The folding mechanism of a beta-sheet: the WW domain.

The folding thermodynamics and kinetics of the Pin WW domain, a three-stranded antiparallel beta-sheet, have been characterized extensively. Folding and activation free energies were determined as a function of temperature for 16 mutants, which sample all strands and turns of the molecule. The mutational phi value (Phi(m)) diagram is a smooth function of sequence, indicating a prevalence of local interactions in the transition state (TS). At 37 degrees C, the diagram has a single pronounced maximum at turn 1: the rate-limiting step during folding is the formation of loop 1. In contrast, key residues for thermodynamic stability are located in the strand hydrophobic clusters, indicating that factors contributing to protein stability and folding kinetics are not correlated. The location of the TS along the entropic reaction coordinate Phi(T), obtained by temperature-tuning the kinetics, reveals that sufficiently destabilizing mutants in loop 2 or in the Leu7-Trp11-Tyr24-Pro37 hydrophobic cluster can cause a switch to a late TS. Phi(m) analysis is usually applied "perturbatively" (methyl truncation), but with Phi(T) to quantitatively assess TS shifts along a reaction coordinate, more severe mutations can be used to probe regions of the free energy surface beyond the TS.

Mapping the transition state of the WW domain beta-sheet.

The folding kinetics of a three-stranded antiparallel beta-sheet (WW domain) have been measured by temperature jump relaxation. Folding and activation free energies were determined as a function of temperature for both the wild-type and the mutant domain, W39F, which modifies the beta(2)-beta(3) hydrophobic interface. The folding rate decreases at higher temperatures as a result of the increase in the activation free energy for folding. Phi-Values were obtained for thermal perturbations allowing the primary features of the folding free energy surface to be determined. The results of this analysis indicate a significant shift from an "early" (Phi(T)=0. 4) to a "late" (Phi(T)=0.8) transition state with increasing temperature. The temperature-dependent Phi-value analysis of the wild-type WW domain and of its more stable W39F hydrophobic cluster mutant reveals little participation of residue 39 in the transition state at lower temperature. As the temperature is raised, hydrophobic interactions at the beta(2)-beta(3) interface gain importance in the transition state and the barrier height of the wild-type, which contains the larger tryptophan residue, increases more slowly than the barrier height of the mutant.

Isolated fetal ascites: prenatal diagnosis and management.

The perinatal outcomes of four patients with isolated fetal ascites were evaluated. The ascites disappeared prior to delivery in 50% of the cases and was resolved shortly after delivery in the remainder. Excellent neonatal outcomes were observed. Thus, isolated fetal ascites may represent a separate condition that significantly differs from the general category of nonimmune hydrops in both perinatal courses and prognoses. The prenatal diagnosis and management of this condition are discussed.

Pigment Changes Associated with Application of Ethephon ((2-Chloroethyl)phosphonic Acid) to Fig (Ficus carica L.) Fruits.

The application of (2-chloroethyl)phosphonic acid (Ethephon) to ;Mission' fig fruits (Ficus carica L.) during late period II of their development stimulated ripening and change in color from green to bluish black within 8 days. Chlorophylls a and b decreased rapidly within 4 days after Ethephon treatment, and degradation continued at a decreasing rate for an additional 4 days, at which time the fruits had attained their maximum diameter and were considered fully ripe. Levels of beta-carotene, lutein, violaxanthin, and neoxanthin decreased in a pattern similar to that of chlorophylls a and b. The rates of beta-carotene and lutein degradation were initially greater than those of the xanthophyll pigments. Degradation rates of the various carotenoids were comparable 4 to 8 days after treatment.There was no measurable anthocyanin synthesis during a 2- to 4-day period following Ethephon treatment. Beyond this lag phase, anthocyanin accumulation was linear, and the amount of pigment synthesized was a function of both light intensity and duration. Although Ethephon promoted the rate of anthocyanin accumulation, it did not increase the total amount of pigment synthesized in treated fruits. Etiolation of fruits from the time of Ethephon treatment until maturity stimulated an increase in growth and completely inhibited anthocyanin production in the skin. Ethephon-treated fruits which ripened while etiolated were larger in diameter and higher in both fresh and dry weights than nonetiolated controls.

Biosynthesis and accumulation of microgram quantities of chlorophyll by developing chloroplasts in vitro.

Developing chloroplasts were incubated under conditions previously shown to induce protochlorophyll and chlorophyll biosynthesis, as well as chloroplast maintenance and partial differentiation in vitro. In the presence of air, delta-aminolevulinic acid, coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate, microgram quantities of chlorophyll accumulated after 1 hour of incubation. Part of the chlorophyll was not extractable in organic solvents; it is referred to as bound chlorophyll. The amount of bound chlorophyll depended on the degree of cotyledon greening at the time of plastid isolation. Etioplasts with or without a lag phase of chlorophyll biosynthesis synthesized nonphototransformable protochlorophyll and smaller amounts of extractable chlorophyll. As the greening of excised cotyledons progressed, more of the chlorophyll became bound before and after in vitro incubation. It is suggested that this increase in the fraction of bound chlorophyll reflects the biosynthesis of membrane-bound chlorophyll receptor sites. In the absence of cofactors, chlorophyll biosynthesis was blocked and porphyrins accumulated, indicating damage of the chlorophyll biosynthetic chain. It is concluded that chlorophyll accumulation constitutes a potentially convenient tool for the study of thylakoid membrane biogenesis in vitro.

Growth and Respiratory Response of Fig (Ficus carica L. cv. Mission) Fruits to Ethylene.

Growth in diameter of the fig (Ficus carica L. cv. Mission) fruit takes place in three distinct periods; two periods (I and III) of rapid growth are separated by a period (II) of slow growth. With respect to exposure to ethylene, the fruit exhibits a two phase response. Ethylene inhibits fruit growth in phase A (period I), the period of cell division, stimulates growth in early phase B (early period II), and stimulates both growth and ripening during the remainder of phase B (late period II and period III). The adverse effect of exogenous ethylene on fruits during phase A is thought to be due to inhibition of cell division. The gradual transition occurring in the response of fruits during phase B was interpreted in terms of carbohydrate level in the fruits.The onset of period III and a respiratory climacteric rise was preceded by or concomitant with a sudden burst of endogenous ethylene synthesis. This, together with the fact that exogenous ethylene applied at the proper stage of fruit growth triggers both ripening and the climacteric rise, leads to the conclusion that ethylene is the causal agent. In other words, the data support the concept that ethylene is a growth hormone that initiates a chain of metabolic and physiological events leading to fig fruit ripening.

Auxins and Gibberellin-like Substances in Parthenocarpic and Non-parthenocarpic Syconia of Ficus carica L., cv. King.

In the King cultivar of fig, the first crop is parthenocarpic, develops on previous year's growth, and a series of supernumerary ovules develops within the original ovules. The second crop, formed on current-season's growth, requires fertilization. To determine whether the 2 crops differed in types, and in patterns of concentrations of total ;free' auxins and acidic gibberellins, they were extracted from weekly fruit samples. Timing of the 3 peaks of total auxins and the single peak of gibberellins was identical in the 2 crops. The first auxin peak in both occurred at the end of fruit growth period I (first rapid growth period), the second shortly before the end of period II (period of slow growth), and the rise and fall in concentrations of the third peak accompanied the rise and fall of the fruit growth rate in period III. The end of period II was marked by the single gibberellin peak. Additional peaks before the first sampling dates, of auxins in the first crop, of gibberellins in the second, were indicated by high concentrations in the first samples and subsequent rapid decline. The same 4 individual auxins appeared in both crops. Auxins I and II were highest in concentration in the first total auxin peak of both crops. In the second peak of the first crop, auxins II and III were highest, whereas in that peak of the second crop auxins II and IV were highest. Qualitative differences in gibberellins occurred in the 2 crops. In general, auxin concentrations were higher in the first than the second crop, and gibberellin concentrations higher in the second. High concentrations of gibberellins coincided with low ones of auxins, and vice versa.

2,4,5-trichlorophenoxyacetic Acid: effect on ethylene production by fruits and leaves of fig tree.

Production of ethylene by fruits and leaves of the fig tree was stimulated by the application of 2,4,5 trichlorophenoxyacetic acid (20 parts per million) to levels that apparently were responsible for hastened matura tion of fruit, epinasty, and senescence of leaves.

Dormancy regulation in peach seeds.

An inhibitor of seed germination was isolated from the integuments of Lovell peach seeds. Evidence from chromatographic analysis, from studies of its absorption of ultraviolet light and from assays on its effects on plant growth, indicate that the inhibitor is, if not identical with, dormin. Termination of rest in peach seeds is correlated with the disappearance of the inhibitor. The effects of the inhibitor are antagonistic to those of gibberellic acid on sections of wheat coleoptiles.