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PubMed1978

Ovarian serotonin content in relation to ovulation.

In the mature cyclic female rat, analysis for ovarian serotonin content reveals comparatively high serotonin content. Fluctuation of serotonin content was observed; peak for serotonin was observed at estrus. In gonadotropin-treated immature rats, there was no detected ovarian serotonin using this procedure. It was concluded that ovaries from gonadotropin-treated immature rats are physiologically different from ovaries taken from mature cyclic rats.

Aging↗
PubMed1993

Sertoli cell cycle: a re-examination of the structural changes during the cycle of the seminiferous epithelium of the rat.

Seminiferous tubules in mammals are composed of cell associations that show a cyclic pattern of renewal and development. The cyclic nature of germ cell development suggests that the cells supporting the spermatogenic process, the Sertoli cells, might also differ structurally during the spermatogenic cycle in terms of the quantity of their constituents. In the present study, cyclic differences in volumes, and surface areas were determined using a sampling technique at the electron microscope level that proportionally samples the Sertoli cell within the seminiferous tubule. Among the many parameters studied, only the surface area of the cell, the volume of lipid, and the volume and surface area of the rough endoplasm reticulum were shown by statistical analysis to vary cyclically. Regarding rough endoplasm reticulum, the volume and surface area of this organelle peaked at mid-cycle and its low was recorded near the end of the cycle, exhibiting an approximate 15-fold difference between extremes. The rough endoplasm reticulum parameters generally correlated with known patterns of protein secretion within the tubule and with the secretion of specific proteins as well as the factors important in controlling protein secretion. Many Sertoli cell structural parameters suggested to be influenced cyclically in the rat in other studies could not be confirmed by the present study. Methodological differences in the present study and past studies are discussed as potential sources of error for these discrepancies.

Animals↗
PubMed2005

Whole cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and in situ structure analysis of streptocidins, a family of tyrocidine-like cyclic peptides.

Streptocidins, a family of tyrocidine-like cyclic decapeptides, are an ideal demonstration object for the detection and in situ structure analysis of natural compounds directly in microbial cells using whole cell matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOFMS), an emerging technique that can be used for rapid sensitive metabolic profiling of microorganisms. Five main members of the streptocidin family (A-E) were detected in Brevibacillus cells picked from agar plates and identified by in situ structure analysis with post-source decay MALDI-TOFMS. This efficient modern method allows the precise detection of metabolites within minutes without the need to isolate and purify the target compounds. The generated mass spectra are of similar quality to those obtained for the purified peptides. In addition, surface extracts were prepared by treating Brevibacillus cells with 70% acetonitrile in the presence of 0.1% trifluoroacetic acid and fractionated by high-resolution reversed-phase high-performance liquid chromatography (HPLC). In this way ten minor streptocidins were detected demonstrating the full biosynthetic variety of streptocidin production on the cellular level. The streptocidins differ from the well-known tyrocidines essentially in position 3 of the decapeptide chain by replacement of the aromatic amino acid (F/W) found in tyrocidines by L-leucine or L-valine.

Amino Acid Sequence↗
PubMed1994

Cocaine alters cyclic motor activity in the fetal rat.

Individual rat fetuses were exposed to cocaine by injection into the cisterna magna on E20 or E21 of gestation. On E20, cocaine elevated motor activity throughout the 20-min session, with activity increasing to double SAL-injected levels by the end of the session. On E21, overall activity also was elevated after cocaine, increasing four- to fivefold in the first 5 min of the session, and decreasing to twice the levels of SAL controls thereafter. Movement time series were subjected to spectral analysis to characterize cyclic motor organization. Cyclicity was evident in 67% of control and 97% of cocaine-injected fetuses. Cocaine increased the number of peaks in the movement spectrum and resulted in greater spectral density at the primary frequency of cyclic movement. Prenatal exposure to cocaine may alter patterns of cyclicity in the fetus, with consequences for the normal development of attention and behavioral state in the neonate.

Animals↗
PubMed1999

Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp.

Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3,7-didesmethylmicrocystin-LR. Three open reading frames (mcyA, mcyB, mcyC) encoding microcystin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an N-methylation domain attached to the first and an epimerization domain attached to the second; McyB (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in mcyA, generated by homologous recombination, abolished the production of both microcystins in M. aeruginosa K-139. Primer extension analysis demonstrated light-dependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes.

Amino Acid Sequence↗
PubMed2006

Electrochemical and homogeneous proton-coupled electron transfers: concerted pathways in the one-electron oxidation of a phenol coupled with an intramolecular amine-driven proton transfer.

Proton-coupled electron transfers currently attract considerable attention in view of their likely involvement in many natural processes. Electrochemistry, through techniques such as cyclic voltammetry, is an efficient way of investigating the reaction mechanism of these reactions, and deciding whether proton and electron transfers are concerted or occur in a stepwise manner. The oxidation of an ortho-substituted 4,6-di (tert-butyl)-phenol in which the phenolic hydrogen atom is transferred during the reaction to the nitrogen atom of a nearby amine is taken as illustrative example. A careful analysis of the cyclic voltammetric responses obtained with this compound and its OD derivative allows, after estimation of the various thermodynamic parameters, ruling out the occurrence of the square scheme mechanism involving the proton-electron and electron-proton sequences. Simulation and comparison of the rate constant and H/D kinetic isotope effect with theoretical predictions show that the experimental value of the preexponential factor is ca. 1 order of magnitude larger than the theoretical value. Detailed calculations suggest that an electric field effect is responsible for this discrepancy.

Journal Article↗
PubMed1997

Single-channel properties of ionic channels gated by cyclic nucleotides.

This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels.

Amino Acid Sequence↗
PubMed1995

Influence of serine in position i on conformation and dynamics of reverse turns.

NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cyclo(-D-Pro1-Ala2-Ser3(Bzl)-Trp4-Orn5(Z)-Tyr 6-) with and without protecting groups on Ser3 and Orn5. This peptide sequence was derived from the active loop sequence of the alpha-amylase inhibitor Tendamistat (HOE 467). The aim was to investigate the role of serine in position i of a standard beta-turn on the conformation and stabilization of this turn. Based on distance and torsion constraints from 2D NMR spectroscopic measurements in DMSO-d6 solution, structure refinement was accomplished by restrained molecular dynamics (MD) simulations in vacuo and in DMSO. The analysis of both structures in solution reveals a considerable effect of the unprotected serine sidechain on the adjacent beta-turn conformation. While in the protected peptide with Ser3(Bzl) a beta II-turn is observed between Trp4 and Orn5, the deprotected compound reveals a beta I-turn in this region. The beta I-turn is stabilized by a backbone-sidechain hydrogen bond from Orn5N alpha H to Ser3O gamma. Comparisons with other NMR-derived solution structures of cyclic model peptides and in some protein structures from literature reveal a general structural motif in the stabilization of beta I-turns by serine in the i position through backbone-sidechain interactions.

Amino Acid Sequence↗
PubMed1993

Direct measurement of cyclic current-voltage responses of integral membrane proteins at a self-assembled lipid-bilayer-modified electrode: cytochrome f and cytochrome c oxidase.

Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic "voltammograms," 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins.

Conductometry↗
PubMed2004

Development of conformationally restricted analogues of bradykinin and somatostatin using constrained amino acids and different types of cyclization.

The structure-based design of peptide drugs requires the knowledge of the bioactive conformation. Studies on this receptor-bound 3D structure require linear or cyclic analogues with strongly reduced flexibility, but high biological activity, since only analogues with retained potency have preserved the bioactive conformation. Constrained amino acids containing double bonds or bulky substituents at the N(alpha)-, C(alpha)- and C(beta)-atom as well as at the aromatic ring atom were successfully applied to obtain potent and stable analogues of bradykinin and somatostatin, which due to their restricted conformation were suitable objects for conformational studies. Besides the generation of constrained cyclic analogues with improved biological and pharmacological properties, cyclic peptides were used as convenient models for the study of turn formations. Cyclization of the linear peptide bradykinin was performed by linking the N-terminus and the C-terminus, and in both bradykinin and somatostatin by cyclization using the amino acid side chains and by backbone cyclization. The later requires the introduction of N(alpha)-functionalised amino acids for ring closure which can be performed either through incorporation of N(alpha)-functionalised amino acids or dipeptide building units. Conformational analysis of a cyclic bradykinin analogue by means of NMR-studies together with molecular dynamics simulation led to a quasicyclic 3D structure with two turns and together with other 3D structures provided a pharmacophore model of bradykinin antagonists.

Amino Acids↗
PubMed2006

Tricyclic antidepressants for chronic vomiting in diabetic patients.

Chronic vomiting in diabetic patients often is unresponsive to prokinetic agents and poorly explained by delayed gastric emptying or neuropathy. This retrospective study examines clinical response to tricyclic antidepressants, a treatment of reported benefit in nondiabetic patients with unexplained vomiting syndromes. Outcomes were studied in 24 diabetic outpatients who had been treated with tricyclic antidepressants specifically for nausea and vomiting after an unsatisfactory response to prokinetic therapy. Symptom patterns and treatment response were determined from chart review and telephone interview. Ten patients (42%) had recurrent, stereotypical vomiting episodes with symptom-free intervals suggesting cyclic vomiting syndrome; 14 (58%) had persistent symptoms. By chart review, at least moderate symptom response to tricyclic antidepressant treatment (median dosage, 50 mg/day) occurred in 88% of subjects, with complete or nearly complete resolution of symptoms in one-third. At follow-up interview, 77% self-reported at least moderate symptom improvement during therapy and 68% rated tricyclic antidepressants the most effective treatment received. Duration of diabetes, presence of neuropathy, and psychiatric status were not predictive of treatment outcome in multivariate analysis, but a cyclical symptom pattern attenuated antidepressant response (P< 0.05). In this retrospective review, the majority of diabetic patients with chronic vomiting and incomplete response to prokinetic therapy benefited from tricyclic antidepressants in low-dose, open-label regimens and rated them the most effective treatment received. This therapeutic option should be further studied in diabetic patients considering the morbidity of chronic vomiting in this population.

Adult↗
PubMed1995

Phosphate excretion during 24 h of hypoxia in conscious rats.

The objective of this study was to investigate renal phosphate excretion during 24 h of hypoxia in conscious rats fed by total parenteral nutrition. Wistar rats weighing 190 g were exposed to hypoxia (inspired oxygen fraction = 0.10) or normoxia (inspired oxygen fraction = 0.21) for 24 h in a normobaric chamber. Renal clearance and hormonal studies were performed. The results showed a greater fractional excretion of phosphate (5.37 +/- 0.07%, P < 0.05) and hypophosphataemia (7.40 +/- 0.12 mg dL-1, P < 0.01) in hypoxic rats (n = 10) than in normoxic rats (n = 13; 3.50 +/- 0.37% and 8.02 +/- 0.16 mg dL-1, respectively). In addition, during hypoxia there was a significant decrease in the excretion of urinary adenosine 3',5'-cyclic monophosphate per glomerular filtrate (2.97 +/- 1.27 nmol dL-1, P < 0.005), a parameter of the renal action of parathyroid hormone, and a stable level of serum parathyroid hormone (10.2 +/- 2.6 ng mL-1) (cf. normoxia: 8.57 +/- 0.70 nmol dL-1 and 8.0 +/- 1.7 ng mL-1, respectively). However, creatinine clearance and the renal adenosine triphosphate level, both of which affect adenosine 3',5'-cyclic monophosphate excretion, were not different between the two groups. These data suggest that exposure of conscious rats to 24 h of hypoxia causes renal hyporesponsiveness to physiological levels of parathyroid hormone, which is manifested as a decrease in adenosine 3',5'-cyclic monophosphate excretion. Phosphaturia is not a direct net effect of hypoxia and secondary hypocapnia on renal phosphate transport, which is known to be regulated by parathyroid hormone through adenosine 3',5'-cyclic monophosphate.

Animals↗
PubMed1975

An analysis of the specificity in pharmacological inhibition of the passive cutaneous anaphylaxis reaction in mice and rats.

An antiserum obtained from mice, immunized to produce an antiovalbumin antibody of the IgE type, was employed in a 48-hour passive cutaneous anaphylaxis (PCA) reaction in both mice and rats. The antiserum contained an antibody which, "fixed" to skin for at least 6 days, was heat labile and eluted from diethylaminoethyl cellulose in the reagin peak. In both rats and mice, the PCA reaction was mediated by a combination of histamine and serotonin and was inhibited by specific antagonists. Various drugs were tested for inhibition of the PCA reaction in recipients also injected with compound 48/80 and histamine. Drugs which have been reported to cause an increase in intracellular cyclic adenosine monophosphate levels [prostaglandins (PG) E1 and E2 and theophylline] all selectively inhibited the PCA reaction at low doses. By varying the length of time of drug administration prior to antigen challenge, the pharmacological half-life of PGE1 was determined to be approximately 9 minutes. At high doses, theophylline also inhibited the 48/80 reaction, and PGE1 inhibited all three reactions, whereas PGE2 only inhibited PCA. Disodium cromoglycate, when given to rats, inhibited only the PCA reaction without effect on the 48/80 or histamine wheal. It was totally ineffective on any parameter measured in the mouse. It is suggested that the PCA reaction in the rodent is induced by an IgE-like antibody and mediator release is, to some extent, sensitive to intracellular levels of cyclic adenosine monophosphate. Analysis of the specificity of drug activity depends upon dose-response studies, species differences and consideration of nonspecific systemic effects.

Animals↗
PubMed1990

Rapid activation of alternative second messenger pathways in olfactory cilia from rats by different odorants.

The molecular mechanisms mediating the chemo-electrical signal transduction in olfactory receptor cells are still elusive. In this study odor induced formation of second messengers in rat olfactory cilia was monitored in a subsecond time range using a rapid kinetic device. Application of micromolar concentration of citralva induced a rapid, transient elevation of the cyclic adenosine monophosphate level, whereas the concentration of inositol trisphosphate was not affected. In contrast, pyrazine caused a rise in the concentration of inositol trisphosphate, not affecting the level of cyclic adenosine monophosphate. Analysis of the kinetic parameter for the odorant induced reaction indicated that apparently two systems are operating simultaneously. The activating effects of odorants appear to be mediated via different G-proteins. Thus, at least two different second messenger pathways appear to be involved in olfactory signal transduction.

Animals↗
PubMed2002

Mediated electrochemistry of horseradish peroxidase. Catalysis and inhibition.

A precise determination of the complex mechanism of catalysis and inhibition involved in the reaction of HRP with H(2)O(2) as substrate and an outersphere single electron donor ([Os(bpy)(2)pyCl](+)) as cosubstrate is made possible by a systematic analysis of the cyclic voltammetric responses as a function of the scan rate and of the substrate and cosubstrate concentrations, complemented by spectrophotometric steady-state and stopped-flow experiments. The bell-shaped calibration curve relating the electrochemical response to the concentration of H(2)O(2) is qualitatively and quantitatively explained by taking into account the conversion of the catalytically active forms of the enzyme into the inactive oxyperoxidase in addition to the primary catalytic cycle. These characteristics should be kept in mind in biosensor applications of HRP. The ensuing analysis and data allow one to predict biosensor amperometric responses in all practical cases. From a mechanistic standpoint, conditions may, however, be defined which render inhibition insignificant, thus allowing an electrochemical characterization of the primary catalytic cycle. At very low concentrations of H(2)O(2), its diffusion tends to control the electrochemical response, resulting in proportionality with H(2)O(2) concentration instead of the square root dependence characteristic of the classical catalytic currents. Intriguing hysteresis and trace crossings behaviors are also quantitatively explained in the framework of the same mechanism. As a consequence of the precise dissection of the rather complex reaction mechanism into its various elementary steps, a strategy may be devised for gaining a better understanding of the mechanism and reactivity patterns of each elementary step.

Catalysis↗
PubMed1996

Electrical transients in the cell-volume response to cyclic AMP of the tsetse fly Malpighian tubule

1. Using cyclic AMP to stimulate perfused tsetse fly Malpighian tubules bathed in SO42- Ringer frequently causes an immediate but transient peak in transtubular potential (Vt), before stabilisation of Vt at an increased value. 2. These transients were investigated by monitoring the associated changes in cable properties and current&shy;voltage (I/V) relationships. Tubules were perfused and bathed in either Cl- Ringer or SO42- Ringer (containing 8 mmol l-1 Cl-). 3. Tubules bathed in Cl- Ringer showed a transient swelling of the cells on exposure to cyclic AMP. Cable analysis confirmed the visually observed narrowing of the tubular lumen and revealed transient increases in core resistance (Rc) and transtubular resistance (Rt). As the cells returned to their initial volume, the lumen became distended, and Rc and Rt fell below their initial levels. These changes were accompanied by an increase, and a subsequent decrease, in the slope of the I/V plot. 4. None of the above changes was apparent in SO42- Ringer, other than a fall in Rt and in the slope of the I/V plot. 5. The results suggest that, in Cl- Ringer, cyclic AMP induces swelling of the tubular cells by promoting increased basolateral solute (and water) entry and that the subsequent rapid return to normal cell volume, with a concomitant progressive increase in the rate of tubular secretion, reflects the operation of a specific cell-volume regulatory mechanism of transepithelial transport. 6. The cyclic-AMP-induced peak that occurs in Vt in SO42- Ringer appears to be primarily due to a transient overshoot in the fall in series resistance (i.e. an increase in basolateral Na+ conductance), accompanied by a proportionately lesser increase in shunt resistance.

Journal Article↗
PubMed1994

Base-catalysed rearrangement of temazepam.

Temazepam undergoes a rearrangement reaction in strongly alkaline media to form a cyclic diamide, 7-chloro-1-methyl-5-phenyl-4,5-dihydro-2H-benzodiazepin-2,3(1H)-di one. Thermodynamic parameters (Eact, delta H++, delta S++, delta G++) involved in the rearrangement reaction, studied using either CH3CN-0.2 N NaOH in H2O (1:1, v/v) or CH3CN-0.2 N NaOD in D2O (1:1, v/v) as the solvent, were similar with an isotope effect (kH/kD) of 0.77 +/- 0.03. Kinetics of the rearrangement reaction were studied as a function of NaOH concentration, temperature and ionic strength. Results indicated that the rate-determining step did not involve proton exchange with solvent. Mass spectral analysis of the cyclic diamides derived by using either D2O or H2(18)O in the solvent mixtures suggested that the formation of the cyclic diamide involved a nucleophilic addition of a hydroxide ion at the C2 carbonyl carbon of temazepam.

Chromatography, High Pressure Liquid↗
PubMed2006

Structural characterization of mono-ruthenium substituted Keggin-type silicotungstates.

We have synthesized the mono-ruthenium substituted Keggin-type silicotungstate [SiW(11)O(39)Ru(III)(H(2)O)](5-) (1a) by reaction of the mono-lacunary silicotungstate precursor [SiW(11)O(39)](8-) with Ru(acac)(3) under hydrothermal conditions and isolated as the caesium salt Cs(5)[SiW(11)O(39)Ru(III)(H(2)O)] (1). The DMSO-coordinated complex [SiW(11)O(39)Ru(III)(DMSO)](5-) (2a) was prepared by reaction of 1a with DMSO in aqueous solution at 353 K and isolated as the caesium-potassium mixed salt Cs(4.9)K(0.1)[SiW(11)O(39)Ru(III)(DMSO)] (2). Both compounds 1 and 2 were characterized by single-crystal X-ray structure analysis, powder X-ray structure analysis, UV-Vis spectroscopy, cyclic voltammetry, IR-spectroscopy and elemental analysis. 1 crystallized in the tetragonal space group P4(2)/ncm with a = 20.9299(4), c = 10.3603(4) Angstrom, Z = 4. The ruthenium atom in the Keggin unit could not be distinguished from the tungsten due to disorder. The structural analysis of 2 (monoclinic, P2(1)/c, a = 13.5850(4), b = 20.2764(7), c = 18.1326(4) Angstrom, beta = 90.8730(10) degrees , Z = 4) successfully revealed that the incorporated ruthenium atom is coordinated by DMSO through a Ru-S bond. Polyanion 2a represents the first mono-substituted Keggin ion in which the ruthenium center is not crystallographically disordered. UV-Vis spectroscopy combined with controlled potential electrolysis confirmed that the incorporated rutheniums in 1 and 2 have a valence state of +3. The IR spectra of both 1 and 2 were very similar. All these data indicate that 1 synthesized by reaction of the mono-lacunary silicotungstate K(8)[SiW(11)O(39)] with Ru(acac)(3) under hydrothermal conditions is truly the mono-ruthenium substituted Keggin-type silicotungstate.

Journal Article↗