The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites.

BACKGROUND: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin-protease complex have been proposed. In the absence of a spatial structure of a serpin-protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial. RESULTS: We used a recently developed method for making precise distance measurements, based on donor-donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1') were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into beta sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease-inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker. CONCLUSIONS: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.

[The efficacy of sublingual specific immunotherapy in children with allergic rhinitis].

Objective:To investigate the efficacy of single-allergen sublingual immunotherapy (SLIT) in children with allergic rhinitis and the potential relationship between sensitization status and efficacy and analyze the possible relationships between sensitized state and clinical efficacy. Method:One hundred and thirty children, aged 4-15 years old, with mites-induced respiratory allergic diseases had been arranged into the treatment group (n=70) or control group (n=50) and received SLIT with standardized dermatophagoides farinae extracts and pharmacotherapy for 1 year. Rhinitis and asthma symptoms and medications, visual analogue scale (VAS), skin prick test (SPT) and peak expiratory flows (PEF) were evaluated. After treatment, patients in the poly-sensitized group who completed the study had been analyzed as subgroup 1 (n=33) and subgroup 2 (n=37) according to the number of coexist allergens. Result:The global clinical parameters had been significantly improved after treatment. The treatment and control group rhinitis symptom score, symptomatic medication score and VAS scores were significantly reduced after 52 weeks treatment (all P<0.05). SLIT group dust mite grade skin reactions decreased after 52 weeks treatment (P<0.05). Dust mite skin reactions grade was greater than before treatment in the control group (P>0.05). Between the two groups, SLIT group rhinitis symptom score at 24 weeks, 36 weeks, 52 weeks were lower than the control group, the difference was statistically significant (P<0.05). SLIT group symptomatic medication score and VAS scores at 36 weeks and 52 weeks compared with the control group, the difference was statistically significant (P<0.05). Dust mite SLIT group grade skin reactions grade was lower than the control group at 52 weeks, the difference was statistically significant (P<0.05). In addition to the 36 weeks poly-sensitized group symptoms of allergic rhinitis score was lower than mono-sensitized group (P＜0.05) and at 24 weeks poly-sensitized group VAS score was lower than single allergy group (P<0.05), the comparison between subgroup 1 and subgroup 2 indicated that, there was no significant difference in symptoms scores, SPT, PEF and VAS at each scheduled follow-up visit. Conclusion:This study shows that SLIT can significantly reduce rhinitis symptoms and drug use, and improve the children with allergies. An equivalent efficacy of single-allergen SLIT is found in poly-sensitized and mono-sensitized children. The number of coexist positive allergens has a limited impact on the efficacy from a long-term perspective.

Extracellular Tau Oligomers Produce An Immediate Impairment of LTP and Memory.

Non-fibrillar soluble oligomeric forms of amyloid-ß peptide (oAß) and tau proteins are likely to play a major role in Alzheimer's disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAß initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aß, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAß levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAß to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and Aß on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aß and tau pathology.

The E3 ubiquitin ligase NEDD4 negatively regulates HER3/ErbB3 level and signaling.

HER3/ErbB3, a member of the epidermal growth factor receptor (EGFR) family, has a pivotal role in cancer and is emerging as a therapeutic antibody target. In this study, we identified NEDD4 (neural precursor cell expressed, developmentally downregulated 4) as a novel interaction partner and ubiquitin E3 ligase of human HER3. Using molecular and biochemical approaches, we demonstrated that the C-terminal tail of HER3 interacted with the WW domains of NEDD4 and the interaction was independent of neuregulin-1. Short hairpin RNA knockdown of NEDD4 elevated HER3 levels and resulted in increased HER3 signaling and cancer cell proliferation in vitro and in vivo. A similar inverse relationship between HER3 and NEDD4 levels was observed in prostate cancer tumor tissues. More importantly, the upregulated HER3 expression by NEDD4 knockdown sensitized cancer cells for growth inhibition by an anti-HER3 antibody. Taken together, our results suggest that low NEDD4 levels may predict activation of HER3 signaling and efficacies of anti-HER3 antibody therapies.

Carboxy-terminal propeptide of human type I collagen and pyridinium cross-links as markers of bone growth in infants 1 to 18 months of age.

Serum carboxy-terminal propeptide of human type I collagen (PICP) concentrations, as a marker for bone formation, and urinary pyridinium (Pyd) cross-link concentrations, as a marker of bone resorption, were determined in 66 healthy infants aged 1-18 months who are being studied longitudinally. We hypothesized that there would be a positive correlation of growth velocity, increase in bone area, and bone mass accretion rates with PICP and Pyd cross-link concentrations. Since osteocalcin is currently used as a marker of bone formation, serum osteocalcin concentrations were also measured. Mean serum PICP and urinary Pyd cross-link concentrations were significantly greater than adult concentrations. Future growth velocity, increase in bone area, and bone mass accretion rates were not associated with PICP, Pyd cross-link, or osteocalcin concentrations. Growth velocity during the 3 months preceding sample collection correlated with serum PICP, Pyd/kg, and osteocalcin concentrations (r = 0.474, p < 0.001; r = 0.379, p < 0.001; and r = 0.516, p < 0.001, respectively). Previous increase in bone area correlated with serum PICP concentrations (r = 0.359, p = 0.01). The relationship between the infant's previous bone mass accretion rate and PICP was of borderline significance (r = 0.281, p = 0.055). In summary, normative data for PICP, Pyd cross-link concentrations, and parameters of bone growth are provided for infants 1-18 months of age and indicate that these markers reflect past and current bone metabolism and may be helpful in monitoring bone disorders in infants.

Cigarette smoke inhalation stimulates dopaminergic neurons in rats.

In humans, nicotine is self administered by inhalation of tobacco smoke as opposed to animal models, where nicotine is administered via systemic injection. The aim of the present study was to clarify whether tobacco smoke inhalation would affect dopaminergic projections differently from the reported activation after the systemic administration of nicotine. For this purpose, tobacco smoke from cigarettes containing 1.0 or 0.1 mg nicotine was delivered by inhalation to rats, while recording from antidromically identified nigrostriatal and mesolimbic dopamine neurons. Smoke inhalation from 1.0 mg nicotine cigarettes caused a peculiar abrupt increase of discharge activity of mesolimbic dopamine neurons, while nigrostriatal cells were less responsive. This activation was promptly antagonized by mecamylamine (2.0 mg/kg, i.v.). In contrast, smoke delivered from 0.1 mg nicotine cigarettes was ineffective. These findings suggest that the boosting activation of mesolimbic dopamine neurons by inhaled nicotine might be relevant for the rewarding properties of tobacco smoking and also for the effectiveness of new treatments to stop smoking.

Microdialysis measurement of cortical and hippocampal acetylcholine release during sleep-wake cycle in freely moving cats.

The variations of Acetylcholine (ACh) release in the cerebral cortex and dorsal hippocampus were monitored by microdialysis during the electroencephalographically recorded sleep-waking cycle in freely moving cats. The results show a state-dependent variation in ACh output in both the cortex and the hippocampus. ACh release increased by approximately 100% during quiet waking (QW) and by 175% during active waking (AW) as referred to slow wave sleep (SWS) baseline. In contrast, a clear difference between the two areas was observed during REM sleep. During this stage ACh release in the cortex reached approximately the same values observed during QW, while in the hippocampus ACh release rose to about 4-fold the level obtained during SWS or twice that of QW. The results support the idea that the increase in ACh release in the cortex reflects the desynchronized EEG of wakefulness and REM sleep, while the marked increase of ACh during REM in the hippocampus may be related to the sustained theta activity in this area.

Fluorescence studies on plasminogen activator inhibitor 1: reactive centre cysteine mutants remain active after fluorophore attachment.

To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1). Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1. The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed. The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration. The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion. The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys. No significant spectral changes could be seen when the P3cys mutant was transferred to latency. In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine. Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.

Gamma-hydroxybutyric acid intake in ethanol-preferring sP and -nonpreferring sNP rats.

Gamma-hydroxybutyric acid (GHB) and ethanol share several pharmacological similarities, suggesting that GHB may exert ethanol-like effects in the central nervous system. The present study was designed to test whether selectively bred ethanol-preferring rats would, unlike ethanol-nonpreferring ones, self-administer GHB, consistent with their higher preference for ethanol. Male ethanol-naive Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) rats were used. In Experiment 1, GHB solution (1% (w/v) in water) was initially offered as the sole fluid available for 14 consecutive days and then presented under the two-bottle, free-choice regimen, one bottle containing water and the other the GHB solution, for an additional 14 consecutive days. During the free-choice phase, high preference for GHB and intake of pharmacologically relevant daily doses of GHB developed in both rat lines, presumably because the 14-day no-choice period would unmask the reinforcing properties of GHB and lead to acquisition of GHB preference also in the supposedly less susceptible sNP rats. In Experiment 2, the forced GHB drinking phase was reduced to 3 days. Under the subsequent free-choice regimen, daily GHB preference and intake were initially low in both sP and sNP rats; however, after approximately 10 days, GHB preference and intake in sP rats rose progressively and then stabilized to significantly higher levels than in sNP rats throughout the entire free-choice phase. It is likely that episodic binges of GHB intake occurring during the first 10 days resulted in experiencing the reinforcing properties of GHB by sP but not sNP rats. The results of the present study suggest that a) sP rats are genetically more sensitive to the reinforcing effects of both ethanol and GHB than sNP rats; and b) disclosure of the higher sensitivity of sP rats to the reinforcing effects of GHB is a function of the length of the induction procedure. The results are also discussed in terms of differences in GHB receptors contributing to the predisposition to ethanol preference and avoidance, respectively.

Sardinian alcohol-preferring rats prefer chocolate and sucrose over ethanol.

The present study was aimed at determining whether the concurrent availability of highly palatable fluids (i.e., a chocolate-flavored drink and a sucrose solution) would alter voluntary ethanol drinking in selectively bred, alcohol-preferring sP and -nonpreferring sNP rats. Ethanol intake occurred under the three-bottle, free choice regimen between 10% (v/v) ethanol solution, tap water, and the palatable fluids for 24 h per day. When rats were given ethanol and water, but no alternative fluids, mean ethanol intake in sP rats ranged between 6 and 7 g/kg per day and mean preference ratio was steadily higher than 80%, whereas mean ethanol intake and preference ratio in sNP rats were constantly lower than 0.3 g/kg and 5%, respectively. In the presence of either the chocolate-flavored drink or sucrose solution, both prepared as isocaloric to the ethanol solution, absolute ethanol intake in sP rats declined by 60-70%; similarly, the preference ratio was reduced by 80-90%. Ethanol intake in sNP rats was unaffected by the simultaneous presentation of either palatable fluids. The results of the present study closely replicate those previously reported in genetically selected, ethanol-preferring HAD rats; however, they differ from those of ethanol-preferring P rats, which were reported to maintain high levels of ethanol intake and preference in the presence of highly palatable fluids. These results are discussed in terms of a) an alternative reinforcement partially substituting for the reinforcing properties of ethanol in sP rats, resulting in a less urgent need of ethanol, and b) genetic animal models of alcoholism diverging in some neurochemical and behavioral traits (e.g., response to the presentation of palatable fluids), which might parallel the different types of alcoholism observed in humans.

Arterial end-to-side grafting in coronary artery bypass grafting: the Tector procedure.

Background. The current treatment of choice in patients with three-vessel coronary disease is coronary artery bypass grafting. The use of the left internal mammary artery in bypass grafting has shown superior long-term outcomes compared with venous grafting. In our study we assess the safety and feasibility of all-arterial coronary artery bypass graft surgery using the procedure as described by Tector et al. in 2001.Methods. Between June 2001 and February 2007, we studied 133 patients eligible for non-emergency surgical revascularisation. Primary endpoints were death or re-infarction within a 30-day period. Secondary endpoints were the need for emergency coronary surgery, angioplasty and mediastinitis. Long-term follow-up had a mean duration of 33 months postoperatively.Results. All 133 patients were successfully revascularised, 98% with the off-pump technique. In 93% of the patients (n=124) full arterial grafting was achieved using both internal mammary arteries. Thirty-day mortality was 1.5% (n=2), ten re-thoracotomies were performed, one myocardial infarction and one case of mediastinitis were reported. In the next four years six additional patients died. Most of these deaths were due to non-cardiovascular causes. Two patients required angioplasty because of distal bypass graft failure and one for new native coronary artery disease. Conclusion. All-arterial bypass grafting using both internal mammary arteries with the technique as described by Tector is safe and feasible without excess deep sternal wound infections. Late major adverse cardiac events are rare and due to distal graft dysfunction, which can be treated by percutaneous coronary intervention. (Neth Heart J 2010;18:7-11.).

The inhibition mechanism of serpins. Evidence that the mobile reactive center loop is cleaved in the native protease-inhibitor complex.

Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1' residues acting as a bait for cognate protease. Current hypotheses are conflicting as to whether the native serpin-protease complex is a tetrahedral intermediate with an intact inhibitor or an acyl-enzyme complex with a cleaved inhibitor P1-P1' peptide bond. Here we show that the P1' residue of the plasminogen activator inhibitor type 1 mutant (P1' Cys) became more accessible to radiolabeling in complex with urokinase-type plasminogen activator (uPA) compared with its complex with catalytically inactive anhydro-uPA, indicating that complex formation with cognate protease leads to a conformational change whereby the P1' residue becomes more accessible. Analysis of chemically blocked NH2 termini of serpin-protease complexes revealed that the P1-P1' peptide bonds of three different serpins are cleaved in the native complex with their cognate protease. Complex formation and reactive center cleavage were found to be rapid and coordinated events suggesting that cleavage of the reactive center loop and the subsequent loop insertion induce the conformational changes required to lock the serpin-protease complex.

Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin.

Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.

The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.

Donor-donor energy migration for determining intramolecular distances in proteins: I. Application of a model to the latent plasminogen activator inhibitor-1 (PAI-1).

A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cysteine residues can be constructed for specific labeling with one or two identical fluorescent probes, so-called donors (d). Fluorescence depolarization experiments on double-labeled Cys mutant monitor both reorientational motions of the d molecules, as well as the rate of intramolecular energy migration. In this report a model that accounts for these contributions to the fluorescence anisotropy is presented and experimentally tested. Mutants of a protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migration, the intramolecular distance between the d groups was calculated by using the Forster mechanism and by accounting for the influence of local anisotropic orientation of the d molecules. The calculated intramolecular distances were compared with those obtained from the crystal structure of PAI-1 in its latent form. To test the stability of parameters extracted from experiments, synthetic data were generated and reanalyzed.

Constant absolute ethanol intake by Sardinian alcohol-preferring rats independent of ethanol concentrations.

The present study was designed to evaluate ethanol drinking behaviour in Sardinian alcohol-preferring (sP) and Sardinian alcohol-non-preferring (sNP) rats in the presence of different ethanol concentrations. Ethanol intake was tested under the two-bottle, free-choice regimen and continuous access schedule. Ethanol-naive sP and sNP rats were initially given ethanol solution at the standard, constant concentration of 10% (v/v) for 8 consecutive days (Phase 1). As expected, daily ethanol intake in sP rats rose from 4 to approximately 6 g/kg; in contrast sNP rats consumed < 10 g/kg/day ethanol. Subsequently, an ascending series of ethanol concentrations, ranging from 3 to 60% (v/v), was presented to sP and sNP rats over a 28-day period (Phase 2). At concentrations varying from 7 to 30%, sP rats consumed constant amounts of absolute ethanol per kg of body weight (approximately 6.0 g/kg/day). Daily ethanol intake in sNP rats remained constantly lower than 1.0 g/kg, irrespective of the ethanol concentration. Data from Phase 2 demonstrate the ability of sP rats to precisely adjust daily ethanol intake and support the hypothesis that voluntary ethanol drinking in sP rats is sustained by specific pharmacological effects of ethanol.

Reduction of voluntary ethanol intake in ethanol-preferring sP rats by the cannabinoid antagonist SR-141716.

The present study assessed the efficacy of the cannabinoid CB1 receptor antagonist, SR-141716, in reducing voluntary ethanol intake in selectively bred Sardinian alcohol-preferring (sP) rats. Ethanol (10%, v/v) and food were available in daily 4 h scheduled access periods; water was present 24 h/day. The acute administration of a 2.5 and a 5 mg/kg dose of SR-141716 selectively reduced ethanol intake, whereas a 10 mg/kg dose of SR-141716 reduced to a similar extent both ethanol and food intake. These results suggest that the cannabinoid CB1 receptor is involved in the mediation of the ethanol-reinforcing effects in sP rats.

Biochemical and biophysical studies of reactive center cleaved plasminogen activator inhibitor type 1. The distance between P3 and P1' determined by donor-donor fluorescence energy transfer.

Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.

Prenatal exposure to a low concentration of carbon monoxide disrupts hippocampal long-term potentiation in rat offspring.

The aim of the present study was to investigate whether functional changes at CA3-CA1 synapses in the hippocampus could underlie learning and memory deficits produced in rat offspring by a prenatal exposure model simulating the carbon monoxide (CO) exposure observed in human cigarette smokers. Electrophysiological endpoints, including long-term potentiation, were examined in 15- to 30-day-old male rats whose mothers were exposed, from day 0 to day 20 of gestation, to 150 ppm of CO resulting in blood levels of carboxyhemoglobin comparable to those found in human cigarette smokers. Evoked field excitatory postsynaptic potentials were measured in the stratum radiatum in hippocampal slices. Results show that before tetanus, input/output functions, presynaptic volley, and paired-pulse facilitation were not affected in CO-exposed offspring, indicating that basal synaptic excitability and terminal Ca(2+) influx were not influenced by prenatal exposure to this gas. Conversely, evoked field excitatory postsynaptic potentials potentiation in response to tetanization was reduced by about 23% and decayed rapidly to baseline values in slices from CO-exposed animals. No changes between and within groups were observed in paired-pulse facilitation after tetanus. The selective impairment of long-term potentiation expression exhibited by CO-exposed rats was paralleled by a significant decrease in heme-oxygenase 2 and neuronal nitric-oxide synthase in the hippocampus. No changes in either enzymatic activity were found in frontal cortex and cerebellum. These electrophysiological and biochemical alterations might account for cognitive deficits previously observed in rats exposed prenatally to CO. Our findings could have clinical implications for the offspring of mothers who smoke during pregnancy.

Prenatal low-level exposure to CO alters postnatal development of hippocampal nitric oxide synthase and haem-oxygenase activities in rats.

The effects of prenatal CO exposure (150 ppm from days 0 to 20 of pregnancy) on the postnatal development of hippocampal neuronal NO synthase (nNOS) and haem-oxygenase (HO-2) isoform activities in 15-, 30- and 90-d-old rats were investigated. Unlike HO-2, hippocampal nNOS activity increased from postnatal days 15-90 in controls. Prenatal CO produced a long-lasting decrease in either nNOS or HO-2. The results suggest that the altered developmental profile of hippocampal nNOS and HO-2 activities could be involved in cognitive deficits and long-term potentiation dysfunction exhibited by rats prenatally exposed to CO levels resulting in carboxyhaemoglobin (HbCO) levels equivalent to those observed in human cigarette smokers.