Cell kinetics of the gastric mucosa of patients treated with omeprazole.
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Adult↗
Biomedical subjects
R Santucci
Publications and source records attributed to R Santucci.
Observer variation in histological assessment of Helicobacter pylori on gastric biopsies.
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Adult↗
A complex of microperoxidase with a synthetic peptide: structural and functional characterization.
This paper reports the kinetic and thermodynamic characterization of the complex obtained by binding to microperoxidase (the heme-containing undecapeptide derived from peptic hydrolysis of cytochrome c) a 13 residues synthetic peptide with some propensity to acquire alpha-helical secondary structure (P13). Our results indicate that P13 binds to the sixth coordination position of the Fe(III) of microperoxidase (Keq = 4.8 x 10(4) M-1 at pH 7.0 and 25 degrees C) via the imidazole of His-12, forming a stable complex. The kinetics of complex formation, its secondary structure and its electrochemical activity are reported. This is a first step towards engineering a miniature-heme complex, for a better understanding of the mechanisms governing electron transfer in hemes and heme-proteins, and possibly for novel biotechnological applications.
Amino Acid Sequence↗
A single mutation (Thr72-->Ile) at the subunit interface is crucial for the functional properties of the homodimeric co-operative haemoglobin from Scapharca inaequivalvis.
The in vivo expression and the functional and spectroscopic properties are reported for a mutant of the homodimeric haemoglobin of the mollusc Scapharca inaequivalvis (HbI), where residue threonine 72 (position 9 in the E helix) at the subunit interface has been substituted by isoleucine. The aim of this study is to test the hypothesis that increasing the hydrophobicity character of the subunit interface may modulate oxygen affinity and co-operativity of this haemoglobin. In fact, X-ray crystal structure studies have shown that the subunit interface, formed by the E and F helices of the two chains, changes its character from hydrophilic to hydrophobic upon oxygenation. This is primarily due to extrusion of Phe97 side-chain from the haem pocket toward the interface, which disrupts a network of ordered water molecules and results in close van der Waals contacts between Phe97 and Thr72 of the partner subunit. Thr72-->Ile HbI was expressed in E. coli after mutation of HbI-DNA and it displays a approximately 40-fold enhancement of oxygen affinity and a marked reduction of co-operativity in oxygen binding, with respect to native HbI. These functional properties and the kinetics of oxygen dissociation and carbon monoxide combination rates, as well as data from EPR and circular dichroism spectroscopy, indicate that indeed the increase of the hydrophobicity at the interface upon mutation stabilizes the "high affinity" conformation of the protein, suggesting that extrusion of Phe97 toward the interface should be facilitated even in the unliganded form.
Animals↗
Colorectal cancer in patients with ulcerative colitis. A prospective cohort study in Italy.
BACKGROUND: The aim of this study was to assess the development of dysplasia or cancer in patients with ulcerative colitis and to determine the effectiveness of colonoscopy and biopsy follow-up in colon cancer surveillance. METHODS: From 1980 to 1986, 65 patients who had ulcerative colitis for 7 years or more participated in a surveillance program of colonoscopy and biopsy. This cohort was followed until December 1992. Forty-nine patients (75.4%) had extensive colitis and 16 (24.6%) left-sided colitis. The mean disease duration was 17.2 years. Three hundred four colonoscopies were performed. During each endoscopy, random biopsies were performed. RESULTS: Seven patients had definite dysplasia of the colorectal mucosa. Four of them had high grade lesions and underwent surgery. In all of these patients, colon cancer (3 Dukes' Stage A, 1 Dukes' Stage B) was found. No cancer was found in the other patients. Pedunculated adenomas were excised from 6 other patients during colonoscopy. When dysplasia was diagnosed, these patients were older than those who were dysplasia free, whereas the age at onset of colitis was significantly higher in the former (P < 0.01). Fifteen patients discontinued follow-up. Two of them developed colon cancer diagnosed at an advanced stage. CONCLUSIONS: Dysplasia, especially of high grade, is a marker of colon cancer risk in patients with longstanding ulcerative colitis. Intensive colonoscopy and biopsy surveillance can lead to the diagnosis of colon cancer at a potentially curable stage. In this series, older age appeared to be an additional risk factor. A careful selection of patients with ulcerative colitis seems mandatory to minimize the cost and optimize the benefit of colon cancer surveillance programs.
Adult↗
Relationship between acrosome reactions and hamster egg penetration after ionophore challenge in absence of teratozoospermia.
OBJECTIVE: To determine the relationship between acrosome reactions and hamster egg penetration after ionophore challenge in nonteratozoospermic semen samples. SETTING: A tertiary care center, the Andrologic Clinic, Department of Internal Medicine, University of L'Aquila. PATIENTS: Twenty-five nonteratozoospermic patients with infertile marriages and nine fertile men. INTERVENTIONS: The hamster egg penetration assay and the acrosome reaction assessment were performed on capacitated sperm suspensions in basal conditions and after ionophore challenge with ionomycin. MAIN OUTCOME MEASURE: The relationship between the acrosome reactions and the hamster egg penetration was analyzed. RESULTS: Although in basal conditions the spontaneous acrosome reaction rate was not correlated with the hamster egg penetration, after ionophore challenge both the induced acrosome reaction rate and the acrosome reaction increase (induced minus spontaneous acrosome reaction rate) correlated with the hamster egg penetration. The highest correlation was observed between the induced acrosome reaction rate and the penetration index. Considering a penetration index = 2 as the threshold of a good response of the hamster egg penetration assay to the ionophore challenge, significantly lower acrosome reaction rates and increases were associated with poor hamster egg penetration. However, different penetration indexes occurred at the same level of induced acrosome reaction rate. CONCLUSIONS: The capability of sperm to react after ionophore challenge reflects to some extent the capability of the acrosome-reacted sperm to undergo the changes of the plasma membrane that are necessary for sperm-oocyte fusion. The simple evaluation of the acrosome reactions may represent a useful complement rather than a substitute for the hamster egg penetration assay in monitoring the responses of human sperm to the ionophore challenge. An impaired inducibility of the acrosome reactions may account for poor hamster egg penetrations exhibited by nonteratozoospermic semen samples.
Acrosome↗
Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative: a spectroscopic and thermodynamic study.
Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated quantitatively by EPR and absorbance spectroscopy. The X-band EPR spectra and the absorption spectra in the Soret region of the nitrosylated derivative of ferrous native and carboxymethylated cytochrome c display the same basic characteristics reported for the beef heart cytochrome a3 in cytochrome c oxidase, and horseradish and baker's yeast cytochrome c peroxidase, as well as the high affinity form of oxygen carrying proteins. Values of the dissociation equilibrium constant for nitrosylation of ferrous native and carboxymethylated cytochrome c are 8.2 x 10(-6) M and < or = 5 x 10(-8) M, respectively, at pH 7.0 and 10 degrees C. The results here reported represent clearcut evidence for the nitric oxide-induced cleavage of the Fe-Met80 bond in ferrous native cytochrome c, and allow estimation of the free energy associated to the heme-iron sixth coordination bond (> 10 kJ mol-1, at 10 degrees C).
Animals↗
Impaired hamster egg penetration by human sperm from ejaculates with low acrosin activity but otherwise normal.
OBJECTIVE: To determine if a low acrosin activity in otherwise normal ejaculate from infertile patients is associated with an impairment of sperm functions involved in the hamster egg penetration. SETTING: A tertiary care center, the Andrology Clinic, Department of Internal Medicine, University of L'Aquila. PATIENTS: Nine infertile patients with low acrosin activity in otherwise normal ejaculate (including normal immunoreactivity for acrosin) were studied; nine fertile men served as a control group. The two groups were homogeneous for seminal parameters. INTERVENTIONS: The hamster egg penetration assay and the acrosome reaction rate assessment were performed on capacitated sperm suspensions in basal conditions and after ionophore challenge. MAIN OUTCOME MEASURES: The penetration rate, the penetration index, and the acrosome reaction rate were measured and compared between the groups. RESULTS: No penetration was achieved in seven patients and a low penetration was achieved in two cases. The difference with the control group was significant. The ionophore challenge was associated to penetration of hamster eggs in seven of nine patients, but the penetration index was significantly lower than the controls. Acrosin activity was correlated to hamster egg penetration. Both spontaneous and induced acrosome reaction rate were similar in the two groups. CONCLUSIONS: A low sperm acrosin activity in otherwise normal ejaculate is associated with an impaired hamster egg penetration. This impairment does not seem to be due to altered dynamics of acrosome reaction.
Acrosin↗
Methodological problems in the use of rectal cell proliferation as a biomarker of colorectal cancer risk.
Clinical and experimental studies have shown that the carcinogenic process of colorectal mucosa is linked to the development of proliferative abnormalities which precede the occurrence of morphological abnormalities such as epithelial dysplasia. In humans, proliferative defects have been demonstrated in the normal rectal mucosa of population groups at risk for colon cancer. Several techniques are available to study cell kinetics in the gastrointestinal mucosa. Each explores different aspects of cell proliferation. We have attempted to evaluate the correlation between various techniques in the normal rectal mucosa of high-risk patients. We found a good correlation between bromodeoxyuridine (BrdU) labeling index and the mucosal activity of the enzyme ornithine decarboxylase, and between tritiated thymidine and the percentage of cells in the S phase of the proliferative cycle as determined by flow cytometry. However, these correlations concern only labeling indices, which can be influenced by physiological events, such as active inflammation or increased cell loss. It may not be the most reliable proliferative marker of cancer risk. Moreover, methods using cells isolated from homogenized tissue do not allow us to evaluate the pool of cells which are examined nor the distribution of proliferating cells within the tissue. For example, an inflamed mucosal specimen is highly infiltrated with inflammatory cells which can interfere with the measurement of epithelial cell proliferation. Nevertheless, the labeling index may be normal even in patients at high risk. For these reasons, we think that the most reliable methods are those using tissue culture, such as tritiated thymidine or BrdU uptake and proliferating cell nuclear antigen (PCNA) immunostaining.
Adenoma↗
Flow cytometry and in vitro tritiated thymidine labeling in normal rectal mucosa of patients at high risk of colorectal cancer.
OBJECTIVES: To compare two different methods to evaluate rectal epithelial cell proliferation as a biomarker of risk of developing colon cancer. METHODS: Samples of normal rectal mucosa from 26 patients at increased risk for colorectal cancer (22 patients with adenoma, three with adenocarcinoma of the large bowel, and one with longstanding ulcerative colitis) were examined by means of in vitro labeling with tritiated thymidine and flow cytometry. RESULTS: We found a significant correlation between thymidine-labeling index and the percentage of cells in S-phase, measured by flow cytometry both in formalin-fixed, paraffin-embedded specimens and in frozen specimens (respectively, r = 0.7647, p < 0.001, and r = 0.4503, p < 0.01). However, using flow cytometry, the percentage of cells in S-phase was significantly higher than the thymidine-labeling index in both fixed-embedded and frozen specimens (p < 0.01). Proliferative parameters were not higher in patients with colon carcinoma, and were not related to the degree of dysplasia, the number of adenomas, or familial occurrence of colorectal cancer. Two specimens taken from normal rectal mucosa of two patients with adenomas showed aneuploidy. No aneuploidy was found in normal rectal specimens of patients with adenocarcinoma. CONCLUSIONS: These results show that the calculation of cells in S-phase with in vitro tritiated thymidine labeling or by flow cytometry produces different results. However, the significant correlation between corresponding parameters obtained with these techniques support the use of either method as "intermediate biomarkers" of colorectal cancer risk and prognosis.
Adenocarcinoma↗
Abnormal rectal cell proliferation and p52/p35 protein expression in patients with ulcerative colitis.
We evaluated the presence of cell proliferation and antigenic abnormalities in rectal biopsies from 37 patients affected by ulcerative colitis and 15 controls. The study was carried out by thymidine labeling and immunochemistry, using antibodies against specific cytoskeletal-associated proteins (p52, p35, alpha-actinin). Among ulcerative colitis patients, 24 had an immunofluorescence pattern similar to that of controls, while 13 showed an abnormal distribution of one or more proteins (p52 alone or p52 and either p35 or alpha-actinin) within the rectal crypts. Patients showed a shift of the proliferative compartment towards the top of the rectal crypts compared with controls. This finding was more evident in patients with p52 or p35 abnormalities. Proliferative and antigenic defects were not related either to age or the duration of colitis. These phenotypic changes might be a biomarker of increased risk of colon cancer in ulcerative colitis.
Adult↗
Reconstitution of horse heart myoglobin with hemins methylated at 6- or 7-positions: a circular dichroism study.
The reconstitution kinetics of horse heart myoglobin, as met-cyano derivative, with two synthetic hemins in which the 6- or the 7-propionate is replaced by a methyl group, has been investigated by circular dichroism, in order to gain information on the heme re-orientation process following the heme insertion into the globin pocket. The results obtained confirm that the preferred heme orientation places the sole propionate into the position occupied by the 6-propionate in the crystal structure, supporting the importance of the salt bridge occurring between this propionate and the basic CD3 residue of the apoprotein. Moreover, they provide new information on the correlations existing between the shape and the intensity of the dichroic bands and the heme orientation inside the reconstituted protein. Our data suggest that positive Soret CD bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'correct' orientation (as in horse heart myoglobin), and negative dichroic bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'reversed' orientation (as for example, in Glycera dibranchiata hemoglobins). Thus, negligible contribution to the CD signal in reconstituted proteins can be associated to a 'wrong' orientation of the heme group, no matter whether the orientation at the equilibrium is the 'correct' or the 'reversed' one. Finally, the results obtained indicate that the perturbations due to the heme re-orientation appear as a local phenomenon, not affecting the distant domains of the macromolecule.
Animals↗
Interaction of inositol hexakisphosphate with liganded ferrous human hemoglobin. Direct evidence for two functionally operative binding sites.
Inositol hexakisphosphate (InsP6) binding to the oxygenated, carbonylated and nitrosylated derivatives of ferrous human hemoglobin (HbO2, HbCO and HbNO, respectively) has been measured at pH 7.0 (0.1 M Bis-Tris buffer, 0.1 M NaCl) and 20 degrees C. The observations indicate the presence of two InsP6 binding sites per tetramer in all the heme liganded hemoglobin derivatives, with different affinities for the polyphosphate. For each binding site, InsP6 interacts with similar affinity constants to HbO2, HbCO and HbNO. Such a finding indicates that different heme ligands do not alter significantly the stereochemistry of the polyphosphate binding cleft. This behaviour seems to indicate that, even though different heme ligands are likely to affect the tertiary conformation of the subunit in a different fashion, the perturbation does not seem to be transmitted to the quaternary arrangement of the whole macromolecule, and, thus, to the InsP6 binding site.
Binding Sites↗
Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.
The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 x 10(-1) M N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 x 10(-1) M NaCl), as well as at 100 K and/or 20 degrees C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb.
Bezafibrate↗
Rectal cell proliferation as an intermediate biomarker of risk of colorectal cancer.
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Biomarkers↗
Effect of ionophore challenge on hamster egg penetration and acrosome reaction of antibody-coated human sperm.
PROBLEM: Following the demonstration that antisperm antibodies do not affect the spontaneous acrosome reactions (AR) of human sperm used for the hamster egg penetration assay (HEPA), we evaluated the effect of the ionophore challenge on HEPA and AR of antibody-coated sperm. METHOD: Motile sperm suspensions from donors were exposed to serum and seminal plasma samples with sperm-head-directed antibodies, washed, capacitated, and challenged with ionomycin. RESULTS: The proportion of acrosome-reacted sperm, assessed by fluoresceinated Pisum sativum agglutinin and supravital stain Hoechst 33258, was significantly higher in the antibody-exposed sperm than in the controls (47.0 +/- 13.2% vs. 39.3 +/- 12.1, respectively; P < 0.05). However, the hamster egg penetration rate and index were not significantly different between antibody-exposed sperm and controls (98.0 +/- 3.7% vs. 85.9 +/- 16.2% and 6.64 +/- 4.1 vs. 6.81 +/- 4.8, respectively). Only one of eight test samples with antisperm antibodies, where the highest increase of AR rate compared with controls was observed (55.0% vs. 35.6%) produced a substantial increase of penetrations per oocyte (7.4 vs. 4.1). CONCLUSION: The ionophore challenge enabled us to recognize a subtle effect of anti-sperm antibodies on the dynamics of acrosomal loss, the clinical relevance of which needs to be elucidated.
Acrosome↗
Effect of bezafibrate and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.
The effect of bezafibrate (BZF) and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of BZF and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of inositol hexakisphosphate (IHP) and 2, 3-diphosphoglycerate (2,3-DPG). Next, in the presence of these allosteric effectors, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced. These findings indicate that BZF and CFA, as already reported for IHP and 2, 3-DPG, induce the stabilization of a low affinity conformation of the ligated hemoprotein (i.e., HbNO). Values of the apparent equilibrium constant for BZF and CFA binding to HbNO (K) are 1.5(+/- 0.2) x 10(-2) M and 2.8(+/- 0.3) x 10(-2) M, respectively, at pH 7.0 (in 0.1 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 0.1 M NaCl) and 20 degrees C. The results reported here represent clearcut evidence for BZF and CFA specific (i.e., functionally relevant) binding to a ligated derivative of Hb (i.e., HbNO).
2,3-Diphosphoglycerate↗
Effect of vitamin A, C, and E supplementation on rectal cell proliferation in patients with colorectal adenomas.
Studies suggest that cell proliferation abnormalities of the colorectal mucosa are associated with risk of neoplasia, and most cancers of the large bowel are thought to arise from adenomas. The results of other studies suggest that vitamins A, C, and E have chemopreventive efficacy against colon cancer in animal models. This study evaluates the effect of dietary vitamin supplementation on cell kinetics in uninvolved rectal mucosa in patients with colorectal adenomas. Twenty patients with colorectal adenomas were given vitamins A, C, and E for 6 months after complete polypectomy, and 21 patients with adenomas received placebo. In each patient, six biopsy specimens were taken from normal-appearing rectal mucosa before treatment and after 3 and 6 months of treatment and were incubated with tritiated thymidine ([3H]thymidine), and the [3H]thymidine-labeled cells were counted by use of autoradiography. Two parameters of cell proliferation were evaluated: 1) the ratio of the number of labeled cells to the total number of cells (thymidine labeling index) and 2) the ratio of the number of labeled cells in the upper 40% of the crypt to the total number of labeled cells in the crypt (phi h). The latter index reflects abnormal expansion of the proliferative compartment and is thought to be an intermediate biomarker of cancer risk. In patients receiving vitamins, phi h decreased progressively from baseline values, with increasing statistical significance (P less than .05 after 3 months, P less than .01 after 6 months). There was a statistically significant decrease in the thymidine labeling index in the 40% of the crypt near the mucosal surface, but the variation in the overall labeling index was not statistically significant. In the placebo group, we observed no statistically significant change in cell kinetics. These findings suggest that vitamin A, C, and E supplementation is effective in reducing abnormalities in cell kinetics that may indicate a precancerous condition. Before larger trials on chemoprevention of colorectal adenoma recurrence are conducted, additional studies are needed (a) to validate that cell kinetics is an intermediate biomarker, (b) to determine active agents, optimal dosage, and the relative efficacy of agents given alone and in combination, and (c) to test toxicity.
Adenoma↗