PilO of Pseudomonas aeruginosa 1244: subcellular location and domain assignment

PilO of Pseudomonas aeruginosa 1244 catalyses the attachment of an O-antigen repeating unit to the beta-carbon of the pilin C-terminal residue, a serine. The present study was conducted to locate the regions of this enzyme important in catalysis and to establish the cellular location of the pilin glycosylation reaction. While PilO was not detectable in extracts of P. aeruginosa or Escherichia coli, even under conditions of overexpression, it was found that an intact MalE-PilO fusion protein was produced in significant amounts. This fusion complemented a P. aeruginosa 1244 mutant containing a pilO deletion and targeted to the cytoplasmic membrane of E. coli. Wzy and WaaL, enzymes that also utilize the O-antigen repeating unit as substrate, were found to share a sequence pattern with PilO even though these proteins have little overall sequence similarity. PilO constructs in which portions of this common sequence were deleted or altered by site-directed mutagenesis lacked pilin glycosylating activity. Deletions of segments downstream from the common region also prevented enzyme activity. Topology studies showed that the two PilO regions associated with enzyme activity were located in the periplasm. These results establish regions of this enzyme important for catalysis and present evidence that pilin glycosylation occurs in the periplasmic space of this organism.     

Limonoids as larvicidal components against mosquito larvae (Aedes aegypti Linn.)

The study focussed on four limonoids (calodendrolide, harrisonin, pedonin and pyroangolensolide) as larvicidal components against mosquito 2nd instar larvae of the species Aedes aegypti Linn. senso stricto. Since pyroangolensolide is close to calodendrolide in structure, it was synthesized through reduction of calodendrolide with chromium(II) chloride in acetone. Harrisonin and pedonin were extracted with cold methanol from the root bark of Harrisonia abyssinica while calodendrolide was extracted with the same solvent from the root bark of Calodendrum capense. The structure of pyroangolensolide was elucidated using physical and spectroscopic techniques. 25, 50, 75, and 100 microM of each compound were tested against the mosquito larvae. Calodendrolide was the most toxic since 100% mortality was registered at all concentrations, while pyroangolensolide showed 100% mortality up to 50 microM and for contents of 25 microM, a mortality of 70% was registered. As a result of this toxicity, lower concentrations (5, 10 and 15 microM) were tested for both calodendrolide and pyroangolensolide. Toxicity of harrisonin and pedonin was lower. The relative toxicity was in the order: calodendrolide > pyroangolensolide > harrisonin > pedonin with LC50 values of 13.2, 16.6, 28.1 and 59.2 microM, respectively.     

Pax7 shows higher satellite cell frequencies and concentrations within intrafusal fibers of muscle spindles

Intrafusal fibers within muscle spindles make up a small subpopulation of muscle fibers. These proprioceptive fibers differ from most extrafusal fibers because, even in maturity, their diameters remain small, and they retain expression of developmental myosins. Although both extrafusal and intrafusal fibers contain satellite cells (SCs), comparatively little is known about intrafusal SCs. Analyzing chicken fast-phasic posterior (PLD) and slow-tonic anterior (ALD) latissimus dorsi muscles, we show that SCs of both intrafusal and extrafusal fibers express Pax7. We further test the hypotheses that intrafusal fibers display parameters reflective of extrafusal immaturity. These hypotheses are that intrafusal fibers contain (a) higher SC frequencies (number of SC nuclei/all nuclei within basal lamina) and concentrations (closer together) and (b) smaller myonuclear domains than do adjacent extrafusal fibers. IHC techniques were applied to PLD and ALD muscles excised at 30 and 138 days posthatch. The hypotheses were validated, suggesting that intrafusal fibers have greater capacities for growth, regeneration, and repair than do adjacent extrafusal fibers. During maturation, extrafusal and intrafusal fibers show similar trends of decreasing SC frequencies and concentrations and increases in myonuclear domains. Thus, extrafusal and intrafusal fibers alike should exhibit reduced capacities for growth, regeneration, and repair during maturation.     

Proteomic analysis of zebrafish caudal fin regeneration

The epimorphic regeneration of zebrafish caudal fin is rapid and complete. We have analyzed the biomechanism of zebrafish caudal fin regeneration at various time points based on differential proteomics approaches. The spectrum of proteome changes caused by regeneration were analyzed among controls (0 h) and 1, 12, 24, 48, and 72 h postamputation involving quantitative differential proteomics analysis based on two-dimensional gel electrophoresis matrix-assisted laser desorption/ionization and differential in-gel electrophoresis Orbitrap analysis. A total of 96 proteins were found differentially regulated between the control nonregenerating and regenerating tissues of different time points for having at least 1.5-fold changes. 90 proteins were identified as differentially regulated for regeneration based on differential in-gel electrophoresis analysis between the control and regenerating tissues. 35 proteins were characterized for its expression in all of the five regenerating time points against the control samples. The proteins identified and associated with regeneration were found to be directly allied with various molecular, biological, and cellular functions. Based on network pathway analysis, the identified proteome data set for regeneration was majorly associated in maintaining cellular structure and architecture. Also the proteins were found associated for the cytoskeleton remodeling pathway and cellular immune defense mechanism. The major proteins that were found differentially regulated during zebrafish caudal fin regeneration includes keratin and its 10 isoforms, cofilin 2, annexin a1, skeletal α1 actin, and structural proteins. Annexin A1 was found to be exclusively undergoing phosphorylation during regeneration. The obtained differential proteome and the direct association of the various proteins might lead to a new understanding of the regeneration mechanism.     

Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.     

Photosynthetic Response of Soybean to Microclimate in 26-Year-Old Tree-Based Intercropping Systems in Southern Ontario,Canada

In order to study the effect of light competition and microclimatic modifications on the net assimilation (NA), growth and yield of soybean (Glycine max L.) as an understory crop, three 26-year-old soybean-tree (Acer saccharinum Marsh., Populus deltoides X nigra, Juglans nigra L.) intercropping systems were examined. Tree competition reduced photosynthetically active radiation (PAR) incident on soybeans and reduced net assimilation, growth and yield of soybean. Soil moisture of 20 cm depth close (< 3 m) to the tree rows was also reduced. Correlation analysis showed that NA and soil water content were highly correlated with growth and yield of soybean. When compared with the monoculture soybean system, the relative humidity (RH) of the poplar-soybean, silver maple-soybean, and black walnut-soybean intercropped systems was increased by 7.1%, 8.0% and 5.9%, soil water content was reduced by 37.8%, 26.3% and 30.9%, ambient temperature was reduced by 1.3°C, 1.4°C and 1.0°C, PAR was reduced by 53.6%, 57.9% and 39.9%, and air CO₂ concentration was reduced by 3.7μmol·mol^-1, 4.2μmol·mol^-1 and 2.8μmol·mol^-1, respectively. Compared to the monoculture, the average NA of soybean in poplar, maple and walnut treatments was also reduced by 53.1%, 67.5% and 46.5%, respectively. Multivariate stepwise regression analysis showed that PAR, ambient temperature and CO₂ concentration were the dominant factors influencing net photosynthetic rate.