Modified Cytoplasmic Ca2+ Sequestration Contributes to Spinal Cord Injury-Induced Augmentation of Nerve-Evoked Contractions in the Rat Tail Artery

In rat tail artery (RTA), spinal cord injury (SCI) increases nerve-evoked contractions and the contribution of L-type Ca²⁺ channels to these responses. In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist) mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca²⁺ entering via L-type Ca²⁺ channels is rapidly sequestered by the sarcoplasmic reticulum (SR) limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca²⁺ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca²⁺ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca²⁺ currents differed between myocytes from sham-operated (control) and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca²⁺ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker) in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca²⁺ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism.     

Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor

The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.     

Angiotensin II is bound to both receptors AT1 and AT2, parallel to the transmembrane domains and in an extended form

We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards the two principal angiotensin II (AnglI) receptors AT1 and AT2 in order to determine contact points between AngII and the two receptors. We have first identified the receptor contact points between an N- and a C-terminal residue of the AngII molecule and the AT1 receptor and constructed with this stereochemical restriction a molecular model of AT1. A similar approach with a modified procedure of photoaffinity labelling has allowed us now to determine contact points also in the AT2 receptor. Molecular modelling of AT2 on the rhodopsin scaffold and energy minimisation of AngII binding into this AT2 model produced a model strikingly similar to the AT11 structure. Superposition of the experimentally obtained contact points of AngII with AT2 upon this model revealed excellent congruence between the experimental and modelling results. Conclusions: (i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.     

The genetics of nitrogen use in hexaploid wheat: N utilisation, development and yield

A genetic study is presented for traits relating to nitrogen use in wheat. Quantitative trait loci (QTLs) were established for 21 traits relating to growth, yield and leaf nitrogen (N) assimilation during grain fill in hexaploid wheat (Triticum aestivum L.) using a mapping population from the cross Chinese Spring × SQ1. Glutamine synthetase (GS) isozymes and estimated locations of 126 genes were placed on the genetic map. QTLs for flag leaf GS activity, soluble protein, extract colour and fresh weight were found in similar regions implying shared control of leaf metabolism and leaf size. Flag leaf traits were negatively associated with days to anthesis both phenotypically and genetically, demonstrating the complex interactions of metabolism with development. One QTL cluster for GS activity co-localised with a GS2 gene mapped on chromosome 2A, and another with the mapped GSr gene on 4A. QTLs for GS activity were invariably co-localised with those for grain N, with increased activity associated with higher grain N, but with no or negative correlations with grain yield components. Peduncle N was positively correlated, and QTLs co-localised, with grain N and flag leaf N assimilatory traits, suggesting that stem N can be indicative of grain N status in wheat. A major QTL for ear number per plant was identified on chromosome 6B which was negatively co-localised with leaf fresh weight, peduncle N, grain N and grain yield. This locus is involved in processes defining the control of tiller number and consequently assimilate partitioning and deserves further examination. Electronic Supplementary Material The online version of this article () contains supplementary material, which is available to authorized users.     

Methionine proximity assay,a novel method for exploring peptide ligand-receptor interaction

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.     

Reduction in phosphoribulokinase activity by antisense RNA in transgenic tobacco: effect on CO2 assimilation and growth in low irradiance

To quantify the importance of the Calvin cycle enzyme phosphoribulokinase (PRK) in photosynthesis and to perturb photosynthesis without large direct reductions in leaf protein content, tobacco plants (Nicotiana tabacum L.) were transformed with an inverted cDNA encoding tobacco PRK. A population of plants expressing antisense RNA and a range of PRK activities from wild-type to less than 5% of wild-type were obtained. CO₂ assimilation under the growing conditions (330 µmol photons m^?2 sec^?1, 350 µbar CO₂, 25°C) was not inhibited until more than 85% of PRK activity had been removed. With reduction in PRK activity of between 85 and 95%, assimilation rates and amounts of chlorophyll compared with wild-type were reduced by up to half. Decreased absorption of light by leaves with less chlorophyll accounte0d for only a small part of the reduction in assimilation rate. When PRK activity was below 15% of wild-type, amounts of ribulose-5-phosphate, ribose-5-phosphate, ATP and fructose-6-phosphate were 1.5- to fivefold higher and levels of ribulose-1,5-bisphosphate, 3-phosphoglyceric acid and ADP 1.5- to fourfold lower than in wild-type. It is estimated that these changes maintained flux through PRK to realise the assimilation rates observed. A possible shift of control within the Calvin cycle towards fructose-1,6-bisphosphatase in plants with low PRK is discussed. Amounts of hexoses and starch in particular were reduced in plants expressing the lowest PRK activities; amounts of sucrose were little affected. Lower CO₂ assimilation in plants with low PRK activity correlated with reduced relative growth rate of shoots and delayed flowering, but there was no effect on specific leaf area. It is concluded that (i) in wild-type plants grown in constant low light, PRK has a flux-control coefficient for CO₂ assimilation of zero, and that even when amounts of PRK are reduced 20-fold relative to wild-type, altered amounts of metabolites compensate for much of the reduction in PRK protein; (ii) in plants where there is a 95% reduction in amounts of PRK, photosynthesis was reduced twofold without large changes in leaf protein content or leaf geometry.     

Conceptual Evaluation of Factors Potentially Affecting Restoration of Habitat Structure within the Channelized Kissimmee River Ecosystem

A critical element of the ongoing effort to restore the ecological integrity of Florida's Kissimmee River ecosystem is the reestablishment of pre-channelization habitat structure and function. Restoration of habitat will form the basis for responses by most biological components of the ecosystem and will provide a key indicator of the success of the restoration effort. This paper evaluates the relative importance of a range of abiotic and biotic habitat parameters in the existing and historic Kissimmee River ecosystem and provides a conceptual framework for predicting expected spatial and temporal responses of river and floodplain habitats to the restoration project. Among the ecological factors and process that influenced the development, dynamics, and maintenance of river and floodplain habitat structure, hydrology is expected to be of central importance in eliciting restoration responses in the Kissimmee River Ecosystem. Based on the assumption that the restoration plan will reestablish historic hydrologic characteristics, predictions are made of expected responses by geomorphic and vegetative components of the Kissimmee River's habitat structure. Recommendations are made regarding key habitat parameters requiring long term tracking and analysis and utilization of a geographic information system(GIS). A hierarchical habitat classification scheme is provided as a foundation for all components of the restoration evaluation program.