Time-dependent distinct roles of Toll-like receptor 4 in a house dust mite-induced asthma mouse model.

House dust mites (HDMs) are a common source of allergens that trigger both allergen-specific and innate immune responses in humans. Here, we examined the effect of allergen concentration and the involvement of Toll-like receptor 4 (TLR4) in the process of sensitization to house dust mite allergens in an HDM extract-induced asthma mouse model.　Intranasal administration of HDM extract induced an immunoglobulin E response and eosinophilic inflammation in a dose-dependent manner from 2.5 to 30 µg/dose. In TLR4-knockout mice, the infiltration of eosinophils and neutrophils into the lung was decreased compared with that in wild-type mice in the early phase of inflammation (total of three doses). However, in the late phase of inflammation (total of seven doses), eosinophil infiltration was significantly greater in TLR4-knockout mice than in wild-type mice. This suggests that the roles of TLR4 signaling are different between the early phase and the later phase of HDM allergen-induced inflammation. Thus, innate immune response through TLR4 regulated the response to HDM allergens, and the regulation was altered during the phase of inflammation.

BUB3 that dissociates from BUB1 activates caspase-independent mitotic death (CIMD).

The cell death mechanism that prevents aneuploidy caused by a failure of the spindle checkpoint has recently emerged as an important regulatory paradigm. We previously identified a new type of mitotic cell death, termed caspase-independent mitotic death (CIMD), which is induced during early mitosis by partial BUB1 (a spindle checkpoint protein) depletion and defects in kinetochore-microtubule attachment. In this study, we have shown that survived cells that escape CIMD have abnormal nuclei, and we have determined the molecular mechanism by which BUB1 depletion activates CIMD. The BUB3 protein (a BUB1 interactor and a spindle checkpoint protein) interacts with p73 (a homolog of p53), specifically in cells wherein CIMD occurs. The BUB3 protein that is freed from BUB1 associates with p73 on which Y99 is phosphorylated by c-Abl tyrosine kinase, resulting in the activation of CIMD. These results strongly support the hypothesis that CIMD is the cell death mechanism protecting cells from aneuploidy by inducing the death of cells prone to substantial chromosome missegregation.

17-AAG, an Hsp90 inhibitor, causes kinetochore defects: a novel mechanism by which 17-AAG inhibits cell proliferation.

The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG), which is currently in clinical trials, is thought to exert antitumor activity by simultaneously targeting several oncogenic signaling pathways. Here we report a novel mechanism by which 17-AAG inhibits cell proliferation, and we provide the first evidence that HSP90 is required for the assembly of kinetochore protein complexes in humans. 17-AAG caused delocalization of several kinetochore proteins including CENP-I and CENP-H but excluding CENP-B and CENP-C. Consistently, 17-AAG induced a mitotic arrest that depends on the spindle checkpoint and induced misalignment of chromosomes and aneuploidy. We found that HSP90 associates with SGT1 (suppressor of G2 allele of skp1; SUGT1) in human cells and that depletion of SGT1 sensitizes HeLa cells to 17-AAG. Overexpression of SGT1 restored the localization of specific kinetochore proteins and chromosome alignment in cells treated with 17-AAG. Biochemical and genetic results suggest that HSP90, through its interaction with SGT1 (SUGT1), is required for kinetochore assembly. Furthermore, time-course experiments revealed that transient treatment with 17-AAG between late S and G2/M phases causes substantial delocalization of CENP-H and CENP-I, a finding that strongly suggests that HSP90 participates in kinetochore assembly in a cell cycle-dependent manner.

GABAA receptor-mediated inhibition by ethanol of long-term potentiation in the basolateral amygdala-dentate gyrus pathway in vivo.

Although ethanol has been reported to inhibit the induction of long-term potentiation in hippocampal CA1 and dentate gyrus synapses of rats, very little is known about the effect of ethanol on synaptic plasticity in other brain regions. Therefore, in the present study, we investigated the effect of ethanol on long-term potentiation in synaptic pathway from the basolateral amygdala to the dentate gyrus by using anesthetized rats in vivo. I.v. (20-40% x 2 ml/kg) or i.c.v. (30-40% x 5 microl) administration of ethanol did not affect the basal amplitude of dentate gyrus field potential evoked by basolateral amygdala stimulation, but significantly inhibited the induction of long-term potentiation following application of tetanic stimulation. Since long-term potentiation in this pathway was independent of N-methyl-d-aspartate receptors, the inhibitory effect of ethanol is unlikely to be caused by suppression of N-methyl-d-aspartate receptor function. Alternatively, long-term potentiation in this pathway was significantly suppressed by the benzodiazepine agonist diazepam (2 mg/kg, i.p.), and the inhibitory effect of ethanol was abolished by the GABAA receptor channel blocker picrotoxin (1 mg/kg, i.p.). The present study demonstrates that ethanol inhibits the induction of long-term potentiation in the basolateral amygdala-dentate gyrus pathway by enhancing GABAA receptor-mediated neurotransmission.

Embryonic development assay with Daphnia magna: application to toxicity of aniline derivatives.

An assay system using Daphnia magna embryos was applied to investigate the adverse effects of aniline derivatives. The data were compared with our previous data for chlorophenols. This new assay provides useful information to evaluate the toxicity of chemicals and the differences in sensitivity between the life stages. The effects of 15 aniline derivatives on embryonic development of D. magna embryos were determined. At the start of exposure, 2-6-h old eggs (between stages 1 and 2, round in shape, diameter approx. 400 microm), were used. In control and solvent control groups, embryonic development from an egg to a free-swimming animal proceeded completely within 3 days with more than 90% hatchability. Median effective concentrations (EC50s) to reduce the numbers hatched were determined and gross morphological abnormalities of hatched animals recorded. Anilines induced no obvious morphological abnormalities and no developmental delay although premature deaths occurred. However, they affected the number of embryos hatching in a dose-dependent manner. In addition, this embryo assay was more sensitive to aniline derivatives (except for aniline) than acute juveniles immobilization assay. Ratios of 48-h EC50 (juvenile)/3-day EC50 (embryo) for eight anilines were greater than 5.0. Particularly, the ratios of 4-methyl-, 4-ethyl- and 3-methylaniline were 77, 23 and 11, respectively. EC50s for embryos and juveniles were poorly correlated (r = 0.41). This indicated that the sensitivities of the two life stages were different to the effects of anilines. EC50s were poorly correlated (r = -0.097) with the log Kow (1-octanol/water partition coefficient). These results were compared with previous results for phenols.

Model-free analysis of a thermophilic Fe(7)S(8) protein compared with a mesophilic Fe(4)S(4) protein.

15N T(1), T(2) and (1)H-(15)N NOE were measured for the thermophilic Fe(7)S(8) protein from Bacillus schlegelii and for the Fe(4)S(4) HiPIP protein from Chromatium vinosum, which is a mesophilic protein. The investigation was performed at 276, 300, and 330 K at 11.7 T for the former, whereas only the 298 K data at 14.1 T for the latter were acquired. The data were analyzed with the model-free protocol after correcting the measured parameters for the effect of paramagnetism, because both proteins are paramagnetic. Both thermophilic and mesophilic proteins are quite rigid, with an average value of the generalized order parameter S2at room temperature of 0.92 and 0.94 for Fe(7)S(8) and Fe(4)S(4) proteins, respectively. The analyzed nitrogens for the Fe(7)S(8) protein showed a significant decrease in S2with increasing temperature, and at the highest temperature >70% of the residues had an internal correlation time. This research shows that subnanosecond rigidity is not related to thermostability and provides an estimate of the effect of increasing temperature on this time scale.

Structural and dynamical properties of a partially unfolded Fe4S4 protein: role of the cofactor in protein folding.

Heteronuclear multidimensional NMR spectroscopy was used to investigate in detail the structural and dynamical properties of a partially unfolded intermediate of the reduced high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum present in 4 M guanidinium chloride solution. After an extensive assignment of 15N and 1H resonances, NOE data, proton longitudinal relaxation times, and 3JHNHalpha coupling constants as well as 15N relaxation parameters (T1, T2, T1rho, and 1H-15N NOE) were obtained and used to build a structural model of the intermediate. The Fe4S4 cluster of the HiPIP plays a decisive role in determining the resulting structure, which is random in the N-terminal half of the protein and partially organized in the loops between the cysteines bound to the cluster. Consistent with the structural data, the backbone mobility is typical of folded proteins in the regions where there are elements of structure and increases with the structural indetermination.

Skin sensitization and photosensitization studies of hydrophobically modified hydroxypropyl methylcellulose in guinea pigs.

Skin sensitization and photosensitization tests of hydrophobically modified hydroxypropyl methylcellulose (HM-HPMC), a new cellulose derivative used as a thickener for topical pharmaceuticals, were conducted using guinea pigs. An aqueous dispersion of HM-HPMC (3 w/v %) was applied in the tests. Skin reaction was not observed in any animal in the HM-HPMC-treated group or control group. In the photosensitization test, no skin reaction was found in any animal in the test-preparation group or the control group. It was concluded that HM-HPMC dispersion does not exhibit skin sensitizing or photosensitizing activity under the condition of this test.

Cloning and characterization of three human cDNAs encoding mRNA (guanine-7-)-methyltransferase, an mRNA cap methylase.

The mRNA cap structure is synthesized by a series of reactions catalyzed by capping enzyme and mRNA (guanine-7-)-methyltransferase. mRNA (guanine-7-)-methyltransferase catalyzes the methylation of GpppN- at the guanine N7 position, which is an essential step for gene expression in eukaryotic cells. Here we isolated three human cDNAs encoding mRNA (guanine-7-)-methyltransferase termed hCMT1a, hCMT1b and hCMT1c. hCMT1a and hCMT1b encode 476 and 504 amino acids, respectively, and differ only at the region coding for the C-terminal portion of the enzyme after amino acid residue 465. The third cDNA hCMT1c seems to encode the same polypeptide as hCMT1a, however, the 3'-noncoding region of hCMT1c contains sequences corresponding to part of the C-terminal coding and noncoding regions of hCMT1b thus consisting of a mosaic of hCMT1a and hCMT1b. RT-PCR showed that all 3 types of mRNAs were expressed in every tissue examined. Comparison of the deduced amino acid sequences with those of other viral and cellular enzymes showed the regions which are highly conserved among mRNA (guanine-7-)-methyltransferases. The recombinant hCMT1a expressed in E. coli exhibited mRNA (guanine-7-)-methyltransferase activity. On the other hand, neither mRNA (guanine-7-)-methyltransferase nor mRNA (nucleoside-2'-O-)-methyltransferase activity was detected with the recombinant hCMT1b protein. Although the biological significance of the expression of these three mRNA (guanine-7-)-methyltransferase mRNA species remains unknown at present, the nucleotide sequences suggest that they are produced by alternative RNA splicing.

Solution structure of the oxidized Fe7S8 ferredoxin from the thermophilic bacterium Bacillus schlegelii by 1H NMR spectroscopy.

The solution structure of the paramagnetic seven-iron ferredoxin from Bacillus schlegelii in its oxidized form has been determined by 1H NMR. The protein, which contains 77 amino acids, is thermostable. Seventy-two residues and 79% of all theoretically expected proton resonances have been assigned. The structure has been determined through torsion angle dynamics calculations with the program DYANA, using 966 meaningful NOEs (from a total of 1305), hydrogen bond constraints, and NMR derived dihedral angle constraints for the cluster ligating cysteines, and by using crystallographic information to build up the two clusters. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.68 A for the backbone atoms and of 1.16 A for all heavy atoms. The contributions to the thermal stability of the B. schlegelii ferredoxin are discussed by comparing the present structure to that of the less stable Azotobacter vinelandii ferredoxin I which is the only other available structure of a bacterial seven-iron ferredoxin. It is proposed that the hydrophobic interactions and the hydrogen bond network linking the N-terminus and the C-terminus together and a high number of salt bridges contribute to the stability.

A repeated-dose dermal toxicity study of hydrophobically modified hydroxypropyl methylcellulose in rats.

A six-month repeated-dose dermal toxicity study followed by a 30-day recovery test of hydrophobically modified hydroxypropyl methylcellulose (HM-HPMC), a new cellulose derivative used as a thickener for topical pharmaceuticals, was conducted using rats. Aqueous paste of HM-HPMC was applied to the skin of rats once daily at dose levels up to 60 mg/kg/day, which was the highest dose that could be administered. Items checked included general signs, urinalysis, hematology, ophthalmology, and histopathology. One rat died during the administration period owing to a malignant tumor in the hemopoietic system, which was not attributed to the test substance. Statistically significant differences were found in some test results, but those were not dose-dependent and were considered to be incidental or spontaneous. It was concluded that the test substance was not toxic upon chronic dermal administration at dose levels up to 60 mg/kg/day.

Chondrocytes as a specific target of ectopic Fos expression in early development.

The Finkel-Biskis-Jinkins murine sarcoma virus, which carries v-fos, induces osteosarcomas, whereas high-level expression of exogenous c-fos in transgenic and chimeric mice leads to postnatal development of osteogenic and chondrogenic tumors, respectively. To test whether such target cell specificity of an oncogene can be detected even in early development, we induced ectopic expression of fos in chicken limb buds by microinjecting replication-competent retrovirus into the presumptive leg field of stage 10 embryos. This caused cartilage truncation of all the long bones of the injected leg, which was mainly attributable to chondrodysplasia due to severe retardation of differentiation of the proliferating chondrocytes into mature or hypertrophic chondrocytes, as well as a slight delay in precartilagenous condensation. Expression of genes for all the other known members of chicken AP-1, which include such transforming genes as c-jun and fra-2, however, caused no macroscopic abnormalities in limb formation, indicating a specific function of Fos proteins in embryonic endochondral bone differentiation. The extent of truncation was stronger with v-Fos than with c-Fos, and comparative analysis of these proteins, as well as v-Fos mutants, revealed that strong transforming activity of Fos protein is necessary to cause dysplasia, suggesting that common molecular mechanisms are involved in both embryonic chondrodysplasia and bone tumor formation in postnatal mice.

High-level expression of exogenous genes by replication-competent retrovirus vectors with an internal ribosomal entry site.

We report the construction of two types of Rous sarcoma virus (RSV)-based replication-competent avian retrovirus vectors, IR1 and IR2 to express an exogenous gene at a very high level. In these vectors, the internal ribosomal entry site (IRES) derived from encephalomyocarditis virus (EMCV) was inserted between the env gene and an exogenous gene. The IR1 vector retains the splicing acceptor site that is present in the downstream of the env gene while the IR2 vector lacks it. Using a v-fos mutant (v-fos-CD3) as an example of exogenous genes, we show here that both IR1 and IR2 vectors expressed the gene product, CD3, at expression levels 5- and 8-fold higher than that of their parental vector without IRES, respectively. These vectors were moderately stable and kept a high-level expression of CD3 for at least three passages through the cells. Analysis of viral transcripts indicate that exogenous genes carried by both IR vectors were translated exclusively from the IRES that is present in all the species of the viral transcripts. High-level expression of exogenous genes was also observed in the case of the Hoxa-13 gene in the IR1 vector or the fra-2 gene in the IR2 vector, indicating that the extremely high-level expression characteristic of these vectors is applicable to several exogenous genes.

Studies on the quantitative autoradiography. III. Quantitative comparison of a novel tissue-mold measurement technique "paste-mold method," to the semiquantitative whole body autoradiography (WBA), using the same animals.

A novel preparation technique, so called "Paste Mold," was devised for organ and tissue distribution studies. This is the most powerful by joining with autoradioluminography (ARLG), which was established and validated recently in the working group of Forum '93 of Japanese Society for study of xenobiotics. A small piece (10-50 mg) of each organ or tissue was available for measuring its radioactive concentration and it was sampled from the remains of frozen carcass used for macroautoradiography (MARG). The solubilization of the frozen pieces was performed with mixing a suitable volume of gelatine and strong alkaline solution prior to mild heating kept at 40 degrees C for a few hours. After that, the tissue paste was molded in template pattern to form the small plates. The molded plates were contacted with Imaging plate (IP) for recording their radioactive concentration. The recorded IP was processed by BAS2000. The molded plate was formed in thickness of 200 microns, so called infinit thickness against soft beta rays, and therefore the resulting relative intensities, represented by (PSL-BG)/S values, indicated practically responsible ratio of the radioactive concentration in organs and tissues, without any calibulation for beta-self absorption coefficiency. On the other hand, the left half body of the frozen carcass was used for making whole body autoradiography (WBA) before the Paste-Mold preparation. Comparison was performed for difference in (PSL-BG)/S values of organs and tissues between frozen and dried sections. A good concordance in relative intensities, (PSL-BG)/S by the Paste-Mold preparation was given with those by the frozen sections rather than dried sections.(ABSTRACT TRUNCATED AT 250 WORDS)