The expression of HIV-1 tat and nef genes induces cell-specific changes in growth properties and morphology of different types of rat cells

Among the viral regulatory genes the tat and nef genes of HIV-1 encode the proteins playing a central role in viral replication and exerting pleiotropic effects on the survival and growth of the cells. These effects differ in various cell types, possibly due to the use of genes from different HIV-1 isolates. In this work, we studied the effects of the tat and nef genes on three types of cultured rat cells: primary embryo fibroblasts, pseudonormal Rat-2, and pheochromocytoma PC12. Both genes affected growth properties and morphology of cells, the effects being cell-specific. The proliferative activity of both Rat-2 and PC12 cells was considerably increased after transfection with the tat gene. In primary rat embryo fibroblasts the tat gene induced multilayered foci. More importantly, it was shown that the efficiency of transformation was higher in cells coexpressing tat and nef. The nef gene caused considerable suppression of Rat-2 cell proliferation, but no changes in their morphology. The nef gene transfection of PC12 cells also led to suppression of their proliferative activity. In addition, cellular agglomerates which were morphologically similar to multinuclear syncytial cells were detected in these cells for the first time.     

Comparative Metabolism of Free‐living Bodo saltans and Parasitic Trypanosomatids

Comparison of the genomes of free‐living Bodo saltans and those of parasitic trypanosomatids reveals that the transition from a free‐living to a parasitic life style has resulted in the loss of approximately 50% of protein‐coding genes. Despite this dramatic reduction in genome size, B. saltans and trypanosomatids still share a significant number of common metabolic traits: glycosomes; a unique set of the pyrimidine biosynthetic pathway genes; an ATP‐PFK which is homologous to the bacterial PP_i‐PFKs rather than to the canonical eukaryotic ATP‐PFKs; an alternative oxidase; three phosphoglycerate kinases and two GAPDH isoenzymes; a pyruvate kinase regulated by fructose‐2,6‐bisphosphate; trypanothione as a substitute for glutathione; synthesis of fatty acids via a unique set of elongase enzymes; and a mitochondrial acetate:succinate coenzyme A transferase. B. saltans has lost the capacity to synthesize ubiquinone. Among genes that are present in B. saltans and lost in all trypanosomatids are those involved in the degradation of mureine, tryptophan and lysine. Novel acquisitions of trypanosomatids are components of pentose sugar metabolism, pteridine reductase and bromodomain‐factor proteins. In addition, only the subfamily Leishmaniinae has acquired a gene for catalase and the capacity to convert diaminopimelic acid to lysine.     

Characterization of p43(ARF), a derivative of the p43 component of multiaminoacyl-tRNA synthetase complex released during apoptosis

In human, nine aminoacyl tRNA synthetases are associated with the three auxiliary proteins, p18, p38, and p43, to form a stable multiprotein complex. The p43 component, which has a potent tRNA binding capacity, is associated to the complex via its N-terminal moiety. This protein is also the precursor of the endothelial monocyte-activating polypeptide II (p43(EMAPII), corresponding to the C-terminal moiety of p43), a cytokine generated during apoptosis. Here we examined the cellular pathway that, starting from the p43 subunit of the complex, leads to this extracellular cytokine. We identified a new intermediate in this pathway, named p43(ARF) for Apoptosis-released Factor. This intermediate is produced in cellulo by proteolytic cleavage of endogenous p43 and is rapidly recovered in the culture medium. This p43 derivative was purified from the medium of human U937 cells subjected to serum starvation. It contains 40 additional N-terminal amino acid residues as compared with the cytokine p43(EMAPII) and may be generated by a member of the matrix metalloproteinase family. Recombinant p43(ARF) is a monomer in solution and binds tRNA with a Kd of approximately 6 nM, 30-fold lower than that of p43. Highly purified p43(ARF) or p43(EMAPII) do not stimulate the expression of E-selectin by human umbilical vein endothelial cells. Our results suggest that the cleavage of p43 and its cellular delocalization, and thus the release of this tRNA binding subunit from the complex, is one of the molecular mechanisms leading to the shut down of protein synthesis in apoptosis.     

A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II

The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water. Therefore protons generated at the catalytic centre of PSII need not be released into the thylakoid lumen as generally thought. The cluster is the beginning of a localized, fast proton transfer conduit on the lumenal side of the thylakoid membrane. Proton-dependent conformational changes of PsbO may play a role in the regulation of both supply of substrate water to the water oxidizing complex and the resultant proton transfer.     

Quantitative proteomic assessment of very early cellular signaling events

Technical limitations have prevented proteomic analyses of events occurring less than 30 s after signal initiation. We developed an automated, continuous quench-flow system allowing quantitative proteomic assessment of very early cellular signaling events (qPACE) with a time resolution of 1 s. Using this technique, we determined that autophosphorylation of the epidermal growth factor receptor occurs within 1 s after ligand stimulation and is followed rapidly by phosphorylation of the downstream signaling intermediates Src homologous and collagen-like protein and phospholipase C gamma 1.     

Free energy gap and statistical thermodynamic fidelity of DNA codes.

DNA nanotechnology often requires collections of oligonucleotides called "DNA free energy gap codes" that do not produce erroneous crosshybridizations in a competitive muliplexing environment. This paper addresses the question of how to design these codes to accomplish a desired amount of work within an acceptable error rate. Using a statistical thermodynamic and probabilistic model of DNA code fidelity and mathematical random coding theory methods, theoretical lower bounds on the size of DNA codes are given. More importantly, DNA code design parameters (e.g., strand number, strand length and sequence composition) needed to achieve experimental goals are identified.     

Identification of β integrin‐like‐ and fibronectin‐like proteins in the bivalve mollusk Mytilus trossulus

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin‐like protein and its presumptive ligand, fibronectin‐like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin‐like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin‐like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin‐like protein was detected firstly at the blastula stage and later, the FN‐LP‐immunoreactive cells were scattered in the trochophore larvae. The fibronectin‐like protein was not expressed in the β integrin‐positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin‐ and fibronectin‐like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.     

Plant-made trastuzumab (herceptin) inhibits HER2/Neu+ cell proliferation and retards tumor growth

Background

Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (mAb), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the mAb. Here we used an Agrobacterium-mediated transient expression system to produce trastuzumab in plant cells.

Methodology/Principal Findings

We describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron-optimized Tobacco mosaic virus- and Potato virus X-based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full-size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK-BR-3, in fluorescence-activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER-2-expressing cancer cell proliferation. We show that plant-made trastuzumab (PMT) bound to the Her2/neu oncoprotein of SK-BR-3 cells and efficiently inhibited SK-BR-3 cell proliferation. Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her2+ cells.

Conclusions/Significance

We conclude that PMT is active in suppression of cell proliferation and tumor growth.