Effects of a Physiotherapeutic Protocol in Respiratory Function, Aerobic Capacity and Quality of Life After Kidney Transplantation.

INTRODUCTION: Chronic kidney disease can lead to dysfunction of the respiratory, cardiac, and musculoskeletal systems, altering the body's metabolism. Renal transplantation and hospital physiotherapy, through specific protocols, can improve these dysfunctions. OBJECTIVES: This study evaluates the impact of a hospital physiotherapeutic protocol in quality of life (QoL), respiratory muscle strength, peak expiratory flow, and 6-minute walk test (6MWT) in the preoperative, first, and fifth days after renal transplantation. METHODS: We evaluated 39 patients who received a renal transplant at Clinics Hospital of University of Campinas for respiratory muscle strength, expiratory peak flow, and functional capacity by the 6MWT. The short form-36 quality of life questionnaire was applied to 12 patients. RESULTS: We observed a significant reduction in respiratory muscle strength and peak expiratory flow in the first postoperative day. On postoperative day 5, there was improvement in respiratory muscle strength and expiratory peak flow. However, aerobic capacity measured by 6MWT remained below predicted. Analysis of QoL showed an improvement in almost all analyzed domains after transplantation. CONCLUSION: A specific physiotherapeutic protocol applied early after transplantation provided recovery of respiratory muscle strength and QoL. However, longer training is necessary to obtain adequate aerobic rehabilitation.

Differential localization of arabinan and galactan side chains of rhamnogalacturonan 1 in cambial derivatives.

The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen (Populus tremula L. x P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues. Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion. Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for identifying the initial cells among their immediate neighbours.

Expression of the ferrioxamine receptor gene of Erwinia amylovora CFBP 1430 during pathogenesis.

Mutants of Erwinia amylovora CFBP 1430 lacking a functional high-affinity iron transport system mediated by desferrioxamine are impaired in their ability to initiate fire blight symptoms (A. Dellagi, M.-N. Brisset, J.-P. Paulin, and D. Expert. Mol. Plant-Microbe Interact. 11:734-742, 1998). In this study, a chromosomal transcriptional lacZ fusion was used to analyze the expression in planta of the E. amylovora ferrioxamine receptor gene foxR. LacZ activity produced by the strain harboring the fusion was highly induced in iron-restricted conditions and in inoculated apple leaf tissues. Microscopic observation revealed differential expression of this gene in relation to the localization and density of bacterial cells within the diseased tissue. Thus, the ability of bacterial cells to express their iron transport system in accordance with environmental conditions is likely important for disease evolution.

Visualization of harpin secretion in planta during infection of apple seedlings by Erwinia amylovora.

Erwinia amylovora is a Gram-negative pathogenic bacterium that infects pear and apple trees as well as other plants from the Rosaceae family. E. amylovora pathogenicity is dependent on a functional Hrp type III secretion system. Harpin, a protein playing a major role in virulence, has been shown to be exported in vitro via the type III secretion apparatus. The data presented here focus on harpin detection in planta after infection of apple seedlings with the wild-type strain CFPB 1430. Using a specific harpin antiserum, harpin was not detected inside the host plant cells, but was found associated with the bacteria and secreted. The extracellular localization of harpin is in agreement with the physiological effects induced by purified harpin when applied as an exogenous elicitor. Harpin was not found associated with the host plant cell wall, a result that weakens its postulated role in cell wall loosening. A differential labelling was observed at the bacterial level: for some bacteria, harpin was exclusively cytoplasmic, whereas in others, it appeared as small patches over the bacterial outer membrane or associated with extracellular linear structures. All the bacteria present within the same area were similarly labelled, suggesting co-ordination in the secretion process. All observations suggest that harpin is synthesized in the bacterial cytoplasm and that secretion occurs from this cytoplasmic pool upon sensing of a plant or bacterial signal.

Fine structure in cellulose microfibrils: NMR evidence from onion and quince.

It has been controversial for many years whether in the cellulose of higher plants, the microfibrils are aggregates of 'elementary fibrils', which have been suggested to be about 3.5 nm in diameter. Solid-state NMR spectroscopy was used to examine two celluloses whose fibril diameters had been established by electron microscopy: onion (8-10 nm, but containing 40% of xyloglucan as well as cellulose) and quince (2 nm cellulose core). Both of these forms of cellulose contained crystalline units of similar size, as estimated from the ratio of surface to interior chains, and the time required for proton magnetisation to diffuse from the surface to the interior. It is suggested that the onion microfibrils must therefore be constructed from a number of cellulose subunits 2 nm in diameter, smaller than the 'elementary fibrils' envisaged previously. The size of these subunits would permit a hexagonal arrangement resembling the cellulose synthase complex.

Liquid crystal order and turbulence in the planar twist of the growing plant cell walls.

During growth, the plant cell wall behaves as a dynamic structure that exemplifies a case of unstable balance between a capability to organize a characteristic order and a permanent tendency to destroy the order. This interpretation allows previous results to be complemented and reappraised; it also integrates the cell wall behavior into a unified frame. The wall thickenings are structured according to a crystal-like pattern (cholesteric mesophase). Laminar helicoids are locally deposited on cell facets and form actual flat and planar twists in which the causes of topological defects are minimized. The helicoidal pattern is short-lived and its occurrence results from a complex interplay between self-assembly and disassembly properties. Two alternative processes destroy this critical state: 1. Externally, the surface extension which produces a progressive degradation and a randomization by shearing and stretching; 2. Internally, aleatory and punctual instabilities which alter the genesis of the system at the plasmalemma/wall interface. As the time goes on, the twisting either resumes or, in contrast, the perturbation increases. The system tends to become unpredictable thus suggesting turbulence.

Enzyme-gold cytochemistry of seed xyloglucans using two xyloglucan-specific hydrolases. Importance of prior heat-deactivation of the enzymes.

Two pure, homogeneous xyloglucan-hydrolyzing enzymes from germinated nasturtium seeds have been used to localize xyloglucans specifically in seed cell walls. The enzymes, a novel endo (1----4)-beta-D-glucanase which shows absolute specificity towards xyloglucans and a beta-D-galactosidase which is capable of removing galactosyl residues from polymeric xyloglucans, were used to stabilize gold sols. The complexes were applied to ultrathin sections of nasturtium (Tropaeolum majus L) and tamarind (Tamarindus indica L) seeds. The gold complexes prepared from the active enzyme proteins retained enzyme activity, and such complexes gave extremely weak section-labelling or no labelling at all. When the enzymes were subjected to heat-deactivation before being used to stabilize the gold sols, gold complexes were obtained which lacked enzyme activity, but which gave strong, specific labelling of xyloglucans in ultrathin sections. The specificity of the labelling was checked by substrate-competition, by pretreatment of sections with the active and heat-denaturated enzymes and by comparing the labelling of xyloglucan-containing storage cells with other cell types in the same section. The labelling was maximal at the pH which was optimal for the active enzyme. We conclude that the enzyme-gold complexes which retain high activity against the substrate to be localized are likely to be unsuitable as cytochemical probes because they may cause in situ substrate modification. In the case of the enzyme complexes described here the specific localization obtained with the gold complexes prepared from heat deactivated enzymes may be attributable to the retention by the heat-treated enzymatically-inactive proteins of substrate recognition.(ABSTRACT TRUNCATED AT 250 WORDS)

Liquid crystal-type assembly of native cellulose-glucuronoxylans extracted from plant cell wall.

In numerous plant cell walls, the cellulose microfibrils are arranged in a helicoidal pattern which has been considered as an analog to a cholesteric order. Here, we report on the spontaneous helicoidal organization which occurs in acellular conditions from aqueous suspensions of cellulose. The cellulosic mucilage of mature seeds of quince (Cydonia oblonga L) was studied both in situ (pre-release mucilage) and after water extraction and in in vitro re-assembly (prolonged high speed ultracentrifugation, further progressive dehydration and embedding in LR White methacrylate or hydrosoluble melamine resin). The cellulosic component was characterized by the use of cellobiohydrolase (CBH1) bound to colloidal gold, and the glucuronic acid residues of the xylan matrix were characterized by the use of cationised gold. Inside the seeds, the pre-release mucilage is mostly helicoidal, with the occurrence of more or less ordered domains, which indicate a fluid organization relevant to an actual liquid crystal state. Cytochemical tests revealed the tight association between cellulose and glucuronoxylans, the latter constituting a charged coat around each microfibril. Following the hydration of the seed, a cellulosic suspension was extracted in which microfibrils were totally dispersed. The progressive dehydration of the suspension gave rise to concentrated viscous drops. Ultrastructural observations revealed the occurrence of multidomain organization, from non-ordered to cholesteric-like regions, revealing that the mucilage is at the same time crystalline and liquid. This constitutes the first demonstration that liquid crystal type assemblies can arise from crystalline and biological cellulose in aqueous suspension. It strengthens the hypothesis that a transient liquid crystal state must occur during the cellulose ordering. The possible morphogenetic role of the glucuronoxylans in the cholesteric organization of the cellulose is discussed.

Cellulose and pectin localization in roots of mycorrhizalAllium porrum: labelling continuity between host cell wall and interfacial material.

Two different types of contacts (or interfaces) exist between the plant host and the fungus during the vesicular-arbuscular mycorrhizal symbiosis, depending on whether the fungus is intercellular or intracellular. In the first case, the walls of the partners are in contact, while in the second case the fungal wall is separated from the host cytoplasm by the invaginated host plasmamembrane and by an interfacial material. In order to verify the origin of the interfacial material, affinity techniques which allow identification in situ of cell-wall components, were used. Cellobiohydrolase (CBH I) that binds to cellulose and a monoclonal antibody (JIM 5) that reacts with pectic components were tested on roots ofAllium porrum L. (leek) colonized byGlomus versiforme (Karst.) Berch. Both probes gave a labelling specific for the host cell wall, but each probe labelled over specific and distinct areas. The CBH I-colloidal gold complex heavily labelled the thick epidermal cell walls, whereas JIM 5 only labelled this area weakly. Labelling of the hypodermis was mostly on intercellular material after treatment with JIM 5 and only on the wall when CBH I was used. Suberin bands found on the radial walls were never labelled. Cortical cells were mostly labelled on the middle lamella with JIM 5 and on the wall with CBH I. Gold granules from the two probes were found in interfacial material both near the point where the fungus enters the cell and around the thin hyphae penetrating deep into the cell. The ultrastructural observations demonstrate that cellulose and pectic components have different but complementary distributions in the walls of root cells involved in the mycorrhizal symbiosis. These components show a similar distribution in the interfacial material laid down around the vesicular-arbuscular mycorrhizal fungus indicating that the interfacial material is of host origin.

Sequential patterns of intramural digestion of galactoxyloglucan in tamarind seedlings.

The structure and breakdown of galactoxyloglucan (GXG)-rich cells was studied from cotyledons of Tamarindus indicus L. The depletion of GXG was followed at different levels: quantitative, histochemical and cytochemical. At the ultrastructural level two probes were used: one general for polysaccharides (periodic acid - thiocarbohydrazide - silver proteinate test), the other specific for the terminal galactosyl residues of GXG (ß-galactosidase-gold complex). They were complemented by water-extraction of the GXG and analysis of the constituting monosaccharides by gas chromatography. Despite their collenchymateous aspect and the chemical similarity of the reserve GXG with the structural xyloglucan of growing walls, the thickened storage walls are not interpretable as being an hypertrophied primary wall. The tamarind cells produce an original type of wall construction in which GXGs are sequestered in a sort of homomolecular bulk. There is no evidence for intussusception of the molecules within a network of cellulose. The bulk of GXG is sandwiched between two thin layers: the outer is comparable to a regular primary wall, the inner behaves like a barrier during GXG withdrawal. Temporal and spatial patterns of GXG-mobilisation lead to the definition of a sequence of stages of cell activities (premobilising, mobilising, postmobilising). They are synchronized with the growth of the seedling axis, the duration and characteristics of the stages being subordinated to the location of the cells within the organ. Cell lysis is initiated in close relationship with intramural cavities. The development of digestion pockets results in a highly digested wall. The barrier prevents any engulfing of the cytoplasm in the wall clefts and creates an increasing free space. The attack front of digestion is always sharp. During all steps, the monosaccharide composition remains stable. At the end of GXG depletion, the storage wall is withdrawn and cells are rendered in a parenchyma-like state. The breakdown is not a complete wall collapse but an original controlled and limited wall-thinning. The data lead to the speculation that the hydrolytic activities result from a complementation between precursors relinquished by the cytoplasm and factors already present in the storage wall.

Use of purified endopolygalacturonase for a topochemical study of elongating cell walls at the ultrastructural level.

Endopolygalacturonase, a fungal enzyme purified by Albersheim and his group which specifically degrades the galacturonosyl linkages of pectic polysaccharides, was used at the ultrastructural level on intact tissues from differentiated organs. Specimens were taken from the elongating zone of mung bean (Vigna radiata) hypocotyls. Incubation with the enzyme solution was performed en bloc, prior to embedding and to ultrastructural cytochemistry (PATAg test for polysaccharides), or by flotation of ultrathin frozen sections. From the morphological viewpoint, the images obtained are sharp and reproducible. They support the plywood model for the organization of expanding walls. The ordering of walls appears to be built up very early, at the very beginning of elongation, in the upper part of the hypocotyl (hook). The specificity of the enzyme allows a topochemical study of the wall. Data indicate an uneven distribution of polysaccharides of pectic type across a single wall and among the different cells. Outer and inner wall areas are highly resistant to extraction by endopolygalacturonase. In the middle lamella the insolubility of amorphous components probably indicates a local concentration of highly methyl-esterified carboxyl groups not susceptible to endopolygalacturonase attack; conversely, adjacent parts of the middle lamella are regularly extracted, indicating a high concentration of galacturonans. An intense extraction occurs in the bow-shaped zone, revealing the occurrence of a massive embedding of unesterified pectic polysaccharides around the fibrillar subunits responsible for the twisted patterns. In the inner and recent part of the wall, the ordering of fibrillar subunits seems progressive and possibly related to the peptic embedding. This incrusting material could play a role in the morphogenesis of the ordered wall by means of specific assembly and interconnections of wall subunits.

Ultrastructure and cytochemistry of Miocene angiosperm leaf tissues.

Angiosperm leaf fossils (16.7-25 x 10(6) years by potassium/argon dating) referable to Zelkova were isolated from pyroclastic deposits in the Succor Creek Formation, Oregon. These fossils reveal a three-dimensional structure in mesophyll cell layers and vascular bundles, while transmission electron microscopy of fixed tissues reveals well-preserved chloroplasts with grana stacks and starch, as well as nuclei with condensed chromatin. Transmission electron microscopy indicates that cell walls retain a cellulosic microfibrillar architecture, while ultrastructural and cytochemical confirmation is presented for chloroplast starch and the presence of native cellulose. The preservation of cell walls and cytologic structures in fossil materials of this age indicates an unusually mild fossilization process attending the post-depositional maturation of associated sediments. The preservation observed appears to be associated with rapid physical dehydration coupled with endogenous fixation by organic acids (possibly tannins and/or chlorophyll derivatives).

A freeze-etching and replication study of wall deposition in elongating plant cells.

The architecture of the expanding wall of mung bean hypocotyl (Phaseolus aureus) and collenchyma of celery (Apium graveolens) was examined using freeze-etching without any cryoprotectant, and surface-replication of frozen-ground and air-dried specimens. The polylamellated organization of the wall was seen. Freeze-etching clearly visualized, within one single fracture plans, the intermediate strata in which the microfibril orientation gradually changes between the main transverse and longitudinal directions. They corresponded to the bow-shaped arcs seen with surface replication and conventional microtomy. The organization of newly-formed microfibrils (periplasmic microfibrils) was seen by their imprints on the plasmalemma. When they were being deposited the microfibrils were loose and sinuous though tightly packed, rigid and parallel on the further layers. Therefore it seems that the fibrillogenesis and the spatial orientation of the microfibrils are two subsequent steps. The role of the periplasm in controlling the three-dimensional arrangement of the wall is emphasized.

Observations with cytochemistry and ultracryotomy on the fine structure of the expanding walls in actively elongating plant cells.

Ultracryotomy with negative staining and cytochemistry (periodic acid - thiocarbohydrazide - silver proteinate test for polysaccharides, in conjunction with mild extractions) were used to study the architecture of the cell wall and its modifications during expansion. Those techniques were applied to the study in situ of the walls of actively elongating parenchyma of mung bean (Phaseolus aureus), and pea (Pisum sativum) root and of collenchyma of celery (Apium graveolens) petioles. These complementary techniques provide information on the 3-dimensional disposition and fine structure of the subunits of the wall. In all the examples examined, the bulk of growing primary wall appears well-ordered and no progressive evolution from a transverse texture near the plasmalemma to a scattered texture near the middle lamella was observed. It seems unlikely that the development of the wall structure in relation to growth could be explained mechanically by a passive shift of the fibrillar elements in response to cellular stress. There is no evidence for an inert change in fibrillar orientation in the major part of the wall. If such occurs the process is limited to the outermost and senescent part of the wall. Thus, the texture observed does not agree with the classical multinet growth hypothesis but rather with the idea of an ordered structure of the primary wall. With the latter, the components should be able to respond in different ways to specific growth regulators and other environmental signals and thus exert a more positive control over the processes of oriented cell growth.