Dietary phytoestrogens and cancer: in vitro and in vivo studies

Thirty postmenopausal women (11 omnivores, 10 vegetarians and 9 apparently healthy women with surgically removed breast cancer) were investigated with regard to the association of their urinary excretion of estrogens, lignans and isoflavonoids (all diphenols) with plasma sex hormone binding globulin (SHBG). A statistically significant positive correlation between urinary total diphenol excretion and plasma SHBG was found which remained statistically significant after elimination of the confounding effect of body mass determined by body mass index (BMI). Furthermore we found a statistically significant negative correlation between plasma SHBG and urinary excretion of 16α-hydroxyestrone and estriol which also remained significant after eliminating the effect of BMI. Furthermore we observed that enterolactone (Enl) stimulates the synthesis of SHBG by HepG2 liver cancer cells in culture acting synergistically with estradiol and at physiological concentrations. Enl was rapidly conjugated by the liver cells, mainly to its monosulfate. Several lignans and the isoflavonoids daidzein and equol were found to compete with estradiol for binding to the rat uterine type II estrogen binding site (the s.c. bioflavonoid receptor). It is suggested that lignans and isoflavonoids may affect uptake and metabolism of sex hormones by participating in the regulation of plasma SHBG levels and in this way influence their biological activity and that they may inhibit cancer cell growth like some flavonoids by competing with estradiol for the type II estrogen binding sites.     

Solubility of sickle hemoglobin measured by a kinetic micromethod.

We have developed a photolytic method to determine the concentration of reactive hemes in a solution in the presence of a trace amount of CO. By measurement of the bimolecular rate of CO binding, and by calibration of the rate constant under equivalent conditions, the concentration of the reactive hemes can be determined. In a solution of sickle hemoglobin, the molecules in the gel contribute negligibly to the recombination rate, allowing the concentration of the molecules in the solution phase to be determined. To optimize signal to noise, modulated excitation methods were employed, although the method could also be used with pulse techniques and suitable signal averaging. Because the optical method employs a microspectrophotometer, only a few microliters of concentrated Hb solution is required to reproduce the entire temperature dependence of the solubility previously determined by centrifugation using milliliter quantities of solutions of the same concentration. This should be especially useful for studies of site-directed mutants, and we present results obtained on one such HbS in which Leu 88 beta has been replaced by Ala. The free energy difference in the polymerization of the Leu 88 beta double mutant is consistent with known differences in the amino acid hydrophobicities. The calibration required for these experiments also provides an excellent determination of the activation energy for binding the first CO to deoxy Hb.     

Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation

The Eph receptors and their ephrin ligands play crucial roles in a large number of cell–cell interaction events, including those associated with axon pathfinding, neuronal cell migration and vasculogenesis. They are also involved in the patterning of most tissues and overall cell positioning in the development of the vertebrate body plan. The Eph/ephrin signaling system manifests several unique features that differentiate it from other receptor tyrosine kinases, including initiation of bi-directional signaling cascades and the existence of ligand and receptor subclasses displaying promiscuous intra-subclass interactions, but very rare inter-subclass interactions. In this review we briefly discuss these features and focus on recent studies of the unique and expansive high-affinity Eph/ephrin assemblies that form at the sites of cell–cell contact and are required for Eph signaling initiation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Cellulase–lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme–lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase–lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM–cellulose interaction were also shown to contribute to lignin-binding.     

Screening of microbes for novel acidic cutinases and cloning and expression of an acidic cutinase from Aspergillus niger CBS 513.88

Isolates from gardening waste compost and 38 culture collection microbes were grown on agar plates at pH 4.0 with the cutinase model substrate polycaprolactone as a carbon source. The strains showing polycaprolactone hydrolysis were cultivated in liquid at acidic pH and the cultivations were monitored by assaying the p-nitrophenyl butyrate esterase activities. Culture supernatants of four strains were analyzed for the hydrolysis of tritiated apple cutin at different pHs. Highest amounts of radioactive hydrolysis products were detected at pHs below 5. The hydrolysis of apple cutin by the culture supernatants at acidic pH was further confirmed by GC–MS analysis of the hydrolysis products. On the basis of screening, the acidic cutinase from Aspergillus niger CBS 513.88 was chosen for heterogeneous production in Pichia pastoris and for analysis of the effects of pH on activity and stability. The recombinant enzyme showed activity over a broad range of pHs with maximal activity between pH 5.0 and 6.5. Activity could be detected still at pH 3.5.     

Biological and Structural Characterization of Glycosylation on Ephrin-A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand''s carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.     

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.     

Protein composition of 6K2‐induced membrane structures formed during Potato virus A infection

The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein‐induced membranous structures from Potato virus A (PVA)‐infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N‐terminal Twin‐Strep‐tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non‐tagged Cerulean‐6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep‐tag‐based affinity chromatography was developed. Both (+)‐ and (–)‐strand PVA RNA and viral protein VPg were co‐purified specifically with the affinity tagged PVA‐SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA‐SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2‐induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co‐purified with PVA‐derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.