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K H Cheng

Publications and source records attributed to K H Cheng.

At least 55 records · Page 3Linked to original sources
PubMed1990

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. I. Theory.

It is shown that fluorescence anisotropy from lipidlike probes in the hexagonal HII phase gives information of (a) orientational order parameters, (b) the wobbling diffusion constant, and (c) the hopping diffusion constant of the probe, DH, equals DL/R2, the lateral diffusion constant over the square of the radius of the hexagonal tubes. Here we consider only lipidlike probes having the absorption transition movement and/or the emission transition moment along the long axis of the molecule. Three models are introduced for analysis of time-resolved data: the "WOBHOP," the "reduced WOBHOP," and the "P2P4HOP" model. The fluorescence anisotropy in response to a very short excitation pulse in each of the three models is a constant plus a number of exponentials. The WOBHOP and reduced WOBHOP models have 3 and 2 exponentials, respectively, and both contain four fitting parameters: r0 (the fundamental anisotropy), (P2) (the second rank orientational order parameter), DW (the wobbling diffusion constant), and DH (the hopping diffusion constant). The P2P4HOP model has eight exponentials and five fitting parameters: the four parameters listed above and (P4) (the fourth rank orientational order parameter). Analysis of fluorescence anisotropy data in the hexagonal HII phase using one of these models allows for obtaining the hopping diffusion constant, and, if the lateral diffusion constant is known, the radius of the hexagonal tubes. Substitution of DH = 0 in each of the three models yields an expression for the fluorescence anisotropy that is used in the literature for lamellar (L alpha or L beta) phases. The fluorescence anisotropy in coexisting L alpha/HII phases is discussed.

Diffusion↗
PubMed1990

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. II. An experimental study.

The polymorphic phase behavior of unsaturated phosphatidylethanolamine (PE)/diacylglycerol (DG) binary lipid mixtures was investigated by the use of time-resolved fluorescence anisotropy. Using a fluorescent lipid, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenylethyl] carbonyl]3-sn-phosphatidyl-choline (DPH-PC), the orientational order and rotational dynamics of the above lipid mixtures in the liquid crystalline lamellar (L alpha) and inverted hexagonal (HII) phases were studied. By employing a one-exponential model (Cheng, K.H. 1989: Biophys. J. 55:1025-1031) to fit the anisotropy decay data, abrupt decreases in the normalized initial anisotropy decay slope and the residual anisotropy of DPH-PC were observed at approximately 6-8% DG, signifying a L alpha/HII phase transition. Using our new theoretical WOBHOP and P2P4HOP models as described in a preceding paper (Van Der Meer, B.W., K.H. Cheng, and S.Y. Chen. 1990. Biophys. J. 58:000-000), two or more rotational correlation times were required to describe the anisotropy decay behavior of DPH-PC in the HII phase. These rotation correlation times were further related to the second and fourth rank order parameters, and the wobbling and hopping diffusion constants of the fluorescent probe in the highly curved lipid cylindrical tubes of the HII phase. The hopping diffusion constant (DH) equals the lateral diffusion constant (DL) divided by R2 (R = radius of the lipid cylindrical tubes). The value of DL was estimated by measuring the excimer formation rate of 1-palmitoyl-2-[10-(1-pyrenl)decanoyl] phosphatidyl choline (py-PC) in the same PE/DG mixtures. Upon comparing the values of DH and DL, the value of R was determined to be approximately 10-15 A, and agreed with that derived from x-ray diffraction (Tate, M.W., and S.M. Gruner, 1989, Biochemistry. 28:4245-4253; Rand, R.P., N.L. Fuller, S.M. Gruner, and V.A. Parsegian. 1990. Biochemistry. 29:76-87).

Diffusion↗
PubMed1990

Selective binding of a 30-kilodalton protein to disposable hydrophilic contact lenses.

To investigate the accumulation of tear proteins on disposable extended-wear contact lenses (42% Etafilcon A and 58% hydration), a technique involving sodium dodecyl sulfate-polyacrylamide minigel electrophoresis combined with a sensitive silver-staining method was used. Besides the binding of large amounts of tear lysozyme the authors found an accumulation of an as yet unidentified 30-kilodalton (kD) protein. Longitudinal experiments showed lysozyme binding after 1 day of lens wear. The 30-kD protein was detected after a 2-day wearing period. The fact that protein deposition occurs during the relatively short wearing period of these lenses (1 week) may explain the unexpectedly high incidence of contact lens-associated conjunctivitis observed with these lenses.

Contact Lenses, Extended-Wear↗
PubMed1989

Role of calcium in the thermal inactivation of calcium transport proteins.

Using purified sarcoplasmic reticulum membranes as model systems, the role of calcium ion in the thermal inactivation of membrane calcium transport was investigated. Hyperthermia induces calcium release from the heavy fraction of sarcoplasmic reticulum. This calcium channel related calcium release was inhibited by the presence of glycerol and enhanced by the presence of ethanol. Calcium was found to protect the thermal-induced calcium transport inactivation of CaATPase in the light fraction of sarcoplasmic reticulum. Both glycerol and cholesterol protect the thermal inactivation of CaATPase. Yet their effects on the calcium-induced protection kinetics were rather different, i.e., the glycerol inserts its protection effect by increasing the degree of cooperativity of calcium binding, while cholesterol increases the calcium-binding affinity. The calcium protection effect was attributed to the ability of calcium to enhance the thermal stability of the protein. This was demonstrated by an upshift (30-39 degrees C) of the transition temperature of the rotational parameter of the native tryptophans of CaATPase in the presence of calcium.

Adenosine Triphosphate↗
PubMed1989

Fluorescence depolarization study on non-bilayer phases of phosphatidylethanolamine and phosphatidylcholine lipid mixtures.

The orientational order and rotational dynamics of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl] carbon yl]-3-sn-phosphatidylcholine (DPH-PC) in dilinoleoylphosphatidylethanolamine (DLPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) binary lipid mixtures were investigated. A previous study (Biochim. Biophys. Acta 731 (1983) 177) indicated that the empirical phase diagram of POPC/DLPE can roughly be divided into three zones. They are the lamellar (15% PC and higher), intermediate (5-15% PC) and inverted hexagonal (0-5% PC) phases. As the lipids changed from the lamellar to intermediate phase, the order parameter increased at all temperatures (1-50 degrees C). On the contrary, the rotational diffusion decreased at high temperatures (20-50 degrees C) but increased at low temperatures (1-10 degrees C). These results indicate that the intermediate phase is in a stressed state at high temperatures but in a highly mobile amorphous state at low temperatures. As the lipid progressed from the intermediate toward hexagonal phase, the order parameter decreased abruptly at all temperatures. The ratio of order parameter in the intermediate phase to that in the hexagonal phase was calculated. This ratio was found to increase linearly with temperature, indicating that a distinct change in the packing symmetry of lipids occurred as temperature increased. From the intermediate to hexagonal phase, the rotational diffusion increased slightly at high temperatures but declined abruptly at low temperatures. These results further agreed with the stressed and amorphous natures of the intermediate phases as described above.

Fluorescence Polarization↗
PubMed1989

Fluorescence depolarization study of lamellar liquid crystalline to inverted cylindrical micellar phase transition of phosphatidylethanolamine.

The orientational order and rotational dynamics of 2-[3-(diphenyl-hexatrienyl) propanoyl]-3-palmitoyl-L-alpha- phosphatidylcholine (DPH-PC) embedded in dioleoylphosphatidyl-ethanolamine (DOPE) were studied by fluorescence depolarization technique. Upon increasing the temperature, the calculated wobbling diffusion constant D perpendicular of the fluorescent probe was found to decrease at the lamellar (L alpha) to inverted cylindrical (H II) phase transition (10 degrees C). This suggested that the increased gauche rotamers of the alkene chains in the HII phase imposes a constraint in the wobbling motion of the fluorophore. The calculated ratio of order parameter in the L alpha phase to that in the HII phase was 1.7 and different from the theoretical value of 2.0 as predicted from the change in packing symmetry. This result can be explained by a slightly higher local order parameter of the fluorophore or by the fast rotational diffusion motion of the fluorophore around the symmetry axis of the cylindrical tubes in the HII phase.

Colloids↗
PubMed1987

Protection of the membrane calcium adenosine triphosphatase by cholesterol from thermal inactivation.

There is correlative evidence that one mechanism of cellular thermoresistance is an increased level of membrane cholesterol. The hypothesis that cholesterol protects membrane proteins from thermal inactivation was tested using Ca-ATPase as a model. The intracellular Ca2+- and Mg2+-dependent ATPase from muscle sarcoplasmic reticulum was reconstituted into lipid mixtures containing different amounts of cholesterol [cholesterol/phospholipid molar ratio (C/PL) = 0.1 or 0.3]. The rate of thermal inactivation of calcium uptake activity of the reconstituted vesicles with C/PL = 0.3 was found to be significantly lower than those with C/PL = 0.1 in the temperature range 43-47 degrees C where hyperthermic cell killing occurs. At 43 degrees C, this is equivalent to a 3 degrees C temperature shift. ATP hydrolysis of Ca-ATPase was found to be substantially heat resistant in reconstituted vesicles with C/PL = 0.1 or 0.3. Glycerol (10%) protects while ethanol (2.5%) and the local anesthetics dibucaine, tetracaine, and procaine sensitize the thermal inactivation of calcium uptake. To investigate the molecular mechanisms of thermal inactivation and cholesterol protection, the responses of the physical state of the lipid and protein conformation to hyperthermic sensitizers and protector were monitored using fluorescent and spin label probes and circular dichroism, respectively. The calcium uptake inactivation appears to be due to a direct thermotropic conformational change (denaturation) of the protein. Cholesterol raises the temperature of inactivation, as does glycerol, while ethanol and the local anesthetics lower it.

Adenosine Triphosphate↗
PubMed1986

The role of cholesterol in the activity of reconstituted Ca-ATPase vesicles containing unsaturated phosphatidylethanolamine.

The effect of cholesterol on the activity of sarcoplasmic reticulum Ca-ATPase, reconstituted in proteoliposomes containing soybean phosphatidylethanolamine (PE), egg phosphatidylcholine (PC), and cholesterol, was examined. The protein incorporation efficiency increased with PE content but appeared to be independent of cholesterol content. At low cholesterol, PE stimulated calcium uptake. The coupling efficiency of the proteoliposomes increased with an increase in cholesterol content at each PC/(PC + PE) ratio and was more pronounced for those proteoliposomes containing high PE. Dynamic fluorescence measurements of the incorporated lipophilic probe, diphenyl-1,3,5-hexatriene, revealed a decrease in the motion and an increase in the order of the phospholipid fatty acyl chains in proteoliposomes with high cholesterol content. A complementary observation was made using electron spin resonance of the spin label, 2,2-dimethyl-5-dodecyl-5-methyloxazolidine N-oxide. Freeze-fracture electron microscopy studies on proteoliposomes containing 0.20 molar ratio of PC/(PC + PE) and cholesterol revealed predominantly vesicular structures with occasional bilayer defects at high cholesterol content. It is postulated that the cholesterol-induced enhancement of the Ca-transport function of the Ca-ATPase is related to the hydration-related bilayer-destabilizing characteristic of the cholesterol molecule as revealed by 31P NMR.

Animals↗
PubMed1986

Correlation between bilayer destabilization and activity enhancement by diacylglycerols in reconstituted Ca-ATPase vesicles.

Using the reconstituted Ca-ATPase vesicles as a model system, we demonstrated that the presence of 1,2-dioleoyl-sn-glycerol (diolein) in the membrane introduces a pronounced enhancement in the Ca-transport function of Ca-ATPase, while the 1,2-dipalmitoyl-sn-glycerol (dipalmitin) does not. We also found by both 31P NMR and freeze-fraction electron microscopy that diolein destabilized lipid bilayers to a greater extent than did dipalmitin. We conclude that the tendency of diacylglycerols to destabilize the phospholipid bilayer is related to their capacity to enhance the activity of the membrane calcium pump.

Animals↗
PubMed1986

Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit.

Changes in the molecular organization of membranes in pericarp cells of ripening tomato fruit were examined by fluorescence depolarization after labeling with fluorescent lipid-soluble probes. The fluorescent labels were partitioned into isolated protoplasts and purified plastids from fruit at various stages of senescence. Values for steady-state anisotropy (r(ss)) of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled protoplasts rose progressively during the early stages of ripening over a time frame that overlapped the climacteric rise in ethylene production. This can be interpreted as reflecting a decrease in the lipid fluidity of primarily plasma membrane. By contrast, there was no significant change during ripening in r(ss) for plastid membranes labeled with DPH, 1-[4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cis- or trans-parinaric acid. Nor was there any change during ripening in the limiting fluorescence anisotropy (r(oo)) and order parameter (S) for plastids labeled with DPH or TMA-DPH, parameters that are corrected for any differences in lifetime. Some degree of lifetime heterogeneity, possibly reflecting structurally distinct domains, was discerned in both young and senescent plastids that had been labeled with DPH or TMA-DPH, but this also did not change as ripening progressed. Thus membranes of the pericarp cells sustain different fates as the tomato fruit ripens, implying that there are distinguishable mechanisms of membrane deterioration in senescing tissues.

Journal Article↗
PubMed1985

Differential polarized phase fluorometric studies of the perturbation of phospholipid packing by BHT.

Butylated hydroxytoluene (BHT) partitions effectively into lipids and modifies cellular function and behavior in numerous ways, particularly at low temperatures. In this study, the modification by BHT of the lifetime and dynamic rotational characteristics of diphenylhexatriene (DPH) in dipalmitoylphosphatidylcholine (DPPC) liposomes was investigated by phase fluorometry. BHT broadens the gel to liquid crystalline transition of DPPC by decreasing the lower end of the transition (T1), leaving the upper end (Th) unaffected. The lifetime of DPH in DPPC does not vary linearly with temperature but declines sharply above the main transition. The average lifetime of DPH is decreased to a greater extent below, compared to above, Th by BHT. This may be due to increased water penetration into the bilayer interior. Non-monoexponential decay also occurs below Th, probably due to a heterogeneous distribution of BHT, which would result in highly perturbed regions of greater than average BHT content. The motional parameter of DPH most affected by BHT is the order parameter (S) which decreased to a considerable extent below Th but is not affected above Th. In contrast, the rotational diffusion coefficient (R) of DPH is increased slightly above Th by BHT.

Butylated Hydroxytoluene↗
PubMed1985

Fluorescence resonance energy transfer study of the associative state of membrane-bound complexes of complement proteins C5b-8.

Human complement protein C8 was labeled with the fluorescent chromophores fluorescein-5-isothiocyanate (FITC), 3-(4-isothiocyanatophenyl)-7-diethylamine-4-methyl coumarin (IPM), eosin-5-isothiocyanate (EOS), or Texas Red (sulforhodamine-101-sulfonyl chloride; TR) with only minor reduction in the specific hemolytic activity of the protein. The distribution of C5b-8 complexes bound to sheep erythrocyte membranes was investigated by monitoring fluorescence resonance energy transfer (RET) between the following RET donor/acceptor pairs of labeled C8: FITC-C8/EOS-C8, IPM-C8/EOS-C8, and FITC-C8/TR-C8. On binding to membranes containing pre-formed C5b67 complexes, specific RET was detected for each of the donor/acceptor pairs of labeled C8 investigated. In contrast, no energy transfer was observed for these RET donor/acceptor pairs of labeled C8 incubated in the presence of control membranes or in membrane-free solution. On the basis of a consideration of the transfer efficiency that would be expected for donor/acceptor pairs of labeled C8 that were uniformly dispersed on the membrane surface, these results suggest that C5b-8 complexes are aggregated into polymeric clusters when membrane-bound. The efficiency of donor-C8 to acceptor-C8 RET--and the hemolytic activity of membrane-bound C5b-8 (in the absence of C9)--are both related to the surface density of membrane-bound C5b67, suggesting that the physical clustering of the membrane-inserted C5b-8 complex may be related to the expression of its cytolytic activity.

Cell Membrane Permeability↗
PubMed1984

Improved yield of plasma membrane from mammalian cells through modifications of the two-phase polymer isolation procedure.

Modifications to the two-phase polymer gradient procedure for isolating plasma membrane from mammalian cells have resulted in greatly increased yields of purified plasma membrane. First, the cells were not treated with a membrane stabilizer (ZnCl2) prior to homogenization. This reduced the severity of homogenization required for disruption and allowed a greater proportion of the surface membrane to form large, flattened sheets that are more easily purified than the smaller fragments formed during more severe homogenization. Second, three crude fractions obtained from the homogenate (600g, 2000g, and 12,000g pellets), rather than a single, low-speed pellet (600g) containing only large sheets of membrane, were subjected to gradient centrifugation to obtain plasma membrane. This modification allowed purification of small as well as large fragments of plasmalemma and greatly increased the yield of purified membrane. Mg+2-dependent, Na+-K+-stimulated ATPase, a marker enzyme for plasma membrane, was enriched in the purified fraction by congruent to 17-fold relative to homogenate on a specific activity basis, and the yield of isolated plasma membrane averaged 70%, and was occasionally as high as 90%.

Animals↗
PubMed1984

A microcomputer-based binaural measurement system.

A microcomputer is used to control a dual-channel, sine-wave generator as the excitation to both the left and right ears in experimental subjects, and for on-line measurement of the neuronal spike counts. The effects of varying the walking pattern and phase angle between the two channels can be investigated. After normalisation of spike counts, the result can be displayed as a 3-D plot on a CRT or an x-y plotter. The system is useful for general steady state and transient binaural measurement.

Biomedical Engineering↗
PubMed1983

Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers.

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a "break" around 7-10 degrees C with optimum storage temperature for unprotected cells being about 10 degrees C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40 degrees C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal "breaks" centered around 8 and 30 degrees C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.

Animals↗
PubMed1983

Rotational diffusion of TEMPONE in the cytoplasm of Chinese hamster lung cells.

The correlation time for rotational diffusion (tau R) of 2,2,6,6-tetramethyl-4-piperidone-N-oxide (TEMPONE) in Chinese hamster lung (V79) cells has been measured. For these cells in an isosmotic solution at 20 degrees C, tau R = 4.18 X 10(-11) s, approximately 3.6 times greater than tau R = 1.17 X 10(-11) s in water. The relationship between tau R and viscosity was investigated in a number of glycerol-water (0-50%) and sucrose-water (20-40%) solutions and a constant Stokes-Einstein volume of 44 A3 was found for TEMPONE in solutions of less than 20% glycerol and sucrose. This gives an average shear viscosity (for rotation of a small molecule) of 0.038 poise for the cytoplasm. When nonsecular terms were used in the calculation of tau R, the activation energies for rotation of TEMPONE in the above solutions correlated well with the activation energies for shear viscosity. The viscosity increases as the cell is shrunk in hypertonic solutions. It also increases with decreasing temperature with an activation energy of 3.7 kcal/mol, about the same as the activation energy for the viscosity of pure water. The rotational correlation times were carefully calculated considering inhomogeneous line broadening, non-Lorentzian line shapes, the need for accurate tensor values and nonsecular terms.

Animals↗
PubMed1983

Thermotropic lipid and protein transitions in chinese hamster lung cell membranes: relationship to hyperthermic cell killing.

Exposure of mammalian cells to hyperthermic temperatures (ca. 41-45 degrees C) appears to act as a direct or triggering effect to produce some later response such as cell death, thermotolerance, or heat-shock protein synthesis. The high activation energy of cell killing indicates that the direct effect of hyperthermia might be a thermotropic transition in some cellular component, for this particular response. Both hyperthermic survival and growth data imply that the temperature for the onset of hyperthermic cell killing is 40-41.5 degrees C for Chinese hamster lung V79 cells. Studies using the electron spin resonance label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene show the existence of lipid transitions at approximately 7-8 and 23-36 degrees C (or a broad transition between these temperatures) in mitochondria and whole cell homogenates, that correlate well with changes in growth and hypothermic killing. No lipid transition was detected near 40-41.5 degrees C that could correlate with hyperthermic killing in either mitochondrial or plasma membranes, but measurements of intrinsic protein fluorescence and protein fluorophore to trans-paranaric acid energy transfer demonstrate the existence of an irreversible transition in protein structure or arrangement above ca. 40 degrees C in both mitochondrial and plasma membranes. This transition is due to protein rearrangement and (or) unfolding such that there is increased exposure of protein tryptophan and tyrosine residues to polar groups and to paranaric acid. The strength of the transition implies that a significant fraction of total membrane protein is involved in this transition, which may be analogous to the heat-induced denaturation of water-soluble proteins. This alteration in membrane structure above ca. 40 degrees C could cause many of the observed changes in plasma membrane and mitochondrial function, which may further be involved in cellular responses to hyperthermia.

Animals↗