Optical third harmonic generation study of the hydration of DNA films.

Optical third harmonic generation (THG) has been observed for the first time from DNA films. The THG signal is observed from NaDNA films exposed to relative humidities (RHs) between 0% and 98%. A strong enhancement (approximately 5x) of the THG signal from NaDNA is observed at 84% RH; no enhancement is observed for RbDNA. The most likely mechanism for such an enhancement is an increased coherence length. A model calculation using estimates of the refractive indices at both the fundamental and third harmonic frequencies supports this interpretation. The observed THG signal has the same polarization as the incident (fundamental) light. For the A conformation, the THG signal polarized perpendicular to the helical axis is approximately twice as strong as the signal polarized parallel to the helical axis. No such anisotropy is observed for either the disordered conformation (below about 50% RH) or the B conformation (above 92% RH).     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes

Hydroxyl group-containing fatty acids play an important role in anti-inflammatory action, neuroprotection, bactericide and anti-cancer defense. However, the mechanism of long-chain hydroxy fatty acids (HFA) transport across plasma membranes is still disputed. Two main hypotheses have been suggested: firstly, that protonated HFAs traverse across the membranes spontaneously and, secondly, that the transport is facilitated by proteinaceous carriers. Here, we demonstrate that the protonated HFA are able to move across planar lipid bilayers without protein assistance. This transport step is accompanied by the acidification of the buffer in receiving compartment and the pH augmentation in the donating compartment. The latter contained liposomes doped with HFA. As revealed by scanning pH-sensitive microelectrodes, the pH shift occurred only in the immediate vicinity of the membrane, while bulk pH remained unchanged. In concurrence with the theoretical model of weak acid transport, the pH value at maximum proton flux was almost equal to the pK of the studied HFA. Intrinsic pK_i values were calculated from the electrophoretic mobilities of HFA-containing liposomes and were 5.4, 6.5, 6.9 and 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, 12-hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic acids, respectively.     

Improved photobiological H2 production in engineered green algal cells

Oxygenic photosynthetic organisms use solar energy to split water (H2O) into protons (H+), electrons (e-), and oxygen. A select group of photosynthetic microorganisms, including the green alga Chlamydomonas reinhardtii, has evolved the additional ability to redirect the derived H+ and e- to drive hydrogen (H2) production via the chloroplast hydrogenases HydA1 and A2 (H2 ase). This process occurs under anaerobic conditions and provides a biological basis for solar-driven H2 production. However, its relatively poor yield is a major limitation for the economic viability of this process. To improve H2 production in Chlamydomonas, we have developed a new approach to increase H+ and e- supply to the hydrogenases. In a first step, mutants blocked in the state 1 transition were selected. These mutants are inhibited in cyclic e- transfer around photosystem I, eliminating possible competition for e- with H2ase. Selected strains were further screened for increased H2 production rates, leading to the isolation of Stm6. This strain has a modified respiratory metabolism, providing it with two additional important properties as follows: large starch reserves (i.e. enhanced substrate availability), and a low dissolved O2 concentration (40% of the wild type (WT)), resulting in reduced inhibition of H2ase activation. The H2 production rates of Stm6 were 5-13 times that of the control WT strain over a range of conditions (light intensity, culture time, +/- uncoupler). Typically, approximately 540 ml of H2 liter(-1) culture (up to 98% pure) were produced over a 10-14-day period at a maximal rate of 4 ml h(-1) (efficiency = approximately 5 times the WT). Stm6 therefore represents an important step toward the development of future solar-powered H2 production systems.     

Protein domains required for formation of stable monomeric Lhca1- and Lhca4-complexes

The peripheral light-harvesting complex of Photosystem I consists of two subpopulations, LHC I-680 and LHC I-730. The latter is composed of the two apoproteins Lhca1 and Lhca4. Recently, reconstitution of monomeric LHC I using bacterially overexpressed Lhca1 or Lhca4 was achieved. In order to obtain insight into the structure requirements for formation of monomeric light-harvesting complexes, we produced a series of N- and C-terminal deletion mutants and used the overexpressed proteins for reconstitution experiments. We found the entire extrinsic N-terminal region dispensable for monomer formation in Lhca1 and Lhca4. Also at the C-terminus, both subunits revealed similarity since all amino acids up to the end of the fourth helix could be removed without abolishing monomer formation. In connection with former corresponding results for Lhcb1, the dispensability of these regions appears to be a general feature in LHC-formation. In LHC I, however, a stabilising effect can be ascribed to these regions since the yield of complexes was decreased. In the majority of the mutant LHC I versions no effect on pigment binding was detected. However, in the LHC with the most extensively N-terminally truncated mutant of Lhca4 a dramatic shift in the 77 K fluorescence emission to shorter wavelengths was observed. This suggests that chlorophylls involved in long wavelength fluorescence emission are located in the chlorophyll array located towards the stromal face of the thylakoid membrane assuming a pigment arrangement corresponding to that in LHC II and CP29. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphology of enkephalin-immunoreactive myenteric neurons in the human gut

The aim of this study was the morphological and further chemical characterisation of neurons immunoreactive for leu-enkephalin (leuENK). Ten wholemounts of small and large intestinal segments from nine patients were immunohistochemically triple-stained for leuENK/neurofilament 200 (NF)/substance P (SP). Based on their simultaneous NF-reactivity and 3D reconstruction of single NF-reactive cells, 97.5% of leuENK-positive neurons displayed the appearance of stubby neurons: small somata; short, stubby dendrites and one axon. Of these leuENK-reactive stubby neurons, 91.3% did not display co-reactivity for SP whereas 8.7% were SP-co-reactive. As to their axonal projection pattern, 50.4% of the recorded leuENK stubby neurons had axons running orally whereas in 29.4% they ran anally; the directions of the remaining 20.2% could not be determined. No axons were seen to enter into secondary strands of the myenteric plexus. Somal area measurements revealed clearly smaller somata of leuENK-reactive stubby neurons (between 259±47 m² and 487±113 m²) than those of putative sensory type II neurons (between 700±217 m² and 1,164±396 m²). The ratio dendritic field area per somal area of leuENK-reactive stubby neurons was between 2.0 and 2.8 reflecting their short dendrites. Additionally, we estimated the proportion of leuENK-positive neurons in comparison to the putative whole myenteric neuron population in four leuENK/anti-Hu doublestained wholemounts. This proportion ranged between 5.9% and 8.3%. We suggest leuENK-reactive stubby neurons to be muscle motor neurons and/or ascending interneurons. Furthermore, we explain why we do not use the term Dogiel type I neurons for this population.     

Potent Kv1.3 inhibitors from correolide-modification of the C18 position

Kv1.3, the voltage-gated potassium channel in human T cells, represents a new target for treating immunosuppression and autoimmune diseases. Correolide (1), a pentacyclic natural product, is a potent and selective Kv1.3 channel blocker. Simplification of correolide via removal of its E-ring generates enone 4, whose modification produced a new series of tetracyclic Kv1.3 blockers. The structure-activity relationship for this class of compounds in two functional assays, Rb_Kv and human T cell proliferation, is presented herein. The most potent analog 43 is 15-fold more potent than correolide as inhibitor of human T cell proliferation.     

A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration

Sodium dodecyl sulfate (SDS) is used to denature and solubilize proteins, especially membrane and other hydrophobic proteins. A quantitative method to determine the concentration of SDS using the dye Stains-All is known. However, this method lacks the accuracy and reproducibility necessary for use with protein solutions where SDS concentration is a critical factor, so we modified this method after examining multiple parameters (solvent, pH, buffers, and light exposure). The improved method is simple to implement, robust, accurate, and (most important) precise.