Biomedical subjects

S Caccia

Publications and source records attributed to S Caccia.

At least 19 recordsLinked to original sources

PubMed2006

Structure and function of the extraembryonic membrane persisting around the larvae of the parasitoid Toxoneuron nigriceps.

The embryo of Toxoneuron nigriceps (Hymenoptera, Braconidae) is surrounded by an extraembryonic membrane, which, at hatching, releases teratocytes and gives rise to a cell layer embedding the body of the 1st instar larva. This cell layer was studied at different developmental times, from soon after hatching up to the first larval moult, in order to elucidate its ultrastructural, immunocytochemical and physiological function. The persisting "larval serosa" shows a striking structural and functional complexity: it is a multifunctional barrier with protective properties, limits the passage of macromolecules and it is actively involved in the enzymatic processing and uptake of nutrients. The reported results emphasizes the important role that the embryo-derived host regulation factors may have in parasitism success in Hymenoptera koinobionts.

Animal Nutritional Physiological Phenomena↗

PubMed2006

Antibodies to tissue-type plasminogen activator (t-PA) in patients with inflammatory bowel disease: high prevalence, interactions with functional domains of t-PA and possible implications in thrombosis.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have an increased prevalence of thromboembolic events. The pathogenetic mechanisms of these events include reduced fibrinolysis, which may be caused by antibodies to tissue-type plasminogen activator (t-PA). OBJECTIVES: To evaluate anti-t-PA antibodies in patients with IBD, considering clinical, biochemical and functional characteristics. PATIENTS AND METHODS: We immunoenzymatically measured anti-t-PA antibodies in plasma from 97 consecutive IBD patients and 97 age- and sex-matched healthy controls. We also assessed the antibody interactions with different epitopes of t-PA, the antibody inhibition on t-PA activity and the correlations with clinical features and other serum antibodies. RESULTS: IBD patients had higher median anti-t-PA antibody levels (5.4 U mL(-1) vs. 4.0 U mL(-1); P < 0.0001): 18 patients were above the 95th percentile of the controls (OR 5.3; 95% CI 1.7-16.3; P < 0.003), and the six with a history of thrombosis tended to have high levels (6.9 U mL(-1)). Anti-t-PA antibody levels did not correlate with IBD type, activity, location or treatment, or with age, sex, acute-phase reactants or other antibodies. The anti-t-PA antibodies were frequently IgG1 and bound t-PA in fluid phase; they recognized the catalytic domain in 10 patients and the kringle-2 domain in six. The IgG fraction from the three patients with the highest anti-t-PA levels slightly reduced t-PA activity in vitro. CONCLUSIONS: The prevalence of anti-t-PA antibodies is high in IBD patients. By binding the catalytic or kringle-2 domains of t-PA, these antibodies could lead to hypofibrinolysis and contribute to the prothrombotic state of IBD.

Adult↗

PubMed2005

Nutrient absorption by Aphidius ervi larvae.

It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) host regulation by the parasitoid larva induces in the aphid haemolymph major changes of the titer of nutritional compounds such as proteins, acylglycerols and free amino acids, in order to meet the stage-specific demands of the developing larva. Since little is known about how the larva absorbs these mobilized nutritional resources, nutrient absorption by larval stages of A. ervi was studied. In 2nd instar larvae, leucine was ten-fold accumulated in the haemocoel, and tyrosine and glutamine two-fold. Glucose and fructose were readily absorbed and fructose was extensively metabolized by larval tissues. In 3rd instars, the presence of a number of larvae that did not ingest the incubation medium enabled us to determine the respective amounts of substrate absorbed by the epidermis and the midgut. An accumulation of leucine in the haemocoel was observed only when midgut cells were involved in absorption, while the amino acid concentration within body fluids never exceeded that of the incubation medium when the uptake was performed only by epidermal cells. The immunofluorescence analysis, the mutual inhibition exerted on labeled glucose or fructose uptakes by a 100-fold excess of the sugars and the strong inhibition of uptakes induced by 0.2mM cytochalasin B support the expression of facilitative GLUT2-like transporters in the apical and basal cell membranes of midgut epithelial cells. Taken together, these results prove that both midgut and epidermis are involved in nutrient absorption throughout the parasitoid development, that GLUT2 transporters are responsible for glucose and fructose uptakes and that the chemical gradient that favors the passive influx of the two sugars is maintained by their conversion to other substrates.

Absorption↗

PubMed2005

High-performance liquid chromatography measurement of hyperforin and its reduced derivatives in rodent plasma.

A reverse-phase high-performance liquid chromatography method was developed for the determination of hyperforin and its reduced derivatives octahydrohyperforin and tetrahydrohyperforin in rodent plasma. The procedure includes solid-phase extraction from plasma using the Baker 3cc C8 cartridge, resolution on the Symmetry Shield RP8 column (150 mm x 4.6 mm, i.d. 3.5 microm) and UV absorbance detection at 300 nm. The assay was linear over a wide range, with an overall coefficient of variation less than 10% for all compounds. The precision and accuracy were within acceptable limits and the limit of quantitation was sufficient for studies preliminarily assessing the disposition of tetrahydrohyperforin and octahydrohyperforin in the mouse and rat.

Animals↗

PubMed2004

Effects of chronic treatment with escitalopram or citalopram on extracellular 5-HT in the prefrontal cortex of rats: role of 5-HT1A receptors.

1 Microdialysis was used to study the acute and chronic effects of escitalopram (S-citalopram; ESCIT) and chronic citalopram (CIT), together with the 5-HT1A receptor antagonist WAY100,635 (N-[2-[methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide trihydrochloride) and the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on extracellular 5-hydroxytryptamine (5-HT) levels in the rat prefrontal cortex. 2 Extracellular 5-HT rose to 234 and 298% of basal values after subcutaneous (s.c.) acute doses of 0.15 and 0.63 mg kg(-1) ESCIT. No further increase was observed at 2.5 mg kg(-1) ESCIT (290%). 3 The effect of 13-day s.c. infusion of 10 mg kg(-1) day(-1) ESCIT on extracellular 5-HT (422% of baseline) was greater than after 2 days (257% of baseline), whereas exposure to ESCIT was similar. In contrast, the increase in extracellular 5-HT induced by the infusion of CIT for 2 (306%) and 13 days (302%) was similar. However, brain and plasma levels of S-citalopram in rats infused with CIT for 13 days were lower than after 2 days. 4 Acute treatment with 2.5 mg kg(-1) ESCIT or 5 mg kg(-1) CIT raised extracellular 5-HT by 243 and 276%, respectively, in rats given chronic vehicle but had no effect in rats given ESCIT (10 mg kg(-1) day(-1)) or CIT (20 mg kg(-1) day(-1)) for 2 or 13 days, suggesting that the infused doses had maximally increased extracellular 5-HT. WAY100,635 (0.1 mg kg(-1) s.c.) increased extracellular 5-HT levels by 168, 174 and 169% of prechallenge values in rats infused with vehicle or ESCIT for 2 or 13 days, respectively. WAY100,635 enhanced extracellular 5-HT levels to 226, 153 and 164% of prechallenge values in rats infused with vehicle or CIT for 2 and 13 days, respectively. 5 8-OH-DPAT (0.025 mg kg(-1)) reduced extracellular 5-HT by 54% in control rats, but had no effect in those given ESCIT and CIT for 13 days. 6 This series of experiments led to the conclusion that chronic treatment with ESCIT desensitizes the 5-HT1A receptors, regulating the release of 5-HT in the prefrontal cortex and enhances the effect of the drug on extracellular 5-HT. They also indicate that chronic treatment with ESCIT and CIT did not prevent WAY100,635 from raising extracellular 5-HT.

Animals↗

PubMed2001

The stereoselective targeting of a specific enzyme-substrate complex is the molecular mechanism for the synergic inhibition of HIV-1 reverse transcriptase by (R)-(-)-PPO464: a novel generation of nonnucleoside inhibitors.

The human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor pyrrolopyridooxazepinone (PPO) derivative, (+/-)-PPO294, was shown to be active toward wild type and mutated HIV-1 RT and to act synergistically in combination with 3'-azido-3'-deoxythymidine (Campiani, G., Morelli, E., Fabbrini, M., Nacci, V., Greco, G., Novellino, E., Ramunno, A., Maga, G., Spadari, S., Caliendo, G., Bergamini, A., Faggioli, E., Uccella, I., Bolacchi, F., Marini, S., (1999) J. Med. Chem. 42, 4462-4470). The (+/-)-PPO294 racemate was resolved into its pure enantiomers, and the absolute configuration was determined by x-ray analysis. Only one enantiomer, (R)-(-)-PPO464, displayed antiviral activity against both the wild type and the K103N mutant HIV-1 RT and was found to interact exclusively with the reaction intermediate formed by RT complexed with both the DNA and the nucleotide substrates. Being the first compound of its class to display this behavior, (R)-(-)-PPO464 is the representative of a novel generation of nonnucleoside inhibitors. (R)-(-)-PPO464 showed significant synergism when tested in combination with other RT inhibitors and efficiently inhibited viral replication when tested against the laboratory strain HIV-1 IIIB or against either wild type or multidrug-resistant clinical isolates. Pharmacokinetic studies in mice and rats showed a more favorable profile for (R)-(-)-PPO464 than for the corresponding racemate. (R)-(-)-PPO464 was also found to easily cross the blood-brain barrier. The coadministration of the HIV-1 protease inhibitor ritonavir increased the bioavailability of (R)-(-)-PPO464, having little effect on its plasma and brain elimination rates.

Animals↗

PubMed2001

Quinoxalinylethylpyridylthioureas (QXPTs) as potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Further SAR studies and identification of a novel orally bioavailable hydrazine-based antiviral agent.

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

Animals↗

PubMed2001

Chronic treatment with reboxetine by osmotic pumps facilitates its effect on extracellular noradrenaline and may desensitize alpha(2)-adrenoceptors in the prefrontal cortex.

1. This study investigated the effect of acute (2 days) and chronic (14 days) treatment with a selective inhibitor of noradrenaline uptake, reboxetine (10 mg kg(-1) day(-1)) by osmotic pumps, on extracellular noradrenaline and the sensitivity of alpha(2)-adrenoceptors in the prefrontal cortex of rats. 2. The effect of continuous infusion of reboxetine for 14 days on cortical extracellular noradrenaline was significantly higher (599% of vehicle levels) than after 2 days (263% of vehicle levels). 3. Brain concentrations of reboxetine after 2 and 14 days of infusion were 37.9+/-17.8 and 37.1+/-7.7 ng g(-1), respectively. 4. Reboxetine infused for 2 and 14 days significantly increased extracellular dopamine in the prefrontal cortex, to a similar extent (257 and 342% of vehicle levels, respectively), whereas extracellular 5-HT was not modified by either treatment. 5. Clonidine (10 and 30 microg kg(-1) i.p.) reduced cortical extracellular noradrenaline similarly in animals treated with reboxetine or vehicle for 2 days whereas the effects in rats infused with reboxetine for 14 days were markedly less than in vehicle-treated animals. 6. Clonidine (0.05 and 0.2 microM), infused through the dialysis probe into the prefrontal cortex, reduced cortical extracellular noradrenaline much less in rats treated with reboxetine for 14 days than in vehicle-treated animals. 7. Reboxetine's effect on extracellular noradrenaline in the prefrontal cortex was greater after chronic treatment and could be associated with desensitization of terminal alpha(2)-adrenoceptors that normally serve to inhibit noradrenaline release.

Adrenergic Uptake Inhibitors↗

PubMed2001

Distribution of the methylpiperazinopyridobenzoxazepine derivative JL13, a potential antipsychotic, in rat brain.

The brain uptake and distribution of the potential antipsychotic 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3][1,5]benzoxazepine fumarate (JL13) was examined in rats after neuropharmacologically active doses. Plasma and brain concentrations of the compound were measured by reversed-phase HPLC with UV detection (210 nm). Clozapine was used as an internal standard. After an intraperitoneal dose of 10 mg kg(-1), the compound attained mean maximum plasma concentrations within 5 min of dosing, then declined with a mean elimination half-life of approximately 1 h. It rapidly crossed the blood-brain barrier and equilibrated with plasma, achieving mean maximum concentrations and area under the curve approximately 20-times those in plasma, with slight regional differences. Disappearance from whole brain almost paralleled its disappearance from plasma. There was a linear relationship between JL13 concentrations in plasma and brain regions, and in all tissues the concentrations of the compound increased almost linearly with the dose over the range of 5-20 mg kg(-1). It thus appears that JL13 brain pharmacokinetics parallels that in plasma, and that plasma concentrations accurately predict brain concentrations in rats.

Animals↗

PubMed2000

The effect of serotonergic agents on haloperidol-induced striatal dopamine release in vivo: opposite role of 5-HT(2A) and 5-HT(2C) receptor subtypes and significance of the haloperidol dose used.

This study investigated, using microdialysis in freely-moving rats, the role of serotonin (5-HT) and 5-HT(2) receptor subtypes in the enhancement of striatal dopamine (DA) release induced by various doses of haloperidol. The subcutaneous injection of 0.01, 0.1 or 1 mg/kg haloperidol dose-dependently increased DA outflow (160, 219 and 230% of baseline, respectively). The effect of 0.01 mg/kg haloperidol was, respectively, potentiated by the 5-HT uptake inhibitor citalopram (1 mg/kg, s.c.; +35%) and reduced by the 5-HT(1A) receptor agonist 8-OH-DPAT (0.025 mg/kg, s.c.; -32%). Also, it was reduced by the 5-HT(2A) antagonist SR 46349B (0.5 mg/kg, s.c. ; -40%) or by the 5-HT(2A/2B/2C) antagonist ritanserin (1.25 mg/kg, i.p.; -34%), and potentiated by the 5-HT(2B/2C) antagonist SB 206553 (5 mg/kg, i.p; +78%). Further, only this latter compound significantly modified basal dopamine release by itself (+26%). Dopamine released by 0.1 mg/kg haloperidol was enhanced (+100%) by citalopram, decreased (-61%) by SR 4634B, but unaltered by SB 206553. Finally, none of the compounds used were able to modify the enhancement of dopamine release induced by 1 mg/kg haloperidol. These results show that central 5-HT(2A) and 5-HT(2C) receptors exert an opposite (respectively excitatory and inhibitory) influence on DA release. Moreover, they suggest that the 5-HT(2A)-dependent modulation depends on the degree of central DA receptor blockade.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2000

Non-nucleoside HIV-1 reverse transcriptase inhibitors: synthesis and biological evaluation of novel quinoxalinylethylpyridylthioureas as potent antiviral agents.

New heterocyclic derivatives of ethylpyridylthiourea, quinoxalinylethylpyridylthiourea (QXPT) and analogues, inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevented HIV-1 cytopathogenicity in T4 lymphocytes. Several of these novel non-nucleoside RT inhibitors, with a substituted pyrroloquinoxalinone heteroaromatic skeleton, showed inhibitory activity against wild-type RT as well as against mutant RTs containing the single amino acid substitutions L1001, K103N, V106A, Y1811 and Y188L that was much greater than other non-nucleoside inhibitors such as nevirapine. Maximum potency in enzymatic assays was achieved with a fluoropyrroloquinoxaline skeleton linked to the ethylpyridylthiourea moiety (FQXPT). In cell-based assays on different cell lines and on human monocyte-macrophages, 6-FQXPT exhibited EC50 values in the nanomolar range, with a promising selectivity index. Moreover, 6-FQXPT showed synergistic antiviral activity with zidovudine.

Amino Acid Substitution↗

PubMed2000

Biotransformation of post-clozapine antipsychotics: pharmacological implications.

The need to develop new antipsychotics that have fewer motor adverse effects and offer better treatment of negative symptoms has led to a new generation of drugs. Most of these drugs undergo extensive first-pass metabolism and are cleared almost exclusively by metabolism, except for amisulpride whose clearance is largely due to urinary excretion. Risperidone has metabolic routes in common with ziprasidone but shows differences in regard to other main pathways: the benzisoxazole moiety of risperidone is oxidised by cytochrome P450 (CYP) 2D6 to the active 9-hydroxyrisperidone, whereas the benzisothiazole of ziprasidone is primarily oxidised by CYP3A4, yielding sulfoxide and sulfone derivatives with low affinity for target receptors in vitro. Olanzapine, quetiapine and zotepine also have some common metabolic features. However, for the thienobenzodiazepine olanzapine a main metabolic route is direct conjugation at the benzodiazepine nucleus, whereas for the dibenzothiazepine quetiapine and the dibenzothiepine zotepine it is CYP3A4-mediated oxidation, leading to sulfoxidation, hydroxylation and dealkylation for quetiapine, but N-demethylation to the active nor-derivative for zotepine. Although the promising benzisoxazole (iloperidone) and benzisothiazole (perospirone) antipsychotics share some metabolic routes with the structurally related available drugs, they too have pharmacologically relevant compound-specific pathways. For some of the new antipsychotics we know the isoenzymes involved in their main metabolic pathways and the endogenous and exogenous factors that, by affecting enzyme activity, can potentially modify steady-state concentrations of the parent drug or its metabolite(s), but we know very little about others (e.g. amisulpride isomers, nemonapride). For yet others, information is scarce about the activity of the main metabolites and whether and how these contribute to the effect of the parent drug. Aging reduces the clearance of most antipsychotics, except amisulpride (which requires further evaluation) and ziprasidone. Liver impairment has little or no effect on the pharmacokinetics of olanzapine, quetiapine, risperidone (and 9-hydroxy-risperidone) and ziprasidone, but information is lacking for amisulpride. Renal impairment significantly reduces the clearance and prolongs the elimination half-life of amisulpride and risperidone. Again, studies are still not available for some drugs (zotepine) and have focused on the parent drug for others (olanzapine, quetiapine, ziprasidone) despite the fact that renal impairment would be expected to lower the clearance of more polar metabolites. Addressing these issues may assist clinicians in the design of safe and effective regimens for this group of drugs, and in selecting the best agent for each specific population.

Antipsychotic Agents↗

PubMed1999

Pyrrolobenzoxazepinone derivatives as non-nucleoside HIV-1 RT inhibitors: further structure-activity relationship studies and identification of more potent broad-spectrum HIV-1 RT inhibitors with antiviral activity.

Pyrrolobenzoxazepinone (PBO) derivatives represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTs) whose prototype is (+/-)-6-ethyl-6-phenylpyrrolo[2,1-d][1,5]benzoxazepin-7(6H)- one (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis and biological evaluation of novel derivatives and analogues of 6 featuring a meta-substituted phenyl or a 2-thienyl ring at C-6 and a pyridine system in place of the fused-benzene ring to yield pyrrolopyridooxazepinones (PPOs). Compared with the lead 6 and nevirapine, several of the synthesized compounds (PBOs 13a-d and PPOs 13i-k) displayed higher inhibitory activity against wild-type RT and clinically relevant mutant RTs containing the single amino acid substitutions L100I, K103N, V106A, Y181I, and Y188L. The most potent inhibitors were further evaluated for in vitro antiviral activity on lymphocytes and monocyte-macrophages, for cytotoxicity on a panel of cell lines, and for potential synergistic antiviral activity with AZT. Pharmacokinetic studies performed on 13b, 13c, and 13i showed that these compounds achieve high concentrations in the brain. The results of the biological and pharmacokinetic experiments suggest a potential clinical utility of analogues such as 13b-d, 13i, and 13j, in combination with nucleoside RT inhibitors, against strains of HIV-1 bearing those mutations that confer resistance to known NNRTI.

Animals↗

PubMed1999

Pyrroloquinoxaline derivatives as high-affinity and selective 5-HT(3) receptor agonists: synthesis, further structure-activity relationships, and biological studies.

The synthesis, pharmacological evaluation, and structure-activity relationships (SARs) of a series of novel pyrroloquinoxalines and heteroaromatic-related derivatives are described. The new pyrroloquinoxaline-related ligands were tested in rat cortex, a tissue expressing high density of 5-HT(3) receptors, and on NG108-15 cells and exhibited IC(50) values in the low nanomolar or subnanomolar range, as measured by the inhibition of [(3)H]zacopride binding. The SAR studies detailed herein delineated a number of structural features required for improving affinity. Some of the ligands were employed as "molecular yardsticks" to probe the spatial dimensions of the lipophilic pockets L1, L2, and L3 in the 5-HT(3) receptor cleft, while the 7-OH pyrroloquinoxaline analogue was designed to investigate hydrogen bonding with a putative receptor site H1 possibly interacting with the serotonin hydroxy group. The most active pyrroloquinoxaline derivatives showed subnanomolar affinity for the 5-HT(3) receptor. In functional studies ([(14)C]guanidinium accumulation test in NG108-15 hybrid cells, in vitro) most of the tested compounds showed clear-cut 5-HT(3) agonist properties, while some others were found to be partial agonists. Several heteroaromatic systems, bearing N-substituted piperazine moieties, have been explored with respect to 5-HT(3) affinity, and novel structural leads for the development of potent and selective central 5-HT(3) receptor agonists have been identified. Preliminary pharmacokinetic studies indicate that these compounds easily cross the blood-brain barrier (BBB) after systemic administration with a brain/plasma ratio between 2 and 20, unless they bear a highly hydrophilic group on the piperazine ring. None of the tested compounds showed in vivo anxiolytic-like activity, but potential analgesic-like properties have been possibly disclosed for this new class of 5-HT(3) receptor agonists.

Animals↗

PubMed1999

Unexpected crucial role of residue 225 in serine proteases.

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

Amino Acid Sequence↗

PubMed1999

The kinetics of the reaction between NO and O2 as studied by a novel approach.

The kinetics of the reaction between NO and O2 was determined by measuring the time course of the decrease in the concentration of NO with a quench-flow technique. NO and O2 were mixed rapidly and reacted for periods of time varying from 10 to 50 s. A second rapid mixing with a solution containing an excess of deoxyhemoglobin and sodium hydrosulfite trapped free NO as nitrosylhemoglobin and reduced O2. The spectrum of the mixture of deoxy- and nitrosylhemoglobin was recorded within 30 s from the second mixing, before any appreciable dissociation of NO from the protein, by means of a flow-cell mounted on-line with the quench-flow apparatus. The amount of NO not consumed in the auto-oxidation reaction was calculated from the proportion of nitrosylhemoglobin in the mixture. As NO and O2 bind deoxyhemoglobin at comparable rates and NO is oxidized to nitrate by oxyhemoglobin, the ratio of hemoglobin/(NO + O2) had to be optimized to avoid the interference of this oxidation reaction. The kinetics was first and second order with respect to O2 and NO, respectively and third order overall with a rate constant k = 4 x kaq = 4 x 2.23 (+/- 0.26) x 10(6) M-2 s-1 at 20 degrees C, invariant in the pH range 7-9, in agreement with published values obtained by different methodologies.

Computer Simulation↗

PubMed1999

Hypericum perforatum L. extract does not inhibit 5-HT transporter in rat brain cortex.

The hydroalcoholic extract of Hypericum perforatum L. is an effective antidepressant, although its mechanism of action is still unknown. It inhibits the synaptosomal uptake of serotonin (5-HT), dopamine and noradrenaline, suggesting a biochemical mechanism similar to the synthetic standard antidepressants. In the present study, further investigating this hypothesis, we confirmed that a hydromethanolic extract of H. perforatum inhibited [3H]5-HT accumulation in rat brain cortical synaptosomes with an IC50 value of 7.9 microg/ml. The IC50 of pure hyperforin was 1.8 microg/ml, so the activity of the total extract is not related only to its hyperforin content (<5%). This inhibitory effect, however, is not due to a direct interaction with, and blockade of, the 5-HT transporters since the extract, like hyperforin, did not inhibit [3H]citalopram binding (IC50 > 100 microg/ml and 10 microg/ml, respectively). We also found that 3-10 microg/ml of the extract, or 0.3-1 microg/ml hyperforin, induced marked tritium release from superfused synaptosomes previously loaded with [3H]5-HT. The releasing effect of the extract resembles the releasing effect of a reserpine-like compound (Ro 04-1284), i.e. it was slightly delayed and was 5-HT carrier- and calcium-independent. These data suggest that the hydromethanolic extract of H. peforatum, similarly to Ro 04-1284, rapidly depletes storage vesicles, raising the cytoplasmic concentration of 5-HT, and this increase is presumably responsible for the apparent inhibition of [3H]5-HT uptake. Therefore, our in vitro data do not confirm that the hydromethanolic extract of H. perforatum acts as a classical 5-HT uptake inhibitor but indicate reserpine-like properties. However, the concentrations of the active component(s) effective in vitro as reserpine-like agent(s) (i.e. corresponding to > or =3 microg/ml of the hydromethanolic extract) do not seem to be achieved in the brain after pharmacologically effective doses of the extract, as indicated by the finding that there were no significant changes of rat brain 5-HT and 5-hydroxyindoleacetic acid levels after a schedule of treatment (3 x 300 mg/kgday, orally) active in an animal model predictive of antidepressant-like activity. These data also suggest that the antidepressant effect of H. perforatum extracts is unlikely to be associated with interaction with GABA, benzodiazepine and 5-HT1 receptors since, in receptor binding studies, we found IC50 values higher than 5 microg/ml. Therefore other, still unknown, mechanisms are possibly involved in H. perforatum antidepressant effects.

Animals↗

PubMed1999

Orally administered ranitidine plasma concentrations before and after biliopancreatic diversion in morbidly obese patients.

BACKGROUND: Patients undergoing biliopancreatic diversion (BPD) may develop gastric ulcers, particularly within the first postoperative year. The prophylactic use of antisecretory compounds at the usual therapeutic doses, mainly conventional H2-receptor antagonists such as ranitidine, may reduce the incidence of this complication, which occurs in approximately 5% of patients after BPD. METHODS: The authors measured the plasma concentrations of ranitidine (300 mg orally) in obese patients, before and 8 months after BPD, and in control subjects of normal weight. The study included 11 obese patients undergoing BPD (age 45+/-14 years; preoperative and postoperative weights 124+/-21 and 92+/-11 kg) and 10 normal-weight subjects (age 37+/-13 years, weight 67+/-9 kg). RESULTS: Postoperative ranitidine plasma concentrations showed only minor differences from preoperative levels, with slightly higher maximum concentrations occurring sooner. The mean area under the curve was on the average 30% higher than preoperatively. All parameters, however, were similar to those in control subjects. CONCLUSIONS: BPD per se does not greatly affect the pharmacokinetic behavior of ranitidine, and therefore a conventional dosage regimen appears adequate for the prophylaxis and therapy of gastric ulcers associated with this operation.

Administration, Oral↗