The ''parallel-up'' packing in cellulose I ␣ and I ␤ unit cells was experimentally demonstrated by a combination of direct-staining the reducing ends of cellulose chains and microdiffraction-tilting electron crystallographic analysis. Microdiffraction investigation of nascent bacterial cellulose microfibrils showed that the reducing end of the growing cellulose chains points away from the bacterium, and this provides direct evidence that polymerization by the cellulose synthase takes place at the nonreducing end of the growing cellulose chains. This mechanism is likely to be valid also for a number of processive glycosyltransferases such as chitin synthases, hyaluronan synthases, and proteins involved in the synthesis of nodulation factor backbones.The polarity of cellulose chains in a microfibril was debated for decades before two groups independently proved the parallel packing by electron microscopic methods. One involved the silver-labeling of the reducing ends of microfibrils (1), while the other was based on the unidirectional degradation of cellulose microfibrils by a cellobiohydrolase (2). In both studies, the cellulose from Valonia was used because of the high crystallinity and large lateral dimension of the microfibrils. Later, the silver-labeling technique was applied to bacterial cellulose and showed the same parallel packing (3). These microscopic analyses confirmed earlier crystallographic proposals that the most probable mode of packing in the unit cell was parallel (4-6).The current knowledge on the crystal structure of cellulose is that the native cellulose is a composite of two distinct crystalline phases called I ␣ and I ␤ (7, 8) corresponding to triclinic and monoclinic unit cells, respectively (9). The existence of the I ␣ and I ␤ structures within a microfibril is another confirmation of the parallel packing, because the triclinic unit accepts only one single chain in a unit cell. Furthermore, the fact that I ␣ and I ␤ coexist in a microfibril suggests that the chains in I ␣ -rich as well as in I ␤ -rich cellulose are parallel. Although the parallel packing of the chains in a cellulose microfibril or a unit cell is now firmly established, the molecular directionality of the chains with respect to the unit cells is not known.Directionality of cellulose chains in a unit cell is frequently defined according to Gardner and Blackwell (4). There are two types of parallel packing, namely, parallel up and parallel down. The parallel-up structure implies that the z coordinate of the O5 atom is greater than that of C5. Two parallel models with opposite molecular directionality thus have been proposed (4) and critically evaluated (10). Molecular dynamics studies recently have suggested that the parallel-up structure was most probable for both cellulose I ␣ and I ␤ (11).The first aim of the current research was to determine experimentally the chain directionality in a unit cell by using electron crystallography in conjunction with the reducing end staining technique. Once established, the deter...

Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry)were tested for their ability to synthesize in vitro sizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (133)-␤-D-glucan (callose). The taurocholate extracts, which did not require the addition of Mg 2؉ , were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IV I , a disorganized form of cellulose I. Besides these similarities, the in vitro microfibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitro microfibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.

Two types of peptide nanotubes, one is prepared from an amphiphilic peptide having a right-handed helix segment and the other from that having a left-handed helix segment, are shown to transform the morphology into a vesicle by membrane fusion due to stereo-complex formation between these helical segments.

The recently developed technique of reductive amination, followed by gold labeling, was applied to visualize the reducing ends of cellulose microcrystals from cellulose I, cellulose II, and cellulose III(I). In these crystals, which were also characterized by electron diffraction, the labeling proved that the chains were organized in a parallel fashion in cellulose I from ramie and Valonia and also in cellulose III(I) from Valonia. In microcrystals of cellulose II from mercerized ramie, the labeling method showed that the chains were packed into an antiparallel mode. These results are discussed in terms of the fine structure of cellulose I where neighboring microfibrils of opposite polarity are visualized. The mercerization process whereby cellulose I is converted into cellulose II is therefore best described in terms of an intermingling of the cellulose chains from neighboring microfibrils of opposite polarity. As opposed to the case of mercerization the conversion of cellulose I into cellulose III(I) does not require the participation of neighboring microfibrils since the crystalline domains are converted individually.

Ultrastructural localization of cellulose Iα and Iβ allomorphs in one microfibril from algal sources was investigated using electron microdiffraction. Both cellulose Iα and Iβ were characterized as one-chain triclinic and two-chain monoclinic unit cells, respectively, in agreement with previous studies. These two structures coexisted in each microfibril, alternating either longitudinally or laterally. The transition zone between the two phases was found to be the interface between adjacent H-bonded molecular sheets (i.e., 0.39-nm lattice planes).

On the basis of the`parallel-up' structure of the cellulose crystal, a crystallographic approach to study the mode of action of cellobiohydrolase Cel7A on Valonia cellulose microcrystal has been carried out. After incubation with Cel7A, most of the initially smooth and well defined Valonia microcrystals displayed fibrillation. However, as the hydrolysis reaction was rather heterogeneous, some microcrystals remained superficially intact. Close investigation on such crystals revealed polar morphology: one end was narrowed extremely or pointed. Electron microdiffraction analysis of these crystals evidenced that the narrowing of the microcrystals occurs at their reducing end side. This was also confirmed by the visualization of selective reducing end labeling at the pointed ends of microcrystals. These lines of investigation are the first demonstration that the processivity of Cel7A action against insoluble highly crystalline celluloses is unambiguously toward non-reducing ends from reducing ends.z 1998 Federation of European Biochemical Societies.

Prokaryotic voltage-gated sodium channels (Na V s) are homotetramers and are thought to inactivate through a single mechanism, named C-type inactivation. Here we report the voltage dependence and inactivation rate of the NaChBac channel from Bacillus halodurans, the first identified prokaryotic Na V , as well as of three new homologues cloned from Bacillus licheniformis (Na V BacL), Shewanella putrefaciens (Na V SheP), and Roseobacter denitrificans (Na V RosD). We found that, although activated by a lower membrane potential, Na V BacL inactivates as slowly as NaChBac. Na V SheP and Na V RosD inactivate faster than NaChBac. Mutational analysis of helix S6 showed that residues corresponding to the "glycine hinge" and "PXP motif" in voltage-gated potassium channels are not obligatory for channel gating in these prokaryotic Na V s, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in Na V BacL (A214G), Na V SheP (A216G), and NaChBac (G219A) accelerated inactivation in these channels, whereas mutation of glycine to alanine in the lower part of helix S6 in NaChBac (G229A), Na V BacL (G224A), and Na V RosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic Na V s does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.Voltage-gated sodium channels (Na V s) 3 generate the rapid upstroke of action potentials in nerve cell axons (1). In mammalian Na V s, the channel is formed by the ␣-subunit, which comprises four repeats of six-transmembrane segments, with each repeat consisting of 300 -400 amino acids. The ␣-subunit carries several glycosylation sites and co-assembles with auxiliary subunits to form the native channel (2, 3). The only structural information on Na V s available to date is a density map of the Na V from the electric organ of the electric eel determined by cryoelectron microscopy (4). Due to its limited resolution of 19 Å, the density map did not provide insights into the gating or sodium selectivity.The first prokaryotic Na V , NaChBac, was cloned from Bacillus halodurans (5). Subsequently, three more prokaryotic sodium channels were cloned and characterized (6, 7). All studied prokaryotic Na V s form homotetramers with a structure thought to be similar to that of some potassium channels with known structures (8 -10). Furthermore, because the proteins could be expressed in large amounts in Escherichia coli and purified by metal chelate affinity chromatography (5, 7, 11), they are promising candidates for high resolution structure determination and structure-function analyses.The physiological role of prokaryotic Na V s may be related to pH homeostasis, motility, and chemotaxis (6, 12). Searching bacterial genomic data bases, we found 26 sequences of putative NaChBac homologues from bacteria living in various environments. We were able to clone the putative Na V genes from three of these bacteria, Bacillus licheniformis, Shewanella pu...

Leaf primordia are generated around the shoot apical meristem. Mutation of the ASYMMETRIC LEAVES2 (AS2) gene of Arabidopsis thaliana results in defects in repression of the meristematic and indeterminate state, establishment of adaxial-abaxial polarity and left-right symmetry in leaves. AS2 represses transcription of meristem-specific class 1 KNOX homeobox genes and of the abaxial-determinant genes ETTIN/ARF3, KANADI2 and YABBY5. To clarify the role of AS2 in the establishment of leaf polarity, we isolated mutations that enhanced the polarity defects associated with as2. We describe here the enhancer-of-asymmetric-leaves-two1 (east1) mutation, which caused the formation of filamentous leaves with abaxialized epidermis on the as2-1 background. Levels of transcripts of class 1 KNOX and abaxial-determinant genes were markedly higher in as2-1 east1-1 mutant plants than in the wild-type and corresponding single-mutant plants. EAST1 encodes the histone acetyltransferase ELONGATA3 (ELO3), a component of the Elongator complex. Genetic analysis, using mutations in genes involved in the biogenesis of a trans-acting small interfering RNA (ta-siRNA), revealed that ELO3 mediated establishment of leaf polarity independently of AS2 and the ta-siRNA-related pathway. Treatment with an inhibitor of histone deacetylases (HDACs) caused additive polarity defects in as2-1 east1-1 mutant plants, suggesting the operation of an ELO3 pathway, independent of the HDAC pathway, in the determination of polarity. We propose that multiple pathways play important roles in repression of the expression of class 1 KNOX and abaxial-determinant genes in the development of the adaxial domain of leaves and, thus, in the establishment of leaf polarity.