Importance of endogenous substrates for cultured adult rat cardiac myocytes.

In Ca-tolerant adult cardiomyocytes the contribution of endogenous substrates (glycogen, tri- and diacylglycerol) to oxidative substrate metabolism was investigated. After 4 h in culture medium (M 199 plus 4% fetal calf serum) the cellular triacylglycerol content is 3.6-fold higher than in fresh myocardium and reflects the free fatty acid composition of the medium. When triacylglycerol is degraded, all long-chain fatty acids are hydrolysed at equal rates. In these quiescent cells, the activity of pyruvate dehydrogenase is low (10% of full activity, in Tyrode solution with 5 mM glucose). Up to 30% of full pyruvate dehydrogenase activity, the contribution of non-lipid substrates (glycogen, glucose, lactate and pyruvate) to oxidative energy production is correlated to pyruvate dehydrogenase activity. At 5 mM medium concentration, glucose, lactate and pyruvate share in energy production the proportions of 15, 36 and 50%, whereas endogenous lipolysis accounts for 78, 61 and 46%. It is concluded that these quiescent cardiomyocytes represent cardiac metabolism in a basal state in which the preference for fatty acids, especially from endogenous lipids, is very pronounced. The utilization of endogenous substrates therefore has to be considered in all studies investigating the oxidative metabolism of these isolated cells.

Formation of proinflammatory cytokines in human term myometrium is stimulated by lipopolysaccharide but not by corticotropin-releasing hormone.

Human term myometrium is poorly characterized as a source of proinflammatory mediators involved in parturition. We have investigated the basal expression of cytokines in myometrium, as well as the effects of CRH and lipopolysaccharide (LPS) on cytokine release. Explants from term myometrium were challenged with CRH or LPS (1 microg/mL each) in short-term tissue culture. Interleukin (IL)-1beta++, IL-6, IL-8, and tumor necrosis factor (TNF)alpha concentrations in the medium were quantified by enzyme immunoassay. The major cytokines released after 24 h were IL-6 and IL-8. All cytokines investigated were stimulated significantly by LPS (P: < 0. 05) but not by CRH. Messenger RNA levels of these cytokines were investigated by RT-PCR. IL-1beta+ and IL-6 messenger RNA were present in preterm and term myometrium before and during labor, whereas IL-8 and TNFalpha were expressed only by myometrium in active labor. Furthermore, myometrial CRH receptors and macrophages were characterized immunohistochemically. We conclude that human term myometrium is a site of production of proinflammatory cytokines and is involved in the inflammation-like reactions mediating the birth process. Cytokine release in term myometrium seems not to be under control of CRH.

Intraamniotic injection of methylene blue leading to methemoglobinemia in one of twins.

The practice of injecting pregnant women with methylene blue to detect membrane rupture and differentiate twin sacs is discussed. The authors recommend caution in the use of substances with potential adverse fetal effects.

Nonimmunologic hydrops fetalis: a review of 19 cases.

Nineteen cases of nonimmunologic hydrops fetalis occurring during a nine-year period were reviewed. The pregnancies were complicated by hydramnios (78%) and preterm delivery (84%). Hydramnios appears to be the most useful indicator of the pregnancy at risk; its occurrence should prompt ultrasonographic investigation for evidence of hydrops. Modalities available for antenatal diagnosis of underlying fetal abnormalities include amniocentesis, serologic tests, fetal cardiac monitoring, radiography, hemoglobin electrophoresis and glucose tolerance testing. A specific cause for the hydrops may not be detectable (42% of our cases were idiopathic). Management of affected pregnancies is influenced by the frequent occurrence of fetal asphyxia and premature delivery. Outcome is poor: only 32% of the babies survived beyond the neonatal period. Symptomatic treatment for the neonate includes fluid restriction, maintenance of blood sugar, support of ventilation and attention to the complications of asphyxia.

Automated and conventional ABR screening techniques in high-risk infants.

Test validity is determined by the proportion of results that are diagnostically confirmed and predicted on the measures used to identify the disease process. This article summarizes the results of a series of 224 stable high-risk infants who were screened by automated (ALGO-1) and conventional (Bio-logics LT) ABR instrumentation. Failure criteria was defined as the absence or prolongation of a replicable wave V response (conventional) or Refer by the automated system. The overall failure rates at a 35 dB screening level were comparable between devices. Sensitivity and specificity measures for the ALGO-1 unit were 100 and 96 percent, respectively. Permanent hearing loss was demonstrated in 5 percent of the newborns screened in this study. Advantages of the automated system include a dual artifact rejection system, attenuating ear couplers, and a battery operated design. These findings suggest that the automated ABR screener is a viable alternative to conventional ABR instrumentation for the limited purpose of neonatal auditory screening.

Common bacterial pneumonitis in infants. Determining the etiology and tailoring the treatment.

In infantile pneumonia, we recommend close attention to the history and physical examination. Baseline studies, including CBC, ESR, blood cultures, and chest film, should be performed at onset and repeated as warranted. Nasopharyngeal secretions or washings should be drawn by means of gentle suction and specimens sent for Gram stain, fluorescent antibody stain for respiratory syncytial virus, and culture for bacteria and for viruses if possible. Acute and convalescent serum specimens should be obtained in serious cases to search for antibodies to RSV, adenovirus, influenza, parainfluenza, cytomegalovirus, and Chlamydia. Serum and urine specimens may be collected for countercurrent immunoelectrophoresis and latex agglutination testing for Hemophilus influenzae type B, Streptococcus pneumoniae, and if indicated, group B streptococcus. If deterioration continues and all tests are negative, the clinician should consider a more invasive procedure such as flexible fiberoptic bronchoscopy, needle aspiration, or open lung biopsy. While awaiting identification of the pathogen, the physician should institute empiric therapy with optimum doses of antimicrobials and monitoring of serum levels of drug. Often the clinician is faced with deterioration and a negative workup. In this situation, other agents may be added, such as antifungal, antiviral, antiprotozoan, and antituberculous agents, as well as various antibiotics, to cover rare and unusual pathogens. Further consultation, even by phone, may at this point provide some insight into an otherwise confusing case.

A non-linear goal programming approach to modeling intraregional economic development.

"This paper provides a one-period normative model that may be used as a guide or benchmark by which the economic planner may develop policies and plans for regional economic development. The model can accomodate a region that could be as large as a small country or as small as a city, provided sufficient relevant data are available." The authors outline "a procedure for modeling and solving economic planning and analysis problems with 'intergoal trade-offs' while retaining the deviational variable as a measure of satisfaction....[They present] a generalized regional polynomial goal programming model with some suggested objectives and constraints [and illustrate] the approach with a hypothetical example."

Hyperbaric oxygen use in neonates. A report of four patients.

We report the use of hyperbaric oxygen in four neonates with delayed wound healing. Three presented with cyanotic congenital heart disease and had wounds associated with surgical procedures; the fourth had a nonhealing wound as a result of a complication of an umbilical-artery catheter. All were treated in a hyperbaric chamber with 100% oxygen at 2 atmospheres absolute. All wounds healed after institution of hyperbaric therapy. There was no evidence of serious side effects in any patient. These observations suggest, but do not prove, the efficacy of hyperbaric oxygen therapy for neonates with delayed wound healing.

Oxidative metabolism of cultured adult cardiomyocytes.

Cultured adult cardiomyocytes from rat exhibit, if unstimulated, low rates of substrate oxidation, corresponding to their mechanical inactivity. They oxidize exogenous fatty acids and lactate preferentially to glucose. Endogenous lipolysis provides the largest portion of oxidative substrates. Pyruvate dehydrogenase is largely in its inactive form. If glucose is the sole exogenous substrate, the quiescent cells produce lactate as a purely aerobic phenomenon probably reflecting the reduced state of the cytosol at low respiration rates.

Catheterization of the posterior tibial artery in the neonate.

Catherization of the posterior tibial artery provides an alternative to cannulation of other arteries. We successfully catheterized this artery 17 times in 15 neonates whose weights ranged from 800 to 3,000 g. The catheters were placed percutaneously 13 times and by cutdown four times, and they remained in place for as long as 12 days (mean, 96 hours). Compromise of distal circulation was found in only two babies, both of whom had had umbilical artery catheters removed because of cyanosis of the toes that had resolved itself prior to posterior tibial artery cannulation. Our patients experienced no short-term complications from this procedure.

Glucose, lactate, and palmitate as substrates for the resting cardiac myocyte.

Cultured adult cardiac myocytes oxidize fatty acids and lactate preferentially to glucose. In contrast to other results obtained with isolated cardiac myocytes the preference for fatty acids is not dependent on carnitine administration, suggesting that the cells of this preparation possess sufficient reserves of carnitine and an intact mechanism for fatty acid activation. It is demonstrated by the substrate oxidation rates that these quiescent cells are in a state of minimal metabolic demand, thus resembling the arrested myocardium.

Substrate utilization of adult cardiac myocytes.

Cultured adult myocytes are in a state of basal metabolism. When glucose is the only exogenous substrate, they produce lactate over CO2 at a constant rate of 2.7 from this substrate. Increase of oxygen tension does not change this behaviour. Insulin preferentially increases lactate formation, dichloroacetate only CO2 production. The fact that the lactate/CO2 ratio can be varied from 0.5 to 16 indicates that there is no close coupling between glycolytic flux and pyruvate oxidation. Both exogenous lactate and fatty acids are used preferentially over glucose. But increase of fatty acid oxidation and inhibition of glucose oxidation are not complementary. Glycolytic flux is only slightly decreased when fatty acid oxidation is already saturated. The results indicate that fatty acids interact with glucose oxidation primarily by inactivation of the pyruvate dehydrogenase. Neither insulin nor dichloroacetate in the presence of glucose inhibit fatty acid oxidation.

2-Fluoroadenosine uptake by erythrocytes and endothelial cells studied by 19F-NMR.

Transport and phosphorylation of 2-fluoroadenosine (F-AR) were studied in human erythrocytes and porcine aortic endothelial cells by 19F-nuclear magnetic resonance (NMR) spectroscopy. F-AR (590 microM) added to a human erythrocyte suspension (15% hematocrit) was rapidly incorporated into adenine nucleotides at a rate of 38 nmol.min-1.ml red blood cells-1. Intracellular F-AR could be distinguished from extracellular F-AR due to a chemical shift difference of 0.43 +/- 0.03 ppm (n = 5 experiments). Compared with F-AR, fluoro-ATP purified by high-performance liquid chromatography (HPLC) exhibited a chemical shift of -0.052 ppm, which was too small to differentiate intracellular F-AR and fluoro-ATP in vivo. F-AR uptake was decreased by inhibition of membrane transport with dipyridamole (25 microM) or blockade of adenosine kinase by iodotubercidin (10 microM). The time course of F-AR uptake suggested that the rate-limiting step was not membrane transport but the intracellular phosphorylation by adenosine kinase. In porcine aortic endothelial cells grown on microcarrier beads and perfused within the magnet, there was a linear relation between the F-AR concentration applied (2, 4, 8, or 32 microM) and net uptake measured (27-827 pmol.min-1.mg-1). Intra- and extracellular fluoroadenine compounds were separated by 0.12 ppm, and HPLC analysis confirmed F-AR conversion to fluoroadenine nucleotides. Our findings demonstrate that cellular transport and metabolism of F-AR can be noninvasively studied and analyzed by 19F-NMR.

Quantitative and kinetic characterization of nitric oxide and EDRF released from cultured endothelial cells.

Endothelial cells (EC) contribute to the control of local vascular diameter by formation of an endothelium derived relaxant factor (EDRF) (1). Whether nitric oxide (NO) is identical with (EDRF) or might represent only one species of several EDRFs has not been decided as yet (2-5). Therefore, we have directly compared in cultured EC the kinetics of NO formation determined in a photometric assay with the vasodilatory effect of EDRF and NO in a bioassay. Basal release of NO was 16, 4 pmol/min/ml packed EC column. After stimulation with bradykinin (BK) and ATP onset of endothelial NO release and maximal response preceded the EDRF-mediated relaxation. Concentrations of NO formed by stimulated EC were quantitatively sufficient to fully explain the smooth muscle relaxation determined in the bioassay. Our data provide convincing evidence that under basal, BK and ATP-stimulated conditions 1. endothelial cells release nitric oxide as free radical, 2. nitric oxide is solely responsible for the vasodilatory properties of EDRF.

Resistance of endothelial cells to anoxia-reoxygenation in isolated guinea pig hearts.

The release of cytosolic enzymes from myocardial and endothelial cells in the anoxic-reoxygenated guinea pig heart was investigated. Isolated hearts were perfused with Tyrode solution in the Langendorff mode. Sixty-minute anoxic perfusion with or without glucose (5 mM) was followed by 15-min normoxic perfusion with glucose. The losses of purine-nucleoside phosphorylase (PNP) from endothelial cells and of lactate dehydrogenase (LDH) and creatine kinase (CK) from the mass of myocardial cells were determined. After 30-min anoxia, the release of LDH and CK but not of PNP increased. Reoxygenation after 60-min anoxia with glucose caused a partial recovery of tissue ATP but also an increase in leakage of LDH (11% of total in 15 min) and CK (10%) and a sudden rise in coronary resistance, indicating contracture development ("oxygen paradox"). PNP release remained low (0.5%). In hearts subjected to glucose-free anoxia, ATP levels did not rise during 15-min reoxygenation, contracture development was delayed, and the release of LDH and CK was diminished (3.1 and 2.7%, respectively). Leakage of PNP was again low (0.5%). The results indicate that cardiomyocytes are more severely injured by anoxia-reoxygenation than the coronary endothelium. The rapidly developing reoxygenation-induced injury of cardiomyocytes seems to be an energy-dependent phenomenon, since it was attenuated in hearts deprived of substrate in anoxia.

Biological activity of des-(B26-B30)-insulinamide and related analogues in rat hepatocyte cultures.

Short-term and long-term biological activities were studied in adult rat hepatocytes cultured in the presence of the insulin analogues des-(B26-B30)-insulinamide, [TyrB25]des-(B26-B30)-insulinamide and [HisB25]des-(B26-B30)-insulinamide. When compared to insulin, full potency of des-(B26-B30)-insulinamide has been reported in rat adipocytes and an enhanced potency has been reported for the other analogues. Steady state binding characteristics of the analogues to hepatocytes were indistinguishable from those of native insulin with half-maximal binding occurring at concentrations of about 0.8 nmol/l. Half-maximal effects for the stimulation of glycolysis and inhibition of basal and glucagon-activated glycogenolysis required identical concentrations for insulin and all 3 analogues. Induction of the key glycolytic enzymes glucokinase and pyruvate kinase as well as the inhibition of glucagon-dependent induction of phosphenolpyruvate carboxy-kinase also required identical concentrations of insulin and the 3 analogues. These data confirm that in cultured hepatocytes the C-terminal amidation of des-(B26-B30)-insulin results in a molecule with full in vitro potency. In contrast to data obtained in adipocytes, the des-(B26-B30)-insulin-amidated analogues with tyrosine or histidine substitutions at position B25 are equally as potent as native insulin in eliciting biological responses in rat hepatocyte culture.

Early enzyme release from myocardial cells is not due to irreversible cell damage.

It is often assumed that the release of enzymes from oxygen deficient heart tissue is due to the irreversible damage of myocardial cells. However, because of diffusion barriers and inhomogeneity of oxygen-deficient tissue this hypothesis cannot be proven in heart tissue. The question whether enzyme release may already occur during reversible injury is of considerable relevance in clinical medicine: first, because the amount of released enzyme activity has been used to estimate the mass of damaged tissue in cardiac infarction and, second, because the stress of some diagnostic interventions may lead to cardiac enzyme release, which according to the irreversibility hypothesis would indicate the death of cells in a cell constant organ.

Ultrastructure of cultured adult myocardial cells during anoxia and reoxygenation.

Cultured heart cells from adult rats were exposed to anoxia in a substrate-free Tyrode's solution at constant pH. In this system the metabolic and the morphologic pattern can be investigated simultaneously. Anoxic changes develop gradually above 2 mumol adenosine triphosphate (ATP)/ gww . Morphometry reveals that the morphologic changes are closely related to the energetic state: creatine phosphate (CP) decay is accompanied by the loss of small mitochondrial matrix granules (r = 0.97). The fall of ATP is coincident with sarcomere shortening (r = 0.95) and, below 4 mumol/ gww , with mitochondrial swelling (r = -0.88). The number of lipid droplets correlates with the ATP level during anoxia and reoxygenation (r = -0.92). The early energetic depletion is accompanied by a moderate release of cytosolic enzymes and morphologic changes: the appearance of sarcolemmal microblebs and an increase in subsarcolemmal vesicles. Below an average ATP level of 2 mumol/ gww an increasing number of individual cells fail to recover when reoxygenated . However, that failure is accompanied neither by massive enzyme release nor by ultrastructural damage regarded as typical for the "oxygen paradox."