Identification of Ten Gibberellins from Sporophytes of the Tree fern, Cyathea australis

Ten gibberellins (GAs) have been identified by Kovats retention indices and full mass spectra from GC-MS analysis of purified extracts of sporophytes of the tree-fern, Cyathea australis. These include the known GA₁, GA₄, GA₉, GA₁₅, GA₂₄, GA₃₅, and GA₅₈ and three new GAs, 12β-hydroxyGA₉ (GA₆₉), 12α-hydroxyGA₉ (GA₇₀) and 12β-hydroxyGA₄ (GA₇₁). The structure of GA₇₁ was established by the preparation and characterization of its methyl ester (as a metabolite of GA₄ methyl ester in a culture of prothallia of Lygodium japonicum).     

Cellular Microcystin Content in N-Limited Microcystis aeruginosa Can Be Predicted from Growth Rate

Cell quotas of microcystin (Q_MCYST; femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q_MCYST and specific growth rate (μ), from which we propose a generalized model that enables Q_MCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q_MCYST from μ, μ_max (maximum specific growth rate), Q_MCYSTmax (maximum cell quota), and Q_MCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict a Q_MCYSTmax of 0.129 fmol cell⁻¹ at μ_max and a Q_MCYSTmin of 0.050 fmol cell⁻¹ at μ = 0. Net MCYST production rate (R_MCYST) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell⁻¹ day⁻¹ at μ_max. MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST/protein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chl a ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.     

Steady-State Growth and Chemical Composition of the Marine Chlorophyte Dunaliella tertiolecta in Nitrogen-Limited Continuous Cultures

The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH₄⁺-N, NO₂⁻-N, NO₃⁻-N, and urea-N limitations. The effect of the nitrogen cell quota (Q_n) on the steady-state growth rate (μ) was the same regardless of the N source. The relationship between μ and Q_n was well described by the Droop equation, but only up to the true maximum growth rate ^μ (= cell washout rate). The ratio between the minimum cell quota (k_Q) and the maximum cell quota (Q_m) was 0.19. Hence, there is no substitute for determining ^μ experimentally. That there was no difference in growth response to different N sources suggests that no internal pooling of inorganic nitrogen occurred. Both the carbon (Q_c) and phosphorus (Q_p) cell quotas under N limitation increased with increasing μ in a threshold fashion: virtually no change in either cell quota up to ~0.8 ^μ, followed by a rapid and large increase up to ^μ. In addition, in the region of low μ, there was an increase in Q_p with a decreasing medium N/P ratio of between 15 and 5 (by atoms). The results generally indicate the physiological limits in cellular constituency under N limitation. The usefulness of this information, however, in describing the response of natural populations of marine phytoplankton to transient nutrient exposures on the temporal and spatial microscales that most likely exist is of limited value.     

Activation of a Reserve Pool of Photosystem II in Chlamydomonas reinhardtii Counteracts Photoinhibition

The effect of strong irradiance (2000 micromole photons per square meter per second) on PSII heterogeneity in intact cells of Chlamydomonas reinhardtii was investigated. Low light (LL, 15 micromole photons per square meter per second) grown C. reinhardtii are photoinhibited upon exposure to strong irradiance, and the loss of photosynthetic functioning is due to damage to PSII. Under physiological growth conditions, PSII is distributed into two pools. The large antenna size (PSII_α) centers account for about 70% of all PSII in the thylakoid membrane and are responsible for plastoquinone reduction (Q_b-reducing centers). The smaller antenna (PSII_β) account for the remainder of PSII and exist in a state not yet able to photoreduce plastoquinone (Q_b-nonreducing centers). The exposure of C. reinhardtii cells to 60 minutes of strong irradiance disabled about half of the primary charge separation between P680 and pheophytin. The PSII_β content remained the same or slightly increased during strong-irradiance treatment, whereas the photochemical activity of PSII_α decreased by 80%. Analysis of fluorescence induction transients displayed by intact cells indicated that strong irradiance led to a conversion of PSII_β from a Q_b-nonreducing to a Q_b-reducing state. Parallel measurements of the rate of oxygen evolution revealed that photosynthetic electron transport was maintained at high rates, despite the loss of activity by a majority of PSII_α. The results suggest that PSII_β in C. reinhardtii may serve as a reserve pool of PSII that augments photosynthetic electron-transport rates during exposure to strong irradiance and partially compensates for the adverse effect of photoinhibition on PSII_α.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C2 to C4n-Alkane-Grown Bacteria

Nineteen new C₂ to C₄n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C₂ to C₄n-alkanes. Cell suspensions of these C₂ to C₄n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60°C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.     

Comparative Effects of Substituted Pyrimidines on Growth and Gibberellin Biosynthesis in Gibberella fujikuroi

The fungicide α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidine methyl alcohol (triarimol) and four other structural analogs of this substance, in which one or more of the substituents were varied, were tested for their comparative effects on growth and gibberellin biosynthesis in the fungus Gibberella fujikuroi. Each of the five analogs tested was capable of inhibiting growth as measured by dry weight in 5-day-old cultures. Three of them [α-(2-chlorophenyl)-α-(4-chlorophenyl)-5-pyrimidine methyl alcohol, fenarimol; α-(2-chlorophenyl)-α-(4-fluorophenyl)-5-pyrimidine methyl alcohol, nuarimol; and triarimol] were effective at appreciably lower concentrations than the other two [α-(4-chlorophenyl)-α-(1-methylethyl)-5-pyrimidine methyl alcohol, experimental compound EL 509; and α-cyclopropyl-α-(4-methoxyphenyl)-5-pyrimidine methyl alcohol, ancymidol].     

Pyrophosphate Fructose-6-P 1-Phosphotransferase from Tomato Fruit : Evidence for Change during Ripening

Three forms of pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) were purified from both green and red tomato (Lycopersicon esculentum) fruit: (a) a classical form (designated Q₂) containing α- (66 kilodalton) and β- (60 kilodalton) subunits; (b) a form (Q₁) containing a β-doublet subunit; and (c) a form (Q₀) that appeared to contain a β-singlet subunit. Several lines of evidence suggested that the different forms occur under physiological conditions. Q₂ was purified to apparent electrophoretic homogeneity; Q₁ and Q₀ were highly purified, but not to homogeneity. The distribution of the PFP forms from red (versus green) tomato was: Q₂, 29% (90%); Q₁, 47% (6%); and Q₀, 24% (4%). The major difference distinguishing the red from the green tomato enzymes was the fructose-2,6-bisphosphate (Fru-2,6-P₂)-induced change in K_m for fructose-6-phosphate (Fru-6-P), the `green forms' showing markedly enhanced affinity on activation (K<sub>m</sub> decrease of 7-9-fold) and the `red forms' showing either little change (Q₀, Q₁) or a relatively small (2.5-fold) affinity increase (Q₂). The results extend our earlier findings with carrot root to another tissue and indicate that forms of PFP showing low or no affinity increase for Fru 6-P on activation by Fru-2,6-P₂ (here Q₁ and Q₀) are associated with sugar storage, whereas the classical form (Q₂), which shows a pronounced affinity increase, is more important for starch storage.     

Cytochrome p-450-dependent hydroxylation of lauric Acid at the subterminal position and oxidation of unsaturated analogs in wheat microsomes

Microsomes from etiolated wheat (Triticum aestivum L. cv Etoile de Choisy) shoots catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation of lauric acid predominantly at the subterminal or (ω-1) position (65%). Minor amounts of 10-hydroxy- (31%) and 9-hydroxylaurate (4%) were also formed. The reaction was catalyzed by cytochrome P-450, since enzyme activity was strongly inhibited by tetcyclacis, carbon monoxide, and antibodies against NADPH-cytochrome c (P-450)-reductase. The apparent K_m for lauric acid was estimated to be 8.5 ± 2.0 μm. Seed treatment with the safener naphthalic acid anhydride or treatment of seedlings with phenobarbital increased cytochrome P-450 content and lauric acid hydroxylase (LAH) activity of the microsomes. A combination of both treatments further stimulated LAH activity. A series of radiolabeled unsaturated lauric acid analogs (8-, 9-, 10-, and 11-dodecenoic acids) was used to explore the regioselectivity and catalytic capabilities of induced wheat microsomes. It has been found that wheat microsomes catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent epoxidation of sp2 carbons concurrently with hydroxylation at saturated positions. The regioselectivity of oxidation of the unsaturated substrates and that of lauric acid were similar. Preincubation of wheat microsomes with reduced nicotinamide adenine dinucleotide phosphate and 11-dodecenoic acid resulted in a partial loss of LAH activity.     

The chemical synthesis and biological oxidation of 7α-hydroxy[26-14C]cholesterol, 7-dehydro[26-14C]cholesterol and 26-hydroxy[26-14C]cholesterol

1. 26-Hydroxycholesterol was obtained by reducing the methyl ester of (±)-3β-hydroxycholest-5-en-26-oic acid, which was synthesized from 25-oxonorcholesterol. 2. Methods for preparing 7α-hydroxycholesterol and 7-dehydrocholesterol were modified to allow the micro-scale preparation of these [¹⁴C]sterols from [26-¹⁴C]-cholesterol. 3. 26-Hydroxycholesterol was oxidized more readily than 7α-hydroxycholesterol, 7-dehydrocholesterol or cholesterol by mitochondrial preparations from livers of mice, rats, guinea pigs, common toads (Bufo vulgaris) and Caiman crocodylus. 4. (±)-3β-Hydroxy[26-¹⁴C]cholest-5-en-26-oic acid was oxidized very rapidly to ¹⁴CO₂ by mouse and guinea-pig mitochondria without evident discrimination between the two optical isomers. 5. An enzyme system that oxidizes 26-hydroxycholesterol to 3β-hydroxycholest-5-en-26-oic acid was identified in the soluble extract of rat-liver mitochondria. This enzyme could use NADP in place of NAD but was not identical with liver alcohol dehydrogenase (EC 1.1.1.1). 6. [26-¹⁴C]Cholesteryl 3β-sulphate was not oxidized by fortified mouse-liver preparations that oxidized [26-¹⁴C]cholesterol to ¹⁴CO₂.     

Pre-Diabetes Augments Neuropeptide Y1- and α1-Receptor Control of Basal Hindlimb Vascular Tone in Young ZDF Rats

Background

Peripheral vascular disease in pre-diabetes may involve altered sympathetically-mediated vascular control. Thus, we investigated if pre-diabetes modifies baseline sympathetic Y₁-receptor (Y₁R) and α₁-receptor (α₁R) control of hindlimb blood flow (Q_fem) and vascular conductance (VC).

Methods

Q_fem and VC were measured in pre-diabetic ZDF rats (PD) and lean controls (CTRL) under infusion of BIBP3226 (Y₁R antagonist), prazosin (α₁R antagonist) and BIBP3226+prazosin. Neuropeptide Y (NPY) concentration and Y₁R and α₁R expression were determined from hindlimb skeletal muscle samples.

Results

Baseline Q_fem and VC were similar between groups. Independent infusions of BIBP3226 and prazosin led to increases in Q_fem and VC in CTRL and PD, where responses were greater in PD (p<0.05). The percent change in VC following both drugs was also greater in PD compared to CTRL (p<0.05). As well, Q_fem and VC responses to combined blockade (BIBP3226+prazosin) were greater in PD compared to CTRL (p<0.05). Interestingly, an absence of synergistic effects was observed within groups, as the sum of the VC responses to independent drug infusions was similar to responses following combined blockade. Finally, white and red vastus skeletal muscle NPY concentration, Y₁R expression and α₁R expression were greater in PD compared to CTRL.

Conclusions

For the first time, we report heightened baseline Y₁R and α₁R sympathetic control of Q_fem and VC in pre-diabetic ZDF rats. In support, our data suggest that augmented sympathetic ligand and receptor expression in pre-diabetes may contribute to vascular dysregulation.     

Comparative studies of bile salts. 5α-Chimaerol, a new bile alcohol from the white sucker Catostomus commersoni Lacépède

1. G.l.c. examination of bile alcohols prepared from the sucker Catostomus commersoni Lacépède (family Catostomidae) showed that although 5α-cyprinol (5α-cholestane-3α,7α,12α,26,27-pentol) was a minor constituent, the principal bile alcohol was an undescribed substance, probably present in the bile as the C-26 sulphate ester, whose i.r., n.m.r. and mass spectra agreed with the structure 5α-cholestane-3α,7α,12α,24,26-pentol. 2. M_D studies suggest that this 5α-chimaerol is the 24(+), 25S enantiomer and that 5β-chimaerol (chimaerol) from Chimaera monstrosa bile also has the 24(+), 25S configuration. These findings imply that bile alcohol biosynthesis in suckers and chimaeras includes stereospecific oxidation of cholesterol at C-26. 3. C. commersoni bile acids (present in minor amounts) probably consist largely of 3α,7α,12α-trihydroxy-5α-cholan-24-oic acid (allocholic acid). 4. 5α-Chimaerol sulphate and 5α-cyprinol sulphate are probably biochemically equivalent as bile salts, and can be considered as arising by parallel evolution.     

Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast Yarrowia lipolytica

Some microorganisms can transform methyl ricinoleate into γ-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C₁₈) to the C₁₀ precursor of γ-decalactone, (ii) accumulation of other lactones (3-hydroxy-γ-decalactone and 2- and 3-decen-4-olide), and (iii) γ-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and γ-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume γ-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-γ-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, β-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the β-oxidation flux. We also identified mutant strains that produced 26 times more γ-decalactone than the wild-type parents.     

2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

2-Butanol and its chemical precursor butanone (methyl ethyl ketone – MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B₁₂-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions.     

Sterculic Acid and Its Analogues Are Potent Inhibitors of Toxoplasma gondii

Toxoplasmosis is a serious disease caused by Toxoplasma gondii, one of the most widespread parasites in the world. Lipid metabolism is important in the intracellular stage of T. gondii. Stearoyl-CoA desaturase (SCD), a key enzyme for the synthesis of unsaturated fatty acid is predicted to exist in T. gondii. Sterculic acid has been shown to specifically inhibit SCD activity. Here, we examined whether sterculic acid and its methyl ester analogues exhibit anti-T. gondii effects in vitro. T. gondii-infected Vero cells were disintegrated at 36 hr because of the propagation and egress of intracellular tachyzoites. All test compounds inhibited tachyzoite propagation and egress, reducing the number of ruptured Vero cells by the parasites. Sterculic acid and the methyl esters also inhibited replication of intracellular tachyzoites in HFF cells. Among the test compounds, sterculic acid showed the most potent activity against T. gondii, with an EC₅₀ value of 36.2 μM, compared with EC₅₀ values of 248-428 μM for the methyl esters. Our study demonstrated that sterculic acid and its analogues are effective in inhibition of T. gondii growth in vitro, suggesting that these compounds or analogues targeting SCD could be effective agents for the treatment of toxoplasmosis.     

Fusion Peptides Promote Formation of Bilayer Cubic Phases in Lipid Dispersions. An X-Ray Diffraction Study

Small angle x-ray diffraction revealed a strong influence of the N-terminal influenza hemagglutinin fusion peptide on the formation of nonlamellar lipid phases. Comparative measurements were made on a series of three peptides, a 20-residue wild-type X-31 influenza virus fusion peptide, GLFGAIAGFIENGWEGMIDG, and its two point-mutant, fusion-incompetent peptides G1E and G13L, in mixtures with hydrated phospholipids, either dipalmitoleoylphosphatidylethanolamine (DPoPE), or monomethylated dioleoyl phosphatidylethanolamine (DOPE-Me), at lipid/peptide molar ratios of 200:1 and 50:1. All three peptides suppressed the H_II phase and shifted the L_α–H_II transition to higher temperatures, simultaneously promoting formation of inverted bicontinuous cubic phases, Q_II, which becomes inserted between the L_α and H_II phases on the temperature scale. Peptide-induced Q_II had strongly reduced lattice constants in comparison to the Q_II phases that form in pure lipids. Q_II formation was favored at the expense of both L_α and H_II phases. The wild-type fusion peptide, WT-20, was distinguished from G1E and G13L by the markedly greater magnitude of its effect. WT-20 disordered the L_α phase and completely abolished the H_II phase in DOPE-Me/WT-20 50:1 dispersions, converted the Q_II phase type from Im3m to Pn3m and reduced the unit cell size from ∼38 nm for the Im3m phase of DOPE-Me dispersions to ∼15 nm for the Pn3m phase in DOPE-Me/WT-20 peptide mixtures. The strong reduction of the cubic phase lattice parameter suggests that the fusion-promoting WT-20 peptide may function by favoring bilayer states of more negative Gaussian curvature and promoting fusion along pathways involving Pn3m phase-like fusion pore intermediates rather than pathways involving H_II phase-like intermediates.     

Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels

The human ether-á-go-go–related gene (hERG) K⁺ channel encodes the pore-forming α subunit of the rapid delayed rectifier current, I_Kr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of I_Kr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q₁ and Q₂, with V_1/2’s of −55.7 (equivalent charge, z = 1.60) and −54.2 mV (z = 1.30), respectively, with the Q₂ charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q₁ and Q₂, decreasing to 4.3 ms for Q₂ at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q₂ movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V_1/2 of −64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q₁ and Q₂ charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.     

Raman dispersion spectroscopy probes heme distortions in deoxyHb-trout IV involved in its T-state Bohr effect

The depolarization ratios of heme protein Raman lines arising from vibrations of the heme group exhibit significant dependence on the excitation wavelength. From the analysis of this depolarization ratio dispersion, one obtains information about symmetry-lowering distortions δQ^Γ of the heme group that can be classified in terms of the symmetry races Γ = A_1g, B_1g, B_2g, and A_2g in D_4h symmetry. The heme-protein interaction can be changed by the protonation of distinct amino acid side chains (i.e., for instance the Bohr groups in hemoglobin derivates), which gives rise to specific static heme distortions for each protonation state. From the Raman dispersion data, it is possible to obtain parameters by fitting to a theoretical expression of the Raman tensor, which provide information on these static distortions and also about the pK values of the involved titrable side chains. We have applied this method to the ν₄ (1,355 cm^-1) and ν₁₀ (1,620 cm^-1) lines of deoxygenated hemoglobin of the fourth component of trout and have measured their depolarization ratio dispersion as a function of pH between 6 and 9. From the pH dependence of the thus derived parameters, we obtain pK values identical to those of the Bohr groups, which were earlier derived from the corresponding O₂-binding isotherms. These are pK_α1 = pK_α2 = 8.5 for the α and pK_β1 = 7.5, pK_β2 = 7.4 for the β chains. We also obtain the specific distortion parameters for each protonation state. As shown in earlier studies, the ν₄ mode mainly probes distortions from interactions between the proximal histidine and atoms of the heme core (i.e., the nitrogens and the C_α atoms of the pyrroles). Group theoretical argumentation allows us to relate specific changes of the imidazole geometry as determined by its tilt and azimuthal angle and the iron-out-of-plane displacement to distinct variations of the normal distortions δQ^Γ derived from the Raman dispersion data. Thus, we found that the pH dependence of the heme distortions δQ^A1g (totally symmetric) and δQ^B1g (asymmetric) is caused by variations of the azimuthal rather than the tilt angle of the Fe-His (F8) bond. In contrast to this, the ν₁₀ line mainly monitors changes resulting from the interaction between peripheral substituents of the porphyrin macrocycle (vinyl). From the pH dependence of the parameters, it is possible to separately identify distortions δQ^Γ affecting the hemes in the α and β chains, respectively. From this, we find that in the α subunit structural changes induced on protonation of the corresponding Bohr groups are mainly transferred via the Fe—N_ε bond and give rise to changes in the azimuthal angle. In the β subunit, however, in addition, structural changes of the heme pocket arise, which most probably result from protonation of the imidazole of the COOH-terminal His (HC3 β). This rearranges the net of H bonds between His HC3 β, Ser (F9 β), and Glu (F7 β).     

Molecular Features Affecting the Biological Activity of the Host-Selective Toxins from Cochliobolus victoriae

The structures of the toxins produced by Cochliobolus victoriae, victorin B, C, D, E, and victoricine, have recently been established. These toxins and modified forms of victorin C were tested for their effect on dark CO₂ fixation in susceptible oat (Avena sativa) leaf slices. Half-maximal inhibition of dark CO₂ fixation occurred with the native toxins in the range of 0.004 to 0.546 micromolar. An essential component for the inhibitory activity of victorin is the glyoxylic acid residue, particularly its hydrated aldehyde group. Removal of glyoxylic acid completely abolished the inhibitory activity of victorin, and the reduction of the aldehydo group transformed the toxin into a protectant. Conversion of victorin to its methyl ester resulted in diminution of inhibitory activity to 10% of the original activity of the toxin, whereas derivatization of the ε-amino group of the β-hydroxylysine moiety resulted in a decrease of inhibitory activity to 1% of that of victorin C. However, the derivatized toxin retained its host selectivity. In addition, the opening of the macrocyclic ring of the toxin drastically reduced the inhibitory activity.     

Antioxidant,inhibition of α-glucosidase and suppression of nitric oxide production in LPS-induced murine macrophages by different fractions of Actinidia arguta stem

In traditional systems of medicine, fruits, leaves, and stems of Actinidia arguta (Sieb. et Zucc.) Planch. ex Miq. have been used to treat various inflammatory diseases. The present study determined the proximate composition, antioxidant, anti-inflammatory, and hypoglycemic potential of A. arguta stem. Phenolic composition of hot water extract and its sub-fractions was determined by Folin–Ciocalteu’s reagent method. In vitro antioxidant activities of the samples were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assays. Anti-inflammatory activity of different fractions was investigated through the inhibition of nitric oxide (NO) production in lipopolysaccharide (1 μg/ml) stimulated RAW 264.7 cells. In addition, inhibition of α-glucosidase activity of hot water extract was determined using p-nitrophenyl-α-d-glucopyranoside (pNPG) as a substrate. Ethyl acetate (557.23 mg GAE/g) fraction contains higher level of total phenolic content. The antioxidant activity evaluated by DPPH radical scavenging assay showed a strong activity for ethyl acetate (IC₅₀ of 14.28 μg/ml) and n-butanol fractions (IC₅₀ of 48.27 μg/ml). Further, ethyl acetate fraction effectively inhibited NO production in RAW 264.7 cells induced by lipopolysaccharide (LPS) than other fractions (nitrite level to 32.14 μM at 200 μg/ml). In addition, hot water extract of A. arguta stem exhibited appreciable inhibitory activity against α-glucosidase enzyme with IC₅₀ of 1.71 mg/ml. The obtained results have important consequence of using A. arguta stem toward the development of effective anti-inflammatory drugs.     

Study on factors influencing stability of whole cell during biodiesel production in solvent-free and tert-butanol system

Utilizing whole cell biocatalyst for biodiesel production has some advantages since it can avoid the complex procedures of isolation, purification and immobilization of extracellular lipase. However, during repeated use of Rhizopus oryzae (R. oryzae) IFO4697 whole cell for biodiesel production in solvent-free system, the whole cell exhibited very poor stability; while the whole cell stability has been found to be significantly improved in tert-butanol system compared to that in solvent-free system. The difference in whole cell stability was found to be due to the difference of product accumulation between solvent-free and tert-butanol system. After 144 h reaction, glycerol and methyl ester accumulated in the cell in solvent-free system came up to about 1000 mg/g and 350 mg/g dry biomass, respectively, while in tert-butanol system, glycerol and methyl ester accumulation was kept at a relatively low level, approximately 100 mg/g and 2 mg/g dry biomass, respectively. The accumulated glycerol influenced whole cell stability through mass transfer limitation only, while the accumulated methyl ester influenced whole cell stability through both mass transfer limitation and product inhibition. Further study showed that a slight loss in enzymatic activity in tert-butanol system was caused by protein leakage.