Are Pesticides Used to Control Thrips Harmonious with Soil-Dwelling Predatory Mite Cosmolaelaps sabelis (Mesostigmata: Laelapidae)?

Edaphic predatory mites could be introduced in pest management programs of pests that live, or spend part of their life cycle, in the soil. Some mesostigmatic mites have been widely used for the management of different species of thrips (Thysanoptera), especially in protected cultivation. The edaphic predator Cosmolaelaps sabelis (Mesostigmata: Laelapidae) was a model species in this study, being exposed to the most applied insecticides for the control of thrips in Brazil. After lethal, sublethal and transgenerational effects were evaluated. The pesticides acephate, acetamiprid + etofenprox, azadirachtin, spinetoram, formetanate hydrochloride, and imidacloprid were classified according to the IOBC/WPRS (International Organization for Biological Control-West Paleartic Regional Section) recommendation, considering the acute toxicity and the effects on adult females' reproduction, in the maternal and first generation. The pesticides acetamiprid + etofenprox and azadirachtin were classified as slightly harmful (Class 2), while spinetoram was classified as moderately harmful (Class 3). Acephate and formetanate hydrochloride were classified as harmful (Class 4). Only imidacloprid didn't cause negative effects on the females. Regarding effects on the first generation, acetamiprid + etofenprox, azadirachtin, and spinetoram caused reduction in the oviposition rates. Therefore, we suggest that complimentary bioassays should be done under semi-field and field conditions using the pesticides that were considered harmful in this study, to assess their effects on this predator in other environments prior to recommending not to use them in integrated programs to manage soil-based pests using chemical and biological tools.

Rendimento do coentro (Coriandrum sativum L) adubado com esterco bovino em diferentes doses e tempos de incorporação no solo / Yield of the coriander [Coriandrum sativum (L)] fertilized with manure at different doses and incorporation times in the soil

RESUMO O uso de esterco bovino é de suma importância para os agricultores familiares que produzem coentro na região de Mossoró-RN, pois esse insumo é amplamente disponível e utilizado pelos agricultores. Objetivando-se avaliar o Rendimento do coentro fertilizado com esterco bovino em diferentes doses e tempos de incorporação ao solo foi conduzido um experimento no período de setembro a novembro de 2011, na Fazenda Experimental Rafael Fernandes, da Universidade Federal Rural do Semi-Árido - UFERSA, Brasil. Os tratamentos consistiram da combinação de quatro doses de esterco bovino incorporadas ao solo: 15,0; 30,0; 45,0 e 60,0 t ha-1 em base seca, com quatro tempos de incorporação: 28; 49; 64 e 80 dias antes da semeadura do coentro - DAS, mais um tratamento controle (ausência de adubação). O delineamento experimental utilizado foi em blocos completos casualizados com os tratamentos arranjados em esquema fatorial 4 x 4 + 1 com 3 repetições. A cultivar de coentro utilizado foi a Verdão e as variáveis determinadas foram altura e número de hastes por planta e rendimento de coentro. O coentro respondeu à aplicação de esterco bovino, produzindo rendimentos máximos de 6453 e 6349 kg ha-1 de massa verde, com a dose de 60 t ha-1 e aos quarenta e seis dias de incorporação antes da semeadura, respectivamente.

ABSTRACT The use of manure is very important to family farmers who produce coriander in the region of Mossoró-RN, because this input is widely available and used by farmers. Aiming to evaluate the yield of the coriander fertilized with manure at different doses and incorporation times in the soil, a trial was conducted from September to November 2011, at the Experimental Farm Rafael Fernandes in the Universidade Federal Rural do Semi-Árido (UFERSA), Mossoró-RN, Brazil. The treatments consisted on the combination of four levels of manure incorporated into the soil: 15.0; 30.0; 45.0 and 60.0 t ha-1 at dry basis, with four incorporation times: 28; 49; 64 and 80 days before the coriander"s sowing - DAS, plus a control treatment (without fertilization). The experimental design was a randomized complete block with treatments arranged in a 4 x 4 + 1, with three replications. The coriander planted was the "Verdão" and the specific variables were height and number of stalks per plant and yield of the coriander. The coriander responded to the application of the manure, producing maximum yields of 6453 and 6349 kg ha-1 of fresh mass, with the dose of 60 t ha-1 and at forty-six days of incorporation before the sowing, respectively.

Body mass index in the first year after kidney transplantation.

INTRODUCTION: Kidney transplant recipients (KTR) experience better appetite, partly due to the use of steroids, and are subjected to less severe dietetic restrictions, hence they tend to increase the uptake of calories, which favors weight gain posttransplantation. In this study, we evaluate the profile of body mass index (BMI) in the first year posttransplantation. METHODS: This was a retrospective study including 131 patients who received transplants between 1991 and 2011. We collected demographic and clinical data such as body weight and height, and calculated BMI pretransplantation and at 6 and 12 months posttransplantation. RESULTS: Mean age was 47.1 ± 13.1 years, 64.9% were male, and 29% of patients were diabetic. Pretransplantation mean BMI was 23.04 ± 4.08 kg/m(2), and at 6 and 12 months posttransplantation it increased to 24.55 ± 4.2 kg/m(2) and 24.65 ± 4.16 kg/m(2), respectively (P < .001). At 6 months, this significant weight gain occurred in all patients, even those malnourished, eutrophic, overweight, and obese at pretransplantation. Looking at pretransplantation malnourished patients, 30.8% remained malnourished 1 year after transplantation. Otherwise, 28.6% of pretransplantation overweight patients and 100% of pretransplantation obese patients could be classified as obese at 1 year posttransplantation. CONCLUSIONS: Increase in BMI is common in obese and nonobese KTR. This study highlights the importance of identifying subjects at risk for excessive weight gain posttransplantation, thus allowing an early nutritional intervention to prevent its complications.

Qualidade fisiológica de sementes de coentro [Coriandrum sativum (L. )] / Physiologic quality of cilantro seeds [Coriandrum sativum (L. )]

A qualidade das sementes esta relacionada de forma direta ao seu potencial fisiológico, representado pela germinação e/ou vigor, expressando sua capacidade de originar plântulas normais. O objetivo do estudo foi a análise dos efeitos da temperatura e do tempo de exposição ao teste de envelhecimento acelerado sobre os parâmetros: germinação, germinação na primeira contagem, emergência, Índice de velocidade de emergência, comprimento de parte aérea e massa seca. O ensaio foi conduzido no delineamento inteiramente casualizado, com quatro repetições. As sementes foram submetidas à dois métodos de envelhecimento acelerado: o tradicional com água destilada e outro com solução saturada de 40 g NaCl 100 mL-1 de água, em duas temperaturas (38 ºC e 41 ºC) e em três períodos de exposição (48, 72 e 96 horas). Melhor se pode verificar o potencial fisiológico das sementes de coentro, quando submetido às condições estressantes: na temperatura de 41 °C com o período de exposição de 96 h (Teste envelhecimento acelerado tradicional) e na temperatura de 41 ºC com período de exposição de 48 h (com solução saturada). Os testes de germinação e primeira contagem evidenciaram diferenças na qualidade fisiológica inicial das sementes em função dos tratamentos aplicados.

The quality quality of the seeds this related of direct form to it physiologic potential, acted by the germination and/or energy, expressing it capacity to originate normal plantules. The objective of the study was the analysis of the effects of the temperature and of the time of exhibition to the test of accelerated aging on the parameters: germination, germination in the first counting, emergency, Index of emergency speed, length of aerial part and mass dries. The rehearsal was driven in the delineamento entirely casualizado, with four repetitions. The seeds were submitted to two methods of accelerated aging: the traditional with distilled water and other with saturated solution of 40 g NaCl 100 mL-1 of water, in two temperatures (38 ºC and 41 ºC) and in three exhibition periods (48, 72 and 96 hours). Better the physiologic potential of the cilantro seeds can be verified, when submitted to the stressful conditions: in the temperature of 41 °C with the period of exhibition of 96 h (it Tests traditional aging) and in the temperature of 41 °C with period of exhibition of 48 h (with saturated solution). The germination tests and first counting evidenced differences in the quality physiologic initial of the seeds in function of the applied treatments.

Eficácia dos coadjuvantes de higiene bucal utilizados na alimentação de cães / Effectiveness of buccal hygiene coadjutant used in the dogs feeding

Avaliou-se a utilização de coadjuvantes na diminuição da placa bacteriana e formação do cálculo dentário em 16 cães. O delineamento experimental foi casualizado com quatro tratamentos (T) e quatro repetições. Os tratamentos foram: T1- controle, T2- coadjuvante com ação mecânica, T3- coadjuvante com tripolifosfato de sódio e T4- coadjuvante com hexametafosfato de sódio. Após sete dias de adaptação, no dia anterior ao início dos tratamentos, os animais foram submetidos à remoção de cálculo dentário. O experimento teve duração de 21 dias e ao final realizaram-se as medições das placas bacterianas formadas com o uso de marcadores (fucsina). O coadjuvante somente com ação mecânica não foi efetivo em retardar o aparecimento da placa bacteriana. Os coadjuvantes com polifosfatos apresentaram uma ação efetiva e significativa na diminuição da formação do calculo dentário

The use of coadjutants in the decrease of the bacterial plate and formation of the dental calculus was evaluated in 16 dogs. The experimental design was randomized with four treatments (T) and four repetitions. The treatments were the following: 1- control; 2- coadjutant 1 (one) with mechanical action; 3- coadjutant 2 (two) with tripolyphosphate of sodium and 4- coadjutant 3 (three) with hexametaphosphate of sodium. After seven days of adaptation, on the day previous to the beginning of the treatments, the animals were submitted to the removal of dental calculus and after twenty-one days of treatment a measurement of the bacterial plaque formed through the use of markers (fucsin). The coadjutant only with mechanical action was not effective in delaying the appearance of the bacterial plaque. The coadjutant containing polyphosphates presented an effective and significant action decreasing the formation of the dental calculus

[Pulmonary function in Portuguese firefighters]. / Função respiratória em bombeiros portugueses.

INTRODUCTION: Portugal has had a high rate of forest fires in recent years. Inhaled wood smoke can have short- and long-term effects on the lung function of people exposed to it. STUDY OBJECTIVES: To assess the lung function of active wildland (forest) firefighters. METHODS: Cross-sectional study. A self-questionnaire on personal and work habits was used and spirometry values were obtained using Piko-6 for a 209 people sample. RESULTS: We found a high rate of smoking (42.9%) and an 11.8% prevalence of obstruction. 41.7% of the obstructed individuals were non-smokers, did not state a knowledge of any respiratory disease, engaged in no other activity that could be related to lung function decrease and did not wear airway protection apparatus. 85.7% did not use any airway protection apparatus when firefighting due to lack of such equipment in their brigades. CONCLUSIONS: Data showed that there is a high pre- valence of smoking habits in this sample of Portuguese firefighters; there is an unsatisfactory usage of airway protection apparatus and the prevalence of airway obstruction is higher than the COPD prevalence in the Portuguese population. We recommend stopping smoking and use of equipment for respiratory protection.

Functional expression of kinin B1 and B2 receptors in mouse abdominal aorta

Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg9BK (DBK). Our aim was to determine the potential expression of kinin B1 and B2 receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD2) calculated from the curves was 7.0 ± 0.1 for BK and 7.3 ± 0.2 for DBK. The efficacy was 51 ± 2 percent for BK and 30 ± 1 percent for DBK when compared to 1 æM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 æM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B1 and B2 subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.

Functional expression of kinin B1 and B2 receptors in mouse abdominal aorta.

Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg(9)BK (DBK). Our aim was to determine the potential expression of kinin B(1) and B(2) receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD(2)) calculated from the curves was 7.0 +/- 0.1 for BK and 7.3 +/- 0.2 for DBK. The efficacy was 51 +/- 2% for BK and 30 +/- 1% for DBK when compared to 1 microM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 microM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B(1) and B(2) subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.

Determination of angiotensin I-converting enzyme activity in cell culture using fluorescence resonance energy transfer peptides.

An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. The advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. The kinetic parameter K(m) for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. The specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. In conclusion, we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture.

Modeling GPCRs.

Many GPCR models have been built over the years for many different purposes, of which drug-design undoubtedly has been the most frequent one. The release of the structure of bovine rhodopsin in August 2000 enabled us to analyze models built before that period to learn things for the models we build today. We conclude that the GPCR modeling field is riddled with "common knowledge". Several characteristics of the bovine rhodopsin structure came as a big surprise, and had obviously not been predicted, which led to large errors in the models. Some of these surprises, however, could have been predicted if the modelers had more rigidly stuck to the rule that holds for all models, namely that a model should explain all experimental facts, and not just those facts that agree with the modeler's preconceptions.

Heavier-than-air flying machines are impossible.

Many G protein-coupled receptor (GPCR) models have been built over the years. The release of the structure of bovine rhodopsin in August 2000 enabled us to analyze models built before that period to learn more about the models we build today. We conclude that the GPCR modelling field is riddled with 'common knowledge' similar to Lord Kelvin's remark in 1895 that "heavier-than-air flying machines are impossible", and we summarize what we think are the (im)possibilities of modelling GPCRs using the coordinates of bovine rhodopsin as a template. Associated WWW pages: www.gpcr.org/articles/2003_mod

Tachyphylactic properties of angiotensin II analogs with bulky and hydrophobic substituents at the N-terminus.

Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimulations with angiotensin II (Ang II), has been shown to be dependent mainly on the N-terminal region of the ligand. To further study the structural requirements for the induction of tachyphylaxis we have synthesized Ang II analogs containing the bulky and very lipophilic substituents 9-fluorenylmethyloxycarbonyl (Fmoc) and 9-fluorenylmethyl ester (OFm) at the alpha-amino (Nalpha-Fmoc-Ang II) or the beta-carboxyl ([Asp(OFm)1]-Ang II) groups of the Asp1 residue, respectively. In binding assays with Chinese hamster ovary cells transfected with the AT1 Ang II receptor, Nalpha-Fmoc-Ang II bound with high affinity, whereas [Asp(OFm)1]-Ang II showed lower affinity. In biological assays, these two analogs were full agonists and showed 30 and 3%, respectively, of the Ang II potency in contracting the guinea-pig ileum smooth muscle. The two analogs induced tachyphylaxis, in spite of the lack of a free amino group in Nalpha-Fmoc-Ang II. Thus, analogs with Fmoc- or OFm-type groups coupled to the Asp1 residue, whether at the amino or carboxyl functions, induce tachyphylaxis through an unreported mechanism. Based in these findings and those available from the literature, an alternate molecular interaction mode between Ang II N-terminal portion and the AT1 receptor is proposed to explain the tachyphylactic phenomenon.

Correlated mutation analyses on very large sequence families.

The 'omics era' (the era of genomics, proteomics, and so forth) is marked by a flood of data that need to be interpreted to become useful information. Thanks to genome sequencing projects, large numbers of sequence families with more than a thousand members each are now available. Novel analytical techniques are needed to deal with this avalanche of sequence data. Sequence entropy is a measure of the information present in an alignment, whereas sequence variability represents the mutational flexibility at a particular position. Entropy versus variability plots can reveal the roles of groups of residues in the overall function of a protein. Such roles can be as part of the main active site, part of a modulator binding site, or transduction of a signal between those sites. Residues that are involved in a common function tend to stay conserved as a group, but when they mutate, they tend to mutate together. Correlated mutation analysis can detect groups of residue positions that show this behaviour. The combination of entropy, variability and correlation is a powerful tool to convert sequence data into useful information. This analysis can, for example, detect the key residues involved in cooperativity in globins, the switch regions in ras-like proteins and the calcium binding and signalling residues in serine proteases. We have extrapolated from these three classes of structurally and functionally well-described proteins to G-protein-coupled receptors (GPCRs). We can detect the residues in the main functional site in GPCRs that are responsible for G-protein coupling, the residues in the endogenous agonist binding site, and the residues in between that transduce the signal to and fro between these sites. The results are discussed in the light of a simple two-step evolutionary model for the development of functional proteins.

Synthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin analogs.

Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50 degrees C, l h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC(1)-AngII and TOAC(0)-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes.

Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization

Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells.

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.

Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid]: a novel protected spin labeled beta-amino acid for peptide and protein chemistry.

The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) is the only spin labeled amino acid that has been used to date to successfully label peptide sequences for structural studies. However, severe difficulty in coupling the subsequent amino acid has been the most serious shortcoming of this paramagnetic marker. This problem stems from the low nucleophilicity of TOAC's amine group towards the acylation reaction during peptide chain elongation. The present report introduces the alternative beta-amino acid 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid (POAC), potentially useful in peptide and protein chemistry. Investigations aimed at addressing the stereochemistry of this cyclic molecule through X-ray diffraction measurements of crystalline and bulk samples revealed that it consists only of the trans conformer. The 9-fluorenylmethyloxycarbonyl group (Fmoc) was chosen for temporary protection of the POAC amine function, allowing insertion of the probe at any position in a peptide sequence. The vasoactive octapeptide angiotensin II (All, DRVYIHPF) was synthesized by replacing Pro7 with POAC. The reaction of Fmoc-POAC with the peptidyl-resin occurred smoothly, and the coupling of the subsequent amino acid showed a much faster reaction when compared with TOAC. POAC7-AII was obtained in good yield, demonstrating that, in addition to TOAC, POAC is a convenient amino acid for the synthesis of spin labeled peptide analogues. The present findings open the possibility of a wide range of chemical and biological applications for this novel beta-amino acid derivative, including structural investigations involving its differentiated bend-inducing characteristics.

Altered neutrophil homeostasis in kinin B1 receptor-deficient mice.

The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.

Mutational analysis of the interaction of the N- and C-terminal ends of angiotensin II with the rat AT(1A) receptor.

1. The role of different residues of the rat AT(1A) receptor in the interaction with the N- and C-terminal ends of angiotensin II (AngII) was studied by determining ligand binding and production of inositol phosphates (IP) in COS-7 cells transiently expressing the following AT(1A) mutants: T88H, Y92H, G196I, G196W and D278E. 2. G196W and G196I retained significant binding and IP-production properties, indicating that bulky substituents in position 196 did not affect the interaction of AngII's C-terminal carboxyl with Lys(199) located three residues below. 3. Although the T88A mutation did not affect binding, the T88H mutant had greatly decreased affinity for AngII, suggesting that substitution of Thr(88) by His might hinder binding through an indirect effect. 4. The Y92H mutation caused loss of affinity for AngII that was much less pronounced than that reported for Y92A, indicating that His in that position can fulfil part of the requirements for binding. 5. Replacing Asp(278) by Glu caused a much smaller reduction in affinity than replacing it by Ala, indicating the importance of Asp's beta-carboxyl group for AngII binding. 6. Mutations in residues Thr(88), Tyr(92) and Asp(278) greatly reduced affinity for AngII but not for Sar(1) Leu(8)-AngII, suggesting unfavourable interactions between these residues and AngII's aspartic acid side-chain or N-terminal amino group, which might account for the proposed role of the N-terminal amino group of AngII in the agonist-induced desensitization (tachyphylaxis) of smooth muscles.

Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors.

Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B1 and B2. The B2 receptor is constitutively expressed, and its targeted disruption leads to salt-sensitive hypertension and altered nociception. The B1 receptor is a heptahelical receptor distinct from the B2 receptor in that it is highly inducible by inflammatory mediators such as bacterial lipopolysaccharide and interleukins. To clarify its physiological function, we have generated mice with a targeted deletion of the gene for the B1 receptor. B1 receptor-deficient animals are healthy, fertile, and normotensive. In these mice, bacterial lipopolysaccharide-induced hypotension is blunted, and there is a reduced accumulation of polymorphonuclear leukocytes in inflamed tissue. Moreover, under normal noninflamed conditions, they are analgesic in behavioral tests of chemical and thermal nociception. Using whole-cell patch-clamp recordings, we show that the B1 receptor was not necessary for regulating the noxious heat sensitivity of isolated nociceptors. However, by using an in vitro preparation, we could show that functional B1 receptors are present in the spinal cord, and their activation can facilitate a nociceptive reflex. Furthermore, in B1 receptor-deficient mice, we observed a reduction in the activity-dependent facilitation (wind-up) of a nociceptive spinal reflex. Thus, the kinin B1 receptor plays an essential physiological role in the initiation of inflammatory responses and the modulation of spinal cord plasticity that underlies the central component of pain. The B1 receptor therefore represents a useful pharmacological target especially for the treatment of inflammatory disorders and pain.