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The search of selective agonists and antagonists of membrane progesterone receptors (mPRs) is a starting point for the study of progesterone signal transduction mechanisms mediated by mPRs, distinct from nuclear receptors. According to preliminary data, the ligand affinity for mPRs differs significantly from that for classical nuclear progesterone receptors (nPRs), which might indicate structural differences in the ligand-binding pocket of these proteins. In the present work, we analyzed the affinity of several progesterone derivatives for mPRs of human pancreatic adenocarcinoma BxPC3 cell line that is characterized by a high level of mPR mRNA expression and by the absence of expression of nPR mRNA. The values were compared with the affinity of these compounds for nPRs. All tested compounds showed almost no affinity for nPRs, whereas their selectivity towards mPRs was different. Derivatives with an additional 19-hydroxyl group and removed 3-keto group had the highest selectivity for mPRs. These results suggest these compounds as the most selective progesterone analogs for studying the mechanisms of progestin action via mPRs.

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Chronic lymphocytic leukemia (CLL) is a malignant disease of small mature lymphocytes. Signals from the CLL microenvironment promote progression of the disease and induce drug resistance. This phenomenon is largely dependent on direct contact between the malignant B cells and stromal cells. CD84 belongs to the signaling lymphocyte activation molecule family of immunoreceptors, which self-associates, forming an orthogonal homophilic dimer. We therefore hypothesized that CD84 may bridge between CLL cells and their microenvironment, promoting cell survival. Our in vitro results show that CD84 expressed on CLL cells interact with CD84 expressed on cells in their microenvironment, inducing cell survival in both sides. Blocking CD84 in vitro and in vivo disrupt the interaction of CLL cells with their microenvironment, resulting in induced cell death. Thus, our findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.

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The cytotoxic activity of synthetic progestins (pregna-D'-pentaranes) II-V full agonists of the progesterone receptor (PR) for PR-positive and PR-negative cells of human breast carcinoma was studied. These compounds were more active in the PR-positive MCF-7 cells than in the PR-negative MDA-MB-453 cells. Cytotoxic effects of tested compounds against normal epithelial MDCK cells were not found. Molecular modeling of studied steroids with PR showed that all progestins with close energy values can bind to the ligand binding domain (LBD) of PR and the magnitude of the energy exceeds the value estimated for the progesterone molecule. Thus, the studied progestins are active against different molecular subtypes of breast cancer and represent a promising class of chemical compounds for oncology.

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Vaccinia virus was treated in a controlled manner with various combinations of nonionic detergents, reducing agents, and proteolytic enzymes, and successive products of the reactions were visualized using atomic force microscopy (AFM). Following removal of the outer lipid/protein membrane, a layer 20 to 40 nm in thickness was encountered that was composed of fibrous elements which, under reducing conditions, rapidly decomposed into individual monomers on the substrate. Beneath this layer was the virus core and its prominent lateral bodies, which could be dissociated or degraded with proteases. The core, in addition to the lateral bodies, was composed of a thick, multilayered shell of proteins of diverse sizes and shapes. The shell, which was readily etched with proteases, was thoroughly permeated with pores, or channels. Prolonged exposure to proteases and reductants produced disgorgement of the viral DNA from the remainders of the cores and also left residual, flattened, protease-resistant sacs on the imaging substrate. The DNA was readily visualized by AFM, which revealed some regions to be "soldered" by proteins, others to be heavily complexed with protein, and yet other parts to apparently exist as bundled, naked DNA. Prolonged exposure to proteases deproteinized the DNA, leaving masses of extended, free DNA. Estimates of the interior core volume suggest moderate but not extreme compaction of the genome.

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Virions of mouse leukemia virus spread on glass substrates were visualized by atomic force microscopy. The size distribution mode was 145 nm, significantly larger than that for human immunodeficiency virus particles. The distribution of particle sizes is broad, indicating that no two particles are likely identical in content or surface features. Virions possess knoblike protrusions, which may represent vestiges of budding from cell membranes. Particles which split open allowed imaging of intact cores with diameters of 65 nm. They also permitted estimation of viral shell thickness (35 to 40 nm) and showed the presence of a distinct trough between the shell and the core surface.

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Moloney murine leukemia virus (M-MuLV) lacking the gene for the envelope glycoprotein (env(-)) was produced in NIH 3T3 cells and investigated using atomic force microscopy (AFM). The particles were compared with similarly produced wild-type virions, some of which had been exposed to a monoclonal antibody against the surface component of the envelope protein (SU protein). The env(-) particles generally exhibit a distinctly different external appearance suggesting only a low density of associated proteins that have an almost fluid, mechanically unstable character. The weakly associated proteins may be host cell membrane proteins that are incorporated into the viral membrane in place of or in addition to virus envelope protein. The amount of this non-viral protein on virion surfaces appears to vary from negligible in most cases to a substantial complement in others. It seems clear that the presence of the envelope protein, in a mechanical sense, significantly strengthens and stabilizes the virion envelope. Binding of monoclonal antibody to wild-type virions indicates that some particles expose a significant amount of antigen while adjacent virions may not. This suggests that the conformation of the envelope glycoprotein or the disposition of oligosaccharides may be different among particles, on some virions exposing the specific epitope, and others little or none.

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Direct visualization of macromolecular crystal growth using atomic force microscopy (AFM) has provided a powerful tool in the delineation of mechanisms and the kinetics of the growth process. It has further allowed us to evaluate the wide variety of impurities that are incorporated into crystals of proteins, nucleic acids, and viruses. It is possible, using AFM, to image the defects and imperfections that afflict these crystals, the impurity layers that poison their surfaces, and the consequences of various factors on morphological development. All of these can be recorded under normal growth conditions, in native mother liquors, over time intervals ranging from minutes to days, and at the molecular level.

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Isolated human immunodeficiency virus (HIV) and HIV-infected human lymphocytes in culture have been imaged for the first time by atomic force microscopy (AFM). Purified virus particles spread on glass substrates are roughly spherical, reasonably uniform, though pleomorphic in appearance, and have diameters of about 120 nm. Similar particles are also seen on infected cell surfaces, but morphologies and sizes are considerably more varied, possibly a reflection of the budding process. The surfaces of HIV particles exhibit "tufts" of protein, presumably gp120, which do not physically resemble spikes. The protein tufts, which number about 100 per particle, have average diameters of about 200 A, but with a large variance. They likely consist of arbitrary associations of small numbers of gp120 monomers on the surface. In examining several hundred virus particles, we found no evidence that the gp120 monomers form threefold symmetric trimers. Although >95% of HIV-infected H9 lymphocytic cells were producing HIV antigens by immunofluorescent assay, most lymphocytes displayed few or no virus on their surfaces, while others were almost covered by a hundred or more viruses, suggesting a dependence on cell cycle or physiology. HIV-infected cells treated with a viral protease inhibitor and their progeny viruses were also imaged by AFM and were indistinguishable from untreated virions. Isolated HIV virions were disrupted by exposure to mild neutral detergents (Tween 20 and CHAPS) at concentrations from 0.25 to 2.0%. Among the products observed were intact virions, the remnants of completely degraded virions, and partially disrupted particles that lacked sectors of surface proteins as well as virions that were split or broken open to reveal their empty interiors. Capsids containing nucleic acid were not seen, suggesting that the capsids were even more fragile than the envelope and were totally degraded and lost. From these images, a good estimate of the thickness of the envelope protein-membrane-matrix protein outer shell of the virion was obtained. Treatment with even low concentrations (<0.1%) of sodium dodecyl sulfate completely destroyed all virions but produced many interesting products, including aggregates of viral proteins with strands of nucleic acid.

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A coupled numerical model of the atmospheric thermo-hydrodynamics and pollutant photochemical transport is described. This model can be used to study the complex relationships between the chemical and thermo-hydrodynamic processes in the atmosphere of urban areas with an emphasis on photochemical ozone formation. Preliminary numerical results of ozone and other key chemical atmospheric pollutant concentrations and distribution across the Houston-Galveston-Brazoria area using virtual emission data from area and mobile sources are presented.

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Brome mosaic virus (BMV), a T = 3 icosahedral plant virus, can be dissociated into coat protein subunits and subunit oligomers at pH 7.5 in the presence of concentrated salts. We have found that during the course of this treatment the coat protein subunits are cleaved, presumably by plant cell proteases still present in the preparation, between amino acids 35 and 36. The truncated protein subunits will then reorganize into T = 1 icosahedral particles and can be crystallized from sodium malonate. Quasi elastic light scattering and atomic force microscopy results suggest that the transition from T = 3 to T = 1 particles can occur by separate pathways, dissociation into coat protein subunits and oligomers and reassembly into T = 1 particles, or direct condensation of the T = 3 virions to T = 1 particles with the shedding of hexameric capsomeres. The latter process has been directly visualized using atomic force microscopy. Native T = 3 virions have been crystallized in several different crystal forms, but neither a rhombohedral form nor either of two orthorhombic forms diffract beyond about 3.4 A. Tetragonal crystals of the T = 1 particles, however, diffract to at least 2.5 A resolution. Evidence suggests that the T = 1 particles are more structurally uniform and ordered than are native T = 3 virions. A variety of anomalous virus particles having diverse sizes have been visualized in preparations of BMV used for crystallization. In some cases these aberrant particles are incorporated into growing crystals where they are frequently responsible for defect formation.

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Helical fibers, presumably proteinaceous and of microbial origin, have been visualized by atomic force microscopy on the surfaces of crystals of satellite tobacco mosaic virus. If the crystals are growing, then the fibers are incorporated intact into the crystal lattice. If broken on the crystal surface, then within a few minutes, the fibers self-reassemble to reestablish continuity. This, we believe, is the first observation of such a crystal surface-catalyzed repair of a biological structure. The surfaces of virus crystals provide ideal workbenches for the visualization and manipulation of nanoscale objects, particularly extended structures such as these fibers.

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Atomic force microscopy (AFM) can be applied both in situ and ex situ to study the growth of crystals from solution. The method is particularly useful for investigating the crystallization of proteins, nucleic acids and viruses because it can be carried out in the mother liquor and in a non-perturbing fashion. Interactions and transformations between various growth mechanisms can be directly visualized as a function of supersaturation, as can the incorporation of diverse impurities and the formation and propagation of defects. Because the crystals can be observed over long periods, it is also possible to obtain precise quantitative measures of the kinetic parameters for nucleation and growth. Finally, AFM has allowed us to identify a number of previously unsuspected phenomena that influence nucleation, rate of growth and the ultimate perfection of macromolecular crystals. These are all features which are important in determining the ultimate resolution and quality of a crystal's diffraction pattern.

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Satellite tobacco mosaic virus (STMV) can undergo at least two physical transitions that significantly alter its mechanical and structural characteristics. At high pH the 17-nm STMV particles expand radially by about 5 A to yield particles having diameters of about 18 nm. This pH-induced transition is further promoted by aging of the virions and degradation of the RNA, so that swollen particles ultimately appear even at neutral pH. While the native 17-nm particles crystallize as orthorhombic or monoclinic crystals which diffract to high resolution (1.8 A), the enlarged 18-nm particles crystallize in a cubic form which diffracts to no better than 5 A. In the transition, not only do the capsid protein subunits move radially outward, but the helical RNA segments with which they interact do as well. This is noteworthy because it demonstrates that the RNA and the protein shell are capable of coordinated movement, and that neither structure is rigidly defined or independent of the other. Using atomic force microscopy, it can be shown that STMV particles, upon drying, lose their mechanical rigidity and undergo deformation. Virions initially 17 nm in diameter shrink to more uniform final sizes than do 18 nm, initially swollen particles. This transition appears to be irreversible, as the particles do not reassume their former size nor structural rigidity upon rehydration. Evidence is also presented that preparations of native virus and their crystals are naturally somewhat heterogeneous and contain a variety of particles of anomalous size.

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The structure of canavalin, the vicilin-class storage protein from jack bean, was refined to 1.7 A resolution in a highly twinned rhombohedral crystal of space group R3 and unit-cell parameters a = b = c = 83.0 A, alpha = beta = gamma = 111.1 degrees. The resulting R and R(free) were 0.176 and 0.245, respectively. The orthorhombic crystal structure (space group C222(1), unit-cell parameters a = 136.5, b = 150.3, c = 133.4 A) was also refined with threefold non-crystallographic symmetry restraints. R and R(free) were 0.181 and 0.226, respectively, for 2.6 A resolution data. No significant difference in the protein structure was seen between these two crystal forms, nor between these two and the hexagonal and cubic crystal forms reported elsewhere [Ko et al. (1993), Acta Cryst. D49, 478-489; Ko et al. (1993), Plant Physiol. 101, 729-744]. A phosphate ion was identified in the lumen of the C-terminal beta-barrel. Lattice interactions showed that the trimeric molecule could be well accommodated in both 'top-up' and 'bottom-up' orientations in a rhombohedral unit cell of the R3 crystal and explained the presence of a high twin fraction. The large inter-trimer stacking interface of the C222(1) crystal may account for its relative stability. Atomic force microscopy (AFM) investigations of the growth of three crystal forms of canavalin indicate the rhombohedral form to be unique. Unlike the other two crystal forms, it contains at least an order of magnitude more screw dislocations and stacking faults than any other macromolecular crystal yet studied, and it alone grows principally by generation of steps from the screw dislocations. The unusually high occurrence of the screw dislocations and stacking faults is attributed to mechanical stress produced by the alternate molecular orientations in the rhombohedral crystals and their organization into discrete domains or blocks. At boundaries of alternate domains, lattice strain is relieved by the formation of the screw dislocations.

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Satellite tobacco mosaic virus (STMV) was probed using a variety of proteases. Consequences of the degradation were analyzed using gel electrophoresis, quasi-elastic light scattering (QELS), and atomic force microscopy (AFM). Proteolysis rates of 30 minutes for complete degradation of the protein capsid, up to many hours, were investigated. With each protease, degradation of virions 17 nm in diameter was shown by QELS to result in particles of 10 nm diameter, which is that of the RNA core observed in the virion by x-ray diffraction analysis. This was verified by direct visualization with atomic force microscopy. Using QELS, it was further shown that freshly prepared RNA cores remain as individual, stable, 10-nm condensed particles for 12 to 24 h. Clusters of particles then formed, followed by very large aggregates of 500 to 1000 nm diameter. AFM showed that the aggregates were composed of groups of the condensed RNA cores and were not due to unfolding of the nucleic acid. No unfolding of the core particles into extended conformation was seen by AFM until the samples were heated well beyond 90 degrees C. Mass spectrometry of RNA core particles revealed the presence of a major polypeptide whose amino acid sequence corresponded to residues 2 through 25 of the coat protein. Amino acids 13 through 25 were previously observed to be in direct contact with the RNA and are presumably protected from protease digestion. Low resolution difference Fourier analyses indicated the courses of the remainders of the amino terminal strands (amino acids 2-12) in intact virions. Any individual strand appears to have several choices of path, which accounts for the observed disorder at high resolution. These positively charged strands, serving as virtual polyamines, engage the helical segments of RNA. The intimate association of amino acid residues 2 through 25 with RNA likely contributes to the stability of the condensed conformation of the nucleic acid cores.

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The parity-violating longitudinal analyzing power, A(z), has been measured in pvectorp elastic scattering at an incident proton energy of 221 MeV. The result obtained is A(z) = [0.84+/-0.29(stat)+/-0.17(syst)]x10(-7). This experiment is unique in that it selects a single parity violating transition amplitude (3P2 - 1D2) and consequently directly constrains the weak meson-nucleon coupling constant h(pp)(rho). When this result is taken together with the existing pvectorp parity violation data, the weak meson-nucleon coupling constants h(pp)(rho) and h(pp)(omega) can, for the first time, both be determined.

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A chimeric human-simian IgG, antigen specific for CD4, when exposed to 0.5 M SO(=)(4) containing 0.4% polyethylene glycol or Jeffamine, self-assembles into discreet, roughly spherical particles 23 nm in diameter. Increasing SO(=)(4) to 1.55 M induces the IgG particles to crystallize in either a hexagonal or a monoclinic form. From X-ray diffraction, the former crystal is of space group P622, with one IgG particle in the unit cell; thus the particle itself must have 622 point group symmetry. Both crystal forms have been imaged using atomic force microscopy. Detailed features of the duodecamer were evident, including the symmetry and a large solvent channel along the sixfold axis. The particles in some ways resemble the hexameric IgG aggregates believed to activate compliment upon antigen binding and, therefore, may have physiological relevance. Investigation of seven other IgGs of diverse origins and subclasses indicates that many, if not most, IgGs form similar particles. To our knowledge, this is the first observation of the assembly of IgG into high symmetry aggregates in the absence of antigen or their crystallization.

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In situ atomic force microscopy (AFM) was used to investigate surface evolution during the growth of single crystals of turnip yellow mosaic virus (TYMV). Growth of the (101) face of TYMV crystals proceeded by two-dimensional nucleation. The molecular structure of the step edges and adsorption of individual virus particles and their aggregates on the crystalline surface were recorded. The surfaces of individual virions within crystals were visualized and seen to be quite distinctive with the hexameric and pentameric capsomers of the T = 3 capsids being clearly resolved. This, so far as we are aware, is the first direct visualization of the capsomere structure of a virus by AFM. In the course of recording the in situ development of the crystals, a profound restructuring of the surface arrangement was observed. This transformation was highly cooperative in nature, but the transitions were unambiguous and readily explicable in terms of an organized loss of classes of virus particles from specific lattice positions. In some cases areas of a single crystal surface were recorded in which were captured successive phases of the transition. We believe this provides the first visual record of a cooperative restructuring of the surface of a supramolecular crystal.

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The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.

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Atomic force microscopy, in contact mode, has been used to image living mammalian cells in culture at both low and high resolutions. The method is shown practical for revealing cytoskeletal features beneath the cell membrane and their restructuring during a variety of cellular activities. Among the processes that we have visualized are locomotion, tissue formation, cell division, transformation by viruses, and cell death. We show that some processes that occur well within cells can, nonetheless, be observed using the atomic force probe. At high resolution, features on the cell surface on the order of 0.5 micron and their changes with time, can be recorded.

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