g<Emphasis FontCategory="NonProportional">raphite</Emphasis> - a Bioconductor package to convert pathway topology to gene network

Background  

Gene set analysis is moving towards considering pathway topology as a crucial feature. Pathway elements are complex entities such as protein complexes, gene family members and chemical compounds. The conversion of pathway topology to a gene/protein networks (where nodes are a simple element like a gene/protein) is a critical and challenging task that enables topology-based gene set analyses.     

Guided cobamide biosynthesis for heterologous production of reductive dehalogenases

Cobamides (Cbas) are essential cofactors of reductive dehalogenases (RDases) in organohalide-respiring bacteria (OHRB). Changes in the Cba structure can influence RDase function. Here, we report on the cofactor versatility or selectivity of Desulfitobacterium RDases produced either in the native organism or heterologously. The susceptibility of Desulfitobacterium hafniense strain DCB-2 to guided Cba biosynthesis (i.e. incorporation of exogenous Cba lower ligand base precursors) was analysed. Exogenous benzimidazoles, azabenzimidazoles and 4,5-dimethylimidazole were incorporated by the organism into Cbas. When the type of Cba changed, no effect on the turnover rate of the 3-chloro-4-hydroxy-phenylacetate-converting enzyme RdhA6 and the 3,5-dichlorophenol-dehalogenating enzyme RdhA3 was observed. The impact of the amendment of Cba lower ligand precursors on RDase function was also investigated in Shimwellia blattae, the Cba producer used for the heterologous production of Desulfitobacterium RDases. The recombinant tetrachloroethene RDase (PceA_Y51) appeared to be non-selective towards different Cbas. However, the functional production of the 1,2-dichloroethane-dihaloeliminating enzyme (DcaA) of Desulfitobacterium dichloroeliminans was completely prevented in cells producing 5,6-dimethylbenzimidazolyl-Cba, but substantially enhanced in cells that incorporated 5-methoxybenzimidazole into the Cba cofactor. The results of the study indicate the utilization of a range of different Cbas by Desulfitobacterium RDases with selected representatives apparently preferring distinct Cbas.     

Photooxidative stress‐induced and abundant small RNAs in Rhodobacter sphaeroides

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (¹O₂) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to ¹O₂. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with ¹O₂ or superoxide (O^–₂). One sRNA was specifically induced by ¹O₂ and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by ¹O₂ and O^–₂ were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH_I and RpoH_II. The most abundant sRNA was processed in the presence of ¹O₂ but not by O^–₂. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of ¹O₂‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by ¹O₂ exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.     

Reorienting the Fab Domains of Trastuzumab Results in Potent HER2 Activators

The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab′)₂-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.     

Role of the microtubule destabilizing proteins SCG10 and stathmin in neuronal growth

The related proteins SCG10 and stathmin are highly expressed in the developing nervous system. Recently it was discovered that they are potent microtubule destabilizing factors. While stathmin is expressed in a variety of cell types and shows a cytosolic distribution, SCG10 is neuron-specific and membrane-associated. It contains an N-terminal targeting sequence that mediates its transport to the growing tips of axons and dendrites. SCG10 accumulates in the central domain of the growth cone, a region that also contains highly dynamic microtubules. These dynamic microtubules are known to be important for growth cone advance and responses to guidance cues. Because overexpression of SCG10 strongly enhances neurite outgrowth, SCG10 appears to be an important factor for the dynamic assembly and disassembly of growth cone microtubules during axonal elongation. Phosphorylation negatively regulates the microtubule destabilizing activity of SCG10 and stathmin, suggesting that these proteins may link extracellular signals to the rearrangement of the neuronal cytoskeleton. A role for these proteins in axonal elongation is also supported by their growth-associated expression pattern in nervous system development as well as during neuronal regeneration.     

Induction of the 72-kilodalton heat shock protein and protection from ultraviolet B-induced cell death in human keratinocytes by repetitive exposure to heat shock or 15-deoxy-delta(12,14)-prostaglandin J2

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.     

Influences of AT1 receptor blockade on tissue metabolism in obese men

ANG II applied to the interstitial space influences carbohydrate and lipid metabolism in a tissue-specific fashion. Thus endogenous ANG II may have a tonic effect on tissue metabolism that could be reversed with ANG II type 1 (AT1) receptor blockade, particularly during adrenergic stimulation. We studied 14 obese men. They were treated for 10 days with the AT1 receptor blocker irbesartan or with placebo in a double-blind and crossover fashion. At the end of each treatment period, we assessed skeletal muscle and adipose tissue metabolism using the microdialysis technique. The ethanol dilution technique was applied to follow changes in tissue blood flow. Measurements were obtained at baseline and during application of incremental isoproterenol concentrations through the microdialysis catheter. Blood pressure decreased from 133 +/- 3/84 +/- 3 to 128 +/- 3/79 +/- 2 mmHg for systolic and diastolic blood, respectively (P = 0.02 and 0.006, respectively) with AT1 receptor blockade. Isoproterenol perfusion caused a dose-dependent increase in dialysate glycerol in adipose tissue and in skeletal muscle. Irbesartan slightly reduced the isoproterenol-induced glycerol response in adipose tissue (P < 0.05 by ANOVA). Ethanol ratio, interstitial glucose supply, and lactate production in adipose tissue and skeletal muscle were similar with placebo and irbesartan. We conclude that AT1 receptor blockade in obese men does not reveal a major tonic ANG II effect on interstitial glucose supply, lipolysis, or glycolysis in skeletal muscle, either at rest or during beta-adrenergic stimulation. Endogeneous ANG II may slightly increase adipose tissue lipolysis. The mechanism may promote the redistribution of triglycerides from adipose tissue toward other organs.