Cannabinoid receptor type 1 modulates excitatory and inhibitory neurotransmission in mouse colon

The effects of cannabinoid receptor agonists and antagonists on smooth muscle resting membrane potentials and on membrane potentials following electrical neuronal stimulation in a myenteric neuron/smooth muscle preparation of wild-type and cannabinoid receptor type 1 (CB1)-deficient mice were investigated in vitro. Double staining for CB1 and nitric oxide synthase (neuronal) was performed to identify the myenteric CB1-expressing neurons. Focal electrical stimulation of the myenteric plexus induced a fast (f) excitatory junction potential (EJP) followed by a fast and a slow (s) inhibitory junction potential (IJP). Treatment of wild-type mice with the endogenous CB1 receptor agonist anandamide reduced EJP while not affecting fIJP and sIJP. EJP was significantly higher in CB1-deficient mice than in wild-type littermate controls, and anandamide induced no effects in CB1-deficient mice. N-arachidonoyl ethanolamide (anandamide), R-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]- 1,4-benzoxazin-6-yl]-1-naphtalenylmethanone, a synthetic CB1 receptor agonist, nearly abolished EJP and significantly reduced the fIJP in wild-type mice. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A), a CB1-specific receptor antagonist, was able to reverse the agonist effects induced in wild-type mice. SR141716A, when given alone, significantly increased EJP in wild-type mice without affecting IJP in wild-type and EJP in CB1-deficient mice. Interestingly, SR141716A reduced fIJP in CB1-deficient mice. In the mouse colon, nitrergic myenteric neurons do not express CB1, implying that CB1 is expressed in cholinergic neurons, which is in line with the functional data. Finally, excitatory and inhibitory neurotransmission in the mouse colon is modulated by activation of CB1 receptors. The significant increase in EJP in CB1-deficient mice strongly suggests a physiological involvement of CB1 in excitatory cholinergic neurotransmission.     

The activation mechanism of class-C G-protein coupled receptors

Class-C G-protein coupled receptors (GPCRs) represent a distant group among the large family of GPCRs. This class includes the receptors for the main neurotransmitters, glutamate and gamma-aminobutyric acid (GABA), and the receptors for Ca(2+), some taste and pheromone molecules, as well as some orphan receptors. Like any other GPCRs, class-C receptors possess a heptahelical domain (HD) involved in heterotrimeric G-protein activation, but most of them also have a large extracellular domain (ECD) responsible for agonist recognition and binding. In addition, it is now well accepted that these receptors are dimers, either homo or heterodimers. This complex architecture raises a number of important questions. Here we will discuss our view of how agonist binding within the large ECD triggers the necessary change of conformation, or stabilize a specific conformation, of the heptahelical domain leading to G-protein activation. How ligands acting within the heptahelical domain can change the properties of these complex macromolecules.     

Preparation of purified lymphoma cells suitable for therapy

BACKGROUND: Very few tumoral Ags have yet been isolated in NHL B cells. It is nevertheless possible to use whole tumor cells as a source of tumor Ags. We describe the purification of large numbers of human NHL B cells directly from lymph node or spleen biopsies, and different preparations allowing their use in a clinical setting. METHODS: The purification procedure consists of the negative selection of tumor B cells: cells to be eliminated are opsonized by CD2 Abs, and then coupled to magnetic beads for separation by the Isolex 300 magnetic separator. RESULTS: The mean yield of the purification was 74% for CD19+ cells, with a mean purity of 87%, dependent on the initial fraction of tumor cells in the biopsy. Using this procedure, a large number of purified tumor cells can be recovered from a biopsy in sterile conditions. We also describe treatments of B cells that can enhance their uptake by APCs, a critical step in anti-tumor immunotherapy strategies. Cells were opsonized by rituximab, or induced in apoptosis by irradiation, or necrosis by heating. Cell lysates were directly prepared from purified tumor cells. DISCUSSION: These procedures were reproducible on every lymphoma cell, and treated cells were phagocytosed by APCs. The methodology described here allows the evaluation of the immunological potential of apoptotic, necrotic, opsonized lymphoma cells, or their lysates, in a clinical setting.     

Human alphaA- and alphaB-crystallins bind to Bax and Bcl-X(S) to sequester their translocation during staurosporine-induced apoptosis

AlphaA- and alphaB-crystallins are distinct antiapoptotic regulators. Regarding the antiapoptotic mechanisms, we have recently demonstrated that alphaB-crystallin interacts with the procaspase-3 and partially processed procaspase-3 to repress caspase-3 activation. Here, we demonstrate that human alphaA- and alphaB-crystallins prevent staurosporine-induced apoptosis through interactions with members of the Bcl-2 family. Using GST pulldown assays and coimmunoprecipitations, we demonstrated that alpha-crystallins bind to Bax and Bcl-X(S) both in vitro and in vivo. Human alphaA- and alphaB-crystallins display similar affinity to both proapoptotic regulators, and so are true with their antiapoptotic ability tested in human lens epithelial cells, human retina pigment epithelial cells (ARPE-19) and rat embryonic myocardium cells (H9c2) under treatment of staurosporine, etoposide or sorbitol. Two prominent mutants, R116C in alphaA-crystallin and R120G, in alphaB-crystallin display much weaker affinity to Bax and Bcl-X(S). Through the interaction, alpha-crystallins prevent the translocation of Bax and Bcl-X(S) from cytosol into mitochondria during staurosporine-induced apoptosis. As a result, alpha-crystallins preserve the integrity of mitochondria, restrict release of cytochrome c, repress activation of caspase-3 and block degradation of PARP. Thus, our results demonstrate a novel antiapoptotic mechanism for alpha-crystallins.     

Involvement of a calcium-dependent dephosphorylation of BAD associated with the localization of Trpc-1 within lipid rafts in 7-ketocholesterol-induced THP-1 cell apoptosis

7-Ketocholesterol is a component of oxidized LDL, which plays a central role in atherosclerosis. It is a potent inducer of cell death towards a wide number of cells involved in atherosclerosis. In this study, it is reported that 7-ketocholesterol treatment induces an increase of cytosolic-free Ca(2+) in THP-1 monocytic cells. This increase is correlated with the induction of cytotoxicity as suggested from experiments using the Ca(2+) channel blockers verapamil and nifedipine. This 7-ketocholesterol-induced apoptosis appears to be associated with the dephosphorylation of serine 75 and serine 99 of the proapoptotic protein Bcl-2 antagonist of cell death (BAD). We demonstrated that this dephosphorylation results mainly from the activation of calcium-dependent phosphatase calcineurin by the oxysterol-induced increase in Ca(2+). Moreover, this Ca(2+) increase appears related to the incorporation of 7-ketocholesterol into lipid raft domains of the plasma membrane, followed by the translocation of transient receptor potential calcium channel 1, a component of the store operated Ca(2+) entry channel, to rafts.     

Incipient allochronic speciation in the pine processionary moth (Thaumetopoea pityocampa, Lepidoptera, Notodontidae)

A plausible case of allochronic differentiation, where barrier to gene flow is primarily attributable to a phenological shift, was recently discovered in Portugal for the pine processionary moth Thaumetopoea pityocampa. Previous results suggested that the observed 'summer population' (SP) originated from the sympatric winter population (WP). Our objectives were to finely analyse these patterns and test their stability in time, through field monitoring and genetic analyses of larvae and adults across different years. Reproductive activity never overlapped between SP and WP. Microsatellites showed a clear differentiation of the SP, consistent with a strong reduction in gene flow owing to the phenological shift. Assignment tests suggested that some individuals shift from the SP to the WP phenology, causing some hybridization. We discuss these patterns and their maintenance over time. This could be a first stage of allochronic speciation, and SP should be considered as a distinct phenological race.     

Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties

One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 microg/mL puromycin for the first 2 days of culture or 3 microg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 x 10(-6) cm/s) and a high electrical resistance (up to 500 Omega x cm(2)). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for cellular and molecular biology studies as well as pharmacological investigations.     

Apoptosis: its functions and control in the ocular lens

The ocular lens is a non-vascular and non-innervated transparent organ that plays an important role in vision processing. This unique organ is derived from the embryonic ectoderm of the brain region through a complicated differentiation process in which apoptosis plays a key role. First, when the committed ectoderm becomes thickened and invaginated, the defined number of cells required to form the lens vesicle is partially determined by apoptosis. Second, separation of lens vesicle from the above corneal ectoderm is executed through apoptosis of the lens stalk cells. Finally, differentiation of the lens epithelial cells is controlled by the regulators, most of which are involved in control of apoptosis at multiple signaling steps. The lens is also characterized by continuous growth and differentiation in the adulthood. Through the different stages of growth and differentiation in the adult lens, various stress conditions can induce apoptosis of the lens epithelial cells, leading to eventual non-congenital cataractogenesis. The present review summarizes the current knowledge on the functions and regulators of apoptosis in the ocular lens.     

Taxa distribution and RAPD markers indicate different origin and regional differentiation of hybrids in the invasive Fallopia complex in central-western Europe

Interspecific hybridization can be a driving force for evolutionary processes during plant invasions, by increasing genetic variation and creating novel gene combinations, thereby promoting genetic differentiation among populations of invasive species in the introduced range. We examined regional genetic structure in the invasive Fallopia complex, consisting of F. japonica var. japonica , F.   sachalinensis and their hybrid F.  ×  bohemica , in seven regions in Germany and Switzerland using RAPD analysis and flow cytometry. All individuals identified as F. japonica var . japonica had the same RAPD phenotype, while F. sachalinensis (11 RAPD phenotypes for 11 sampled individuals) and F.  ×  bohemica (24 RAPD phenotypes for 32 sampled individuals) showed high genotypic diversity. Bayesian cluster analysis revealed three distinct genetic clusters. The majority of F . ×  bohemica individuals were assigned to a unique genetic cluster that differed from those of the parental species, while the other F . ×  bohemica individuals had different degrees of admixture to the three genetic clusters. At the regional scale, the occurrence of male-fertile F. sachalinensis coincided with the distribution of F . ×  bohemica plants showing a high percentage of assignment to both parental species, suggesting that they originated from hybridization between the parental species. In contrast, in regions where male-fertile F. sachalinensis were absent, F . ×  bohemica belonged to the non-admixed genetic group, indicating multiple introductions of hybrids or sexual reproduction among hybrids. We also found regional differentiation in the gene pool of F.  ×  bohemica , with individuals within the same region more similar to each other than to individuals from different regions.