Monika Mueller

Oregano: a source for peroxisome proliferator-activated receptor gamma antagonists.

Peroxisome proliferator-activated receptors (PPARs) are drug targets for several perturbations of metabolic syndrome, defined as the coexistence of obesity, hyperglycemia, hypertension, and hyper/dyslipidemia. In this study, PPAR activation by oregano (e.g., Origanum vulgare) and its components was tested. Oregano extracts bind but do not transactivate PPARgamma, and binding affinity differs among different oregano extracts. The extracts contain PPARgamma antagonists (e.g., quercetin, luteolin, rosmarinic acid, and diosmetin), selective PPARgamma modulators (e.g., naringenin and apigenin), and PPARgamma agonists (e.g., biochanin A). Oregano extract and isolated compounds in the extract antagonize rosiglitazone-mediated DRIP205/TRAP220 recruitment to PPARgamma, pointing to oregano extracts as putative food supplements for weight reduction. Rosmarinic acid and biochanin A, PPARalpha agonists, may ameliorate the lipid profile. By endothelial nitric oxide synthase activation, oregano extract could prevent atherosclerosis. The results warrant further investigation of oregano extract for its potential to prevent and ameliorate metabolic syndrome and its complications.

Potential Health-modulating Effects of Isoflavones and Metabolites via Activation of PPAR and AhR

Isoflavones have multiple actions on cell functions. The most prominent one is the activation of estrogen receptors. Other functions are often overlooked, but are equally important and explain the beneficial health effects of isoflavones. Isoflavones are potent dual PPARα/γ agonists and exert anti-inflammatory activity, which may contribute to the prevention of metabolic syndrome, atherosclerosis and various other inflammatory diseases. Some isoflavones are potent aryl hydrocarbon receptor (AhR) agonists and induce cell cycle arrest, chemoprevention and modulate xenobiotic metabolism. This review discusses effects mediated by the activation of AhR and PPARs and casts a light on the concerted action of isoflavones.

Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability.

Immunoglobulin G (IgG), the most common class of commercial monoclonal antibodies (mAbs), exists as multimers of two identical light chains (LC) and two identical heavy chains (HC) assembled together by disulfide bridges. Due to the kinetics of mAb assembly, it is suggested that expression of LC and HC in equal amounts is not optimal for IgG production. We designed a set of vectors using internal ribosome entry site (IRES) elements to control LC and HC expression. The intracellular LC:HC ratio of stable IgG expressing Chinese hamster ovary (CHO) cell pools can be controlled effectively at four different ratios of 3.43, 1.24, 1.12, and 0.32. The stable pools were used to study the impact of LC:HC ratio on mAb expression and quality. Gene amplification was most effective for pools with excess LC and generated the highest mAb titers among the transfected pools. When LC:HC ratio was greater than one, more than 97% of the secreted products were IgG monomers. The products also have similar N-glycosylation profiles and conformational stabilities at those ratios. For pools presented a lower LC:HC ratio of 0.32, monomers only constituted half of the product with the other half being aggregates and mAb fragments. High mannose-type N-glycans increased while fucosylated and galactosylated glycans decreased significantly at the lowest LC:HC ratio. Product stability was also adversely affected. The results obtained provide insights to the impact of different LC:HC ratios on stable mAb production and useful information for vector design during generation of mAb producing cell lines.

Protein precipitation by polyethylene glycol: a generalized model based on hydrodynamic radius.

PEGs for protein precipitation are usually classified by molecular weight. The higher molecular weight precipitants are more efficient but result in higher viscosity. Following empirical evidence that the precipitation efficiency is more comprehensively characterized by PEG hydrodynamic radius (r(h,PEG)) than molecular weight, this paper proposes a model to explicate the significance of r(h,PEG). A general expression was formulated to characterize the PEG effect exclusively by r(h,PEG). The coefficients of a linearized form were then fitted using empirical solubility data. The result is a simple numerical relation that models the efficiency of general-shaped PEG precipitants as a function of r(h,PEG) and protein hydrodynamic radius (r(h,prot)). This equation also explains the effects of environmental conditions and PEG branching. While predictions by the proposed correlation agree reasonably well with independent solubility data, its simplicity gives rise to potential quantitative deviations when involving small proteins, large proteins and protein mixtures. Nonetheless, the model offers a new insight into the precipitation mechanism by clarifying the significance of r(h,PEG). This in turn helps to refine the selection criterion for PEG precipitants.

Red clover extract: a source for substances that activate peroxisome proliferator―activated receptor α and ameliorate the cytokine secretion profile of lipopolysaccharide-stimulated macrophages

Objective: Inflammation and hyperlipidemia or dyslipidemia contribute to an increased risk of atherosclerosis and cardiovascular disease. Cardiovascular disease represents one of the major causes of premature death worldwide. The activation of peroxisome proliferator-activated receptor (PPAR) &agr;, a drug target for hyperlipidemia and dyslipidemia, leads to an improved blood lipid profile. In this study, we determined the putative anti-inflammatory and PPAR&agr; stimulatory activities of red clover, an alternative to the classic hormone therapy used currently to treat menopausal symptoms. Methods: Lipopolysaccharide-induced macrophages were used as a model for anti-inflammatory activity, and a chimeric GAL4-PPAR&agr; system was used as a model for putative hypolipidemic activity. Results: Red clover extract and the isoflavones genistein and biochanin A were moderate PPAR&agr; activators. Daidzein only slightly activated PPAR&agr;, but its metabolite 6-hydroxydaidzein exerted a much higher PPAR&agr; activity. Similarly, the metabolite 3&vprime;-hydroxygenistein achieved higher activation efficiency than its precursor, genistein, did. In lipopolysaccharide-stimulated macrophages, red clover extract and its compounds reduced the secretion of proinflammatory cytokines, interleukin-6 and tumor necrosis factor &agr;, increased the secretion of the anti-inflammatory interleukin-10, and/or reduced the expression of nuclear factor-&kgr;B, inducible nitric oxide synthase, and/or cyclooxygenase 2. Tumor necrosis factor &agr; production was most efficiently reduced by biochanin A and genistein. Interleukin-6 levels were most efficiently reduced by genistein and equol. Conclusions: Owing to its PPAR&agr; activation and modulation of the secreted cytokine profile, red clover extract is a putative candidate for preventing atherosclerosis and, thus, cardiovascular disease.

Red clover extract: a putative source for simultaneous treatment of menopausal disorders and the metabolic syndrome.

Objective:Currently, red clover extract is used to treat menopausal disorders as an alternative to classic hormone therapy. Several human and animal studies have attributed hypolipidemic, hypoglycemic, or antiatherosclerotic effects to red clover extract or isoflavones. This study was designed to determine the peroxisome proliferator-activated receptor (PPAR) &ggr; activation by red clover extract. Design:The PPAR&ggr; binding affinities and the transactivation activities of red clover extracts, isoflavones, and their metabolites were analyzed. The presence of specific substances in the extracts was proved by high-performance liquid chromatography/electrospray ionization/mass spectrometry. Results:The red clover extracts and the compounds genistein and biochanin A were potent PPAR&ggr; ligands and activators. Several metabolites exerted higher binding affinities or transactivational activities than their precursor molecules. 6-Hydroxydaidzein exerted a more than 100-fold higher binding affinity than its precursor daidzein. The maximal transactivational activity of 6-hydroxydaidzein and 3′-hydroxygenistein exceeded even that of rosiglitazone, a known PPAR&ggr; agonist. Equol and O-desmethylangolensin showed an approximately fivefold higher binding affinity and, in the case of O-desmethylangolensin, a fourfold higher PPAR&ggr; agonistic activity than the precursor. The daily dose of Menoflavon forte, a widely used red clover extract for treatment of menopausal disorders, provides theoretically 15% to 30% of the daily recommended dose of rosiglitazone. Considering the more active metabolites formed, activity must be higher in vivo. Conclusions:This study shows that red clover extracts, the major compounds, and especially several main metabolites exert significant PPAR&ggr; binding and transactivational activity. Red clover extract, which is currently used for treating menopausal disorders, could be simultaneously used for ameliorating the metabolic syndrome.

Comparison of hormonal activity of isoflavone-containing supplements used to treat menopausal complaints.

Objectives: The isoflavones present in red clover and soy are used as an alternative treatment for menopausal complaints and are commercially available as high-dose food supplements. These preparations contain varying amounts of active ingredients, often without detailed specifications. Thus, it is difficult to derive a recommended daily dose, and the reliability of these products is rather low. Methods: We quantified the isoflavone content of 19 different isoflavone-containing preparations and compared their binding and transactivational activities with regard to estrogen receptor &agr;, estrogen receptor &bgr;, androgen receptor, progesterone receptor, peroxisome-proliferator-activated receptor, and aryl hydrocarbon receptor. Results: The food supplements that we tested bound to and transactivated both the estrogen receptors and the other receptors. After comparing the isoflavone content quantified by us with the isoflavone content specified on the package labels, we found that at least the specified isoflavone content or more could be detected in only 5 of the 19 food supplements that we tested. Conclusions: Preparations containing isoflavones should be standardized for the isoflavone aglycone content to facilitate the prediction of theoretical hormonal activity, facilitate the intake of a controlled amount of isoflavones, and ensure greater product reliability.

Cytotoxic antibody fragments for eliminating undifferentiated human embryonic stem cells

Human embryonic stem cells (hESC) possess great potential for applications in regenerative medicine due to their ability to differentiate into any cell type in the body. However, it is crucial to remove residual undifferentiated hESC from the differentiated population to avoid teratoma formation in vivo. The monoclonal antibody, mAb 84, has been shown to bind and kill undifferentiated hESC and is very useful for the elimination of contaminating undifferentiated hESC prior to transplantation. As mAb 84 is an IgM, its large size may impede penetration into embryoid bodies (EB) or cell clumps. To improve penetration, four antibody fragment formats of mAb 84 were engineered and expressed in Escherichia coli: Fab 84, scFv 84, scFv 84-diabody and scFv 84-HTH. All 4 fragments bound specifically to hESC, but only scFv 84-HTH, a single chain variable fragment with a dimerizing helix-turn-helix motif, could recapitulate the cytotoxicity of mAb 84 on multiple hESC lines. The results suggest that multivalency and flexibility between the antigen-binding sites may be essential features required for killing of hESC by mAb 84 and its derivatives. Imaging of EB treated with scFv 84-HTH or mAb 84 showed an even distribution of scFv 84-HTH throughout the EB whereas mAb 84 was localized more to the periphery.

Branched polyethylene glycol for protein precipitation

The use of linear PEGs for protein precipitation raises the issues of high viscosity and limited selectivity. This paper explores PEG branching as a way to alleviate the first problem, by using 3‐arm star as the model branched structure. 3‐arm star PEGs of 4,000 to 9,000 Da were synthesized and characterized. The effects of PEG branching were then elucidated by comparing the branched PEG precipitants to linear versions of equivalent molecular weights, in terms of IgG recovery from CHO cell culture supernatant, precipitation selectivity, solubility of different purified proteins, and precipitation kinetics. Two distinct effects were observed: PEG branching reduced dynamic viscosity; secondly, the branched PEGs precipitated less proteins and did so more slowly. Precipitation selectivity was largely unaffected. When the branched PEGs were used at concentrations higher than their linear counterparts to give similar precipitation yields, the dynamic viscosity of the branched PEGs were noticeably lower. Interestingly, the precipitation outcome was found to be a strong function of PEG hydrodynamic radius, regardless of PEG shape and molecular weight. These observations are consistent with steric mechanisms such as volume exclusion and attractive depletion. Biotechnol. Bioeng. 2012; 109:736–746.

Physical properties and biological activities of hesperetin and naringenin in complex with methylated β-cyclodextrin

Summary The aim of this work is to improve physical properties and biological activities of the two flavanones hesperetin and naringenin by complexation with β-cyclodextrin (β-CD) and its methylated derivatives (2,6-di-O-methyl-β-cyclodextrin, DM-β-CD and randomly methylated-β-CD, RAMEB). The free energies of inclusion complexes between hesperetin with cyclodextrins (β-CD and DM-β-CD) were theoretically investigated by molecular dynamics simulation. The free energy values obtained suggested a more stable inclusion complex with DM-β-CD. The vdW force is the main guest–host interaction when hesperetin binds with CDs. The phase solubility diagram showed the formation of a soluble complex of AL type, with higher increase in solubility and stability when hesperetin and naringenin were complexed with RAMEB. Solid complexes were prepared by freeze-drying, and the data from differential scanning calorimetry (DSC) confirmed the formation of inclusion complexes. The data obtained by the dissolution method showed that complexation with RAMEB resulted in a better release of both flavanones to aqueous solution. The flavanones-β-CD/DM-β-CD complexes demonstrated a similar or a slight increase in anti-inflammatory activity and cytotoxicity towards three different cancer cell lines. The overall results suggested that solubilities and bioactivities of both flavanones were increased by complexation with methylated β-CDs.