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Biomedical subjects

F Palma

Publications and source records attributed to F Palma.

At least 19 recordsLinked to original sources

Rheological characterization of an artificial synovial fluid.

Rheological measurements on two classes of artificial synovial fluids have been carried out in the attempt to get a suitable but cheap lubricant for wear tests of prosthetic materials. Fluids of both classes are solutions of hyaluronic acid (HA) that, for one class, is dissolved into a simple Ringer solution whereas, for the other class, into a mixture of human serum and Ringer solution. Similar rheological properties have been observed for both classes of fluids. Experimental results have been interpreted by two classical models that are commonly used in the literature to describe the rheological behavior of colloidal systems and of polymer solutions with high entanglement density, respectively. The quality of correlations shows that, at high HA concentrations, entangled structures are largely present and cannot be neglected.

Biocompatible Materials↗

Characterization of the Tuber borchii nitrate reductase gene and its role in ectomycorrhizae.

The nitrate assimilation pathway represents a useful model system in which to study the contribution of a mycorrhizal fungus to the nitrogen nutrition of its host plant. In the present work we cloned and characterized the nitrate reductase gene (tbnr1) from Tuber borchii. The coding region of tbnr1 is 2,787 nt in length, and it encodes a protein of 929 amino acids. Biochemical and Northern-blot analyses revealed that nitrate assimilation in T. borchii is an inducible system that responds mainly to nitrate. Furthermore, we cloned a nitrate reductase cDNA (tpnr1) from Tilia platyphyllos to set up a quantitative real-time PCR assay that would allow us to determine the fungal contribution to nitrate assimilation in ectomycorrhizal tissue. Using this approach we demonstrated that the level of tbnr1 expression in ectomycorhizae is eight times higher than in free-living mycelia, whereas tpnr1 transcription was found to be down-regulated after the establishment of the symbiosis. Enzymatic assays showed that NADPH-dependent nitrite formation markedly increases in ectomycorrhizae. These findings imply that the fungal partner plays a fundamental role in nitrate assimilation by ectomycorrhizae. Amino acid determination by HPLC revealed higher levels of glutamate, glutamine and asparagine in symbiotic tissues compared with mycelial controls, thus suggesting that these amino acids may represent the compounds that serve to transfer nitrogen to the host plant.

Amino Acid Sequence↗

Identification of differentially expressed cDNA clones in Tilia platyphyllos-Tuber borchii ectomycorrhizae using a differential screening approach.

No information is presently available on the molecular mechanisms that control the morphogenesis of the truffle, an ectomycorrhizal ascomycetous fungus of great economic interest not only for forestry and agronomy but also for the organoleptic properties of its hypogeous fruitbodies. A Tilia platyphyllos- Tuber borchii model system was used in order to identify genes induced or up-regulated during symbiosis, since their isolation is a prerequisite for the understanding of the molecular bases of mycorrhizal development and regulation. The strategy applied involved the construction of an ectomycorrhizal cDNA library and random selection of clones, followed by a differential screening procedure to analyse cDNA expression in uninfected roots, ectomycorrhizae and free-living mycelia. The results revealed that many genes - and more plant genes than fungal genes - are expressed at higher levels during the symbiotic phase. Several clones were also investigated in order to understand their biological function. This study represents the first attempt to extend our knowledge of the molecular mechanisms underlying the establishment of ectomycorrhiza in Tuber species.

Ascomycota↗

Cloning, expression, and characterization of the hxk-1 Gene from the white truffle Tuber borchii vittad.: A first step toward understanding sugar metabolism.

Recent biochemical investigations of Tuber borchii Vittad. mycelium have demonstrated the presence of three distinct forms of hexokinase (HK(M1), HK(M2), and HKM3). In the investigation described here, a gene coding for hexokinase (hxk-1) from T. borchii was isolated and characterized. The hxk-1 gene is characterized by an ORF of 1494 nucleotides and codes for a polypeptide of 497 aa. The gene was overexpressed in Escherichia coli, and the recombinant protein was kinetically characterized. The K(cat) value for fructose is in agreement with the data reported for the hexokinase of Yarrowia lipolytica, the Km for ATP is not dependent on the sugar used, and the enzyme is not inhibited by trehalose 6-phosphate or glucose 6-phosphate. The biochemical characteristics confirm that this enzyme is a hexokinase, as suggested by the Pileup results, and it corresponds to the HKM1 isoform. This work represents the first characterization of the key enzyme of the glycolytic pathway and the related gene in a Tuber species.

Amino Acid Sequence↗

One-step purification of a fully active hexahistidine-tagged human hexokinase type I overexpressed in Escherichia coli.

The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa. Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate. In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions. In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg. The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation.

Chromatography, Affinity↗

Recipient factors as determinants of mortality after adult liver transplantation.

The factors that can influence the outcome of orthotopic liver transplantation (OLT) are numerous. The purpose of this study was to determine the effects of recipient preoperative factors on patient mortality. Between April 1986 and April 1998 a total of 600 OLTs were performed in our institution. We retrospectively reviewed our first 203 consecutive primary adult OLTs with at least 4 years of follow-up. A case-control comparison was performed between survivors and nonsurvivors, and differences in recipient variables were studied for their correlation with patient mortality. A logistic regression analysis was also performed. Mortality was significantly increased among those with fulminant hepatic failure (FHF) (66.6%, p = 0.003), primary cancer (63.1%, p = 0.018), females (46.1%, p = 0. 043), encephalopathy grade IV (72.7%, p = 0.012), recipients under respiratory support (69.2%, p = 0.031), and ABO-incompatible transplants (80%, p = 0.05). FHF, primary cancer, and female gender were the only variables that had a significant association with mortality in the logistic regression analysis. A higher incidence of prolonged respiratory support, bacterial and fungal infections, pneumonia, and chronic rejection contributed to the lower outcome observed in females. These results stress the need for continuous evaluation of the selection criteria of candidates for OLT suffering from primary cancer and FHF. The impact of recipient gender on mortality warrants further analysis but suggests that in the future more attention must be paid to the influence of this factor on the final outcome of OLT.

Adult↗

Expression, purification, and characterization of a recombinant erythroid-specific hexokinase isozyme.

Hexokinase type I (HK I; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the predominant glucose-phosphorylating enzyme in red blood cells, exists in human erythrocytes in multiple molecular forms that differ in isoelectric point and are separable by ion-exchange chromatography. The major forms, designated HK Ia, Ib and Ic, have similar kinetic properties but are characterized by different age-dependent decay and different intracellular distribution in reticulocytes. HK Ib, which elutes between HK I and HK II in the DEAE ion-exchange chromatography, appears to be unique to RBCs and different from any other hexokinase isozyme previously described. Indeed, Murakami and Piomelli recently reported the presence of a specific HK isozyme (named HKr) expressed in K562 cells and in human reticulocytes and, moreover, the resolution of the human HK I gene structure provided the direct evidence of an erythroid-specific exon 1. To further investigate the microheterogeneity of HK I in human RBCs we established a prokaryotic expression system for the HKr isozyme, using the pET plasmid, inducible with IPTG. The recombinant HKr, expressed in bacterial cells as a catalytically active enzyme, was purified to homogeneity by a combination of DEAE ionexchange chromatography followed by hydrophobic interaction chromatography and dye-ligand affinity chromatography. The kinetic and chromatographic properties of the homogeneous recombinant HKr suggest that this erythroid-specific HK isozyme in fact corresponds to the HK isoform previously described in human RBCs and referred to as HK Ib.

Erythrocytes↗

Comparative study of the cytoplasmic domain of band 3 from human and rabbit erythrocyte membranes.

The cytoplasmic domain of band 3 (cdb3) is thought to play an important role in human erythrocyte aging. In order to investigate the role of cdb3 during rabbit erythrocyte aging, we compared rabbit cdb3 with the corresponding protein from human erythrocyte membranes. We describe a purification procedure for rabbit cdb3 comparing rabbit and human cdb3 on sodium dodecyl sulphate-polyacrylamide electrophoresis, we found fragments of different molecular weights, implying different chymotryptic cuts in the two species. Anti-human antibodies did not bind to the rabbit cdb3; we also noticed structural differences in the protein between the two species, which may also play a role in the aging processes. Rabbit erythrocyte membranes have a higher capacity of binding hemichromes, due to the higher content of band 3. While in rabbit erythrocyte membranes only one binding site for hemichromes (corresponding to cdb3) was found, we confirmed the existence of two binding sites in human membranes. The second binding site probably corresponds to glycophorin, a protein not present in rabbit membranes.

Animals↗

Role of dehydroascorbate in rabbit erythrocyte hexokinase inactivation induced by ascorbic acid/Fe(II).

In this study we investigated the species involved in the process of hexokinase inactivation induced by ascorbic acid/Fe(II) in rabbit erythrocytes. Our results suggest a model in which divalent iron is first oxidized to the trivalent state and then triggers the oxidation of ascorbic acid. The H202 formed during this process accelerates the formation of dehydroascorbic acid, which appears to be necessary and sufficient to induce hexokinase inactivation. This model was validated by showing that: (a) H202-decomposing enzymes, unlike scavengers of the hydroxyl radicals, reduced the extent of hexokinase inactivation; (b) when H202 was used instead of ascorbate/Fe(II), it was unable, even at very high concentrations, to inhibit hexokinase activity; (c) replacing Fe(II) with either Fe(III) or H202 resulted in comparable levels of ascorbic acid-induced hexokinase inactivation; (d) expression of maximal hexokinase inhibiting activity was also triggered via enzyme-catalyzed oxidation of ascorbic acid or direct addition of dehydroascorbic acid; (e) the level of dehydroascorbic acid, which was actively generated in the external medium upon addition of ascorbic acid/Fe(II), increased as a function of time. Taken together, these results demonstrate that the process of hexokinase inactivation induced by ascorbic acid/Fe(II) is mediated by dehydroascorbate and that iron and H202 have the sole function of accelerating its formation.

Animals↗

Purification and characterization of the carboxyl-domain of human hexokinase type III expressed as fusion protein.

In mammalian tissues hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) exists as four isoenzymes encoded by distinct genes. These proteins are homologous and are organized in two homologous domains, with the exception of hexokinase type IV which has only one. This organization is believed to be the result of a duplication and tandem fusion event involving the gene encoding for the ancestral hexokinase. In this study, we cloned the carboxyl-domain of human hexokinase type III and expressed it in Escherichia coli as a glutathione S-transferase fusion protein, using the pGEX-2T expression vector. The recombinant protein showed catalytic activity. A comparative study of the kinetic properties of the expressed carboxyl-domain and the enzyme partially purified from human lymphocytes is also shown. The results now allow a better understanding of the role of the carboxyl-domain in determining the catalytic properties of the enzyme.

Amino Acid Sequence↗

High-performance capillary electrophoretic separation of proteins and peptides using a bonded hydrophilic phase capillary.

High performance capillary electrophoresis (HPCE) was applied to the separation of protein and peptide mixtures with molecular masses ranging from 1300 to 96000 Da using a new bonded hydrophilic phase capillary, CElect-P150. This coated capillary reduces the interaction between proteins and silanol groups in capillary walls, allowing a complete recovery of the proteins and peptides of interest. HPCE was also used for the analysis of a complex mixture of tryptic fragments and to monitor the process of enzymatic digestion. Moreover, using a CElect-P150 capillary, highly reproducible analysis was possible without preconditioning the capillary with acid or basic solutions before each new analysis.

Animals↗

Mitochondria-bound hexokinase from rabbit reticulocytes is resistant to the inactivation induced by Fe(II)/ascorbate.

Exposure of rabbit reticulocytes to Fe(II)/ascorbate induced a pronounced decay in hexokinase activity. In reticulocytes, this enzyme is present in at least three different molecular forms, Ia, Ia* and Ib, sub-types of hexokinase type I, which show different intracellular distribution. Hexokinase Ia and Ib are soluble, whereas hexokinase Ia* is almost entirely bound to the mitochondria. Anion exchange chromatography of hexokinase from intact reticulocytes exposed to Fe(II)/ascorbate revealed a selective inactivation of forms Ia and Ib, whereas the form Ia* did not show any decay. Binding to the mitochondrial membrane seems to be responsible for the observed resistance of the form Ia* to the inactivation elicited by Fe(II)/ascorbate. Indeed, by using a cell-free system in which hexokinase Ia* was solubilized using Triton X-100, the decay in hexokinase activity induced by iron/ascorbate involved all three enzymatic forms.

Animals↗

Simultaneous high-performance capillary electrophoretic determination of reduced and oxidized glutathione in red blood cells in the femtomole range.

This paper describes a high-performance capillary electrophoretic (HPCE) method which allows a quick, simultaneous and quantitative determination of reduced (GSH) and oxidized (GSSG) glutathione in mammalian red blood cells using a Supelco-bonded hydrophilic phase capillary CElect-P150. The extraction procedure of GSH and GSSG from erythrocytes using Microcon-10 membranes is very simple and allows a correct evaluation of these compounds present in the red blood cells. Furthermore, the HPCE method does not require removal of the excess N-ethylmaleimide used to block the glutathione in its reduced state, making the simultaneous evaluation of GSH and GSSG possible in a very short time (ca. 4 min), with a sensitivity at femtomole level.

Animals↗

Inactivation of rabbit red blood cell hexokinase activity promoted in vitro by an oxygen-radical-generating system.

Rabbit red blood cell hexokinase (EC 2.7.1.1) has been shown to be inactivated in vitro by incubating intact erythrocytes in the presence of an oxygen-radical-generating system represented by ascorbate and iron. It was interesting to note that among the glycolytic enzymes, only hexokinase was found to be susceptible to the action of oxygen radicals, suggesting that the loss of activity of this enzyme may be one of the first signals of cellular damage in rabbit red blood cells. This statement is supported by the fact that, under the experimental conditions used, we did not observe any significant plasma membrane lipid peroxidation nor intracellular proteolysis. Furthermore, mature erythrocytes are unable to synthesize hexokinase as well as other proteins de novo; therefore, the inactivation of this enzyme, which is both the first and one of the rate-limiting enzymes of the glycolytic pathway, could play an important role in determining metabolic impairment of red blood cells, with possible physiological implications. We also investigated the effect of various radical scavengers and antioxidants (glucose, vitamin E, dithiothreitol, flavonoids) which are able to influence the inactivation of hexokinase activity to different extents. Finally, under the experimental conditions used (90 min of incubation at 37 degrees C), we did not observe any difference in the hemolysis of rabbit red blood cells incubated in the presence or absence of ascorbate and iron (hemolysis was about 1% after 90 min of incubation), suggesting that the system used was able to furnish information about the cellular damage produced by oxygen radicals without provoking cell lysis.

Adenosine Triphosphatases↗