Quantitative trait loci mapping of polyphenol metabolites from a 'Latham' x 'Glen Moy' red raspberry (Rubus idaeus L) cross.

OBJECTIVE: The objective of this study was to investigate the genetic control of polyphenol accumulation in red raspberry (Rubus idaeus L). METHODS: The levels of total anthocyanins and 37 individual polyphenol metabolites were measured over three years in a raspberry biparental mapping population. Quantitative trait loci (QTLs) for these traits were mapped onto a high-density SNP linkage map. RESULTS: At least one QTL was detected for each trait, with good consistency among the years. On four linkage groups (LG), there were major QTLs affecting several metabolites. On LG1, a QTL had large effects on anthocyanins and flavonols containing a rutinoside or rhamnose group. On LG4, a QTL had large effects on several flavonols and on LG5 and LG6 QTLs had large effects on ellagic acid derivatives. Smaller QTLs were found on LG2 and LG3. CONCLUSION: The identification of robust QTLs for key polyphenols in raspberry provides great potential for marker-assisted breeding for improved levels of potentially health beneficial components.

Assessing the influence of genotype and temperature on polyphenol composition in cloudberry (Rubus chamaemorus L.) using a novel mass spectrometric method.

A high-throughput abbreviated liquid chromatography mass spectrometric (ACMS) method was used to assess the relative influence of genotype and temperature on polyphenol composition in cloudberries. Principal component analysis (PCA) plots of the collated ACMS data showed a separation between crosses based on their female parents (Nyby or Fjellgull). Crosses with Nyby as the female parent had higher relative levels of masses assignable to certain ellagitannin derivatives. Crosses with Fjellgull had higher levels of distinctive masses assignable to quercetin derivatives (including a hydroxy-3-methylglutaroyl hexose derivative not previously identified in cloudberry) and anthocyanin derivatives. There was also a separation between samples grown at lower and higher temperatures, which was driven by m/z signals associated with ellagitannins and notably a major component, sanguiin H-6. Therefore, abbreviated MS techniques can discern genetic and/or environmental influences in polyphenol composition and can quickly assess quality in breeding programmes or in response to environmental changes.

Optoelectronic information encryption with phase-shifting interferometry.

A technique that combines the high speed and the high security of optical encryption with the advantages of electronic transmission, storage, and decryption is introduced. Digital phase-shifting interferometry is used for efficient recording of phase and amplitude information with an intensity recording device. The encryption is performed by use of two random phase codes, one in the object plane and another in the Fresnel domain, providing high security in the encrypted image and a key with many degrees of freedom. We describe how our technique can be adapted to encrypt either the Fraunhofer or the Fresnel diffraction pattern of the input. Electronic decryption can be performed with a one-step fast Fourier transform reconstruction procedure. Experimental results for both systems including a lensless setup are shown.

Error-reduction techniques and error analysis for fully phase- and amplitude-based encryption.

The performance of fully phase- and amplitude-based encryption processors is analyzed. The effects of noise perturbations on the encrypted information are considered. A thresholding method of decryption that further reduces the mean-squared error (MSE) for the fully phase- and amplitude-based encryption processes is provided. The proposed thresholding scheme significantly improves the performance of fully phase- and amplitude-based encryption, as measured by the MSE metric. We obtain analytical MSE bounds when thresholding is used for both decryption methods, and we also present computer-simulation results. These results show that the fully phase-based method is more robust. We also give a formal proof of a conjecture about the decrypted distribution of distorted encrypted information. This allows the analytical bounds of the MSE to be extended to more general non-Gaussian, nonadditive, nonstationary distortions. Computer simulations support this extension.

The thrombin receptor second cytoplasmic loop confers coupling to Gq-like G proteins in chimeric receptors. Additional evidence for a common transmembrane signaling and G protein coupling mechanism in G protein-coupled receptors.

Thrombin activates human platelets and other cells in part by cleaving an unusual G protein-coupled receptor. Thrombin cleavage of this receptor's amino-terminal exodomain unmasks a new amino terminus. This then binds intramolecularly to the body of the receptor to trigger transmembrane signaling and activation of Gi- and Gq-like G proteins. Toward identifying the domains responsible for thrombin receptor-G protein interactions, we examined the signaling properties of chimeric receptors in which thrombin receptor cytoplasmic sequences replaced the cognate sequences in the Gs-coupled beta2-adrenergic receptor (beta2AR) or the Gi-coupled dopamine D2 receptor (D2R). In Xenopus oocytes, a chimeric beta2AR bearing the thrombin receptor second cytoplasmic (C2) loop gained the ability to trigger intracellular Ca2+ release in response to adrenergic agonist, whereas a beta2AR bearing the cognate C2 loop from the D2R did not. Similarly, in COS-7 cells, a chimeric D2R bearing the thrombin receptor C2 loop gained the ability to trigger phosphoinositide hydrolysis in response to dopaminergic agonist, apparently by coupling to a Gq-like G protein. No detectable Gs coupling was seen. Thus, the thrombin receptor C2 loop was able to confer Gq-like coupling in several different receptor contexts. These observations suggest that the thrombin receptor C2 loop specifies Gq coupling by directly contacting Gq or by contributing to a structure required for Gq coupling. The ability of the thrombin receptor C2 loop to function in the context of the D2R and beta2AR strongly suggests that the transmembrane switching and G protein activation strategies used by the thrombin receptor must be very similar to those used by the D2R and beta2AR despite the thrombin receptor's strikingly different liganding mechanism.

Expression of tissue plasminogen activator in cerebral capillaries: possible fibrinolytic function of the blood-brain barrier.

Previous work has shown that tissue plasminogen activator (tPA) is a key enzyme in the control of fibrinolysis within the vascular system. The sources of brain tPA and the mechanisms by which tPA secretion and production occur within cerebral microcirculation are not well established. In this study, expression of tPA was investigated in cerebral capillaries and capillary-depleted brain isolated from cortices of 4- to 5-week-old rats and guinea pigs. In both species, a single tPA band of M(r) 67,000 was detected in cerebral capillaries by Western blot analysis. The tPA signal was absent from capillary-depleted brain. These results were corroborated at the messenger ribonucleic acid level. Reverse transcription-polymerase chain reaction analysis revealed the presence of tPA complementary deoxyribonucleic acid in samples derived from cerebral microvessels and demonstrated very low or undetectable tPA expression in capillary-depleted brain. Immunohistochemical analysis confirmed tPA localization in endothelial cells of brain capillaries. We conclude that microvascular endothelium, i.e., the blood-brain barrier, may have a role in promoting plasmin-dependent fibrinolysis in brain microcirculation. Delineation of the molecular mechanisms of blood-brain barrier-mediated fibrinolysis will likely contribute to future stroke prevention efforts.

The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly.

Ligand-gated ion channels are oligomeric membrane proteins in which homologous subunits specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we used a dominant-negative assay in which truncated subunits of the mouse muscle acetylcholine receptor (AChR) were coexpressed with the four wild-type subunits in transfected COS cells. Fragments of the alpha, delta, and gamma subunits consisting solely of the extracellular N-terminal domain blocked surface expression of the AChR and the formation of alpha delta heterodimers, an early step in the assembly pathway of the AChR. Immunoprecipitation and sucrose gradient sedimentation experiments showed that an N-terminal fragment of the alpha subunit forms a specific complex with the intact delta subunit. Thus the extracellular N-terminal domain of the alpha, delta, and gamma subunits contains the information necessary for specific subunit association.

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells.

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.

Characterization of 125I-tissue plasminogen activator binding to cerebellar granule neurons.

Mouse cerebellar cells in culture secrete tissue plasminogen activator (tPA) into the culture medium. Fibrin overlays have shown tPA to be associated with granule neurons in these cultures. This cell associated tPA can be displaced by extensive washing of the cells or by a brief lowering of the pH (less than 4), which leads to a loss of fibrinolytic activity by the cells. Incubation of these fibrinolytically inactive cells with exogenously added murine tPA leads to the restoration of the fibrinolytic activity, indicating the presence of tPA binding sites on these granule neurons. Using 125I-tPA, the binding to cerebellar granule neurons is rapid, saturable, specific, high affinity (Kd = 50 pM) and reversible. Both murine and human tPA compete with 125I-tPA for binding, with both murine and human urokinase (uPA) as well as human thrombin and plasminogen fail to compete. Neither the catalytic site nor the carbohydrate moiety of tPA appear to be involved in the binding, since both diisopropyl-fluorophosphate-treated tPA and endoglycosidase-H-treated tPA compete with 12I-tPA for binding. Furthermore, epidermal growth factor does not compete well with tPA for binding even at a 10:1 molar excess, suggesting that the epidermal growth factor-like (EGF) domain of tPA may not be involved in the binding mechanism. Autoradiography and antibody immunofluorescence show the specific tPA binding is to granule neurons in these cultures. Thus, granule neurons possess tPA receptors on their surface, where this protease binds retaining is functional activity and may play a role in cell and axon migration.

Tissue plasminogen activator binding to mouse cerebellar granule neurons.

Cultures of dissociated neonatal mouse cerebellar cells secrete primarily tissue plasminogen activator (tPA) and to a lesser extent urokinase plasminogen activator (uPA) into the culture medium. Fibrin overlays have localized plasminogen activator to granule neurons in these cultures; furthermore, this granule cell plasminogen activator activity is blocked by an antibody to tPA. Developmental studies indicate that maximal levels of soluble plasminogen activator in the culture medium preceed the peak of fibrinolytic activity by these cultures, suggesting that secreted PA may bind back to the surface of these granule neurons. Here we show that granule cell-associated tPA can be displaced by a brief pH shock. However, incubation of these fibrinolytically inactive cultures with exogenously added mouse tPA leads to a specific binding of active tPA to granule neurons as visualized by subsequent fibrin overlay. In similar studies mouse uPA, human uPa, and human tPA fail to show fibrinolytic activity associated with the cerebellar culture, whereas mouse tPA fails to bind to cerebellar glial cell cultures. These findings suggest that granule neurons possess binding sites for tPA on their surface, where this protease can retain its functional activity and may play an important role in cell migration or other cell activities.

Plasminogen activator secretion in relation to Schwann cell activities.

The molecular characterization of neural secretory plasminogen activators and their possible association with two well-defined Schwann cell activities (mitosis and migration) were investigated. Schwann cells from peripheral sensory ganglion roots were established as primary cultures of high purity. Conditioned culture medium was fractionated by gel electrophoresis and assayed for plasminogen-dependent in situ caseinolytic activity. Both tissue-type and urokinase-type plasminogen activators were detected early in culture. The amount of the tissue-type form decreased with increasing time in culture. Proliferative ability measured as [3H]thymidine incorporation into the nucleus varied under several culture conditions and was lowest in defined serum-free medium; however, these serum-free cultures expressed the highest level of plasminogen activator secreting cells. Combined autoradiography/fibrin overlay assays allowed for a direct analysis at the single cell level; interestingly the findings did not support the idea of an obligatory expression of the enzyme in replicating cells. Cell motility was assayed in combined gold particle clearing/fibrin overlay assays and likewise no obligatory association of protease secretion and cell movement could be demonstrated. The results are discussed in relation to previous studies in this field.