Characterization of thermal shock damage in cordierite-mullite refractory materials by non-destructive methods.

Ultrasonic pulse velocity testing and image anal. were used to predict thermal stability of refractories. Two cordierite-mullite compns. used as substrates in fast firing of porcelain whiteware characterized by different microstructure morphologies and crack propagation behavior were investigated. The measurement of the ultrasonic velocity was used to assess the material degrdn. with increasing thermal shock cycles and specimen damage was monitored using image anal. and further results of material degrdn. were obtained. A brief discussion about the correlation between thermomech. properties, microstructure, crack propagation behavior and thermal shock resistance is presented. Moreover, empirical models are developed to predict thermomech. properties from ultrasonic velocity and surface damage measurements. Then, service life prediction models of refractory plates from measured values of ultrasonic velocities in plates in the as-received state are presented

Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion

Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles, i.e., Sulfolobus solfataricus, S. acidocaldarius and S. tokodaii, indicates that only few proteins are freely secreted into the growth medium and that the majority originates from cell envelope bound forms. In S. acidocaldarius both cell-associated and secreted α-amylase activities are detected. Inactivation of the amyA gene resulted in a complete loss of activity, suggesting that the same protein is responsible for the a-amylase activity at both locations. It is concluded that protein secretion in Sulfolobus is a limited process, and it is suggested that the S-layer may act as a barrier for the free diffusion of folded proteins into the medium

A few amino acid substitutions are responsible for the higher thermostability of a novel NAD+-dependent bacillar alcohol dehydrogenase.

The gene adh-hT encoding a thermostable and thermophilic NAD+-dependent alcohol dehydrogenase (ADH) from the novel and more thermophilic Bacillus stearothermophilus LLD-R strain was cloned and its nucleotide sequence determined. The deduced protein sequence shows remarkable amino acid substitutions when compared to the sequence of the protein isolated from strain NCAl503 and significant similarity with the highly thermostable ADH from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The alignment of these sequences led to the identification of three amino acid replacements probably responsible for the higher thermostability of the novel bacillar ADH. Adh-hT gene expression in Escherichia coli, a fast purification procedure and the characterization of the recombinant enzyme are also describe

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

Decreasing the Stability and Changing the Substrate Specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single aminoacid 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

A cell-type specific and enhancer-dependent silencer in the regulation of the expression of the human urokinase plasminogen activator gene.

A transcriptional silencer has been identified in the 5' regulatory region of the human urokinase plasminogen activator (uPA) gene. This region is able to block transcription from the human u-PA as well as the rabbit beta-globin promoters in a cell type specific and orientation independent way. The silencer is enhancer dependent and is active in two cell lines (HeLa and CV-1) which produce little if any uPA, but not in the high uPA producer PC3. Silencing activity and enhancer dependence can be separated: the silencing activity has been localized to the DNA fragment -660 to -536, while the enhancer dependence is located in the -536 to -308 fragment. The DNA sequence of the silencer region contains an element that closely resembles the TGF-beta responsive negative element TIE

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 (hphmut) gene from Escherichia coli, isolated by directed evolution at 75°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°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 hphmut 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 MAR-like transcriptional regulator of the archaeaon Sulfolobus solfataricus binds to the alcohol dehydrogenase promoter

The understanding of basic processes in the third domain of life, Archaea, has been greatly deepened by the completion of several genome sequencing projects like that of S. solfataricus.The conversion of a sequence database into a functional one, favours the understanding of networks controlling fundamental cellular processes, like DNA repair systems, and regulatory networks. Transcription studies in Archaea have revealed that the archaeal basal machinery resembles its eukaryotic counterpart. Intriguingly, sequencing projects have evidenced the existence of homologues of bacterial transcriptional regulators To date, a few of them have been studied, but a model explaining their role in the regulation of specific metabolic genes still needs to be elucidated. In this context, to investigate mechanisms of transcription regulation in the hyperthermophilic archaeon S. solfataricus, we focused on the expression of a putative bacterial-like transcription factor that in Bacteria is involved in the regulation of genes for the degradation of aromatic compounds. The recombinant protein has been purified to homogeneity, and is a highly thermostable DNA binding protein. The encoding gene is transcriptionally regulated in response to aromatic compounds. Mobility shift assays and DNA footprinting showed that this protein has a specific target, that is an aromatic alcohol dehydrogenase (adh) gene. The correlation between adh gene regulation and the binding of a specific factor in its promoter sequence suggests that this specific interaction could be responsible for the switch on of the aromatic aldehyde metabolism in response to environmental changes