Biomedical subjects

R Santucci

Publications and source records attributed to R Santucci.

At least 37 records · Page 2Linked to original sources

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

Nitric oxide synthase inhibition in human sperm affects sperm-oocyte fusion but not zona pellucida binding.

There is recent evidence that mouse and human spermatozoa contain constitutive nitric oxide synthase (cNOS) and can synthesize nitric oxide. The aim of this study was to investigate whether the inhibition of human sperm cNOS could affect sperm-oocyte fusion and sperm binding to the zona pellucida (ZP). N(G)-nitro-L-arginine methyl ester (L-NAME) was used as cNOS inhibitor. Sperm-oocyte fusion was evaluated using the hamster egg penetration test (HEPT). The ZP binding was evaluated using the hemizona assay. L-NAME added from the onset of capacitation strongly inhibited sperm-oocyte fusion. This inhibitory effect was dose dependent, stereospecific, and suppressed by L-arginine in a dose-dependent manner. L-NAME also inhibited sperm-oocyte fusion in the HEPT enhanced with progesterone (P), where P (5 microM) was added for 15 min to capacitated sperm. A lesser but significant inhibition was also observed when sperm suspensions were exposed to L-NAME following capacitation in both versions of HEPT. On the contrary, L-NAME did not affect ZP binding. In conclusion, the present study provides the evidence that cNOS plays a role in the human sperm's capacity to fuse with oocyte but not in the ZP binding.

Animals↗

PubMed1999

Coupling of the oxygen-linked interaction energy for inositol hexakisphosphate and bezafibrate binding to human HbA0.

The energetics of signal propagation between different functional domains (i.e. the binding sites for O2, inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to -3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO2 = 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T- and R-like tertiary conformation(s) within a quaternary R-like structure).

Allosteric Regulation↗

PubMed1999

Naturally-occurring antisperm antibodies in men: interference with fertility and implications for treatment.

Naturally-occurring antisperm antibodies in men are a relative cause of infertility, being the fertility impairment related with the degree of sperm autoimmunization. The impairment of sperm penetration through the cervical mucus represents the best established mechanism of the antibody interference with fertility. Another mechanism may involve complement-mediated sperm injury and opsonizing effect through the female genital tract. Finally, sperm-bound antibodies can interfere with sperm functions involved in the fertilization process, mainly in the sperm-zona pellucida interaction. While some mechanisms of the antibody-interference with fertility depend only on the degree of sperm autoimmunization (e.g., inhibition of cervical mucus penetration), other mechanisms (e.g., interference with gametes interaction) could or could not occur depending on the relevance in the fertilization process of the specific antigen(s) recognized by antisperm antibodies, which are policlonal in nature. Intrauterine insemination is an effective treatment when sperm autoimmunization is low or moderate, mainly if combined with corticosteroid treatment and superovulated cycles. On the contrary, its effectiveness in cases of high degree of sperm autoimmunization is controversial. The resort to "high tech" procedures is mandatory when other less invasive approaches have failed or they may also be chosen as a first-choice method in cases of high degree of sperm autoimmunization. Since in most reports the fertilization rate with in vitro fertilization and embryo transfer (IVF-ET) was significantly lower in the presence of sperm-bound antibodies than in the case of other indications, the likelihood of fertilization is higher with intracytoplasmatic sperm injection (ICSI), where the reported fertilization rates are similar to those in other indications, or even higher.

Adrenal Cortex Hormones↗

PubMed1999

Heterotropic effectors exert more significant strain on monoligated than on unligated hemoglobin.

The effect of allosteric effectors, such as inositol hexakisphosphate and/or bezafibrate, has been investigated on the unliganded human adult hemoglobin both spectroscopically (employing electronic absorption, circular dichroism, resonance Raman, and x-ray absorption near-edge spectroscopies) and functionally (following the kinetics of the first CO binding step up to a final 4% ligand saturation degree). All data indicate that the unliganded T-state is not perturbed by the interaction with either one or both effectors, suggesting that their functional influence is only exerted when a ligand molecule is bound to the heme. This is confirmed by the observation that CO dissociation from partially liganded hemoglobin ( </= 0.04) is strongly altered by the presence of either effector, and the effect is enhanced whenever the two effectors are simultaneously present. Altogether, these data are a direct demonstration of the occurrence of a strain induced by the presence of a ligand molecule bound to the heme, and for the first time there is a clear indication that the expression of the functional heterotropic effect by these non-heme ligands requires this strain, which is not present in the unliganded molecule.

Adult↗

PubMed1999

Formation of a molten-globule-like state of cytochrome c induced by high concentrations of glycerol.

The effect of glycerol on the structure of cytochrome c was investigated by circular dichroism, absorbance and EPR spectroscopy. The results obtained show that an increasing concentration of the organic solvent (70-99.2%, v/v) in aqueous-polyalcohol mixtures converts native cytochrome c into a new, low spin form through a fully reversible, two-state transition. The glycerol-stabilized form (that we call here the G state) retains native-like amounts of alpha-helix structure while rigid tertiary structure and native Fe(III)-Met(80) axial bond are lost. Analysis of data suggests a molten globule character of the G state; support to this view is afforded by the striking similarities between the spectroscopic (and, thus, structural) properties of the G state with those of the acidic molten globule of the protein (A state).

Animals↗

PubMed1999

Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time.

In this study, we investigated the optical features of the redox metal-dependent proteins cytochrome-c, horseradish peroxidase (HRP), and ascorbate oxidase embedded in a sol-gel-processed silica matrix as a function of gelation time. Circular dichroism, absorbance, and fluorescence spectroscopies revealed that the sol-gel process affects the complex structure of the dimeric ascorbate oxidase (although the prosthetic coppers still remain bound to the enzyme) but not that of monomeric cytochrome-c and HRP. Any modifications in ascorbate oxidase occurred in the initial gelation phase; the drying process induced no further alterations and the enzyme remained stable for months. Unfolding-refolding experiments on cytochrome-c revealed severely restricted motility in the protein moiety in the xerogel, the concentrated matrix that forms after drying. The diffusion time of the solvent within the matrix, which regulated the enzyme-substrate reaction rate, depended on the thickness of the monolith, not on the dryness of the specimen.

Ascorbate Oxidase↗

PubMed1998

Unmediated heterogeneous electron transfer reaction of ascorbate oxidase and laccase at a gold electrode.

The unmediated electrochemistry of two large Cu-containing proteins, ascorbate oxidase and laccase, was investigated by direct-current cyclic voltammetry. Rapid heterogeneous electron transfer was achieved in the absence of promoters or mediators by trapping a small amount of protein within a solid, electrochemically inert, tributylmethyl phosphonium chloride membrane coating a gold electrode. The problems typical of proteins in solution, such as adsorption on the electrode surface, were avoided by this procedure. In anaerobic conditions, the cyclic voltammograms, run at a scan rate of up to 200 mV/s, showed the electron transfer process to be quasi-reversible and diffusion-controlled. The pH-dependent redox potentials (+360 mV and +400 mV against a normal hydrogen electrode at pH7.0 for ascorbate oxidase and laccase respectively and +390 mV and +410 mV at pH5.5) were similar to those of the free proteins. The same electrochemical behaviour was recorded for the type 2 Cu-depleted derivatives, which contain reduced type 3 Cu, whereas the apoproteins were electrochemically inactive. Under aerobic conditions the catalytic current intensity of holoprotein voltammograms increased up to approx. 2-fold at a low scanning rate, with unchanged redox potentials. The voltammograms of type 2 Cu-depleted proteins and of apoproteins were unaffected by the presence of oxygen. This suggests that electron uptake at the electrode surface involves type 1 Cu and that only in the presence of oxygen is the intramolecular electron transfer to other protein sites rapid enough to be observed. The analogy with available kinetic results is discussed.

Ascorbate Oxidase↗

PubMed1998

A prospective analysis of the accuracy of the TEST-yolk buffer enhanced hamster egg penetration test and acrosin activity in discriminating fertile from infertile males.

The aim of this study, based on a prospective analysis, was to evaluate the accuracy of the TEST-yolk buffer enhanced hamster egg penetration test (HEPT) and acrosin activity in discriminating fertile from infertile males as compared with the conventional semen profile. The incidence of spontaneous pregnancies was monitored during a follow-up period ranging between 18-24 months in 62 couples without untreatable female infertility. The diagnostic accuracy of the HEPT was significantly higher than that of the conventional semen profile, as evaluated with fitted ROC curves. The best discriminatory ability was exhibited by a penetration index (penetrations/oocyte) of 2 as cut-off value. In our series it gave a positive predictive value of 77.8% and a negative predictive value of 92.3%. Acrosin activity did not show a discriminating power significantly higher than the conventional semen profile, but an acrosin activity <20 microIU/10(6) spermatozoa was found only among patients who did not achieve a pregnancy. In conclusion, this prospective analysis indicates that the TEST-yolk buffer enhanced HEPT constitutes a significant improvement over the conventional semen profile in discriminating fertile from infertile males. Acrosin activity could provide a useful adjunctive test because of the high negative predictive value of its poor results.

Acrosin↗

PubMed1997

Covalent complex of microperoxidase with a 21-residue synthetic peptide as a maquette for low-molecular-mass redox proteins.

Here we report the structural and functional characterization of a covalent complex (MKP) obtained by cross-linking microperoxidase (Mp), the haem-undecapeptide obtained by the peptic digestion of cytochrome c, with a 21-residue synthetic peptide (P21) analogous to the S-peptide of the RNase A. The covalent complex has been prepared by introducing a disulphide bond between Cys-1 of P21 and Lys-13 of Mp, previously modified with a thiol-containing reagent. On formation of the complex (which is a monomer), the helical content of P21 increases significantly. The results obtained indicate that His-13 of P21 co-ordinates to the sixth co-ordination position of the haem iron, thus leading to the formation of a complex characterized by an equilibrium between an 'open' and a 'closed' structure, as confirmed by molecular dynamics simulations. Under acidic pH conditions, where His-13 of P21 is loosely bound to the haem iron ('open' conformation), MKP displays appreciable, quasi-reversible electrochemical activity; in contrast, at neutral pH ('closed' conformation) electrochemical behaviour is negligible, indicating that P21 interferes with the electron-transfer properties typical of Mp. On the whole, MKP is a suitable starting material for building a miniature haem system, with interesting potential for application to biosensor technology.

Amino Acid Sequence↗

PubMed1997

The Soret circular dichroism spectrum as a probe for the heme Fe(III)-Met(80) axial bond in horse cytochrome c.

A spectroscopic signal sensitive to the strength of the heme iron(III)-Met(80) bond in cytochrome c represents a useful tool that will provide valuable information on the heme pocket region and redox properties of the protein. At present, the 695-nm absorption band is perhaps the simplest diagnostic signal for the axial bond; this band disappears when the Fe(III)-Met(80) bond is disrupted. From the analysis of the Soret region circular dichroism spectrum of cytochrome c under conditions that gradually induce disruption of the Fe(III)-Met(80) bond, we present evidence that the 416-nm spectral dichroic band provides independent information addressing the strength of the axial bond between Fe(III) and Met(80) in cytochrome c. Further, this study demonstrates extension of the diagnostic application to very dilute protein samples based on the useful sample concentration of 5-10 microM vs 200-300 microM required for 695-nm absorbance measurements.

Animals↗

PubMed1997

Interference of antisperm antibodies with the induction of the acrosome reaction by zona pellucida (ZP) and its relationship with the inhibition of ZP binding.

OBJECTIVE: To determine whether antisperm antibodies can interfere with the induction of the acrosome reaction (AR) by the zona pellucida (ZP) and whether this interference also can occur in the absence of an inhibitory effect on ZP binding. DESIGN: Prospective in vitro study. SETTING: A tertiary care center, the Andrologic Clinic, University of L'Aquila. PATIENT(S): Sera from 12 infertile patients with high titers of circulating antibodies directed against the sperm head were studied. INTERVENTION: None MAIN OUTCOME MEASURE(S): The effect of antisperm antibodies on ZP binding was evaluated by matching antibody-exposed and nonexposed donor sperm suspensions labeled with fluorescein or rhodamine, respectively, and incubated with the same salt-stored human ZPs. The effect of antibodies on ZP-induced AR was determined by challenging antibody-exposed and nonexposed donor sperm suspensions with human ZPs disaggregated with acidic NaH2PO4. Acrosomal status was evaluated using fluorescein-labeled Pisum sativum agglutinin and supravital stain Hoechst 33258. In some selected cases, the effect of antisperm antibodies on the acrosomal status of sperm bound to intact ZP also was evaluated using transmission electron microscopy. RESULT(S): Five of 12 sera exhibited an inhibitory effect on ZP binding. An inhibition of AR induction by disaggregated ZPs (ranging from 64% to 98%) was produced by all 5 sera with an inhibitory effect on ZP binding and by 2 of 7 sera without an inhibitory effect on ZP binding. The different effects of antisperm antibodies on AR induction by disaggregated ZP were confirmed by comparing with ultrastructural evaluations on the acrosomal status of sperm bound to intact ZP. CONCLUSION(S): Antisperm antibodies can interfere with the induction of AR by ZP. This inhibition can occur even in the absence of an inhibitory effect on ZP binding. Neither effect may occur.

Acrosome↗

PubMed1997

Cell proliferation of the duodenal mucosa in patients affected by familial adenomatous polyposis.

BACKGROUND & AIMS: Patients affected by familial adenomatous polyposis (FAP) are at increased risk of developing duodenal adenomas. In this study, cell proliferation of duodenal mucosa was analyzed to detect kinetic abnormalities related to cancer risk. METHODS: Duodenal biopsy specimens were collected in three groups of patients: group A, 9 hospital controls; group B, 14 patients with FAP without duodenal adenomas; and group C, 6 patients with FAP and duodenal adenomas. Proliferative cell nuclear antigen was assessed through immunohistochemistry. The main labeling parameters were (1) overall labeling index (LI) and (2) LI in the upper 40% of the crypts. RESULTS: Overall LI in groups B and C was higher than in group A (both P < 0.01). LI in group C was also significantly higher than in group B (P < 0.01). Similarly, the upper 40% LI was higher in groups B and C than in group A (both P < 0.01). This value was higher in group C than in group B (P < 0.05). CONCLUSIONS: These data suggest the presence of cell kinetics abnormalities in FAP and the existence of two subgroups of patients with FAP at different risks of duodenal neoplasia. These abnormalities could be used as an intermediate biomarker for chemoprevention studies of duodenal cancer in FAP.

Adenomatous Polyposis Coli↗

PubMed1997

Occurrence of the interference of sperm-associated antibodies on sperm fertilizing ability as evaluated by the sperm-zona pellucida binding test and by the TEST-yolk buffer enhanced sperm penetration assay.

PROBLEM: This study was performed to evaluate the occurrence as well as the level of the interference of sperm-associated antibodies on fertilization process. METHOD: Motile sperm suspensions from 28 infertile patients with high degree of autoimmunization against the sperm head were tested with the zona pellucida (ZP) binding test and with the sperm penetration assay (SPA) enhanced with TEST-yolk buffer. Both tests were also performed using donor sperm exposed and non-exposed to the patients' circulating sperm antibodies. RESULTS: A low ZP-binding was exhibited by sperm from 50% of patients with normal semen profile. All normozoospermic patients with low ZP-binding showed circulating sperm-antibodies with inhibitory effect on ZP-binding, while no patient with normal ZP-binding showed circulating sperm-antibodies with inhibitory effect. No normozoospermic patient exhibited a negative SPA result, and only in 16% of cases the penetration index was slightly less than 2 (the lowest value exhibited by fertile controls). Circulating antisperm-antibodies did not significantly affect the hamster egg penetration. CONCLUSION: Even in the presence of high degree of autoimmunization against the sperm head, sperm fusion with oolemma is not impaired after sperm preincubation with TEST-yolk buffer, while an impairment of the ZP-binding is demonstrable in half cases, when non-immunologic factors are excluded. A substantial role in this interference is likely exerted by IgG antibodies transuded from the blood into the genital tract.

Autoantibodies↗

PubMed1997

Folic acid supplementation and cell kinetics of rectal mucosa in patients with ulcerative colitis.

It has been suggested that colon cancer risk in ulcerative colitis (UC) is correlated to a reduced bioavailability of folate. We studied the effects of folate supplementation on the pattern of rectal cell proliferation in patients affected by long-standing UC. In the rectal mucosa of these patients, an expansion of proliferating cells to the crypt surface is found frequently. This abnormality is considered an intermediate biomarker in chemoprevention trials. Twenty-four patients (13 males; age, 26-70 years; UC duration, 7-34 years) with UC in remission for 1 month at least were assigned randomly to one of the following treatments: (a) folinic acid (15 mg/day) or (b) placebo. Cell proliferation was analyzed through immunohistochemistry on sections of rectal biopsies incubated for 1 hour in a culture medium containing bromodeoxyuridine. Fragments were taken at admission to the study and after 3 months of treatment. As compared to the baseline values, after 3 months of therapy in patients treated with folinic acid, a significant reduction of the frequency of occurrence of labeled cells in the upper 40% of the crypts (phi h value) was observed (0.1836 +/- 0.0278 versus 0.1023 +/- 0.0255; P < 0.01). On the contrary, no significant proliferative changes were observed in the placebo group. These results suggest that folate supplementation contributes to regulating rectal cell proliferation in patients with long-standing UC. These findings may be significant for chemoprevention of colon cancer in these patients.

Adult↗

PubMed1996

Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c.

This paper reports a voltammetric and spectroscopic investigation of the guanidine-induced unfolding of cytochrome c at neutral pH and 25 degrees C. Electrochemical data provide direct evidence for the presence of an equilibrium intermediate (form I) strictly dependent on the denaturant concentration. The midpoint potential of form I has been determined (E1/2 = +0.010 V vs. NHE) and its structural features defined from analysis of the circular dichroism and absorbance spectroscopy data obtained under the same experimental conditions. From the correlation of electrochemical and spectroscopic data, we propose that the features detected by the intermediate conform to the molten globule state.

Animals↗

PubMed1996

Influence of glycerol on the structure and stability of ferric horse heart myoglobin: a SAXS and circular dichroism study.

The influence of glycerol on the structural properties of Fe(III)-horse heart myoglobin has been investigated by absorbance, CD and SR-SAXS spectroscopy. The results obtained indicate that both the tertiary and the secondary (alpha-helix) conformations of the protein are influenced by glycerol; in particular, an increase of approx. 8% in helical content was observed. Further, analysis of both the acid- and guanidine-induced denaturation transitions points to a glycerol-induced decreased stability of the tertiary structure; conversely, the alpha-helix conformation is found to be stabilized by the organic solvent. Finally, the SR-SAXS data show that gyration radius, cross-section and thickness of the protein increase in the presence of the organic solvent; however, the protein maintains a compact state.

Animals↗

PubMed1996

Influence of glycerol on the structure and redox properties of horse heart cytochrome c. A circular dichroism and electrochemical study.

The effect of glycerol on the structure and redox properties of horse heart cytochrome c was investigated by absorption spectroscopy, circular dichroism, and dc cyclic voltammetry techniques. The results show that the organic solvent increases the alpha-helix structure of the protein and induces slight changes at the active-site environment: however, the overall tertiary structure does not appear to be significantly perturbed. Glycerol stabilizes cytochrome c, the free energy of denaturation (delta G0) being approximately 0.7 kcal/mol larger than that determined in phosphate buffer under the same conditions, and influences the heterogeneous electron transfer kinetics at a chemically modified gold electrode: on the other hand, the redox potential of the protein is unaltered. On the whole, the results obtained indicate that glycerol acts as a suitable stabilizing agent of cytochrome c, which is of interest for application in biotechnology: the organic solvent does not alter the tertiary structure significantly or the redox properties of the protein. This has to be interpreted not only in terms of the glycerol-induced solvent ordering around the protein surface, but also as due to the specific features of the protein matrix.

Animals↗