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R Morimoto

Publications and source records attributed to R Morimoto.

At least 19 recordsLinked to original sources
PubMed2003

Hypothyroidism induces type 2 iodothyronine deiodinase expression in mouse heart and testis.

In the present study we show the expression profiles of both type 1 and type 2 iodothyronine deiodinases (D1 and D2) in a wide spectrum of mouse tIssues, and D2 regulation by thyroid status. A characteristic tIssue-specific expression for each isoform was observed. D2 transcripts were detected in most tIssues with variable levels of expression. The observed D2 mRNA tIssue distribution was similar to that described in rats and is in agreement with the view of different patterns of expression between rodents and humans. However, it is interesting to note that despite the low levels of D2 transcripts in mouse heart and testis in the euthyroid state, the induction of hypothyroidism caused a significant increase in D2 activity in these tIssues. Similar results were also obtained in adult rats. These results suggest a previously unrecognized role for type 2 deiodinase in controlling intracellular triiodothyronine levels in rodent heart and testis during states of thyroid hormone deficiency.

Animals↗
PubMed2001

Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development.

The unfolded protein response (UPR) is a transcriptional and translational intracellular signaling pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER). We have used C. elegans as a genetic model system to dissect UPR signaling in a multicellular organism. C. elegans requires ire-1-mediated splicing of xbp-1 mRNA for UPR gene transcription and survival upon ER stress. In addition, ire-1/xbp-1 acts with pek-1, a protein kinase that mediates translation attenuation, in complementary pathways that are essential for worm development and survival. We propose that UPR transcriptional activation by ire-1 as well as translational attenuation by pek-1 maintain ER homeostasis. The results demonstrate that the UPR and ER homeostasis are essential for metazoan development.

Animals↗
PubMed2001

Differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI) is a vesicular glutamate transporter in endocrine glutamatergic systems.

Vesicular glutamate transporter is present in neuronal synaptic vesicles and endocrine synaptic-like microvesicles and is responsible for vesicular storage of L-glutamate. A brain-specific Na(+)-dependent inorganic phosphate transporter (BNPI) functions as a vesicular glutamate transporter in synaptic vesicles, and the expression of this BNPI defines the glutamatergic phenotype in the central nervous system (Bellocchio, E. E., Reimer, R. J., Fremeau, R. T., Jr., and Edwards, R. H. (2000) Science 289, 957-960; Takamori, S., Rhee, J. S., Rosenmund, C., and Jahn, R. (2000) Nature 407, 189-194). However, since not all glutamatergic neurons contain BNPI, an additional transporter(s) responsible for vesicular glutamate uptake has been postulated. Here we report that differentiation-associated Na(+)-dependent inorganic phosphate cotransporter (DNPI), an isoform of BNPI (Aihara, Y., Mashima, H., Onda, H., Hisano, S., Kasuya, H., Hori, T., Yamada, S., Tomura, H., Yamada, Y., Inoue, I., Kojima, I., and Takeda, J. (2000) J. Neurochem. 74, 2622-2625), also transports L-glutamate at the expense of an electrochemical gradient of protons established by the vacuolar proton pump when expressed in COS7 cells. Molecular, biological, and immunohistochemical studies have indicated that besides its presence in neuronal cells DNPI is preferentially expressed in mammalian pinealocytes, alphaTC6 cells, clonal pancreatic alpha cells, and alpha cells of Langerhans islets, these cells being proven to secrete L-glutamate through Ca(2+)-dependent regulated exocytosis followed by its vesicular storage. Pancreatic polypeptide-secreting F cells of Langerhans islets also expressed DNPI. These results constitute evidence that DNPI functions as another vesicular transporter in glutamatergic endocrine cells as well as in neurons.

Adenosine Triphosphate↗
PubMed1997

Function and regulation of heat shock factor 2 during mouse embryogenesis.

The spontaneous expression of heat shock genes during development is well documented in many animal species, but the mechanisms responsible for this developmental regulation are only poorly understood. In vertebrates, additional heat shock transcription factors, distinct from the heat shock factor 1 (HSF1) involved in the stress response, were suggested to be involved in this developmental control. In particular, the mouse HSF2 has been found to be active in testis and during preimplantation development. However, the role of HSF2 and its mechanism of activation have remained elusive due to the paucity of data on its expression during development. In this study, we have examined HSF2 expression during the postimplantation phase of mouse development. Our data show a developmental regulation of HSF2, which is expressed at least until 15.5 days of embryogenesis. It becomes restricted to the central nervous system during the second half of gestation. It is expressed in the ventricular layer of the neural tube which contains mitotically active cells but not in postmitotic neurons. Parallel results were obtained for mRNA, protein, and activity levels, demonstrating that the main level of control was transcriptional. The detailed analysis of the activity of a luciferase reporter gene under the control of the hsp70.1 promoter, as well as the description of the protein expression patterns of the major heat shock proteins in the central nervous system, show that HSF2 and heat shock protein expression domains do not coincide. This result suggests that HFS2 might be involved in other regulatory developmental pathways and paves the way to new functional approaches.

Animals↗
PubMed1997

Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

BACKGROUND: The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. RESULTS: The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. CONCLUSIONS: The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes in protein structure as a result of calcium binding may facilitate phosphorylation. A small, but significant, movement of metal ions and sidechains could position catalytically important threonine residues for phosphorylation. The second calcium site represents a new calcium-binding motif that can play a role in the stabilization of protein structure. We discuss how the information about catalytic events in the active site could be transmitted to the peptide-binding domain.

Adenosine Diphosphate↗
PubMed1997

HSF1 granules: a novel stress-induced nuclear compartment of human cells.

Heat shock factor 1 (HSF1) is the ubiquitous stress-responsive transcriptional activator which is essential for the inducible transcription of genes encoding heat shock proteins and molecular chaperones. HSF1 localizes within the nucleus of cells exposed to heat shock, heavy metals, and amino acid analogues, to form large, irregularly shaped, brightly staining granules which are not detected during attenuation of the heat shock response or when cells are returned to their normal growth conditions. The kinetics of detection of HSF1 granules parallels the transient induction of heat shock gene transcription. HSF1 granules are also detected using an HSF1-Flag epitope tagged protein or a chimeric HSF1-green fluorescent protein which reveals that these nuclear structures are stress-induced and can be detected in living cells. The spatial organization of HSF1 granules in nuclei of stressed cells reveals that they are novel nuclear structures which are stress-dependent and provides evidence that the nucleus undergoes dynamic reorganization in response to stress.

Amino Acids↗
PubMed1997

HSF1 transcription factor concentrates in nuclear foci during heat shock: relationship with transcription sites.

In this paper, we show that upon heat shock, HSF1 concentrates in the nucleus of diploid human fibroblasts in two large foci. The relative distribution of HSF1 nuclear foci and active heat shock protein (hsp) genes was investigated by combining fluorescence in situ hybridization (FISH) for the detection of hsp nuclear transcripts and immunofluorescence for the detection of HSF1. We show that the HSF1 foci are distinct from the sites of hsp70 and hsp90 genes transcription. This is the second report of ploidy-dependent foci of transcription factors that are independent of their specific transcription sites. However, the correlation between the number of HSF1 foci and the ploidy of the cells strongly supports the existence of a specific chromosomal target for HSF1 foci.

Apoptosis↗
PubMed1996

[Hypertonic saline induced bronchoconstriction in sensitized rabbits].

Hypertonic saline is a potent stimulus to airway narrowing in most asthmatic patients. However, the mechanism of airway narrowing induced by a change in osmolarity is not clearly understood. In ovalbumin-sensitized rabbits, we found that bronchoconstriction occurred after inhalation of hypertonic saline, and then studied the mechanisms responsible for this bronchoconstriction. Eighteen anesthetized, paralyzed, mechanically ventilated (40 breath/min, TV 7 ml/kg) ovalbumin-sensitized rabbits were exposed to aerosols of hypertonic saline (ultrasonic nebulizer, 0.5 ml/min, 1 min). Total lung resistance (RL) and dynamic compliance (Cdyr) of the lung were measured before and after the exposure. The concentration of NaCl was increased from 0.9% to 7.2% in 0.9% steps. RL increased and Cdyr decreased as the dose of NaCl rose and they reached plateaus at doses of 6.3% and 7.2%, respectively. These responses were markedly inhibited by treatment with atropine (5 mg/kg i.v., p < 0.05 vs. control group), but treatment with chlorpheniramine (1 mg/kg iv.) suppressed the responses only at low concentrations of NaCl. In contrast, treatment with indomethacin, did not significantly change the responses. We conclude that inhalation of hypertonic saline can cause bronchoconstriction in sensitized rabbits, and that vagal stimulation plays a major role in this bronchoconstriction.

Animals↗
PubMed1995

Neural networks as an aid in the diagnosis of lymphocyte-rich effusions.

Neural network (NN) technology was applied to digital image analysis data for 112 Papanicolaou-fixed and -stained smears of lymphocyte-rich effusions (LREs). The smears were analyzed with an inexpensive image analysis system assembled in our laboratory. Several models were developed using backpropagation NN development software in an effort to optimize classification of the LREs as reactive lymphocytosis or malignant lymphoma and to analyze the effects of various parameters on classification rates. The greatest specificity and sensitivity of LRE classification were achieved with NN models that consisted of 7 input neurons, including 5 morphometric and 2 densitometric variables, 10 hidden-layer neurons and 1 output neuron. This NN architecture with a sigmoidal transfer function provided a true cross-validation rate of 89.3% of testing data, with a sensitivity of 76.9%, specificity of 93.0% and shrinkage of 10.7%. The same NN architecture with a step transfer function provided a true cross-validation rate of 95.3%, sensitivity of 85.7%, specificity of 97.6% and shrinkage of 0%. The effects of various parameters, such as network size, shrinkage and ratio of sample size to input layer size, on NN accuracy are discussed.

Ascitic Fluid↗
PubMed1993

Computerized interactive morphometry and the diagnosis of lymphoid-rich effusions.

An experimental computerized interactive morphometry system for the classification of lymphoid-rich effusions is presented. The relatively inexpensive microcomputer-based system was assembled in our laboratory with commercially available hardware and software. One hundred twenty-two effusions (86 benign lymphocytoses, 26 lymphomas, and 10 chronic lymphocytic leukemias) were studied. Lymphoid cells were selected randomly by the system from real-time images of Papanicolaou-fixed and stained cytospin smears of pleural, peritoneal, and pericardial effusions. Parameters of nuclear shape, area, and optical density were measured automatically. Multiparameter statistical procedures of discriminant classificatory analysis analyzed the distribution of lymphoid nuclear profile integrated optical density to yield three groups of effusions, each with a predictive value of diagnosis of 100%. Neither these statistical procedures nor a simple rule-based expert system accurately classified chronic lymphocytic leukemia effusions based on the distribution of lymphoid nuclear profile area. When chronic lymphocytic leukemia effusions were excluded, however, the predictive values of a diagnosis of lymphoma by statistical analysis and by rule-based expert system were 92.3% and 88.9%, respectively, whereas the predictive values of a diagnosis of benign lymphocytosis were 97.7% and 91.3%, respectively. Potential applications and limitations of this technology for the diagnosis of lymphoid-rich effusions are discussed.

Cell Nucleus↗
PubMed1991

Sequential activation of genes for heme pathway enzymes during erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells.

Changes in the level of transcripts encoding enzymes of the heme biosynthetic pathway as well as those encoding ubiquitous proteins were examined in murine Friend virus-transformed erythroleukemia cells during erythroid cell differentiation induced by chemicals including dimethyl sulfoxide (DMSO). Early changes following DMSO treatment were marked decreases in mRNAs for three ubiquitous proteins, i.e., a 70 kDa heat shock protein (less than 6 h), heme oxygenase and nonspecific delta-aminolevulinate synthase (ALAS) (less than 12 h). These changes were followed by sequential increases in mRNAs for enzymes in the heme biosynthetic pathway. Namely, mRNAs for the erythroid-specific ALAS, delta-aminolevulinate dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase started to increase at 12, 18, 18-24 and 24 h, respectively. Nuclear runoff studies revealed that these changes are largely transcriptional. Treatments with other inducers of erythroid differentiation, e.g., hexamethylene bisacetamide, n-butyric acid and N'-methylnicotinamide, also showed similar effects on mRNAs as those following DMSO. These findings suggest that both suppression of ubiquitous genes and activation of heme pathway enzyme genes are associated with erythroid differentiation, and the former occurs preceding changes in the latter.

Animals↗
PubMed1991

Nuclear colocalization of cellular and viral myc proteins with HSP70 in myc-overexpressing cells.

The c-myc oncogene and its viral counterpart v-myc encode phosphoproteins which have been located within cell nuclei, excluding nucleoli. We have expressed the c-myc gene under the simian virus 40 early promoter and studied the distribution of its protein product in transient expression assays in COS, HeLa, and 293 cells. We found three distinct patterns of c-myc immunofluorescence in the transfected cells: one-third of the c-myc-positive cells displayed a diffuse nuclear distribution, and in two-thirds of the cells the c-myc fluorescence was accumulated either in small amorphous or in large multilobed phase-dense nuclear structures. Unexpectedly, these structures also stained for the HSP70 heat shock protein in both heat-shocked and untreated cells. Our results indicate that both transient and stable overexpression of either the c-myc or v-myc protein induces translocation of the endogenous HSP70 protein from the cytoplasm to the nucleus, where it becomes sequestered in structures containing the myc protein. Interestingly, the closely related N-myc protein does not stimulate substantial nuclear expression of the HSP70 protein. Studies with chimeric myc proteins revealed that polypeptide sequences encoded by the second exon of c-myc are involved in colocalization with HSP70.

Animals↗
PubMed1991

Cardiac rupture due to severe fatty infiltration in the right ventricular wall.

An extremely rare case of sudden death caused by cardiac rupture due to severe fatty infiltration in the right ventricular myocardium is presented. The patient, a 74-year-old woman, had no history of chest trauma, hypertension, or pulmonary disease. The autopsy showed a small tear in the right ventricle and cardiac tamponade, but no coronary artery lesion. In the right ventricular myocardium, muscle fibers were definitely atrophic or absent, with massive fatty replacement. Fatty infiltration of the myocardium, if severe, can be a cause of serious cardiac dysfunction or, occasionally, sudden death.

Adipose Tissue↗
PubMed1989

Evaluation of the involvement of a male specific cytochrome P-450 isozyme in senescence-associated decline of hepatic drug metabolism in male rats.

The major "male specific" species of cytochrome P-450 (P-450ml) was purified and an antibody against it used to evaluate the involvement of this isozyme in alterations of drug metabolism in senescence. P-450ml exhibited strikingly high imipramine (IM) N-demethylase activity while it showed no IM 2-hydroxylation, which is an alternate pathway of IM metabolism in rat liver microsomes. The antibody to P-450ml inhibited 80% of imipramine N-demethylation in young male rats. In old male rats, which have been shown to have lower IM N-demethylase activity, a 60% inhibition was observed. The inhibitable portion of this activity in old male rats is about one third of that in young rats, but the remaining portion not inhibited by this antibody is almost identical in young and old rats. IM 2-hydroxylation on the other hand was not inhibited by this antibody at all. It also inhibited about 30% of diazepam(DZ) N-demethylation in young rats but showed no inhibition in old rats, resulting in the loss of the age difference in the remaining portion. DZ 3-hydroxylation was not inhibited by this antibody, in spite of the fact that it showed a markedly higher activity in young male than in young female rats with a subsequent reduction in old age in male rats. This study provides the first direct evidence that differences in the amount of the major male specific P-450 isozyme (P-450ml) are responsible for the age- and sex-associated differences in some of the drug metabolizing activities. It also became apparent that P-450ml may not be the only isozyme responsible for these differences.

Aging↗
PubMed1986

Pharmacological effects of nalorphine and nalorphine-7,8-oxide (nalorphine-epoxide): interaction of the intrinsic activity, affinity and pharmacological responses.

We examined the relationship between the pharmacological effects and the interactions of the receptors of nalorphine and its epoxide. The abilities of nalorphine-epoxide to displace [3H]-dihydromorphine (mu-site) and [3H]-ethylketocyclazocine (kappa-site) were practically equal to those of the parent compound, nalorphine, using binding assay to the rat brain membrane preparations. Furthermore, the affinities of mu- and kappa-receptors are virtually uninfluenced by epoxidation of the 7,8-double bond of nalorphine using electrically stimulated mouse and rabbit vasa deferentia. The intrinsic activity of nalorphine, however, is considerably decreased by epoxidation. Moreover, the antagonistic effect of nalorphine to the morphine-induced antinociception (via mu-receptors) was little influenced by epoxidation, but the antinociceptive effect of nalorphine using the acetic acid writhing test was considerably reduced by epoxidation. These results suggest the presence of a higher receptor capacity for the antinociception mediated through kappa-receptors and that the differences between the pharmacological responses of nalorphine and its epoxide are due to the differences of their intrinsic activities.

Analgesics↗
PubMed1985

Structure and expression of the human gene encoding major heat shock protein HSP70.

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.

Acetyltransferases↗
PubMed1984

Cell-specific expression of heat shock proteins in chicken reticulocytes and lymphocytes.

We have found that chicken reticulocytes respond to elevated temperatures by the induction of only one heat shock protein, HSP70, whereas lymphocytes induce the synthesis of all four heat shock proteins (89,000 mol wt, HSP89; 70,000 mol wt, HSP70; 23,000 mol wt, HSP23; and 22,000 mol wt, HSP22). The synthesis of HSP70 in lymphocytes was rapidly induced by small increases in temperature (2 degrees-3 degrees C) and blocked by preincubation with actinomycin D. Proteins normally translated at control temperatures in reticulocytes or lymphocytes were not efficiently translated after incubation at elevated temperatures. The preferential translation of mRNAs that encode the heat shock proteins paralleled a block in the translation of other cellular proteins. This effect was most prominently observed in reticulocytes where heat shock almost completely repressed alpha- and beta-globin synthesis. HSP70 is one of the major nonglobin proteins in chicken reticulocytes, present in the non-heat-shocked cell at approximately 3 X 10(6) molecules per cell. We compared HSP70 from normal and heat-shocked reticulocytes by two-dimensional gel electrophoresis and by digestion with Staphylococcus aureus V8 protease and found no detectable differences to suggest that the P70 in the normal cell is different from the heat shock-induced protein, HSP70. P70 separated by isoelectric focusing gel electrophoresis into two major protein spots, an acidic P70A (apparent pl = 5.95) and a basic P70B (apparent pl = 6.2). We observed a tissue-specific expression of P70A and P70B in lymphocytes and reticulocytes. In lymphocytes, P70A is the major 70,000-mol-wt protein synthesized at normal temperatures whereas only P70B is synthesized at normal temperatures in reticulocytes. Following incubation at elevated temperatures, the synthesis of both HSP70A and HSP70B was rapidly induced in lymphocytes, but synthesis of only HSP70B was induced in reticulocytes.

Anemia↗