Influence of method of application of paclobutrazol on soil residues and growth retardation in a ‘Starkrimson-Delicious’ apple orchard

A 3-year field study was conducted to determine the influence of mode of application of the gibberellin-inhibitor paclobutrazol (PP333), [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4 triazol-1-yl)pentan-3-ol], on PP333 soil residue levels and vegetative growth retardation of 10-year-old Starkrimson Delicious (Malus Domestica Borkh.) spur-type apple trees. Treatments were applied in March, 1986 and consisted of foliar or soil sprays (200 ppm, 7 × applications at petal fall (+) 2, 4, 6, 8, 10 and 12 weeks) or a single soil drench (8.2 g A.I./tree) applied to the collar at petal fall. Foliar sprays were applied with and without a plastic ground cover to evaluate the influence of foliar runoff on the degree of soil absorption and its subsequent effect on vegetative growth. PP333 was extracted over a 3 year period (1986–88) from 400 cm² soil patches located at the drip line of each tree, with the exception of soil drenches which were sampled near the collar. PP333 soil extracts were purified and quantitatively analyzed by HPLC. PP333 soil residue levels following foliar sprays were comparable to the soil spray treatment for each year and decreased at a rate of 50% per year from 1986–1988. Foliar sprays retarded terminal growth in the year of application, whereas the soil spray did not inhibit growth until the following year. PP333 residue levels were highest in the soil drench where growth retardation was evident in 1987 and 1988. The greatest carry-over effect occurred in the soil treatments, especially the soil drench application which resulted in the highest soil residue rates throughout the 3 year period.     

Replacement of cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c with threonine: improved stability of the mutant protein

Site-directed mutagenesis has been used to change the codon for cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c to a threonine codon. The resulting protein is active in vivo, is methylated as in the wild-type protein and has optical properties indistinguishable from those of the wild-type protein. The threonine-107 iso-1-cytochrome c demonstrated fully reversible electrochemical behaviour and a mid-point reduction potential of 272 mV versus NHE. In addition, this mutant does not demonstrate a tendency to autoreduce or to dimerize as does the wild-type protein. These properties of the threonine-107 mutant establish that it will provide a useful background in which to make subsequent mutations for mechanistic and physical studies of yeast iso-1-cytochrome c.     

Physiology of Tuberization in Solanum tuberosum L: cis-Zeatin Riboside in the Potato Plant: Its Identification and Changes in Endogenous Levels as Influenced by Temperature and Photoperiod

Using high pressure liquid chromatography, the cucumber cotyledon bioassay, and mass spectrometry a cytokinin isolated from Solanum tuberosum L. cv. Katahdin plant tissues has been identified as cis-zeatin riboside. Zeatin riboside (ZR) levels in plants grown under inducing conditions (28 C day and 13 C night with a 10-hour photoperiod) were significantly higher than those in plants grown under noninducing conditions (30 C day and 28 C night with an 18-hour photoperiod). The highest level of ZR was noted in below-ground tissue after 4 days exposure to inducing conditions, with tuber initiation observed after 8 days. A companion study conducted to determine the effect of ZR on in vitro tuberization of noninduced rhizomes revealed that after 1 month in culture, controls exhibited 0% tuberization, while ZR treatments of 0.3 and 3.0 milligrams per liter showed 39 and 75% tuberization, respectively.     

Structural model for the trialkyltin binding site on cat hemoglobin

The binding site for trialkyltin complexes on the alpha- chain of cat oxyhemoglobins is proposed to involve the SG and NE2 atoms of Cys-13 and His-113 respectively. On deoxygenation, the conformation of this region changes substantially, allowing complexation only through the ND1 nitrogen atom of His-113, a much less favorable interaction. Thus the model presented explains the preferential binding of trialkyltin complexes to R-state cat hemoglobin and suggests the type of interaction that is likely to occur between these compounds and a variety of less well-characterized enzymes to produce the metabolic effects that trialkyltin complexes are known to produce in vivo.     

Structural and kinetic properties of Rhodobacter sphaeroides photosynthetic reaction centers containing exclusively Zn-coordinated bacteriochlorophyll as bacteriochlorin cofactors

The Zn-BChl-containing reaction center (RC) produced in a bchD (magnesium chelatase) mutant of Rhodobacter sphaeroides assembles with six Zn-bacteriochlorophylls (Zn-BChls) in place of four Mg-containing bacteriochlorophylls (BChls) and two bacteriopheophytins (BPhes). This protein presents unique opportunities for studying biological electron transfer, as Zn-containing chlorins can exist in 4-, 5-, and (theoretically) 6-coordinate states within the RC. In this paper, the electron transfer perturbations attributed exclusively to coordination state effects are separated from those attributed to the presence, absence, or type of metal in the bacteriochlorin at the H_A pocket of the RC. The presence of a 4-coordinate Zn^2 + ion in the H_A bacteriochlorin instead of BPhe results in a small decrease in the rates of the P* → P⁺H_A⁻ → P⁺Q_A⁻ electron transfer, and the charge separation yield is not greatly perturbed; however coordination of the Zn^2 + by a fifth ligand provided by a histidine residue results in a larger rate decrease and yield loss. We also report the first crystal structure of a Zn-BChl-containing RC, confirming that the H_A Zn-BChl was either 4- or 5-coordinate in the two types of Zn-BChl-containing RCs studied here. Interestingly, a large degree of disorder, in combination with a relatively weak anomalous difference electron density was found in the H_B pocket. These data, in combination with spectroscopic results, indicate partial occupancy of this binding pocket. These findings provide insights into the use of BPhe as the bacteriochlorin pigment of choice at H_A in both BChl- and Zn-BChl-containing RCs found in nature.     

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

The role of the heme propionate groups in determining the electron transfer and electrostatic properties of myoglobin have been studied by thermodynamic, kinetic, and spectroscopic studies of horse heart myoglobin in which the heme propionate groups are esterified. Spectroelectrochemical analysis has established that the E_m,7 of dimethylester heme-substituted Mb (DME-Mb) (E_m,7 = 100.2(2) mV vs. NHE (Normal Hydrogen Electrode) (25 °C) is increased  40 mV relative to that of the native protein with ΔH° = −12.9(2) kcal/mol and ΔS° = −51.0(8) cal/mol/deg (pH 7.0, μ = 0.1 M (phosphate)). The second order rate constant for reduction of DME-metMb by Fe(EDTA)²⁻ is increased  > 400-fold relative to that for reduction of native metMb to a value of 1.34(2) × 10³ M⁻¹ s⁻¹ with ΔS^‡ = −13(1) cal/mol/deg and ΔH^‡ = 9.2(3) (pH 7.0, μ = 0.1 M (phosphate)). Analysis of the pH dependences of the reduction potential and rate constant for reduction by Fe(EDTA)²⁻ demonstrates that heme propionate esterification introduces significant changes into the electrostatic interactions in myoglobin. These changes are also manifested by differences in the pH dependences of the ¹H NMR spectra of native and DME-metMb that reveal shifts in pK_a values for specific His residues as the result of heme propionate esterification. In sum, the current results establish that heme propionate esterification not only affects the electron transfer properties of myoglobin but also influences the titration behavior of specific His residues.     

Inhibitors of human indoleamine 2,3-dioxygenase identified with a target-based screen in yeast

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan degradation enzyme that is emerging as an important drug target. IDO is expressed by many human tumors to help them escape immune detection, and it has been implicated in depression and in the formation of senile nuclear cataracts. There is a need for potent and selective IDO inhibitors for use in research and as lead compounds for drug development. We show that expression of human IDO in a Saccharomyces cerevisiae tryptophan auxotroph restricts yeast growth in the presence of low tryptophan concentrations and that inhibition of IDO activity can restore growth. We use this assay to screen for IDO inhibitors in collections of pure chemicals and crude natural extracts. We identify NSC 401366 (imidodicarbonimidic diamide, N-methyl-N'-9-phenanthrenyl-, monohydrochloride) as a potent nonindolic IDO inhibitor (Ki=1.5 +/- 0.2 microM) that is competitive with respect to tryptophan. We also use this assay to identify the active compound caulerpin from a crude algal extract. The yeast growth restoration assay is simple and inexpensive. It combines desirable attributes of cell- and target-based screens and is an attractive tool for chemical biology and drug screening.     

Cofacial heme binding is linked to dimerization by a bacterial heme transport protein

Campylobacter jejuni is a leading bacterial cause of food-borne illness in the developed world. Like most pathogens, C. jejuni requires iron that must be acquired from the host environment. Although the iron preference of the food-borne pathogen C. jejuni is not established, this organism possesses heme transport systems to acquire iron. ChaN is an iron-regulated lipoprotein from C. jejuni proposed to be associated with ChaR, an outer-membrane receptor. Mutation of PhuW, a ChaN orthologue in Pseudomonas aeruginosa, compromises growth on heme as a sole iron source. The crystal structure of ChaN, determined to 1.9 A resolution reveals that ChaN is comprised of a large parallel beta-sheet with flanking alpha-helices and a smaller domain consisting of alpha-helices. Unexpectedly, two cofacial heme groups ( approximately 3.5 A apart with an inter-iron distance of 4.4 A) bind in a pocket formed by a dimer of ChaN monomers. Each heme iron is coordinated by a single tyrosine from one monomer, and the propionate groups are hydrogen bonded by a histidine and a lysine from the other monomer. Sequence analyses reveal that these residues are conserved among ChaN homologues from diverse bacterial origins. Electronic absorption and electron paramagnetic resonance (EPR) spectroscopy are consistent with heme binding through tyrosine coordination by ChaN in solution yielding a high-spin heme iron structure in a pH-dependent equilibrium with a low-spin species. Analytical ultracentrifugation demonstrates that apo-ChaN is predominantly monomeric and that dimerization occurs with heme binding such that the stability constant for dimer formation increases by 60-fold.     

Metal Ions and Electrolytes Regulate the Dissociation of Heme from Human Hemopexin at Physiological pH

The stability of the hemopexin-heme (Hx-heme) complex to dissociation of the heme prosthetic group has been examined in bicarbonate buffers in the presence and absence of various divalent metal ions. In NH₄HCO₃ buffer (pH 7.4, 20 mm, 25 °C) containing Zn²⁺ (100 μm), 14% of the heme dissociates from this complex (4.5 μm) within 10 min, and 50% dissociates within 2 h. In the absence of metal ions, the rate of dissociation of this complex is far lower, is decreased further in KHCO₃ solution, and is minimal in NaHCO₃. In NH₄HCO₃ buffer, dissociation of the Hx-heme complex is accelerated by addition of divalent metals with decreasing efficiency in the order Zn²⁺ > Cu²⁺ ≫ Ni²⁺ > Co²⁺≫Mn²⁺. Addition of Ca²⁺ prior to addition of Zn²⁺ stabilizes the Hx-heme complex to dissociation of the heme group, and addition of Ca²⁺ after Zn²⁺-induced dissociation of the Hx-heme complex results in re-formation of the Hx-heme complex. These effects are greatly accelerated at 37 °C and diminished in other buffers. Overall, the solution conditions that promote formation of the Hx-heme complex are similar to those found in blood plasma, and conditions that promote release of heme are similar to those that the Hx-heme complex should encounter in endosomes following endocytosis of the complex formed with its hepatic receptor.