Biomedical subjects

R Scandurra

Publications and source records attributed to R Scandurra.

At least 19 recordsLinked to original sources

PubMed2002

The selective estrogen receptor modulator raloxifene regulates osteoclast and osteoblast activity in vitro.

Raloxifene is a selective estrogen receptor modulator (SERM) that prevents bone loss. Although it is largely used for the treatment of osteoporosis, the mechanisms by which this compound modulates the activity of bone cells are still poorly understood. In this study we investigate whether raloxifene affects osteoclast and osteoblast activity in vitro. Bone marrow cultures were established from neonatal mice and treated with 1,25(OH)(2) vitamin D(3) (VitD(3), 10(-8) mol/L) to induce osteoclast generation. Similar to 17beta-estradiol, raloxifene significantly reduced the number of osteoclasts in a concentration-dependent manner, with maximal inhibition at 10(-11) mol/L (-48%). However, as for 17beta-estradiol, at a high concentration (10(-7) mol/L), the inhibitory effect of raloxifene was abolished. In a pit assay, raloxifene inhibited bone resorption. A maximal effect was observed at 10(-9) mol/L, and maintained at a high concentration, indicating that inhibition of osteoclast formation and inhibition of bone resorption may be due to activation of, at least in part, different pathways. Osteoblasts from neonatal mice calvariae were also exposed to raloxifene. In these cells, this compound induced a concentration-dependent increase of proliferation, which was blocked by the estrogen-receptor antagonist ICI 164,384. Raloxifene also increased the osteoblast-specific transcription factor Cbfa1/Runx2 and alpha2 procollagen type I chain mRNAs, with a pattern that only partially coincided with that of 17beta-estradiol. Consistent with decreased osteoclastogenesis, raloxifene inhibited the mRNA expression of interleukin (IL)-1beta and IL-6 at a low concentration, but not at a high concentration, whereas 17beta-estradiol had similar effects on IL-6 and inhibited IL-1beta at both concentrations. Furthermore, both compounds were able to inhibit tumor necrosis factor (TNF)-alpha-induced IL-1beta, but not IL-6, increase. In conclusion, these data show that raloxifene negatively modulates osteoclasts, and positively affects osteoblasts, suggesting not only an antiresorptive role, but also an osteoblast stimulatory role.

Animals↗

PubMed2001

Crystallization and X-ray diffraction measurements of a thermophilic archaeal recombinant amidase from Sulfolobus solfataricus MT4.

Recombinant amidase is a 55.8 kDa enzyme from the thermophilic archaeon Sulfolobus solfataricus MT4 that catalyses the hydrolysis of aliphatic amides of 2-6 C atoms as well as many aromatic amides. Single crystals of purified amidase were obtained by the hanging-drop method at 294 K. Diffraction data for the native protein (2.55 A resolution) and a putative derivative (2.20 A) have been collected at low temperature using synchrotron radiation. The crystals belong to the rhombohedral space group R3. Structure determination by multiple isomorphous replacement is in progress. It is expected that structural information from this signatured thermostable amidase will increase our knowledge of the molecular mechanisms employed to maintain high-temperature stability in thermophilic proteins.

Amidohydrolases↗

PubMed2001

Contribution of an aspartate residue, D114, in the active site of clostridial glutamate dehydrogenase to the enzyme's unusual pH dependence.

Glutamate dehydrogenase from Clostridium symbiosum displays unusual kinetic behaviour at high pH when compared with other members of this enzyme family. Structural and sequence comparisons with GDHs from other organisms have indicated that the Asp residue at position 114 in the clostridial enzyme may account for these differences. By replacing this residue by Asn, a mutant protein has been created with altered functional properties at high pH. This mutant protein can be efficiently overexpressed in Escherichia coli, and several criteria, including mobility in non-denaturing electrophoresis, circular dichroism (CD) spectra and initial crystallisation studies, suggest a folding and an assembly comparable to those of the wild-type protein. The D114N mutant enzyme shows a higher optimum pH for activity than the wild-type enzyme, and both CD data and activity measurements show that the distinctive time-dependent reversible conformational inactivation seen at high pH in the wild-type enzyme is abolished in the mutant.

Aspartic Acid↗

PubMed2001

Molecular and biochemical characterization of the recombinant amidase from hyperthermophilic archaeon Sulfolobus solfataricus.

We have cloned, sequenced, and overexpressed in Escherichia coli the amidase gene from the hyperthermophilic archaeon Sulfolobus solfataricus (strain MT4). The recombinant thermophilic protein was expressed as a fusion protein with an N-terminus six-histidine-residue affinity tag. The enzyme, the first characterized archaeal amidase, is a monomer of 55,784 daltons, enantioselective, and active on 2- to 6-carbon aliphatic amides and on many aromatic amides, over the pH range 4-9 and at temperatures from 60 degrees to 95 degrees C. The S. solfataricus amidase belongs to the class of amidases that share a characteristic signature, GGSS(S/ G)GS, located in the central region of the protein, and which show remarkable variability in their individual substrate specificities, can hydrolyze aliphatic or aromatic substrates, and share a large invariance of their primary structure.

Amidohydrolases↗

PubMed2000

Protein stability in extremophilic archaea.

Extremophilic microorganisms have adapted their molecular machinery to grow and thrive under the most adverse environmental conditions. These microorganisms have found their natural habitat at the boiling and freezing point of water, in high salt concentration and at extreme pH values. The extremophilic proteins, selected by Nature to withstand this evolutionary pressure, represent a wide research field for scientists from different disciplines and the study of the determinants of their stability has been an important task for basic and applied research. A surprising conclusion emerges from these studies: there are no general rules to achieve protein stabilization. Each extremophilic protein adopts various strategies and the outstanding adaptation to extreme temperature and solvent conditions is realized through the same weak electrostatic and hydrophobic interactions among the ordinary amino acid residues which are also responsible for the proper balance between protein stability and flexibility in mesophilic proteins.

Adaptation, Physiological↗

PubMed2000

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima.

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme. The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.

Bacterial Proteins↗

PubMed2000

Genetic potential of interfacial guided osteogenesis in implant devices.

The purpose of this review is to summarise recent advances in the design and composition of bioactive surface layers of implantabile biomaterials, and thus the genetic potential of osteoprogenitor cells to recognize and respond to these diverse implanted biomaterials. Changes applied to a biomaterial's surface, in general, could improve its biocompatibility, osseointegration and durability properties, which are required for long-term implantation in the living body. In this review, the implant-bone interface was evaluated and interpreted on the basis of osteoblast cell cultures, i.e., on the genetic potential of osteoblasts to express different phenotype markers depending on the type of biomaterials used. The interface formed by in vitro-grown osteoblasts may be used to identify components of the in vivo implant-bone interface. Over the years, a large number of implant systems consisting of many different biomaterials have been introduced in dentistry and orthopaedics. This paper discusses the performance of currently used metals and other biomaterials, by focusing on the events which occur immediately after implantation and on their impact on the bone-implant interface. The review demonstrates that continuous improvements in composition, surface modality and design of implants may benefit osseointegration and clinical longevity of such implants. No load-bearing conditions or clinical status are discussed. Titanium (Ti) and calcium phosphate ceramics are regarded as the most biocompatible synthetic substances known to be used in hard tissue implantation. These biomaterials are osteoconductive, and do not induce ectopic bone formation. Nonetheless, they provide a physical matrix which is suitable for the deposition of new bone and may guide both the growth and extension of the bone. Comparative investigation evaluated that Ti implant systems appear to be apposed by more bone than ceramic systems, although alternatives concerning the type of Ti alloy and bioactive surface layer engineering, generate extremely diverse osseointegration results. Manufacturers have created an extensive range of inorganic or ceramic coatings on Ti implants in order to achieve better bone healing and osteoconduction. Biologically active molecules, added to the implant surface, represent breakthroughs in guided interfacial osteogenesis. This methodology offers an enormous potential of genetic controlling and promoting osteogenesis. The bone growth factors are not fully understood, but most researchers agree that the contact between the bioactive surface layer of the implant and bone is not static but dynamic and that the above factors may maximise the implant osseointegration.

Animals↗

PubMed1999

Heat-stable pullulanase from Bacillus acidopullulyticus: characterization and refolding after guanidinium chloride-induced unfolding.

Heat-stable pullulanase from Bacillus acidopullulyticus was characterized with respect to its stability against thermal and chemical denaturation and its reactivation after complete chemical unfolding. The enzyme was quite thermostable and retained 55% of activity after heating at 60 degrees C for 30 min at pH 5.5. At pH 6.0, only 9% residual activity was observed. The addition of sucrose, polyols, and Na2SO4 strongly stabilized the enzyme against thermal inactivation. The processes of chemical unfolding by guanidinium chloride (GdmCl) and refolding were studied by enzymological and spectroscopic criteria. B. acidopullulyticus pullulanase was very sensitive to GdmC1 denaturation and had a transition midpoint at 1.2M GdmCl. Reactivation after complete unfolding in 5 M GdmCl was initiated by dilution of the unfolding mixture: 67% reactivation was observed under standard conditions. The influence of some chemical and physical parameters (pH, chemical agents, temperature, and unfolding and refolding time) on refolding was investigated. Of the additives tested to assist reactivation, only bovine serum albumin (BSA) increased the yield of activity to 80%. The full regain of structure and activity was proven by comparing the enzymological, physicochemical, and spectroscopic properties of the native and refolded pullulanase.

Animals↗

PubMed1999

Stability, refolding and Ca2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei.

The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +/- 0.29 M for intrinsic fluorescence and 4.90 +/- 0.31 M for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 +/- 0.010 microM and a Hill coefficient H of 1.00 +/- 0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DeltaGH2OD increased from 6.83 +/- 0.43 to 8.42 +/- 0.55 kcal.mol-1. The refolding of the pullulanase, on the other hand, was not affected by Ca2+.

Acrylamide↗

PubMed1998

Cloning and sequencing of ISC1041 from the archaeon Sulfolobus solfataricus MT-4, a new member of the IS30 family of insertion elements.

A genomic fragment containing the insertion sequence ISC1041 has been cloned by PCR from the archaeon Sulfolobus solfaricus MT-4, an extremophilic microorganism which grows at 87 degrees C. The 1038 bp ISC1041 element contains an imperfect 18 nt repeat and a long open reading frame which encodes a polypeptide of 311 amino acid residues. The translated amino acid sequence shows a significant similarity to IS30-like transposases. Structural analysis indicates that ISC1041 is a novel member of the IS30 family and displays the DDE motif not previously seen in Archaea. This motif is believed to be involved in the integration mechanism of many mobile elements. As this motif is present in several integrases and transposases which, despite the lack of overall protein homologies, share topological homologies to the DDE motif, a common ancestor has been proposed. The finding of an IS30-like transposase in the archaeal kingdom may have relevance for horizontal gene transfer.

Amino Acid Sequence↗

PubMed1998

Protein thermostability in extremophiles.

Thermostability of a protein is a property which cannot be attributed to the presence of a particular amino acid or to a post synthetic modification. Thermostability seems to be a property acquired by a protein through many small structural modifications obtained with the exchange of some amino acids and the modulation of the canonical forces found in all proteins such as electrostatic (hydrogen bonds and ion-pairs) and hydrophobic interactions. Proteins produced by thermo and hyperthermophilic microorganisms, growing between 45 and 110 degrees C are in general more resistant to thermal and chemical denaturation than their mesophilic counterparts. The observed structural resistance may reflect a restriction on the flexibility of these proteins, which, while allowing them to be functionally competent at elevated temperatures, renders them unusually rigid at mesophilic temperatures (10-45 degrees C). The increased rigidity at mesophilic temperatures may find a structural determinant in increased compactness. In thermophilic proteins a number of amino acids are often exchanged. These exchanges with some strategic placement of proline in beta-turns give rise to a stabilization of the protein. Mutagenesis experiments have confirmed this statement. From the comparative analysis of the X-ray structures available for several families of proteins, including at least one thermophilic structure in each case, it appears that thermal stabilization is accompanied by an increase in hydrogen bonds and salt bridges. Thermostability appears also related to a better packing within buried regions. Despite these generalisations, no universal rules can be found in these proteins to achieve thermostability.

Adenylate Kinase↗

PubMed1997

Acid-induced disassembly of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus occurs below pH 2.0.

The stability of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus at low pH values has been studied by activity assay, spectroscopic methods, size-exclusion chromatography and ultracentrifugation analysis. The enzyme is exceptionally stable and at pH 2.0 its hexameric assembly is preserved despite the changes observed in its tertiary structure. Below pH 1.7 dissociation into monomers starts and is accompanied by a progressive loss of tertiary interactions. Dissociation intermediate(s) were not detectable. At pH 2.0 the addition of NaCl causes the same structural changes observed upon further addition of protons. The monomeric state of the enzyme at pH 1.0 shows a significant content of native secondary structure and can be unfolded by guanidinium chloride. The role of electrostatic interactions in the high stability of the enzyme structure at low pH values is discussed.

Archaea↗

PubMed1996

Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis.

By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue. Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots. The antibody was used to monitor purification of the inactive protein. F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD+ completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients S20,W of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280000 +/- 5000 g/mol. consistent with the hexameric structure for the wild-type protein and 135000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98000) and a trimer (147000). To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations. M(r,app) showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution. This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 l/g. An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 l/g. Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species. Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis. On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers. This issue can only be resolved by making alternative dimeric mutants.

Aspartic Acid↗

PubMed1996

Refolding pathway and association intermediates of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

The denaturation and renaturation processes of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus have been investigated using guanidinium chloride as denaturant. The enzyme is highly stable and the transition midpoint for guanidinium chloride denaturation is 6.1 M. The recovery of enzyme structure occurs after dilution of the denaturant at 20 degrees C through the formation of structured monomers. Concentration of the structured monomers leads to the formation of higher association states with a tertiary structure different from that of the native enzyme. Activity is observed only in the presence of the hexamers, although a heating step at 70 degrees C is required to fully reactivate the hexamer formed at 20 degrees C. The refolding process and the intermediate(s) were studied by activity assay, spectroscopic methods, size-exclusion chromatography, and ultracentrifugation analysis.

Archaea↗

PubMed1995

Molecular properties of glutamate dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

This study is concerned with the structural characterization in solution of the glutamate dehydrogenase from the Archaeon Sulfolobus solfataricus. At neutral pH both alpha-helix and beta-sheet constitute the secondary structure of this enzyme, on the basis of circular dichroism. A complex, temperature dependent self-association equilibrium regulates the formation of the enzyme quaternary structure, which seems to be accompanied by a reversible structural change. At 25 degrees C the enzyme is mostly represented by monomeric subunits at concentrations lower than 0.02 mg/ml, while oligomers are predominant at concentrations higher than 0.12 mg/ml. The mid-point of the association curve shifts from 0.05 mg/ml at 25 degrees C to about 0.1 mg/ml at 45 degrees C. Only the oligomeric form appears to be temperature resistant. Monomeric and oligomeric enzyme show distinct behaviour on guanidine hydrochloride perturbation at neutral pH. The monomer denaturation, although complex, is reversible. Two fluorescent tryptophan classes are detectable in the monomer, monitoring the independent unfolding of two regions through a multistate transition. Instead, the oligomeric protein shows a complex denaturation pattern with the tendency to aggregate irreversibly at high denaturant concentration.

Circular Dichroism↗

PubMed1995

Insights into thermal stability from a comparison of the glutamate dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis.

In the light of the solution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from the mesophile Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequences for the thermophilic glutamate dehydrogenases from Thermococcus litoralis and Pyrococcus furiosus against the sequence and the molecular structure of the glutamate dehydrogenase from C. symbiosum, to provide insights into the molecular basis of their thermostability. This homology-based modelling is simplified by the relatively small number of amino acid substitutions between the two thermophilic glutamate dehydrogenase sequences. The most frequent amino acid exchanges involve substitutions which increase the hydrophobicity and sidechain branching in the more thermostable enzyme; particularly common is the substitution of valine to isoleucine. Examination of the sequence differences suggests that enhanced packing within the buried core of the protein plays an important role in maintaining stability at extreme temperatures. One hot spot for the accumulation of exchanges lies close to a region of the molecule involved in its conformational flexibility and these changes may modulate the dynamics of this enzyme and thereby contribute to increased stability.

Amino Acid Sequence↗

PubMed1995

Crystallization of the NAD(P)-dependent glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

The NAD(P)-dependent glutamate dehydrogenase from Pyrococcus furiosus has been crystallized by the hanging-drop method of vapour diffusion using lithium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group P4(2)2(1)2 with unit-cell dimensions of a = b = 167.2, c = 172.9 A. Consideration of the values of V(m) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a trimer. P. furiosus belongs to the family of Archaea and is one of the most thermostable organisms known, having an optimal growth temperature of 376 K. The glutamate dehydrogenase isolated from this organism has a half-life of 12 h at 373 K and, therefore, the determination of the structure of this enzyme will be important in advancing our understanding of how proteins are adapted to enable them to survive at such extreme temperatures.

Journal Article↗

PubMed1995

Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability.

The glutamate dehydrogenase gene from the hyperthermophilic archaeon Pyrococcus furiosus has been functionally expressed in Escherichia coli under the control of the lambda PL promoter. The P. furiosus glutamate dehydrogenase amounted to 20% of the total E. coli cell protein, and the vast majority consisted of hexamers. Following activation by heat treatment, an enzyme could be purified from E. coli that was indistinguishable from the glutamate dehydrogenase purified from P. furiosus. Hybrid genes, that consisted of the coding regions for the homologous glutamate dehydrogenases from P. furiosus and the mesophilic bacterium Clostridium difficile, were constructed and successfully expressed in E. coli. One of the resulting hybrid proteins, containing the glutamate binding domain of the C. difficile enzyme and the cofactor binding domain of the P. furiosus enzyme, did not show a detectable activity. In contrast, the complementary hybrid containing the P. furiosus glutamate and the C. difficile cofactor binding domain was a catalytically active hexamer that showed a reduced substrate affinity but maintained efficient cofactor binding with the specificity found in the Clostridium symbiosum enzyme. Compared with the C. difficile glutamate dehydrogenase, the archaeal-bacterial hybrid is slightly more thermoactive, less thermostable but much more stable towards guanidinium chloride-induced inactivation and denaturation.

Archaea↗