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Biomedical subjects

R H Notter

Publications and source records attributed to R H Notter.

At least 73 records · Page 4Linked to original sources
PubMed1986

Phospholipid binding and biophysical activity of pulmonary surfactant-associated protein (SAP)-35 and its non-collagenous COOH-terminal domains.

Surfactant-associated protein of Mr = 35,000, SAP-35, is the major glycoprotein present in mammalian pulmonary surfactants. In this study, canine SAP-35 and several of its COOH-terminal peptides were purified and characterized by amino acid composition and NH2-terminal sequencing analysis. These proteins were then studied in terms of their specific lipid-binding characteristics and surface activity when combined with a synthetic phospholipid mixture, SM, chosen as an approximation of lung surfactant phospholipids. Purified, delipidated SAP-35 bound SM strongly. In contrast, SAP-21 (a non-collagenous fragment generated by collagenase digestion) bound phospholipid weakly; SAP-18 (an acidic COOH-terminal fragment comprising residues Gly-118 to Phe-231) did not bind phospholipid, demonstrating the importance of hydrophobic amino acid residues Gly-81 to Val-117 and the NH2-terminal collagenous domain in interaction of the SAP-35 with phospholipids. In surface activity experiments, purified SAP-35 enhanced the adsorption of SM phospholipids in terms of both rate and overall surface tension lowering. However, the adsorption facility of the SM-SAP-35 mixture did not approach that of either whole surfactant or the surfactant extract preparations, calf lung surfactant extract or surfactant-TA, used in exogenous surfactant replacement therapy for the neonatal respiratory distress syndrome. In addition, the dynamic surface activity of the SM-SAP-35 mixture was well below that of natural surfactant or surfactant extracts. This was also true of mixtures of SM phospholipids combined with the SAP-18 and SAP-21 fragments of SAP-35.

Amino Acid Sequence↗
PubMed1986

A quantitative study of the development of type II pneumocytes in fetal lung.

Cell populations dissociated from fetal rabbit lungs were analyzed by laser flow cytometry for the presence of type II pneumocytes. These cells are distinguishable by the staining of their lamellar bodies with the fluorescent lipophilic dye, phosphine-3R and by their intensity of low-angle light scatter. Lung cells were obtained by enzymatic dissociation from fetal rabbits at gestational ages of 24 d, 27 d, and from 2-d newborn rabbits. Flow cytometric analysis was sufficiently sensitive to discriminate between fetuses. Quantitative analysis of type II pneumocytes showed that newborn rabbits had a distinct cell subpopulation in a region of low-angle light scatter and phosphine-3R fluorescence intensity similar to that previously reported on type II cells from adult rabbits. By contrast, 24-d gestation rabbits had a negligible type II cell subpopulation. Fetuses of 27 and 30 d gestation showed a slow but progressive increase in the numbers of cells in the type II region. Mathematical analyses of light scatter and fluorescence intensity distributions were used to define statistically significant (P less than .05) boundaries that characterize the development of the type II cell subpopulation in fetal rabbit lung. The methods employed offer new possibilities for quantification of developing lung cell subpopulations of particular interest to the problem of respiratory distress syndrome in human neonates.

Age Factors↗
PubMed1986

Adsorption of natural lung surfactant and phospholipid extracts related to tubular myelin formation.

The microstructure of aqueous dispersions of pulmonary surfactant lipids extracted from bovine lung lavage was investigated by electron microscopic analysis. Following organic solvent extraction (chloroform/methanol) from natural lung surfactant, the mixed lipids (CLL), with 1% residual protein, were dispersed in water by two techniques, probe sonication at 4 degrees C and mechanical vortexing. Surface pressure-time adsorption isotherms were defined for the CLL dispersions, followed by staining with tannic acid, uranyl acetate, and lead citrate for electron microscopy of microstructure. CLL extract dispersions adsorbed in seconds to surface pressures near 45 dynes/cm (surface tension 25 dynes/cm) at low concentrations less than or equal to 0.25 mg/ml after dispersion by sonication of 4 degrees C and by mechanical vortexing. Ultrastructurally, the CLL dispersions were somewhat heterogeneous, but large thin-walled phospholipid vesicles, both intact and fragmented, predominated. No tubular myelin was formed. By contrast, natural lung surfactant (LS) from bronchoalveolar lavage had characteristic regions of tubular myelin when it adsorbed well at low concentrations (less than or equal to 0.25 mg phospholipid/ml). When divalent cations were removed from solution with 5 mM EDTA, this distinctive microstructure was not present and natural LS adsorbed less rapidly; higher concentrations of 0.63 mg phospholipid/ml were necessary for maximal adsorption of natural LS without tubular myelin. These results suggest that while tubular myelin is associated with optimal adsorption for natural LS, it is not a required configuration for rapid adsorption facility at low phospholipid concentrations in general. Specifically, for dispersions of surfactant extracts, other microstructures allow adsorption facility equivalent to that of natural LS with tubular myelin.

Adsorption↗
PubMed1986

Hydrophobic surfactant-associated protein in whole lung surfactant and its importance for biophysical activity in lung surfactant extracts used for replacement therapy.

Hydrophobic protein of 6,000 and 14,000 daltons was isolated from mammalian pulmonary surfactant obtained from canine, human, and bovine alveolar lavage material. Low molecular weight, hydrophobic, surfactant-associated protein (SAP), herein referred to as SAP 6-14, was distinguished from SAP-35, the major glycoprotein in mammalian surfactants (the 35,000 dalton glycoprotein A or apolipoprotein A) by amino acid composition, peptide mapping, and by resistance of SAP 6-14 to digestion by endoglycosidase F, collagenase, trypsin, and other proteases. The amino acid composition of SAP 6-14 was found to be highly enriched in leucine and other hydrophobic amino acids. The characteristics of protein isolated from bovine replacement surfactant extracts utilized for the treatment of hyaline membrane disease in humans were also studied. SAP 6-14 isolated from calf lung surfactant replacement extracts (CLSE) and surfactant-TA were found to be identical to SAP 6-14 isolated from ether/ethanol extracts of various mammalian surfactants. By contrast, SAP-35, the major surfactant-associated glycoprotein of molecular weight = 35,000, and other higher molecular weight proteins were not detected in significant quantities in the CLSE or surfactant-TA replacement surfactants, either by highly sensitive silver stain analysis or by immunoblot using monospecific antisera generated against bovine SAP-35. Biophysical studies of the CLSE replacement surfactant containing only SAP 6-14 and native phospholipids demonstrated full surface activity compared to natural lung surfactant. Dynamic surface tension lowering and adsorption properties of CLSE were essentially identical to those of freshly isolated bovine whole surfactant. Thus, hydrophobic SAP 6-14 is the only protein detected in bovine lung extract surfactants with full biophysical activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Surface property changes from interactions of albumin with natural lung surfactant and extracted lung lipids.

These experiments characterize the effects of albumin on the dynamic surface activity of natural lung surfactant (LS), and an extracted mixed lipid fraction (CLL), at physiologic temperature, humidity, and film cycling rate on an oscillating bubble apparatus. Measurements of albumin effects on the surface pressure-time (pi-t) adsorption isotherms of CLL and LS are also reported. Results show that albumin in concentrations greater than or equal to 20 mg/ml increased the minimum dynamic surface tension of LS suspensions (0.4 mg phospholipid/ml) from less than 1 dyne/cm to 21 dynes/cm at 37 degrees C. Albumin in low concentrations (2 mg/ml) had a similar detrimental effect on the dynamic surface activity of extracted surfactant lipids, CLL. In addition, albumin also inhibited the isolated adsorption facility of LS and CLL; instead of adsorbing rapidly to their maximum spreading pressures of 45 dynes/cm, both surfactant mixtures (at 0.063 and 0.125 mg phospholipid/ml) adsorbed more slowly or reached lower final surface pressures in the presence of plasma protein. A striking finding was that albumin inhibition of surface activity was moderated or abolished at high lipid concentrations. For example, minimum dynamic surface tensions less than 1 dyne/cm were reached on the oscillating bubble for natural LS at concentrations greater than 0.75 mg/ml and CLL at concentrations greater than 1.5 mg/ml, even in the presence of very large amounts of albumin (100 mg/ml). Similarly, LS and CLL adsorption facility was protected from albumin inhibition at sufficiently high phospholipid concentrations. Albumin inhibition of natural LS adsorption was also moderated by the presence of 1.4 mM Ca2+ ions. These results show that albumin in plasma transudates has the potential to seriously impair alveolar surfactant activity in vivo. However, the detrimental effect will be mitigated if a critical threshold of phospholipid is present.

Adsorption↗
PubMed1985

Pulmonary physiological and surfactant changes during injury and recovery from hyperoxia.

The time course of lung injury and recovery from a sublethal exposure to 100% O2 was investigated in adult rabbits. Animals were exposed to 100% O2 for 64 h and then returned to room air for varying periods of time up to 200 h. By the end of the exposure period, the alveolar permeability to solute increased significantly, and biochemical analyses of bronchoalveolar lavages showed a 30% decline in phospholipid content and a threefold increase in protein levels. However, other parameters such as wet-to-dry lung weight ratios, blood gas values, and pressure-volume mechanics were not significantly different from control levels after 64 h of hyperoxia. Twenty-four hours postexposure, alveolar phospholipid levels had declined even further (51% of control), and mean protein levels in lavage increased to eight times control values. These lavages exhibited severely impaired dynamic surface activity at 37 degrees C and 100% humidity in an oscillating bubble apparatus. In addition, total lung capacity, lung compliance, and arterial O2 partial pressure declined greatly at this time. Between 12 and 48 h postexposure, animal mortality was 35%; the remaining animals survived, and physiological parameters returned to normal by 200 h postexposure. Bronchoalveolar lavages from the recovered animals contained protein levels equal to those of controls and phospholipid levels approximately twice those in control lavages. Lavage surface activity also returned to normal by the 200 h postexposure time point.

Animals↗
PubMed1985

Lung surfactant replacement in premature lambs with extracted lipids from bovine lung lavage: effects of dose, dispersion technique, and gestational age.

Extracted bovine calf lung lipids (CLL) with minimal protein (approximately 1%) were instilled prior to ventilation in groups of premature lambs of average gestational ages of 127 and 133 days. Aqueous dispersions of CLL were prepared by two techniques prior to instillation: sonication in an ice bath (S) and mechanical vortexing at room temperature (V). A low surfactant dose (15 mg CLL/kg animal weight) and a high dose (100 mg/kg) were investigated for each dispersion technique. Following tracheal instillation of surfactant, lambs were ventilated with 100% oxygen for 2 h with umbilical circulation intact, and for up to an additional 10 h after separation. A clear improvement in blood oxygenation and lung compliance was found over controls for lambs given 15 mg/kg and 100 mg/kg CLL(V), and 100 mg/kg CLL(S). Lambs treated with 15 mg/kg CLL(S) failed to improve over controls. Experimental groups treated with equal doses of CLL(V) and CLL(S) had similar amounts of lung lavage phospholipid, with values progressively declining during ventilation. Analyses of in vitro surface properties showed that both vortexed and sonicated CLL dispersions adsorbed to equilibrium surface pressures of 45-47 dynes/cm in seconds at concentrations greater than or equal to 0.25 mg CLL/ml. Both dispersions also lowered surface tension to less than 1 dyne/cm under dynamic compression at 37 degrees C in 100% humidity, although CLL(V) showed some enhancement over CLL(S) in dynamic surface activity at low subphase concentration (0.5 mg/ml). Moreover, CLL(V) and CLL(S) differed markedly in their effects on pressure-volume mechanics in a surfactant-deficient excised rat lung model. Instilled CLL(V) dispersions improved excised lung pressure-volume mechanics at significantly lower concentrations than CLL(S) dispersions.

Animals↗
PubMed1985

Double-blind clinical trial of calf lung surfactant extract for the prevention of hyaline membrane disease in extremely premature infants.

A prospective, double-blind, controlled trial was conducted to determine whether instillation of an exogenous surfactant into the lungs before the first breath could prevent hyaline membrane disease. The surfactant is calf lung lipid extracted from saline lung lavage. Entry was limited to infants who were 24 to 28 weeks' gestation, who were born at Children's Hospital of Buffalo, and whose mothers had not received betamethasone for more than 24 hours before birth. Treated infants received 3 mL (90 mg) of calf lung surfactant extract instilled into their trachea before the first breath; control infants received 3 mL of normal saline. A prospective scoring system and respiratory support variables were used to compare the groups. At 48 hours of age, only two of 14 calf lung surfactant extract-treated infants (14%) had hyaline membrane disease compared with seven of 13 control infants (54%) (P = .033). Inspired oxygen, mean airway pressure, ventilator rate and ventilator efficiency index were also lower in the treated group during the first 48 hours of life (P less than .01 to P less than .001). Calf lung surfactant extract instillation at birth appears to be an effective material and method of preventing hyaline membrane disease in extremely premature infants.

Animals↗
PubMed1985

Double-blind, randomized trial of a calf lung surfactant extract administered at birth to very premature infants for prevention of respiratory distress syndrome.

Organic solvent extraction of surfactant obtained by lavage of calf lungs yields a highly surface-active material. A double blind, randomized clinical trial to determine the effect of this material on respiratory distress syndrome in premature infants was initiated in the Neonatal Intensive Care Unit at the University of Rochester in December 1983. Infants 25 to 29 weeks gestational age were eligible for entry into the trial. At the time of this interim analysis 32 patients had been randomly selected and entered into the trial, 16 surfactant-treated patients and 16 in a control group who received only saline. At birth, intrapulmonary instillation of the calf lung surfactant extract dispersed in saline or saline alone occurred in the delivery room immediately after intubation and prior to ventilation; infants were then ventilated and treated as usual. At 6, 12, 24, 48, and 72 hours after birth, the severity of respiratory distress was categorized as either minimal, intermediate, or severe based on oxygen and mean airway pressure requirements. Differences observed at six hours after birth were of marginal significance, but at 12 and 24 hours the surfactant-treated group had significantly (P less than .01) less severe respiratory distress compared with the control group. Differences between treated and control infants were not statistically significant at 48 and 72 hours after birth. In four surfactant-treated infants the severity of respiratory distress worsened between 24 and 48 hours after birth, suggesting that one dose of surfactant at birth may not be sufficient for some infants.

Clinical Trials as Topic↗
PubMed1984

Characterization of the plasma and mitochondrial membrane potentials of alveolar type II cells by the use of ionic probes.

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.

Animals↗
PubMed1984

Lysophosphatidylcholine cell depolarization: increased membrane permeability for use in the determination of cell membrane potentials.

Current techniques for the determination of cellular membrane potentials based on the uptake of a radiolabeled lipophilic cation, [3H]triphenylmethylphosphonium, and the cyanine dye, DiOC5(3), were analyzed in terms of the proportions of these probes which are accumulated due to potential-dependent and potential-independent forces. Measurements were made of probe uptake in two model systems: rabbit type II pneumocytes and human promyelocytic HL60 cells. For both cell types, the membrane potential-independent component of triphenylmethylphosphonium uptake was found to be a function of several variables, including the length of exposure of the cells to the transport facilitator tetraphenylboron, the concentration of tetraphenylboron, and the integrity of the cell membrane. To accurately determine the magnitude of the potential-independent component of probe uptake by type II and HL60 cells, the cell-permeabilizing agent lysophosphatidylcholine was used. The ability of lysophosphatidylcholine to depolarize cell membranes and accurately predict membrane potential-independent accumulation was found to be equal to or superior to several other techniques commonly used to achieve membrane depolarization (e.g. gramicidin, valinomycin plus high external potassium). Lysophosphatidylcholine cell treatment was found to be a simple, rapid, and accurate technique to increase cell membrane permeability and allow equilibration of intra- and extracellular ions. The method is shown to be useful for determining membrane potential-independent accumulation of both radiolabeled and fluorescent probes of membrane potential.

Animals↗
PubMed1984

Pulmonary surfactant: an interdisciplinary approach.

Pulmonary surfactant research is considered from the perspective of interdisciplinary correlations based on information acquired from biophysics and physical chemistry, biochemistry, cell biology, physiology, and medicine. Current views of the important molecular constituents of the pulmonary surfactant system are described and related to the biophysical surface properties necessary to generate appropriate effects on respiratory physiology. The fundamental importance of multidisciplinary characterizations of lung surfactant, together with correlations between these descriptions, is stressed throughout. The primary advantage of such an approach is that it provides broad but coordinated principles and data with which to test hypotheses of lung surfactant function and roles in respiratory physiology and pathophysiology. This perspective is used to examine available information about the functional composition of lung surfactant, its surface tension-lowering properties at physiological temperature and humidity, and considerations relevant to the formulation of effective exogenous surface-active mixtures in the treatment of surfactant-deficient states. Also discussed is the biophysical state in vivo of pulmonary surfactant at the alveolar level, including current knowledge of the alveolar hypophase as well as the concept of functional surfactant acting in predominantly dry alveoli in the normal lung.

Animals↗
PubMed1983

Comparative adsorption of natural lung surfactant, extracted phospholipids, and artificial phospholipid mixtures to the air-water interface.

Adsorption to the air-water interface of natural lung surfactant obtained by bovine lung lavage is compared and contrasted with the adsorption of mixtures of synthetic phospholipids and of extracted mixed lung lipids containing minimal protein. Surface pressure-time (pi-t) adsorption isotherms are measured at 35 degrees C for the surfactant mixtures as a function of the presence or absence of divalent metal cations (Ca2+ and Mg2+) and of heating to 45 degrees C or 90 degrees C. The effect of aqueous dispersion technique (sonication or mechanical vortexing) on the adsorption process is also studied for the extracted or synthetic phospholipid mixtures. The results imply that the protein component is necessary for the optimal adsorption of natural lung surfactant. However, by taking advantage of different methods available for phospholipid dispersion in an aqueous phase in vitro, it is possible to formulate dispersions of extracted lung phospholipids containing of order 1% protein which adsorb as well as the complete surfactant system. These results suggest that protein concentrations in surfactant mixtures can be minimized for applications such as exogenous lung surfactant replacement for the neonatal Respiratory Distress Syndrome (RDS). However, for situations which may involve alterations in endogenous surfactant function such as in lung injury, effects involving pulmonary surfactant protein and protein-lipid interactions may be of functional significance.

Adsorption↗
PubMed1983

Natural and artificial lung surfactant replacement therapy in premature lambs.

The effect of tracheal instillation of surface-active mixtures in premature lambs was studied as an animal model of exogenous surfactant replacement therapy for the respiratory distress syndrome (RDS). Specific mixtures studied were 7:3 (molar ratio) dipalmitoyl phosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) and extracted mixed lipids (with 1% protein) from cow lung lavage (CLL). Preventilatory tracheal instillation of greater than 15 mg/kg of CLL in 10 ml 0.15 M NaCl to premature lambs gave improved alveolar-arterial O2 gradient and blood gases and increased lung compliance, compared with control lambs over a 15-h period. Lambs receiving 7:3 DPPC:PG dispersions were not improved over controls with regard to pressure-volume characteristics and were worse than controls in arterial oxygenation. In terms of in vitro surface properties, both extracted natural CLL and 7:3 DPPC:egg PG were able to lower aqueous surface tension to 1 dyn/cm under dynamic compression. However, the dynamic respreading of CLL films on successive surface cycles was superior to that of 7:3 DPPC:PG. Moreover, after dispersal in 0.15 M NaCl by vortexing (5 mg/80 ml), CLL adsorbed to surface pressure (tau values of 45 dyn/cm within 10 min. 7:3 DPPC:PG adsorbed to significantly lower tau values after subphase dispersal by a variety of methods.

Animals↗
PubMed1983

Ultrasonic and jet aerosolization of phospholipids and the effects on surface activity.

Surface active aerosols were produced from aqueous dispersions of mixed lipids (CLL), extracted from bovine lung lavage. Particle size distributions were measured as a function of humidity for two types of aerosol generators: ultrasonic and jet. Lipid dispersions before aerosolization were prepared by sonication in an ice bath and by mechanical vortexing. Over a range of high humidity greater than 60-70%, ultrasonic nebulization gave CLL aerosols with mass median aerodynamic diameters (MMAD) of 1.4 +/- 0.1 micron, compatible with predicted alveolar deposition fractions of 0.2-0.3 according to current deposition models. For humidities of 30-95%, jet nebulization gave MMAD values of 0.4-0.5 micron, which have lower predicted alveolar deposition. The surface pressure-time (pi - t) adsorption characteristics at 35 +/- 2 degrees C of CLL dispersions prepared initially by vortexing or sonication were not significantly affected by ultrasonic nebulization over a 1-2 h time period. In addition, the dynamic surface tension lowering of both kinds of CLL dispersion was not affected by ultrasonic nebulization (minimum surface tension less than 1 dyne/cm at 37 degrees C and 100% humidity). Current interest in the treatment of the respiratory distress syndrome (RDS) with exogenous surfactant replacement has focused largely on the delivery of surfactant replacement has focused largely on h delivery of surfactants to infants by tracheal instillation at birth. However, the ability to form multi-component surfactant aerosols with appreciable alveolar deposition fractions and high surface activity may help to expand the utility of replacement therapy to patients with aerated lungs.

Adsorption↗
PubMed1982

Isolation of type II pneumocytes by laser flow cytometry.

The isolation of a pure population of type II pneumocytes from adult rabbit lung by the technique of multiparameter laser flow cytometry is reported here. Lipophilic phosphine-3R was used to stain cell dissociated from lungs by elastase and trypsin. Type II cells were then sorted by simultaneous measurement of high phosphine-3R fluorescent staining of lamellar bodies and intermediate intensity of low-angle light scatter. We were able to obtain an essentially pure population (98%) at sorting rates of 5 X 10(5) lamellar-body-containing cells per hour. The cell sorter technique offers an attractive alternative to the density gradient isolation of type II cells, particularly in terms of detailed population analysis. In addition, refinements in cell sorter methods offer the potential to discriminate differentiating type II cells with few lamellar bodies that do not segregate on the basis of buoyant density.

Acridines↗
PubMed1982

Bilirubin interactions with phospholipid components of lung surfactant.

This work examines the dynamic surface pressure-area behavior of films of unconjugated bilirubin spread from chloroform solution at 22 degrees C on 0.15 M NaCl and buffered phosphate subphases. Film behavior is examined at pH 5.5, 7.4 and 8.0. The interactions of bilirubin in mixed films with dipalmitoyl phosphatidylcholine and with 9:1 dipalmitoyl phosphatidylcholine:dioleolyl phosphatidylcholine are examined at similar temperature and pH values. It is found that unconjugated bilirubin modifies the dynamic surface pressure-area behavior of phospholipid films in both the high and low surface pressure regimes, with bilirubin exerting its greatest effect at low pH where its subphase solubility is low. Because many premature infants suffering from the Respiratory Distress Syndrome (RDS) have accompanying hyperbilirubinemia, with possible bilirubin transport to the alveolar space, the interactions of bilirubin with phospholipid films are discussed in terms of potential effects on pulmonary surfactant in vivo.

Bilirubin↗