Biomedical subjects

V Bocchini

Publications and source records attributed to V Bocchini.

At least 37 records · Page 2Linked to original sources

PubMed1997

Heterologous expression in Escherichia coli of the gene encoding an archaeal thermoacidophilic elongation factor 2. Properties of the recombinant protein.

The gene encoding the elongation factor 2 from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-2) was expressed in Escherichia coli using the pT7-7 expression vector. The synthesis of the heterologous product did not increase upon addition of isopropyl-beta-thiogalactopyranoside. The amount of purified intact recombinant SsEF-2 (SsEF-2rec) was about 3 mg from 60 g of transformed wet cells. Recombinant and naturally occurring SsEF-2 showed identical electrophoretic mobility, immunological properties and the N-terminal amino acid sequence; both were lacking the initial methionine. Differently from SsEF-2, SsEF-2rec did not undergo post-translational modification of His603 into diphthamide, as indicated by its inability to be ADP-ribosylated. SsEF-2rec appeared indistinguishable from SsEF-2 in the fulfillment of its biological functions; in fact, it was fully capable to support poly(Phe) synthesis, to bind GDP and to display either the intrinsic or the ribosome-dependent GTPase. Finally, SsEF-2rec was endowed with the same heat stability as SsEF-2. Altogether these findings proved that SsEF-2rec was functionally active as SsEF-2. The used expression system could allow to produce mutated forms of SsEF-2 obtained by mutagenesis of the corresponding gene.

Bacterial Proteins↗

PubMed1997

A time-dependent increase in glial fibrillary acidic protein expression and glutamine synthetase activity in long-term subculture of the GL15 glioma cell line.

1. Astrocytes are the most numerous cellular elements in the central nervous tissue, where they play a critical role in physiological and pathological events. The biological signals regulating astrocyte growth and differentiation are relevant for both physiology and pathology, but they are still little understood. 2. Using a poorly differentiated glioma cell line, GL15, we investigated whether, in long-term subculture, this could upregulate the expression of glial fibrillary acidic protein (GFAP), as described in some rodent astrocyte cell lines. Under the same culture conditions, we investigated glutamine synthetase (GS) activity, growth-associated protein (GAP)-43 expression, and expression of several neutrotrophic factors. 3. A dramatic increase in GFAP expression was evidenced by Western blotting during progressive in vitro growth of GL15 cells. GS specific activity was also upregulated in long-term culture. The time spent in vitro by GL15 cells did not affect GAP-43 and neutrophic factor BDNF and NT3 expression as revealed by RT-PCR analysis. 4. Our results suggest that, in GL15, GFAP and GS genes may have common or integrated regulatory mechanisms elicited at the cell confluency which could be relevant for both astrocyte physiology and astrocyte pathology. These mechanisms are not involved in GAP-43 and neutrophic factor BDNF and NT3 expression.

Astrocytes↗

PubMed1996

Protein-encoding genes in the sulfothermophilic archaea Sulfolobus and Pyrococcus.

A number of unrelated protein-encoding genes from sulfothermophilic archaea, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Pyrococcus furiosus and Pyrococcus woesei, has been analyzed. In the Sulfolobus genus, the content of A + T is significantly higher than that of C + G and the base usage follows the order, A > T > G > C. In Pyrococcus, the A + T content is also higher than that of C + G, but with lower values; in the order of base usage, G precedes T. The codon usage of these sulfothermophiles has been determined; alternative start codons are frequently used in both genera; codon preferences reflect the rich A + T composition of the corresponding genomes; for both genera the codon bias is particularly evident within the different arginine triplets, where AGA and AGG are predominant. From the similarities in the codon usage, close taxonomic relationships become evident within the Sulfolobus or the Pyrococcus genus; a lower, but significant similarity is also clear between these genera. The synonymous codon usage of these sulfothermophiles shows similarities with that of Saccharomyces cerevisiae and bovine mitochondria, whereas clear divergences are observed with the halophilic archaeal genus, Halobacterium, or the eubacterium, Escherichia coli. The unrelated proteins of the considered sulfothermophiles have been analyzed for the content of hydrophobic residues; the comparison with mesophiles reveals a significant increase in the average hydrophobicity of amino acid residues. This finding could indicate a mechanism of adaptation of proteins in organisms living under extreme environments. It is noteworthy that an opposite trend, i.e. a decreased average hydrophobicity, occurs in unrelated halophilic proteins.

Animals↗

PubMed1996

Archaeal elongation factor 1 beta is a dimer. Primary structure, molecular and biochemical properties.

The elongation factor 1 beta (EF-1 beta), that in eukarya and archaea promotes the replacement of GDP by GTP on the elongation factor 1 alpha x GDP complex, was purified to homogeneity from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1 beta). Its primary structure was established by sequenced Edman degradation of the entire protein or its proteolytic peptides. The molecular weight of SsEF-1 beta was estimated as about 10000 or 20000 under denaturing or native conditions respectively; this finding suggests that the native protein exists as a dimer. The peptide chain of SsEF-1 beta is much shorter than that of its eukaryotic analogues and homology is found only at their C-terminal region; no homology exists between SsEF-1 beta and eubacterial EF-Ts. At 50 degrees C, at a concentration of SsEF-1 beta 5-fold higher than that of SsEF-1 alpha x [3H]GDP the rate of the exchange of [3H]GDP for GTP becomes about 160-fold faster. An analysis of the values of the energetic parameters indicates that in the presence of SsEF-1 beta the GDP/GTP exchange is entropically favoured. At 100 degrees C the half-life of SsEF-1 beta is about 4 h.

Biopolymers↗

PubMed1996

Large T antigen coding sequences of two DNA tumor viruses, BK and SV40, and nonrandom chromosome changes in two glioblastoma cell lines.

The T antigen (TAg) coding sequences of two DNA tumor viruses, BKV and SV40, were detected by Polymerase Chain Reaction (PCR) amplification followed by Southern-blot hybridization in two human glioblastoma multiforme derived cell lines. RT-PCR analysis indicated that these two TAg coding sequences were expressed in both tumor cell lines carrying the viral early region DNAs. Moreover, analytical polyacrylamide gel electrophoresis (PAGE) and DNA sequence analyses showed that the amplified PCR products are indistinguishable from the TAg coding sequences of BKV and SV40 wildtype strains. Cytogenetic study performed in the two cell lines showed unbalanced changes, mainly gains of chromosomes 3p, 5, 6, 7, and 19 and losses of chromosomes 3, 3q, 16, 9p22-->pter, 18, and 20. Excess of chromosomes 6 and 7 are common to the two cell lines. The putative role of the TAg of the two DNA tumor viruses in transformation and karyotype changes is discussed.

Antigens, Viral, Tumor↗

PubMed1996

The site for GTP hydrolysis on the archaeal elongation factor 2 is unmasked by aliphatic alcohols.

An appropriate mixture of ethylene glycol and BaCl2 enhanced the otherwise very low intrinsic GTPase activity of the elongation factor 2 isolated from the archaeon Sulfolobus solfataricus (SsEF-2). The enzymatic activity became up to 300-fold higher than that of the SsEF-2 GTPase measured in the absence of any stimulator, but remained 20-fold lower than that stimulated by ribosome. The stimulatory effect of ethylene glycol/Ba2+ was attributed to the increased affinity for GTP, probably related to a conformational change occurring in a hydrophobic region near the catalytic site.

Adenosine Diphosphate Ribose↗

PubMed1996

Expression in Escherichia coli of thermostable elongation factor 1 alpha from the archaeon Sulfolobus solfataricus.

The elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was expressed in Escherichia coli and purified. The SsEF-1 alpha gene was amplified by PCR and cloned in the Ndel site of the pT7-7 expression vector, under the control of the promoter of T7 RNA polymerase. Upon induction with isopropyl beta-D-thiogalactopyranoside, the recombinant SsEF-1 alpha (recSsEF-1 alpha) was purified from the E. coli S-100 extract by a two-step procedure. From 1 litre of cell culture, about 2 mg of purified recSsEF-1 alpha was obtained. The N-terminal sequence of the first 30 amino acid residues of recSsEF-1 alpha was identical with that translated from the nucleotide sequence of the corresponding gene, except for the initial residue, which in recSsEF-1 alpha was Ser instead of Met. The M(r) of recSsEF-1 alpha (determined by electrospray MS) was almost coincident with that of the naturally occurring SsEF-1 alpha (SsEF-1 alpha). The thermal-inactivation and thermophilicity profiles of SsEF-1 alpha and recSsEF-1 alpha were identical. Concerning the functional properties, recSsEF-1 alpha was able to support poly(Phe) synthesis in vitro, to bind GDP and GTP and to elicit an NaCl-dependent GTPase activity [Masullo, De Vendittis and Bocchini (1994) J. Biol. Chem. 269, 20376-20379] with the same efficiency as that displayed by SsEF-1 alpha.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of NADH oxidase from the archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus.

The enzyme NADH oxidase (EC 1.6.99.3) has been isolated from the two thermoacidophilic archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus and characterized. In both organisms the enzyme oxidizes specifically beta-NADH in the presence of molecular oxygen and requires the presence of a flavin cofactor, showing a high specificity for FAD. A stoicheiometric amount of hydrogen peroxide to NADH is formed as the end product of the reaction, indicating that both enzymes are two-electron donors. The purified enzymes exhibit quite different molecular properties. S. acidocaldarius NADH oxidase is a monomeric protein with an estimated molecular mass of about 27 kDa, whereas S. solfataricus NADH oxidase is a dimeric protein with a molecular mass of 35 kDa per subunit; S. solfataricus NADH oxidase is purified as an FAD-containing protein, whereas S. acidocaldarius NADH oxidase does not contain a flavin molecule. Furthermore, a comparison of the N-terminal amino acid sequence shows no similarities either between the two proteins or to any other NADH oxidases. Both enzymes are essentially thermophilic. In the temperature range 20-80 degrees C, the energy of activation is almost the same for both activities, suggesting that similar energetic parameters are required. Also both oxidases display a great stability to heat. The half-life of heat inactivation is about 180 min at 90 degrees C for S. acidocaldarius NADH oxidase and 77 min at 98 degrees C for the S. solfataricus enzyme. The activity of the two enzymes is inhibited by urea and guanidine and are regulated very differently by several organic solvents.

Amino Acid Sequence↗

PubMed1995

Studies on the polypeptide elongation factor 2 from Sulfolobus solfataricus. Interaction with guanosine nucleotides and GTPase activity stimulated by ribosomes.

The elongation factor 2 from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-2) binds [3H]GDP at 1:1 molar ratio. The bound [3H]GDP is displaced by GTP or its nonhydrolyzable analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by ATP, thus indicating that only the two guanosine nucleotides compete for the same binding site. The affinity of SsEF-2 for [3H]GDP is higher than that for GTP and Gpp(NH)p. On the contrary, in the presence of ribosomes the affinity of SsEF-2 for GDP is lower than that for Gpp(NH)p. SsEF-2 is endowed with an intrinsic hardly detectable GTPase activity that is stimulated by ribosomes up to 2000-fold. The ribosome-stimulated SsEF-2 GTPase (GTPaser) reaches a maximum at pH 7.8 and is not affected by ATP but is competitively inhibited by either GDP or Gpp(NH)p. Both Km for [gamma-32P]GTP and kcat of GTPaser increase with increasing temperature, and the highest catalytic efficiency is reached at 80 degrees C. The ADP-ribosylation of SsEF-2 does not significantly affect either the binding of GDP and GTP or the kinetics of the GTPaser. A hypothesis on the stimulation by ribosome of SsEF-2 GTPase is proposed.

Adenosine Diphosphate Ribose↗

PubMed1995

The first nucleotide sequence of an archaeal elongation factor 1 beta gene.

An archaeal elongation factor 1 beta gene has been isolated for the first time from a Sulfolobus solfataricus genomic library. The sequenced clone (869 bp) contained two open reading frames, one coding for a protein made of 91 amino acid residues (SsEF-1 beta), the other one encoding a nonidentified product (ORF 115). The amino acid sequences of segments at the N- and C-terminal of the translated SsEF-1 beta were identical to those determined for the native protein. Northern and Southern analyses showed that the SsEF-1 beta gene is represented in S. solfataricus by a unique sequence. Compared to eubacterial or eukaryal corresponding genes the SsEF-1 beta is much shorter.

Amino Acid Sequence↗

PubMed1995

Relevance of histidine-84 in the elongation factor Tu GTPase activity and in poly(Phe) synthesis: its substitution by glutamine and alanine.

Substitution of His-84 (-->Gln and -->Ala), a residue of the switch II region of E. coli elongation factor (EF) Tu, hardly affected the binding of GTP or GDP. The activity in poly(Phe) synthesis and GTP hydrolysis of EF-Tu H84Q were both reduced to about 35%, as compared to EF-Tu wt, whereas EF-Tu H84A was inactive in poly(Phe) synthesis but still showed a 10% residual GTPase activity. Phe-tRNAPhe exerted a similar inhibitory effect on the GTPase activity of EF-Tu wt and EF-Tu H84Q while abolishing that of EF-Tu H84A. Ribosomes enhanced the GTPase activity of EF-Tu H84Q, but not that of EF-Tu H84A, on which they even seemed to exert an inhibitory effect. The one-round GTP hydrolysis associated with the EF-TuH84Q-dependent binding of Phe-tRNAPhe to poly(U)-programmed ribosomes was less efficient than with EF-Tu wt. Kirromycin stimulated the GTPase activities of both mutants less than EF-Tu wt. The results of this work do not support a catalytic role of His-84 in the intrinsic GTPase of EF-Tu, but they emphasize the importance of its side-chain for polypeptide synthesis and GTP hydrolysis.

Alanine↗

PubMed1995

Macrophage inflammatory protein 1-alpha mRNA expression in an immortalized microglial cell line and cortical astrocyte cultures.

Macrophage inflammatory protein 1 (MIP-1) is a recently characterized inflammatory and chemokinetic cytokine. Proinflammatory stimuli have been shown to induce expression of MIP-1 by macrophages. We hypothesized that microglia and astrocytes express MIP-1 alpha because of their many immunologic similarities to macrophages. MIP-1 alpha mRNA was examined with quantitative reverse transcription and polymerase chain reaction in an immortalized mouse microglial cell line (BV-2) and in mouse cortical astrocyte cultures. We found that in both the BV-2 microglial cell line and in astrocyte cultures, MIP-1 alpha mRNA was strongly induced by lipopolysaccharide and the phorbol ester PMA. MIP-1 alpha mRNA was reduced by dBcAMP, interferon-gamma, and PGE1. Dexamethasone decreased MIP-1 alpha mRNA levels in astrocyte cultures, but not in BV-2 microglial cells. Interleukin-1 beta, tumor necrosis factor alpha, and MIP-1 alpha had no effect on MIP-1 alpha mRNA expression. These findings demonstrate that MIP-1 alpha mRNA is expressed by cultured glial cells and is regulated by proinflammatory and anti-inflammatory stimuli. MIP-1 alpha may be expressed by microglia and astrocytes in vivo, and may help modulate cerebral inflammation.

Animals↗

PubMed1995

PKA and PKC activation induces opposite glial fibrillary acidic protein (GFAP) expression and morphology changes in a glioblastoma multiform cell line of clonal origin.

Possible differentiation mechanisms were investigated in a glioblastoma multiform cell line (GL15) presenting an undifferentiated phenotype with weak glial fibrillary acidic protein (GFAP) and strong vimentin (VIM) expression. Serum-free conditions induced time-dependent increases of GFAP-mRNA and GFAP protein levels, associated with a process-bearing astrocytic morphology. Activation of protein kinase C (PKC) by tumor promoter phorbol 12-myrystate 13-acetate (PMA) induced a rapid morphological differentiation and a decrease in GFAP mRNA, whereas the GFAP level remained unchanged. Such parameters were shown to characterize a physiological differentiation stage in astroglial cultures. Treatment of process-bearing GL15 cells with dibutyryl cyclic AMP (dbcAMP), a protein kinase A (PKA) activator, induced a time-dependent decrease in the GFAP mRNA and GFAP protein levels and reverted morphological changes induced by serum-free conditions. Neither PMA nor dbcAMP influenced the VIM mRNA expression. In GL15 cells, PKC and PKA activation have opposite effects. Understanding the role of these kinases in malignant transformation and in the in vitro differentiation process is of both basic and clinical interest.

Blotting, Northern↗

PubMed1995

Leukemia inhibitory factor mRNA is expressed in cortical astrocyte cultures but not in an immortalized microglial cell line.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine synthesized by a variety of cell types. In the nervous system LIF affects neuronal differentiation, and may be important during cerebral infection and inflammation. To clarify the cellular source of LIF in the brain, we examined the expression of LIF mRNA by primary cortical astrocyte cultures and an immortalized microglial cell line. The microglial cell line did not express LIF mRNA in response to pro-inflammatory agents such as lipopolysaccharide (LPS) that induced expression of other cytokine mRNAs. In contrast, primary astrocyte cultures grown in serum-containing medium expressed LIF mRNA constitutively, and this expression was regulated by pro-inflammatory and anti-inflammatory stimuli. Agents which activate the cAMP and protein kinase C second messenger systems also increased LIF mRNA in astrocyte cultures. These results suggest that astrocytes, but not microglia, may be an important source of LIF during cerebral inflammation and infection.

Animals↗

PubMed1995

Glutamine synthetase gene expression in a glioblastoma cell-line of clonal origin: regulation by dexamethasone and dibutyryl cyclic AMP.

We investigated the expression of glutamine synthetase (GS), an enzyme involved in astroglial metabolism and marker of astroglial functional maturity, in a glioblastoma cell-line (GL-15) of clonal origin. In spite of their phenotypic immaturity, evidenced in a mosaic fashion by a poor glial fibrillary acidic protein (GFAP) expression, the level of GS-mRNA is high in GL15 cells and the considerable amount of GS biological activity can be further induced and stabilized by glucocorticoids. A correlation between the induction by dexamethasone of the GS-mRNA level and the GS biological activity suggests a transcriptional regulation of GS expression by the aforesaid hormone. Under this hormonal action, changes in cell morphology occur and they are correlated with an overexpression of the GFAP, a marker of astroglial differentiation. On the contrary, dibutyryl cyclic AMP (dbc AMP) down-regulates the GS-mRNA expression and decreases GS activity. These results suggest that GL-15 cells have a common glucocorticoid dependent mechanism able to induce GS and GFAP as well as morphological changes. However in these cells AMPc responsive elements are involved in the negative modulation of the GS expression, contrary to what occurs in normal astroglial cells.

Astrocytes↗

PubMed1995

Glyceraldehyde-3-phosphate dehydrogenase in the hyperthermophilic archaeon Sulfolobus solfataricus: characterization and significance in glucose metabolism.

Glyceraldehyde-3-phosphate dehydrogenase in the archaeon Sulfolobus solfataricus (SsGAPD) has been purified 232 fold with an overall recovery of about 25%. The enzyme is a homomeric tetramer with an M(r) of 41 kDa/subunit. It utilizes either NAD+ or NADP+ as coenzyme but its affinity for the latter is about 50 fold higher. SsGAPD activity is maximum at 87 degrees C. In the range 45-87 degrees C the Arrhenius plot is linear and the activation energy is 55 kJ/mol. The enzyme is thermostable, with a half-life of 45 min at 87 degrees C. The primary structure of SsGAPD shows 35% identity with that of other archaeal GAPDs. Its N-domain shows sequence motifs typical of the dinucleotide binding proteins while the catalytic C-terminal region contains a cysteine residue (C140), required for catalysis, that is conserved in all the archaeal, eukaryal and bacterial GAPDs. These remarks suggest that archaeal GAPDs show a convergent molecular evolution to the eukaryal and eubacterial enzymes in the catalytic region.

Amino Acid Sequence↗

PubMed1994

Crystallization of a hyperthermophilic archaeal elongation factor 1 alpha.

Intact and fully active elongation factor aEF-1 alpha from the hyperthermophilic archaeon Sulfolobus solfataricus has been crystallized as a complex with GDP. Crystals were stable at temperatures below 8 degrees C and showed significant diffraction beyond 3.0 A. The orthorhombic lattice parameters were a = 62.9 A, b = 81.3 A, c = 115.6 A with one molecule per asymmetric unit.

Bacterial Proteins↗

PubMed1994

Archaebacterial elongation factor 1 alpha carries the catalytic site for GTP hydrolysis.

The elongation factor 1 alpha from the archaebacterium Sulfolobus solfataricus (aEF-1 alpha) possesses an intrinsic GTPase activity that is triggered by NaCl up to 5.2 M and requires the presence of at least 1 mM MgCl2 or MnCl2. Chloride salts of other monovalent cations are inefficient, whereas other sodium salts are much less efficient or not efficient at all as compared with NaCl. This aEF-1 alpha GTPase (GTPaseNa) reaches a maximum in a broad pH range and is not affected by other nucleoside triphosphates but is competitively inhibited by GDP. The turnover of GTPaseNa is rate limited by the breakdown of GTP. The Km for GTP is in the range of 2.2-9.3 microM, depending on the NaCl concentration and temperature. The highest catalytic efficiency is reached at 87 degrees C, which is the optimum temperature for growth of S. solfataricus. The energetic parameters of GTPaseNa are similar to those reported in the literature for the GTPase of Escherichia coli elongation factor Tu (EF-Tu) triggered by 2 M KCl, thus suggesting that the GTPase activity supported by either EF-Tu or aEF-1 alpha undergoes a similar mechanism of activation by salt at high concentration. A molecular mechanism for this activation is proposed.

Catalysis↗