Endocannabinoids and cannabinoid analogues block human cardiac Kv4.3 channels in a receptor-independent manner

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K⁺ channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K⁺ current (I_to1). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I_Kv4.3 in a concentration-dependent manner, AEA and 2-AG being the most potent (IC₅₀ ∼ 0.3-0.4 µM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I_to1 recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.     

Analysis of kinesin-2 function in photoreceptor cells using synchronous Cre-loxP knockout of Kif3a with RHO-Cre

PURPOSE: To determine the relationship between the presence of kinesin-2 and photoreceptor cell viability and opsin transport, by generating RHO-Cre transgenic mice and breeding them to mice with a floxed kinesin-2 motor gene. METHODS: Different lines of RHO-Cre transgenic mice were generated and characterized by transgene expression, histology, and electrophysiology. Mice from one line, showing uniform transgene expression, were crossed with Kif3a(flox)/Kif3a(flox) mice. The time courses of photoreceptor Cre expression, KIF3A loss, ectopic opsin accumulation, and photoreceptor cell death were determined by Western blot analysis and microscopy. RESULTS: One of the RHO-Cre lines effected synchronous expression of Cre and thus uniform excision of Kif3a(flox) in rod photoreceptors across the retina. After the neonatal production of CRE and the initiation of KIF3A loss, ectopic accumulation of opsin was detected by postnatal day (P)7, and ensuing photoreceptor cell death was evident after P10 and almost complete by P28. Of importance, the photoreceptor cilium formed normally, and the disc membranes of the nascent outer segment remained normal until P10. CONCLUSIONS: The RHO-Cre-8 mice provide an improved tool for studying gene ablation in rod photoreceptor cells. Regarding kinesin-2 function in photoreceptor cells, the relatively precise timing of events after CRE excision of Kif3a(flox) allows us to conclude that ectopic opsin is a primary cellular lesion of KIF3A loss, consistent with the hypothesis that opsin is a cargo of kinesin-2. Moreover, it demonstrates that KIF3A loss results in very rapid photoreceptor cell degeneration.     

Compliance with methadone-based substitutive treatment: a proposed model based on immunoassay urinary sample screening

Methadone (MTD) maintenance treatment is a recognized method to reduce illicit opiate abuse. Because of the difficulties of collecting 24-hour urines routinely, the monitoring of MTD compliance is currently done with random urinary screening. However, monitoring of MTD compliance by random urinary screening lacks accuracy because of its highly variable pharmacokinetics, leading to false positive or negative results. This study's objective was to identify factors influencing the reliability of urinary screening of methadone for MTD compliance monitoring in a field setting involving usual care for opiate-dependent patients. In a cross-sectional population-based study, 1981 urine samples obtained from 68 patients in parallel with drug dose, gender, and weight were analyzed by MTD enzyme immunoassay (EMIT). Urinary pH was measured, and positive threshold was determined experimentally by box-plot analysis. Multivariate determinants of MTD excretion were established with stepwise multiple regression analysis. On this basis, adjusted values for MTD excretion were proposed and verified with an (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) assay from independent urine samples that were negative or doubtful by the MTD assay. MTD excretion was higher in men, decreased with increased urinary pH, and increased with daily dosage of MTD; these factors explain 32% of the total variance of urinary MTD. Adjustment on these 3 variables (urinary pH, sex, daily dosage) improved the prediction of compliance to MTD treatment. Threshold was stable across pH values and in agreement with EDDP results. The influence of simple variables such as gender, urinary pH, and daily dosage on urinary MTD excretion could be put in evidence and accounted for. Adjusted values of urinary MTD are more reliable than the raw values for monitoring compliance to MTD treatment.     

Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse

PURPOSE: To test the hypothesis that Nrl(-)(/)(-) photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS: The photoreceptor layer of fixed retinal tissue of 4- to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single- and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the a-wave of the ERG. RESULTS: Outer segments of Nrl(-/-) photoreceptors ( approximately 7 mum) are shorter than those of wild-type (WT) rods ( approximately 25 mum) and cones ( approximately 15 mum); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl(-/-) photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl(-/-) a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the lambda(max) of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was approximately 50 ms, more than two times faster than that of rods. CONCLUSIONS: Many morphological, molecular, and electrophysiological features of the Nrl(-/-) photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease.     

Survivin as prognostic factor in squamous cell carcinoma of the oral cavity

A series of 78 cases of oral squamous cell carcinoma was analysed by immunohistochemistry for expression of survivin, a recent apoptosis inhibitor. All cases were positive for survivin expression and were divided into two groups using a system of scores. Disease-specific survival curves were calculated according to Kaplan-Meier algorithm, and log rank test was used to compare survival curves. Then, Cox regression analysis was applied to determine the single contribution of covariates on survival rate. So, Cox analysis allowed us to detect the variables most associated to survival. Among the studied variables, such as grade of differentiation, tumor size, stage, recurrence of disease, lymph node presence, only stage and recurrence of disease were predictors of outcome; however, when we analyzed the survival without considering recurrence (that was the stronger predictor of death), a stepwise Cox analysis showed that Survivin, stage and grade of differentiation are significantly associated to survival, with a higher value for Survivin. These data suggest that survivin expression may identify cases of oral squamous cell carcinoma with more aggressive and invasive phenotype and, therefore, could influence the decision for the therapy at the time of diagnosis.     

P-cadherin expression and survival rate in oral squamous cell carcinoma:an immunohistochemical study

Background  

P-cadherin (P-cad) is a transmembrane molecule involved in the cell-cell adhesion and similar to E-cadherin (E-cad), but less investigated in oncology, especially in in vivo studies. Aims of the present study were to assess the prevalence of P-cad expression in oral squamous cell carcinoma (OSCC) and to verify whether P-cad can be considered a marker of prognosis in patients with OSCC.     

Two new HLA class I alleles recognised by PCR sequence-specific primer and sequencing based typing: B*3805 and Cw*0408

Two new HLA class I alleles have been recognised by molecular-based typing. B*3805 was initially identified by polymerase chain reaction using sequence-specific primers (PCR-SSP) and afterwards confirmed by sequencing based typing (SBT) studies in a Spanish Caucasian blood cord unit. A unique nucleotide change throughout exons 2, 3 and 4, leading to the amino acid replacement Ser11Ala, differentiates B*3801 and *3805. This position behaves as a dimorphic residue in HLA-B and -C loci, and seems to be structurally unrelated to peptide and TcR recognition. Cw*0408 was first detected by SBT in two African American bone marrow donors in combination with its most structurally related allele, Cw*04011. The single amino acid change found between Cw*04011 and Cw*0408 was Thr163Leu, a residue involved in pocket A of the peptide-binding cleft. This new allele could be the result of a gene conversion event between Cw*04011 and any of the Cw*03 alleles.     

Identification by sequencing based typing and complete coding region analysis of three new HLA class II alleles: DRB3*0210, DRB3*0211 and DQB1*0310

The study of HLA class II polymorphism by direct exon 2 DNA sequencing analysis has been established to be a reliable and accurate high-resolution typing procedure. This approach shows some advantages in relation to previous methods, polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO) and sequence-specific primers (PCR-SSP), basically due to the capability of analysis for the complete sequenced genomic region, including non-polymorphic motifs. DRB3 and DQB1 sequencing based typing (SBT) in unrelated bone marrow donor searching allowed us to detect three new alleles. The complete coding region sequences were characterised from cDNA. Two new DRB3 alleles, DRB3*0210 and DRB3*0211, were described in two Caucasian bone marrow donors. Both sequences showed single point mutations regarding DRB3*0202, producing amino acid replacements at positions 51 (Asp to Thr) and 67 (Leu to Ile), respectively. These two point mutations can be found in other DRB alleles, and suggest that gene conversion would be involved in the origin of both alleles. A new DQB1 sequence was found in a Spanish patient that showed two nucleotide differences, positions 134 and 141, with regard to its close similar DQB1*03011 allele. Only substitution at position 134 provoked amino acid replacement at residue 45, Glu to Gly. This single amino acid change would be involved in the lack of serologic recognition of this new molecule by DQ7-specific reagents.     

Characterization of a new HLA-B18 allele, B*1806, which lacks expression of the Bw6 epitope

Abstract: HLA-B18 is a well defined Bw6-associated serologic specificity. Up to now, four different sequences have been characterised in Caucasian populations (B*1801,3,4,5), and one in Orientals (B*1802). We report a new HLA-B18 subtype (B*1806) which was serologically detected in a Spanish Caucasian individual as a B18 Bw4-associated antigen. Complete coding region sequencing showed that B*1806 differs from B*1801 in a unique nu-cleotide at position 299 (A to T), giving rise to an amino acid replacement in residue 76 (glutamic acid to valine) placed at the α1 domain. Therefore, in contrast to the serologic results, B*1806 possesses the canonical Bw6 motif at position 77–83. Subsequent flow cytometric assays proved that B*1806 evidences neither Bw4 nor Bw6 epitopes. Only three additional HLA-B alleles encode valine at codon 76, B*4601, B*7301 and B*5503, and like B*1806, all of them would include a Bw6 motif associated to the negative recognition by Bw6 antibodies. These findings support that valine at position 76 will modify the Bw6 epitope drastically, and suggest that this group of HLA-B alleles would define a third, Bw4 and Bw6-negative, lineage of molecules. Furthermore, valine 76 will also prevent the binding of Bw6 antibodies to those HLA-C antigens with the canonical Bw6 epitope (Cw*1,3,7,8,12,13,14,16).