The use of a combinatorial library method to isolate human tumor cell adhesion peptides

Summary Tumor cell progression is dependent in part on the successful adhesive interactions of the cells with the extracellular matrix. In this study, a new approach is described to isolate linear peptide ligand candidates involved in cellular adhesion. A synthetic combinatorial peptide library based on the one-bead-one-peptide concept was incubated with live human prostate cancer cells for 90 min at 37 °C. The peptide bead coated with a monolayer of cells was then isolated for micro sequencing. The DU145 (DU-H) cells were chosen since they have been previously characterized as containing elevated levels of a laminin receptor for cell adhesion, the ₆₁ integrin on the cell surface. The use of a function-blocking antibody (GoH3) allows for the detection of peptides which are ₆-specific ligand candidates. From two different libraries (linear 9-mer and 11-mer) of a total of 1 500 000 beads, 68 peptide beads containing attached cells were isolated. These positive beads were then retested to determine the ability of the GoH3 antibody to block binding of the cells to the peptide beads. The ₆ integrin candidate peptide beads (five in total) were recovered and two of the beads were microsequenced. These two peptides, RU-1 (LNIVSVNGRHX) and RX-1 (DNRIRLQAKXX), resemble the previously reported active peptide sequences (GD-2 and AG-73) from native laminin. The RU-1, RX-1 and AG-73 peptides were tested for their ability to support cell attachment and to bind the cell surface of DU-H prostate carcinoma cells in suspension using fluorescence-activated cell-sorting (FACS) analysis. Both RU-1 and AG-73 peptides supported cellular attachment within 1 h. In contrast, after 1 h, EHS laminin supported both cellular attachment and spreading. The RX-1 peptide exhibited only weak binding to the DU-H prostate carcinoma cells. FACS analysis indicated that AG-73 peptide attached to tumor cell surfaces over a range of concentrations, whereas the RU-1 peptide showed a homogeneous concentration required for attachment. The described strategy for screening a random peptide library offers three advantages: (i) ligands for conformationally sensitive receptors of adhesion can be isolated using live cells; (ii) specific binding can be selected for using function-blocking antibodies; and (iii) peptides supporting adhesion independent of spreading properties can be distinguished. In principle, specific adhesive peptides without prior knowledge of the sequence could be isolated for any epithelial cell surface receptor for which a function-blocking reagent is available.     

On the critical exponent for random walk intersections

The exponent _d for the probability of nonintersection of two random walks starting at the same point is considered. It is proved that 1/2<₂3/4. Monte Carlo simulations are done to suggest ₂=0.61 and ₃0.29.     

Mapping protein-protein contact sites using cellulose-bound peptide scans

Summary We have characterized the interaction of two monoclonal antibodies with their respective antigens using cellulose-bound sets of overlapping peptides (peptide scans). Both antibodies CB/RS/5 and CB/MT/1 recognize discontinuous epitopes present in human interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-). In addition, the interaction between TNF-a and its 55-kDa receptor (TNF-R) was investigated by the same approach. Both antibodies, as well as TNF-, interacted with two or more regions of the peptide scans. Antibody-binding competition studies between the native antigens and peptides, covering single parts of the binding regions, enabled us to distinguish between binding to the paratope or other regions of the antibody. The combination of these experimental approaches allowed the identification of short antigen-derived sequences that are separated on the primary sequence but close in space on the surface of IL-10 and TNF-, thus representing putative discontinuous epitopes. In the case of the TNF-R-derived peptide scans, two of the identified regions interact with the structurally similar TNF- in the TNF--TNF-R complex. These data indicate that this approach should be generally applicable for mapping nonlinear protein-protein contact sites.     

Comparative conformational analysis of peptide libraries

Six computer-based combinatorial libraries,including tetrapeptide sequences (generated with fiveamino acids) and conformations (generated with fivemain chain and three side chain rotamers), wereobtained and sequence-conformation probabilities werecalculated with a molecular and statistical mechanicsprocedure. The structural motifs -helix,-sheet, 3₁₀-helix, reverse turn I and-turn were focused in these calculations. Itis shown that sequence-conformation-probabilitysurfaces provide a broad view of structural changesaccompanying changes in sequence. Numerical indicesare defined to enable comparisons between frequenciesof occurrence of these structural motifs in peptidelibraries and in a database of low sequence identityprotein structures. Fine details ofsequence-conformation-probability surfaces show theeffect of point mutations. Broad comparisons betweendifferent regions of these surfaces indicate how toselect the occurrence of structural motifs in thecombinatorial synthesis of peptide chains.     

Positivity of entropy production in the presence of a random thermostat

We study nonequilibrium statistical mechanics in the presence of a thermostat acting by random forces, and propose a formula for the rate of entropy productione() in a state . When is a natural nonequilibrium steady state we show thate()0, and sometimes we can provee()>0.     

Interfacial properties in solid-stabilized emulsions

We prepared concentrated monodisperse oil-in-water emulsions stabilized by solid particles. The osmotic resistance, , of the emulsions was measured for different oil volume fractions above the random close packing ( ). The dimensionless osmotic resistance, /(/R) ( being the interfacial tension and R being the undeformed drop radius), was always substantially higher than the corresponding values obtained for surfactant-stabilized emulsions. It can be concluded that droplet deformation in solid-stabilized emulsions is not controlled by the capillary pressure, /R, of the non-deformed droplets but rather by 0/R, 0 being a parameter characterizing the rigidity of the droplets surfaces. The data can be interpreted considering that the interfacial layers are elastic at small deformations and exhibit plasticity at intermediate deformations. 0 corresponds to the surface yield stress, i.e. the transition between elastic and plastic regimes. We discuss the origin of the surface behavior considering the strong lateral interactions that exist between the adsorbed solid particles. We propose an independent measurement of 0 based on the critical bulk stress that produces droplet fragmentation in dilute emulsions submitted to shear. Finally, the bulk shear elastic modulus was measured as a function of and confirms many of the features revealed by the osmotic resistance.     

Analysis of novel streptavidin-binding peptides,identified using a phage display library,shows that amino acids external to a perfectly conserved consensus sequence and to the presented peptides contribute to binding

Summary Streptavidin-binding peptides containing the consensus amino acid sequence motif EPDW were identified using a phage display library. Phage presenting peptides containing these sequences bound streptavidin in a biotin-sensitive fashion and could be eluted with biotin. The previously identified streptag peptide sequence (AWRHPQGG) competed with phage presenting the EPDW consensus sequence for streptavidin binding. Furthermore, the EPDW sequence has two amino acids in common with yet another previously identified streptavidin-binding sequence, GDWVFI, which has similar biochemical properties. Binding inhibition studies revealed that residues flanking EPDW, as well as residues of the modified phage pIII product to which displayed peptides are fused, contributed to streptavidin binding. The derivation of small molecules based on the structure of peptides selected using display methods is a potentially important application of phage display technology. The relevance of the observations made here for that application are discussed. Finally, a group of nuisance peptides of the consensus sequence WHWWXW, whose binding specificity has not been fully elucidated, but which have been isolated in a number of biopanning experiments, including those that do not utilize streptavidin, are also described.     

Random aggregation and random-walking center of mass

Two random aggregation models are used in demonstrating the properties of the random displacementsr _i of the center of mass of aggregating particles. It is found that r _i is a randomly decreasing sequence that scales with the cluster size (steps)s and _i ^=1/s r _i s ^1/D , whereD is the fractal dimension. The center-of-mass random walk is a consistent representation of the dynamics of aggregation.     

Photostability of Molecules of Aromatic Amines in a Polymeric Matrix

In the temperature range T = 5–295 K, the spectralluminescent properties and curves of thermostimulated luminescence (TSL) of molecules of triphenylamine (TPA), trirtolylamine (TTA), triranisolamine (TAA), and N,N'diphenylbis(3methylphenyl)(1,1'biphenyl)4,4'diamine (TPD) in polystyrene and 4Brpolystyrene matrices have been investigated. It has been found that photoirradiation at room temperature in the region of the lowest electron transition of molecules leads to a decrease in the intensities of the luminescence, photoluminescence, and TSL bands, as well as to the formation of new deep traps for charge carriers. At equal irradiation doses these changes in TPD are noticeably less pronounced than in TPA, TTA, and TAA. The higher photochemical stability of TPD molecules compared to TPA, TTA, and TAA is attributed to the difference in the mechanisms of nonradiative deactivation of the triplet states of molecules.     

More inequalities for critical exponents

A variety of rigorous inequalities for critical exponents is proved. Most notable is the low-temperature Josephson inequalitydv +2 2–. Others are 1 1 +v, 1 1 , 1,d 1 + 1/ (for d),dv, ₃ + (for d), ₄ , and _{2m 2m+2} (form 2). The hypotheses vary; all inequalities are true for the spin-1/2 Ising model with nearest-neighbor ferromagnetic pair interactions.NSF Predoctoral Fellow (1976–1979). Research supported in part by NSF Grant PHY 78-23952.     

Contribution to the thermodynamics of the isomorphous γ ⇆ α-transition in metallic cerium

Using the method of the theory of thermodynamic stability and the form of the lines of the phase equilibrium of the isomorphous -transition in metallic cerium, the authors investigate the behavior of the basic thermodynamic characteristics of this transition in the two-phase region and in a neighborhood of the critical point. Attention is focused primarily on the little-studied adiabatic quantities (T/C_V, (–P/V)_S, (T/V)_S). It is shown that along the phase tie-lines these quantities do not depend on V and S; in the limit of the critical point all adiabatic quantities have nonzero minima, and all isodynamic quantities (T/Cp, (–P/V)_T, (T/V)_P) approach zero according to the same law. The obtained thermodynamic results are compared with existing experimental data and models which can be interpreted thermodynamically. It is concluded that critical phenomena in cerium correspond to critical behavior of the first type.Dnepropetrovsk State University Dedicated to the 300th Anniversary of the Unification of the Ukraine with Russia.Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Fizika, No. 5, pp. 66–70, May, 1991.     

The temperature vibrations of urea molecules

The structural and dynamic parameters of urea at 112°K and 295°K were determined by the least squares method. The characteristic temperature of the torsional optical vibrations of a molecule about a C-O bond was determined and is in good agreement with the value determined by Raman scattering. The fractional X-coordinate of the nitrogen atom corrected for torsional vibrations was determined and it was found that the magnitude of the projection of the C-N bond in the given temperature range changes only within the limits of observational errors. A new method, called temperature difference synthesis, is described and it is shown that it is suitable for rapid qualitative determination of the thermal anisotropy of the vibrations of atoms in a crystal lattice.

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112°K 295°K. C-O , , . X- , C-N . , , , .

     

A theory of 1/f noise

Letu() be an absolutely integrable function and define the random process where thet _i are Poisson arrivals and thes _i, are identically distributed nonnegative random variables. Under routine independence assumptions, one may then calculate a formula for the spectrum ofn(t), S _n(), in terms of the probability density ofs, p_s(). If any probability density p_s() having the property p_s() I for small is substituted into this formula, the calculated S_n() is such that S_n() 1 for small . However, this is not a spectrum of a well-defined random process; here, it is termed alimit spectrum. If a probability density having the property p_s() for small , where > 0, is substituted into the formula instead, a spectrum is calculated which is indeed the spectrum of a well-defined random process. Also, if the latter p_s is suitably close to the former p_s, then the spectrum in the second case approximates, to an arbitrary, degree of accuracy, the limit spectrum. It is shown how one may thereby have 1/f noise with low-frequency turnover, and also strict 1/f ^1– noise (the latter spectrum being integrable for > 0). Suitable examples are given. Actually, u() may be itself a random process, and the theory is developed on this basis.     

К ВОПРОСУ O КРИТИЧЕСКО Й ЧАСТОТЕ ПРИ ПРЕРЫВИСТОМ ОСВЕ ЩЕНИИ

c , c n , n AgBr. .     

On the electromagnetic structure of elementary particles

Using modern similarity and dimensionality methods, criteria of similarity are derived and used as transformations, which effect the conversion from one natural system of units to another. The exclusion principles thus defined are used to determine the powers of the similarity criteria in quantitative relations.Systems of units of the fermion and boson types are used in the simplest identification of the parameters corresponding to elementary particles.A set of electric and magnetic physical constants with dimensionality length, area, and volume, is obtained and successfully unified within the limits of a vortex ring, the maximum dimensions of which are defined by the Compton wavelength, and the minimum by the classical radius of the particle. The vortex ring model is in accordance with the latest experimental data, and it enables the behavior of the incident and target particles in the scattering process to be predicted.In modern theoretical physics the elementary particles are still considered as essentially structureless point formations, and hence it is impossible to give a purely theoretical treatment of the structure of the particles. Thus the various attempts in this direction (Hofstadter, Blokhintsev) have a polyphenomenological character and are internally inconsistent. (The search for the structure of an elementary particle is carried out on the assumption that it is not elementary, since truly elementary particles are defined as point size.) The author recognizes the need for an original approach to the structure of elementary particles, based on a method of study adequate for the problem. Such a method is the theory of dimensionality and similarity (Sedov, Gukhman, and Kirpichev), which serves as a scientific basis of a physical experiment (Kirpichev), or as the scientific basis for a model of the phenomena, insofar as the criteria of similarity are a reflection of the physical model of the process (Gukhman).It is a pleasure to thank Academician L. I. Sedov and Professor K. A. Putilov for valuable criticism and advice, and Professor A. S. Irisov and V. V. Lokhin for useful discussions.     

Particle velocities faster than the speed of light

In connection with another article by the author, we show how it might be possible to travel faster than the speed of light. We show that for clocks and rods moving faster than the speed of light, we get instead of time dilation and Lorentz contraction, respectively, time contraction and Lorentz expansion, respectively. It is shown that this paper is in confirmation with earlier articles dealing with this subject.     

Magnetic susceptibility and electrical conductivity of the highest chromium germanide

Results are given of measurements over the temperature range 80–1000 ° K of the magnetic susceptibility () and the electrical resistivity () of the highest chromium germanide Cr₁₁Ge₁₉. A ferromagnetic spin ordering is indicated at T < 86 ° K. From the (T) dependence and the values of the resistivity, thermo-emf and Hall constant, it is concluded that the compound studied is a semimetal with p-type conductivity.     

New two-color dimer models with critical ground states

We define two new models on the square lattice in which each allowed configuration is a superposition of a covering by white dimers and one by black dimers. Each model maps to a solid-on-solid (SOS) model in which the height field is two dimensional. Measuring the stiffness of the SOS fluctuations in the rough phase provides critical exponents of the dimer models. Using this height representation, we have performed Monte Carlo simulations. They confirm that each dimer model has critical correlations and belongs to a new universality class. In the dimer-loop model (which maps to a loop model) one height component is smooth, but has unusual correlated fluctuations; the other height component is rough. In the noncrossing-dimer model the heights are rough, having two different elastic constants; an unusual form of its elastic theory implies anisotropic critical correlations.     

Long-wave optical absorption in electron-bombarded GaAs

The absorption spectra of n- and p-type GaAs bombarded with 2-MeV electrons at T=300K were studied in the spectral range from 0.2 eV to Eg. It was found that shallow radiation-defect levels E_c – 0.01 eV and E_c + (0.06–0.1) eV were formed. The structureless character of the absorption in the region h < Eg in electron-bombarded gallium arsenide specimens was shown to be due to the distinctive features of photoionization of deep levels and the strong electron-phonon interaction in this material.Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Fizika, No. 7, pp. 93–97, July, 1981.     

The surface layer on a ferromagnetic. The energy of some periodic domain structures

Explicit solutions are found for a number of special cases of the potential problem of periodic charge distribution. The effect of uneven distribution of the charges of a Bloch wall on the magnetostatic energy is studied, the effective width of the Bloch wall is determined, an analytical expression is found for this distribution of poles and finally the effective height of the surface layer is also determined. An expression is derived for the orientation of the vectors of magnetization in the surface layer with respect to the easy directions of magnetization. The stability of the basic domain structure up to an inclination of 6°of the plane under observation to the direction of easy magnetization and the dimensions of the domains in periodic closure structures follow from the calculations.

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