Biomedical subjects
Publications and source records attributed to M Nishimura.
A cDNA for thylakoid-bound ascorbate peroxidase of pumpkin was cloned and characterized. Thylakoid-bound ascorbate peroxidase had a high similarity to cytosolic ascorbate peroxidases, and the precursor contained a transit peptide to chloroplasts at its amino-terminus and a putative membrane-spanning region at its carboxy-terminus.
A cDNA encoding the plastid omega-3 fatty acid desaturase was isolated from a tobacco (Nicotiana tabacum cv. SR1) leaf cDNA library. The amino terminal extension of the deduced amino acid sequence of this clone had a characteristic feature of the transit peptides of plastid-destined proteins. Northern analysis indicated that the mRNA corresponding to this cDNA was present in leaves, but was not detected in roots. Responses to wounding of the plastid and microsome omega-3 desaturase genes were investigated in tobacco leaves. The mRNA level of the plastid omega-3 desaturase gene increased to about 2-fold that of unwounded controls at 12 h after a wounding treatment. On the other hand, the mRNA level of the microsome omega-3 desaturase gene remained constant in the wounded leaves. Linolenic acid contents of major leaf polar lipids increased by wounding. These results indicate that wounding enhances the accumulation of the plastid omega-3 desaturase mRNA, and increases the conversion of linoleic acid to linolenic acid in leaf polar lipids.
A cDNA clone for isocitrate lyase (ICL) was isolated from a cDNA library prepared from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA encoded a polypeptide with 576 amino acids, whose sequence is more than 79% identical to those of ICL from other higher plants and contains the C-terminal tripeptide, Ser-Arg-Met, which is a putative targeting signal to microbodies. Immunogold analysis revealed that ICL protein is exclusively localized in microbodies in etiolated pumpkin cotyledons. Double labeling experiments with protein A-gold particles of different sizes showed that ICL protein coexists with a leaf-peroxisomal enzyme, glycolate oxidase, in the microbodies of greening and senescing pumpkin cotyledons, indicating that transformation between glyoxysomes and leaf peroxisomes occurs directly during greening and senescence of the cotyledons. Immunoblot analysis showed that the amount of ICL protein increased markedly during germination and decreased rapidly when seedlings were exposed to illumination, which induced the microbody transition from glyoxysomes to leaf peroxisomes. In senescing cotyledons, the level of the ICL protein and ICL mRNA increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes. Changes in the amount of ICL protein did not correspond to the changes in the level of ICL mRNA during greening and senescence of the cotyledons, an indication that post-transcriptional regulation plays an important role in the microbody transition.
Glyoxysomal citrate synthase in pumpkin is synthesized as a precursor that has a cleavable presequence at its N-terminal end. To investigate the role of the presequence in the transport of the protein to the microbodies, we generated transgenic Arabidopsis plants that expressed beta-glucuronidase with the N-terminal presequence of the precursor to the glyoxysomal citrate synthase of pumpkin. Immunogold labeling and cell fractionation studies showed that the chimeric protein was transported into microbodies and subsequently was processed. The chimeric protein was transported to functionally different microbodies, such as glyoxysomes, leaf peroxisomes, and unspecialized microbodies. These observations indicated that the transport of glyoxysomal citrate synthase is mediated by its N-terminal presequence and that the transport system is functional in all plant microbodies. Site-directed mutagenesis of the conserved amino acids in the presequence caused abnormal targeting and inhibition of processing of the chimeric protein, suggesting that the conserved amino acids in the presequence are required for recognition of the target or processing.
In the present study, intestinal apo A-IV synthesis was investigated using a carbon tetrachloride (CCl4)-induced cirrhosis rat model. Triglyceride (TG) content in rat cirrhotic liver was increased markedly by 170% (P < 0.001) and apo B was increased by 20% (P < 0.05) compared with control levels. These results reflected the steatotic change in the liver. In contrast, TG levels in the small intestine of cirrhotic rats decreased significantly (P < 0.01). In addition, intestinal apo A-IV (jejunum P < 0.001; ileum P < 0.01) and its mRNA levels (jejunum P < 0.01; ileum P < 0.05) were also reduced. The decreased apo A-IV content in the jejunum was confirmed by immunohistochemical analysis. These results indicate that intestinal apo A-IV synthesis in cirrhosis is suppressed, at least under the condition of an overnight fast. Therefore, decreased intestinal apo A-IV synthesis may relate to the decreased ability to absorb fat in cirrhosis, but a fat-loading study will be necessary to confirm this hypothesis. It is unknown from the present study why serum apo A-IV level is not significantly decreased, despite a reduction in apo A-IV synthesis. The clearance of apo A-IV by the liver may be delayed or apo A-IV synthesis may be rather markedly enhanced during fat absorption in liver cirrhosis.
1. The frequencies (F, s-1) of miniature endplate potentials and the quantal content (m) of endplate potentials were measured intracellularly and simultaneously at mouse diaphragm endplates in a bathing solution that contained 0.6 mM Ca2+ ions and 5 mM Mg2+ ions. 2. Twin pulses at 4 ms intervals gave the quantal contents of the first (m1) and second (m2) responses. The ratio of m2/m1 was taken as an indicator of the temporal facilitation of the release of transmitter. 3. Dinitrophenol (DNP, 10 microM) increased the values of F and m at 36 degrees C. This effect did not depend on extracellular Ca2+ ions. 4. The potentiating effect of DNP disappeared at 24 degrees C but the value of m2/m1 remained constant. 5. These results suggest that the effect of DNP is modifiable by temperature which can affect systems that control the intracellular metabolism of Ca2+ ions.
PROBLEM: The immunological mechanism of an effective immunotherapy with paternal lymphocytes for unexplained recurrent spontaneous abortion (RSA) is not yet clear. Previous studies revealed that progesterone plays an important role in maintaining normal pregnancy and lower expression of progesterone receptor (PGR) on lymphocytes was found in RSA. Therefore, it was of interest to investigate whether immunotherapy for RSA would be able to enhance the expression of PGR on lymphocytes of RSA. METHOD: PGR expression on lymphocytes was analyzed with indirect immunofluorescence using flow cytometry. RESULTS: There was no change of PGR expression on PBL of RSA between pre- and post-immunotherapy (P > 0.05), while in the presence of 10.0 micrograms/ml progesterone for 24 h, PGR expressed on PBL on post-immunotherapy was increased significantly as compared with that of pre-immunotherapy in successful cases (P < 0.05) and decreased in abortive cases (P < 0.05). Most PGR was expressed on both CD4+ and CD8+ lymphocyte subsets. In successful cases, CD8+PGR+ subset of post-immunotherapy was found to be increased significantly (P < 0.05) in comparison with that of pre-immunotherapy. CONCLUSION: The data in the present study suggest that immunotherapy for RSA induced a higher expression of PGR on progesterone-treated lymphocytes, which may be involved in successful pregnancy.
We examined the mesenteric lymphatic and portal venous transport of triglyceride (TG), free fatty acids (FFA), apolipoproteins (apo)A-IV and B in response to a bolus duodenal infusion of a TG-free control solution, and long-chain (18:1) and medium-chain (8:0) TG (LCT and MCT, respectively) emulsions in the rat. Additionally, intestinal and hepatic apo A-IV and apo B mRNA levels were also measured. Lymph apo A-IV, apo B (B-48), FFA, and TG output increased after LCT infusion, whereas only apo A-IV and FFA outputs increased after MCT infusion. On the other hand, portal FFA and apo A-IV transports increased at 15 min after MCT infusion but not after LCT infusion. Portal TG and apo B transports were not altered by either MCT or LCT infusion. Jejunal apo A-IV mRNA was increased after both MCT and LCT infusions. Hepatic apo A-IV mRNA levels increased only after MCT infusion. Conversely, neither LCT nor MCT had any effect on apo B mRNA levels in intestine or liver. These results indicate that apo A-IV is regulated by MCT absorption and that fatty acid reesterification and lipoprotein assembly are not prerequisite for such regulation. Conversely, it is likely that apo B-48 participates only in the formation and/or transport of chylomicrons after LCT absorption.
20-Hydroxyeicosatetraenoic acid (20-HETE) is a major arachidonate metabolite in the kidney of the spontaneously hypertensive rat (SHR). The increase in its synthesis has been associated with the elevation of blood pressure in the SHR. The omega-hydroxylation of arachidonic acid is an activity associated with members of the CYP4A gene family which, in the rat, comprises three major isoforms: 4A1, 4A2 and 4A3. 20-HETE displays potent and diverse biological activities which can affect pro- and anti-hypertensive mechanisms dependent upon where, when and by which isoform it has been produced. Therefore, it is important to identify and characterize its biosynthetic system. We compared CYP4A mRNA and protein expression to patterns of 20-HETE synthesis in the SHR kidney. The reverse transcription/polymerase chain reaction (RT/PCR) technique was used to amplify CYP4A mRNA in microdissected nephron segments. Southern blot hybridization of PCR products obtained from nephron segments with the CYP4A1 cDNA probe demonstrated strong signals in S2 and S3 segments of the proximal tubule. Immunoblots of nephron segments using a polyclonal anti-rat liver CYP4A1 antibody which cross-reacts with CYP4A2 and CYP4A3, and 14C-arachidonic acid metabolism, confirmed that arachidonic acid omega-hydroxylation, i.e., 14C-20HETE formation, and CYP4A proteins were also localized mainly in the S2 and S3 segments. Correlation also existed between the age-dependent increase in arachidonate omega-hydroxylation in the kidney and CYP4A mRNA levels as measured by Northern hybridization of total RNA using the CYP4A1 cDNA probe. Immunoblot analysis revealed that at 7 weeks, where 20-HETE production is at its maximum, all three proteins are expressed. CYP4A3 and 4A1 immunoreactive proteins appeared at 3 weeks, showed maximum levels at 5 and 7 weeks, respectively, and gradually decreased to lower levels at 13 and 20 weeks, whereas CYP4A2 levels were undetectable at 3, 5 and 7 weeks but appeared at 13-20 weeks. Additional immunoblots indicated that renal cortical CYP4A1 protein levels were higher in SHR compared to Sprague-Dawley and Wistar-Kyoto rats. The increased levels of CYP4A1-immunoreactive band at 7 weeks corresponded to the maximal activity of arachidonate omega-hydroxylation. Thus, CYP4A1 might play a significant role in contributing to the increased cortical/proximal production of 20-HETE seen in 7-week-old SHR. However, given the high homology among members of the CYP4A gene family and the lack of specific tools to discern among these isoforms, additional studies have to be carried out to substantiate our findings.
OBJECTIVES: The relation between pulmonary function and low attenuation areas (LAAs) on high-resolution computed tomography (HRCT) is not clear in subclinical pulmonary emphysema. Accordingly we examined pulmonary function and HRCT in asymptomatic community-based volunteers. DESIGN: The existence of LAAs on CT was evaluated independently by three respiratory physicians who were blind to the pulmonary function test data and smoking histories of the subjects. The LAA grade was assessed by a visual scoring method from 0 to 5, and the individual LAA score and the values of pulmonary function tests were compared. PARTICIPANTS: 57 subjects aged from 32 to 82 years [mean 62 +/- (SD) 11] years were enrolled in the study. RESULTS: LAAs were found in 20 of the 49 subjects who were current or ex-smokers but in none of the 8 who never smoked. LAAs observed were mostly less than 25% of the total areas of the lungs. Although all mean values were within normal limits, the forced expiratory volume in 1 s (%pred), maximal expiratory flow at 50% of forced vital capacity (%pred), diffusing capacity of the lungs for carbon monoxide (DLCO; %pred) and diffusing capacity for carbon monoxide per unit alveolar volume (DLCO/VA; %pred) were all significantly less in those with LAAs than in those without LAAs. There was also a significant tendency for DLCO %pred and DLCO/VA %pred to decrease as the LAA scores increased. CONCLUSION: Those who have LAAs on CT have a small but significant deterioration in pulmonary function tests even if the LAAs observed are minimal or mild in degree. Longitudinal studies will be necessary to determine whether LAAs on CT identify the subjects who will develop airflow obstruction over time.
To investigate angiotensinogen regulation in high-renin hypertension, we infused porcine renin intravenously at either a low (4 mU/kg per hour, n = 6) or high (20 mU/kg per hour, n = 9) dose into male Sprague-Dawley rats (225 to 250 g) for 5 days using osmotic minipumps. Control rats received 0.9% NaCl. In renin-infused rats, mean arterial pressure and plasma renin activity were significantly elevated. Both low- and high-renin infusions lowered plasma angiotensinogen levels. Plasma angiotension II was elevated in rats given renin but reached statistical significance only at the higher dose. Angiotensinogen mRNA isolated from the liver, adrenal gland, kidney, and brain was measured by slot blot analysis. Both renin doses were associated with significant decreases in the levels of liver and hypothalamic angiotensinogen mRNA. In the medulla oblongata, angiotensinogen mRNA was reduced only by the higher renin dose. The lower dose increased angiotensinogen mRNA in the adrenal gland, and in kidney, angiotensinogen mRNA level was unchanged by renin infusion. Angiotensinogen mRNA visualized on Northern blots showed that the number of mRNA species in liver decreased from three in control rats to a single mRNA species after renin infusion. Tissue differences in the size of the major angiotensinogen mRNA species were also apparent. This, together with changes in the total hybridization signal of angiotensinogen mRNA in tissues, suggests that renin differentially affects the different angiotensinogen mRNA transcripts. Results of this study indicate that angiotensinogen gene expression is regulated not only by alterations in levels of circulating angiotensin II but also by other mechanisms, presently unidentified, that are activated by renin infusions.
BACKGROUND AND PURPOSE: The underlying cause of white-matter lesions, which are frequent findings in cerebrovascular disease (CVD) and Alzheimer's disease (AD), remains uncertain. We performed immunohistochemical analysis of serum protein extravasation to investigate the function of the blood-brain barrier in white-matter lesions. METHODS: White-matter lesions were estimated by use of Kluver-Barrera staining in patients diagnosed clinicopathologically as having ischemic CVD (n = 14) and AD (n = 12) and from nonneurological control subjects (n = 6). Axonal damages were investigated by use of immunohistochemistry for amyloid protein precursor. Alteration of the blood-brain barrier was examined with fibrinogen and immunoglobulins used as markers. The numbers of HLA-DR-positive microglia and glial fibrillary acidic protein-positive astroglia were examined comparatively. RESULTS: White-matter lesions were graded as normal (grade 0) in 14 of the 32 cases (44%), slight (grade I) in 10 cases (31%), moderate (grade II) in 6 cases (19%), and severe (grade III) in 2 cases (6%). Amyloid precursor protein was accumulated most frequently in grade II white-matter lesions. Immunohistochemistry for serum proteins labeled astroglial cell bodies and their processes, which seemed to have sequestered extravasated proteins. The groups with detectable white-matter lesions had significantly higher grading scores for fibrinogen and immunoglobulins than the control group (P < .05). Although the higher scores for serum protein extravasation were statistically significant in ischemic CVD cases (P < .05), there was no significant increase in AD cases. Activated microglia and astroglia were more numerous in the groups with white-matter lesions in both ischemic CVD and AD cases, although this increase in the number of astroglia was not evident in regions with clasmatodendrosis. CONCLUSIONS: Dysfunction of the blood-brain barrier is more prominent in white-matter lesions seen in ischemic CVD than in AD and may have a role in the pathogenesis of cerebrovascular white-matter lesions.
To evaluate effects of current smoking on the breakdown of lung elastin, we measured levels of elastin-derived peptides (EDP) in unconcentrated bronchoalveolar lavage (BAL) fluid from 42 community-based older asymptomatic volunteers (60 +/- 11 yr [mean +/- SD]) and examined the relationships of the concentrations of EDP with immunologically detected neutrophil elastase (NE) bound with alpha(1)-proteinase inhibitor and with esterolytic activity against the NE-sensitive synthetic substrate, methoxysuccinyl-alanyl-alanyl-prolyl-valyl paranitroanilide (MEOSAAPVNA). The measurement of EDP levels was carried out by an indirect, competitive ELISA, using a sheep IgG fraction generated against insoluble human lung elastin as a primary antibody. EDP concentrations were significantly elevated in current smokers (n = 24) compared with age-matched noncurrent smokers (n = 18) (29.9 +/- 3.5 [mean +/- SE] versus 17.0 +/- 1.8 ng/mg BAL fluid albumin, p < 0.01). We found weak but significant correlations of EDP levels with NE-alpha(1)-proteinase inhibitor complex (r = 0.38, p < 0.05) and with the esterolytic activity against the NE-sensitive synthetic substrate in BAL fluid (r = 0.65, p < 0.001). In addition, EDP levels in BAL fluid had a significant positive relationship with plasma cotinine concentrations (r = 0.53, p < 0.001), though not with pack-years of smoking (r = 0.1, NS). These data provide further evidence that the concentrations of EDP increase in BAL fluid from current smokers compared with noncurrent smokers and that enhanced breakdown of the lung elastin is associated with the increased load of NE in the lung.
To gain insight into the source of exhaled nitric oxide (NO) in normal humans, we examined the effects of respiratory pattern on the concentration of NO in exhaled air while subjects were wearing a noseclip and then under endotracheal intubation, using a specially designed gas sampling system to separate exhaled air into two fractions: the initially exhaled 200 ml (Fraction 1; F1), and the remainder (Fraction 2; F2). We also examined the effect of hypoxic gas inhalation (F(I)O2 = 0.1, 3 min) on the concentration of exhaled NO. The concentration of exhaled NO measured with a chemiluminescence NO analyzer was significantly lower with intubation, by 59.2 +/- 10.6% (mean +/- SD) (F1) and 54.4 +/- 8.0% (F2), than without intubation. The concentration of NO in F1 was consistently higher than that in F2 with or without intubation. With breath holding, the concentration of NO increased markedly only in F1. In contrast, prolongation of the expiratory phase slightly but significantly increased the concentration of NO only in F2. Inhalation of hypoxic gas did not cause any significant change in NO concentration in either fraction. These data indicate that in normal humans wearing a noseclip, about 40 to 45% of NO in exhaled air originates from the lungs, particularly from intrathoracic airways. The concentration of exhaled NO collected from subjects wearing a noseclip is not affected by hypoxic gas inhalation.
Although the ventilatory and heart rate responses to hypoxia are known to vary widely among subjects, it is not known how exercise or hypercapnia influence the intersubject variability of these responses. If the intersubject variability increases under such conditions, the inherent response of individuals will have more impact on ventilation and heart rate under a variety of hypoxic conditions during exercise or with hypercapnia than at rest or with normocapnia. Seventeen healthy male volunteers underwent tests to measure ventilatory response to isocapnic progressive hypoxia three times respectively: at rest; during CO2 inhalation (end-tidal carbon dioxide tension (PET,CO2) raised by 5 torr from the baseline level); and during mild exercise with a cycle ergometer (12.5 W) in a supine position. The mean (SEM) value of hypoxic ventilatory response (HVR) (delta minute ventilation (V'E)/delta arterial oxygen saturation (Sa,O2) was significantly increased both in the exercise and hypercapnic runs compared with that in the control run (0.45 +/- 0.12, 0.34 +/- 0.08, respectively, vs 0.12 +/- 0.02 L.min-1/% fall), although the respiratory pattern was different under the two loaded conditions. The intersubject variation in HVR was also significantly increased during the two loaded conditions compared with the control, although a significant correlation remained between the control value and that obtained during either loaded condition (r = 0.66 and r = 0.60, respectively). The heart rate (HR) response evaluated by the slope factor (delta HR/delta Sa,O2) was not significantly different either in the mean value or in the intersubject variability among the three experimental conditions. In conclusion, exercise or CO2 inhalation not only increase the slope value of HVR but also amplify the intersubject variability of the response. In contrast, the HR response to hypoxia evaluated as a slope factor does not change with exercise or CO2 inhalation.
The purpose of this study is to examine the relationship between breathlessness and the ventilatory response to hypercapnia or hypoxia in patients with chronic obstructive pulmonary disease (COPD). Fifteen male patients (mean forced expiratory volume in one second (FEV1): 1.13 L) underwent tests to determine hyperoxic hypercapnic ventilatory response (HCVR) and isocapnic hypoxic ventilatory response (HVR) with simultaneous quantification of breathlessness by modified Borg scale. The ventilatory output was evaluated by the ratio of minute ventilation (V'E) divided by measured maximal voluntary ventilation (MVV). The magnitude of HCVR or HVR was assessed as the slope value of the V'E/MVV-end-tidal carbon dioxide pressure (PET,CO2) or arterial oxygen saturation (Sa,O2) regression line, respectively. The breathlessness during the tests was evaluated not only linearly in relation to V'E/MVV, but also at given levels of PET,CO2 or Sa,O2. The mean value of the breathlessness at two different levels of ventilation was greater during HVR than during HCVR, suggesting that hypoxia is dyspnogenic independently of ventilatory stimulation. The HCVR was inversely correlated with the breathlessness response to ventilation, while similar correlation was partly present for HVR. The HVR was positively correlated with the breathlessness at an Sa,O2 of 80%, whilst there was no such correlation between the HCVR and the breathlessness related to PET,CO2. Therefore, patients with a higher breathlessness related to increased ventilation had a lower HCVR and HVR, whilst those with a higher breathlessness with desaturation, which might include a direct influence of hypoxia, had a higher HVR. These findings suggest an interaction between ventilatory response and breathlessness during the test, which may partly include behavioural modulation of HCVR and HVR through the breathlessness in various ways, depending on the origin and nature of the sensation.
To investigate whether the histaminergic neurons degenerate in Parkinson's disease (PD) and multiple system atrophy (MSA), we studied the number of large-sized neurons in the tuberomammillary nucleus in patients with PD, patients with MSA, and age-matched controls. The number of large-sized neurons in the tuberomammillary nucleus in PD patients was not altered compared with controls, and Lewy bodies were rarely present in the tuberomammillary nucleus. In contrast, the number of large-sized neurons in the tuberomammillary nucleus in MSA patients was significantly decreased compared with controls. Thus, the central histaminergic neurons are affected in MSA and preserved in PD.
In germinating fatty seedlings, microbodies are functionally transformed to leaf peroxisomes from glyoxysomes during greening, and then converted to glyoxysomes from leaf peroxisomes during senescence. Immunocytochemical studies revealed that glyoxysomes can exchange directly into leaf peroxisomes during greening and leaf peroxisomes are once again directly converted to glyoxysomes during senescence. The reversible transformations of microbodies are regulated at various levels, such as gene expression, splicing of the mRNA and degradation of microbody proteins. The regulatory mechanisms underlying this organelle differentiation are described.