Improved quantification of perfusion in patients with cerebrovascular disease.

In recent years measurements of cerebral perfusion using bolus-tracking MRI have become common clinical practice in the diagnosis and management of patients with stroke and cerebrovascular disease. An active area of research is the development of methods to identify brain tissue that is at risk of irreversible damage, but amenable to salvage using reperfusion treatments, such as thrombolysis. However, the specificity and sensitivity of these methods are limited by the inaccuracies in the perfusion data. Accurate measurements of perfusion are difficult to obtain, especially in patients with cerebrovascular diseases. In particular, if the bolus of MR contrast is delayed and/or dispersed due to cerebral arterial abnormalities, perfusion is likely to be underestimated using the standard analysis techniques. The potential for such underestimation is often overlooked when using the perfusion maps to assess stroke patients. Since thrombolysis can increase the risk of haemorrhage, a misidentification of 'at-risk' tissue has potentially dangerous clinical implications. This thesis presents several methodologies which aim to improve the accuracy and interpretation of the analysed bolus-tracking data. Two novel data analysis techniques are proposed, which enable the identification of brain regions where delay and dispersion of the bolus are likely to bias the perfusion measurements. In this way true hypoperfusion can be distinguished from erroneously low perfusion estimates. The size of the perfusion measurement errors are investigated in vivo, and a parameterised characterisation of the bolus delay and dispersion is obtained. Such information is valuable for the interpretation of in vivo data, and for further investigation into the effects of abnormal vasculature on perfusion estimates. Finally, methodology is presented to minimise the perfusion measurement errors prevalent in patients with cerebrovascular diseases. The in vivo application of this method highlights the dangers of interpreting perfusion values independently of the bolus delay and dispersion

Cell wall arabinan is essential for guard cell function

Stomatal guard cells play a key role in the ability of plants to survive on dry land, because their movements regulate the exchange of gases and water vapor between the external environment and the interior of the plant. The walls of these cells are exceptionally strong and must undergo large and reversible deformation during stomatal opening and closing. The molecular basis of the unique strength and flexibility of guard cell walls is unknown. We show that degradation of cell wall arabinan prevents either stomatal opening or closing. This locking of guard cell wall movements can be reversed if homogalacturonan is subsequently removed from the wall. We suggest that arabinans maintain flexibility in the cell wall by preventing homogalacturonan polymers from forming tight associations

Non-cellulosic polysaccharides from cotton fibre are differently impacted by textile processing.

Cotton fibre is mainly composed of cellulose, although non-cellulosic polysaccharides play key roles during fibre development and are still present in the harvested fibre. This study aimed at determining the fate of non-cellulosic polysaccharides during cotton textile processing. We analyzed non-cellulosic cotton fibre polysaccharides during different steps of cotton textile processing using GC-MS, HPLC and comprehensive microarray polymer profiling to obtain monosaccharide and polysaccharide amounts and linkage compositions. Additionally, in situ detection was used to obtain information on polysaccharide localization and accessibility. We show that pectic and hemicellulosic polysaccharide levels decrease during cotton textile processing and that some processing steps have more impact than others. Pectins and arabinose-containing polysaccharides are strongly impacted by the chemical treatments, with most being removed during bleaching and scouring. However, some forms of pectin are more resistant than others. Xylan and xyloglucan are affected in later processing steps and to a lesser extent, whereas callose showed a strong resistance to the chemical processing steps. This study shows that non-cellulosic polysaccharides are differently impacted by the treatments used in cotton textile processing with some hemicelluloses and callose being resistant to these harsh treatments

High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

<p>Abstract</p> <p>Background</p> <p>Molecular probes are required to detect cell wall polymers <it>in-situ </it>to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure.</p> <p>Results</p> <p>Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types.</p> <p>Conclusion</p> <p>These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.</p

Metabolism of polysaccharides in dynamic middle lamellae during cotton fibre development

Main conclusion: Evidence is presented that cotton fibre adhesion and middle lamella formation are preceded by cutin dilution and accompanied by rhamnogalacturonan-I metabolism. Cotton fibres are single cell structures that early in development adhere to one another via the cotton fibre middle lamella (CFML) to form a tissue-like structure. The CFML is disassembled around the time of initial secondary wall deposition, leading to fibre detachment. Observations of CFML in the light microscope have suggested that the development of the middle lamella is accompanied by substantial cell-wall metabolism, but it has remained an open question as to which processes mediate adherence and which lead to detachment. The mechanism of adherence and detachment were investigated here using glyco-microarrays probed with monoclonal antibodies, transcript profiling, and observations of fibre auto-digestion. The results suggest that adherence is brought about by cutin dilution, while the presence of relevant enzyme activities and the dynamics of rhamnogalacturonan-I side-chain accumulation and disappearance suggest that both attachment and detachment are accompanied by rhamnogalacturonan-I metabolism

Pollen grain development is compromised in Arabidopsis agp6 agp11 null mutants

Arabinogalactan proteins (AGPs) are structurally complex plasma membrane and cell wall proteoglycans that are implicated in diverse developmental processes, including plant sexual reproduction. Male gametogenesis (pollen grain development) is fundamental to plant sexual reproduction. The role of two abundant, pollen-specific AGPs, AGP6, and AGP11, have been investigated here. The pollen specificity of these proteoglycans suggested that they are integral to pollen biogenesis and their strong sequence homology indicated a potential for overlapping function. Indeed, single gene transposon insertion knockouts for both AGPs showed no discernible phenotype. However, in plants homozygous for one of the insertions and heterozygous for the other, in homozygous double mutants, and in RNAi and amiRNA transgenic plants that were down-regulated for both genes, many pollen grains failed to develop normally, leading to their collapse. The microscopic observations of these aborted pollen grains showed a condensed cytoplasm, membrane blebbing and the presence of small lytic vacuoles. Later in development, the generative cells that arise from mitotic divisions were not seen to go into the second mitosis. Anther wall development, the establishment of the endothecium thickenings, the opening of the stomium, and the deposition of the pollen coat were all normal in the knockout and knockdown lines. Our data provide strong evidence that these two proteoglycans have overlapping and important functions in gametophytic pollen grain development

Arabinogalactan-protein and pectin epitopes in relation to an extracellular matrix surface network and somatic embryogenesis and callogenesis in Trifolium nigrescens Viv

The formation of an extracellular matrix surface network (ECMSN), and associated changes in the distribution of arabinogalactan-protein and pectin epitopes, have been studied during somatic embryogenesis (SE) and callogenesis of Trifolium nigrescens Viv. Scanning electron microscopy observations revealed the occurrence of an ECMSN on the surface of cotyledonary-staged somatic embryos as well as on the peripheral, non-regenerating callus cells. The occurrence of six AGP (JIM4, JIM8, JIM13, JIM16, LM2, MAC207) and four pectin (JIM5, JIM7, LM5, LM6) epitopes was analysed during early stages of SE, in cotyledonary-staged somatic embryos and in non-embryogenic callus using monoclonal antibodies. The JIM5 low methyl-esterified homogalacturonan (HG) epitope localized to ECMSN on the callus surface but none of the epitopes studied were found to localize to ECMSN over mature somatic embryos. The LM2 AGP epitope was detected during the development of somatic embryos and was also observed in the cell walls of meristematic cells from which SE was initiated. The pectic epitopes JIM5, JIM7, LM5 and LM6 were temporally regulated during SE. The LM6 arabinan epitope, carried by side chains of rhamnogalacturonan-I (RG-I), was detected predominantly in cells of embryogenic swellings, whilst the LM5 galactan epitope of RG-I was uniformly distributed throughout the ground tissue of cotyledonary-staged embryoids but not detected at the early stages of SE. Differences in the distribution patterns of low and high methyl-esterified HG were detected: low ester HG (JIM5 epitope) was most abundant during the early steps of embryo formation and highly methyl-esterified form of HG (JIM7 epitope) became prevalent during embryoid maturation