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Biomedical subjects

S Marini

Publications and source records attributed to S Marini.

At least 19 recordsLinked to original sources
PubMed2006

Double-contrast MRI (DC-MRI) in the study of the cirrhotic liver: utility of administering Gd-DTPA as a complement to examinations in which SPIO liver uptake and distribution alterations (SPIO-LUDA) are present and in the identification and characterisation of focal lesions.

PURPOSE: The aim of this study was to compare the performance of double-contrast magnetic resonance imaging (DC-MRI) with the sequential use of superparamagnetic iron oxide (SPIO) and gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) contrast agents compared with unenhanced MRI and SPIO-enhanced MRI (SPIO-MRI) in the study of the cirrhotic liver. Special attention was paid to cases in which alterations of liver uptake and distribution of the SPIO contrast medium [SPIO-liver uptake and distribution alterations (SPIO-LUDA)] could lead to diagnostic errors at SPIO-MRI. MATERIALS AND METHODS: We used DC-MRI to study 67 patients suffering from hepatic cirrhosis and on a waiting list for liver transplant. The study was performed with a 1.5-Tesla device and characterised by three phases: the first phase without contrast material (unenhanced MRI), the second after the administration of ferumoxides (SPIO-MRI), and the third, a double-contrast study following the injection of a bolus of paramagnetic contrast material (DC-MRI). The sensitivity of unenhanced MRI, SPIO-MRI and DC-MRI in identifying and characterising hepatic focal lesions was assessed, together with the diagnostic increment of one technique with respect to the others. The gold standard was histological confirmation in 38 cases and clinical-radiological follow-up in all cases. Liver function, kidney function, blood tests and urinalysis were performed in all patients 24-48 h before and after the MRI examination. RESULTS: In 14/67 cases (20.8%), SPIO-LUDA were present, which posed a limitation to the SPIO-MRI examination. Focal lesions were absent in 44 patients, and the action of the ferumoxides was reduced by the presence of SPIO-LUDA in nine cases. There were five cases of confluent fibrosis, two of decompensated cirrhosis, one of vascular thrombosis, and one of scarring in a patient who had undergone hepatic resection for hepatocellular carcinoma (HCC). In all these cases, completion of the MR examination with the DC technique clarified the MR picture, confirming the absence of focal lesions. Twenty-three patients had a total of 68 lesions, which consisted of 37 dysplastic nodules (DN), 19 HCC nodules, two relapses of HCC following chemoembolisation, two HCC associated with portal thrombosis, one cancer-cirrhosis, two angiomas and five small cysts. SPIO-LUDA were present in five patients, thus limiting the identification, characterisation or assessment of the real size of the lesions. SPIO-LUDA were the result of vascular thrombosis in one case and fibrosis in four cases. In all of these cases, DC-MRI proved useful for diagnosis. The sensitivity of unenhanced MRI, SPIO-MRI and DC-MRI for lesion detection was 57.3%, 67.6% and 75%, respectively. The results obtained in the characterisation of the lesions were 20.5%, 63.2% and 73.5% for unenhanced MRI, SPIO-MRI and DC-MRI, respectively. The diagnostic increment of SPIO-MRI over unenhanced MRI for lesion identification and characterisation was 9% and 42.7%, respectively, whereas the diagnostic increment of DC-MRI over SPIO-MRI was 7.4% and 10.3%, respectively. CONCLUSIONS: In our study, the combined use of two contrast agents, negative and positive, provided greater diagnostic confidence and caused no side effects in the patients.

Contrast Media↗
PubMed2004

Effects of beta-naphthoflavone, phenobarbital and dichlobenil on the drug-metabolizing system of liver and nasal mucosa of Italian water frogs.

In this study, we have examined the presence and inducibility of phase I and II drug-metabolizing enzymes in the liver and nasal mucosa of Italian water frogs of control and pretreated with beta-naphthoflavone, phenobarbital and dichlobenil by using typical substrates for these enzymes along with polyclonal antibodies mainly raised against mammalian enzymes. The CYP content and various monooxygenase and phase II enzyme activities in the liver of this frog were found similar, when reported, to those of largely aquatic and semiaquatic frogs. The treatment with beta-naphthoflavone resulted in an induction in the liver of a CYP1A and the induction was manifested by (a) immunoblot analysis using anti-rat CYP1A1, (b) an increase of CYP1A-mediated methoxyresorufin-O-demethylase and ethoxyresorufin-O-deethylase activities. The treatments with both phenobarbital and dichlobenil did not produce in the liver any effect on the assayed enzymes. When the nasal mucosa of water frogs was analyzed, various monooxygenase and phase II enzymatic activities, generally comparable to those of liver, were determined. However, by using antibodies anti-three GST different classes, we found a different reactivity into the cytosol of the two tissues indicating a differential tissue susceptibility to toxic effects of xenobiotics. In the nasal mucosa, a protein immunorelated to CYP2A and monooxygenase activities (i.e. ethoxycoumarin-O-deethylase and coumarin-7-hydroxylase) linked in mammals to this isoform have also been found. The treatment of water frogs with the herbicide dichlobenil decreased both the above-mentioned activities and the immunoreactive CYP2A apoprotein. The pretreatment with metyrapone, a CYP inhibitor, protected the CYP2A apoprotein and its linked activities from toxic effect of dichlobenil indicating a key role of this enzyme in the bioactivation of this herbicide. The findings of the present work suggest that the hepatic CYP1A induction and the nasal CYP2A-like inhibition profiles might provide two potential biomarkers of the Italian water frogs exposure to environmental and aquatic pollutants.

7-Alkoxycoumarin O-Dealkylase↗
PubMed2004

Cervicovaginal infections during pregnancy: epidemiological and microbiological aspects.

OBJECTIVE: The assessment of the association of cervicovaginal infections during pregnancy with preterm (pPROM) and term (PROM) premature rupture of membranes, preterm delivery, mid-trimester miscarriage and intrauterine death, and the definition of the risk factors that identify pregnant women who should have a cervicovaginal culture. METHODS: We retrospectively studied the relationship between pregnancy outcomes and cervicovaginal infections in 3217 pregnant women between January 1998 and December 1999. Microbiological assessment included Gram staining and specific cultures; bacterial vaginosis was diagnosed by Amsel's criteria. We also studied the medical, obstetric, sexual, demographic and social history of 11,212 pregnant women who underwent cervicovaginal culture between January 1992 and December 2001. RESULTS: Overall, 1425 of the 3217 cultures (44.3%) were positive. The micro-organisms most frequently found were: yeasts (44%), Ureaplasma urealiticum (29%); group B streptococcus (15%); and bacterial vaginosis (11%). Cervicovaginal cultures were found positive in 84.6% of pPROM, 55.0% of PROM, 50.8% of preterm deliveries, 43.8% of mid-trimester miscarriages, 31.4% of intrauterine deaths and in 33.5% of controls. Among the 11 212 cervicovaginal cultures considered in the second study, an overall 6301 (56.2%) were positive, 2711 (43%) in asymptomatic women. Cervicovaginal infections were associated with country of origin, age under 25 years, age at first intercourse under 15 years, more than ten partners, more than one partner in the past 6 months, prior abortions, past sexually transmitted diseases (STDs) and HIV infection. CONCLUSION: Cervicovaginal infections were significantly associated with PROM (p<0.0001), pPROM (p<0.0001) and preterm delivery (p<0.0001), but not with intrauterine death. The association with mid-trimester miscarriage approached statistical significance (p=0.06). The main risk factors for cervicovaginal infections were country of origin, age under 25 years, age at first intercourse under 15 years, more than ten partners, more than one partner in the past 6 months, prior abortions, past STDs and HIV infection.

Adult↗
PubMed2003

Respiratory inhibition of isolated mammalian mitochondria by salivary antifungal peptide histatin-5.

Histatin-5 is a peptide secreted in the human saliva, which possesses powerful antifungal activity. Previous studies have shown that this peptide exerts its candidacidal activity, through the inhibition of both mitochondrial respiration and the formation of reactive oxygen species. The purpose of the present study was to investigate the biological consequences of histatin-5 action on mammalian mitochondria to verify if the toxic mechanism exerted on mitochondria from Candida albicans is an exclusive for fungal cells. Moreover, hypothesising that the damage exerted on mitochondria may induce programmed cellular death pathways, we evaluated two main markers of apoptosis: the mitochondrial membrane potential (DeltaPsi) and the release of cytochrome c. The results obtained show that exposure of isolated mammalian mitochondria to histatin-5 determines: (i) a large inhibition of the respiratory chain at the level of complex I, (ii) a slight decrease in the mitochondrial membrane potential, and (iii) no release of cytochrome c.

Animals↗
PubMed2003

4-Biphenylaldehyde and 9-anthraldehyde: two fluorescent substrates for determining P450 enzyme activities in rat and human.

1. 4-Biphenylaldehyde (4-BA) and 9-anthraldehyde (9-AA) were examined as substrates for cytochrome P450 (CYPs) enzymes in rat and human. Both aldehydes were oxidized by CYPs to fluorescent carboxylic acids, which can be assayed with a high sensitivity by an easy fluorimetric method. 2. With liver microsomes from control and induced rats, the oxidation of both 9-AA and 4-BA followed simple Michaelis-Menten kinetics. Only microsomes from rats pretreated with phenobarbital (a strong inducer of P4502B1/2) could increase (about threefold) the oxidation rates (V(max)) of both aldehydes above the control values, which were 6.7+/-1.1 and 3.3+/-0.6 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively. On the other hand, the (K)(m)'s, which were similar for both aldehydes (about 25 micro M), did not change significantly with any inducer. The use of purified rat CYP1A1, 2E1, 2B1 and 2C11 in a reconstituted system showed that only 2B1 and 2C11 could oxidize both substrates with a high turnover. 3. In human liver microsomes, the oxidation rates of these aldehydes (1.6+/-0.2 and 0.42+/-0.1 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively) were lower than those of rat but with similar K(m)'s(20-26 microm). 4. The oxidation of these aldehydes was also determined with cDNA-expressed CYP1A1, 1A2, 2A6, 2B6, 2C9, 2D6, 2E1 and 3A4 and with a characterized bank of 14 human liver microsomes. In a reconstituted system, only CYP2B6, 2A6, 3A4 and with a lower turnover 2C9 oxidized both substrates. 5. Among the CYP marker activities of the 14 human samples, good correlations were only observed between CYP3A-dependent 6 beta-testosterone hydroxylase and the oxidation of 4-BA (r = 0.74) or 9-AA (r = 0.80) and between the oxidation of 4-BA versus 9-AA (r = 0.74). Weak correlations were also found between the 2B6-linked S-mephenytoin N- demethylase and the oxidation of 4-BA (r = 0.58) or 9-AA (r = 0.65). 6. Inhibition experiments revealed that the oxidation of these aldehydes was inhibited by ketoconazole, 8-methoxypsoralene and sulphophenazole, selective inhibitors for P4503A6, 2A6 and 2C9, respectively. 7. In summary, based on the use of cDNA-expressed CYPs, correlation analysis and chemical inhibition, the metabolism in human liver microsomes of these aldehydes appears primarily catalysed by CYP3A, although CYP2A6, 2B6 and 2C9 may play a role. 9-AA and particularly 4-BA, owing to the high rate of its metabolism, may be two novel useful fluorescent probe substrates for assaying CYP activities in various species.

Adolescent↗
PubMed2003

l-Deprenyl metabolism by the cytochrome P450 system in monkey (Cercopithecus aethiops) liver microsomes.

1. The aim was to clarify the kinetic and cytochrome P450 (CYP) enzymes involved in l-deprenyl metabolism by liver microsomal preparations from African green monkeys, an animal model extensively used in the study of Parkinson's disease. 2. CYP levels and monoxygenase activities were similar to those observed in microsomes from other monkey strains. The enzyme kinetics of both l-methamphetamine and l-nordeprenyl formation were characterized by a high- and low-affinity component. For l-methamphetamine, the apparent K(m1) and K(m2) were 1.07 +/- 0.01 and 350 +/- 2.7 micro M, and V(max1) and V(max2) were 4.70 +/- 0.01 and 8.9 +/- 0.02 nmol min(-1) mg protein(-1), respectively. For l-nordeprenyl, K(m1) and K(m2) were 0.96 +/- 0.05 and 168 +/- 15 micro M, and V(max1) and V(max2) were 3.34 +/- 0.02 and 3.91 +/- 0.02 nmol min(-1) mg protein(-1), respectively The ratio V(max)/K(m) for both metabolites was 2 orders of magnitude higher for the low K(m) component than for the high K(m), suggesting that the former component is the major determinant of l-deprenyl N-dealkylation. At 15 micro M l-deprenyl, both ketoconazole and 8-methoxypsoralen significantly inhibited l-methamphetamine and l-nordeprenyl formation, indicating that CYP3A and CYP2A enzymes were involved in both reactions. At 500 micro M l-deprenyl, however, inhibition studies suggest the involvement of CYP1A and 2D enzymes. 3. The metabolism of l-deprenyl by monkey liver microsomes is very efficient, indicating that CYP-dependent metabolism is relevant and could contribute to neuroprotection in primate models of Parkinson's disease.

Animals↗
PubMed2002

Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis.

In this paper the production of antibodies against saxitoxin (STX) is described, as is the optimization and comparison of two competitive ELISA formats (direct and indirect) for the detection of this toxin. Tests were performed in a 96-well microplate using the toxin-specific polyclonal antibodies produced in our laboratory, obtained from rabbits immunized with saxitoxin-keyhole limpet hemocyanin (STX-KLH). In indirect ELISA format saxitoxin, conjugated to bovine serum albumin (STX-BSA) was coated onto the microtitre plate and incubated with standard toxin and anti-STX antibody. A goat anti-rabbit IgG Peroxidase conjugate was used to enable detection. In the direct ELISA format, STX standard, STX conjugate to horseradish peroxidase (STX-HRP), and enzyme substrate/chromogen solution were sequentially added to the microplate after antibody coating.Results showed the saxitoxin detection limit to be 3 and 10 pg mL(-1) for direct and indirect ELISA formats, respectively. The suitability of the assay for quantification of saxitoxin in mussels was also studied. Samples were spiked with saxitoxin before and after sample treatment to study the extraction efficiency and matrix effect, respectively. After treatment, samples were analysed at 1:1000 v/v dilution in PBS to minimize the matrix effect and to detect the regulatory limit of 40-80 micro g saxitoxin per 100 g mussels as stipulated by the Food and Drug Administration. The efficiency of extraction of saxitoxin was from 72 to 102%. These data were confirmed by liquid chromatography coupled with fluorimetric detection, the technique currently used for quantitative determination of toxins in seafood.

Animals↗
PubMed2001

Selective inactivation of rat and bovine olfactory cytochrome P450 by three haloethanes.

The effects of halothane, 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) and 1,1-dichloro-1-fluoroethane (HCFC-141b) on the P450 system in olfactory and hepatic microsomes of bovine and rat have been investigated. In the in vitro experiments, all three compounds decreased olfactory CYP-dependent activities in microsomes from both species, especially under anaerobic conditions, halothane showing the greatest effect. Hepatic activities were not affected. A selective olfactory CYP depletion was also observed in vivo after treatment with halothane, but not with HCFC-123 or HCFC-141b. A loss of olfactory ethoxycoumarin-O-deethylase activity was also found both in vitro and in vivo experiments, suggesting that a CYP2A isoform may be the main target of inactivation. The present results therefore suggest that CYP2A, the major isoform expressed in the olfactory tissue of mammals, may be particularly prone to catalyze the reductive metabolism of halothane both in anaerobic and aerobic conditions.

Anesthetics, Inhalation↗
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

Immunodetection of lactosylated proteins as a useful tool to determine heat treatment in milk samples.

This paper reports the optimisation of a competitive immunoassay (ELISA) to detect lactosylated proteins in milk samples. The assay employs monoclonal antibodies for lactosylated proteins produced in our laboratory and requires no pre-treatment of the samples other than a dilution step. Monoclonal antibodies were fully characterised in terms of selectivity and cross-reactivity with structurally related molecules and used in a competitive assay format with lactosylated standard proteins (lactosylated ovalbumin). The detection limit for lactosylated ovalbumin was 0.015 microgram ml-1 and the working range was from 0.010 to 40 micrograms ml-1. The data obtained indicate that the ELISA developed is applicable to diluted milk samples and is able to distinguish between milk samples that have undergone different heat treatments (UHT and pasteurised milk).

Animals↗
PubMed2001

Thoracic disk herniation presenting as abdominal and pelvic pain: a case report.

We report the case of a young woman who presented with a 2-month history of severe abdominal and pelvic pain. The past history was significant for a fall from a bicycle 1 week before the onset of her pain. Physical examination was remarkable for periumbilical tenderness. Work-up including pelvic sonogram and diagnostic laparoscopy suggested endomyometritis. The pain was minimally relieved by nonsteroidal anti-inflammatory drugs and narcotic analgesics. Thoracic spine magnetic resonance imaging (MRI) revealed a large disk herniation at the T9-10 level compressing the spinal cord. The patient subsequently underwent T9-10 diskectomy and laminectomy with dramatic relief of her symptoms. Postoperative rehabilitation hastened her functional improvement. This is a rare case of symptomatic thoracic disk herniation after trauma presenting as abdominal and pelvic pain. Physicians should be aware of this unusual presentation of thoracic disk herniation to avoid invasive diagnostic procedures.

Abdominal Pain↗
PubMed2000

Cytochrome P450-dependent monooxygenase activities and their inducibility by classic P450 inducers in the liver, kidney, and nasal mucosa of male adult ring-necked pheasants.

In this study, several P450-dependent monoxygenase activities in the liver, kidney, and nasal mucosa of ring-necked pheasants were examined. In addition, the presence and inducibility of P450 isoenzymes in the hepatic and renal tissues of pheasants were examined by using typical substrates and inducers of P450s along with polyclonal antibodies raised against mammalian isoforms. Anti-rat P450 1A1 recognized in microsomes of both pheasant liver and kidney a protein that was markedly induced by beta-naphthoflavone and accompanied by an increase of various monooxygenases, in particular, methoxyresorufin-O-demethylase (MROD) activity. Anti-rat P450 2E1 revealed in microsomes of the pheasant liver but not in kidney an immunoreactive protein that was slightly induced by acetone but not accompanied by an increase of para-nitrophenol hydroxylase activity. On the other hand, acetone treatment caused an induction of other hepatic monoxygenases including MROD, erythromycin N-demethylase, and 6beta-testosterone hydroxylase. These two latter activities, known to be markers for 3A isoenzymes in rodents, were also enhanced in pheasant liver by phenobarbital but not by dexamethasone. The treatment with these two inducers also lacked to point out hepatic and renal proteins immunorelated to P450 3A or 2B subfamily, suggesting that these isoforms may be not expressed in pheasant. On the other hand, anti-rat P450 2C11 recognized two immunorelated proteins in the liver of both control and treated pheasants. The treatment with clofibrate, a mammalian inducer of 4A subfamily, induced both in liver and kidney of pheasant: i) a protein that cross-reacted with anti rat P450 4A1 and ii) the (omega) and (omega-1) lauric acid hydroxylase activities, known to be associated in mammals to this P450 subfamily. In the nasal mucosa of pheasant, a protein immunorelated to P450 2A and some monooxygenase activities (i.e., 7-ethoxycoumarin O-deethylase) linked, in mammals, to this isoform have been found; by contrast a protein immunoreactive with anti P450 2G1 was not found. In conclusion, the immunochemical properties and monooxygenase activities of constitutive and inducible P450s in pheasants were different not only from those of mammals but also from those of chickens. The findings of the present work also suggest that the P450 induction profiles might provide a potential biomarker of pheasant exposure to chemicals or environmental pollutants in the wild-field or in the stock-farm.

Acetone↗
PubMed2000

Redox equilibria of manganese peroxidase from Phanerochaetes chrysosporium: functional role of residues on the proximal side of the haem pocket.

Redox potentials of recombinant manganese peroxidase from Phanerochaetes chrysosporium have been measured by cyclic voltammetry as a function of pH, between pH 4.5 and pH 10.5. They display a bimodal behaviour (characterized by an 'alkaline' and an 'acid' transition), which indicates that (at least) two protonating groups change their pK(b) values upon reduction (and/or oxidation) of the iron atom in haem. Analogous measurements have been carried out on four site-directed mutants involving residues in close proximity to the proximal ligand, His(173), in order to investigate the role played by residues of the proximal haem pocket on the redox properties of this enzyme. Results obtained suggest that the protonation state of N(delta) of the proximal imidazole group is redox-linked and that it is crucial in regulating the 'alkaline' transition. On the other hand, none of the proximal mutants alters the 'acid' transition, suggesting that it is modulated by groups located in a different portion of the protein.

Electrochemistry↗
PubMed2000

Cleavage of bovine collagen I by neutrophil collagenase MMP-8. Effect of pH on the catalytic properties as compared to synthetic substrates.

The enzymatic processing of bovine collagen I by neutrophil collagenase (MMP-8) has been monitored at 37 degrees C, envisaging the occurrence of multiple intermediate steps, following the initial cleavage, which leads to the formation of (1/4) and (3/4) fragments. Further, the first cleavage event has been investigated at 37 degrees C as a function of pH, and catalytic parameters have been obtained through a global analysis of steady-state kinetic data, such as to get an overall consistent picture of k(cat)/K(m), k(cat), and K(m). These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimental conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK(a) values for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymatic activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natural substrates.

Animals↗
PubMed2000

pH- and temperature-dependence of functional modulation in metalloproteinases. A comparison between neutrophil collagenase and gelatinases A and B.

Metalloproteases are metalloenzymes secreted in the extracellular fluid and involved in inflammatory pathologies or events, such as extracellular degradation. A Zn(2+) metal, present in the active site, is involved in the catalytic mechanism, and it is generally coordinated with histidyl and/or cysteinyl residues of the protein moiety. In this study we have investigated the effect of both pH (between pH 4.8 and 9.0) and temperature (between 15 degrees C and 37 degrees C) on the enzymatic functional properties of the neutrophil interstitial collagenase (MMP-8), gelatinases A (MMP-2) and B (MMP-9), using the same synthetic substrate, namely MCA-Pro-Leu-Gly approximately Leu-DPA-Ala-Arg-NH(2). A global analysis of the observed proton-linked behavior for k(cat)/K(m), k(cat), and K(m) indicates that in order to have a fully consistent description of the enzymatic action of these metalloproteases we have to imply at least three protonating groups, with differing features for the three enzymes investigated, which are involved in the modulation of substrate interaction and catalysis by the enzyme. This is the first investigation of this type on recombinant collagenases and gelatinases of human origin. The functional behavior, although qualitatively similar, displays significant differences with respect to what was previously observed for stromelysin and porcine collagenase and gelatinase (Stack, M. S., and R. D. Gray. 1990. Arch. Biochem. Biophys. 281:257-263; Harrison, R. K., B. Chang, L. Niedzwiecki, and R. L. Stein. 1992. Biochemistry. 31:10757-10762). The functional characterization of these enzymes can have some relevant physiological significance, since it may be related to the marked changes in the environmental pH that collagenase and gelatinases may experience in vivo, moving from the intracellular environment to the extracellular matrix.

Amino Acid Sequence↗
PubMed2000

Anion- and pH-linked conformational transition in horseradish peroxidase.

In a previous study we have shown that bringing horseradish peroxidase to pH 3.0 induces a spectroscopic transition (G. Smulevich et al., Biochemistry 36 (1997) 640). We have extended the investigation on this pH-linked conformational change to different experimental conditions, such as (i) in phosphate alone, (ii) in HCl alone and (iii) in phosphate + NaCl. The data obtained allow a number of conclusions to be drawn, namely: (a) the exposure to pH 3.0 under all conditions brings about an alteration of the distal portion of the heme pocket, leading to the rapid formation of a hexa-coordinated species; (b) only in the presence of phosphate is the hexa-coordination followed by a slow cleavage (or severe weakening) of the proximal Fe-His bond, and (c) the rate of this second process is speeded up in the presence of Cl- ions. Such observations underline the presence of a communication pathway between the two opposite sides of the heme pocket, such that any alteration of the structural arrangement on one side of the heme cavity is transmitted to the other, inducing a corresponding conformational change.

Anions↗
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↗