Complementary Screens Research Articles

The 19-Amino Acid Insertion in the Tumor-associated Splice Isoform Rac1b Confers Specific Binding to p120 Catenin

The Rac1b splice isoform contains a 19-amino acid insertion not found in Rac1; this insertion leads to decreased GTPase activity and reduced affinity for GDP, resulting in the intracellular predominance of GTP-bound Rac1b. Here, using co-precipitation and proteomic methods, we find that Rac1b does not bind to many common regulators of Rho family GTPases but that it does display enhanced binding to SmgGDS, RACK1, and p120 catenin (p120(ctn)), proteins involved in cell-cell adhesion, motility, and transcriptional regulation. We use molecular modeling and structure analysis approaches to determine that the interaction between Rac1b and p120(ctn) is dependent upon protein regions that are predicted to be unstructured in the absence of molecular complex formation, suggesting that the interaction between these two proteins involves coupled folding and binding. We also find that directed cell movement initiated by Rac1b is dependent upon p120. These results define a distinct binding functionality of Rac1b and provide insight into how the distinct phenotypic program activated by this protein may be implemented through molecular recognition of effectors distinct from those of Rac1.

Almiramides A−C: Discovery and Development of a New Class of Leishmaniasis Lead Compounds

Leishmaniasis is a debilitating disease caused by protozoan parasites of the genus Leishmania, which affects an estimated 12 million people worldwide. The discovery of new lead compounds for leishmaniasis is therefore a pressing concern for global health programs. The organic extract of a Panamanian collection of the marine cyanobacterium Lyngbya majuscula showed strong in vitro activity in two complementary screens against the tropical parasite Leishmania donovani, the causative agent of visceral leishmaniasis. Chromatographic separation of this complex mixture led to the isolation of the highly N-methylated linear lipopeptides, almiramides A-C (1-3). Comparison with the biological activities of a number of related metabolites and semisynthetic derivatives revealed key features required for activity and afforded one new compound (11) with superior in vitro activity. Subsequent synthesis of a library of simplified analogues led to the discovery of several compounds with improved therapeutic indices to the natural products.

Dynamic Host Energetics and Cytoskeletal Proteomes in Human Immunodeficiency Virus Type 1-Infected Human Primary CD4 Cells: Analysis by Multiplexed Label-Free Mass Spectrometry

We report on a proteomic analysis of ex vivo human immunodeficiency virus (HIV) type 1 infection in human primary CD4 cells by shotgun liquid chromatography-tandem mass spectrometry analysis, revealing two distinct proteomic profiles at two phases of virus replication. Relative to mock-infected cells, 168 signature proteins exhibited abundance changes at the first sign of Gag p24 production (8 h postinfection [p.i.]) or the peak of virus replication (24 h p.i.); interestingly, most of the changes were exclusive to only one phase of virus replication. Based on characterization by functional ontology and known human-HIV protein interactions, we observed the enrichment for protein abundance increases pertaining to protein synthesis and nucleasomal reorganization amid an otherwise placid cellular proteome at the first sign of HIV replication. In contrast, we observed indications of decreased protein turnover, concomitant with heightened DNA repair activities and preludes to apoptosis, in the presence of robust virus replication. We also observed hints of disruptions in protein and small molecule trafficking. Our label-free proteomic strategy allowed us to perform multiplexed comparisons-we buttressed our detection specificity with the use of a reverse transcriptase inhibitor as a counterscreen, enabling highlighting of cellular protein abundance changes unique to robust virus replication as opposed to viral entry. In conjunction with complementary high-throughput screens for cellular partners of HIV, we put forth a model pinpointing specific rerouting of cellular biosynthetic, energetic, and trafficking pathways as HIV replication accelerates in human primary CD4 cells.

Special issue of Neurotoxicology and Teratology on “Emerging high throughput and complementary model screens for neurotoxicology”

Special issue of Neurotoxicology and Teratology on “Emerging high throughput and complementary model screens for neurotoxicology”

Temporal regulation of embryonic M-phases.

Temporal regulation of M-phases of the cell cycle requires precise molecular mechanisms that differ among different cells. This variable regulation is particularly clear during embryonic divisions. The first embryonic mitosis in the mouse lasts twice as long as the second one. In other species studied so far (C. elegans, Sphaerechinus granularis, Xenopus laevis), the first mitosis is also longer than the second, yet the prolongation is less pronounced than in the mouse. We have found recently that the mechanisms prolonging the first embryonic M-phase differ in the mouse and in Xenopus embryos. In the mouse, the metaphase of the first mitosis is specifically prolonged by the unknown mechanism acting similarly to the CSF present in oocytes arrested in the second meiotic division. In Xenopus, higher levels of cyclins B participate in the M-phase prolongation, however, without any cell cycle arrest. In Xenopus embryo cell-free extracts, the inactivation of the major M-phase factor, MPF, depends directly on dissociation of cyclin B from CDK1 subunit and not on cyclin B degradation as was thought before. In search for other mitotic proteins behaving in a similar way as cyclins B we made two complementary proteomic screens dedicated to identifying proteins ubiquitinated and degraded by the proteasome upon the first embryonic mitosis in Xenopus laevis. The first screen yielded 175 proteins. To validate our strategy we are verifying now which of them are really ubiquitinated. In the second one, we identified 9 novel proteins potentially degraded via the proteasome. Among them, TCTP (Translationally Controlled Tumor Protein), a 23-kDa protein, was shown to be partially degraded during mitosis (as well as during meiotic exit). We characterized the expression and the role of this protein in Xenopus, mouse and human somatic cells, Xenopus and mouse oocytes and embryos. TCTP is a mitotic spindle protein positively regulating cellular proliferation. Analysis of other candidates is in progress.

Expression of RAB4B, a protein governing endocytic recycling, is co-regulated with MHC class II genes

The small GTPase RAB4 regulates endocytic recycling, a process that contributes to Major Histocompatibility Complex (MHC)-mediated antigen presentation by specialized antigen presenting cells (APC) of the immune system. The gene encoding the RAB4B isoform of RAB4 was singled out by two complementary genome-wide screens. One of these consisted of a computer scan to identify genes containing characteristic MHC class II-related regulatory sequences. The second was the use of chromatin immunoprecipitation coupled to microarrays (ChIP-on-chip) to identify novel targets of a transcriptional co-activator called the MHC class II transactivator (CIITA). We show that the RAB4B gene is regulated by a typical MHC class II-like enhancer that is controlled directly by both CIITA and the multiprotein transcription factor complex known as the MHC class II enhanceosome. RAB4B expression is thus activated by the same regulatory machinery that is known to be essential for the expression of MHC class II genes. This molecular link between the transcriptional activation of RAB4B and MHC class II genes implies that APC boost their antigen presentation capacity by increasing RAB4-mediated endocytic recycling.

Resonance and Cross-Polarization Effects in Conventional and Complementary Split Ring Resonator Periodic Screens

The behavior of periodic plane arrays of split ring resonators (SRRs) and of complementary split ring resonators (CSRRs) when they are illuminated by a plane wave is analyzed. The effects of the angle of incidence and polarization are considered. A surface admittance approach is used for the analysis. Experimental results in free space are provided in order to check the theory. Good qualitative agreement is observed between theory and experiment, although some quantitative disagreements still remain and need explanation. Application of the studied devices as frequency selective surfaces and polarization converters can be envisaged as a consequence of the high variety of the reported resonant and cross-polarization effects

Ab initioanalysis of frequency selective surfaces based on conventional and complementary split ring resonators

Frequency selective surfaces (FSSs) made up of periodic arrays of split ring resonators (SRRs) are analysed. This analysis includes complementary screens, or complementary SRR-FSSs (CSRR-FSSs). It is shown that these FSSs show a dual behaviour, with a stop/pass band behaviour at the frequency of resonance of the SRRs/CSRRs. Cross-polarization effects in the SRR and the CSRR are considered, and it is shown that they permit resonance to occur for normally incident plane wave excitation. This latter property of SRRs and CSRRs also implies that the FSSs considered may act as polarizers and polarization converters as well. An analytical theory, valid for perfectly conducting and infinitely thin screens, is proposed for the SRR-FSSs and CSRR-FSSs. These approximations are valid in the microwave and millimetre-wave range, and up to the terahertz range.

A one-bead, one-stock solution approach to chemical genetics: part 2

Background: Chemical genetics provides a systematic means to study biology using small molecules to effect spatial and temporal control over protein function. As complementary approaches, phenotypic and proteomic screens of structurally diverse and complex small molecules may yield not only interesting individual probes of biological function, but also global information about small molecule collections and the interactions of their members with biological systems. Results: We report a general high-throughput method for converting high-capacity beads into arrayed stock solutions amenable to both phenotypic and proteomic assays. Polystyrene beads from diversity-oriented syntheses were arrayed individually into wells. Bound compounds were cleaved, eluted, and resuspended to generate ‘mother plates’ of stock solutions. The second phase of development of our technology platform includes optimized cleavage and elution conditions, a novel bead arraying method, and robotic distribution of stock solutions of small molecules into ‘daughter plates’ for direct use in chemical genetic assays. This library formatting strategy enables what we refer to as annotation screening, in which every member of a library is annotated with biological assay data. This phase was validated by arraying and screening 708 members of an encoded 4320-member library of structurally diverse and complex dihydropyrancarboxamides. Conclusions: Our ‘one-bead, multiple-stock solution’ library formatting strategy is a central element of a technology platform aimed at advancing chemical genetics. Annotation screening provides a means for biology to inform chemistry, complementary to the way that chemistry can inform biology in conventional (‘investigator-initiated’) small molecule screens.

Babinet’s principle for elastic waves: A numerical test

Babinet’s principle states that the diffracted fields from complementary screens are the negative of each other. In electromagnetics, Babinet’s principle for infinitely thin perfectly conducting complementary screens implies that the sum, beyond the screen plane, of the electric and the magnetic fields (adjusting physical dimensions) equals the incident (unscreened) electric field. A test of the principle for the elastodynamic case was made using numerical calculations, and the results demonstrate that Babinet’s principle holds quite well for complementary plane screens with contrasting boundary conditions; that is, the complementary screen of a stress-free screen is a rigid screen with openings where the original stress-free screen existed, and vice versa. The results are exact in an anisotropic SH case; for the P–SV case, the diffracted waves, PdP, SdS, PdS, and SdP satisfy the principle exactly, while the refracted waves, PdPrSc and SdPrSc, do not satisfy the principle at all (these waves are generally much smaller than the PdS and SdP waves). Diffracted surface waves also do not satisfy the principle. The numerical method is based on a domain-decomposition technique that assigns a different mesh to each side of the screen plane. The effects of the screens on wave propagation are modeled through the boundary conditions, requiring a special boundary treatment based on characteristic variables. The algorithm solves the velocity/stress wave equations and is based on a Fourier/Chebyshev differential operator.

Extension of the babinet principle to scatterers with lumped impedance loads

It is shown that the Babinet principle for scattering by complementary screens can be extended to complementary screens with lumped impedance loads. The complementary loads need to be in a relationship of the same form as the well known relationship between input impedances of Booker.

An Extension of Babinet's Principle

Abstract It is shown that the diffraction pattern produced by any of two complementary screens and the one produced by a slit having the shape of their boundary, oscillates in opposite phase. This extension of Babinet's principle is illustrated with examples from nuclear scattering and classical optics.

Some extensions of the concept of complementary electromagnetic structures

The well known Booker's concept of complementary electromagnetic structures is valid for plane, infinitely thin, perfectly conducting complementary screens situated in a homogeneous lossless medium. It is shown in the paper that the concept can be extended to include pairs of arbitrarily shaped dielectric and/or magnetic bodies, possibly lossy, of arbitrary values of complex permittivity and permeability, positioned symmetrically with respect to the plane of the structure. In this manner, further practical problems can be solved by reducing them to problems the solution of which is either known or can be obtained more easily.Particular attention is devoted to quasi-TEM transmission lines. The complementary concept is extended to include the surrounding medium as well. It is shown that many novel exact relationships can be derived between parameters of such complementary lines, which can aid in determining some parameters, the expressions for which are not known, in terms of known parameters.

Elemental derivation of the Babinet principle in electromagnetic form

Two complementary, thin screens treated in the Babinet principle have one thing in common, the line of their edges. Employing the assumption that scattering of electromagnetic waves from a thin, conducting screen is solely from the elements of the edges, we have made a simple derivation of the Babinet principle in electromagnetic form. We needed to treat but one element of edge with the aperture and conductor interchanged. The factors which affect the amplitudes and phases of the electric and magnetic scattered wavelets reaching any point of observation from the common element of edge of the complementary screens are identical except for phase factors of 0° or 180° caused by polarization effects at the conducting edge. Symmetry conditions from electromagnetic theory yield those factors. We have illustrated the Babinet principle in electromagnetic form by tabularly expressing the scattered waves from two complementary half-planes. These results also were based on the assumption that scattering by a thin screen is solely from the edges.

Babinet's principle for elastic waves

Babinet's principle for elastic waves gives a relationship between the displacement fields due to complementary diffracting screens. The relationship has been derived using the seismic representation theorem as the starting point. The relationship for rigid complementary screens, for stress-free complementary screens, and for mixed rigid-stress-free complementary screens in each case has the same form. In deriving the relationship it is necessary to assume that the “jumps” across the complementary screens are the same as those that would exist when both complementary screens are present. This assumption has been tested in a seismic modeling experiment that utilized both stress-free and rigid screens. The experiments show that the assumption is valid within the accuracy of the experiment and, consequently, that Babinet's principle for elastic waves is valid to the same degree of accuracy.

On the Validity of Babinet's Principle for Fraunhofer Diffraction

Some misconceptions concerning Babinet's principle are pointed out. The Fraunhofer diffraction patterns of complementary screens are not similar unless the detail in the screens obeys certain conditions, which are not easily fulfilled. Some examples of the diffraction patterns of complementary screens are given, illustrating the extent to which Babinet's principle is valid in practice. A way in which the principle can be used to give the diffraction patterns of three-dimensional crystal-structure models is indicated.

Babinet's principle in an anisotropic medium

The results of the theory of diffraction in an isotropic medium are used to derive relations between the vectors of electromagnetic fields produced by diffraction on complementary screens, for the case of two-dimensional diffraction in an electrically anisotropic medium.