Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.     

Role of ultrasonography in diagnosing early rheumatoid arthritis and remission of rheumatoid arthritis - a systematic review of the literature

Introduction

Ultrasonography (US) might have an added value to clinical examination in diagnosing early rheumatoid arthritis (RA) and assessing remission of RA. We aimed to clarify the added value of US in RA in these situations performing a systematic review.

Methods

A systematic literature search was performed for RA, US, diagnosis and remission. Methodological quality was assessed; the wide variability in the design of studies prohibited pooling of results.

Results

Six papers on the added value of US diagnosing early RA were found, in which at least bilateral metacarpophalangeal (MCP), wrists and metatarsophalangeal (MTP) joints were scanned. Compared to clinical examination, US was superior with regard to detecting synovitis and predicting progression to persistent arthritis or RA. Eleven papers on assessing remission were identified, in which at least the wrist and the MCP joints of the dominant hand were scanned. Often US detected inflammation in patients clinically in remission, irrespective of the remission criteria used. Power Doppler signs of synovitis predicted X-ray progression and future flare in patients clinically in remission.

Conclusions

US appears to have added value to clinical examination for diagnosing of RA when scanning at least MCP, wrist and MTP joints, and, when evaluating remission of RA, scanning at least wrist and MCP joints of the dominant hand. For both purposes primarily power Doppler US might be used since its results are less equivocal than those of greyscale US.     

Arabinose-rich polymers as an evolutionary strategy to plasticize resurrection plant cell walls against desiccation

A variety of Southern African resurrection plants were surveyed using high-throughput cell wall profiling tools. Species evaluated were the dicotyledons, Myrothamnus flabellifolia and Craterostigma plantagineum; the monocotyledons, Xerophyta viscosa, Xerophyta schlecterii, Xerophyta humilis and the resurrection grass Eragrostis nindensis, as well as a pteridophyte, the resurrection fern, Mohria caffrorum. Comparisons were made between hydrated and desiccated leaf and frond material, with respect to cell wall composition and polymer abundance, using monosaccharide composition analysis, FT-IR spectroscopy and comprehensive microarray polymer profiling in combination with multivariate data analysis. The data obtained suggest that three main functional strategies appear to have evolved to prepare plant cell walls for desiccation. Arabinan-rich pectin and arabinogalactan proteins are found in the resurrection fern M. caffrorum and the basal angiosperm M. flabellifolia where they appear to act as ‘pectic plasticizers’. Dicotyledons with pectin-rich walls, such as C. plantagineum, seem to use inducible mechanisms which consist of up-regulating wall proteins and osmoprotectants. The hemicellulose-rich walls of the grass-like Xerophyta spp. and the resurrection grass E. nindensis were found to contain highly arabinosylated xylans and arabinogalactan proteins. These data support a general mechanism of ‘plasticising’ the cell walls of resurrection plants to desiccation and implicate arabinose-rich polymers (pectin-arabinans, arabinogalactan proteins and arabinoxylans) as the major contributors in ensuring flexibility is maintained and rehydration is facilitated in these plants.     

Immunoprofiling of pectic polysaccharides

An assay is described for the rapid identification of unbranched homogalacturonan and branched components occurring in samples of pectic polysaccharides using anti-pectin monoclonal antibodies. The assay involves the immunodetection of pectic polysaccharides after separation into two components during the application in small volumes to nitrocellulose. In this system, homoglacturonan-rich components migrate further on the nitrocellulose in contrast to pectic components with abundant side chains, resulting in a clear separation and discrete rings of distinct polysaccharides that can be identified using specific antibodies. This procedure is also directly applicable to preparations of plant material without the need for isolation of pectic polysaccharides.     

Phage display: practicalities and prospects

Phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles. Such phage thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but also have the capacity to replicate. Using phage display vast numbers of variant nucleotide sequences may be converted into populations of variant peptides and proteins which may be screened for desired properties. It is now some seventeen years since the first demonstration of the feasibility of this technology and the intervening years have seen an explosion in its applications. This review discusses the major uses of phage display including its use for elucidating protein interactions, molecular evolution and for the production of recombinant antibodies.     

Differential retrotranslocation of mitochondrial Bax and Bak

The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death.     

The relation between cartilage biomarkers (C2C,C1,2C,CS846, and CPII) and the long-term outcome of rheumatoid arthritis patients within the CAMERA trial

Introduction  

The aim of this study was to investigate whether serum biomarker levels of C2C, C1,2C, CS846, and CPII can predict the long-term course of disease activity and radiographic progression early in the disease course of rheumatoid arthritis (RA).     

Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension‐cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence and fluorochrome labelling of resin‐embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls analysed showed calcofluor‐stained appositions. However, in habituated and dehabituated cells, appositions were not recognized by an anticallose antibody. This finding suggested the accumulation of an extracellular polysaccharide different to callose, probably a 1,4‐β‐glucan in these cell lines. Appositions in habituated cells also contained homogalacturonan (HG) with a high degree of methyl esterification (DE), rhamnogalacturonan (RG) and xyloglucan. Habituated cell walls were also enriched in pectins, particularly HG, with a low DE, and RG. The levels of extensin epitope that colocalized with RG in habituated cells also diminished with the increasing number of subcultures. Habituated cells also liberated less extensin into the medium. In habituated cells, a decrease in the cell wall arabinogalactan protein (AGP) labelling was observed both in cell walls and in the culture medium. The increase in the number of subcultures in 0.3 µM dichlobenil was accompanied by an increment in some pectic epitopes (JIM5 and LM5) and a decrease in other pectic and in protein epitopes (JIM7, PAM1, LM6, LM2 and MAC207), indicating a re‐structuring of cell walls throughout the habituation procedure. Dehabituated cells showed an overall composition similar to that of non‐habituated cells, with exception of an increase in glucose in hemicellulosic fractions tightly bound to cellulose. However, these cells also showed reduced levels of extensin and AGP labelling. These differences could be related to the high tolerance to dichlobenil observed in dehabituated cells.     

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.