Rat brain serotonin receptors in Xenopus oocytes are coupled by intracellular calcium to endogenous channels.

Serotonin activates chloride currents in Xenopus oocytes injected with a subfraction of rat brain poly(A)+ mRNA. Patch-clamp recordings from cell-attached patches showed that serotonin, applied locally outside the patch, caused the opening of channels of approximately equal to 3 pS conductance and an average lifetime of approximately equal to 100 msec. The extrapolated reversal potential indicated that the channels are chloride-selective. Single-channel currents with similar characteristics were observed in inside-out patches from native oocytes in response to elevated calcium concentrations on the cytoplasmic side. Measurements of intracellular calcium concentration ([Ca2+]i) by fura-2 fluorescence showed approximately equal to 10-fold increases in [Ca2+]i in response to serotonin application in both normal and calcium-free Ringer solution in mRNA-injected oocytes. Little or no response to serotonin was observed in native oocytes. These results suggest that serotonin activation of receptors that are inserted into the oocyte membrane following injection of rat brain poly(A)+ mRNA can induce calcium release from intracellular stores. The increase in [Ca2+]i subsequently activates calcium-dependent chloride channels. Because calcium-dependent chloride channels and a receptor-controlled mechanism of internal calcium release have been shown to exist in native oocytes, we conclude that the newly inserted serotonin receptors utilized the endogenous second-messenger-mediated calcium release to activate endogenous calcium-dependent chloride channels.     

Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I

Synaptotagmin I is a synaptic vesicle-associated protein essential for synchronous neurotransmission. We investigated its impact on the intracellular Ca(2+)-dependence of large dense-core vesicle (LDCV) exocytosis by combining Ca(2+)-uncaging and membrane capacitance measurements in adrenal slices from mouse synaptotagmin I null mutants. Synaptotagmin I-deficient chromaffin cells displayed prolonged exocytic delays and slow, yet Ca(2+)-dependent fusion rates, resulting in strongly reduced LDCV release in response to short depolarizations. Vesicle recruitment, the shape of individual amperometric events, and endocytosis appeared unaffected. These findings demonstrate that synaptotagmin I is required for rapid, highly Ca(2+)-sensitive LDCV exocytosis and indicate that it regulates the equilibrium between a slowly releasable and a readily releasable state of the fusion machinery. Alternatively, synaptotagmin I could function as calcium sensor for the readily releasable pool, leading to the destabilization of the pool in its absence.     

Regulation of a subset of release-ready vesicles by the presynaptic protein Mover

Neurotransmitter release occurs by regulated exocytosis from synaptic vesicles (SVs). Evolutionarily conserved proteins mediate the essential aspects of this process, including the membrane fusion step and priming steps that make SVs release-competent. Unlike the proteins constituting the core fusion machinery, the SV protein Mover does not occur in all species and all synapses. Its restricted expression suggests that Mover may modulate basic aspects of transmitter release and short-term plasticity. To test this hypothesis, we analyzed synaptic transmission electrophysiologically at the mouse calyx of Held synapse in slices obtained from wild-type mice and mice lacking Mover. Spontaneous transmission was unaffected, indicating that the basic release machinery works in the absence of Mover. Evoked release and vesicular release probability were slightly reduced, and the paired pulse ratio was increased in Mover knockout mice. To explore whether Mover’s role is restricted to certain subpools of SVs, we analyzed our data in terms of two models of priming. A model assuming two SV pools in parallel showed a reduced release probability of so-called “superprimed vesicles” while “normally primed” ones were unaffected. For the second model, which holds that vesicles transit sequentially from a loosely docked state to a tightly docked state before exocytosis, we found that knocking out Mover selectively decreased the release probability of tight state vesicles. These results indicate that Mover regulates a subclass of primed SVs in the mouse calyx of Held.

Synaptic transmission is initiated by exocytosis of neurotransmitters from presynaptic nerve terminals. Exocytosis involves tethering of synaptic vesicles (SVs) at release sites, priming to make SVs release-competent, and membrane fusion mediated by Synaptotagmins and SNAREs (1, 2). SVs in the release-competent state constitute the readily releasable pool (RRP) (3). Presynaptic proteins, such as Munc13s, Munc18s, and CAPS, are essential for generating the RRP and for regulating replenishment of this pool during short-term plasticity (4).Historically, the RRP had been regarded as a uniform set of primed SVs. But more recent evidence suggested that a fraction of SVs in the RRP might be more prone to undergoing exocytosis than others, and these SVs were dubbed “superprimed.” In this view, both primed and superprimed SVs are release-competent, albeit with different kinetics (5–7). More recently, evidence emerged that the primed states may be dynamic, reversible, and activity-dependent (8, 9) and they may correlate with morphological features of the synapse (10, 11). Furthermore, molecular evidence indicates that priming proceeds in distinct, reversible steps (12). These reports gave rise to a model which holds that primed SVs fluctuate between a loosely docked state (LS), in which SNARE complexes are partially zippered, and a tightly docked state (TS), in which SNARE zippering has progressed further. In this model, release predominantly occurs from TS vesicles; the transition from LS to TS is slow at rest but can occur rapidly in the presence of Ca²⁺, on a millisecond time scale (13).Exocytosis is abolished when components of the core machinery are perturbed, such as Munc13s, Munc18s, or Synaptotagmin-1. However, it is unknown whether there are proteins specifically fine-tuning parts of the priming process. Unlike most presynaptic proteins, the SV protein Mover is not expressed in all species and all synapses (14, 15). Mover is thus a prime candidate for modulating the ubiquitous release machinery. Here, we have tested the role of Mover for modulating synaptic transmission at the calyx of Held. Our data indicate that Mover regulates the release probability (p_r) of a subset of primed SVs.     

Ras-associated small GTPase 33A, a novel T cell factor, is down-regulated in patients with tuberculosis

Ras-associated small GTPases (Rabs) are specific regulators of intracellular vesicle trafficking. Interference with host cell vesicular transport is a hallmark of many intracellular pathogens, including the notable example Mycobacterium tuberculosis. We performed, by quantitative polymerase chain reaction, gene-expression analyses for selected Rab molecules in peripheral-blood mononuclear cells from patients with tuberculosis (TB) and healthy control subjects, to identify candidate genes that are critically involved in the host immune response. Comparison revealed significant differences in the expression of genes for Rab13, Rab24, and Rab33A. Rab33A gene expression was down-regulated in patients with TB and was predominantly expressed in CD8+ T cells. We excluded possible influences of differences in T cell percentages between the 2 study groups, demonstrating that Rab33A gene expression changes on the single-cell level. In vitro, Rab33A RNA expression was induced in T cells on activation and by dendritic cells infected with M. tuberculosis. Our findings identify Rab33A as a T cell regulatory molecule in TB and suggest its involvement in disease processes.     

Single channel activity associated with the calcium dependent outward current inHelix pomatia

A recently improved version of the extracellular patch clamp technique (9, 13) was used to record currents from microscopic membrane areas of Helix neurons with predominant Ca²⁺ dependent outward currents. Current fluctuations in the patches consisted mainly of frequently interrupted, one-sided steps indicating discrete open-closed state changes of single channels with an ohmic conductance of approximately 19 pS. Frequency of occurrence of the elementary events compares with amplitudes of macroscopic currents during depolarizing voltage steps of varied amplitude. Average delays in appearance of the events vary in line with delayed time courses of the cell's outward current.     

Antitumor activity of CTFB, a novel anticancer agent, is associated with the down-regulation of nuclear factor-kappaB expression and proteasome activation in head and neck squamous carcinoma cell lines

This study aimed to characterize the antitumor activity of 5-Chloro-N-[2-[2-(4-chloro-phenyl)-3-methyl-butoxy]-5-trifluoromethyl-phenyl]-2-hydroxy-benzamide (CTFB), a novel anticancer agent, in head and neck cancer cell lines, FaDu, SCC-25 and cisplatin-resistant CAL-27. CTFB was generated as a result of an extensive medicinal chemistry effort on a lead compound series discovered in a high-throughput screen for inducers of apoptosis. All cell lines showed significant growth delay in response to CTFB treatment at a concentration of 1 micromol/L with 17.16 +/- 2.08%, 10.92 +/- 1.22%, and 27.03 +/- 1.86% of cells surviving at 120 h in FaDu, CAL-27, and SCC-25, respectively. To define proteins involved in the mechanism of action of CTFB, we determined differences in the proteome profile of cell lines before and after treatment with CTFB using two-dimensional difference gel electrophoresis followed by computational image analysis and mass spectrometry. Eight proteins were found to be regulated by CTFB in all cell lines. All these proteins are involved in cytoskeleton formation and function and/or in cell cycle regulation. We showed that CTFB-induced cell growth delay was accompanied by cell cycle arrest at the G(0)-G(1) phase that was associated with the up-regulation of p21/WAF1 and p27/Kip1 expression and the down-regulation of cyclin D1. Furthermore, we showed that activity of CTFB depended on the down-regulation of nuclear factor-kappaB (NF-kappaB) and NF-kappaB p65 phosphorylated at Ser(536). The level of proteasome activity correlated with the response to CTFB treatment, and the down-regulation of NF-kappaB is accompanied by enhanced proteasome activity in all investigated head and neck cancer cell lines. In this report, we show that CTFB reveals multiple effects that lead to delayed cell growth. Our data suggest that this compound should be studied further in the treatment of head and neck cancer.     

Doctor-patient discussions of alternative medicine for back pain

OBJECTIVE: To document the frequency of conversations about alternative medicine during primary care consultations for back pain in diverse settings. DESIGN: "Exit interview" type patient survey. SETTINGS: General practices in Seattle, Washington; rural Israel; and Birmingham, England. PATIENTS: A convenience sample of 218 adults completing a doctor visit for back pain. MAIN OUTCOME MEASURES: Frequencies of doctor-patient discussions of alternative medicine. RESULTS: Alternative medicine was discussed in a minority of visits (US site 40%, Israel site 37%, UK site 14%, p < 0.05). At each site, patients initiated at least half of the discussions. Users were five to six times more likely to discuss alternative medicine with their doctor than non-users (p < 0.05 for comparison at each site). The percentage of patients who used alternative medicine but left the consultation without discussing it was similar at all sites (US site 17%, Israel site 23%, UK site 15%). CONCLUSIONS: Discussions of alternative medicine occurred in a minority of consultations for back pain although the rate varied considerably by site. Discussions were initiated primarily by patients who use it.