Multiple conformational transitions of hen egg-white lysozyme in aqueous acetic acid solutions

Circular dichroism measurements revealed that hen egg-white lysozyme underwent multiple conformational transitions upon the addition of acetic acid. The transitions were reversible as judged from complete recoveries of enzymatic activity, electrophoretic mobility in SDS-polyacrylamide gel, and of ellipticity. Two transitions, with the mid-concentrations of 26 and 38% (v/v), were observed with the CD spectra in the amide absorption region. The two transitions were essentially athermal in the temperature ranges, 0 to 25 degrees C for the former and -10 to 10 degrees C for the latter. The trough ellipticity for the product of the transition at the higher acetic acid concentration (DII form) very closely approached the value for the synthetic polypeptides in the beta-conformation as the temperature was lowered. Molecular weight measurements by sedimentation equilibrium indicated that the products were both monomeric. Measurements of CD spectra in the aromatic absorption region showed another transition, whose mid-concentration varied with temperature from 26% (v/v) (at about 25 degrees C) to 38% (v/v) (at -10 degrees C). A change in the hydrodynamic volume detectable by exclusion chromatography was associated with this transition only.     

The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.     

FTIR study of state and phase transitions of low moisture sucrose and lactose

Mid-infrared spectra of freeze-dried sucrose and lactose systems were acquired over a range of temperatures (30-200 degrees C) and water contents (0-6.3%). Starting from the glassy state, the experimental conditions were selected to cover the main thermal transitions: the glass-rubber transition, the crystallisation and, for some samples, the subsequent melting. The FTIR spectra were very sensitive to the physical state. While subtle but systematic spectral differences between the glassy and rubbery states were detectable throughout the spectrum, a very pronounced increase in spectral resolution was observed as crystallisation occurred and was followed by the expected spectral broadening during melting. The temperatures at which these changes occurred were in satisfactory agreement with the transition temperatures measured by differential scanning calorimetry (DSC). The increase in molecular mobility as a result of increasing temperature or plasticisation by water led to a significant shift of the O-H stretching band to higher wavenumbers indicating a weakening of hydrogen bonding. This shift reached a maximum as the DSC measured crystallisation temperature range was approached. As expected, the crystallisation led to a highly effective hydrogen bonding network. This was more significant for lactose than for sucrose. No significant step change in hydrogen bonding was observed at Tg. As anticipated, the temperature at which these transitions occurred decreased with increasing water content but overlapped when observed in the context of the shifted temperature (T-Tg).     

Circular dichroism of two conformations of poly[d(G-C)] induced by low pH.

Circular dichroism (CD) and UV absorption data showed that poly[d(G-C)] (at 0.09M NaCl, 0.01M Na+ (phosphate), 20 degrees C) underwent two conformational transitions upon lowering of the pH by the addition of HCl. The first transition was complete at about pH 3.0. The second transition was complete upon lowering the pH to 2.6 or upon raising the temperature, at pH 3.0, to about 40 degrees C. There was no indication of denaturation during either transition. The CD spectrum for the second acid conformation had large CD bands including a positive one at 288nm, a characteristic associated with C X C+ base-pairs. Electron microscopy showed no significant formation of condensed supramolecular aggregates corresponding to the first or second acid forms of poly[d(G-C)]. On the basis of spectral data, electron microscopy, and proton-uptake measurements, we propose models for the secondary structures that poly[d(G-C)] adopts in its two acid conformations.     

Polymorphic phases of galactocerebrosides: spectroscopic evidence of lamellar crystalline structures

Fourier transform infrared spectroscopy was applied to study the structural and thermal properties of bovine brain galactocerebroside (GalCer) containing amide linked non-hydroxylated or alpha-hydroxy fatty acids (NFA- and HFA-GalCer, respectively). Over the temperature range 0-90 degrees C, both GalCer displayed complex thermal transitions, characteristic of polymorphic phase behavior. Upon heating, aqueous dispersions of NFA- and HFA-GalCer exhibited high order-disorder transition temperatures near 80 and 72 degrees C, respectively. En route to the chain melting transition, the patterns of the amide I band of NFA-GalCer were indicative of two different lamellar crystalline phases, whereas those of HFA-GalCer were suggestive of lamellar gel and crystalline bilayers. Cooling from the liquid-crystalline phase resulted in the formation of another crystalline phase of NFA-GalCer and a gel phase of HFA-GalCer, with a phase transition near 62 and 66 degrees C, respectively. Prolonged incubation of GalCer bilayers at 38 degrees C revealed conversions among lamellar crystalline phases (NFA-GalCer) or between lamellar gel and crystalline bilayer structures (HFA-GalCer). Spectral changes indicated that the temperature and/or time induced formation of the lamellar crystalline structures of NFA- and HFA-GalCer was accompanied by partial dehydration and by rearrangements of the hydrogen bonding network and bilayer packing mode of GalCer.     

Poly(rA).poly(rU) with Ni(2+) ions at different temperatures: infrared absorption and vibrational circular dichroism spectroscopy

Phase transitions were studied of the sodium salt of poly(rA).poly(rU) induced by elevated temperature without Ni(2+) and with Ni(2+) in 0.07 M concentration in D(2)O (approximately 0.4 [Ni]/[P]). The temperature was varied from 20 degrees C to 90 degrees C. The double-stranded conformation of poly(rA).poly(rU) was observed at room temperature (20 degrees C-23 degrees C) with and without Ni(2+) ions. In the absence of Ni(2+) ions, partial double- to triple-strand transition of poly(rA).poly(rU) occurred at 58 degrees C, whereas only single- stranded molecules existed at 70 degrees C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni(2+) ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45 degrees C and 70 degrees C with maximum stability around 53 degrees C. Triple- to single-stranded transition of poly(rA).poly(rU) occurred around 72 degrees C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86 degrees C. Hysteresis took place in the presence of Ni(2+) during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.     

Thermal and Alkaline Denaturation of Bovine β-Casein

The secondary structure of bovine beta-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in beta-casein in addition to a wide range of secondary structure elements, such as 10-20% alpha-helix, approximately 30% turns, 32-35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78 degrees C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40 degrees C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (< or = 30 degrees C) occur primarily in the monomer. It is hypothesized that the transition at 10 degrees C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40 degrees C are more likely the reflection of hydrophobic changes in the core of beta-casein. As beta-casein undergoes self-association and increases in size, the transition at higher temperature (78 degrees C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that beta-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.     

Temperature induced denaturation of collagen in acidic solution

The denaturation of collagen solution in acetic acid has been investigated by using ultra-sensitive differential scanning calorimetry (US-DSC), circular dichroism (CD), and laser light scattering (LLS). US-DSC measurements reveal that the collagen exhibits a bimodal transition, i.e., there exists a shoulder transition before the major transition. Such a shoulder transition can recover from a cooling when the collagen is heated to a temperature below 35 degrees C. However, when the heating temperature is above 37 degrees C, both the shoulder and major transitions are irreversible. CD measurements demonstrate the content of triple helix slowly decreases with temperature at a temperature below 35 degrees C, but it drastically decreases at a higher temperature. Our experiments suggest that the shoulder transition and major transition arise from the defibrillation and denaturation of collagen, respectively. LLS measurements show the average hydrodynamic radius R(h), radius of gyration R(g)of the collagen gradually decrease before a sharp decrease at a higher temperature. Meanwhile, the ratio R(g)/R(h) gradually increases at a temperature below approximately 34 degrees C and drastically increases in the range 34-40 degrees C, further indicating the defibrillation of collagen before the denaturation.     

Binding effect of Cu(2+) as a trigger on the sol-to-gel and the coil-to-helix transition processes of polysaccharide, gellan gum

The binding effect of divalent cation Cu(2+) on the gelation process with a coil-helix transition in Cu(2+)/gellan aqueous solutions has been successfully elucidated by EPR, CD, and viscoelasticity measurements. Generally, Na-type gellan gum in aqueous solution can make gel when accompanied by an intrinsic coil-helix formation induced by hydrogen bonding between chains without any additional cations at T(ch)(-)(in) ( approximately 29 degrees C) with cooling temperature. An extrinsic coil-helix transition, induced by additional divalent cations in advance of the intrinsic sol-gel transition of gellan gum, is separately detected by CD measurement. The extrinsic coil-helix transition temperatures T(ch)(-)(ex) (>47 degrees C), which increased with the Cu(2+) concentration added, were nearly identical to the sol-gel transition temperature, T(sg), determined by the viscoelasticity measurement. Judging from the molar ellipticity by CD measurement and quantitative analysis of EPR spectra, it was elucidated that the helix forming process via divalent cations is composed of two steps ascribed to the different origins, i.e., a chemical binding effect via Cu(2+) ions in the initial stage and hydrogen bonds subsequently. Finally, we propose the coil-helix and the sol-gel transition mechanism initiated by the binding effect with the divalent cation, in which the partial chelate formation can cause local formation of helices and junction zones in the vicinity of the chelates at the initial stage of the process and stabilize the helices and the junction zones. On the other hand, the stabilized helices and junction zones can induce further formation and further stabilization of the Cu(2+)-gellan chelates. The mutual stabilization promotes the formation of three-dimensional network structure at the higher temperature than the intrinsic temperature for network formation.     

Differential membrane packing of stereoisomers of bis(monoacylglycero)phosphate

Bis(monoacylglycero)phosphate (BMP) reveals an unusual sn-1,sn-1' stereoconfiguration of glycerophosphate. We synthesized sn-(3-myristoyl-2-hydroxy)glycerol-1-phospho-sn-1'-(3'-myristoyl-2'-hydroxy)glycerol (1,1'-DMBMP) and characterized the thermotropic phase behavior and membrane structure, in comparison with those of the corresponding sn-3:sn-1' stereoisomer (3,1'-DMBMP), by means of differential scanning calorimetry (DSC), small- and wide-angle X-ray scattering (SAXS and WAXS, respectively), pressure-area (pi-A) isotherms, epifluorescence microscopy of monolayers, and molecular dynamics (MD) simulations. In DSC, these lipids exhibited weakly energetic broad peaks with an onset temperature of 9 degrees C for 1,1'-DMBMP and 18 degrees C for 3,1'-DMBMP. In addition, a highly cooperative, strongly energetic transition peak was observed at approximately 40 degrees C for 1,1'-DMBMP and approximately 42 degrees C for 3,1'-DMBMP. These results are supported by the observation that 1,1'-DMBMP exhibited a larger phase transition pressure (pi(c)) than 3,1'-DMBMP. Small- and wide-angle X-ray scattering measurements identified these small and large energetic transitions as a quasi-crystalline (L(c1))-quasi-crystalline with different tilt angle (L(c2)) phase transition and an L(c2)-L(alpha) main phase transition, respectively. X-ray measurements also revealed that these DMBMPs undergo an unbinding at the main phase transition temperature. The MD simulations estimated stronger hydrogen bonding formation in the 3,1'-DMBMP membrane than in 1,1'-DMBMP, supporting the experimental data.     

Redox-dependent structural reorganization in putidaredoxin, a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida

Putidaredoxin (Pdx), a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida, transfers electrons from NADH-putidaredoxin reductase to cytochrome P450cam. Pdx exhibits redox-dependent binding affinities for P450cam and is thought to play an effector role in the monooxygenase reaction catalyzed by this hemoprotein. To understand how the reduced form of Pdx is stabilized and how reduction of the [2Fe-2S] cluster affects molecular properties of the iron-sulfur protein, crystal structures of reduced C73S and C73S/C85S Pdx were solved to 1.45 angstroms and 1.84 angstroms resolution, respectively, and compared to the corresponding 2.0 angstroms and 2.03 angstroms X-ray models of the oxidized mutants. To prevent photoreduction, the latter models were determined using in-house radiation source and the X-ray dose received by Pdx crystals was significantly decreased. Structural analysis showed that in reduced Pdx the Cys45-Ala46 peptide bond flip initiates readjustment of hydrogen bonding interactions between the [2Fe-2S] cluster, the Sgamma atoms of the cysteinyl ligands, and the backbone amide nitrogen atoms that results in tightening of the Cys39-Cys48 metal cluster binding loop around the prosthetic group and shifting of the metal center toward the Cys45-Thr47 peptide. From the metal center binding loop, the redox changes are transmitted to the linked Ile32-Asp38 peptide triggering structural rearrangement between the Tyr33-Asp34, Ser7-Asp9 and Pro102-Asp103 fragments of Pdx. The newly established hydrogen bonding interactions between Ser7, Asp9, Tyr33, Asp34, and Pro102, in turn, not only stabilize the tightened conformation of the [2Fe-2S] cluster binding loop but also assist in formation of a specific structural patch on the surface of Pdx that can be recognized by P450cam. This redox-linked change in surface properties is likely to be responsible for different binding affinity of oxidized and reduced Pdx to the hemoprotein.     

Thermally induced coil-to-helix transition of sodium gellan gum with different molar masses in aqueous salt solutions

Using 5 samples of well-purified Na-gellans (Na-gellans G1-G5, weight-average molar mass M(w) = 120 x 10(3)-32 x 10(3) at 40 degrees C), the effects of molar mass on the coil-to-double-helix transition in aqueous solutions with 25 mM NaCl were studied by light scattering and circular dichroism (CD) measurements, viscometry, and differential scanning calorimetry (DSC). From the temperature dependence of M(w), molar ellipticity at 201 nm [theta]201, intrinsic viscosity [eta], and DSC exothermic curves, it was found that the coil-to-double-helix transitions for G1-G5 samples took place at almost the same temperature. The [eta] and M(w) obtained in the temperature range from 40 to 25 degrees C can be explained by a simple coil/double-helix equilibrium model using the double-helix contents determined from CD data. The van't Hoff's transition enthalpy deltaH(vH) of Na-gellans depended on M(w). It is concluded that the coil-to-double-helix transitions of Na-gellans are all-or-none type transitions, and are accelerated with increasing M(w).     

Circular dichroism and UV-resonance Raman investigation of the temperature dependence of the conformations of linear and cyclic elastin

We used electronic circular dichroism (CD) and UV resonance Raman (UVRR) spectroscopy at 204 nm excitation to examine the temperature dependence of conformational changes in cyclic and linear elastin peptides. We utilize CD spectroscopy to study global conformation changes in elastin peptides, while UVRR is utilized to probe the local conformation and hydrogen bonding of Val and Pro peptide bonds. Our results indicate that at 20 degrees C cyclic elastin predominantly populates distorted beta-strand, beta-type II and beta-type III turn conformations. At 60 degrees C, the beta-type II turn population increases, while the distorted beta-strand population decreases. Linear elastin predominantly adopts distorted beta-strand and beta-type III turn conformations with some beta-type II turn population at 20 degrees C. Increasing temperature to 60 degrees C results in a small increase in the turn population.     

Differential scanning calorimetry studies of the inverse temperature transition of the polypentapeptide of elastin and its analogues

Differential scanning calorimetry studies have been carried out on the sequential polypeptide of elastin, (L-Val1-L-Pro2-Gly3-L-Val4-Gly5)n, abbreviated as PPP, and its more hydrophobic analogues (L-Leu1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Leu1-PPP, and (L-Ile1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Ile1-PPP Consistent with inverse temperature transitions, the temperatures of the transitions for which maximum heat absorption occurs are inversely proportional to the hydrophobicities of the polypentapeptides (31 degrees C for PPP, 16 degrees C for Leu1-PPP, and 12 degrees C for Ile1-PPP), and the endothermic heats of the transitions are small and increase with increasing hydrophobicity, i.e., 1.2, 2.9, and 3.0 kcal/mol pentamer for PPP, Leu1-PPP, and Ile1-PPP, respectively. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order, as the temperature is raised above that of the transition, has been repeatedly observed using different physical characterizations. Furthermore, the studies demonstrated identical conformations for PPP and Il21-PPP above and below the transition. Both heats and temperatures of the transitions vary with hydrophobicity, but not in simple proportionality.     

Preheat treatment for Mycobacterium tuberculosis Hsp16.3: correlation between a structural phase change at 60 degrees C and a dramatic increase in chaperone-like activity.

The in vitro chaperone-like activity of Mycobacterium tuberculosis small heat shock protein Hsp16.3 was found to be dramatically enhanced to the same extent after preheat treatment at or over 60 degrees C. Structural analysis using gel filtration, native pore-gradient PAGE, nondenaturing PAGE, and far-UV CD spectroscopy consistently revealed no significant difference between the native and the preheated Hsp16.3 proteins. However, near-UV CD spectroscopy clearly demonstrated that the tertiary structure of preheated Hsp16.3 is quite similar to its native conformation, with a minor but significant difference. Further analysis using differential scanning calorimetry indicated that Hsp16.3 exhibited a structural transition near 60 degrees C. All these results together indicate that Hsp16.3 suffers a phase change at approximately 60 degrees C, which seem to remove a structural energy barrier for the protein to refold to a conformational status with increased chaperone-like activity.     

Crystal structure of putidaredoxin, the [2Fe-2S] component of the P450cam monooxygenase system from Pseudomonas putida

Stability of the [2Fe-2S]-containing putidaredoxin (Pdx), the electron donor to cytochrome P450cam in Pseudomonas putida, was improved by mutating non-ligating cysteine residues, Cys73 and Cys85, to serine singly and in combination. The increasing order of stability is Cys73Ser/Cys85Ser>Cys73Ser>Cys85Ser>WT Pdx. Crystal structures of Cys73Ser/Cys85Ser and Cys73Ser mutants of Pdx, solved by single-wavelength anomalous dispersion phasing using the [2Fe-2S] iron atoms to 1.47 A and 1.65 A resolution, respectively, are nearly identical and very similar to those of bovine adrenodoxin (Adx) and Escherichia coli ferredoxin. However, unlike the Adx structure, no motion between the core and interaction domains of Pdx is observed. This higher conformational stability of Pdx might be due to the presence of a more extensive hydrogen bonding network at the interface between the two structural domains around the conserved His49. In particular, formation of a hydrogen bond between the side-chain of Tyr51 and the carbonyl oxygen atom of Glu77 and the presence of two well-ordered water molecules linking the interaction domain and the C-terminal peptide to the core of the molecule are unique to Pdx. The folding topology of the NMR model is similar to that of the X-ray structure of Pdx. The overall rmsd of Calpha positions between the two models is 1.59 A. The largest positional differences are observed for residues 18-21 and 33-37 in the loop regions and the C terminus. The latter two peptides display conformational heterogeneity in the crystal structures. Owing to flexibility, the aromatic ring of the C-terminal Trp106 can closely approach the side-chains of Asp38 and Thr47 (3.2-3.9 A) or move away and leave the active site solvent-exposed. Therefore, Trp106, previously shown to be important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions is in a position to regulate and/or mediate electron transfer to or from the [2Fe-2S] center of Pdx.     

The Alzheimer beta-peptide shows temperature-dependent transitions between left-handed 3-helix, beta-strand and random coil secondary structures

The temperature-induced structural transitions of the full length Alzheimer amyloid beta-peptide [A(beta)(1-40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy. The full length peptide undergoes an overall transition from a state with a prominent population of left-handed 3(1) (polyproline II; PII)-helix at 0 degrees C to a random coil state at 60 degrees C, with an average DeltaH of 6.8 +/- 1.4 kJ.mol(-1) per residue, obtained by fitting a Zimm-Bragg model to the CD data. The transition is noncooperative for the shortest N-terminal fragment A(beta)(1-9) and weakly cooperative for A(beta)(1-40) and the longer fragments. By analysing the temperature-dependent 3J(HNH(alpha)) couplings and hydrodynamic radii obtained by NMR for A(beta)(1-9) and A(beta)(12-28), we found that the structure transition includes more than two states. The N-terminal hydrophilic A(beta)(1-9) populates PII-like conformations at 0 degrees C, then when the temperature increases, conformations with dihedral angles moving towards beta-strand at 20 degrees C, and approaches random coil at 60 degrees C. The residues in the central hydrophobic (18-28) segment show varying behaviour, but there is a significant contribution of beta-strand-like conformations at all temperatures below 20 degrees C. The C-terminal (29-40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures. These results on structural preferences and transitions of the segments in the monomeric form of A(beta) may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques.     

The crystal structure and thermotropic liquid-crystal properties of N-n-undecyl-D-gluconamide

N-n-Undecyl-D-gluconamide, C17H35O6, crystallizes in space group P1, with one molecule in a unit cell a = 5.2267(6), b = 19.628(9), c = 4.7810(4) A, alpha = 93.23(2), beta = 95.60(1), gamma = 89.58(2) degrees, V = 487.35 A3, Dx = 1.19 g.cm-3. The crystal lattice is isostructural with N-n-heptyl-D-gluconamide having monolayer head-to-tail molecular packing. The molecules have a V-shaped conformation. The hydrogen bonding of the gluconamide moieties includes a four-link homodromic cycle. The transition to a smectic A liquid-crystal phase at 156.7 degrees is preceded by two crystal-to-crystal phase transitions at 77.2 degrees and 99.4 degrees. The long d-spacing of the intermediate crystal phase of 39 A, and the d-spacing of the liquid-crystal phase of 32 A, are consistent with a transition to a bilayer head-to-head molecular packing.     

Changes in chromatin structure during the aging of cell cultures as revealed by differential scanning calorimetry

Nuclei from cultured human cells were examined by differential scanning calorimetry. Their melting profiles revealed four structural transitions at 60, 76, 88, and 105 degrees C (transitions I-IV, respectively). In immortalized (i.e., tumor) cell cultures and in normal cell cultures of low passage number, melting profiles were dominated by the 105 degrees C transition (transition IV), but in vitro aging of normal and Werner syndrome cells was associated with a marked decrease in transition IV followed by an increase in transition III at the expense of transition IV. At intermediate times in the aging process, much DNA melted at a temperature range (95-102 degrees C) intermediate between transitions III and IV, and this is consistent with the notion that aging of cell cultures is accompanied by an increase in single-strand character of the DNA. Calorimetric changes were observed in the melting profile of nuclei from UV-irradiated tumor cells that resembled the age-induced intermediate melting of chromatin. It is suggested that aging is accompanied by an increase in single-stranded character of the DNA in chromatin, which lowers its melting temperature, followed by strand breaks in the DNA that destroy its supercoiling potential.     

Thermoperiodic stem elongation involves transcriptional regulation of gibberellin deactivation in pea

The physiological basis of thermoperiodic stem elongation is as yet poorly understood. Thermoperiodic control of gibberellin (GA) metabolism has been suggested as an underlying mechanism. We have investigated the influence of different day and night temperature combinations on GA levels, and diurnal steady-state expression of genes involved in GA biosynthesis (LS, LH, NA, PSGA20ox1, and PsGA3ox1) and GA deactivation (PsGA2ox1 and PsGA2ox2), and related this to diurnal stem elongation in pea (Pisum sativum L. cv Torsdag). The plants were grown under a 12-h light period with an average temperature of 17 degrees C. A day temperature/night temperature combination of 13 degrees C/21 degrees C reduced stem elongation after 12 d by 30% as compared to 21 degrees C/13 degrees C. This was correlated with a 55% reduction of GA1. Although plant height correlated with GA1 content, there was no correlation between diurnal growth rhythms and GA1 content. NA, PsGA20ox1, and PsGA2ox2 showed diurnal rhythms of expression. PsGA2ox2 was up-regulated in 13 degrees C/21 degrees C (compared to 21 degrees C/13 degrees C), at certain time points, by up to 19-fold. Relative to PsGA2ox2, the expression of LS, LH, NA, PSGA20ox1, PsGA3ox1, and PsGA2ox1 was not or only slightly affected by the different temperature treatments. The sln mutant having a nonfunctional PsGA2ox1 gene product showed the same relative stem elongation response to temperature as the wild type. This supports the importance of PsGA2ox2 in mediating thermoperiodic stem elongation responses in pea. We present evidence for an important role of GA catabolism in thermoperiodic effect on stem elongation and conclude that PsGA2ox2 is the main mediator of this effect in pea.