Changes in Cell Wall Polysaccharides of Green Bean Pods during Development

The changes in cell wall polysaccharides and selected cell wall-modifying enzymes were studied during the development of green bean (Phaseolus vulgaris L.) pods. An overall increase of cell wall material on a dry-weight basis was observed during pod development. Major changes were detected in the pectic polymers. Young, exponentially growing cell walls contained large amounts of neutral, sugar-rich pectic polymers (rhamnogalacturonan), which were water insoluble and relatively tightly connected to the cell wall. During elongation, more galactose-rich pectic polymers were deposited into the cell wall. In addition, the level of branched rhamnogalacturonan remained constant, while the level of linear homogalacturonan steadily increased. During maturation of the pods, galactose-rich pectic polymers were degraded, while the accumulation of soluble homogalacturonan continued. During senescence there was an increase in the amount of ionically complexed pectins, mainly at the expense of freely soluble pectins. The most abundant of the enzymes tested for was pectin methylesterase. Peroxidase, beta-galactosidase, and alpha-arabinosidase were also detected in appreciable amounts. Polygalacturonase was detected only in very small amounts throughout development. The relationship between endogenous enzyme levels and the properties of cell wall polymers is discussed with respect to cell wall synthesis and degradation.     

Analysis of retted and non retted flax fibres by chemical and enzymatic means

Flax fibres (Linum usitatissimum L.) were subjected to chemical and enzymatic analysis in order to determine the compositional changes brought about by the retting process and also to determine the accessibility of the fibre polymers to enzymatic treatment. Chemical analysis involved subjecting both retted and non retted fibres to a series of sequential chemical extractions with 1% ammonium oxalate, 0.05 M KOH, 1 M KOH and 4 M KOH. Retting was shown to cause minimal weight loss from the fibres but caused significant changes to the pectic polymers present. Retted fibres were shown to have significantly lower amounts of rhamnogalacturonan as well as arabinan and xylan. In addition the average molecular mass of the pectic extracts was considerably lowered. Enzyme treatment of the 1 M KOH extracts with two different enzymes demonstrated that the non retted extract contained a relatively high molecular weight xylan not found in the retted extract. Treatment of the 1 M KOH extracts and the fibres with Endoglucanase V from Trichoderma viride demonstrated that while this enzyme solubilised cellulose as well as xylan and xyloglucan oligomers from the extract, it had limited access to these polymers on the fibre. MALDI-TOF MS analysis of the material solubilised from the extract suggested that the xylan was randomly substituted with 4-O-methyl glucuronic acid moieties. The xyloglucan was shown to be of the XXXG type and was substituted with galactose and fucose units. The enzyme treatments of the fibres demonstrated that the xylan and xyloglucan polymers in the fibres were not accessible to the enzyme but that material which was entrapped by the cellulose could be released by the hydrolysis of this cellulose.     

Metabolic Profiling of the Response to an Oral Glucose Tolerance Test Detects Subtle Metabolic Changes

Background

The prevalence of overweight is increasing globally and has become a serious health problem. Low-grade chronic inflammation in overweight subjects is thought to play an important role in disease development. Novel tools to understand these processes are needed. Metabolic profiling is one such tool that can provide novel insights into the impact of treatments on metabolism.

Methodology

To study the metabolic changes induced by a mild anti-inflammatory drug intervention, plasma metabolic profiling was applied in overweight human volunteers with elevated levels of the inflammatory plasma marker C-reactive protein. Liquid and gas chromatography mass spectrometric methods were used to detect high and low abundant plasma metabolites both in fasted conditions and during an oral glucose tolerance test. This is based on the concept that the resilience of the system can be assessed after perturbing a homeostatic situation.

Conclusions

Metabolic changes were subtle and were only detected using metabolic profiling in combination with an oral glucose tolerance test. The repeated measurements during the oral glucose tolerance test increased statistical power, but the metabolic perturbation also revealed metabolites that respond differentially to the oral glucose tolerance test. Specifically, multiple metabolic intermediates of the glutathione synthesis pathway showed time-dependent suppression in response to the glucose challenge test. The fact that this is an insulin sensitive pathway suggests that inflammatory modulation may alter insulin signaling in overweight men.     

Characterization of a rabbit polyclonal antibody against threonine-AMPylation

An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.