Multiple Neuronal Connexins in the Mammalian Retina

Gap junctions are abundant in the mammalian retina and many neuronal types form neural networks. Several different neuronal connexins have now been identified in the mammalian retina. Cx36 supports coupling in the AII amacrine cell network and is essential for processing rod signals. Cx36 is probably also responsible for photoreceptor coupling. Horizontal cells appear to be extensively coupled by either Cx50 or Cx57. These results indicate that multiple neuronal connexins are expressed in the mammalian retina and that different cell types express different connexins.     

Presynaptic Localization and Possible Function of Calcium-Activated Chloride Channel Anoctamin 1 in the Mammalian Retina

Calcium (Ca²⁺)-activated chloride (Cl⁻) channels (CaCCs) play a role in the modulation of action potentials and synaptic responses in the somatodendritic regions of central neurons. In the vertebrate retina, large Ca²⁺-activated Cl⁻ currents (I_Cl(Ca)) regulate synaptic transmission at photoreceptor terminals; however, the molecular identity of CaCCs that mediate I_Cl(Ca) remains unclear. The transmembrane protein, TMEM16A, also called anoctamin 1 (ANO1), has been recently validated as a CaCC and is widely expressed in various secretory epithelia and nervous tissues. Despite the fact that tmem16a was first cloned in the retina, there is little information on its cellular localization and function in the mammalian retina. In this study, we found that ANO1 was abundantly expressed as puncta in 2 synaptic layers. More specifically, ANO1 immunoreactivity was observed in the presynaptic terminals of various retinal neurons, including photoreceptors. I_Cl(Ca) was first detected in dissociated rod bipolar cells expressing ANO1. I_Cl(Ca) was abolished by treatment with the Ca²⁺ channel blocker Co²⁺, the L-type Ca²⁺ channel blocker nifedipine, and the Cl⁻ channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and niflumic acid (NFA). More specifically, a recently discovered ANO1-selective inhibitor, T16A_inh-A01, and a neutralizing antibody against ANO1 inhibited I_Cl(Ca) in rod bipolar cells. Under a current-clamping mode, the suppression of I_Cl(Ca) by using NPPB and T16A_inh-A01 caused a prolonged Ca²⁺ spike-like depolarization evoked by current injection in dissociated rod bipolar cells. These results suggest that ANO1 confers I_Cl(Ca) in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission.     

Flavin Mononucleotide-Based Fluorescent Proteins Function in Mammalian Cells without Oxygen Requirement

Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions.     

Discrete Distributions of Adenosine Receptors in Mammalian Retina

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyladenosine and the mixed A1-A2 agonist N-[3H]ethylcarboxamidoadenosine [( 3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer. The binding of [3H]NECA was observed primarily over the retinal pigmented epithelium and the outer and inner segments of photoreceptors. [3H]NECA labeling was not affected either by including a low concentration of unlabeled A1 agonist or by pretreating tissue with N-ethylmaleimide to inhibit ligand binding at A1 sites. While virtually all of the [3H]NECA binding was displaced by an excess of unlabeled NECA, displacement with antagonist or a large excess of cyclohexyladenosine revealed that approximately 30% of the [3H]NECA binding was at non-A1,A2 sites. The majority of the binding in the outer retina thus labeled A2 receptor sites. The unique localizations of the two classes of adenosine receptors suggest different functions in visual processing.     

Spikes in Mammalian Bipolar Cells Support Temporal Layering of the Inner Retina

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Highlights? Light-driven calcium changes were recorded in mouse bipolar cell axon terminals ? Bipolar cells cluster into ≥eight functional types, which match anatomical types ? Fast and slow bipolar cells project to the inner retina in an organized manner ? The fastest bipolar cells generate clear all-or-nothing spikes     

Differential RNA Sequence Requirement for Dengue Virus Replication in Mosquito and Mammalian Cells

Dengue virus cycles between mosquitoes and humans. Each host provides a different environment for viral replication, imposing different selective pressures. We identified a sequence in the dengue virus genome that is essential for viral replication in mosquito cells but not in mammalian cells. This sequence is located at the viral 3′ untranslated region and folds into a small hairpin structure. A systematic mutational analysis using dengue virus infectious clones and reporter viruses allowed the determination of two putative functions in this cis-acting RNA motif, one linked to the structure and the other linked to the nucleotide sequence. We found that single substitutions that did not alter the hairpin structure did not affect dengue virus replication in mammalian cells but abolished replication in mosquito cells. This is the first sequence identified in a flavivirus genome that is exclusively required for viral replication in insect cells.     

Phosphodiesterase Activities for Cyclic Nucleotides in Nerve Endings from the Bovine Posterior Pituitary Gland

Abstract: The cyclic nucleotide phosphodiesterase (PDE) activities were studied in a nerve ending fraction from bovine neural lobes. Most of the activity was particulate and unaffected by calcium. Lineweaver-Burk plots for this fraction showed negative cooperativity with apparent K _m values for cyclic AMP of 11 μ M and for cyclic GMP of 4 μ M . The soluble activities for both cyclic nucleotides were activated by calcium and inhibited by calmodulin-binding drugs (trifluoperazine and calmidazolium). The apparent K _m values were 50 μ M for cyclic AMP and 20 μ M for cyclic GMP for the soluble activities. Sucrose density gradients resolved the soluble activities into two peaks. The activity with the higher sedimentation rate (MW 122,000 daltons) hydrolysed both cyclic nucleotides and was calcium-calmodulin-dependent. The other peak (MW 47,000 daltons) had a higher affinity for cyclic AMP than for cyclic GMP and was calcium-independent. Solubilized particulate activities gave two main peaks on the density gradient, both calcium-independent. One was mainly for cyclic AMP (MW 47,000 daltons) and the other mainly for cyclic GMP (MW 133,000 daltons). The function of PDEs in relation to secretion was discussed.     

Combined Test for Assessment of Anterior Pituitary Function

A combined test consisting of the simultaneous administration of insulin, thyrotrophin-releasing hormone (TRH), and luteinizing hormone and follicle stimulating hormone-releasing hormone (LH/FSH-RH) was performed in 24 people. Eleven of these also had the three individual tests performed separately, and the remaining 13 had a separate test of either LH/FSH-RH and TRH together or singly at a later date. In both normal people and patients, whether the tests were performed alone or in combination, no difference was found between the hormone responses (growth hormone, cortisol, LH, FSH, thyroid-stimulating hormone) seen to these stimuli.It is proposed that combined administration of insulin and the hypothalamic releasing hormones may be used as a single test for the assessment of anterior pituitary function. The test is convenient and time saving, and with care can be performed in outpatients.     

Cyclic Nucleotides and the Release of Vasopressin from the Rat Posterior Pituitary Gland

Abstract: The effect of ATP, Mg²⁺, or MgATP on the release of luteinizing hormone-releasing hormone (LH-RH) from hypothalamic granules was examined under in vitro conditions. Granules, isolated from adult male hypothalami, were incubated at 37°C in a buffered (pH 7.8) medium containing 0.15 m -KCl. The addition of ATP to the incubation mixture did not stimulate the release of LH-RH. In contrast, the addition of MgATP stimulated the release of LH-RH, the release being 62% greater than control. The addition of Mg²⁺ to the incubated granules also stimulated the release of LH-RH. However, the magnitude of this Mg²⁺-stimulated release of LH–RH was significantly ( P < 0.01) lower than that of the MgATP-stimulated release, indicating that ATP stimulates LH-RH release in a Mg²⁺-dependent manner. As both MgATP and Mg²⁺ alone stimulated LH-RH release, we characterized further these two release processes by incubating the granules under one of the following conditions: incubation at 4°C in a buffered medium containing 0.15 m -KCl or incubation at 37°C in a medium that does not contain KCl. Under these two incubation conditions, the MgATP-stimulated release of LH-RH was not manifested, whereas the Mg²⁺-stimulated release of LH-RH was manifested. On the basis of these differences, we propose that two different processes can lead to the release of LH-RH from isolated hypothalamic granules: one process involves ATP and Mg²⁺ (MgATP) and another process involves Mg²⁺ alone.     

ADAMs与哺乳动物受精

ADAMs是近几年发现的一类具有多个结构区和广泛生物学功能的糖蛋白,它们在哺乳动物受精中的作用日益得到实验结果的支持,本文简要总结了近几年ADAMs在哺乳动物受精中作用的研究进展。