Carbenoxolone-sensitive and cesium-permeable potassium channel in the rod cells of frog taste discs

The rod cells in frog taste discs display the outward current and maintain the negative resting potential in the condition where internal K+ is replaced with Cs+. We analyzed the properties of the Cs+-permeable conductance in the rod cells. The current-voltage (I/V) relationships obtained by a voltage ramp were bell-shaped under Cs+ internal solution. The steady state I/V relationships elicited by voltage steps also displayed the bell-shaped outward current. The activation of the current accelerated with the depolarization and the inactivation appeared at positive voltage. The gating for the current was maintained even at symmetric condition (Cs+ external and internal solutions). The wing cells did not show the properties. The permeability for K+ was a little larger than that for Cs+. Internal Na+ and NMDG+ could not induce the bell-shaped outward current. Carbenoxolone inhibited the bell-shaped outward Cs+ current dose dependently (IC50: 27μM). Internal arachidonic acid (20μM) did not induce the linear current-voltage (I-V) relationship which is observed in two-pore domain K+ channel (K2P). The results suggest that the resting membrane potentials in the rod cells are maintained by the voltage-gated K+ channels

Reconstituted Ion Channels of Frog Fungiform Papilla Cell Membrane.

We identified a Cl^- channel, two K^+ channels and a cAMP-gated channel which were isolated from bullfrog fungiform papilla cell membranes and incorporated into phospholipid bilayers using the tip-dip method. The 156 pS channels were inhibited by 100μM 4, 4\u27-diisothiocyanostilbene-2, 2\u27-disulfonic acid (DIDS) and displayed the reversal potential identical to the equilibrium potential of Cl^-, it was identified as a Cl^- channel. Two types of K^+ channel had unitary conductances of 79 and 43 pS, which may correspond to those of Ca^-activated and cAMP-blockable K^+ channels observed in isolated intact frog taste cell membranes, respectively. These results suggest that the tip-dip method is useful for stable investigation of the properties of ion channels already identified in the taste cell. Furthermore, the 23 pS channels were newly found and were activated directly by internal cAMP as cyclic nucleotide-gated (CNG) nonselective cation channels established in olfactory receptor cells. Thus, our results suggest the possibility that besides Cl^- and K^+ channels, the cAMP-gated channels contribute to taste transduction

High Extracellular Ca2+ Stimulates Ca2+-Activated Cl− Currents in Frog Parathyroid Cells through the Mediation of Arachidonic Acid Cascade

Elevation of extracellular Ca2+ concentration induces intracellular Ca2+ signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca2+ pathways, but the direct mechanism responsible for the rise of intracellular Ca2+ concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca2+ signaling in frog parathyroid cells and show that Ca2+-activated Cl− channels are activated by intracellular Ca2+ increase through an inositol 1,4,5-trisphophate (IP3)-independent pathway. High extracellular Ca2+ induced an outwardly-rectifying conductance in a dose-dependent manner (EC50∼6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca2+-induced and Ca2+ dialysis-induced currents reversed at the equilibrium potential of Cl− and were inhibited by niflumic acid (a specific blocker of Ca2+-activated Cl− channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca2+-induced current, suggesting the change of intracellular Cl− concentration in a few minutes. Extracellular Ca2+-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca2+-induced current. IP3 dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG), arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE) dialysis increased the conductance identical to extracellular Ca2+-induced conductance. These results indicate that high extracellular Ca2+ raises intracellular Ca2+ concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl− conductance

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Wing (Ib) cells in frog taste discs detect dietary unsaturated fatty acids

The effects of unsaturated fatty acids on membrane properties were studied using conventional whole-cell patch-clamp recording of isolated wing (Ib) cells in bullfrog (Lithobates catesbeianus) taste discs. Applying arachidonic acid to the bath induced monophasic inward currents in 60% of wing cells and biphasic inward and outward currents in the other cells. The intracellular dialysis of arachidonic acid did not induce an inward current; however, it enhanced a slowly developing Ba2+-sensitive outward current. The effects of various unsaturated fatty acids were explored under the condition of Cs+ internal solution. Linoleic and α-linolenic acids induced large inward currents. Oleic, eicosapentaenoic and docosahexaenoic acids elicited the same inward currents as those of arachidonic acid. Wing cells, under the basal condition with Cs+ internal solution, displayed a small inward current of -1.1±0.1pA/pF at -50mV (n=40), in which the peak existed at a membrane potential of -49mV. Removing external Ca2+ further increased the inward current by -2.9±0.3pA/pF at -50mV (n=4) from the basal current and the peak was located at -55mV. External linoleic acid (50μM) also induced a similar inward current of -5.6±0.6pA/pF at -50mV (n=19) from the basal current and the peak was located at -61mV. External Ca2+-free saline and linoleic acid induced similar current/voltage (I/V) relationships elicited by a ramp voltage as well as voltage steps. Linoleic acid-induced currents were not influenced by replacing internal EGTA with BAPTA, whereas inward currents disappeared under the elimination of external Na+ and addition of flufenamic acid. These results suggest that dietary unsaturated fatty acids may depolarize wing (Ib) cells, which affects the excitability of these cells

Ictal central apnea and bradycardia in temporal lobe epilepsy complicated by obstructive sleep apnea syndrome

We describe the case of a 12-year-old boy who developed temporal lobe epilepsy (TLE) with daily complex partial seizures (CPS) and monthly generalized seizures. Moreover, he frequently snored while asleep since early childhood. Polysomnography (PSG) revealed severe obstructive sleep apnea with apnea–hypopnea index (AHI) of 37.8/h. Video-PSG with simultaneous electroencephalography (EEG) recording captured two ictal apneic episodes during sleep, without any motor manifestations. The onset of rhythmic theta activity in the midtemporal area on EEG was preceded by the onset of apnea by several seconds and disappeared soon after cessation of central apnea. One episode was accompanied by ictal bradycardia of <48 beats/min which persisted for 50 s beyond the end of epileptic activity. After treatment with carbamazepine and tonsillectomy/adenoidectomy, the seizures were well controlled and AHI decreased to 2.5/h. Paroxysmal discharges also disappeared during this time. Uncontrolled TLE complicated by sleep apnea should be evaluated for the presence of ictal central apnea/bradycardia

Intracellular free calcium concentration in human taste bud cells increases in response to taste stimuli

AbstractWe examined changes of intracellular free calcium concentration [Ca2+]i elicited by taste stimuli of sucrose, denatonium and NaCl in the taste buds of seven human fungiform papillae. In one taste bud we observed an increase in [Ca2+]i induced by only NaCl. In another bud an increase of [Ca2+]i in response to both NaCl and sucrose was found. The Ca2+ responses to NaCl and sucrose occurred in differential areas within the one taste bud. In the other five fungiform papillae [Ca2+]i was not changed by the taste stimuli. These results suggest that an increase of [Ca2+]i participates in taste transduction mechanisms for sucrose and NaCl, and that taste cells in one taste bud may respond to differential stimuli

Effect of additives on protein solubility in HeLa cell TRIzol lysates.

<p>The solubility of proteins remaining after addition of nucleic acids (<b>A</b>), sodium chloride (<b>B</b>) or carbohydrates (<b>C</b>) was determined.</p

Analysis of total cell protein lysates.

<p><b>A.</b> SDS-PAGE of total cell proteins lysed with 8 M urea or precipitated from the organic phase of TRIzol homogenates (an equivalent number of HeLa cells were used in each case). Endogenous proteins were also analyzed using western blotting. <b>B.</b> SDS-PAGE of total cell protein TRIzol and GdnHCl lysates. Equivalent amounts of protein in soluble fractions (S) and precipitates (P) following centrifugation were loaded. <b>C.</b> SDS-PAGE of TRIzol lysates prepared from <i>E. coli</i> BL21(DE3) expressing or not expressing human β-actin.</p