The cellular pathway of photosynthate transfer in the developing wheat grain. II. A structural analysis and histochemical studies of the pathway from the crease phloem to the endosperm cavity

In the developing wheat grain, photosynthate is transferred longitudinally along the crease phloem and then laterally into the endosperm cavity through the crease vascular parenchyma, pigment strand and nucellar projection. In order to clarify this cellular pathway of photosynthate unloading, and hence the controlling mechanism of grain filling, the potential for symplastic and apoplastic transfer was examined through structural and histochemical studies on these tissue types. It was found that cells in the crease region from the phloem to the nucellar projection are interconnected by numerous plasmodesmata and have dense cytoplasm with abundant mitochondria. Histochemical studies confirmed that, at the stage of grain development studied, an apoplastic barrier exists in the cell walls of the pigment strand. This barrier is composed of lignin, phenolics and suberin. The potential capacity for symplastic transfer, determined by measuring plasmodesmatal frequencies and computing potential sucrose fluxes through these plasmodesmata, indicated that there is sufficient plasmodesmatal cross-sectional area to support symplastic unloading of photosynthate at the rate required for normal grain growth. The potential capacity for membrane transport of sucrose to the apoplast was assessed by measuring plasma membrane surface areas of the various cell types and computing potential plasma membrane fluxes of sucrose. These fluxes indicated that the combined plasma membrane surface areas of the sieve element–companion cell (se–cc) complexes, vascular parenchyma and pigment strand are not sufficient to allow sucrose transfer to the apoplast at the observed rates. In contrast, the wall ingrowths of the transfer cells in the nucellar projection amplify the membrane surface area up to 22-fold, supporting the observed rates of sucrose transfer into the endosperm cavity. We conclude that photosynthate moves via the symplast from the se–cc complexes to the nucellar projection transfer cells, from where it is transferred across the plasma membrane into the endosperm cavity. The apoplastic barrier in the pigment strand is considered to restrict solute movement to the symplast and block apoplastic solute exchange between maternal and embryonic tissues. The implications of this cellular pathway in relation to the control of photosynthate transfer in the developing grain are discussed.     

Roles of external and cellular Cl− ions on the activation of an apical electrodiffusional Cl− pathway in toad skin

Summary This study is concerned with the short-circuit current,I _sc, responses of the Cl^–-transporting cells of toad skin submitted to sudden changes of the external Cl^– concentration. [Cl]₀. Sudden changes of [Cl]₀, carried out under apical membrane depolarization, allowed comparison of the roles of [Cl]₀ and [Cl]_cell on the activation of the apical Cl^– pathways. Equilibration of shortcircuited skins symmetrically in K-Ringer's solutions of different Cl^– concentrations permitted adjustment of [Cl]_cell to different levels. For a given Cl^– concentration (in the range of 11.7 to 117mm) on both sides of a depolarized apical membrane, this structure exhibits a high Cl^– permeability,P _(Cl)apical. On the other hand, for the same range of [Cl]_cell but with [Cl]₀=0,P _(Cl)apical is reduced to negligible values. These observations indicate that when the apical membrane is depolarizedP _(Cl)apical is modulated by [Cl]₀; in the absence of external Cl^– ions, intracellular Cl^– is not sufficient to activateP _(Cl)apical. Computer simulation shows that the fast Cl^– currents induced across the apical membrane by sudden shifts of [Cl]₀ from a control equilibrium value strictly follow the laws of electrodiffusion. For each experimental group, the computer-generatedI _sc versus ([Cl]_cell–[Cl]₀) curve which best fits the experimental data can only be obtained by a unique pair ofP _(Cl)apical andR _b (resistance of the basolateral membrane), thus allowing the calculation of these parameters. The electrodiffusional behavior of the net Cl^– flux across the apical membrane supports the channel nature of the apical Cl^– pathways in the Cl^–-transporting cell. Cl^– ions contribute significantly to the overall conductance of the basolateral membrane even in the presence of a high K concentration in the internal solution.     

LptB-LptF coupling mediates the closure of the substrate-binding cavity in the LptB2FGC transporter through a rigid-body mechanism to extract LPS

Lipopolysaccharides (LPS) are essential envelope components in many Gram-negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP-binding cassette (ABC) transporter LptB₂FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF’s coupling helix being important in coordinating cavity collapse with LptB dimerization.     

Cell sheet technology: a promising strategy in regenerative medicine

Regenerative medicine is a burgeoning field that is important to combat challenging diseases and functional impairments. Compared with traditional cell therapies with evident shortcomings (e.g., cell suspension injection or tissue engineering with scaffolds), scaffold-free cell sheet technology enables transplanted cells to be grafted and fully maintain their viability on target sites. Clinical and experimental studies have advanced the application of cell sheet technology to numerous tissues and organs (e.g., liver, cornea and bone). However, previous reviews have failed to discuss vital aspects of this rapidly developing technology, and many new challenges are gradually emerging. This review aims to provide a comprehensive introduction to cell sheet technology from cell selection to the ultimate applications of cell sheets, and challenges and future visions are also described.     

Centrosomes and cancer.

The centrosome functions as the major microtubule organizing center (MTOC) of the cell and as such it determines the number, polarity, and organization of interphase and mitotic microtubules. Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division are all dependent on the normal function of the centrosome and on its orderly duplication, once and only once, in each cell cycle. Malignant tumor cells show characteristic defects in cell and tissue architecture and in chromosome number that can be attributed to inappropriate centrosome behavior during tumor progression. In this review, we will summarize recent observations linking centrosome defects to disruption of normal cell and tissue organization and to chromosomal instability found in malignant tumors.     

Senescence and N release from clover roots following permanent excision of the shoot

Six to seven week old red clover plants (Trifolium pratense L. cv Merviot) were used to investigate the time-course of root senescence following complete and permanent excision from the shoot. Plants were grown in sand culture watered with nutrient solution. After excision of the shoots, roots were left in situ and sampled over a period of up to 42 days. Respiration rate began to decrease immediately after excision, reaching 50% of its initial value after 24 h. The decline involved a reduction in the capacity of the respiratory pathways as measured in the presence of an uncoupler (FCCP) and exogenous glucose. The reduction in respiration could be prevented by supplying 100 mM sucrose to excised roots incubated in nutrient solution at the time of excision, but not 4–5 days after excision. There was a steady reduction in the protein and soluble sugar concentrations from the time of excision and a smaller reduction in starch. Free amino acid concentrations increased immediately after excision, but the temporal dynamics differed between individual amino acids. The total concentration of free amino acids rose to a maximum value 6–13 days after excision, before declining. Under these conditions roots survived for a remarkably long period of time. Depending on the experiment, cell viability, measured as the percentage of cells with positive turgor, was unchanged for at least 20 days, and complete loss of viability was not observed until 34–42 days after excision. There was no appreciable loss of N from the roots until cell viability declined significantly. The potential implications of these results for modelling and management of N cycling in cropping systems is discussed briefly.     

Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

Metabolic modeling of single Th17 cells reveals regulators of autoimmunity

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ERα is an RNA-binding protein sustaining tumor cell survival and drug resistance

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