Conservation of Hox gene clusters in the self-fertilizing fish Kryptolebias marmoratus (Cyprinodontiformes; Rivulidae).

In this study, whole Hox gene clusters in the self-fertilizing mangrove killifish Kryptolebias marmoratus (Cyprinodontiformes; Rivulidae), a unique hermaphroditic vertebrate in which both sex organs are functional at the same time, were identified from whole genome and transcriptome sequences. The aim was to increase the understanding of the evolutionary status of conservation of this Hox gene cluster across fish species.

Deviated nasal septum hinders intranasal sprays: a computer simulation study.

BACKGROUND: This study investigates how deviated nasal septum affects the quantity and distribution of spray particles, and examines the effects of inspiratory airflow and head position on particle transport. METHODS: Deposition of spray particles was analysed using a three-dimensional computational fluid dynamics model created from a computed tomography scan of a human nose with leftward septal deviation and a right inferior turbinate hypertrophy. Five simulations were conducted using FluentTM software, with particle sizes ranging from 20-110 μm, a spray speed of 3 m/s, plume angle of 68(deg), and with steady state inspiratory airflow either present (15.7 L/min) or absent at varying head positions. RESULTS: With inspiratory airflow present, posterior deposition on the obstructed side was approximately four times less than the contralateral side, regardless of head position, and was statistically significant. When airflow was absent, predicted deposition beyond the nasal valve on the left and right sides were between 16% and 69% lower and positively influenced by a dependent head position. CONCLUSION: Simulations predicted that septal deviation significantly diminished drug delivery on the obstructed side. Furthermore, increased particle penetration was associated with presence of nasal airflow. Head position is an important factor in particle deposition patterns when inspiratory airflow is absent.

Modulation of cognition and neuronal plasticity in gain- and loss-of-function mouse models of the schizophrenia risk gene Tcf4.

The transcription factor TCF4 was confirmed in several large genome-wide association studies as one of the most significant schizophrenia (SZ) susceptibility genes. Transgenic mice moderately overexpressing Tcf4 in forebrain (Tcf4tg) display deficits in fear memory and sensorimotor gating. As second hit, we exposed Tcf4tg animals to isolation rearing (IR), chronic social defeat (SD), enriched environment (EE), or handling control (HC) conditions and examined mice with heterozygous deletion of the exon 4 (Tcf4Ex4δ+/-) to unravel gene-dosage effects. We applied multivariate statistics for behavioral profiling and demonstrate that IR and SD cause strong cognitive deficits of Tcf4tg mice, whereas EE masked the genetic vulnerability. We observed enhanced long-term depression in Tcf4tg mice and enhanced long-term potentiation in Tcf4Ex4δ+/- mice indicating specific gene-dosage effects. Tcf4tg mice showed higher density of immature spines during development as assessed by STED nanoscopy and proteomic analyses of synaptosomes revealed concurrently increased levels of proteins involved in synaptic function and metabolic pathways. We conclude that environmental stress and Tcf4 misexpression precipitate cognitive deficits in 2-hit mouse models of relevance for schizophrenia.

Mutations in a cyclic nucleotide-gated channel lead to abnormal thermosensation and chemosensation in C. elegans.

The C. elegans tax-4 mutants are abnormal in multiple sensory behaviors: they fail to respond to temperature or to water-soluble or volatile chemical attractants. We show that the predicted tax-4 gene product is highly homologous to vertebrate cyclic nucleotide-gated channels. Tax-4 protein expressed in cultured cells functions as a cyclic nucleotide-gated channel. The green fluorescent protein (GFP)-tagged functional Tax-4 protein is expressed in thermosensory, gustatory, and olfactory neurons mediating all the sensory behaviors affected by the tax-4 mutations. The Tax-4::GFP fusion is partly localized at the sensory endings of these neurons. The results suggest that a cyclic nucleotide-gated channel is required for thermosensation and chemosensation and that cGMP is an important intracellular messenger in C. elegans sensory transduction.

Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming.

Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering, priming to fusion competence, and Ca2+-triggered fusion are taking place in a highly coordinated manner. We show that the active zone components Munc13-1, an essential vesicle priming protein, and RIM1, a Rab3 effector with a putative role in vesicle tethering, interact functionally. Disruption of this interaction causes a loss of fusion-competent synaptic vesicles, creating a phenocopy of Munc13-1-deficient neurons. RIM1 binding and vesicle priming are mediated by two distinct structural modules of Munc13-1. The Munc13-1/RIM1 interaction may create a functional link between synaptic vesicle tethering and priming, or it may regulate the priming reaction itself, thereby determining the number of fusion-competent vesicles.

Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2).

Glutamate is the major excitatory neurotransmitter in mammalian CNS. In the presynaptic nerve terminal, glutamate is stored in synaptic vesicles and released by exocytosis. Previously, it has been shown that a transport protein originally identified as a brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) functions as vesicular glutamate transporter and thus has been renamed VGLUT1. Recently, a protein highly homologous to VGLUT1, "differentiation-associated BNPI" (DNPI), has been discovered. Northern blot and in situ hybridization analyses indicate that DNPI mRNA is expressed in some brain regions in which VGLUT1 mRNA is not expressed. We now show that DNPI functions as vesicular glutamate transporter with properties very similar to VGLUT1 and propose to rename the protein VGLUT2. VGLUT2 is highly enriched in synaptic vesicles. Furthermore, VGLUT2 resides on a vesicle population that is distinct from vesicles containing the vesicular GABA transporter or VGLUT1, showing that the expression of VGLUT1 and VGLUT2 do not overlap. When VGLUT2 was expressed in BON cells, membrane fractions displayed ATP-dependent, carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive glutamate uptake. Overexpression of VGLUT2 in cultured autaptic GABAergic neurons yielded postsynaptic currents that were insensitive to the GABA(A) receptor antagonist bicuculline but blocked by the AMPA-receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[F]quinoxaline. Thus, expression of VGLUT2 suffices to cause GABAergic neurons to release glutamate in addition to GABA in a manner very similar to that reported previously for VGLUT1.

Substance P abolishes the facilitatory effect of ATP on spontaneous glycine release in neurons of the trigeminal nucleus pars caudalis.

Glycine release was facilitated by the activation of presynaptic ATP receptors (P(2X)-type) in a preparation of dissociated trigeminal nucleus pars caudalis neurons in which the native synaptic boutons were preserved. The action of ATP was completely blocked by substance P (SP) without alteration of the miniature IPSC (mIPSC) amplitude distribution. SP itself had no effect on mIPSC frequency or amplitude. The inhibitory effect of SP on ATP action was blocked by CP99994, indicating that the SP receptors are of the neurokinin-1 type. The ATP-induced facilitation of the mIPSC frequency was unaffected by Cd(2+). Moreover, SP did not inhibit the increase in mIPSC frequency induced high K(+) application, suggesting that SP did not modulate voltage-dependent calcium channels or subsequent steps in the release process. KT5720 and phorbol 12-myristate 13-acetate did not block SP action, indicating that neither the cAMP-protein kinase A nor the protein kinase C pathway mediates the SP effects. However, in the presence of N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7), SP was no longer able to inhibit the ATP-induced stimulation of mIPSC frequency. 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine also suppressed the SP action, suggesting that SP modulates P(2X) receptors via a Ca(2+)/calmodulin-dependent protein kinase II-mediated pathway. In conventional whole-cell mode, the presence of W-7 in the patch pipette did not affect the SP inhibitory action. Thus, SP is not likely to be generating its modulation through the production of a retrograde signal (involving calmodulin) from the postsynaptic cell to the presynaptic boutons. These results are the first demonstration of the modulation of one presynaptic receptor by another. Because SP inhibits the ATP stimulation of glycine release, SP may play a significant role in hyperalgesia or chronic pain.

Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution.

We attempted to apply the directed evolution approach to enhancing enzyme properties in the presence of organic solvents, in which enzyme stability and activity were often drastically reduced. Stability and catalytic activity of phospholipase A(1) in the presence of an organic solvent were enhanced by error-prone polymerase chain reaction (PCR) and DNA shuffling followed by a filter-based visual screening. Three mutants (SA8, SA17 and SA20) were isolated on indicator plates (i.e., 1% phosphatidylcholine gels containing 30% dimethyl sulfoxide (DMSO)) after a second mutant library was treated in 50% DMSO for 36 h. The half-life values of the three mutants exhibited an approximately 4-fold increase. The three mutants also exhibited increased stability in all organic solvents tested compared with the wild-type enzyme. Thus, an enzyme variant having superior catalytic efficiency in most of the organic solvents could be obtained by using any solvent suitable for designing the efficient screening system, regardless of the properties of the particular solvent.

Purification and characterization of Pseudomonas fluorescens SIK W1 lipase expressed in Escherichia coli.

Pseudomonas fluorescens SIK W1 lipase was expressed as a form of inclusion bodies in Escherichia coli, which was equivalent to 46% of total cell protein. The inclusion bodies isolated from other cell components were solubilized in the buffer containing 8 M urea and then refolded by diluting urea. The lipase with active conformation was purified by hydrophobic interaction chromatography, gel filtration, anion-exchange chromatography and hydroxyapatite chromatography from the refolded sample. By these purification steps, a single band for active lipase was detected on non-reducing SDS-PAGE and 10-fold purification was attained on the basis of specific activity. Specific activity of the purified lipase toward olive oil emulsion was found to be 7395 units per mg protein. The optimum pH and temperature of the lipase were pH 8.5 and 45-55 degrees C, respectively. The lipase showed higher lipolytic activity toward tricaproin (C6) and tricaprylin (C8) among the triacylglycerols examined and preferentially hydrolyzed ester bond of 1- and 3-position of triolein. Lipase activity was greatly increased by approx. 6-fold and stability for pH was shifted to alkaline pH by Ca2+ ion. The lipase was inhibited by Hg2+, Ag2+, p-chloromercuribenzoate, diethylpyrocarbonate and sodium dodecyl sulfate.

Structure and function of the photoreceptor stentorins in Stentor coeruleus. I. Partial characterization of the photoreceptor organelle and stentorins.

The unicellular ciliary protozoan, Stentor coeruleus, exhibits photophobic and phototactic responses to visible light stimuli. The pigment granule contains the photoreceptor chromoproteins (stentorins). Stentorin localized in the pigment granules of the cell serves as the primary photoreceptor for the photophobic and phototactic responses in this organism. An initial characterization of the pigment granules has been described in terms of size, absorbance spectra and ATPase activity. Two forms of the stentorin pigments have been isolated from the pigment granules. Stentorin I has an apparent molecular weight of 68,600 and 52,000 by SDS-PAGE (at 10 and 13% gel, respectively) or 102,000 by steric exclusion HPLC, whereas stentorin II is a larger molecular assembly probably composed of several proteins (mol. wt. greater than 500,000). Stentorin I is composed of at least two heterologous subunits corresponding to apparent mol. wts. of 46,000 (fluorescent, Coomassie blue negative) and 52,000 (fluorescent, Coomassie blue positive) on SDS-PAGE (13% gel). However, these values were found to be strongly dependent on the degree of crosslinking in the acrylamide gel. Stentorin II appears to be the primary photoreceptor whose absorption and fluorescence properties are consistent with the action spectra for the photoresponses of the ciliate to visible light.

Serotonin modulates high-voltage-activated Ca2+ channels in rat ventromedial hypothalamic neurons.

The modulation of high-voltage-activated (HVA) Ca2+ channels by serotonin (5-HT) was studied in ventromedial hypothalamic (VMH) neurons acutely dissociated from 12-14-day-old Wistar rats using the whole-cell and nystatin perforated-patch recording configurations. 5-HT inhibited the HVA Ca2+ channels in a concentration-, time- and voltage-dependent manner. This inhibition was mimicked by the 5-HT1A agonist 8-hydroxy-dipropylaminotetralin and was prevented by pretreatment with pertussis toxin (PTX). omega-Conotoxin-GVIA, omega-agatoxin-IVA, nicardipine and omega-conotoxin-MVIIC blocked each fraction of HVA Ca2+ channel currents, suggesting the existence of N-, P-, L- and Q-types of HVA Ca2+ channels. A component of the current resistant to these Ca2+ channel antagonists also existed in the VMH neurons. Among these five components of HVA Ca2+ channel currents, the N- and Q-type currents were significantly inhibited by 5-HT. These findings suggest that the activation of 5-HT1A receptors produces the selective inhibition of N- and Q-type Ca2+ channels through a PTX-sensitive G-protein in rat VMH neurons.

Alpha2-adrenoceptor-mediated enhancement of glycine response in rat sacral dorsal commissural neurons.

The effect of noradrenaline on the glycine response was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus using nystatin perforated patch recording configuration under voltage-clamp conditions. Noradrenaline reversibly potentiated the 10(-5)M glycine-induced Cl- current in a concentration-dependent manner. Single channel recordings in a cell-attached mode revealed that noradrenaline decreased the closing time of the glycine-activated channel activity. Noradrenaline neither changed the reversal potential of the glycine response nor affected the affinity of glycine to its receptor. Clonidine mimicked and yohimbine blocked the noradrenaline action on glycine response. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride, protein kinase A inhibitor, mimicked the effect of noradrenaline on glycine response. Noradrenaline failed to affect the glycine response in the presence of these intracellular cyclic AMP and protein kinase A modulators. However, noradrenaline further enhanced the glycine response even in the presence of phorbol-12-myristate-13-acetate and chelerythrine, a protein kinase C inhibitor. Pertussis toxin treatment for 6-8 h blocked the noradrenaline facilitatory effect on the glycine response. In addition, noradrenaline potentiated the strychnine-sensitive postsynaptic currents evoked in a slice preparation of sacral dorsal commissural nucleus. These results suggest that the activation of alpha2-adrenoceptor by noradrenaline coupled with pertussis toxin-sensitive G-proteins reduces intracellular cyclic AMP formation through the inhibition of adenyl cyclase. The reduction of cyclic AMP decreases the protein kinase A activity, thus resulting in the potentiation of the glycinergic inputs to the sacral dorsal commissural neurons. It is thus feasible that the noradrenergic input to the sacral dorsal commissural nucleus modulates such nociceptive signals as pain by intracellular enhancing the glycine response.

Mu-opioid agonist-induced activation of G-protein-coupled inwardly rectifying potassium current in rat periaqueductal gray neurons.

The characteristics of the inwardly rectifying K+ current activated by a mu-type opioid agonist, D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO), were examined in the acutely dissociated rat periaqueductal gray neurons using the nystatin-perforated and the conventional whole-cell recording modes under voltage-clamp conditions. DAMGO activated inward currents in a concentration- and voltage-dependent manner. The DAMGO-induced current was an inwardly rectifying K+ current (I(DAMGO)) which was sensitive to K+ channel blockers, quinine and Ba2+ but insensitive to Cs+ and tetraethylammonium. In the conventional whole-cell clamp mode, guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDPbetas, 0.4 mM) inhibited the amplitude of I(DAMGO) to 28% of that of the initial current. After the intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTPgammas, 0.4 mM) for 1 min, the first application of DAMGO irreversibly activated I(DAMGO). By the extracellular application of N-ethylmaleimide at a concentration of 50 microM for 2 min, I(DAMGO) was completely abolished. When a conventional whole-cell patch was made with a patch-pipette containing 1 microg/ml of pertussis toxin together with 1 mM of beta-nicotinamide adenine dinucleotide, I(DAMGO) gradually declined to about 41% of its initial amplitude. The extracellular application of second messenger modulators including protein kinase inhibitor (staurosporin), protein kinase A activators (forskolin, 3-isobutyl-l-methyl-xanthine and dibutyryladenosine 3'5'-cyclic monophosphate) and protein kinase C activators (phorbol-12-myristate-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol) had no effect on I(DAMGO). These results suggest that (i) DAMGO-activated inwardly rectifying K+ current is mediated by pertussis toxin-sensitive guanine nucleotide binding proteins (G-proteins); (ii) the types of G protein involved in I(DAMGO) are Gi and/or Go; and (iii) the G-proteins exert their roles in I(DAMGO) without any mediation of the second messenger systems.

Effect of nilvadipine on high-voltage activated Ca2+ channels in rat CNS neurons.

The effect of nilvadipine, a dihydropyridine derivative, on high-voltage activated (HVA) Ca2+ channels was investigated in freshly dissociated rat frontal cortical neurons. The cortical neurons were observed to have five pharmacologically distinct HVA Ca2+ channel subtypes consisting of the L-, N-, P-, O- and R-types. Nilvadipine selectively inhibited the L-type Ca2+ channel current which comprised 23% of the total HVA Ca2+ channel current. A reversible inhibitory effect of nilvadipine on the L-type Ca2+ channel current was also observed in a concentration-dependent fashion without affecting the current-voltage relationship. The half-maximum inhibitory concentration was 3 x 10(-8) M. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca2+ influx, in combination with the cerebral vasodilatory action, may prevent neuronal damage during brain ischemia.

Regional difference of high voltage-activated Ca2+ channels in rat CNS neurones.

The diversity of high voltage-activated (HVA) Ca2+ channels in rat CNS neurones was investigated with the nystatin perforated patch recording configuration. The neurones were freshly dissociated from rat substantia nigra, ventromedial hypothalamus, tuberomammillary nucleus, nucleus tractus solitarius, hippocampal CA1 region and cerebellum. Five different types of HVA Ca2+ channels were distinguished pharmacologically; dihydropyridine sensitive L-type, omega-conotoxin-GVIA sensitive N-type, omega-agatoxin-IVA sensitive P-type, omega-conotoxin-MVIIC sensitive Q-type, and R-type which is insensitive to these organic Ca2+ antagonists. The results showed clearly that the five subtypes of HVA Ca2+ channels differ considerably in their distribution among various CNS regions.

Interaction between the intracellular Ca2+ stores in rat dissociated hippocampal neurones.

The interaction between the Ca(2+)-activated K+ currents (IK(Ca)) induced by acetylcholine (ACh), 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R)-ACPD) and caffeine in rat hippocampal CA1 neurones was investigated to examine the functional independence between IP3- and Ca(2+)-sensitive Ca2+ stores. Pretreatment with either ACh or (1S,3R)-ACPD almost completely suppressed the subsequent IK(Ca) induced by another. Pretreatment with caffeine partially suppressed the IK(Ca) induced by either ACh or (1S,3R)-ACPD, although the inhibitory effect varied among the neurones, while pretreatment with either ACh or (1S,3R)-ACPD also partially inhibited the caffeine-induced IK(Ca). Continuous treatment with ryanodine and thapsigargin nonselectively decreased both the caffeine- and ACh-induced IK(Ca). These results thus indicate a close interaction between IP3- and Ca(2+)-sensitive stores and also suggest that the relative size of both stores and the closeness between them differ among neurones.

Developmental changes of GABA(A) receptor-chloride channels in rat Meynert neurons.

The developmental changes of GABA(A) receptors were investigated in Meynert neurons freshly dissociated from day 0, 2 week-, and 6 month-old rats using both nystatin and gramicidin perforated patch recording modes under voltage-clamp conditions. The age-related changes in the current amplitude and threshold concentration in the concentration-response relationships for GABA indicated the developmental alteration of the GABA(A) receptor subunits and the channel density. The GABA-induced E(Cl-) measured by the gramicidin perforated patch mode shifted to more negative with development. The decay time constant of GABAergic inhibitory postsynaptic spontaneous currents (sIPSCs) in the synaptic active zone accelerated with aging. The GABA-induced currents were potentiated in a concentration dependent manner in the presence of benzodiazepine (BZP) agonists, diazepam (DZP) and zolpidem (ZPM). The potentiation rate of DZP on the GABA(A) response decreased with aging, but not in the case of ZPM, which demonstrated a stronger action in the aging rat neurons. These results suggested that the GABA(A) receptor x Cl- channel complexes may thus change both the assembly and interaction of subunits as well as their functional roles with aging.

Modulation of glycine-induced chloride current in acutely dissociated rat periaqueductal gray neurons by mu-opioid agonist DAGO.

Effect of a mu-opioid agonist (D-Ala2,N-MePhe4,Gly5-ol-enkephalin, DAGO), on glycine (Gly)-induced chloride current (IGly) was investigated in the periaqueductal gray (PAG) neurons acutely dissociated 1-2-week-old Wistar rats by the use of nystatin-perforated patch recording configuration under voltage-clamp condition. At a holding potential (VH) of -40 mV, DAGO caused a sustained potentiation of IGly at the low concentrations (10(-6)-10(-5) M) but reduced slightly the Gly response at the high concentration (10(-4) M). The reversal potential of IGly was equal to the Cl- equilibrium potential (ECl) and was not changed in the presence of 10(-6) M DAGO. The 10(-5) M Gly response was inhibited by the simultaneous treatment of forskolin and 3-isobutyl-1-methylxanthine (IBMX). H-89, a protein kinase A (PKA) inhibitor, increased the 10(-5) M Gly response but had little effect on the 10(-4) M Gly response. DAGO increased 10(-5) M Gly response in the presence of forskolin and IBMX but, not more than in the absence of forskolin and IBMX. The 10(-5) M Gly response augumented by DAGO was not affected by adding H-89. The present results suggest that the glycine-induced chloride current is cAMP dependent and is inhibited by PKA, and that the potentiation of the glycine response by DAGO is also cAMP dependent and is due to the inhibition of PKA as that of H-89. We conclude that the potentiation of glycine response by DAGO is mediated by an inhibition of cAMP-dependent PKA in the PAG neurons.

Excitatory effect of Cd2+ on cat adrenal chromaffin cells.

To understand the mechanism(s) underlying the Cd2+- and Co2+-induced increases in the cytosolic free Ca2+ concentration ([Ca]i) in cat adrenal chromaffin cells, we used nystatin-perforated patch recording method and fura-2 microfluorometry. Under the current-clamp conditions, the external application of 5x10(-7) M Cd2+ slowly depolarized the cells resulting in the bursting of action potentials. Under the voltage-clamp conditions, Cd2+ evoked a slow inward current accompanied by a decrease of K+ conductance at a holding potential of -40 mV, and Co2+ mimicked Cd2+ action. In some cells (16%), Cd2+ evoked an additional rapid transient outward current associated with an increased K+ conductance and a successive slow inward current. The Cd2+-induced inward current was activated in a concentration-dependent manner with a half-maximum concentration of 9.3x10(-8) M. The Cd2+- and Co2+-induced [Ca]i increases measured with fura-2 microfluorometry were maximal at 10(-6) and 10(-5) M, respectively, and the higher concentrations of both cations caused the smaller responses. Additional transient increase in [Ca]i was often evoked upon the removal of relatively higher concentrations of these metals. It was concluded that the Cd2+-induced membrane depolarization due to the decrease in K+ conductances evoked the bursting firings resulting in the increase in [Ca]i, and consequently might stimulate the catecholamine secretion.