The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.     

Carbon dioxide increase: the implications at the ecosystem level*

Abstract Possible effects of increased atmospheric concentrations of CO₂ on forest ecosystems are discussed and as an example a simulation case study using a set of mixed-age and mixed-species forest stand models is presented. The responses of the models to a simple scenario (uniform growth increase of all trees as a response to CO₂ enrichment) include increases in biomass that are considerably less than the increases in growth rate of the trees. These simulations and more general discussion of the possible effects of increased photosynthetic production identify the problem of scaling-up small time-scale and space-scale measurements of plant responses to CO₂ enrichment to the ecosystem level.     

The Fine Structure of the Scopula-Stalk Region of Vorticella convallaria

ABSTRACT. We examined by SEM and TEM the stalk-scopular junction, the stalk, and stalk formation in Vorticella convallaria Linnaeus, 1767. The stalk sheath is anchored to the walls of the scopular lip and to the scopular cilia by thin fibrils. Experimental extraction of these fibrils weakens this junction enough to separate the stalk from the cell body. Telotrochs escape from the stalk by means of violent contractions of the cell body, accelerated beating of the trochal band cilia, and twisting of the cell body against the stalk. The edges of the scopular lip spread over the scopular cilia after escape and, in some cases, fuse to enclose the entire, aboral scopular surface in a cupola-like structure. The sessile cells contain fewer and smaller scopular granules than telotrochs. The presence of disintegrating scopular granules in the stalk matrix of some sessile cells suggests that they contain material which is secreted over a period of time to form the stalk. Eruptive formation of the initial adhesive pad and quick elongation of the distal part of the stalk suggests a rapid exocytosis of the larger, more numerous granules of the telotroch. The stalk sheath is formed of fibrils making up complete and incomplete compartments peripherally arranged along the major stalk axis.     

Kinetics of Root Elongation of Maize in Response to Short-Term Exposure to NaCl and Elevated Calcium Concentration

To study the effect of salt (NaCl) on root elongation we developeda device that measures this effect by means of a Linear VariableDifferential Transformer (LVDT). To test the efficacy of thedevice we performed experiments demonstrating that (a) ratesof elongation of primary maize (Zea mays L.) roots were comparableto elongation rates of primary roots growing freely in solutionculture; and (b) chilling and low O₂ concentrations of the solutionelicited the expected responses. Inhibition of root elongation by 75 mol m^–3 NaCl was gradual.At an iso-osmotic concentration, mannitol did not inhibit rootgrowth, suggesting that the inhibition was not due to osmoticfactors but rather to effects of salt on metabolism. The additionof supplemental Ca (10 mol m^–3) ameliorated this stressfulcondition. Timing of the application of Ca was critical. Treatmentwith Ca after addition of NaCl only partially restored growth,but pretreatment with Ca completely prevented the inhibitionof growth by salt stress. Key words: Root growth, Zea mays L., salinity     

Nuclear magnetic resonance characterization of metabolite disorder in orange trees caused by citrus sudden death disease

Citrus sudden death (CSD) is a new disease of sweet orange and mandarin trees grafted on Rangpur lime and Citrus volkameriana rootstocks. It was first seen in Brazil in 1999, and has since been detected in more than four million trees. The CSD causal agent is unknown and the current hypothesis involves a virus similar to Citrus tristeza virus or a new virus named Citrus sudden death-associated virus . CSD symptoms include generalized foliar discoloration, defoliation and root death, and, in most cases, it can cause tree death. One of the unique characteristics of CSD disease is the presence of a yellow stain in the rootstock bark near the bud union. This region also undergoes profound anatomical changes. In this study, we analyse the metabolic disorder caused by CSD in the bark of sweet orange grafted on Rangpur lime by nuclear magnetic resonance (NMR) spectroscopy and imaging. The imaging results show the presence of a large amount of non-functional phloem in the rootstock bark of affected plants. The spectroscopic analysis shows a high content of triacylglyceride and sucrose, which may be related to phloem blockage close to the bud union. We also propose that, without knowing the causal CSD agent, the determination of oil content in rootstock bark by low-resolution NMR can be used as a complementary method for CSD diagnosis, screening about 300 samples per hour.     

REFOCUSING THE RESPONSIVENESS REQUIREMENT

Many guidelines for international research require that studies be responsive to host community health needs or health priorities. Although responsiveness possesses great intuitive and rhetorical appeal, existing conceptions are confusing and difficult to apply. Not only are there few examples of what research the responsiveness requirement permits and what it rejects, but its application can lead to contradictory results. Because of the practical difficulties in applying responsiveness and the danger that misapplying responsiveness could harm the interests of developing countries, we argue that responsiveness should be refocused in three ways: in terms of (1) who enforces it, (2) under what standard, and (3) in what cases. We conclude that responsiveness should be applied by host country officials at the policy level with the exercise of judgment when externally funded research threatens to displace scarce local resources.     

Content and De Novo Synthesis of Cocaine in Embryos and Endosperms from Fruit of Erythroxylum coca Lam

There is controversy as to whether the mature fruit of Erythroxylumcoca var. coca Lam. contains the cocaine alkaloid (benzoylmethylecgonine).In the present study, cocaine was monitored to determine ifit was present in embryos and endosperms of mature fruit ofE. coca var. coca Lam., and if present, the time required forde novo synthesis in imbibing seed. Seeds from mature fruitof E. coca were dissected to separate the embryos from the endosperms.The separated embryos and endosperms were analysed for cocaine.Subsequently, endosperms and embryos from seed imbibed. undera light and dark treatment were separated on days 3, 6, 9, 12and 15 and analysed for cocaine. Cocaine was present in embryos(0.005% of d. wt) and endosperms (0–001% of d. wt) ofmature fruit of E. coca. De novo synthesis of cocaine occurredonly in embryos of seed imbibed under light after day 9 of imbibition. Erythroxylum coca, alkaloid, benzoylmethylecgonine, cocaine, embryo, endosperm, seed imbibition     

A review of the genus Semele (Ruscaceae) systematics in Madeira

The present study attempts to review the systematics of Semele (Ruscaceae) in Madeira, based on phenotypic diversity. The variation in some vegetative (climbing shoot, second-order branches or 'phylloclades') and sexual (inflorescence and flowers) characters was analysed in 115 plant specimens from 30 field populations, herbaria of the Costa collection and Madeira Botanical Garden (MADJ) and certain gardens. Thirty-one quantitative and qualitative characters have been utilized in the analysis. Kaiser–Meyer–Olkin (KMO) analysis indicates adequate sampling. Principal component analysis (PCA) reveals that the spatial distribution of individuals has a discontinuous behaviour. Principal coordinate analysis (PCO) utilizing the Gower coefficient on standardized data revealed a significantly discontinuous distribution of individuals, such that two different clusters can be defined. The Student's t -test and Tukey test on separate characters, when individuals were classified according to the Costa classification, confirms the significant differences between grouping accessions. This leads to the recognition of two species within the genus in Madeira. Literature and herbarium studies show that these two taxa are conspecific with Semele androgyna (L.) Kunth sensu stricto (s.s.) and Semele menezesi Costa sensu lato (s.l.) . A separated statistical analysis of the S. androgyna cluster shows the possible existence of additional subgroups. Based on field population distribution, ecological behaviour and variation in features, we propose the recognition of two species, S. androgyna (L.) Kunth and S. menezesi (Costa) Pinheiro de Carvalho, and two subspecies S. androgyna (L.) Kunth androgyna Pinheiro de Carvalho and S. androgyna (L.) Kunth pterygophora Pinheiro de Carvalho.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 483–497.     

Synthesis of an Agonistic,Difluoro-Azido Photolabel of Angiotensin II and Labeling of the AT1 Receptor: Transmembrane Domains 3, 6, and 7 Form the Ligand-Binding Pocket

p-Azido-phenylalanine has been frequently used for photoaffinity labeling of target proteins such as the angiotensin receptors. However, chemical studies showed that simple aryl nitrenes first react intramolecularly, forming a semistable cyclic keteneimine and then reacting with nucleophile residues in the target structure like those of lysine and arginine. We synthesized 3,5-difluoro-4-azidophenylalanine where the formation of the keteneimine is prevented and where photoincorporation should be due to nonselective nitrene insertion only. This new amino acid was introduced in position 8 of angiotensin II and compared with the corresponding azidophenylalanine peptide using human AT₁ receptor as target. The new photolabel maintained full agonist activity and a similar yield of photolabeling but without the previously observed gradual hydrolysis. Several selective proteolyses of the labeled receptor indicate that the new photolabel forms three simultaneous contact regions on the hAT₁ receptor, suggestive of a nonselective behavior of the photolabel. A major contact was established in the sixth transmembrane domain but also in the third and seventh domain. Our results are in excellent agreement with those recently obtained from methionine proximity assay studies.