Efficient protocols for CAPS-based mapping inArabidopsis

Positional cloning continues to be an essential method for gene identification and characterisation. The introduction of PCR-based techniques such as Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Length Polymorphisms (SSLP) and Cleaved Amplified Polymorphic Sequences (CAPS) has greatly increased the efficiency of gene mapping in arabidopsis. To develop the CAPS marker approach further, we have altered several critical mapping parameters. Efficiency was improved by using a small volume of dry seed for DNA extraction instead of the commonly used vegetative tissue. Reproducibility of PCR reactions was enhanced by faster and reduced protocols for PCR and restriction enzyme digestion and optimisation of PCR conditions for over 50 CAPS primer pairs. Finally, the density of genetic markers was increased by providing polymorphic information for all CAPS markers in arabidopsis ecotypes Wassilewskija (Ws), Columbia (Col) and Cape Verde Islands (Cvi).     

Potocytosis

Potocytosis represents a mechanism by which small and large molecules as well as macromolecular complexes are sequestered and transported by caveolae. Caveolae are flask-shaped plasma membrane specializations characterized by a filamentous coat consisting of caveolins that decorates the inside surface of each caveola membrane. They have endocytotic functions that differ from the clathrin-coated pit pathway. Ligands bound to receptors that are internalized by caveolae can be delivered to four different locations in the cell bypassing the lysosome and at least four different caveolae membrane traffic patterns during potocytosis can be distinguished. Hence, cells have two endocytic machines and each is designed to accomplish different tasks. This review provides a brief summary of the discovery of caveolae and of potocytosis, and focuses on recent discoveries of the unique endocytic capabilities of caveolae in a variety of different cells.     

Footprint analysis of gait using a pressure sensor system.

The purpose of this study was to investigate if the detailed pressure data of the footprints of normal gait add essential information to the spatio-temporal variables of gait. The gait of 62 healthy adult subjects was investigated using GAITRite pressure sensor system. Each footprint was divided into 12 equal trapezoids and after that the hindfoot, midfoot and forefoot analysis was developed. A typical activation pattern of the sensors with two peaks of active area and peak pressure distribution during normal walking was obtained. The first peak reflected the heel strike, and the second peak reflected push-off at the end of the stance phase. The lowest pressure values were in the midfoot, where the lateral part of the foot activated sensors more than the medial part. The footprint patterns of right and left legs were symmetrical and corresponded with the symmetry found in the spatio-temporal variables of gait. The variability for the active area and the peak pressure were more pronounced for the lateral part of the midfoot and a smaller variation was seen in areas with concentrated observations (e.g. 1st, 2nd and 5th lateral trapezoids). Increasing active area in the forefoot was associated with decreasing pressure sensor activity in the midfoot. The footprint patterns identified the symmetry between the legs and at the same time revealed the velocity performance.     

beta-Hydroxyergothioneine, a new ergothioneine derivative from the mushroom Lyophyllum connatum, and its protective activity against carbon tetrachloride-induced injury in primary culture hepatocytes

A new ergothioneine derivative named beta-hydroxyergothioneine was isolated from the mushroom Lyophyllum connatum. Ergothioneine,N-hydroxy-N',N'-dimethylurea, and connatin (N-hydroxy-N',N'-dimethylcitrulline) were also isolated. All the compounds displayed the ability to scavenge free radicals, based on a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Structural determination, including the absolute stereochemistry of beta-hydroxyergothioneine, was achieved by spectroscopic analysis and X-ray crystallography. The radical scavenging activity of beta-hydroxyergothioneine was almost the same as that of ergothioneine. beta-Hydroxyergothioneine showed the greatest protective activity against carbon tetrachloride-induced injury in primary culture hepatocytes.     

Nutrient-dependent mTORC1 Association with the ULK1–Atg13–FIP200 Complex Required for Autophagy

Autophagy is an intracellular degradation system, by which cytoplasmic contents are degraded in lysosomes. Autophagy is dynamically induced by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation. Although it has been suggested that mTOR is a major negative regulator of autophagy, how it controls autophagy has not yet been determined. Here, we report a novel mammalian autophagy factor, Atg13, which forms a stable ~3-MDa protein complex with ULK1 and FIP200. Atg13 localizes on the autophagic isolation membrane and is essential for autophagosome formation. In contrast to yeast counterparts, formation of the ULK1–Atg13–FIP200 complex is not altered by nutrient conditions. Importantly, mTORC1 is incorporated into the ULK1–Atg13–FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the ~3-MDa ULK1–Atg13–FIP200 complex.     

Revisiting the Ring-opening Bulk Polymerization of ϵ -caprolactones using Commercial Lipases

The lipase-catalyzed ring-opening polymerization of &#107 -caprolactone ( &#107 -CL) and its derivatives was revisited using seven commercial enzymes. Lipases from Pseudomonas fluorescens (AK) and porcine pancreatic lipase (PPL) gave the best results, in both reaction conversion and degree of polymerization. Dependency on temperature and added concentration of enzyme was investigated, and there was a linear correlation between M n and the conversion ratio. The reaction proceeded rather slowly and the residual activity of these enzymes after prolonged incubation in &#107 -CL was studied. There was a negative correlation between the conversion ratio in the polymerization reaction and the tolerance of the enzymes for the solvent (monomer). Accordingly, a mechanism involving enzymatic ring-opening and non-enzymatic (but catalytic) polymerization was proposed.     

Leaf Size and Leaf Area Index in <Emphasis Type="Italic">Fagus sylvatica</Emphasis> Forests: Competing Effects of Precipitation,Temperature, and Nitrogen Availability

Plants across diverse biomes tend to produce smaller leaves and a reduced total leaf area when exposed to drought. For mature trees of a single species, however, the leaf area–water supply relationship is not well understood. We tested the paradigm of leaf area reduction upon drought by a transect study with 14 mature Fagus sylvatica forests along a steep precipitation gradient (970–520 mm y⁻¹) by applying two independent methods of leaf size determination. Contrary to expectation, average leaf size in dry stands (520–550 mm y⁻¹) was about 40% larger and SLA was higher than in moist stands (910–970 mm y⁻¹). As a result of increased leaf sizes, leaf area index significantly increased from the high- to the low-precipitation stands. Multiple regression analyses suggested that average leaf size was primarily controlled by temperature, whereas the influence of soil moisture and soil C/N ratio was low. Summer rainfall of the preceding year was the most significant predictor of total leaf number. We assume that leaf expansion of beech was independent of water supply, because it takes place in May with ample soil water reserves along the entire transect. In contrast, bud formation, which determines total leaf number, occurs in mid-summer, when droughts are severest. We conclude that leaf expansion and stand leaf area of beech along this precipitation gradient are not a simple function of water availability, but are controlled by several abiotic factors including spring temperature and possibly also nitrogen supply, which both tend to increase toward drier sites, thus overlaying any negative effect of water shortage on leaf development.