Gene/Protein Disease Symptom Drug Enzyme Compound
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Phospholipase A2 (PLA2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra (N. naja naja) and bee venom PLA2 exhibit hydrolytic activity towards 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m-1). Pancreatic PLA2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m-1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA2 is bound to these monolayer microstructures. Very little PLA2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA2 monolayer hydrolysis electrostatically driving PLA2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.
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PMID:Phospholipase A2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface. 775 50

To study ultrastructurally the mechanisms of lysosome reactions to cell membrane-derived intracellular membranes we developed a cell free system using small inside-out and rightside-out cell membrane vesicles (IOVs and ROVs) as a target of the reactions. The IOVs were generated from rat erythrocyte ghosts in a low ionic strength alkaline solution in the absence of divalent cations after erythrocytes were reacted with wheat germ agglutinin-coated colloidal gold [WGA (CG)], while ROVs were from ghosts homogenized in a buffer with MgSO4 and bovine serum albumin-coated CG [BSA (CG)]. WGA (CG)s bound to the cell surface were rearranged on the membrane and distributed irregularly on the inner surface of generated small IOVs. A coat structure derived from the ghost's submembranous coat was almost depleted from their outer surface. By contrast, BSA (CG)-binding to the membranes was negligible in the process of ROV formation. When isolated rat liver lysosomes were incubated with these WGA (CG)-binding small IOVs at 37 degrees C, CG particles were found in several lysosomes under electron microscopy. Some lysosomes adhered to the IOVs, and their limiting membranes were found to collapse and disappear partially at the adhering region, suggesting their fusion. This reaction seems to occur even in cytosol-free solution. By contrast, the lysosomes indicated very low reaction to BSA (CG)-containing ROV, and to WGA (CG) or BSA (CG) alone. Therefore, it is suggested that isolated liver lysosomes react, at least to fuse, in a cytosol-independent fashion, with surface coat-depleted IOVs derived from WGA (CG)-bound and then -rearranged erythrocyte membranes.
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PMID:Reactivity of lysosomes to inside-out cell membrane vesicles in a cell-free system. 806 43

The air-filled microspheres of the ultrasound-contrast agent Albunex are unique in that the walls consist of human serum albumin molecules which have been made insoluble by sonication of the albumin solution. The microspheres were isolated by flotation, and the washed microspheres were labelled with 125I. The labelled material was cleared from the circulation mainly as particles, not as soluble albumin molecules. In rats, 80% of intravenously injected microspheres were cleared from the blood within 2 min. Nearly 60% of the dose was recovered in the liver, only 5% in the lungs, 9% in the spleen, and negligible quantities in kidneys, heart and brain. Of the radioactivity in the liver, more than 90% was taken up by Kupffer cells (liver macrophages). The protein in the liver was degraded apparently with first-order kinetics (half-life 40 min). In pigs, over 90% of the intravenously injected dose was recovered in the lungs. The vastly increased recovery in pig lungs, compared with that in rats, is probably due to the pulmonary intravascular macrophages of the pig; macrophages are not normally found in this location in rats (or humans). In a separate series of experiments in rats, the biodistribution of shell material from the microspheres was examined. The microspheres were made to collapse by applying external pressure on the suspension, leaving sedimentable protein material consisting of layers of insoluble albumin from the 'shells' surrounding the air bubble. The 'shells' and the microspheres were cleared from the circulation and taken up by the liver with the same kinetics. In the lungs, a higher proportion (15%) of shells than of microspheres was recovered.
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PMID:Biodistributions of air-filled albumin microspheres in rats and pigs. 817 4

To assess the role of hemofiltration (HF) among different treatment modalities, we reviewed our clinical material from 37 patients that consecutively underwent the treatment from 1981 on. A number of 12 patients on HF for at least 1 year deliberately switched to hemodialysis (HD) or hemodiafiltration (HDF) were studied retrospectively. Biochemical and nutritional parameters, cardiovascular aspects and morbidity data were collected during one year before and after the treatment change. A sodium balance study was performed in 9 patients during HF as well. No significant differences in plasma urea, creatinine, phosphate, body weight, serum albumin, transferrin, hemoglobin and PCR were found. BUN tended to be lower during HD-HDF because of the more efficient removal of urea with these treatments. Indeed, the Kt/V index was 0.91 during HF and it was 1.15 with HD-HDF. There were no differences in hypotensive episodes and morbidity. Sodium loss was strictly related to body fluid removal during HF session with a net sodium loss (NSL) of 128 mEq per liter of fluid removal (FR) (NSL = 6.44 + 122 FR; r:0.83; p < 0.01). Adapting sodium concentration of substitution fluid to patients weight gain, cardiovascular stability improved in those subjects more prone to collapse. With equivalence in PCR during the 2 periods, although Kt/V was 20% lower during HF, it seems reasonable to assume that the lower urea clearance might be compensated by the more efficient removal of higher molecular weight substances and/or by the improved biocompatibility of HF.
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PMID:The contribution of hemofiltration among the treatment modalities of chronic uremia. 817 95

The localized concentration of energy during a single bubble collapse is manifested in two forms, which are the emission of an acoustic pulse, and the emission of a light pulse. Through precise control of experimental parameters, one can levitate a single bubble in a standing wave field and measure the magnitude of the acoustic and light pulses resulting from the violent collapse of the cavity. The information acquired from such measurements provides better understanding of the mechanisms that are responsible for the emissions, which may lead to the practical application of controlled bubble implosions. An experimental apparatus was developed to measure the acoustic and light emissions from a single, stable sonoluminescing bubble. Two surfactant additives were studied to determine the effects on the bubble emissions. Triton X-100, which has previously been shown to provide free interfacial motion, reduced the magnitude of both the acoustic and light pulses from the bubble. The protein bovine serum albumin (BSA) which has been shown to hinder interfacial motion, allowed the bubble to be driven to higher acoustic pressures, and resulted in an increase in the magnitude of the light pulses from the bubble. Images of the sonoluminescing bubble indicate that the collapse remains spherical in the cases presented, and that bubble translation can be correlated with weak acoustic and light emissions.
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PMID:The effects of surfactant additives on the acoustic and light emissions from a single stable sonoluminescing bubble. 930 Oct 48

Novel temperature-sensitive polymers containing glucose units in their backbone were synthesized and covalently conjugated to trypsin. A series of copolymers based on N-isopropylacrylamide (NIPAAm) and glucosyoxylethyl methacrylate (GEMA) were prepared by using 4,4'-azobis(4-cyanovaleric acid) as an initiator, which resulted in one terminal carboxylic acid group per polymer chain. The polymers were conjugated to primary amine groups of trypsin with water-soluble carbodiimide as a coupling agent, which led to a star-shaped conformation. The polymer-enzyme conjugation was confirmed and characterized by size exclusion and reversed-phase chromatography. Almost of all amine groups in trypsin available for the conjugation were consumed and, consequently, a very dense layer of copolymers was actually coated around the enzyme surface. The conjugated enzymes exhibited reversible precipitation/resolubilization behaviors over a wide range of temperatures, depending on the content of GEMA in the copolymer. They also demonstrated no detectable self-digestion (autolysis) process, but the unconjugated enzyme showed very severe autolysis that led to a rapid inactivation in aqueous solution. When bovine serum albumin was used as a substrate, the protein substrate was not attacked by the conjugated enzyme, but completely digested by the unconjugated enzyme. This result was presumably caused by a steric repulsion process of the attached polymer chains around the enzyme toward the protein substrate. However, the enzyme retained sufficient activity against a low molecular weight substrate. Interestingly, the conjugated enzymes demonstrated very peculiar enzyme activity-temperature profiles, with two apparent optimal temperatures, indicating that a temperature-controlled collapse and flocculation of the copolymers around the enzyme surface modulated the mass transfer rates of substrate to the active site of the enzyme. The conjugated enzymes also exhibited improved thermal stability with increasing the amount of carbohydrate units in the polymer chain.
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PMID:Conjugation of trypsin by temperature-sensitive polymers containing a carbohydrate moiety: thermal modulation of enzyme activity. 962 35

The effect of probe sonication during microsphere processing on the stability of various aliphatic polyesters based on lactic acid (PLA) and lactic/glycolic acid (PLGA) was investigated. The weight average molecular weight (Mw) of the polymers dissolved in dichloromethane (DCM) generally decreased with an increase in duration and/or intensity of the sonication process. The extent of the Mw-reduction was more pronounced with polymers of high initial Mw and high GA content. Polydispersity indices (PD=Mw/Mn) were nearly unchanged indicating that random chain cleavage is the likely degradation mechanism. From the observation that ultrasound-induced polymer degradation slightly increased in the presence of suspended drug particles acting as cavitation nuclei, it can be concluded that the mechanical stress induced by the implosive collapse of cavitation bubbles is at least partly responsible for the observed effects in PLA/ PLGA solutions. The use of ultrasound for the preparation of W/O, O/W and W/O/W emulsions exhibited different effects depending on the formulation and the type of emulsion. The preparation of W/O emulsions generally lead to Mw-changes comparable to those observed for the corresponding polymer solutions. Fatty acid free bovine serum albumin (BSAff) was found to protect PLA and PLGA against ultrasound-induced degradation in W/O-emulsions due to the formation of a semisolid interfacial film. A tremendous effect not only on the polymer Mw, but also on its PD could be observed, when ultrasound was used to emulsify an organic polymer solution or W/O-emulsion in an external aqueous phase. As this last finding was found to have rather important implications on the drug loading efficiency, the hydration, the degradation and the initial release characteristics of the resulting microspheres, it can be concluded that probe sonication can be a rather critical process step during the preparation of microspheres.
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PMID:Ultrasound-induced degradation of PLA and PLGA during microsphere processing: influence of formulation variables. 970 13

Poly(D,L-lactide-co-glycolide) (PLG, 65:35) was used to encapsulate bovine serum albumin (BSA) using a water-in-oil-in-water (W/O/W) double emulsion solvent extraction technique. To investigate the effects of an inner water/oil ratio on microsphere characteristics, microspheres were fabricated using four different formulations with a fixed oil volume of 12ml and the inner aqueous phase volume of 0.2ml, 0.3 ml, 0.4ml or 0.5 ml, respectively. Spherical microspheres were obtained after collection by filtration for formulations employing any of the four different inner water/oil ratios. However, microspheres with smaller inner water volumes tend to collapse after vacuum drying. The surface of the formulation with a higher inner water/oil ratio was shown to possess many more pores than that of the formulations with lower inner water/oil ratios. These pores may facilitate the water withdrawal during vacuum drying. Furthermore, microspheres with the lowest inner water/oil ratio (1/60) had higher initial burst release due to its larger surface area. However, microspheres with the highest inner water volume yield a faster release profile of BSA due to interconnected voids within microspheres and more pores on the surface. Therefore, the inner water/oil ratio is a crucial factor in the W/O/W double emulsion technique affecting the morphology and release kinetics of the resulting microspheres.
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PMID:Effects of inner water volume on the peculiar surface morphology of microspheres fabricated by double emulsion technique. 1150 69

The buffer requirements to maintain mitochondrial intactness and membrane potential in in vitro studies were investigated, using gradient purified yeast mitochondria. It was found that the presence of phosphate is crucial for generation of a stable membrane potential and for preserving the intactness of the outer membrane, as assessed by probing the accessibility of Tom40p to trypsin and the leakage of cytochrome b2 from the intermembrane space. Upon addition of respiratory substrate in the absence of phosphate, mitochondria generate a membrane potential that collapses within 1 min. Under the same conditions, the mitochondrial outer membrane is disrupted. The presence of phosphate prevents both phenomena. The DeltapH component of the proton motive force appears to be responsible for the compromised outer membrane integrity. The collapse of the membrane potential is reversible to a limited extent. Only when phosphate is added soon enough after the addition of exogenous respiratory substrate can a stable membrane potential be obtained again. Within a few minutes, this capacity is lost. The presence of Mg(2+) prevents rupture of the outer membrane, but does not prevent rapid dissipation of the membrane potential. Similar results were obtained for mitochondria isolated and stored in the presence of dextran or bovine serum albumin.
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PMID:Phosphate is required to maintain the outer membrane integrity and membrane potential of respiring yeast mitochondria. 1174 88

The potency of various uncouplers for collapsing the light-induced pH gradient across thylakoid membranes in intact chloroplasts was investigated by time-resolved optical spectroscopy. The thylakoid transmembrane pH gradient ([delta]pH) was monitored indirectly by measuring the rate of cytochrome (Cyt) f reduction following a light flash of sufficient duration to create a sizable [delta]pH. The results show that the rate of Cyt f reduction is controlled in part by the internal pH of the thylakoid inner aqueous space. At pH values from 6.5 to 8.0, the Cyt f reduction rate was maximal, whereas at lower pH values from 6.5 to 5.5 the reduction rate decreased to 25% of the maximal rate. The ability of three uncouplers, nigericin, carbonylcyanide m-chlorophenylhydrazone, and gramicidin, to accelerate the rate of Cyt f reduction was determined for intact chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of the uncouplers for collapsing the [delta]pH was determined using the empirical relationship between the [delta]pH and the Cyt f reduction rate. For intact chloroplasts, nigericin was the most effective uncoupler, followed by carbonylcyanide m-chlorophenylhydrazone, which interacted strongly with bovine serum albumin. Gramicidin D, even at high gramicidin:chlorophyll ratios, did not completely collapse the pH gradient, probably because it partitions in the envelope membranes and does not enter the intact chloroplast.
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PMID:Dissipation of the Proton Electrochemical Potential in Intact Chloroplasts (II. The pH Gradient Monitored by Cytochrome f Reduction Kinetics). 1223 69


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