Herbicide-binding Sites Revealed in the Structure of Plant Acetohydroxyacid Synthase

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 angstrom resolution) and with the imidazolinone, imazaquin (IQ; 2.8 angstrom). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 angstrom of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 angstrom from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG(4) monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 mu g 10(6) cells(-1) mL(-1) at a PEI nitrogen: DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from similar to 13 to similar to 39 mg L-1. Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended activated hypothermic synthesis.

Diurnal polyp expansion behavior in stony corals may enhance carbon availability for symbionts photosynthesis

Photosynthesis of zooxanthellate stony corals may be limited by inorganic carbon at high irradiances. We demonstrated that oxygen consumption of expanded corals is higher than that of contracted corals in both night-expanding and day-expanding corals. It is assumed that at the single-polyp level, the expansion of tentacles increases the surface area for solute exchange with the surrounding water, which may alleviate potential carbon limitation and excess oxygen levels in the tissue under high irradiance. We investigated this hypothesis using stable carbon isotope (613 C) analysis of coral species from the Red Sea exhibiting different morphologies. delta C-13 ratios in zooxanthellae of branched coral colonies with small polyp size that extend their tentacles during daytime (diurnal morphs) showed lower delta C-13 values in their zooxanthellae - 13.83 +/- 1.45 parts per thousand, compared to corals from the same depth with large polyps, which are usually massive and expand their tentacles only at night (nocturnal morphs). Their algae delta C-13 was significantly higher, averaging - 11.33 +/- 0.59 parts per thousand. Carbon isotope budget of the coral tissue suggests that branched corals are more autotrophic, i.e., that they depend on their symbionts for nutrition compared to massive species, which are more heterotrophic and depend on plankton predation. (c) 2005 Elsevier B.V. All rights reserved.

Meganema perideroedes gen. nov., sp nov., a filamentous alphaproteobacterium from activated sludge

An industrial wastewater treatment plant at Grindsted, Denmark, has suffered from bulking problems for several years caused by filamentous bacteria. Five strains were isolated from the sludge by micromanipulation, Phylogenetic analysis of the 16S rRNA gene sequences showed that the strains formed a monophyletic cluster in the Alphaproteobacteria, and they were phenotypically different from their closest relatives and from all hitherto known filamentous bacteria described (closest relative Brevundimonas vesicularis ATCC 11426(T), 89(.)8% sequence similarity). In pure culture, the cells (1(.)5-2(.)0 mu m) in filaments are Gram-negative and contain polyphosphate and polyhydroxyalkanoates. The optimum temperature for growth is 30 degrees C and the strains grow in 2 % NaCl and are oxidase- and catalase-positive. Ubiquinone 10 is the major quinone. The major fatty acid (C-18: 1 omega 7c) and smaller amounts of unsaturated fatty acids, 3-hydroxy fatty acids with a chain length of 16 and 18 carbon atoms and small amounts of 10-methyl-branched fatty acids with 18 carbon atoms (C-19: 0 10-methyl) affiliated the strains with the Methylobacterium/Xanthobacter group in the Alphaproteobacteria. The G + C content of the DNA is 42(.)9 mol% (for strain Gr1(T)). The two most dissimilar isolates by 16S rRNA gene comparison (Gr1(T) and Gr10; 97(.)7 % identical) showed 71(.)5 % DNA-DNA relatedness. Oligonucleotide probes specific for the pure cultures were designed for fluorescence in situ hybridization and demonstrated that two filamentous morphotypes were present in the Grindsted wastewater treatment plant. It is proposed that the isolates represent a new genus and species, Meganema perideroedes gen. nov., sp. nov. The type strain of Meganema perideroedes is strain Gr1(T) (=DSM 15528(T) =ATCC BAA-740(T)).

Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP

Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic sub unit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs front that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required mgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator.

Specializations of the granular prefrontal cortex of primates: Implications for cognitive processing

The biological underpinnings of human intelligence remain enigmatic. There remains the greatest confusion and controversy regarding mechanisms that enable humans to conceptualize, plan, and prioritize, and why they are set apart from other animals in their cognitive abilities. Here we demonstrate that the basic neuronal building block of the cerebral cortex, the pyramidal cell, is characterized by marked differences in structure among primate species. Moreover, comparison of the complexity of neuron structure with the size of the cortical area/region in which the cells are located revealed that trends in the granular prefrontal cortex (gPFC) were dramatically different to those in visual cortex. More specifically, pyramidal cells in the gPFC of humans had a disproportionately high number of spines. As neuron structure determines both its biophysical properties and connectivity, differences in the complexity in dendritic structure observed here endow neurons with different computational abilities. Furthermore, cortical circuits composed of neurons with distinguishable morphologies will likely be characterized by different functional capabilities. We propose that 1. circuitry in V1, V2, and gPFC within any given species differs in its functional capabilities and 2. there are dramatic differences in the functional capabilities of gPFC circuitry in different species, which are central to the different cognitive styles of primates. In particular, the highly branched, spinous neurons in the human gPFC may be a key component of human intelligence. (C) 2005 Wiley-Liss, Inc.

Visualisation of macropinosome maturation by the recruitment of sorting nexins

We report that phosphoinositol-binding sorting nexin 5 ( SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome; these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.

A windows program for the derivation of steady-state equations in enzyme systems

Despite the number of computer-assisted methods described for the derivation of steady-state equations of enzyme systems, most of them are focused on strict steady-state conditions or are not able to solve complex reaction mechanisms. Moreover, many of them are based on computer programs that are either not readily available or have limitations. We present here a computer program called WinStes, which derives equations for both strict steady-state systems and those with the assumption of rapid equilibrium, for branched or unbranched mechanisms, containing both reversible and irreversible conversion steps. It solves reaction mechanisms involving up to 255 enzyme species, connected by up to 255 conversion steps. The program provides all the advantages of the Windows programs, such as a user-friendly graphical interface, and has a short computation time. WinStes is available free of charge on request from the authors. (c) 2006 Elsevier Inc. All rights reserved.

Solution properties of star and linear poly(N-isopropylacrylamide)

The LCST transitions of novel N-isopropylacrylamide ( NIPAM) star polymers, prepared using the four-armed RAFT agent pentaerythritoltetrakis(3-(S-benzyltrithiocarbonyl) propionate) (PTBTP) and their hydrolyzed linear arms were studied using H-1 NMR, PFG-NMR, and DLS. The aim was to determine the effect of polymer architecture and the presence of end groups derived from RAFT agents on the LCST. The LCST transitions of star PNIPAM were significantly depressed by the presence of the hydrophobic star core and possibly the benzyl end groups. The effect was molecular weight dependent and diminished once the number of repeating units per arm >= 70. The linear PNIPAM exhibited an LCST of 35 degrees C, regardless of molecular weight; the presence of both hydrophilic and hydrophobic end groups after hydrolysis from the star core was suggested to cancel effects on the LCST. A significant decrease in R-H was observed below the LCST for star and linear PNIPAM and was attributed to the formation of n-clusters. Application of a scaling law to the linear PNIPAM data indicated the cluster size n = 6. Tethering to the hydrophobic star core appeared to inhibit n-cluster formation in the lowest molecular weight stars; this may be due to enhanced stretching of the polymer chains, or the presence of larger numbers of n-clusters at temperatures below those measured.

Growth of crystals in optical tweezers

We report here on the use of optical tweezers in the growth and manipulation of protein and inorganic crystals. Sodium chloride and hen egg-white lysozyme crystals were grown in a batch process, and then seeds from the solution were introduced into the optical tweezers. The regular and controllable shape and the known optical birefringence in these structures allowed a detailed study of the orientation effects in the beam due to both polarization and gradient forces. Additionally, we determined that the laser tweezers could be used to suspend a crystal for three-dimensional growth under varying conditions. Studies included increasing the protein concentration, thermal cycling, and a diffusion-induced increase in precipitant concentration. Preliminary studies on the use of the tweezers to create a localized seed for growth from polyethylene oxide solutions are also reported.

The motor neurite terminal determines where neuromuscular synapses form in the absence of agrin

Agrin is a proteoglycan secreted by motor neurite terminals that functions to initiate and maintain AChR clusters at the nerve terminal. This led to the theory that neurite terminals decide where neuromuscular synapses form by secreting agrin. However, initiation of AChR clustering occurs in the absence of the innervating motoneuron and in the absence of agrin. In this instance, the muscle, not the nerve, is deciding the location of neuromuscular synapses by drawing neurite terminals towards pre-existing AChR clusters. If this were true, one would expect the initial innervation patterns to be the same in agrin-deficient mice and wild-type mice. To test this we quantified the intramuscular axonal branching and synapse formation in the diaphragm at E14.5 in agrin-deficient mice and wild-type mice. Heterozygote mothers were anaesthetised with Nembutal (30 mg) and killed via cervical dislocation. In the diaphragm, the nerve trunk runs down the centre of the muscle and extends branches primarily toward the lateral side. In agrin-deficient mice however, we found significantly more branches exited the phrenic nerve trunk, branched in the periphery and extended further on the medial side. Moreover, we found that the percentage α-bungarotoxin/synaptophysin colocalisations, markers of pre- and postsynaptic differentiation, respectively, was the same in agrin-deficient mice and wild-type mice. These results show that initial innervation patterns are not the same in agrin-deficient mice and wild-type mice indicating neurite terminals, not muscle, decide the placement of neuromuscular synapses in the absence of agrin.