Lipophilic Metabolites from Five‐Needle Pines,Pinus armandii and Pinus kwangtungensis,Exhibiting Antibacterial Activity

Lipophilic extractive metabolites from needles and defoliated twigs of Pinus armandii and P. kwangtungensis were studied by GC/MS. Needles of P. armandii contained predominantly 15‐O‐functionalized labdane type acids (anticopalic acid), fatty acids, nonacosan‐10‐ol, sterols, nonacosan‐10‐ol and sterol saponifiable esters, and acylglycerols, while P. kwangtungensis needles contained no anticopalic acid, but more trinorlabdane (14,15,16‐trinor‐8(17)‐labdene‐13,19‐dioic acid) and other labdane type acids, nonacosan‐10‐ol and its saponifiable esters. The major compounds in the P. armandii defoliated twig extract were abietane and isopimarane type acids, fatty acids, sterols, labdanoids (cis‐abienol), cembranoids (isocembrol and 4‐epi‐isocembrol), saponifiable sterol esters, and acylglycerols. The same extract of P. kwangtungensis contained larger quantities of fatty acids, caryophyllene oxide, serratanoids, sterols, saponifiable sterol esters, and acylglycerols, but lesser amounts of abietane and isopimarane type acids, cis‐abienol, and lacked cembranoids. Both twig and needle extracts of P. armandii and P. kwangtungensis, as well as the extracts’ fractions, significantly inhibited the growth of Gram‐negative bacteria Serratia marcescens with MIC of 0.1 mg ml^?1, while in most cases they slightly stimulated the growth of Gram‐positive bacteria Bacillus subtilis at the same concentrations. Thus, lipophilic extractive compounds from the needles and defoliated twigs of both pines are prospective for the development of antiseptics against Gram‐negative bacteria.     

Nonequivalence of transketolase active centers with respect to acceptor substrate binding

The interaction of transketolase with its acceptor substrate, ribose 5-phosphate, has been studied. The active centers of the enzyme were shown to be functionally nonequivalent with respect to ribose 5-phosphate binding. Under the conditions where only one out of the two active centers of transketolase is functional, their affinities for ribose 5-phosphate are identical. The phenomenon of nonequivalence becomes apparent when the substrate interacts with one of the two active centers. As a consequence of such interaction, the affinity of the second active center for ribose 5-phosphate decreases.     

Interleukin-7-aggravated joint inflammation and tissue destruction in collagen-induced arthritis is associated with T-cell and B-cell activation

Introduction

We sought to investigate the capacity of interleukin (IL)-7 to enhance collagen-induced arthritis and to study by what mechanisms this is achieved.

Methods

Mice received multiple injections with IL-7 or phosphate-buffered saline (PBS) as a control. Arthritis severity and incidence were determined by visual examination of the paws. Joint destruction was determined by assessing radiographs and immunohistochemistry of the ankle joints. Total cellularity and numbers of T-cell and B-cell subsets were assessed, as well as ex vivo production of interferon-γ (IFN-γ), IL-17, and IL-4. Proinflammatory mediators were measured in serum with multianalyte profiling.

Results

IL-7 increased arthritis severity and radiology-assessed joint destruction. This was consistent with IL-7-increased intensity of cell infiltrates, bone erosions, and cartilage damage. Splenic CD19⁺ B cells and CD19⁺/GL7⁺ germinal center B cells, as well as CD4 and CD8 numbers, were increased by IL-7. IL-7 expanded memory T cells, associated with increased percentages of IFN-γ-, IL-4-, and IL-17-producing CD4⁺ T cells. On antigen restimulation of draining lymph node cells in vitro IL-7 treatment was found to increase IFN-γ and IL-17 production, whereas IL-4 was reduced. IL-7 also increased concentrations of proinflammatory mediators, indicative of T-cell activation (sCD40L), vascular activation (VCAM-1, VEGF), tissue destruction (fibroblast growth factor-basic (FGF-b), LIF), and chemotaxis (MIP-1γ, MIP-3β, lymphotactin, MDC, and MCP-5).

Conclusions

In arthritic mice, IL-7 causes expansion of T and B cells, associated with increased levels of proinflammatory mediators. IL-7 intensifies arthritis severity and joint destruction, accompanied by increased Th1 and Th17 activity. These data indicate that IL-7 could be an important mediator in arthritic conditions and that targeting IL-7 or its receptor represent novel therapeutic strategies.     

New evidence for cofactor's amino group function in thiamin catalysis by transketolase

Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4′-methylamino-thiamin diphosphate. We demonstrated the kinetic stability of the dihydroxyethyl carbanion/enamine intermediate to be dependent on the functionality of the 4′-aminopyrimidine moiety of thiamin diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. König, R. Kluger, G.A. Kochetov, G. Schneider, G. Hübner, Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisae, FEBS J. (2005) 272 1326-1342]. This paper extends these investigations of the function of the coenzyme’s aminopyrimidine in transketolase catalysis exemplified for the 4′-monomethylamino-thiamin diphosphate analogue. Here, we report near UV circular dichroism data and NMR-based analysis of reaction intermediates that give evidence for a strong destabilisation of the carbanion/enamine of DHE-4’-monomethylamino-thiamin diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to the conjugate acid of the carbanion/enamine intermediate, an assignment additionally corroborated by ¹H NMR-based intermediate analysis. As opposed to the kinetically stabilized carbanion/enamine intermediate in transketolase when reconstituted with the native cofactor, DHE-4′-monomethylamino-thiamin diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale.     

Effect of bivalent cations on the interaction of transketolase with its donor substrate

The effect of the type of the cation cofactor of transketolase (i.e., Ca2+ or Mg2+) on its interaction with xylulose 5-phosphate (donor substrate) has been studied. In the presence of magnesium, the active centers of the enzyme were functionally equivalent with respect to xylulose 5-phosphate binding and exhibited identical affinities for the donor substrate. Substitution of Ca2+ for Mg2+ results in the loss of the equivalence. In particular, this becomes apparent on binding of xylulose 5-phosphates to one of the two active centers of the enzyme, which caused the second center to undergo a several fold decrease in the affinity for the donor substrate.     

Can Nep1-like proteins form oligomers?

Nep1-like proteins (NLPs) are a novel family of microbial elicitors of plant necrosis that induce a hypersensitive-like response in dicot plants. The spatial structure and role of these proteins are yet unknown. In a paper published in BMC Plant Biology (2008; 8:50) we have proposed that the core region of Nep1-like proteins (NLPs) belong to the Cupin superfamily. Based on what is known about the Cupin superfamily, in this addendum to the paper we discuss how NLPs could form oligomers.Key words: quaternary structure, necrosis and ethylene inducing proteins, NLPs, MpNEP1, MpNEP2, NPP1, Moniliophthora perniciosa, Phytophthora parasiticaCupins may be organized as monomers, dimers, hexamers and octamers of β-barrel domains.¹ To the best of our knowledge trimers have not been detected yet. The interaction of two monomers building up a dimeric structure is basically performed by three types of interactions: hydrophobic interactions between β-strands in different subunits, salt bridges and hydrogen bonds between β-strands. In cupin dimers, the hydrophobic interactions occur between two βI strands in different subunits (Fig. 1A and B). This strand represents the central axis of rotation of the dimer as one residue in βI interacts with the corresponding residue in the other subunit (Fig. 1B). Therefore, all residues in βI must be hydrophobic, as one residue interacts with the other subunit and the next one in the sequence interacts with the interior of the protein. Charged residues in βI would disrupt such interactions. Most cupin dimers have strong hydrophobic residues such as tryptophan (W), phenylalanine (F) and methionine (M) pointing towards the own subunit (↓), while small hydrophobic residues such as leucine (L), isoleucine (I), and valine (V) point to the other subunit (↑). A particular case is leucine that interacts with other subunits, for instance, βI = liaW (positions 217–220 in Fig. 1B) and βI = LVsw of type I and II NLP consensuses, respectively. Therefore, the pattern of hydropathicity suggests that the side chain orientation is βI = l217 ↑ i218 ↓ a219 ↑ W220 ↓ d221 ↑. However we observe that just after βI there is a charged residue (aspartate D221) which would point outwards disrupting the dimer or at least making it less stable. It is interesting to observe that the requirement for a negatively charged residue at this last position is very high: 96% of all type I NLPs contains an aspartate (D) or glutamate (E) indicating an important role for it, maybe in avoiding dimerization of the NLPs. A second interesting hypothesis is as follows: several cupins are oxygenases, decarboxylases, etc. and use a negatively charged residue, such as aspartate or glutamate as proton donor.¹ Now, if the alternate pattern of side chains of the residues is βI = l217 ↓ i218 ↑ a219 ↓ W220 ↑ d221 ↓, instead of the previous one, then the aspartate or glutamate residue would point to the hydrophobic pocket and would be positioned to interact with the metal ion, as in cupins with enzymatic activity. However, there are no experimental evidences that the NLPs have enzymatic activity.Open in a separate windowFigure 1(A) Three-dimensional structure prediction for type I NLP consensus, (B) Interface between two βI strands in type I NLP consensus. From the left to the right: EF-coil with the conserved residue H162, βC and βH strands (superposed) with the conserved histidines H133 and H135 in βC, H193 and leucine L195 in βH, W220 in βI and W118 in βB. The strands in the right subunit follow the same pattern but rotated.The second type of interaction is salt bridges between charged residues in different subunits. Analyzing all interacting side chains in the 1VJ2 protein (dimer), we verify that the charged side chains of N35 and E57 (numbers in original structure) are only 2.72 Å apart. In the NLPs, this corresponds to N108^36% (Q108^60%) at the border of βB and E138^98%. The negatively charged residue D125 helps to correct the orientation of the subunits in relation to each other avoiding any disorientation. The high conservation level of these residues suggests that NLPs are dimeric structures. However, as we will see next, only hydrophobic and charged interactions are not enough to build a dimer.Garcia et al. (2007)² have used small angle X-ray scattering (SAXS) to show that, in solution, at low concentrations (<2 mg/ml) the two copies of the NLPs of Moniliophthora perniciosa, MpNEP1 and MpNEP2, exist as dimers and monomers, respectively. The same technique showed that at higher concentrations, >5 mg/ml, both proteins exist as dimers, as is the case for PpNPP1.² They also reported, based on electrophoresis analysis, that PpNPP1 and MpNEP1 exist as oligomers and MpNEP2 as monomers.² However, experiments with the PpNPP1 in size exclusion chromatography using myoglobin as size standard suggest that PpNPP1 is a monomer.³ Figure 2 compares MpNEP1, MpNEP2 and PpNPP1, where the most relevant differences in sequence are marked with asterisks (*) and are possibly related to the differences in oligomeric properties between MpNEP1 and PpNPP1 with MpNEP2. These positions are methionine M27 and leucine L35, which occur only in MpNEP2, glycine G250, which occurs only in MpNEP2 and NEP1 (Fusarium oxysporum) and lysine K31, which occurs only MpNEP2, {"type":"entrez-protein","attrs":{"text":"BAB04114","term_id":"10173008"}}BAB04114 (Bacillus halodurans) and {"type":"entrez-protein","attrs":{"text":"AAU23136","term_id":"52003194"}}AAU23136 (Bacillus licheniformis). The other residues are aspartate D28, which occurs 9 times and alanine A37 which occurs 7 times of all investigated NLPs. Thus, the sequence mdHDkiakl at the start of the NLPs seems to explain the monomeric state of MpNEP2, although at higher concentrations they form dimers. Besides the weak hydrophobic interactions, dimeric cupins and bicupins (two β barrels in the same sequence building up a dimeric-like 4d-structure) are stable structures (see Fig. 1 in ref. ⁴). By aggregating the first β-strand in the start domain of one β-barrel to the ABIDG β-sheet of the other β-barrel, composing a big ABIDGY β-sheet (Y is the first β-strand). For instance, using the bicupin 1L3J (oxalate decarboxylase) as template, the low confidence level β-strand at position 26–33 (v in H29D30 avv) in type I NLPs corresponds to the first β-strand. Since this proceeds from both barrels they can build a stable structure (see Fig. 1 in ref. ⁴). The quaternary structure is related to the presence of interaction residues in the BID β-sheet of the cupin structure. These are present in the NLPs and would enable them to form dimers.Open in a separate windowFigure 2Alignment of type I NLP consensus, PpNPP1, MpNEP1 and MpNEP2. Solid line boxes are β-strands, double line boxes are α-helices. The sequence positions marked with asterisks (*) are possibly related to the differences in oligomeric properties between MpNEP1 and PpNPP1 with MpNEP2.     

A prototype of an electric-discharge gas flow oxygen−iodine laser: I. Modeling of the processes of singlet oxygen generation in a transverse cryogenic slab RF discharge

The existing kinetic model describing self-sustained and electroionization discharges in mixtures enriched with singlet oxygen has been modified to calculate the characteristics of a flow RF discharge in molecular oxygen and its mixtures with helium. The simulations were performed in the gas plug-flow approximation, i.e., the evolution of the plasma components during their motion along the channel was represented as their evolution in time. The calculations were carried out for the O₂: He = 1: 0, 1: 1, 1: 2, and 1: 3 mixtures at an oxygen partial pressure of 7.5 Torr. It is shown that, under these conditions, volumetric gas heating in a discharge in pure molecular oxygen prevails over gas cooling via heat conduction even at an electrode temperature as low as ~100 K. When molecular oxygen is diluted with helium, the behavior of the gas temperature changes substantially: heat removal begins to prevail over volumetric gas heating, and the gas temperature at the outlet of the discharge zone drops to ~220–230 K at room gas temperature at the inlet, which is very important in the context of achieving the generation threshold in an electric-discharge oxygen?iodine laser based on a slab cryogenic RF discharge.