CNV-like potentials on the cortical surface associated with conditioning in head-restrained rats

Head-restrained rats were conditioned to perform a CNV task: to press a lever in response to an imperative auditory stimulus (S2) given 1.5 sec after a warning stimulus (S1) for a drop of jelly food. With an electrode on the surface of the forelimb cortex, (1) sharp wave complexes immediately after S1 and S2, and (2) a negative slow potential (SP) between S1 and S2, on which early and late components were discernible, were recorded in association with performance of this task. With the electrode at a depth of 2 mm in the same cortical area, the corresponding field potential showed a long-lasting positive shift in addition to the components of the surface potential. These monopolar recordings were obtained with respect to a common reference at the frontal sinus. The surface-minus-depth potential (the transcortical potential), consequently, showed a surface-negative tonic wave, confirming Pirch's report (1980). During extinction of this conditioning, the SP between S1 and S2 disappeared, while the sharp waves following S1 and S2 remained with little modification, suggesting that the sharp waves are a kind of evoked potential (EP) elicited by the stimuli.Recording from 5 surface electrodes set in an array over the left hemisphere contralateral to the used forelimb during development of the conditioning revealed not only a spatial distribution of the SP but also a transition of the potentials. As the conditioning progressed, the negativity of the early SP component tended to increase, while that of the late component tended to decrease and was confined to the sensorimotor cortex. The similarities of the rat cortical surface potentials to the human and monkey CNV in their wave form and function suggests that the rat brain can produce electrical activity analogous to the human CNV.     

Motor response information influence on CNV shape and resolution time

This study systematically investigates changes in CNV waveform shape and resolution time that result from the presentation of facilitatory, inhibitory, or no motor response (MR) information simultaneously with the warning (S1) or imperative (S2) stimulus of the S1-S2-MR CNV paradigm. Analyses indicate that the simultaneous presentation of S1 and information to produce or inhibit a MR attenuates initial CNV development. Further, when the S1 information is inhibitive, CNV development is retarded throughout. The contribution of an inhibitory psychological process during CNV development is proposed. The data also indicate that CNV resolution time is not dependent on the presence of a motor response. It is suggested that CNV resolution time is indicative of psychological completion or closure.     

R1 in the Shaker S4 occupies the gating charge transfer center in the resting state

During voltage-dependent activation in Shaker channels, four arginine residues in the S4 segment (R1-R4) cross the transmembrane electric field. It has been proposed that R1-R4 movement is facilitated by a "gating charge transfer center" comprising a phenylalanine (F290) in S2 plus two acidic residues, one each in S2 and S3. According to this proposal, R1 occupies the charge transfer center in the resting state, defined as the conformation in which S4 is maximally retracted toward the cytoplasm. However, other evidence suggests that R1 is located extracellular to the charge transfer center, near I287 in S2, in the resting state. To investigate the resting position of R1, we mutated I287 to histidine (I287H), paired it with histidine mutations of key voltage sensor residues, and determined the effect of extracellular Zn(2+) on channel activity. In I287H+R1H, Zn(2+) generated a slow component of activation with a maximum amplitude (A(slow,max)) of ～56%, indicating that only a fraction of voltage sensors can bind Zn(2+) at a holding potential of -80 mV. A(slow,max) decreased after applying either depolarizing or hyperpolarizing prepulses from -80 mV. The decline of A(slow,max) after negative prepulses indicates that R1 moves inward to abolish ion binding, going beyond the point where reorientation of the I287H and R1H side chains would reestablish a binding site. These data support the proposal that R1 occupies the charge transfer center upon hyperpolarization. Consistent with this, pairing I287H with A359H in the S3-S4 loop generated a Zn(2+)-binding site. At saturating concentrations, A(slow,max) reached 100%, indicating that Zn(2+) traps the I287H+A359H voltage sensor in an absorbing conformation. Transferring I287H+A359H into a mutant background that stabilizes the resting state significantly enhanced Zn(2+) binding at -80 mV. Our results strongly support the conclusion that R1 occupies the gating charge transfer center in the resting conformation.     

Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.     

Adaptation changes in dynamic postural control and contingent negative variation during backward disturbance by transient floor translation in the elderly

ABSTRACT: BACKGROUND: We investigated adaptation changes in dynamic postural control and contingent negative variation (CNV) in 13 young and 12 elderly adults. Subjects repeatedly underwent backward postural disturbance by a forward floor translation (S2) 2 s after an auditory warning signal (S1). Initial and second sets were conducted, each set with 20 trials. Posterior peak position of the center of pressure in the anteroposterior direction (CoPy) after S2 was identified. Electroencephalograms from Cz were averaged for each set, and the CNV negative peak was identified. RESULTS: Compared with the first trial, the posterior peak position of CoPy changed significantly forward from the 12th trial in the young and from the 19th trial in the elderly during the initial set. The mean of the posterior peak position was more forward in second set than in the initial set for both groups and was significantly backward in the elderly compared to the young for both sets. These findings indicate that subjects in both groups adapted better to the postural disturbance in the second set than in the initial set, and the adaptation was later in the elderly. Late CNV in the young started to increase negatively from the middle of the S1-S2 period and peaked just before S2. Peak CNV amplitude was larger in the second set than in the initial set. In contrast, late CNV in the elderly exhibited no negative increase as in the young and peaked in the middle of the S1-S2 period, which was followed by gradual decreasing toward S2. No adaptive changes were found in late CNV for the elderly. CONCLUSIONS: It is conceivable that reduced activation of the frontal lobe may be one of the factors contributing to the decrease in postural adaptability in the elderly. The elderly may use various brain regions for the adaptation of dynamic postural control compared with the young.     

Mechanisms for Bt Toxin Resistance and Increased Chemical Pesticide Susceptibility in Cry1Ac10-resistant Cultured Insect Cells

Cabbage looper moth (Trichoplusia ni) cell line BTI-Tn-5B1-4 (TnH5) has developed high-level resistance (>1000 fold) by the selection of Bt Cry1Ac10 toxin. In order to examine mechanisms of resistance to Cry1Ac10 toxin (biological pesticide), both general esterase activities and cell tolerance to osmotic lysis were compared between non-selected Cry1Ac10-susceptible Trichoplusia ni cell line TnH5-S and Cry1Ac10-resistant Trichoplusia ni cell line TnH5-R selected by Bt Cry1Ac10. The Cry1Ac10-resistant TnH5-R cells had lower general esterase activity than the non-selected TnH5-S cells, and the esterase isozyme bands for the Cry1Ac10-resistant TnH5-R cells were much weaker than that for the non-selected TnH5-S cells. Both activated Cry1Ac10 toxin and multi-toxin from Bacillus thuringiensis subsp. aizawai GC-91 (an engineering bacterium) could not inhibit the esterase activity both in the Cry1Ac10-susceptible and Cry1Ac10-resistant cells, but two chemical pesticides, chlopyrifos and methomyl, could greatly inhibit the esterase activities both in the TnH5-R and TnH5-S cells. On the other hand, cell tolerance to osmotic lysis caused by hypotonic solution for the Cry1Ac10-resistant TnH5-R cells was higher than that for the non-selected TnH5-S cells (2.5×). Based on these results, we made the following conclusions. The general esterase activities in the Cry1Ac10-resistant TnH5-R cells was not related to Bt Cry1Ac10 resistance, but the susceptibility to the two tested chemical pesticides increased in TnH5-R cells because of their lower esterase activity. The increase of cell tolerance to osmotic lysis for the Cry1Ac10-resistant TnH5-R cells may be one of the mechanisms for Bt toxin resistance because midgut cells of insects are also disrupted by an osmotic lysis caused by Bt toxin.     

Asymmetric ATP binding and hydrolysis activity of the Thermus aquaticus MutS dimer is key to modulation of its interactions with mismatched DNA

Prokaryotic MutS and eukaryotic Msh proteins recognize base pair mismatches and insertions or deletions in DNA and initiate mismatch repair. These proteins function as dimers (and perhaps higher order oligomers) and possess an ATPase activity that is essential for DNA repair. Previous studies of Escherichia coli MutS and eukaryotic Msh2-Msh6 proteins have revealed asymmetry within the dimer with respect to both DNA binding and ATPase activities. We have found the Thermus aquaticus MutS protein amenable to detailed investigation of the nature and role of this asymmetry. Here, we show that (a) in a MutS dimer one subunit (S1) binds nucleotide with high affinity and the other (S2) with 10-fold weaker affinity, (b) S1 hydrolyzes ATP rapidly while S2 hydrolyzes ATP at a 30-50-fold slower rate, (c) mismatched DNA binding to MutS inhibits ATP hydrolysis at S1 but slow hydrolysis continues at S2, and (d) interaction between mismatched DNA and MutS is weakened when both subunits are occupied by ATP but remains stable when S1 is occupied by ATP and S2 by ADP. These results reveal key MutS species in the ATPase pathway; S1(ADP)-S2(ATP) is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1(ATP)-S2(ATP) and S1(ATP)-S2(ADP) are formed preferentially in the presence of mismatched DNA. These MutS species exhibit differences in interaction with mismatched DNA that are likely important for the mechanism of MutS action in DNA repair.     

Cauda equina syndrome and nitric oxide synthase immunoreactivity in the spinal cord of the dog

The development of the cauda equina syndrome in the dog and the involvement of spinal nitric oxide synthase immunoreactivity (NOS-IR) and catalytic nitric oxide synthase (cNOS) activity were studied in a pain model caused by multiple cauda equina constrictions. Increased NOS-IR was found two days post-constriction in neurons of the deep dorsal horn and in large, mostly bipolar neurons located in the internal basal nucleus of Cajal seen along the medial border of the dorsal horn. Concomitantly, NOS-IR was detected in small neurons close to the medioventral border of the ventral horn. High NOS-IR appeared in a dense sacral vascular body close to the Lissauer tract in S1-S3 segments. Somatic and fiber-like NOS-IR appeared at five days post-constriction in the Lissauer tract and in the lateral and medial collateral pathways arising from the Lissauer tract. Both pathways were accompanied by a dense punctate NOS immunopositive staining. Simultaneously, the internal basal nucleus of Cajal and neuropil of this nucleus exhibited high NOS-IR. A significant decrease in the number of small NOS immunoreactive somata was noted in laminae I-II of L6-S2 segments at five days post-constriction while, at the same time, the number of NOS immunoreactive neurons located in laminae VIII and IX was significantly increased. Moreover, high immunopositivity in the sacral vascular body persisted along with a highly expressed NOS-IR staining of vessels supplying the dorsal sacral gray commissure and dorsal horn in S1-S3 segments. cNOS activity, based on a radioassay of compartmentalized gray and white matter regions of lower lumbar segments and non-compartmentalized gray and white matter of S1-S3 segments, proved to be highly variable for both post-constriction periods.     

Slow potentials of the prefrontal cortex in dogs and the classical secretory conditioned reflex

Slow potentials (CNV and component P300) were recorded in the medial part of the prefrontal cortex of dogs trained to classical secretory conditioned reflex and its differentiation. CNV increased when the conditioned stimulus was preceded by a signal of different meaning, as compared with CNV to the same conditioned signal following the stimulus of the same meaning; the greatest values of CNV and P300 were observed in response to differential stimulus preceded by a positive signal.     

Discovery, synthesis and SAR analysis of novel selective small molecule S1P4-R agonists based on a (2Z,5Z)-5-((pyrrol-3-yl)methylene)-3-alkyl-2-(alkylimino)thiazolidin-4-one chemotype

High affinity and selective S1P(4) receptor (S1P(4)-R) small molecule agonists may be important proof-of-principle tools used to clarify the receptor biological function and effects to assess the therapeutic potential of the S1P(4)-R in diverse disease areas including treatment of viral infections and thrombocytopenia. A high-throughput screening campaign of the Molecular Libraries-Small Molecule Repository was carried out by our laboratories and identified (2Z,5Z)-5-((1-(2-fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)-3-methyl-2-(methylimino) thiazolidin-4-one as a promising S1P(4)-R agonist hit distinct from literature S1P(4)-R modulators. Rational chemical modifications of the hit allowed the identification of a promising lead molecule with low nanomolar S1P(4)-R agonist activity and exquisite selectivity over the other S1P(1-3,5)-Rs family members. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the effects of the S1P(4)-R signaling cascade and elucidate the molecular basis of the receptor function.     

Neutralizing monoclonal antibody specific for alpha-bungarotoxin: preparation and characterization of the antibody, and localization of antigenic region of alpha-bungarotoxin

We prepared an alpha-bungarotoxin-specific monoclonal antibody that neutralizes the biological activity of the toxin in vivo. The antigenic determinant combining specifically with this antibody was determined on the basis of cross-reaction experiments using three other long neurotoxins and peptide fragments of alpha-bungarotoxin. The antigenic determinant was located on the peptide fragment containing S34-S35-R36-G37-K38, which forms a part of the expected site that binds to the acetylcholine receptor proteins.     

Identification of nonprotein ligands to the metal ions bound to glutamine synthetase

Electron paramagnetic resonance (EPR) was used to study the environment of Mn2+ bound to the tight (n1) metal ion binding site of glutamine synthetase in the presence of analogues of the tetrahedral adduct, L-methionine (S)-sulfoximine [Met(O)(NH)-S] and L-methionine (R)-sulfoximine [Met(O)(NH)-R]. The Mn2+ EPR spectrum in the presence of Met(O)(NH)-S is identical with the previously published spectrum obtained from a mixture of isomers [Met(O)(NH)-RS] [Villafranca, J. J., Ash, D. E., & Wedler, F. C. (1976) Biochemistry 15, 544] and is characteristic of a highly octahedral metal ion environment with a small zero field splitting. The presence of Met(O)(NH)-R produces an EPR spectrum that appears characteristic of a more distorted metal ion environment, with a larger zero field splitting. These data demonstrate that the two isomers interact differently with the enzyme-bound Mn2+. Broadening of the Mn2+ EPR spectrum in the presence of Met(O)(NH) is observed in 17O-enriched water due to superhyperfine coupling of water to the metal ion. Deconvolution of the spectrum demonstrates the presence of at least a single water molecule in the inner coordination sphere of the metal ion. Superhyperfine coupling due to the 14N nucleus of the imine nitrogen of the sulfoximine moiety of Met(O)(NH)-S but not of Met(O)(NH)-R has been detected by electron spin-echo envelope modulation spectroscopy. Two intense peaks are evident in the presence of Met(O)(NH)-S with frequencies at 1.7 and 3.3 MHz. These peaks are absent when [15N]imine-labeled Met(O)(NH) is used, indicating the presence of the sulfoximine nitrogen of Met(O)(NH)-S in the inner coordination sphere of the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations of arginine 64 within the putative Ca(2+)-binding lumenal interhelical a-b loop of the photosystem II D1 protein disrupt binding of the manganese stabilizing protein and cytochrome c(550) in Synechocystis sp. PCC6803

Mutations D1-R64E, D1-R64Q, and D1-R64V in the putative calcium-binding lumenal interhelical a-b loop of the photosystem II (PSII) D1 protein were characterized in terms of impact on growth, extrinsic protein binding, photoactivation, and properties of the H(2)O-oxidation complex. The D1-R64E charge reversal mutation greatly weakened the binding of the extrinsic manganese-stabilizing protein (MSP) and, to a considerably lesser extent, weakened the binding of cytochrome c(550) (c550). Both D1-R64Q and D1-R64E exhibited an increased requirement for Ca(2+) in the cell growth medium. Bare platinum electrode measurements of O(2)-evolving membranes showed a retarded appearance of O(2) following single turn-over flashes, especially in the case of the D1-R64E mutant. The D1-R64E mutant also had a pronounced tendency to lose O(2) evolution activity in the dark and exhibited an increased relative quantum yield of photoactivation, which are characteristics shared by mutants that lack extrinsic proteins. S(2) and S(3) decay measurements in the isolated membranes indicate that D1-R64E and D1-R64Q have faster decays of these higher S-states as compared to the wild-type. However, fluorescence decay in the presence of DCMU, which monitors primarily Q(A)(-) charge recombination with PSII donors, showed somewhat slower decays. Taken together, the fluorescence and S-state decay indicate that the midpoint of either Q(B)(-) has been modified to be more negative in the mutants or that a recombination path presumably involving either Q(B)(-) or Y(D) has become kinetically more accessible.     

Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.     

Adaptation changes in dynamic postural control and contingent negative variation during repeated transient forward translation in the elderly

Background

Adaptation changes in postural control and contingent negative variation (CNV) for the elderly were investigated during repeated forward floor translation.

Methods

Fifteen healthy elderly persons, living in the suburban area of Kanazawa City, Japan, underwent backward postural disturbance by a forward-floor translation (S2) 2 s after an auditory warning signal (S1). A set with 20 trials was repeated until a negative peak of late CNV was recognized in the 600-ms period before S2, and the last set was defined as the final set. Electroencephalograms, center of foot pressure in the anteroposterior direction (CoPap), and electromyograms of postural muscles were analyzed.

Results

CoPap displacement generated by the floor translation was significantly decreased until the twelfth trial in the first set, and mean CoPap displacement was smaller in the second and final sets than in the first set. The mean displacement was significantly smaller in the final set than the previous set. A late CNV with a negative peak was not recognized in the first and second sets. However, most subjects (13/15) showed a negative peak by the fourth set, when the late CNV started to increase negatively from about 1,000 ms after S1 and peaked at about 300 ms before S2. At about 160 ms before the CNV peak, the CoPap forward shift started. The increase in timing of the gastrocnemius activity related to the CoPap shift was significantly correlated with the CNV peak timing (r = 0.64). After S2, peak amplitudes of the anterior postural muscles were significantly decreased in the final set compared to the first set.

Conclusions

It was demonstrated that even for the elderly, with so many repetitions of postural disturbance, a late CNV with a negative peak was recognized, leading to accurate postural preparation. This suggests the improvement of frontal lobe function (e.g., anticipatory attention and motor preparation) in the elderly.     

Activity of the 30-S CsCl core in elongation-factor-dependent GTP hydrolysis.

The activity of a 30-S CsCl core lacking proteins S1, S2, S3, S5, S9, S10, S14, S20 and S21 has been studied in the ribosome-dependent FTPase reactions in the presence of the 50-S subunit with and without methanol. Without methanol, the 30-S CsCl core was unable to sustain GTPase activity dependent on elongation factor G (EF-G), while it was only slightly active in the presence of elongation factor T (EF-T). With EF-T, addition of methanol induced in the presence of either 30-S subunits or 30-S CsCl cores an activity which was more than 10-fold higher than that observed with the 30-S subunit in the absence of methanol. Methanol lowered the Mg2+ optimum of the EF-T-dependent GTPase reaction from approximately 20 mM to approximately 10 mM. In the absence of methanol the EF-G-dependent (GTPase reaction at low concentration of monovalent cations depends on the 50-S subunit alone (30-S-uncoupled EF-G GTPase). Addition of the intact 30-S subunit but not of its CsCl core abolished inhibition of the 30-S-uncoupled EF-G-GTPase by NH4+. The 30-S CsCl core caused the same effect as the 30-S subunit when methanol was present. 30-S-uncoupled EF-G GTPase activity was lower than the GTPase activity dependent on 30-S plus 50-S subunits at [EF-G]/[50-S] below 5 but was considerably higher in the presence of a large excess of EF-G. In the presence of methanol the 30-S CsCl core behaved similarly to the 30-S subunit. Our results indicate that the action of the 30-S subunit in elongation-factor-dependent GTPases is supported by structural features that are preserved in the 30-S CsCl core. The 30-S split proteins are therefore not essential for EF-G and EF-T activities in the hydrolysis of GTP. With EF-T, in all conditions tested association of the ribosomal subunits appeared to accompany GTPase activity. Association seems also to be a prerequisite of the EF-G GTPase activity that depends on both ribosomal subunits.     

Discovery,design and synthesis of a selective S1P3 receptor allosteric agonist

Potent and selective S1P₃ receptor (S1P₃-R) agonists may represent important proof-of-principle tools used to clarify the receptor biological function and assess the therapeutic potential of the S1P₃-R in cardiovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide was identified by a high-throughput screening of MLSMR library as a promising S1P₃-R agonist. Rational chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide, a S1P₃-R agonist endowed with submicromolar activity and exquisite selectivity over the remaining S1P_1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide is an allosteric agonist and binds to the S1P₃-R in a manner that does not disrupt the S1P₃-R–S1P binding. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the molecular basis of the receptor function, and provides the bases for further rational design of more potent and drug-like S1P₃-R allosteric agonists.     

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating

Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.     

Postimperative negativity of the contigent negative variations: a critical index of a stress reaction in the normal subject?]

21 subjects were recorded during two experimental sessions: 1st session: 2 CNVs in control conditions; 2nd session: 1) control CNV, 2) CNV and arithmetic calculation, 3) CNV and labyrinthine stimulation, 4) CNV, arithmetic calculation and labyrinthine stimulation, 5) control CNV. The results obtained show a post-imperative extension of the CNV in the situation 2: arithmetic calculation (P less than 0.025) and 4: double interference (P less than 0.05).     

The separation, properties and possible subunit composition of adenosine 3',5'-monophosphate-dependent protein kinases in brown adipose tissue.

Two 8.5-S protein kinases (ATP : protein phosphotransferase EC 2.7.1.37) and one 6.6-S protein kinase were purified 500--1000-fold from the acid-soluble fraction of brown adipose tissue. The catalytic properties of the kinases were similar. Each kinase was activated by cyclic AMP and had two components of cyclic AMP binding. In the presence of 200 nM cyclic AMP, undissociated kinase activity sedimented at 7.7 or 5.5 S. Free catalytic activity (3.2 S) could be detected but was unstable. Free regulatory units could not be detected. The 8.5-S protein kinase was dissociated by freezing and thawing to a 7.7-S variety with loss of the higher affinity component of binding. The 7.7-S kinase was sedimented through linear gradients of sucrose containing different concentrations of cyclic AMP. At each concentration, kinase activity lost from the holoenzyme peak (% of original) was identical with the amount of cyclic AMP bound at equilibrium (% oof maximum). Similar experiments on the 8.5-S kinase showed that the binding component with higher affinity was not associated with the release of catalytic activity. The results were consistent with the propostal that the kinases isolated contained one more cyclic AMP binding subunit than catalytic subunit (3 : 2 for 8.5 S and 2 : 1 for 6.6 S) and that this extra subunit was released to give an equal number of subunits of each type before catalytic activity was liberated.