Function and formation of $\beta$1,4-galactosyltransferase-specific adhesions during growth and differentiation of F9 embryonal carcinoma cells

Galactosyltransferase (GalTase) is localized in the Golgi, where it functions in oligosaccharide synthesis, as well as on the cell surface where it serves as a cell adhesion molecule. GalTase-specific adhesions are functional in a number of important biological events, including F9 embryonal carcinoma (EC) cell adhesions. GalTase-based adhesions are formed by recognition and binding to terminal N-acetylglucosamine (GlcNAc) residues on its glycoprotein counterpart on adjacent cell surfaces. The object of this work has been to investigate the formation and function of GalTase-specific adhesions during F9 cell growth and differentiation. We initially investigated GalTase synthesis during differentiation and found that the increase in GalTase activity was specific for the Golgi compartment; surface GalTase levels remained constant during differentiation. These data indicated that the increase in cell adhesions expected with increased cell-matrix interaction in differentiated F9 cells is not the consequence of increased surface GalTase expression and, more interestingly, that the two pools of GalTase are under differential regulation. Synthesis and recognition of the consociate glycoprotein component was next investigated. Surface GalTase recognized several surface glycoproteins in a pattern that changes with differentiation. Uvomorulin, lysosome-associated membrane protein-1 (LAMP-1), and laminin were recognized by surface GalTase and are, therefore, potential components in GalTase-specific adhesions. Furthermore, these interactions were aberrant in an adhesion-defective F9 cell line that results, at least in part, from abnormal oligosaccharide synthesis. The function played by surface GalTase in growth and induction of differentiation was examined. Inhibition of surface GalTase function by a panel of reagents inhibited F9 cell growth. GalTase expression at both the transcription and protein levels were differentially regulated during the cell cycle, with surface expression greatest in the G1 phase. Disruption of GalTase adhesion by exposure to anti-GalTase antibodies during this period resulted in extension of the G2 phase, a result similar to that seen with agents known to inhibit growth and induce differentiation. Finally, other studies have suggested that a subset of cell adhesion molecules have the capability to induce differentiation in EC cells systems. We have determined in F9 cells that dissociating GalTase adhesion by galactosylation of and release of the consociate glycoproteins induces differentiation, as defined by increased laminin synthesis. The ability to induce differentiation by surface galactosylation was greatest in cells grown in cultures promoting cell-cell adhesions, relative to cultures with minimal cell-cell interactions. ^

$\beta\sb2$-adrenergic receptor desensitization, internalization and phosphorylation in response to full and partial agonists

The objective of this study is to test the hypothesis that partial agonists produce less desensitization because they generate less of the active conformation of the $\beta\sb2$-adrenergic receptor ($\beta$AR) (R*) and in turn cause less $\beta$AR phosphorylation by beta adrenergic receptor kinase ($\beta$ARK) and less $\beta$AR internalization. In the present work, rates of desensitization, internalization, and phosphorylation caused by a series of $\beta$AR agonists were correlated with a quantitative measure, defined as coupling efficiency, of agonist-dependent $\beta$AR activation of adenylyl cyclase. These studies were preformed in HEK-293 cells overexpressing the $\beta$AR with hemagglutinin (HA) and 6-histidine (6HIS) epitopes introduced into the N- and C-termini respectively. Agonists chosen provided a 95-fold range of coupling efficiencies, and, relative to epinephrine, the best agonist, (100%) were fenoterol (42%), albuterol (4.9%), dobutamine (2.5%) and ephedrine (1.1%). At concentrations of these agonists yielding $>$90% receptor occupancy, the rate and extent of the rapid phase (0-30 min) of agonist induced desensitization of adenylyl cyclase followed the same order as coupling efficiency, that is, epinephrine $\ge$ fitnoterol $>$ albuterol $>$ dobutamine $>$ ephedrine. The rate of internalization, measured by a loss of surface receptors during desensitization, with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like desensitization and internalization, $\beta$AR phosphorylation exhibited a dependency on agonist strength. The two strongest agonists epinephrine and fenoterol provoked 11 to 13 fold increases in the level of $\beta$AR phosphorylation after just 1 min, whereas the weakest agonists dobutamine and ephedrine caused only 3 to 4 fold increases in phosphorylation. With longer treatment times, the level of $\beta$AR phosphorylation declined with the strong agonists, but progressively increased with the weaker partial agonists. The major conclusion drawn from this study is that the occupancy-dependent rate of receptor phosphorylation increases with agonist coupling efficiencies and that this is sufficient to explain the desensitization, internalization, and phosphorylation data obtained.^ The mechanism of activation and desensitization by the partial $\beta$AR agonist salmeterol was also examined in this study. This drug is extremely hydrophobic and its study presents possibly unique problems. To determine whether salmeterol induces desensitization of the $\beta$AR its action has been studied using our system. Employing the use of reversible antagonists it was found that salmeterol, which has an estimated coupling efficiency near that of albuterol caused $\beta$AR desensitization. This desensitization was much reduced relative to epinephrine. Consistent with its coupling efficiency, it was found to be similar to albuterol in its ability to induce internalization and phosphorylation of the $\beta$AR. (Abstract shortened by UMI.) ^

Velocity of early BCR-ABL transcript elimination as an optimized predictor of outcome in chronic myeloid leukemia (CML) patients in chronic phase on treatment with imatinib

UNLABELLED Early assessment of response at 3 months of tyrosine kinase inhibitor treatment has become an important tool to predict favorable outcome. We sought to investigate the impact of relative changes of BCR-ABL transcript levels within the initial 3 months of therapy. In order to achieve accurate data for high BCR-ABL levels at diagnosis, beta glucuronidase (GUS) was used as a reference gene. Within the German CML-Study IV, samples of 408 imatinib-treated patients were available in a single laboratory for both times, diagnosis and 3 months on treatment. In total, 301 of these were treatment-naïve at sample collection. RESULTS (i) with regard to absolute transcript levels at diagnosis, no predictive cutoff could be identified; (ii) at 3 months, an individual reduction of BCR-ABL transcripts to the 0.35-fold of baseline level (0.46-log reduction, that is, roughly half-log) separated best (high risk: 16% of patients, 5-year overall survival (OS) 83% vs 98%, hazard ratio (HR) 6.3, P=0.001); (iii) at 3 months, a 6% BCR-ABL(IS) cutoff derived from BCR-ABL/GUS yielded a good and sensitive discrimination (high risk: 22% of patients, 5-year OS 85% vs 98%, HR 6.1, P=0.002). Patients at risk of disease progression can be identified precisely by the lack of a half-log reduction of BCR-ABL transcripts at 3 months.

Measurement of isotopic uranium in water for compliance monitoring by liquid scintillation counting with alpha/beta discrimination

A simple and inexpensive method is described for analysis of uranium (U) activity and mass in water by liquid scintillation counting using $\alpha$/$\beta$ discrimination. This method appears to offer a solution to the need for an inexpensive protocol for monitoring U activity and mass simultaneously and an alternative to the potential inaccuracy involved when depending on the mass-to-activity conversion factor or activity screen.^ U is extracted virtually quantitatively into 20 ml extractive scintillator from a 1-$\ell$ aliquot of water acidified to less than pH 2. After phase separation, the sample is counted for a 20-minute screening count with a minimum detection level of 0.27 pCi $\ell\sp{-1}$. $\alpha$-particle emissions from the extracted U are counted with close to 100% efficiency with a Beckman LS6000 LL liquid scintillation counter equipped with pulse-shape discrimination electronics. Samples with activities higher than 10 pCi $\ell\sp-1$ are recounted for 500-1000 minutes for isotopic analysis. Isotopic analysis uses events that are automatically stored in spectral files and transferred to a computer during assay. The data can be transferred to a commercially available spreadsheet and retrieved for examination or data manipulation. Values for three readily observable spectral features can be rapidly identified by data examination and substituted into a simple formula to obtain $\sp{234}$U/$\sp{238}$U ratio for most samples. U mass is calculated by substituting the isotopic ratio value into a simple equation.^ The utility of this method for the proposed compliance monitoring of U in public drinking water supplies was field tested with a survey of drinking water from Texas supplies that had previously been known to contain elevated levels of gross $\alpha$ activity. U concentrations in 32 samples from 27 drinking water supplies ranged from 0.26 to 65.5 pCi $\ell\sp{-1}$, with seven samples exceeding the proposed Maximum Contaminant Level of 20 $\mu$g $\ell\sp{-1}$. Four exceeded the proposed activity screening level of 30 pCi $\ell\sp{-1}$. Isotopic ratios ranged from 0.87 to 41.8, while one sample contained $\sp{234}$U activity of 34.6 pCi $\ell\sp{-1}$ in the complete absence of its parent, $\sp{238}$U. U mass in the samples with elevated activity ranged from 0.0 to 103 $\mu$g $\ell\sp{-1}$. A limited test of screening surface and groundwaters for contamination by U from waste sites and natural processes was also successful. ^

The binding site of karyopherin alpha for karyopherin beta overlaps with a nuclear localization sequence.

By using proteolysis, recombinant mutant proteins, or synthetic peptides and by testing these reagents in liquid phase binding or nuclear import assays, we have mapped binding regions of karyopherin alpha. We found that the C-terminal region of karyopherin alpha recognizes the nuclear localization sequence (NLS), whereas its N-terminal region binds karyopherin beta. Surprisingly, karyopherin alpha also contains an NLS. Thus, karyopherin alpha belongs to a group of proteins that contain both a ligand (NLS) and a cognate receptor (NLS recognition site) in one molecule with a potential for autologous ligand-receptor interactions. The NLS of karyopherin alpha overlaps with the binding site of karyopherin alpha for karyopherin beta. Hence, binding of karyopherin beta to karyopherin alpha covers the NLS of karyopherin alpha. This prevents autologous ligand receptor interactions and explains the observed cooperative binding of karyopherin alpha to a heterologous NLS protein in the presence of karyopherin beta.

Identification and characterization of an immunophilin expressed during the clonal expansion phase of adipocyte differentiation.

Mouse 3T3-L1 cells differentiate into fat-laden adipocytes in response to a cocktail of adipogenic hormones. This conversion process occurs in two discrete steps. During an early clonal expansion phase, confluent 3T3-L1 cells proliferate and express the products of the beta and delta members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. The cells subsequently arrest mitotic growth, induce the expression of the alpha form of C/EBP, and acquire the morphology of fully differentiated adipocytes. Many of the genes induced during the terminal phase of adipocyte conversion are directly activated by C/EBP alpha, and gratuitous expression of this transcription factor is capable of catalyzing adipose conversion in a number of different cultured cell lines. The genetic program undertaken during the clonal expansion phase of 3T3-L1 differentiation, controlled in part by C/EBP beta and C/EBP delta, is less clearly understood. To study the molecular events occurring during clonal expansion, we have identified mRNAs that selectively accumulate during this phase of adipocyte conversion. One such mRNA encodes an immunophilin hereby designated FKBP51. In this report we provide the initial molecular characterization of FKBP51.

Hippocampal long-term depression and depotentiation are defective in mice carrying a targeted disruption of the gene encoding the RI beta subunit of cAMP-dependent protein kinase.

The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). We have combined pharmacologic and genetic approaches to demonstrate that PKA activity is required for both homosynaptic LTD and depotentiation and that a specific neuronal isoform of type I regulatory subunit (RI beta) is essential. Mice carrying a null mutation in the gene encoding RI beta were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CA1 synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RI beta mutant mice. Despite a compensatory increase in the related RI alpha protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RI beta. Since the late phase of CA1 LTP also requires PKA but is normal in RI beta mutant mice, our data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits.

Transforming growth factor beta mediates the progesterone suppression of an epithelial metalloproteinase by adjacent stroma in the human endometrium.

Unlike most normal adult tissues, cyclic growth and tissue remodeling occur within the uterine endometrium throughout the reproductive years. The matrix metalloproteinases (MMPs), a family of structurally related enzymes that degrade specific components of the extracellular matrix are thought to be the physiologically relevant mediators of extracellular matrix composition and turnover. Our laboratory has identified MMPs of the stromelysin family in the cycling human endometrium, implicating these enzymes in mediating the extensive remodeling that occurs in this tissue. While the stromelysins are expressed in vivo during proliferation-associated remodeling and menstruation-associated endometrial breakdown, none of the stromelysins are expressed during the progesterone-dominated secretory phase of the cycle. Our in vitro studies of isolated cell types have confirmed progesterone suppression of stromal MMPs, but a stromal-derived paracrine factor was found necessary for suppression of the epithelial-specific MMP matrilysin. In this report, we demonstrate that transforming growth factor beta (TGF-beta) is produced by endometrial stroma in response to progesterone and can suppress expression of epithelial matrilysin independent of progesterone. Additionally, we find that an antibody directed against the mammalian isoforms of TGF-beta abolishes progesterone suppression of matrilysin in stromal-epithelial cocultures, implicating TGF-beta as the principal mediator of matrilysin suppression in the human endometrium.

Liquid-phase combinatorial synthesis.

A concept termed liquid-phase combinatorial synthesis (LPCS) is described. The central feature of this methodology is that it combines the advantages that classic organic synthesis in solution offers with those that solid-phase synthesis can provide, through the application of a linear homogeneous polymer. To validate this concept two libraries were prepared, one of peptide and the second of nonpeptide origin. The peptide-based library was synthesized by a recursive deconvolution strategy [Erb, E., Janda, K. D. & Brenner, S. (1994) Proc. Natl. Acad. Sci. USA 91, 11422-11426] and several ligands were found within this library to bind a monoclonal antibody elicited against beta-endorphin. The non-peptide molecules synthesized were arylsulfonamides, a class of compounds of known clinical bactericidal efficacy. The results indicate that the reaction scope of LPCS should be general, and its value to multiple, high-throughput screening assays could be of particular merit, since multimilligram quantities of each library member can readily be attained.

Efeitos da Imunocastração e de agonistas beta-adrenérgicos sobre a qualidade da carne de bovinos

Os agonistas beta-adrenérgicos (βAA) são conhecidos por aumentar a hipertrofia muscular e lipólise, neste caso uma maneira de se reduzir o efeito da lipólise seria a imunocastração. Dessa forma, o objetivo deste projeto foi avaliar o efeito dos βAA e da imunocastração sobre a qualidade da carne de bovinos Nelore. Foram utilizados noventa e seis bovinos Nelore, sendo que metade dos animais (n=48) receberam uma dose da vacina de imunocastração, e após 30 dias receberam a segunda dose. A outra metade dos animais (n=48) não recebeu nenhuma dose da vacina. Durante 70 dias os animais foram alimentados com uma dieta padrão composta de 24% volumoso e 76% de concentrado. Após 70 dias de confinamento os animais foram divididos em três grupos, dentro de bloco (peso inicial) e condição sexual e foram alimentados por 30 dias, com umas das seguintes dietas: CON - dieta padrão utilizada na fase anterior, sem a adição de βAA; ZIL - dieta padrão acrescida de 80 mg/dia Cloridrato de Zilpaterol; RAC - dieta padrão acrescida de 300 mg/dia Cloridrato de Ractopamina. Ao final desse período os animais foram abatidos e colhidas amostras do músculo Longissimus dorsi para as avaliações de qualidade de carne, lipídeos totais, perfil de ácidos graxos, análise sensorial do consumidor, perfil morfométrico muscular, expressão dos genes calpaína e calpastatina, comprimento de sarcômero. Para a maioria das características avaliadas não foram observadas interações entre os tratamentos. Ao avaliar o efeito da condição sexual, os animais imunocastrados apresentaram maiores intensidades de cor L, a e b, lipídios totais, ácidos oleico, palmítico e total de monoinsaturados e maior frequência para as fibras oxidativas (FO) e glicolíticas (FG) em relação aos não-castrados. Contudo, os animais não-castrados tiveram uma tendência a apresentarem uma carne mais macia na análise sensorial e obtiveram maior frequência das fibras oxidativasglicolíticas (FOG) em relação aos imunocastrados. Quanto ao efeito dos βAA, o grupo ZIL apresentaram uma carne menos macia na força de cisalhamento, maiores concentrações de ácidos heptadecanoico, linoleico, araquidonico ácido C20:3 N6C8C11C14, ômega 6, maior frequência para as FO e menor para FG em comparação ao grupo RAC e CON. No entanto, os animais do grupo CON e ZIL apresentaram maior área para as FO em comparação ao grupo RAC, enquanto que para as FOG, os animais do grupo CON tiveram maior área do que os animais do grupo RAC e ZIL. Na análise sensorial, os grupos RAC e ZIL receberam menores notas para os atributos textura e qualidade global em relação ao CON. Não foi observado efeito da condição sexual e dos βAA sobre a expressão dos genes e comprimento de sarcômero. Conclui-se que a condição sexual e a suplementação com os βAA podem alterar a qualidade da carne, perfil de ácidos graxos e morfométrico muscular, sem, contudo, alterar a expressão dos genes e do comprimento de sarcômero.

The origin of the difference in the superconducting critical temperatures of the beta(H) and beta(L) phases of (BEDT-TTF)(2)I-3

Incommensurate lattice fluctuations are present in the beta(L) phase (T-c similar to 1.5 K) of ET2I3 (where ET is BEDT-TTF - bis(ethylenedithio)tetrathiafulvalene) but are absent in the beta(H) phase (T-c similar to 7 K). We propose that the disorder in the conformational degrees of freedom of the terminal ethylene groups of the ET molecules, which is required to stabilise the lattice fluctuations, increases the quasiparticle scattering rate and that this leads to the observed difference in the Superconducting critical temperatures, T-c, of the two phases. We calculate the dependence of T-c on the interlayer residual resistivity. Our theory has no free parameters. Our predictions are shown to be consistent with experiment. We describe experiments to conclusively test our hypothesis.

Adaptive phase measurements in linear optical quantum computation

Photon counting induces an effective non-linear optical phase shift in certain states derived by linear optics from single photons. Although this non-linearity is non-deterministic, it is sufficient in principle to allow scalable linear optics quantum computation (LOQC). The most obvious way to encode a qubit optically is as a superposition of the vacuum and a single photon in one mode-so-called 'single-rail' logic. Until now this approach was thought to be prohibitively expensive (in resources) compared to 'dual-rail' logic where a qubit is stored by a photon across two modes. Here we attack this problem with real-time feedback control, which can realize a quantum-limited phase measurement on a single mode, as has been recently demonstrated experimentally. We show that with this added measurement resource, the resource requirements for single-rail LOQC are not substantially different from those of dual-rail LOQC. In particular, with adaptive phase measurements an arbitrary qubit state a alpha/0 > + beta/1 > can be prepared deterministically.

The spatial patterns of beta/A4 deposit subtypes and blood vessels in Alzheimer's disease cases with pronounced congophilic angiopathy

The spatial patterns of diffuse, primitive and classic beta/A4 deposits was studied in relation to blood vessels in 24 cortical tissues from five elderly cases of Alzheimer's disease with pronounced congophilic angiopathy (CA). Beta/A4 deposit subtypes and beta/A4 stained blood vessels were clustered in the tissue. In many instances, the clusters of beta/A4 deposits and blood vessels were regularly spaced along the cortical strip. Total beta/A4 deposits were positively correlated with blood vessels in five tissues only. Similarly, clusters of diffuse and primitive beta/A4 subtypes were each positively correlated with blood vessels in two brain regions. By contrast, clusters of classic beta/A4 deposits were positively correlated with blood vessels in 62% of the cortical tissues examined. These results suggest that in patients with significant CA, initial deposition of beta/A4 protein was unrelated to blood vessels. However, clusters of classic beta/A4 deposits appeared to be in phase with clusters of blood vessels along the cortex.