How to move with no rigid skeleton? The octopus has the answers

The octopus is amazingly flexible and shows exceptional control and coordination in all its movements. It seems remarkable to us skeletal creatures that the octopus achieves all this without a single bone.     

Regulation of light-dependent Gqalpha translocation and morphological changes in fly photoreceptors

Heterotrimeric G-proteins relay signals between membrane-bound receptors and downstream effectors. Little is known, however, about the regulation of Galpha subunit localization within the natural endogenous environment of a specialized signaling cell. Here we show, using live Drosophila flies, that light causes massive and reversible translocation of the visual Gqalpha to the cytosol, associated with marked architectural changes in the signaling compartment. Molecular genetic dissection together with detailed kinetic analysis enabled us to characterize the translocation cycle and to unravel how signaling molecules that interact with Gqalpha affect these processes. Epistatic analysis showed that Gqalpha is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicated that Gqbeta is essential for targeting of Gqalpha to the membrane and suggested that Gqbeta is also needed for efficient activation of Gqalpha by rhodopsin. Our results support the 'two-signal model' hypothesis for membrane targeting in a living organism and characterize the regulation of both the activity-dependent Gq localization and the cellular architectural changes in Drosophila photoreceptors.     

Screening in vivo for RNA-binding peptides from combinatorial libraries.

We have modified a previously developed genetic assay system for RNA-polypeptide interactions in a attempt to more readily identify RNA-binding peptides. The first modification involved the design of a "complex" library that would contain a variety of RNA-binding polypeptides. The second modification involved the use of neomycin phosphotransferase (NPT II) as the reporter gene, therefore allowing "selection" of RNA-binding peptides by kanamycin resistance. The improved screening system should allow the identification of peptides that bind to a variety of RNA structures.     

Inhibition of Papaya latex papain by photosensitive inhibitors. 1-(4,5-dimethoxy-2-nitrophenyl)-2-nitroethene and 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene

1-(4,5-Dimethoxy-2-nitrophenyl)-2-nitroethene (1) was shown to be an irreversible inhibitor of papain (EC 3.4.22.2), causing a complete inhibition (120 min preincubation, pH 8.0), assuming that it attached to Cys-25 at the active site of the enzyme (while a short preincubation time caused activation). Only partial inhibition of papain was achieved, however, with 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (2), a compound synthesized in this work, which is also an irreversible inhibitor of papain. Since both compounds 1 and 2, and in each case of the inhibited enzyme, were 2-nitrobenzyl derivatives, they and the modified enzyme were expected to be photosensitive. Indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in a full recovery of the enzyme activity following inactivation with compound 1 (similar to our previous finding with -galactosidase) and up to 67% recovery following inhibition with compound 2.     

Synthetic Peptide-Based Vaccines Against Influenza

Advances have been made in the development of vaccines based on synthetic peptides representing protective epitopes of the influenza virus. The study reviewed here evaluates the capacity of three epitopes in different delivery systems to induce specific immune response and protect mice against viral challenge infection. Although peptide vaccines are not in use yet, they continue to be explored as is discussed herewith.     

The Middle Subunit of Replication Protein A Contacts Growing RNA-DNA Primers in Replicating Simian Virus 40 Chromosomes

The eukaryotic single-stranded DNA binding protein replication protein A (RPA) participates in major DNA transactions. RPA also interacts through its middle subunit (Rpa2) with regulators of the cell division cycle and of the response to DNA damage. A specific contact between Rpa2 and nascent simian virus 40 DNA was revealed by in situ UV cross-linking. The dynamic attributes of the cross-linked DNA, namely, its size distribution, RNA primer content, and replication fork polarity, were determined. These data suggest that Rpa2 contacts the early DNA chain intermediates synthesized by DNA polymerase α-primase (RNA-DNA primers) but not more advanced products. Possible signaling functions of Rpa2 are discussed, and current models of eukaryotic lagging-strand DNA synthesis are evaluated in view of our results.     

Human embryonic stem cells from aneuploid blastocysts identified by pre-implantation genetic screening

Human embryonic stem cells are derived from the inner cell mass of pre-implantation embryos. The cells have unlimited proliferation potential and capacity to differentiate into the cells of the three germ layers. Human embryonic stem cells are used to study human embryogenesis and disease modeling and may in the future serve as cells for cell therapy and drug screening. Human embryonic stem cells are usually isolated from surplus normal frozen embryos and were suggested to be isolated from diseased embryos detected by pre-implantation genetic diagnosis. Here we report the isolation of 12 human embryonic stem cell lines and their thorough characterization. The lines were derived from embryos detected to have aneuploidy by pre-implantation genetic screening. Karyotype analysis of these cell lines showed that they are euploid, having 46 chromosomes. Our interpretation is that the euploid cells originated from mosaic embryos, and in vitro selection favored the euploid cells. The undifferentiated cells exhibited long-term proliferation and expressed markers typical for embryonic stem cells such as OCT4, NANOG, and TRA-1-60. The cells manifested pluripotent differentiation both in vivo and in vitro. To further characterize the different lines, we have analyzed their ethnic origin and the family relatedness among them. The above results led us to conclude that the aneuploid mosaic embryos that are destined to be discarded can serve as source for normal euploid human embryonic stem cell lines. These lines represent various ethnic groups; more lines are needed to represent all populations.     

Modeling DNA polymerase μ motions: subtle transitions before chemistry

To investigate whether an open-to-closed transition before the chemical step and induced-fit mechanism exist in DNA polymerase μ (pol μ), we analyze a series of molecular-dynamics simulations with and without the incoming nucleotide in various forms, including mutant systems, based on pol μ's crystal ternary structure. Our simulations capture no significant large-scale motion in either the DNA or the protein domains of pol μ. However, subtle residue motions can be distinguished, specifically of His³²⁹ and Asp³³⁰ to assemble in pol μ's active site, and of Gln⁴⁴⁰ and Glu⁴⁴³ to help accommodate the incoming nucleotide. Mutant simulations capture a DNA frameshift pairing and indicate the importance of Arg⁴⁴⁴ and Arg⁴⁴⁷ in stacking with the DNA template, and of Arg⁴⁴⁸ and Gln⁴⁴⁰ in helping to stabilize the position of both the DNA template and the incoming nucleotide. Although limited sampling in the molecular-dynamics simulations cannot be ruled out, our studies suggest an absence of a large-scale motion in pol μ. Together with the known crystallization difficulties of capturing the open form of pol μ, our studies also raise the possibility that a well-defined open form may not exist. Moreover, we suggest that residues Arg⁴⁴⁸ and Gln⁴⁴⁰ may be crucial for preventing insertion frameshift errors in pol μ.     

Chromatin ionic atmosphere analyzed by a mesoscale electrostatic approach

Characterizing the ionic distribution around chromatin is important for understanding the electrostatic forces governing chromatin structure and function. Here we develop an electrostatic model to handle multivalent ions and compute the ionic distribution around a mesoscale chromatin model as a function of conformation, number of nucleosome cores, and ionic strength and species using Poisson-Boltzmann theory. This approach enables us to visualize and measure the complex patterns of counterion condensation around chromatin by examining ionic densities, free energies, shielding charges, and correlations of shielding charges around the nucleosome core and various oligonucleosome conformations. We show that: counterions, especially divalent cations, predominantly condense around the nucleosomal and linker DNA, unburied regions of histone tails, and exposed chromatin surfaces; ionic screening is sensitively influenced by local and global conformations, with a wide ranging net nucleosome core screening charge (56-100e); and screening charge correlations reveal conformational flexibility and interactions among chromatin subunits, especially between the histone tails and parental nucleosome cores. These results provide complementary and detailed views of ionic effects on chromatin structure for modest computational resources. The electrostatic model developed here is applicable to other coarse-grained macromolecular complexes.