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Biomedical subjects

A Raggi

Publications and source records attributed to A Raggi.

At least 19 recordsLinked to original sources
PubMed2004

A calpain-like proteolytic activity produces the limited cleavage at the N-terminal regulatory domain of rabbit skeletal muscle AMP deaminase: evidence of a protective molecular mechanism.

On storage at 4 degrees C, rabbit skeletal muscle AMP deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.

AMP Deaminase↗
PubMed2003

Cytokine-regulated expression of collagenase-2 (MMP-8) is involved in the progression of ovarian cancer.

Matrix metalloproteinases (MMPs) have been implicated in ovarian cancer progression. Among them, MMP-8 that degrades type I collagen may play a crucial role. The aim of our study was to determine MMP-8 expression and regulation in ovarian cancer and its association with other MMPs and tissue inhibitors of metalloproteinases (TIMPs). Tissue microarrays (TMAs) containing tissue cylinders from 302 patients were used for immunohistochemical studies. In addition, MMP-8 expression in vitro was analysed by a specific immunoassay and PCR-analysis. MMP-7 (81%), MMP-8 (95%), MT3-MMP (100%), TIMP-2 (100%), and TIMP-3 (96%) were expressed in all the OVCAs, but the staining intensities varied. MMP-3 (6%), MMP-9 (57%) and TIMP-1 (43%) expressions were more rarely detected. Only MMP-8 expression levels correlated with tumour grade (P<0.01), tumour stage (P<0.01), and a poor prognosis (P<0.05). MMP-8 protein and gene expression in vitro was found to be significantly upregulated by interleukin-1beta (IL-1beta, P<0.01). The data indicate that MMP-8 overexpression in OVCAs is regulated by IL-1beta and that pro-inflammatory cytokines may promote the invasive potential of ovarian cancer.

Adult↗
PubMed2003

From durum wheat to pasta: effect of technological processing on the levels of arsenic, cadmium, lead and nickel--a pilot study.

A study was undertaken to investigate the effect of technological processing in pasta-making on the content of arsenic, cadmium, lead and nickel. Milling of durum wheat as well as further processing were carried out in a pilot plant. Commercial pasta samples purchased from the local market were also included for comparison. Furthermore, the effect of cooking was investigated to gain information on the actual content of the selected elements in the final ready-to-eat product. Analyte concentrations in whole grain, semolina, pasta and cooked pasta were determined by inductively coupled plasma-mass spectrometry. Milling reduced the element content in the order nickel > arsenic > cadmium > lead. In the experimental conditions of this study, slight contamination by atmospheric lead in milling/pasta-making and release of nickel during pasta-making were observed. These issues have evidently been effectively dealt with in industrial processing given that remarkably lower levels of lead and nickel were found in commercial pastas compared with the experimental samples. On the whole, commercial pasta samples showed low average levels of all the elements included in this study. Cooking caused a significant decrease of the element content in all pasta samples, with average losses of 50-60% on a dry weight basis.

Arsenic↗
PubMed2001

Regulation of skeletal muscle AMP deaminase: lysine residues are critical for the pH-dependent positive homotropic cooperativity behaviour of the rabbit enzyme.

Reaction of rabbit skeletal muscle AMP deaminase with a low molar excess of trinitrobenzene sulfonic acid (TNBS) results in conversion of the enzyme into a species with about six trinitrophenylated lysine residues per molecule which no longer manifests positive homotropic cooperativity at pH 7.1 or at the optimal pH value of 6.5 in the presence of low K+ concentrations. Substitution of the reactive thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) does not protect the enzyme from the TNBS-induced changes of the catalytic properties, indicating that cysteine residues modification is not at the basis of the effects of TNBS treatment on AMP deaminase and strongly suggesting the obligatory participation of lysine residues to the constitution of a regulatory anionic site to which AMP must bind to stimulate the enzyme at alkaline pH. The TNBS-treated enzyme is also completely desensitized to inhibition by ATP, but not to inhibition by GTP and stimulation by ADP. This observation suggests a connection between the operation of the hypothesized anionic activating site, responsible for positive homotropic cooperativity, and the inhibition exerted by anionic compounds that compete for the same site, among them the most efficient metabolite being probably ATP.

AMP Deaminase↗
PubMed2001

Influence of laboratory homogenization procedures on trace element content of food samples: an ICP-MS study on soft and durum wheat.

Sample contamination as a consequence of abrasion of grinding tools during the homogenization of food materials to be analysed for trace elements was addressed. The possible release of 15 trace elements (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, Sn, V, Zn) from six different grinding and milling devices, operating either continuously or discontinuously, was evaluated. All the devices were commercially available and were representative of models usually employed in food and agricultural laboratories. Wheat grains belonging to one soft and one durum cultivar were used as test material. The determination of the analyte concentrations in subsamples submitted to the different preparation treatments was performed by quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS). Accordingly, a suitable digestion method was developed and the ArC+ interference affecting Cr determination was evaluated and corrected. Statistical differences with respect to the control were detected for 10 elements (Al, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb) and in most cases contamination of the samples was traced back to the composition of the grinding equipment. None of the investigated devices was contamination-free with respect to all of the quantified elements. Abrasion of the grinding tools was higher with durum wheat than with soft wheat as a consequence of their different hardiness.

Analysis of Variance↗
PubMed1999

Presence in human skeletal muscle of an AMP deaminase-associated protein that reacts with an antibody to human plasma histidine-proline-rich glycoprotein.

Histidine-proline-rich glycoprotein (HPRG) is a protein that is synthesized by parenchimal liver cells. The protein has been implicated in a number of plasma-specific processes, including blood coagulation and fibrinolysis. We have recently reported the association of an HPRG-like protein with rabbit skeletal muscle AMP deaminase (AMPD). The results of the immunological analysis reported here demonstrate that an antibody against human plasma HPRG reacts with an AMPD preparation from human skeletal muscle. To probe the localization of the putative HPRG-like protein in human skeletal muscle, serial sections from frozen biopsy specimens were processed for immunohistochemical and histoenzymatic stains. A selective binding of the anti-HPRG antibody to Type IIB muscle fibers was detected, suggesting a preferential association of the novel protein to the AMPD isoenzyme contained in the fast-twitch glycolytic fibers.

AMP Deaminase↗
PubMed1997

Association of purified skeletal-muscle AMP deaminase with a histidine-proline-rich-glycoprotein-like molecule.

Denaturation of rabbit skeletal-muscle AMP deaminase in acidic medium followed by chromatography on DEAE-cellulose in 8 M urea atpH 8.0 allows separation of two main peptide components of similar apparent molecular mass (75-80 kDa) that we tentatively assume correspond to two different enzyme subunits. Whereas the amino acid composition of one of the two peptides is in good agreement with that derived from the nucleotide sequence of the known rat and human AMPD1 cDNAs, the second component shows much higher contents of proline, glycine and histidine. N-Terminal sequence analysis of the fragments liberated by limited proteolysis with trypsin of the novel peptide reveals a striking similarity to the fragments produced by plasmin cleavage of the rabbit plasma protein called histidine-proline-rich glycoprotein (HPRG). However, some divergence is observed between the sequence of one of the fragments liberated from AMP deaminase by a more extensive trypsinization and rabbit plasma HPRG in the region containing residues 472-477. A fragment with a blocked N-terminus, which was found among those liberated by proteolysis with pepsin of either whole AMP deaminase or the novel component of the enzyme, shows an amino acid composition quite different from that of the N-terminus of the known subunit of AMP deaminase. By coupling this observation with the detection in freshly prepared AMP deaminase of a low yield of the sequence (LTPTDX) corresponding to that of HPRG N-terminus, it can be deduced that in comparison with HPRG, the putative HPRG-like component of AMP deaminase contains an additional fragment with a blocked N-terminus, which is liberated by a proteolytic process during purification of the enzyme. The implications of the association to rabbit skeletal-muscle AMP deaminase of a HPRG-like protein species are discussed.

AMP Deaminase↗
PubMed1995

Regulation of skeletal-muscle AMP deaminase: involvement of histidine residues in the pH-dependent inhibition of the rabbit enzyme by ATP.

Reaction of rabbit skeletal-muscle AMP deaminase with a low molar excess of diethyl pyrocarbonate results in conversion of the enzyme into a species with one or two carbethoxylated histidine residues per subunit that retains sensitivity to ATP at pH 7.1 but, unlike the native enzyme, it is not sensitive to regulation by ATP at pH 6.5. This effect mimics that exerted on the enzyme by limited proteolysis with trypsin, which removes the 95-residue N-terminal region from the 80 kDa enzyme subunit. These observations suggest involvement of some histidine residues localized in the region HHEMQAHILH (residues 51-60) in the regulatory mechanism which stabilizes the binding of ATP to its inhibitory site at acidic pH. Carbethoxylation of two histidine residues per subunit abolishes the inhibition by ATP of the proteolysed enzyme at pH 7.1, suggesting the obligatory participation of a second class of histidine residues, localized in the 70 kDa subunit core, in the mechanism of the pH-dependent inhibition of the enzyme by ATP. At a slightly acidic pH, these histidine residues would be positively charged, resulting in a desensitized form of the enzyme similar to that obtained with the carbethoxylation reaction.

AMP Deaminase↗
PubMed1994

Evidence of a species-differentiated regulatory domain within the N-terminal region of skeletal muscle AMP deaminase.

Rabbit skeletal muscle AMP deaminase was submitted to limited proteolysis by trypsin that converts the native 80 kDa enzyme subunit to a stable product of approx. 70 kDa, which, in contrast to the native enzyme, is not sensitive to regulation by ATP at pH 6.5. Tryptic peptide mapping indicates that proteolysis is confined to the N-terminal region of the molecule, identifying in this region of AMP deaminase a non-catalytic, 95 residue regulatory domain that stabilises the binding of ATP to a distant site in the molecule. Protein sequence analysis reveals a marked degree of divergence between rat and rabbit skeletal muscle AMP deaminases in the regions containing residues 7-12 and 51-52, giving molecular basis to the hypothesis of the existence of isoenzymes of AMP deaminase in the mature skeletal muscle of the mammals.

AMP Deaminase↗
PubMed1990

Regulation of skeletal-muscle AMP deaminase. Evidence for a highly pH-dependent inhibition by ATP of the homogeneous derivative of the rabbit enzyme yielded by limited proteolysis.

Limited proteolysis of rabbit skeletal-muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) with trypsin results in conversion of the enzyme into a species which over the pH range 6.5-7.1 exhibits hyperbolic kinetics at low K+ concentration even in the absence of ADP, but shows a 20% decrease in activity at saturating substrate concentration. Analysis by sedimentation-equilibrium techniques reveals the proteolysed enzyme to be homogeneous and to have a molecular mass of 222,000 Da, indicative of a trimeric structure with a subunit molecular mass of 72,000 Da, in contrast with the tetrameric structure of the native enzyme, composed of four 79,000-Da subunits. These observations suggest a role of the 7,000-Da fragment which is removed by proteolysis in the maintenance of the three-dimensional structure of the subunit that causes the enzyme at low K+ concentration to show homotropic positive co-operativity. Study of the influence of pH, isolated from that of K+, on the kinetics of AMP deaminase reveals a highly pH-dependent inhibitory effect by ATP which is completely absent at acid pH values and abruptly manifests itself just above neutrality. This phenomenon may have significance in the metabolism of exercising muscle, in connection with the pH-dependent interaction of AMP deaminase with the thick filament.

AMP Deaminase↗
PubMed1989

Structure-function relationships in cardiac troponin T.

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.

Animals↗
PubMed1987

Regulatory properties of AMP deaminase isoenzymes from rabbit red muscle.

We examined the kinetic and regulatory properties of the two isoenzymes of red muscle AMP deaminase, forms A and B, corresponding respectively to the single isoenzymes present in the heart and white skeletal muscle. At the optimal pH value, 6.5, both enzymes show hyperbolic substrate-velocity curves and are inhibited by GTP, inducing sigmoid kinetics. An effect similar to that of GTP is exerted on form B by ATP, whereas form A is almost insensitive to this nucleotide. At pH 7.1 both enzymes follow sigmoid kinetics. ATP enhances the sigmoidicity of the substrate-velocity curve of form B, but it stimulates form A, reverting sigmoidal to hyperbolic kinetics shown by the enzyme at optimal pH. At pH 7.1, form A is also less sensitive to the inhibitory action of Pi and GTP. These results suggest that, owing to the presence of form A, AMP deamination occurs in red muscle also at moderate work intensity. A possible role of this process in counteracting the production of adenosine by 5'-nucleotidase is hypothesized.

AMP Deaminase↗
PubMed1985

Interaction with troponin T from white skeletal muscle restores in white skeletal muscle AMP deaminase those allosteric properties removed by limited proteolysis.

Limited proteolysis of rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) with trypsin results in conversion of the enzyme to a form which is no longer inhibited by ATP and exhibits hyperbolic kinetics even at low K+ concentration and in the absence of ADP. The interaction with troponin T from white skeletal muscle or with the phosphorylated 42-residue N-terminal peptide of troponin T restores in the trypsin-treated AMP deaminase the sensitivity to adenine nucleotides and increases the KA for K+ activation of the enzyme from 1 mM to 12 mM, this effect being diametrically opposite to that exerted by limited proteolysis on the native enzyme. Treatment of the N-terminal peptide of troponin T with alkaline phosphatase abolishes the modulating properties of the peptide, suggesting that phosphorylation-dephosphorylation processes may be involved in the regulation of the enzyme.

AMP Deaminase↗
PubMed1983

pH-dependent cold lability of rabbit skeletal muscle AMP deaminase.

Rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) at low temperature and pH value above 7 undergoes inactivation, with a half-time of the order of several minutes. The loss of activity becomes more extensive at lower enzyme concentrations and higher pH values. It is reversible, since cold-inactivated AMP deaminase can be reactivated by raising the temperature, but not by lowering the pH, of the incubation mixture. The residual activity at the end of the inactivation process at various temperatures, reflecting the equilibrium between active and inactive forms of the enzyme, has been studied as a function of pH to determine the apparent pK and heat of ionization of the process. A general mechanism of reversible inactivation of AMP deaminase is postulated which assumes that deprotonation of the enzyme is followed by isomerization to a form which at low temperature slowly dissociates into the less-active subunits. Cold-inactivated AMP deaminase no longer shows the pH-dependent sigmoidal behaviour of the native enzyme, but regains this property along with the reactivation process. This suggests that allosteric kinetics at basic pH are probably produced by the same isomerization process which is involved in the mechanism for cold lability of the enzyme.

AMP Deaminase↗
PubMed1981

Inactivation of rat muscle 5'-adenylate aminohydrolase by tyrosine nitration with tetranitromethane.

Reaction of rat muscle AMP deaminase with low molar excess of tetranitromethane results in a rapid loss of free thiol groups and a concomitant decrease in enzyme activity at high, but not at low, AMP concentration. This modification appears to be limited to the same non-essential thiol groups reactive towards specific reagents in non-denaturing conditions. On incubation with higher molar excess of tetranitromethane, a loss of enzyme activity is observed, which correlates with nitration of tyrosine residues. By amino acid analysis, approximately there tyrosine residues per subunit are estimated to be nitrated in the completely inactivated enzyme. The kinetic properties of the partially inactivated AMP deaminase reveal a negative co-operatively behaviour at approximately half saturation. This suggests that modification of tyrosine residues is also responsible for alteration of the binding properties of the hypothesized activating site of AMP deaminase.

AMP Deaminase↗
PubMed1979

Adenylate metabolism in the heart. Regulatory properties of rabbit cardiac adenylate deaminase.

The kinetic properties of a 300-fold purified cardiac AMP deaminase were studied and compared with those of the corresponding enzyme from skeletal muscle. The heart enzyme is activated by ATP and less efficiently by ADP, and is inhibited by Pi, phosphocreatine and GTP. ATP, even at micromolar concentrations, is able to abolish the effects of the inhibitors. The affinity of the enzyme for AMP is low in the absence of activators (Km 3.1 mM), but, in the presence of ATP, becomes as high as that of skeletal-muscle AMP deaminase (Km 0.4 mM). The maximal activation by ATP is observed at alkaline pH (pH 7.5-8.0). Under the same conditions ATP is maximally inhibitory for skeletal-muscle enzyme. These results suggest that AMP deaminase in the heart is always in the activated state, whereas in skeletal muscle the enzyme is active only during exhaustive contractions.

AMP Deaminase↗