Cuticular Membrane Fine Structure of Nicotiana tabacum L. Leaves

The cuticular membrane (CM) ofNicotiana tabacumL., includingthe cellin wall (CW), was examined to gain more informationabout the nature and chemical constitution of its fine structurefor possible inclusion in a model system, as recent literaturequestions its function as a major water permeability barrier.Different preparation techniques were used and the results evaluatedto select a method for future studies on tobacco leaf cuticles.Fixation with OsO₄included in the primary fixative, either asa vapour or in combination with other agents, followed by OsO₄aspost-fixative, gave good contrast of the CM. The lamellar structureof the tobacco cuticle proper (CP) was revealed by contrastingwith uranyl acetate and lead citrate. The fine lamellar structureof the CP was very clearly contrasted when KMnO₄was includedin the primary fixative. This was interpreted as indicatingthe tobacco CP to be polar. The reticulate fibrillar patternof the tobacco cuticular layer (CL) containing polysaccharideswas well contrasted when either OsO₄or paraformaldehyde wereincluded in the primary fixative. Cold fixation with glutaraldehydeand dimethyl sulphoxide and post-fixation with OsO₄revealedelectron-opaque material in the outer cutinized, irregularlyoutlined, region of the CW. These ultrahistochemical reactionsare discussed in relation to the known chemical compositionand possible water permeability of the CM. Cuticular fine structure; cuticular transpiration; Nicotiana tabacumL.     

A dual function for Deep orange in programmed autophagy in the Drosophila melanogaster fat body

Lysosomal degradation of cytoplasm by way of autophagy is essential for cellular amino acid homeostasis and for tissue remodeling. In insects such as Drosophila, autophagy is developmentally upregulated in the larval fat body prior to metamorphosis. Here, autophagy is induced by the hormone ecdysone through down-regulation of the autophagy-suppressive phosphoinositide 3-kinase (PI3K) signaling pathway. In yeast, Vps18 and other members of the HOPS complex have been found essential for autophagic degradation. In Drosophila, the Vps18 homologue Deep orange (Dor) has previously been shown to mediate fusion of multivesicular endosomes with lysosomes. A requirement of Dor for ecdysone-mediated chromosome puffing has also been reported. In the present report, we have tested the hypothesis that Dor may control programmed autophagy at the level of ecdysone signaling as well as by mediating autophagosome-to-lysosome fusion. We show that dor mutants are defective in programmed autophagy and provide evidence that autophagy is blocked at two levels. First, PI3K activity was not down-regulated correctly in dor larvae, which correlated with a decrease in ecdysone reporter activity. The down-regulation of PI3K activity was restored by feeding ecdysone to the mutant larvae. Second, neither exogenous ecdysone nor overexpression of PTEN, a silencer of PI3K signaling, restored fusion of autophagosomes with lysosomes in the fat body of dor mutants. These results indicate that Dor controls autophagy indirectly, via ecdysone signaling, as well as directly, via autolysosomal fusion.     

Protein kinase Czeta attenuates hypoxia-induced proliferation of fibroblasts by regulating MAP kinase phosphatase-1 expression

We have previously found that hypoxia stimulates proliferation of vascular fibroblasts through Galphai-mediated activation of ERK1/2. Here, we demonstrate that hypoxia also activates the atypical protein kinase Czeta (PKCzeta) isozyme and stimulates the expression of ERK1/2-specific phosphatase, MAP kinase phosphatase-1 (MKP-1), which attenuates ERK1/2-mediated proliferative signals. Replication repressor activity is unique to PKCzeta because the blockade of classical and novel PKC isozymes does not affect fibroblast proliferation. PKCzeta is phosphorylated upon prolonged (24 h) exposure to hypoxia, whereas ERK1/2, the downstream kinases, are maximally activated in fibroblasts exposed to acute (10 min) hypoxia. However, PKCzeta blockade results in persistent ERK1/2 phosphorylation and marked increase in hypoxia-induced replication. Similarly prolonged ERK1/2 phosphorylation and increase in hypoxia-stimulated proliferation are also observed upon blockade of MKP-1 activation. Because of the parallel suppressive actions of PKCzeta and MKP-1 on ERK1/2 phosphorylation and proliferation, the role of PKCzeta in the regulation of MKP-1 expression was evaluated. PKCzeta attenuation reduces MKP-1 expression, whereas PKCzeta overexpression increases MKP-1 levels. In conclusion, our results indicate for the first time that hypoxia activates PKCzeta, which acts as a terminator of ERK1/2 activation through the regulation of downstream target, MKP-1 expression and thus serves to limit hypoxia-induced proliferation of fibroblasts.     

Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression

The class III phosphatidylinositol 3-kinase (PI3K-III) complex and its phosphorylated lipid product phosphatidylinositol 3-phosphate (PtdIns3P) control the three topologically related membrane-involution processes autophagy, endocytosis, and cytokinesis. The activity of the catalytic unit of PI3K-III complex, the Vacuolar sorting protein 34 (VPS34), depends on the membrane targeting unit Vacuolar sorting protein 15 (VPS15), and the tumor suppressor protein Beclin 1. It is established that the overall activity of VPS34 is positively regulated by Beclin 1, whose positive influence is further controlled through the association with a set of Beclin1 interacting components, which stimulate or inhibit VPS34. The interaction between Beclin 1 and Beclin 1-associated components are controllable and is regulated by phosphorylation in a context-dependent manner. Here, we focus on an emerging concept whereby the activity of the PI3K-III complex is controlled by ubiquitination of Beclin 1 or Beclin 1-associated molecules. In summary, at least three different ubiquitin ligases can affect the positive regulatory function of Beclin 1 towards VPS34, suggesting that ubiquitination is an important and physiologically relevant event in tuning the tumor suppressor function of Beclin 1.     

Crystal structure of the P2 C-repressor: a binder of non-palindromic direct DNA repeats

As opposed to the vast majority of prokaryotic repressors, the immunity repressor of temperate Escherichia coli phage P2 (C) recognizes non-palindromic direct repeats of DNA rather than inverted repeats. We have determined the crystal structure of P2 C at 1.8 Å. This constitutes the first structure solved from the family of C proteins from P2-like bacteriophages. The structure reveals that the P2 C protein forms a symmetric dimer oriented to bind the major groove of two consecutive turns of the DNA. Surprisingly, P2 C has great similarities to binders of palindromic sequences. Nevertheless, the two identical DNA-binding helixes of the symmetric P2 C dimer have to bind different DNA sequences. Helix 3 is identified as the DNA-recognition motif in P2 C by alanine scanning and the importance for the individual residues in DNA recognition is defined. A truncation mutant shows that the disordered C-terminus is dispensable for repressor function. The short distance between the DNA-binding helices together with a possible interaction between two P2 C dimers are proposed to be responsible for extensive bending of the DNA. The structure provides insight into the mechanisms behind the mutants of P2 C causing dimer disruption, temperature sensitivity and insensitivity to the P4 antirepressor.     

Screening of different algae for green synthesis of gold nanoparticles

The cyanobacteria Phormidium valderianum, P. tenue and Microcoleus chthonoplastes and the green algae Rhizoclonium fontinale, Ulva intestinalis, Chara zeylanica and Pithophora oedogoniana were exposed to hydrogen tetrachloroaurate solution and were screened for their suitability for producing nano‐gold. All three cyanobacteria genera and two of the green algae (Rhizoclonium fontinale and Ulva intestinalis) produced gold nanoparticles intracellularly, confirmed by purple colouration of the thallus within 72?h of treatment at 20°C. Extracted nanoparticle solutions were examined by UV‐vis spectroscopy, transmission electron microscopy (TEM) and X‐ray diffractometry (XRD). XRD confirmed the reduction of Au (III) to Au (0). UV‐vis spectroscopy and TEM studies indicated the production of nanoparticles having different shapes and sizes. Phormidium valderianum synthesized mostly spherical nanoparticles, along with hexagonal and triangular nanoparticles, at basic and neutral pHs (pH 9 and pH 7, respectively). Medicinally important gold nanorods were synthesized (together with gold nanospheres) only by P. valderianum at acidic pH (pH 5); this was initially determined by two surface plasmon bands in UV‐vis spectroscopy and later confirmed by TEM. Spherical to somewhat irregular particles were produced by P. tenue and Ulva intestinalis (TEM studies). The UV‐vis spectroscopy of the supernatant of other algal extracts indicated the formation of mostly spherical particles. Production of gold nanoparticles by algae is more ecofriendly than purely chemical synthesis. However, the choice of algae is important: Chara zeylanica and Pithophora oedogoniana were found to be unable to produce nanoparticles.     

A microstructurally driven model for pulmonary artery tissue

A new constitutive model for elastic, proximal pulmonary artery tissue is presented here, called the total crimped fiber model. This model is based on the material and microstructural properties of the two main, passive, load-bearing components of the artery wall, elastin, and collagen. Elastin matrix proteins are modeled with an orthotropic neo-Hookean material. High stretch behavior is governed by an orthotropic crimped fiber material modeled as a planar sinusoidal linear elastic beam, which represents collagen fiber deformations. Collagen-dependent artery orthotropy is defined by a structure tensor representing the effective orientation distribution of collagen fiber bundles. Therefore, every parameter of the total crimped fiber model is correlated with either a physiologic structure or geometry or is a mechanically measured material property of the composite tissue. Further, by incorporating elastin orthotropy, this model better represents the mechanics of arterial tissue deformation. These advancements result in a microstructural total crimped fiber model of pulmonary artery tissue mechanics, which demonstrates good quality of fit and flexibility for modeling varied mechanical behaviors encountered in disease states.     

The role of phosphoinositides in membrane transport.

Phosphoinositides serve as intrinsic membrane signals that regulate intracellular membrane trafficking. Recently, phosphoinositides have been found to direct the localization and activity of effector proteins containing consensus sequence motifs such as FYVE, PH and ENTH domains. In addition, recent results show that regulated synthesis and turnover of phosphoinositides by membrane-associated phosphoinoside kinases and phosphatases spatially restrict the location of effectors critical for cellular transport processes, such as clathrin-mediated endocytosis, autophagy, phagocytosis, macropinocytosis and biosynthetic trafficking.     

Deciphering the rules governing assembly order of mammalian septin complexes

Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9-17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals interdependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six- to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.     

A tumor-associated mutation of FYVE-CENT prevents its interaction with Beclin 1 and interferes with cytokinesis

The tumor suppressor activity of Beclin 1 (BECN1), a subunit of class III phosphatidylinositol 3-kinase complex, has been attributed to its regulation of apoptosis and autophagy. Here, we identify FYVE-CENT (ZFYVE26), a phosphatidylinositol 3-phosphate binding protein important for cytokinesis, as a novel interacting protein of Beclin 1. A mutation in FYVE-CENT (R1945Q) associated with breast cancer abolished the interaction between FYVE-CENT and Beclin 1, and reduced the localization of these proteins at the intercellular bridge during cytokinesis. Breast cancer cells containing the FYVE-CENT R1945Q mutation displayed a significant increase in cytokinetic profiles and bi-multinuclear phenotype. Both Beclin 1 and FYVE-CENT were found to be downregulated in advanced breast cancers. These findings suggest a positive feedback loop for recruitment of FYVE-CENT and Beclin 1 to the intercellular bridge during cytokinesis, and reveal a novel potential tumor suppressor mechanism for Beclin 1.