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1.
Purpose: The intent of this work was to assess the impact of lyophilization on the encapsulation of salmon calcitonin (sCT) into liposomes. Methods: Four different liposomal formulations were investigated, i.e. DPPC:Chol:DSPE-PEG2000 (75:20:5 and 65:30:5) and DPPC:Chol (80:20 and 66.7:33.3). Lipid films were prepared and hydrated with loading buffer containing sCT and different concentrations of the cryoprotectant, trehalose dihydrate. The liposomes were lyophilized, reconstituted and extruded to obtain small unilamellar vesicles. Non-encapsulated sCT was separated by gel filtration. Non-lyophilized formulations and liposomes lyophilized without the cryoprotectant were used as controls. Liposomes were analyzed for particle size, polydispersity index, zeta-potential and encapsulation efficiency. 31P-NMR (phosphorous nuclear magnetic resonance spectroscopy) was performed on selected formulations. Results: Post-lyophilization, no significant change in particle sizes and zeta-potentials were noted, regardless of the presence or absence of the cryoprotectant. Encapsulation efficiencies, however, increased following lyophilization, in both PEGylated (lyophilization control batch) and non-PEGylated liposomes (cryoprotectant batches only). 31P-NMR revealed the presence of two distinct vesicle populations – liposomes and micelles – in PEGylated formulation. The presence of micelles might be responsible for the observed encapsulation enhancement of sCT in the PEGylated formulation. Conclusions: Lyophilization resulted in an increase in encapsulation efficiency of sCT in PEGylated liposomes, even in the absence of a cryoprotectant, due to presence of micellar vesicles. 相似文献
2.
Surface areas and fluctuations evaluated from 50 ns molecular dynamics simulations of fully hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers in a 1:2 trehalose:lipid ratio carried out at surface tensions 10, 17 and 25 dyn/cm/leaflet are compared with those of pure bilayers under the same conditions. Trehalose increases the surface area, as consistent with the surface tension lowering observed in simulations at constant area. The system bulk elastic modulus K b = 1.5 ± 0.3 × 10 10 dyn/cm 2. It is independent of bilayer surface area and trehalose content within statistical error. In contrast, the area elastic modulus K a shows a strong area dependence. At 64 Å 2/lipid (the experimental surface area), K a = 138 ± 26 dyn/cm for a pure DPPC bilayer and 82 ± 10 dyn/cm for one with trehalose; i.e. trehalose increases fluidity of the bilayer surface at this area per lipid. 相似文献
3.
The structure and thermal behavior of hydrated and lyophilized dipalmitoylphosphatidylcholine (DPPC) multilayers in the presence of trehalose were investigated by differential scanning calorimetry and X-ray diffraction methods. Trehalose enters the aqueous space between hydrated bilayers and increases the interbilayer separation (from 0.36 to 1.37 nm in the different DPPC phases at 1 M trehalose). It does not affect the lipid chain packing and also the slow isothermal conversion at 4 degrees C of the metastable L beta' phase into the equilibrium crystalline Lc phase. Addition of trehalose leads to a slight upward shift (about 1 degrees C at 1 M trehalose) of the three phase transitions (sub-, pre-, and main transition) in fully hydrated DPPC while their other properties (enthalpy, excess specific heat, and transition width) remain unchanged. The effect of trehalose on the thermal behavior of DPPC multilayers freeze-dried from an initially completely hydrated state is qualitatively similar to that of water. These data support the "water replacement" hypothesis about trehalose action. It is suggested that trehalose prevents the formation of direct interbilayer hydrogen bonds in states of low hydration. 相似文献
4.
Summary The cell coat picture effect which is usually obtained with the conventional RR method, that is with the RR/OsO 4 coupled reaction, is investigated. In this first paper, each of conceivable events which might take place between RR, OsO 4 and cell surface membrane is discussed or studied. Various tests are carried out on ascites Ehrlich carcinoma cells and Zajdela ascites hepatoma cells treated with numerous chemical reagents, as also on a few pure proteins. The set of data supports the concept that the staining pattern is due to the combination in surface membranes of RR with a colloidal-like form of OsO 2. The latter might occur during the formation of stable cyclic osmic acid diesters between OsO 4 and membrane unsaturated lipids. A possibility by which the resulting marker is though also to be in a colloidal-like state is put forward. A next report will deal with this problem. 相似文献
5.
The endophytic cyanobacterium, Anabaena azollae, isolated from laboratory cultures of Azolla caroliniana Willd., contains three spectroscopically distinct biliproteins. About 70% of the biliprotein is c-phycocyanin ( max 610 nm) and 13% is allophycocyanin ( max 647 nm, shoulder 620 nm). A third pigment corresponds to phycoerythrocyanin ( max 570 nm, shoulder 590 nm). In very dilute solutions of allophycocyanin, at constant pH and buffer strength, the 647 nm maximum disappears and a single max occurs at 615–620 nm. The 647 nm absorption maximum reappears upon concentrating the dilute solution. Very dilute solutions of phycoerythrocyanin exhibit a broad peak between 570 and 590 nm. Absorption spectra of c-phycocyanin are not significantly altered upon dilution. Fluorescence emission maxima of phycoerythrocyanin, c-phycocyanin, and allophycocyanin occur at 630 nm, 643 nm and 660 nm respectively, using 540 nm excitation. Two subunits, of molecular weight 16,500 () and 20,600 (), are seen in c-phycocyanin upon dissociation with SDS. Dissociation of allophycocyanin and phycoerythrocyanin with SDS yields one sizeclass of subunits, with a molecular weight of about 17,500 for allophycocyanin and 18,000 for phycoerythrocyanin.Contribution No. 684
Offprint requests to: G. A. Peters 相似文献
6.
By means of contact angle determinations with two liquids, on hydrated as well as on dried protein layers, the long-range and the short-range contributions to the protein surface tensions, and from these the protein ( G
131) and the protein-ligand ( G
132) free energies of interaction in aqueous media, were determined. For human serum albumin (HSA), human IgG, and human IgA, the differences between G
131
HYDRATED
and G
131
DRY
were connected with the behavior of these proteins in low concentrations of (NH 4) 2SO 4 versus saturated (NH 4) 2SO 4 solutions. By interpolation, intermediate points are found that correlate well with the known salting-out properties of these three proteins. On the basis of the data, it is predicted that the precipitation of IgG by 1/3 saturated (NH 4) 2SO 4 is preventable, or reversible, by the admixture of 15% dimethylsulfoxide; both predictions are confirmed experimentally. From the G
132 values found, it is shown that HSA and IgG should attach to phenyl ligands under physiological conditions, but that IgA is so hydrophilic that it only can adhere to phenyl ligands after partial dehydration brought about when admixed with 1 M (NH 4) 2SO 4. Closer analysis of the values obtained for the long-range and short-range components of the surface tensions of HSA, IgG, and IgA allow deeper insight into their functional, chemical, and physicochemical properties. 相似文献
7.
A systematic study is presented of the effects of trehalose on the physical properties of extruded DPPC–cholesterol unilamellar vesicles. Particular emphasis is placed on examining how the interactions present in the hydrated state translate into those in the dehydrated state. Observations from HSDSC and DSC are used to examine the phase behavior of hydrated and dehydrated vesicles, respectively. The concentration of trehalose inside and outside the vesicles is manipulated, and is shown to affect the relative stability of the membranes. Our results show for the first time that a combination of high inner and low outer trehalose concentration is able to decrease the gel-to-liquid crystalline phase temperature ( Tm), while any other combination will not. Upon dehydration, the Tm of all lipid mixtures increases. The extent of the increase depends on the trehalose distribution across the bilayer. The Tm changes in the same direction with trehalose concentration for both freeze-dried and fully hydrated samples, suggesting that the trehalose distribution across the vesicle membrane, as well as the trehalose–phospholipid interaction, is maintained upon lyophilization. The results presented in this work may aid in the formulation of systems to be used in the lyophilization of liposomes for drug delivery applications. 相似文献
8.
Summary The in situ growth of microorganisms in Berea sandstone cores preferentially plugged the larger pore entrances. The largest pore entrance sizes after microbial plugging ranged from 20 to 38 m, compared with 59 to 69 m before plugging. The pore entrance size distribution of plugged cores was shifted to smaller sizes. A mathematical model based on Poiseuille's equation was found to adequately predict permeability reductions (greater than 90%) caused by microbial growth in the large pore entries.Nomenclature
Q
volumetric flow rate ( L
3/ t)
-
C
orifice constant (dimensionless)
-
A
cross-sectional area ( L
2)
-
g
gravity ( L/t
2)
-
h
pieziometric head ( L)
- s
transmittivity ( L
2)
-
R
e
Reynolds number (dimensionless)
-
a
constant (dimensionless)
-
density ( M/ L
3)
-
viscosity ( M/Lt)
-
d
diameter ( L)
-
L
length ( L)
- P
pressure change ( M/L
2) 相似文献
9.
The influence of phosphorus (P) and nitrogen (N) supply on biomass, leaf area, photon saturated photosynthetic rate ( Pmax), quantum yield efficiency (), intercellular CO 2 concentration ( Ci), and carboxylation efficiency (CE) was investigated in Vicia faba. The influence of P on N accumulation, biomass, and leaf area production was also investigated. An increase in P supply was consistently associated with an increase in N accumulation and N productivity in terms of biomass and leaf area production. Furthermore, P increased the photosynthetic N use efficiency (NUE) in terms of Pmax and . An increase in P supply was also associated with an increase in CE and a decrease in Ci. Under variable daily meteorological conditions specific leaf nitrogen content (N L), specific leaf phosphorus content (P L), specific leaf area ( L), root mass fraction (R f), Pmax, and remained constant for a given N and P supply. A monotonic decline in the steady-state value of R f occurred with increasing N supply. L increased with increasing N supply or with increasing N L. We tested also the hypothesis that P supply positively affects both N demand and photosynthetic NUE by influencing the upper limit of the asymptotic values for Pmax and CE, and the lower limit for Ci in response to increasing N.This revised version was published online in March 2005 with corrections to the page numbers. 相似文献
10.
Photoinhibition of photosynthesis is manifested at the level of the leaf as a loss of CO 2 fixation and at the level of the chloroplast thylakoid membrane as a loss of photosystem II electron-transport capacity. At the photosystem II level, photoinhibition is manifested by a lowered chlorophyll a variable fluorescence yield, by a lowered amplitude of the light-induced absorbance change at 320 nm (A 320) and 540-minus-550 nm (A 540–550), attributed to inhibition of the photoreduction of the primary plastoquinone Q A molecule. A correlation of the kinetics of variable fluorescence yield loss with the inhibition of Q A photoreduction suggested that photoinhibited reaction centers are incapable of generating a stable charge separation but are highly efficient in the trapping and non-photochemical dissipation of absorbed light. The direct effect of photoinhibition on primary photochemical parameters of photosystem II suggested a permanent reaction center modification the nature of which remains to be determined.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement 相似文献
11.
Both Alcaligenes eutrophus JMP 134 and its plasmid-free derivative Alcaligenes eutrophus JMP 222 utilize 2,6-dinitrophenol as sole source of carbon, energy, and nitrogen. In the presence of ammonia resting cells of these strains release two mol of nitrite per mol of 2,6-dinitrophenol. Alcaligenes eutrophus JMP 222-1D, a mutant of strain JMP 222 obtained by transposon (Tn5) mutagenesis, is able to use 2,6-dinitrophenol as nitrogen source but not as source of carbon and energy. Resting cells of this mutant liberate only one mol of nitrite per mol of 2,6-dinitrophenol. A single metabolite was detected by high-pressure liquid chromatography and identified as 2-hydroxy-5-nitropenta-2,4-dienoic acid from the mass spectrum, the 1H-, and 13C-NMR spectra. Strain JMP 222-1S, a spontaneous mutant of strain JMP 222-1D, accumulates 4-nitropyrogallol which was identified as the initial metabolite of 2,6-dinitrophenol degradation.Non-standard abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- 2,6-DNP
2,6-dinitrophenol
- HNMA
2-hydroxy-5-nitromuconic acid
- HNPA
2-hydroxy-5-nitropenta-2,4-dienoic acid
- NB
nutrient broth
- NMR
nuclear magnetic resonance
- NPG
4-nitropyrogallol
- O.D.
optical density
- t R
retention time
- UV/Vis
ultraviolet/visible 相似文献
12.
Summary Phloretin and other neutral phloretin-like molecules are able to decrease the electrostatic potential within neutral lipid bilayers and monolayers. The relationship between the change in the dipole potential and the aqueous concentration of the molecule is well described by a Langmuir isotherm. From the Langmuir isotherm, the apparent dissociation constants ( K
D
A
) and the maximum dipole potential change (
max) are obtained for the different phloretin-like molecules tested. Considering the phloretin analogs as derivatives of acetophenone containing two kinds of substituents, one on the benzene ring and another on the carbon chain, it is found that (a) K
D
A
is related to the hydrophobicity of the compound and is also a function of the position of the hydroxyl substituent in the ring; (b) from the dependence of K
D
A
on the length of the acyl chain, it is estimated that the free-energy change is 650 cal/mole CH 2; (c)
max is not a simple function of the dipole moment of the molecule but depends on the substituent on the carbon chain and on the position and number of hydroxyl groups on the benzene ring; (d) phloretin adsorption parameters are a function of membrane lipid composition. The results are discussed in terms of the effect of these compounds on chloride transport in red blood cells. 相似文献
13.
Fluorescence recovery after photobleaching was used to investigate the translational diffusion of a fluorescent derivative of a membrane-spanning lipid in L
phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine prepared in water and in glycerol. The translational diffusion coefficient in hydrated bilayers ( D
w) ranged between 2 and 5x10 –8 cm 2/s and in glycerinated bilayers ( D
g) the range was between 3 and 24×10 –10 cm 2/s between 10° and 40°C. These results are discussed in terms of models for diffusion in membranes. 相似文献
14.
Summary Measurements were made of the difference in the electrochemical potential of protons (
) across the membrane of vesicles reconstituted from the ATPase complex ( TF
0
·F
1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential () and pH difference across the membrane ( pH), respectively.In the presence of Tris buffer the maximal and no pH were produced, while in the presence of the permeant anion NO
3
–
the maximal pH and a low were produced by the addition of ATP. When the ATP concentration was 0.24 mm, the was 140–150 mV (positive inside) in Tris buffer, and the pH was 2.9–3.5 units (acidic inside) in the presence of NO
3
–
. Addition of a saturating amount of ATP produced somewhat larger and pH values, and the
attained was about 310 mV.By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4–5 during ATP hydrolysis.The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied. 相似文献
15.
Kinetics were determined for the four transients K 590, L 540, M 410, O 660 of the photochemical cycle of bacteriorhodopsin (BR 570) both in 1H 2O and in 2H 2O over a wide temperature range. Breaks in the Arrhenius plots, observed at 25–32 for the longest-lived transients coincide with a transition point in the microviscosity of the membrane as measured by depolarization of an added fluorescent probe. The earliest isotope effect occurs in the decay of L 540, and is present in the subsequent formation and decay of M 410 and O 660. Thus in the light-driven proton pump of BR 570, proton ejection from the Schiff base correlates with decay of L 504 and reprotonation occurs with the decay of both M 410 and O 660 back to BR 570. 相似文献
16.
Summary The addition of calcium to suspensions of egg phosphatidylcholine and cardiolipin converts multiwalled liposomes to the hexagonal (H II) phase (Rand, R.P., Sengupta, S. (1972) Biochim. Biophys. Acta
255:484–492). We have studied this lamellar to hexagonal phase transition by freeze-fracture, thin-section electron microscopy, and X-ray diffraction and have morphologically characterized the intermediate stages. The first step in the transition involves the invagination and fusion of bilayers, marked by the appearance of lipidic intramembrane particles and crater-like indentations, as the large liposomes are converted to smaller flattened and elongated vesicles. The next step is the formation of tightly packed hexagonal arrays of tubules, each tubule being about 11 to 15 nm in diameter. These tubules are filled with fluid and a lipid bilayer forms the wall of each cylinder. Finally this tubular bilayer phase is converted to the hexagonal (H II) phase, where the distance between tubes is 5.5 to 7.5 nm. 相似文献
17.
1. |
Underwater downwelling quantal irradiance spectra were measured in estuarine and coastal areas under various tidal and rainfall conditions. At midday the available spectrum near the bottom has maximal irradiance in the region of about 570 to 700 nm in the estuary, whereas in offshore coastal areas greatest irradiance occurs between 500 and 570 nm. At twilight in an estuary, maximal underwater downwelling irradiance shifts to the 490–520 nm region.
| 2. |
The visual pigment absorption maxima of 27 species of benthic crustaceans from semi-terrestrial, estuarine and coastal areas have values ranging from 483 to 516 nm. There is no obvious shift in the
max from long wavelengths in estuarine species to shorter wavelengths in coastal species. The only match between
max and midday spectrum was for a continental shelf species,Geryon quinquedens.
| 3. |
The Sensitivity Hypothesis is predicted to account for the visual sensitivity of benthic crabs from estuarine and coastal areas. To assess the match between visual spectral sensitivity and environmental spectra, photon capture effectiveness was calculated for a range of idealized visual pigment absorption functions operating in the measured environmental spectra.
| 4. |
All crab species are poorly adapted for maximal photon capture at midday, since pigments having
max longer than 540 nm function best under all daytime spectral conditions. Photon capture of visual pigments with
max near 500 nm improves dramatically at twilight, particularly at lower visual pigment densities and shallow depths. However, pigments having
max at wavelengths longer than those for the crabs are equally or more efficient at photon capture. Therefore the Sensitivity Hypothesis is not supported for crustaceans.
| 相似文献
18.
A bacterium isolated from a petal of Casa Blanca Lily (ST26 strain) produced a marked amount of extracellular trehalose (- d-glucopyranosyl-[1,1]-- d-glucopyranose) in culture medium containing glucose. 16S rDNA-based phylogeny showed that ST26 belongs to, or is related to, Cellulosimicrobium cellulans, a close relative of Cellulomonas spp. Various Cellulomonas strains obtained from culture collections also showed extracellular trehalose productivity, suggesting that trehalose production is a common property of this bacterial genus. ST26 accumulated trehalose in medium supplied with glucose but not with sucrose, glycerol or maltose. Effective extracellular trehalose production by ST26 was achieved by supplying 0.5–1% ammonium sulfate and 0.5–1% CaCO 3. The addition of CaCO 3 adjusted the pH of the culture to around 5.0. The optimized culture conditions yielded trehalose from glucose at a conversion rate of 61%. The addition of ammonium sulfate greatly reduced the dry cell weight of ST26 and intracellular content of trehalose, which suggests that the addition of ammonium sulfate makes ST26 cells leak trehalose into the medium. ST26 effectively propagated in minimal medium containing trehalose as a sole carbon source, which suggests that trehalose serves as a carbohydrate reserve of this organism.The nucleotide sequence of 16S rDNA of ST26 has been submitted to the DDBJ databank under accession number AB109293 相似文献
19.
The relationship between the electrochemical proton gradient,
H+
–
, and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference () across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a pH of 0.6 units (interior acid) and a of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H + diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m -3 MgATP the pH was increased to about 1.0 unit and the to about-10 mV. Under these conditions the proton-motive force (
p
H+
–
/ F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg 2+ having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg 2+ again had no effect. Nigericin greatly increased pH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H +-translocating ATPase. The
H+
–
generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.Abbreviations
H+
–
electrochemical proton gradient
-
electrical potential difference across membrane
- p
proton-motive force (
H+
–
/ F)
- FCCP
p-trifluoromethoxycarbonylcyanidephenylhydrazone
- TPMP +
triphenylmethylphosphonium ion 相似文献
20.
Summary The membrane permeability coefficients for the homologous monocarboxylic acids, formic through hexanoic, as well as benzoic and salicylic, were determined for egg phosphatidylcholine-decane planar bilayer membranes. The permeabilities of formic, acetic and propionic acid were also determined for solvent-free phosphatidylethanolamine bilayers. Permeability coefficients were calculated from tracer fluxes measured under otherwise symmetrical conditions, and precautions were taken to ensure that the values were not underestimated due to unstirred layer effects. The relation between the nonionic (HA) permeability ( P
m
) and the hexadecane/water partition coefficient ( K
p
) was: log
m
=0.90 log K p+0.87 (correlation coefficient=0.996). Formic acid was excluded from the analysis because its permeability was sixfold higher than predicted by the other acids. The permeabilities for solvent-free membranes were similar to those for decanecontaining membranes. The exceptionally high permeability of formic acid and the high correlation of the other permeabilities to the hexadecane/water partition coefficient is a pattern that conforms with other nonelectrolyte permeabilities through bilayers. Similarly, the mean incremental free energy change per methylene group ( G-CH 2-) was –764 cal mol –1, similar to other homologous solutes in other membrane systems. However, much less negative G values (–120, to –400 cal mol –1) were previously reported for fatty acids permeating bilayers and biological membranes. These values are due primarily to unstirred layer effects, metabolism and binding to membranes and other cell components. 相似文献
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