Biomedical subjects

M Hirasawa

Publications and source records attributed to M Hirasawa.

At least 19 recordsLinked to original sources

PubMed1999

Oxidation-reduction properties of chloroplast thioredoxins, ferredoxin:thioredoxin reductase, and thioredoxin f-regulated enzymes.

Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins f, for spinach and Chlamydomonas reinhardtii thioredoxins m, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin f, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (Em) values at pH 7.0 of -290 mV for both spinach and pea thioredoxin f, -300 mV for both C. reinhardtii and spinach thioredoxin m, -320 mV for spinach FTR, -290 mV for spinach PRK, -315 mV for pea FBPase, and -330 mV for spinach FBPase were obtained. With the exception of spinach FBPase, titrations showed a single two-electron component at all pH values tested. Spinach FBPase exhibited a more complicated behavior, with a single two-electron component being observed at pH values >/= 7.0, but with two components being present at pH values <7.0. The slopes of plots of Em versus pH were close to the -60 mV/pH unit value expected for a process that involves the uptake of two protons per two electrons (i. e., the reduction of a disulfide to two fully protonated thiols) for thioredoxins f and m, for FTR, and for pea FBPase. The slope of the Em versus pH profile for PRK shows three regions, consistent with the presence of pKa values for the two regulatory cysteines in the region between pH 7.5 and 9.0.

Animals↗

PubMed1999

Serum levels of Clara cell 10-kDa protein are decreased in patients with asthma.

Clara cell 10-kDa protein (CC10), the predominant product from nonciliated cells in the epithelial lining of bronchioles (Clara cells), has been shown to have immunomodulatory and antiinflammatory activity and may play a role in controlling airway inflammation. This study was designed to measure serum CC10 concentrations in healthy and asthmatic nonsmokers. Serum CC10 concentrations in asthmatic nonsmokers were significantly lower than in healthy nonsmokers. Asthmatic patients with a long duration of the disease (>/=10 years) had significantly lower serum CC10 levels than those with a short duration of the disease (<10 years). There was no significant difference in serum CC10 levels in asthmatic patients between the time of the asthmatic attack and the stable condition. Serum CC10 levels may reflect decreased production of CC10 caused by remodeling of the small airways in asthma.

Asthma↗

PubMed1999

Three kinds of antibacterial substances from Lentinus edodes (Berk.) Sing. (Shiitake, an edible mushroom).

Three kinds of antibacterial substances were extracted by chloroform, ethylacetate or water from dried Shiitake mushrooms (Lentinus edodes). These substances possess efficient antibacterial activities against Streptococcus spp., Actinomyces spp., Lactobacillus spp., Prevotella spp., and Porphyromonas spp. of oral origin. In contrast, other general bacteria, such as Enterococcus spp., Staphylococcus spp., Escherichia spp., Bacillus spp., and Candida spp. were relatively resistant to these substances. Chloroform extracts had bactericidal activity against both growing and resting bacterial cells of S. mutans and P. intermedia, whereas the other two extracts showed bacteriostatic activity against both growing and resting bacterial cells of S. mutans and resting bacterial cell of P. intermedia. The antibacterial activity of chloroform extracts and ethylacetate extracts were relatively heat-stable. The water extract was heat-labile.

Anti-Bacterial Agents↗

PubMed1998

The single mutation Trp35-->Ala in the 35-40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36-C39 1H-13C NMR.

The role of the invariant Trp residue at the redox site of thioredoxins was investigated by site-directed mutagenesis of a Chlamydomonas reinhardtii thioredoxin h. Though being still redox active with NADPH-thioredoxin reductase and chemical substrates [dithiothreitol and 5,5'-dithio-bis(2-nitrobenzoic acid)] the Trp35-->Ala-mutated protein completely lost the capacity to activate the thiol-regulated NADPH-dependent malate dehydrogenase. However, it was able to activate a mutant malate dehydrogenase where only the most exposed disulfide was retained. The pH dependence of the redox-site Cys beta 1H/13C-NMR frequencies of the wild-type and mutated proteins, in both the reduced and oxidised states, were compared over the pH range 5.8-10. The mutation does not affect the conserved buried Asp30, which titrates with a pKa of 7.5 in the oxidised proteins in agreement with previous studies. However, for the reduced forms of the proteins, the pH dependence of resonances of both Cys was strongly affected by the mutation. In the case of the wild-type thioredoxin, two apparent pKa values were found around 7.0 and 9.5 and could be assigned to the titration of Cys36 and Cys39 thiol, respectively, similar to the case of Escherichia coli thioredoxin. For the mutated thioredoxin a single pKa was found around 8.3. This result can be interpreted as a single pKa of either Cys36 or Cys39 or both. While the mutation clearly affects ionisations, the measured redox potentials of the active-site Cys pair are not significantly affected by the Trp35-->Ala mutation. Possible roles of an aromatic side chain on the reactivity of the catalytic Cys residues in thioredoxins are proposed.

Animals↗

PubMed1998

Activity of ventromedial hypothalamic neurons suppressing heart rate is associated with paradoxical sleep in the rat.

Cardiovascular change is one of the common features of paradoxical sleep. Our study offers evidence that one of the central areas regulating the circulation during sleep is the ventromedial nucleus of the hypothalamus (VMH). We found a group of neurons in this hypothalamic nucleus of rats whose electrical activity was exclusively increased during paradoxical sleep, and was associated with a reduction in heart rate. The onset of this neural activity usually followed that of paradoxical sleep. The incidence and duration of paradoxical sleep was increased by means of microinjection of carbachol, a cholinergic agonist, into the pontine reticular formation, and the neural activity of the VMH still appeared in synchrony with carbachol-induced paradoxical sleep. These results suggest that the cholinergic paradoxical sleep-inducing mechanism in the pons facilitate the excitability of these neurons. We have previously shown that these VMH neurons suppress blood pressure and heart rate via inhibition of the vasomotor neurons in the medulla oblongata. Taken together, our findings suggest that a group of neurons in the VMH suppresses the circulatory system during paradoxical sleep.

Animals↗

PubMed1998

A conserved tryptophan at the ferredoxin-binding site of ferredoxin:nitrite oxidoreductase.

Treatment of spinach leaf ferredoxin-dependent nitrite reductase with N-bromosuccinimide (NBS), under conditions where slightly less than 1 mol of tryptophan is modified per mole of nitrite reductase, inhibits the catalytic activity of the enzyme by ca. 80% without any effect on substrate binding or other enzyme properties. Complex formation between nitrite reductase and ferredoxin completely protects the enzyme against this inhibition. Transient kinetic measurements show that the second-order rate constant for reduction of NBS-modified nitrite reductase by reduced ferredoxin is approximately four-fold larger than that observed for the native, unmodified enzyme. Also, reduction of NBS-modified nitrite reductase by the 5-deazariboflavin radical shows a different kinetic pattern than that observed with the native enzyme, suggesting that tryptophan modification increases access of the radical to the low-potential [4Fe-4S] cluster of the enzyme, decreases the accessibility to the siroheme group of the enzyme, or both. The tryptophan that is modified has been identified as the absolutely conserved W92. A methionine, M73, that is also modified by NBS, has been identified. The ferredoxin-binding site on spinach nitrite reductase thus appears to include W92 and perhaps M73, in addition to the previously identified R375, R556, and K436.

Amino Acid Sequence↗

PubMed1998

The role of aromatic and acidic amino acids in the electron transfer reaction catalyzed by spinach ferredoxin-dependent glutamate synthase.

Treatment of the ferredoxin-dependent, spinach glutamate synthase with N-bromosuccinimide (NBS) modifies 2 mol of tryptophan residues per mol of enzyme, without detectable modification of other amino acids, and inhibits enzyme activity by 85% with either reduced ferredoxin or reduced methyl viologen serving as the source of electrons. The inhibition of ferredoxin-dependent activity resulting from NBS treatment arises entirely from a decrease in the turnover number. Complex formation of glutamate synthase with ferredoxin prevented both the modification of tryptophan residues by NBS and inhibition of the enzyme. NBS treatment had no effect on the secondary structure of the enzyme, did not affect the Kms for 2-oxoglutarate and glutamine, did not affect the midpoint potentials of the enzyme's prosthetic groups and did not decrease the ability of the enzyme to bind ferredoxin. It thus appears that the ferredoxin-binding site(s) of glutamate synthase contains at least one, and possibly two, tryptophans. Replacement of either phenylalanine at position 65, in the ferredoxin from the cyanobacterium Anabaena PCC 7120, with a non-aromatic amino acid, or replacement of the glutamate at ferredoxin position 94, decreased the turnover number compared to that observed with wild-type Anabaena ferredoxin. The effect of the change at position 65 was quite modest compared to that at position 94, suggesting that an aromatic amino acid is not absolutely essential at position 65, but that glutamate 94 is essential for optimal electron transfer.

Amino Acid Oxidoreductases↗

PubMed1998

Oxidation-reduction properties of the regulatory site of spinach phosphoribulokinase.

The oxidation-reduction midpoint potential (Em) of the regulatory disulfide, formed between Cys16 and Cys55, of spinach chloroplast phosphoribulokinase has been determined both for the wild-type enzyme and for a C244S-C250S double mutant, using enzymatic activity to monitor the oxidation-reduction state of the regulatory disulfide. At pH 7.0, Em values for the two-electron reduction of the regulatory disulfide of -295 +/- 10 and -290 +/- 10 mV were measured for the wild-type and mutant, respectively. In contrast to the dependence of activity on ambient potential (Eh) observed for the wild-type enzyme and the double mutant, which both followed the Nernst equation for a two-electron process, high and constant activity was exhibited by a C16S-C244S-C250 triple mutant of the enzyme at all Eh values tested. Em values for the wild-type enzyme were also measured at pH values of 6.7, 7.5, 7.7, and 8.2 and the Em vs pH data in this region give a good fit to a straight line with a slope of -60 mV/pH unit.

Chloroplasts↗

PubMed1998

Spontaneous myocarditis in DBA/2 mice. Light microscopic study with transmission and X-ray analytical electron microscopic studies.

DBA/2 inbred mice spontaneously develop myocarditis and a unique form of subepicardial inflammation of the right ventricle characterized by a prominent eosinophilic infiltrate with calcinosis. We studied this myocarditis using light microscopy and both transmission and analytical X-ray electron microscopy, paying particular attention to eosinophil-associated cardiocyte injury. At 5 weeks of age, many eosinophils and mononuclear cells (MNCs) were seen in the subepicardium of the right ventricle. Electron microscopy showed that cardiocytes underwent degenerative changes, including myofibrillar lysis, accumulation of Z-band material and mitochondrial inclusions, and rupture of plasma membranes. The infiltrating eosinophils appeared to be activated, and cells with cytoplasmic vacuoles, suggestive of degranulation, were noted. The myocardial injury was most severe in the 7th week and healed with myocardial fibrosis and calcinosis by the 8th week. Analytical X-ray electron microscopy showed that the calcinosis was initiated in mitochondrial inclusions of injured cardiocytes. The peripheral eosinophil count did not increase during the course of the disease, but there was a positive correlation between the ratio of eosinophils to infiltrated white blood cells (Eo/WBCs) in the right ventricle and the severity of myocardial damage. Eosinophils may play a significant part in subepicardial cardiocyte injury seen in DBA/2 mice.

Animals↗

PubMed1998

Immunolocalization of extracellular matrix proteins and integrins in sarcoid lymph nodes.

To improve our understanding of the role of extracellular matrix (ECM) proteins and integrins during the processes of granuloma formation in sarcoidosis, we examined the distribution of ECM proteins and the expression of integrins in sarcoid lymph nodes by immunohistochemical methods. We also examined the expression of transforming growth factor-beta1 (TGF-beta1), which is one of major regulators for synthesis of ECM proteins. Most ECM proteins were detected in the periphery of the granulomas in a concentric pattern, and fibronectin was diffusely detected from an early to a regressive stage. Compared with normal lymph nodes, most beta1-integrin subfamilies (alpha1, alpha4, alpha5 and alpha6) were more strongly expressed on lymphocytes around the granulomas. Epithelioid cells exhibited strong expression of the alpha5 molecule. Fibroblasts exhibited the expression of the alpha2 and alpha5 molecules surrounding ECM proteins. The alpha5beta1 molecule had a distribution similar to that of fibronectin. TGF-beta1 was detected in epithelioid cells throughout the various evolutional stages and its expression was especially marked in mature granulomas. Interaction of fibronectin and the alpha5beta1 molecule may have an important role in the process of formation of sarcoid granuloma. The expression of TGF-beta1 may be involved in the regression of sarcoid granuloma by initiating fibrosis and atrophy of epithelioid cells.

Extracellular Matrix Proteins↗

PubMed1998

Arginine vasopressin, fever and temperature regulation.

While central administration of arginine vasopressin (VP) to the non-febrile rat at high doses can cause hypothermia, there is little evidence for a role for endogenous VP in normal thermoregulation. In contrast, VP arising from cell bodies in the bed nucleus of the stria terminalis and innervating the ventral septal areas and possibly the amygdala appears to be an endogenous antipyretic, i.e. a substance capable of reducing fever. As the synthesis of VP in bed nucleus neurons is dependent upon circulating androgens, female rats have much less VP in these cells and their projections than do male rats. In keeping with this, females may make use of VP to a lesser extent than do males to bring about antipyresis. The phenomenon whereby the VP receptor can become sensitized by previous exposure to VP may be responsible for some states of endogenous antipyresis, in which fevers are suppressed through overactivity of the vasopressinergic system. States of endogenous antipyresis can be revealed around the time of parturition in both the neonate and the mother.

Animals↗

PubMed1998

Clinical and microbiological effects of controlled-release local delivery of minocycline on periodontitis in dogs.

OBJECTIVE: To evaluate the clinical and microbiological efficacy of minocycline in a subgingival local delivery system as an adjunct to tooth scaling and root planing in dogs with periodontal disease. ANIMALS: Nine 4- to 7-year-old Beagles with periodontitis. PROCEDURE: After scaling of teeth and root planing, 2 treatment and 1 or 2 control sites were selected for each dog: treated sites (n = 18) received minocycline hydrochloride periodontal formulation and control sites (n = 12) received ointment base (no minocycline). Gingival crevicular fluid was collected at a baseline (prior to treatment) and at week 4. Clinical and microbiological effects were evaluated and compared among sites. RESULTS: In minocycline-treated sites, clinical indices were significantly decreased at week 4, compared with those at baseline. Minocycline-treated sites were associated with a significant decrease in gingival crevicular fluid, probing depth, and bleeding on probing values, compared with those for control sites at week 4. Compared with that for control sites, total bacteria count in periodontal pockets of minocycline-treated sites had an obvious tendency to decrease by week 4. Proportions of Porphyromonas and Fusobacterium spp were significantly decreased at week 4, compared with proportions at control sites and with pretreatment (baseline) values. CONCLUSIONS: When used as an adjunct to tooth scaling and root planing, minocycline periodontal formulation stimulated favorable clinical and antimicrobial responses.

Animals↗

PubMed1998

Tn4351-generated non-haemolytic and/or non-pigmented mutants of Porphyromonas gingivalis.

Escherichia coli-Porphyromonas gingivalis plasmid-shuttle vectors R751::* omega 4, pVOH1, and pVAL-1 were used for isolation of non-pigmented, defective protease, or non-haemolytic activity phenotypes in P. gingivalis. Transfer frequencies for R751::* omega 4, pVOH1, and pVAL-1 varied from 1.7 x 10(-9) to 5.3 x 10(-11) depending on the P. gingivalis 381 and ATCC 33277 strains. Two erythromycin-resistant transconjugants were screened from P. gingivalis 381 and eighteen were screened from ATCC 33277. Among isolated transconjugants, two from ATCC 33277 contained only one transposon insertion, while others included both Tn4351 and R751 sequences. The one transconjugant which contained a single insertion of Tn4351, designated NUM-T14, demonstrated defective pigmentation, and defective protease and haemolytic activities. The other transconjugant, designated NUM-T29, demonstrated defective haemolytic activity, but had black pigmentation and protease activity. Cell surface protein analysis by SDS-PAGE indicated that 40 and 18 kD proteins were lost or reduced and that 45 and 36 kD proteins were made to appear in both NUM-T14 and NUM-T29 transconjugants.

Blotting, Southern↗

PubMed1997

The ferredoxin-binding site of ferredoxin: Nitrite oxidoreductase. Differential chemical modification of the free enzyme and its complex with ferredoxin.

Spinach (Spinacea oleracea) leaf ferredoxin (Fd)-dependent nitrite reductase was treated with either the arginine-modifying reagent phenyl-glyoxal or the lysine-modifying reagent pyridoxal-5'-phosphate under conditions where only the Fd-binding affinity of the enzyme was affected and where complex formation between Fd and the enzyme prevented the inhibition by either reagent. Modification with [14C]phenylglyoxal allowed the identification of two nitrite reductase arginines, R375 and R556, that are protected by Fd against labeling. Modification of nitrite reductase with pyridoxal-5'-phosphate, followed by reduction with NaBH4, allowed the identification of a lysine, K436, that is protected by Fd against labeling. Positive charges are present at these positions in all of the Fd-dependent nitrite reductase for which sequences are available, suggesting that these amino acids are directly involved in electrostatic binding of Fd to the enzyme.

Amino Acid Sequence↗

PubMed1997

Passive immunization against dental plaque formation in humans: effect of a mouth rinse containing egg yolk antibodies (IgY) specific to Streptococcus mutans.

Passive immunization involving the delivery of antibodies specific to pathogens of infectious diseases to the host has been an attractive approach to establish protective immunity against a variety of microbial pathogens, including Streptococcus mutans, which is the principal etiologic agent of dental caries in humans. The overall purpose of the present study was to determine the effectiveness of a mouth rinse containing antibodies to S. mutans in preventing the establishment of this bacterium in dental plaque of humans. The antibodies were derived from egg yolks obtained from hens immunized with whole cells of S. mutans grown in sucrose-containing medium. The immunoglobulin derived from the yolks (IgY) of immunized hens was characterized in vitro and in vivo in human volunteers. Cross-reactivity tests showed that immune IgY reacted with every serotype, except serotype b, which had lost its GTase activity, when the bacteria were cultured in sucrose-containing medium. Immune IgY inhibited S. mutans adherence to saliva-coated hydroxyapatite discs by 59.2%, while control IgY caused an inhibition of only 8.2%. In the short-term (4-hour) test using a mouth rinse containing 10% sucrose, immune IgY decreased the ratio of the percentage of S. mutans per total streptococci in saliva. In the long-term (7-day) test using a mouth rinse without sucrose, the ratio in saliva was not significantly reduced in the volunteers using the immune IgY due to the large standard deviation. However, comparing the ratios of the percentage of S. mutans per total streptococci in plaque of individual subjects, there was a tendency for a reduction of the ratios in the volunteers receiving the mouth rinse containing immune IgY. These results support the effectiveness of IgY with specificity to S. mutans grown in the presence of sucrose as an efficient method to control the colonization of mutans streptococci in the oral cavity of humans.

Antibodies, Bacterial↗

PubMed1997

Serum and BAL Clara cell 10 kDa protein (CC10) levels and CC10-positive bronchiolar cells are decreased in smokers.

Cigarette smoking has diverse effects on the structure and function of the lung. Smoking appears to reduce the levels of Clara cell 10 kDa protein (CC10) in the alveolar lining fluid, but the influence of smoking serum on CC10 levels is still debated, and it has not been clear whether smoking reduces the number of CC10-producing lung cells. The aims of this study were to clarify the influence of smoking on CC10 levels in the alveolar lining fluid and bloodstream, and on the number of CC10-producing lung cells. CC10 concentrations were measured in sera and bronchoalveolar lavage (BAL) fluids, by means of enzyme-linked immunosorbent assay using monoclonal and polyclonal antibody, and the immunohistochemical expression of CC10 was examined in the lungs of nonsmokers and smokers using the monoclonal antibody, TY-5, against CC10/human urinary protein-1. CC10 concentrations in sera and in BAL fluids from healthy smokers were significantly lower than in healthy nonsmokers. Immunohistochemical expression of CC10 was found exclusively in nonciliated bronchiolar epithelial cells. As compared to that of nonsmokers, the mean percentage of CC10-positive bronchiolar epithelial cells was significantly decreased in lung tissue specimens obtained from smokers who had normal results in pulmonary function tests. It was concluded that smoking reduces the proportion of Clara cell 10 kDa protein-producing bronchiolar epithelial cells, resulting in decreased levels of Clara cell 10 kDa protein in the lower respiratory tract and in the bloodstream. The protein is a new blood biochemical and immunohistochemical marker, reflecting structural changes in peripheral airways induced by cigarette smoking.

Adult↗

PubMed1997

Protection by trehalose of DNA from radiation damage.

The most serious damage to cells exposed to radiation is attributed mostly to effects on the structure of cellular DNA. We found that trehalose protects DNA from irradiation. In the presence of 10 mM trehalose, DNA can be protected from about 4 times higher doses of beta- and gamma-ray irradiation. The protective effect increases with the amount of the sugar. Other disaccharides, sucrose, and maltose had similar effects.

Beta Particles↗

PubMed1997

Intracellular viral localization in murine coxsackievirus-B3 myocarditis. Ultrastructural study by electron microscopic in situ hybridization.

Group B Coxsackieviruses are a common cause of myocarditis. To detect the viral genome and its localization in the myocardium, we examined C3H/He mice with Coxsackievirus B3 (CVB3) myocarditis on days 5, 8, and 14 after inoculation by the reverse transcriptase polymerase chain reaction and by in situ hybridization. Sense and antisense CVB3 RNA were detected in the myocardium of all mice up to day 14 by reverse transcriptase polymerase chain reaction. Light microscopic in situ hybridization with a cDNA probe for CVB3 showed clusters of positive signals in the areas of myocardial necrosis and cell infiltration. With electron microscopic in situ hybridization, CVB3 RNA was detected in the cytoplasm of cardiocytes, between the myofibrils, near the mitochondria, and in tubular or vesicular structures. Viral RNA was also detected in necrotic debris, in the cytoplasm of macrophages, and in the cytoplasm of interstitial fibroblasts. These findings suggest that CVB3 RNA is replicated in the cytoplasm of cardiocytes, transferred into tubular or vesicular structures, released into the interstitium, and phagocytosed by macrophages. Some positive signals were also detected in the cytoplasm of cardiocytes showing close contact with infiltrating lymphocytes, suggesting that the lymphocytes recognized virus-infected cardiocytes and caused cell-mediated immune cardiocyte damage.

Animals↗