Effect of non-steroidal anti-inflammatory drugs on behavioral actions of diazepam in mice.

OBJECTIVE: To investigate pharmacological interactions of diazepam with non steroidal anti-inflammatory drugs. METHODS: Non selective cyclooxygenase enzyme inhibitors (100 mg/kg acetylsalicylic acid, 10 mg/kg inhibitors (100 mg/kg acetylsalicylic acid, 10 mg/kg indomethacin, and 10 mg/kg diclofenac), a selective cyclooxygnase-1 inhibitor (10 mg/kg acetylsalicylic acid), and a selective cyclooxygnase-2 inhibitor (10 mg/kg celecoxib) of non steroidal anti-inflammatory drugs were individually pretreated to 15 and 24 groups of Albino mice for dose and time dependent models (n = 8, each treatment) before sleeping induced by diazepam (20 mg/kg, intraperitoneally). In 6 groups using an open field and 4 groups using traction test models (n = 10), 5 and 10 mg/kg of diazepam, intraperitoneally were given to induce sedation and muscle relaxation, and 2 mg/kg to induce anxiolytic action after treatment with acetylsalicylic acid (10 mg/kg) to 4 groups (n = 6). This study was carried out at the Al-Fateh Medical Science University, Tripoli, Libya between February and May 2009. RESULTS: In dose and time dependent models non selective cyclooxygenase and selective cyclooxygnase-1 inhibitors significantly reduced the duration of sleep induced by diazepam in mice by 60-75%, while the selective cyclooxygnase-2 inhibitor did not (p > 0.05). However, anxiolytic, muscle relaxant, and sedative effects of diazepam were unchanged by acetylsalicylic acid. CONCLUSION: Non-steroidal anti-inflammatory drugs, most likely cyclooxygenase selective-1 inhibitors reduced the duration of sleep induced by diazepam, and this interaction could be of a pharmacodynamic type.

Correlation between photosynthesis and the transthylakoid proton gradient.

In isolated intact chloroplasts, maximal rates of photosynthetic O2 evolution (in saturating HCO3) are associated with a critical transthylakoid proton gradient as a result of the stoichiometric consumption of 2 mol NADPH and 3 mol ATP/mol CO2 fixed. Studies with the fluorescent probe 9-aminoacridine reveal that in the illuminated steady state the critical delta pH is 3.9. CO2-dependent O2 evolution is inhibited by increases of 0.1-0.2 in delta pH that occur when catalase is omitted from the medium, NO2- is included as an electron acceptor, or when chloroplasts are illuminated under low partial pressures of O2. Low concentrations of antimycin (0.33 microM) or NH4Cl (0.33 mM)decrease delta pH and relieve this inhibition of electron flow. The energy transfer inhibitor quercetin lowers the high ATP/ADP ratio associated with these conditions, but does not lower delta pH or relieve the inhibition. A decrease of delta pH below 3.9 by weaker illumination, millimolar levels of NH4Cl or micromolar levels of antimycin, results in lower rates of photosynthesis owing to limitation by the phosphorylation rate. These findings show that in absence of rate limitation by the carbon cycle, the extent of thylakoid energization is related to the ratio of ATP to NADPH production and in turn, the rate of CO2 assimilation.

Light-induced Mg2+ ATPase activity of coupling factor in intact chloroplasts.

Intense illumination isolated, intact, spinach chloroplasts triggers the well known proton-pumping Mg2+ ATPase activity of coupling factor, which can be assayed in subsequently lysed chloroplasts by monitoring ATP-driven quenching of 9-aminoacridine fluorescence. The light-triggered ATPase activity decays slowing in the dark and is inhibited by N,N'-dicyclohexylcarbodiimide. After osmotic lysis and washing of the chloroplasts, preillumination no longer triggers maximal proton-pumping ATPase until methylviologen and dithiothreitol are added to the medium. It is suggested that intact organelles contain soluble or loosely bound cofactors necessary for light-triggering of coupling factor ATPase. On osmotic lysis, these endogenous cofactors are diluted or inactivated and must be replaced by addition of a dithiol reagent and an electron acceptor.

The involvement of ferredoxin-NADP+ reductase in cyclic electron transport in chloroplasts.

The sites of action, in spinach thylakoid, of known inhibitors of electron transport at the reducing end of photosystem I have been more accurately located by parallel investigation of effects on three partial reactions: photo-reduction (from water) of added NADP+, photoreduction of added cytochrome c, and dark reduction of cyto-chrome c by added NADPH. Comparison with inhibitory effects on cyclic electron flow (registered by the slow phase of the electrochromic response during repetitive flash excitation) permitted assessment of the role of ferredoxin and ferredoxin-NADP+ reductase (ferredoxin: NADP+ oxidoreductase, EC 1.18.1.3) in the cyclic electron transport pathway around photosystem I. Disulfodisalicylidenepropane-1,1-diamine inhibited all the above partial reactions except the ferredoxin-dependent photoreduction of cytochrome C. thereby indicating its interference with the reductase or the complexation between reductase and ferredoxin. Studies with purified ferredoxin-NADP+ reductase established it as the sensitive component. Cyclic flow is also sensitive to the above inhibitor and thus presumably involves the reductase. Supporting evidence for this came from studies of inhibition by substituted maleimides, which are inhibitors of electron transfer through the isolated reductase; these also inhibited the slow phase of the electrochromic response and all partial reactions except the photoreduction of cytochrome c. In contrast, an antiserum against the reductase affected only reactions involving NADP. The conclusion is drawn that the pathway of cyclic electron transport includes both ferredoxin and ferredoxin-NADP+ reductase, but not the NADP-binding site on the reductase.

Cytochrome function in the cyclic electron transport pathway of chloroplasts.

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the alpha-bands of cytochrome b6 and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b6 and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 microM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH4Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift with formation half-time of about 2 ms. Ths inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isoprophy-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b6 and f by factors of ten or more. These observations indicate that cytochrome b6, plastoquinone and cytochrome f participated in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 mumol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO2 fixation.

Relative activities of linear and cyclic electron flows during chloroplast CO2-fixation.

Evolution of oxygen and turnover of cytochromes b-563 and f were measured upon illumination of isolated intact spinach chloroplasts with a series of flashes. The flash yield of cytochrome f oxidation approximated the sum of the yields of cytochrome b-563 reduction and electron transfer through Photosystem II, regardless of whether HCO(-3), 3-phosphoglycerate or O2 served as the terminal electron acceptor. No absorbance contribution form cytochrome b-559 was discerned within the time range studied. Some pseudocyclic electron flow occurred when both HCO(-3) and 3-phosphoglycerate were omitted, and possibly also during induction of photosynthesis; however, the flash yield data suggest tht O2 is not reduced at a significant rate during steady state photosynthesis. The maximum rate of cytochrome f turnover (1000 microequiv./mg chlorophyll per h) was adequate to support the highest rates of photosynthesis observed in isolated chloroplasts. These results agree with the concept that cytochrome f is a component both of the linear and cyclic pathways whereas cytochrome b-563 functions only in the cyclic pathway. NH4Cl decreased the half time of cytochrome b-563 oxidation fro 11.6 to 8.2 ms and decreased the half time of cytochrome f reduction from 7.2 to 2.8 ms. The cyclic and linear pathways thus seem to be jointly regulated by a transthylakoid H+ gradient through a common control point on the reducing side of cytochrome f. Cyclic turnover also increased during the induction phase of photosynthesis, when linear throughput is limited by the rate of utilization of NADPH. The slow rise in the P-518 transient correlated with increased cyclic activity under the above conditions. It is proposed that flexibility in the utilization of linear and cyclic pathways allows the chloroplast to generate ATP and NADPH in ratios appropriate to varying needs.

Cyclic electron transport in isolated intact chloroplasts. Further studies with antimycin.

Antimycin has been used to study the role of cyclic electron transport in isolated intact chloroplasts maintained under aerobic conditions. At all light intensities, antimycin inhibits CO2 fixation when assay conditions are optimal. When turnover of the Calvin cycle is inhibited, antimycin stimulates bicarbonate-dependent O2 evolution. Energy-dependent processes such as chlorophyll a and 9-aminoacridine fluorescence quenching, and light-scattering (apparent absorption) changes are inhibited by antimycin. The results suggest that cyclic electron transport contributes to photophosphorylation under aerobic conditions and is obligatory as a source of ATP during the most active periods of CO2 fixation in vivo. Cyclic electron transport can be stimulated either by inhibiting Photosystem II activity or increasing the turnover of Photosystem I relative to Photosystem II. These effects are interpreted in terms of the need for correct redox poising of carriers in the pathway in order to sustain maximum rates of cyclic electron flow. Binding studies indicate the presence of a high affinity antimycin binding site on chloroplast membranes. The stoichiometry and dissociation constant of the high affinity site are consistent with the idea that antimycin inhibits cyclic electron transport by binding to a b-type cytochrome in the thylakoid membrane.

Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue.

Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner G.J. and Siegelman, H.W. (1975) Science 190, 1298--1299). The ATPase activity of fresh vacuole suspensions was found to be 2--3 times that of protoplasts from the same tissue. 70--80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6mug/10(6) vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N'-dicyclohexylcarbodiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits. Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tuplipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for (Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K+, Na+, Mg2+, Cl-, and Ca2+ respectively, which are about the same as those in protoplasts.

Evidence for an alpha-helical epitope on outer surface protein A from the Lyme disease spirochete, Borrelia burgdorferi: an application of steady-state and time-resolved fluorescence quenching techniques.

Outer surface protein A (OspA) is a major antigen of Borrelia burgdorferi, the etiological agent of Lyme disease. A recombinant form of OspA (OspA-257) from B. burgdorferi, strain B31, contains 257 amino acids and a single tryptophan residue at position 216 (Trp-216). Mapping studies indicate that Trp-216 is involved in the epitope for the agglutinating monoclonal antibody 105.5. However, the fluorescence emission maximum of the native protein is 330 nm, indicating that Trp-216 is not solvent-exposed. Primary structure analysis suggests an alpha-helical conformation for residues approx. 204-217, which, if located on the protein surface, would allow Trp-216 to be buried, while leaving hydrophilic residues on the opposite side of the helix exposed. This helix would place Lys-212 within approx. 6 A of Trp-216; the presence of such a positively-charged residue can, in principle, be ascertained from fluorescence quenching studies. Stern-Volmer plots confirm that Trp-216 is indeed buried in the native protein, but is readily accessible to the small polar quencher, acrylamide. Furthermore, the dominant component of the fluorescence emission shows only weak dynamic quenching by the positively-charged quencher, Cs+, while the minor component undergoes static quenching by I-, indicating the proximity of a positively-charged residue. These data are consistent with the existence of an alpha-helix from residues 204-217 in the predicted orientation at the protein surface, hence indicating the structure of the antigenic determinant.

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark.

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg2+. The reaction is inhibited by (I) an antibody to ferredoxin-NADP+ reductases (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q. It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.

Structural analysis of an outer surface protein from the Lyme disease spirochete, Borrelia burgdorferi, using circular dichroism and fluorescence spectroscopy.

The etiological agent of Lyme disease is the tick-borne spirochete, Borrelia burgdorferi. A major antigen of B. burgdorferi is a 31 kDa lipoprotein called outer surface protein A (OspA). Recently, a truncated form of OspA (lacking 17 amino acids at the N-terminus) was cloned, expressed and purified in large quantities (Dunn, J.J., Lade, B.A. and Barbour, A.G. (1990) Protein Expression and Purification 1, 159-168). The truncated protein (OspA-257) is water-soluble, retains the ability to bind antibodies from the sera of Lyme disease patients and may prove useful in development of a vaccine against Lyme disease. We have used far UV circular dichroism (CD) and fluorescence spectroscopy to characterize the secondary structure of and to study conformational changes in OspA-257. CD spectra from 260 to 178 nm predict five classes of secondary structure: alpha-helix (11%), anti-parallel beta-sheet (32%), parallel beta-sheet (10%), beta-turns (18%) and aperiodic structures (including 'random coil') (30%). Analysis of the primary sequence of OspA yielded the most likely sites for alpha-helical regions (residues 100-107, 121-134, 253-273) and for antigenic determinants (Lys-46, Asp-82, Lys-231). CD spectra of the native protein show little change from pH 3 to 11. Thermal denaturation curves, indicate that 'salt bridges' play a role in stabilizing the native protein. Both thermal and chemical denaturations that eliminate all secondary structure as judged by CD or fluorescence are reversible. Denaturation by guanidine-HCl (gdn-HCl) appears to be a cooperative, two-state transition, as indicated by a sudden change in the CD spectrum at approximately 0.75 M gdn-HCl, and an isodichroic point at 208 nm in all CD spectra measured from 0.0-1.75 M gdn-HCl.