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F Dallocchio

Publications and source records attributed to F Dallocchio.

At least 19 recordsLinked to original sources
PubMed2006

Cerebrospinal fluid and serum levels and intrathecal production of active matrix metalloproteinase-9 (MMP-9) as markers of disease activity in patients with multiple sclerosis.

In this study, we employed a sensitive activity assay system to measure cerebrospinal fluid (CSF) and serum levels of active matrix metalloproteinase-9 (MMP-9) in 37 relapsing-remitting (RR), 15 secondary progressive (SP) and nine primary progressive (PP) multiple sclerosis (MS) patients, grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also studied, as neurological controls, 48 patients with other inflammatory neurological disorders (OIND) and 48 with non-inflammatory neurological disorders (NIND). To assess active MMP-9/TIMP-1 circuit, CSF and serum levels of MMP-9 tissue inhibitor TIMP-1 were quantified by ELISA in the same patient population. CSF mean levels of active MMP-9, CSF active MMP-9/TIMP-1 ratios and intrathecal active MMP-9 synthesis, as indicated by specific index, were more elevated in MS than in NIND (P < 0.05, < 0.02 and < 0.02, respectively), serum active MMP-9/TIMP-1 ratio was higher in MS (P < 0.01) and OIND (P < 0.02) than in NIND, and serum TIMP-1 concentrations were lower in MS than in NIND (P<0.05). More importantly, serum active MMP-9 mean levels, serum active MMP-9/TIMP-1 ratio and intrathecal production of active MMP-9 were increased in MS patients with clinical (P < 0.001, < 0.001 and < 0.05, respectively) and MRI (P < 0.001, < 0.001 and < 0.02, respectively) disease activity, whereas CSF mean concentrations of active MMP-9 and CSF active MMP-9/TIMP-1 ratio were enhanced only in MS patients with MRI evidence of disease activity (P < 0.02 and < 0.01, respectively). Altogether, these findings suggest that a shift in MMP-9/TIMP-1 balance towards proteolytic activity of MMP-9 could be relevant in MS immune dysregulation. In addition, our results indicate that CSF and serum levels of active MMP-9 may represent a potential surrogate biomarker for monitoring MS disease activity. In particular, serum active MMP-9/TIMP-1 ratio seems to be a very appropriate indicator of ongoing MS inflammation, since it is easily measurable.

Adult↗
PubMed2001

Thermal stability of the hemagglutinin-neuraminidase from Sendai virus evidences two folding domains.

The domain structure of hemagglutinin-neuraminidase from Sendai virus (cHN) was investigated by studying the thermal stability in the 20-100 degrees C range. Differential scanning calorimetry evidences two conformational transitions. The first transition is apparently a reversible two-state process, with Tm 48.3 degrees C, and is shifted to 50.1 degrees C in the presence of the substrate analogue 2,3-dehydro-2-deoxy-N-acetyl neuraminic acid, meaning that the substrate binding domain is involved in the transition. The second transition, with apparent Tm 53.2 degrees C, is accompanied by irreversible loss of enzymatic activity of the protein, and the presence of the substrate analogue does not affect the Tm. The data indicate that cHN is composed of two independent folding domains, and that only one domain is involved in the binding of the substrate. Our results suggest that the paramyxovirus neuraminidases have the folding properties of a two-domain protein.

Calorimetry↗
PubMed2000

Thermal stabilities of lupin seed conglutin gamma protomers and tetramers.

Various experimental approaches have been used in this work to assess the thermal stabilities of lupin seed conglutin gamma at two pH values, 4.5 and 7.5, at which the protein exists as a protomer and a tetramer, respectively. The patterns of thermal unfolding at the two pH values differed significantly; the tetramer aggregated and became insoluble, whereas the protomer was still soluble after thermal treatment. Also, the midpoint transition temperatures were dramatically different, being 60.3 and 75.1 degrees C for the protomer and tetramer, respectively. The behavior of conglutin gamma at neutral pH was also affected by disulfide formation/interchange, in that some unfolded protein molecules became covalently stabilized. More detailed analyses by differential scanning calorimetry and indirect fluorescence measurements, using 8-anilino-1-naphthalenesulfonic acid as a probe, confirmed the remarkable differences observed in the thermal stabilities of the two protein forms and allowed models for their unfolding patterns to be drawn.

Calorimetry, Differential Scanning↗
PubMed1999

Anomeric specificity and protein-substrate interactions support the 3D model for the hemagglutinin-neuraminidase from sendai virus.

The 3D structure of paramyxovirus hemagglutinin-neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the hemagglutinin-neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase.

Catalytic Domain↗
PubMed1998

6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a comparison of the enzyme from different species.

The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP+ reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.

Allosteric Regulation↗
PubMed1998

Mild proteolysis induces a ready-to-fuse state on Sendai virus envelope.

The Sendai virus fuses with host cell membranes in a pH-independent manner through an unknown mechanism. Here we report that mild trypsin pre-treatments of Sendai virions, for example 15 min at 4 degrees C, give Sendai virions the ability to fuse at a rate up to 10-fold higher than control. By using human erythrocytes as host cell membranes, viral fusion was assessed by hemolysis as well as fluorescence dequenching of octadecyl rhodamine B chloride. The mild protease treatment strikingly shortens the lag time taken by the virus to start the fusion process. Similar data were obtained on reconstituted Sendai virus envelope. Among proteases, tested as fusion enhancer, trypsin is more effective than either endoproteinase Lys-C, chymotrypsin, or endoproteinase Arg-C. After removal of trypsin from treated virions the fusion rate enhancement remains for hours at room temperature. The lack of protease specificity, together with the impossibility to detect any new N-terminal products, suggests that only a small percentage of viral envelope components are cleaved, still a large enough number to set the envelope in a ready-to-fuse state.

Chymotrypsin↗
PubMed1997

The cross-linking by o-phthalaldehyde of two amino acid residues at the active site of 6-phosphogluconate dehydrogenase.

o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.

Animals↗
PubMed1996

6-Phosphogluconate dehydrogenase from Trypanosoma brucei. Kinetic analysis and inhibition by trypanocidal drugs.

The kinetics of 6-phosphogluconate dehydrogenase from Trypanosoma brucei was examined and compared to those of the same enzyme from lamb's liver. Variation of kinetic parameters as a function of pH suggests a chemical mechanism similar to other 6-phosphogluconate dehydrogenases. The comparison extended to a detailed analysis of the effect on enzyme activity by several inhibitors including the trypanocidal drugs suramin, melarsoprol and analogues of these compounds. The T. brucei enzyme differs significantly from its mammalian counterpart with respect to several inhibitors, particularly the substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue adenosine 2',5'-bisphosphate which have respectively 170-fold and 40-fold higher affinity for the parasite enzyme.

Animals↗
PubMed1995

Activation of the Sendai virus fusion protein by receptor binding.

2,3 Dehydro-2-deoxy-N-acetyl-neuraminic acid (DNANA) competitively inhibits the neuraminidase activity of Hemagglutinin-neuraminidase (HN) from Sendai virus. The inhibition constant depends on the presence of the Fusion (F) protein, which is 30 microM in the presence of active F protein and 50 microM when the F protein is inactivated. These data correlate with previously reported evidence of interaction of the F protein with HN (Dallocchio, F., Tomasi, M., & Bellini, T. (1994) Biochem. Biophys. Res. Comm. 201, 988-993). Desialyzation of erythrocytes, by Clostridium neuraminidase, lowers the hemolytic activity of SV to < 0.1% of that observed on untreated erythrocytes. However, addition of DNANA causes a concentration-dependent increase of hemolytic activity. Both HN and the F protein are required for the activation of hemolytic activity by DNANA. The affinity constant for DNANA, calculated from the activation of hemolytic activity on desialyzed erythrocytes, is 35 microM, very close to the Ki for neuraminidase activity. These data suggest that the binding of the F protein to HN, induced by the binding to HN of a substrate or a substrate analogue, causes a conformational change which activates the F protein.

Allantois↗
PubMed1995

Bromopyruvate for the affinity labelling of a single cysteine residue near the carboxylate binding site of lamb liver 6-phosphogluconate dehydrogenase.

Bromopyruvate inactivates 6-phosphogluconate dehydrogenase by affinity labeling. Kinetic analyses, stoichiometry and isolation of a single labelled tryptic peptide of the modified protein indicate that inactivation is due to the affinity labeling of a single cysteine residue, identified as cysteine 401. It thus appears that this cysteine is within a short distance from the protein site involved in the binding of the carboxylate group of the substrate. These results suggest that the carboxylate binding site of proteins could be used as an anchorage point for affinity labeling, and that bromopyruvate can be used to individuate an amino acid residue within few A from this site.

Affinity Labels↗
PubMed1994

Inhibition of Sendai virus hemagglutinin neuraminidase by the fusion protein.

The Sendai virus envelope contains two glycoproteins: the fusion (F) protein and the hemagglutinin-neuraminidase (HN). Inactivation of F causes the loss of fusogenic activity and an increase of the neuraminidase activity of HN. After inactivation of F, HN can be inhibited by fetuin or asialofetuin, as already observed on the water-soluble, C-terminal fragment of HN (Dallocchio, F., Bellini, T., Martuscelli, G., Baiocchi, M., & Tomasi, M. (1991) Biochem. Int. 25, 663-668). Disruption of viral envelopes by detergents does not affect the neuraminidase activity of virions containing inactive F, while it causes an increase of the neuraminidase activity in native virions. Reconstitution of HN into liposomes is accompanied by a decrease of enzymatic activity, due to the random inside-outside distribution of the protein. However, the decrease of the neuraminidase activity is higher in liposomes containing both HN and F. These data suggest that F inhibits the neuraminidase activity of HN.

Animals↗
PubMed1993

Selective modification of Sendai virus hemagglutinin neuraminidase by pyridoxal 5'-phosphate: evidence for an allosteric modulation of neuraminidase activity.

Incubation of Sendai virus with pyridoxal 5'-phosphate (PLP) causes inhibition of hemolytic activity, a slight reduction of hemagglutinating activity, and an increase in neuraminidase activity. The effects on hemagglutination and neuraminidase are prevented by the presence in the incubation mixture of sialyl lactose, a substrate of hemagglutinin-neuraminidase. Incubation with PLP of the water-soluble enzymatic domain of the neuraminidase has no effect on enzymatic activity, while the allosteric inhibition (Dallocchio et al. (1991) Biochem. Int. 25, 663-668) disappears. Both virus-bound and solubilized neuraminidase are selectively modified by PLP at the lysine-553. Our data suggest that PLP inactivates a previously undetected inhibitory site on the viral neuraminidase, and that a physiological effector is present on the viral envelope.

Allosteric Regulation↗
PubMed1993

Use of trinitrobenzensulfonate for affinity labeling of lysine residues at phosphate binding sites of some enzymes.

Trinitrobenzensulfonate, a reagent for lysine residues, inactivates lamb liver 6-phosphogluconate dehydrogenase through affinity labeling. Complete inactivation is due to the binding of only one residue of reagent per enzyme subunit. Other enzymes with a phosphate binding site are also inactivated by affinity labeling. It appears that trinitrobenzensulfonate, when used at low concentrations, first binds to a phosphate binding site, then reacts with a nearby lysine residue. This reagent presents some advantages over pyridoxal phosphate, which has similar characteristics.

Affinity Labels↗
PubMed1993

Simple purification and secondary structure evaluation of Sendai virus neuraminidase water soluble fragment.

We have developed a quick and simple method to purify the water soluble fragment of Sendai virus neuraminidase (cHN). We used the trifluoperazine (TFP) as detergent because of its ability to selectively solubilize the HN (Baiocchi et al., (1988) FEBS Lett. 238, 171-174). Here we describe conditions by which trypsin produces the cHN from the TFP treated virions. The cHN is further purified by size exclusion chromatography and it fully retains the neuraminidase activity and the allosteric inhibitory site as described in (Dallocchio et al., (1991) Biochem. Int. 25, 663-668). Both circular dichroism (CD) spectra, analyzed by deconvolution methods and secondary structure prediction were used to assess the secondary structure of cHN.

Amino Acid Sequence↗
PubMed1992

Subunits asymmetry in the ternary complex of lamb liver 6-phosphogluconate dehydrogenase detected by a NADP analogue.

Incubation of lamb liver 6-phosphogluconate dehydrogenase, a dimeric enzyme with periodate-oxidized NADP causes the inactivation of the enzyme due to the covalent binding of 2 mol of inhibitor/mol of dimer. In the presence of substrate, the inactivation is faster and is almost complete after the labelling of only one subunit. These results not only confirm the hypothesis of a 'half-of-the-sites' mechanism of action of the enzyme, but also suggest that the formation of the ternary complex (enzyme-substrate-coenzyme) in one subunit causes a conformational change that makes the other subunit unable to bind the coenzyme (and even the adenylic part of it) and, thus, this subunit becomes inactive. It appears that while one subunit catalyses the oxidation of 6-phosphogluconate the other is inactive in this reaction.

Animals↗
PubMed1992

NADPH activates a decarboxylation reaction catalysed by lamb liver 6-phosphogluconate dehydrogenase.

NADP-dependent lamb liver 6-phosphogluconate dehydrogenase catalyses the oxidative decarboxylation of 2-deoxy-6-phosphogluconate, an analogue of the natural substrate. The first products of the reaction are NADPH and 3-keto-2-deoxy-6-phosphogluconate. The NADPH, released from the enzyme, binds to the coenzyme site of the same or the other subunit, activating the decarboxylation reaction in which has not a redox role, since it can be substituted by an analogue devoid of enzymatic redox power. These findings are compared to those obtained with other NADP-dependent decarboxylating dehydrogenases.

Animals↗