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The growth factor receptor-bound protein 2 (Grb2) is an SH2 domain-containing docking module that participates in the signaling of numerous oncogenic growth factor receptor protein-tyrosine kinases (PTKs). Presented herein is a 5-methylindolyl-containing macrocyclic tetrapeptide mimetic (5) that binds to Grb2 SH2 domain protein with K(d)=75 pM. This represents the highest affinity yet reported for a synthetic inhibitor against any SH2 domain. In whole cell assays this novel analogue is able to effectively block the association of Grb2 to cognate cytoplasmic erbB-2 at IC(50)10nM without prodrug derivatization or the addition of carrier peptide motifs. Anti-mitogenic effects against erbB-2-dependent breast cancers are achieved at non-cytotoxic concentrations (IC(50)=0.6 microM). Macrocycle 5 may be representative of a new class of therapeutically relevant Grb2 SH2 domain-directed agents.

A proteomics-based method using stable isotope labeling to assess the relative abundance of peptides or proteins is described. Bradykinin and carbonic anhydrase were labeled with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate, a membrane impermeant reagent that is reactive with primary amines. Specificity of the label to primary amines was demonstrated using tandem mass spectrometry. Also, relative quantitation was achieved by secondary labeling with natural isotopic abundance and stable isotope-labeled methyl iodide. We believe this to be an effective stable isotope-labeling method for quantitative proteomics.

Crosslinked hyaluronic acid (HA) hydrogels were evaluated for their ability to elicit new microvessel growth in vivo when preloaded with one of two cytokines, vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF). HA film samples were surgically implanted in the ear pinnas of mice, and the ears retrieved 7 or 14 days post implantation. Histologic analysis showed that all groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p0.01). Moreover, aqueous administration of either growth factor produced substantially more vessel growth than an HA implant with no cytokine. However, the most striking result obtained was a dramatic synergistic interaction between HA and VEGF. Presentation of VEGF in crosslinked HA generated vessel density of NI = 6.7 at day 14, where NI is a neovascularization index defined below, more than twice the effect of the sum of HA alone (NI = 1.8) plus VEGF alone (NI=1.3). This was twice the vessel density generated by co-addition of HA and bFGF (NI=3.4, p0.001). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, synergistic angiogenic response produced by release of VEGF from crosslinked HA films.

The DNA polymerase (POL) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral DNA replication and, thus, may be considered as a viable target for anti-KSHV therapeutics. To produce large quantities of homogeneous and pure POL required for high-throughput screening (HTS) for inhibitors, we generated a recombinant baculovirus vector encoding a hexahistidine (His6)-tagged POL and infected Spodoptera frugiperda Sf-9 insect cells. High expression of recombinant POL (rPOL) was achieved for up to 72h post-infection. The rPOL was solubilized in lysis buffer containing 0.3% Cymal-5 detergent, purified by metal-chelating and dsDNA-cellulose affinity chromatography, and analyzed by anti-His antibody Western blot and mass spectrometry. The functionality of rPOL was confirmed by its DNA synthesis activity in vitro, which was effectively blocked by the anti-herpetic DNA polymerase inhibitors, foscarnet and cidofovir diphosphate, in a dose-dependent manner. The POL expressed and purified from the recombinant baculovirus-infected insect cells may be useful toward the development of HTS of large chemical libraries to identify novel KSHV DNA polymerase inhibitors.

The crucial functions of HIV-1 nucleocapsid-p7 protein (NC-p7) at different stages of HIV replication are dependent on its nucleic acid binding properties. In this study, a search has been made to identify antagonists of the interaction between NC-p7 and d(TG)(4). A chemical library of approximately 2000 small molecules (the NCI Diversity Set) was screened, of the 26 active inhibitors that were identified, five contained a xanthenyl ring structure. Further analysis of 63 structurally related compounds led to the identification of 2,3,4,5-tetrachloro-6-(4('),5('),6(')-trihydroxy-3(')-oxo-3H-xanthen-9(')-yl)benzoic acid, which binds to NC-p7 stoichiometrically. This compound exerted a significant anti-HIV activity in vitro with an IC(50) of 16.6+/-4.3 microM (means+/-SD). Synthetic variants lacking the two hydroxyls at positions 4(') and 5(') in the xanthenyl ring system failed to bind NC-p7 and showed significantly less protection against HIV infection. Molecular modeling predicts that these hydroxyl groups would bind to the amide nitrogen of Gly(35) with other contacts at the carbonyl oxygens of Gly(40) and Lys(33).

An alternative method to enzymatic digestion for protein identification by mass spectrometry has been developed that is based on chemical cleavage by formic acid. This method was tested on gel-purified apomyoglobin and BSA, as well as unknown proteins that cofractionate with Ty1-virus-like particles from Saccharomyces cerevisiae. Cleavage at aspartyl residues was found to be efficient and specific, and this specificity of cleavage lent itself easily to database searches. Parallel digestions using trypsin were also performed. The formic acid cleavage method generated comparable or better results than tryptic digestion for protein identification.

Protein kinase C (PKC)-theta, a member of the ‘novel’ subfamily of PKC isoforms, is of singular importance in transducing signals in T-lymphocytes. Since understanding of regulatory phosphorylation of novel PKCs is fragmentary and inconsistent with findings for ‘classical’ PKC isoforms, we investigated three potential phosphorylation sites on PKC-theta; in the activation loop (Thr538), turn motif (Ser676) and hydrophobic motif (Ser695). Combined evidence from phospho-specific antisera and MS demonstrates phosphorylation at all three sites. Unlike its closest paralogue, PKC-delta, lack of negative charge in the activation loop of PKC-theta results in a profound catalytic defect (>100-fold reduction in the T538A mutant); the high sequence similarity between PKC-theta and -delta assists in the formulation of structural hypotheses to account for this major difference. In contrast with mechanisms proposed for other PKC isoforms, phosphorylation at the other two sites does not reconstitute catalytic activity. Activation loop phosphorylation is critical invivo, since the T538A mutant completely lost its capacity to mediate T-cell receptor-stimulation of nuclear factor κB (NF-κB) activation in Jurkat T-cells. Hydrophobic motif phosphorylation also substantially influences PKC-theta catalytic activity (5-fold reduction in the S695A mutant), but does not impair NF-κB activation in Jurkat T-cells. Its mechanism is independent of secondary effects on activation loop phosphorylation and cannot be explained by thermal instability. Turn motif phosphorylation has a limited effect on kinase activity, but negatively regulates other aspects of PKC-theta function, since the S676A mutant is more efficient than wild-type in inducing NF-κB activation in Jurkat T-cells. These findings expand our understanding of the roles of phosphorylation in novel PKCs, and indicate that PKC-theta is a constitutively competent kinase as a consequence of constitutive phosphorylation of its activation loop.

Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.

HIV-1 Gag protein assembles into 100- to 120-nm diameter particles in mammalian cells. Recombinant HIV-1 Gag protein assembles in a fully defined system in vitro into particles that are only 25–30 nm in diameter and that differ significantly in other respects from authentic particles. However, particles with the size and other properties of authentic virions were obtained in vitro by addition of inositol phosphates or phosphatidylinsitol phosphates to the assembly system. Thus, the interactions between HIV-1 Gag protein molecules are altered by binding of inositol derivatives; this binding is apparently essential for normal HIV-1 particle assembly. This requirement is not seen in a deleted Gag protein lacking residues 16–99 within the matrix domain.

The hepatocyte growth factor (HGF/SF) receptor, Met, regulates mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. Activation of Met via autocrine, paracrine, or mutational mechanisms can lead to tumorigenesis and metastasis and numerous studies have linked inappropriate expression of this ligand-receptor pair to most types of human solid tumors. To prepare mAbs to human HGF/SF, mice were immunized with native and denatured preparations of the ligand. Recloned mAbs were tested in vitro for blocking activity against scattering and branching morphogenesis. Our results show that no single mAb was capable of neutralizing the in vitro activity of HGF/SF, and that the ligand possesses a minimum of three epitopes that must be blocked to prevent Met tyrosine kinase activation. In vivo , the neutralizing mAb combination inhibited s.c. growth in athymic nu/nu mice of tumors dependent on an autocrine Met-HGF/SF loop. Importantly, growth of human glioblastoma multiforme xenografts expressing Met and HGF/SF were markedly reduced in the presence of HGF/SF-neutralizing mAbs. These results suggest interrupting autocrine and/or paracrine Met-HGF/SF signaling in tumors dependent on this pathway is a possible intervention strategy.

The human endogenous retrovirus, type K (HERV-K) represents the most biologically active form of known retroelements present in the human genome. Several HERV-K genomes have transcriptionally active open reading frames and encode their own protease (PR). The HERV-K PR has been shown to authentically cleave human immunodeficiency virus type 1 (HIV-1) matrix-capsid peptide in the presence of HIV-1 PR inhibitors. This raised the possibility that HERV-K PR could complement HIV-1 PR function in HIV-1-infected individuals. To investigate this possibility, we fused the HIV-1 vpr gene to the HERV-K PR gene (vpr-PR). The vpr-PR expression plasmid and a PR-defective HIV-1 clone were cotransfected into 293T cells. Progeny virions were assayed for processing of the HIV-1 polyproteins by Western blot and for changes in infectivity. HERV-K PR fused to Vpr was incorporated into HIV-1 virions at a high concentration and cleaved the Gag and Pol precursor proteins. However, neither Gag nor Pol polyproteins were correctly processed. Moreover, the HERV-K PR did not restore virus infectivity. While these results do not exclude the possibility that the HERV-K PR could complement an HIV-1 PR whose function is impaired due to drugs or drug-resistant mutations, they clearly demonstrate that the HERV-K PR cannot substitute for the function of the wild-type HIV-1 PR.

War, violence, and population: making the body count James A. Tyner, 2009 Guilford Press, New York and London 226 + i–xi pp. Notes, references and index. Foreword by Chris Philo ISBN 978-1-60623-03...

We have developed a complete system for discovery of lead compounds as inhibitors of creatine kinase B. In this article, we describe production and purification of the recombinant protein, conditions and features of an optimized high-throughput screening assay, and results of our implementation of the system using a diverse compound library.

A subline of mesoderm-derived mouse NIH3T3 fibroblasts was selected for its ability to proliferate in serum-free media. This cell line (SFDH) grows as a monolayer at low density and spontaneously forms dense, multicellular spheroids at high density. Spheroid formation can also be induced by the addition of dexamethasone, polybrene, or heparin. Spheroids eventually detach from the substrate, but will reattach and re-form monolayers when transferred to fresh culture vessels and media, repeating the cycle again upon reaching high density. Thin section analysis of spheroids shows morphologically-distinct regions of cells, including an attenuated outer surface and a cuboidal interior with occasional lumen-like areas. Over time in culture, spheroids express increasing levels of met, the Met ligand-SF/HGF and cytokeratin, an epithelial marker, in comparison to monolayers. Both monolayer and spheroid-derived cells are rapidly tumorigenic in nude mice. Media conditioned by SFDH cells contain factors that stimulate growth and attachment of a variety of tumorigenic and non-tumorigenic cell lines, inducing cells to divide in serum-free media for up to 14 days when plated on tissue culture-treated and nontreated plastic surfaces pre-coated with SFDH conditional media. The growth-stimulating activity fractionates as a single peak over a sepharose column in the presence of 6 m urea, and sediments as a high molecular weight complex. Growth-stimulating activity can be neutralized by several antisera specific for hepatocyte growth factor, and the same sera recognize a novel ∼37 kD protein in active supernatants. The cyclic, continuous nature of alternating monolayer and spheroid forms makes this cell line appropriate for studying changing gene expression patterns in progressive cell–cell/cell–matrix interactions.

Examination of the literature for the period of this review revealed nearly two hundred citations that employed surface plasmon resonance (SPR) spectroscopy using BIAcore technology to evaluate biospecific interactions, demonstrating the increasing popularity of this powerful technique. Among these we noted the development of several new applications/modifications of standard techniques. In general, we find the qualitative aspects of the reported experiments to be excellent but the quantitative descriptions (kT, kon, koff, and keq) as well as the binding models still lagging behind.

We have analyzed the binding of recombinant human immunodeficiency virus type 1 nucleocapsid protein (NC) to very short oligonucleotides by using surface plasmon resonance (SPR) technology. Our experiments, which were conducted at a moderate salt concentration (0.15 M NaCl), showed that NC binds more stably to runs of d(G) than to other DNA homopolymers. However, it exhibits far more stable binding with the alternating base sequence d(TG)n than with any homopolymeric oligodeoxyribonucleotide; thus, it shows a strong sequence preference under our experimental conditions. We found that the minimum length of an alternating d(TG) sequence required for stable binding was five nucleotides. Stable binding to the tetranucleotide d(TG)2 was observed only under conditions where two tetranucleotide molecules were held in close spatial proximity. The stable, sequence-specific binding to d(TG)n required that both zinc fingers be present, each in its proper position in the NC protein, and was quite salt resistant, indicating a large hydrophobic contribution to the binding. Limited tests with RNA oligonucleotides indicated that the preferential sequence-specific binding observed with DNA also occurs with RNA. Evidence was also obtained that NC can bind to nucleic acid molecules in at least two distinct modes. The biological significance of the specific binding we have detected is not known; it may reflect the specificity with which the parent Gag polyprotein packages genomic RNA or may relate to the functions of NC after cleavage of the polyprotein, including its role as a nucleic acid chaperone. A single protein species, the Gag polyprotein, is sufficient for assembly of retrovirus particles. Since this process includes the selective encapsidation of viral RNA, this protein is evidently capable of specific interactions with nucleic acids. The nature of these interactions is not well understood as yet. After the virion is released from the cell, the polyprotein is cleaved by the virus-encoded protease; one of the cleavage products, termed the nucleocapsid protein (NC), then binds to the genomic RNA, forming the ribonucleoprotein core of the mature particle (21, 35, 41). The interaction between Gag and the genomic RNA is known to involve the NC domain of the polyprotein, since mutants within this domain of Gag are defective in RNA packaging (e.g., references 2, 16, 17, 24‐27, 31, 36, 37, and 39) and since the specificity of encapsidation tends to be determined by the NC domain in chimeric Gag molecules (9, 18, 49). However, NC is a basic protein and has frequently been described as binding to single-stranded DNA or RNA in a sequenceindependent manner. Indeed, it is probably capable of binding to any single-stranded nucleic acid under appropriate conditions. This binding activity appears to be crucial at several stages of virus replication (13, 19, 28, 46). In the experiments described here, we have analyzed the binding of recombinant human immunodeficiency virus type 1 (HIV-1) NC to short oligonucleotides. These studies were performed at moderate ionic strengths, at which the nonspecific electrostatic interaction between NC and nucleic acids is minimized. We find that under these conditions, the protein exhibits profound sequence preferences. This sequence-specific binding is dependent upon the zinc fingers of the protein and has a strong hydrophobic component. The biological significance of this sequence specificity is not clear at present, but the results suggest that studies with very short oligonucleotides may provide important insights into NC function and perhaps functions of Gag as well.

Sharkskin melt fracture (SSF) refers to a fine surface distortion found on extradates of certain polymers at shear stress levels around 0.1 MPa. SSF can limit the productivity of extrusion lines. Two schools of thought concerning the mechanism of SSF have developed: one assigns the initiation of fracture to slip in the die land [e. g., Ramamurthy (1986)] whereas the other attributes the fracture to excessive stress at the exit as the flow profile redistributes [e. g., Howells and Benbow (1962), Kurtz (1984)]. Both may contribute to the problem. The former is supported strongly by the seeming invariant shear stress required for SSF and by the reported kink in the flow curve at this stress.

Background : Nutrient deficiencies affect the health and wellness of large populations around the world. For example, the majority suffer from vitamin, essential fatty acid (such as omega-3), dietary fiber, and other important ingredient deficiencies due to their limited supply in the human food chain. Current trends in the nutraceutics industry to place these substances in higher. more-efficiently dispersed quantities in our food have become critically essential to their business plans. Nutrients in the form of small solids or droplets improve bioavailability. However, there remain numerous barriers to successful implementation of cost effective manufacturing processes. These challenges are addressed in the work presented here with particular focus on stability, bioavailability, and consumer acceptance. The goal is to develop large scale manufacturing systems that implement efficient platform technologies, with their respective operational maps, to produce functional food formulations, with particle sizes of these specially formulated nutraceutical ingredients in the micron-and nano- range. Objective : Demonstrating that stable micron- and nano-size emulsions, liposomes, and aqueous suspensions of functional food formulations can be produced using both “top down” and “bottom up” methods is our main objective. Addressing the challenges associated with the incorporation of these ingredients into large scale manufacturing systems, mainly mechanical stability and related shelf-life issues, is also a focus. That is, to develop proper processing protocols providing improved quality foods enriched with ingredients that are in limited supply in our food chain; to enhance human health and wellness world-wide. Methods : The formulations considered here typical of those used for increasing bioavailability of the infused, specially formulated ingredients with anti-cancer, anti-aging, and in-general wellness properties, lowering fat content and enhancing the shelf-life stability. Included are (a) an oil-in-water (fish oil/omega-3) emulsion, (b) liposome chaperones to vitamin C, and (c) aqueous suspensions (curcumin crystals, lutein/carotenoids, and fiber in soy milk). The production techniques include both “top-down” particle size reduction and “bottom-up” formation of crystals/precipitates via solubility adjustments. Both techniques are based on high shear processing of multiple liquid feeds. Using an impinging jet system, micro-mixing scales less than 100 nm were obtained. Results : (a) All nano-emulsion types, single, double and larger, either as oil-in-water and water-in-oil, can effectively be produced from various formulations using “top-down” methods. Illustrated here are single, oil-in-water systems; concentrations of 12-14 wt. % fish oil/omega-3 were mixed with water containing food grade surfactants. The high shear processing produced stable, submicron particles; with median particle sizes of 119-163 nm, no particles larger than 1 micron, and the “fish” odor was suppressed. Pertinent discussions related to the other types are also given as suggested path forward approaches for the development of nutrient enriched functional foods. This includes water-in-oil formulations for reduced fat content and the delivery of multiple species via double and triple emulsions, as compared to liposome configurations. (b) Although liposomes may be used to encapsulate both hydrophobic and hydrophilic substances, we selected liposomal vitamin C as our initial proof-of-concept system since it is absorbed into the body over four times more easily than its non-encapsulated form. After top down processing, the median size was 200 nm, compared to a median size of about 5 microns obtained by traditional self-assembly protocols. (b) Aqueous suspensions of micron- and nano- size formulations were also accomplished. The top down size reduction technique was used for processing soy bean fibers and lutein and the bottom-up method used for curcumin crystals. The fibers initially had a median size of 150 microns and a bi-modal distribution was obtained after processing; 99% of the particles were smaller than 15 microns with median sizes at 10 microns and the larger peak at about 200 nm. The curcumin submicron particles were formed via anti-solvent crystallization; with stable particles in the range of 300-500 nm. Conclusions : Our study demonstrates that stable micron- and nano-size emulsions, liposomes, and aqueous suspensions can be produced using both “top down” and “bottom up” methods. The formulation properties, in terms of particle size and stability, strongly depend on the processing parameters used in terms of energy input and temperature history. The energy requirements of the “bottom up” methods may be substantially lower than those of “top down” methods. Although some of the processes presented here have been scaled up to commercial levels, more work is needed in terms of fully assessing the bioavailability of the produced formulations and optimizing the processes to minimize cost. Key words : nano-emulsion, nano-suspension, high-shear processing, crystallization, curcumin, fish oil, liposomal vitamins: C and E, lutein, nutraceuticals, omega-3, soybean fiber

We have developed a facile means for the refolding of milligram quantities of purified proteins that employs gel filtration chromatography. We demonstrate by electrophoretic mobility shift and NMR spectroscopy that human ETS-1 protein, bovine ribonucelase A and E. coli integration host factor can be refolded into the native conformation using this technique. We have extended this strategy to the preparation of miligram quantities of macromolecular complexes suitable for structural analysis by NMR spectroscopy or X-ray crystallography. The diverse challenges to overcome in refolding these proteins illustrates the potential of this technique as a general approach for recovery of recombinant proteins produced as insoluble inclusion bodies.

Six monoclonal antibodies were prepared from mice immunized with a bacterially expressed human ets-2 protein. These antibodies specifically recognize the two human ets-2-encoded proteins p56 and p54 but failed to react with chicken, mouse, rat, bovine, or monkey proteins, suggesting that the antibodies recognize epitopes specific to the human ets-2 protein. Differential reactivities of these monoclonal antibodies with the peptide fragments generated by partial proteolytic digestion of the bacterially expressed ets-2 protein indicated that the six antibodies recognize at least three distinct epitopes in the B domain of the ets-2 protein. Immunoprecipitation experiments comparing native and denaturing conditions suggested that the ets-2 domain detected by the monoclonal antibodies is masked in the native condition by either protein folding or interacting proteins. The biochemical analysis of the ets-2 protein will be facilitated by the development of these monoclonal antibodies, which may be useful as both domain-specific probes and tools for specifically detecting the human ets-2 protein in heterologous expression systems.

Phosphorylation of the human ets-2 protein in response to mitogenic signals to T lymphocytes was investigated in Jurkat cells. Activation of the cells by antibodies against the T-cell antigen receptor-CD3 complex or by concanavalin A was followed within 5 min by increased phosphorylation of the protein, as shown by a mobility shift of the protein from 54 to 56 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased incorporation of 32P. The Ca2+ ionophores A23187 and ionomycin were able to mimic this effect, suggesting that this phosphorylation is mediated by Ca2+.

The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well. We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n. We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). Our results show that the interactions between these two molecules are surprisngly complex: while the K(d) for binding of a single d(TG)4 molecule to NC is only approximately 5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule. The strengths of these additional binding reactions are quantitated. The implications of this multivalency for the functions of NC in virus replication are discussed.

Using research in social psychology, the authors illustrate how two key routes to improve communication between marketing and engineering are dependent on the strength of managers’ psychological connection to their functional area compared to the firm as a whole (i.e., relative functional identification). In particular, they argue that relative functional identification moderates the relationship between strategies traditionally used to affect interfunctional relationships (information-sharing norms and integrated goals) and the frequency, bidirectionality, and coerciveness of interfunctional communication behaviors. In turn, these communication behaviors are linked to information use and perceived relationship effectiveness. The authors empirically test predictions of the framework in two studies. Study 1 results suggest that (1) the efficacy of the traditional strategies depends on marketing managers’ relative functional identification and (2) the traditional strategies can have negative and unintended effects on communication behaviors. In Study 2, the authors replicate and extend Study 1 by illustrating that bidirectional communication is as important as frequency in increasing both information use by engineering personnel and the perceived effectiveness of interfunctional relationships. The authors conclude with implications for theory building and managerial practice.

Although consumer researchers have investigated impulse buying for nearly 50 years, almost no research has empirically examined its normative aspects. This article presents conceptual and empirical evidence that consumers' normative evaluations (i.e., judgments about the appropriateness of engaging in impulse buying behavior) moderate the relationship between the impulse buying trait and consumers' buying behaviors. Specifically, the relationship between the buying impulsiveness trait and realted buying behaviors is significant only when consumers believe that acting on impulse is appropriate. The findings from two studies across student and retail customer samples converge and support the hypothesized moderating role of consumers' normative evaluations. Copyright 1995 by the University of Chicago.

Ultrasound frequencies ranging from 26 kHz–10 MHz are used in a number of applications varying from cell disruptions to therapeutic and diagnostic procedures. Previous results from our laboratory have demonstrated that 1‐MHz ultrasound at therapeutic intensities causes significant changes in the electrophysiological properties of epithelial tissues. The nature of these alterations indicated that, among other effects, the Na/K‐dependent ATPase activity of the tissue was reduced. In this study, the effects of ultrasonic irradiation (30 kHz) on enzyme activities in two model systems have been examined: one membrane incorporated (Na/K‐dependent ATPase in inverted erythrocyte ghosts from rat blood) and the other in ‘‘free’’ solution (ATP‐dependent firefly luciferin/luciferase reaction). Ultrasonic irradiation produced irreversible decreases in the activity of the enzyme in each system. At present, the analysis leads one to conclude that sono‐chemical (including free radical) and not thermal effects on the enzy...

The mechanics of isothermal melt spinning are studied for a viscoelastic liquid with a power law viscosity and a constant shear modulus. Steady state velocities and stresses are in agreement with experiment. The onset of the draw resonance instability, the magnitude of diameter fluctuations in the unstable region, and a second stable region at high draw ratio are predicted accurately.

ATP and respiration (NADH)-driven NAD(P) + transhydrogenase (EC 1.6.1.1) activities are low in membranes from Escherichia coli cultured on yeast extract medium (17 and 21 nmol/min × mg) but high on glucose (82 and 142 nmol/min × mg). The ATPase and respiratory activities in both cases appeared comparable. Growth of the bacteria in yeast extract medium followed by washing and replacement into a glucose medium showed that after 3 h the energy-linked and energy-independent NAD(P) + transhydrogenase (reduction of acetylpyridine NAD + by NADPH) activities had appeared simultaneously. Incorporation of chloramphenicol or omission of glucose in the induction medium resulted in no increase in these activities indicating that de novo protein synthesis is required for the induction of energy-linked and -independent NAD(P) + transhydrogenase. It was found that the K m values for acetylpyridine NAD + and NADPH for the energy-independent reaction in membranes from glucose grown cells (143 and 62 μ m ) were similar to those in membranes from cells grown on glucose-yeast extract (135 and 45 μ m ), respectively, but the maximum velocity at infinite acetyl pyridine NAD + and NADPH increased from 353 to 2175 nmol/min × mg. Furthermore, the membrane-bound NAD(P) + transhydrogenase in glucose-yeast extract grown cells showed substrate inhibition at high NADPH and low acetyl pyridine NAD + levels. Further kinetic data demonstrate that the mechanism of the energy-independent NAD(P) + transhydrogenase in E. coli is similar to that of the mitochondrial enzyme and exhibits similar responses to competitive inhibitors at the NAD + and NADPH sites.

The human ets-2 gene is a homolog of the v-ets oncogene of the E26 virus and codes for a 56-kilodalton nuclear protein. The ets-2 protein is phosphorylated and has a rapid turnover, with a half-life of 20 min. When human lymphocytic CEM cells were treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the level of the ets-2 protein was quickly elevated 5- to 20-fold. This effect of TPA was mimicked by a synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol, and was blocked by the protein kinase C inhibitor H7, indicating that protein kinase C is involved in the induction. The increase in the ets-2 protein was due to stabilization of the protein, because the protein had a half-life of more than 2 h in the presence of TPA and the ets-2 mRNA level did not increase significantly upon TPA treatment. The protein synthesis inhibitor cycloheximide enhanced the effect of TPA on the ets-2 protein and could itself slow turnover of the protein. Properties of the ets-2 protein, such as nuclear localization, phosphorylation, rapid turnover, and response to protein kinase C, indicate that this protein belongs to a group of oncogene proteins which are generally thought to have regulatory functions in the nucleus (e.g., myc, fos, myb, and p53). Our results suggest that protein kinase C, either directly or indirectly, regulates the level of the ets-2 protein by posttranslational mechanisms.

The spinning of fibers from polymer melts is sometimes limited at high takeup rates by the onset of a flow instability known as “draw resonance,” which is characterized by large amplitude fluctuations in the takeup area and the force. In this work, the onset and growth of the spinning instability is analyzed for an isothermal Newtonian liquid by examining the nonlinear dynamics of the most unstable spatial mode. At draw ratios below the critical value D R = 20·21 computed from linear theory the system is stable to finite-amplitude perturbations. At higher draw ratios all disturbances approach a stable finite-amplitude oscillation which agrees in period and magnitude with experimental observations of draw resonance.

Modern Food Microbiology, Third Edition. J.M. Jay. Van Nostrand Reinhold, 115 Fifth Ave., New York, NY 10003. 642 pp. 1986. $29.95 (US). Title: Fermentation and Biochemical Engineering Handbook; Principles, Process Design, and Equipment Author(s): Henry C. Vogel (Editor) Publisher: Noyes Publications, Park Ridge, NJ. 1986 Principles of Fermentation Technology P.F. Stanbury and A. Whitaker Pergamon Press Inc., Elm3ford, NY, USA 225 pp., 1984, $19.25 (soft cover), $48.00 (hard cover) Biotechnology in Agricultural Chemistry H.M. Le Baron, R.O. Mumma, R.C. Honeycutt and J.H. Duesing (eds) ACS Symposium Series 33t, American Chemical Society, Washington, DC 367 pp., 1987, $64.95 (US) Supercritical Fluids ‐ Chemical and Engineering Principles and Applications T.G. Squires and M.E. Paulaitis (Editors) ACS Symposium Series #329, 294pp., $59.95 (US & Canada), $71.95 (Export) Title: Biogeneration of aromas. Authors: Parliment, T. H. and R. Croteau (Editors) Publishers: ACS Symposium Series, Washington, DC, 1986 Titl...

Active transport vesicles of Escherichia coli were shown to possess low levels of energy-independent and energy-dependent nicotinamide nucleotide transhydrogenase activities. Breakage of such vesicles in a French pressure cell resulted in a fraction which had an 8-10-fold increased respiration- and ATP-driven transhydrogenase activities. Stimulation of the ATPase activity in vesicles with Triton X-100 was also paralledled by a 2-fold increase in the energy-independent transhydrogenase. Disruption of the vesicles similarly resulted in increases in the energy-independent transhydrogenase, NADH and succinate oxidase activities but a decrease in succinate supported proline uptake. In the light of these findings, the "sidedness' of the vesicle membranes is discussed.

Spin line tension, diameter attenuation, and the onset of the draw resonance instability are determined for a viscoelastic polymer melt, taking into account the changes in physical properties resulting from cooling of the filament. The theory predicts the experimentally observed stabilization of very short and very long filaments.

We are now, by means of remarkable genetic engineering techniques, able to utilize retroviral oncogenes to probe for and define a number of homologously associated genes. These genes, the proto-oncogenes, are thought to be involved in the conversion of normal cells to neoplasia. These genes are known to be of normal cellular origin, but many were initially identified in retroviruses. Proto-oncogenes are highly conserved throughout the animal kingdom and are believed to play significant roles in cellular growth and/or differentiation. These proto-oncogenes have been associated recently with a number of chromosomal abnormalities involving certain malignant diseases. Cytogenetic data is accumulating that correlates specific proto-oncogene alterations with known chromosomal insults, such as breaks, rearrangements, and translocation, each associated with certain leukemias and malignancies. These studies have great potential for new approaches in treatment modalities, employing oncogenes as markers in predictive and pathologic diagnosis. For now the goal to use oncogenes as markers of neoplasia is certainly sufficient to justify current research efforts.

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of Escherichia coli by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of E. coli , was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD+ and NADP+, but NADPH served to accelerate the rate of inhibition.

Summary The stimulation of the energy dependent transhydrogenase (reduction of NADP by NADH) driven by oxidative energy was linear with oligomycin in the ammonia-EDTA depleted (AE) submitochondrial particle but sigmoidal in the more severely depleted ammonia-Sephadex-urea (ASU) and silicotungstate (STA) particles. The optimal activities were quite high even in the more depleted particles. The mitochondrial energy transfer factor C (Factor C) stimulated this activity of the AE particle alone and in combination with oligomycin. Oligomycin also induced substantial activity in the ASU particle in NAD reduction by succinate driven by ascorbatetetramethyl-p-phenylenediamine oxidation in the presence of antimycin. The results show that these particles are not significantly depleted in components involved in the production of the non-phosphorylated high energy intermediate from respiration.