Activation of estrogen receptor-beta by a special extract of Rheum rhaponticum (ERr 731), its aglycones and structurally related compounds

The special extract ERr 731^® from the roots of Rheum rhaponticum is the major constituent of Phytoestrol^® N which is used for the treatment of climacteric symptoms in menopausal women. However, the molecular mode of action of ERr 731^® was unknown. For the first time, ERr 731^® and its aglycones trans-rhapontigenin and desoxyrhapontigenin were investigated with regard to the activation of the estrogen receptor- or estrogen receptor-β (ER, ERβ). The related hydroxystilbenes cis-rhapontigenin, resveratrol and piceatannol were studied as comparators. As controls, 17β-estradiol or the selective ER-(propylpyrazoltriol) or ERβ-agonists (diarylpropionitril) were used. Neither in ER-expressing yeast cells, in the ER-responsive Ishikawa cells, nor in human endometrial HEC-1B cells transiently transfected with the ER an activation of ER by ERr 731^® or the other single compounds was detected. Furthermore, an antiestrogenic effect was not observed. In contrast in human endometrial HEC-1B cells transiently transfected with the ERβ, 100 ng/ml ERr 731^® and the single compounds significantly induced the ERβ-coupled luciferase activity in a range comparable to 10⁻⁸ M 17β-estradiol. All effects were abolished with the pure ER antagonist ICI 182780, indicating an ER-specific effect. The ERβ agonistic activity by ERr 731^® could be of importance for its clinical use, as central functions relevant to climacteric complaints are proposed to be mediated via ERβ activation.     

Wnt5a induces a tolerogenic phenotype of macrophages in sepsis and breast cancer patients

A well-orchestrated inflammatory reaction involves the induction of effector functions and, at a later stage, an active downregulation of this potentially harmful process. In this study we show that under proinflammatory conditions the noncanonical Wnt protein, Wnt5a, induces immunosuppressive macrophages. The suppressive phenotype induced by Wnt5a is associated with induction of IL-10 and inhibition of the classical TLR4-NF-κB signaling. Interestingly, this phenotype closely resembles that observed in reprogrammed monocytes in sepsis patients. The Wnt5a-induced feedback inhibition is active both during in vitro LPS stimulation of macrophages and in patients with sepsis caused by LPS-containing, gram-negative bacteria. Furthermore, using breast cancer patient tissue microarrays, we find a strong correlation between the expression of Wnt5a in malignant epithelial cells and the frequency of CD163(+) anti-inflammatory tumor-associated macrophages. In conclusion, our data point out Wnt5a as a potential target for an efficient therapeutic modality in severe human diseases as diverse as sepsis and malignancy.     

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase‐2

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase‐2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase‐2 also regulates autophagy, genomic stability and ageing. Caspase‐2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase‐2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase‐2 and impedes dimerization and activation of caspase‐2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase‐2. Depletion of endogenous API5 leads to an increase in caspase‐2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase‐2‐dependent apoptotic cell death. These results establish API5/AAC‐11 as a direct inhibitor of caspase‐2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.     

Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion

RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B″ proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the β-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B″ proteins. However, unlike U1A and U2B″, some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an α-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.     

Production of epoxide hydrolases in batch fermentations of <Emphasis Type="Italic">Botryosphaeria rhodina</Emphasis>

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.     

Markers of stemness in equine mesenchymal stem cells: a plea for uniformity

Mesenchymal stromal cells (MSC) are a very promising subpopulation of adult stem cells for cell-based regenerative therapies in veterinary medicine. Despite major progress in the knowledge on adult stem cells during recent years, a proper identification of MSC remains a challenge. In human medicine, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) recently proposed three criteria to define MSC. Firstly, cells must be plastic-adherent when maintained under standard culture conditions. Secondly, MSC must express CD73, CD90 and CD105, and lack expression of CD34, CD45, CD14 or CD11b, CD79α or CD19 and MHC class II antigens. Thirdly, MSC must be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro. Successful isolation and differentiation of equine MSC from different sources such as bone marrow, fat tissue, umbilical cord blood, Wharton's Jelly or peripheral blood has been widely reported. However, their unequivocal immunophenotyping is hampered by the lack of a single specific marker and the limited availability of monoclonal anti-horse antibodies, which are two major factors complicating successful research on equine MSC. Detection of gene expression on mRNA level is hereby a valuable alternative, although the need still exists to test several antibody clones in search for cross-reactivity. To date, commercial antibodies recognizing equine epitopes are only available for CD13, CD44 and MHC-II. Moreover, as the expression of certain adult stem cell markers may differ between species, it is mandatory to define a set of CD markers which can be uniformly applied for the identification of equine MSC.     

Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor,Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.     

The Larval Eye of Nannochoristid Scorpionflies (Insecta,Mecoptera)

Abstract The stemmata of last–instar Nannochoristalarvae are compound eyes composed of 10 or more ommatidia. Each ommatidium has four Semper cells, four distal and four proximal retinula cells which form a cruciform and layered rhabdom. The ommatidia are separated by epidermal cells (possibly rudimentary pigment cells). Corneal lenses are lacking. At the posterior edge, aberrant stemma units may be present which lack a dioptric apparatus and have a star–shaped rhabdom composed of at least six retinula cells. The stemmata of Nannochoristaappear to be derived from stemmata of the Panorpa-type (Mecoptera-Panorpidae). Differences between the stemmata of Nannochoristaand Panorpacan be explained as adaptations to aquatic life (flat cornea) or as regression. A compound larval eye is ascribed to the ground plan of the Mecoptera sensu latoand is considered a genuine plesiomorphy. The identical basic number (seven) of stemmata in the Neuropteroid/Coleoptera assemblage, Amphiesmenoptera and some Mecoptera (Bittacidae, Boreidae) is attributed to parallel evolution.     

Risk stratification by residual enzyme activity after newborn screening for medium-chain acyl-CoA dehyrogenase deficiency: Data from a cohort study

ABSTRACT: BACKGROUND: Since the introduction of medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency in population newborn bloodspot screening (NBS) programs, subjects have been identified with variant ACADM (gene encoding MCAD enzyme) genotypes that have never been identified in clinically ascertained patients. It could be hypothesised that residual MCAD enzyme activity can contribute in risk stratification of subjects with variant ACADM genotypes. METHODS: We performed a retrospective cohort study of all patients identified upon population NBS for MCAD deficiency in the Netherlands between 2007-2010. Clinical, molecular, and enzymatic data were integrated. RESULTS: Eighty-four patients from 76 families were identified. Twenty-two percent of the subjects had a variant ACADM genotype. In patients with classical ACADM genotypes, residual MCAD enzyme activity was significantly lower (median 0%, range 0-8%) when compared to subjects with variant ACADM genotypes (range 0-63%; 4 cases with 0%, remainder 20-63%). Patients with (fatal) neonatal presentations before diagnosis displayed residual MCAD enzyme activities <1%. After diagnosis and initiation of treatment, residual MCAD enzyme activities <10% were associated with an increased risk of hypoglycaemia and carnitine supplementation. The prevalence of MCAD deficiency upon screening was 1/8,750 (95% CI 1/7,210-1/11,130). CONCLUSIONS: Determination of residual MCAD enzyme activity improves our understanding of variant ACADM genotypes and may contribute to risk stratification. Subjects with variant ACADM genotypes and residual MCAD enzyme activities <10% should be considered to have the same risks as patients with classical ACADM genotypes. Parental instructions and an emergency regimen will remain principles of the treatment in any type of MCAD deficiency, as the effect of intercurrent illness on residual MCAD enzyme activity remains uncertain. There are, however, arguments in favour of abandoning the general advice to avoid prolonged fasting in subjects with variant ACADM genotypes and 10% residual MCAD enzyme activity.