Interactions of Transthyretin (TTR) and Retinol-Binding Protein (RBP) in the Uptake of Retinol by Primary Rat Hepatocytes

The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP–transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP–TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [³H]retinol–RBP or the [³H]retinol–RBP–TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. Analysis by HPLC showed that the incorporated [³H]retinol in NPC was mainly converted to retinyl ester, although in PC it remained mainly as unesterified retinol. However, the amount of retinol taken up from the RBP–TTR complex was nearly twofold greater than that from RBP alone. The uptake of [³H]retinol from protein-bound retinol was inhibited by an excess of either retinol–RBP or retinol–RBP–TTR complex. Moreover, retinol uptake through the RBP–TTR complex was inhibited by an excess of free TTR. From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP–TTR complex into both PC and NPC. The uptake of [³H]retinol (2 μM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.     

Thermal transitions in dimyristoylphosphatidyleholine foam bilayers

Thermal transitions in the system dimyristoylphosphatidylcholine/water/ethanol/sodium chloride were studied in the temperature range 10–31 °C. The water-ethanol dispersions were investigated by differential scanning calorimetry and the foam bilayers by the microinterferometric method for investigation of thin liquid films. Calorimetry showed that an increase in ethanol content (up to 47.5 vol.% — the concentration used in the experiments with foam bilayers) did not significantly influence the temperature of the main phase transition and led to the disappearance of the pretransition. The microinterferometric study of the foam bilayer thickness showed that there were two thermal transitions — at 13 and 23 °C. An Arrhenius type dependence was obtained for the critical concentration of dimyristoylphosphatidylcholine (DMPC) in the solution, which was necessary for the formation of the foam bilayer. A steep change in the slope of the linearized Arrhenius dependence was found at 23 °C. Values of the binding energy of a DMPC molecule in the foam bilayers were calculated using the hole-nucleation theory of stability and permeability of bilayers. It was proved that the phase transition at 23 °C was due to melting of the hydrocarbon tails of phospholipid molecules. The low-temperature phase transition was assumed to be due to a change in the tilt of the hydrocarbon tails. These experiments demonstrate for the first time the occurrence of phase transitions in foam bilayers. Correspondence to: D. Exerowa     

Thermal stability of bovine-brain myelin membrane

The thermal behaviour of bovine-brain myelin membrane has been studied by high-sensitivity differential scanning calorimetry, Fourier-transform infrared spectroscopy and thermal gel analysis. Spectroscopic results indicate that protein transitions take place between 60°C and 90°C, while thermal gel analysis has provided the thermal denaturation profiles of myelin proteolipid, DM-20 protein and the Wolfgram Fraction. An irreversible calorimetric transition centred at 80.3 ± 0.2°C with a specific enthalpy of 4.7 ± 0.6 J/g of total protein has been assigned to the thermal denaturation of myelin proteolipid and DM-20 protein. The effects of the myelin storage conditions, scan rate, ionic strength and pH on this calorimetric transition have also been investigated. The thermal transition of the proteolipid practically disappears after treatment of the myelin with different amounts of chloroform-methanol 2:1 (v/v), a treatment which is generally used in proteolipid purification. On the other hand, the addition of several detergents to myelin only causes minor modifications to this transition, which then occurs at about 70°C, with a specific enthalpy of between 2.5 and 3.6 J/g of total protein. These results appear to show that detergents preserve the native conformation of the proteolipid far more than do organic solvents. Hence the use of detergents would seem to be the appropriate method for proteolipid purification.Abbreviations DSC Differential scanning calorimetry - TGA Thermal gel analysis - FTIR Fourier-transform infrared spectroscopy - PLP Proteolipid protein - MBP Myelin basic protein - DM-20 Protein DM-20 - WF Wolfgram fraction - BSA Bovine serum albumine - SDS Sodium dodecyl sulfate - ANSA 4-amino-3-hydroxynaphthalene-1-sulphonic acid - OG -d-glucopyranoside - PAGE Polyacrylamide gel electrophoresis - Chaps 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CNS Central nervous system Correspondence to: P. L. Mateo     

Conformation of the abortifacient protein pinellin: A circular dichroic study

The conformation of pinellin was studied by circular dichroism, which showed a minimum at 223 nm and a double maximum at 198–200 nm. The protein was rich in -sheet (about 40%) with little -helix, based on current CD analyses. It was stable betweenpH 4 and 10 beyond which it unfolded reversibly, but in alkaline solution, prolongly stored at, say,pH 12, it became irreversibly denatured. Thermal denaturation indicated a transition between 55° and 68°C; the solution at 80°C was partially renatured upon air-cooling back to room temperature. Addition of sodium dodecyl sulfate caused a sharp increase in -helix, which leveled off at 0.25 mM surfactant.     

Thermal stability and thermodynamic analysis of native and methoxypolyethylene glycol modified trypsin

Four methoxypolyethylene glycols (MPEG, molecular masses 350, 750, 2000 and 5000 Da), each activated by nitrophenyl chloroformate, were used to modify trypsin. Compared with the native trypsin, the MPEG-modified trypsin was more stable against temperature between 30°C and 70°C, longer chain of MPEG moiety corresponding to higher thermal stability. The T for the native and the modified trypsin (0.4 mg ml^–1) was increased from 47°C to 66°C. The stabilization effect caused by MPEG modification was the result of decreasing in both the autolysis rate and the thermal denaturation rate. The thermodynamic analysis of the thermal denaturation process showed that the activation free energy (G^*) of the native and the modified trypsin at 60°C was increased from 102.9 to 109.3 kJ mol^–1; the activation enthalpy (H^*) was increased from 57.4 to 86.9 kJ mol^–1; the activation entropy (S^*) was increased from –136 to –67 J molK^–1. A possible explanation for the decreased thermal denaturation rate caused by MPEG modification was also discussed.     

Role of lipids in theNeurospora crassa membrane. I. Influence of fatty acid composition on membrane lipid phase transitions

Summary The relationship between lipid composition and phase transition was investigated by differential scanning calorimetry for intact and membrane phospholipid extracts of wild-type (w/t) and thecel ^– (Tw 40) mutant ofNeurospora crassa. Thecel ^– (Tw 40) mutant (grown on minimal, sucrose medium supplemented with Tween 40 at 34 °C) had approximately twice the saturated fatty acid content ofw/t organisms grown at 22 °C. The gel-liquid crystal phase transitions of ergosterol-free extracts derived fromw/t andcel ^– (Tw 40) occur at –31 and –11 °C, respectively. The heats of transition (H) of these extracts were 1 and 13 cal/g, respectively. The addition of ergosterol (the predominant sterol inNeurospora) to the phospholipid extracts decreased the observed heats of transition, but did not alter the transition temperature. IntactNeurospora, whetherw/t orcel ^– (Tw 40) did not manifest similar gel-liquid crystal phase transitions in the differential scanning calorimeter. However, an endothermic peak at approximately 30 °C was observed in intact cells and extracted phospholipids of bothw/t andcel ^– (Tw 40) organisms. This peak was insensitive to the addition of ergosterol, had a low heat content (H1 cal/g), and was reversible.     

Effect of Neurotensin Peptidomimetics on Thermoregulation in Rats

The effects of the tripeptide analogues of neurotensin, GZR123 and GZR125, on thermoregulation was studied in rats that were kept at different ambient temperatures ( _c): in the cold ( _c = 4–6°C), thermoneutral ( _c = 27–28°C), and hot ( _c = 31–32°C) environment, as well as at room temperature ( _c = 20–21°C). In the cold environment, the injection of GZR123 disturbed the vegetative mechanisms of heat emission, leading to peripheral vasoconstriction and possibly changing heat production. Similar to neurotensin, GZR125 disturbed the development of compensatory vasoconstriction in the cold environment and at room temperature, which resulted in a decrease in body temperature. At high temperature, this peptide induced vasodilation.     

Critical analysis of factors influencing sphaeroplast generation from Saccharomyces cerevisiae

Efficient synthesis of large numbers of viable sphaeroplast from Saccharomyces cerevisiae has been found to be influenced by a number of factors. In this case, Trichoderma harzianum, NCIM 1185, culture filtrate has been used to prepare sphaeroplast from Saccharomyces cerevisiae, NCIM 3288. A method has been devised to isolate large number of viable sphaeroplast from the cell. Detailed analysis of various factors affecting the formation of sphaeroplasts from Saccharomyces cerevisiae has not yet been reported. This study showed critical analysis of various factors which influenced sphaeroplast formation. Most suitable conditions were: Age of the organism in slant — 1 d, cell age in liquid medium — 24 h, time of incubation of cell with 0.3% -mercaptoethanol — 30 min, level of lytic ezyme concentration — 79.2 ml, concentration of cell (dry wt. equivalent) — 0.1262 g, time of contact with lytic enzyme — 25 min, temperature of sphaeroplast formation — 30 °C, phosphate buffer — 25 mM of pH 6.5 and KCl as osmotic stabilizer — 0.7 M.     

Gln54Leu of the conserved polar residue inthe interfacial coiled coil position (d) results in significant stabilization of the original structure of the COMP pentamerization domain

The assembly domain of cartilage oligomeric matrixprotein (COMP) forms an -helical coiled coilhomopentamer with a conserved polar glutamine in theinterior (d) position. We substituted Gln⁵⁴ forapolar Leu in the recombinant fragment of the rat COMPdomain. Biochemical studies and circular dichroism(CD) spectroscopy showed that the mutant, similarly tothe wild-type (w.t.) peptide, forms spontaneously an-helical pentamer. Thermal transitions of thew.t. and mutant pentamers were analyzed by CDspectroscopy and differential scanning calorimetry.The Gln⁵⁴Leu mutation increased the thermal stabilityof the pentamer with reduced disulfide bonds from73 °C to 104 °C. The denaturation of thedisulfide bonded w.t. pentamer was observed at108 °C while the mutant pentamer cannot bedenatured up to 120 °C (the apparatus limit).Thus, by Gln⁵⁴Leu mutation we found a way tosignificantly stabilize the coiled coil pentamer,making this peptide even more attractive as an oligomerization tool for various biotechnological applications.     

Triggering of cryoprotectant synthesis by the initiation of ice nucleation in the freeze tolerant frog,Rana sylvatica

Summary The triggering of cryoprotectant synthesis was examined in the freeze tolerant wood frog,Rana sylvatica. A slow decrease in ambient temperature (1°C every 2 days) from 3° to –2.1 °C was used to search for a specific trigger temperature. None was found. Instead it was found that, despite subzero temperature, animals which remained in a supercooled unfrozen state had low blood glucose (1.66±0.44 mol/ml) while those which had frozen had high blood glucose (181±16 mol/ml). These results indicate that it is the initiation of ice nucleation, rather than a specific subzero temperature, which triggers cryoprotectant glucose synthesis. This was confirmed by monitoring the freezing curves for individual frogs with sampling of blood and tissues at various times relative to the initiation of nucleation (detected as an instantaneous temperature jump from –3 to –1°C). Animals sampled before nucleation had low blood and liver glucose contents and a low percentage of liver phosphorylase in thea form. Within 4 min of the initiation of freezing, however, blood glucose had jumped to 16 mol/ml and liver glucose to 39.5 mol/g wet weight. Glucose in both compartments continued to increase as the time of freezing increased correlated with an increase in liver phosphorylasea content from 47% before nucleation to 100% after 50 min of freezing. The results clearly demonstrate that freeze tolerant frogs have no anticipatory synthesis of cryoprotectant as a preparation for winter but rather can translate the initiation of extracellular ice formation into a signal which rapidly activates cryoprotectant production by liver.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

The influence of metal cations and pH on the heat sensitivity of photosynthetic oxygen evolution and chlorophyll fluorescence in spinach chloroplasts

The heat-sensitivity of photosynthetic oxygen evolution of thylakoids isolated from spinach increases by increasing the pH above neutral value. The temperature for inactivation (transition temperature) is lowered from about 45° C (pH 6.0–7.4) to 33°C (pH 8.5). Similar results are obtained with intact chloroplasts. At pH 7.0 the transition temperature of washed thylakoids decreases by lowering the salt concentration below 20 mM with monovalent cations (Li⁺, Na⁺, K⁺) and below 3–4 mM with divalent cations (Mg²⁺, Ca²⁺, Sr²⁺). Illumination decreases the heat-sensitivity of oxygen evolution in intact chloroplasts, but even increases the heat-sensitivity in uncoupled chloroplasts. In intact chloroplasts the transition temperature of the heat-induced rise in chlorophyll fluorescence yield (F_o; see Schreiber and Armond 1978) decreases from 44° C to 38° C when the pH of the suspending medium is increased from 6.5 to 8.5. At 20° C, F_o is almost insensitive to pH (6.0–8.5). At 40° C, however, F_o is constant between 6.0 and 7.0, but strongly increases by increasing the pH above neutral value. The results are discussed in terms of a close relation between electrostatic forces at the thylakoid membrane and thermal sensitivity of photosynthetic apparatus. It is suggested that the heat-sensitivity of the photosystem II complex partially depends on the ionization state of fixed groups having alkaline pK. The packed volume of thylakoids suspended in a low salt medium increases when the temperature is increased above 30° C (pH 7.0) and above 20° C (pH 8.0), respectively. This result suggests a heat-induced increase in surface charge density of the thylakoid membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES morpholinoethane sulfonic acid - MOPS 2-N-morpholinopropane sulfonic acid - TRICIN N-[tris(hydroxymethyl)-methyl] glycine     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

We have studied the pigment arrangement in purified cytoplasmic membranes of the thermophilic green bacterium Chloroflexus aurantiacus. The membranes contain 30–35 antenna bacteriochlorophyll a molecules per reaction center; these are organized in the B808–866 light-harvesting complex, together with carotenoids in a 2:1 molar ratio. Measurements of linear dichroism in a pressed polyacrylamide gel permitted the accurate determination of the orientation of the optical transition dipole moments with respect to the membrane plane. Combination of linear dichroism and low temperature fluorescence polarization data shows that the Q_y transitions of the BChl 866 molecules all lie almost perfectly parallel to the membrane plane, but have no preferred orientation within the plane. The BChl 808 Q_y transitions make an average angle of about 44° with this plane. This demonstrates that there are clear structural differences between the B808–866 complex of C. aurantiacus and the B800–850 complex of purple bacteria. Excitation energy transfer from carotenoid to BChl a proceeds with about 40% efficiency, while the efficiency of energy transfer from BChl 808 to BChl 866 approaches 100%. From the minimal energy transfer rate between the two spectral forms of BChl a, obtained by analysis of low temperature fluorescence emission spectra, a maximal distance between BChl 808 and BChl 866 of 23 was derived.Abbreviations BChl bacteriochlorophyll - BPheo bacteriopheophytin - CD circular dichroism - LD linear dichroism - Tris Tris(hydroxymethyl)aminomethane     

Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus

The rotenone sensitive NADH: menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50°C and pH 8.1, and the enzyme presents a K _M of 9 M for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 ± 3.5 iron atoms and 3.7 menaquinone per FMN. At least five iron—sulfur centers are observed by EPR spectroscopy: two [2Fe–2S]^2+/1+ and three [4Fe–4S]^2+/1+ centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.     

Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry,Circular Dichroism,and Turbidity Measurements

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).     

Phase transitions in plant cuticles

The effect of temperature on wet plant cuticles has been investigated with the following techniques: Calorimetry, densitometry, spin-label electron-spin-resonance-(ESR)-spectroscopy, photo bleaching, and light and electron microscopy. At low temperatures cuticles ofCitrus aurantium L. andHedera helix show, at 16.3°C, a sharp transition (T0.5°C) with a latent heat of 4.7±0.5 J g^-1-cuticle. Below transition: The main orientation of the polymer matrix is parallel to the normal of the cuticle and the main orientation of the layer with soluble lipids is perpendicular to the normal. The cuticle is in a rigid state. Above transition (between 16.3°C and 38°C): Only the orientation of the polymer matrix has changed (tilted in parts). There exist several very sharp (T0.1°C) transitions (38°C, 41°C, 45°C, 49°C, ...) with a latent heat in the order of 0.4 J g^-1-cuticle. Above 38°C: The lamella of the soluble lipids is in a fluid state. Above 45°C there is a change in the molecular orientation of the soluble lipids as well as in the polymer matrix. The soluble lipids are mainly oriented parallel to the normal. The dry cuticles show no phase transition between 0°C and 200°C. At room temperature a dry/wet transition can be observed.Abbreviations ESR-spectroscopy electron-spin-resonance-spectroscopy     

Levels of the upper forest boundary in northern Asia

On the Ural range the elevation of upper timberline changes at grade 71 m per degree of latitude in linear regression. Much lengthy cross-section—for the semi-arid regions of middle Siberia and adjacent Kazakhstan, and for the regions of eastern Siberia dominated by larch forests—exhibit parabolic regression of timberline levels upon geographic latitude. The longitudinal gradient of timberlines presumably depends on radiation balance related with the amount of precipitation. The arctic boundary of taiga in eastern Europe and Siberia lies mostly on average latitude 69° 36 E. It correlates with mean July temperature 11.2 °C, or with duration of the growing season 128 days with stable temperature of air exceeding 0°C which amounts to 876°. Daily temperatures exceeding 5° and especially 10 °C are seemingly less influential there. The value of 11.2 °C deviates by about 1 °C from the value of above 10 °C for three summer month reported by Langlet 1935, which shows the close environmental control regulating the northern and upper boundary of the northern, mostly coniferous forest on the northern hemisphere.     

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

Comparative study on the thermostability of collagen I of skin and bone: Influence of posttranslational hydroxylation of prolyl and lysyl residues

Pepsin-solubilized collagen I from skin and bone was analyzed with regard to its thermal stability as a triple helical molecule in solution and afterin vitro fibril formation. Collagen I from human control bone was compared with samples showing deficiencies or surplus in the degree of hydroxylation of lysine. The helix to coil transitions were studied by circulardichroism measurements and limited trypsin digestion. Melting of fibrils from standardizedin vitro self-assembly was investigated turbidimetrically. Human control bone collagen I has a maximum transition rate (T _m ) at 43.3°C in 0.05% acetic acid. This is 1.9°C above control skin (T _m =41.4°C), most likely, due to a higher degree of prolyl hydroxylation—0.48 in bone vs. 0.41 in skin collagen I. Lysyl overhydroxylation of human and mouse bone collagen I appears to reduce theT _m slightly (1°C). Underhydroxylated bone collagen has aT _m which is 2°C below control. Melting temperatures ofin vitro formed fibrils are an indication for higher thermostability in parallel with an increase of lysyl hydroxylation. Accordingly, the melting temperature of such fibrils from human control skin, 49.3°C, exceeds control bone by 1.4°C. The degree of lysyl hydroxylation in these samples is 0.14 and 0.10, respectively. Further underhydroxylation (0.06) reduced it down to 45.4°C, while extensive overhydroxylation did not continue to increase the thermal stability of fibrils.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.