Glycosidase-catalyzed synthesis of glycosylated nutraceutical ingredients

Hydroxyphenyl propenoic acids (hydroxycinnamic acids) and their alcohol derivatives are common components of the human diet which often occur in plants in the form of various glycosides. As the diets rich in polyphenols have repeatedly been related to low incidence of cardiovascular, neurodegenerative, and oncological diseases, various food supplements containing these compounds are becoming increasingly popular among the general population. In quest of a biocatalytic route to structurally complex phenolic glycosides, we built a sustainable and convenient, one-pot two-enzyme method for the glucosylation of arylalkyl alcohols based on the synthetic exploitation of a fungal rutinosidase from A. niger and rhamnosidase from A. terreus. Both these enzymes were available to us as heterologous proteins produced by a recombinant strain of P. pastoris. As an example, the -glucoside salidroside, a compound endowed with various pharmacological effects and commercialized in Rhodiola rosea nutraceutical formulations, was obtained in high isolated yield and purity from tyrosol thanks to our one-pot enzymatic process. Furthermore, during the course of our investigation, we found that the rutinosidase from A. niger not only efficiently converted hydroxylated aromatic acids (e.g. coumaric and ferulic acids) into the respective phenolic rutinosides, but surprisingly could also catalyze the formation of the respective glycosyl esters. Here the results of our systematic study about the glycosidase-based biocatalytic preparation of lycosylated nutraceutical ingredients, which lead us to the discovery of a unique enzymatic entry to naturally occurring glycosyl esters, are reported

Anisotropy in the pion angular distribution of the reaction pp -> pp pi0 at 400 MeV

The reaction pp -> pp pi0 was studied with the WASA detector at the CELSIUS storage ring. The center of mass angular distribution of the pi0 was obtained by detection of the gamma decay products together with the two outgoing protons, and found to be anisotropic with a negative second derivative slope, in agreement with the theoretical predictions from a microscopic calculation.Comment: Revtex 4 style, 5 pages 7 figures, PACS numbers:13.60.Le, 13.75.Cs, 21.45.+v, 25.10.+

Molecular Defects in Cardiac Myofilament Ca2+-Regulation Due to Cardiomyopathy-Linked Mutations Can Be Reversed by Small Molecules Binding to Troponin

The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca2+ regulation via troponin. HCM is usually linked to higher myofilament Ca2+-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential. HIGHLIGHTS - Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable. - We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. - We have identified a new class of drugs that are capable of both reducing Ca2+-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca2+-sensitivity. - The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable. - Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers

Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested

Interleukin-23 engineering improves CAR T cell function in solid tumors

Cytokines that stimulate T cell proliferation, such as interleukin (IL)-15, have been explored as a means of boosting the antitumor activity of chimeric antigen receptor (CAR) T cells. However, constitutive cytokine signaling in T cells and activation of bystander cells may cause toxicity. IL-23 is a two-subunit cytokine known to promote proliferation of memory T cells and T helper type 17 cells. We found that, upon T cell antigen receptor (TCR) stimulation, T cells upregulated the IL-23 receptor and the IL-23α p19 subunit, but not the p40 subunit. We engineered expression of the p40 subunit in T cells (p40-Td cells) and obtained selective proliferative activity in activated T cells via autocrine IL-23 signaling. In comparison to CAR T cells, p40-Td CAR T cells showed improved antitumor capacity in vitro, with increased granzyme B and decreased PD-1 expression. In two xenograft and two syngeneic solid tumor mouse models, p40-Td CAR T cells showed superior efficacy in comparison to CAR T cells and attenuated side effects in comparison to CAR T cells expressing IL-18 or IL-15

Exclusive Measurements of pp -> dpi+pi0: Double-Pionic Fusion without ABC Effect

Exclusive measurements of the reaction pp -> dpi+pi0 have been carried out at T_p = 1.1 GeV at the CELSIUS storage ring using the WASA detector. The isovector pi+pi0 channel exhibits no enhancement at low invariant pipi masses, i. e. no ABC effect. The differential distributions are in agreement with the conventional t-channel Delta-Delta excitation process, which also accounts for the observed energy dependence of the total cross section. This is an update of a previously published version -- see important note at the end of the article