Efficacy and specificity of antisense laminin chain-specific expression vectors in blocking laminin induction by TGFβ1: Effect of laminin blockade on TGFβ1-mediated cellular responses

Transforming growth factorβ1 (TGFβ1) elicits a multitude of cellular responses from the epithelial-derived human colon cancer Moser cells. TGFβ1 induces the expression of laminin and fibronectin, and previous studies show that the induction of fibronectin is functionally associated with the regulation of carcinoembryonic antigen (CEA) expression by TGFβ1 (Huang and Chakrabarty, 1994, J Biol Chem 269:28764–28768). In this study we constructed antisense laminin chain-specific expression vectors and determined their efficacy in blocking the expression and the induction of the large multichain laminin molecule by TGFβ1. We also determined the functional role of laminin in several TGFβ1-mediated responses: growth inhibition, downmodulation of anchorage-independent growth, and cellular invasion. Expression of either antisense laminin chain A, B1, or B2 RNA resulted in a downmodulation of endogenous laminin mRNA expression and blocked the induction of laminin protein by TGFβ1 without affecting the induction of other adhesion molecules such as fibronectin or CEA. It is concluded that antisense RNA directed to only one of the laminin chains was sufficient to disrupt the induction of the complex laminin molecule in quite a specific manner. Expression of antisense laminin RNA downregulated cellular adhesion to extracellular matrix (ECM) laminin and blocked the ability of TGFβ1 to upmodulate adhesion to ECM laminin. Expression of antisense laminin RNA, however, did not alter the downregulating effect of TGFβ1 on cellular proliferation, anchorage-independent growth, or cellular invasion, suggesting that the induction of laminin did not play a significant functional role in these TGFβ1-mediated cellular responses. It is likely that other adhesion pathways may be involved in mediating the action of TGFβ1 in this cell line. J. Cell. Physiol. 178:296–303, 1999. © 1999 Wiley-Liss, Inc.     

A Review on Design Strategies for Carbon Based Metal Oxides and Sulfides Nanocomposites for High Performance Li and Na Ion Battery Anodes

Carbon‐oxide and carbon‐sulfide nanocomposites have attracted tremendous interest as the anode materials for Li and Na ion batteries. Such composites are fascinating as they often show synergistic effect compared to their singular components. Carbon nanomaterials are often used as the matrix due to their high conductivity, tensile strength, and chemical stability under the battery condition. Metal oxides and sulfides are often used as active material fillers because of their large capacity. Numerous works have shown that by taking one step further into fabricating nanocomposites with rational structure design, much better performance can be achieved. The present review aims to present and discuss the development of carbon‐based nanocomposite anodes in both Li ion batteries and Na ion batteries. The authors introduce the individual components in the composites, i.e., carbon matrices (e.g., carbon nanotube, graphene) and metal oxides/sulfides; followed by evaluating how advanced nanostructures benefit from the synergistic effect when put together. Particular attention is placed on strategies employed in fabricating such composites, with examples such as yolk–shell structure, layered‐by‐layered structure, and composite comprising one or more carbon matrices. Lastly, the authors conclude by highlighting challenges that still persist and their perspective on how to further develop the technologies.     

Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope-labeled concatenated peptide standards

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the alpha, beta, and gamma protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits alpha, beta, and gamma was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies.     

Extended polypeptide linkers establish the spatial architecture of a pyruvate dehydrogenase multienzyme complex

Icosahedral pyruvate dehydrogenase (PDH) enzyme complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of approximately 75-90 A from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.     

Differential effects of p63 mutants on transactivation of p53 and/or p63 responsive genes

p63, known to play a role in development, has more recently also been implicated in cancer progression. Mutations in p63 have been shown to be responsible for several human developmental diseases. Differential splicing of the p63 gene gives rise to p63 isoforms, which can act either as tumor suppressors or as oncogene. In this report, we studied the effects of naturally occurring TAp637 mutants on the regulation of p53/p63 and p63 specific target genes. We observed significant differences among p63 mutants to regulate the p53/p63 and p63 specific target genes. Additionally, we observed a differential effect of p63 mutants on wildtype-p63-mediated induction ofp53/p63 and p63 specific target genes. We also demonstrated that these mutants differentially regulate the binding of wildtype p63 to the promoter of target genes. Furthermore, the effects of these mutants on cell death and survival were consistent with their ability to regulate the downstream targets when compared to wildtype TAp63T. In summary, our data demonstrate that p63 mutants exhibit differential effects on p63 and p53/p63 specific target genes and on the induction of apoptosis, and provide further insight into the function of p63.     

Three-dimensional imaging of the highly bent architecture of Bdellovibrio bacteriovorus by using cryo-electron tomography

Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria, including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformation of the outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highly bent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We propose that rearrangements of these structures, in combination with the unique properties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey.     

Chemoreceptors in Caulobacter crescentus: trimers of receptor dimers in a partially ordered hexagonally packed array

Chemoreceptor arrays are macromolecular complexes that form extended assemblies primarily at the poles of bacterial cells and mediate chemotaxis signal transduction, ultimately controlling cellular motility. We have used cryo-electron tomography to determine the spatial distribution and molecular architecture of signaling molecules that comprise chemoreceptor arrays in wild-type Caulobacter crescentus cells. We demonstrate that chemoreceptors are organized as trimers of receptor dimers, forming partially ordered hexagonally packed arrays of signaling complexes in the cytoplasmic membrane. This novel organization at the threshold between order and disorder suggests how chemoreceptors and associated molecules are arranged in signaling assemblies to respond dynamically in the activation and adaptation steps of bacterial chemotaxis.