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Biomedical subjects

H Shimokawa

Publications and source records attributed to H Shimokawa.

At least 109 records · Page 6Linked to original sources
PubMed1997

Transforming growth factor-beta 1 modulates myofibroblastic phenotype of rat palatal fibroblasts in vitro.

The effects of transforming growth factor-beta 1 (TGF-beta 1) on normal rat palatal fibroblasts in vitro were investigated in the present study in order to unravel the precise mechanisms by which the phenotypic modulation of fibroblasts occurs during the scar formation process. TGF-beta 1 dramatically changed the morphology of normal palatal fibroblasts from polygonal into an elongated shape, which was very similar to that of fibroblasts derived from experimental immature scar tissue in rat palatal mucosa. This morphological transition was concomitant with an increase in the expression of alpha-smooth muscle (alpha-SM) actin protein, a marker for myofibroblasts, when determined by immunocytochemistry. An immunoblot study also revealed that alpha-SM actin expression in palatal fibroblasts became evident after 24 h of TGF-beta 1 treatment and increased time-dependently up to 72 h. Northern blot analysis showed that TGF-beta 1 stimulated endogenous TGF-beta 1 mRNA expression in palatal fibroblasts within 24 h. Neither epidermal growth factor nor basic fibroblast growth factor had any effect on either alpha-SM actin expression or TGF-beta 1 mRNA expression. Pretreatment of palatal fibroblasts with TGF-beta 1 significantly increased the contractile capacity in a three-dimensional collagen gel culture, even when the culture medium was deprived of TGF-beta 1 for 72 h of the experimental period. Moreover, the contractility of scar fibroblasts, which highly expressed alpha-SM actin protein and TGF-beta 1 mRNA, was significantly lowered by a neutralizing antibody to TGF-beta 1. These data strongly suggest that TGF-beta 1 is a potential inducer of phenotypic expression of myofibroblasts in palatal fibroblasts and that auto-induction of TGF-beta 1 mRNA expression may play an important role in the scar formation process in palatal mucosa.

Actins↗
PubMed1997

Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats.

The long-term administration of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, produces coronary vascular remodeling and myocardial hypertrophy in animals. This study used a rat model to investigate the role of angiotensin I converting enzyme (ACE) in the pathogenesis of such changes. We studied the following groups, all of which received drug treatment in their drinking water: untreated controls, and those administered L-NAME, L-NAME, and an ACE inhibitor (ACEI), and L-NAME and hydralazine. Cardiovascular structural changes and tissue ACE activities were evaluated after the first, fourth, and eighth week of treatment. In rats treated with L-NAME alone, vascular remodeling was evident at the fourth and eighth week, and myocardial hypertrophy was present at the eighth week of treatment. The vascular and myocardial remodeling were characterized by increased tissue ACE activities and immunodetectable ACE in those tissues. These changes were markedly reduced by ACEI, but not by hydralazine treatment. Increased local ACE expression may thus be important in the pathogenesis of cardiovascular remodeling in this model.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1997

Bradykinin-induced vasodilation of human coronary arteries in vivo: role of nitric oxide and angiotensin-converting enzyme.

OBJECTIVES: The present study aimed to determine the role of nitric oxide (NO) and angiotensin-converting enzyme (ACE) in bradykinin (BK)-induced dilation of human coronary arteries in vivo. BACKGROUND: BK, produced by way of the kinin-kallikrein system, causes endothelium-dependent vasodilation. However, little is known about the mechanism of BK-induced dilation of coronary arteries in humans in vivo. METHODS: The effects of an inhibitor of NO synthesis and of an ACE inhibitor on BK-induced coronary vasodilation were examined in 20 patients who had no significant atherosclerotic stenosis in the artery under study. Lumen diameters of the large epicardial coronary arteries and coronary blood flow (CBF) were measured by quantitative coronary arteriography and intracoronary Doppler technique. RESULTS: Intracoronary infusion of BK (0.6 and 2.0 micrograms/min) increased coronary artery diameter and CBF with no change in arterial pressure or heart rate. The BK-induced increases in coronary artery diameter and CBF were significantly reduced (p < 0.01) after pretreatment with NG-monomethyl-L-arginine (200 mumol) and were significantly increased (p < 0.01) after pretreatment with enalaprilat (50 micrograms). CONCLUSIONS: BK-induced dilation of human large epicardial and resistance coronary arteries is mediated by NO and increased by prior ACE inhibition.

Aged↗
PubMed1997

Inflammatory cytokines cause coronary arteriosclerosis-like changes and alterations in the smooth-muscle phenotypes in pigs.

We recently developed a porcine model in which chronic, local treatment with interleukin-1 beta (IL-1 beta) causes coronary arteriosclerosis-like changes and hyperconstrictive responses. This study was designed to examine whether or not other major inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) might also cause similar coronary responses and whether those responses are associated with alterations in the smooth-muscle phenotypes. A segment of the porcine coronary artery was aseptically wrapped with cotton mesh, absorbing IL-1 beta, TNF-alpha, and IL-1 alpha. Two weeks after the operation, coronary arteriography showed the development of mild stenotic lesions at the cytokine-treated sites, where hyperconstrictive responses were repeatedly induced by intracoronary serotonin or histamine. Histologically mild intimal thickening was noted at those cytokine-treated sites. Immunostaining and immunoblotting demonstrated that all three myosin heavy chain isoforms, SM1, SM2 (smooth-muscle type), and SMemb (nonmuscle type), were noted in the normal coronary segments, whereas in the segments treated with inflammatory cytokines, SM1 and SM2 were markedly reduced, and only SMemb was noted. These results indicate that inflammatory cytokines all have a similar ability to induce coronary arteriosclerosis-like changes and hyperconstrictive responses, which are associated with alterations in smooth-muscle phenotypes toward dedifferentiation.

Animals↗
PubMed1997

Tyrosine kinase inhibitor markedly suppresses the development of coronary lesions induced by long-term treatment with platelet-derived growth factor in pigs in vivo.

Platelet-derived growth factor (PDGF) plays an important role in the development of coronary atherosclerosis. However, it remains to be examined what morphologic and functional changes are induced in vivo by the long-term treatment with PDGF itself or what pharmacologic interventions could suppress those changes in vivo. Our study was designed to address these points. We examined the effects of long-term treatment with PDGF on the porcine coronary artery in vivo. Under aseptic conditions, the proximal segments of the left porcine coronary artery were gently wrapped with cotton mesh absorbing sepharose beads either with or without recombinant human PDGF-AA or -BB. Two weeks after the operation, coronary hyperconstrictions to intracoronary serotonin or histamine were noted at the sites treated with PDGF-AA or -BB. Histologically, neointimal formation and geometric remodeling (reduction of the total vessel area) were noted at the PDGF-treated sites. These functional and histologic changes of the coronary artery induced by PDGF were markedly inhibited by cotreatment with ST 638, a specific inhibitor of tyrosine kinases. A Western blot analysis showed that ST 638 markedly suppressed the PDGF-induced tyrosine phosphorylations in the coronary segment. These results indicate that long-term treatment with PDGF induces neointimal formation, geometric remodeling, and vasospastic responses in vivo, for all of which, activation of tyrosine kinases is substantially involved.

Absorption↗
PubMed1997

Role of nitric oxide in substance P-induced vasodilation differs between the coronary and forearm circulation in humans.

It has been shown that substance P causes endothelium-dependent vasodilation in the human coronary and forearm vessels. However, the precise mechanism whereby substance P dilates the coronary and peripheral vasculatures is unknown in humans. The aim of this study was to examine whether the vasodilator effect of substance P is mediated by nitric oxide in the human coronary and forearm vessels. Eight patients with normal coronary angiograms were studied for the measurements of coronary blood flow (intracoronary Doppler guide wire and quantitative coronary arteriography) and forearm blood flow (strain-gauge plethysmograph). Intracoronary acetylcholine (10 micrograms/min for 2 min) and substance P (30 and 90 ng/min for 2 min) increased coronary blood flow from the baseline value. Intracoronary infusion of NG-monomethyl-L-arginine (L-NMMA) at 200 mumol significantly attenuated the magnitudes of increase in coronary blood flow induced by both acetylcholine (p < 0.01) and substance P (p < 0.01). Acetylcholine (4, 8, and 16 micrograms/min for 2 min) and substance P (0.8, 1.6, and 3.2 ng/min for 2 min) also increased forearm blood flow in a dose-dependent manner. Intraarterial L-NMMA (8 mumol/min for 5 min) decreased the magnitudes of increase in forearm blood flow induced by acetylcholine (p < 0.01). L-NMMA at the same dosage decreased the increase in forearm blood flow induced by substance P, but the magnitude of the inhibitory effect of L-NMMA on blood-flow responses to substance P was significantly smaller in the forearm than in coronary vessels. It is suggested that endothelium-derived nitric oxide contributes to substance P-induced vasodilation, and that the contribution of nitric oxide to substance P-induced vasodilation is smaller in the forearm than in coronary circulation.

Acetylcholine↗
PubMed1997

Glibenclamide, a specific inhibitor of ATP-sensitive K+ channels, inhibits coronary vasodilation induced by angiotensin II-receptor antagonists.

The purpose of our study was test the hypothesis that endogenous angiotensin II contributes to the basal coronary artery tone by acting at vascular ATP-sensitive K+ (K+ATP) channels. Coronary blood flow (CBF) and other hemodynamic parameters were measured in anesthetized dogs. Intracoronary infusion of the selective antagonists of angiotensin II AT1 receptors (L-158,809 and E4177) increased CHF without affecting other hemodynamic parameters, indicating that endogenous angiotensin II caused coronary vaso-constriction through the AT1 subtype receptors. Coronary vasodilation in response to AT1 receptor antagonists was blunted by pretreatment with glibenclamide (a specific inhibitor of K+ATP channels; p < 0.01) but not by either an adenosine-receptor antagonist or an inhibitor of nitric oxide synthesis. Coronary vasodilation in response to AT1-receptor antagonists was partly reduced (p < 0.01) by PD-123319 (the AT2-receptor antagonist). Glibenclamide had no effect on coronary vasodilation induced by sodium nitroprusside. These results indicate that in dogs in vivo, coronary vasodilation in response to AT 1-receptor antagonists inhibited markedly by glibenclamide and partly by PD-123319, suggesting that endogenous angiotensin II contributes to the maintenance of basal coronary vascular tone by acting at K+ATP channels through its receptors.

Angiotensin Receptor Antagonists↗
PubMed1997

Short-term estrogen augments both nitric oxide-mediated and non-nitric oxide-mediated endothelium-dependent forearm vasodilation in postmenopausal women.

Estrogen is known to improve in the short term the impaired endothelium-dependent vasodilating responses in postmenopausal women, which may account in part for the beneficial cardiovascular effects of the female hormone. Endothelium-dependent vasodilation is achieved by combined effects of endothelium-derived prostacyclin, nitric oxide (NO), and hyperpolarizing factor. In this study, we investigated our hypothesis that short-term estrogen improves both NO-mediated and non-NO-mediated endothelium-dependent vasodilation in postmenopausal women. The study included 12 postmenopausal women (aged 64 +/- 3 years). The forearm blood flow was measured by strain-gauge plethysmography. The forearm vascular responses to the endothelium-dependent vasodilators, acetylcholine and substance P, were examined before and after intravenous administration of conjugated estrogen and subsequently after intraarterial infusion of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis. Short-term estrogen augmented the forearm vasodilating responses to both acetylcholine and substance P. The treatment with L-NMMA almost abolished the augmented response to acetylcholine but did not affect that to substance P. The forearm vascular response to sodium nitroprusside was unchanged by the estrogen administration. These results indicate that estrogen augments (in the short-term) both NO-mediated and non-NO-mediated endothelium-dependent forearm vasodilation in postmenopausal women. Thus the beneficial effect of estrogen on endothelial vasodilator function appears to extend to non-NO-dependent mechanism(s).

Aged↗
PubMed1997

Central role of vascular smooth muscle hyperreactivity in coronary hyperconstriction after balloon injury in miniature pigs.

BACKGROUND: Coronary constrictive responses to autacoids become augmented 1 week after balloon injury in our swine model. The present study aimed to elucidate the mechanisms of this effect. METHODS: In 12 hypercholesterolaemic miniature pigs, the coronary constrictive response to serotonin was examined angiographically 1 week after injury. After the angiographic study, organ chamber experiments using excised coronary artery were performed to clarify whether functional changes in endothelial cells or in vascular smooth muscle cells contributed to the hyperconstriction. RESULTS: The coronary constrictive response to serotonin in vivo was significantly greater at the previously injured site than at the non-injured site. The degree of the hyperconstriction at the previously injured site exceeded that predicted from a geometric theory. Histological examination demonstrated that the previously injured site was almost covered with regenerated endothelial cells. In vitro studies demonstrated that serotonin caused significantly greater contraction in coronary artery strips, whether with or without endothelium, from the previously injured site than in those from the non-injured site. Endothelium-dependent relaxation in response to serotonin was comparable at the injured and non-injured sites. CONCLUSIONS: These results suggest that the coronary hyperconstriction response to serotonin 1 week after injury results primarily from hyperreactivity of vascular smooth muscle. Whereas any contribution of endothelial dysfunction or the geometric effect may be minimal.

Animals↗
PubMed1997

Platelet activating factor causes hyperconstriction at the inflammatory coronary lesions in pigs in vivo.

BACKGROUND: Although platelet activating factor (PAF) is an important vasoactive substance released from activated leukocytes, platelets and endothelial cells, little is known about its effect at the inflammatory coronary lesions in vivo. OBJECTIVE: To examine the coronary vasomotor responses to PAF at the inflammatory lesions in our swine model with interleukin-1 beta (IL-1 beta) in vivo. METHODS: Under aseptic conditions, the proximal segment of the porcine left coronary artery was dissected and wrapped with cotton mesh absorbing IL-1 beta. Two weeks after the operation, coronary vasomotion in response to intracoronary administration of 0.3 and 1 microgram/kg PAF, 1, 3, and 10 micrograms/kg serotonin, 1, 3 and 10 micrograms/kg histamine, and 5 and 50 micrograms/kg prostaglandin F2 alpha was examined by coronary arteriography. RESULTS: At the IL-1 beta-treated site, PAF, serotonin and histamine, but not prostaglandin F2 alpha, caused hyperconstriction (n = 8). A synergy of the vasoconstricting effects of PAF and serotonin was also noted (n = 6). Administration of TCV-309, a selective PAF receptor antagonist, abolished the hyperconstrictive responses to PAF but not those to other agonists (n = 6). The PAF-induced coronary hyperconstrictions were significantly inhibited by administrations of the protein kinase C inhibitors staurosporine and sphingosine, but not by administrations of ryanodine, thapsigargin, or indomethacin (n = 4 each). CONCLUSIONS: These results indicate that PAF causes hyperconstriction at the inflammatory coronary lesions in vivo by itself as well as in a synergistic manner with serotonin and that the PAF-induced hyperconstrictions are substantially mediated by a protein kinase C-dependent pathway in vivo.

Animals↗
PubMed1997

Characterization of the inner enamel epithelium in the enamel-free area based on the ability to secrete enamel protein demonstrated by in situ hybridization and immunohistochemistry.

Both the expression of amelogenin mRNA and secretion of amelogenin were investigated in rat molars by in situ hybridization and immunohistochemistry. Probes were designed by multiple-labeling of oligonucleotide probes for in situ hybridization. Amelogenin mRNA first appeared in differentiating ameloblasts of the distal region and some inner enamel epithelial cells of enamel-free area (EFA cells) of the second cusp at postnatal day 0. At the same time, amelogenin protein was detected in the extracellular matrix between dentin and differentiating ameloblasts and in some EFA cells of the second cusp. At postnatal day 1-3, amelogenin was expressed in the secretory ameloblasts, and in the matrix beneath these cells. Both amelogenin mRNA and amelogenin were detected in the EFA cells and their extracellular matrix. After postnatal day 5, amelogenin mRNA and amelogenin were detected in the secretory ameoloblasts and extracellular matrix in the enamel-forming region, respectively. At this time, amelogenin mRNA was not detected in the EFA cells, but a small amount of amelogenin was found in the matrix beneath the EFA cells. These findings suggest that EFA cells differentiate into amelogenin-secreting cells, i.e. ameloblasts, but that the secretion lasts for only a short period at the early stage of tooth development.

Amelogenin↗
PubMed1997

Basal release of nitric oxide is decreased in the coronary circulation in patients with heart failure.

It is unknown whether basal release of endothelium-derived nitric oxide in the coronary artery is altered in heart failure in humans. The aim of the present study was to evaluate the effect of inhibition of nitric oxide synthesis on basal tone of the conduit and resistance coronary arteries in awake patients. Coronary blood flow velocity (Doppler guide wire) and coronary arterial diameter (quantitative coronary angiography) were measured in 14 patients with heart failure caused by nonischemic left ventricular dysfunction (7 idiopathic dilated cardiomyopathy and 7 valvular insufficiency) and 7 patients with normal ventricular function (controls). Intracoronary N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthesis, at graded doses decreased coronary blood flow in both groups. However, the magnitude of flow reduction was smaller in patients with heart failure than in control patients (P<.0001). The magnitude of coronary blood flow reduction in response to L-NMMA inversely correlated to indexes of left ventricular contractile function (P<.01) but was not affected by the cause of heart failure. Constriction of the large epicardial coronary artery with L-NMMA also tended to be attenuated in patients with heart failure. In summary, vasoconstricting response to L-NMMA was blunted in the coronary resistance artery in heart failure in vivo. These findings suggest that basal release of nitric oxide in the coronary circulation is decreased in patients with heart failure.

Adult↗
PubMed1997

Chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade: effects on cardiovascular remodeling in rats induced by the long-term blockade of nitric oxide synthesis.

We have shown previously that angiotensin-converting enzyme (ACE) inhibitors prevent coronary vascular remodeling (medial thickening and perivascular fibrosis) and myocardial remodeling (fibrosis and hypertrophy) in rats induced by long-term inhibition of nitric oxide (NO) synthesis with oral administration of N omega-nitro-L-arginine methyl ester (L-NAME). ACE inhibitors inhibit both the formation of angiotensin II and the catabolism of bradykinin. In this study, we aimed to determine the relative contribution of the latter two mechanisms to the beneficial effects of an ACE inhibitor on structural remodeling. First, we examined the effects of the ACE inhibitor temocapril and the angiotensin II AT1 subtype receptor antagonist CS-866 on the structural remodeling induced by administering L-NAME for 8 weeks. Temocapril and CS-866 were equally effective in preventing remodeling. Second, we examined whether the effect of temocapril on the remodeling induced by L-NAME was reduced by the bradykinin receptor antagonist HOE140. The latter drug did not alter the beneficial effect of temocapril on remodeling. In conclusion, although species differences must be considered to apply our conclusion to clinical conditions, the present results suggest that the inhibition of angiotensin II activity, mediated via the AT1 receptors, is responsible for the beneficial effects of an ACE inhibitor in our animal model of coronary vascular and myocardial remodeling induced by the long-term inhibition of NO synthesis.

Angiotensin Receptor Antagonists↗
PubMed1997

Correlations between recruitable coronary collateral flow velocities, distal occlusion pressure, and electrocardiographic changes in patients undergoing angioplasty.

Direct assessment of coronary collateral flow has been difficult in humans. The goal of this study was to correlate the magnitudes and waveform characteristics of recruitable coronary collateral flow velocity measured with Doppler guidewire with other hemodynamic and functional indexes of collateral circulation in patients during angioplasty. Twenty-six patients [age 60 +/- 10 (SD) years] were studied for measurements of flow velocity at the distal segment of the dilated vessel. Collateral flow signals were demonstrated in 18 patients (69%) during balloon inflation. There was a weak yet significant positive correlation between the magnitude of collateral flow velocity and distal occlusion pressure (p < 0.01). The sum of ischemic ST elevation on the 12-lead electrocardiogram was inversely correlated with distal occlusion pressure, but not with the magnitude of collateral flow velocity. Subgroup analysis between patients with (n = 18) and without (n = 8) ST elevation during balloon inflation revealed higher collateral flow velocity signals (p < 0.0001) and greater systolic components of collateral flow in the latter group (p < 0.0001). Thus, functionally significant coronary collaterals showed greater velocity signals and systolic predominance in flow pattern. The functional capacity of human coronary collaterals may be semiquantitated using the Doppler guidewire technique.

Angina Pectoris↗
PubMed1996

Altered serotonin receptor subtypes mediate coronary microvascular hyperreactivity in pigs with chronic inhibition of nitric oxide synthesis.

BACKGROUND: We previously reported that chronic inhibition of nitric oxide synthesis by administration of N omega-nitro-L-arginine methyl ester (L-NAME) causes microvascular hyperreactivity to 5-hydroxytryptamine (5-HT) and vascular structural changes in pigs in vivo. In the present study, we investigated the relative contributions of 5-HT receptor subtypes to microvascular hyper-reactivity in this animal model. METHODS AND RESULTS: Coronary vasomotor response was studied in 16 pigs treated with oral L-NAME for 4 weeks (L group) and in 11 control pigs (C group). Intracoronary administration of 5-HT at 30 micrograms/kg decreased coronary blood flow (CBF) in the two groups. The decrease in CBF by 5-HT was greater (P < .01) in the L group than in the C group. The decrease in CBF by 5-HT in the C group was blocked completely by pretreatment with ketanserin, a 5-HT2 antagonist. In contrast, the augmented decrease in CBF by 5-HT in the L group was only partly inhibited by ketanserin alone and was blocked completely by ketanserin and methiothepin, a 5-HT1/5-HT2 antagonist. The decrease in CBF caused by prostaglandin F2 alpha and the increase in CBF caused by nitroglycerin were comparable between the two groups and were not affected by the 5-HT antagonists. CONCLUSIONS: These results suggest that the 5-HT-induced microvascular hyperreactivity may be mediated by relative changes in affinity for 5-HT receptors or de novo expression of 5-HT1 receptors in microvascular smooth muscle cells in our animal model.

Animals↗
PubMed1996

Coronary artery spasm does not depend on the intracellular calcium store but is substantially mediated by the protein kinase C-mediated pathway in a swine model with interleukin-1 beta in vivo.

BACKGROUND: The intracellular mechanism for coronary artery spasm is still unknown. Since the protein kinase C (PKC)-mediated pathway and Ca2+ release from sarcoplasmic reticulum (SR) are important intracellular mechanisms of vascular smooth muscle contraction, we examined the possible role of these two mechanisms in the pathogenesis of coronary spasm in our swine model in vivo. METHODS AND RESULTS: In 25 pigs, interleukin-1 beta (IL-1 beta) was applied chronically to the coronary arteries from the adventitia to induce an inflammatory/proliferative lesion. Two weeks after the operation, either intracoronary serotonin or histamine repeatedly induced coronary spasm at the IL-1 beta-treated site. At those spastic sites, phorbol-12, 13-dibutyrate, a PKC-activating phorbol ester, also induced coronary spasm, which was blocked by pretreatment with the PKC inhibitors staurosporine and sphingosine. Serotonin- and histamine-induced coronary spasm was also significantly inhibited by pretreatment with staurosporine, sphingosine, or nifedipine (an L-type Ca2+ channel antagonist) but not by ryanodine (an inhibitor of Ca(2+)-induced Ca2+ release from SR) or thapsigargin (an inhibitor of Ca(2+)-ATPase of SR). Bay K 8644 (an L-type Ca2+ channel agonist) also induced coronary spasm at the IL-1 beta-treated site, which was significantly inhibited by pretreatment with staurosporine, sphingosine, and nifedipine. In contrast, coronary vasoconstriction induced by prostaglandin F2 alpha was not affected by pretreatment with staurosporine or sphingosine but was significantly inhibited by pretreatment with ryanodine, thapsigargin, or nifedipine. CONCLUSIONS: These results suggest that (1) PKC activation largely accounts for the serotonin- and histamine-induced coronary spasm; (2) at the spastic site, the calcium influx through L-type Ca2+ channels may be augmented via the PKC-mediated pathway; and (3) the Ca2+ release from the SR into the cytosol may not play a primary role in coronary spasm.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

An improved quantitative RT-PCR fluorescent method for analysis of gene transcripts in the STS-65 space shuttle experiment.

We describe a reverse transcription polymerase chain reaction (RT-PCR) technique using fluorescent dUTP to examine changes in mRNA level in samples. In this procedure, the amplified product is identified by the fluorescent detection system in an automated DNA sequencer, and if the MW of the DNA/RNA or the fluorescent dye is different, several samples can be analyzed in a single lane. The basis for this technique is similar to that of radiolabeled methods, and we applied this technique for the comparison of the expression level of the rat c-myc gene in osteoblasts exposed to microgravity and unit gravity conditions. Using the fluorescent- and radiolabeled methods, the level of rat c-myc mRNA were compared quantitatively and the results demonstrated that the c-myc expression level was not altered by microgravity. Therefore, this fluorescent RT-PCR technique is useful for gene expression analysis particularly when the samples are limited, such as in space flight experiments. The method also allows for rapid assessment of mRNA changes in many samples simultaneously.

Animals↗