A quantitative treatment of the kinetics of the folding transition of ribonuclease A.

New experimental data and a quantitative theoretical treatment are given for the kinetics of the thermal folding transition of ribonuclease A at pH 3.0. A three-species mechanism is used as a starting point for the analysis: U1 (slow) in equilibrium U2(fast) in equilibrium N, where U1 and U2 are two forms of the unfolded enzyme with markedly different rates of refolding and N is the native enzyme. This mechanism is based on certain facts established in previous studies of refolding. The kinetics of unfolding and refolding show two phases a fast phase and a slow phase, over a range of temperatures extending above the transition midpoint, Tm. The three-species mechanism can be used in this range. At higher temperatures a new much faster kinetic phase is also observed corresponding to the transient formation of a new intermediate (I). Although the general solution for a four-species mechanism is complex it is not difficult to extend the three-species analysis for the special case found here, in which the fast reaction (I in equilibrium N) is well separated from the other two reactions. At temperatures below the transition zone the slow phase of refolding becomes kinetically complex. No attempt has been made to extend the analysis to include this effect. The basic test of the three-state analysis is the prediction as a function of temperature of alpha2, the relative amplitude of the fast phase, both for unfolding and refolding. At temperatures above Tm for which the three-state analysis must be extended to include the new intermediate I, a crresponding quanitity alpha2(cor) is predicted and compared with measured values. Data used in the three-state prediction are values of tau2 and tau1, the time constants of the fast and slow kinetic phases, plus a single value of alpha2 measured when tau2 and tau1 are well separated. The observed and predicted values of alpha2 agree within experimental error. The analysis predicts correctly that, for these experiments, alpha2 should have the same value in unfolding as in refolding in the final conditions. The analysis also predicts satisfactorily the equilibrium transition curve from kinetic data alone. Four striking properties of the kinetics are explained or correlated by the analysis: (a) the drop in alpha2 to a minimum near Tm as well as the delayed rise in alpha2 above Tm;(b) the vanishing of alpha1 above the transition zone; (c) the sharp drop in tau1 inside the transition zone followed by a partial leveling off outside this zone; and (d) the passage of tau2 through a maximum near Tm. Through a comparison of observed and predicted values of alpha2, the analysis also rules out the alternative three-species mechanism U1 (slow) in equilibrium N (fast) in equilibrium U2. Finally, the temperature dependence of the amplitude for the fast reaction (I in equilibrium N) is discussed; the behavior of I is like that of U2 and I may be an unfolded species populated at equilibrium...     

Polyphenols in litter from tropical montane forests across a wide range in soil fertility

In nutrient-poor ecosystems high polyphenol concentrations in plant litter have been proposed to influence soil nutrient availability in benefit of the plants. We addressed the question whether litter polyphenol concentrations vary across a soil chronosequence of almost identical geology, climate and plant species composition, but of a wide range in nitrogen (N) and phosphorus (P) availability in the Hawaiian Islands. Concentrations of total phenolics (TPh) and proanthocyanidins (PA) in leaf litter of the dominant tree species Metrosideros polymorpha were higher at the oldest, P-limited site compared to the youngest, N-limited site, with intermediate values at the two relatively fertile sites co-limited by N and P. Polyphenol concentrations in fine root litter differed considerably from those observed in leaf litter and varied differently across the soil age gradient. Long-term fertilization did not significantly alter polyphenol concentrations in Metrosideros litter at either site. Moreover, green leaves and leaf litter of Metrosideros showed similar relative differences among sites when compared between natural populations and plants from the same populations but grown in a common garden. These results suggest that polyphenol concentrations inherently vary among populations of the dominant tree species in Hawaiian montane forests possibly indicating an adaptation to ecosystem properties such as substrate age related differences in soil fertility. The combined above- and below-ground input rate of TPh ranged from 62.4 to 170.8 g/m²/yr and was significantly higher at the P-limited than at the N-limited site. Root-derived polyphenols contributed a much higher absolute and relative amount of phenolic input at the N-limited than at the P-limited site. The differences in amount, quality, and pathways of input might suggest specific interactions with soil processes and nutrient cycling among the Hawaiian rainforests studied here.     

Radical chemistry of epigallocatechin gallate and its relevance to protein damage

The radical chemistry of the plant polyphenolics epigallocatechin gallate (EGCG) and epigallocatechin (EGC) were investigated using electron paramagnetic resonance spectroscopy. Radical species formed spontaneously in aqueous solutions at low pH without external oxidant and were spin stabilized with Zn(II). The spectra were assigned to the gallyl radical and the anion gallyl radical, with only 10% of the signal assigned to a radical from the galloyl ester. Spectral simulations were used to establish a pK(a) of 4.8 for the EGCG radical and a pK(a) of 4.4 for the EGC radical. The electrochemical redox potentials of EGCG and EGC varied from 1000 mV at pH 3 to 400 mV at pH 8. The polyphenolics did not produce hydroxyl radicals unless reduced metal ions such as iron(II) were added to the system. Zinc(II)-stabilized EGCG radicals were more effective protein-precipitating agents than unoxidized EGCG and produced irreversibly complexed protein. EGCG and other naturally occurring polyphenolics are effective radical scavengers but their radical products have the potential to damage biological molecules such as proteins.     

Transformation and normalization of oligonucleotide microarray data

MOTIVATION: Most methods of analyzing microarray data or doing power calculations have an underlying assumption of constant variance across all levels of gene expression. The most common transformation, the logarithm, results in data that have constant variance at high levels but not at low levels. Rocke and Durbin showed that data from spotted arrays fit a two-component model and Durbin, Hardin, Hawkins, and Rocke, Huber et al. and Munson provided a transformation that stabilizes the variance as well as symmetrizes and normalizes the error structure. We wish to evaluate the applicability of this transformation to the error structure of GeneChip microarrays. RESULTS: We demonstrate in an example study a simple way to use the two-component model of Rocke and Durbin and the data transformation of Durbin, Hardin, Hawkins and Rocke, Huber et al. and Munson on Affymetrix GeneChip data. In addition we provide a method for normalization of Affymetrix GeneChips simultaneous with the determination of the transformation, producing a data set without chip or slide effects but with constant variance and with symmetric errors. This transformation/normalization process can be thought of as a machine calibration in that it requires a few biologically constant replicates of one sample to determine the constant needed to specify the transformation and normalize. It is hypothesized that this constant needs to be found only once for a given technology in a lab, perhaps with periodic updates. It does not require extensive replication in each study. Furthermore, the variance of the transformed pilot data can be used to do power calculations using standard power analysis programs. AVAILABILITY: SPLUS code for the transformation/normalization for four replicates is available from the first author upon request. A program written in C is available from the last author.     

Phenolic acclimation to ultraviolet-A irradiation in <Emphasis Type="Italic">Eucalyptus nitens</Emphasis> seedlings raised across a nutrient environment gradient

We investigated the effects of long-term acclimation of Eucalyptus nitens seedlings to ultraviolet-A (UV-A) irradiation (320–400 nm) on phenolic compounds (gallotannins, stilbenes, and flavonols), photochemical efficiency, and chlorophyll and carotenoid contents. Seedlings were raised under four nutrient regimes, ranging from low to high application rates, in an environment that included or excluded UV-A irradiance. Our aims were: to classify phenolic compounds that absorb in the UV-A and their relative contribution to total UV-A absorption; to identify how phenolic compounds respond to UV-A exposure and exclusion, and to determine how plant nutrient status affects acclimation of photo-and pigment-chemistry to UV-A exposure and exclusion. Gallotannins contributed to only a minor fraction of total absorption within the lower range (320–360 nm) of the UV-A spectrum. Stilbene and flavonol compounds dominated absorption within the 320–360 and 360–400 nm ranges, respectively. Contents of gallotannin were generally high in UV-A-exposed seedlings. Although there was a significant effect of UV-A on contents of stilbenes, a general response (across nutrient treatment comparisons) was not evident. Contents of flavonols were not affected by UV-A exposure. Contents of gallotannin, stilbene, and flavonols decreased from low to high nutrient-application treatments. There were no effects of UV-A on photochemical efficiency or pigment-chemistry.     

The effects of short-term resistance training on endocrine function in men and women

This investigation examined hormonal adaptations to acute resistance exercise and determined whether training adaptations are observed within an 8-week period in untrained men and women. The protocol consisted of a 1-week pre-conditioning orientation phase followed by 8 weeks of heavy resistance training. Three lower-limb exercises for the quadriceps femoris muscle group (squat, leg press, knee extension) were performed twice a week (Monday and Friday) with every other Wednesday used for maximal dynamic 1 RM strength testing. Blood samples were obtained pre-exercise (Pre-Ex), immediately post-exercise (IP), and 5 min post-exercise (5-P) during the first week of training (T-1), after 6 weeks (T-2) and 8 weeks (T-3) of training to determine blood concentrations of whole-blood lactate (LAC), serum total testosterone (TT), sex-hormone binding globulin (SHBG), cortisol (CORT) and growth hormone (GH). Serum TT concentrations were significantly (P ≤ 0.05) higher for men at all time points measured. Men did not demonstrate an increase due to exercise until T-2. An increase in pre-exercise concentrations of TT were observed both for men and women at T-2 and T-3. No differences were observed for CORT between men and women; increases in CORT above pre-exercise values were observed for men at all training phases and at T-2 and T-3 for women. A reduction in CORT concentrations at rest was observed both in men and women at T-3. Women demonstrated higher pre-exercise GH values than men at all training phases; no changes with training were observed for GH concentrations. Exercise-induced increases in GH above pre-exercise values were observed at all phases of training. Women demonstrated higher serum concentrations of SHBG at all time points. No exercise-induced increases were observed in men over the training period but women increased SHBG with exercise at T-3. SHBG concentrations in women were also significantly higher at T-2 and T-3 when compared to T-1 values. Increases in LAC concentrations due to exercise were observed both for men and women for all training phases but no significant differences were observed with training. These data illustrate that untrained individuals may exhibit early-phase endocrine adaptations during a resistance training program. These hormonal adaptations may influence and help to mediate other adaptations in the nervous system and muscle fibers, which have been shown to be very responsive in the early phase of strength adaptations with resistance training. Accepted: 11 December 1997     

Stimulation of rat testis microsomal 17alpha-hydroxyprogesterone side-chain cleavage enzyme by luteinizing hormone added in vitro

The addition of purified luteinizing hormone to isolated rat testis microsomes causes an increase in the activity of the enzyme system that converts 17α-hydroxyprogesterone into C₁₉ steroids in these tissue organelles. The gonadotrophin does not act in this system by altering the Michaelis constants of the enzyme for either steroid or nicotinamide nucleotide, but rather appears specifically to increase the catalytic capability of the protein.