Biomedical subjects

I Messana

Publications and source records attributed to I Messana.

At least 19 recordsLinked to original sources

PubMed2005

Two proline-rich peptides from pig (Sus scrofa) salivary glands generated by pre-secretory pathway underlying the action of a proteinase cleaving ProAla bonds.

The primary structures of two salivary proline-rich peptides (PRP-SP-A, M 6156.0 amu and PRP-SP-B, M 1905.0 amu), from pig (Sus scrofa) were determined. The PRP-SP-B peptide, 21 residues long, overlaps with a sequence repeated 43 times in three deposited cDNAs coding for PRP proteins cloned from porcine parotid glands (Swiss-Prot codes: Q95JC9, Q95JD1, Q95JD0). PRP-SP-A peptide, 56 amino acid residues long, overlaps with the N-terminus repeats of Q95JC9 and Q95JD1 and it is phosphorylated at Ser 12 and 14. The two peptides were found both in whole saliva and in granules from pig parotid glands. The biosynthesis of the two peptides implies the action of a proteinase responsible for Pro downward arrow Ala cleavage in the pre-secretory process.

Alanine↗

PubMed2005

Statherin levels in saliva of patients with precancerous and cancerous lesions of the oral cavity: a preliminary report.

OBJECTIVE: The aim of this study was to measure concentration of human salivary statherin in patients with oral cavity pathologies and salivary gland diseases. SUBJECTS AND METHODS: Levels of statherin were analysed with High Performance Liquid Chromatography (HPLC) in following groups of subjects: group A: 24 patients with neoplastic diseases of salivary glands, group B: 13 patients with inflammatory lesions of salivary glands, group C: 13 patients with precancerous and cancerous lesions of the oral cavity excluding salivary gland tumors, group D: 20 healthy volunteers (control group). RESULTS: Our preliminary data indicated a sensible reduction of the statherin level in the saliva of patients with precancerous and cancerous lesions of the oral cavity (group C) compared with the healthy subjects (group D). The statherin levels are not significantly reduced either in the inflammatory (group B) or in the salivary glands tumours (group A), compared with the healthy subjects (group D). CONCLUSION: Statherin could play a protective effect in oral cavity in association with its other functions.

Adolescent↗

PubMed2003

Bezafibrate induces a mitochondrial derangement in human cell lines: a PPAR-independent mechanism for a peroxisome proliferator.

Bezafibrate is a hypolipidemic drug that belongs to the group of peroxisome proliferators because it binds to peroxisome proliferator-activated receptors type alpha (PPARs). Peroxisome proliferators produce a myriad of extraperoxisomal effects, which are not necessarily dependent on their interaction with PPARs. An investigation on the peculiar activities of bezafibrate could clarify some of the molecular events and the relationship with the biochemical and pharmacological properties of this class of compounds. In this view, the human acute promyelocytic leukemia HL-60 cell line and human rabdomiosarcoma TE-671 cell line were cultured in media containing bezafibrate and a number of observations such as spectrophotometric analysis of mitochondrial respiratory chain enzymes, NMR metabolite determinations, phosphofructokinase enzymatic analysis, and differentiation assays were carried on. Bezafibrate induced a derangement of NADH cytochrome c reductase activity accompanied by metabolic alterations, mainly a shift to anaerobic glycolysis and an increase of fatty acid oxidation, as shown by NMR analysis of culture supernatants where acetate, lactate, and alanine levels increased. On the whole, the present results suggest a biochemical profile and a therapeutic role of this class of PPARs ligands more complex than those previously proposed.

Acetates↗

PubMed2003

Human salivary peptides derived from histatins.

Human saliva from a healthy donor was subjected to fractionation by gel chromatography and six pools were collected and analysed by MALDI-TOF-MS and HPLC-ESI-MS. Three peptides, corresponding to 888.3, 687.3, and 524.1 amu and SNYLYDN, YLYDN, and LYDN sequences (determined by automated Edman sequencing), were isolated from pool 4. YLYDN was not previously described in human saliva. The peptides show the common C-terminal sequence of histatin 3 and histatin 1. To exclude the possibility that the three peptides were an artifact of the purification procedure, nine samples of human saliva were collected from healthy donors, immediately acidified with 0.2% TFA, and analysed by RP-HPLC-ESI-MS. The three peptides were detected in all the analyzed samples. SNYLYDN was always found at a concentration higher than that of YLYDN and LYDN. A correlation analysis performed on quantitative data indicated that the three peptides derive only from histatin 3. Other already known histatins also were searched for in the chromatogram. Histatins 1, 2, 3, 5, 6, 7, 8, and 10 were observed, although not in all samples analyzed, whereas other minor histatins were not detected.

Amino Acid Sequence↗

PubMed2003

The coupling of RP-HPLC and ESI-MS in the study of small peptides and proteins secreted in vitro by human salivary glands that are soluble in acidic solution.

The aim of this study was the development of a method based on the coupling of RP-HPLC and ESI-MS for identifying and quantifying proteins and peptides secreted by human salivary glands in vitro. Salivary gland specimens, obtained from informed patients undergoing surgical resection, were incubated in an optimized medium. Incubation media of glandular specimens, selected on the basis of cytomorphological and ultrastructural analysis, were investigated by HPLC-MS. Several salivary peptides/proteins, previously recognized in human whole saliva, were searched for along the chromatogram by the selected ion monitoring (SIM) strategy. Analysis of the incubation media of parotid glands revealed the presence of basic PRPs PC, PD, PH, IB-1, II-2, and acidic PRP-1 and PRP-3 in all of the investigated samples. Basic PRPs PB and PA, acidic PRPs, and cystatins SN and S1 were detected in all of the incubation media of submandibular glands, whereas histatin 1 was detected in only one sample. Moreover, the method allowed detection of some post-translational derivatives of known salivary proteins, as well as of several previously unidentified small peptides. The present method represents a sensitive and powerful instrument to detect peptides and proteins secreted by human salivary glands in vitro.

Adult↗

PubMed2001

Role of ergothioneine on S-nitrosoglutathione catabolism.

Ergothioneine (ESH) is a low-molecular-mass thiol present in millimolar concentrations in a limited number of tissues, including erythrocytes, kidney, seminal fluid and liver; however, its biological function is still unclear. In the present study we investigated the role of ESH in the catabolism of S-nitrosoglutathione (GSNO). The results show that: (1) GSNO decomposition is strongly influenced by ESH (k"=0.178+/-0.032 M(-1) x s(-1)); (2) ammonia is the main nitrogen-containing compound generated by the reaction; and (3) nitrite is practically absent under both aerobic and anaerobic conditions. These findings are markedly different from those reported for the GSH-induced decomposition of GSNO, in which the nitrogen-containing end products are nitrite, ammonia and nitrous oxide (N(2)O) under aerobic conditions but nitrite, ammonia, nitric oxide (NO) and small quantities of hydroxylamine under anaerobic conditions. Considering the high concentration of ESH in specific cells, the reaction with GSNO should be considered as an important molecular event occurring in the cell.

Ergothioneine↗

PubMed2001

Determination of the human salivary peptides histatins 1, 3, 5 and statherin by high-performance liquid chromatography and by diode-array detection.

A reversed-phase high-performance liquid chromatography (HPLC) method with diode-array detection for the quantification of several human salivary peptides is described. Sample pretreatment consisted of the acidification of whole saliva by phosphate buffer. This treatment produced precipitation of mucins, alpha-amylases and other high-molecular-mass salivary proteins, simultaneous inhibition of intrinsic protease activities and reduction of sample viscosity. Direct HPLC analysis by diode-array detection of the resulting acidic sample allowed one to quantify histatin 1, histatin 3, histatin 5, statherin, as well as uric acid, in normal subjects. Moreover, the groups of peaks pertaining to proline-rich proteins and cystatins were tentatively identified. The method can be useful in assessing the concentration of salivary peptides from normal subjects and from patients suffering oral and/or periodontal diseases.

Acids↗

PubMed2000

Determination of biophysical parameters of polypeptide retro-inverso isomers and their analogues by capillary electrophoresis.

The relationship between the electrophoretic mobility, microobs, Stokes radius, rs, ionization state, and solution conformation of the all L-alpha-polypeptide, 1, the corresponding retro-all D-alpha-polypeptide, 2, and several truncated analogues, 3-5, has been investigated under low pH buffer conditions by high-performance capillary zonal electrophoresis (HPCZE) with coated capillaries. The results confirm that, under these conditions, the all L-alpha-polypeptide, 1, and its retro-inverso isomer, 2, exhibit nonidentical electrophoretic mobilities and thus different Stokes radii. At higher pH values, i.e., pH 5.0, the electrophoretic behavior of this retro-inverso isomer pair, however, converges. These results indicate that variations in the dipole characteristics of the polypeptide main chain and subtle differences introduced by the spatial constraints of the L-alpha-Pro-->D-alpha-Pro residue replacement lead to differences in the Stokes radii and electrophoretic mobilities of these polypeptides. Since the observed electrophoretic mobilities, microobs, reflect the mean of the mobilities of each charge species participating according to their Stokes radius or their intrinsic charge and mole fraction abundances, the results confirm that polypeptide retro-inverso isomers with unmodified amino and carboxy termini are resolvable. This outcome was achieved despite their notional topographical and conformational similarities as assessed from high-field proton nuclear magnetic resonance (1H NMR) spectroscopy and circular dichroism (CD) spectroscopy.

Amino Acid Sequence↗

PubMed2000

Determination of S-nitrosoglutathione in erythrocytes by capillary zone electrophoresis.

A method for separation and quantification of S-nitrosoglutathione in red cell extracts by capillary electrophoresis is reported. The method is based on the direct analysis of the metaphosphoric acid erythrocyte extract containing diethylenetriaminepentaacetic acid. Optimization of the method is briefly discussed. Best results in the shortest time were obtained at 25 degrees C, using a coated capillary, 7 kV applied voltage and phosphate sodium 40 mmol/L (pH 2.2) as running buffer. Reproducibility, detection limits, and recoveries of S-nitrosoglutathione analyses were checked. The results evidenced that S-nitrosoglutathione is formed in erythrocytes treated with S-nitrosocysteine, a transnitrosating agent. Under our experimental conditions, the contemporaneous detection and quantification of reduced and oxidized glutathione present in cell extract could also be performed.

Electrophoresis, Capillary↗

PubMed2000

Separation of diadenosine polyphosphates by capillary electrophoresis.

The influence of buffer composition and pH on the electrophoretic behavior of diadenosine polyphosphates with a phosphate chain ranging from two to five phosphate groups has been examined. The electrophoretic mobility in carbonate buffer increases according to the number of phosphates, whereas in borate buffer the mobility changes in an irregular way as a function of pH. This finding can be rationalized by a well-known interaction of borate with ribose rings, which modifies the charge and the hydrodynamic radius of each diadenosine polyphosphate in a different way. Our study shows that the best separation of diadenosine polyphosphates can be achieved at the highest pH values of the range examined both in borate and carbonate buffers.

Borates↗

PubMed2000

Blood bank conditions and RBCs: the progressive loss of metabolic modulation.

BACKGROUND: Human RBC metabolism is modulated by the cell oxygenation state. Among other mechanisms, competition of deoxyhemoglobin and some glycolytic enzymes for the cytoplasmic domain of band 3 is probably involved in modulation. This metabolic modulation is connected to variations in intracellular NADPH and ATP levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. The present study investigates the effect of storage on this metabolic modulation and its relationship with the alteration of membrane protein composition. STUDY DESIGN AND METHODS: RBCs stored in CPD-saline-adenine-glucose-mannitol were assayed for glucose uptake and partition between glycolysis and the pentose phosphate pathway at high and low oxygen saturation by nuclear magnetic resonance spectroscopy after 1, 14, 21, 35, and 42 days of storage. Membrane protein composition was determined by SDS-PAGE on Days 1, 14, 35, and 42. Metabolic values and 2,3 DPG concentration were also measured after rejuvenation for 1 hour at 37 degrees C with pyruvate-inosine-phosphate-adenine solution on Day 21. RESULTS: Metabolic differences between RBCs incubated at high and low oxygen saturation decreased during storage, and, on Day 35, the two groups did not have significant differences (p = 0.111). SDS-PAGE showed that membrane protein composition was concurrently modified. The percentage of unmodified band 3 decreased during storage, principally between Days 14 and 35. In rejuvenated RBCs, oxygen-dependent modulation was not restored. CONCLUSIONS: RBCs stored in CPD-saline-adenine-glucose-mannitol do show a progressive loss of oxygen-dependent metabolic modulation, which is not restored after rejuvenation and which seems partly related to modifications in membrane proteins, mainly band 3.

Adenine↗

PubMed1999

Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13C- and 1H-MR spectroscopy.

Information displayed by homonuclear and heteronuclear spin-coupling patterns in 13C- and 1H-MR spectra allowed us to identify the major lactate isotopomers produced either from [1-(13)C]-glucose or from [2-(13)C]-glucose by human erythrocytes. Relative concentrations of detectable isotopomers were determined by integrating the corresponding MR signals. The interpretation of these data in terms of the fractional glucose metabolised through glycolysis and pentose phosphate pathway was performed by a computer simulation of the metabolism that took into account metabolic schemes pertaining to glycolysis and to the F-type of pentose phosphate pathway. The simulation was organised in a way to anticipate the populations of the isotopomers produced from any precursor at a priori established metabolic steady state. By the simulation, isotopomer populations were determined according to different values of pentose cycle, defined as the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. The populations of the isotopomers originating from [2-(13)C]-glucose were described by polynomials, and ratios between the polynomials were used in conjunction with 13C- and 1H-MR data to determine pentose cycle values. The knowledge of glucose uptake and of pentose cycle value allowed us to perform accurate measurement of the pentose phosphate pathway flux, of the hexokinase and phosphofructokinase fluxes as well as, indirectly, of the carbon dioxide production.

Erythrocytes↗

PubMed1999

Low-temperature sensitivity and enhanced Bohr effect in red deer (Cervus elaphus) haemoglobin: a molecular adaptive strategy to life at high altitude and low temperature.

A study of the functional properties of haemoglobin from red deer (Cervus elaphus) whose habitat varies over a wide range of latitude, was performed. The oxygen-binding properties of the most common haemoglobin phenotype from the species living in Sardinia were examined with particular attention to the effect of pH, chloride, 2, 3-bisphosphoglycerate and temperature. Results indicate that red deer haemoglobin, like all haemoglobins from ruminants so far examined, is characterized by a low intrinsic oxygen affinity, with chloride being its main physiological modulator in vivo. The functional results and the low temperature sensitivity of the oxygen affinity are discussed in the light of the amino acid sequence of closely related ruminant haemoglobins.

Adaptation, Physiological↗

PubMed1999

Study of the antinociceptive action of the ethanolic extract and the triterpene 24-hydroxytormentic acid isolated from the stem bark of Ocotea suaveolens.

We describe here the antinociceptive action of the crude extract (CE), the chemical isolation and characterisation and preliminary pharmacological analysis of 24-hydroxytormentic acid, isolated from the stem bark of Ocotea suaveolens (Lauraceae). The CE given by i.p. or p.o. routes, 30 min and 1 h prior, produced significant inhibition of abdominal constrictions caused by acetic acid and also inhibited both phases of formalin-induced licking in mice. The antinociception caused by the CE, given by i.p. and p.o. routes, lasted up to 4 and 2h, respectively. When assessed in the hot-plate test, the CE was inactive. Its antinociceptive action was not associated with non-specific effects such as muscle relaxation or sedation. The antinociception of CE was not influenced by naloxone, L-arginine or DL-p-chlorophenylalanine methyl ester, when assessed against the formalin assay. The triterpene 24-hydroxytormentic acid, given i.p. 30 min before testing, produced significant, dose-related and equipotent antinociceptive action against both phases of formalin-induced licking in mice. These results demonstrate, for the first time, the occurrence of the triterpene 24-hydroxytormentic acid in the stem bark of Ocotea suaveolens, and show that the CE and 24-hydroxytormentic acid exhibit marked antinociception against the neurogenic and the inflamamtory algesic responses induced by formalin in mice. The mechanism by which this compound and CE produces antinociception still remains unclear, but is unlikely to involve the activation of opioid, nitric oxide or serotonin systems or non-specific peripheral or central depressant actions.

Analgesics↗

PubMed1998

Circe's haemoglobins, pig-human hybrids: functional characterization and structural considerations.

We report the isolation and the functional characterization of alpha and beta chains from pig (Sus scropha domesticus) haemoglobin, as well as of the pig-human hybrid haemoglobins, alpha2(h)beta2(p) and alpha2(p)beta2(h) (i.e. Circe's haemoglobins), obtained by mixing the purified alpha and beta pig chains respectively with the corresponding partner human chains. Their functional properties have been compared with those of both parental haemoglobins in order to obtain information on the role of the different subunits and of their inter-relationships, both at the structural and functional levels. The results indicate that the functional properties of both hybrids are closer to those of the parental haemoglobin that provides the beta chains, confirming the major role of the beta chains in determining the oxygen affinity and the modulation mechanisms of the tetrameric molecule. This is supported by the thermodynamic properties, since the very low DeltaH of oxygen binding that characterizes pig haemoglobin and the alpha2(h)beta2(p) hybrid haemoglobin may be taken as the reflection of specific structural properties of pig beta chain.

Amino Acids↗

PubMed1998

Thermodynamics of inositol hexakisphosphate interaction with human oxyhemoglobin.

The interaction of inositol hexakisphosphate (IHP) with oxygenated human adult hemoglobin (Hb) was investigated at 25 degreesC. The affinity of IHP for oxygenated Hb is strongly pH-dependent, and potentiometric measurements of proton uptake and release upon IHP addition have shown that over the range between pH 8.0 and pH 6.0 in oxygenated Hb there are three groups of residues that change their pKa values after IHP addition, likely because of their interaction with negative charges of the heterotropic effector. On the basis of previous calculations on the electrostatic properties of human Hb (Matthew, J. B., Hanania, G. I. H., and Gurd, F. R. N. (1979) Biochemistry 18, 1919-1928; Lee, A. W.-m., Karplus, M., Poyart, C., and Bursaux, E. (1988) Biochemistry 27, 1285-1301), two of these groups might be Val1beta and His143beta, which are located in the beta1beta2 dyad axis, where they have been also proposed to interact with 2,3-diphosphoglycerate, whereas the third group does not appear easily identifiable. Calorimetric measurements of the heat associated with IHP binding at different pH values over the same range indicate that IHP binding is mostly enthalpy-driven at pH < 7 and mostly entropy-driven at pH > 7.

Adult↗

PubMed1998

Glycated human hemoglobin (HbA1c): functional characteristics and molecular modeling studies.

A minor hemoglobin component of human red blood cell hemolysate, HbA1c, is the result of the non-enzymatic reaction of glucose with the alpha-amino groups of the valine residues at the N-terminus of the beta-chains of human hemoglobin. In this paper, the effect of protons, chloride and 2,3-diphosphoglycerate (DPG) on the functional properties of HbA1c has been investigated in some details. Moreover, the structural modifications induced on the native molecule by the sugar moieties, studied by computer modeling, do agree with the observed functional alterations. In particular, the functional results indicate that: (a) the low-affinity conformation (or T-state) of HbA1c is destabilized by the chemical modification per se; (b) the Bohr effect is reduced with respect to that of native HbA0; (c) the affinity of the T-state of HbA1c for 2,3-diphosphoglycerate is about 2.6 x lower than that of the corresponding conformational state of HbA0, while the R-state is less affected with, the affinity being 1.7 x lower. At the structural level, computer modeling studies show that the two sugar moieties are asymmetrically disposed within the 2,3-diphosphoglycerate binding site. In addition, molecular mechanics and dynamics calculations concerning the interaction with 2,3-diphosphoglycerate indicate that while in HbA0 the effector can assume two different stable orientations, in glycated Hb only one orientation is possible. All together, the results show that glycation of the Val 1 residues of both beta-chains does not impair the binding of DPG but imposes a different mode of binding by changing the internal geometry of the complex and the surface distribution of the positive electrostatic potential within the binding pocket.

Chemical Phenomena↗

PubMed1998

Predictive model for capillary electrophoretic peptide mobility in 2,2,2-trifluoroethanol-water solution.

Using capillary electrophoresis (CE) on a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da, the Stokes radii at different protonation stages and the acidic dissociation constants in water and in a 2,2,2-trifluoroethanol (TFE) water mixture (30% v/v) were determined. These results permitted us to establish separately the reliability of semiempirical models utilized for the prediction of peptide size and charge at different acidic pHapp (pHapp range: 2.00-4.25). The data obtained on size and charge were utilized in order to provide suitable mobility predictions on the basis of the charge-to-size ratio. The best predictive conditions for size and charge were found at the most acidic range of pHapp studied (2.00-2.25), either in water or a TFE-water mixture, and reliable predictive equations for peptide mobility were established at this pHapp.

Electrophoresis, Capillary↗