Thermoregulated Expression and Characterization of an NAD(P)H-Dependent 2-Cyclohexen-1-one Reductase in the Plant Pathogenic Bacterium Pseudomonas syringae pv. glycinea

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of Pseudomonas putida M10. The respective P. syringae gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated ncr for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding morB gene of P. putida M10. The ncr gene was overexpressed in Escherichia coli, and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the ncr gene in different Pseudomonas species and various strains of P. syringae was analyzed by PCR and Southern blot hybridization. The results indicated that the ncr gene is widespread among P. syringae pv. glycinea strains but not in other pathovars of P. syringae or in any of the other Pseudomonas strains tested.     

Force-generating capacities and fatigability of the quadriceps femoris in relation to different exercise modes

In this study, we examined whether different exercise modes provoke functional differences in maximal and explosive force-generating capacities and fatigability of the quadriceps femoris (QF). Additionally, the interaction of different functional capacities was studied in competitive athletes. Ten competitive tennis players and 10 endurance athletes participated in the study. Pre-exercise force-generating capacities were determined during maximal voluntary isometric knee extensions (MVC). Fatigability of the QF was studied using sustained isometric contractions with target loads of 20% and 40% of pre-exercise MVC. Postexercise MVCs were conducted 20 seconds, 1 minute, and 3 minutes post task failure. Muscle activation of the QF during the fatiguing exercises and postexercise MVCs was estimated using surface electromyography. Higher explosive force-generating capacities, but no differences in absolute moments, were detected in tennis players compared with endurance athletes. Fatigability of the QF during both fatiguing tasks was approximately the same in both athletic populations. This was indicated by minor group differences in endurance time, postexercise MVC production, and electromyography (EMG)-estimated muscle activation during fatigue. Variability in endurance time was not significantly associated with pre-exercise force-generating capacities in these competitive athletes. In both athletic populations, recovery of MVC was significantly slower after the fatiguing contraction with 20% of MVC compared with that with 40% of MVC. These results may enhance understanding of plasticity of the neuromuscular system and yield interesting information for the optimization of athletic training programs. Explosive strength training might enhance endurance athletes' explosiveness without decreasing muscle fatigue resistance. The exercise profile of competitive tennis is suggested to act as a sufficient trigger to reach high neuromuscular fatigue resistance but may be inadequate to cause significant gains in absolute muscle strength.     

Tumor stress, cell death and the ensuing immune response

A cornucopia of physiological and pathological circumstances including anticancer chemotherapy and radiotherapy can induce cell death. However, the immunological consequences of tumor cell demise have remained largely elusive. The paradigm opposing 'apoptosis versus necrosis' as to their respective immunogenicity does not currently hold to predict long-term immunity. Moreover, the notion that tumor cells may be 'stressed' before death to be recognized by immune cells deserves to be underlined. 'Eat-me', 'danger' and 'killing' signals released by stressed tumor under the pressure of cytotoxic compounds may serve as links between the chemotherapy-elicited response of tumor cells and subsequent immune responses. This review will summarize the state-of-the-art of cancer immunity and describe how tumor cell death dictates the links between innate and acquired immunity.     

Dendritic cells and innate defense against tumor cells

Tumor growth results from a delicate balance between intrinsic dysregulation of oncogenes, tumor suppressor and stability genes counteracted by extrinsic defenses composed of immune cells shaping tumor immunogenicity. Although immune subversion might be the ultimate outcome of this process, a complex network of cellular interactions take place eventually leading to tumor specific cognate immune responses. The links between innate and cognate antitumor immunity eliciting protective T cell responses are instigated by cytokines, chemokines and damage associated molecular patterns. The intricate differentiation pathway whereby dendritic cells could undergo an efficient maturation program in the tumor microenvironment appears crucial. We will discuss the role of innate effectors and cancer therapies in the process of defense against tumor cells.     

Genetic analysis of preharvest sprouting in a six-row barley cross

Preharvest sprouting (PHS) can be a problem in barley (Hordeum vulgare L.) especially malting barley, since rapid, uniform, and complete germination are critical. Information has been gained by studying the genetics of dormancy (measured as germination percentage, GP). The objective of this study was to determine if the quantitative trait loci (QTLs) discovered in previous research on dormancy are related to PHS. PHS was measured as sprout score (SSc) based on visual sprouting in mist chamber-treated spikes and as alpha-amylase activity (AA) in kernels taken from mist chamber-treated spikes that showed little or no visible sprouting. GP was also measured. All traits were measured at 0 and 14 days after physiological maturity. Evaluation of the spring six-row cross, Steptoe (dormant)/Morex (non-dormant) doubled haploid mapping population grown in greenhouse and field environments revealed QTL regions for SSc, AA, and GP on five, four, and six of the seven barley chromosomes, respectively. In total, seven and eight regions on five and six chromosomes had effects ranging from 4 to 31% and 3 to 39% on PHS and dormancy, respectively. One chromosome 3H and three chromosome 5H QTLs had the greatest effects. All PHS QTLs coincide with known dormancy QTLs, but some QTLs appear to be more important for PHS than for dormancy. Key QTLs identified should benefit breeding of barley for a suitable balance between PHS and dormancy.     

Extraction of high-quality bacterial RNA from infected leaf tissue for bacterial in planta gene expression analysis by multiplexed fluorescent Northern hybridization

Plant pathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. In planta gene expression analysis is important to understand the impact of these genes on bacterial virulence. A new mRNA-based approach using multiplexed Northern hybridization was developed. High-quality bacterial and plant total RNA was successfully isolated from leaf tissue infiltrated with Pseudomonas syringae . The procedure employs a new extraction buffer formulation containing glycine, sodium dodecylsulphate, cetyltrimethylammonium bromide, high-molecular-weight polyethylene glycol and β-mercaptoethanol. Cell lysis and classical acid–phenol extraction steps followed by LiCl precipitation yielded large amounts of total RNA of high purity and integrity. Multiplexing of DIG and chemically fluorescently labelled RNA probes was developed and expression data were normalized using the 23S rRNA gene as reference. The method was validated by studying in planta expression of the P. syringae genes mucD , cmaA , cfl , corR , corS and corP comprising a selection of highly expressed biosynthetic and low-expressed regulatory genes. The method was assessed regarding its sensitivity and might by useful for studying a variety of plant–microbe interactions.     

Site-directed mutagenesis of the temperature-sensing histidine protein kinase CorS from Pseudomonas syringae

Several plant pathogenic bacteria belonging to the species Pseudomonas syringae produce the phytotoxin coronatine to enhance their virulence. Pseudomonas syringae pv. glycinea PG4180 synthesizes coronatine at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C, its optimal growth temperature. In contrast, temperature has virtually no effect on coronatine synthesis in P. syringae pv. tomato strain DC3000. A modified two-component system controlling coronatine synthesis and consisting of the histidine protein kinase (HPK), CorS, the response regulator, CorR, and a third essential component, CorP, had been identified previously in both strains. CorS had been identified previously as a potential thermo-sensor. Comparison of the amino acid sequences of the HPKs from the two organisms revealed distinct differences. Site-directed mutagenesis of CorS from PG4180 was used to identify amino acyl residues potentially important for temperature signal perception. Point mutations and combinations of these were introduced into corS of PG4180 to generate corS variants with increased similarities to the respective allele from strain DC3000. These mutations resulted in either loss of activity, increase of thermoresponsiveness, or had no effect on CorS activity. Although none of the introduced mutations resulted in a clear conversion of CorS activity from thermo-responsive to temperature-independent, amino acyl residues important for temperature-dependent CorS activity and coronatine biosynthesis were identified.     

Immunogenicity of HLA-A1-restricted peptides derived from S100A4 (metastasin 1) in melanoma patients

S100A4 (metastasin 1) belongs to the S100 family of Ca²⁺ binding proteins. While not present in most differentiated adult tissues, S100A4 is upregulated in the micromilieu of tumors. It is primarily expressed by tumor-associated macrophages, fibroblasts, and tumor endothelial cells. Due to its strong induction in tumors S100A4 is a promising target for cancer immunotherapy. By reverse immunology, using epitope prediction programs, we identified 3 HLA-A1-restricted peptide epitopes (S100A4 A1-1, A1-2, and A1-3) which are subject to human T cell responses as detected in peripheral blood of melanoma patients by means of IFN-γ ELISPOT and cytotoxicity assays. In addition, IFN-γ responses to S100A4 A1-2 can not only be induced by stimulation of T cells with peptide-loaded DC but also by stimulation with S100A4 protein-loaded DC, indicating that this epitope is indeed generated by processing of the endogenously expressed protein. In addition, S100A4 A1-2 reactive T cells demonstrate lysis of HLA-A1⁺ fibroblasts in comparison to HLA-A1⁻ fibroblasts. In summary, this HLA-A1-restricted peptide epitope is a candidate for immunotherapeutical approaches targeting S100A4-expressing cells in the tumor stroma.     

Hydroxylation of naphthalene by aromatic peroxygenase from <Emphasis Type="Italic">Agrocybe aegerita</Emphasis> proceeds via oxygen transfer from H<Subscript>2</Subscript>O<Subscript>2</Subscript> and intermediary epoxidation

Agrocybe aegerita peroxidase/peroxygenase (AaP) is an extracellular fungal biocatalyst that selectively hydroxylates the aromatic ring of naphthalene. Under alkaline conditions, the reaction proceeds via the formation of an intermediary product with a molecular mass of 144 and a characteristic UV absorption spectrum (A _max 210, 267, and 303 nm). The compound was semistable at pH 9 but spontaneously hydrolyzed under acidic conditions (pH <7) into 1-naphthol as major product and traces of 2-naphthol. Based on these findings and literature data, we propose naphthalene 1,2-oxide as the primary product of AaP-catalyzed oxygenation of naphthalene. Using ¹⁸O-labeled hydrogen peroxide, the origin of the oxygen atom transferred to naphthalene was proved to be the peroxide that acts both as oxidant (primary electron acceptor) and oxygen source.