen (C-Ag) and lipopolysaccharide antigen (L-Ag). The use of S-Ag for ELISA resulted in the most sensitive detection of the antibody to B. bronchiseptica from guinea pig sera immunized with killed bacteria and sera derived from naturally infected guinea pigs. Like C-Ag, S-Ag was highly specific, showing no cross-reactivity with Pasteurella multocida. Assessment of antibody formations in animals with experimentally induced infection using the three antigen preparations revealed that the antibody to S-Ag was formed earlier than antibodies to the other two antigen preparations following growth of the bacterium in the lungs. These results indicate that ELISA with S-Ag as an antigen is a useful tool for the serological diagnosis of infection by B. bronchiseptica.     

Possible involvement of nitroglycerin converting step in nitroglycerin tolerance.

Nitroglycerin (GTN) produces a dilation of vascular smooth muscle by releasing NO through a putative GTN-converting step. However, the response to GTN is markedly attenuated after prolonged or repeated exposure, resulting in tolerance. We investigated the mechanisms of GTN tolerance, employing exogenous and endogenous NO in rat aorta. In endothelium-denuded rat aortic strips, the GTN-induced relaxation response was attenuated by preceding exposure to either GTN or sodium nitroprusside (SNP). In contrast, the SNP-induced relaxation response was not affected by this protocol of GTN or SNP preexposure. Preincubation of aortic strips with lipopolysaccharide (LPS) +/- L-arginine for 12 h also caused attenuation of GTN-induced responses such as relaxation, cGMP production and nitrite/nitrate formation. The attenuating effect of LPS abolished in aortic strips co-incubated with LPS and cycloheximide or N(G)-nitro-L-arginine. These results suggest that GTN tolerance is predominantly associated with the reduction of NO release from GTN, which is caused through inhibition of a GTN-converting step due to preceding exposure to NO itself.     

Modulation of porcine granulosa cell functions by interleukin-1

Increasing evidence suggests that functions of the immune system and gonads are closely related with each other. In cultures of granulosa and luteal cells, macrophages have been shown to modulate steroidogenic functions. In this paper we present the modulatory effects of interleukin-1, a cytokine produced predominantly by activated macrophages, on gonadotropin-induced differentiation, as well as growth of cultured porcine granulosa cells.     

EPR studies of 5-bromouracil crystal after irradiation with X rays in the bromine K-edge region

Radicals induced in a single crystal of 5-bromouracil (BrUra) by synchrotron soft X rays in the bromine K-edge region (13.461-13.482 keV) were investigated using the X-band EPR method. The crystal was irradiated at three peak energies of the absorption spectrum at room temperature or at 80 K. A hydrogen abstraction radical derived from N1 of the pyrimidine ring was commonly observed for all of the energies used, though with some variation in quantity. Similar characteristics were also observed in the EPR signal for the off-K-edge low-energy (13.42 keV) and (60)Co gamma rays used for comparison. When irradiated at 80 K, a much larger exposure (roughly 10 times) of soft X rays was needed to obtain the same signal intensity as that observed at room temperature. EPR signals were not detectable with gamma irradiation at liquid nitrogen temperature.