Development of a lab-on-chip electrochemical biosensor for water quality analysis based on microalgal photosynthesis.

The present work was dedicated to the development of a lab-on-chip device for water toxicity analysis and more particularly herbicide detection in water. It consists in a portable system for on-site detection composed of three-electrode electrochemical microcells, integrated on a fluidic platform constructed on a glass substrate. The final goal is to yield a system that gives the possibility of conducting double, complementary detection: electrochemical and optical and therefore all materials used for the fabrication of the lab-on-chip platform were selected in order to obtain a device compatible with optical technology. The basic detection principle consisted in electrochemically monitoring disturbances in metabolic photosynthetic activities of algae induced by the presence of Diuron herbicide. Algal response, evaluated through oxygen (O2) monitoring through photosynthesis was different for each herbicide concentration in the examined sample. A concentration-dependent inhibition effect of the herbicide on photosynthesis was demonstrated. Herbicide detection was achieved through a range (blank - 1 µM Diuron herbicide solution) covering the limit of maximum acceptable concentration imposed by Canadian government (0.64 µM), using a halogen white light source for the stimulation of algal photosynthetic apparatus. Superior sensitivity results (limit of detection of around 0.1 µM) were obtained with an organic light emitting diode (OLED), having an emission spectrum adapted to algal absorption spectrum and assembled on the final system.

Integrated electrochemical biosensor based on algal metabolism for water toxicity analysis.

An autonomous electrochemical biosensor with three electrodes integrated on the same silicon chip dedicated to the detection of herbicides in water was fabricated by means of silicon-based microfabrication technology. Platinum (Pt), platinum black (Pt Bl), tungsten/tungsten oxide (W/WO3) and iridium oxide (Pt/IrO2) working ultramicroelectrodes were developed. Ag/AgCl and Pt electrodes were used as reference and counter-integrated electrodes respectively. Physical vapor deposition (PVD) and electrodeposition were used for thin film deposition. The ultramicroelectrodes were employed for the detection of O2, H2O2 and pH related ions H3O(+)/OH(-), species taking part in photosynthetic and metabolic activities of algae. By measuring the variations in consumption-production rates of these electroactive species by algae, the quantity of herbicides present at trace level in the solution can be estimated. Fabricated ultramicroelectrodes were electrochemically characterized and calibrated. Pt Black ultramicroelectrodes exhibited the greatest sensitivity regarding O2 and H2O2 detection while Pt/IrO2 ultramicroelectrodes were more sensitive for pH measurement compared to W/WO3 ultramicroelectrodes for pH measurement. Bioassays were then conducted to detect traces of Diuron herbicide in water samples by evaluating disturbances in photosynthetic and metabolic activities of algae caused by this herbicide.

Impacts of agriculture on the parasite communities of northern leopard frogs (Rana pipiens) in southern Quebec, Canada.

Given that numerous amphibians are suffering population declines, it is becoming increasingly important to examine the relationship between disease and environmental disturbance. Indeed, while many studies relate anthropogenic activity to changes in the parasitism of snails and fishes, little is known of the impact on the parasites of amphibians, particularly from agriculture. For 2 years, the parasite communities of metamorphic northern leopard frogs from 7 agricultural wetlands were compared with those from 2 reference wetlands to study differences in parasite community diversity and abundance of various species under pristine conditions and 3 categories of disturbance: only agricultural landscape, only pesticides, and agricultural landscape with pesticides. Agricultural (and urban) area was negatively related to species richness, and associated with the near absence of adult parasites and species that infect birds or mammals. We suggest that agriculture and urbanization may hinder parasite transmission to frogs by limiting access of other vertebrate hosts of their parasites to wetlands. The only parasite found at all localities was an unidentified echinostome infecting the kidneys. This parasite dominated communities in localities surrounded by the most agricultural land, suggesting generalist parasites may persist in disrupted habitats. Community composition was associated with dissolved organic carbon and conductivity, but few links were found with pesticides. Pollution effects may be masked by a strong impact of land use on parasite transmission.

Rapid chlorophyll a fluorescence transients of Lemna minor leaves as indication of light and exogenous electron carriers effect on photosystem II activity.

By using saturating flash, we investigated the change in the rapid fluorescence rise when Lemna minor leaf was exposed to different light conditions and treated with exogenous electron acceptors (methyl viologen and duroquinone) and electron donor (hydroxylamine). Investigation was carried out by using combined pulse amplitude modulated fluorometer and plant efficiency analyzer system, which were employed simultaneously to provide different light conditions and to induce rapid fluorescence rise respectively. We have shown that when leaf of L. minor was exposed to different conditions of illumination, rapid fluorescence rise was greatly influenced by the electron transport functions beyond quinone A-plastoquinone reduction. This was indicated by the change in both fluorescence yield and appearance time of the different transients. When exogenous electron donor (hydroxylamine) and acceptors (methyl viologen and duroquinone) were applied in in vivo condition, we showed that rapid fluorescence rise represented a reliable indicator of PSII-PSI electron transport state and energy dissipation process.

Surgically induced osteoarthritis in the rat results in the development of both osteoarthritis-like joint pain and secondary hyperalgesia.

OBJECTIVE: In the present study, we sought to develop/characterize the pain profile of a rat model of surgically induced osteoarthritis (OA). METHODS: OA was surgically induced in male Lewis rats (200-225 g) by transection of the medial collateral ligament and medial meniscus of the femoro-tibial joint. In order to characterize the pain profile, animals were assessed for a change in hind paw weight distribution (HPWD), development of mechanical allodynia, and the presence of thermal and mechanical hyperalgesia. Rofecoxib and gabapentin were examined for their ability to decrease change in weight distribution and tactile allodynia. RESULTS: Transection of the medial collateral ligament and medial meniscus of male Lewis rats resulted in rapid (<3 days) changes in hind paw weight bearing and the development of tactile allodynia (secondary hyperalgesia). There was, however, no appreciable effect on thermal hyperalgesia or mechanical hyperalgesia. Treatment with a single dose of rofecoxib (10 mg/kg, PO, day 21 post surgery) or gabapentin (100mg/kg, PO, day 21 post surgery) significantly attenuated the change in HPWD, however, only gabapentin significantly decreased tactile allodynia. CONCLUSION: The rat medial meniscal tear (MMT) model mimics both nociceptive and neuropathic OA pain and is responsive to both a selective cylooxygenase-2 (COX-2) inhibitor commonly utilized for OA pain (rofecoxib) and a widely prescribed drug for neuropathic pain (gabapentin). The rat MMT model may therefore represent a predictive tool for the development of pharmacologic interventions for the treatment of the symptoms associated with OA.

Comparison by PAM fluorometry of photosynthetic activity of nine marine phytoplankton grown under identical conditions.

The photosynthetic activity of marine phytoplankton from five algal classes (Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira oceanica, Thalassiosira weissflogii, Dunaliella tertiolecta, Mantoniella squamata, Emiliania huxleyi, Pavlova lutheri and Heterosigma akashiwo) was investigated under identical growth conditions to determine interspecies differences. Primary photochemistry and electron transport capacity of individual species were examined by pulse amplitude-modulated (PAM) fluorescence. Although few differences were found in maximal photosystem II (PSII) photochemical efficiency between various species, large differences were noticed in their PSII-photosystem I (PSI) electron transport activity. We found that species such as T. oceanica and M. squamata have much lower photochemical activity than H. akashiwo. It appeared that processes involved in electron transport activity were more susceptible to change during algal evolution compared with the primary photochemical act close to PSII. Large variations in the nonphotochemical energy dissipation event among species were also observed. Light energy required to saturate photosynthesis was very different between species. We have shown that M. squamata and H. akashiwo required higher light energy (>1300 micromol m(-2) s(-1)) to saturate photosynthesis compared with S. costatum and E. huxleyi (ca 280 micromol m(-2) s(-1)). These differences were interpreted to be the result of variations in the size of light-harvesting complexes associated with PSII. These disparities in photosynthetic activity might modulate algal community structure in the natural environment where light energy is highly variable. Our results suggest that for an accurate evaluation of primary productivity from fluorescence measurements, it is essential to know the species composition of the algal community and the individual photosynthetic capacity related to the major phytoplankton species present in the natural phytoplankton assemblage.

Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis.

OBJECTIVE: To describe an in vivo model in the rat in which change in weight distribution is used as a measure of disease progression and efficacy of acetaminophen and two nonsteroidal anti-inflammatory drugs (NSAIDs) in a model of monosodium iodoacetate (MIA)-induced osteoarthritis (OA). METHODS: Intra-articular injections of MIA and saline were administered to male Wistar rats (175-200 g) into the right and left knee joints, respectively. Changes in hind paw weight distribution between the right (osteoarthritic) and left (contralateral control) limbs were utilized as an index of joint discomfort. Acetaminophen and two archetypal, orally administered NSAIDs, naproxen and rofecoxib, were examined for their ability to decrease MIA-induced change in weight distribution. RESULTS: A concentration-dependent increase in change in hind paw weight distribution was noted after intra-articular injection of MIA. Both naproxen and rofecoxib demonstrated the capacity to significantly (P<0.05) decrease hind paw weight distribution in a dose-dependent fashion, indicating that the change in weight distribution associated with MIA injection is susceptible to pharmacological intervention. CONCLUSION: The determination of differences in hind paw weight distribution in the rat MIA model of OA is a technically straightforward, reproducible method that is predictive of the effects of anti-inflammatory and analgesic agents. This system may be useful for the discovery of novel pharmacologic agents in human OA.

Validation of a flow cytometric acridine orange micronuclei methodology in rats.

Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to evaluation of much larger numbers of cells clearly demonstrate the usefulness of flow cytometry method in the routine micronucleus evaluation.

PAM fluorometry in the determination of the sensitivity of Chlorella vulgaris, Selenastrum capricornutum, and Chlamydomonas reinhardtii to copper.

The pulse-amplitude-modulation (PAM) fluorometric method was used in the past as a sensitive and rapid method for assessing toxic effect of pollutants in plants. Here, we used the advantages of this method to evaluate the difference in sensitivity of Chlorella vulgaris, Selenastrum capricornutum, and Chlamydomonas reinhardtii to copper toxicity. We found by using the PAM-fluorescence parameters (phiM, phi'M, QP, and QN), that C. reinhardtii is the most sensitive species, followed by S. capricornutum and C. vulgaris. Indeed, the copper 5-, 48-, and 96-h EC50 for phiM, phi'M, and QP vary between 11 and 34 microg/L for C. reinhardtii, while for S. capricornutum this value is between 25 and 50 microg/L. However, the same parameters obtained from C. vulgaris did not show susceptibility to copper, even at very high concentrations (100 microg/L). In this study, the estimated 5-h EC50 for C. reinhardtii and S. capricornutum are lower than the 24-h EC50 reported earlier for these species with other bioassays, indicating that the use of PAM fluorometry in copper bioassay provides a more sensitive method than the other biotests in aquatic toxicology.

The antithrombotic effects of CI-1031 (ZK-807834) and enoxaparin in a canine electrolytic injury model of arterial and venous thrombosis.

Factor Xa is a serine protease positioned at the convergence point of the intrinsic and extrinsic coagulation pathways and is therefore an attractive target in the development of novel anticoagulant drugs. The objective of this study was to evaluate the efficacy of CI-1031 (N-[2-[5-amidino-2-hydroxyphenoxy]-6-[3-(1-methyl-1H-imidazolin-2-yl)-phenoxy]-3,5-difluoropyrid), a potent and selective inhibitor of Factor Xa, in a canine electrolytic injury model of arterial and venous thrombosis. Enoxaparin (enoxaparin sodium), a low molecular weight heparin currently approved for treatment and prevention of deep vein thrombosis and unstable angina, was also tested for efficacy in this model. CI-1031 was administered intravenously to anesthetized dogs at three doses: 1.25, 2.5 and 5 microg/kg/min (n=5 for each group) as a continuous infusion for 5.5 h. The control group (n=5) received a continuous infusion of vehicle (3.69 mmol citric acid and 0.9% sodium chloride solution) at a rate of 1 ml/kg/h. Ninety minutes after administration of CI-1031 prothrombin times increased 1.2-, 1.6- and 2.0-fold over baseline values in the 1.25, 2.5 and 5 microg/kg/min groups, respectively. The time to formation of an occlusive thrombus in the femoral arteries averaged 69+/-5 min in the control group compared to 127+/-19, 192+/-33 and 219+/-15 min in the low-, mid- and high-dose CI-1031 groups. In the femoral veins, occlusion time in the controls averaged 56+/-11 min compared to 153+/-22, 137+/-30 and 214+/-26 min in the three treatment groups. Thrombus weights in the control arteries averaged 51+/-4 mg compared to 45+/-5, 28+/-10 and 15+/-3 mg in the CI-1031 treated groups. On the venous side, control thrombus weights averaged 96+/-18 mg compared to 75+/-16, 51+/-16 and 25+/-4 mg in the low-, mid- and high-dose CI-1031 groups. A plasma CI-1031 concentration of approximately 400 ng/ml was associated with a 50% reduction in thrombus weight relative to control animals. Enoxaparin was administered intravenously at a loading dose of 50, 100 or 200 IU/kg for 1 h followed by a maintenance infusion of 25, 50 or 100 IU/kg/h for 4.5 h. The most dramatic changes in coagulation parameters were observed in thrombin time with virtually no changes in prothrombin time. Enoxaparin elicited a dose-dependent increase in time to thrombotic occlusion and a dose-dependent decrease in thrombus weight similar to that observed with CI-1031. Time to occlusion in the enoxaparin-treated groups averaged 117+/-33, 188+/-32 and 217+/-22 min in the low-, mid- and high-dose groups in the femoral arteries and 84+/-22, 171+/-31 and 133+/-33 min in the femoral veins. Thrombus weights averaged 33+/-10, 12+/-5 and 10+/-4 mg in the arteries and 32+/-9, 13+/-2 and 21+/-6 mg in the veins in the low-, mid- and high-dose groups. Blood loss with CI-1031 tended to be less than enoxaparin at doses that provided comparable efficacy. These results demonstrate that CI-1031, like enoxaparin, is an effective antithrombotic agent in an established canine model of arterial and venous thrombosis. CI-1031 provided dose-dependent efficacy with minimal changes in ex vivo coagulation parameters, suggesting it may be a safe and effective antithrombotic agent for both arterial and venous indications.

Evaluation of different algal species sensitivity to mercury and metolachlor by PAM-fluorometry.

In this study, the pulse-amplitude-modulation (PAM)-fluorometric method was used to evaluate the difference in the sensitivity to mercury (Hg) and metolachlor of six algal species: Ankistrodesmus falcatus, Selenastrum capricornutum, Chlorella vulgaris, Nannoplankton (PLS), Microcystis aeruginosa and Pediastrum biwae. We found that the fluorescence parameters (phiM, the maximal photosystem II (PSII) quantum yield, phi'M, the operational PSII quantum yield at steady state of electron transport, Q(P), the photochemical quenching value, and Q(N), the non-photochemical quenching value) were appropriate indicators for inhibitory effects of mercury but only phi'M and Q(N) were useful for metolachlor. The examined algal species showed very different levels of sensitivity to the effect of Hg and of metolachlor. The most sensitive species to Hg and metolachlor were respectively M. aeruginosa and A. falcatus, while the least sensitive were C. vulgaris and P. biwae. We interpreted these differences by the action mode of pollutants and by the different metabolism properties and morphological characteristics between algal species. These results related to fluorescence parameters may offer useful tool to be used in bioassay for different pollutants. Heterogeneous algal sensitivity to the same pollutant suggests the need to use a battery of species to evaluate the effects of mixtures of pollutants in aquatic systems.

Spectroscopic analysis of desiccation-induced alterations of the chlorophyllide transformation pathway in etiolated barley leaves.

Effects of water deficit on the chlorophyllide (Chlide) transformation pathway were studied in etiolated barley (Hordeum vulgare) leaves by analyzing absorption spectra and 77-K fluorescence spectra deconvoluted in components. Chlide transformations were examined in dehydrated leaves exposed to a 35-ms saturating flash triggering protochlorophyllide (Pchlide) and Chlide transformation processes. During the 90 min following the flash, we found that dehydration induced modifications of Chlide transformations, but no effect on Pchlide phototransformation into Chlide was observed. During this time, content of NADPH-Pchlide oxydoreductase in leaves did not change. Chlide transformation process in dehydrated leaves was characterized by the alteration of the Shibata shift process, by the appearance of a new Chlide species emitting at 692 nm, and by the favored formation of Chl(ide) A(668)F(676). The formation of Chl(ide) A(668)F(676), so-called "free Chlide," was probably induced by disaggregation of highly aggregated Chlide complexes. Here, we offer evidence for the alteration of photoactive Pchlide regeneration process, which may be caused by the desiccation-induced inhibition of Pchlide synthesis.

Repeated analysis of semen parameters in beagle dogs during a 2-year study with the HMG-CoA reductase inhibitor, atorvastatin.

Sperm analyses are often incorporated into reproductive toxicity studies in rats. Due to the relative ease of collecting multiple samples throughout a study, semen analysis in non-rodents such as dogs offers the opportunity to assess potential development of functional effects of compounds on male reproduction over time. In the present study, semen parameters were evaluated in beagle dogs during and at termination of a chronic toxicity study with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin. Male dogs received 0, 10, 40, or 120 mg/kg orally in gelatin capsules for up to 104 weeks (n = 10/group). After 52 weeks of dosing, 3 dogs/group were euthanized, and 2/group were withdrawn from treatment for a 12-week reversal period and euthanized at Week 64. The remaining 5/group continued treatment until Week 104. Semen was collected from all animals for 3 consecutive weeks prior to termination of the 52-week animals (Weeks 50, 51, 52) for analysis of sperm parameters, using manual methods of evaluation. Semen was collected from the remaining animals at Weeks 64, 78, 91, and 104, and was analyzed. At necropsy, testes, epididymides, and prostates were weighed and evaluated histologically, and epididymal sperm counts were determined. Serum cholesterol was decreased 25--60% at all doses during the study. There were no drug-related differences in semen volume and color, total sperm count, and sperm concentration, morphology, progressiveness, and percent motility during treatment with atorvastatin. There were also no effects on reproductive organ weights or histopathology, and no effects on epididymal sperm count. Thus, incorporation of semen analyses into this study allowed the evaluation of potential male reproductive effects in dogs at multiple time points during the study. Statistical power calculations demonstrated acceptable statistical power (> 80%) for semen sperm count, concentration, morphology, and motility with group sizes of 8--10 animals, and for semen sperm count and concentration or epididymal sperm count with group sizes of 3--5 animals, using the methodology described in this paper.

Antithrombotic effect of LB-30057 (CI-1028), a new synthetic thrombin inhibitor, in a rabbit model of thrombosis: comparison with inogatran.

LB-30057 (CI-1028) is a novel, orally bioavailable, direct thrombin inhibitor with a Ki of 0.38 nM against human thrombin. The effects of LB-30057 on thrombus formation and hemostasis were evaluated in a veno-venous shunt model of thrombosis in rabbits, and compared with inogatran, another direct inhibitor of thrombin. Each compound was studied at 5 or 6 different doses with 5 or 6 rabbits in each group. After administration as a bolus i.v. injection followed by continuous infusion, both LB-30057 and inogatran dose-dependently inhibited thrombus formation, which was measured as an increase in time to occlusion (TTO) and a decrease in thrombus weight. Both compounds also improved vena caval blood flow and reduced the overall incidence of thrombotic occlusion. LB-30057 significantly prolonged TTO from 23 +/- 4 min (before dose) to 110 +/- 10 min at the highest dose (0.7 mg/kg + 47 microg/kg/min) (p < 0.001), and reduced thrombus weight from 57 +/- 2 mg to 15 +/- 5 mg (p < 0.001). Occlusive thrombus formed in only one of six rabbits that received the highest dose of LB-30057 (vs. 13/13 in the control group, p < 0.01). At the dose that produced the maximum antithrombotic effect (0.7 mg/kg + 47 microg/kg/min), LB-30057 increased aPTT and bleeding time approximately 2-and 2.5-fold above baseline, respectively. On a gravimetric basis, LB-30057 and inogatran displayed comparable in vivo antithrombotic efficacy. When compared to equally effective anti thrombotic doses of inogatran, LB-30057 caused less prolongation in aPTT, had no effect on PT, and tended to have less of effect on bleeding time. These results indicate that LB-30057 is an effective antithrombotic compound and it appears to have a better benefit/risk profile than inogatran in this experimental model.

The antithrombotic effects of CI-1028, an orally bioavailable direct thrombin inhibitor, in a canine model of venous and arterial thrombosis.

Direct thrombin inhibitors represent a new class of drug that may offer a therapeutic alternative for the treatment and prevention of thrombembolic conditions, especially on the venous side of the systemic circulation. CI-1028 (PD 172524/LB30057) is a potent, highly selective inhibitor of thrombin that is orally bioavailable. The efficacy of this compound has been demonstrated in animal models in which intra-venous administration was used. The objective of this study was to evaluate the efficacy of CI-1028 after oral administration in a canine electrolytic injury model of venous and arterial thrombosis. CI-1028 was administered via oral gavage, and animals received either saline or 10, 15, 20, or 30 mg/kg of drug. Fifteen minutes later, the dogs were anesthetized and a femoral artery and vein were exposed and instrumented to induce electrolytic injury and thrombosis while continuously monitoring blood flow in the vessels. Maximum blood CI-1028 concentrations of 0.88+/-0.27, 1.8+/-0.3, 2.2+/-0.5, and 3.2+/-0.5 microg/mL were generally achieved 15 to 30 minutes after administering the compound in the 10-, 15-, 20-, and 30-mg/kg groups, respectively. Administration of CI-1028 increased the time to occlusion (TTO), the principal efficacy end point, in a dose-dependent manner in both arteries and veins. The TTO in the control group (n=8) averaged 66+/-11 minutes in the arteries and 69+/-6 minutes in the veins. In dogs treated with 10 mg/kg (n=8), the TTO was not significantly different from that of the control group. In the 15-mg/kg group (n=9) TTO averaged 140+/-27 minutes in the arteries (p=not significant) and 125+/-15 minutes (p<0.05) in the veins. In the 20-mg/kg group (n=8), TTO was significantly longer than controls in both types of vessels, averaging 168+/-30 minutes in the arteries (p=0.05) and 155+/-21 minutes (p<0.05) in the veins. Likewise, at 30 mg/kg (n=8) both the arterial (179+/-17 minutes) and venous (188+/-15 minutes) TTO was significantly prolonged compared with controls. Surgical blood loss and template bleeding times tended to increase in a dose-dependent manner but a statistically significant elevation was detected for template bleeding time only at the highest dose. Dramatic changes in thrombin time were detected, consistent with the CI-1028 mechanism of action. Virtually no changes were detected in prothrombin time. Maximum activated partial thromboplastin time (aPTT) and activated clotting time changes were detected approximately 30 minutes after dosing, and they were approximately twofold and fivefold baseline values, respectively, at the highest dose. In conclusion, these results demonstrate that CI-1028 provides dose-dependent antithrombotic efficacy after oral administration in a canine model of venous and arterial thrombosis.

Purine-induced alterations of dopamine metabolism in rat pheochromocytoma PC12 cells.

Studies with cerebrospinal fluid from subjects with Parkinson's disease suggest that purine abnormalities may be present in this disorder. The effects of purines on dopamine metabolism have not been characterized, though adenosine is known to inhibit dopaminergic neurotransmission. In this study, dopamine, its precursor 3,4-dihydroxyphenylalanine (DOPA), and its degradation products 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured in rat pheochromocytoma PC12 cells following 24-h incubation with 5, 50, and 500 microM adenosine, adenine, guanosine, guanine, hypoxanthine, xanthine, and uric acid. Incubation with adenosine increased DOPA, DOPAC, and HVA, while adenine treatment decreased DOPA. Guanosine (500 microM) decreased DOPA, dopamine, and DOPAC, while lower concentrations increased DOPAC and HVA. Incubation with guanine decreased dopamine, and xanthine decreased dopamine and DOPAC. Hypoxanthine and uric acid exerted minimal effects. These results indicate that purines exert a variety of effects on dopamine metabolism. The influence of purine metabolism on the dopaminergic deficit in the Parkinsonian brain merits further investigation.

Evidence of chlorophyll synthesis pathway alteration in desiccated barley leaves.

In etiolated leaves, saturating flash of 200 ms induces phototransformation of protochlorophyllide (Pchlide) F655 into chlorophyllide (Chlide), then into Chl through reactions which do not need light sensibilisation. The synthesis of Chl is known to be slowed down in etiolated leaves exposed to desiccation stress. In order to analyse the intensity and time-course of Chlide transformation into Chl, we used the fluorescence emission of etiolated leaves previously exposed to a 200 ms saturating flash. We used low-temperature fluorescence spectroscopy to reveal the inhibition site of Chl synthesis in etiolated barley leaves exposed to water stress. Shibata shift appears as the main target point of the water deficit. It was found that water deficit inhibits partially active Pchlide F655 regeneration. Also, esterification of Chlide into Chl is impaired. It appears that these inhibitory effects alter the appearance of PSII active reaction centres.

Analysis of a mutation in phosphodiesterase type 4 that alters both inhibitor activity and nucleotide selectivity.

Cyclic nucleotide phosphodiesterase type 4 (PDE4) is a cAMP-specific phosphodiesterase that is found as four distinct genes in the mammalian genome (PDE4A, 4B, 4C, and 4D). Mutation analysis was done to identify the amino acids involved in activity and inhibitor selectivity. Mutations at Asp333 were made in HSPDE4D3 based on mutations that affect rolipram sensitivity in RNPDE4B1. The PDE4D3 Asp-Asn mutant was resistant to inhibition by rolipram as well as several other PDE4 inhibitors tested. These results suggest that this residue is near the inhibitor binding pocket in PDE4D3. Sequence comparison of PDE4 with cGMP-specific PDE proteins shows a conserved aspartic acid at position 333 in PDE4D3 and a conserved asparagine at this position in PDE enzymes that hydrolyze cGMP. Therefore, cGMP hydrolysis by PDE4D3 Asp-Asn was measured. PDE4D3 Asp-Asn hydrolyzes cGMP with kinetic constants similar to those observed for this protein with cAMP (K(m) approximately 20 microM, V(max) approximately 2 micromol AMP/min/mg recombinant protein). Under identical conditions, the K(m) value for cAMP hydrolysis by wild-type PDE4D3 is 3 microM and the V(max) value is 1 micromol AMP/min/mg recombinant protein. In addition, the PDE4D3 Asp-Ala mutant protein could hydrolyze cGMP. Finally, the analogous mutation in HSPDE4B1 (Asp413Asn) also allows hydrolysis of cGMP. These results show that this aspartic acid residue is important in inhibitor binding and nucleotide discrimination and suggest this residue is in the active site of PDE4.