Pressure perturbation and differential scanning calorimetric studies of bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) were used to characterize thermal phase transitions, membrane packing, and volumetric properties in multilamellar vesicles (MLVs) composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at different temperatures. For PLFE MLVs derived from cells grown at 78 degrees C, the first DSC heating scan exhibits an endothermic transition at 46.7 degrees C, a small hump near 60 degrees C, and a broad exothermic transition at 78.5 degrees C, whereas the PPC scan reveals two transitions at approximately 45 degrees C and 60 degrees C. The endothermic peak at 46.7 degrees C is attributed to a lamellar-to-lamellar phase transition and has an unusually low DeltaH (3.5 kJ/mol) and DeltaV/V (0.1%) value, as compared to those for the main phase transitions of saturated diacyl monopolar diester lipids. This result may arise from the restricted trans-gauche conformational changes in the dibiphytanyl chain due to the presence of cyclopentane rings and branched methyl groups and due to the spanning of the lipid molecules over the whole membrane. The exothermic peak at 78.5 degrees C probably corresponds to a lamellar-to-cubic phase transition and exhibits a large and negative DeltaH value (-23.2 kJ/mol), which is uncommon for normal lamellar-to-cubic phospholipid phase transformations. This exothermic transition disappears in the subsequent heating scans and thus may involve a metastable phase, which is irreversible at the scan rate used. Further, there is no distinct peak in the plot of the thermal expansion coefficient alpha versus temperature near 78.5 degrees C, indicating that this lamellar-to-cubic phase transition is not accompanied by any significant volume change. For PLFE MLVs derived from cells grown at 65 degrees C, similar DSC and PPC profiles and thermal history responses were obtained. However, the lower growth temperature yields a higher DeltaV/V ( approximately 0.25%) and DeltaH (14 kJ/mol) value for the lamellar-to-lamellar phase transition measured at the same pH (2.1). A lower growth temperature also generates a less negative temperature dependence of alpha. The changes in DeltaV/V, DeltaH, and the temperature dependence of alpha can be attributed to the decrease in the number of cyclopentane rings in PLFE at the lower growth temperature. The relatively low DeltaV/V and small DeltaH involved in the phase transitions help to explain why PLFE liposomes are remarkably thermally stable and also echo the proposal that PLFE liposomes are generally rigid and tightly packed. These results help us to understand why, despite the occurrence of thermal-induced phase transitions, PLFE liposomes exhibit a remarkably low temperature sensitivity of proton permeation and dye leakage.     

Differential scanning calorimetric study of the thermotropic phase behavior of a polymerizable, tubule-forming lipid

A comparative study of the polymorphism exhibited by the polymerizable, tubule-forming phospholipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3- phosphocholine (DC23PC) and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC) in aqueous suspension is reported. Differential scanning calorimetry (DSC), as well as freeze-fracture electron microscopy and Raman spectroscopy, have been used to study the influence on phase behavior of rigid diacetylene groups in the fatty acyl chains of a phosphatidylcholine. DTPC large multilamellar vesicle (MLV) and small unilamellar vesicle (SUV) suspensions were found to retain liposome morphology after chain crystallization had occurred. In marked contrast, diacetylenic DC23PC suspensions do not maintain liposomal morphology in converting to the low temperature phase. Large MLVs of DC23PC with outer diameters in excess of 1 micron convert to a gel phase with cylindrical or tubular morphology at 38 degrees C, just a few degrees below the lipid's chain melting temperature (TM(H), i.e. temperature of an endothermic event observed during a heating scan) of 43.1 degrees C. Unlike the large MLVs, small MLVs or SUVs of DC23PC, with diameters of 0.4 +/- 0.3 micron and 0.04 +/- 0.02 micron, respectively, exhibit metastability in the liquid-crystalline state for several tens of degrees below the chain melting temperature prior to converting to a gel phase which, by electron microscopy, manifests itself as extended multilamellar sheets. Raman data collected at TM(H) -40 degrees C demonstrate that the gel state formed by DC23PC is very highly ordered relative to that of DTPC, suggesting that special chain packing requirements are responsible for the novel phase behavior of DC23PC.     

Phase and surface properties of lipid bilayers containing neoglycolipids

The physical properties conferred to DPPC bilayers by including neoglycolipids composed by two different trisaccharides: mannose-mannose-mannose (3M) and glucose-mannose-glucose (GMG) attached to a cholesterol (cho) and a distearylglycerol (diC18) lipid moiety by a spacer were evaluated by means of the measurement of the electrokinetic potential and interfacial fluorescent probes. The phase properties measured with diphenylhexatriene (DPH) were correlated with the surface properties measured with merocyanine 540, dansyl, and Laurdan probes. The results show that the surface properties of large unilamellar vesicles depend on the sugar exposure to the water phase and also on the hydrocarbon moiety by which it is anchored to the bilayer. The combination of the cholesterol moiety with the saccharide attenuates the cooperativity decrease induced by the cholesterol moiety without the sugar portion. The neoglycolipid GMG-diC18 promotes opposite effects affecting slightly the cooperativity at the hydrocarbon core of DPPC and displacing the phase transition temperature to higher values. The presence of neoglycolipid with diC18 introduces defects in the packing at the interface of the membrane in the gel state. It is concluded that a relatively low proportion of neoglycolipids affects significantly the interfacial properties of DPPC bilayers in large unilamellar vesicles in the absence of changes at the membrane bulk at 25 degrees C.     

Control of haemoglobin synthesis. The effects of iron deprivation, cobalt and temperature on the rate and extent of globin synthesis in reticulocytes.

A detailed examination of the kinetics of protein synthesis in rabbit reticulocytes in the presence of the iron chelating agent 2,2'-dipyridyl showed that between 30 degrees C and 42 degrees C there were characteristically two distinct phases of protein synthesis. An initial phase (I), in which no inhibition of protein synthesis was apparent, was followed by a gradual decline in the rate of protein synthesis leading to the second phase (II) in which protein synthesis occurred at a linear but inhibited rate for extended periods. In contrast, below 30 degrees C, incubation in the presence of dipyridyl caused no inhibition of protein synthesis. Between 30 degrees C and 42 degrees C the duration and amount of protein synthesis occurring in phase I before the onset of inhibition were inversely related of the inhibition as was the final rate of incorporation in phase II. During phase II, a partial reversal of the inhibition caused by dipyridyl was obtained by lowering the incubation temperature. This resulted in a burst of protein synthesis at the uninhibited rate until the amount of protein synthesis reached the same level as that in reticulocytes maintained continuously with dipyridyl at the lower incubation temperature. This burst of synthesis was observed in reticulocytes which had been held in phase II for as long as 90 min. It was also possible to reverse the inhibition by addition of haemin to cells in phase II. At any particular incubation temperature, a fixed number of rounds of protein synthesis had to occur before the onset of phase II became apparent. By the use of puromycin we showed that this was not a requirement for the synthesis of globin or of any other protein. We believe that this critical amount of protein synthesis reflects the residual ability of reticulocytes to initiate new protein chains in the absence of concurrent haem synthesis. Reticulocytes preincubated in the presence of cobaltous ions showed almost no inhibition of protein synthesis upon subsequent incubation with dipyridyl. The results are compared to those obtained in reticulocyte lysates and are discussed in terms of current theories to account for control of protein chain initiation by haemin.     

A calorimetric study of the thermotropic behavior of pure sphingomyelin diastereomers

The phase-transition properties of sphingomyelins were investigated in detail with totally synthetic, chemically and stereochemically pure (2S,3R)-(N-stearoylsphingosyl)-1-phosphocholine (D-erythro-C18-SPM) (1) and the corresponding 2S,3S isomer (L-threo-C18-SPM) (2). Heating scans of an unsonicated dispersion of 1 right after hydration showed a main transition (I) at 44.7 degrees C (delta H = 6.8 kcal/mol). Upon incubation at 20-25 degrees C a second transition (II) appeared at 36.0 degrees C (delta H = 5.7 kcal/mol). The two gel phases were designated as G alpha and G beta phases, respectively. The G beta phase was also metastable and relaxed to a third gel phase (G gamma) upon incubation below 10 degrees C. Conversion of the G gamma phase to the liquid-crystalline phase occurred via two new endotherms at 33.4 degrees C (2.6 kcal/mol) (III) and 43.6 degrees C (8.0 kcal/mol) (IV) as well as a main transition at 44.7 degrees C (9.5 kcal/mol). Possible interpretations have been proposed to account for the observed phase transitions. The L-threo isomer 2 showed similar thermotropic behavior to dipalmitoylphosphatidylcholine (DPPC): a "main transition" at 44.2 degrees C (6.0 kcal/mol), a "pretransition" at 43.1 degrees C (1.8 kcal/mol), and upon incubation at 7 degrees C for 2 weeks, a very broad "subtransition" at ca. 35 degrees C. The results are substantially different from previous studies of sphingomyelins using mixtures of stereoisomers. Mixing of 1 with 2, 1 with DPPC, and 2 with DPPC removed the metastability of the gel phase and resulted in a single transition.     

Spin labeling EPR studies of the properties of oxidized phospholipid-containing lipid vesicles

This study aims at characterizing the structure and some properties of phospholipid multi-lamellar vesicles (MLVs) containing the oxidized species γ-palmitoyl-β-(9-hydroperoxy-10,12-octadecanedienoyl)-lecithin (HPPLPC), γ-palmitoyl-β-(9-hydroxy-10,12-octadecanedienoyl)-lecithin (HOPLPC), γ-palmitoyl-β-glutaroyl-lecithin (GlPPC) and γ-palmitoyl-β-azelaoyl-lecithin (AzPPC). Sepharose 4B gel-chromatography was used to ensure and check that only MLVs are used in EPR measurements. Gel-solid to gel-liquid transition temperature (T_m), lateral phase separation, fluidity gradient and polarity profile were studied by use of EPR spectroscopy of enclosed n-doxylstearoyl lecithin spin labels. Contrarily to conjugate dienes and normal phospholipids, pure carboxyacyl species yielded aqueous suspensions showing gel-chromatography elution profile resembling that of lysolecithin micelles. Conjugate dienes/DPPC MLVs showed lateral phase separation at room temperature and T_m value lower than pure DPPC MLVs. Pure conjugate dienes MLVs resembled more PLPC MLVs and displayed free miscibility with PLPC in mixed MLVs. Pure HPPLPC MLV bilayer appeared to be slightly more rigid, while that of HOPLPC and the polarity profile of MLVs made of the pure conjugate dienes species were similar to those of normal PLPC. It is concluded that carboxyacyl lecithins in MLVs tend to disrupt vesicle structure, while conjugated dienes lecithins are more able to affect some physical properties of the bilayer, and that DPPC in MLVs enhances these effects while PLPC shows a better compatibility with the lipoperoxides.     

Viability loss of neem (Azadirachta indica) seeds associated with membrane phase behaviour.

Storage of neem (Azadirachta indica) seeds is difficult because of their sensitivity to chilling stress at moisture contents (MC) > or =10% or imbibitional stress below 10% MC. The hypothesis was tested that an elevated gel-to-liquid crystalline phase transition temperature (Tm) of membranes is responsible for this storage behaviour. To this end a spin probe technique, Fourier transform infrared microspectroscopy, and electron microscopy were used. The in situ Tm of hydrated membranes was between 10 degrees C and 15 degrees C, coinciding with the critical minimum temperature for germination. During storage, viability of fresh embryos was lost within two weeks at 5 degrees C, but remained high at 25 degrees C. The loss of viability coincided with an increased leakage of K+ from the embryos upon imbibition and with an increased proportion of cells with injured plasma membranes. Freeze-fracture replicas of plasma membranes from chilled, hydrated axes showed lateral phase separation and signs of the inverted hexagonal phase. Dehydrated embryos were sensitive to soaking in water, particularly at low temperatures, but fresh embryos were not. After soaking dry embryos at 5 degrees C (4 h) plus 1 d of further incubation at 25 degrees C, the axis cells were structurally disorganized and did not become turgid. In contrast, cells had a healthy appearance and were turgid after soaking at 35 degrees C. Imbibitional stress was associated with the loss of plasma membrane integrity in a limited number of cells, which expanded during further incubation of the embryos at 25 degrees C. It is suggested that the injuries brought about by storage or imbibition at sub-optimal temperatures in tropical seeds whose membranes have a high intrinsic Tm (10-15 degrees C), are caused by gel phase formation.     

Lipid lateral heterogeneity in phosphatidylcholine/phosphatidylserine/diacylglycerol vesicles and its influence on protein kinase C activation.

To test the hypothesis that the activation of protein kinase C (PKC) is influenced by lateral heterogeneities of the components of the lipid bilayer, the thermotropic phase behavior of dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO) vesicles was compared with the activation of PKC by this system. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to monitor the main transition (i.e., the gel-to-fluid phase transition) as a function of mole fraction DO (chi(DO)) in DMPC/DO, DMPS/DO, and [DMPC/DMPS (1:1, mol/mol)]/DO multilamellar vesicles (MLVs). In each case, when chi(DO) < or approximately 0.3, DO significantly broadened the main transition and shifted it to lower temperatures; but when chi(DO) > approximately 0.3, the main transition became highly cooperative, i.e., narrow, again. The coexistence of overlapping narrow and broad transitions was clearly evident in DSC thermograms from chi(DO) approximately 0.1 to chi(DO) approximately 0.3, with the more cooperative transition growing at the expense of the broader one as chi(DO) increased. FTIR spectroscopy, using analogs of DMPC and DMPS with perdeuterated acyl chains, showed that the melting profiles of all three lipid components in [DMPC/DMPS (1:1, mol/mol)]/DO MLVs virtually overlay when chi(DO) = 0.33, suggesting that a new type of phase, with a phospholipid/DO mole ratio near 2:1, is formed in this system. Collectively, the results are consistent with the coexistence of DO-poor and DO-rich domains throughout the compositions chi(DO) approximately 0.1 to chi(DO) approximately 0.3, even at temperatures above the main transition. Comparison of the phase behavior of the binary mixtures with that of the ternary mixtures suggests that DMPS/DO interactions may be more favorable than DMPC/DO interactions in the ternary system, especially in the gel state. PKC activity was measured using [DMPC/DMPS (1:1, mol/mol)]/DO MLVs as the lipid activator. At 35 degrees C (a temperature above the main transition of the lipids), PKC activity increased gradually with increasing chi(DO) from chi(DO) approximately 0.1 to chi(DO) approximately 0.4, and activity remained high at higher DO contents. In contrast, at 2 degrees C (a temperature below the main transition), PKC activity exhibited a maximum between chi(DO) approximately 0.1 and chi(DO) approximately 0.3, and at higher DO contents activity was essentially constant at 20-25% of the activity at the maximum. We infer from these results that the formation of DO-rich domains is related to PKC activation, and when the lipid is in the gel state, the coexistence of DO-poor and DO-rich phases also contributes to PKC activation.     

Interleukin-2-induced small unilamellar vesicle coalescence

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19 degrees C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (相似文献   

Influence of salt on the structure of DMPG studied by SAXS and optical microscopy

Aqueous dispersions of 50 mM dimyristoylphosphatidylglycerol (DMPG) in the presence of increasing salt concentrations (2-500 mM NaCl) were studied by small angle X-ray scattering (SAXS) and optical microscopy between 15 and 35 degrees C. SAXS data show the presence of a broad peak around q approximately 0.12 A(-1) at all temperatures and conditions, arising from the electron density contrasts within the bilayer. Up to 100 mM NaCl, this broad peak is the main feature observed in the gel and fluid phases. At higher ionic strength (250-500 mM NaCl), an incipient lamellar repeat distance around d=90-100 A is detected superimposed to the bilayer form factor. The data with high salt were fit and showed that the emergent Bragg peak is due to loose multilamellar structures, with the local order vanishing after approximately 4d. Optical microscopy revealed that up to 20 mM NaCl, DMPG is arranged in submicroscopic vesicles. Giant (loose) multilamellar vesicles (MLVs) start to appear with 50 mM NaCl, although most lipids are arranged in small vesicles. As the ionic strength increases, more and denser MLVs are seen, up to 500 mM NaCl, when MLVs are the prevailing structure. The DLVO theory could account for the experimentally found interbilayer distances.     

Acyl chain orientational order in large unilamellar vesicles: comparison with multilamellar liposomes: a 2H and 31P nuclear magnetic resonance study.

Large unilamellar vesicles (LUVs) composed of 1-[2H31]palmitoyl-2-oleoyl phosphatidylcholine (POPC-d31), with diameters of approximately 117 +/- 31 and 180 +/- 44 nm, were prepared by extrusion through polycarbonate filters with pore sizes of 0.1 and 0.2 microns, respectively. The 2H nuclear magnetic resonance (NMR) spectra obtained at 21 degrees C contain two components: a broad component (approximately 17 kHz linewidth) corresponding to the methylene groups and a narrower component originating from the methyl groups. Spectra with increasing powder pattern characteristics were obtained by reducing the rate of phospholipid reorientations by addition of glycerol (to increase the solvent viscosity) and by lowering the temperature. Full powder spectra, characteristic of liquid-crystalline bilayers, were obtained for both LUV samples at 0 degrees C in the presence of 50 wt% glycerol. Individual quadrupolar splittings were not resolved in these spectra, due to broader linewidths in the LUVs, which have significantly shorter values for spin-spin relaxation time T2 measured from the decay of the quadrupolar echo (90 microseconds) than the multilmellar vesicles (MLVs; 540 microseconds). Smoothed order parameter profiles (OPPs) were obtained for these samples by integration of the dePaked spectra. The OPPs were very similar to the OPP of POPC-d31 MLVs in 50 wt% glycerol at the same temperature, indicating that orientational order in MLVs and LUVs with a diameter of > or = 100 nm is essentially the same. The presence of 80 wt% glycerol was found to have a disordering effect on the vesicles.     

Time-temperature analyses of cell killing of synchronous G1 and S phase Chinese hamster cells in vitro

Time-temperature analyses of durations of heating required to achieve isosurvival were used to compare hyperthermic cell killing of synchronous Chinese hamster ovary (CHO) cells heated in G1 or S at temperatures of 42 to 45.5 degrees C. G1 populations were obtained by incubation of mitotic cells for 90 min at 37 degrees C. S phase populations were obtained by incubation of mitotic cells for 12 h at 37 degrees C in medium supplemented with 2 micrograms/ml aphidicolin, a reversible inhibitor of DNA alpha polymerase; S phase survival was also determined in an aphidicolin-free system by using high specific activity [3H]thymidine. In both systems, the thermosensitivity was similar and decreased as the cells progressed from early S phase, in agreement with earlier studies (R. A. Read, M. H. Fox, and J. S. Bedford. Radiat. Res. 98, 491-505 (1984]. A comparison of Arrhenius plots of the inverse of durations of heating required to achieve isosurvival for cells heated in G1 or S phase showed similar temperature dependence above 43.5 degrees C, yet the plots for heat-sensitive S phase cells were offset from those for heat-resistant G1 cells by about 1.5 degrees C, i.e., S phase cells respond to 43 degrees C with a rate similar to that observed in G1 cells heated at 44.5 degrees C. Using least-squares regression of the semilog plots, the curves were analyzed either as continually bending curves or as two straight lines with a break at 43.5 degrees C. When the data were analyzed using two straight lines, no significant differences in the slopes of the time-temperature plots of G1 or S phase cells were observed. A quantitative comparison between the two methods of data analysis demonstrated that in both phases the data were better fit with a continuously curving line, rather than two straight lines.     

Effect of temperature on spore germination and vegetative cell growth of Clostridium botulinum.

Spore germination and vegetative growth of Clostridium botulinum type E strain VH at 2 to 50 degrees C were studied. At all of these temperatures, germination began immediately after the addition of the spores to the germination medium. Microscopic observations during germination revealed three types of spores: phase bright (ungerminated), phase variable (partially germinated), and phase dark (fully germinated). At all temperatures except 50 degrees C, there was a pronounced lag between the initial appearance of phase-variable spores and their eventual conversion to phase-dark spores. The number of partially germinated spores increased steadily, reaching 40 to 60% by 18 to 21 h of incubation. During this time, phase-dark, fully germinated spores developed slowly and did not exceed 28% in any of the samples. At 18 to 26 h of incubation, the rate of full germination increased abruptly four-fold. There was extensive and relatively rapid germination at 2 degrees C, the lowest temperature tested, yielding about 60% phase-variable spores by 18 h, which became phase-dark by 26 h of incubation. The optimum temperature for partial and full germination was consistently 9 degrees C. Germination at 50 degrees C was exceptionally rapid and was completed within 1 to 2 h, although 40% remained phase bright. Vegetative cells showed detectable growth at 6 to 41 degrees C, with a distinct optimum at 32.5 degrees C. No growth occurred at 50 degrees C, and only marginal growth was observed at 6 to 14 degrees C. The psychrophilic nature of the germination process coupled with the cold tolerance of vegetative growth appears to give C. botulinum type E an advantage in cold climates as well as in cold-stored foods.     

Characterization of DNA polymerase alpha activity from a mouse DNA temperature-sensitive mutant, strain tsFT20, which shows a defect in DNA polymerase alpha activity at restrictive temperatures

tsFT20 cells derived from mouse FM3A cells are DNA temperature-sensitive mutants, which have heat-labile DNA polymerase alpha activity. When tsFT20 cells were incubated at restrictive temperatures, intracellular levels of DNA polymerase alpha activity changed biphasically, showing an initial fast decrease (phase I) and a subsequent slow decrease (phase II). The activity of DNA polymerase alpha from tsFT20 cells cultured at a permissive temperature (33 degrees C) was greatly increased by the addition of glycerol or ethylene glycol to the reaction mixture, while little increase in enzyme activity was observed at any concentration of glycerol or ethylene glycol tested with the enzyme from the cells cultured at a restrictive temperature (39 degrees C) for 8 h (phase II). The activity of DNA polymerase alpha from wild-type cells was also increased by the addition of glycerol but the increase was much less than that in the tsFT20 cells. An in vitro preincubation experiment showed that DNA polymerase alpha from tsFT20 cells cultured at 33 degrees C very rapidly lost its ability to be stimulated by glycerol. Furthermore, the experiment using the extracts prepared from tsFT20 cells cultured at 39 degrees C for various periods showed that the ability to be stimulated by glycerol decreased with the duration of incubation time at 39 degrees C. DNA polymerase alpha from the revertants, which can grow at 39 degrees C and exhibit a partial recovery in heat stability of DNA polymerase alpha activity, showed an intermediate response to glycerol, between those of DNA polymerase alpha from tsFT20 and from the wild-type cells. Finally, it was observed that the level of enzyme activity that can be stimulated by glycerol correlated well with the DNA synthesizing ability of tsFT20 cells.     

A Fourier-transform infrared spectroscopic study of the polymorphic phase behavior of 1,2- bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine; a polymerizable lipid which forms novel microstructures

We have investigated the phase characteristics of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC23PC), a phosphatidylcholine with diacetylenic groups in the acyl chains, and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC), using Fourier-transform infrared spectroscopy (FTIR). Previous studies on the phase behavior of DC23PC in H2O have shown that DC23PC exhibits: (1) formation of cylindrical structures ('tubules') by cooling fluid phase multilamellar vesicles (MLVs) through Tm (43 degrees C), and 2) metastability of small unilamellar vesicles (SUVs) in the liquid-crystalline state some 40 degrees C below Tm, with subsequent formation of a gel phase comprised of multilamellar sheets at 2 degrees C. The sheets form tubules when heated and cooled through Tm. FTIR results presented here indicate that as metastable SUVs are cooled toward the transition to bilayer sheets, spectroscopic changes occur before the calorimetric transition as measured by a reduction in the CH2 symmetric stretch frequency and bandwidth. In spite of the vastly different morphologies, the sheet gel phase formed from SUVs is spectroscopically similar to the tubule gel phase. The C-H stretch region of DC23PC gel phase shows bands at 2937 and 2810 cm-1 not observed in the saturated analog of DC23PC, which may be related to perturbations in the acyl chains introduced by the diacetylenic moiety. The narrow CH2 scissoring mode at 1470 cm-1 and the prominent CH2 wagging progression indicate that DC23PC gel phase was highly ordered acyl chains with extended regions of all-trans methylene segments. In addition, the 13 cm-1 reduction in the C = O stretch frequency (1733-1720 cm-1) during the induction of DC23PC gel phase indicates that the interfacial region is dehydrated and rigid in the gel phase.     

Cell cycle dependence of transformation expression in mouse cells transformed by a thermosensitive mutant (Ts-121) of polyoma virus.

Change in division capability as a phenotypic expression of cellular transformation was investigated by using one of the temperature-sensitive (ts) mutants of the polyoma virus-transformed cell line, the 121-6-5 cells of BALB/3T3. When contact -inhibited cells were treated with hyaluronidase at 39 degrees C, a single round of cell division was induced after which cell growth was inhibited by cell density. However, if the cells were incubated at 35 degrees C, after the enzyme treatment, density-inhibition block disappeared and the cells entered a second division. This indicates that the release of cells from density-inhibition depends on the low temperature incubation. The ability of cells to complete a second division was examined by shifting the cells from 39 degrees C to 35 degrees C during different phases of the first division cycle after the enzyme-treatment. A 6-hour incubation of S phase cells at 35 degrees C resulted in a second cycle of division, while the 24-hour incubation of G1 cells at 35 degrees C did not induce a second round of division. These results suggest that expression of the transformed phenotype in 121-6-5 cells is clearly dependent upon both the temperature and the phase of the division cycle.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

31P NMR and AFM studies on the destabilization of cell and model membranes by the major bovine seminal plasma protein, PDC-109

The effect of PDC-109 binding to dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles (MLVs) and supported membranes was investigated by (31)P NMR spectroscopy and atomic force microscopy. Additionally, the effect of cholesterol on the binding of PDC-109 to phosphatidylcholine (PC) membranes was studied. Binding of PDC-109 to MLVs of DMPC and DPPG induced the formation of an isotropic signal in their (31)P NMR spectra, which increased with increasing protein/lipid ratio and temperature, consistent with protein-induced disruption of the MLVs and the formation of small unilamellar vesicles or micelles but not inverse hexagonal or cubic phases. Incorporation of cholesterol in the DMPC MLVs afforded a partial stabilization of the lamellar structure, consistent with previous reports of membrane stabilization by cholesterol. AFM results are consistent with the above findings and show that addition of PDC-109 leads to a complete breakdown of PC membranes. The fraction of isotropic signal in (31)P NMR spectra of DPPG in the presence of PDC-109 was less than that of DMPC under similar conditions, suggesting a significantly higher affinity of the protein for PC. Confocal microscopic studies showed that addition of PDC-109 to human erythrocytes results in a disruption of the plasma membrane and release of hemoglobin into the solution, which was dependent on the protein concentration and incubation time.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme.

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma

The specific monoclonal antibody productivity (q(Mab)) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33 degrees C to 39 degrees C. q(Mab) was constant at each temperature and was independent of the phase of culture. The q(Mab) increased 97% at 39 degrees C and decreased by 21% at 33 degrees C compared with controls at 37 degrees C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, Y(Lac/Llc') was constant for 33 degrees C to 39 degrees C and Y(Amm/Gin) was constant for 37 degrees C to 39 degrees C. Y(Amm/Gin) at 33 degrees C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of q(Mab) observed at the higher temperatures. (c) 1994 John Wiley & Sons, Inc.