Surfactant protein C: a review of its unique properties and metabolism. Traditionally, our thinking about surfactant proteins has centered around their effects on the biophysical properties of surfactant phospholipids. It is now apparent that the three major surfactant proteins (SP-A, SP-B, and SP-C) are a biochemically and functionally diverse group of mammalian peptides. Accumulated data suggest that they have roles beyond modulation of alveolar surface tension. SP-C is a 33-35 amino acid peptide found in organic extracts of pulmonary surfactant. In part, because of its extreme hydrophobicity, a full understanding of SP-C is presently incomplete. Progress to date has included evaluation of the biophysical properties and investigations of the SP-C gene, including studies of the SP-C promoter. This review describes the unique structural and functional properties of the SP-C molecule and summarizes available data on its molecular biology and metabolism. Studies from literature show that SP-C represents a physiologically important peptide with novel structural properties; namely, extreme hydrophobicity, an alpha-helical membrane spanning region, and a unique posttranslational modification: palmitoylation. From data on similarly modified proteins, we propose that the properties of SP-C, including the covalent addition of palmitic acid, render it capable of being targeted to and interacting with specific cell membranes. A complete understanding of SP-C, especially with regard to its metabolism and function, may require a reorientation of our thinking to consider SP-C as a membrane peptide and not just as a "surfactant protein."


Cigarette smoke-induced airway goblet cell secretion: dose-dependent differential nerve activation. We studied the effect of acute inhalation of middle-tar cigarette smoke on airway goblet cell secretion in anesthetized guinea pigs. Secretion induced by a low dose of smoke (10 breaths diluted 1:10 in air) was blocked by either hexamethonium or by filtering out the particulate phase of the smoke. The response was partially inhibited by atropine but was not inhibited by propranolol, phentolamine, or capsaicin pretreatment. Cutting the nerve supply to the airways did not inhibit the response to low-dose smoke. In contrast, goblet cell secretion induced by a high dose of cigarette smoke (20 breaths undiluted) was inhibited by capsaicin pretreatment but not by autonomic receptor blockade nor by filtering out the particulate phase. Secretion induced by the vapor phase of the high dose of cigarette smoke was blocked by capsaicin pretreatment but was not inhibited by hexamethonium. We conclude that in guinea pig airways the particulate phase of low doses of smoke activates cholinergic nerves via stimulation of parasympathetic ganglia, whereas the vapor phase of high doses of smoke activates capsaicin-sensitive sensory nerves.


Prostaglandin D2 production and identification of prostaglandin H synthase within canine mast cell granule. We have identified the presence of functional prostaglandin H synthase (PGH synthase, E.C. 1.14.99.1, or cyclooxygenase) within canine mast cell granules by demonstrating the generation of prostaglandin (PG) D2 from isolated and purified granules incubated with substrate as arachidonic acid or stimulated with calcium ionophore, A23187. This confirms the presence of both enzyme and substrate within the granule. Localization of PGH synthase to the granule was confirmed by immunoblotting of the pure granule preparation and by immunocytochemistry using the whole cell. In functional studies, colchicine, a microtubule polymerization inhibitor, caused a fall of up to 70%, both in the amount of histamine released and in the amount of PGD2 generated. This suggests either that functional PGH synthase is closely associated and coactivated with granules or that there is an independent association of this enzyme with the microtubule system. Release of the preformed and newly formed mediators of the mast cell appear to be closely linked, and prevention of degranulation may therefore attenuate the effects of both classes of mediators.


Structural and functional impairment of surfactant protein A after exposure to nitrogen dioxide in rats. The major surfactant protein A (SP-A) determines the dynamics of surfactant metabolism and function. We now for the first time studied the in vivo exposure of the SP-A to nitrogen dioxide (NO2) and compared it to in vitro exposure effects. Several properties that are believed to possess physiological functions were investigated. Exposure of rat SP-A to NO2 showed 1) a higher alveolar pool size after in vivo exposure, 2) a slightly decreased mannose binding capacity for the in vivo exposed form but a highly decreased binding after in vitro exposure, 3) an affected protein-lipid aggregation that was impaired most by the in vitro exposed protein, 4) a low inhibition for the secretion of phosphatidylcholine in isolated type II pneumocytes, and 5) no difference in apparent molecular weight and immunological reactivity of the differently exposed SP-A. The NO2-induced alterations of the SP-A may contribute to the pulmonary toxicity of this oxidant. It is clear from this study that the extend of the effects from in vivo and in vitro exposure are different. Therefore both forms of exposure are necessary to assess the biological significance of NO2 exposure.


Spontaneous production of PDGF A-chain homodimer by rat lung fibroblasts in vitro. Platelet-derived growth factor (PDGF) is considered a decisive mediator of fibroblast growth and phenotype within the lung. The cellular sources of PDGF within the lung remain undefined. The ability of lung fibroblasts themselves to produce PDGF in vitro was therefore investigated. Northern and Western blot analyses revealed the expression of PDGF-A mRNA and secretion of A-chain containing proteins by fibroblasts derived from adult and fetal rat lung. PDGF-A gene or protein expression were below the limits of detection in two human lung fibroblast lines examined in a similar manner. PDGF-B transcripts or proteins were not detected in any lung fibroblast line examined. Conditioned medium (CM) was collected from these same lung fibroblast lines and tested for its ability to promote cell growth using human fetal lung fibroblasts as targets. Both adult and fetal rat lung fibroblasts were found to produce a potent and efficacious stimulus for cell growth. Growth-promoting activity in rat fibroblast-derived CM functioned as a "competence" factor and was partially inhibited by anti-PDGF antibody. Thus rat lung fibroblasts in vitro produce potent growth factors of which at least one appears to be PDGF-AA. Differences in the expression of PDGF-AA between rat and human lung fibroblasts exist. Growth factor-producing fibroblasts may play a role in lung repair and remodeling through production PDGF-AA in vivo.


L-arginine restores endothelium-dependent relaxation in pulmonary circulation of chronically hypoxic rats. We investigated whether loss of endothelial-derived relaxing factor (EDRF) activity in the pulmonary vessels of chronically hypoxic rats could be restored by pretreatment with L-arginine. We measured vasodilation to acetylcholine (ACh), calcium ionophore A23187, or linsidomine (Sin-1) under conditions of increased vascular tone induced by U-46619 (50 pmol/min), as well as vasoconstriction to endothelin-1 (ET) in isolated lungs pretreated with meclofenamate (3 microM). In lungs from normoxic (N) rats, in vitro L- or D-arginine (10(-3) M) did not alter vasodilation to the endothelium-dependent agents ACh (10(-9)-10(-6) M) and A23187 (10(-9)-10(-7) M), but NG-monomethyl-L-arginine (10(-3) M) completely abolished it. In lungs from rats exposed to 3 wk of hypoxia (H), vasodilation to ACh or A23187 was fully restored after in vitro L-arginine (10(-3) M) or N alpha-benzoyl-L-arginine (5 x 10(-5) M) but remained abolished after D-arginine, L-citrulline, L-ornithine, or L-argininosuccinic acid. In vivo pretreatment of H rats with L-arginine (300 mg/kg iv) 30 min before isolating the lung also restored vasodilation to A23187. Vasodilation to the endothelium-independent agent Sin-1 was similar in both groups of lungs and was not altered by in vitro L-arginine. L-arginine attenuated the increased pressor response to ET (300 pmol) of H rat lungs but had no effect in N rats. Our results demonstrate that loss of EDRF activity associated with hypoxic pulmonary hypertension may be reversed by supplying L-arginine.


Localization of alveolar surfactant clearance in rabbit lung cells. Localization of surfactant phospholipid clearance in lung cells was investigated in vivo in rabbits using radiolabeled dipalmitoylphosphatidylcholine (DPPC) and 1,2-dihexa-decyl-sn-glycero-3-phosphocholine (DPPC-ether), a phospholipase A1- and A2-resistant analogue of DPPC. After intratracheal injection of liposomes of the labeled lipids associated with unlabeled surfactant, adult rabbits were killed in groups of three to five at 0, 4, 12, and 24 h with recovery of bronchoalveolar lavages for alveolar macrophages and surfactant. Type II cells and tissue-associated macrophages were isolated on Percoll gradients following elastase and trypsin digestion of the lungs. Radiolabel recoveries as saturated phosphatidylcholine were measured in alveolar wash, alveolar macrophages, lung tissue, and the type II cell and mixed cell bands from the Percoll gradients. Cost accounting of label demonstrated similar recoveries at 0 h, but significantly more DPPC-ether compared with DPPC in cells at later times, indicating ineffective degradation of the DPPC-ether. Internalization of the lung tissue-associated labels into cells was time dependent. At all times, greater than 65% of the cell-associated labels were recovered in type II cells, indicating the primary role for these cells in clearing alveolar surfactant phospholipid in vivo. The total contribution of alveolar macrophages to the overall clearance was approximately 20%.


Pulmonary SP-A enhances adsorption and appears to induce surface sorting of lipid extract surfactant. The effect of surfactant concentration and supplementation with surfactant-associated protein A (SP-A) on the surface activity of lipid extract surfactant (LES) was examined using a captive bubble technique. Adsorption of LES is strongly concentration dependent over the range of 50-1,000 micrograms/ml. Addition of SP-A to LES at low concentrations in the presence of calcium dramatically increases the rate of adsorption. In quasistatic cycling experiments, samples containing SP-A require less compression to achieve low surface tensions even during the first compression cycle. Calculated film compressibilities at 15 mN/m indicate that SP-A alters the surfactant monolayer behavior such that in a small number of cycles the compressibility is indistinguishable from that of pure DPPC. Furthermore, SP-A reduces the incidence of bubble "clicking," suggesting a stabilization of the monolayer at low surface tensions. In dynamic cycling experiments, SP-A reduces compression of the film area required to achieve a low surface tension of approximately 1 mN/m. SP-A eliminated the plateau just below 25 mN/m normally observed during the compression phase with low concentrations of LES and the shoulder observed at approximately 35 mN/m during expansion. In the presence of SP-A and, to a lesser extent with high concentrations of LES, there is a marked lag in the increase in surface tension during the initial part of the dynamic expansion loop, with surface tensions remaining near 1 mN/m for approximately 10% of the increase in bubble area. The results indicate that SP-A enhances phospholipid adsorption during dynamic cycling and may enhance elimination of non-DPPC lipids during cycling.(ABSTRACT TRUNCATED AT 250 WORDS)


Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+i and inositol polyphosphates. The temporal relationship between the alpha-thrombin-induced increase in transendothelial permeability and the alpha-thrombin-mediated changes in several key transmembrane signaling events was examined in confluent monolayers of bovine pulmonary artery endothelial cells (BPAEC). The time courses of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] generation, changes in cytosolic [Ca2+] ([Ca2+]i), and reorganization of cytoskeletal F-actin were determined to assess the relationship between these events and the onset of the alpha-thrombin-induced increase in endothelial permeability. alpha-Thrombin (10(-7) M) increased the transendothelial 125I-albumin clearance rate half-maximally by approximately 1 min and maximally by approximately 2 min (160% over control level). The increase in permeability occurred concomitantly with reorganization of F-actin cytoskeleton (i.e., loss of peripheral band and increased stress fiber density) and increased actin polymerization. Stimulation of fura-2-loaded BPAEC with 10(-7) M alpha-thrombin produced a typical biphasic rise in [Ca2+]i. The initial rapid increase in [Ca2+]i peaked by approximately 16 s after thrombin challenge and the [Ca2+]i response showed a slow decrease to half-maximal within 50 s. The alpha-thrombin-induced increase in permeability as well as the increase in [Ca2+]i were consistently preceded by increased Ins(1,4,5)P3 generation detectable within 10 s after thrombin challenge. These results indicate that alpha-thrombin triggers a cascade of events (i.e., Ins(1,4,5)P3 generation and the ensuing rise in [Ca2+]i), which may comprise the second messengers that mediate F-actin reorganization and the increase in endothelial permeability.


Filter paper equilibration as a novel technique for in vitro studies of the composition of airway surface fluid. We describe techniques which we developed to study the composition and regulation of airway surface fluid (ASF). ASF from isolated equine tracheal mucosa was absorbed onto or equilibrated with small strips of ashless filter paper after timed incubations in a water-saturated, 37 degrees C environment. After expression of the fluid from the paper, constant-volume aliquots of the samples were pipetted with a microvolumetric pipette on a filmed grid, together with standards containing known concentrations for Na, Cl, K, Ca, S, and P. Analysis of the microdroplets by dispersive X-ray microanalysis on a scanning electron microscope demonstrated that ASF from "unstimulated" tracheal tissue is significantly altered compared with the Ringer solution which was used to rinse the mucosal surface before incubation, in that Na is significantly decreased (approximately 130 mM), whereas K is significantly increased (approximately 16 mM). Furthermore, when a beta-adrenergic agonist (isoproterenol 10(-4) M) was added to the serosal surface, the Na concentration significantly increased to values which approached Ringer solution (146 +/- 5.3 mM) and were significantly different compared with the Na values obtained in unstimulated tissues. In contrast, Na remained unchanged after addition of a cholinergic agonist (metacholine, 10(-4) M) to the serosal side of the tissue; however, as with beta-adrenergic stimulation, most other minor elements and especially K significantly decreased compared with their values in unstimulated tissue. These results clearly suggest that ASF composition is clearly controlled by active transport of the airway epithelium and may be influenced by neurohumoral stimulation.


Influences of endogenous and exogenous TGF-beta on elastin in rat lung fibroblasts and aortic smooth muscle cells. The factors that regulate elastin production during neonatal lung development have not been elucidated. Previous investigations suggested that transforming growth factor-beta (TGF-beta) increases elastin production by neonatal rat lung fibroblasts (LF). We examined whether this effect of TGF-beta was unique to these cells or was evident in other neonatal cells, which constitutively produce elastin, or in cells from adults, whose constitutive elastin production is low. We have quantitated soluble elastin, elastin mRNA, and TGF-beta production in primary cultures of smooth muscle cells (SMC) and LF from neonatal and adult rats and have examined the alterations in soluble elastin and elastin mRNA that result from adding 100 pM exogenous TGF-beta 1 to these cultures. Unsupplemented cultures of LF and SMC obtained from neonatal rats exhibited higher steady-state levels of elastin mRNA and contained more soluble elastin in their culture medium than did cells from adult animals. When neonatal LF were supplemented with 100 pM TGF-beta 1, they showed a significant increase in the soluble elastin content of their culture medium and their steady-state elastin mRNA. Neither LF obtained from adults nor SMC obtained from neonatal or adult rats significantly increased their soluble elastin or steady-state elastin mRNA after the addition of exogenous TGF-beta. When neonatal LF were supplemented with an anti-TGF-beta neutralizing antibody, the soluble elastin content of the culture medium decreased significantly. These data suggest that the responsiveness of elastin expression to TGF-beta is limited to neonatal LF and that endogenous TGF-beta influences elastin production by neonatal LF.


Surfactant proteins and lipids are regulated independently during hyperoxia. Adult hamsters were exposed to 100% oxygen for up to 8 days. At time of death lung tissue was analyzed for the expression of surfactant protein (SP) genes, and surfactant was isolated from alveolar lavage fluid. Surfactant was analyzed for the composition of proteins and phospholipids and for its surface properties. We found, over the 8 days of exposure, that an alveolitis composed of polymorphonuclear leukocytes (PMNs) and alveolar macrophages, accompanied by exudation of edema fluid, appeared in the alveolar spaces. The steady-state levels of SP mRNAs declined after 8 days of exposure to 100% oxygen, but the patterns indicated individual genetic control. SP-A was elevated early in the course of the hyperoxic exposure but decreased significantly by day 8; SP-B decreased continuously; SP-C was unchanged (or slightly elevated) through day 2 and then declined. The amounts of recoverable lavage surfactant increased by greater than threefold, and the phospholipid composition showed increasing percentages of disaturated phosphatidylcholine. All surfactants lowered surface tension to less than 10 dyn/cm, but the adsorption rates decreased as exposure progressed. The results indicate that lung injury induced by 100% oxygen is accompanied by altered patterns of surfactant metabolism, possibly because of a changing type II cell phenotype or alterations in Clara cell-derived surfactant. These changes may result in perturbed physiological function contributing to decreased lung compliance.


Near-infrared spectroscopy in newborn infants. Neonatal encephalopathy of early onset, plausibly related to hypoxia and ischemia remains one of the main problems in perinatal medicine. Efforts are necessary to find new non-invasive methods for assessing brain oxygenation. Near-infrared spectroscopy (NIRS) provides information on the concentrations of the oxygenated and reduced forms of hemoglobin, as well as the redox state of cytochrome aa3. Different important variables can be derived through hemoglobin measurement, such as cerebral blood volume and flow, and the responses of these to changes in pCO2. Changes in cytochrome aa3 may provide immediate information on intracellular oxygen utilization. Various studies have shown the feasibility of NIRS in preterm infants. Methodological and technical problems of this method are discussed.


Bromide treatment of pharmaco-resistant epilepsies with generalized tonic-clonic seizures: a clinical study. In a retrospective controlled clinical study we investigated the efficacy of bromides (BR) as to the frequency of generalized tonic-clonic seizures (GTCS) as a monotherapy (n = 5) or combined with other antiepileptic drugs (AEDs) (n = 55), and compared the results statistically with those achieved with a treatment based on phenobarbitone (PB) and/or phenytoin (PHT) in a very similar group of another 60 patients. All patients treated with BR had previously proved to be pharmaco-resistant. In most of the cases the epilepsies were based on early cerebral lesions, and were partly associated with severe mental and physical handicaps. Most of the patients were between three and 14 years old. The percentage of responders was 58%, which means a reduction in the GTCS-frequency of more than 50%. The positive result was observed for 28 months on the average. 27% of all patients became free of GTCS for 18 months on the average (range, 44 days to 62 months), and 32% were improved (reduction in GTCS-frequency of more than 50% while not GTCS-free) for nearly three years on the average (range, 79 days to 110 months). Compared to PHT or PB, BR were slightly more efficient and similarly tolerable, but no statistically significant difference was seen (48% responders in the control group versus 58% responders in the BR-group). Thus, the statistical correlation with competitive therapies confirmed the good efficacy of BR against GTCS, which was previously demonstrated by clinical tests without control studies. We recommended BR in the case of every pharmaco-resistant epilepsy with GTCS as a therapeutic alternative.


Occipital deep white matter hyperintensity as seen by MRI: 1. Clinical significance. Magnetic resonance imaging was performed in 270 patients with various neurologic complaints (1-15Y) with a 0.5 tesla superconducting imaging system using a field echo T1-weighted sequence and spin echo T2-weighted and PD-weighted sequences. Twenty-seven of them had deep white matter hyperintensity (DWMH) in the occipital lobe on T2-weighted images. The frequency of mild DWMH differed in different age groups, suggesting that mild DWMH may result from delayed myelination in the central nervous system. However, the frequency of severe DWMH, which was revealed as isointense relative to cerebrospinal fluid, did not differ in different age groups and it was significantly more common in severely retarded patients. Classification of DWMH based on the signal intensity is valuable to distinguish white matter abnormalities in the occipital lobe from delayed myelination in the same site.


Multimodality evoked potentials in severe athetoid cerebral palsy: correlation with clinical features and all-night polygraphical data. We examined the correlation between the clinical and electrophysiological features, short latency somatosensory evoked potentials (SSEPs), auditory brainstem responses (ABRs), electrically elicited blink reflexes (BRs) and all-night polysomnographical examination (PSG) data in eight patients with severe athetoid cerebral palsy (ACP). Absence ABRs were observed in cases who had suffered from severe neonatal hyperbilirubinemia (posticteric ACP), and in most of them gaze abnormalities and a significant reduction in rapid eye movements during REM sleep, as observed on PSG, coexisted. Prolongation of the interpeak latency, N13-N20, of SSEPs existed concurrently with disturbed late components of BRs in two cases of posticteric ACP. The phasic contractions of the submental muscle during sleep were impaired in most of the patients. Multimodality evoked potentials together with PSGs seem to be useful for assessing brainstem dysfunctions in ACP and might also be of use for elucidating the pathogenesis of the episodic sudden death in ACP.


Usefulness of PET scan in a child with mesial frontal lobe epilepsy. Positron emission tomography (PET) scan with 18F-fluorodeoxyglucose (18F-FDG) was performed in a 14-year-old boy who had seizures suspected to have originated in mesial frontal lobe. The seizures occurred in clusters and were characterised by a change in the facial expression at seizure onset and complex motor manifestations consisting of kicking, swaying and screaming. Ictal EEG showed rhythmic alpha-waves in the left frontal area association with the ictus. Cerebral CT, MRI and SPECT revealed nothing of significance, but the PET brain scans showed frontal and parietal hypometabolism, which was most prominent in the left mesial frontal lobe. The present case suggests that FDG-PET scanning may be useful for the diagnosis of the mesial frontal epilepsy, when other imaging studies fail to show abnormalities.


A case of myasthenia gravis complicated with hyperthyroidism and thymic hyperplasia in childhood. We report here a case of myasthenia gravis complicated with hyperthyroidism and thymic hyperplasia. The patient was a 13-year-old girl with struma and hyperthyroidism which began at age 12. Two weeks following the initiation of treatment against hyperthyroidism she developed left blepharoptosis, diplopia, and dysphagia, which responded promptly to edrophonium administration. An increase of the anti-acetylcholine receptor antibody was found in the serum. A chest CT showed a large soft tissue mass in front of the ascending aorta, which was proven histopathologically as thymic hyperplasia. The patient underwent an extensive thymectomy and was placed on combination therapy with an anti-thyroid drug, glucocorticosteroid, and an anti-cholinesterase drug. Her symptoms and signs have been well controlled by this treatment. Coexistence of myasthenia gravis, hyperthyroidism, and thymic hyperplasia in childhood have never been documented in literature.


Infantile polymyositis: a case report.

Infantile polymyositis: a case report. A 10-month-old Japanese boy developed progressive muscle weakness and hypotonia at 3 months of age. Because of striking inflammatory cellular infiltration in his muscle biopsy, he was diagnosed as having infantile polymyositis and was placed on steroid and immunosuppressive medication when he was 10-month-old. His physical condition was not significantly altered, though serum creatine kinase (CK) level was normalized (1,500 iu/l----90 iu/l). These findings contrast with previous reports documenting improvement with steroid administration.


Microgyric and necrotic cortical lesions in twin fetuses: original cerebral damage consecutive to twinning? Extensive cortical necrosis associated with malformative microgyric-like lesions and with necrotic lesions of the white matter was observed in two male 25 week fetuses. These cases differed from previously reported cases of brain damage in monozygotic twins: both fetuses were affected and the lesions occurred early in pregnancy, before the end of neuronal migration, thus resulting in a cortical malformation associated with destructive lesions.


Abnormal metabolism of carnitine and valproate in a case of acute encephalopathy during chronic valproate therapy. We analyzed the urinary metabolic profiles of valproate (VPA) and carnitine metabolism in an epileptic patient who died of acute encephalopathy during VPA therapy. On admission, the serum free carnitine level was greatly decreased and gas chromatographic mass spectrometric analysis of organic acids in urine showed a complete lack of beta-oxidation metabolites of VPA, while omega-oxidation was markedly increased. After administration of L-carnitine, the levels of acylcarnitine in both serum and urine, and of serum free carnitine increased, and the metabolites of beta-oxidation appeared in urine, while there was no improvement in the liver and renal functions. This is not a typical case of VPA-induced hepatotoxicity and the main cause of the disease is not clear. But the results show that the mitochondrial beta-oxidation of VPA was greatly disturbed in this patient, which may be related to the carnitine deficiency induced by the chronic VPA-therapy.


The more unusual sleep disturbances of childhood. The sleep patterns of children often cause anxiety to their parents. Some disturbances are unusual, and therefore may cause diagnostic difficulties. Sleep walking and night terrors can be confused with epileptic seizures. The sudden sleep of narcolepsy can lead to false accusations, when in fact the episodes are beyond the child's control. The associated phenomena of cataplexy, hypnogogic hallucinations and sleep paralysis can be particularly alarming, especially if they occur in the absence of narcolepsy. The overlap between narcolepsy and the Kleine-Levin syndrome is confirmed. Although of a different nature the sleep apnoea syndrome is equally important from the point of view of diagnosis and treatment.


Electrophysiological study on hydranencephaly. An electrophysiological study was performed on 2 children with hydranencephaly diagnosed by CT and/or MRI. Case 1 was a 4-month-old boy who had no rostral tissues above the midbrain. Case 2 was a 5-year-old boy in whom CT showed retention of the thalamus. Short latency somatosensory evoked potentials (SSEP) in both cases exhibited the absence of cortical activity (N1 and P4) with the preservation of waves of brain stem origin. However, in case 1, wave component No was not observed, while No was seen in case 2. It was postulated, thus, that the No component of SSEP on median nerve stimulation in children, which corresponds to N16 in adults, may originate in the thalamus.


A comparative study of four different bronchial challenge tests. The correlation between 4 commonly used tests for assessment of airway hyperresponsiveness was studied in 10 asthmatics by performing bronchial challenges with histamine, ultrasonically nebulised distilled water (UNDW), exercise, and a relevant allergen. All tests were performed within 4 weeks for each individual and at least 48 h apart. A significant correlation was found between histamine challenge and exercise challenge (r = -0.74). Other correlations were substantial (0.4-0.5) but did not reach statistical significance. This suggests that the challenges have different mechanisms or pathways leading to bronchoconstriction, and assessment of the pattern of individual patients' bronchial hyperresponsiveness may require application of a number of different bronchial challenge tests. For routine use histamine challenge seems the most appropriate.


Enhanced production of platelet activating factor by peripheral granulocytes from children with asthma. Granulocytes from 23 asthmatic children aged 4-15 years and 32 age-matched healthy children were studied. Cells were purified by Dextran sedimentation and Percoll gradient centrifugation from heparinized blood. After in vitro stimulation by ionophore A23187 the amount of newly synthesized PAF and LTC4 was assessed by radio receptor assay or radioimmunoassay respectively. Eight patients had symptoms of asthma within the last 3 weeks before examination. Granulocytes from the symptomatic patients showed a significantly higher PAF generation (median 125 ng/10(6) cells, range 7-189 ng/10(6) cells) when compared to asymptomatic patients (p less than 0.001, median 14 ng/10(6) cells, range 6-33 ng/10(6) cells) or controls (p less than 0.001, median 11 ng/10(6) cells, range 3-26 ng/10(6) cells). In contrast, LTC4 generation was increased in both patient groups. The results suggest a regulatory role of PAF in the exacerbation of asthma.


Natural history of bronchial asthma in childhood. A prospective study from birth up to 12-14 years of age. In a cohort of 1654 consecutively born children followed from birth, the cumulated incidence of asthma up to 11 years was 5.3% and the prevalence 3% between 10 and 11 years of age. The asthma début was prior to 1.5 years in 1/3 and before 3 years in 1/2 of the children. Among 59 children examined at 11.5-14.5 years of age, 16 (27%) had no prevalent asthma. On the other hand, inadequate medication was found in 13, undiagnosed chest deformity in four, and wheezing in seven children. All 54 tested children including those with no prevalent asthma had a PC20 histamine less than 8 mg/ml. Tread mill test gave a significant reaction in 15 children. Animal danders and pollens were the most common allergens giving reactions at skin prick test. Compared to one earlier Swedish study an increase in mite sensitivity was found. Cord-blood IgE concentration and a positive immediate family history of atopic disease had no predictive value for the severity of asthma at this follow-up. A new total asthma score including number of days with functional impact of asthma during the last year and present medication was used for group comparisons. Children with a high score at 11.5-14.5 years had more exercise-induced asthma, more concomitant allergic diseases, earlier asthma start, more chest deformity, hyperreactivity both on tread mill and histamine challenge tests, elevated IgE, positive Phadiatop and more reactions at skin prick test, especially to animals and mites. Bronchial asthma was found more often in children born during August through October, possibly due to unsuitable indoor climate and more virus infections during their first 6 months of life. The severity of the asthma was, however, not influenced by the month of birth. No significant differences were found between boys and girls regarding the age at début, asthma severity at follow-up, or bronchial histamine threshold levels.


Effect of inhaled formoterol versus terbutaline on respiratory function in moderate bronchial asthma. In a double-blind cross-over study comparing the duration of action of 12 micrograms and 24 micrograms formoterol, 500 micrograms terbutaline and placebo, lung function tests were performed in 12 never-smokers with non-allergic bronchial asthma. All the patients were hyperreactive to methacholine, had normal serum IgE level, and negative skin or RAST results. The lung function tests were carried out over an 11-h period and included measurements of lung volumes, airway resistance, dynamic spirometry, nitrogen single breath wash-out test and single breath diffusion capacity for CO. The airway resistance and maximal forced expiratory flow tests indicated a pronounced bronchodilator effect without adverse effects of 12 micrograms and 24 micrograms formoterol on both large and peripheral airways. Improved intrapulmonary gas distribution measured by the nitrogen wash-out test and more even distribution of the lung volumes indicating more homogeneous ventilation was seen for approx. 3 h after terbutaline and approx. 11 h after 24 micrograms formoterol. Measurement of the CO diffusion capacity indicates more effective gas diffusion and a better ventilation-perfusion ratio for at least 11 h, especially after inhalation of 24 micrograms formoterol.


Genetic control of eosinophilia in guinea pig strains inbred for high or low bronchial allergic reactivity. 2. A genetic study of spontaneous and immunization-induced eosinophilia. In 4 inbred strains of guinea pig the tendency to develop peripheral high-eosinophilia was shown to be genetically controlled. The development of eosinophilia with age and following immunization was examined in high- and low-eosinophilic parental strains, in F1-hybrids and in backcross offspring. The results show that probably only one or a very few segregating genes control eosinophilia, and they also indicate that different genes are involved in the determination of spontaneous (age-related) and immunization-induced eosinophilia.


Histamine-induced bronchoconstriction in conscious guinea pigs measured by strain-gauge transduction. A new method. A new objective method for measuring histaminedihydrochloride-induced bronchoconstriction in conscious guinea pigs has been developed. The aim of the present work was to investigate whether identical results could be obtained when the clinical observation of conscious guinea pigs with symptoms of respiratory distress (bronchoconstriction) was compared to an objective measuring technique of this parameter. An evaluation of the repeatability of the method was made and the respiration frequence (fR) and histaminedihydrochloride challenge concentration were compared to see whether there was a correlation between the two. Consequently, an apparatus was built that allowed simultaneous recording of breathing pattern and clinical observation of the animal being challenged. The breathing pattern was recorded by a strain-gauge transducer, connected to a measuring bridge, and the curves obtained on a jet ink x-y writer were used for calculating changes in duration of expiratory phase (Te) and fR. During the attacks of histaminedihydrochloride-induced bronchoconstriction a significantly prolonged Te could be calculated from the respiration curves. A high degree of agreement was found between this objective measure and clinically observed respiratory distress. The repeatability of the method was comparable to that of corresponding methods used for histaminedihydrochloride challenge in man. No obvious correlation was found between changes in fR and histaminedihydrochloride challenge concentration.


High-dose inhaled steroids in the management of asthma. A comparison of the effects of budesonide and beclomethasone dipropionate on pulmonary function, symptoms, bronchial responsiveness and the adrenal function. The efficacy of budesonide (800 micrograms b.d.) and beclomethasone dipropionate (750 micrograms b.d.) in controlling the symptoms of asthma, pulmonary function, bronchial responsiveness to histamine, and adrenal function, was assessed in a double-blind, double-dummy cross-over study of 36 adult chronic asthmatic patients. The patients, the majority of whom were assessed to be affected to a severe degree, were insufficiently controlled in their current regimen of inhaled steroids and/or inhaled and oral bronchodilators. A 2 weeks baseline period preceded 6 weeks of treatment with each of the study drugs. Both treatment groups showed improvements from baseline in clinical assessment of lung function carried out after the first 6 weeks of treatment. No significant differences were seen throughout the entire 12 weeks study, when comparing the effects of the treatments on FEV1, FVC, PEF or the histamine PC20. Asthma severity, symptom score and inhaled bronchodilator use showed the same results after both treatments. It is concluded that inhalations of budesonide and beclomethasone dipropionate in high doses are equally potent in the treatment of severe asthma. There is no significant influence on the adrenal function and no significant side effects during a period equal to that of the present study.


Bronchial asthma induced by chick pea and lentil. Allergic reactions to legumes through inhalation have rarely been described. We report the case of a 20-year-old man who experienced asthmatic attacks when exposed to the steam from cooking either chick pea or lentil. Type I hypersensitivity to the antigens in these legumes was demonstrated by means of immediate skin reactivity, histamine release tests, RAST and RAST inhibition. Specific bronchial challenges with the heated (75 degrees for 30 min) extracts of chick pea and lentil elicited isolated immediate responses.


Involvement of arachidonate cyclooxygenase products in bronchial hyperresponsiveness induced by subthreshold concentration of aerosolized thromboxane A2 analogue (STA2) in guinea pigs. Effects of a thromboxane synthetase inhibitor (OKY-046) and a cyclooxygenase inhibitor (indomethacin) on bronchial hyperresponsiveness induced by subthreshold concentration of aerosolized thromboxane A2 analogue (STA2) were investigated in anesthetized, artificially ventilated guinea pigs in order to examine the role of the cyclooxygenase pathway in bronchial hyperresponsiveness. Pretreatment with aerosolized OKY-046 significantly inhibited the bronchial hyperresponsiveness to histamine, but pretreatment with intravenous indomethacin showed a tendency to potentiate bronchial hyperresponsiveness. These results suggest that subthreshold concentration of thromboxane A2 contributes to bronchial hyperresponsiveness through activating the cyclooxygenase pathway including thromboxane A2 synthesis, and that the released cyclooxygenase products have an inhibitory effect on the bronchial hyperresponsiveness in guinea pigs.


Development of increased airway responsiveness in two nurses performing methacholine and histamine challenge tests. Two nurses who frequently examined patients with methacholine or histamine challenge tests developed increased airway responsiveness and symptoms of asthma. It is important to use a good system for evacuating the test aerosol during challenge tests.


Occupational asthma in the confectionary industry caused by sensitivity to egg. We report on a patient with asthma induced by occupational exposure to egg used to spray cakes before baking. A type I hypersensitivity to egg white was demonstrated by means of skin test, immunoassay for specific IgE, and immediate bronchial provocation test response to an egg white extract.


Optimization and validation of analytical conditions for bovine serum albumin using capillary electrophoresis. A quick and reproducible capillary electrophoresis method was optimized and validated for the assay of bovine serum albumin (BSA). The effects of various parameters such as pH of buffer, concentration of buffer, capillary dimensions, use of coated capillaries, and additives such as surfactants and protein solubilizers were evaluated. The capillary coatings or additives did not give any advantage in reducing the surface adsorption of BSA on the capillary walls. The optimized conditions include use of borate buffer, pH 8.5 having a concentration of 150 mM in a 27 cm capillary with an aperture window of 100 x 200 microns for detection. The optimized method for the detection of BSA was validated. The interday and intraday coefficient of variation was not greater than 7.59% at BSA concentrations of 25-1000 micrograms/ml. The method developed was reproducible and accurate.


Coomassie blue protein dye-binding assays measure formation of an insoluble protein-dye complex. The solubility of the protein-Coomassie brilliant blue (CBB) complex formed upon Bradford (Anal. Biochem. 72, 248-254, 1976) or Sedmak and Grossberg (Anal. Biochem. 79, 544-552, 1977) protein assay has been investigated by centrifugation or filtration of the assay mix within 10 min of adding dye reagent. The results show complete loss of color yield in the respective supernates and filtrates. This indicates that the protein-CBB complexes are insoluble at the time of absorbance measurement. Protein solubility in the dye reagent may dictate the relative response of the assay to an individual protein and the requirement for macromolecular structure.


Fluorescent rotors and their applications to the study of G-F transformation of actin. New fluorescent rotor molecules having hydrophilic functional groups, which are derivatives of p-(N,N-dialkylamino)benzylidenemalononitrile, were synthesized. Their properties as fluorescent rotors were confirmed by an observation of solvent viscosity-dependent fluorescence. Incorporation of hydrophilic groups into the molecules increased the solubility of fluorescent rotors in aqueous media; the application of the compounds to biochemical systems became feasible as a consequence. To demonstrate this applicability, we attempted to monitor the G-F transformation of rabbit skeletal muscle actin with these newly synthesized compounds. All the compounds carrying a malononitrile moiety showed greater fluorescence in F-actin. Among them, 1-(2-hydroxyethyl)-6-[(2,2-dicyano)vinyl]-2,3,4-trihydroquinoli ne gave the best result by the criteria of the difference in fluorescence quantum yield for G- and F-actin, solubility, and stability of the compound. The method has the major advantage of not requiring covalent modification of actin.


A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. Glucose metabolism in yeast can be stopped within 0.1 s by spraying the cells in 60% methanol at -40 degrees C. With this procedure the integrity of the cells is not damaged. Using stopped-flow equipment for the incubation with glucose, major changes within a second are shown to occur in intracellular glucose-6-phosphate whereas the fructose-1,6-bisphosphate concentration remains constant. After quenching, the cells can be separated from the medium, washed with cold methanol when required, and extracted using chloroform at -40 degrees C at neutral pH, ensuring minimal degradation of labile metabolites. With partly automated enzymatic methods, a large variety of metabolites, including all glycolytic intermediates, can be determined in the neutral extracts. During the first second after addition of glucose, a significant increase in free intracellular glucose is found.


High-performance liquid chromatography determination of polydeoxyribonucleotides in plasma: its application to the determination of defibrotide's pharmacokinetics in the rabbit. We describe an HPLC method for the determination of whole polydeoxyribonucleotides in animal plasma. This method was compared to a colorimetric method, which evaluates the sugar moiety of polydeoxyribonucleotides, and to an agarose gel electrophoresis method, which evaluates the whole polydeoxyribonucleotides as does the HPLC method, and was found to give results very close to those obtained with these two other methods. A pharmacokinetic study of the antithrombotic, profibrinolytic, polydeoxyribonucleotidic drug defibrotide was carried out by evaluating the plasma drug levels by these three methods. The pharmacokinetic parameters calculated from the data are very similar.


Electroporation-mediated gene transfer into hepatocytes: preservation of a growth hormone response. An electroporation procedure is described which allows the introduction of foreign genes into freshly isolated rat hepatocytes while preserving their growth hormone responsiveness. A single-pulse procedure performed at low voltage (150-200 V) but with high capacitance (960 microF), conditions which caused minimal cell damage and increased hepatocyte survival in culture (greater than 80%), was found to be optimal for both the basal and the hormone-stimulated expression of transfected genes. Transfection of the cells suspended in a phosphate buffer at high concentrations (20-25 x 10(6)/ml) with large amounts of plasmid (30 micrograms/assay) gave the best results. Raising the temperature up to 25 or 37 degrees C (instead of 4 degrees C) decreased about twofold basal CAT expression but appeared to increase the magnitude (i.e., fold induction) of hormonal effects. Expression of the reporter gene driven by either a viral or a liver gene promoter reached a maximum after 24 h, a situation especially favorable when studying liver-specific gene expression known to decay rapidly in cultured hepatocytes. This procedure was successfully applied to the study of a growth hormone-dependent serine protease inhibitor gene promoter.


The characteristic negative Cotton effect of ganglioside lactones observed by circular dichroism spectrometry. Circular dichroism spectrometry was applied to gangliosides and their lactones and revealed that the lactones have a characteristic strong negative Cotton effect around 235 nm. Four monolactones and two dilactones, which were formed from GM4, GD3, and GD1b, gave molar ellipticities at the wavelength in magnitude of 10(4), while their parent gangliosides, along with other gangliosides such as GM3, GM1, GD1a, GT1b, and GQ1b, showed no distinct feature. Two ganglioside esters, GM4-methyl ester and O-Ac-GT1b did not show the Cotton effect. The molar ellipticities had an additivity with respect to the number of lactone rings. The Cotton effect was attributed to the carbonyl group on the lactone ring.


Monosaccharide determination of glycoconjugates by reverse-phase high-performance liquid chromatography of their phenylthiocarbamyl derivatives. A method for the determination of neutral sugars and hexosamines present in glycoconjugates by reverse-phase high-performance liquid chromatography (HPLC) of their phenylthiocarbamyl (PTC) derivatives has been developed. After acid hydrolysis, neutral sugars are converted to glycamines by reaction with ammonium acetate in the presence of sodium cyanoborohydride and are subsequently derivatized with phenylisothiocyanate, while the hexosamines present in the same hydrolysate, after separation on Dowex 50, are treated directly with this reagent. HPLC of the PTC-glycamines of the neutral sugars is performed on Microsorb C18 in an isocratic manner while chromatography of the PTC-hexosamines employs a Pico-Tag column with gradient elution to achieve separation from the PTC-amino acids. The procedure has proven to be highly sensitive, requiring as little as picomole amounts for the chromatographic step; monosaccharide compositions determined on glycoproteins and glycopeptides by this method were found to compare favorably to those previously obtained by other techniques.


Profiling assay for lipoxygenase products of linoleic and arachidonic acid by gas chromatography-mass spectrometry. A method for determination of the lipoxygenase products of linoleic acid (9- and 13-hydroxyoctadecadienoic acid; 9-HODE, 13-HODE) and of arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acid; 5-, 8-, 9-, 11-, 12-, and 15-HETE) is described. The method combines solid-phase extraction, derivatization to the corresponding fully hydrogenated methylester/trimethylsilylether derivatives and capillary gas chromatography coupled with electron impact mass spectrometry. Each regioisomeric HODE and HETE shows a unique pair of mass spectrometric fragment ions originating from fission of the fatty acid carbon chain at the hydroxylated position. The carboxyl-terminal fragment is used for quantification relative to a carboxyl-18O2-labeled analogue added as internal standard and the methyl-terminal fragment is monitored for confirmation. The assay can be extended for quantification of the complete hydroxylation profile of linoleic and arachidonic acid. Applications of this assay are demonstrated for the quantification of HODEs and HETEs in normal, hyperplastic, and neoplastic mouse epidermis. In mouse epidermis papilloma, the tissue levels of 8- and 12-HETE were found to be increased by one to two orders of magnitude compared to levels in normal epidermis.


Triple-label beta liquid scintillation counting. The detection of radioactive compounds by liquid scintillation has revolutionized modern biology, yet few investigators make full use of the power of this technique. Even though multiple isotope counting is considerably more difficult than single isotope counting, many experimental designs would benefit from using more than one isotope. The development of accurate isotope counting techniques enabling the simultaneous use of three beta-emitting tracers has facilitated studies in our laboratory using the multiple tracer indicator dilution technique for assessing rates of transmembrane transport and cellular metabolism. The details of sample preparation, and of stabilizing the liquid scintillation spectra of the tracers, are critical to obtaining good accuracy. Reproducibility is enhanced by obtaining detailed efficiency/quench curves for each particular set of tracers and solvent media. The numerical methods for multiple-isotope quantitation depend on avoiding error propagation (inherent to successive subtraction techniques) by using matrix inversion. Experimental data obtained from triple-label beta counting illustrate reproducibility and good accuracy even when the relative amounts of different tracers in samples of protein/electrolyte solutions, plasma, and blood are changed.


Chromatographic separation of oligodeoxynucleotides with identical length: application to purification of oligomers containing a modified base. A general procedure is described for separation and purification of oligodeoxynucleotides of identical length but different base composition, in particular, of oligomers containing modified bases such as 4-substituted thymines and 6-substituted guanines, using an anion-exchange column (either Mono Q or NucleoPac). The modified oligomers can be well separated from the analogous oligomers containing unmodified thymine or guanine under the basic conditions of the chromatography. The effects of oligomer length, base composition, and lipophilicity on the separation are discussed. A general rule which can be used for prediction of the order of elution of different oligomers and for estimation of tautomeric form of a modified base in the oligomer is presented.


Analysis of human synovial fluid phospholipase A2 on short chain phosphatidylcholine-mixed micelles: development of a spectrophotometric assay suitable for a microtiterplate reader. The development of a reliable assay for human synovial fluid phospholipase A2 (HSF PLA2) is important for the kinetic characterization of the enzyme and for the identification of enzyme inhibitors. This enzyme behaves differently from other extracellular PLA2s in many standard phospholipase assays and is generally assayed using radiolabeled, autoclaved Escherichia coli as a substrate. We have now developed a nonradioactive, continuous, spectrophotometric assay for this enzyme that is adaptable for use with a microtiterplate reader and is suitable for screening enzyme inhibitors. The assay uses a thioester derivative of diheptanoyl phosphatidylcholine as a substrate, with which the enzyme displays a specific activity of about 25 mumol min-1 mg-1. The substrate concentration curve fits a Hill equation with an apparent Km of 500 microM and a Hill coefficient of two. The enzyme has a pH optimum of 7.5 in this assay and requires about 10 mM Ca2+ for maximal activity. The presence of 0.3 mM Triton X-100 was necessary to solubilize the substrate; however, higher concentrations of the detergent inhibited enzyme activity. Using this spectrophotometric assay, inhibition of HSF PLA2 by a thioether phosphonate phosphatidylethanolamine analog was observed with an IC50 of 18 microM.


Development of screening methods for detection of carbohydrate-binding proteins by use of soluble glycosylated polyacrylamide-based copolymers. Mammalian endogenous carbohydrate-binding proteins (lectins) play fundamental roles in a variety of mechanisms of interactions both at the molecular and cellular levels. We have investigated the binding of one of them (human brain lectin) to soluble acrylamide copolymerized with derivatives of either lactose (O-beta-lactosyloxyallylallylaminoacrylamide copolymer) or D-mannose (D-alpha-mannosyloxyallylallylaminoacrylamide copolymer) in direct enzyme affinoassays, in an attempt to develop simple procedures for detection and estimation of its carbohydrate-binding activity. Biotinylated plant lectins were utilized as reference standards. Affinoassays employed the polymer dotted on nitrocellulose and the polymer coated on microtiter plates as well as detection of bound biotinylated lectin by streptavidin/horseradish peroxidase reagent. Both assays provided reproducible binding, inhibitable by specific sugars. The microtiter plate assay is well suited to sensitive detection of the negative endogenous lectin by competition with biotinylated brain lectin. We conclude that the use of derivatized acrylamide in dotting and microtiter plate assays may prove practical for detection of endogenous lectins and that such polymers may serve as model substances in the study of biological partners of these carbohydrate-binding proteins.


Continuous spectrophotometric assay for restriction endonucleases using synthetic oligodeoxynucleotides and based on the hyperchromic effect. A continuous spectrophotometric assay for the EcoRV restriction endonuclease has been developed. The synthetic self-complementary oligonucleotide d(GACGATATCGTC) (which is double stranded under the assay conditions) is used as the substrate. The EcoRV endonuclease recognizes d(GATATC) sequences cutting between the central T and dA bases. Thus d(GACGATATCGTC) is converted to d(GACGAT) and d(pATCGTC) during catalysis. Both of the hexameric products are single stranded under the assay conditions. The conversion of the dodecameric substrate to the two hexameric products and the concomitant change from double- to single-stranded DNA is associated with an increase in absorbance at 254 nm due to the hyperchromic effect. This change can be used to monitor column effluents for endonuclease activity and also for Km and kcat determination under steady-state kinetic conditions.


Assay of alpha 1,3 N-acetyl-D-galactosaminyl transferase by affinity chromatography. A high-performance liquid affinity chromatography column that contains immobilized anti-A monoclonal antibody specifically retards blood group A-active oligosaccharides and can be used to detect the product(s) of the reaction catalyzed by alpha-1,3-N-acetyl-D-galactosaminyltransferase: [formula: see text] After a brief incubation (15 min) of an assay mixture containing 1-100 microliters human serum, the sugar nucleotide donor UDP-GalNAc, and radiolabeled oligosaccharide acceptors 2'-fucosyllactose and/or lacto-N-fucpentaose I blood group A-active products are isolated and quantitated in a single affinity chromatographic step that takes less than 30 min. Kinetic studies to determine the pH optima for serum alpha-3-GalNAc transferase from individuals of blood groups A1 and A2 and the Km value for UDP-GalNAc for the A1 transferase agree with previous determinations. As monoclonal antibodies against many different complex carbohydrate antigens are now available, the method described could be adapted to give rapid, inexpensive assays for a variety of glycosyltransferases.


Allele-specific and asymmetric polymerase chain reaction amplification in combination: a one step polymerase chain reaction protocol for rapid diagnosis of familial defective apolipoprotein B-100. We have combined the asymmetric polymerase chain reaction (PCR) with allele-specific PCR to detect a single point mutation. A set of two priming oligonucleotides and a third allele-specific primer were used to identify heterozygotes for a G to A mutation at nucleotide 10,708 in the apolipoprotein B (apo B) gene. The system requires neither restriction enzyme digestion nor allele-specific oligonucleotides as conventionally applied for allele-specific hybridization of slot blots. This method clearly allows for the detection of the mutant allele by inspection, after agarose gel electrophoresis of a single PCR reaction. DNA from 40 patients with familial defective apo B-100 due to the G to A mutation at nucleotide 10,708 in the apo B gene and their normal relatives was analyzed. Complete agreement with allele-specific hybridization of slot blots confirms supposition that the system is effective to screen a larger population.


Immunodetection of biotinylated lymphocyte-surface proteins by enhanced chemiluminescence: a nonradioactive method for cell-surface protein analysis. Biotinylation and radioiodination have been compared for labeling lymphocyte-surface proteins and the labeled proteins symmetrically immunoprecipitated with antibodies recognizing major lymphocyte markers such as the murine Thy-1, CD25 (the alpha subunit of the interleukin-2 receptor), CD45, and human CD2 glycoproteins. The detection of biotinylated proteins by enhanced chemiluminescence after transfer to nitrocellulose was found to be fast and as efficient as the detection of iodinated proteins by autoradiography. The vectoriality of cell-surface biotinylation was ascertained by two-dimensional electrophoresis of the cellular extract in which the major cytoplasmic proteins were not found biotinylated. This nonradioactive labeling procedure offers a convenient and efficient alternative to radiolabeling of cell surfaces for the biochemical analysis of extracellular domains of membrane proteins.


A method for the evaluation of the efficiency of signal sequences for secretion and correct N-terminal processing of human parathyroid hormone produced in Escherichia coli. Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1-37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1-37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.


Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase. A simple two-step purification of Vibrio harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. Acyl-ACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20 degrees C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acyl-ACP preparation and analysis.


Quantitative fluorometric analysis of plant and microbial chitosanases. A quantitative fluorometric assay for chitosanase activity in bacterial and plant tissues was developed. The assay can be conducted with either finely milled preparations of chitosan in suspension or dissolved chitosan; activity is based on measurements of glucosamine (GlcN) or oligomers of GlcN. GlcN is detected fluorometrically after reaction with fluorescamine with detection in the nanomole range. Fluorescence measurements of chitosanase activity and radioassay of chitinase in commercial preparations of chitinase from Streptomyces griseus revealed that both activities were present. Specific activities for the S. griseus chitosanase using suspended and soluble chitosans were respectively 1.24 and 6.4 mumol GlcN.min-1.mg protein-1. Specific activity of the S. griseus chitinase was 0.98 mumol GlcN.min-1.mg protein-1. Sweet orange callus tissue was tested for chitosanase and chitinase activity. It was necessary to remove small amine-containing molecules from the callus preparations before chitosanase activity could be assayed. The specific activity for chitinase and chitosanase in desalted extracts of nonembryogenic Valencia sweet orange callus tissue was determined to be 18.6 and 89.4 nmol GlcN.min-1.mg protein-1, respectively.


Adenine and guanine nucleotide content of Triton-extracted cytoskeletal fractions of nonmuscle cells. Determination of the adenine and guanine nucleotides in Triton X-100-extracted cytoskeletal fractions was utilized to estimate the actin and tubulin content of the assembled cytoskeletons in nonmuscle cells. Results with stable cell lines (i.e., rat pheochromocytoma PC12 and neuroblastoma NB41A3) and with primary cultures (i.e., human foreskin fibroblasts and chick embryonic dorsal root ganglion neurons) exhibited levels of cytoskeletal fraction ADP and GDP consistent with their assembly-induced nucleoside-5'-triphosphatase activities only previously analyzed in vitro. Likewise, estimates of actin and tubulin content fall in the range of values obtained by other experimental approaches. In contrast, analysis of whole cell nucleotides showed high [ATP]/[ADP] and [GTP]/[GDP] ratios, suggesting there is little, if any, contamination of the cytoskeletal nucleotide pool by other cellular nucleotides.


Comparison of spectrum-shifting intracellular pH probes 5'(and 6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7, 1'(3'H)-isobenzofuran]-3'-one and 2',7'-biscarboxyethyl-5(and 6)-carboxyfluorescein. The dyes carboxy-SNARF-1 and BCECF are fluorescent probes of intracellular pH that exhibit changes in spectral shape upon proton binding which allow one to use measurements of fluorescence at two or more wavelengths in order to measure pH without artifacts associated with variability in dye loading, etc. In evaluating these dyes for this study, whole spectra, rather than measurements at two wavelengths, were analyzed. For BCECF, the effects of the intracellular milieu were minimal: both the pH-sensitive excitation spectrum and the pKa agreed closely with values found in extracellular solution. In contrast, both the spectra and the pKa for the emission spectrum-shifting carboxy-SNARF-1 showed significant differences between intracellular and extracellular dye. As a result, extremely misleading values for intracellular pH will be obtained if one attempts to use extracellular dye to calibrate intracellular carboxy-SNARF-1 measurements. Multiple origins were found for the discrepancy: (i) the intracellular dye was found to be significantly quenched, with the deprotonated form being more strongly quenched than the protonated form; and (ii) the pKa for the equilibrium with intracellular hydrogen ions was shifted by +0.2 pH units. These effects were readily reversed by disruption of the cell, but were not due to sequestering of dye in an acidic cell compartment.


Quantitation of lysophosphatidylcholine molecular species in rat cardiac tissue. We have developed a rapid and sensitive procedure for isolation and measurement of 1-acyllysophosphatidylcholine (LPC) species in rat myocardial tissue. Tissues were spiked with heptadecanoyl-LPC internal standard and extracted with chloroform/methanol. The chloroform phase was dried, resuspended in chloroform/propan-2-ol (2/1, v/v), and applied to an aminopropyl-bonded phase (Bond Elut) column. Following stepwise elution with several solvent mixtures, the LPC fraction (ethyl acetate/methanol, 4/6, v/v) was separated by HPLC with direct quantitation of palmitoyl-LPC (P-LPC), oleoyl-LPC (O-LPC), and stearoyl-LPC (S-LPC), using an evaporative light scattering mass detector. Calibration curves were generated for each individual LPC species. Recoveries of added [14C]LPC and of heptadecanoyl-LPC internal standard after extraction and chromatography were 85.8 +/- 1.9% (mean +/- SE, N = 10) and 83.4 +/- 1.8% (N = 15), respectively. This assay showed satisfactory sensitivity, reproducibility, and accuracy for measurement of LPC species in rat myocardial tissue. The major molecular species of LPC in rat myocardium were found to be P-LPC and S-LPC, which were two- to sixfold as abundant as O-LPC. In isolated, crystalloid-perfused rat hearts the time of perfusion was found to significantly influence the content of P-LPC (0 min, 252 +/- 10; 15 min, 178 +/- 10, P less than 0.001, compared with 0 min; 40 min, 131 +/- 4, P less than 0.001; and 70 min, 129 +/- 4, P less than 0.001; nmol/g dry weight), but not the content of O-LPC and S-LPC. The method will be useful for studying the participation of LPC species in physiology, pathophysiology, and therapeutics.


The human platelet as a reproducible and sensitive cell for the detection and assay of platelet-activating factor. A new procedure for the preparation of human platelets consistently sensitive to platelet-activating factor (PAF) in the low nanomolar range has been developed. Key to the success of this approach was the addition of adenosine during the isolation phase, providing an excellent recovery of stable cells, and the inclusion of ADP in the aggregation assay, providing increased sensitivity to PAF. Examination of the binding profile of tritium-labeled PAF to these platelets in the presence or absence of ADP revealed significant difference in the Kd values but not in the number of specific binding sites. Other reagents having an influence on the reactivity and stability of the human platelets, as regards its interaction with PAF, are described.


Preparative labeling of proteins with [35S]methionine. The most common technique for preparative labeling of proteins with radioisotopes for experimental purposes utilizes 125I. This isotope has certain limitations, including the emission of gamma- and X-irradiation, the release of gaseous 125I2 from solutions of Na 125I, and the potential for concentration of 125I in thyroid glands. We have discovered a means for labeling proteins rapidly and simply with [35S]methionine. The technique is applicable to a wide variety of proteins. Antibodies labeled by our technique remain functional.


Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase. A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The Km for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-gamma-aminobutyryl-Ile-His-Pro - Phe-His-Leu-Val-Ile-His-Thr- [5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was found to be 4.3 microM at the optimum pH of 4.5. Reaction products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis or by 252Cf plasma desorption mass spectrometry. Cleavage of the fluorogenic substrate was between the histidine-threonine residues, releasing the fluorescent product, threonine-[5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid. Proteolytic activity was expressed as nanomoles of fluorescent product released at 22 degrees C/60 min, pH 4.5, and the release of 0.9 nmol product was equivalent to one hemoglobin proteolytic unit (O.D.A700 increase of 0.100) produced at 37 degrees C/60 min, pH 3.5. The aspartic proteinase inhibitor pepstatin had an IC50 of 27 nM when tested in a dose-response study with the purified enzyme. The apparent Ki for pepstatis was 2.9 nM. Several synthetic inhibitors of the enzymes were identified with IC50's in the nanomolar range. The most potent compound, A70450, was characterized as a fast, tight-binding inhibitor having an IC50 of 1.3 nM and apparent Ki of 0.17 nM.


Interdigital chondrogenesis and extra digit formation in the duck leg bud subjected to local ectoderm removal. In the chick embryo the interdigital tissue in the stages previous to cell death exhibits in vitro a high chondrogenic potential, and forms extra digits when subjected in vivo to local ectodermal removal. In the present work we have analyzed the chondrogenic potential both in vivo and in vitro of the interdigital mesenchyme of the duck leg bud. As distinct from the chick, the interdigital mesenchyme of the duck leg bud exhibits a low degree of degeneration, resulting in the formation of webbed digits. Our results show that duck interdigital mesenchyme exhibits also a high chondrogenic potential in vitro until the stages in which cell death starts. Once cell death is finished chondrogenesis becomes negative and the interdigital mesenchyme forms a fibroblastic tissue. In vivo the interdigital mesenchyme of the duck leg bud subjected to ectoderm removal forms ectopic foci of chondrogenesis with a range of incidence similar to that in the chick. Unlike those of the chick the ectopic cartilages of the duck are rounded and smaller, and appear to be located at the distal margin of the interdigital mesenchyme. Formation of extra digits in the duck occurs with a lower incidence than in the chick. It is concluded that ectopic chondrogenesis and formation of extra digits is related to the intensity of interdigital cell death. The non-degenerating interdigital mesenchymal cells destined to form the interdigital webs of the duck appear to contribute very little to the formation of interdigital cartilages.


Scanning electron microscopy of esophageal microvasculature in human infants and rabbits. The microvasculature of the esophagus was studied by scanning electron microscopy of vascular corrosion casts in human infants and rabbits. In both species, segmental circumferential arteries arise from main longitudinal arteries, the latter giving off numerous perforating arteries. The tunica muscularis is supplied by branches of circumferential and perforating arteries, the submucosa and its glands by branches of perforatings. Terminal arborizations of perforating arteries feed a subepithelial capillary network. These capillaries are drained by a venous plexus in the lamina propria which is connected to a submucosal venous plexus. Perforating veins, running parallel to the corresponding arteries, connect the submucosal plexus with circumferential veins, and finally empty into main longitudinal veins. Valves were not present in any of the veins. Submucosal veins were less numerous in man than in rabbit. The number and caliber of equivalent vessels in human submucosal plexus decreased from the pharyngoesophageal to the gastroesophageal junction, suggesting the latter to be at particular risk in portal hypertension. The subepithelial capillary network reveals a longitudinal arrangement in rabbits, while the same network shows no preferential organization in human infants. The microvascular architecture of the esophagus in humans and rabbits is comparable, especially in the lay-out of the venous plexuses and the absence of venous valves. Therefore the rabbit could serve as an experimental model for studies on portal hypertension. The present results strongly suggest particular significance of the venous plexus in the lamina propria for the genesis of esophageal varices.


Embryonic development of the house shrew (Suncus murinus). I. Embryos at stages 9 and 10 with 1 to 12 pairs of somites. The embryonic development during the period from 1 to 12 pairs of somites was observed in an insectivore species, the house shrew (Suncus murinus), which has been bred within a closed colony. Embryos were staged by the number of somite pairs. Each stage was punctuated at every addition of three pairs of somites and numbered after the Carnegie system. The first somite became apparent between 8 and 9.0 days after fertilization, and the 12th somite appeared between 9.5 and 10.0 days. The rate of somite formation was one pair in every 3-4 h on average. The embryonic events during this period were as follows: 1. From the beginning of stage 9, the embryonic body consistently displayed a kyphosis, and as development progressed, the caudal portion of the embryo spiralled clockwise. 2. The first and second pharyngeal arches formed; their development was precocious among mammalian embryos in relation to somitic count. 3. The segmental pattern of the neural fold was similar to that of laboratory rodents and primates. The first fusion of the cranial neural folds took place in the occipital somite region, the second fusion in the diencephalic region, and the third at the end of the neural plate, thus leaving two neuropores in the cephalic region. 4. The timing of appearance of the optic sulcus was similar to that of human embryos but was delayed in comparison with that of laboratory rodents. 5. The heart always showed a more advanced state than that of other mammalian embryos. From the beginning of stage 9, an unpaired endocardial tube was seen in the bulbo-ventricular region, and deflection from a symmetrical appearance soon took place. 6. The differentiation of foregut was also precocious, and the thyroid and respiratory primordia appeared earlier than in other mammals. The present study emphasizes that there are considerable variations in timing and manner of morphogenesis among early mammalian embryos.


The regulative potential of the limb region in 11.5-day rat embryos following the amputation of the fore-limb bud. The regulative potential of the fore-limb region following the removal of the limb bud was investigated in 11.5-day rat embryos. Fore-limb buds were amputated from a total of 54 embryos. Five embryos were immediately examined via scanning electron microscopy (SEM) to assess the quality of the operation and the reproducibility of the technique. In all cases, the forelimb bud and adjacent tissues extending down to the celomic cavity at the same level were completely removed. The remaining 49 operated embryos were cultured in vitro and examined at different time intervals. Gross inspection of embryos which had been cultured for 24 h, revealed that 24 out of 37 had developed a pair of limb bud-like protrusions at the operation site. Twelve formed only a single protrusion, while nothing was found in the remaining embryo. These protrusions were examined in greater detail under SEM and light microscopy. SEM observations showed that these protrusions were covered with an intact layer of ectoderm. In embryos with a pair of protrusions, these outgrowths developed opposite somites 7 to 13. The failure of either one of these two outgrowths to form, produced our second type of experimental embryo, those which had just a single protrusion. Histological examination revealed that an apical ectodermal ridge (AER) was discernible on the protrusions of 36% of the embryos. Finally, we have established how these protrusions were constructed from SEM observations of operated embryos cultured for 6 h and 10 h.


Regeneration in the Xenopus tadpole optic nerve is preceded by a massive macrophage/microglial response. Changes in the optic nerve following a crush lesion and during axonal regeneration have been studied in Xenopus tadpoles, using ultrastructural and immunohistological methods. Degeneration of both unmyelinated and myelinated axons is very rapid and leads to the formation, within 5 days, of a nerve which consists largely of degeneration debris and cells. Immunohistological analysis with monoclonal antibody 5F4 shows that there is a rapid and extensive microglial/macrophage response to crush of the nerve. Regenerating axons have begun to enter the distal stump by 5 days and grow along the outer part of the nerve in close approximation to the astrocytic glia limitans. Between 5 and 10 days after nerve crush, regenerating axons reach and pass the chiasma. Macrophages are seen in the nerve at the site of the lesion within 1 h, and the response peaks between 3-5 days, just before axonal regeneration gets under way.


Development and hormonal regulation of mast cells in the Harderian gland of Syrian hamsters. The morphological features and relative number of mast cells per mm2 were studied in the Harderian glands of male and female Syrian hamsters (Mesocricetus auratus) under different experimental conditions. The structural and ultrastructural characteristics of Harderian mast cells corresponded to those of connective tissue mast cells. The Harderian glands from female hamsters contained more mast cells than those of male hamsters. A subcutaneous implant of testosterone (2 mg/24 mg beeswax) resulted in a rapid decrease in the number of recognizable mast cells 6 h after the implantation. Neither orchidectomy nor ovariectomy significantly altered the relative number of mast cells. However, the daily subcutaneous injection of 20 IU of human chorionic gonadotropin during 20 days resulted in a significant decrease of identifiable mast cells. The administration of another steroid such as progesterone or the induction of states of hypo- and hyperthyroidism did not alter the distribution of mast cells in the Harderian glands of female Syrian hamsters.


Clinical application of magnetic resonance imaging of the heart and great vessels. This report reviews the clinical applications of magnetic resonance imaging, (MRI) for the heart and great vessels based on the first 120 patients studied with 1.5 Tesla scanner. Cine scans were obtained in 85% of patients studied with the remainder having T1 spin-echo imaging. MRI provides high-resolution multiplanar images for defining abnormalities in cardiac structure and is especially useful for congenital heart disease. Cine MR evaluates cardiac dynamic functions such as left ventricular volumes and ejection fraction, left ventricular segmental wall motion, and valvular function. It is also useful for detection of aortic and pulmonary arterial disease and diseases of the pericardium. It is concluded that MR has broad applications for diagnosis of cardiac and great-vessel disorders.


Morphometrical variations of prolactin cells in response to prolonged and systemic administration of Met-enkephalin in female rats. A stimulatory effect on prolactin secretion had been describe after acute and systemic administration of met-enkephalin, but the effects of this opioid after chronic administration has not been reported, and the response of mammotroph cells is not clear. As a complement to previous studies, a morphometric analysis (light and electron microscopy) was carried out on prolactin cells from female rats treated chronically with met-enkephalin. Clear features of cellular hyperactivity appeared after chronic and systemic administration of the opioid, and these persisted for two weeks. The changes consisted in increases of cellular, cytoplasmic and nuclear areas, volume and surface densities of the Golgi complex and rough endoplasmic reticulum, as well as the numbers of exocytotic figures. These morphological alterations were paralleled by an increase in serum prolactin levels as detected by RIA. It is concluded that the increase in the synthesis and secretory activity of prolactin cells following chronic and systemic administration of met-enkephalin is very similar to those observed after acute and intraventricular administration.


Iomeprol vs iopamidol in intraarterial peripheral digital subtraction angiography. The aim of this research was to compare the efficacy and tolerability of iomeprol, 150 mg iodine/mL, a new nonionic contrast medium, and iopamidol, 150 mg iodine/mg in intraarterial (IA) peripheral digital subtraction angiography (DSA) in 100 patients; a group of 40 patients were also submitted to a complete coagulation screening to check the influence of contrast media on blood clotting. The study was a comparative, double-blind clinical trial. The compound was assigned to each patient according to a randomization list. Small size (4-5 French) catheters were used in all patients to minimize arterial trauma and bedding time and to assess the quality of x-ray pictures in this condition. Vital signs, EKG tracings and laboratory parameters were monitored before and after the angiographic procedure; the coagulation screening included: thrombin time, prothrombin time, partial thromboplastin time, euglobulin lysis time, plasma thromboplastin antecedent, and plasminogen activator inhibitor (PAI). Both contrast media did not produce any adverse reaction or clinically significant alteration of studied parameters; in the 40-patient group subjected to massive coagulative screening, no important alteration after contrast media administration was reported. The score for contrastographic efficacy was very good with both media with a prevalence of better results in the iomeprol group.


Effects of intravenous streptokinase and CLS 2210 (calcium dobesilate) on the biochemical markers of early acute myocardial infarction: a historic comparison with both studies. In a previous double-blind, randomized study, CLS 2210 (a new formulation of calcium dobesilate) or placebo was administered by intravenous infusion to 41 patients having their first acute myocardial infarction. In the present study 19 comparable patients were treated intravenously with streptokinase under identical conditions and the results compared with those from the previous study. In all patients administration was begun within three hours of onset of symptoms and continued over seventy-two hours. Blood samples were taken for the measurement of serum activity of creatine kinase and its isoenzyme MB, and the serum and urinary concentrations of myoglobin and glycosaminoglycans were also measured. The purpose of this study was to determine the effects of CLS 2210, placebo, and streptokinase on these biochemical markers of acute myocardial infarction, thereby assessing their actions in limiting myocardial necrosis. In the CLS 2210-treated patients, the levels of serum creatine kinase and serum and urinary myoglobin were significantly lower than in the placebo patients throughout the seventy-two hours (p = 0.01, 0.005, 0.004 respectively). The levels of creatine kinase MB and serum glycosaminoglycan in the CLS 2210 patients were initially higher than in the placebo patients but fell below placebo levels between the fortieth and fifty-fifth hours, respectively (p = 0.89, 0.02). Only the glycosaminoglycan urinary concentrations were higher in the CLS 2210 group than in the placebo group throughout (p = 0.0005). The values for the six variables investigated showed no statistically significant difference between placebo and streptokinase.(ABSTRACT TRUNCATED AT 250 WORDS)


Double-blind comparison of captopril with nifedipine in hypertension complicated by intermittent claudication. In a double-blind, crossover trial 12 patients with hypertension and peripheral arterial disease were randomized to three months' treatment with captopril 25-50 mg twice daily or nifedipine SR 20-40 mg twice daily. While both treatments were equally effective at lowering blood pressure, postexercise calf blood flow availability was greater during treatment with captopril (P less than 0.04). This was not reflected in walking capacity assessed by treadmill exercise. The results suggest that both captopril and nifedipine are appropriate antihypertensive agents in patients with peripheral arterial disease.


Effects of alternately aligned static micromagnetic fields on intravascular endothelial lining. Effect of alternately aligned static micromagnetic fields on intravascular endothelial linings were investigated in 32 common carotid arteries of mongrel dogs. Endothelia of bilateral carotid arteries were mechanically denuded 5 cm in length, and as a general rule, 1 denuded artery was wrapped by a 1- or 5-cm-long film of alternately aligned static micromagnetic fields with 250 gauss/cm2 and the artery on other side (control) was not wrapped. The arteries were excised at weeks 1, 2, 3, 4, 5, 6, and 8 after endothelial denudation in order to examine the intraluminal surfaces of the distal, intermediate, and proximal portions of the specimen by light and scanning electron microscopy. Histopathologically, all the distal and proximal portions in each group were well lined with endothelium. There was, however, a significant difference between the experimental and control groups in the endothelial regeneration of the intermediate portion. Endothelial coverage of the serial specimens of the experimental group was found to occur earlier than that of the control group. Alternately aligned static micromagnetic fields accelerated the intravascular endothelial linings.


Double-blind comparison of the clinical, hemodynamic, and electrocardiographic effects of sodium meglumine ioxaglate or iohexol during diagnostic cardiac catheterization. The clinical effects and the maximal hemodynamic and electrocardiographic effects of two low-osmolality radiographic contrast media (ioxaglate and iohexol) were directly compared during diagnostic cardiac catheterization in a double-blind, randomized study in 80 patients. Because small changes were expected after injection of both of these agents, sensitive ECG and intracardiac-pressure-monitoring methods were used, and maximal changes, as well as mean changes in variables, were analyzed. Symptoms were absent, mild, or moderate in 67-77% of patients after left ventriculography and in 97-100% of patients after coronary arteriography. After left ventriculography, maximum and minimum left ventricular systolic pressure and end-diastolic pressure, the first derivative of left ventricular pressure (dp/dt), heart rate, were significantly altered over the two-minute observation period but were not different from the preinjection values at two minutes after both agents. Small but significant increases in mean aortic pressure, cardiac output, and pulmonary arterial wedge pressures were seen at two minutes after both agents.


Use of cardiopulmonary bypass, high-dose epinephrine, and standard-dose epinephrine in resuscitation from post-countershock electromechanical dissociation. To determine the effects of cardiopulmonary bypass with standard-dose epinephrine, high-dose epinephrine, and standard-dose epinephrine on perfusion pressures, myocardial blood flow, and resuscitation from post-countershock electromechanical dissociation. Prospective, controlled laboratory investigation using a canine cardiac arrest model randomized to receive one of three resuscitation therapies. After the production of post-countershock electromechanical dissociation, 25 animals received ten minutes of basic CPR and were randomized to receive cardiopulmonary bypass with standard-dose epinephrine, high-dose epinephrine, or standard-dose epinephrine. Myocardial blood flow was measured using a colored microsphere technique at baseline, during basic CPR, and after intervention. Immediate and two-hour resuscitation rates were determined for each group. Return of spontaneous circulation was achieved in eight of eight cardiopulmonary bypass with standard-dose epinephrine compared with four of eight high-dose epinephrine and three of eight standard-dose epinephrine animals (P less than .04). One animal was resuscitated with CPR alone and was excluded. Survival to two hours was achieved in five of eight cardiopulmonary bypass with standard-dose epinephrine, four of eight high-dose epinephrine, and three of eight standard-dose epinephrine animals (NS). Coronary perfusion pressure increased significantly in the cardiopulmonary bypass with standard-dose epinephrine group when compared with the other groups (cardiopulmonary bypass with standard-dose epinephrine, 76 +/- 45 mm Hg; high-dose epinephrine, 24 +/- 12 mm Hg; standard-dose epinephrine, 3 +/- 14 mm Hg; P less than .005). Myocardial blood flow was higher in cardiopulmonary bypass with standard-dose epinephrine and high-dose epinephrine animals compared with standard-dose epinephrine animals but did not reach statistical significance. Cardiac output increased during cardiopulmonary bypass with standard-dose epinephrine (P = .001) and standard-dose epinephrine (NS) compared with basic CPR but decreased after epinephrine administration in the high-dose epinephrine group (NS). Resuscitation from electromechanical dissociation was improved with cardiopulmonary bypass and epinephrine compared with high-dose epinephrine or standard-dose epinephrine alone. However, there was no difference in survival between groups. Cardiopulmonary bypass with standard-dose epinephrine resulted in higher cardiac output, coronary perfusion pressure, and a trend toward higher myocardial blood flow. A short period of cardiopulmonary bypass with epinephrine after prolonged post-countershock electromechanical dissociation cardiac arrest can re-establish sufficient circulation to effect successful early resuscitation.


Selective aortic arch perfusion during cardiac arrest: a new resuscitation technique. To demonstrate the technique of selective aortic arch perfusion during cardiac arrest and to observe the hemodynamic effects of volume infusion and aortic epinephrine administration. Sequential series, nonrandomized, noncontrolled. Fourteen mongrel dogs weighing 21 to 36 kg. Animals had midaortic arch pressure, right atrial pressure, and descending aortic arch balloon occlusion catheters placed. After ten minutes of ventricular fibrillation, balloon inflation and aortic arch infusions were initiated as follows: group 1 (six), 30 mL/kg/min of 0.9% NaCl for two minutes; group 2 (four), 30 mL/kg/min of oxygenated lactated Ringer's with 2 mg/L epinephrine for two minutes, followed by CPR; and group 3 (four), 20 mL/kg/min of oxygenated perfluorochemicals with 4 mg/L epinephrine for one minute, then CPR. Midaortic arch pressure, right atrial pressure, and coronary perfusion pressure each rose significantly in all groups. Midaortic arch pressure and coronary perfusion pressure increases were greater in groups 2 and 3 than in group 1. In groups 1 and 2, right atrial pressure increases at end-selective aortic arch perfusion were excessive as midaortic arch pressure and right atrial pressure increased linearly and similarly after 20 to 30 seconds. In groups 2 and 3, CPR-diastolic midaortic arch pressure and coronary perfusion pressure after selective aortic arch perfusion were good and similar to midaortic arch pressure and coronary perfusion pressure at end-selective aortic arch perfusion. Selective aortic arch perfusion is technically feasible, but excessive right atrial pressure increases limit maximal infusion rates and volumes. Selective aortic arch perfusion infusates with epinephrine produce greater midaortic arch pressure and coronary perfusion pressure during infusion than infusate without epinephrine. Controlled studies are needed to determine if selective aortic arch perfusion improves resuscitation outcome.


Minimal coronary stenoses and left ventricular blood flow during CPR. To assess the effect of a 33% coronary stenosis on myocardial blood flow during normal sinus rhythm and CPR. Prospective, before and after cardiac arrest and CPR; before and after creation of a 33% stenosis. The University of Arizona Resuscitation Research Laboratory. Ten domestic closed-chest swine with patent coronary stenoses. A Teflon cylinder was placed in the mid-left anterior descending coronary artery to create a 33% stenosis. Myocardial blood flow was measured with colored microspheres both proximal and distal to the stenosis during normal sinus rhythm and during CPR. During normal sinus rhythm, the stenosis did not alter the amount of myocardial blood flow distribution or quantity. Proximal to the stenosis the endocardial/epicardial flow ratio was 1.49 +/- 0.33, and distal to the stenosis it was 1.50 +/- 0.50. Likewise, during normal sinus rhythm, blood flow proximal and distal to the stenosis did not differ for either the epicardium (79 +/- 9 versus 66 +/- 13 mL/min/100 g) or the endocardium (111 +/- 27 versus 83 +/- 19 mL/min/100 g). However, the distribution of myocardial blood flow was markedly altered during CPR. The resultant endocardial/epicardial flow ratios were significantly less than during normal sinus rhythm, 0.49 +/- 0.11 (three minutes of CPR) and 0.74 +/- 0.07 (eight minutes of CPR) proximal to the stenosis and 0.39 +/- 0.15 (three minutes of CPR) and 0.49 +/- 0.14 (eight minutes of CPR) distal to the stenosis (P less than .05 versus normal sinus rhythm). In the presence of a 33% mid-left anterior descending coronary artery stenosis, endocardial blood flow at eight minutes of CPR was significantly lower distal to the stenosis compared with proximal to the stenosis (23 +/- 7 mL/min/100 g versus 74 +/- 18 mL/min/100 g; P less than .02). Minimal coronary lesions that do not diminish myocardial perfusion during normal physiologic conditions appear to significantly decrease subendocardial blood flow during cardiac arrest and CPR.


The esophageal detector device: a rapid and accurate method for assessing tracheal versus esophageal intubation in a porcine model. To assess time and accuracy of the esophageal detector device (EDD), disposable end-tidal CO2 monitor (ETCO2), and standard clinical methods for detection of endotracheal tube placement. Prospective, randomized, single-blinded, controlled laboratory investigation. Thirty airway managers (physicians, nurse anesthetists, and paramedics) used one pig (Sus scrofa) as the intubated, respiratory depressed/arrest model. Part 1: A standard 7.5-mm endotracheal tube was placed in either the esophagus or the trachea of the anesthetized swine. Anatomic location was verified by bronchoscopy. Airway managers blinded to the endotracheal tube location were assigned randomly to identify tube position by one of three methods (EDD, ETCO2, or clinical methods). Speed and accuracy of the assessment were recorded. Part 2: A second identical tube was placed, so that both the esophagus and the trachea were intubated; then, the esophageal tube was bag-ventilated for one minute. Each blinded airway manager, using only the EDD, determined placement site of both tubes. Part 1: Mean time to determine tube placement for group A (EDD) was 13.8 seconds; group B (ETCO2), 31.5 seconds; and group C (clinical methods), 39 seconds. Comparison by analysis of variance yielded a value of P less than .001. Both groups A and B were 100% accurate, whereas 30% of the subjects from group C mistakenly assessed an esophageal tube as in the trachea. Part 2: The EDD remained 100% sensitive and specific despite prior ventilation of the esophageal tube. In this porcine model, the EDD and ETCO2 were more accurate than clinical methods in determining endotracheal tube placement. The EDD demonstrated a significant time advantage over both ETCO2 and clinical methods. Prior ventilation of the esophageal tube does not interfere with the accuracy of the EDD.