Allyl sulfur compounds and cellular detoxification system: effects and perspectives in cancer therapy.

Natural organosulfur compounds (OSCs) have been shown to have chemopreventive effects and to suppress the proliferation of tumor cells in vitro through the induction of apoptosis. The biochemical mechanisms underlying the antitumorigenic and anti-proliferative effects of garlic-derived OSCs are not fully understood. Several modes of action of these compounds have been proposed, and it seems likely that the rate of clearance of allyl sulfur groups from cells is a determinant of the overall response. The aim of this review is to focus attention on the effects of natural allyl sulfur compounds on the cell detoxification system in normal and tumor cells. It has been already reported that several natural allyl sulfur compounds induce chemopreventive effects by affecting xenobiotic metabolizing enzymes and inducing their down-activation. Moreover, different effects of water- and oil-soluble allyl sulfur compounds on enzymes involved in the detoxification system of rat tissues have been observed. A direct interaction of the garlic allyl sulfur compounds with proteins involved in the detoxification system was studied in order to support the hypothesis that proteins possessing reactive thiol groups and that are involved in the detoxification system and in the cellular redox homeostasis, are likely the preferential targets of these compounds. The biochemical transformation of the OSCs in the cell and their adducts with thiol functional groups of these proteins, could be considered relevant events to uncover the anticancer properties of the allyl sulfur compounds. Although additional studies, using proteomic approaches and transgenic models, are needed to identify the molecular targets and modes of action of these natural compounds, the allyl sulfur compounds can represent potential ideal agents in anticancer therapy, either alone or in association with other antitumor drugs.

Characterization of toad liver glutathione transferase.

The major form of glutathione transferase from the toad liver previously designed as Bufo bufo liver GST-7.6 (A. Aceto, B. Dragani, T. Bucciarelli, P. Sacchetta, F. Martini, S. Angelucci, F. Amicarelli, M. Miranda and C. Di Ilio, Biochem. J. 289 (1993) 417-422) has been characterized. According to its partial amino acid sequence, the toad enzyme may be included in the pi class GST and named bbGST P2-2. However, bbGST P2-2 appears to be immunologically, structurally and kinetically distinct from any other members of pi family, including bbGST P1-1, suggesting that it may constitute a subset of pi class GST. The data support the hypothesis that the transition from aquatic to terrestrial life causes a switch of the GST amphibian pattern promoting the expression of a GST form (bbGST P2-2) able to counteract, with higher efficiency, the toxic effects of reactive metabolites of oxidative metabolism and those of hydrophobic xenobiotics.

Identification of a conserved N-capping box important for the structural autonomy of the prion alpha 3-helix: the disease associated D202N mutation destabilizes the helical conformation.

Peptides corresponding to three alpha helices present in the C-terminal region of the human prion protein have been synthesized and their structural autonomy analyzed by circular dichroism (CD) and NMR spectroscopy. The results obtained indicate that the protein fragment corresponding to the alpha 3-helix, in contrast to alpha 1 and alpha 2 peptides, shows a complete structural autonomy. The chemical shifts values found for NH and CHalpha resonance of the isolated alpha 3 peptide, formed by 30 aminoacid residues, were markedly and surprisingly similar to the corresponding values of the alpha 3-helix in the protein. The structural autonomy of the alpha 3-helix is profoundly determined by the presence of the conserved capping box and, in part, by the ionic bond formed between Glu200 and Lys204. On the basis of these observations a novel PrP consensus pattern, centered on the alpha 3-helix region, has been defined. The data indicate that this autonomous and highly conserved region of the PrPc likely plays a critical role in folding and stability. This gives an explanation of why many of pathogenic mutations occur in this part of the molecule, sharing relevant effects on the overall protein conformation. In particular the D202N capping mutation almost completely destabilizes the isolated alpha 3 peptide. While it is well known that the D202N substitution is associated with a GSS disease, the possible structural basis of this fatal pathology has never been investigated. We propose that a lower alpha 3-helical propensity leading to a major destabilization of the PrPc molecule initiates the pathogenic process associated with D202N capping mutation.

Purification and characterization of a novel glutathione transferase from Ochrobactrum anthropi.

Glutathione transferase was purified from Ochrobactrum anthropi and its N-terminal sequence was determined to be MKLYYKVGACSLAPHIILSEAGLPY. The apparent molecular mass of the protein (24 kDa) was determined by SDS-polyacrylamide gel electrophoresis analysis. The amino acid sequence obtained showed similarities with known bacterial glutathione transferases in the range of 72-64%. Immunoblotting experiments performed with antisera raised against glutathione transferase from O. anthropi did not show cross-reactivity with two bacterial glutathione transferases belonging to Serratia marcescens and Proteus mirabilis.

Purification and characterization of three Pi class glutathione transferase from monkey (Macaca fascicularis) placenta.

Three forms of glutathione transferase (GST) with an apparent isoelectric point of pH 4.65 (GST I), 4.75 (GST II) and 4.9 (GST III) were resolved from the monkey (Macaca fascicularis) placenta after GSH-affinity chromatography followed by chromatofocusing. Substrate specificity, immunological reactivity, as well as N-terminal aminoacid sequences indicate that the three enzymes belongs to the pi class of GST. Reverse phase HPLC analysis indicates that the three GST arise from the combination of two different subunits eluting respectively at 29.60 +/- 0.10 min and 32.43 +/- 0.13 min. GST I is an homodimer of the 29.60 +/- 0.10 min subunit, GST III is an homodimer of the 32.43 +/- 0.13 min subunit, whereas the GST II is an heterodimer of the 29.60 +/- 0.10 min and 32.43 +/- 0.13 min subunits. Our results strongly suggest that unlike human, multiple forms of pi class GST exist in monkey placenta.

Unfolding and inactivation of monomeric superoxide dismutase from E. coli by SDS.

The inactivation and the unfolding of the naturally monomeric Cu, Zn, superoxide dismutase from E. coli upon addition of sodium dodecylsulphate have been studied. In contrast to the bovine enzyme, CD, EPR, NMR spectroscopy and pulsed low resolution NMR measurements found an unfolding transition followed by inactivation of the enzyme. During this transition the active site becomes accessible to the bulk water. The unfolding is reversible and both, the tridimensional structure of the protein and the active site, can be restored upon dialysis. In addition, unfolding occurs without loss of metals in the solution.

A computerized guideline for pressure ulcer prevention.

This paper illustrates the implementation of a computerized guideline for pressure ulcer prevention. In particular, it describes the aspects related to the site-specification of a guideline delivered by the Agency for Health Care Policy Research (AHCPR), to its integration with the electronic patient record, and to its implementation within the clinical routine. The primary goal of the system is both to facilitate nurses assessing the risk of ulcer development, and to manage patients at risk by producing daily prevention work-plans. Concerning this functionality, particular attention has been paid to manage nurse's non-compliance with the guideline suggestions and to collect data for evaluating the guideline impact. Moreover, since it is well known that nurses are often over-loaded, the human computer interaction has been studied in such a way to optimise the time spent for data input. An additional functionality of the system is the novice nurses' education - they can browse a graphical representation of the guideline, asking details about the different tasks, and they can simulate patients to obtain real-time advice. The educational tool is written in Java and it is based on a representation of the guideline as a relational database. A preliminary evaluation of the system has been performed and the results are presented on the management of about 40 patients.

A computerised guideline for pressure ulcer prevention.

This work illustrates the implementation of a computerised guideline for the pressure ulcers prevention. In particular, we describe the site-specification of a guideline delivered by the Agency for Health Care Policy Research, its integration with the electronic patient record, and its introduction within the clinical routine. The system facilitates trained nurses in the patient management by producing daily workplans, and novice nurses by running as an educational tool.

FtsQ interaction mutants: a way to identify new antibacterial targets.

FtsQ is a highly conserved component of the divisome that plays a central role in the assembly of early and late cell division proteins. The biological activity of this protein is still largely unknown, but its ability to interact with many components of the divisome was described by both two-hybrid assays and co-immunoprecipitation experiments. This paper describes the behaviour of ftsQ point mutants, created by random mutagenesis without regard to their phenotype, in which FtsQ is impaired in its ability to interact with its Escherichia coli division partners. Our results allow the identification of FtsQ residues involved in the interaction with other partner proteins and the determination of the biological significance of these interactions. The knowledge derived by this study could constitute not only the basis for understanding how these proteins assemble in the divisome, but also a starting point for the design of new antibacterial drugs that disrupt the bacterial division machinery.

Acetylation of RTN-1C regulates the induction of ER stress by the inhibition of HDAC activity in neuroectodermal tumors.

Reticulons are a family of highly conserved proteins, localized in the endoplasmic reticulum (ER) and involved in different cellular functions, such as intracellular membrane trafficking, apoptosis and nuclear envelope formation. The reticulon protein family consists of four members, but their specific functions are presently poorly understood. RTN-1C overexpression triggers apoptosis, regulating ER stress versus DNA damage-induced cell death in a mutually exclusive way. The different RTN isoforms share a C-terminal reticulon homology domain containing two hydrophobic segments and a 66-amino acid hydrophilic loop. In the C-terminal region of RTN-1C, a unique consensus sequence (GAKRH) has recently been identified, showing 100% identity with the DNA-binding domain of histone H4. In this study, we show that this sequence is essential for RTN-1C-mediated apoptosis. It is noteworthy that the lysine 204 present in this region is post-translationally modified by acetylation and that this event is associated with a significant decrease in histone deacetylase activity and contributes to RTN-1C binding to DNA. These data demonstrate a molecular mechanism by which RTN-1C controls apoptosis and indicate this protein to be a novel potential target for cancer therapy.

Structural characterization of human glyoxalase II as probed by limited proteolysis.

Human glyoxalase II is partially proteolyzed by trypsin, under non denaturing conditions, only at the level of the C-terminal region. The proteolytic cleavage resulted in an inactivation of the enzyme without loss of the secondary structure. Sodium dodecyl sulphate polyacrylamide gel-electrophoresis and microsequence analysis showed that the glyoxalase II is proteolyzed at the level of Arg 184 and Lys 230 and undergoes a third cleavage in a region located at the beginning of the supposed C-terminal domain. The proteolysis occurs either in the presence or in the absence of specific inhibitors. Our limited proteolysis experiments and secondary structure prediction give evidence for the presence of two domains characterized by different pattern of secondary structure.

Zn(2+) ions selectively induce antimicrobial salivary peptide histatin-5 to fuse negatively charged vesicles. Identification and characterization of a zinc-binding motif present in the functional domain.

The salivary antimicrobial peptide histatin-5 is able to aggregate and fuse negatively charged small unilamellar vesicles, and this fusogenic activity is selectively induced by the presence of zinc ions. Circular dichroism spectroscopy shows that histatin-5, in the presence of negatively charged vesicles and zinc ions, undergoes a conformational change leading to the stabilization of an alpha-helical secondary structure. We attribute the specific action of the zinc ions to the presence of a consensus sequence, HEXXH, located in the C-terminal functional domain of histatin-5, a recognized zinc-binding motif in many proteins. Two-dimensional proton NMR spectroscopy of histatin-5 in a trifluoroethanol/water mixture (a membrane mimetic environment) has been performed and the results analyzed by means of distance geometry and restrained molecular dynamics simulations. Our results reveal that the peptide chain, including the Zn-binding consensus sequence corresponding to residues 15-19, is in a helicoidal conformation. Comparison of the chemical shifts of the individual amino acids in histatin-5 with those recently reported in other solvents indicates that trifluoroethanol/water has a structuring capability somewhere between water and dimethyl sulfoxide. The mechanism of action of this antimicrobial peptide is discussed on the basis of its structural characteristics with particular attention to the Zn-binding motif.

The conserved N-capping box in the hydrophobic core of glutathione S-transferase P1-1 is essential for refolding. Identification of a buried and conserved hydrogen bond important for protein stability.

The second domain of cytosolic glutathione S-transferases (GSTs) contains a strictly conserved N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) at the beginning of alpha6-helix in the hydrophobic core of the molecule. Considering the specific function attributed to capping box residues in the helix nucleation, we decided to investigate, by site-directed mutagenesis, the role that this motif could have in the folding and stability of human GSTP1-1. Both capping box mutants, S150A and D153A, were significantly more thermolabile than wild-type GSTP1-1, indicating that the local destabilization of the alpha6-helix determined by a single capping residue mutation affects the overall stability of the protein. The results also show that, in addition to capping interactions, an important role in the stability of the final structure of the protein is played by a buried and conserved hydrogen bond formed between the side chain of Asp-153 and the amide NH of Ile-144 located in the long loop preceding alpha6-helix. Reactivation experiments in vitro indicate that the N-capping box is essential for refolding of the denatured protein at a physiological temperature. The results suggest that during folding this buried and conserved motif, making a definite set of native-like contacts, determines the formation of a specific folding nucleus that probably represents a transition state of the folding process.

Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies.

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya (wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3',4'-trihydroxychalcone and 4, 3',4'-trihydroxy-3-methoxychalcone to the corresponding 3, 3'-biflavanones, as mixtures of racemic and meso forms.

Identification of an N-capping box that affects the alpha 6-helix propensity in glutathione S-transferase superfamily proteins: a role for an invariant aspartic residue.

We have identified an N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) that is strictly conserved, at the beginning of alpha 6 helix, in all glutathione S-transferases (GSTs) and most of the related superfamily proteins. By using CD and peptide modelling we have demonstrated that the capping box residues have an important role in determining the helical conformation adopted by this fragment in the hydrophobic environment of the protein. This is an example in which a local motif, contributing to nucleation of a structural element essential to the global folding of the protein, is strictly conserved in a superfamily of homologous proteins.

Purification and characterization of glutathione transferases from the sea bass (Dicentrarchus labrax) liver.

Two forms of glutathione transferase were purified from liver cytosol of the sea bass (Dicentrarchus labrax) by GSH-Sepharose affinity chromatography followed by chromatofocusing. The major enzyme (DL-GST-6.7; 75% of total activity bound to the column) has a pI value of 6.7 and is composed of two subunits of apparent molecular mass 26.5 kDa. The minor enzyme (DL-GST-8.2; 25% of total activity bound to the column) has a pI value of 8.2 and is composed of two subunits of molecular mass 23.5 kDa. Both isoenzymes appear to have blocked N-terminal. The purified proteins were characterized with respect to substrate specificity, CD spectra, TNS binding properties (with 2-toluidinylnaphthalene 6-sulfonate), and immunological reactivity. Partial internal amino acid sequence was also determined for each isoenzyme. The results obtained suggest that DL-GST-6.7 and DL-GST8.2 are novel GSTs belonging, respectively, to theta and alpha classes.

Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi.

The gene coding for a novel glutathione S-transferase (GST) has been isolated from the bacterium Ochrobactrum anthropi. A PCR fragment of 230 bp was obtained using oligonucleotide primers deduced from N-terminal and 'internal' sequences of the purified enzyme. The gene was obtained by screening of a genomic DNA partial library from O. anthropi constructed in pBluescript with a PCR fragment probe. The gene encodes a protein (OaGST) of 201 amino acids with a calculated molecular mass of 21738 Da. The product of the gene was expressed and characterized; it showed GST activity with substrates 1-chloro-2, 4-dinitrobenzene (CDNB), p-nitrobenzyl chloride and 4-nitroquinoline 1-oxide, and glutathione-dependent peroxidase activity towards cumene hydroperoxide. The overexpressed product of the gene was also confirmed to have in vivo GST activity towards CDNB. The interaction of the recombinant GST with several antibiotics indicated that the enzyme is involved in the binding of rifamycin and tetracycline. The OaGST amino acid sequence showed the greatest identity (45%) with a GST from Pseudomonas sp. strain LB400. A serine residue in the N-terminal region is conserved in almost all known bacterial GSTs, and it appears to be the counterpart of the catalytic serine residue present in Theta-class GSTs. Substitution of the Ser-11 residue resulted in a mutant OaGST protein lacking CDNB-conjugating activity; moreover the mutant enzyme was not able to bind Sepharose-GSH affinity matrices.