Interfacial structure of sugar beet pectin studied by atomic force microscopy.

Unlike pectins from other origins, sugar beet pectin (SBP) acts as an emulsifier, a property which has been correlated to its more hydrophobic character and high protein content. In this work, we have investigated the structure of SBP at interfaces by atomic force microscopy (AFM). Three situations were studied: the mica/water, graphite/water, and air/water interface. For the latter, the interfacial film was transferred onto mica using the Langmuir-Blodgett method. While the adsorption of individual pectin chains on mica requires the addition of divalent cations, on graphite a thin layer containing amorphous areas and rodlike chains forms spontaneously. We suggest that the layer contains proteins and pectin chains which are bound to the graphite via CH-pi interactions. SBP adsorbed at the air/water interface forms an elastic layer, as evidenced by pendant drop and surface shear rheology measurements. AFM Images reveal the layer is crippled with holes and contains rodlike chains, suggesting that the pectin chains prevent the formation of a densely packed protein layer. Nevertheless, we show that the interfacial pectin film is more resistant to displacement by surfactants than a pure protein film, possibly because of the formation of linkages between the pectin chains. In contrast, alkali treatment of the pectin appears to remove the pectin chains from the air/water interface and leaves a film that behaves similarly to pure protein. This work gives a new insight into the nanoscale organization of polysaccharides and polysaccharide-protein mixtures at macroscopic surfaces. The results gathered from the different interfaces studied permit a better understanding of the likely structure of SBP at the interface of emulsion droplets. Such knowledge might be used to modify rationally the pectin in order to improve its emulsifying properties, leading to broader commercial applications.

Comparative imaging of a bacterial surface-located GFP fusion protein by epifluorescence and scanning near-field optical microscopy.

IcsA is an autotransporter protein that plays a role in the virulence of Shigella bacteria. We have examined the cellular localization of a fusion of an IcsA fragment to the green fluorescent protein (GFP) expressed in Escherichia coli using a dual epifluorescence and scanning near-field optical microscope. By combining the data obtained from far-field with near-field microscopy of the same sample, discrimination between surface-bound fusion proteins and fusion proteins located in the cellular cytoplasm becomes possible. Furthermore, and for the first time, the inherent advantages in resolution of the near-field images provides highly specific details of the location of a GFP fusion protein on a bacterial cell surface.

Watching molecular processes with the atomic force microscope: dynamics of polymer adsorption and desorption at the single molecule level.

The formation of networks is an important step in the synthesis of many biological assemblies. For example, during the synthesis of plant cell walls the factors which dictate the arrangement of the polymeric constituents that make up the cell wall are not yet understood. Factors such as site-directed binding provide a possible theoretical background for beginning to understand the assembly of complex biological structures, but modelling of this process is difficult, time consuming and lacks experimental methods for verification. Through the use of atomic force microscopy (AFM) it has been demonstrated that changes in the binding of a single heterogeneous cell wall polysaccharide to a charged substrate can be followed in real time. Furthermore, subsequent image analysis allows the probability of binding of the molecule to be mapped to produce a real data set which is comparable with those obtained in simulation studies. In addition, these AFM studies have provided new mechanistic clues to the adsorption/desorption process of this polysaccharide.

Atomic force microscopy of plant cell walls, plant cell wall polysaccharides and gels.

Methods developed for the routine imaging of polysaccharides by atomic force microscopy (AFM) have been used to image plant polysaccharides from higher plants (pectin) and algae (carrageenan). These methods have been extended to image K-carrageenan association in hydrated films. Finally, AFM has been used to image polysaccharide architecture in moist plant cell walls. Simple experimental and image processing methods have been used to enhance molecular structure in 'rough' cell wall surfaces.

Visualization of plant cell walls by atomic force microscopy.

Atomic force microscopy has been used to visualize the ultrastructure of hydrated plant cell wall material from prepared apple (Malus pumila MILL; Cox orange pippin), water chestnut (Eleocharis dulcis L.), potato (Solanum tuberosum L.; Bintje), and carrot (Daucus carota L.; Amsterdamse bak) parenchyma. Samples of cell wall material in aqueous suspension were deposited onto freshly cleaved mica. Excess water was blotted away and the moist samples were imaged in air at ambient temperature and humidity. The three-dimensional images obtained highlighted the layered structure of the plant cell walls and revealed features interpreted as individual cellulose microfibrils and plasmodesmata.

Imaging polysaccharides by atomic force microscopy.

Techniques have been developed for the routine reliable imaging of polysaccharides by atomic force microscopy (AFM). The polysaccharides are deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged under alcohols. The rationale behind the development of the methodology is described and data is presented for the bacterial polysaccharides xanthan, acetan, and the plant polysaccharides l-carrageenan and pectin. Studies on uncoated polysaccharides have demonstrated the improved resolution achievable when compared to more traditional metal-coated samples or replicas. For acetan the present methodology has permitted imaging of the helical structure. Finally, in addition to data obtained on individual polysaccharides, AFM images have also been obtained of the network structures formed by kappa-carrageenan and gellan gum.

Observation of the helical structure of the bacterial polysaccharide acetan by atomic force microscopy.

A method has been developed that has been found to give reproducible images of uncoated polysaccharides by Atomic Force Microscopy (AFM). Aqueous solutions of the polysaccharide are deposited as drops onto freshly cleaved mica surfaces, air dried, and then imaged under butanol. The method has been used to obtain images of the bacterial polysaccharide acetan. In regions within the deposited sample, where the molecules are aligned side-by-side, it has been possible to observe a periodic structure along the polysaccharide chain, attributable to the helical structure of acetan.

A possible neurochemical basis for the neuropsychiatric aspects of baclofen therapy.

Baclofen, commonly used to reduce severe muscle spasms in patients with spinal cord injuries, is also active in the brain. A patient with pre-existing bipolar affective disorder developed increased depression while on baclofen, which progressed to a delusional depression when baclofen and haloperidol were rapidly decreased. When the dose of haloperidol was increased to a previously well tolerated dose to deal with the depressive delusion, a pseudoparkinson's state developed. This case demonstrates the interactive effects of baclofen and haloperidol on central noradrenergic and dopaminergic systems and suggests a possible neurochemical basis for the difference between delusional and nondelusional depression that is consistent with the different therapeutic response to psychotropic drugs of patients with these illnesses. The paradoxical appearance of the pseudoparkinson state in this patient when much higher doses of haloperidol had been free of such side effects, may reflect baclofen-induced alterations in receptor sensitivity. It appears that baclofen should be used with caution in patients with neuropsychiatric problems and that, when used, the withdrawal of baclofen should be continued over several weeks to allow receptor sensitivity to return to normal levels.