Supercooling of the A phase of 3He.

Because of the extreme purity, lack of disorder, and complex order parameter, the first-order superfluid 3He A-B transition is the leading model system for first order transitions in the early universe. Here we report on the path dependence of the supercooling of the A phase over a wide range of pressures below 29.3 bar at nearly zero magnetic field. The A phase can be cooled significantly below the thermodynamic A-B transition temperature. While the extent of supercooling is highly reproducible, it depends strongly upon the cooling trajectory: The metastability of the A phase is enhanced by transiting through regions where the A phase is more stable. We provide evidence that some of the additional supercooling is due to the elimination of B phase nucleation precursors formed upon passage through the superfluid transition. A greater understanding of the physics is essential before 3He can be exploited to model transitions in the early universe.

Fragility of surface states in topological superfluid 3He.

Superfluid 3He, with unconventional spin-triplet p-wave pairing, provides a model system for topological superconductors, which have attracted significant interest through potential applications in topologically protected quantum computing. In topological insulators and quantum Hall systems, the surface/edge states, arising from bulk-surface correspondence and the momentum space topology of the band structure, are robust. Here we demonstrate that in topological superfluids and superconductors the surface Andreev bound states, which depend on the momentum space topology of the emergent order parameter, are fragile with respect to the details of surface scattering. We confine superfluid 3He within a cavity of height D comparable to the Cooper pair diameter ξ0. We precisely determine the superfluid transition temperature Tc and the suppression of the superfluid energy gap, for different scattering conditions tuned in situ, and compare to the predictions of quasiclassical theory. We discover that surface magnetic scattering leads to unexpectedly large suppression of Tc, corresponding to an increased density of low energy bound states.

Thermal transport of helium-3 in a strongly confining channel.

The investigation of transport properties in normal liquid helium-3 and its topological superfluid phases provides insights into related phenomena in electron fluids, topological materials, and putative topological superconductors. It relies on the measurement of mass, heat, and spin currents, due to system neutrality. Of particular interest is transport in strongly confining channels of height approaching the superfluid coherence length, to enhance the relative contribution of surface excitations, and suppress hydrodynamic counterflow. Here we report on the thermal conduction of helium-3 in a 1.1 µm high channel. In the normal state we observe a diffusive thermal conductivity that is approximately temperature independent, consistent with interference of bulk and boundary scattering. In the superfluid, the thermal conductivity is only weakly temperature dependent, requiring detailed theoretical analysis. An anomalous thermal response is detected in the superfluid which we propose arises from the emission of a flux of surface excitations from the channel.

Fabrication of microfluidic cavities using Si-to-glass anodic bonding.

We demonstrate the fabrication of ∼1.08 µm deep microfluidic cavities with characteristic size as large as 7 mm × 11 mm or 11 mm diameter, using a silicon-glass anodic bonding technique that does not require posts to act as separators to define cavity height. Since the phase diagram of 3He is significantly altered under confinement, posts might act as pinning centers for phase boundaries. The previous generation of cavities relied on full wafer-bonding which is more prone to failure and requires dicing post-bonding, whereas these cavities are made by bonding a pre-cut piece of Hoya SD-2 glass to a patterned piece of silicon in which the cavity is defined by etching. Anodic bonding was carried out at 425 °C with 200 V, and we observe that pressurizing the cavity to failure (>30 bars pressure) results in glass breaking, rather than the glass-silicon bond separation. In this article, we discuss the detailed fabrication of the cavity, its edges, and details of the junction between the coin silver fill line and the silicon base of the cavity that enables a low internal-friction joint. This feature is important for mass coupling torsional oscillator experimental assays of the superfluid inertial contribution where a high quality factor (Q) improves frequency resolution. The surface preparation that yields well-characterized smooth surfaces to eliminate pinning sites, the use of transparent glass as a cover permitting optical access, low temperature capability, and attachment of pressure-capable ports for fluid access may be features that are important in other applications.

Effect of Dinitrosyl Iron Complexes (NO Donors) on the Metabolic Processes in Human Fibroblasts.

The results of the study of the effect of mononuclear dinitrosyl iron complexes (DNICs) with functional sulfur-containing ligands (NO donors) on the cell viability and metabolism of human lung fibroblasts are presented, and the efficiency of their action is evaluated. It was shown that cationic DNICs increased the cell viability of fibroblasts and demonstrated the cytoprotective properties. Fluorescent analysis revealed that the DNICs compounds decrease the mitochondrial membrane potential but do not have a significant effect on the level of glutathione and reactive oxygen species in fibroblasts. It is assumed that the DNICs have the therapeutic potential for treating cardiovascular diseases.

The A-B transition in superfluid helium-3 under confinement in a thin slab geometry.

The influence of confinement on the phases of superfluid helium-3 is studied using the torsional pendulum method. We focus on the transition between the A and B phases, where the A phase is stabilized by confinement and a spatially modulated stripe phase is predicted at the A-B phase boundary. Here we discuss results from superfluid helium-3 contained in a single 1.08-µm-thick nanofluidic cavity incorporated into a high-precision torsion pendulum, and map the phase diagram between 0.1 and 5.6 bar. We observe only small supercooling of the A phase, in comparison to bulk or when confined in aerogel, with evidence for a non-monotonic pressure dependence. This suggests that an intrinsic B-phase nucleation mechanism operates under confinement. Both the phase diagram and the relative superfluid fraction of the A and B phases, show that strong coupling is present at all pressures, with implications for the stability of the stripe phase.

Observation of a new superfluid phase for 3He embedded in nematically ordered aerogel.

In bulk superfluid 3He at zero magnetic field, two phases emerge with the B-phase stable everywhere except at high pressures and temperatures, where the A-phase is favoured. Aerogels with nanostructure smaller than the superfluid coherence length are the only means to introduce disorder into the superfluid. Here we use a torsion pendulum to study 3He confined in an extremely anisotropic, nematically ordered aerogel consisting of ∼10 nm-thick alumina strands, spaced by ∼100 nm, and aligned parallel to the pendulum axis. Kinks in the development of the superfluid fraction (at various pressures) as the temperature is varied correspond to phase transitions. Two such transitions are seen in the superfluid state, and we identify the superfluid phase closest to Tc at low pressure as the polar state, a phase that is not seen in bulk 3He.

Transfer printing of CVD graphene FETs on patterned substrates.

We describe a simple and scalable method for the transfer of CVD graphene for the fabrication of field effect transistors. This is a dry process that uses a modified RCA-cleaning step to improve the surface quality. In contrast to conventional fabrication routes where lithographic steps are performed after the transfer, here graphene is transferred to a pre-patterned substrate. The resulting FET devices display nearly zero Dirac voltage, and the contact resistance between the graphene and metal contacts is on the order of 910 ± 340 Ω µm. This approach enables formation of conducting graphene channel lengths up to one millimeter. The resist-free transfer process provides a clean graphene surface that is promising for use in high sensitivity graphene FET biosensors.

Modification of the 3He phase diagram by anisotropic disorder.

Motivated by the recent prediction that uniaxially compressed aerogel can stabilize the anisotropic A phase over the isotropic B phase, we measure the pressure dependent superfluid fraction of (3)He entrained in 10% axially compressed, 98% porous aerogel. We observe that a broad region of the temperature-pressure phase diagram is occupied by the metastable A phase. The reappearance of the A phase on warming from the B phase, before superfluidity is extinguished at T(c), is in contrast to its absence in uncompressed aerogel. The phase diagram is modified from that of pure (3)He, with the disappearance of the polycritical point (PCP) and the appearance of a region of A phase extending below the PCP of bulk (3)He, even in zero applied magnetic field. The expected alignment of the A phase texture by compression is not observed.

Saponins from Tribulus terrestris L are less toxic for normal human fibroblasts than for many cancer lines: influence on apoptosis and proliferation.

The objective of the study was to explore the influence of saponins derived from Tribulus terrestris L. (TT) on normal human skin fibroblasts and to compare it with their anticancer properties. In this study, [3H]thymidine incorporation and MTT to assess cell proliferation and viability, respectively, and immunoblotting and HPLC analysis to explore intracellular signal transduction pathways have been used. We found that TT caused a dose-dependent decrease in [3H]thymidine incorporation into the DNA of treated fibroblast compared to the untreated controls. Viability of treated cells remained within the control levels with treatment of up to 5 micro g TT/ml medium. It was significantly depressed with incubation in > or =6 micro g TT/ml medium with an IC50 of 12.6 micro g TT/ml of cultivating media. ERK1/2 was significantly dephosphorylated at 5 mins of incubation with TT until the 48th hour, when phosphorylation slightly recovered, but was still below the control levels. In contrast, p38 and JNK phosphorylation was positively influenced, with peaks at 1 hr and 24 hrs of incubation respectively. Phosphorylation/dephosphorylation events of SAPK/MAPK clearly correlated with Mkp-1 induction. Procaspase 3 was activated after 5 mins of incubation and coincided with a rapid actin cleavage. There was a significant decrease of putrescine concentration and a concomitant increase of spermidine and spermine at 2 mins of treatment. According to our results, TT is less toxic for normal human skin fibroblasts in comparison to many cancer lines investigated in previous studies. The molecular mechanism of this cytotoxicity involves up- and downregulation of polyamines' homeostasis, suppression of proliferation, and induction of apoptosis. Further research in this field using animal models would help to explore and interpret the potential properties of TT as an anticancer supplement.

Highly potent p21(WAF1)-derived peptide inhibitors of CDK-mediated pRb phosphorylation: delineation and structural insight into their interactions with cyclin A.

The tumour suppressor protein p21(WAF1) plays a central role in regulating eukaryotic cell-cycle progression. Through its association with G1- and S-phase CDK complexes it regulates activation of the retinoblastoma protein (pRb) and E2F transcription factors. Recognition of CDK/cyclin complexes by p21 occurs, at least in part, through a protein-protein interaction with a binding groove on the cyclin subunit. The same groove has been shown to be involved in the recruitment of macromolecular CDK substrates, including pRb and E2F. Blocking of this recruitment site therefore prevents recognition and subsequent phosphorylation of CDK substrates and offers a therapeutic approach towards restoration of p21-like tumour suppression. Starting from the C-terminal cyclin-binding domain of p21 we have identified the minimal and optimized bioactive (152)HAKRRLIF(159) peptide sequence with respect to CDK protein kinase inhibition where pRb is the substrate. The phosphorylation of histone H1, however, which does not contain a recognizable cyclin-binding motif, was unaffected. Detailed structure-activity relationship investigations revealed that the determinants within this sequence are residues Arg(155), Leu(157) and Phe(159) and more completely define the composition of the cyclin-binding motif. A marked increase in potency was obtained upon replacement of the native Ser(153) with an Ala residue in the context of short synthetic peptide inhibitors and significantly, this mutation resulted in comparable affinity with CDK2/cyclin A as does the full-length recombinant p21 (which has CDK2 and cyclin A binding sites). Peptides derived from various proteins known to interact with cyclins were compared for potency and selectivity. A molecular model of the complex between the cyclin groove and the HAKRRLIF peptide was constructed. This model accounts for the observed peptide structure-activity relationships, including the potency enhancement of the LIF sequence occupying the hydrophobic pocket. Furthermore, it provides generic insights into molecular interactions governing cyclin groove recognition and lays the foundation for the development of peptidomimetic inhibitors of CDKs.

A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: affinity, stoichiometry, and thermodynamics.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Waf1/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 10(7) M(-)(1), corresponding to a K(d) of 87.7 nM. Measurement of the interaction of truncation and substitution analogues based on the p21 (141-160) sequence with PCNA revealed that the N-terminal part (residues 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153-160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141-160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNA-p21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.

Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin.

Peptides derived from the third alpha-helix of the homeodomain (residues 43-58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N- and C-terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C-terminal segment 52Arg-Arg-Met-Lys-Trp-Lys-Lys58 of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide's basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.

Proteomics approach in classifying the biochemical basis of the anticancer activity of the new olomoucine-derived synthetic cyclin-dependent kinase inhibitor, bohemine.

The aim of this study was to use two-dimensional electrophoresis (2-DE) coupled with multivariate principal component analysis (PCA) to characterize the quantitative changes in the protein composition of the CEM T-lymphoblastic leukemia cell line after treatment with bohemine (BOH), a synthetic olomoucin-derived cyclin-dependent kinase inhibitor (CDKI). Cell classification, reflecting protein patterns, clearly distinguished two main groups: one group consists of 9, 12 and 24 h treated BOH cells while the second is represented by the 0 and 24 h control untreated cells and the 6 h BOH-exposed CEM lymphoblasts. Discriminant protein spots differentially expressed in the BOH-treated CEM cells were selected for identification by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or electrospray ionization-tandem MS (ESI-MS/MS). Five of the selected protein spots were unequivocally identified as alpha-enolase, triosephosphate isomerase, eukaryotic initiation factor 5A, and alpha- and beta-subunits of Rho GDP-dissociation inhibitor 1. These proteins, all significantly downregulated in CEM T-lymphoblast leukemia in the course of BOH treatment, are known to play an important role in cellular functions such as glycolysis, protein biosynthesis, and cytoskeleton rearrangement. These results indicate that the cellular effects of olomoucine-derived CDKIs are not dependent on their ability to inhibit CDKs and could be mediated by several factors such as a decrease in protein synthesis and/or glycolysis which in turn diminishes the ability of cancer cells to function.

Use of reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene columns for the rapid separation and purification of acid-soluble nuclear proteins.

The suitability of polystyrene-divinylbenzene reversed-phase HPLC columns for rapid separation and purification of acid-soluble nuclear proteins was evaluated. We used a polystyrene-divinylbenzene reversed-phase HPLC column (PLRP-S) for purification of nuclear proteins extracted with 0.3 M HCl or 5% HClO4. We are able to obtain electrophoretically pure fractions for a number of nuclear proteins including HMG14, HMG17 and variants of histone H3. The identity of proteins in these fractions was confirmed by immunochemical analysis, protein sequencing, mass spectrometry and migration on two-dimensional polyacrylamide gel electrophoresis. These methods do not require special preparation of the sample and are quicker than similar published methods.

A mitogen- and anisomycin-stimulated kinase phosphorylates HMG-14 in its basic amino-terminal domain in vivo and on isolated mononucleosomes.

The rapid, transient induction of 80-100 immediate-early (IE) genes upon mitogenic stimulation occurs irrespective of protein synthesis and is mediated by modification of existing proteins. Two mechanisms, not mutually exclusive, involving modification either of sequence-specific transcription factors or of structural chromatin proteins primed by pre-association with responsive effectors are conceivable. Here, we show that upon IE gene induction, the non-histone high-mobility-group protein HMG-14, but not the related protein HMG-17, becomes serine phosphorylated in its basic, amino-terminal region close to where it binds nucleosomal DNA. Phosphorylation, normally transient, occurs independent of transcription and is quantitative and prolonged during superinduction. Brief micrococcal nuclease digestion substantially releases HMG-14 from nuclei in the mononucleosome-bound state. Finally, mononucleosomes prepared from mitogen-stimulated, but not control, cells contain a mitogen-activated kinase that phosphorylates HMG-14 in vitro on the same site(s) as in intact cells. The association of HMG-14 and its mitogen-activated kinase with nuclease-sensitive mononucleosomes has implications for models of mitogen-stimulated IE gene induction.

Anisomycin and rapamycin define an area upstream of p70/85S6k containing a bifurcation to histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction.

Anisomycin, a translational inhibitor, synergizes with growth factors and phorbol esters to superinduce c-fos and c-jun by a number mechanisms, one of which is its ability to act as a potent signalling agonist, producing strong, prolonged activation of the same nuclear responses as epidermal growth factor or tetradecanoyl phorbol acetate. These responses include the phosphorylation of pp33, which exists in complexed and chromatin-associated forms, and of histone H3 and an HMG-like protein. By peptide mapping and microsequencing, we show here that pp33 is the phosphoprotein S6, present in ribosomes and in preribosomes in the nucleolus. Ablation of epidermal growth factor-, tetradecanoyl phorbol acetate-, or anisomycin-stimulated S6 phosphorylation by using the p70/85S6k inhibitor rapamycin has no effect on histone H3 and HMG-like protein phosphorylation or on the induction and superinduction of c-fos and c-jun. Further, [35S]methionine-labelling and immunoprecipitation studies show that the ablation of S6 phosphorylation has no discernible effect on translation in general or translation of newly induced c-fos transcripts. Finally, we show that anisomycin augments and prolongs S6 phosphorylation not by blocking S6 phosphatases but by sustained activation of p70/85S6k. These results suggest the possible use of anisomycin and rapamycin to define upstream and downstream boundaries of an area of signalling above p70/85S6k which contains a bifurcation that produces histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction in the nucleus.

Phosphorylation-related accumulation of the 125K nuclear matrix protein mitotin in human mitotic cells.

The preparation of mammalian cells for entry into mitosis is related to a cascade of G2 phase phosphorylations of several nuclear proteins driven by mitosis-specific protein kinases. Using a monoclonal antibody we have identified previously in mammalian cells a 125K/pI6.5 protein, associated with the nuclear matrix, and markedly increased in mitotic cells, which was named 'mitotin'. Here, we show by short-term [35S]methionine labeling of cell cycle synchronized cells that this protein is synthesized at comparable rates throughout interphase. However, upon cycloheximide block of protein synthesis mitotin labeled during S phase is rapidly degraded, while the degradation of mitotin labeled during late G2 phase is abolished, resulting in its net and marked increase. The accumulation of mitotin in premitotic and mitotic cells is related to its phosphorylation and the metabolic stability of its two phosphorylated forms. The metabolic stabilization and accumulation of a nuclear matrix protein upon phosphorylation suggests the operation of a novel mechanism among the complex events preparing the cell for mitosis.