Biomedical subjects
Publications and source records attributed to N Sugimoto.
In sum, our results indicate that LPS shock-associated hypothermia involves the following major mechanisms: 1) a decrease in the threshold Tb for activation of cold thermogenesis; 2) the resultant widening of the interthreshold zone; and 3) cold-seeking behavior. We speculate that, in severe systemic inflammation, this hypothermia constitutes an adaptive response.
We recently demonstrated that Bordetella bronchiseptica dermonecrotizing toxin (DNT) modifies a small GTP-binding protein rho and causes assembly of actin stress fibers and focal adhesions in MC3T3-E1 cells, indicating that DNT activates the function of rho protein (Horiguchi et al., 1995, J. Cell Sci. 108, 3243-3251). In this study, we examined by electron microscopy ultrastructural changes in DNT-treated MC3T3-E1 cells under conditions in which DNT elicited the above morphological changes. We found that DNT induced proliferation of cytoplasmic membrane organelles, such as Golgi apparatus, smooth and rough endoplasmic reticulum, and mitochondria, and formation of plasmalemmal caveolae. Therefore we examined also the effect of Clostridium botulinum C3 exoenzyme (C3), which has been known to ADP-ribosylate and inactivate rho protein, and found that C3 exoenzyme inhibited the development of membrane organelles in the MC3T3-E1 cells. These findings show that proliferation of membrane organelles and caveolae formation are controlled by the function of rho, which is the target of DNT action.
The Duffy blood group system consists of three alleles, FYA, FYB, and FY. To study the molecular evolution of the three alleles, we established the polymorphism of a dinucleotide (GT) repeat sequence (designated FyGT/C) in the 3' flanking region of the Duffy gene, and studied the relationship between FyGT/C and Duffy polymorphism in Japanese, people of African origin, and chimpanzee. By single-strand conformation polymorphism and sequence analysis, five and two alleles were identified in Japanese and Africans, respectively. In 110 random Japanese, the FyGT/C genotypes observed were in agreement with Hardy-Weinberg law. From the sequence of the chimpanzee Duffy gene, including both flanking regions, FYB was identified as the ancestral gene of the human alleles. The FyGT/C sequences associated with the FY allele of Africans were distinct from those of Duffy positives, whereas the FYB and FYA alleles shared common FyGT/C sequences. Thus, it is suggested that the first split took place between the FYB and FY alleles, and the second between the FYB and FYA alleles.
Several timed daily environmental cues alter the pattern of nycthemeral variations in body core temperature in rodents. The present study investigated the effect of timed exercise on variations of daily body core temperature. Male rats were housed in cages with a running wheel at an ambient temperature of 24 degrees C with a 12:12 h light/dark cycle. Timed daily exercise rats (TEX) were allowed access to the wheel for 6 h in the last half of the dark phase, freely exercising rats (FEX) could run at any time, and sedentary rats (NEX) were not allowed to run. After a 3-week exercise period, all animals were denied access to the wheel. The intraabdominal temperatures (Tab) and spontaneous activities of rats were measured for 6 days after the exercise period. The Tab values of the TEX rats were significantly higher than those of the other two groups only in the last half of the dark phase, while Tab in the FEX and NEX rats showed no significant difference. The specific Tab changes in the TEX rats lasted for 2 days after the exercise period. Spontaneous activity levels were higher in the TEX rats than the FEX and NEX rats in the last half of the dark phase for 1 day after the exercise period. The results suggest that daily exercised limited to a fixed time per day modifies nycthemeral variations of body core temperature in rats so that the temperature increases during the period when the animals had previously exercised. Such a rise in body core temperature is partly attributed to an increase in the spontaneous activity level.
The effects of botulinolysin (Blyn), a thiol-activated hemolysin produced by Clostridium botulinum, on contractility of rat aortic ring were studied in order to clarify an underlying mechanism of vasoconstriction by the toxin observed previously as an increase in perfusion pressure in isolated rat organs. Blyn (30 hemolytic units/ml; HU/ml) itself did not elicit any apparent change in resting tension of the ring. Contractile tension elicited by a high concentration of phenylephrine in endothelium-intact rings increased significantly after treatment with Blyn (30 HU/ml), while phenylephrine-induced contraction of endothelium-denuded rings was not influenced by toxin treatment. In rings with intact endothelium, acetylcholine (ACh)-induced relaxation was significantly inhibited after treatment with Blyn (30, 10, 1 HU/ml). In contrast, relaxation of denuded rings by sodium nitroprusside was not affected by toxin treatment (30 HU/ml). Arginine (10(-4) M) partly reversed the inhibition of ACh-induced relaxation by the toxin (1 HU/ml). Endothelium-dependent relaxation by histamine or adenosine triphosphate was also inhibited by Blyn (1 HU/ml), but the relaxation elicited by calcium ionophore A23187 was not influenced by the toxin. The results indicate that Blyn acts on endothelium and inhibits agonist-induced endothelium-dependent relaxation of blood vessels.
The repeatedly observed attenuation of fever in vagotomized rats has been accepted as evidence of an essential role of vagal afferents in the transduction of pyrogenic signals from the periphery to the brain. If, however, the general condition of a vagotomized animal is poor (the usual case) and accompanied by malnutrition and body mass loss (common complications of vagotomy), the febrile responsiveness can be suppressed not because of the lack of vagal afferentation, but rather secondarily to a malnutrition-associated thermogenic incompetence. In the present study, we addressed this dilemma. Male Wistar rats were subjected to subdiaphragmatic vagotomy (or sham surgery) and, 24 days later, catheterized in the jugular vein. Postsurgically, the rats were closely watched and fed highly palatable food. Their febrile responsiveness [colonic (Tc) and tail skin (Tsk) temperature responses] to Escherichia coli lipopolysaccharide (LPS: 1 microgram/kg i.v.) was tested on day 27 postvagotomy. To verify the completeness of vagotomy, each rat was food deprived for 24 h and then euthanized; its stomach's evacuatory function was assessed by weighing the organ. One month postsurgery, both food consumption and body mass of the vagotomized rats (33 +/- 2 g/day and 313 +/- 4 g, respectively) were similar to the control values (30 +/- 1 g/day and 315 +/- 8 g). In the sham rats, LPS induced a monophasic Tc rise of 0.5 +/- 0.3 degree C at 70 min postinjection (peak), preceded by a fall in Tsk. Neither this Tsk fall (tail skin vasoconstriction) nor the resultant fever occurred in the vagotomized rats; at 70 min, Tc change was -0.1 +/-0.1 degree C. The gastric mass (4.1 +/- 0.5 g in the vagotomized vs. 1.8 +/- 0.1 g in sham rats) indicated the effectiveness of vagotomy. In sum, although the vagotomy-associated malnutrition was successfully prevented with special perioperative care, the vagotomized animals still did not respond to LPS with fever. Malnutrition is, therefore, unlikely to constitute the main reason of the febrile irresponsiveness of vagotomized rats.
Subdiaphragmatically vagotomized rats cannot mount a febrile response to pyrogens and are believed to have severe thermoregulatory deficiencies. We addressed the issue of thermoeffector competence of vagotomized rats by asking three questions. In Expt. 1 we asked, can vagotomized rats readily recruit tail skin vasoconstriction in the course of a moderate cold exposure? In Expt. 2 the question was, can brown adipose tissue (BAT) thermogenesis readily be activated in vagotomized rats (e.g., in response to a tail pinch)? In Expt. 3, we investigated the question: can vagotomized rats elevate their body temperature in response to ephedrine (a drug of high hyperthermizing potential) to the same extent as sham-operated controls? Rats were vagotomized or sham operated and implanted with a catheter into the jugular vein and a thermocouple into the interscapular BAT. To prevent the common complications of vagotomy, special perioperative care was given. During experiments, colonic, tail skin, and BAT temperatures (Tc, Tsk, and TBAT, respectively) were measured. The vagotomized animals were well nourished and had a body mass (325 +/- 6 g) similar to that of the controls (338 +/- 6 g). In Expt. 1, in response to external cooling (15 degrees C, 1 h), the vagotomized (n = 30) and sham-operated (n = 31) rats recruited tail skin vasoconstriction at close values of both Tc (37.84 +/- 0.08 and 37.97 +/- 0.07 degrees C) and Tsk (33.16 +/- 0.17 and 33.18 +/- 0.18 degrees C, respectively). In Expt. 2, tail pinch-associated stress in vagotomized rats resulted in a sharp rise in the TBAT-Tc gradient by 0.3-1.0 degree C. In Expt. 3, ephedrine administered intravenously (whether in a 5 or 35 mg/kg dose) evoked similar hyperthermic responses in the vagotomized and sham-operated rats: a moderate (approximately 2.5 degrees C) Tc rise in the low dose and a "supramaximal" (approximately 5.0 degrees C) rise in the high dose. In sum, the answer to all three questions asked is yes. Vagotomized rats, at least when well nourished, exhibit no signs of thermoeffector deficiency. It is, therefore, not effector incompetence but rather vagal deafferentation per se that can explain the febrile irresponsiveness of vagotomized rats.
We studied the effect of an abdominal prick with a needle on LPS-induced fever in freely-moving rats. LPS was injected intraperitoneally by the following 3 methods: 1) through a hypodermic needle pricked into the abdominal cavity, 2) through a catheter chronically indwelt in the abdominal cavity, and 3) through a catheter chronically indwelt in the abdominal cavity immediately after an abdominal prick was made. In the second method, core body temperature (Tb) began to rise about 1 h after the injection, reaching a maximal level at around 2.5 h and decreasing gradually thereafter. In the first and third methods, Tb rose again to make a second peak after making the first peak of fever. This was the same when LPS was injected through a hypodermic needle pricked into the abdominal cavity under restrained condition. These results suggest that the abdominal prick with a needle is responsible for the development of the second peak (or prolongation) of LPS-fever in rats.
An 86-year-old woman was admitted with hemoptysis and rapid deterioration of renal function. Renal biopsy disclosed necrotizing crescentic glomerulonephritis. Based on positivity for serum myeloperoxidase antineutrophil cytoplasmic autoantibody (MPO-ANCA), MPO-ANCA-associated glomerulonephritis was diagnosed. Steroid pulse therapy was performed, but the patient died after the second course. Autopsy revealed renal vasculitis with fibrinoid necrosis extending to the level of the arcuate arteries. Vasculitis was also observed in the liver, adrenal gland, uterus, and spleen, suggesting the presence of microscopic polyarteritis. This case demonstrates the broad spectrum of MPO-ANCA-positive vasculitis and suggests the need for a more effective therapy suitable for very elderly patients.
Cation effect on DNA/DNA, RNA/RNA, and RNA/DNA stabilities was investigated quantitatively. Plots of predicted delta G degree 37 values vs. measured delta G degree 37 ones showed the same relationship for DNA/DNA, RNA/RNA, and RNA/DNA. The number of sodium ion concerned in a double helix formation was estimated, and the result indicated that the number of bound sodium ion may determine the duplex stability regardless of DNA/DNA, RNA/RNA, and RNA/DNA. Since these duplex stability can be predicted with the nearest-neighbor model, it is considered that the number of bound sodium ions is affected by the nearest-neighbor bases. The importance of the screening effect of the electrostatic repulsion by bound cations was also indicated for determining the duplex stability as well as the hydrogen bonding and stacking interactions.
We report stabilities of internal non-Watson-Crick rU-dG and rG-dT pairs in RNA/DNA hybrids. UV melting curves revealed that a hybrid containing internal rG-dT pair had about 2 kcal mol-1 greater stability in free energy change at 37 degrees C than hybrids with other mismatches, rA-dA and rU-dT, at the same position. We discuss why internal rG-dT as well as rU-dG is stable nearly Watson-Crick base pairs rather than the other mismatches.
We carried out an in vitro selection of aptamers from an RNA library with phosphorothioate linkages instead of normal phosphodiesters. This "thio-RNA" library had smaller dissociation rate constant (kd) to a target protein than a natural RNA library. On the basis of the results, an improved selection procedure to obtain ideal nucleic acid drugs will be discussed.
Leadzyme, CUGGGAGUCC, is a ribozyme that requires Pb2+. We have investigated effects of target sequences and metal ions on a catalytic reaction of the leadyzme. The leadzyme plays as a catalyst for one site cleavage of a mutant substrate, GGAC decrease GAGCCAG, and a wild-type substrate, GGACC decrease GAGCCAG. The addition of 25 microM Nd3+ in the presence of 25 microM Pb2+ led to significant increase in the cleavage yield for the mutant substrate. But this increase of the yield is less than that for the wild-type substrate. The result suggests that the effect of Nd3+ on the leadzyme reaction depends on substrate sequence.
To improve the previous DNA/DNA nearest-neighbor parameters, thermodynamic parameters (deltaH degrees, deltaS degrees and deltaG degrees) of 50 DNA/DNA duplexes were measured. Enthalpy change of a helix initiation factor is also considered though the parameters reported recently did not contain the factor. A helix initiation factor for DNA/DNA duplex determined here was the same as that of RNA/RNA duplex (deltaG degrees(37) = 3.4 kcal/mol). The improved nearest-neighbor parameters reproduced not only these 50 experimental values used here but also 15 other experimental values obtained in different studies. Comparing deltaG degrees(37) values of DNA/DNA nearest-neighbor parameters obtained here with those of RNA/RNA and RNA/DNA, RNA/RNA duplex was generally the most stable of the three kinds of duplexes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA duplexes is sequence dependent.
Lead-dependent ribozyme (leadzyme) is a ribozyme working with Pb2+. In this paper, we have investigated the combined effect of metal ions, especially rare earth ions, on the cleavage reaction by the leadzyme. As a result, it was observed that although only a rare earth ion or another divalent ion except Pb2+ did not play a role as the catalyst, the addition of a rare earth ion in the presence of Pb2+ increased significantly the yield of the cleavage reaction. The result suggests that the complex between the leadzyme and the substrate should have two classes of metal ion binding sites.
We have previously identified a novel first exon of Duffy gene and two inverse GATA motifs in the 600 bp 5' flanking region. The proximal GATA is positioned downstream from the start position of endothelium and upstream from that of erythroid. One base substitution (-365T --> C) was found in the proximal GATA motif from three black Fy(a-b-) individuals, and was regarded as a common polymorphic mutation in black Fy(a-b-) individuals. The upstream sequence of the novel first exon was inserted in the upstream of chloramphenicol acetyltransferase (CAT) gene and transfected in human erythroleukemia cell line (HEL) and human microvascular endothelial cells (HMvEC). The black type mutation abolished the CAT transcription in HEL cells but not in HMvEC. Deletion mutagenesis study revealed that the proximal GATA motif represent the erythroid regulatory core region for Duffy gene. Gel shift assay showed that the proximal GATA motif is the target sequence of GATA-1. These studies indicate that the black type mutation abolishes Duffy gene expression in erythroid but not in postcapillary venule endothelium, which is compatible with the Northern blot and immunohistochemical observation in black Fy(a-b-) individuals.
A simple model system is described which mimics the second step of splicing and reverse cyclization reactions of the self-splicing intron from Tetrahymena thermophila. This model is based on the L-21 Sca I catalyzed ligation reaction between exogenously added oligomers: cucu + UCGa L-21 Sca I cucua + UCG. Steady-state kinetics for the forward and reverse direction were measured at 15 degrees C to find oligonucleotides that exhibit Michaelis-Menten behavior with acceptable KMS. CUCU and UCGA fit both criteria and were chosen for further studies. Steady-state kinetics reveal a lag that appears to be an RNA folding step that is eliminated by preincubation of the ribozyme with 2 mM and higher [Mg2+] and by UCGA. At constant ionic strength, the Mg2+ dependence of steady-state rates exhibits a sharp maximum near 5 mM Mg2+. Pre-steady-state and steady-state kinetics, along with active-site titrations, explain the Mg2+ profile: the rate of reaction up to and including chemistry increases with Mg2+ concentration, while the fraction of active ribozyme and the rate of postchemistry steps decrease with Mg2+ concentration. The rate-limiting step at 5 mM Mg2+ for the reaction mimicking the second step of splicing is either chemistry or a conformational change before chemistry involving ribozyme bound with substrates. The rate-limiting step at 50 mM Mg2+ appears to be a postchemistry conformational change of the ribozyme or product release. At 50 mM Mg2+, single-turnover experiments support ordered binding of substrates with 5'-exon mimic binding before 3'-splice site mimic. Moreover, the 3'-splice site mimic binds and reacts in the presence of 5'-exon mimics predocked into the catalytic core. Results also indicate that Mg2+ ions associate with the ribozyme upon docking.
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