Dielectric behavior of wild-type yeast and vacuole-deficient mutant over a frequency range of 10 kHz to 10 GHz.

Dielectric behavior of Saccharomyces cerevisiae wild-type and vacuole-deficient mutant cells has been studied over a frequency range of 10 kHz to 10 GHz. Both types of cells harvested at the early stationary growth phase showed dielectric dispersion that was phenomenologically formulated by a sum of three separate dispersion terms: beta 1-dispersion (main dispersion) and beta 2-dispersion (additional dispersion) and gamma-dispersion due to orientation of water molecules. The beta 1-dispersion centered at a few MHz, which has been extensively studied so far, is due to interfacial polarization (or the Maxwell-Wagner effect) related to the plasma membrane. The beta 2-dispersion for the vacuole-deficient mutant centered at approximately 50 MHz was explained by taking the cell wall into account, whereas, for the wild-type cells, the beta 2-dispersion around a few tens MHz involved the contributions from the vacuole and cell wall.     

New approach to reveal genes that control cell wall biogenesis of the yeast Saccharomyces cerevisiae

The Mcd4 protein of Saccharomyces cerevisiae is probably involved in addition of the phosphoethanolamine moiety to the first mannose residue of the glycosylphosphatidylinositol precursor(s). However, significance of this modification is unclear. Besides, functions of the MCD4 gene also is not completely clear, since mutations in this gene may have pleiotropic manifestations, which are not obviously related to the glycosylphosphatidylinositol biosynthesis. To clarify the functions of Mcd4p we have performed a search for genes whose mutations are lethal or semilethal in combination with the ssu21 mutation in MCD4. In total, we have isolated six mutations some of which cause sensitivity to SDS and/or calcofluor white. Genes which are able to complement two of these mutations were cloned. They were MNN9 which encodes protein involved in formation of outer chains of the N-linked glycans of secretory proteins and GWT1, encoding the protein of the endoplasmic reticulum involved in the glycosylphosphatidylinositol biosynthesis. The results obtained indicate that in both cases growth inhibition was caused by defect of cell wall biogenesis and alteration of folding of secretory proteins. Search for mutations that lethal in combination with the ssu21 is an effective approach to reveal genes involved in the control of cell wall biogenesis.     

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.     

Inhibitory activity of 1-farnesylpyridinium on the spatial control over the assembly of cell wall polysaccharides in Schizosaccharomyces pombe

The modes of actions of 1-farnesylpyridinium (FPy) on yeast cell growth were investigated on the basis of its effects on cell cycle progression, morphogenesis and the related events for construction of cell wall architecture in Schizosacchromyces pombe. FPy predominantly inhibited the growth of the yeast cells after various cycles of cell division so that cells were arrested at the phase of separation into daughter cells accompanying morphological changes to swollen spherical cells at 24 h of incubation. FPy-treated cells were osmotically stable but were susceptible to the lytic action of (1, 3) beta-D-glucanases, and characterized by serious damages to the cell wall architecture as represented by a rough and irregular surface outlook. The isolated cell wall fraction gave a similar hexose composition with or without FPy treatment, suggesting that FPy did not inhibit the synthesis of each cell wall polysaccharide. FPy was permissive for the extracellular accumulation of amorphous cell wall materials and septum development in protoplasts, but absolutely interfered with the following morphogenetic process for construction of the rod-shaped cell wall architecture. Our results suggest the inhibitory activity of FPy on the spatial control over the assembly of cell wall polysaccharides.     

Ascorbate and plant cell growth

Ascorbate and related enzymes are involved in the control of several plant growth processes. Ascorbate modulates cell growth by controlling (i) the biosynthesis of hydroxyproline-rich proteins required for the progression of G1 and G2 phases of the cell cycle, (ii) the cross-linking of cell wall glycoproteins and other polymers, and (iii) redox reactions at the plasma membrane involved in elongation mechanisms. The effect of ascorbate on onion root elongation is reviewed here. The ascorbate free radical induces a high vacuolization responsible for elongation. This effect may be dependent on the activity of the redox system linked to the plasma membrane. Current data are discussed on the basis of the modulation of the plasma membrane energetic state derived from the ascorbate-induced hyperpolarization and the activity of an intrinsic transplasmalemma ascorbate-regenerating enzyme.     

Identification and culture of proliferative cells in abnormal Taenia solium larvae: Role in the development of racemose neurocysticercosis

Racemose neurocysticercosis is an aggressive disease caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord resulting in a mass effect and chronic inflammation. Although expansion is likely caused by the proliferation and growth of the parasite bladder wall, there is little direct evidence of the mechanisms that underlie these processes. Since the development and growth of cysts in related cestodes involves totipotential germinative cells, we hypothesized that the expansive growth of the racemose larvae is organized and maintained by germinative cells. Here, we identified proliferative cells expressing the serine/threonine-protein kinase plk1 by in situ hybridization. Proliferative cells were present within the bladder wall of racemose form and absent from the homologous tissue surrounding the vesicular form. Cyst proliferation in the related model species Taenia crassiceps (ORF strain) occurs normally by budding from the cyst bladder wall and proliferative cells were concentrated within the growth buds. Cells isolated from bladder wall of racemose larvae were established in primary cell culture and insulin stimulated their proliferation in a dose-dependent manner. These findings indicate that the growth of racemose larvae is likely due to abnormal cell proliferation. The different distribution of proliferative cells in the racemose larvae and their sensitivity to insulin may reflect significant changes at the cellular and molecular levels involved in their tumor-like growth. Parasite cell cultures offer a powerful tool to characterize the nature and formation of the racemose form, understand the developmental biology of T. solium, and to identify new effective drugs for treatment.     

Growth-phase-dependent gene expression profiling of poplar (Populus alba x Populus tremula var. glandulosa) suspension cells

Complex sequences of morphological and biochemical changes occur during the developmental course of a batch plant cell culture. However, little information is available about the changes in gene expression that could explain these changes, because of the difficulties involved in isolating specific cellular events or developmental phases in the overlapping phases of cell growth. In an attempt to obtain such information we have examined the global growth phase-dependent gene expression of poplar cells in suspension cultures by cDNA microarray analysis. Our results reveal that significant changes occur in the expression of genes with functions related to protein synthesis, cell cycling, hormonal responses and cell wall biosynthesis, as cultures progress from initiation to senescence, that are highly correlated with observed developmental and physiological changes in the cells. Genes encoding protein kinases, calmodulin and proteins involved in both ascorbate metabolism and water-limited stress responses also showed strong stage-specific expression patterns. Our report provides fundamental information on molecular mechanisms that control cellular changes throughout the developmental course of poplar cell cultures.     

Lymphoma models for B cell activation and tolerance. VI. Reversal of anti-Ig-mediated negative signaling by T cell-derived lymphokines

We have recently described three "immature" B cell lymphomas which are exquisitely sensitive to growth inhibition by anti-Ig reagents and may serve as models for tolerance induction in normal B cells. These cells are inhibited from cell cycle progression into S after receiving a negative signal in early G1. In this paper, we demonstrate that the growth inhibition by anti-Ig can be prevented and reversed by the addition of supernatants from T cell lines. One such line, called Tova, produces factors which restore normal levels of DNA synthesis in the presence of concentrations of anti-Fab or anti-kappa immunoglobulins which cause up to a 90% inhibition of thymidine incorporation in a 2- to 3-day culture period. This factor is at least partially effective when added up to 24 hr after anti-Ig to unsynchronized lymphoma cells and it does not alter the growth of control cultures. Studies using synchronized lymphoma cells indicated that the T cell factor permitted cycle progression into S when added during the early G1 exposure to anti-kappa and was less effective when added late in G1. Preliminary characterization suggests that both B cell growth factor II (interleukin 5) and B cell stimulatory factor 1 (interleukin 4) have additive activity in this system, although another unidentified lymphokine may also be involved. The relevance of T cell reversal of Ig receptor-mediated negative signaling to neonatal B cell tolerance is emphasized.     

过表达漆酶基因Lelcc1的香菇菌株构建及表型分析

真菌漆酶（laccase）是一种多酚氧化酶,在真菌生长发育中具有重要作用。本研究采用根癌农杆菌介导转化的方法,以香菇Lentinula edodes菌株W1为受体菌株,在Leactin基因启动子调控下过表达Lelcc1基因;对其中7个单拷贝插入的转化子进行qRT-PCR分析,7个Lelcc1基因表达量较出发菌株W1均上调了1.5-8倍。进一步分析了这7个转化子的遗传稳定性;挑取了3个稳定的转化子进行表型分析,主要包括不同培养基中的生长速度、代料栽培过程中菌棒转色程度、以及透射电镜观察菌丝细胞的超微结构。发现转化子和受体菌株W1的生长速度无显著差异,但代料栽培过程中转化子较W1转色更快,菌棒表面颜色更深;透射电镜观察菌丝细胞的超微结构发现在细胞壁厚度、细胞膜形态等方面,其中两个超量表达转化子与W1间存在显著差异。结果表明,香菇Lelcc1基因可能参与了转色过程中色素合成或积累,也可能与细胞壁形成有关。     

Genome-Wide Screening of Aluminum Tolerance in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genome-wide screening has identified 37 Al-tolerance genes in Saccharomyces cerevisiae. These genes can be roughly categorised into three groups on the basis of function, i.e., genes related to vesicle transport processes, signal transduction pathways, and protein mannosylation. The largest group is composed of genes related to vesicle transport processes; severe Al sensitivity was found in yeast strains lacking these genes. The retrograde transport of endosome-derived vesicles back to the Golgi apparatus is an important factor in determining the Al tolerance of the vesicle transport system. The PKC1-MAPK cascade signalling pathway is important in the Al tolerance of signal transduction. The lack of the gene implicated in this process leads to weakened cell wall architecture, rendering the yeast Al-sensitive. Alternatively, Al might attack the cell wall and/or plasma membrane, and, as signalling is prevented in cells devoid of the genes related to signalling processes, the cells may be unable to alleviate the damage. The genes for protein mannosylation are also associated with Al tolerance, demonstrating the importance of cell wall architecture. These genes are involved in cell integrity processes. An erratum to this article is available at .     

Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities

The insulin-like growth factor (IGF)-binding proteins (IGFBPs) are a family of six homologous proteins with high binding affinity for IGF-I and IGF-II. Information from NMR and mutagenesis studies is advancing knowledge of the key residues involved in these interactions. IGF binding may be modulated by IGFBP modifications, such as phosphorylation and proteolysis, and by cell or matrix association of the IGFBPs. All six IGFBPs have been shown to inhibit IGF action, but stimulatory effects have also been established for IGFBP-1, -3, and -5. These generally involve a decrease in IGFBP affinity and may require cell association of the IGFBP, but precise mechanisms are unknown. The same three IGFBPs have well established effects that are independent of type I IGF receptor signaling. IGFBP-1 exerts these effects by signaling through alpha(5)beta(1)-integrin, whereas IGFBP-3 and -5 may have specific cell-surface receptors with serine kinase activity. The regulation of cell sensitivity to inhibitory IGFBP signaling may play a role in the growth control of malignant cells.     

Arabidopsis formin AtFH6 is a plasma membrane-associated protein upregulated in giant cells induced by parasitic nematodes

Plant-parasitic nematodes Meloidogyne spp induce an elaborate permanent feeding site characterized by the redifferentiation of root cells into multinucleate and hypertrophied giant cells. We have isolated by a promoter trap strategy an Arabidopsis thaliana formin gene, AtFH6, which is upregulated during giant cell formation. Formins are actin-nucleating proteins that stimulate de novo polymerization of actin filaments. We show here that three type-I formins were upregulated in giant cells and that the AtFH6 protein was anchored to the plasma membrane and uniformly distributed. Suppression of the budding defect of the Saccharomyces cerevisiae bni1Delta bnr1Delta mutant showed that AtFH6 regulates polarized growth by controlling the assembly of actin cables. Our results suggest that AtFH6 might be involved in the isotropic growth of hypertrophied feeding cells via the reorganization of the actin cytoskeleton. The actin cables would serve as tracks for vesicle trafficking needed for extensive plasma membrane and cell wall biogenesis. Therefore, determining how plant parasitic nematodes modify root cells into giant cells represents an attractive system to identify genes that regulate cell growth and morphogenesis.     

The role of pectin in plant morphogenesis

The presence of a polysaccharidic cell wall distinguishes plant cells from animal cells and is responsible for fundamental mechanistic differences in organ development between the two kingdoms. Due to the presence of this wall, plant cells are unable to crawl and contract. On the other hand, plant cell size can increase by several orders of magnitude and cell shape can change from a simple polyhedron or cube to extremely intricate. This expansive cellular growth is regulated by the interaction between the cell wall and the intracellular turgor pressure. One of the principal cell wall components involved in temporal and spatial regulation of the growth process is pectin. Through biochemical changes to pectin composition and biochemical configuration, the properties of this material can be altered to trigger specific developmental processes. Here, the roles of pectin in three systems displaying rapid growth - the elongation zone of the root, the tip region of the pollen tube, and organ primordia formation at the shoot apical meristem - are reviewed.     

Rice plants response to the disruption of OsCSLD4 gene

The plant cell wall is a complex polysaccharide network and performs important developmental and physiological functions far beyond supplying the physical constrains. Plant cells have the ability to react to cell wall defects as exhibited by changes in gene expression, accumulation of ectopic lignin, stress responses and growth arrest. It is a major challenge to understand how plants sense and respond to wall integrity since very little is known about the signaling involved in the responses. Cellulose synthase-like D (CSLD) proteins mediating the biosynthesis of a wall polysaccharide polymer make up a common subfamily to all plants. Recently, we have reported the functional characterization of CSLD4 in rice. Mutations in OsCSLD4 show morphological alterations and pleiotropic effects on wall compositions and structure. Our study demonstrates that OsCSLD4 play a critical role in cell wall formation and plant growth. Here we show the subtle wall alterations by separating the culm residues into five fractions. Quantitative RT-PCR analysis further revealed that the expression of various genes involved in xylan synthesis and cell cycle regulation was altered in mutant plants, as the responses to OsCSLD4 disruption. Therefore, plants may have fine sensory machinery to react to wall defects and modulate growth for adapting to the changes.Key words: OsCSLD4, cell wall biosynthesis, plant development, wall integrity, rice     

Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIF5A domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIF5A may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.     

Arabidopsis VASCULAR-RELATED NAC-DOMAIN6 directly regulates the genes that govern programmed cell death and secondary wall formation during xylem differentiation

Xylem consists of three types of cells: tracheary elements (TEs), parenchyma cells, and fiber cells. TE differentiation includes two essential processes, programmed cell death (PCD) and secondary cell wall formation. These two processes are tightly coupled. However, little is known about the molecular mechanisms underlying these processes. Here, we show that VASCULAR-RELATED NAC-DOMAIN6 (VND6), a master regulator of TEs, regulates some of the downstream genes involved in these processes in a coordinated manner. We first identified genes that are expressed downstream of VND6 but not downstream of SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), a master regulator of xylem fiber cells, using transformed suspension culture cells in microarray experiments. We found that VND6 and SND1 governed distinct aspects of xylem formation, whereas they regulated a number of genes in common, specifically those related to secondary cell wall formation. Genes involved in TE-specific PCD were upregulated only by VND6. Moreover, we revealed that VND6 directly regulated genes that harbor a TE-specific cis-element, TERE, in their promoters. Thus, we found that VND6 is a direct regulator of genes related to PCD as well as to secondary wall formation.     

Cytoskeleton and morphogenesis in brown algae

BACKGROUND: Morphogenesis on a cellular level includes processes in which cytoskeleton and cell wall expansion are strongly involved. In brown algal zygotes, microtubules (MTs) and actin filaments (AFs) participate in polarity axis fixation, cell division and tip growth. Brown algal vegetative cells lack a cortical MT cytoskeleton, and are characterized by centriole-bearing centrosomes, which function as microtubule organizing centres. SCOPE: Extensive electron microscope and immunofluorescence studies of MT organization in different types of brown algal cells have shown that MTs constitute a major cytoskeletal component, indispensable for cell morphogenesis. Apart from participating in mitosis and cytokinesis, they are also involved in the expression and maintenance of polarity of particular cell types. Disruption of MTs after Nocodazole treatment inhibits cell growth, causing bulging and/or bending of apical cells, thickening of the tip cell wall, and affecting the nuclear positioning. Staining of F-actin using Rhodamine-Phalloidin, revealed a rich network consisting of perinuclear, endoplasmic and cortical AFs. AFs participate in mitosis by the organization of an F-actin spindle and in cytokinesis by an F-actin disc. They are also involved in the maintenance of polarity of apical cells, as well as in lateral branch initiation. The cortical system of AFs was found related to the orientation of cellulose microfibrils (MFs), and therefore to cell wall morphogenesis. This is expressed by the coincidence in the orientation between cortical AFs and the depositing MFs. Treatment with cytochalasin B inhibits mitosis and cytokinesis, as well as tip growth of apical cells, and causes abnormal deposition of MFs. CONCLUSIONS: Both the cytoskeletal elements studied so far, i.e. MTs and AFs are implicated in brown algal cell morphogenesis, expressed in their relationship with cell wall morphogenesis, polarization, spindle organization and cytokinetic mechanism. The novelty is the role of AFs and their possible co-operation with MTs.