A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.     

Respiratory Chains from Aerobic Thermophilic Prokaryotes

Thermophiles are organisms that grow optimally above 50 degrees C and up to approximately 120 degrees C. These extreme conditions must have led to specific characteristics of the cellular components. In this paper we extensively analyze the types of respiratory complexes from thermophilic aerobic prokaryotes. The different membrane-bound complexes so far characterized are described, and the genomic data available for thermophilic archaea and bacteria are analyzed. It is observed that no specific characteristics can be associated to thermophilicity as the different types of complexes I-IV are present randomly in thermophilic aerobic organisms, as well as in mesophiles. Rather, the extensive genomic analyses indicate that the differences concerning the several complexes are related to the organism phylogeny, i.e., to evolution and lateral gene transfer events.     

Dissimilatory Oxidation and Reduction of Elemental Sulfur in Thermophilic Archaea

The oxidation and reduction of elemental sulfur and reduced inorganic sulfur species are some of the most important energy-yielding reactions for microorganisms living in volcanic hot springs, solfataras, and submarine hydrothermal vents, including both heterotrophic, mixotrophic, and chemolithoautotrophic, carbon dioxide-fixing species. Elemental sulfur is the electron donor in aerobic archaea like Acidianus and Sulfolobus. It is oxidized via sulfite and thiosulfate in a pathway involving both soluble and membrane-bound enzymes. This pathway was recently found to be coupled to the aerobic respiratory chain, eliciting a link between sulfur oxidation and oxygen reduction at the level of the respiratory heme copper oxidase. In contrast, elemental sulfur is the electron acceptor in a short electron transport chain consisting of a membrane-bound hydrogenase and a sulfur reductase in (facultatively) anaerobic chemolithotrophic archaea Acidianus and Pyrodictium species. It is also the electron acceptor in organoheterotrophic anaerobic species like Pyrococcus and Thermococcus, however, an electron transport chain has not been described as yet. The current knowledge on the composition and properties of the aerobic and anaerobic pathways of dissimilatory elemental sulfur metabolism in thermophilic archaea is summarized in this contribution.     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

Production of highly purified hydroxytyrosol from Olea europaea leaf extract biotransformed by hyperthermophilic beta-glycosidase

A large amount of highly purified hydroxytyrosol (91-94% in weight) is obtained in short time by a simple biotransformation of Olea europaea leaf extract by a partially purified hyperthermophilic beta-glycosidase immobilized on chitosan support. The biotransformation conditions have been modulated for increasing the hydroxytyrosol yield, whilst chitosan and chitin matrices are used as adsorbent materials in liquid phase hydroxytyrosol extraction from the biotransformed mixtures. Natural and non-toxic hydroxytyrosol has been by this way produced from a vegetal source, and this compound appeared for the first time highly purified by natural and biocompatible safe biopolymers in comparison to previous results. Moreover, the GC analyses have displayed that the eluates from a two-step bioreactor have qualitative composition very similar to that of the extra-virgin olive oil polar fraction. The proposed bioreactor could also find application in the utilization of olive mill waste waters (OMWW), medium rich in large amounts of oleuropein, which can be converted in pharmacologically active compounds.     

Effects induced by mono- and divalent cations on protein regions responsible for thermal adaptation in β-glycosidase from<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>

The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus -glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1) an extra segment (83–124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2) a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the -glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg²⁺. Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 °C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107–R245 and E109–R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A–C intermolecular interface of Sgly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474–K72 and D473–R402, with consequent partial dissociation of the tetrameric structure.Abbreviations Amide I amide I band in a ²H₂O medium - EM energy minimization - ONPG o-nitrophenyl--d-galactopyranoside - Sgly Escherichia coli expressed Sulfolobus solfataricus -glycosidase     

Structural and functional characterization of N-terminally blocked peptides isolated from the venom of the social wasp Polybia paulista

Two novel peptides were isolated from the crude venom of the social wasp Polybia paulista, by using RP-HPLC under a gradient of MeCN from 5 to 60% (v/v) and named Polybine-I and -II. Further purification of these peptides under normal phase chromatography, rendered pure enough preparations to be sequenced by Edman degradation chemistry. However, both peptides did not interact with phenylisothiocyanate reagent, suggesting the existence of a chemically blocked N-terminus. Therefore, the sequences of both peptides were assigned by ESI-MS/MS under CID conditions, as follows: Polybine-I Ac-SADLVKKIWDNPAL-NH₂ (Mr 1610 Da) and Polybine-II Ac-SVDMVMKGLKIWPL-NH₂ (Mr 1657 Da). During the tandem mass spectrometry experiments, a loss of 43 a.m.u. was observed from the N-terminal residue of each peptide, suggesting the acetylation of the N-terminus. Subsequently, the peptides with and without acetylation were synthesized on solid phase and submitted to functional characterizations; the biological activities investigated were: hemolysis, chemotaxis of polymorphonucleated leukocytes (PMNL), mast cell degranulation and antibiosis. The results revealed that the acetylated peptides exhibited more pronounced chemotaxis of PMNL cells and mast cell degranulation than the respective non-acetylated congeners; no hemolytic and antibiotic activities were observed, irrespective to the blockage or not of the -amino groups of the N-terminal residues of each peptide. Therefore, the N-terminal acetylation may be related to the increase of the inflammatory activity of both peptides.     

Octreotide represses secretory-burst mass and nonpulsatile secretion but does not restore event frequency or orderly GH secretion in acromegaly

Octreotide is a potent somatostatin analog that inhibits growth hormone (GH) release and restricts somatotrope cell growth. The long-acting octreotide formulation Sandostatin LAR is effective clinically in approximately 60% of patients with acromegaly. Tumoral GH secretion in this disorder is characterized by increases in pulse amplitude and frequency, nonpulsatile (basal) release, and irregularity. Whether sustained blockade by octreotide can restore physiological secretion patterns in this setting is unknown. To address this question, we studied seven patients with GH-secreting tumors during chronic receptor agonism. Responses were monitored by sampling blood at 10-min intervals for 24 h, followed by analyses of secretion and regularity by multiparameter deconvolution and approximate entropy (ApEn). The somatostatin agonist suppressed GH secretory-burst mass, nonpulsatile (basal) GH release, and pulsatile secretion, thereby decreasing total GH secretion by 86% (range 70-96%). ApEn decreased from 1.203 +/- 0.129 to 0.804 +/- 0.141 (P = 0.032), denoting greater regularity. None of GH pulse frequency, basal GH secretion rates, or ApEn normalized. In summary, chronic somatostatin agonism is able to repress amplitude-dependent measures of excessive GH secretion in acromegaly. Presumptive tumoral autonomy is inferred by continued elevations of event frequency, overall pattern disruption (irregularity), and nonsuppressible basal GH secretion.     

Cell cycle time and rate of entry of cells into mitosis in the small intestine of young rats

Cell cycle time (T(C)) and the rate of entry of cells into mitosis (r(M)) in the jejunum and duodenum of young rats were investigated using the stathmokinetic method. The cell cycle times in the jejunum were 24.3 and 28.3 h in light and dark periods, respectively. Cell cycle times in the duodenum were 17.1 and 21.5 h in light and dark periods, respectively. Rates of entry of cells into mitosis in the jejunum were 1.2 and 1.1 cells/cell/h in light and dark periods and rates of entry of cells into mitosis in the duodenum were 1.4 and 1.8 cells/cell/h in light and dark periods, respectively. Although these changes to cell cycle time values are not statistically significant, the variation between the two periods should be considered in relation to its possible biological effects.     

Production of oligosaccharides by autohydrolysis of brewery's spent grain

Brewery's spent grain was treated with water in a process oriented towards the production of xylo-oligosaccharides (XOS). A wide range of temperatures and reaction times were tested and the effects of these operational variables on hemicellulose solubilization and reaction products were investigated. The maximal XOS yield (61% of the feedstock xylan) was obtained at 190 degrees C after 5 min of reaction. Several oligosaccharide mixtures with different molecular weight distributions were obtained depending on temperature and reaction time. Longer reaction times led to decreased oligosaccharide production and enhanced concentrations of monosaccharides, sugar decomposition products and acetic acid. With reaction times leading to the maximal yields of XOS, little decomposition into organic acids and aldehydes was found at all the temperatures assayed. From the composition of processed solids, it was calculated that 63-77% of the initial xylan was selectively solubilized in autohydrolysis treatments.