Exploring the pros and cons of new approaches for gamete cross-border donation based on fresh and vitrified oocytes.

As highlighted by European statistics, the employment of donor oocytes is a growing option for women who cannot make use of their own gametes. As the potential recipients are continuously increasing in number, a donor programme which satisfies this demand is mandatory. Improvements in cryopreservation techniques, like oocyte and embryo vitrification, have led to the overcoming of the sequence of stimulation-retrieval-transfer both from a spatial and a temporal point of view, with the development of cryobanks of oocytes permitting crossborder donation. However, while some studies report comparable success when using vitrified and fresh oocytes we still need to investigate whether the use of fresh oocytes give higher live birth rate than cryopreserved ones, when the same number of oocytes are given. The performance of embryo cryopreservation, conversely, seems to be more reliable. A novel approach based on the shipment of frozen sperm from the recipient's country to the oocyte donor's one, where fresh oocytes are inseminated and the resulting embryos frozen and transported back to the referring IVF centre to perform a frozen embryo transfer may be a good strategy. We believe that the use of frozen embryos from fresh oocytes could be associated with a higher cumulative live birth rate per cycle, while favouring personalised oocyte recipient care with a flexible number of oocytes assigned and limiting the burden of travelling abroad.

A simple, modular synthesis of C4-substituted tryptophan derivatives.

The modular and versatile synthesis of C4-substituted tryptophan derivatives by direct functionalization of easily available N-acetyl 4-boronate tryptophan methyl ester via transition metal-catalyzed and metal-mediated cross coupling reactions is described. The versatility of the chemistry is highlighted by the gram-scale synthesis of 4-boronated N-acetyl-tryptophan methyl ester and the rapid synthesis of C4-aryl, C4-alkyl, C4-cyano, C4-trifluoromethyl, C4-azido, and C4-hydroxy tryptophan derivatives. The utility of our methodology is illustrated through the quick approach to the tricyclic azepino indole skeleton embedded in many natural products.

Development of a genetic system for hyperthermophilic Archaea: expression of a moderate thermophilic bacterial alcohol dehydrogenase gene in Sulfolobus solfataricus.

The Escherichia coli/Sulfolobus solfataricus shuttle vector pEXSs was used as a cloning vehicle for the gene transfer and expression of two bacterial genes in Sulfolobus solfataricus. The alcohol dehydrogenase (adh) from the moderate thermophilic Bacillus stearothermophilus (strain LLDR) and a mutagenised version encoding a less thermostable ADH enzyme were the selected genes. S. solfataricus adh promoter and aspartate aminotransferase terminator were used to drive the heterologous gene expression and to guarantee the correct termination of the transcripts, respectively. The constructed vectors were found to be able to carry these 'passenger' genes without undergoing any rearrangements. The active transcription of bacillar mRNAs was ascertained in vivo by RT-PCR. Transformed S. solfataricus expressed functional exogenous ADHs that showed unaffected kinetic and chemical-physical features.

Identification and molecular characterization of an endoglucanase gene, celS, from the extremely thermophilic archaeon Sulfolobus solfataricus.

A genomic region upstream of the alcohol dehydrogenase (Ssadh) gene was cloned and sequenced from a library of Sulfolobus solfataricus MT4 strain. The isolated 4,040-bp DNA fragment revealed an open reading frame (celS), lying in the opposite direction to Ssadh, which showed significant similarity to endo-beta-1,4-glucanases from Pyrococcus furiosus, Thermotoga maritima, and Thermotoga neapolitana. celS was shown to be a functional gene in vivo: a specific celS mRNA was detected by primer extension analysis showing a unique initiation transcription site coinciding with the ATG translation initiation codon. The specific gene product was detected as an extracellular cellulase after enzyme staining by carboxymethyl cellulose (CMC) SDS-PAGE, showing a molecular weight in agreement with that deduced from the open reading frame. Depending on growth conditions, different levels of cellulase activity and specific celS transcript were detected, revealing an inductive effect of CMC and suggesting a repressive role of glucose.

Thermoadaptation of a mesophilic hygromycin B phosphotransferase by directed evolution in hyperthermophilic Archaea: selection of a stable genetic marker for DNA transfer into Sulfolobus solfataricus.

A mutated version of the hygromycin B phosphotransferase (hph(mut)) gene from Escherichia coli, isolated by directed evolution at 75 degrees C in transformants of a thermophilic strain of Sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. This gene was demonstrated to be able to express the hygromycin B resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic strains of S. solfataricus, i.e., up to 82 degrees C. Furthermore, it may be transferred to S. solfataricus cells by cotransformation with pKMSD48, another extrachromosomal element derived from the virus SSV1 of Sulfolobus shibatae, without any loss of stability and without affecting the replication and infectivity of this viral DNA. The hph(mut) and the wild-type gene products were expressed at higher levels in E. coli and purified by specific affinity chromatography on immobilized hygromycin B. Comparative characterization revealed that the mutant enzyme had acquired significant thermoresistance and displayed higher thermal activity with augmented catalytic efficiency.

A single point mutation (Glu85Arg) increases the stability of the thioredoxin from Escherichia coli.

Glu85 in the Escherichia coli thioredoxin, which is localized in the loop between beta4 and beta5, was substituted with the Arg present in the corresponding position in Bacillus acidocaldarius thioredoxin. This suggested that it could play an important role in the structure and thermostability of this protein owing to its involvement in numerous interactions. The effects of the mutation on the biophysical properties were analysed by circular dichroism, spectrofluorimetry and limited proteolysis, supported by molecular dynamics data. As modelling predicted, an increase in stability for E85R due to additional H-bonds between the beta5 and alpha4 regions was observed.

Oxygen: friend or foe? Archaeal superoxide dismutases in the protection of intra- and extracellular oxidative stress.

Both "environmental chemistry" and metabolic biochemical reactions can constantly generate in vivo free radicals and other oxygen-derived species that can cause severe damage to almost all biomolecules, especially to DNA, proteins, and lipids. The superoxide anion has been shown to be the most readily generated and spread radical among organisms and it is a common intermediate of oxidative stress processes in the cells. The antioxidant defense system of superoxide dismutases (SOD) scavenges and minimizes the formation of this radical, and thus plays a major role in reducing cumulative oxidative damage in different cell compartments both in aerobic and anaerobic cells. In the cell, cytosol SODs are constitutively present and induced by many oxidative agents able to raise the superoxide concentrations. Presence of SODs, however, in extracellular cell-associated locations demonstrates how valuable they are in maintaining the integrity of cells against oxidative stress generated by the cell environment, particularly upon increased oxygenation. Because SODs have recently been found in Archaea, which are prokaryotes, sometimes living in extreme environments, even in anaerobic ones, these enzymes can be considered essential: they may have allowed the evolution of aerobic respiration starting from an ancient form of oxygen-insensitive life.

NMR solution structure of a novel thioredoxin from Bacillus acidocaldarius possible determinants of protein stability.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx), an eubacterium growing optimally at 333 K, is the first Trx described to date from a moderate thermophilic source. To understand the molecular basis of its thermostability, the three-dimensional structure in the oxidized form was determined by NMR methods. A total of 2276 1H-NMR derived distance constraints along with 23 hydrogen-bonds, 72 phi and 27 chi1 torsion angle restraints, were used in a protocol employing simulated annealing followed by restrained molecular dynamics and restrained energy minimization. BacTrx consists of a well-defined core region of five strands of beta-sheet, surrounded by four exposed alpha-helices, features shared by other members of the thioredoxin family. The BacTrx 3D structure was compared with the Escherichia coli Trx (EcTrx) determined by X-ray crystallographic diffraction, and a number of structural differences were observed that may contribute to its thermostabilty. The results of structural analysis indicated that protein stability is due to cumulative effects, the main factor being an increased number of ionic interactions cross-linking different secondary structural elements and clamping the C-terminal alpha-helix to the core of the protein.

A superoxide dismutase from the archaeon Sulfolobus solfataricus is an extracellular enzyme and prevents the deactivation by superoxide of cell-bound proteins.

An oxygen-induced iron superoxide dismutase was found in the culture fluid of the thermoacidophilic crenarchaeon Sulfolobus solfataricus during growth on glucose-rich media. This protein was also identified as being associated with the cell-surface, with the amount of the released and cell-bound protein fractions depending on the growth phase of the cells. The steady decrease in cell-associated superoxide dismutase during continued growth correlated with the increase of free superoxide dismutase in the medium. Both enzyme fractions were purified to homogeneity and found to be active with different catalytic efficiency, with the released superoxide dismutase showing a fourfold lower specific activity. Characterization in comparison with the cytosolic superoxide dismutase revealed identical N-terminal sequences, electrophoretic mobility, isoelectric point, and molecular mass for all three differently located enzymes. In order to clarify the physiological role of the cell-associated superoxide dismutase, the prevention of cell-bound protein deactivation by oxyradicals was also investigated. Glucose dehydrogenase, which was chosen as a model enzyme, was demonstrated to be located on the cell surface and to be inactivated by potassium superoxide by in vivo assays. The direct protective effect of superoxide dismutase on glucose dehydrogenase was demonstrated by in vitro assays on the free released enzyme. Similarly, the prevention of deactivation by potassium superoxide was also demonstrated for the integral membrane protein succinate dehydrogenase by intact cell assay. Superoxide dismutase added to cells was shown to moderately reduce the critical damaging peroxidation and hence play a major role in maintaining the integrity of the outer cell envelope components.

Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase.

Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65 degrees C; this is analogous to previous findings with mesophilic ADH at 25 degrees C. Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

Prediction and experimental testing of Bacillus acidocaldarius thioredoxin stability.

In order to investigate further the determinants of protein stability, four mutants of thioredoxin from Bacillus acidocaldarius were designed: K18G, R82E, K18G/R82E, and D102X, in which the last four amino acids were deleted. The mutants were constructed on the basis of molecular dynamic studies and the prediction of the structure of thioredoxin from B. acidocaldarius, performed by a comparative molecular modelling technique using Escherichia coli thioredoxin as the reference protein. The mutants obtained by PCR strategy were expressed in E. coli and then characterized. CD spectroscopy, spectrofluorimetry and thermodynamic comparative studies permitted comparison of the relative physicochemical behaviour of the four proteins with that of the wild-type protein. As predicted for the molecular dynamic analysis at 500 K in vacuo, the wild-type structure was more stable than that of the mutants; in fact the Tm of the four proteins showed a decrease of about 15 degrees C for the double and the truncated mutants, and a decrease of about 12 degrees C for the single mutants. A difference in the resistance of the proteins to denaturants such as guanidine HCl and urea was revealed; the wild-type protein always proved to be the most resistant. The results obtained show the importance of hydrogen bonds and ion pairs in determining protein stability and confirm that simulation methods are able to direct protein engineering in site-directed mutagenesis.

Asn249Tyr substitution at the coenzyme binding domain activates Sulfolobus solfataricus alcohol dehydrogenase and increases its thermal stability.

A mutant of the thermostable NAD+-dependent homotetrameric alcohol dehydrogenase from Sulfolobus solfataricus (SsADH), which has a single substitution, Asn249Tyr, located at the coenzyme binding domain, was obtained by error prone PCR. The mutant enzyme, which was purified from Escherichia coli to homogeneous form, exhibits a specific activity that is more than 6-fold greater than that of the wild type enzyme, as measured at 65 degrees C with benzyl alcohol as the substrate. The oxidation rate of aliphatic alcohols and the reduction rate of aromatic aldehydes were also higher. The dissociation constants for NAD+ and NADH determined at 25 degrees C and pH 8.8 were 3 orders of magnitude greater compared to those of the wild type enzyme. It is thought that the higher turnover of the mutant SsADH is due to the faster dissociation of the modified enzyme-coenzyme complex. Spectroscopic studies showed no relevant changes in either secondary or tertiary structure, while analysis with fluorescent probes revealed a significant increase in surface hydrophobicity for the mutant, with respect to that of the wild type molecule. The mutant SsADH displays improved thermal stability, as indicated by the increase in Tm from 90 to 93 degrees C, which was determined by the apparent transition curves. Kinetic thermal stability studies at pH 9.0 for mutant SsADH showed a marked increase in activation enthalpy compensated by an entropy gain, which resulted in a higher activation barrier against thermal unfolding of the enzyme. Ammonia analysis showed that the Asn249Tyr substitution produced the effect of markedly reducing the extent of deamidation during thermoinactivation, thus suggesting that Asn249 plays a significant role in the mechanism of irreversible thermal denaturation of the archaeal ADH. Furthermore, the decrease in the activating effect by moderate concentrations of denaturants and studies with proteases and chelating agents point to an increase in structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme.

The alcohol dehydrogenase gene: distribution among Sulfolobales and regulation in Sulfolobus solfataricus.

The distribution of the alcohol dehydrogenase gene (adh) among different Archaea was investigated by Southern blot analysis revealing the potentiality of the adh gene as a specific marker for the genus Sulfolobus. Moreover, the in vivo expression of the adh gene from a new isolate of Sulfolobus solfataricus, G theta, was studied to investigate gene regulation in Archaea. Primer extension analysis allowed the identification of a single initiation site and the TATA box element. Comparison of the G theta adh promoter with the corresponding Ssadh (adh from S. solfataricus) and RC3adh (adh from Sulfolobus RC3) also revealed the presence of two putative regulatory inverted repeats at the 5' of the TATA element. Northern blot analysis and enzymatic assays demonstrated that the transcription and expression of the G theta adh gene is regulated by different carbon and energy sources or by the natural substrate of the ADH enzyme.

Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements.

The gene encoding the alcohol dehydrogenase (adh-hT) from the thermophilic bacterium Bacillus stearothermophilus LLD-R strain has been overexpressed in Escherichia coli and the corresponding recombinant protein purified to homogeneity. Two putative structural determinants contributing to the higher stability of ADH-hT had been identified by comparison with the less thermostable ADH (ADH-T) from the less thermophilic B. stearothermophilus NCA 1503. In order to ascertain their role, mutations were designed to eliminate in ADH-hT a salt bridge at the N-terminus and a proline residue in the coenzyme binding domain replacing the amino acids located at the same positions in ADH-T. Three mutants--Glu11Lys, Pro242Ala, and Glu11Lys/Pro242Ala--were expressed at high level and the proteins purified and characterized. In general, the mutations had little effect on the activity, indicating that they were not disruptive. The thermal resistance was changed displaying quite additive effects.

Computational analysis of the thermal stability in thioredoxins: a molecular dynamics approach.

The knowledge of the relationship between the three-dimensional structure of a protein and its biological and stability is one of the most challenging problem in protein chemistry, since offers the possibility of changing both the specific action of a protein and its stability. In this work, we have approached the problem with studies on a protein family, the thioredoxins, using homology procedures, molecular dynamics simulations in vacuo at 300 K and 500 K and in water solution at 300 K, to determine the relationship between the three-dimensional structure of these proteins and their thermal stability. A comparative analysis, using computational approach, was performed between two thioredoxins with different resistance to temperature. Results obtained using the molecular dynamics techniques and minimization procedures give explanations of the experimental data, underlining that these techniques are able to correlate the increase in protein stabilization with the conformational and structural changes caused by single amino acid replacement. In addition, we report the factors that can be used as a guide in protein engineering and in site-directed mutagenesis to increase or decrease thermal stabilization for this protein family.

A protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units.

Protein disulfide bond formation is a rate limiting step in protein folding and is catalyzed by enzymes belonging to the protein disulfide oxidoreductase superfamily, including protein disulfide isomerase (PDI) in eucarya and DsbA in bacteria. The first high resolution X-ray crystal structure of a protein disulfide oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus reveals structural details that suggest a relation to eukaryotic PDI. The protein consists of two homologous structural units with low sequence identity. Each unit contains a thioredoxin fold with a distinct CXXC active site motif. The accessibilities of both active sites are rather different as are, very likely, their redox properties. The protein shows the ability to catalyze the oxidation of dithiols as well as the reduction of disulfide bridges.

An autonomously replicating transforming vector for Sulfolobus solfataricus.

A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E. coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C.

Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-dependent reduction of bovine insulin disulphides. The protein has a molecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edman degradation after cleavage with trypsin and cyanogen bromide. The sequences of known bacterial Trxs were aligned at the active site: BacTrx has an identity ranging from 45 to 53% with all sequences except that of the unusual Anabaena strain 7120 Trx (37% identity). The gene coding for BacTrx was isolated by a strategy based on PCR gene amplification and cloned in a plasmid downstream of a lac-derived promoter sequence; the recombinant clone was used as the expression vector for Escherichia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl beta-d-thiogalactoside; expressed BacTrx represents approx. 5% of the total cytosolic protein. CD spectra and differential scanning calorimetry measurements demonstrated that BacTrx is endowed with a higher conformational heat stability than the Trx from E. coli. Nanogravimetry experiments showed a lower content of bound water in BacTrx than in E. coli Trx, and a transition temperature approx. 10 degrees C higher for BacTrx. The three-dimensional model of the oxidized form of BacTrx was constructed by a comparative molecular modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as reference proteins. Increased networks of ion-pairs and shorter loops emerged as major features of the BacTrx structure compared with those of the template proteins. The findings are discussed in the light of the current knowledge about molecular determinants of protein stability.

Stabilization of Enzymes against Thermal Stress and Freeze-Drying by Mannosylglycerate.

2-O-(beta)-Mannosylglycerate, a solute that accumulates in some (hyper)thermophilic organisms, was purified from Pyrococcus furiosus cells, and its effect on enzyme stabilization in vitro was assessed. Enzymes from hyperthermophilic, thermophilic, and mesophilic sources were examined. The thermostabilities of alcohol dehydrogenases from P. furiosus and Bacillus stearothermophilus and of glutamate dehydrogenases from Thermotoga maritima and Clostridium difficile were improved to a significant extent when enzyme solutions were incubated at supraoptimal temperatures in the presence of 2-O-(beta)-mannosylglycerate, but no effect on the thermostability of glutamate dehydrogenase from P. furiosus was detected. On the other hand, there was a remarkable effect on the thermal stabilities of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and bovine liver glutamate dehydrogenase, which were used as model systems to evaluate stabilization of enzymes of mesophilic origin. For all of the enzymes examined and at the highest temperatures tested, 2-O-(beta)-mannosylglycerate was a better thermoprotectant than trehalose. The stabilizing effect exerted by 2-O-(beta)-mannosylglycerate on enzymes suggests a role for this compound as a protein thermostabilizer under physiological conditions. 2-O-(beta)-Mannosylglycerate was also effective in the protection of enzymes against stress imposed by freeze-drying, with its protecting effect being similar to or better than that exerted by trehalose. The data show 2-O-(beta)-mannosylglycerate to be a potential enzyme stabilizer in biotechnological applications.