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.

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