Biomedical subjects

A Aoyama

Publications and source records attributed to A Aoyama.

At least 37 records · Page 2Linked to original sources

PubMed1993

Cloning of the mouse hsp25 gene and an extremely conserved hsp25 pseudogene.

A genomic clone of the murine gene encoding the small heat-shock protein, Hsp25, was isolated. The coding region is interrupted by two introns of 128 bp and approximately 600 bp at identical positions as the human hsp27 gene. The 5' flanking regions of the mouse and human genes are very strongly conserved and contain several sequence motives for the transcription factors, HSF and Sp1. In the same screen we also isolated a hsp25 pseudogene. The sequence conservation between this pseudogene and hsp25 cDNA is very high (99%) indicating that this pseudogene emerged very recently.

Amino Acid Sequence↗

PubMed1993

Expression of the murine small heat shock proteins hsp 25 and alpha B crystallin in the absence of stress.

Stress induces the synthesis of several large and small heat shock proteins (hsp's). Two related small hsp's, hsp25 and alpha B crystallin exist in mice. alpha B crystallin is an abundant protein in several tissues even in the absence of stress. Particularly high amounts accumulate in the eye lens. Here we show that hsp25 is likewise constitutively expressed in many normal adult tissues. In the absence of stress the protein is most abundant in the eye lens, heart, stomach, colon, lung, and bladder. The stress-independent expression pattern of the two small hsp's is distinct. In several tissues the amount of hsp25 exceeds that accumulating in NIH 3T3 fibroblasts in response to heat stress. hsp25, like alpha B crystallin, exists in a highly aggregated form in the eye lens. The expression of hsp25 and alpha B crystallin in normal tissues suggests an essential, but distinct function of the two related proteins under standard physiological conditions.

3T3 Cells↗

PubMed1993

Alpha B-crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection.

alpha B-crystallin, a major soluble protein of vertebrate eye lenses, is a small heat shock protein which transiently accumulates in response to heat shock and other kinds of stress in mouse NIH 3T3 fibroblasts. Ectopic expression of an alpha B-crystallin cDNA clone renders NIH 3T3 cells thermoresistant. alpha B-crystallin accumulates in response to the synthetic glucocorticoid hormone dexamethasone. Dexamethasone-treated NIH 3T3 cells become thermoresistant to the same extent as they accumulate alpha B-crystallin. A cell clone in which alpha B-crystallin is superinduced upon heat shock acquires augmented thermotolerance. Expression of the ras oncogene causes a rapid but transient accumulation of alpha B-crystallin within 1 day. Later, sustained ras oncogene expression suppresses the dexamethasone-mediated alpha B-crystallin accumulation. Thus, oncogenic transformation triggered by the ras oncogene interferes with hormone-mediated accumulation of alpha B-crystallin and concomitant acquisition of thermoresistance. Other known heat shock proteins do not accumulate in response to ectopic alpha B-crystallin expression or to dexamethasone treatment. These results indicate that alpha B-crystallin can protect NIH 3T3 fibroblasts from thermal shock.

3T3 Cells↗

PubMed1993

[Maternal and cord blood thyroid hormones in Graves' disease and fetal states].

Serum concentrations of thyroid hormones (T3, T4, fT3 and fT4), TSH and TRAb in 14 mothers with Graves' disease at 1-4 weeks before delivery were comparably studied with cord blood hormone levels and various fetal states. Twelve mothers were on anti-thyroid drugs and 2 were drug-free, with 8 being euthyroid and 6 being hyperthyroid. Serum concentrations of T3, T4 and fT3 in cord blood were all the same regardless of maternal thyroidal function. Though cord blood TRAb levels correlated with maternal levels, cord fT4 levels were low in those who given antithyroid drugs. TSH levels in cord blood were low in Cesarean section delivery and babies with low birth weight. Birth weight and gestational weeks were both inversely correlated with maternal T4, fT4, fT3 and TRAb concentrations. Thus, maternal hormone levels at late stage of pregnancy in Graves' disease could be useful indices for perinatal risk.

Adult↗

PubMed1993

Oncogene-mediated effects on cellular gene expression.

The expression of oncogenes results in alterations of the morphology and growth properties of the affected cells. The oncogenes encode proteins which interfere with mitotic signal transduction pathways and ultimately cause changes in the pattern of gene expression. It must be the products of at least some of these oncoprotein-affected genes which catalyze the altered cell properties. The study of such genes should help our understanding of the biochemistry of malignant growth and transformed cell morphology. The molecular mechanisms which underlie the oncoprotein-mediated gene induction should lead to the elucidation of the signal transduction pathway on which the oncoproteins impinge.

Base Sequence↗

PubMed1991

Striking homology of the 'variable' N-terminal as well as the 'conserved core' domains of the mouse and human TATA-factors (TFIID).

A complementary DNA (cDNA) encoding a mouse TFIID (mIID) was isolated from mouse brain cDNA libraries. The 316 amino acid sequence deduced from cDNA sequences revealed the presence of an amino-terminal region enriched in serine, threonine, and proline (STP-cluster), an uninterrupted stretch of 13 glutamine residues (Q-run), a second STP-cluster, and a conserved carboxy-terminal region. Amino acid sequences of the first STP-cluster and the conserved carboxy-terminal region were identical to those of the human TFIID (hIID). However, the Q-run was considerably shorter than that in hIID and sequences in the second STP-cluster diverged from those of the hIID. The murine TFIID transcript is expressed as a 2 kilobase poly(A)+ RNA in the mouse brain. Southern blot analysis identified a single gene copy per haploid mouse genome.

Amino Acid Sequence↗

PubMed1991

Alpha B-crystallin is a small heat shock protein.

Sequence similarity between alpha B-crystallin and small heat shock proteins (HSPs) has prompted us to investigate whether alpha B-crystallin expression is induced by heat shock. Indeed, accumulation of alpha B-crystallin was detected immunologically in NIH 3T3 cells after incubation at elevated temperatures and after addition of Cd2+ or sodium arsenite to these cells. Two-dimensional gel electrophoresis revealed identity between alpha B-crystallin from eye lenses and from heat-treated fibroblasts. The promoter of the alpha B-crystallin gene was fused to the bacterial chloramphenicol acetyltransferase gene and was shown to confer heat inducibility on this reporter gene in transient transfection assays. A perfect heat shock element within the promoter region is likely to mediate this response. Small HSPs and alpha B-crystallin were shown to share the following two physical properties: (i) they form supramolecular structures with sedimentation values around 17 S and (ii) they are associated with the nucleus at high temperatures and are localized in the cytoplasm under normal conditions. We conclude that alpha B-crystallin has to be considered a member of the class of small HSPs.

Animals↗

PubMed1991

Alpha B crystallin accumulation is a specific response to Ha-ras and v-mos oncogene expression in mouse NIH 3T3 fibroblasts.

The conditional expression of the v-mos and Ha-ras(EJ) oncogenes in NIH 3T3 cells leads to the accumulation of a 23-kDa protein (p23) (R. Klemenz, S. Hoffmann, R. Jaggi, and A.-K. Werenskiold, Oncogene 4:799-803, 1989). We purified p23 to homogeneity and determined part of the amino acid sequence. The obtained sequence is identical with that of the eye lens protein alpha B crystallin. Northern (RNA) blot and Western immunoblot experiments were performed to demonstrate that alpha B crystallin mRNA and protein do indeed accumulate as a consequence of v-mos and Ha-ras oncogene expression. Comparison of cDNA clones obtained from the mRNA of eye lenses and of oncogene-expressing fibroblasts revealed identity between them. The major transcription initiation site of the alpha B crystallin gene in our experimental system was shown by primer extension experiments to be identical with the one used in eye epithelial cells. In addition, we identified a second minor initiation site 49 nucleotides further upstream. Serum growth factors did not stimulate alpha B crystallin expression in growth-arrested cells.

Amino Acid Sequence↗

PubMed1990

Regulation of brain-specific transcription of the mouse myelin basic protein gene: function of the NFI-binding site in the distal promoter.

We investigated brain-specific transcription elements in the distal region (-253 to -54) of the mouse myelin basic protein (MBP) promoter by in vitro transcription using mouse brain nuclear extracts. Using deletion and base substitution mutants, we identified one tissue-specific transcription element at the downstream core of the NFI-site between -115 and -111. Foot-printing assay demonstrated that three transcription factors bind around this element; NFI-related (-130 to -111), M1 (-110 to -97) and Sp1 (-92 to -84). NFI-related factor(s) in brain extracts consisted of a characteristic population which was different from that in other tissues. Methylation interference experiments revealed that this factor(s) interacted with the NFI-downstream core in a brain-specific manner. We suggest the existence of a brain-specific NFI-related factor(s).

Animals↗

PubMed1990

Characterization of cell fusion in XC cells induced by Suncus murinus mammary tumor virus.

Syncytium formation in a rat tumor cell line (XC) induced by Suncus murinus mammary tumor virus (Sm-MTV) was studied. Multinucleate giant cells containing 20-30 nuclei were formed in a monolayer of XC cells by cocultivation with X-ray-irradiated Sm-MTV producing cells (Sm-MT-1). By fluorescent antibody staining. Sm-MTV antigens were demonstrated in the cytoplasm of syncytia, and budding particles and intracytoplasmic A particles were found in syncytial giant cells by electron microscopy. Cell-free supernatant of Sm-MT-1 was capable of inducing syncytia at a much lower incidence than cocultivation of Sm-MT-1 cells. Syncytium formation was completely inhibited when anti-Sm-MTV bovine serum was added to the coculture medium. Pretreatment of XC cells with actinomycin-D caused a partial reduction of Sm-MTV-induced cell fusion, but syncytium formation did occur at a reduced rate even when cellular RNA synthesis was completely inhibited. Dexamethasone increased virus production in Sm-MT-1 cells, resulting in the enhancement of Sm-MTV mediated syncytium formation. Sm-MTV was found to have a unique characteristic of cell fusion activity on XC cells with striking enhancement by dexamethasone.

Animals↗

PubMed1990

Antimetastatic effect of defibrinogenation with batroxobin depends on the natural killer activity of host in mice.

Using batroxobin, a thrombin-like enzyme found in snake venom, the effects of defibrinogenation on artificial lung metastasis in mice were studied. The role of natural killer (NK) cells in the inhibitory effects of defibrinogenation on metastasis was also investigated. Artificial lung metastasis experiments were performed by inoculating either B16-F10 cells or B16-BL/6 cells, highly metastatic strains of B16 melanoma cells, into C57BL/6 mice via the tail vein. The administration of batroxobin significantly inhibited lung metastasis, as did NK activity augmented by poly (I).poly (C) were administered, lung metastasis was more markedly inhibited. When NK activity was suppressed by administration of anti-(asialo GM1) antibody, lung metastasis was markedly increased. When batroxobin was administered with anti-(asialo GM1) antibody, no inhibitory effects on lung metastasis, such as those seen with batroxobin alone, were observed. The administration of batroxobin had no effect at all on spleen lymphocyte NK activity. These results indicated that defibrinogenation due to batroxobin inhibits lung metastasis, and these effects depend on NK activity of the host.

Animals↗

PubMed1990

A 170-kDa membrane-bound protease is associated with the expression of invasiveness by human malignant melanoma cells.

Malignant spreading of cancer cells requires cell surface proteases that cleave the crosslinked collagenous matrix of connective tissues. From correlating the morphologically defined invasiveness of tumor cells with the presence of specific membrane-associated proteases, we have identified a malignant human melanoma cell line, LOX, that invades crosslinked gelatin films in vitro and contains uniquely a neutral 170-kDa gelatinase in the cell membrane. A similar gelatinase was found in membranes recovered from culture media conditioned with LOX. The 170-kDa gelatinase is a wheat germ agglutinin-binding protein. The proteolytic activity is maximal at neutral pH, enhanced by EDTA and dithiothreitol, inhibited by the cysteine protease inhibitors N-ethylmaleimide, HgCl2, and phenylmethylsulfonyl fluoride, and can bind to an organomercurial adsorbent, suggesting that it is a neutral sulfhydryl-sensitive protease. This 170-kDa gelatinase of LOX cells was not found in a control melanoma cell line, SK-MEL28, or in 32 other tumor cell lines that did not show extracellular gelatin degradation. Thus, we have identified a large membrane-bound protease that may be a specific marker molecule for melanoma cell invasiveness.

Cell Fractionation↗

PubMed1990

A new transcription element in the JC virus enhancer.

The enhancer of JC virus restricts its gene expression to the brain. In a previous study, we demonstrated an enhancer element, nuclear factor I (NFI) motif, in the middle of the enhancer. Here, we demonstrate that the NFI motif is a tissue-specific element. We further present a new tissue-specific element, SacI motif, just upstream from the NFI motif. These motifs showed transcriptional enhancement both in vitro and in vivo and acted upon a heterologous adenovirus major late promoter. DNase I footprint analyses demonstrated that the SacI motif bound to a brain nuclear factor, and that its binding region overlapped with the NFI motif. Gel shift experiments with the SacI motif revealed that the populations of SacI motif factors in the brain and HeLa cell extracts were different. Together with our previous findings about tissue-specific NFI-like factor(s), cooperation of NFI motif and SacI motif factors may be required for the strong brain specificity of the viral gene expression.

Animals↗

PubMed1990

[The effects of diclofenac sodium on thyroid function tests in vivo and in vitro].

In order to investigate the effects of the non-steroidal antiinflammatory drug, diclofenac sodium on thyroid function, 8 male and 2 female volunteers were given single dose of 50 mg diclofenac sodium. Ninety minutes after the administration of the drug, the concentrations of total T 3, total T 4 and free T 4 were decreased significantly, but the concentrations of free T 3 and the levels of T 3 uptake, % free T 3 and % free T 4 were increased. When diclofenac sodium was added to serum in vitro, the levels of free T 3, free T 4 and T 3 uptake were increased. These results showed that diclofenac sodium inhibited the binding of T 3 and T 4 competitively to the binding protein. The apparent decline in the concentrations of total T 3 and total T 4 after diclofenac sodium administration suggested that the drug altered distribution and metabolism of T 3 and T 4 mainly by increasing their free forms.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1989

The promoter elements of the mouse myelin basic protein gene function efficiently in NG108-15 neuronal/glial cells.

We measured transiently-expressed beta-galactosidase activity by introducing the mouse myelin basic protein (MBP)-lacZ chimeric gene (MBP-lacZ) into the NG108-15 neuronal/glial hybrid cell line. Deletion studies of the promoter region of the MBP gene showed that the promoter region between -1318 bp and -254 bp might contain sequences that repress MBP promoter activity. Fine deletion analysis using BAL 31 exonuclease revealed sequences between bp -208 and -140, -139 and -118, and -89 and -75 which were critical for promoter activity in NG 108-15 cells. DNaseI footprinting analysis revealed a cellular factor(s) that bind to the promoter region between bp -127 and -106 with NG108-15 whole cell extracts. The SV40 promoter was activated by insertion of the sequences around the region protected in footprinting experiments, in a manner independent of its orientation in NG108-15 cells. This protected region is thought to be one of the critical cis-acting DNA elements for efficient transcription.

Animals↗

PubMed1989

Tissue-specific in vitro transcription from the mouse myelin basic protein promoter.

The mouse myelin basic protein promoter was transcribed in brain nuclear extracts. The distal promoter region from -253 to -54 directed preferential transcription in brain extracts, whereas the same region repressed transcription activity in liver extracts. Stimulation of transcription was observed when the distal region was located only in a native orientation. The proximal region downstream from -53 alone still directed preferential transcription. It is suggested that cooperative function by the two promoter regions may be required for higher specificity.

Animals↗

PubMed1989

[The effects of exercise training on metabolic phases. With special reference to intraorganic fat and amino acids in Sprague-Dawley rats with streptozotocin diabetes].

We attempted to clarify the effects of aging and the difference between heavy and mild exercise training on the fat and amino acid (AA) systems of streptozotocin diabetic Sprague-Dawley rats in vivo. We exercised two groups of rats, both composed of juvenile and aged rats. The first group, however, consisted of diabetic rats only, the second of normal rats only. Both groups underwent mild training, 20 cm/s, or heavy training, 33 cm/s, for an hour a day, five times a week for four weeks. We looked for fat and amino acids in the serum, liver, red skeletal muscles and heart muscles at the pre- and post-training stages. The results were as follows: Although we observed decreased triglyceride (TG) levels in the serum, liver and intraskeletal muscles in the normal control group, similar decreases were also observed in the diabetic group undergoing insulin treatment with mild exercise training. On the other hand, we observed a clear increase in the rats subjected to heavy exercise training. This increase was particularly significant in the aged rats. There was, however, no significant change in the TG levels in the intraheart muscles of either the juvenile or aged rats, including the diabetic ones. Exercise training in the normal control group produced decreases in total AA, glycogenic AA, branched chain AA and ketogenic AA. Although there was no significant decrease in the TG levels in the serum, we could see clear signs of a decrease in the liver and the intraskeletal muscles. By contrast, the more the rats undergoing insulin treatment for diabetes were exercised, the greater the increase in the amount of AA in the livers and intraskeletal muscles. These changes were especially significant in the aged group. Consistent with previous reports, the diabetic groups produced different results for fat and AA in proportion to the intensity of exercise training and age of the rats. We also demonstrated that the differences were very small in the serum, but significant in the organs, livers and skeletal muscles. From the above findings, we have concluded that exercise training should play a major role in the treatment of diabetes in consideration not only of serum, but also transitions of the whole body's metabolic phases, including the intraorganic systems.

Aging↗

PubMed1989

Unique male mammary tumors developing in the inbred soft-furred field rats Millardia meltada.

Inbred lines of the soft-furred rat, Millardia meltada, were studied with special reference to spontaneous male mammary tumors. Adult males had the hyperplastic, pigmented inguinal mammary tissues and frequently developed bilateral mammary tumors. The tumors, no longer pigmented, were histologically well-differentiated adenocarcinomas associated with the myoepithelial cells and showed a variety of growth patterns depending on the stage of progression. They were transplantable to male, but not to female or castrated male nude mice. A significant number of androgen as well as estrogen binding sites were demonstrated in the nuclei of the transplanted tumor cells by in situ autoradiography. Neither virus particle nor mouse mammary tumor virus-specific antigen expression was detected in the tumor cells by electron microscopic and immunohistochemical studies. Normal females were generally free of pigment and tumor, although the androgen treatment induced marked pigmentation (deposit of nonfluorescent lipofuscin and hemosiderin) and hyperplasia of the female mammary epithelial cells, without a tumor. Possible relationships between pigmentation and tumor development were considered.

Animals↗