Nanopropulsion by biocatalytic self-assembly.

A number of organisms and organelles are capable of self-propulsion at the micro- and nanoscales. Production of simple man-made mimics of biological transportation systems may prove relevant to achieving movement in artificial cells and nano/micronscale robotics that may be of biological and nanotechnological importance. We demonstrate the propulsion of particles based on catalytically controlled molecular self-assembly and fiber formation at the particle surface. Specifically, phosphatase enzymes (acting as the engine) are conjugated to a quantum dot (the vehicle), and are subsequently exposed to micellar aggregates (fuel) that upon biocatalytic dephosphorylation undergo fibrillar self-assembly, which in turn causes propulsion. The motion of individual enzyme/quantum dot conjugates is followed directly using fluorescence microscopy. While overall movement remains random, the enzyme-conjugates exhibit significantly faster transport in the presence of the fiber forming system, compared to controls without fuel, a non-self-assembling substrate, or a substrate which assembles into spherical, rather than fibrous structures upon enzymatic dephosphorylation. When increasing the concentration of the fiber-forming fuel, the speed of the conjugates increases compared to non-self-assembling substrate, although directionality remains random.

Functional connectivity between the thalamus and postsubiculum: analysis of evoked responses elicited by stimulation of the laterodorsal thalamic nucleus in anesthetized rats.

The laterodorsal nucleus (LDN) of the thalamus provides a prominent afferent projection to the postsubiculum (dorsal presubiculum). To characterize synaptic transmission in this pathway, we placed stimulating electrodes in the LDN and recorded fEPSPs elicited in the postsubiculum of urethane-anesthetized rats. LDN stimulation elicited a source-sink dipole between the deep and superficial layers of the postsubiculum, respectively, consistent with anatomical evidence for the termination of thalamic afferents in the superficial layers of the structure, and the existence of deep layer neurons with apical dendrites extending into these layers. Postsubicular fEPSPs were typically 0.5-1.0 mV in amplitude, with a peak latency of approximately 6 ms. Consistent with anatomical observations, the short onset latency of fEPSPs elicited by LDN stimulation, and their ability to follow a 60-Hz train of stimulation, indicate that the projection is monosynaptic. Paired-pulse stimulation revealed pronounced paired-pulse depression that was maximal at 100 ms, suggesting that initial release probabilities are high at LDN-postsubiculum synapses, in common with many neocortical pathways. A conventional tetanus protocol that yields LTP in hippocampal pathways had no effect on postsubicular fEPSPs, but long-term depression could be induced by 60-Hz stimulation. Drug infusion studies revealed that synaptic transmission in the LDN-postsubiculum projection is predominantly AMPA-receptor mediated. Rats were implanted with indwelling infusion cannulae targeting the postsubiculum, and, after a recovery period, were anaesthetized withurethane, and implanted with stimulating and recording electrodes. Infusion of CNQX almost completely abolished postsubicular fEPSPs, whereas D-AP5 had little effect. However, 60-Hz LTD was blocked by D-AP5 infusion, revealing that this form of synaptic plasticity is NMDA-receptor dependent.

Differential effects of severe water stress on linear and cyclic electron fluxes through Photosystem I in spinach leaf discs in CO(2)-enriched air.

Linear and cyclic electron fluxes through Photosystem I in 1% CO(2) were quantified in spinach leaf tissue under severe water stress. Using actinic light with a peak at 697 nm for preferential light absorption by Photosystem I while also stimulating Photosystem II to improve redox poising, the cyclic electron flux after 60 s of illumination was a substantial proportion (33-44%) of the total electron flux through PSI at irradiances up to ~1,070 micromol photons m(-2) s(-1). At the maximum irradiance, the cyclic electron flux changed little with the progressive water loss from leaf tissue up to ~60%; by contrast, the linear electron flux was approximately halved. A reason for this differential effect of water stress on the capacity for cyclic and linear electron flow could be the increased crowding of soluble proteins in the stroma due to chloroplast shrinkage. Indeed the confinement of soluble proteins to a smaller chloroplast volume was indicated by cryo-scanning electron microscopy. It is known that the diffusion coefficient of large proteins is decreased when the background concentration of small proteins is raised; by contrast, the diffusion coefficient of small proteins is not affected by increasing the concentration of a large protein (Muramatsu and Minton in Proc Natl Acad Sci USA 85:2984-2988, 1988). Therefore, we suggest that linear electron flow, being coupled to the Calvin-Benson cycle, is limited by the diffusion of large macromolecules, especially the ribulose 1, 5-bisphosphate carboxylase/oxygenase complex. By contrast, cyclic electron flow, involving relatively small macromolecules such as ferredoxin, is less susceptible to inhibition by crowding in the stroma.

Separation of light-induced linear, cyclic and stroma-sourced electron fluxes to P700+ in cucumber leaf discs after pre-illumination at a chilling temperature.

Pre-illumination of cucumber leaf discs at 4 degrees C with low-irradiance white light (i) led to a marked decrease in the extent of photo-oxidation of P700 (the special chlorophyll pair in the PSI reaction center) in actinic light at room temperature and (ii) hastened the post-illumination re-reduction of P700+. Quantifying the linear, cyclic and stroma-sourced electron fluxes to P700+ in two actinic light regimes, we found that there was no increase in cyclic or linear electron fluxes to account for these changes. Rather, we observed a decrease in the maximum extent of P700 photo-oxidation assayed by a strong flash superimposed on continuous, background light of wavelength 723 nm, which we interpret to represent a loss of stable charge separation in PSI due to enhanced charge recombination as a result of the pre-illumination treatment. The funneling of electrons towards fewer non-damaged PSI complexes could explain the hastened post-illumination re-reduction of P700+, aided by a slight increase in a stroma-sourced electron flux after prolonged pre-illumination at 4 degrees C. Quantifying the separate fluxes to P700+ helps to elucidate the effects of chilling of cucumber leaf discs in the light and the reasons for the hastened post-illumination re-reduction of P700+.

A rapid, whole-tissue determination of the functional fraction of PSII after photoinhibition of leaves based on flash-induced P700 redox kinetics.

Assaying the number of functional PSII complexes by the oxygen yield from leaf tissue per saturating, single-turnover flash, assuming that each functional PSII evolves one oxygen molecule after four flashes, is one of the most direct methods but time-consuming. The ratio of variable to maximum Chl fluorescence yield (F(v)/F(m)) in leaves can be correlated with the oxygen yield per flash during a progressive loss of PSII activity associated with high-light stress and is rapid and non-intrusive, but suffers from being representative of chloroplasts near the measured leaf surface; consequently, the exact correlation depends on the internal leaf structure and on which leaf surface is being measured. Our results show that the average F(v)/F(m) of the adaxial and abaxial surfaces has a reasonable linear correlation with the oxygen yield per flash after varied extents of photoinactivation of PSII. However, we obtained an even better linear correlation between (1) the integrated, transient electron flow (Sigma) to P700+, the dimeric Chl cation in PSI, after superimposing a single-turnover flash on steady background far-red light and (2) the relative oxygen yield per flash. Leaves of C3 and C4 plants, woody and herbaceous species, wild-type and a Chl-b-less mutant, and monocot and dicot plants gave a single straight line, which seems to be a universal relation for predicting the relative oxygen yield per flash from Sigma. Measurement of Sigma is non-intrusive, representative of the whole leaf tissue, rapid and applicable to attached leaves; it may even be applicable in the field.

The stoichiometry of the two photosystems in higher plants revisited.

The stoichiometry of Photosystem II (PSII) to Photosystem I (PSI) reaction centres in spinach leaf segments was determined by two methods, each capable of being applied to monitor the presence of both photosystems in a given sample. One method was based on a fast electrochromic (EC) signal, which in the millisecond time scale represents a change in the delocalized electric potential difference across the thylakoid membrane resulting from charge separation in both photosystems. This method was applied to leaf segments, thus avoiding any potential artefacts associated with the isolation of thylakoid membranes. Two variations of this method, suppressing PSII activity by prior photoinactivation (in spinach and poplar leaf segments) or suppressing PSI by photo-oxidation of P700 (the chlorophyll dimer in PSI) with background far-red light (in spinach, poplar and cucumber leaf segments), each gave the separate contribution of each photosystem to the fast EC signal; the PSII/PSI stoichiometry obtained by this method was in the range 1.5-1.9 for the three plant species, and 1.5-1.8 for spinach in particular. A second method, based on electron paramagnetic resonance (EPR), gave values in a comparable range of 1.7-2.1 for spinach. A third method, which consisted of separately determining the content of functional PSII in leaf segments by the oxygen yield per single turnover-flash and that of PSI by photo-oxidation of P700 in thylakoids isolated from the corresponding leaves, gave a PSII/PSI stoichiometry (1.5-1.7) that was consistent with the above values. It is concluded that the ratio of PSII to PSI reaction centres is considerably higher than unity in typical higher plants, in contrast to a surprisingly low PSII/PSI ratio of 0.88, determined by EPR, that was reported for spinach grown in a cabinet under far-red-deficient light in Sweden [Danielsson et al. (2004) Biochim. Biophys. Acta 1608: 53-61]. We suggest that the low PSII/PSI ratio in the Swedish spinach, grown in far-red-deficient light with a lower PSII content, is not due to greater accuracy of the EPR method of measurement, as suggested by the authors, but is rather due to the growth conditions.

Quantification of cyclic electron flow around Photosystem I in spinach leaves during photosynthetic induction.

The variation of the rate of cyclic electron transport around Photosystem I (PS I) during photosynthetic induction was investigated by illuminating dark-adapted spinach leaf discs with red + far-red actinic light for a varied duration, followed by abruptly turning off the light. The post-illumination re-reduction kinetics of P700+, the oxidized form of the photoactive chlorophyll of the reaction centre of PS I (normalized to the total P700 content), was well described by the sum of three negative exponential terms. The analysis gave a light-induced total electron flux from which the linear electron flux through PS II and PS I could be subtracted, yielding a cyclic electron flux. Our results show that the cyclic electron flux was small in the very early phase of photosynthetic induction, rose to a maximum at about 30 s of illumination, and declined subsequently to <10% of the total electron flux in the steady state. Further, this cyclic electron flow, largely responsible for the fast and intermediate exponential decays, was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, suggesting an important role of redox poising of the cyclic components for optimal function. Significantly, our results demonstrate that analysis of the post-illumination re-reduction kinetics of P700+ allows the quantification of the cyclic electron flux in intact leaves by a relatively straightforward method.