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Biomedical subjects

C E Smith

Publications and source records attributed to C E Smith.

At least 181 records · Page 10Linked to original sources
PubMed1989

Affinity purification of active plasminogen activator inhibitor-1 (PAI-1) using immobilized anhydrourokinase. Demonstration of the binding, stabilization, and activation of PAI-1 by vitronectin.

Human Hep G2 hepatoma and HT 1080 fibrosarcoma cells were cultured in large scale under conditions which allowed enhanced secretion of plasminogen activator inhibitor-1 (PAI-1). A modified urokinase was obtained by reacting urokinase with phenylmethylsulfonyl fluoride followed by alkali treatment. The resulting product, called anhydrourokinase, was found to reversibly bind the PAI-1 when immobilized on cyanogen bromide-activated Sepharose 4B beads. Using this affinity absorbent, we have purified PAI-1 from the cell-conditioned media. A number of differences have been observed during Hep G2 and HT 1080 PAI purification. 1) The PAI activity in Hep G2 medium concentrate is more stable, and the concentrate depleted of active PAI-1 showed spontaneous regeneration of PAI-1 activity. In contrast, the PAI activity in HT 1080 medium concentrate declines rapidly on standing. 2) Hep G2 PAI-1 invariably copurified with an adhesive protein, vitronectin or its NH2-terminal fragment, while pure HT 1080 PAI-1 alone was obtained by affinity purification on anhydrourokinase-Sepharose 4B. 3) Based on specific activity measurement and complex formation analysis using a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis technique, the purified Hep G2 PAI-1 appears completely active while the HT 1080 PAI-1 is only one-fourth as active. SDS was found to exert dual effects on purified PAI-1s. SDS treatment partially inactivated a fully active Hep G2 PAI-1 and a moderately active HT 1080 PAI-1 but partially activated an HT 1080 PAI-1 whose activity had previously been allowed to decay to a very low level. Purified vitronectin was found to enhance and stabilize the PAI-1 activity of the partially active HT 1080 PAI-1. It is concluded that fully active PAI-1 in association with vitronectin can be isolated by anhydrourokinase-Sepharose 4B chromatography and that vitronectin is a binding protein for PAI-1 which activates and stabilizes PAI-1.

Carcinoma, Hepatocellular↗
PubMed1989

Ba2+ stimulation of luteinizing-hormone release demonstrates two mechanisms of Ca2+ entry in gonadotrope cells.

Kinetic studies on gonadotropin-releasing-hormone (gonadoliberin, GnRH)-stimulated luteinizing-hormone (lutropin, LH) release in the cultured rat gonadotrope demonstrated a biphasic pattern of LH release. The first rapid phase of release was unaffected by the voltage-gated Ca2+-channel blockers methoxyverapamil (D600) and nifedipine [a dihydropyridine (DHP)], whereas the later second phase was partially inhibited by both drugs. These results suggested that the initial phase of LH release is independent of Ca2+ entry through dihydropyridine (DHP)-sensitive Ca2+ channels and might depend on entry of extracellular Ca2+ by another mechanism. These mechanisms were further studied by utilizing Ba2+ as a Ca2+ substitute. Ba2+, which freely permeates DHP-sensitive Ca2+ channels in the absence of GnRH, induced LH release which was sensitive to blockade by D600 and nifedipine. However, in the presence of the channel blockers, Ba2+-induced LH release could be elicited when GnRH was added to the system. This indicates that GnRH stimulates LH release by initially activating a DHP-insensitive Ca2+-entry mechanism and then a DHP-sensitive mechanism. The DHP-sensitive mechanism freely allows Ba2+ entry in the absence of GnRH-receptor occupancy, whereas the DHP-insensitive mechanism requires GnRH-receptor activation for Ba2+ entry.

Animals↗
PubMed1989

Sodium dodecyl sulfate enhancement of quantitative immunoenzyme dot-blot assays on nitrocellulose.

Treating proteins with low concentrations of sodium dodecyl sulfate (SDS) and boiling for 2-3 min increased the linear range and total amount of protein that could be bound to nitrocellulose. Human serum albumin (HSA) and cathepsin G (Cat G) were both optimally bound at an SDS concentration of 10 micrograms/ml, while bronchial leukocyte proteinase inhibitor (BLPI) required 50 micrograms/ml SDS for optimum binding, corresponding to SDS-to-protein weight ratios of 0.5 and 2.5, respectively. Ionic strength and pH of the blotting buffers had a greater effect on the binding of SDS-treated proteins than on native proteins, with the linear binding range and total capacity for SDS-treated proteins being increased. Boiling SDS-treated human leukocyte extracts inactivated endogenous peroxidases, eliminating their interference with peroxidase-linked secondary antibodies in immunoassays. The nonionic detergents, Tween 20 and Nonidet P-40, were shown to rapidly wash both native and SDS-treated HSA off the filters, but these HSA samples were stable to washing with SDS. Although SDS-treated Cat G was more stable with nonionic detergents than was native Cat G, it was less resistant to washing with SDS. The substitution of SDS for nonionic detergents improved the response of immunoassays with native and SDS-treated proteins. Affinity-purified antibodies to human mast cell tryptase cross-reacted with native Cat G, but not with SDS-treated Cat G, indicating that SDS treatment can improve the specificity of immunoassays employing polyclonal antisera. These effects appear to be the result of partial denaturation and increases in the hydrophobicity of SDS-treated relative to native proteins.

Buffers↗
PubMed1989

Biosynthesis and secretion of enamel proteins in the rat incisor.

The synthesis and secretion of enamel proteins (EPs) in rat incisors was examined using cytochemical and biochemical methods. Radioautography after injection of 3H-methionine showed that ameloblasts in the presecretory, secretory, and maturation stages of amelogenesis actively synthesized and secreted proteins. Immunocytochemistry with an antibody to mouse amelogenins revealed the presence of EPs in the protein synthetic and secretory organelles of these cells at all three stages. Labeling was also found in elements of the endosomal/lysosomal compartment. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of proteins extracted from enamel and enamel organ showed several protein bands. However, transfer to nitrocellulose paper and immunoblotting revealed that most of the proteins recognized by the antibody were situated between approximately 14 and 32 kDa. EPs were further characterized by using lectins to examine their carbohydrate content. Lectin-gold cytochemistry on sections showed the binding of wheat germ agglutinin and Helix pomatia lectin to secretory stage enamel. Lectin blotting indicated that the amelogenins were heterogeneously glycosylated and contained the sugars N-acetyl-glucosamine/N-acetyl-neuraminic acid and N-acetyl-D-galactosamine. Fluorography at 6 and 10 min and 1 h after injection of 35S-methionine revealed four labeled bands in the main amelogenin group near 22, 28, 30, and 32 kDa. A short-lived protein of approximately 58 kDa was also observed primarily in cells. The appearance of labeled proteins in enamel was paralleled by their disappearance from cells and the intensity of the radiolabeled protein bands, both, in enamel and in cells, decreased towards the maturation stage. These data are consistent with the concept that ameloblasts produce multiple amelogenins throughout amelogenesis.

Ameloblasts↗
PubMed1989

Degradation and loss of matrix proteins from developing enamel.

The pattern and timing of the breakdown and loss of matrix proteins were studied in developing rat incisor enamel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography, radioautography, and in vitro incubations of proteins isolated from freshly dissected, crushed pieces of enamel. For biochemical studies, the technique of Robinson et al. (1974, 1977, 1983) was used to transect the enamel organ and enamel into a series of strips at 1 mm intervals along the length of the tooth. The proteins in each strip were extracted and either quantified by Lowry analysis or applied to 12% slab (enamel) or 5-15% continuous gradient (enamel organ) SDS-polyacrylamide gels and separated by electrophoresis. The biochemical studies indicated that the amount of protein contained within an enamel strip increased gradually by volume across the secretory stage, reached a peak early during the maturation stage, and then declined rapidly thereafter. The distribution of enamel proteins on SDS-polyacrylamide gels changed markedly throughout this period. These changes included increases and decreases in the intensity of staining of proteins at certain molecular weights (e.g., 18 kDa) and the appearance and disappearance of some proteins not seen clearly near the start of the secretory stage of amelogenesis (e.g., 32 and 10 kDa). Labeling studies with 35S-methionine suggested that the "stacked" arrangement of proteins typical of forming enamel (secretory stage) actually represented a very dynamic association of proteins, with new ones being added at the top of the stack and then breaking down with time to become those seen at lower molecular weights. Across the secretory stage, new proteins were always added to the top of the stack, but during early maturation this activity slowed dramatically, allowing the breakdown of aging proteins to be visualized more clearly. Radioautographic studies with 3H-methionine indicated that the breakdown of newly secreted proteins also was correlated with a movement of label from the site of secretion into deeper, previously unlabeled, areas of forming enamel. In vitro studies revealed that the rate and degree of breakdown of enamel proteins varied markedly, depending on the stage of amelogenesis from which the proteins were extracted. Secretory stage enamel proteins showed slow in vitro degradation with accumulation of proteins near 18 kDa. Early maturation stage enamel proteins showed more rapid breakdown with little accumulation of proteins near 18 kDa, whereas late maturation stage enamel proteins showed complete degradation by 2 days of incubation in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Ameloblasts↗
PubMed1989

A method for sampling the stages of amelogenesis on mandibular rat incisors using the molars as a reference for dissection.

A method for locating specific stages of amelogenesis on continuously erupting incisors was devised for rats weighing 101 +/- 5 g (n = 32). The technique is based on reflecting reference lines from the mandibular molars as perpendiculars to the labial surface of mandibular incisors. From these reference lines additional measurements are then made along the midline of the labial surface of the incisor in an apical or incisal direction to find the site desired for sampling. Histological studies on 24 decalcified incisors split into segments by using such reference lines and reconstructed by morphometry indicated that a reference line reflected from the contact point between the 2nd and 3rd molars crossed the enamel organ and adjacent enamel at 3,181 +/- 329 microns incisal to the start of the secretory zone of amelogenesis. A reference line from the 2nd and 1st molars crossed the enamel organ and enamel at 1,238 +/- 424 microns incisal to the start of the maturation zone of amelogenesis, while a reference line from the mesial side of the 1st molar crossed the enamel organ and enamel almost exactly where the enamel becomes completely soluble following prolonged decalcification in EDTA. Although reference lines were reproducible within a group of male rats having similar body weights, the linear distance between the apical end of the incisor and the point at which they crossed the tooth increased at a rate of 1 mm per 159 g for rats between 50 and 300 g body weight. This suggests that molars do not maintain a fixed relationship to incisors over time, and extreme care must be taken to standardize an experiment to a specific body weight when using this method.

Amelogenesis↗
PubMed1989

Cumulative dose-response curves for atracurium in patients with myasthenia gravis.

The potency of atracurium was determined in five patients with moderate to severe generalized myasthenia gravis undergoing thymectomy. Train-of-four stimulation was applied to the ulnar nerve and the force of contraction of the adductor pollicis was measured. Cumulative dose-response curves were obtained during thiopentone-nitrous oxide-fentanyl anaesthesia. The average time to complete the dose-response studies was 12.7 +/- 1.5 minutes. The ED50, ED90 and ED95 of atracurium were (mean +/- SEM) 0.07 +/- 0.01, 0.12 +/- 0.22, and 0.14 +/- 0.04 mg.kg-1, respectively. The time to 25 per cent first twitch recovery was 35 +/- 4 min following maximum blockade. Ten normal patients were studied in the same manner. Their ED50, ED90 and ED95 were 0.13 +/- 0.01, 0.21 +/- 0.02 and 0.24 +/- 0.03 mg.kg-1, respectively. These results demonstrated that, in patients with moderate to severe generalized myasthenia gravis, atracurium was 1.7-1.9 times as potent as in normal individuals.

Adult↗
PubMed1989

"Priming" with neostigmine: failure to accelerate reversal of single twitch and train-of-four responses.

Train-of-four stimulation can shorten the apparent onset time of neuromuscular blocking drugs. This study was designed to verify whether the same occurred with neostigmine-assisted recovery, and whether this apparent acceleration could explain the previously reported effectiveness of the priming technique for reversal agents. Fourteen adults received atracurium, 0.5 mg.kg-1, during a thiopentone-nitrous oxide-enflurane anaesthetic. The ulnar nerves of both arms were stimulated with train-of-four stimulation every 12 seconds until 1 per cent recovery of first twitch, at which time stimulation in one arm was switched to single twitch. When mean first twitch height reached 10 per cent of control, neostigmine, 0.04 mg.kg-1, was administered either as a single bolus, or as a "priming" dose of 0.01 mg.kg-1, followed 3 min later by 0.03 mg.kg-1. No statistically significant differences were observed between single twitch in one arm and first twitch height of the train-of-four in the other arm for the next 10 min. With priming, first twitch height was 45 +/- (SEM) 5 per cent at 5 min and 85 +/- 6 per cent at 10 min, compared with 72 +/- 5 per cent (p less than 0.05) and 91 +/- 2 per cent (NS) respectively without priming. Train-of-four ratio was 28 +/- 3 per cent at 5 min and 65 +/- 5 per cent at 10 min with priming, versus 53 +/- 4 per cent (P less than 0.05) and 73 +/- 3 per cent (NS) respectively without priming.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia Recovery Period↗
PubMed1989

The effect of a random initial value in neural first-passage-time models.

The effect of a random initial value is examined in several stochastic integrate-and-fire neural models with a constant threshold and a constant input. The three models considered are approximations of Stein's model, namely: (1) a leaky integrator with deterministic trajectories, (2) a Wiener process with drift, and (3) an Ornstein-Uhlenbeck process. For model 1, different distributions for the initial value lead to commonly observed interspike interval distributions. For model 2, a discrete and a uniform distribution for the initial value are examined along with some parameter estimation procedures. For model 3, with a truncated normal distribution for the initial value, the coefficient of variation is shown to be greater than 1, and as the threshold becomes large the first-passage-time distribution approaches an exponential distribution. The relationships among the models and between them and previous models are also discussed, along with the robustness of the model assumptions and methods of their verification. The effects of a random initial value are found to be most pronounced at high firing rates.

Action Potentials↗
PubMed1989

Circulatory effects of acute normovolaemic haemodilution in rats with healed myocardial infarction.

This study evaluated the haemodynamic response to different degrees of acute normovolaemic haemodilution with hetastarch at rest and during preload and afterload stress in 123 anaesthetised Sprague-Dawley rats divided according to cardiac status (sham operated, n = 46; no myocardial infarction, n = 47; large myocardial infarction, n = 30). Histological infarct size was 41 (SEM 2)%. Cardiac output was recorded using an electromagnetic flow probe. The preload test was achieved by rapid infusion of blood adjusted for packed cell volume, while the afterload test was achieved by an aortic clamp technique. Prior to haemodilution, rats with large myocardial infarction had lower (p less than 0.05) cardiac index, stroke volume index, left ventricular dp/dt, and oxygen delivery, and higher left ventricular end diastolic pressure compared with sham operated rats and rats with no myocardial infarction. Haemodilution to a haematocrit of 30% in all three groups resulted in a similar significant (p less than 0.05) rise in cardiac index and stroke volume index such that blood pressure was maintained despite the decrease (p less than 0.05) in peripheral resistance. Compared with sham operated rats, rats with large myocardial infarcts haemodiluted to packed cell volume of 20% had a more limited rise in cardiac index of 14 (SEM 6) v 45(12)%, p less than 0.05, and a greater fall in oxygen delivery of 50(5) v 28(9)%, p less than 0.05 which was associated with a higher left ventricular end diastolic pressure of 6.6(1.0) v 3.7(0.4) mm Hg, p less than 0.05. Following preload and afterload stress, rats with large myocardial infarcts had lower (p less than 0.05) left ventricular developed pressure, peak cardiac index and peak stroke volume index compared with sham operated rats and rats with no myocardial infarction, independent of packed cell volume. The data suggest that large myocardial infarctions impaired the ability of the left ventricle to pump blood and generate pressure. This diminished left ventricular performance was most apparent in response to preload and afterload stress and at the lowest level of haemodilution employed (packed cell volume 20%).

Animals↗
PubMed1989

The incidence of myocardial ischemia during anesthesia for coronary artery bypass surgery in patients receiving pancuronium or vecuronium.

This study was performed to compare the incidence of prebypass myocardial ischemia in patients receiving fentanyl and enflurane for anesthesia along with either pancuronium or vecuronium. Ninety-eight patients with normal left ventricular function were randomly allocated to receive either pancuronium 0.15 mg.kg-1 or vecuronium 0.15 mg.kg-1 in a double-blind manner after fentanyl 40 micrograms.kg-1 for induction of anesthesia for elective coronary artery bypass grafting (CABG). Premedication included diazepam 0.15 mg.kg-1 po, morphine 0.10 mg.kg-1, and scopolamine 0.005 mg.kg-1 im. Two lead Holter monitor recordings (leads V6 and V9) from the time of arrival in the operating suite to institution of cardiopulmonary bypass were analyzed for ischemia by a cardiologist blinded to the choice of muscle relaxant. Intraoperatively, heart rates greater than 90 beats.min-1 and systolic blood pressure +/- 20% of ward values were treated with propranolol, enflurane, or phenylephrine. Nitroglycerin was infused for ECG signs of ischemia or pulmonary hypertension. After induction of anesthesia the heart rate and cardiac index were consistently decreased in patients receiving vecuronium and also lower in these patients compared with those receiving pancuronium. Thirty-two per cent of patients receiving pancuronium received propranolol for heart rates greater than 90 beats.min-1 versus 7% of those who received vecuronium (P approximately 0.01). Eight patients developed 13 episodes of ischemia after administration of the muscle relaxant: four who received pancuronium (n = 44; 9%) and four receiving vecuronium (n = 54; 7%). Four episodes occurred at induction or tracheal intubation, two in each group. There were four perioperative myocardial infarctions as determined by ECG and CPK-MB levels, two in each group.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, Inhalation↗
PubMed1989

Dose-response relationships for edrophonium and neostigmine as antagonists of atracurium and vecuronium neuromuscular blockade.

To determine the potencies of edrophonium and neostigmine as antagonists of nondepolarizing neuromuscular blockade produced by atracurium and vecuronium, dose-response curves were constructed for both antagonists when given at 10% spontaneous recovery of first twitch height. Ninety ASA physical status 1 and 2 adults were given either 0.4 mg/kg atracurium or 0.08 mg/kg vecuronium during thiopental-nitrous oxide-enflurane anesthesia. Train-of-four stimulation was applied to the ulnar nerve every 12 s, and the force of contraction of the adductor pollicis muscle was recorded. When spontaneous recovery of first twitch height reached 10% of its initial control value, edrophonium (0.1, 0.2, 0.4, or 1 mg/kg) or neostigmine (0.005, 0.01, 0.02, or 0.05 mg/kg) was administered by random allocation. Neuromuscular function in another ten subjects was allowed to recover spontaneously. Assisted recovery was defined as actual recovery minus mean spontaneous recovery observed in patients who were not given antagonists. First twitch recovery was initially more rapid when vecuronium was antagonized compared with atracurium, but no difference was detected after 10 min. At 10 min the neostigmine ED80 was 0.022 +/- 0.003 (SEM) mg/kg after atracurium and 0.024 +/- 0.003 mg/kg after vecuronium. The edrophonium ED80 was 0.44 +/- 0.11 mg/kg with atracurium and 0.46 +/- 0.12 mg/kg with vecuronium, giving a neostigmine:edrophonium potency ratio of 20. Atracurium train-of-four fade could be antagonized more easily with edrophonium, whereas that of vecuronium was more easily antagonized by neostigmine. It is concluded that edrophonium and neostigmine are not equally effective against atracurium and vecuronium.

Atracurium↗
PubMed1989

Differential effects of pancuronium on masseter and adductor pollicis muscles in humans.

The sensitivity of the masseter, one of the muscles of the upper airway, to pancuronium was measured in ten adults undergoing elective surgery and compared with that of the adductor pollicis. During thiopental-nitrous oxide-enflurane (end-tidal concentration less than 0.25%) anesthesia, supramaximal nerve stimulation was applied to the ulnar nerve at the elbow and to the nerve to the masseter, at a point inferior to the zygomatic arch, anterior to the mandibular condyle. Jaw closure was measured by a force transducer system attached to both an oral airway and a metal frame fixed to the operating table 10 cm caudad to the chin. Cumulative dose-response curves for pancuronium (initial dose = 0.02 mg/kg, incremental doses = 0.01 mg/kg) were determined. Control twitch tensions were (mean +/- SEM) 473 +/- 75 g at the masseter and 660 +/- 118 g at the adductor pollicis. The masseter was slightly more sensitive to pancuronium, the ED50 being 0.024 +/- 0.001 mg/kg compared with 0.028 +/- 0.001 mg/kg for the adductor pollicis (P less than 0.05). Corresponding values for the ED90 were 0.038 +/- 0.004 and 0.043 +/- 0.002 mg/kg, respectively (P less than 0.05). The time from injection of the first dose of pancuronium to maximum blockade was 3.2 +/- 0.2 min at the masseter and 3.8 +/- 0.2 min at the adductor pollicis (P less than 0.01). Following incremental doses, this time was 1.8 +/- 0.1 and 2.6 +/- 0.1 min, respectively (P less than 0.01). It is concluded that after injection of pancuronium, neuromuscular blockade is greater at the masseter and occurs sooner than at the adductor pollicis. Jaw relaxation can be achieved with relatively small doses of pancuronium. This suggests that return of adductor pollicis function may not imply complete masseter muscle recovery.

Adult↗
PubMed1989

Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor.

Biochemical and histochemical studies have shown the presence of various carbohydrates in enamel. Using lectin-gold cytochemistry, we have examined the distribution of glycoconjugates containing N-acetyl-D-galactosamine (GalNAc) and/or N-acetyl-glucosamine (GlcNAc)/N-acetyl-neuraminic acid (NeuNAc) residues in rat incisor ameloblasts and in forming and maturing enamel embedded in Lowicryl K4M, LR Gold, and LR White resins. The enamel proteins that contain these carbohydrate moieties were further characterized by lectin blotting. All three resins allowed, albeit to a variable degree, detection of the binding sites for Helix pomatia agglutinin (HPA) and wheat germ agglutinin (WGA) GalNAc, and GlcNAc/NeuNAc, respectively. In general, Lowicryl K4M permitted more intense reactions with both lectins. Lectin binding was observed over the rough endoplasmic reticulum (weak labeling with WGA), the Golgi apparatus, lysosomes, secretory granules, and the enamel matrix. These compartments were shown by double labeling with WGA and anti-amelogenin antibody, and by previous immunocytochemical studies, to contain enamel proteins. Furthermore, WGA binding was more concentrated at the growth sites of enamel. Lectin blotting showed that several proteins in the amelogenin group were glycosylated and contained the sugars GalNAc and GlcNAc/NeuNAc. Fewer proteins were stained by HPA than by WGA, and the staining pattern suggested that the extracellular proteins recognized by these two lectins are processed differently. The HPA-reactive proteins were lost by or during the early maturation stage, whereas many of the WGA-reactive proteins persisted into the mid maturation stage. The heterogeneous staining of certain protein bands observed with WGA suggests that they contain more than one component. Two distinct glycoproteins containing GlcNAc/NeuNAc also appeared during the maturation stage. These results are consistent with the notion that ameloblasts produce an extracellular matrix composed mainly of glycosylated amelogenins which are differently processed throughout amelogenesis.

Acetylgalactosamine↗
PubMed1989

Distal first metatarsal epiphysis. A common pediatric variant.

While most anatomists have referred to a distal epiphyseal growth plate in the first metatarsal as rare or occasional, one definitive study has shown this accessory growth center to be extremely common in children under the age of 12 years. The authors discuss the significance of this phenomenon, and present a literary review, a clinical case report, and a retrospective radiographic study of 217 pediatric feet, confirming the incidence of the distal growth plate.

Child↗