Biomedical subjects

F Bossa

Publications and source records attributed to F Bossa.

At least 19 recordsLinked to original sources

PubMed2003

Stability and oligomerization of recombinant GadX, a transcriptional activator of the Escherichia coli glutamate decarboxylase system.

One of the most important strategies that enteric bacteria adopt for maintaining the cytoplasmic pH neutral under acid stress involves the glutamate decarboxylase (Gad) system. The system works by the concerted action of a cytoplasmic, pyridoxal 5'-phosphate-dependent glutamate decarboxylase and a transmembrane antiporter, which imports glutamate and exports gamma-aminobutyrate (GABA), the decarboxylation product, thereby providing local buffering of the extracellular environment. Herein, we provide a preliminary biochemical characterization of GadX, an activator of the Gad system belonging to the AraC/XylS family of bacterial transcriptional regulators. The GadX protein has been purified as a chimeric MalE-GadX with a yield of 15-20 mg/l of bacterial culture. The fusion protein is fairly stable, although a conformational change occurs upon storage, which reduces the binding affinity by a factor of 2, without affecting the binding pattern. Partial removal of the MalE moiety from the fusion protein triggers the formation of a species which is likely to be a heterodimer, or a higher oligomer, of the type GadX/MalE-GadX. This experimental evidence is in line with the well-known tendency of AraC/XylS-like proteins to dimerize via their N-terminal domain.

AraC Transcription Factor↗

PubMed2003

Colorectal cancer and high grade dysplasia complicating ulcerative colitis in Italy. A retrospective co-operative IG-IBD study.

BACKGROUND: Ulcerative colitis is a well-known risk factor for colorectal cancer. AIM: To take a census of the cases of colorectal cancer in ulcerative colitis patients observed in Italy and to evaluate the clinical presentation of neoplastic complication. PATIENTS AND METHODS: Experts from 28 Italian centres specialised in the management of inflammatory bowel disease or malignancies participated to the study. They were invited to send clinical data of patients with ulcerative colitis complicated by colorectal cancer or high-grade dysplasia consecutively observed between 1985 and 2000. One hundred and twelve patients (92 with cancer and 20 with high-grade dysplasia) were collected. Fourteen of them had undergone colectomy and ileo-rectal anastomosis for ulcerative colitis. Data of surgical patients were analysed separately. RESULTS: The mean age at diagnosis of ulcerative colitis and colorectal cancer patients was 39.3 and 53.2 years, respectively, and the mean duration between diagnosis of ulcerative colitis and cancer was 13.9 years (range 0-53). Inflammation was proximal to the splenic flexure in 71 cases (76.3%). One hundred and three colorectal cancers were registered (93 patients with single lesion and five patients with two synchronous cancers), with 76.7% of cancers being located in the left colon. As to the surgical patients, the mean age at diagnosis of ulcerative colitis and cancer was 28.9 and 47.0 years, respectively, and the mean diagnostic interval for ulcerative colitis and cancer was 18.1 years. Only 51 out of 112 patients were in follow-up. An early diagnosis of neoplasia (high grade dysplasia, stage A or B sec. Dukes) occurred in 72.5% of patients who were subjected to endoscopic surveillance and in 48.0% of patients who did not undergo endoscopic surveillance (p=0.02). CONCLUSIONS: These data show an earlier diagnosis of cancer in patients who had undergone endoscopic surveillance. The poor compliance to the follow-up program, however, reduces its effectiveness. Moreover, total colectomy allows an easier follow-up, with only the rectum being controlled. Colectomy with ileo-rectal anastomosis or proctocolectomy with ileo-anal anastomosis, could represent a valid alternative in patients at high risk of cancer who refuse endoscopic surveillance.

Adolescent↗

PubMed2001

l-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase. A subgroup of strictly related enzymes specialized for different functions.

Serine hydroxymethyltransferase (SHMT) is a member of the fold type I family of vitamin B6-dependent enzymes, a group of evolutionarily related proteins that share the same overall fold. The reaction catalysed by SHMT, the transfer of Cbeta of serine to tetrahydropteroylglutamate (H4PteGlu), represents in the cell an important link between the breakdown of amino acids and the metabolism of folates. In the absence of H4PteGlu and when presented with appropriate substrate analogues, SHMT shows a broad range of reaction specificity, being able to catalyse at appreciable rates retroaldol cleavage, racemase, aminotransferase and decarboxylase reactions. This apparent lack of specificity is probably a consequence of the particular catalytic apparatus evolved by SHMT. An interesting question is whether other fold type I members that normally catalyse the reactions which for SHMT could be considered as 'forced errors', may be close relatives of this enzyme and have a catalytic apparatus with the same basic features. As shown in this study, l-threonine aldolase from Escherichia coli is able to catalyse the same range of reactions catalysed by SHMT, with the exception of the serine hydroxymethyltransferase reaction. This observation strongly suggests that SHMT and l-threonine aldolase are closely related enzymes specialized for different functions. An evolutionary analysis of the fold type I enzymes revealed that SHMT and l-threonine aldolase may actually belong to a subgroup of closely related proteins; fungal alanine racemase, an extremely close relative of l-threonine aldolase, also appears to be a member of the same subgroup. The construction of three-dimensional homology models of l-threonine aldolase from E. coli and alanine racemase from Cochliobolus carbonum, and their comparison with the SHMT crystal structure, indicated how the tetrahydrofolate binding site might have evolved and offered a starting point for further investigations.

Alanine Racemase↗

PubMed2001

[Extended total gastrectomy: indications in the 3rd millennium].

BACKGROUND: Total extended gastrectomy (TEG) is indicated in the treatment of gastric cancer for necessity or to achieve an oncologic radicality. By this surgical treatment the stomach and other organs or a part of them involved by primitive tumor are removed. METHODS: The authors report a study about 15 patients, out of 116 cases of gastric cancer, operated by TEG between 1990-1998. The middle-age of this patients was 63 years (range 45-76) and their general conditions were good in 9 cases and not-good in 6. The postoperative total parenteral nutrition (TPN) was carried out in all the patients, while preoperatively only in the most compromised patients. The surgical treatments were: 2 TG (total gastrectomy)+splenecomy; 3 TG+splenectomy+pancreatic resection; 4 TG+splenectomy+pancreatic resection+distal esophageal resection; 1 TG+distal esophageal resection; 2 TG+atypic hepatic resection; 1 TG+ atypic hepatic resection+duodenum resection; 2 TG+large intestine resection. While 10 patients were operated on to obtain radicality, 5 patients had a palliative treatment. RESULTS: There was not perioperative mortality, but we have observed: one dehiscence of the duodenal stump and one pancreatic fistula treated with conservative therapy; one left subfrenic abscess treated with surgical therapy. The survival has been higher in the patients treated with radicality. On the basis of these cases, the authors consider: 1) the possibility to obtain radicality by TEG; 2) the gastric localizations more often associated to extravisceral neoplastic localization; 3) the role of extensive lymph node resection (III and IV level) to obtain oncological radicality or neoplastic reduction. CONCLUSIONS. On the basis of their personal experience and related literature, the authors conclude that TEG is indicated to: 1) obtain a better lymphadenectomy; 2) obtain an oncologic radicality; 3) reduce the neoplastic mass in order to facilitate adjuvant therapy; 4) avoid or treat neoplastic complications; 5) improve the quality of life.

Aged↗

PubMed2000

Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase.

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.

Animals↗

PubMed2000

The contribution of a conformationally mobile, active site loop to the reaction catalyzed by glutamate semialdehyde aminomutase.

The behavior of glutamate semialdehyde aminomutase, the enzyme that produces 4-aminolevulinate for tetrapyrrole synthesis in plants and bacteria, is markedly affected by the extent to which the central intermediate in the reaction, 4,5-diaminovalerate, is allowed to dissociate. The kinetic properties of the wild-type enzyme are compared with those of a mutant form in which a flexible loop, that reversibly plugs the entrance to the active site, has been deleted by site-directed mutagenesis. The deletion has three effects. The dissociation constant for diaminovalerate is increased approximately 100-fold. The catalytic efficiency of the enzyme, measured as k(cat)/K(m) in the presence of saturating concentrations of diaminovalerate, is lowered 30-fold to 2.1 mM(-1) s(-1). During the course of the reaction, which begins with the enzyme in its pyridoxamine form, the mutant enzyme undergoes absorbance changes not seen with the wild-type enzyme under the same conditions. These are proposed to be due to abortive complex formation between the pyridoxal form of the enzyme (formed by dissociation of diaminovalerate) and glutamate semialdehyde itself.

Amino Acids, Diamino↗

PubMed1999

Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase.

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively. Asn(5) is deamidated equally to Asp and isoAsp, while Asn(220) is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted in the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 50-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t(1/2) for deamidation of Asn(220) was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn(5) and Asn(220) are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.

Amides↗

PubMed1999

Purification of a 76-kDa iron-binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin.

A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 ml of seminal plasma. Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate. It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin. When tested for its iron binding capacity, with Fe-NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm.

Amino Acid Sequence↗

PubMed1999

The response to stationary-phase stress conditions in Escherichia coli: role and regulation of the glutamic acid decarboxylase system.

Inducible bacterial amino acid decarboxylases are expressed at the end of active cell division to counteract acidification of the extracellular environment during fermentative growth. It has been proposed that acid resistance in some enteric bacteria strictly relies on a glutamic acid-dependent system. The Escherichia coli chromosome contains distinct genes encoding two biochemically identical isoforms of glutamic acid decarboxylase, GadA and GadB. The gadC gene, located downstream of gadB, has been proposed to encode a putative antiporter implicated in the export of gamma-aminobutyrate, the glutamic acid decarboxylation product. In the present work, we provide in vivo evidence that gadC is co-transcribed with gadB and that the functional glutamic acid-dependent system requires the activities of both GadA/B and GadC. We also found that expression of gad genes is positively regulated by acidic shock, salt stress and stationary growth phase. Mutations in hns, the gene for the histone-like protein H-NS, cause derepressed expression of the gad genes, whereas the rpoS mutation abrogates gad transcription even in the hns background. According to our results, the master regulators H-NS and RpoS are hierarchically involved in the transcriptional control of gad expression: H-NS prevents gad expression during the exponential growth whereas the alternative sigma factor RpoS relieves H-NS repression during the stationary phase, directly or indirectly accounting for transcription of gad genes.

Acids↗

PubMed1998

Crystallization and preliminary X-ray analysis of the beta-isoform of glutamate decarboxylase from Escherichia coli.

Glutamate decarboxylase (GAD) is a vitamin B6 enzyme which catalyzes the alpha-decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Escherichia coli cells coexpress two highly homologous enzyme isoforms, GADalpha and GADbeta. Well diffracting crystals of GADbeta were obtained by taking advantage of the possibility of expressing each isoform separately. They belong to space group P31 or P32 with the unit-cell dimensions a = b = 115.6 and c = 206.6 A and contain one GAD hexamer in the asymmetric unit. High-resolution synchrotron data were collected at 100 K for the native protein and a potential heavy-atom derivative.

Bacterial Proteins↗

PubMed1998

Easy method to predict solvent accessibility from multiple protein sequence alignments.

An easy and uncomplicated method to predict the solvent accessibility state of a site in a multiple protein sequence alignment is described. The approach is based on amino acid exchange and compositional preference matrices for each of three accessibility states: buried, exposed, and intermediate. Calculations utilized a modified version of the 3D_ali databank, a collection of multiple sequence alignments anchored through protein tertiary structural superpositions. The technique achieves the same accuracy as much more complex methods and thus provides such advantages as computational affordability, facile updating, and easily understood residue substitution patterns useful to biochemists involved in protein engineering, design, and structural prediction. The program is available from the authors; and, due to its simplicity, the algorithm can be readily implemented on any system. For a given alignment site, a hand calculation can yield a comparative prediction.

Amino Acid Sequence↗

PubMed1998

The roles of His-167 and His-275 in the reaction catalyzed by glutamate decarboxylase from Escherichia coli.

Two histidine residues in glutamate decarboxylase from Escherichia coli, potential participants in catalysis because they are conserved among amino acid decarboxylases and because they are at the active site in the homologous enzyme ornithine decarboxylase, were mutated. His-275 is shown to bind the cofactor pyridoxal 5'-phosphate but not to contribute directly to catalysis. The H275N enzyme was unable to bind the cofactor whereas the H275Q mutant contained 50% of the normal complement of cofactor and its specific activity (expressed per mole of cofactor) was 70% of that of the wild-type enzyme. The H167N mutant bound the cofactor tightly, its specific activity was approximately half that of the wild-type enzyme and experiments in D2O showed that it catalyzed replacement of the carboxyl group with retention of configuration as does the wild-type enzyme. Comparison of reaction profiles by observing changes in the absorbance of the cofactor after stopped-flow mixing, revealed that a slow reaction, in which approximately one-third of the wild-type enzyme is converted to an unreactive complex during catalysis, does not occur with the H167N mutant enzyme. This reaction is attributed to a substrate-induced conformational change, a proposal that is supported by differential scanning calorimetry.

Amino Acid Sequence↗

PubMed1998

The structure of serine hydroxymethyltransferase as modeled by homology and validated by site-directed mutagenesis.

We describe a model for the three-dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of the alpha-family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of site-directed mutants. New active site features are also proposed for further experimental testing.

Amino Acid Sequence↗

PubMed1998

Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase.

A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked by 12 bp at the 5' end and 287 bp at the 3' end. The full-length cDNA was then cloned into a pET22b vector as a NdeI-EcoRI insert. HMS174(DE3) cells were transformed with this plasmid and, after induction with isopropyl beta-D-thiogalactopyranoside, expressed a catalytically active serine hydroxymethyltransferase. The enzyme was purified and shown to be the expressed rabbit enzyme lacking the first methionine residue. Spectral characteristics of the bound pyridoxal phosphate and kinetic constants for the natural substrates L-serine and tetrahydrofolate were essentially identical to the values obtained previously for the rabbit cytosolic enzyme. The pattern of bands shown by the pure recombinant enzyme on an isoelectric focusing gel containing 6 M urea showed a major band and a minor band representing about 15-20% of the protein. Upon incubation of the recombinant enzyme at pH 7.3 and 37 degreesC, three new bands were observed on isoelectric focusing with the concomitant formation of isoaspartyl residues, as determined by reactivity with protein isoaspartyl methyltransferase. These results are consistent with deamidation of Asn residues to isoaspartyl during the in vitro incubation. The enzyme purified from rabbit liver has previously been shown to contain isoaspartyl residues.

Animals↗

PubMed1998

Characterization and analysis of D-amino acids.

The need to screen a large number of natural extracts, with the aim of detecting D-amino acids or isolating and characterizing peptides containing them, has stimulated the development of novel and improved procedures for the analysis of amino acid enantiomeric mixtures, with special attention paid to automation. Different methods for the analysis of D-amino acids are described and discussed.

Amino Acids↗

PubMed1997

Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis.

The cytosolic and mitochondrial alcohol dehydrogenases from Kluyveromyces lactis (KlADHs) were purified and characterised. Both the N-terminally blocked cytosolic isozymes, KlADH I and KlADH II, were strictly NAD-dependent and exhibited catalytic properties similar to those previously reported for other yeast ADHs. Conversely, the mitochondrial isozymes, KlADH III and KlADH IV, displayed Ala and Asn, respectively, as N-termini and were able to oxidise at an increased rate primary alcohols with aliphatic chains longer than ethanol, such as propanol, butanol, pentanol and hexanol. Interestingly, the mitochondrial KlADHs, at variance with cytosolic isozymes and the majority of ADHs from other sources, were capable of accepting as a cofactor, and in some case almost equally well, either NAD or NADP. Since Asp-223 of horse liver ADH, thought to be responsible for the selection of NAD as coenzyme, is strictly conserved in all the KlADH isozymes, this amino-acid residue should not be considered critical for the coenzyme discrimination with respect to the other residues lining the coenzyme binding pocket of the mitochondrial isozymes. The relatively low specificity of the mitochondrial KlADHs both toward the alcohols and the cofactor could be explained on the basis of an enhanced flexibility of the corresponding catalytic pockets. An involvement of the mitochondrial KlADH isozymes in the physiological reoxidation of the cytosolic NADPH was also hypothesized. Moreover, both cytosolic and KlADH IV isozymes have an additional cysteine, not involved in zinc binding, that could be responsible for the increased activity in the presence of 2-mercaptoethanol.

Alcohol Dehydrogenase↗

PubMed1997

Stability of aspartate aminotransferase from Sulfolobus solfataricus.

Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is an extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C. The structural stability of this protein was investigated by coupling isothermally and thermally induced denaturation studies to molecular modeling. Gel filtration analysis indicated that SsAspAT unfolds with an N2 reversible 2D mechanism. In the molecular model, a cluster of hydrophobic residues was shown at the interface between the subunits of SsAspAT and suggested this cluster as a structural feature stabilizing the enzyme quaternary structure. At 25 degrees C, SsAspAT is less resistant to guanidinium chloride-induced denaturation than the cytosolic aspartate aminotransferase from pig heart (cpAspAT), which was chosen as a mesophilic counterpart in the thermodynamic analysis since it shares with SsAspAT the two-state unfolding mechanism. Therefore, in the case of aspartate aminotransferases, thermal stability does not correlate with the stability against chemical denaturants. Isothermal denaturation curves at 25 degrees C and melting profiles recorded in the presence of guanidinium chloride showed that the delta G degrees (H2O) at 25 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; the parameter delta n, related to the number of binding sites for the denaturant differentially exposed in unfolded and folded states, is higher for SsAspAT than for cpAspAT; and delta Cp is lower for the thermophilic enzyme than for the mesophilic one by 8 kJ/K.mol. These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT. In agreement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network.

Amino Acid Sequence↗

PubMed1997

Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.

Archaeal Proteins↗