Iron oxidation in sheep, horse and recombinant human apoferritins.
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Animals↗
Biomedical subjects
S Sun
Publications and source records attributed to S Sun.
Protein sequence and structure relationship ARMA spectral analysis: application to membrane proteins.
If it is assumed that the primary sequence determines the three-dimensional folded structure of a protein, then the regular folding patterns, such as alpha-helix, beta-sheet, and other ordered patterns in the three-dimensional structure must correspond to the periodic distribution of the physical properties of the amino acids along the primary sequence. An AutoRegressive Moving Average (ARMA) model method of spectral analysis is applied to analyze protein sequences represented by the hydrophobicity of their amino acids. The results for several membrane proteins of known structures indicate that the periodic distribution of hydrophobicity of the primary sequence is closely related to the regular folding patterns in a protein's three-dimensional structure. We also applied the method to the transmembrane regions of acetylcholine receptor alpha subunit and Shaker potassium channel for which no atomic resolution structure is available. This work is an extension of our analysis of globular proteins by a similar method.
Amino Acid Sequence↗
Inhibition of neointimal hyperplasia by blocking alpha V beta 3 integrin with a small peptide antagonist GpenGRGDSPCA.
PURPOSE: Neointimal hyperplasia is a leading cause of restenosis after vascular procedures. Recent findings showed that smooth muscle cell (SMC) migration from the media into the neointima is a critical step in the development of the hemodynamically compromising neointimal lesion. Moreover, integrins are believed to play a role in SMC motility. Therefore we studied the role of one ubiquitous integrin, alpha V beta 3, in SMC migration. METHODS: Transwell assay was used to study in vitro migration of human and rabbit SMCs after stimulation with platelet-derived growth factor (PDGF). A neutralizing monoclonal antibody to alpha V beta 3, LM609, and a specific arginine-glycine-aspartic acid (RGD) antagonist, GpenGRGDSPCA, were used in the migration assay to inhibit alpha V beta 3-mediated SMC migration. In addition, GpenGRGDSPCA was administered locally to rabbit carotid artery after balloon angioplasty to determine the effect of blocking alpha V beta 3 on neointimal hyperplasia. RESULTS: We showed that PDGF-induced human SMC migration is mediated by the alpha V beta 3 integrin by use of LM609 to inhibit migration and that SMC migration is RGD dependent by use of GpenGRGDSPCA to inhibit migration. We have also inhibited rabbit SMC migration with GpenGRGDSPCA to demonstrate the cross-species preservation of the RGD peptide sequence in SMC mortality. Finally, when we administered GpenGRGDSPCA locally to rabbit carotid artery after balloon angioplasty, there was a statistically significant reduction in neointimal lesion formation compared with arteries administered an inactive peptide or saline solution. CONCLUSIONS: We have demonstrated the important role of the alpha V beta 3 integrin in SMC migration in vitro and in neointimal hyperplasia in vivo.
Amino Acid Sequence↗
An antigenic recombinant fusion protein from Trichinella spiralis induces a protective response in BALB/c mice.
A previously reported recombinant lambda Ts39 fusion protein (FP) derived from Trichinella spiralis larvae was purified by affinity chromatography using an anti-beta-galactosidase antibody conjugated column and the effect of FP on induction of a protective response in mice was studied. BALB/c mice were injected three times at weekly intervals with FP emulsified in an equal volume of Freund's complete adjuvant and one week after the last injection mice were each challenged with a lethal dose of 1200 L1 larvae of Trichinella spiralis. As a result, the FP induced significant protection. Mice were also infected orally with 250 L1 larvae each after injection of the FP. On the 35th day of infection, immunized mice with the FP harboured 78% fewer muscle larvae than saline controls. Also, the numbers of adult worms in the small intestine were smaller in FP injected mice than saline controls on day 6 and 9 of the infection. These results suggest that the recombinant lambda Ts39 FP is a potentially valuable antigen for vaccine development.
Animals↗
Complete structure of the gene encoding an immunodominant antigen of Dirofilaria immitis and larva-specific synthesis of primary transcript.
The complete gene encoding an immunodominant antigen of Dirofilaria immitis was isolated from a Charomid 9-36 genomic DNA library. This genomic DNA clone termed 'Dg2' was characterized by restriction mapping, DNA sequencing of the 5' flanking region, the exon/intron boundaries and the polyadenylation addition site. The Dg2 with 4872 bp in length consisted of five exons interspersed with four introns. These exons reveal a single open reading frame followed by a long 3' non-coding region of 1383 bp. The open reading frame of 969 bp encodes a polypeptide of 322 amino acids with a molecular weight of 34,400. The ATG translation initiation codon starts 22 nucleotides downstream from the 5' end of the first exon. The polyadenylation signal sequence. AATAAA, is located at the 3' end of the last exon. The transcription initiation site was determined by primer extension technique. S1 nuclease mapping analysis demonstrated that the primary transcript derived from Dg2 is synthesized in microfilariae but not in male or female adult worms. The result suggests that the stage-specific expression of Dg2 is regulated at the level of primary transcript.
Amino Acid Sequence↗
Gastric intramural PCO2 as monitor of perfusion failure during hemorrhagic and anaphylactic shock.
Indirect measurement of gastric intramural pH (pHG) utilizing a luminal tonometer in the stomach has been proposed for monitoring the severity and progression of perfusion failure. In the present study, we investigated gastric PCO2 and pHG as indicators and quantitators of the severity of perfusion failure in the experimental rodent model of both hemorrhagic and anaphylactic shock. Gastric intramural PCO2 (PGCO2) and pHG were directly measured with miniaturized sensors inserted into the anterior wall of the stomach. In hemorrhagic shock, animals were bled into a reservoir maintained at a pressure of 35 mmHg. pHG decreased from 7.39 +/- 0.08 to 6.67 +/- 0.11 (P < 0.01), and PGCO2 increased from 53 +/- 4 to 136 +/- 3 Torr (P < 0.01). Anaphylactic shock was induced in animals that had been sensitized 21 days before with crystallized ovalbumin. Antigen challenge produced an immediate reduction in mean aortic pressure from 144 to 60 mmHg. pHG decreased from 7.40 +/- 0.05 to 6.99 +/- 0.07 (P < 0.01), and PGCO2 increased from 48 +/- 5 to 133 +/- 9 Torr (P < 0.01). The increases in PGCO2 were highly correlated with decreases in gastric blood flow in both hemorrhagic (r = 0.96) and anaphylactic shock (r = 0.92). The correlations with pHG were more moderate. These experiments demonstrated prominent increases in PGCO2 and H+ during both hemorrhagic and anaphylactic shock. We further noted that the estimation of pHG based on the assumption that HCO3-concentrations of the stomach wall and arterial blood are the same was not fully sustained.
Anaphylaxis↗
Spontaneous gasping during cardiopulmonary resuscitation without mechanical ventilation.
Spontaneous gasping is frequently observed during cardiac arrest, especially when mechanical ventilation is withheld during precordial compression. We related spontaneous gasping to pulmonary gas exchange and cardiac resuscitability in a rodent model of cardiac arrest. Ventricular fibrillation was electrically induced in 15 Sprague-Dawley rats. After 4 min untreated ventricular fibrillation, precordial compression was initiated. Coronary perfusion pressure was maintained between 25 and 30 mm Hg. Oxygen was supplied at the tracheal tube port coincident with start of precordial compression in 10 animals. Five additional control animals were identically treated except they were mechanically ventilated coincident with start of precordial compression. After 6 min precordial compression, defibrillation was attempted and five of 10 nonventilated animals, and all control animals, were resuscitated by direct current countershock. In the successfully resuscitated, nonventilated animals, the frequency of spontaneous gasping during precordial compression progressively increased to an average of 19 gasps/min but it was < 6 gasps/min in nonresuscitated animals. More frequent gasping was associated with correspondingly greater arterial PO2 (110 versus 51 mm Hg, p < 0.01) and lesser PCO2 (55 versus 91 mm Hg, p < 0.01). In control animals, no spontaneous gasping was observed during precordial compression. Arterial PO2 and PCO2 of mechanically ventilated animals was more like that of spontaneously gasping rats. According, the frequency of spontaneous gasping in absence of mechanical ventilation is predictive of cardiac resuscitation success and associated with improved arterial oxygenation and CO2 removal.
Analysis of Variance↗
Cardiopulmonary resuscitation by precordial compression but without mechanical ventilation.
It is widely held that mechanical ventilation is essential for cardiopulmonary resuscitation (CPR). However, cardiac output and therefore pulmonary blood flow is reduced to less than one-third of normal during CPR. We therefore reasoned that ventilatory requirements are correspondingly reduced and postulated that gas exchange may be maintained during precordial compression with oxygen passively delivered to the airway in the absence of mechanical ventilation. After tracheal intubation, Sprague-Dawley rats were randomized. Fifteen animals were maintained on positive-pressure ventilation with room air and an additional 15 animals breathed spontaneously. Cardiac arrest was induced by electrical fibrillation. The inspired gas concentration of oxygen was then increased to 100% in both groups. Precordial compression was begun after 4 min of untreated ventricular fibrillation. After an additional 6 min of precordial compression, resuscitation was attempted by DC countershock. During cardiac resuscitation, there were no significant differences in coronary perfusion pressure between mechanically ventilated and spontaneously breathing animals, but arterial PO2 was significantly lower and arterial PCO2 was significantly higher in the absence of positive-pressure ventilation. However, neither resuscitability nor 24-h survival were affected. Postresuscitation myocardial contractility, reflected in the maximally generated dP/dt40, was also not adversely affected. In the unventilated group, only resuscitated animals developed spontaneous gaspings at an average frequency of 17 +/- 2/min-1. The current emphasis on mechanical ventilation as the highest priority for cardiopulmonary resuscitation is therefore not fully supported under the experimental conditions of this study.
Animals↗
Vascular ring: unusual cause of dysphagia in an adult with right-sided aortic arch and mirror-image branching.
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Adult↗
Hypertension-related coronary thrombosis: prothrombic role of angiotensin II.
Although hypertension is a major risk factor in acute myocardial infarction, concomitant hypercoagulability causing thrombosis leading to myocardial infarction remains unproven for lack of an appropriate coagulation test. This study was devised to determine whether a modified recalcification time (MRT) test can demonstrate that angiotensin II, a potent vasoconstrictor, also accelerates coagulation to promote thrombosis. The MRT incorporates blood cells and chemical coagulants for maximizing sensitivity. Four groups (A, B, C, and D) of aliquots of citrated human blood were incubated for 2 hours at 37 degrees C after adding to A--20 microL saline, to B--10 micrograms Escherichia coli endotoxin, to C--20 micrograms angiotensin II, and to D--a combination of E coli endotoxin and angiotensin II. The experiment was repeated with nonincubated aliquots. Modified recalcification time values +/- standard deviation in minutes were: A--5.5 +/- 1.5, B--4.6 +/- 1.1, C--4.9 +/- 1.0, and D--3.9 +/- 1.0. Significance (Student's t test) was as follows: B versus A P < .001; C versus A, P < .05; C versus D, P < .001; B versus C, P < .05; and B versus D, P < .001. No significant changes occurred in nonincubated blood. We conclude that angiotensin II has a hypercoagulable effect, as does endotoxin. The hypercoagulability in concert with vasospasm can explain the role of hypertension in acute myocardial infarction. This in vitro study excludes the role of other in vivo mechanisms in the development of angiotensin II-induced hypercoagulability.
Angiotensin II↗
Ventricular fibrillation voltage as a monitor of the effectiveness of cardiopulmonary resuscitation.
Ventricular fibrillation (VF) voltage was previously identified as a predictor of the success of cardiopulmonary resuscitation. In the present study we investigated the mechanism by which VF voltage predicts the success of cardiac resuscitation in a well-established rodent model of cardiac arrest. After 4 minutes of untreated VF, precordial compression was initiated and maintained for 6 minutes. Increases in coronary perfusion pressure during precordial compression were associated with concomitant increases in VF voltage (r = 0.61, p = 0.013). Significantly greater coronary perfusion pressure (24 vs 17 mm Hg) and VF voltage (0.17 vs 0.12 mV) were observed in resuscitated animals. To obviate electrical artifacts produced by precordial compression, boluses of oxygenated blood were injected into the ascending aorta in another 5 animals as an alternative method of cardiac resuscitation. This restored myocardial perfusion before defibrillation. Increases in VF voltage from 0.04 mV to 0.47 mV during aortic infusions were again correlated with coronary perfusion pressure (r = 0.62, p < 0.01) and predicted the success of cardiac resuscitation. Greater VF voltages after initiation of cardiac resuscitation were associated with increases in myocardial creatine phosphate, from 0.23 to 0.70 mmol/kg wet weight, and significant decreases in lactate content, from 22.8 to 13.9 mmol/kg wet weight. Increases in creatine phosphate were highly correlated with increases in VF voltage (r = 0.99, p < 0.01). Accordingly, increases in VF voltage during cardiac resuscitation reflect increases in myocardial perfusion and favorable changes in myocardial energy metabolism. As such, VF voltage, like coronary perfusion pressure, serves as a quantitative predictor of the success of cardiopulmonary resuscitation.
Action Potentials↗
A new hypothesis about the relationship between free radical reactions and hemorheological properties in vivo.
This paper is concerned with a hypothesis that disturbance of free radical reactions may lead to abnormality of hemorheological properties in vivo, and so the free radicals generated in vivo may damage certain tissue cells indirectly by reducing the supply of oxygen and nutrients to these cells through slowing the circulation of blood. This hypothesis is based on the following evidence: A. We have found that the whole blood viscosity at low shear rate correlates to the lipid peroxidation in the patients suffering from certain cardio- or cerebrovascular diseases, and in dogs during liver ischemia reperfusion or hemorrhagic pancreatitis. B. Reports have shown that several alterations of hemorheological properties may take place as a result of free radical reactions, such as lipid peroxidation. For instance, lipid peroxidation may lead to decrease of deformability of red cells, increase of aggregation of red cells, formation of liquid thrombin, etc. C. We have demonstrated that some alterations of hemorheological properties involve the role of free radicals in rats suffering from intestinal ischemia/reperfusion. As evidence for this conclusion, superoxide dismutase (SOD) used as a specific scavenger of superoxide anion radical (O2-) can significantly prevent the intestinal ischemia/reperfusion induced changes of lipid peroxidation, red cell aggregation, Cassion's viscosity and whole blood viscosity at low shear rate in rats.
Animals↗
Effect of endothelin-1 infusion on the development of intimal hyperplasia after balloon catheter injury.
Previous studies have shown endothelin-1 (ET-1) to be mitogenic for smooth-muscle cells. We explored in vivo the ability of high ET-1 levels to worsen angioplasty restenosis. Left carotid artery balloon endothelial denudation was performed on 14 rats. ET-1 was delivered via osmotic pump at a rate of 5 pmol/kg/min. Intimal development and plasma ET-1 levels were assessed at 2 weeks. Blood pressure and heart rate were measured throughout the study. For analysis, the animals were divided into three groups based on ET-1 levels at harvest: control, 4.3 +/- 0.5 pmol/ml (n = 6); low ET-1, 5.2 +/- 0.9 pmol/ml (n = 4); and high ET-1, 23.1 +/- 5.9 pmol/ml (n = 4). Although ET-1 infusion caused blood pressure elevation in both ET-1 groups, this was more marked and prolonged in the group with a high ET-1 level at study conclusion. Evaluation of the intimal:medial area ratio showed a marked increase in intimal thickness in the high ET-1 group versus control (1.13 +/- 0.23 versus 0.35 +/- 0.11; p < 0.05). We conclude that ET-1 infusion in responsive animals can cause worsening of the intimal hyperplastic response after mechanical injury. Further study is required to elucidate whether this is entirely caused by a direct effect of ET-1 on smooth-muscle cell mitogenesis or is also by the hemodynamic effects of ET-1-induced hypertension, or an effect of another mediator released in response to the ET-1 (e.g., angiotensin II).
Animals↗
Ferroxidase kinetics of horse spleen apoferritin.
Protein ferroxidase site(s), which catalyze the reaction between ferrous ion and dioxygen, have long been thought to play a role in core formation in ferritin; however, the mechanism of the reaction has never been studied in detail. In the present work, the enzymatic activity of ferritin was examined using oximetry, the net Fe2+ oxidation reaction being as follows. [formula: see text] The reaction exhibits saturation kinetics with respect to both Fe2+ and O2 (apparent Michaelis constants: Km,Fe = 0.35 +/- 0.01 mM and Km,O2 = 0.14 +/- 0.03 mM). The enzyme has a turnover number kcat = 80 +/- 3 min-1 at 20 degrees C with maximal activity at pH 7. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe(II) oxidation occurs in 1-electron steps. Zinc(II) is a competitive inhibitor of iron(II) oxidation at Zn2+/apoprotein ratios > or = 6 (inhibitor constant KI,Zn = 0.067 +/- 0.011 mM) but appears to be a noncompetitive inhibitor at lower ratios (< or = 2), indicating the presence of more than one type of zinc binding site on the protein. At increments of 50 Fe2+/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. However, when larger increments are employed, some iron oxidation appears to occur on the surface of the mineral core. The results of these studies emphasize the role of the protein shell in all phases of core growth and confirm the presence of a functionally important catalytic site in ferritin in addition to other binding sites on the protein for iron.
Animals↗
Chimaeric protein A/protein G and protein G/alkaline phosphatase as reporter molecules.
The IgG binding domains of staphylococcal protein A and streptococcal protein G were expressed as a chimaera using the pGEX vector which has been advocated because its fusion proteins tend to be soluble and easily isolated on immobilised glutathione. This chimaera was soluble and abundant (yield = 18 mg/l of bacterial culture) and was tested by double diffusion in agarose and by ELISA. It was found to bind IgG of all species that either parent could bind. It was superior to protein A or protein G in binding mouse Ig. A chimaera of protein G and alkaline phosphatase was also constructed and found to be soluble and abundant (yield = 20 mg/l of bacterial culture). This protein could be used as a secondary reagent in ELISA at 5 micrograms/ml for human, rabbit and mouse and at 25 micrograms/ml for sheep.
Alkaline Phosphatase↗
Positive and negative cis-acting regulatory elements mediate expression of the mouse vascular smooth muscle alpha-actin gene.
Segments of the 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene were assayed for promoter activity in transfected mouse BC3H1 myogenic cells and AKR-2B embryonic fibroblasts. The region between -150 and -191 that functions as a positive transcriptional element in myogenic and fibroblastic cells contains a mammalian-specific inverted CC(A/T)6GG-type consensus sequence. Expression was restricted to fully differentiated myogenic cells when an additional sequence spanning -191 to -224 was included in reporter gene constructs. This 33-base pair (bp) negative regulatory element is 70% conserved between the mouse and human genes and contains a 10-bp motif at its 3' end that only partially resembles a CC(A/T)6GG element. Retention of a GGGA motif at the 3' boundary of the 33-bp region is sufficient to maintain full transcriptional repression in fibroblasts and is partly responsible for repression in undifferentiated myoblasts. Complete muscle tissue-restrictive expression requires an additional 8 bp from the CC(A/T)6GG-like element immediately 5' to the GGGA motif, since replacement of this region with an unrelated 10-bp sequence completely eliminated restrictive transcriptional behavior in undifferentiated myoblasts. The distal portion of the 5'-flanking region between -224 and -1074 contains six E-box motifs (CANNTG) and mediates high level transcription only in postconfluent BC3H1 myoblasts. Analysis of reporter gene constructs including either the proximal E-box at -240 or all six E-boxes indicate that the five distal E-boxes are not required for high level transcription. A 724-bp segment of the 5'-flanking region consisting of the proximal E-box flanked upstream by a mammalian-specific 352-bp region was sufficient for maximal transcriptional activation in postconfluent BC3H1 myoblasts. Deletion of the 352-bp region restricts the early transcriptional response to high cell density in temporal studies of promoter activity during BC3H1 myogenic cell differentiation.
Actins↗
Protein structure prediction based on statistical potential.
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Biometry↗
Molecular cloning of the cDNA encoding a 42 kDa antigenic polypeptide of Anisakis simplex larvae.
The gene encoding an antigenic polypeptide of Anisakis simplex larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from A. simplex larvae was ligated into phage vector lambda gtll DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gtll expression library was constructed. A cDNA clone encoding a 42 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing cDNA for a 42 kDa protein was isolated. The gene encoding this 42 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 42 kDa polypeptide. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.
Amino Acid Sequence↗