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PubMed2006

Involvement of STAT3 in bladder smooth muscle hypertrophy following bladder outlet obstruction.

We examined the involvement of the signal transducer and activator of transcription 3 (STAT3) in bladder outlet obstruction (BOO)-induced bladder smooth muscle hypertrophy using a rat in vivo and in vitro study. BOO induced increases in bladder weight and bladder smooth muscle thickness 1 week after the operation. By using antibody microarrays, 64 of 389 proteins blotted on the array met our selection criteria of an INR value between > or = 2.0 and < or = 0.5. This result revealed up-regulation of transcription factors, cell cycle regulatory proteins, apoptosis-associated proteins and so on. On the other hand, down-regulation (INR value < or = 0.5) of proteins was not found. In a profiling study, we found an increase in the expression of STAT3. A significant increase in nuclear phosphorylated STAT3 expression was confirmed in bladder smooth muscle tissue by immunohistochemistry and Western blot analysis. Cyclical stretch-relaxation (1 Hz) at 120% elongation significantly increased the expression of STAT3 and of alpha-smooth muscle actin in primary cultured bladder smooth muscle cells. Furthermore, the blockade of STAT3 expression by the transfection of STAT3 small interfering RNA (siRNA) significantly prevented the stretch-induced increase in alpha-smooth muscle actin expression. These results suggest that STAT3 has an important role in the induction of bladder smooth muscle hypertrophy.

Animals↗
PubMed2005

The relationship between focal seizures and sleep: an analysis of the cyclic alternating pattern.

PURPOSE: The aim of this study was to examine the relationship between focal epileptic seizures and sleep through analysis of the cyclic alternating pattern (CAP). METHODS: We analyzed the recordings of a total of 56 nocturnal partial seizures (13 occurring as isolated events and 43 "in clusters" during a full-night ambulatory polysomnography) in 12 adult patients affected by localization-related focal epilepsy (7 males, 5 females; mean age 35+12.8 years; range 25-71). RESULTS: On exact binomial distribution analysis, seizures were more frequent in CAP than in non-CAP sleep, and in CAP phase A than in CAP phase B (p<0.001). Seizures occurring in clusters were more frequently associated with CAP sleep (p<0.05) than isolated seizures and first seizures of seizure clusters. Increase of CAP rate during the 30-min of sleep period after the occurrence of a seizure was documented in both cluster and isolated seizures. CONCLUSIONS: Our data indicate that an intra-sleep condition of highly fluctuating vigilance constitutes the real substrate for the occurrence of epileptic seizures regardless of the NREM stage in which they occur. The occurrence of partial seizures during sleep appears to induce sleep instability, which may in turn result in the facilitation of clusters of seizures. Together, these data highlight the potential of sleep fragmentation to modulate intra-sleep seizure occurrence.

Adult↗
PubMed1993

Conformational mobility in cyclic oligopeptides.

Analysis of two isomeric cyclic hexapeptides of composition (Asp, Arg, Gly2, Pro, D-Pro) by a nuclear Overhauser effect constrained distance geometry conformation search yielded a narrowly defined backbone conformation for one and considerable ambiguity about the conformation in part of the other. Preliminary 13C relaxation studies of these peptides suggest that it is possible that this difference may correspond to a physical difference in internal mobility. In connection with this observation, other experimental evidence bearing on the backbone conformational mobility of cyclic oligopeptides with 4-10 residues, frequently considered to have well-defined backbones, is reviewed. Conformational heterogeneity involving rotation of a peptide bond plane relative to the overall ring plane is identified as a common phenomenon. Nuclear magnetic resonance line-shape studies at temperatures down to 200 K can detect backbone motions with activation free energy barriers down to about 10 kcal/mole, but conformational exchange with lower barriers, though detectable in other ways, will not be obvious from nmr spectra alone.

Amino Acid Sequence↗
PubMed1997

Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 microm porous microspherical silica gel.

A high-performance liquid chromatographic method has been developed for the forensic analysis of eleven frequently used cyclic antidepressant drugs (ADSs) (amitriptyline, amoxapine, clomipramine, desipramine, dosulepine, doxepin, imipramine, maprotiline, melitracen, mianserine and nortriptyline) using a recently developed reversed-phase column with 2 microm particles for the analysis of biological samples. The separation was carried out using two different C8 reversed-phase columns (column 1: 100 mm X 4.6 mm I.D., particle size 2 microm, TSK gel Super-Octyl; column 2: 100 mm X 4.6 mm I.D., particle size 5 microm, Hypersil MOS-C8) for comparison. The mobile phase was composed of methanol-20 mM KH2PO4 (pH 7) (60:40, v/v) and the flow-rate was 0.6 ml/min for both columns. The absorbance of the eluent was monitored at 254 nm. When the eleven drugs were determined, the sensitivity with the 2 microm particles was about five times greater than with the 5 microm particles. Retention times on column 1 were shorter than those on column 2. These results show that the new ODS column packing with a particle size of 2 microm gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.

Antidepressive Agents, Tricyclic↗
PubMed2002

Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD.

We studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G-->T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC.

Adenocarcinoma↗
PubMed1991

Cyclic-ratio schedule analysis of a serotonin agonist and depletor on consummatory behaviour.

Four male Wistar rats, 180 days old at the start of the experiment, at 85% of their free-feeding body weight were trained to respond on a geometric cyclic-ratio schedule comprised of the following ratio values: 2, 4, 8, 16, 32 and 64, for 0.1 ml of 5% sucrose reinforcement. The response functions (response rates plotted against reinforcement rates) were linear and of negative slope over the range of ratio values from 2 to 16. IP administration of 1.0 mg/kg 5-MeODMT reduced the x- and y-intercepts of the linear portion of the response function without altering the slope relative to 2.0 ml/kg 0.85% saline IP. This was interpreted as a perceived palatability effect. IP administration of 100 mg/kg pCPA elevated the reinforcement rate intercept but also decreased the slope of the response function. This finding was interpreted as an increase in the perceived palatability of the reinforcer, coupled with a decrease in motivation at higher schedule conditions, possibly due to peripheral effects of pCPA.

Animals↗
PubMed2006

A critical analysis of the cyclic and open alternatives of the transmetalation step in the stille cross-coupling reaction.

The transmetalation step of the Stille cross-coupling reaction catalyzed by PdL(2) (L = PH(3), AsH(3)) has been analyzed by means of DFT methods for PhBr as the electrophile and CH(2)=CHSnMe(3) as the nucleophile. Both experimentally proposed mechanisms (cyclic and open) were theoretically studied. For the case of the cyclic mechanism, the associative and dissociative ligand substitution alternatives were both analyzed. For the case of the open mechanism, the cis and the trans pathways were evaluated. All the reaction pathways were also studied taking into account the solvent effects by means of continuum models, for THF and PhCl as solvents. In selected cases, explicit solvent molecules were introduced to account for their potential role as ligands. Theoretical analysis indicates that the open reaction mechanism is preferred for organotriflate systems, whereas the cyclic mechanism is favored for the reaction with organohalide systems.

Journal Article↗
PubMed2002

Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels.

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K(+) and other monovalent cations, but exclude Na(+); this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K(+)-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K(+) currents.

Amino Acid Sequence↗
PubMed1999

Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme.

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (tauc) decreased with increasing [ATP] to a minimum value of approximately 0.43 s at [ATP] >1.00 mM. The open time constant (tauo) increased with increasing [ATP] with a minimal tauo of approximately 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of approximately 250 ms is dominant at low ATP concentrations (10 microM) and a much longer open state with a time constant of approximately 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent.

3T3 Cells↗
PubMed2006

LC/ESI/MS method development for the analysis of hepatotoxic cyclic peptide microcystins in animal tissues.

Microcystins (MCYSTs) are a family of related cyclic heptapeptides produced by several genera and species of blue-green algae (cyanobacteria). MCYSTs are potent and specific inhibitors of the serine threonine family of protein phosphatases, especially PP1 and PP2A. MCYSTs inhibit a liver's protein phosphatase by forming a covalent linkage between MCYSTs' Mdha residue and the phosphatase's cysteine residue. Due to the covalent linkage, analysis of MCYSTs in animal tissues has been limited to determination of unbound MCYST concentration. The MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) oxidation procedure allows for the detection of total MCYST burden by releasing the carboxylic acid MMPB from MCYST's Adda amino acid. An internal standard 4-phenylbutyric acid (4PB) accounts for losses during the method. LC/MS conditions were developed using a ThermoFinnigan LCQDuo ion trap in negative electrospray ionization (ESI). Since both compounds produce the [M-H](-) ion, analysis occurs in selected ion monitoring (SIM) mode for both MMPB (m/z 207.1) and 4PB (m/z 163.1). Complete oxidation of MCYST-LR in liver tissues occurs in 3h. A solid phase extraction (SPE) cartridge removes MMPB and 4PB from the oxidant solution. The process efficiency for the SPE procedure is only 51.3%; however, suppression experiments indicate a 41.8% loss in signal strength due to matrix interferences. Therefore, the extraction efficiency for the SDB-XC cartridge procedure is 93.1%. This research has been successful in developing an LC/MS method for the analysis of total MCYST burden in animal tissues.

Animals↗
PubMed1999

Electrospray ionization mass spectrometric analysis of microcystins, cyclic heptapeptide hepatotoxins: modulation of charge states and [M + H]+ to [M + Na]+ ratio.

Electrospray ionization mass spectrometry was used to develop a rapid, sensitive, and accurate method for determination and identification of hepatotoxic microcystins, cyanobacterial cyclic heptapeptides. To optimize the electrospray ionization conditions, factors affecting charge state distribution, such as amino acid components of sample, proton affinity of the additives, and additive concentration, were investigated in detail and a method for controlling charge states was developed to provide molecular-related ions for assignment of molecular weight and reasonably abundant precursor ions for MS/MS analysis. A procedure for identification of microcystins consisting of known amino acids was proposed: for microcystins giving abundant [M + 2H]2+ ions, the addition of nitrogen-containing bases to the aqueous sample solution is effective to obtain an increased intensity of [M + H]+ ions, whereas the addition of Lewis acids containing nitrogen can produce increased abundances of [M + 2H]2+ ions for microcystins giving weak [M + 2H]2+ ions. Microcystins possessing no arginine residue always give sodium adduct ions [M + Na]+ as the base peak, and these are difficult to fragment via low energy collision-induced dissociation to yield structurally informative products; the addition of oxalic acid increases [M + H]+ ion abundances, and these fragment readily.

Acetates↗
PubMed1996

Alternating lever cyclic-ratio schedule analysis of the effects of atropine sulfate.

Cholinergic dysfunction has been implicated in the behavioral and memory impairment that is the hallmark of conditions such as delirium and Alzheimer's Disease. Anticholinergic drugs have been widely used in procedures designed to mimic aspects of the pathology of these conditions in rats. Procedures in use vary widely in sensitivity and behavioral specificity and may be confounded by administration of high drug doses that may not be physiologically relevant. The current study proposes the use of rats responding on an alternating lever cyclic-ratio schedule to study the effects of the anticholinergic compound atropine sulfate. This procedure enables simultaneous measurement of anticipatory ratio tracking (postreinforcement pause durations), perseverations (lever switching errors), and nonspecific peripheral drug effects (running response rates). Results of this study suggest that the schedule is sensitive to low drug doses (0.1-1.0 mg/kg atropine), measures the ability to track changing ratio conditions and to execute lever alternation, and allows for monitoring of peripheral drug effects during behavioral testing. The procedure's sensitivity and low effective dose range may make it useful in the study of behaviors related to anticholinergic effects.

Animals↗
PubMed2005

All-night EEG power spectral analysis of the cyclic alternating pattern components in young adult subjects.

OBJECTIVE: To analyze in detail the frequency content of the different EEG components of the Cyclic Alternating Pattern (CAP), taking into account the ongoing EEG background and the nonCAP (NCAP) periods in the whole night polysomnographic recordings of normal young adults. METHODS: Sixteen normal healthy subjects were included in this study. Each subject underwent one polysomnographic night recording; sleep stages were scored following standard criteria. Subsequently, each CAP A phase was detected in all recordings, during NREM sleep, and classified into 3 subtypes (A1, A2, and A3). The same channel used for the detection of CAP A phases (C3/A2 or C4/A1) was subdivided into 2-s mini-epochs. For each mini-epoch, the corresponding CAP condition was determined and power spectra calculated in the frequency range 0.5-25 Hz. Average spectra were obtained for each CAP condition, separately in sleep stage 2 and SWS, for each subject. Finally, the first 6h of sleep were subdivided into 4 periods of 90 min each and the same spectral analysis was performed for each period. RESULTS: During sleep stage 2, CAP A subtypes differed from NCAP periods for all frequency bins between 0.5 and 25 Hz; this difference was most evident for the lowest frequencies. The B phase following A1 subtypes had a power spectrum significantly higher than that of NCAP, for frequencies between 1 and 11 Hz. The B phase after A2 only differed from NCAP for a small but significant reduction in the sigma band power; this was evident also after A3 subtypes. During SWS, we found similar results. The comparison between the different CAP subtypes also disclosed significant differences related to the stage in which they occurred. Finally, a significant effect of the different sleep periods was found on the different CAP subtypes during sleep stage 2 and on NCAP in both sleep stage 2 and SWS. CONCLUSIONS: CAP subtypes are characterized by clearly different spectra and also the same subtype shows a different power spectrum, during sleep stage 2 or SWS. This finding underlines a probable different functional meaning of the same CAP subtype during different sleep stages. We also found 3 clear peaks of difference between CAP subtypes and NCAP in the delta, alpha, and beta frequency ranges which might indicate the presence of 3 frequency components characterizing CAP subtypes, in different proportion in each of them. The B component of CAP differs from NCAP because of a decrease in power in the sigma frequency range. SIGNIFICANCE: This study shows that A components of CAP might correspond to periods in which the very-slow delta activity of sleep groups a range of different EEG activities, including the sigma and beta bands, while the B phase of CAP might correspond to a period in which this activity is quiescent or inhibited.

Adult↗
PubMed1991

Conformational analysis of two cyclic disulfide peptides.

Complete nmr and CD studies of two cyclic tetrapeptides with disulfide bonds, Ac-L-Pen-L-Pro-D-Val-L-Cys-NH2 (1) and Ac-L-Cys-L-Pro-D-Val-L-Cys-NH2 (2) bonds have been carried out in different solvents to investigate the formation and stabilization of beta-turn structures and to determine the stereochemistry of the disulfide linkage. Both peptides have three-dimensional structures with a type II beta-turn, as derived from quantitative nuclear Overhauser effect data. The combined use of CD and nmr indicates that the dihedral angle of the disulfide bridge is different in the two peptides, although the chirality is maintained.

Amino Acid Sequence↗
PubMed2004

Conformational and biochemical analysis of the cyclic peptides which modulate serine protease activity.

Prostate-specific antigen (PSA), a member of the kallikrein sub-group of the trypsin serine protease family, is a widely used marker for prostate cancer. Several sequences with specific binding to PSA have been identified by using phage display peptide libraries. The GST-fusion proteins of the characterized sequences have been shown to increase the enzyme activity of PSA to a synthetic substrate. The corresponding three cyclic synthetic analogues CVFTSNYAFC (A-1), CVFAHNYNYLVC (B-2) and CVAYCIEHHCWTC (C-4) have similar PSA promoting activity. Despite differences in the amino acid sequences, all three peptides bind to the same region of PSA. The conformation of the peptides was investigated by proton NMR spectroscopy. In addition, alanine replacement was used to characterize the prerequisites for binding. It is proposed that interactions with PSA are based on the aromatic and hydrophobic features of the amino acid side chains. Furthermore, it is suggested that peptides form beta-turn structures forced by cysteine bridges directing important aromatic side chains to the same side of the turn-structure.

Amino Acid Sequence↗