Elevated α-Linolenic Acid Content in Extra-plastidial Membranes of Tomato Accelerates Wound-Induced Jasmonate Generation and Improves Tolerance to the Herbivorous Insects Heliothis peltigera and Spodoptera littoralis

Journal of Plant Growth Regulation - In tomato, desaturation of linoleic acid (18:2) to α-linolenic acid (18:3) is mediated in the plastidial membranes by the ω-3 fatty acid desaturases 7...     

ATP-sensitive K+ channels in cardiac muscle from cold-acclimated goldfish: characterization and altered response to ATP

ATP-sensitive potassium channels (K(ATP)) play an important, if incompletely defined, role in myocardial function in mammals. With the discovery that K(ATP) channels are also present at high densities in the hearts of vertebrate ectotherms, speculation arises as to their function during periods of cold-acclimation and depressed ATP synthesis. We used single-channel and intracellular recording techniques to examine the possibility that channel activity would be altered in cardiac muscle from goldfish (Carassius auratus) acclimated at 7+/-1 degrees C relative to control (21+/-1 degrees C). As previously observed in mammals, K(ATP) channels in isolated ventricular myocytes were inwardly rectified with slope conductances of 63 pS. However, channel mean open-time and overall open-state probability (Po) were significantly increased in cells from the cold-acclimated animals. In addition, K(ATP) channels in cells from fish acclimated at 7 degrees were nearly insensitive to the inhibitory effects of 2 mM ATP, whether studied at 7 or at 21 degrees C. Transmembrane action potential duration (APD) in hearts of cold-acclimated fish studied at 21 degrees was significantly shorter than that observed in hearts of warm-acclimated fish at the same temperature; this difference was eliminated by the K(ATP) channel antagonist glibenclamide (5 microM). These data suggest that K(ATP) channels in the hearts of cold-acclimated animals are more active and less sensitive to ATP-inhibition than those in warm-acclimated fish, possibly reflecting a functional adaptation to promote tolerance of low temperatures in this species.     

Identification, characterization, and expression of a unique secretory lipase from the human pathogen Leishmania donovani

Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a ~33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.     

Resonance Raman analysis of the mechanism of energy storage and chromophore distortion in the primary visual photoproduct

The vibrational structure of the chromophore in the primary photoproduct of vision, bathorhodopsin, is examined to determine the cause of the anomalously decoupled and intense C(11)=C(12) hydrogen-out-of-plane (HOOP) wagging modes and their relation to energy storage in the primary photoproduct. Low-temperature (77 K) resonance Raman spectra of Glu181 and Ser186 mutants of bovine rhodopsin reveal only mild mutagenic perturbations of the photoproduct spectrum suggesting that dipolar, electrostatic, or steric interactions with these residues do not cause the HOOP mode frequencies and intensities. Density functional theory calculations are performed to investigate the effect of geometric distortion on the HOOP coupling. The decoupled HOOP modes can be simulated by imposing approximately 40 degrees twists in the same direction about the C(11)=C(12) and C(12)-C(13) bonds. Sequence comparison and examination of the binding site suggests that these distortions are caused by three constraints consisting of an electrostatic anchor between the protonated Schiff base and the Glu113 counterion, as well as steric interactions of the 9- and 13-methyl groups with surrounding residues. This distortion stores light energy that is used to drive the subsequent protein conformational changes that activate rhodopsin.     

The phospholamban phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme.

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic protein phosphatase activity, which can dephosphorylate phospholamban and regulate calcium transport. This phosphatase has been suggested to be a mixture of both type 1 and type 2 enzymes (E. G. Kranias and J. Di Salvo, 1986, J. Biol. Chem. 261, 10,029-10,032). In the present study the sarcoplasmic reticulum phosphatase activity was solubilized with n-octyl-beta-D-glucopyranoside and purified by sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, and DEAE-Sephadex. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. The partially purified phosphatase could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase(s). Enzymatic activity was inhibited by inhibitor-2 and by okadaic acid (I50 = 10-20 nM), using either phosphorylase a or phospholamban as substrates. The sensitivity of the phosphatase to inhibitor-2 or okadaic acid was similar for the two sites on phospholamban, phosphorylated by the cAMP-dependent and the calcium-calmodulin-dependent protein kinases. Phospholamban phosphatase activity was enhanced (40%) by Mg2+ or Mn2+ (3 mM) while Ca2+ (0.1-10 microM) had no effect. These characteristics suggest that the phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme, and this activity may participate in the regulation of Ca2+ transport through dephosphorylation of phospholamban in cardiac muscle.     

Caribbean damselfish with varying territory quality: correlated behaviors but not a syndrome

The behavioral syndrome hypothesis suggests that individualanimals within a population behave differently due to specificbehavioral types, and these should be consistent across behaviorsor in different contexts. In contrast, for animals that livewithin an environment in which territory quality can changeover time, natural selection should have favored behavioralflexibility and modulation of the cost of defense in relationto territory quality. This would require assessment of the territoryfollowed by displays of appropriate types and intensities ofbehavior. We examined the territorial behavior of male beaugregorydamselfish (Stegastes leucostictus) by enhancing territory qualityusing artificial breeding sites and comparing their behaviorto males on lower quality natural sites. When male fish weredefending high-quality artificial territories, they had higherlevels of aggression toward male conspecifics and courtshiptoward females than when on low-quality natural territories.We also found that aggression and courtship behaviors were correlatedon natural sites but not on artificial sites. Behaviors werenot correlated within individuals when males switched from naturalto artificial territories or from artificial to natural territories.These results indicate that males assess their current territoriesand adjust behaviors accordingly and that courtship and aggressivebehaviors are not linked within a permanent behavioral syndrome.     

Single-Molecule Vibrational Spectroscopy Adds Structural Resolution to the Optical Trap

The ability to apply forces on single molecules with an optical trap is combined with the endogenous structural resolution of Raman spectroscopy in an article in this issue, and applied to measure the Raman spectrum of ds-DNA during force-extension.The resounding success of single-molecule biophysical techniques has encouraged the development of additional tools for more detailed exploration. The unique ability of single-molecule methods to apply force and torque, to disentangle heterogeneity, and to watch equilibrium kinetics would pair beautifully with the ultrafast time resolution and atomistic structural sensitivity of vibrational spectroscopy. However, the weak signal levels endemic to vibrations have left them mostly in the domain of bulk spectroscopy; cross-sections for Raman scattering are typically 10¹⁴ times smaller than for fluorescence emission. In this issue, Rao et al. (1) overcome this gap using surface-enhanced Raman spectroscopy (SERS) (2,3) to add vibrational spectroscopic resolution to their optical trap. In this experiment, a single DNA strand is brought into the near-field vicinity of a silver nanoparticle-coated silica bead that enhances its Raman scattering, and the spectrum is recorded as the DNA is extended in the optical trap. The authors find that applying force shifts the phosphate-stretching vibrational frequency. Molecular dynamics and density functional theory calculations were used to explain these results by showing that external load applied to the DNA backbone induces Ångstrom-level displacements in the P-O bonds.This work is immediately relevant to the communities interested in DNA mechanics and single-molecule Raman spectroscopy. While the authors’ results may refine our structural models for DNA in the low-force regime (1–9 pN), the ongoing debate about the molecular nature of the transition into overstretched DNA (≥65 pN) (4) would be well served by additional structural resolution. For the SERS community, the optical trap provides a fantastic control as it allows one to unambiguously verify that a single-molecule is probed and systematically control its distance and orientation to the metal surface, which may finally resolve long-standing mysteries about the mechanism of SERS. Ideally, both methods will be advanced in concert at the expense of coercing as much information as possible out of a single molecule.While this work is groundbreaking, the real excitement is in its potential. One limitation in most implementations of both single-molecule force and fluorescence spectroscopy is acute sensitivity to distance changes >5 nm, which diminishes upon approaching the subnanometer scale. Raman scattering and infrared absorption vibrational spectroscopies offer a complementary distance sensitivity as molecular oscillators sense their local environment and couple to one another on scales of ∼0.1 nm; see Fig. 1 for a comparison. The optical trap can now be used to initiate specific structural changes to be probed by SERS. In such mechanistic studies, one benefits from the fact that the vibrational spectrum is an endogenous probe, arising from oscillations in all the different bonds present (enzyme as well as substrate), that directly encodes the kinetics and dynamics of structural changes. Such a detailed view of hydrogen-bond rearrangements, covalent-bond formation/breaking, and symmetry changes can offer subtle details that are impossible to tag with fluorophores or directly monitor via a force measurement. As vibrational spectroscopy is rapidly approaching the molecular fingerprinting level with DNA base resolution (5) and protein identification (6), there is an optimistic future for this apparently new multiplexed technique across the various divisions of biophysics.Open in a separate windowFigure 1(A) Examples of mesoscopic structural changes typically underlying single-molecule experiments, such as unfolding of DNA and proteins; translocation of enzymes on a scaffold such as the motor proteins, dynein and kinesin, and replication proteins; and binding of substrates such as ATP and FAD (7,8). (B) Examples of microscopic structural changes probed by bulk vibrational spectroscopy, which may complement single-molecule studies such as hydrogen bonding, isomerization, subtle secondary structural changes such as α-helix rotation and β-sheet reordering, and ligand-binding geometry and kinetics (9–12).     

Spectral signatures of heterogeneous protein ensembles revealed by MD Simulations of 2DIR spectra

A model for the calculation of amide I FTIR and 2DIR spectra taking into account fluctuations in hydrogen bonding and structure from molecular dynamics (MD) simulations is tested on three systems. It is found that although the homogeneous lineshape approximation yields satisfactory FTIR spectra, 2DIR spectra are sensitive to the inhomogeneity naturally present in any solvated protein and the common approximations of a static structure and averaged-effect solvent are invalid. By building on the local amide Hamiltonian and incorporating site energy variation with electrostatic-based models and disorder from MD trajectories, good agreement is obtained between calculated and measured 2DIR spectra. The largest contribution to the observed inhomogeneity is found to be the fluctuating site energies, which in turn are most sensitive to the water solvent. With the ability to accurately predict 2DIR spectra from atomistic simulations, new opportunities to test force fields and mechanistic predictions from MD are revealed.