Biomedical subjects
Publications and source records attributed to T Noguchi.
We examined the diuretic effects of phosphodiesterase inhibitors in heart failure patients with and without renal failure. We found that, despite the improvement in central hemodynamics, phosphodiesterase inhibitors do not necessarily facilitate diuresis in heart failure in patients with concomitant renal failure.
FTIR difference spectra due to light-induced formation were measured in control PS II membrane fragments and in samples where the magnetic interaction with the non-heme iron center in its high-spin Fe2+ state is eliminated by three different methods, i.e., extraction of the non-heme iron center, treatment with cyanide, and incubation at high pH (pH = 11). The results obtained reveal that (i) the most pronounced band at 1479 cm-1 reflecting the C../--O stretching mode of in the semiquinone anion radical remains invariant to "iron depletion" while it shifts by 4 and 2 cm-1 to lower frequencies upon cyanide and high pH treatments, respectively, (ii) peaks observed in the 2600-3000 cm-1 region which arise from Fermi resonance of harmonics and combinations of the imidazole ring modes with the hydrogen-bonding NH stretching vibrations are not affected upon iron depletion but are lost in cyanide and high pH-treated samples, and (iii) all three treatments give rise to some similar changes in the amide I bands of the protein backbones and in imidazole ring modes of the coupled histidine. These results show that the hydrogen-bonding interaction of is virtually unaffected upon non-heme iron depletion; in particular, the strong hydrogen bond between QA and a histidine side chain (most likely His 215 of the D2 subunit) is not changed. In marked contrast, drastic changes take place in the hydrogen bonding between QA and His upon CN- and high pH treatments. The straightforward interpretation is that the hydrogen bond is lost upon these treatments. Despite the striking difference in the effect of hydrogen-bonding interaction, all three treatments lead to similar structural pertubations on the protein conformational changes due to formation and ring vibrations of the coupled histidine side chain. On the basis of the data presented in the study, it is inferred that, concerning the hydrogen bond interaction, the microenvironment of is close to the native state when a suitable iron depletion is performed. Accordingly, the previously reported conclusion on the hydrogen-bonding pattern of in PS II [MacMillan, F., Lendzian, F., Renger, G., and Lubitz, W. (1995) Biochemistry 34, 8144-8156] studied by using iron-depleted preparations most likely reflects the situation in an intact PS II.
The L-III transcriptional regulatory element of the rat pyruvate kinase L gene is located between -170 and -150 base pairs upstream of the hepatocyte-specific transcription initiation site. As the L-III element is not only necessary for cell type-specific expression but also for transcriptional stimulation by carbohydrates, it is also referred to as a carbohydrate-response element. Electrophoretic mobility shift assays using rat liver nuclear extract showed that L-III element-binding protein (L-IIIBP) was observed as multiple bands. These bands disappeared when the nuclear extract was preincubated at 60 degrees C for 5 min and were competed with unlabeled L-III oligonucleotide but not with unlabeled adenovirus major late promoter E box oligonucleotide. In addition, these bands were not affected in the presence of antiserum against upstream stimulating factor (USF). Thus, we conclude that L-IIIBP is different from USF. Then, heat-labile L-IIIBP was purified from rat liver nuclear extracts. Purified L-IIIBP exhibited two bands on sodium dodecyl sulfate/polyacrylamide gel electrophoresis by silver staining. Ultraviolet crosslinking experiment showed that both bands had binding activity to the L-III oligonucleotide.
Dok, a 62-kDa Ras GTPase-activating protein (rasGAP)-associated phosphotyrosyl protein, is thought to act as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induced marked tyrosine phosphorylation of Dok. This adhesion-dependent phosphorylation of Dok was mediated, at least in part, by Src family tyrosine kinases. The maximal insulin-induced tyrosine phosphorylation of Dok required a Src family kinase. A mutant Dok (DokDeltaPH) that lacked its pleckstrin homology domain failed to undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild-type protein, DokDeltaPH did not localize to subcellular membrane components. Insulin promoted the association of tyrosine-phosphorylated Dok with the adapter protein NCK and rasGAP. In contrast, a mutant Dok (DokY361F), in which Tyr361 was replaced by phenylalanine, failed to bind NCK but partially retained the ability to bind rasGAP in response to insulin. Overexpression of wild-type Dok, but not that of DokDeltaPH or DokY361F, enhanced the cell migratory response to insulin without affecting insulin activation of mitogen-activated protein kinase. These results identify Dok as a signal transducer that potentially links, through its interaction with NCK or rasGAP, cell adhesion and insulin receptors to the machinery that controls cell motility.
We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of alpha-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.
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Brassinosteroids (BRs) are steroidal plant hormones essential for normal plant growth and development. Mutants in the biosynthesis or perception of BRs are usually dwarf. The tomato Dwarf gene (D), which was predicted to encode a cytochrome P450 enzyme (P450) with homology to other P450s involved in BR biosynthesis, was cloned previously. Here, we show that DWARF catalyses the C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate precursor of brassinolide. To do this, we first confirmed that the D cDNA complemented the mutant light- and dark-grown phenotypes of the extreme dwarf (dx) allele of tomato. To identify a substrate for the DWARF enzyme, exogenous application of BR intermediates to dx plants was carried out. C-6 oxoBR intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR, 6-deoxoCS, had little effect. Quantitative analysis of endogenous BR levels in tomato showed mainly the presence of 6-deoxoBRs. Furthermore, dx plants were found to lack CS and had a high level of 6-deoxoCS in comparison to D plants that had CS and a lower level of 6-deoxoCS. Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS was obtained by functional expression of DWARF in yeast. In these experiments, the intermediate 6alpha-hydroxycastasterone was identified, indicating that DWARF catalyzes two steps in BR biosynthesis. These data show that DWARF is involved in the C-6 oxidation in BR biosynthesis.
Receptors coupled to the inhibitory G protein Gi, such as that for lysophosphatidic acid (LPA), have been shown to activate MAP kinase through a RAS-dependent pathway. However, LPA (but not insulin) has now been shown to activate MAP kinase in a RAS-independent manner in CHO cells that overexpress a dominant-negative mutant of the guanine nucleotide exchange protein SOS (CHO-DeltaSOS cells). LPA also induced the activation of MAP kinase kinase (MEK), but not that of RAF1, in CHO-DeltaSOS cells. The RAS-independent activation of MAP kinase by LPA was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) or by overexpression of a dominant-negative mutant of the gamma isoform of PI3K. Furthermore, LPA induced the activation of the atypical zeta isoform of protein kinase C (PKC-zeta) in CHO-DeltaSOS cells in a manner that was sensitive to wortmannin or to the dominant-negative mutant of PI3Kgamma, and overexpression of a dominant-negative mutant of PKC-zeta inhibited LPA-induced activation of MAP kinase. These observations indicate that Gi protein-coupled receptors induce activation of MEK and MAP kinase through a RAS-independent pathway that involves PI3Kgamma-dependent activation of atypical PKC-zeta.
Interactions of the primary quinone acceptor QA of photosystem II (PS II) with surrounding amino acid residues were studied by analysis of FTIR difference spectra of QA upon its photoreduction (QA-/QA). Structural coupling with a His side chain was revealed by identifying the imidazole bands in the QA-/QA spectrum using the PS II core complexes from Synechocystis PCC 6803 in which both of the two imodazole nitrogens of His side chains were specifically labeled with 15N. Strong hydrogen bonding of the imidazole NH was shown by (i) the presence of several peaks at 2600-3000 cm-1, which arise from Fermi resonance of harmonics or combinations of imidazole ring modes with the hydrogen bonding NH stretching vibration, and (ii) the 1179 cm-1 band, which can be assigned to the mode including NH deformation, is at a frequency significantly higher than the corresponding 1151 cm-1 band of model compounds 4- and 5-methylimidazole in aqueous solution. Also, the presence of the bands specific to the Npi-protonated state at 1109/1102/1090 and 1359 cm-1 suggests that the QA-coupled His is protonated at the Npi site. These results are in good agreement with the model of QA interaction in which His215 (D2), which coordinates to the non-heme iron at Ntau, is hydrogen bonded to the QA carbonyl through the Npi-H bond. In contrast, no bands of Trp side chains were detected in the QA-/QA spectrum upon labeling of the indole ring of Trp residues with indole-d5. This result indicates that Trp254 (D2), which corresponds to Trp252 (M) of the bacterial reaction center that is located in van der Waals contact with QA, is not strongly coupled with QA in PS II. Probably, the predicted pi-pi interaction is not strong enough to influence the vibrations of the indole ring of Trp upon QA reduction, or Trp254 (D2) is located rather far from QA in PS II.
Mammalian pyruvate kinase (PK), a key glycolytic enzyme, has two genes named PKL and PKM, which produce the L- and R-type isoenzymes by means of alternative promoters, and the M1-and M2-types by mutually exclusive alternative splicing respectively. The expression of these genes is tissue-specific and under developmental, dietary and hormonal control. The L-type isoenzyme (L-PK) gene contains multiple regulatory elements necessary for regulation in the 5' flanking region, up to position -170. Both L-II and L-III elements are required for stimulation of L-PK gene transcription by carbohydrates such as glucose and fructose, although the L-III element is itself responsive to carbohydrates. The L-II element is also responsible for the gene regulation by polyunsaturated fatty acids. Nuclear factor-1 proteins and hepatocyte nuclear factor 4, which bind to the L-II element, may also be involved in carbohydrate and polyunsaturated fatty acid regulation of the L-PK gene respectively. However, the L-III-element-binding protein that is involved in carbohydrate regulation remains to be clarified, although involvement by an upstream stimulating factor has been proposed. Available evidence suggests that the carbohydrate signalling pathway to the L-PK gene includes a glucose metabolite, possibly glucose 6-phosphate or xylulose 5-phosphate, as well as phosphorylation and dephosphorylation mechanisms. In addition, at least five regulatory elements have been identified in the 5' flanking region of the PKM gene up to position -279. Sp1-family proteins bind to two proximal elements, but the binding of proteins to other elements have not yet been clarified. Glucose may stimulate the transcription of the PKM gene via hexosamine derivatives. Sp1 may be involved in this regulation via its dephosphorylation, although the carbohydrate response element has not been determined precisely in the PKM gene. Thus glucose stimulates transcription of the PKM gene by the mechanism which is probably different from the L-PK gene.
The FHIT (fragile histidine triad) gene has been recently identified and cloned at chromosome 3p14.2 including FRA3B, the most common fragile site in the human genome. FHIT is suggested to be a candidate tumour suppressor gene in gastrointestinal tract tumours. To elucidate the role of the FHIT gene in gastric cancer, a total of 133 curatively R0-resected gastric carcinomas were investigated for loss of heterozygosity (LOH) at 3p14.2, using four polymorphic microsatellite loci (D3S1300, D3S1313, D3S1481, and D3S1234). LOH of the FHIT gene affecting at least one of the investigated loci was observed in 20 of 123 informative tumours (16.3 per cent). The presence of LOH was correlated neither with major prognostic factors such as pT category, pN category or vascular invasion, nor with histological type or grade of differentiation of the tumours. In addition, there were no differences in the prognosis between patients with gastric carcinomas showing LOH at the FHIT gene and patients with tumours lacking LOH at the FHIT gene. These findings suggest that LOH of the FHIT gene represents an event in the tumourigenesis of only a small subset of gastric carcinomas and does not correlate with tumour progression or prognosis.
The expression and prognostic role of cyclin D1, cyclin E, and p21 (WAF1/CIP1) were immunohistochemically investigated in 413 curatively resected gastric carcinomas. p21 was expressed in 65.4 per cent (n=270), cyclin D1 in 23.7 per cent (n=98), and cyclin E in 13.6 per cent ( n=56) of the tumours. The expression of p21, cyclin D1, and cyclin E was positively associated with the papillary or tubular type of the WHO classification, as well as with the intestinal type according to the Lauren classification. No significant correlation could be found between the expression of p21, cyclin D1 and cyclin E and the parameters pT category, lymph node involvement, and blood vessel and lymphatic vessel invasion. Concerning survival, no prognostic impact of p21, cyclin D1, and cyclin E expression could be verified, even when different subgroups of patients were analysed separately according to the pT and pN category as well as to the Lauren classification. The present data suggest that neither cyclin D1, cyclin E nor their inhibitor p21 can predict the survival of gastric cancer patients, nor is their immunohistochemical detection a suitable tool for identifying subgroups of patients who may be at higher risk.
We investigated the blocking effect of the conformationally restricted analogs of milnacipran on NMDA receptors by recording the whole-cell currents of Xenopus oocytes injected with rat brain mRNA and the single channel currents of cultured hippocampal neurons under voltage-clamp conditions. Their protective effect against excitotoxicity was also investigated on cultured cortex neurons. All conformationally restricted analogs examined blocked activated NMDA receptors, though their structures were quite different from known NMDA receptor blockers. The analogs with a (1S, 2R, 1'S)-configuration such as PPDC ((1S, 2R)-1-phenyl-2[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide+ ++) had lower IC50 values than those with other configurations. The empirical Hill coefficients for each compound were close to unity, indicating a 1:1 stoichiometry for the block. PPDC decreased the maximum responses to both N-methyl D-aspartate (NMDA) and glycine without altering their dissociation constants. The blocking effect was enhanced on hyperpolarization. PPDC had no effects on other glutamate receptor subtypes (AMPA, kainate, and metabotropic glutamate receptors) or other neurotransmitter receptors (GABA(A), 5HT2C, and AChM1 receptors) produced by the oocytes. PPDC decreased the mean open time of NMDA receptors without decreasing their elementary conductance. The microscopic blocking rate constant was 2.8x10(7) M(-1)s(-1). The macroscopic unblocking rate constant of PPDC was much faster than that of MK-801. Only the analogs with the (1S, 2R, 1'S)-configuration protected the cultures against NMDA-induced neurotoxicity, though they failed to protect against kainate-induced neurotoxicity. These results show that conformationally restricted analogs, at least PPDC, selectively blocked open channels of NMDA receptors.
We investigated the effects of circulating inflammatory cytokines and adhesion molecules induced by ortho-topic liver transplantation (OLT) on pulmonary function. Although the plasma interleukin-8 (IL-8) levels increased gradually, peaking at the end of the operation, these increases were considered minimal. The baseline endothelial adhesion molecule (E-selectin) level was several times higher than the normal value, but after reperfusion of the new transplanted liver, the plasma E-selectin concentrations decreased to within the normal range and remained almost normal during the postoperative period. Similar changes were observed in the plasma levels of other types of adhesion molecules. Although PaO(2)/FIO(2) showed a significant inversed correlation with the peak IL-8 concentration, after the exclusion of two patients, one of whom died and one of whom rejected the transplanted liver, no correlation was able to be found between the PaO(2)/FIO(2) ratio and the maximum IL-8 concentration. Furthermore, there was no correlation between the adhesion moleclues and PaO(2)/FIO(2). These results suggest that IL-8 exerts only a slight effect on respiratory function following successful pediatric liver transplantation, and that circulating adhesion molecules do not affect perioperative lung function.
It is known that bone resorption is mediated by osteoclasts, and lipopolysaccharide (LPS) and inflammatory mediators such as interleukin-1 (IL-1) and prostaglandin E2 (PGE2) induce osteoclast differentiation from haemopoietic cells, 2-aminoethanesulphonic acid, which is known as taurine, is an important nutrient and is added to most synthetic human infant milk formulas. In this study, it was found that 2-aminoethanesulphonic acid inhibits the stimulation of bone resorption mediated by LPS of the periodontopathic microorganism Actinobacillus actinomycetemcomitans Y4 in organ cultures of newborn mouse calvaria. The effect of 2-aminoethanesulphonic acid on the development and survival of osteoclast-like multinucleated cells produced in a mouse bone-marrow culture system was also examined. 2-aminoethanesulphonic acid (100 microg/ml) suppressed the formation of these osteoclast-like cells in the presence of LPS of A. actinomycetemcomitans Y4, IL-1alpha or PGE2 in mouse marrow cultures. On the other hand, 2-aminoethanesulphonic acid did not inhibit 1alpha, 25-dihydroxyvitamin D3-mediated osteoclast differentiation. Although IL-1alpha elongated the survival of the osteoclast-like cells, 2-aminoethanesulphonic acid blocked the supportive effect of IL-1alpha on osteoclast survival. 2-aminoethanesulphonic acid showed no effect on the growth of mouse osteoblasts. Finally, it was found that 2-aminoethanesulphonic acid inhibited alveolar bone resorption in experimental periodontitis in hamsters. These results suggest that 2-aminoethanesulphonic acid is an effective agent in preventing inflammatory bone resorption in periodontal diseases.
For production of monoclonal antibodies against domoic acid, a causative agent of amnesic shellfish poisoning, three immunogens, domoic acid conjugated with bovine serum albumin (BSA), ovalbumin (OVA) and human gamma globulin (HGG), were prepared. The antiserum obtained from BALB/c mice immunized with domoic acid-BSA showed the highest affinity for domoic acid. The monoclonal antibody, DA-3, obtained from the mice was highly specific for domoic acid and showed a minor cross-reactivity with the isomers of domoic acid (isodomoic acids B, E, F, G and H), except for isodomoic acid A. Using DA-3 antibody, an indirect competitive enzyme immunoassay (idc-EIA) was developed for measurement of domoic acid. The working range for quantitative measurement of domoic acid and the quantification limit for domoic acid in shellfish were estimated to be 0.15-10 ng/ml and less than 0.04 microg/g, respectively. The mean recovery of domoic acid added to extracts of shellfish at toxin levels of 0.02 to 0.2 microg/ml was 103% with a coefficient of variation of 4.5%. The newly developed idc-EIA seems to be a useful method for monitoring domoic acid in shellfish.
A subclone HL60/DOX was selected from a human leukemic HL60 cell line for resistance to doxorubicin (DOX) by exposure to stepwise increasing concentrations of the drug and coexposure to a potential P-glycoprotein (P-gp) inhibitor, cepharanthine (a biscoclaurine alkaloid). Compared with the parent HL60 cells, the HL60/DOX cells were 13.0-fold more resistant to DOX and showed multidrug-resistant (MDR) phenotype characterized by 4.6-fold, 2.3-fold, and 5.7-fold cross-resistance to vincristine, pirarubicin, and etoposide, respectively, but no cross-resistance to alkylating agent, cisplatin. Immunocytochemical analyses using the specific monoclonal antibody, MRPr1, and quantitative analyses using a competitive reverse transcription-polymerase chain reaction (CRT-PCR) confirmed overexpression of MRP gene products (about 8-fold determined by CRT-PCR) in this resistant clone. The P-gp expression was not detectable by the monoclonal antibody, C219, in the HL60/DOX cells, and that was consistent with extremely low levels of mdr1 mRNA expression determined by CRT-PCR in this clone. Drug accumulation and efflux studies demonstrated the significantly increased efflux rate of DOX compared to the parent HL60 cells. This enhancement of DOX efflux was reversed by the addition of 10 microM verapamil. To investigate the additional underlying mechanisms contributing to MDR phenotype in the HL60/DOX cells, the levels of DNA topoisomerases (Topo) including Topo I, Topo IIalpha, and Topo IIbeta, and gamma-glutamylcystein synthetase (y-GCS) expression were determined using CRT-PCR techniques. Normal expression of each enzyme at the transcriptional level was demonstrated in this resistant clone. Southern blot analysis of the gene organization in the HL60/DOX cells revealed the amplification of MRP gene. These results indicate that alteration of the drug accumulation from enhanced efflux appears to be a major mechanism(s) of MDR phenotype and attributable to high levels of MRP expression in the HL60/DOX cells. Overexpression of MRP in this clone is regulated by the genomic amplification of DNA and increased levels of the MRP mRNA, independently with the normal expression of Topo I, Topo IIalpha, Topo IIbeta, or gamma-GCS.
A 73-year-old woman who suffered from progressive hoarseness for 6 years and dysphagia without pain for 1 year presented with a soft tissue deposition on the posterior region of the vocal cords and narrowing in the subglottic area. Biopsy of this soft tissue and histological examination revealed laryngeal amyloidosis. A tracheostomy and partial removal of the amyloid were performed with general anesthesia. The airway was secured with a smaller diameter endotracheal tube, which was inserted atraumatically with Magill's forceps. The larynx is a rare site for amyloidosis. Laryngeal amyloidosis is fragile and hemorrhagic. Therefore, massive bleeding may occur during intubation. Anesthetists should take care in intubating the tracheas of these patients and be aware of other systemic diseases in laryngeal amyloidosis.