Therapeutic modulation of cannabinoid lipid signaling: Metabolic profiling of a novel antinociceptive cannabinoid-2 receptor agonist

AimsAM-1241, a novel, racemic cannabinoid-2 receptor (CB2) ligand, is the primary experimental agonist used to characterize the role of CB2-mediated lipid signaling in health and disease, including substance abuse disorders. In vivo pharmacological effects have been used as indirect proxies for AM-1241 biotransformation processes that could modulate CB2 activity. We report the initial pre-clinical characterization of AM-1241 biotransformation and in vivo distribution.Main methodsAM-1241 metabolism was characterized in a variety of predictive in vitro systems (Caco-2 cells; mouse, rat and human microsomes) and in the mouse in vivo. Liquid chromatography and mass spectrometry techniques were used to quantify AM-1241 tissue distribution and metabolic conversion.Key findingsAM-1241 bound extensively to plasma protein/albumin. A pharmacological AM-1241 dose (25 mg/kg, i.v.) was administered to mice for direct determination of its plasma half-life (37 min), following which AM-1241 was quantified in brain, spleen, liver, and kidney. After p.o. administration, AM-1241 was detected in plasma, spleen, and kidney; its oral bioavailability was ~ 21%. From Caco-2 permeability studies and microsomal-based hepatic clearance estimates, in vivo AM-1241 absorption was moderate. Hepatic microsomal metabolism of AM-1241 in vitro generated hydroxylation and demethylation metabolites. Species-dependent differences were discovered in AM-1241's predicted hepatic clearance. Our data demonstrate that AM-1241 has the following characteristics: a) short plasma half-life; b) limited oral bioavailability; c) extensive plasma/albumin binding; d) metabolic substrate for hepatic hydroxylation and demethylation; e) moderate hepatic clearance.SignificanceThese results should help inform the design, optimization, and pre-clinical profiling of CB2 ligands as pharmacological tools and medicines.     

A study of the cambial zone and conductive phloem of common beech (Fagus sylvatica L.) using an image analysis method

Quantification is a major problem when using histology to study the influence of ecological factors on tree structure. This paper presents a method to prepare and to analyse transverse sections of cambial zone and of conductive phloem in bark samples. The following paper (II) presents the automated measurement procedure. Part I here describes and discusses the preparation method, and the influence of tree age on the observed structure. Highly contrasted images of samples extracted at breast height during dormancy were analysed with an automatic image analyser. Between three young (38 years) and three old (147 years) trees, age-related differences were identified by size and shape parameters, at both cell and tissue levels. In the cambial zone, older trees had larger and more rectangular fusiform initials. In the phloem, sieve tubes were also larger, but their shape did not change and the area for sap conduction was similar in both categories. Nevertheless, alterations were limited, and demanded statistical analysis to be identified and ascertained. The physiological implications of the structural changes are discussed.     

Laser Raman studies of conformational variations of poly-L-lysine

The frequencies and intensities of the laser Raman spectra of poly-L -lysine (PLL) have been observed in the following studies: (1) the thermally induced α-to-β transition which occurs with increasing temperature at high pH; (2) the ionized form to α transition at 10°C by increasing pH; and (3) the ionized form to α transition by ionic strength at low pH. The frequency-dependent bands which have been observed are the amide I (in H₂O), amide I′ (in D₂O), amide III, and C–C stretch. It has been found possible to assign an unique set of frequencies and intensities to each conformation of PLL of α, β, and ionized form. In this way the nature of the conformations intermediate in the transitions can be determined. The frequencies of the amide III and amide III′ are very weak in the α-helix and somewhat higher than usual in the β form. Hence it appears the amide III and amide III′ bands may differ from one type of polypeptide to another with the same backbone conformation.     

Cross Talk between CD3 and CD28 Is Spatially Modulated by Protein Lateral Mobility

Functional convergence of CD28 costimulation and TCR signaling is critical to T-cell activation and adaptive immunity. These receptors form complex microscale patterns within the immune synapse, although the impact of this spatial organization on cell signaling remains unclear. We investigate this cross talk using micropatterned surfaces that present ligands to these membrane proteins in order to control the organization of signaling molecules within the cell-substrate interface. While primary human CD4⁺ T cells were activated by features containing ligands to both CD3 and CD28, this functional convergence was curtailed on surfaces in which engagement of these two systems was separated by micrometer-scale distances. Moreover, phosphorylated Lck was concentrated to regions of CD3 engagement and exhibited a low diffusion rate, suggesting that costimulation is controlled by a balance between the transport of active Lck to CD28 and its deactivation. In support of this model, disruption of the actin cytoskeleton increased Lck mobility and allowed functional T-cell costimulation by spatially separated CD3 and CD28. In primary mouse CD4⁺ T cells, a complementary system, reducing the membrane mobility increased the sensitivity to CD3-CD28 separation. These results demonstrate a subcellular reaction-diffusion system that allows cells to sense the microscale organization of the extracellular environment.     

Anterior mediastinal paraganglioma: A case for preoperative embolization

ABSTRACT: BACKGROUND: Paraganglioma is a rare but highly vascular tumor of the anterior mediastinum. Surgical resection is a challenge owing to the close proximity to vital structures including heart, trachea and great vessels. Preoperative embolization has been reported once to facilitate surgical treatment. CASE PRESENTATION: We report a case of anterior mediastinal paraganglioma that was embolized preoperatively and was resected without the need for cardiopulmonary bypass and without major bleeding complications. CONCLUSION: We make a case to further the role of preoperative embolization in the treatment of mediastinal paragangliomas.     

Spliceosome structure: Piece by piece

Processing of pre-mRNAs by RNA splicing is an essential step in the maturation of protein coding RNAs in eukaryotes. Structural studies of the cellular splicing machinery, the spliceosome, are a major challenge in structural biology due to the size and complexity of the splicing ensemble. Specifically, the structural details of splice site recognition and the architecture of the spliceosome active site are poorly understood. X-ray and NMR techniques have been successfully used to address these questions defining the structure of individual domains, isolated splicing proteins, spliceosomal RNA fragments and recently the U1 snRNP multiprotein·RNA complex. These results combined with extant biochemical and genetic data have yielded important insights as well as posing fresh questions with respect to the regulation and mechanism of this critical gene regulatory process.     

The use of human artificial gametes and the limits of reproductive freedom

Recent developments in generating gametes via in vitro gametogenesis (IVG) from induced pluripotent stem cells (iPSCs) and their successful use for reproductive purposes in animals strongly suggest that soon these methods could also be used in human reproduction. At least two questions emerge in this context: (a) if a legislator should permit their use and (b) if ethical claims emerge that support their provision, e.g., by public health care systems. This urges an ethical reflection of the new reproductive options this technique might offer. Since the concept of reproductive freedom is a key aspect for the ethical evaluation of artificial reproductive technologies (ARTs), it is necessary to analyze if the new possibilities emerging from IVG fall within the scope of this concept. The results may constitute a morally relevant difference between different imaginable applications of IVG and potentially justify differences in claims to access this technology.     

THE MAINTENANCE OF SPERM VARIABILITY: CONTEXT‐DEPENDENT SELECTION ON SPERM MORPHOLOGY IN A BROADCAST SPAWNING INVERTEBRATE

Why are sperm so variable despite having a singular, critical function and an intimate relationship with fitness? A key to understanding the evolution of sperm morphology is identifying which traits enable sperm to be successful fertilizers. Several sperm traits (e.g., tail length, overall size) are implicated in sperm performance, but the benefits of these traits are likely to be highly context dependent. Here, we examined phenotypic selection on sperm morphology of a broadcast spawning tube worm (Galeolaria gemineoa). We conducted laboratory experiments to measure the relationship between average sperm morphology and relative fertilization success across a range of sperm environments that were designed to approximate the range of sperm concentrations and ages encountered by eggs in nature. We found that the strength and form of multivariate selection varied substantially across our environmental gradients. Sperm with long tails and small heads were favored in high‐concentration environments, whereas sperm with long heads were favored at low concentrations and old ages. We suggest variation in the local fertilization environment and resulting differences in selection can preserve variability in sperm morphology both within and among males.