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1H-nuclear magnetic resonance studies on the conformational changes related to the foaming properties of beta-lactoglobulin.

beta-Lactoglobulin (beta-LG) is a whey protein with foaming ability that can be used as a food ingredient. The structural changes that occur during foaming cannot be easily assessed. In combination with deuterium exchange, 1H-NMR allows new insight into the structural features of beta-LG during foaming. beta-Lactoglobulin was dissolved in D2O and forced to foam. During foaming, the amide protons of exposed residues were exchanged for deuterium atoms, which do not appear in the 1H-NMR spectrum. Protein in solutions that had produced unstable foams showed no exchange beyond that found in unfoamed controls, indicating that the structure had remained intact. Protein in solutions that had produced stable foams showed extensive exchange. Protons of both Trp19 and Met107 exchanged with deuterium, indicating that most of the interior had been exposed to solvent. Most of the beta-LG structure was recovered after collapse of the foam. In the early steps of foaming, apparently only random coil or other exposed regions are involved in foam stabilization. More vigorous shear stress forces induce further unfolding of the beta-strands. This unfolding is suggested to be a dynamic, reversible equilibrium between an open and closed conformation of beta-strands that allows not only for interaction of the protein with the air phase but also for some secondary structure to be retained and the original structure to be recovered. Freeze-dry foaming is also discussed.

Chemical Phenomena↗

Accumulation of surfactant protein D in human pulmonary alveolar proteinosis.

Surfactant protein D (SP-D) is a collagenous calcium-dependent carbohydrate-binding protein that is structurally related to the serum mannose-binding proteins and pulmonary surfactant protein A. SP-D was initially characterized as a biosynthetic product of freshly isolated rat type II cells and first purified in chemical amounts from bronchoalveolar lavage of rats with silica-induced alveolar lipoproteinosis. The present studies describe the characterization of human SP-D isolated from therapeutic bronchoalveolar lavage of patients with pulmonary alveolar proteinosis. Human proteinosis SP-D was extracted from the 10,000 x g pellet of bronchoalveolar lavage with 100 mmol/L glucose or ethylenediamine tetraacetic acid, and specifically bound to and eluted from maltosyl-agarose. The protein cross-reacted with monospecific antibodies to rat SP-D by enzyme-linked immunosorbent assay and immunoblot and eluted near the position of rat SP-D on reverse-phase high performance liquid chromatography. When chromatographed on 4% agarose (A-15M) in the presence of ethylenediamine tetraacetic acid, the solubilized human proteinosis SP-D eluted near the void volume and earlier than rat SP-D dodecamers or human SP-D multimers in the lavage supernatant. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of proteins in the lavage pellet with antibodies to the carbohydrate-binding domain of proteinosis human SP-D demonstrated covalently cross-linked multimers of SP-D monomers (43 kd, reduced) and multimers of trimeric components stabilized by disulfide and non-disulfide bonds. These studies describe the isolation and biochemical characterization of human SP-D and demonstrate the abnormal accumulation of this protein in the air spaces of patients with alveolar proteinosis.

Amino Acid Sequence↗

Maturational differences in hyperoxic AP-1 activation in rat lung.

Immature organisms (neonates; <12 h old) have vastly differing responses to hyperoxic injury than adults. A common feature of hyperoxic gene regulation is involvement of activator protein (AP)-1. We evaluated lung AP-1 binding as well as that of the AP-1 subunit proteins c-Fos, c-Jun, phosphorylated c-Jun, Jun B, and Jun D after exposure to >95% O(2) for 3 days. Unlike adults, neonates showed no increased AP-1 binding in hyperoxia despite a high affinity of the AP-1 binding complexes for phosphorylated c-Jun and Jun D as demonstrated by supershift of these antibodies with the AP-1 complexes. Moreover, neonatal lungs exhibited two distinguishable AP-1 binding complexes, whereas adult lungs had one. In neonates, sequential immunoprecipitation revealed that the lower AP-1 complex was composed of proteins from both the Fos and Jun families, whereas the upper complex consisted of Jun family proteins, with predominance of Jun D. In adults, the single AP-1 complex appeared to involve other Fos or non-Fos or non-Jun family proteins as well. Neonatal lungs showed a higher level of Jun B and Jun D immunoreactive proteins in both air and hyperoxia compared with those in adult lungs. These results suggest that significant maturational differences in lung AP-1 complexes exist and that these may explain transcriptional differences in hyperoxic gene regulation.

Aging↗

Effect of a novel 5-lipoxygenase activating protein inhibitor, BAYx 1005, on asthma induced by cold dry air.

BACKGROUND: Leukotrienes have been implicated in the mediation of airway obstruction induced by hyperventilation of cold dry air in asthmatic subjects. The effect of a novel inhibitor of 5-lipoxygenase activating protein, BAYx 1005, on the bronchospastic response to cold dry air hyperventilation was investigated in asthmatic patients. METHODS: After a screening cold dry air hyperventilation challenge to document cold air responsiveness, 16 asthmatic subjects (baseline forced expiratory volume in one second (FEV1) > 60% of predicted) underwent cold air challenge three hours after receiving 750 mg of BAYx 1005 or placebo using a randomised, double blind, crossover design. Leukotriene synthesis inhibition was estimated by measuring the concentration of leukotriene B4 in whole blood stimulated with calcium ionophore A21387. RESULTS: Treatment with BAYx 1005 produced a 34% (95% CI 11 to 63) increase in the amount of cold air minute ventilation required for a 10% decrease in FEV1 (PD10VE) compared with placebo (mean (SE) 37.6 (1.12) 1/min compared with 28.0 (1.13) 1/min, p < 0.006). The PD20VE increased 19% (95% CI 8 to 31) after treatment with BAYx 1005 compared with placebo (57.3(1.10)1/min versus 48.1 (1.10) 1/min, p < 0.002). Treatment with BAYx 1005 produced a 15.4% decrease in ionophore-stimulated LTB4 production, while treatment with placebo produced a 7.1% increase in ex vivo LTB4 (p < 0.02). CONCLUSIONS: Treatment with BAYx 1005, a novel inhibitor of leukotriene synthesis, produced a significant blunting of cold dry air responsiveness consistent with the hypothesis that leukotrienes mediate part of the bronchoconstriction induced by hyperventilation of cold dry air.

Adolescent↗

Effect of processing on the displacement of whey proteins: applying the orogenic model to a real system.

Atomic force microscopy (AFM) has been used to investigate the displacement of a commercial whey protein system and the behavior as compared to that of beta-lactoglobulin (Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Orogenic displacement of protein from the air-water interface by competitive adsorption. J. Colloid Interface Sci. 1999, 210, 157-166). The whey protein isolate (WPI) was displaced from an air-water interface by the surfactants Tween 20 and Tween 60. Displacement data obtained were compared with data obtained for pure beta-lactoglobulin and have shown that WPI was more resistant to displacement from the air-water interface than native beta-lactoglobulin. This was related to the greater surface elasticity of WPI at higher surface stresses. In the presence of Tween 20, WPI was observed to remain on the interface at surface pressures up to 8 mN/m greater than the surface pressure at which complete displacement of beta-lactoglobulin was observed. Displacement of WPI and beta-lactoglobulin films by the surfactant Tween 60 showed similar results. However, because of the lower surface activity of Tween 60, it was not possible to reach surface tension values similar to those obtained for Tween 20. Despite the lower surface activity of Tween 60, WPI was still observed to be present at the interface at surface pressure values greater than those by which beta-lactoglobulin had been completely displaced.

Chemical Phenomena↗

Generation of carbon monoxide and iron from hemeproteins in the presence of 7,8-dihydroneopterin.

7,8-Dihydroneopterin and neopterin are secreted by human and primate macrophages after activation by interferon-gamma in a ratio of 2:1. 7,8-Dihydroneopterin is known to suppress radical-mediated processes, but it is also able in the presence of iron ions to generate superoxide radical anion and hydroxyl radicals from molecular oxygen. Effects of 7,8-dihydroneopterin were investigated on (met)myoglobin and (met)hemoglobin. Addition of 7,8-dihydroneopterin to heme proteins in air-saturated solution resulted in dose-dependent cleavage of the porphyrin moiety. The liberation of non-heme iron and carbon monoxide originating from the cleaved porphyrin was quantified. Both were generated at equimolar concentrations with a linear correlation coefficient of 0.9. Addition of ferrous iron significantly accelerated the pteridine-mediated cleaving of the porphyrin. However, the total yield of porphyrin cleaved was controlled by the pterin rather than by the ferrous ion concentration. 7,8-Dihydroneopterin is assumed to reduce the heme iron in intact protein molecules, thereby preparing the conditions for binding of oxygen and carbon monoxide as ligands. Beyond that, it is concluded that hydroxyl radicals might be generated via reduction of molecular oxygen to superoxide anion in the autoxidation process and dismutation to hydrogen peroxide and subsequent Fenton reaction.

Animals↗

Differential gene transcription following intravenous injection of air bubbles in rats with varying resistance to decompression sickness.

Decompression sickness (DCS) is a pathology caused by the appearance of gas emboli in the bloodstream and tissues. However, the weak correlation between the amount of venous gas emboli (VGE) and the development of DCS, as well as the considerable interindividual variability in DCS susceptibility, suggests that a higher DCS resistance could be associated with a better management of VGE-induced stress. To study the effects of VGE independently of the hyperbaric stress induced by diving, Wistar and DCS-resistant male and female rats received 5 mL/kg of a 0.9% NaCl solution containing air microbubbles through the tail vein. After 120 min, the liver and lungs were harvested. Wet-to-dry weight ratio was determined in the lungs. Gene expression was quantified by reverse transcription-polymerase chain reaction in the liver. Compared with standard Wistar, DCS-resistant rats exhibited a lower lung wet-to-dry weight ratio after air microbubble injection, suggesting lower pulmonary fluid accumulation. In the liver, DCS-resistant rats showed higher tissue factor transcription at the basal state and post-air microbubble injection. Tissue factor pathway inhibitor was lower in DCS-resistant rats at the basal state but higher following air microbubble injection. Levels of heat shock protein 70 (HSP70), heat shock protein 27 (HSP27), and early growth response 1 (Egr-1) were higher in DCS-resistant rats after air microbubble injection. At the basal state, only HSP27 was higher in DCS-resistant rats, with HSP70 lower and Egr-1 not different. These results help clarify the pathways involved in the response to VGE and highlight potential mechanisms underlying resistance to DCS, including enhanced anticoagulant pathways and improved cellular stress responses.NEW & NOTEWORTHY This study suggests for the first time that DCS resistance may be associated with a better tolerance to VGE. This greater DCS resistance could be achieved through improved control of the procoagulant effects of bubbles via TFPI-dependent inhibitory mechanisms and an enhanced cellular stress response to VGE by HSP70, HSP27, and EGR-1. It also suggests that it may be possible to stratify the individual DCS risk based on the thromboinflammatory response to bubbles.

Animals↗

SP-B deficiency causes respiratory failure in adult mice.

Targeted deletion of the surfactant protein (SP)-B locus in mice causes lethal neonatal respiratory distress. To assess the importance of SP-B for postnatal lung function, compound transgenic mice were generated in which the mouse SP-B cDNA was conditionally expressed under control of exogenous doxycycline in SP-B-/- mice. Doxycycline-regulated expression of SP-B fully corrected lung function in compound SP-B-/- mice and protected mice from respiratory failure at birth. Withdrawal of doxycycline from adult compound SP-B-/- mice resulted in decreased alveolar content of SP-B, causing respiratory failure when SP-B concentration was reduced to <25% of normal levels. Decreased SP-B was associated with low alveolar content of phosphatidylglycerol, accumulation of misprocessed SP-C proprotein in the air spaces, increased protein content in bronchoalveolar lavage fluid, and altered surfactant activity in vitro. Consistent with surfactant dysfunction, hysteresis, maximal tidal volumes, and end expiratory volumes were decreased. Reduction of alveolar SP-B content causes surfactant dysfunction and respiratory failure, indicating that SP-B is required for postnatal lung function.

Age Factors↗

A protein-based three terminal electronic device.

Because of their natural functional characteristics, involving inter- and intramolecular electron transfer, metalloproteins are good candidates for biomolecular nanoelectronics. In particular, blue copper proteins, such as azurin, can bind gold via a disulfide site present on its surface and they have a natural electron transfer activity that can be exploited for the realization of molecular switches whose conduction state can be controlled by tuning their redox state through an external voltage source. We report on the implementation of a prototype of protein transistor operating in air and in the solid state, based on this class of proteins. The three terminal devices exhibit various functions depending on the relative source-drain and gate-drain voltages bias, opening a way to the implementation of a new generation of logic architectures.

Azurin↗

An outbreak of streptococcal sore throat and rheumatic fever in a Royal Air Force training camp; significance of serum antibody to M-associated protein.

A large outbreak of streptococcal sore throat in a Royal Air Force Training Camp resulted in five cases of rheumatic fever among the 16- to 18-year-old apprentices, and one case in a 33-year-old airman. The most prevalent type of group A streptococcus isolated from throat swabs was M-type 5 and there was serological evidence that at least four of the rheumatic fever (R.F.) cases were due to this type.Among the patients with uncomplicated throat infection the anti-streptolysin O (ASO) and anti-deoxyribonuclease B (anti-DNAase B) responses were in general rather low, even where there was evidence of protective antibody against type 5. However, a combination of the results of the ASO and anti-DNAase B tests gave an estimate of the extent of streptococcal infection 15-25% higher than did either test alone.The titres of antibody to M-associated protein (MAP) were >/= 60 in all the R.F. patients, and in about 50% of the other patients with ASO titres >/= 200. This figure is unusually high compared with data from several other outbreaks of streptococcal infection due to different serotypes and also greatly exceeds comparable figures for cases of sporadic sore throat and acute glomerulonephritis.

Adolescent↗

Enhancement of the foaming properties of protein dried in the presence of trehalose.

Surface tension, foamability, and foam stability kinetics have been measured for the pure proteins bovine serum albumin (BSA) and beta-lactoglobulin, before and after aqueous solutions of the proteins had been subjected to different drying conditions, and also for whey protein concentrate (WPC). Pure proteins were air-dried, at 78 or 88 degrees C, in the presence and absence of sucrose or trehalose, at a mass ratio of 5:1 sugar/protein. WPC was spray-dried in the presence of various sugars: trehalose, sucrose, lactose, and lactitol. Spray-drying WPC without sugars resulted in a dramatic decrease in the foam stability, whereas drying in the presence of sugars gave better retention of the original foaming properties. Trehalose in particular resulted in almost complete retention of the foam stability observed for the nondried WPC. Pure beta-lactoglobulin showed similar behavior, but trehalose did not seem to afford the same protection to BSA.

Animals↗

In vitro acute effects of tobacco smoke on tumor necrosis factor alpha and interleukin-6 production by alveolar macrophages.

Tobacco smoke is a usual form of oxidant aggression present in the domestic environment. In the present study, the in vitro acute effects of a 2-cigarette smoke gas phase were evaluated on cell viability and cytokine secretion by alveolar macrophages (AM) from guinea pigs and human healthy subjects. Cell injury was estimated immediately after smoke exposure by evaluation of ATP cell content (measured by bioluminescence) and lactic dehydrogenase (LDH) release in the culture medium. LDH release was also measured when the interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF) activities were evaluated. No cytotoxic effect was found: The ATP cell content of both guinea pig AM and human AM did not significantly change after tobacco smoke exposure. Similarly, the LDH release in the culture medium was unchanged both immediately after tobacco smoke exposure and at the time of the cytokine evaluation (18-20 h later) compared to cells cultured in the air. The total protein synthesis by the guinea pig AM evaluated by 35S-L-methionine labeling was unaffected by tobacco smoke exposure. The production of IL-6 and TNF activities was evaluated 18-20 h after smoke exposure. The IL-6 activity was measured by the proliferation test of 7TD1 hybridoma cell line; the TNF activity was evaluated by the L929 mouse fibroblast cytotoxic test and by an immunoradiometric assay (for human AM). A 2-cigarette smoke exposure decreased both activities significantly. The exposure of the guinea pig AM reduced IL-6 activity by 24.3 +/- 6.7%, 42.4 +/- 7.8%, and 39.7 +/- 9.6% and TNF activity by 33.8 +/- 10.4%, 35.1 +/- 10.7%, and 38.8 +/- 9.9% (respectively unstimulated cells and AM activated by 0.1 and 10 micrograms LPS/mL). The decrease in monokine production by the human AM was, respectively, 57.8 +/- 8.8%, 59.7 +/- 11.4%, and 49.9 +/- 10.5% of IL-6 activity and 37.4 +/- 14.6%, 17.6 +/- 9.6%, and 37.2 +/- 6.3% of TNF activity. The possible release of cytokine inhibitors was also investigated. The inhibitory activity against recombinant TNF and IL-6 was evaluated in culture medium from unstimulated AM exposed to tobacco smoke and did not significantly differ from that of AM exposed to air, demonstrating that the decrease of monokine levels could not be explained by the release of inhibitory factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Rheological properties of human saliva.

From measurements with a Couette-type viscometer provided with a guard ring it was shown that at the saliva-air interface a protein layer is adsorbed. Measurements of the surface shear modulus of this layer on saliva of 7 healthy subjects were performed at a frequency of about 70 Hz and a temperature of 25 degrees C. For a surface age of about 1.5 h the surface shear modulus and the surface viscosity were in the order of 1 Nm-1 and 10(-3) Nm-1 s, respectively. From ellipsometric measurements it was found that the thickness of the protein layer was approx. 100 nm and, using this value, it could be concluded that the shear modulus and the dynamic viscosity were in the order of 10(7) Pa and 10(4) Pa s, respectively. The layer appeared to be fragile. Even shear deformation amplitudes of 4 x 10(-5) are too high to assure linearity. The complex viscosity (eta = eta' - i eta") of the bulk liquid of human submandibular saliva below the absorbed layer was measured in the frequency range 70 Hz-200 kHz with 3 torsional resonators, each for a different frequency, and a Ni-tube resonator. It was concluded, that the real part of the complex viscosity (eta') decreases from 1.1 mPa s at 70 Hz to a value of 0.95 mPa s at high frequencies. Except at the lowest frequency (70 Hz), the value of eta" was too small to be detected.

Adult↗

Large-scale insect cell culture: methods, applications and products.

The primary development in large-scale insect cell culture over the past year has been the continuing accumulation of documented evidence (fundamental and applied) that conventional aerated stirred-tank and air-lift bioreactors may be employed for insect cell cultivation and recombinant protein production, provided that air sparging, agitation, and the addition to the medium of Pluronic F-68 and methyl cellulose polymers are carefully controlled.

Animals↗

TAC-1, a regulator of microtubule length in the C. elegans embryo.

Regulation of microtubule growth is critical for many cellular processes, including meiosis, mitosis, and nuclear migration. We carried out a genome-wide RNAi screen in Caenorhabditis elegans to identify genes required for pronuclear migration, one of the first events in embryogenesis requiring microtubules. Among these, we identified and characterized tac-1 a new member of the TACC (Transforming Acidic Coiled-Coil) family [1]. tac-1(RNAi) embryos exhibit very short microtubules nucleated from the centrosomes as well as short spindles. TAC-1 is initially enriched at the meiotic spindle poles and is later recruited to the sperm centrosome. TAC-1 localization at the centrosomes is regulated during the cell cycle, with high levels during mitosis and a reduction during interphase, and is dependent on aurora kinase 1 (AIR-1), a protein involved in centrosome maturation. tac-1(RNAi) embryos resemble mutants of zyg-9, which encodes a previously characterized centrosomal protein of the XMAP215 family and was also found in our screen. We show that TAC-1 and ZYG-9 are dependent on one another for their localization at the centrosome, and this dependence suggests that they may function together as a complex. We conclude that TAC-1 is a major regulator of microtubule length in the C. elegans embryo.

Animals↗

Critical care management of the patient with acute respiratory distress syndrome (ARDS). Part 1: Pathophysiology and implications for mechanical ventilation.

Acute respiratory distress syndrome (ADRS) is a severe, life-threatening consequence of certain pulmonary and systemic insults. It is thought to result from a dramatic change in the permeability of the alveolar-capillary membrane, allowing the movement of fluid and proteins into alveolar air spaces. These changes are followed by inactivation of surfactant, bringing about a significant alteration in lung compliance. It is common for the devastating changes to lung function in ARDS to necessitate the patient being supported by mechanical ventilation. However, the poor compliance of the ARDS-affected lung can greatly increase the risk of ventilator induced lung injury. This has led to a concern that traditional ventilation strategies may in fact be perpetuating the very conditions they attempt to compensate for.

Adult↗

Latex aeroallergen quantification in hospitals of Moscow, Russia.

BACKGROUND: Health care workers (HCWs) who use latex gloves regularly are at a significant risk for developing allergic sensitization to natural rubber latex (NRL) proteins. The airborne route of exposure presents a hazard for sensitization and subsequent allergic symptoms. OBJECTIVE: The purpose of this study was to measure the content of the NRL proteins in the air of work areas (laboratories, treatment rooms, operating rooms, examination rooms, and dentist's treatment rooms) in eight Moscow hospitals where the employees used powdered latex gloves. METHODS: The air samples were collected by a volumetric filtration method using a static air sampler at a flow rate of 3 L/second. Airborne particles were collected onto polytetrafluoroethylene filters rated 99.9% efficient at 0.3 microm. The NRL allergens were extracted from the air filter samples in phosphate-buffered saline and quantified by an inhibition immunoassay using a five-person pool of sera containing latex-specific human immunoglobulin E. RESULTS: The airborne latex allergen concentrations in 11 areas (including positive control) where latex gloves were used varied from 5 to 26 ng/m3. Conversely, in 12 areas where latex gloves were seldom used or where powdered-free latex gloves were used allergen levels were not detectable (including two negative controls). The highest concentration of latex allergen (26 ng/m3) was found in an examination room of a gynecology department. Low and moderate concentrations were detected in most operating rooms, and low concentrations in the laboratories and treatment rooms. CONCLUSIONS: This study indicates that, in general, the airborne NRL concentrations in the work areas of Moscow hospitals are rather low compared with NRL concentrations reported from American hospitals and are dependent on the intensity of latex glove usage.

Air Pollution, Indoor↗