Geranyl diphosphate synthase is required for biosynthesis of gibberellins

Geranyl diphosphate synthase (GPS) is generally considered to be responsible for the biosynthesis of monoterpene precursors only. However, reduction of LeGPS expression in tomato (Lycopersicon esculentum) by virus-induced gene silencing resulted in severely dwarfed plants. Further analysis of these dwarfed plants revealed a decreased gibberellin content, whereas carotenoid and chlorophyll levels were unaltered. Accordingly, the phenotype could be rescued by application of gibberellic acid. The dwarfed phenotype was also obtained in Arabidopsis thaliana plants transformed with RNAi constructs of AtGPS. These results link geranyl diphosphate (GPP) to the gibberellin biosynthesis pathway. They also demand a re-evaluation of the role of GPS in precursor synthesis for other di-, tri-, tetra- and/or polyterpenes and their derivatives.     

Biphenyls as potent vitronectin receptor antagonists. Part 3: Squaric acid amides

Vitronectin receptor (alpha(V)beta(3)) antagonists have been implicated as a possible new treatment of restenosis following balloon angioplasty. In this work we investigate a series of novel arginine mimetic scaffolds leading to new insight of the alpha(V)beta(3)/ligand interaction. Squaric acid amide 10 is a subnanomolar alpha(V)beta(3) antagonist with improved potency on human smooth muscle cell migration.     

High-throughput flow cytometry-based assay to identify apoptosis-inducing proteins

After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry-based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers.     

The contribution of TRPV4-mediated calcium signaling to calcium homeostasis in endothelial cells

A large variety of cation transport systems are involved in the regulation of calcium homeostasis in endothelial cells. The focus of the present study is to determine the contribution of nonselective cation channels from the TRP (transient receptor potential) family to cellular calcium homeostasis of porcine aortic endothelial cells (PAEC). One member of the TRPV (vanniloid) subfamily, TRPV4, has previously been shown to be involved in cation transport induced by a large variety of stimulations including osmolarity, temperature, mechanical stress, and phosphorylation. Here, we demonstrate the existence of several TRP proteins, including TRPV4, in PAEC using RT-PCR. To test whether this channel is functional, we performed FURA-2 calcium measurements and whole-cell patch-clamp experiments. We observed the induction of large calcium signals following mechanical stress, altered extracellular temperature, and the selective TRPV4 activator 4-alpha -PDD. These effects were diminished in the presence of the TRPV4 inhibitor miconazole, suggesting the involvement of this channel in mediating endothelial calcium signals. The large amounts of transported calcium and the short signaling ways suggest a potentially important role of this channel in many physiological processes.     

Small-molecule inhibitors of the Rce1p CaaX protease

The Rce1p protease is required for the maturation of the Ras GTPase and certain other isoprenylated proteins and is considered a chemotherapeutic target. To identify new small-molecule inhibitors of Rce1p, the authors screened the National Cancer Institute Diversity Set compound library using in vitro assays to monitor the proteolytic processing of peptides derived from Ras and the yeast a-factor mating pheromone. Of 46 inhibitors initially identified with a Ras-based assay, only 9 were effective in the pheromone-based assay. The IC(50) values of these 9 compounds were in the low micromolar range for both yeast (6-35 microM) and human Rce1p (0.4-46 microM). Four compounds were somewhat Rce1p selective in that they partially inhibited the Ste24p protease and did not inhibit Ste14p isoprenylcysteine carboxyl methyltransferase, 2 enzymes also involved in the maturation of isoprenylated proteins. The remaining 5 compounds inhibited all 3 enzymes. The 2 most Rce1p-selective agents were ineffective trypsin inhibitors, further supporting the specificity of these agents for Rce1p. The 5 least specific compounds formed colloidal aggregates, a proposed common feature of promiscuous inhibitors. Interestingly, the most specific Rce1p inhibitor also formed a colloidal aggregate. In vivo studies revealed that treatment of wild-type yeast with 1 compound induced a Ras2p delocalization phenotype that mimics observed effects in rce1 ste24 null yeast. The 9 compounds identified in this study represent new tools for understanding the enzymology of postisoprenylation-modifying enzymes and provide new insight for the future development of Rce1p inhibitors.     

Coping with Stress: The Effectiveness of Different Types of Music

Listening to classical and self-selected relaxing music after exposure to a stressor should result in significant reductions in anxiety, anger, and sympathetic nervous system arousal, and increased relaxation compared to those who sit in silence or listen to heavy metal music. Fifty-six college students, 15 males and 41 females, were exposed to different types of music genres after experiencing a stressful test. Several 4 x 2 mixed design analyses of variance were conducted to determine the effects of music and silence conditions (heavy metal, classical, or self-selected music and silence) and time (pre-post music) on emotional state and physiological arousal. Results indicate listening to self-select or classical music, after exposure to a stressor, significantly reduces negative emotional states and physiological arousal compared to listening to heavy metal music or sitting in silence.     

Presence of virulence and fitness gene modules of enterohemorrhagic Escherichia coli in atypical enteropathogenic Escherichia coli O26

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O26 cause hemolytic-uremic syndrome (HUS) whereas atypical enteropathogenic E. coli (aEPEC) O26 typically cause uncomplicated diarrhea but have been also isolated from HUS patients. To gain insight into the virulence of aEPEC O26, we compared the presence of O island (OI) 122, which is associated with enhanced virulence in EHEC strains, among aEPEC O26 and EHEC O26 clinical isolates. We also tested these strains for the high pathogenicity island (HPI) which is a fitness island. All 20 aEPEC O26 and 20 EHEC O26 investigated contained virulence genes located within OI-122 (efa1/lifA, nleB, nleE, ent). In both aEPEC O26 and EHEC O26, OI-122 was linked to the locus for enterocyte effacement, forming a mosaic island which was integrated in pheU. Moreover, strains of these two pathotypes shared a conserved HPI. These data support a close relatedness between aEPEC O26 and EHEC O26 and have evolutionary implications. The presence of OI-122 in aEPEC O26 might contribute to their pathogenic potential.     

Differential effect of HOE642 on two separate monovalent cation transporters in the human red cell membrane.

Residual K(+) fluxes in red blood cells can be stimulated in conditions of low ionic strength. Previous studies have identified both the non-selective, voltage-dependent cation (NSVDC) channel and the K(+)(Na(+))/H(+) exchanger as candidate pathways mediating this effect, although it is possible that these pathways represent different modes of operation of a single system. In the present study the effects of HOE642, recently characterised as an inhibitor of the K(+)(Na(+))/H(+) exchanger, on NSVDC has been determined to clarify this question. Radioisotope flux measurements and conductance determinations showed that HOE642 exerted differential effects on the NSVDC channel and the K(+)(Na(+))/H(+) exchanger, confirming that the salt loss observed in low ionic strength solutions represents contributions from at least two independent ion transport pathways. The findings are discussed in the context of red blood cell apoptosis (eryptosis) and disease.     

Molecular phylogeny of the Heterotrichea (Ciliophora, Postciliodesmatophora) based on small subunit rRNA gene sequences

A comprehensive molecular analysis of the phylogenetic relationships within the Heterotrichea including all described families is still lacking. For this reason, the complete nuclear small subunit (SSU) rDNA was sequenced from further representatives of the Blepharismidae and the Stentoridae. In addition, the SSU rDNA of a new, undescribed species of the genus Condylostomides (Condylostomatidae) was sequenced. The detailed phylogenetic analyses revealed a consistent branching pattern: while the terminal branches are generally well resolved, the basal relationships remain unsolved. Moreover, the data allow some conclusions about the macronuclear evolution within the genera Blepharisma, Stentor, and Spirostomum suggesting that a single, compact macronucleus represents the ancestral state.     

Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium

We identified Shiga toxin gene (stx)-negative Escherichia coli O26:H11 and O26:NM (nonmotile) strains as the only pathogens in the stools of five patients with hemolytic-uremic syndrome (HUS). Because the absence of stx in E. coli associated with HUS is unusual, we examined the strains for potential virulence factors and interactions with microvascular endothelial cells which are the major targets affected during HUS. All five isolates possessed the enterohemorrhagic E. coli (EHEC)-hlyA gene encoding EHEC hemolysin (EHEC-Hly), expressed the enterohemolytic phenotype, and were cytotoxic, in dose- and time-dependent manners, to human brain microvascular endothelial cells (HBMECs). Significantly reduced cytotoxicity in an EHEC-Hly-negative spontaneous derivative of one of these strains, and a dose- and time-dependent cytotoxicity of recombinant E. coli O26 EHEC-Hly to HBMECs, suggest that the endothelial cytotoxicity of these strains was mediated by EHEC-Hly. The toxicity of EHEC-Hly to microvascular endothelial cells plausibly contributes to the virulence of the stx-negative E. coli O26 strains and to the pathogenesis of HUS.