Thermal and high hydrostatic pressure inactivation of myrosinase from green cabbage: a kinetic study

Myrosinase, a family of enzymes which coexist with glucosinolates in all Brassica vegetables, catalyses the hydrolysis of glucosinolates to yield compounds that can have beneficial effects on human health. In this study, the thermal and pressure inactivation of myrosinase from green cabbage was kinetically investigated. Thermal inactivation started at 35 C and inactivation kinetics was studied in the temperature range 35–55 C. Thermal inactivation of green cabbage myrosinase followed the well known consecutive step model. Pressure inactivation started at 300 MPa, even at 10 C, and the consecutive step model effectively described pressure inactivation in the range 300–450 MPa at 10 C. The combined effects of applying various pressures and temperatures on myrosinase inactivation kinetics were studied in the ranges 35–50 C and, 100–400 MPa. The inactivation followed first-order kinetics at all of the applied combinations. This study demonstrates that myrosinase from green cabbage is highly susceptible to both thermal and high pressure processing. Furthermore, it is also noted that myrosinase stability during processing appears to vary widely between different Brassica species.

Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7

P>Type III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.

Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130-kd transmembrane glycoprotein and a member of the growing family of receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 is expressed on platelets, certain T cells, monocytes, neutrophils, and vascular endothelial cells and is involved in a range of cellular processes, though the role of PECAM-1 in platelets is unclear. Cross-linking of PECAM-1 results in phosphorylation of the ITIM allowing the recruitment of signaling proteins that bind by way of Src-homology domain 2 interactions. Proteins that have been implicated in the negative regulation of cellular activation by ITIM-bearing receptors include the tyrosine phosphatases SHP-1 and SHP-2. Tyrosine phosphorylation of immunoreceptor tyrosine-based activatory motif (ITAM)-bearing receptors such as the collagen receptor GPVI-Fc receptor gamma-chain complex on platelets leads to activation. Increasing evidence suggests that ITIM- and ITAM-containing receptors may act antagonistically when expressed on the same cell. In this study it is demonstrated that cross-linking PECAM-1 inhibits the aggregation and secretion of platelets in response to collagen and the GPVI-selective agonist convulxin. In these experiments thrombin-mediated platelet aggregation and secretion were also reduced, albeit to a lesser degree than for collagen, suggesting that PECAM-1 function may not be restricted to the inhibition of ITAM-containing receptor pathways. PECAM-1 activation also inhibited platelet protein tyrosine phosphorylation stimulated by convulxin and thrombin; this was accompanied by inhibition of the mobilization of calcium from intracellular stores. These data suggest that PECAM-1 may play a role in the regulation of platelet function in vivo.

The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activitor of T cells (LAT) in platelets stimulated by collagen and convulxin.

There is extensive evidence to show that phosphatidylinositol 3-kinase plays an important role in signaling by the immune family of receptors, which has recently been extended to include the platelet collagen receptor, glycoprotein VI. In this report we present two potential mechanisms for the regulation of this enzyme on stimulation of platelets by collagen. We show that on stimulation with collagen, the regulatory subunit of phosphatidylinositol 3-kinase associates with the tyrosine-phosphorylated form of the adapter protein linker for activator of T Cells (LAT) and the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif of the Fc receptor gamma-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor gamma-chain and LAT with p85 are rapid and supported by the Src-homology 2 domains of the regulatory subunit. We did not obtain evidence to support previous observations that the regulatory subunit of phosphatidylinositol 3-kinase is regulated through association with the tyrosine kinase Syk. The present results provide a molecular basis for the regulation of the p85/110 form of phosphatidylinositol 3-kinase by GPVI, the collagen receptor that underlies activation.

Occurrence of Poecilochirus austroasiaticus (Acari: Parasitidae) in forensic autopsies and its application on postmortem interval estimation

Despite the fact that mites were used at the dawn of forensic entomology to elucidate the postmortem interval, their use in current cases remains quite low for procedural reasons such as inadequate taxonomic knowledge. A special interest is focused on the phoretic stages of some mite species, because the phoront-host specificity allows us to deduce in many occasions the presence of the carrier (usually Diptera or Coleoptera) although it has not been seen in the sampling performed in situ or in the autopsy room. In this article, we describe two cases where Poecilochirus austroasiaticus Vitzthum (Acari: Parasitidae) was sampled in the autopsy room. In the first case, we could sample the host, Thanatophilus ruficornis (Küster) (Coleoptera: Silphidae), which was still carrying phoretic stages of the mite on the body. That attachment allowed, by observing starvation/feeding periods as a function of the digestive tract filling, the establishment of chronological cycles of phoretic behavior, showing maximum peaks of phoronts during arrival and departure from the corpse and the lowest values in the phase of host feeding. From the sarcosaprophagous fauna, we were able to determine in this case a minimum postmortem interval of 10 days. In the second case, we found no Silphidae at the place where the corpse was found or at the autopsy, but a postmortem interval of 13 days could be established by the high specificity of this interspecific relationship and the departure from the corpse of this family of Coleoptera.

GTP binding and intramolecular regulation by the ROC domain of Death Associated Protein Kinase 1

The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases.

Induction of modular classification rules: using Jmax-pruning

The Prism family of algorithms induces modular classification rules which, in contrast to decision tree induction algorithms, do not necessarily fit together into a decision tree structure. Classifiers induced by Prism algorithms achieve a comparable accuracy compared with decision trees and in some cases even outperform decision trees. Both kinds of algorithms tend to overfit on large and noisy datasets and this has led to the development of pruning methods. Pruning methods use various metrics to truncate decision trees or to eliminate whole rules or single rule terms from a Prism rule set. For decision trees many pre-pruning and postpruning methods exist, however for Prism algorithms only one pre-pruning method has been developed, J-pruning. Recent work with Prism algorithms examined J-pruning in the context of very large datasets and found that the current method does not use its full potential. This paper revisits the J-pruning method for the Prism family of algorithms and develops a new pruning method Jmax-pruning, discusses it in theoretical terms and evaluates it empirically.

Jmax-pruning: a facility for the information theoretic pruning of modular classification rules

The Prism family of algorithms induces modular classification rules in contrast to the Top Down Induction of Decision Trees (TDIDT) approach which induces classification rules in the intermediate form of a tree structure. Both approaches achieve a comparable classification accuracy. However in some cases Prism outperforms TDIDT. For both approaches pre-pruning facilities have been developed in order to prevent the induced classifiers from overfitting on noisy datasets, by cutting rule terms or whole rules or by truncating decision trees according to certain metrics. There have been many pre-pruning mechanisms developed for the TDIDT approach, but for the Prism family the only existing pre-pruning facility is J-pruning. J-pruning not only works on Prism algorithms but also on TDIDT. Although it has been shown that J-pruning produces good results, this work points out that J-pruning does not use its full potential. The original J-pruning facility is examined and the use of a new pre-pruning facility, called Jmax-pruning, is proposed and evaluated empirically. A possible pre-pruning facility for TDIDT based on Jmax-pruning is also discussed.

Computationally efficient induction of classification rules with the PMCRI and J-PMCRI frameworks

In order to gain knowledge from large databases, scalable data mining technologies are needed. Data are captured on a large scale and thus databases are increasing at a fast pace. This leads to the utilisation of parallel computing technologies in order to cope with large amounts of data. In the area of classification rule induction, parallelisation of classification rules has focused on the divide and conquer approach, also known as the Top Down Induction of Decision Trees (TDIDT). An alternative approach to classification rule induction is separate and conquer which has only recently been in the focus of parallelisation. This work introduces and evaluates empirically a framework for the parallel induction of classification rules, generated by members of the Prism family of algorithms. All members of the Prism family of algorithms follow the separate and conquer approach.

Mononuclear palladium and heterodinuclear palladium–ruthenium complexes of semicarbazone ligands: synthesis, characterization, and application in C–C cross-coupling reactions

Reaction of salicylaldehyde semicarbazone (L-1), 2-hydroxyacetophenone semicarbazone (L-2), and 2-hydroxynaphthaldehyde semicarbazone (L-3) with [Pd(PPh3)(2)Cl-2] in ethanol in the presence of a base (NEt3) affords a family of yellow complexes (1a, 1b and 1c, respectively). In these complexes the semicarbazone ligands are coordinated to palladium in a rather unusual tridentate ONN-mode, and a PPh3 also remains coordinated to the metal center. Crystal structures of the 1b and 1c complexes have been determined, and structure of 1a has been optimized by a DFT method. In these complexes two potential donor sites of the coordinated semicarbazone, viz. the hydrazinic nitrogen and carbonylic oxygen, remain unutilized. Further reaction of these palladium complexes (1a, 1b and 1c) with [Ru(PPh3)(2)(CO)(2)Cl-2] yields a family of orange complexes (2a, 2b and 2c, respectively). In these heterodinuclear (Pd-Ru) complexes, the hydrazinic nitrogen (via dissociation of the N-H proton) and the carbonylic oxygen from the palladium-containing fragment bind to the ruthenium center by displacing a chloride and a carbonyl. Crystal structures of 2a and 2c have been determined, and the structure of 2b has been optimized by a DFT method. All the complexes show characteristic H-1 NMR spectra and, intense absorptions in the visible and ultraviolet region. Cyclic voltammetry on all the complexes shows an irreversible oxidation of the coordinated semicarbazone within 0.86-0.93 V vs. SCE, and an irreversible reduction of the same ligand within -0.96 to -1.14 V vs. SCE. Both the mononuclear (1a, 1b and 1c) and heterodinuclear (2a, 2b and 2c) complexes are found to efficiently catalyze Suzuki, Heck and Sonogashira type C-C coupling reactions utilizing a variety of aryl bromides and aryl chlorides. The Pd-Ru complexes (2a, 2b and 2c) are found to be better catalysts than the Pd complexes (1a, 1b and 1c) for Suzuki and Heck coupling reactions.

Synthesis, structure and electrochemical properties of some thiosemicarbazone complexes of ruthenium

Reaction of salicylaldehyde thiosemicarbazone (L-1), 2-hydroxyacetophenone thiosemicarbazone (L-2) and 2-hydroxynapthaldehyde thiosemicarbazone (L-3) with [Ru(dmso)(4)Cl-2] affords a family of three dimeric complexes (1), (2) and (3) respectively. Crystal structure of the complex (3) has been determined. In these complexes, each monomeric unit consists of one ruthenium center and two thiosemicarbazone ligands, one of which is coordinated to ruthenium as O,N,S-donor and the other as N,S-donor forming a five-membered chelate ring. Two such monomeric units remain bridged by the sulfur atoms of the O,N,S-coordinated thiosemicarbazones. Due to this sulfur bridging, the two ruthenium centers become so close to each other, that a ruthenium-ruthenium single bond is also formed. All the complexes are diamagnetic in the solid state and in dimethylsulfoxide solution show intense absorptions in the visible and ultraviolet region. Origin of these spectral transitions has been established from DFT calculations. Cyclic voltammetry on the complexes shows two irreversible ligand oxidations on the positive side of SCE and two irreversible ligand reductions on the negative side.

Syntheses, structures and efficient catalysis for C–C coupling of some benzaldehyde thiosemicarbazone complexes of palladium

Reaction of the 4-R-benzaldehyde thiosemicarbazones (denoted in general as L-R; R = OCH(3), CH(3), H, Cl and NO(2)) with trans-[Pd(PPh(3))(2)Cl(2)] afforded a group of mixed-ligand complexes (denoted in general as 1-R) incorporating a N,S-coordinated thiosemicarbazone. a triphenylphosphine and a chloride. Similar reaction with Na(2)[PdCl(4)] afforded a family of bis-thiosemicarbazone complexes (denoted in general as 2-R), where each ligand is N,S-coordinated. Crystal structures of 1-CH(3), 1-NO(2), 2-OCH(3), 2-NO(2) and L-NO(2) have been determined. In all the complexes the thiosemicarbazones are coordinated to the metal center, via dissociation of the acidic proton, as bidentate N,S-donors forming five-membered chelate rings. With reference to the structure of the uncoordinated thiosemicarbazone, this coordination mode is associated with a conformational change around the C=N bond. All the 1-R and 2-R complexes display intense absorptions in the visible region. Catalytic activity of the 1-R and 2-R complexes towards some C-C coupling reactions (e.g. Suzuki, Heck and Sonogashira) has been examined and while both are found to be efficient catalysts, 1-R is much better catalyst than 2-R.