Scaling properties of glycine-rich sequences in guanidine hydrochloride solutions

The intrinsic polymer properties of glycine-rich sequences are evaluated with a set of iso-1-cytochrome c variants with N-terminal inserts of the sequence (GGGGGK)(n) for n = 1-5. The thermodynamics and kinetics of His-heme loop formation are measured as a function of guanidine hydrochloride (GdnHCl) concentration for loop sizes ranging from 22 to 46 residues. The scaling exponent for loop formation, ν(3), evaluated using the Jacobson-Stockmayer equation is near 1.8, at 1.5 and 3.0 M GdnHCl, but it increases to 2.2 in 6.0 M GdnHCl. Previous work on a set of iso-1-cytochrome c variants with (AAAAAK)(n) inserts gave ν(3) = 2.2 for alanine-rich sequences in both 3.0 and 6.0 M GdnHCl. Chain stiffness was evaluated from the relative magnitude of Flory's characteristic ratio, C(n), for alanine-rich versus glycine-rich sequences. In 3.0 M GdnHCl, C(n)(Ala)/C(n)(Gly) is 1.6, decreasing to 1.3 in 6.0 M GdnHCl. The data suggest that solvent-backbone interactions dominate polypeptide conformational properties under good solvent conditions whereas side-chain-dependent properties are more important under poor solvent conditions. The results provide a direct experimental assessment in terms of polymer properties of the distinct roles of Gly versus Ala in the folding code.     

Hydration-State Change of Horse Heart Cytochrome c Corresponding to Trifluoroacetic-Acid-Induced Unfolding

We investigate the hydration state of horse-heart cytochrome c (hh cyt c) in the unfolding process induced by trifluoroacetic acid (TFA). The conformation of hh cyt c changes from the native (N) state (2.9 < pH < 6.0) to the acid-unfolded (U_A) state (1.7 < pH < 2.0) to the acid-induced molten globule (A) state (pH ∼1.2). Hydration properties of hh cyt c during this process are measured at 20°C by high-resolution dielectric relaxation (DR) spectroscopy, UV-vis absorbance, and circular dichroism spectroscopy. Constrained water of hh cyt c is observed at every pH as an ∼5-GHz Debye component (DC) (DR time, τ_D ∼30 ps) and its DR amplitude (DRA) is increased by 77% upon N-to-U_A transition, when pH changes from 6.0 to 2.0. Even in the N state, the DRA of the constrained-water component is found to be increased by 22% with decreasing pH from 6.0 to 2.9, suggesting an increase in the accessible surface area of native hh cyt c. Moreover, hypermobile water around native hh cyt c is detected at pH 6.0 as a 19-GHz DC (τ_D ∼ 8.4 ps < τ_DW = 9.4 ps), but is not found at other pH values. The DRA signal of constrained water is found to return to the pH 2.9 (N-state) level upon U_A-to-A transition. Fast-response water (slightly slower than bulk) around A-state hh cyt c is detected at pH 1.2, and this suggests some accumulation of TFA⁻ ions around the peptide chain. Thus, this high-resolution DR spectroscopy study reveals that hh cyt c exhibits significant hydration-state change in the TFA-unfolding process.     

Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase

The human pathogen Shigella flexneri subverts host function and defenses by deploying a cohort of effector proteins via a type III secretion system. The IpaH family of 10 such effectors mimics ubiquitin ligases but bears no sequence or structural homology to their eukaryotic counterpoints. Using rates of ¹²⁵I-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B∼¹²⁵I-ubiquitin thioester co-substrate in the nanomolar range ([S]_½ = 140 ± 32 nm; n = 1.8 ± 0.1) and cooperative substrate inhibition at micromolar concentrations ([S]_½ = 740 ± 240 nm; n = 1.7 ± 0.2), requiring ordered binding to two functionally distinct sites per subunit. The isosteric substrate analog Ubc5BC85S-ubiquitin oxyester acts as a competitive inhibitor of wild-type Ubc5B∼¹²⁵I-ubiquitin thioester (K_i = 117 ± 29 nm), whereas a Ubc5BC85A product analog shows noncompetitive inhibition (K_i = 2.2 ± 0.5 μm), consistent with the two-site model. Re-evaluation of a related IpaH3 crystal structure (PDB entry 3CVR) identifies a symmetric dimer consistent with the observed cooperativity. Genetic disruption of the predicted IpaH9.8 dimer interface reduces the solution molecular weight and significantly ablates the k_cat but not [S]_½ for polyubiquitin chain formation. Other studies demonstrate that cooperativity requires the N-terminal leucine-rich repeat-targeting domain and is transduced through Phe³⁹⁵. Additionally, these mechanistic features are conserved in a distantly related SspH2 Salmonella enterica ligase. Kinetic parallels between IpaH9.8 and the recently revised mechanism for E6AP/UBE3A (Ronchi, V. P., Klein, J. M., and Haas, A. L. (2013) E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites. J. Biol. Chem. 288, 10349–10360) suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases.     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO₄ to NAD⁺ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD⁺. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD⁺-bound states. ITC measurements of RslTpt1 binding to NAD⁺ (K_D ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD⁺-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP⁺, which contains an adenylate 2′-PO₄ (mimicking the substrate RNA 2′-PO₄), binds with lower affinity (K_D ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD⁺ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD⁺ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.     

Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.

     

Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm⁻¹) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 k_BT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.     

In Intact Cone Photoreceptors,a Ca2+-dependent,Diffusible Factor Modulates the cGMP-gated Ion Channels Differently than in Rods

We investigated the modulation of cGMP-gated ion channels in single cone photoreceptors isolated from a fish retina. A new method allowed us to record currents from an intact outer segment while controlling its cytoplasmic composition by superfusion of the electropermeabilized inner segment. The sensitivity of the channels to agonists in the intact outer segment differs from that measured in membrane patches detached from the same cell. This sensitivity, measured as the ligand concentration necessary to activate half-maximal currents, K _1/2, also increases as Ca²⁺ concentration decreases. In electropermeabilized cones, K _1/2 for cGMP is 335.5 ± 64.4 μM in the presence of 20 μM Ca²⁺, and 84.3 ± 12.6 μM in its absence. For 8Br-cGMP, K _1/2 is 72.7 ± 11.3 μM in the presence of 20 μM Ca²⁺ and 15.3 ± 4.5 μM in its absence. The Ca²⁺-dependent change in agonist sensitivity is larger in extent than that measured in rods. In electropermeabilized tiger salamander rods, K _1/2 for 8Br-cGMP is 17.9 ± 3.8 μM in the presence of 20 μM Ca²⁺ and 7.2 ± 1.2 μM in its absence. The Ca²⁺-dependent modulation is reversible in intact cone outer segments, but is progressively lost in the absence of divalent cations, suggesting that it is mediated by a diffusible factor. Comparison of data in intact cells and detached membrane fragments from cones indicates that this factor is not calmodulin. At 40 μM 8Br-cGMP, the Ca²⁺-dependent change in sensitivity in cones is half-maximal at K _Ca = 286 ± 66 nM Ca²⁺. In rods, by contrast, K _Ca is ∼50 nM Ca²⁺. The difference in magnitude and Ca²⁺ dependence of channel modulation between photoreceptor types suggests that this modulation may play a more significant role in the regulation of photocurrent gain in cones than in rods.     

Microsecond Time-Resolved Absorption Spectroscopy Used to Study CO Compounds of Cytochrome bd from Escherichia coli

Cytochrome bd is a tri-heme (b ₅₅₈, b ₅₉₅, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O₂ with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ<1.5 µs) electron transfer from heme d to heme b ₅₉₅ in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b ₅₅₈ (τ∼16 µs). It returns from the b-hemes to heme d with τ∼180 µs. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (τ∼16 µs) yielding a transient hexacoordinate state (CO-Fe²⁺-L). Then the ligand slowly (τ∼30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (τ∼20 µs), some heme b ₅₅₈ (τ∼0.2–3 ms), and finally migrates from heme d to heme b ₅₉₅ (τ∼24 ms) in ∼5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions.     

Effect of Sarcoplasmic Reticulum (SR) Calcium Content on SR Calcium Release Elicited by Small Voltage-Clamp Depolarizations in Frog Cut Skeletal Muscle Fibers Equilibrated with 20 mM EGTA

Cut muscle fibers from Rana temporaria (sarcomere length, 3.5–3.9 μm; 14–16°C) were mounted in a double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium– gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were stimulated with a voltage-clamp pulse protocol that consisted of pulses to −70, −65, −60, −45, and −20 mV, each separated by 400-ms periods at −90 mV. The change in total Ca that entered into the myoplasm (Δ[Ca_T]) and the Ca content of the SR ([Ca_SR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259–336). Fibers were stimulated with the pulse protocol, usually every 5 min, so that the resting value of [Ca_SR] decreased from its initial value of 1,700–2,300 μM to values near or below 100 μM after 18–30 stimulations. Three main findings for the voltage pulses to −70, −65, and −60 mV are: (a) the depletion-corrected rate of Ca release (release permeability) showed little change when [Ca_SR] decreased from its highest level (>1,700 μM) to ∼1,000 μM; (b) as [Ca_SR] decreased below 1,000 μM, the release permeability increased to a maximum level when [Ca_SR] was near 300 μM that was on average about sevenfold larger than the values observed for [Ca_SR] > 1,000 μM; and (c) as [Ca_SR] decreased from ∼300 μM to <100 μM, the release permeability decreased, reaching half its maximum value when [Ca_SR] was ∼110 μM on average. It was concluded that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.     

Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (_ΔΨ_m), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]_m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²⁺]_m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²⁺ indicators with a 20-fold difference in Ca²⁺ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²⁺ indicator (D4cpv) calibrated against known Ca²⁺ levels. Matrix pH (pH_m) and _ΔΨ_m were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²⁺]_m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²⁺]_m coincided with a rapid elevation in pH_m and was followed by an after-stimulus _ΔΨ_m hyperpolarization. However, pH_m decreased and no _ΔΨ_m hyperpolarization was observed in response to lower levels of [Ca²⁺]_m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca²⁺]_m are required to stimulate presynaptic mitochondrial energy metabolism.     

Methemoglobin formation in mutant hemoglobin α chains: electron transfer parameters and rates

Hemoglobin-mediated transport of dioxygen (O₂) critically depends on the stability of the reduced (Fe²⁺) form of the heme cofactors. Some protein mutations stabilize the oxidized (Fe³⁺) state (methemoglobin, Hb M), causing methemoglobinemia, and can be lethal above 30%. The majority of the analyses of factors influencing Hb oxidation are retrospective and give insights only for inner-sphere mutations of heme (His58, His87). Herein, we report the first all-atom molecular dynamics simulations on both redox states and calculations of the Marcus electron transfer (ET) parameters for the α chain Hb oxidation and reduction rates for Hb M. The Hb wild-type (WT) and most of the studied α chain variants maintain globin structure except the Hb M Iwate (H87Y). The mutants forming Hb M tend to have lower redox potentials and thus stabilize the oxidized (Fe³⁺) state (in particular, the Hb Miyagi variant with K61E mutation). Solvent reorganization (λ_solv 73–96%) makes major contributions to reorganization free energy, whereas protein reorganization (λ_prot) accounts for 27–30% except for the Miyagi and J-Buda variants (λ_prot ∼4%). Analysis of heme-solvent H-bonding interactions among variants provide insights into the role of Lys61 residue in stabilizing the Fe²⁺ state. Semiclassical Marcus ET theory-based calculations predict experimental k_ET for the Cyt b5-Hb complex and provide insights into relative reduction rates for Hb M in Hb variants. Thus, our methodology provides a rationale for the effect of mutations on the structure, stability, and Hb oxidation reduction rates and has potential for identification of mutations that result in methemoglobinemia.     

Forward Modeling of Fluctuating Dietary 13C Signals to Validate 13C Turnover Models of Milk and Milk Components from a Diet-Switch Experiment

Isotopic variation of food stuffs propagates through trophic systems. But, this variation is dampened in each trophic step, due to buffering effects of metabolic and storage pools. Thus, understanding of isotopic variation in trophic systems requires knowledge of isotopic turnover. In animals, turnover is usually quantified in diet-switch experiments in controlled conditions. Such experiments usually involve changes in diet chemical composition, which may affect turnover. Furthermore, it is uncertain if diet-switch based turnover models are applicable under conditions with randomly fluctuating dietary input signals. Here, we investigate if turnover information derived from diet-switch experiments with dairy cows can predict the isotopic composition of metabolic products (milk, milk components and feces) under natural fluctuations of dietary isotope and chemical composition. First, a diet-switch from a C₃-grass/maize diet to a pure C₃-grass diet was used to quantify carbon turnover in whole milk, lactose, casein, milk fat and feces. Data were analyzed with a compartmental mixed effects model, which allowed for multiple pools and intra-population variability, and included a delay between feed ingestion and first tracer appearance in outputs. The delay for milk components and whole milk was ∼12 h, and that of feces ∼20 h. The half-life (t_½) for carbon in the feces was 9 h, while lactose, casein and milk fat had a t_½ of 10, 18 and 19 h. The ¹³C kinetics of whole milk revealed two pools, a fast pool with a t_½ of 10 h (likely representing lactose), and a slower pool with a t_½ of 21 h (likely including casein and milk fat). The diet-switch based turnover information provided a precise prediction (RMSE ∼0.2 ‰) of the natural ¹³C fluctuations in outputs during a 30 days-long period when cows ingested a pure C3 grass with naturally fluctuating isotope composition.     

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K_s, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K_d, ≤3 nM) and prothioconazole-desthio the lowest (K_d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC₅₀s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC₁₀₀, >256 μg ml⁻¹). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC₁₀₀ [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml⁻¹) with the exception of clotrimazole, which was as potent as malachite green (MIC₁₀₀, ∼1 μg ml⁻¹). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC₅₀, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC₁₀₀, ∼1 to 2 μg ml⁻¹) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.     

Plant regeneration from organogenic callus and assessment of clonal fidelity in Elephantopus scaber Linn., an ethnomedicinal herb

An efficient callus induction and plant regeneration system has been standardized for an ethnomedicinal plant, Elephantopus scaber Linn. Two explants i. e. seeds and leaf segments were used for callus induction. Murashige and Skoog (MS) medium supplemented with 5.0 μM 2, 4-dichlorophenoxy acetic acid (2, 4-D) and 0.5 μM kinetin (Kn) gave the optimum frequency (89 %) of callus induction from seed explant. The results showed that the highest response in terms of percent callus regenerating (91 %) and number of shoots (56) per culture was recorded on MS medium supplemented with 6.0 μM N₆-benzylaminopurine (BA) and 1.5 μM α naphthalene acetic acid (NAA). The best rooting of regenerated shoots was obtained on half strength MS medium supplemented with 6.0 μM indole-3- butyric acid (IBA). On this medium, 100 % of the shoots produced roots with a mean number of 3.2 roots per shoot. The positive role of vesicular arbuscular mycorrhizae (VAM) along with potting mix has been well established in the present study. Of the various potting mix employed for plant acclimatization, the highest response of 100 % plant survival was noticed when autoclaved garden soil, sand (2:1) and VAM was utilized as potting mix. Inter-simple sequence repeats (ISSR) were used to establish the clonal fidelity of regenerated plantlets and the banding profiles from callus derived plants were monomorphic and similar to those of mother plant, thus ascertaining the true-to-type nature of these plants.     

Elastic-Mathematical Theory of Cells and Mitochondria in Swelling Process: II. Effect of Temperature upon Modulus of Elasticity of Membranous Material of Egg Cells of Sea Urchin, Strongylocentrotus purpuratus, and of Oyster, Crassostrea virginica

The elastic behavior of the cell wall as a function of the temperature has been studied with particular attention being given to the swelling of egg cells of Strongylocentrotus purpuratus and Crassostrea virginica in different sea water concentrations at different temperatures. It was found that the modulus of elasticity is a nonlinear function of temperature. At about 12-13°C the modulus of elasticity (E) is constant, independent of the stress (σ) and strain (ε_ν) which exist at the cell wall; the membranous material follows Hooke's law, and E ≈ 3 × 10⁷ dyn/cm² for S. purpuratus and C. virginica. When the temperature is higher or lower than 12-13°C, the modulus of elasticity increases, and the membranous material does not follow Hooke's law, but is almost directly proportional to the stresses existing at the cell wall. On increasing the stress, the function E_σ = E(σ) approaches saturation. The corresponding stress-strain diagrams, σ = σ(ε_ν), and the graphs, E_σ = E(σ) and E_σ = E(t) are given. The cyto-elastic phenomena at the membrane are discussed.     

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase: STRUCTURE OF THE ENZYME·PROGESTERONE SUBSTRATE COMPLEX AND RATE-LIMITING C–H BOND CLEAVAGE*

Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both k_cat and k_cat/K_m, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (k_on 2.4 × 10⁷ m⁻¹ s⁻¹) is only ∼2-fold greater than the catalytic efficiency (k_cat/K_m = 1.3 × 10⁷ m⁻¹ s⁻¹) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.     

The Dynamics of Finite Haploid Populations with Overlapping Generations. II. the Diffusion Approximation

The dynamics of a gene in a haploid population can be explained approximately by considering the average reproductive value of the gene. The dynamics of the average reproductive value are similar to those of a gene in a population with nonoverlapping generations with the following modifications: The effective population size, N_e, replaces N; the average mutation rates µ* and ν* replace µ and ν; the average overall selection r*+(T-1)s** replaces s; and time is measured in terms of generations, T. The implications of the average selection coefficient to adaptive life histories are discussed.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.