Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors

Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10(-9) cm(2)/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein's expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand-integrin-cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties.     

Selective modification of cell surface proteins and thymidine transport in hamster cells exposed to cholera toxin.

The increased adherence and morphological response which occurs in Chinese hamster ovary cells as a result of exposure to cholera toxin is paralleled by modification in the relative exposure of outer proteins. Mild proteolysis treatment of the cells prelabeled with [3H] glucosamine reveals a markedly different kinetics of release of external glycopeptides as a result of exposure to cholera toxin. Selective alterations in external tyrosyl-rich proteins can also be detected by lactoperoxidase-catalyzed radioiodination. The above modifications are accompanied by a decrease in the rate of thymidine uptake by toxin-treated cells.     

Unraveling the role of membrane proteins Notch, Pvr, and EGFR in altering integrin diffusion and clustering

The role of three membrane proteins in altering the diffusion and clustering of integrin receptors has been measured. Integrins are membrane proteins responsible for integrating intracellular and extracellular signaling events and anchoring cells to the extracellular matrix. The methodology used to elucidate the role of other membrane proteins in altering integrin diffusion and clustering combines fluorescence microscopy with RNA interference (RNAi), which is a technique to reduce the expression of a target protein. The three RNAi-targeted membrane proteins were epidermal growth factor receptor (EGFR), platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr), and Notch. Real-time polymerase chain reaction or quantitative immunocytochemistry was used to measure a reduction in mRNA or protein concentration after RNAi treatment, respectively. Fluorescence recovery after photobleaching showed that reducing the concentration of EGFR or Notch results in less constrained integrin diffusion and, in the case of Notch RNAi, 4?% more mobile integrins. Fluorescence resonance energy transfer measurements performed before and after RNAi treatments indicate that clustering decreases for wild-type integrin, but increases for a high-ligand-affinity integrin mutant after reducing the expression of EGFR, Pvr, or Notch. A model to explain the measured changes after reducing the expression of these three membrane proteins involving cholesterol-enriched nanodomains is proposed.

A possible role of histidine residues in long-range electron transfer in proteins

Metal–histidine complexes are recurrent structural motifs in proteins exhibiting long-range electron transfer (ET), being involved both as electron donor or acceptor groups and as bridges which mediate ET between other cofactors. That observation suggests that the histidine residue could play an active role in ET, beyond that of simply binding the metal ion. Density functional theory and ab initio computations, performed on a simplified model system of the ET chain in semisynthetic Zinc cytochromes, confirm that expectation, suggesting that the nitrogen site of the histidine ring can exchange both a proton and a whole hydrogen atom with its redox partners. This finoling indicates that proton-assisted ET appears to be a plausible mechanism in this system.Contribution to Jacopo Tomasi Honorary Issue     

Inhibition of the binding of low-density lipoprotein to its cell surface receptor in human fibroblasts by positively charged proteins.

A group of proteins and polyamino acids with positively charged domains were shown to inhibit the binding of 125I-LDL to its receptor on the surface of human fibroblasts. The list of inhibitory proteins included platelet factor 4 (which has a cluster of lysine residues at its carboxyl terminus), two lysine-rich histones, poly-L-lysines of chain length greater than 4, and protamine. These proteins were effective in the concentration range of 5--10 microgram/ml. Two other positively charged proteins, lysozyme and avidin, did not inhibit 125I-LDL binding. Kinetic studies suggested that protamine was not acting simply as a competitive inhibitor with regard to the LDL receptor. In light of previous data showing that polyanions such as heparin and polyphosphates also inhibit 125-I-LDL binding to its cell surface receptor, the current findings suggest that charge interactions are important in this binding reaction. In a related series of studies, a number of glycoproteins and their asialo derivatives as well as a number of sugar phosphates failed to inhibit 125I-LDL binding to its receptor in fibroblasts.     

Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of UVR and UVR-induced cytokines on production of extracellular matrix proteins and proteases by dermal fibroblasts cultured in collagen gels%

Synthesis of extracellular matrix (ECM) proteins and their degradation by matrix metalloproteinases (MMP) are part of the dermal remodeling resulting from chronic exposure of skin to ultraviolet radiation (UVR). We have compared two alternative mechanisms for these responses, namely, a direct mechanism in which UV-B or UV-A is absorbed by fibroblasts and an indirect mechanism in which cytokines, produced in skin in response to UVR, stimulate production of the ECM proteins and MMP. These studies were carried out on human dermal fibroblasts grown in contracted, free-floating 9 day old collagen gels as a dermal equivalent. Synthesis of tropoelastin, collagen, fibrillin, MMP-1, -2, -3 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 were measured. Tropoelastin, collagen and fibrillin levels were stable between days 4 and 10, and MMP and TIMP decreased by day 10. Neither UV-B (2.5-50 mJ/cm2) nor UV-A (2-12 J/cm2) altered synthesis of ECM proteins, but UV-A increased MMP-1 and -3 production. Tropoelastin synthesis increased in response to transforming growth factor-beta1 (5 ng/mL) treatment. Both interleukin-1beta and tumor necrosis factor-alpha (10 ng/mL) decreased fibrillin messenger RNA levels but increased MMP-1, -3 and -9 synthesis markedly. Collagen synthesis was not modulated by UV-B, UV-A or cytokine treatment. These results indicate that certain cytokines may have greater effects on production of ECM proteins and MMP than absorption of UV-B and UV-A by fibroblasts grown in dermal equivalents and suggest that the former pathway may play a role in the dermal remodeling in photoaged skin.     

On the possible role of adparticles spillover on a heterophase surface in causing superadditive activity

It is shown that for a two-stage reaction on the surface involving two types of surface patches with adsorption heats lower and higher than q_opt migration of adparticles between surface patches (spillover) can both increase and decrease the reaction rate.     

On the possible role of the nitrogen atom in living matter.

Molecules of NH3 are capable of emitting stimulated radiation (MASER). Their organic derivates could have a similar effect, as is suggested by the new results published in Electron Correlations in Molecules and Solids [P. Fulde, Electron Correlations in Molecules and Solids, Springer-Verlag, Berlin, Heidelberg, 1993].     

Photo-oxidation of proteins and its role in cataractogenesis.

Proteins comprise approximately 68% of the dry weight of cells and tissues and are therefore potentially major targets for photo-oxidation. Two major types of processes can occur with proteins. The first of these involves direct photo-oxidation arising from the absorption of UV radiation by the protein, or bound chromophore groups, thereby generating excited states (singlet or triplets) or radicals via photo-ionisation. The second major process involves indirect oxidation of the protein via the formation and subsequent reactions of singlet oxygen generated by the transfer of energy to ground state (triplet) molecular oxygen by either protein-bound, or other, chromophores. The basic principles behind these mechanisms of photo-oxidation of amino acids, peptides and proteins and the potential selectivity of damage are discussed. Emphasis is placed primarily on the intermediates that are generated on amino acids and proteins, and the subsequent reactions of these species, and not the identity or chemistry of the sensitizer itself, unless the sensitizing group is itself intrinsic to the protein. A particular system is then discussed--the cataractous lens--where UV photo-oxidation may play a role in the aetiology of the disease, and tryptophan-derived metabolites act as UV filters.     

Adsorption of benzene-1,4-dithiol on the Au(111) surface and its possible role in molecular conductance

It is a consensus in the field of molecular electronics that the transport of charge across a single molecule depends sensitively on the details of the interaction between the molecule and the metallic leads, such as the molecular orientation. To advance the design of complex molecular devices, it is crucial to have a detailed understanding of these many aspects that influence the electron transport. A simple system that has been used as a paradigm of the class of conjugated aryl molecules is the benzene-1,4-dithiol (BDT). However, we still do not have a full understanding of the BDT transport experiments. Usually the geometries considered in transport calculations assumed that the BDT was connected to the two Au leads via the S atoms, and that the molecule was either perpendicular or close to a perpendicular configuration relative to the Au surfaces. Using ab initio calculations, we show that, for an isolated molecule, the configuration with largest adsorption energy has the BDT phenyl ring closer to being parallel to the surface, and we then argue, based on nonequilibrium Green's function-density functional theory calculations, that, depending on the experimental procedure, this may be the relevant configuration to be used in the transport calculations.     

On the electronic structure of neutral and ionic azobenzenes and their possible role as surface mounted molecular switches

We report quantum chemical calculations, mostly based on density functional theory, on azobenzene and substituted azobenzenes as neutral molecules or ions, in ground and excited states. Both the cis and trans configurations are computed as well as the activation energies to transform one isomer into the other and the possible reaction paths and reaction surfaces along the torsion and inversion modes. All calculations are done for the isolated species, but results are discussed in light of recent experiments aiming at the switching of surface mounted azobenzenes by scanning tunneling microscopes.     

Comparative analysis of the electrophoretic composition of proteins from native and transformed strains of the mung-beanBradyrhizobium sp.Vigna radiata L.

An experiment on the transfer into the mung-bean rhizobia gene of a recombinant plasmid containing an insect toxin gene (pCaVItoxneo) was performed in order to protect the root nodules from parasitic insects. Transformed strains (MTL) are obtained. The frequency of transformation was 10⁻⁶. A study of the protein composition showed that the transformed strain MTL 12 contained an additional three polypeptides with MM 83, 93, and 98 kDa; the transformed strain MTL 17, one additional polypeptide with MM 83 kDa. Institute of Microbiology, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 41 71 29. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 651–653, September–October, 1999.     

Investigations into the role of activated carbon in a moisture-blocking cement formulation

Physical deterioration is a natural consequence of moisture attack in finished concrete. Moisture is retained in open voids in prepared cement during mixing and hydration stages. Further atmospheric moisture uptake occurs following dehydration under use conditions in polymer-containing cement formulations, since polymers act as conduits for moisture ingress. The initial attribute of added strength from the polymer in improved cement formulations is thus nullified. Customary solution to the moisture problem has centered around improving moisture resistance by significant reduction of inherent macro defects. A more complete method of moisture blocking by the reaction of activated carbon with a vinyl polymer included in the cement formulation is described here. The moisture blocking action is due to the mitigation of open voids by the occlusion of a carbon-vinyl additive, essentially resulting in filled voids or no voids in the prepared cement, producing a durable finished concrete product. Supporting evidence for the above by thermal, polarization, X-ray and SEM are reported in this paper. This revised version was published online in July 2006 with corrections to the Cover Date.     

The role of hydrophobic surfaces in altering water-mediated peptide-peptide interactions in an aqueous environment

Using Born-Oppenheimer molecular dynamics within the density functional framework, we calculated the effective force acting on water-mediated peptide-peptide interaction between antiparallel β-sheets in an aqueous environment and also in the vicinity of a hydrophobic surface. From the magnitude of the effective force (corresponding to the slope of the free energy as a function of the interpeptide distance) and its sign (a negative value indicates an effective attraction, whereas a positive value indicates an effective repulsion) we can elucidate the fundamental differences of the water-mediated peptide-peptide interactions in those two environments. The computed effective forces indicate that the water-mediated interaction between peptides in an aqueous environment is attractive in the range of interpeptide distance d = 7-8 ? when hydrophobic surfaces are not nearby. Due to the stabilization of the water molecules bridging between the two β-sheets, a free energy barrier exists between the direct and indirect (water-mediated) interpeptide interactions. However, when the peptides are in the proximity of hydrophobic surfaces, this free energy barrier decreases because the hydrophobic surfaces enhance the interpeptide attraction by the destabilization and ease-to-libration of the bridging water molecules between them.     

An investigation on the possible role of melatonin in melanogenesis

The possible role of melatonin in melanogenesis was investigated by performing reactions of melatonin with peroxidase + H2O2 or H2O2 only, in the presence or absence of UV irradiation. Samples of the reaction mixtures were drawn at different times (from 15 to 480 min), the enzyme (when present) was removed by ultrafiltration and the samples so obtained were analyzed by MALDI/MS.The results show that melatonin undergoes oligomerization reaction with peroxidase + H2O2, leading to heptameric species. For high reaction times the MALDI/MS data do not show the formation of larger oligomers, but UV-vis spectroscopy indicates that the oligomerization processes proceed. The failure of MALDI-TOF in the identification of larger oligomers was related to the chemical-physical and morphological behavior of melanins.In the case of UV irradiation, the formation of species originating from the O- and O2 addition to melatonin, which activate new oligomerization channels, has been observed.     

On the possible role of montmorillonites in prebiotic peptide formation

Summary The ability of montmorillonite clay minerals for catalyzing peptide formation from amino acids in aqueous solution has been investigated using glycine and Cu²⁺ and Ca²⁺ containing montmorillonites as reaction systems. Evaporation cycle experiments showed that minor amounts of di- and tripeptide are formed from the amino acid. Further polymerization of dipeptide, however, seems to be more favoured by this reaction system than the initial step of dipeptide formation, and a possible role of clays in prebiotic peptide evolution could be seen therefore in the prolongation of peptide chains. Cu²⁺ ions in the clay matrix did not show any advantage over the usual Ca²⁺ ions embedded in natural montmorillonite.

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Über die mögliche Rolle von Montmorilloniten bei der präbiotischen Peptidbildung

Zusammenfassung Die Fähigkeit von Tonmineralien der Montmorillonitklasse zur Katalyse von Peptidbildungsreaktionen aus Aminosäuren in wäßriger Lösung wurde am Beispiel von Glyzin und Kupfer sowie Kalzium und Morillonit untersucht. Experimente mit Verdampfungszyklen haben gezeigt, daß kleinere Mengen von Di- und Tripeptiden aus der Aminosäure gebildet werden. Die weitere Polymerisation von Dipeptiden hingegen scheint wesentlich leichter in diesem Reaktionssystem zu verlaufen als der Anfangsschritt der Bildung des Dipeptides. Eine mögliche Rolle von Tonmineralien in der präbiotischen Peptidevolution kann daher in der Verlängerung von Peptidketten gesehen werden. Kupferionen in der Tonmatrix zeigen keinerlei Vorteile gegenüber den üblichen Kalziumionen, die in natürlichem Montmorillonit vorkommen.

     

Methods for rapidly altering the permeability of mammalian cells.

Various agents alter mammalian cells so that they rapidly become nonspecifically permeable to substances that ordinarily do not penetrate intact cells. Thus, toluene renders liver cells permeable to nucleotides and macromolecules. Tween 80 and Tween 60 act on similar fashion, and the effect is reversible. Dextran sulfate reversibly alters the permeability of Ehrlich ascites tumor cells, which offers a tool for studying the control of macromolecular syntheses and other processes. Brief exposure to external ATP alters the permeability of certain transformed mouse cells but not of untransformed cells. The effect of ATP is rapidly reversible.     

A possible role for extraterrestrial vanadium in the encounter of life

Extraterrestrial vanadium compounds typically contain vanadium in low oxidation states (V^II, V^III), reflecting anoxic environments (low oxygen fugacities) during genesis. In meteorites, most of which are fragments from asteroids and thus represent samples from the very beginning of our solar system, are found the pyroxenes which are typical V^II bearing minerals. In calcium- and aluminium-rich inclusions of dust particles collected from the coma of comet Wild 2, osbornite (TiN) with titanium replaced by up to 63% vanadium has been found. The atmospheres of “hot Jupiter” type exoplanets, as well as the atmospheres of early M type stars (red dwarfs) contain vanadium(II)oxide, which is also likely a constituent in interstellar clouds. The relevance of these vanadium occurrences for the generation of complex molecules out of simple ones under primordial conditions is briefly discussed in light of the catalytic potential of vanadium nitrides and -oxides. At higher oxygen fugacities, oxidic V^IV and V^V species are available. In this context, the ability of higher valent vanadium oxides to form nano-structured webs of protocells, and the potential of decavanadate and VO²⁺ to interfere with chemical processes in micellar structures and lipid vesicles is addressed.