Distinct interactions between actin and essential myosin light chain isoforms

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein–protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin^ala3) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (K_D = 575 nM) was significantly (p < 0.01) lower compared with the affinity of hVLC-1 to α-actin (K_D = 186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p < 0.01) lower association rate (k_on: 1018 M⁻¹ s⁻¹) compared with k_on of the hVLC-1/α-actin complex interaction (2908 M⁻¹ s⁻¹). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.     

Single-molecule measurement of the stiffness of the rigor myosin head

The force-extension curve of single myosin subfragment-1 molecules, interacting in the rigor state with an actin filament, has been investigated at low [ATP] by applying a slow triangle-wave movement to the optical traps holding a bead-actin-bead dumbbell. In combination with a measurement of the overall stiffness of the dumbbell, this allowed characterization of the three extensible elements, the actin-bead links and the myosin. Simultaneously, another method, based on an analysis of bead position covariance, gave satisfactory agreement. The mean covariance-based estimate for the myosin stiffness was 1.79 pN/nm (SD = 0.7 pN/nm; SE = 0.06 pN/nm (n = 166 myosin molecules)), consistent with a recent report (1.7 pN/nm) from rabbit muscle fibers. In the triangle-wave protocol, the motion of the trapped beads during interactions was linear within experimental error over the physiological range of force applied to myosin (±10 pN), consistent with a Hookean model; any nonlinear terms could not be characterized. Bound states subjected to forces that resisted the working stroke (i.e., positive forces) detached at a significantly lower force than when subjected to negative forces, which is indicative of a strain-dependent dissociation rate.     

Hydrolysis of the soluble fluorescent molecule carboxyumbelliferyl-beta-D-glucuronide by E. coli beta-glucuronidase as applied in a rugged, in situ optical sensor

Techniques utilizing β-glucuronidase (GUS) activity as an indicator of Escherichia coli (E. coli) presence use labeled glucuronides to produce optical signals. Carboxyumbelliferyl-β-d-glucuronide (CUGlcU) is a fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs and temperatures and, therefore, may be an ideal reagent for use in an in situ optical sensor. This paper reports for the first time the Michaelis-Menten kinetic parameters for the binding of E. coli GUS with CUGlcU as K_m = 910 μM, V_max = 41.0 μM min⁻¹, V_max/K_m 45.0 μmol L⁻¹ min⁻¹, the optimal pH as 6.5 ± 1.0, optimal temperature as 38 °C, and the Gibb's free energy of activation as 61.40 kJ mol⁻¹. Additionally, it was found CUGlcU hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps. Comparison studies were made with the more common fluorescent molecule methylumbelliferyl-β-d-glucuronide (MUGlcU). Experiments showed GUS preferentially binds to MUGlcU in comparison to CUGlcU. CUGlcU was also demonstrated in a prototype optical sensor for the detection of E. coli. Initial bench testing of the sensor produced detection of low concentrations of E. coli (1.00 × 10³ CFU/100 mL) in 230 ± 15.1 min and high concentrations (1.05 × 10⁵ CFU/100 mL) in 8.00 ± 1.01 min.     

Characterization of Tightly Associated Smooth Muscle Myosin-Myosin Light-Chain Kinase-Calmodulin Complexes

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ∼ 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at ∼ 1 nM free [Ca²⁺]. There were two MLCK pools that bound unphosphorylated SMM with K_d ∼ 10 and 0.2 μM and phosphorylated SMM with K_d ∼ 20 and 0.2 μM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca²⁺ and phosphorylation of SMM occurred at a pCa₅₀ of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.     

Effects of UV radiation and nitrate limitation on the production of biogenic sulfur compounds by marine phytoplankton

We tested the effects of UV radiation (UVR) and nitrate limitation on the production of dimethylsulfide (DMS), particulate dimethylsulfoniopropionate (DMSPp), and particulate dimethylsulfoxide (DMSOp) in natural seawater from the Gulf of Mexico and in phytoplankton cultures. DMS/Chl a ratios in PAR-only and PAR + UV-exposed seawater were 0.44–2.0 and 0.46–1.9 nmol DMS μg⁻¹ Chl a, respectively, whereas the ratios in cultures of Amphidinium carterae were 1.0–2.2 nmol μg⁻¹ in PAR-exposed samples and 0.91–2.1 nmol μg⁻¹ in PAR + UV-exposed samples. These results suggested that UVR did not substantially affect DMS/Chl a ratios in seawater and A. carterae culture samples. Similarly, UVR had no significant effect on DMSOp/Chl a in seawater samples (0.83–1.6 nmol DMSO μg⁻¹ Chl a for PAR + UV vs. 0.70–1.5 nmol μg⁻¹ for PAR-exposed seawater samples, respectively) or in A. carterae cultures (0.20–1.3 and 0.19–0.88 nmol DMSO μg⁻¹ Chl a in PAR + UV- and PAR-exposed cultures, respectively). In an experiment with the diatom, Thalassiosira oceanica, the culture was grown in high nitrate (30 μM) or low nitrate (6 μM) media and exposed to PAR-only or PAR + UV. The low nitrate, PAR-only samples showed an increase of intracellular dimethylsulfoniopropionate (DMSP) concentration from 2.1 to 15 mmol L⁻¹ in 60 h, but the increase occurred only after cultures reached the stationary phase. Cultures of T. oceanica grown under UVR had lower growth rates than those under PAR-only (μ′ = 0.17 and 0.32 d⁻¹, respectively) and perhaps did not experience severe nitrate limitation even in the low nitrate treatment. These results suggest that the elevated UVR in low nitrate environments could result in reduction of DMSP in some species, whereas DMSP concentrations would not be affected in eutrophic areas.     

Effect of inorganic phosphate on the force and number of myosin cross-bridges during the isometric contraction of permeabilized muscle fibers from rabbit psoas

The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12°C, sarcomere length 2.5 μm). Changes in the hs strain were measured by imposing length steps or small 4 kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3-20 mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8 ± 1.1 kPa nm⁻¹. Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7 ± 3.7 kPa nm⁻¹. An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (r_F) is 23.5 ± 1.0 s⁻¹ in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6 ± 0.9 s⁻¹ at 20 mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (>1 ms) delay between generation of stiffness and generation of force by the cross-bridges.     

Plasmodium falciparum: In vitro interaction of quassin and neo-quassin with artesunate, a hemisuccinate derivative of artemisinin

Quassia amara L. (Family Simaroubaceae) is known to have several medicinal properties including the activity against malaria. An HPLC method was employed for purification of the biologically active quassinoids; quassin (Q) and neo-quassin (NQ), further characterized by MALDI-TOF analyses. Purified Q, NQ and the crude bark extract (S1) along with artesunate (AS) were studied for their in vitro anti-plasmodial activity. The in vivo toxicity studies at intraperitoneal doses with higher concentrations of the crude bark extract (S1) in Balb/C mice ruled out the apprehension of toxicity. Interaction studies between the test compounds among themselves (Q + NQ) and individually with artesunate (AS + Q, AS + NQ), were carried out in vitro at four ratios (1:5, 1:2, 2:1 and 5:1) on chloroquine sensitive (MRC-pf-20) and resistant (MRC-pf-303) strains of Plasmodium falciparum. The crude bark extracts of Q. amara exhibited higher P. falciparum inhibitory activity (IC₅₀ = 0.0025 μg/ml) as compared to that of the isolated compounds, quassin (IC₅₀ = 0.06 μg/ml, 0.15 μM), neo-quassin (IC₅₀ = 0.04 μg/ml, 0.1 μM) and also to the positive control, artesunate (IC₅₀ = 0.02 μg/ml, 0.05 μM). The in vitro drug interaction study revealed the compounds, quassin and neo-quassin to be additive to each other. At lower ratios, artesunate was found to be a potential combination partner with both the compounds. It was interesting to note that none of the combinations exhibited antagonistic interactions. This phenomenon offers the opportunity for further exploration of novel therapeutic concentrations and combinations.     

Bicarbonate use in three aquatic plants

Our study aimed to test the ability of aquatic plants to use bicarbonate when acclimated to three different bicarbonate concentrations. To this end, we performed experiments with the three species Ceratophyllum demersum, Egeria densa, Lagarosiphon major to determine photosynthetic rates under varying bicarbonate concentrations. We measured bicarbonate use efficiency, photosynthetic performance and respiration. For all species, our results revealed that photosynthetic rates were highest in replicates grown at low alkalinity. Thus, E. densa had approx. five times higher rates at low (264 ± 15 μmol O₂ g⁻¹ DW h⁻¹) than at high alkalinity (50 ± 27 μmol O₂ g⁻¹ DW h⁻¹), C. demersum had three times higher rates (336 ± 95 and 120 ± 31 μmol O₂ g⁻¹ DW h⁻¹), and L. major doubled its rates at low alkalinity (634 ± 114 and 322 ± 119 μmol O₂ g⁻¹ DW h⁻¹). Similar results were obtained for bicarbonate use efficiency by E. densa (136 ± 44 and 43 ± 10 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹) and L. major (244 ± 29 and 82 ± 24 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹). As to C. demersum, efficiency was high but unaffected by alkalinity, indicating high adaptation ability to varied alkalinities. A pH drift experiment supported these results. Overall, our results suggest that the three globally widespread worldwide species of our study adapt to low inorganic carbon availability by increasing their efficiency of bicarbonate use.     

Cardiomyopathy Mutations Reveal Variable Region of Myosin Converter as Major Element of Cross-Bridge Compliance

The ability of myosin to generate motile forces is based on elastic distortion of a structural element of the actomyosin complex (cross-bridge) that allows strain to develop before filament sliding. Addressing the question, which part of the actomyosin complex experiences main elastic distortion, we suggested previously that the converter domain might be the most compliant region of the myosin head domain. Here we test this proposal by studying functional effects of naturally occurring missense mutations in the β-myosin heavy chain, 723Arg → Gly (R723G) and 736Ile → Thr (I736T), in comparison to 719Arg → Trp (R719W). All three mutations are associated with hypertrophic cardiomyopathy and are located in the converter region of the myosin head domain. We determined several mechanical parameters of single skinned slow fibers isolated from Musculus soleus biopsies of hypertrophic cardiomyopathy patients and healthy controls. Major findings of this study for mutation R723G were i), a >40% increase in fiber stiffness in rigor with a 2.9-fold increase in stiffness per myosin head (S^∗_{rigor R723G} = 0.84 pN/nm S^∗_{rigor WT} = 0.29 pN/nm); and ii), a significant increase in force per head (F^∗_10°C, 1.99 pN vs. 1.49 pN = 1.3-fold increase; F^∗_20°C, 2.56 pN vs. 1.92 pN = 1.3-fold increase) as well as stiffness per head during isometric steady-state contraction (S^∗_active10°C, 0.52 pN/nm vs. 0.28 pN/nm = 1.9-fold increase). Similar changes were found for mutation R719W (2.6-fold increase in S^∗_rigor; 1.8-fold increase in F^∗_10°C, 1.6-fold in F^∗_20°C; twofold increase in S^∗_active10°C). Changes in active cross-bridge cycling kinetics could not account for the increase in force and active stiffness. For the above estimates the previously determined fraction of mutated myosin in the biopsies was taken into account. Data for wild-type myosin of slow soleus muscle fibers support previous findings that for the slow myosin isoform S^∗ and F^∗ are significantly lower than for fast myosin e.g., of rabbit psoas muscle. The data indicate that two mutations, R723G and R719W, are associated with an increase in resistance to elastic distortion of the individual mutated myosin heads whereas mutation I736T has essentially no effect. The data strongly support the notion that major elastic distortion occurs within the converter itself. Apparently, the compliance depends on specific residues, e.g., R719 and R723, presumably located at strategic positions near the long α-helix of the light chain binding domain. Because amino acids 719 and 723 are nonconserved residues, cross-bridge stiffness may well be specifically tuned for different myosins.     

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin

Background

Studies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin.

Methods

To measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (F_unb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin.

Results

F_unb from unregulated actin (0.10 ± 0.01 pN) was significantly increased in the presence of CaD (0.17 ± 0.02 pN), tropomyosin (0.17 ± 0.02 pN) or both regulatory proteins (0.18 ± 0.02 pN). ERK phosphorylation of CaD significantly reduced the F_unb (0.06 ± 0.01 pN). Inspection of the traces of the F_unb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment.

Conclusions

CaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels.

General significance

This study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.     

Sorgoleone,a sorghum root exudate: Algicidal activity and acute toxicity to the ricefish Oryzias latipes

The discovery of natural and natural-based compounds has resulted in its application as an alternative to synthetic algicides to control harmful algae in aquatic systems. Of the many natural-product-based algicides, sorgoleone, a natural plant product from Sorghum bicolor root exudates has been investigated for its controlling effect on different algal species and its acute fish toxicity. Growth of the blue green algal species Microcystis aeruginosa Kützing was completely inhibited by the crude methanol extract of sorghum root at 20 μg mL⁻¹. The most noticeable inhibition was observed in the bioassay of n-hexane soluble extract, where 98% growth inhibition occurred in M. aeruginosa at the concentration of 1.25 μg mL⁻¹. Sorgoleone very effectively controlled blue green algae inhibiting 97% of M. aeruginosa at 0.5 μg mL⁻¹ and 99% of Anabaena affinis Lemmermann at 4 μg mL⁻¹. In contrast, inhibition of the green algae species Chlorella vulgaris Beijerinck and Scenedensmus spp. at 16 μg mL⁻¹ sorgoleone was 87 and 68%, respectively. There were no mortalities or adverse effects observed in any of the fish exposed to water control, solvent control, and a nominal concentration of 1 μg mL⁻¹ during the test period. The no observed effect concentration (NOEC) value was 1.5 μg mL⁻¹ for the tested fish (O. latipes). Sorgoleone can be considered as an effective and an ecologically and environmentally sustainable approach to controlling harmful algae.     

Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

Synthesis of [I]iodoDPA-713: A new probe for imaging inflammation

[¹²⁵I]IodoDPA-713 [¹²⁵I]1, which targets the translocator protein (TSPO, 18 kDa), was synthesized in seven steps from methyl-4-methoxybenzoate as a tool for quantification of inflammation in preclinical models. Preliminary in vitro autoradiography and in vivo small animal imaging were performed using [¹²⁵I]1 in a neurotoxicant-treated rat and in a murine model of lung inflammation, respectively. The radiochemical yield of [¹²⁵I]1 was 44 ± 6% with a specific radioactivity of 51.8 GBq/μmol (1400 mCi/μmol) and >99% radiochemical purity. Preliminary studies showed that [¹²⁵I]1 demonstrated increased specific binding to TSPO in a neurotoxicant-treated rat and increased radiopharmaceutical uptake in the lungs of an experimental inflammation model of lung inflammation. Compound [¹²⁵I]1 is a new, convenient probe for preclinical studies of TSPO activity.     

Mechanism of the Ca-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA

The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca²⁺ binds to the EF2 domain of S100A4 with micromolar affinity and that the K_d value for Ca²⁺ is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in K_d results from a reduced dissociation rate constant (from 16 s^− 1 to 0.3 s^− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca²⁺]. However, for Ca²⁺ transients to be effective in further promoting dissociation, the elevated Ca²⁺ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein-myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data.     

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin

Background

Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin.

Methods

To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (F_unb) in the absence and presence of calponin or phosphorylated calponin.

Results

F_unb from F-actin alone (0.12 ± 0.01 pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02 pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01 pN). To further verify that this enhancement of F_unb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the F_unb obtained at a [KCl] of 25 mM (0.21 ± 0.02 pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01 pN).

Conclusions

This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.

General significance

This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.     

A simple bacterial transformation method using magnesium- and calcium-aminoclays

An efficient and user-friendly bacterial transformation method by simple spreading cells with aminoclays was demonstrated. Compared to the reported transformation approaches using DNA adsorption or wrapping onto (in)organic fibers, the spontaneously generated clay-coated DNA suprastructures by mixing DNA with aminoclay resulted in transformants in both Gram-negative (Escherichia coli) and Gram-positive cells (Streptococcus mutans). Notably, the wild type S. mutans showed comparable transformation efficiency to that of the E. coli host for recombinant DNA cloning. This is a potentially promising result because other trials such as heat-shock, electroporation, and treatment with sepiolite for introducing DNA into the wild type S. mutans failed. Under defined conditions, the transformation efficiency of E. coli XL1-Blue and S. mutans exhibited ~ 2 × 10⁵ and ~ 6 × 10³ CFU/μg of plasmid DNA using magnesium-aminoclay. In contrast, transformation efficiency was higher in S. mutans than that in E. coli XL1-Blue for calcium-aminoclay. It was also confirmed that each plasmid transformed into E. coli and S. mutans was stably maintained and that they expressed the inserted gene encoding the green fluorescent protein during prolonged growth of up to 80 generations.     

Characterization of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase multigene family of Malus domestica Borkh

Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990 bp) and MD-ACO3 (966 bp) have been compared with a previously cloned gene of apple, MD-ACO1 (with an ORF of 942 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3′ untranslated regions (3′-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-ACO1) and 71% (MD-ACO2) in the 3′-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants (K_m) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) for the substrate ACC, catalytic constants (K_cat) of 6.6 × 10⁻² (MD-ACO1), 3.44 × 10⁻² (Md-ACO2) and 9.14 × 10⁻² (MD-ACO3) and K_cat/K_m (μM s⁻¹) values of 7.38 × 10⁻⁴ μM s⁻¹ (MD-ACO1), 0.86 × 10⁻⁴ M s⁻¹ (MD-ACO2) and 3.8 × 10⁻⁴ μM s⁻¹ (MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.     

Inter-population variability of leaf morpho-anatomical and terpenoid patterns of Pistacia atlantica Desf. ssp. atlantica growing along an aridity gradient in Algeria

Three Algerian populations of female Pistacia atlantica shrubs were investigated in order to check whether their terpenoid contents and morpho-anatomical parameters may characterize the infraspecific variability. The populations were sampled along a gradient of increasing aridity from the Atlas mountains into the northwestern Central Sahara.As evidenced by Scanning Electron Microscopy, tufted hairs could be found only on seedling leaves from the low aridity site as a population-specific trait preserved also in culture. Under common garden cultivation seedlings of the high aridity site showed a three times higher density of glandular trichomes compared to the low aridity site. Increased aridity resulted also in reduction of leaf sizes while their thickness increased. Palisade parenchyma thickness also increases with aridity, being the best variable that discriminates the three populations of P. atlantica.Analysis of terpenoids from the leaves carried out by GC-MS reveals the presence of 65 compounds. The major compounds identified were spathulenol (23 μg g⁻¹ dw), α-pinene (10 μg g⁻¹ dw), verbenone (7 μg g⁻¹ dw) and β-pinene (6 μg g⁻¹ dw) in leaves from the low aridity site; spathulenol (73 μg g⁻¹ dw), α-pinene (25 μg g⁻¹ dw), β-pinene (18 μg g⁻¹ dw) and γ-amorphene (16 μg g⁻¹ dw) in those from medium aridity and spathulenol (114 μg g⁻¹ dw), α-pinene (49 μg g⁻¹ dw), germacrene D (29 μg g⁻¹ dw) and camphene (23 μg g⁻¹ dw) in leaves from the high aridity site. Terpene concentrations increased with the degree of aridity: the highest mean concentration of monoterpenes (136 μg g⁻¹ dw), sesquiterpenes (290 μg g⁻¹ dw) and total terpenes (427 μg g⁻¹ dw) were observed in the highest arid site and are, respectively, 3-, 5- and 4-fold higher compared to the lower arid site. Spathulenol and α-pinene can be taken as chemical markers of aridity. Drought discriminating compounds in low, but detectable concentrations are δ-cadinene and β-copaene. The functional roles of the terpenoids found in P. atlantica leaves and principles of their biosynthesis are discussed with emphasis on the mechanisms of plant resistance to drought conditions.     

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2 ± 75.7 μg/ml. Nifedipine, a well known L-type Ca²⁺ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1 ± 3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4 ± 56.2 μg/ml. Whole-cell L-type Ca²⁺ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I_Ca,L by 32.6 ± 9.2% and at 250 μg/ml it decreased by 89.3 ± 7.4%. Thus, inhibition of L-type Ca²⁺ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.     

Accumulation of lead by free and immobilized cyanobacteria with special reference to accumulation factor and recovery

Lead accumulation by free and immobilized cyanobacteria, Lyngbya majuscula and Spirulina subsalsa was studied. Exponentially growing biomass was exposed to 1-20 mg L⁻¹ of Pb(II) solution at pH 6, 7 and 8 for time periods ranging from 10 min to 48 h. L. majuscula accumulated 10 times more Pb (13.5 mg g⁻¹) than S. subsalsa (1.32 mg g⁻¹) at pH 6 within 3 h of exposure to 20 mg L⁻¹ Pb(II) solution and 76% of the Pb could be recovered using 0.1 M EDTA. This chelator (2 μM) did not influence Pb accumulation whereas 100 μM citrate increased that of S. subsalsa 6- to 8-fold. L. majuscula filaments enmeshed in a glass wool packed in a column removed 95.8% of the Pb from a 5 mg L⁻¹ Pb solution compared to free and dead biomass which removed 64 and 33.6% Pb respectively. A 92.5% recovery of accumulated Pb from the immobilized biomass suggests that repeated absorption-desorption is possible.