Direct visualization of changes in deacylated Na(+) gellan polymer morphology during the sol-gel transition.

Changes in gellan polymer morphology during the sol-gel transition were directly visualized by transmission electron microscopy and a model incorporating these changes and existing physical data is proposed. Our observations suggest that the most thermodynamically stable conformations of gellan polymers in solution, in the absence of added cations, are the double helix and double-helical duplexes. We have demonstrated two forms of lateral aggregation of gellan helices in the presence of Ca(2+) and K(+) ions. One type forms junction zones that lead to network formation and gelation, while the second type leads to the formation of isolated fibers of aggregated helices and inhibition of gelation. The proposed model of gellan gelation is based on these observations where thermoreversibility, gel strength, and endothermic transitions of gellan gels can be explained.

Ultrastructural evidence for intramolecular double stranding in iota-carrageenan.

Kinetic studies of primary processes of conformational ordering in gel-forming biopolymers have suggested that a change in mechanism from intermolecular to intramolecular multistrand formation occurs on lowering the concentration of biopolymer. We report here ultrastructural observations consistent with intramolecular double stranding in a carbohydrate polymer, iota-carrageenan, by arresting this process of primary conformational ordering by an ultra-rapid freeze fixation technique. High-resolution transmission electron microscopy (TEM) revealed isolated iota-carrageenan chains showing a range of morphologies (linear, circular, and hairpin) consistent with intramolecular stranding. Control experiments in which iota-carrageenan was frozen in the disordered form revealed longer and thinner strands.

Improved visualization of folded collagen alpha-chains by ultra-rapid freezing.

Transmission electron microscopy techniques are commonly employed to examine the folded polymer structure of collagen polypeptides. These techniques include deposition of a sample by spraying, slow freeze-fixation, air drying and vacuum drying the specimen at room temperature, and using additives such as glycerol in the collagen preparation. Here we report preliminary observations of type I collagen alpha-chains, folded in water, at a concentration of 35 micrograms ml-1 and 10 micrograms ml-1, visualized by an ultra-rapid, freeze-fixation technique designed to minimize structural deformation caused by spraying, additives and poor freeze-fixation. The technique also allows the use of submicrolitre sample volumes of known concentrations with negligible loss and shearing, while at the same time providing excellent contrast to the collagen polymer for electron microscopy. This technique can be employed to study the structure of a wide range of macromolecules (proteins and carbohydrates).

Hydrodynamic radius alone governs the mobility of molecules through plasmodesmata.

Various fluorescent molecular probes have been injected into the cytoplasm of nectary trichome cells of Abutilon striatum to ascertain the conductivity of the plasmodesmata. Most of the probes were based on fluorescein conjugated to a range of amino acids and peptides. The probes are not broken down by cytoplasmic enzymes during the period of observation. The results indicate that there are no specific effects of aromatic amino acids, either polar or hydrophobic types, on the conductivity of the Abutilon plasmodesmata, contrary to reports for other plants. The conductivity of the plasmodesmata in the trichomes is slightly greater than for any that have been studied in the tissues of other plants. It is proposed that in Abutilon the mobility of a probe is determined solely by the effective Stokes radius of the molecule, and that the radius of the molecule is governed by the molecular weight and, in particular, by the nature of the side groups in the peptide chain attached to the fluorochrome. Calculations are presented which indicate that channels between material in the plasmodesmatal annulus are the most likely route for the diffusion of the probes, and that the width of individual channels in the annulus is close to 3 nm.

Suberin lamellae in the hypodermis of maize (Zea mays) roots; development and factors affecting the permeability of hypodermal layers.

The development of suberin lamellae in the hypodermis of Zea mays cv. LG 11 was observed by electron microscopy and the presence of suberin inferred from autoliuorescence and by Sudan black B staining in nodal (adventitious) and primary (seminal) root axes. Suberin lamellae were evident at a distance of 30-50 mm from the tip of roots growing at 20°C and became more prominent with distance from the tip. Both oxygen deficiency and growth at 13°C produced shorter roots in which the hypodermis was suberized closer to the root tip. There were no suberin lamellae in epidermal cells or cortical collenchyma adjacent to the hypodermis. Plasmodesmata were not occluded by the suberin lamellae: there were twice as many of them in the inner tangential hypodermal wall (1,14 µn-2 ) as in the junction between the epidermis and hypodermis (0.54 µm-2 ). Water uptake by seminal axes (measured by micropotometry) was greater at distances more than 100 mm from the root lip than in the apical zone where the hypodermis was unsuberized. In the more mature zones of roots grown at 13°C rates of water uptake were greater than in roots grown at 20°C even though hypodermal suberization was more marked. Sleeves of epidermal/hypodermal cells (plus some accessory collenchyma) were isolated from the basal 60 mm of nodal axes by enzymatic digestion (drisclase). The roots were either kept totally immersed in culture solution or had the basal 50 mm exposed to moist air above the solution surface. In both treatments the permeabilities to tritiated water and 86 Rb were low (circa 10-5 mms-1 ) in sleeves isolated from the extreme base. In roots grown totally immersed, however, the permeability of sleeves increased 10 to 50-fold over a distance of 40 mm. In roots exposed to moist air the permeability remained at a low level until the point where the root entered the culture solution and then increased rapidly (> 50-fold in a distance of 8 mm). Growth of roots in oxygen depleted (5% O2 ) solutions promoted the development of extensive cortical aerenchymas. These developments were not associated with any reduction in permeability of sleeves isolated from the basal 40 mm of the axis. It was concluded that the presence of suberin lamellae in hypodermal walls does not necessarily indicate low permeability of cells or tissues to water or solutes. The properties of the walls (lamellae?) can be greatly changed by exposure to moist air, perhaps due to increased oxygen availability.

Understanding the artefact problem in freeze-fracture replication: a review.

Freeze-fracture and freeze-etching techniques do not provide artefact-free images of native in vivo or in vitro cells and tissues. Each preparation stage can produce specific artefacts which must be recognized and understood if these methods are to contribute meaningful information to cell biology, This paper reviews the latest information available on artefacts in freeze-fracture replication (and etching) methods and points to possibilities for avoiding some of them. Different specimens show different sensitivity to artefactual changes and the final images must be interpreted carefully with regard to the multi-event process that has led to their production.

Specimen heating during sputter-coating.

It is possible to sputter thin films of gold on to surfaces of frozen biological specimens at very low temperatures (less than 120 K) without untoward effects from heating. This is achieved by using permanent magnets to confine the plasma and thus to minimize the energy required to give a reasonable sputtering yield. The system described uses only 250 V at 12-15 mA to give 15 nm films within 2-3 min. It is shown, from theory portraying 'worst-case' conditions, that the specimen temperature could not increase by more than 6.0 K at equilibrium. Practical results support the theoretical assumptions. Similar considerations have been applied to sputtering at normal ambient temperature where it is shown that appropriate design of simple apparatus and selection of operational conditions can give adequate films in a reasonable time with negligible (less than 3 K) temperature rise above the starting temperature.

A device for the rapid freezing of biological specimens under precisely controlled and reproducible conditions.

The construction and preliminary testing of a device is described which can be used to freeze biological specimens in any cryogenic liquid at temperatures down to the nitrogen freezing point (63 K) and which can operate in the pressure range 1.3 kNm-2 to 1 MNm-2. Ultra-rapid freezing can be carried out in a subcooled cryogenic liquid either hyperbarically or at atmospheric pressure. Slow freezing rates can be achieved by cooling the specimens in a controlled manner in the vapour phase above the liquid bath.

The entry of ions and molecules into roots: an investigation using electron-opaque tracers.

Various solutions containing ions or molecules which may be visualized in the electron microscope have been presented to barley (Hordeum vulgare L.) roots. Large molecules such as gold sol particles (<6.0 nm) or ferritin (approx. 12.0 nm) were not found to be taken into the cytoplasm of any cells. Very dilute solutions of uranyl acetate or lanthanum nitrate resulted in the presence of typical electron-opaque crystals in the cortical apoplasm, as well as in cytoplasmic vesicles of cortical and some endodermal cells, but not in the cytoplasm or cell walls of stellar cells. Colloidal lanthanum hydroxide, however, while also impeded by the Casparian band, accumulated in vesicles in endodermal cells, and also penetrated into the stele.These results support the concept that different pathways exist for the movement of water and different ions across barley roots. They also indicate the relevance of the Casparian bands, the suberin lamellae, the formation of endocytotic vesicles, and the plasmodesmata, in studies on water and ion uptake.

Ultrastructural study of poly- -hydroxybutyrate granules from Bacillus cereus.

The freeze-etching technique was used to examine the effects of fracturing and etching on the appearance of poly-beta-hydroxybutyrate granules from Bacillus cereus. These granules were examined in extracts isolated by hypochlorite or by sonic treatment, and also in fixed and unfixed intact cells; in the latter case they were compared with granules in thin sections of intact cells. After freeze-fracturing, the diameter of the granules in intact cells was between 240 and 720 nm. The granules consisted of a central core, of diameter between 140 and 370 nm, which occupied less than 50% of the volume of the granule and which was either stretched or removed on fracturing; the core was surrounded by an outer coat which may be bounded by a membrane.

Uptake and binding of uranyl ions by barley roots.

After undergoing the processing for electron microscopy, bound uranyl ions are revealed as characteristic electron-opaque crystals. Meristematic walls and associated vesicles become heavily labeled, while pinocytotic accumulation into vacuoles seems probable in cap cells and those just behind the meristem. The endodermal Casparian strip and suberinized lamella are effective barriers to the passage of uranyl ions.

The tertiary endodermis in barley roots: Fine structure in relation to radial transport of ions and water.

The presence of numerous pits containing plasmodesmata in the inner tangential wall of the tertiary endodermis in barley roots is demonstrated by electron microscopy. The pit floor is covered by a thin layer of material which is continuous with and resembles the tertiary wall. The plasmodesmatal pore is constricted at its ends so that the plasmalemma lining the pore is appressed to the desmotubule. The frequency of plasmodesmata and their cross-sectional area is estimated, and phosphate and water fluxes through them are calculated on the assumption that they represent the only communication between the cortex and the vascular tissue. The pressure gradient across the ends of the plasmodesmata necessary to support the observed water flux is calculated for limiting cases of the pore radius and the viscosity of the fluid passing through the pore.