Photoactivatable opiate derivatives as irreversible probes of the mu-opioid receptor

The synthesis of aryldiazonium and arylazido derivatives of carfentanil, etonitazene, and naltrexone and of a triazaspirodecane derivative is described. The chemical stability and the spectral characteristics of these compounds were verified, and their binding affinity constants for the different opioid receptor classes were determined, in the absence of light, from competition experiments. With the exception of the naltrexyl derivatives, which remained nonselective, all compounds tested displayed a pronounced mu-binding selectivity with mu/delta and mu/kappa ratios ranging from 12 to 1000. After irradiation, only the arylazido probes led to an irreversible mu-binding-site inactivation. This inactivation fulfilled the criteria for photoaffinity labeling such as protection against inactivation by other opiate ligands and absence of an effect of scavengers on the extent of the inactivation. Most of the photoactivatable probes formed long-lasting reversible complexes with the opioid binding sites: an efficient dissociation procedure was thus required to discriminate between pseudoirreversible and covalent complexes. The marked differences in labeling efficacy between aryldiazonium salts and their corresponding arylazido derivatives are discussed.     

Anti-actin antibodies. Detection and quantitation of total and skeletal muscle actin in human plasma using a competitive ELISA

A competitive ELISA has been used to titrate skeletal muscle and total actins in human plasma. Specific antibodies directed against the variable N-terminal 1-7 sequence and conserved sequences respectively were used. The N-terminus of actin appears to be accessible in native and brevin-complexed actins. The skeletal muscle actin isoform represents about 1% of the total circulating actin (mean: 50 micrograms/ml plasma), but is markedly increased after severe muscle tissue injuries.     

Stability of lipid and protein composition of very low-density lipoproteins during the storage after freezing of whole serum and isolated lipoproteins (author's transl)]

The lipid and protein composition of lipoprotein is now regarded with great interest for the identification of hyperlipoproteinemias. Because this relatively time-consuming study cannot be envisaged currently in clinical services, it seems useful to analyse a storage procedure which does not affect the composition of very-low density lipoproteins, whose proportion is very important among subjects with type IV hyperlipoproteinemia. Two studies were considered, one using whole sera stored at -20 degrees C and one using very low-density lipoproteins held in the same conditions. The lipid composition was determined for triglycerides and cholesterol concentrations of total proteins, proteins soluble in tetramethylurea (apoprotein C, "arginine-rich fraction", and apoprotein D), and peptides CII, CIII1 and CIII2 of apoprotein C. For both procedures considered, no significant changes were seen in lipid and protein composition for periods as long as 8 weeks.     

Patients with renal cell carcinoma nodal metastases can be accurately identified: external validation of a new nomogram

Outcome of patients with renal cell carcinoma nodal metastases (NM) is substantially worse than that of patients with localized disease. This justifies more thorough staging and possibly more aggressive treatment in those at risk of or with established NM. We developed and externally validated a nomogram capable of highly accurately predicting renal cell carcinoma NM in patients without radiographic evidence of distant metastases. Age, symptom classification, tumour size and the pathological nodal stage were available for 4,658 individuals. The data of 2,522 (54.1%) individuals from 7 centers were used to develop a multivariable logistic regression model-based nomogram predicting the individual probability of NM. The remaining data from 2,136 (45.9%) patients from 5 institutions were used for external validation. In the development cohort, 107/2,522 (4.2%) had lymph node metastases vs. 100/2,136 (4.7%) in the external validation cohort. Symptom classification and tumour size were independent predictors of NM in the development cohort. Age failed to reach independent predictor status, but added to discriminant properties of the model. A nomogram based on age, symptom classification and tumour size was 78.4% accurate in predicting the individual probability of NM in the external validation cohort. Our nomogram can contribute to the identification of patients at low risk of NM. This tool can help to risk adjust the need and the extent of nodal staging in patients without known distant metastases. More thorough staging can hopefully better select those in whom adjuvant treatment is necessary. (c) 2007 Wiley-Liss, Inc.     

A mu-opioid receptor-filter assay. Rapid estimation of binding affinity of ligands and reversibility of long-lasting ligand-receptor complexes

A filter-associated binding technique, originally described by Leysen and Gommeren [J. Receptor Res. 4, 817 (1984); Drug Dev. Res. 8, 119 (1986)], was focused on the study of mu-opioid receptor sites. The interesting binding features of 3H-Sufentanil, a mu-selective radioligand, permitted such a filter procedure to be performed. Its application was 3-fold. A 5-min binding assay allowed us to verify that specific 3H-Sufentanil binding to filter-absorbed rat brain membranes was endowed with the known kinetic and equilibrium binding properties of membrane-bound mu-opioid receptor sites. Moreover, the filtration technique allowed us to get rapid information about the dissociation rates of unlabelled compounds from the receptor sites. In practice, membranes, preincubated with high concentrations of cold drugs, were absorbed to filters. Dissociation was achieved by repeatedly applying buffer samples on the filter, and monitored by the recovery in free specific 3H-Sufentanil binding sites. Finally, such a dissociation procedure, improved by a washing buffer at high ionic strength, was found to be much more efficient and attractive than the classical dilution-centrifugation procedure, especially for slowly-dissociating compounds. Special attention was paid to the discrimination between pseudo-irreversible binding of drugs and stable covalent labelling of mu-opioid receptor sites (either by affinity or photoaffinity probes), particularly when unlabelled ligands were the only tools available.     

Single graft loss in dual renal transplant recipients: impact of graft placement on recipient outcomes

We aimed to assess the impact of graft placement in dual renal transplantation on the risk for single graft loss and to report recipient outcomes. Between 2004 and 2007, 55 dual renal transplants were performed at our institution. Allografts were placed bilaterally (one in each iliac fossa) in 42 patients and unilaterally (both in the same iliac fossa) in 14 patients. Nine recipients (16.4%) underwent explantation of a single graft as a consequence of vascular thrombosis designated as the SINGLE group, whereas 46 had two functional allografts (DUAL group). There was a higher rate of graft loss in case of unilateral placement (n = 5/14) compared with bilateral placement (n = 4/41) (35.7% vs. 9.8%, P = 0.035). One‐year glomerular filtration rate was significantly lower in the SINGLE group (29.4 ml/min/1.73 m² vs. 49.4 ml/min/1.73 m² in the DUAL group, P < 0.05). Significantly, none of the nine recipients of the SINGLE group returned to dialysis with a mean follow‐up of 34.1 months. Graft survival at 1 year was 100% and 97.9% in SINGLE and DUAL groups, respectively. Unilateral placement of both allografts is associated with an increased risk of single graft loss and therefore lower renal function at 1 year. However, this strategy is safe in selected indications.