Diel patterns of leaf C export and of main shoot growth for Flaveria linearis with altered leaf sucrose-starch partitioning

Diel C export from source leaves of two Flaveria linearis lines [85-1: high cytosolic fructose-1,6-bisphosphatase (cytFBPase) and 84-9: low cytFBPase] were estimated using three methods, including leaf steady-state (14)CO(2) labelling, leaf metabolite analysis, and leaf dry mass analysis in conjunction with leaf CO(2) exchange measurements. Synthesis and accumulation of starch during the daytime were much higher in 84-9. Relative (14)C-export (export as a % of photosynthesis) in the light was 36% higher in 85-1. The diel export patterns from (14)C-analyses correlated with those based on metabolite or dry weight/gas exchange analyses during the daytime, but not during the night. Night-time export estimated from (14)C-disappearance was 3.6 times lower than those estimated using the other methods. Even though the starch degradation at night was greater for 84-9, night-time export in 84-9 was similar to 85-1, since 84-9 showed both higher respiration and accumulation of soluble sugars (i.e. glucose) at night. Patterns of (14)C allocation to sink organs were also different in the two lines. Main stem growth was less in 84-9, being reduced most in the light when leaf export was lower relative to 85-1. Supplementation with sucrose for 1 h daily via the roots at a time when leaf export in 84-9 was low relative to 85-1 increased the stem growth rate of 84-9 to a level similar with that of 85-1. This study provides evidence that diel C availability predicted by source strength (e.g. C-export rate) influences main stem extension growth and the pattern of sink development in F. linearis.     

Rosmarinic acid failed to suppress hydrogen peroxide-mediated apoptosis but induced apoptosis of Jurkat cells which was suppressed by Bcl-2

Rosmarinic acid (RosA), frequently found as a secondary metabolite in herbs and medicinal plants, has exhibited antioxidative and anti-inflammatory activities. RosA was shown to inhibit the proliferation and induce apoptosis of Jurkat T cells but the mechanism of action of RosA in apoptosis remains elusive. RosA inhibited the proliferation of Jurkat cells in a dose-dependent manner by suppressing the expression of cyclin D₃ and p21^Cip1/Waf1 and up-regulating p27^Kip1. RosA induced apoptosis of Jurkat cells in a dose-dependent manner and failed to protect them from hydrogen peroxide (H₂O₂)-mediated apoptosis. Induction of apoptosis by RosA correlated with suppression of Bcl-2 but not of Bak or PUMA. Overexpression of Bcl-2 protected Jurkat cells from both H₂O₂- and RosA-induced apoptosis by altering the ratio of anti- to pro-apoptotic members of the Bcl-2 family. In conclusion, RosA inhibited Jurkat cell proliferation by altering the expression of cyclins and cyclin-dependent kinase inhibitors and induced apoptosis most likely acting through the mitochondrial pathway and possessed no anti-oxidant properties.     

Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.     

Hydrolysis by phospholipase D of phospholipids in solution state or adsorbed on a silica matrix

Phospholipases D (PLD) catalyse hydrolysis and transphosphatidylation reactions in phospholipids. In the present study, the hydrolytic activity for cabbage PLD was investigated with five different substrates (dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), didecanoylphosphatidylcholine (DDPC), 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine and lyso-phosphatidylcholine (lyso-PC)) in solution or adsorbed on a silica matrix. In the specific buffer solutions, where the substrates were proved to form large multilamellar polydisperse aggregates, PLD showed preference for DPPC > DPPE > DDPC > 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine > lyso-PC. When the substrates were adsorbed on the silica matrix, PLD hydrolysed 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine and lyso-PC, DDPC, but not DPPC or DPPE. Theoretical studies of the simplest possible adducts between the phospholipids and the silica matrix were performed. Examination of local geometries of DPPC showed a significant blocking of the P-O-X bond-prone to hydrolysis, which could possibly block the access of PLD. Immobilization of phospholipids could be applied for improving the yield of reactions catalysed by PLD as well as for performing a targeted production of short-chain length phosphatidic acid analogs.     

Bcl-2 but not clusterin/apolipoprotein J protected human diploid fibroblasts and immortalized keratinocytes from ceramide-induced apoptosis: role of p53 in the ceramide response

The role of clusterin/apolipoprotein J (Clu/ApoJ) and Bcl-2 on C(2)-ceramide-induced apoptosis of embryonic human diploid fibroblasts, MRC-5 and immortalized adult skin keratinocytes, HaCaT was investigated. C(2)-ceramide-induced apoptosis of HaCaT in a time- and dose-dependent manner, while in MRC-5 only at higher concentrations. There was a dose-dependent accumulation of Clu/ApoJ and downregulation of Bcl-2 which correlated with C(2)-ceramide-induced apoptosis of MRC-5. While overexpression of Bcl-2 suppressed C(2)-ceramide-mediated apoptosis in both cell types, Clu/ApoJ failed to do so, accessed by morphological changes, DNA fragmentation and PARP cleavage. There was no change in the expression of endogenous p53 or p21(Waf1/Cip1) upon C(2)-ceramide treatment of MRC-5. However, mutant p53(143ala) increased the sensitivity of MRC-5 to C(2)-ceramide-induced apoptosis by markedly downregulating Bcl-2, pointing to a role for p53. These results suggested that whereas downregulation of Bcl-2 may be a crucial factor involved in C(2)-ceramide-induced apoptosis, accumulation of Clu/ApoJ may be a signal of stress response. Moreover, the ceramide-activated apoptotic pathway may be regulated by p53.     

Clinical evaluation of three denture cushion adhesives by complete denture wearers

doi: 10.1111/j.1741‐2358.2010.436.x Clinical evaluation of three denture cushion adhesives by complete denture wearers Objective: The aim of this study was the clinical evaluation of three denture cushion adhesives and whether the results were correlated to Kapur Index for denture‐supporting tissues. Background: Various types of denture adhesives are used among denture patients. However, information on the clinical behaviour of denture cushions is limited. Materials and methods: Thirty edentulous patients had their denture‐supporting tissues scored by Kapur Index and their old dentures replaced. They received three brands of denture cushion adhesives (Fittydent^®, Protefix^® and Seabond^®) and were instructed to use them in a sequence according to the group they were randomly assigned to. Each brand of adhesive was used for 48 h on the lower denture according to the manufacturer’s suggestions. After each brand was used, participants spent 24 h without applying any sort of adhesive. Finally, a questionnaire evaluating and comparing the performance of each brand was filled out. Results: Denture adhesives generally improved patient satisfaction and masticatory ability, especially in participants with poor Kapur Index and those who reported a poor retention of their old dentures. Conclusion: Fittydent^® was the most preferred adhesive, showing the best retention and the longest duration of its effect, but also reported as difficult to remove from the denture‐bearing area.     

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS-mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.     

De-ubiquitinating proteases USP2a and USP2c cause apoptosis by stabilising RIP1

Dynamic ubiquitination impacts on the degradation of proteins by the proteasome as well as on their effects as signalling factors. Of the many cellular responses that are regulated by changes in ubiquitination, apoptosis has garnered special attention. We have found that USP2a and USP2c, two isoforms of the ubiquitin-specific protease USP2, cause cell death upon ectopic expression. We show that both USP2 isoforms can control the ubiquitination status of many proteins but from a panel of potential targets only the protein level of RIP1 was increased by these enzymes. This effect is responsible for the activity of USP2a and USP2c to cause cell death. Both enzymes likewise de-ubiquitinate TRAF2, a ubiquitin-ligase in the TNFR1 complex. Whilst this and the similar sub-cellular localisations of both enzyme isoforms indicate a substantial overlap of activities, inactivation by RNAi revealed that only the knock-down of USP2c resulted in apoptosis, whilst targeting USP2a did not have any consequence on the cells' survival. Consequently, we focussed our studies on USP2a and found that TRAF2 inhibits USP2a's effect on K48- but not on K63-linked ubiquitin chains. Hence, the ratio between USP2a and TRAF2 protein levels determines the cells' sensitivity to cell death.     

Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli

The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K(a)≥10(17)M(-1) at 23°C, pH 7.0).