Isolation and identification of alectrol as (+)-orobanchyl acetate, a germination stimulant for root parasitic plants

Alectrol, a germination stimulant for root parasitic plants, was purified from root exudates of red clover (Trifolium pratense L.) and identified as a strigolactone, (+)-orobanchyl acetate [(3aS,4S,8bS,E)-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2-oxo-3,3a,4,5,6,7,8,8b-octahydro-2H-indeno[1,2-b]furan-4-yl acetate], by 1D and 2D NMR spectroscopy and ESI- and EI-MS spectrometry. Orobanchyl acetate afforded an [M-42](+) ion in EI-MS and thus had been recognized as an isomer of strigol. Orobanchyl acetate was detected in root exudates of soybean (Glycine max L.) and cowpea (Vigina unguiculata L.) along with orobanchol.     

Basal and stimulated lactate fluxes in primary cultures of astrocytes are differentially controlled by distinct proteins

Lactate release by astrocytes is postulated to be of importance for neuroenergetics but its regulation is poorly understood. Basigin, a chaperone protein for specific monocarboxylate transporters (MCTs), represents a putatively important regulatory element for lactate fluxes. Indeed, basigin knockdown by RNA interference in primary cultures of astrocytes partially reduced both proton-driven lactate influx and efflux. But more strikingly, enhancement of lactate efflux induced by glutamate was prevented while the effect of sodium azide was significantly reduced by treatment of cultured astrocytes with anti-basigin small interfering RNA. Enhancement of glucose utilization was unaffected under the same conditions. Basal lactate uptake and release were significantly reduced by MCT1 knockdown, even more so than with basigin knockdown, whereas glutamate-driven or sodium azide-induced enhancement of lactate release was not inhibited by either MCT1, 2, or 4 small interfering RNAs. In conclusion, MCT1 plays a pivotal role in the control of basal proton-driven lactate flux in astrocytes while basigin is only partly involved, most likely via its interaction with MCT1. In contrast, basigin appears to critically regulate the enhancement of lactate release caused by glutamate (or sodium azide) but via an effect on another unidentified transporter at least present in astrocytes in vitro.     

Prepubertal onset of slipped capital femoral epiphysis associated with hypothyroidism: a case report and literature review

Background

Slipped capital femoral epiphysis (SCFE) is a common hip disorder characterized by displacement of the capital femoral epiphysis from the metaphysic through the femoral epiphyseal plate. SCFE usually occurs during puberty, with obesity a common risk factor. We experienced a rare case of SCFE associated with hypothyroidism in a prepubescent patient who was not obese.

Case presentation

The patient was an 8-year-old boy suffering from bilateral SCFE with hypothyroidism. The patient’s growth had started to slow at 4 years of age, and at 8 years he was of short stature. During his evaluation for SCFE management, primary hypothyroidism was diagnosed due to the presence of anti-thyroid peroxidase and anti-thyroglobulin antibodies. After the patient was treated for hypothyroidism, which improved his thyroid function, surgery was performed for bilateral SCFE.

Conclusions

Among the 42 patients with SCFE associated with hypothyroidism in the literature, most SCFE occurred during puberty or in adults with delayed epiphyseal closure. Only two patients (4.8%), including the present patient, were ≤9 years old. Although being overweight or obese is common for patients with SCFE associated with hypothyroidism (76.0%), it was not observed in the present case. Persistent hypothyroidism, however, may be a risk factor for SCFE even before puberty and without obesity.

     

A third-generation mouse model of Alzheimer's disease shows early and increased cored plaque pathology composed of wild-type human amyloid β peptide

We previously developed single App knock-in mouse models of Alzheimer''s disease (AD) harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (App^NL-G-F and App^NL-F mice, respectively). These models showed Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner. The former model exhibits extensive pathology as early as 6 months, but is unsuitable for investigating Aβ metabolism and clearance because the Arctic mutation renders Aβ resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aβ antibodies. The latter model may take as long as 18 months for the pathology to become prominent, which leaves an unfulfilled need for an Alzheimer''s disease animal model that is both swift to show pathology and useful for antibody therapy. We thus utilized mutant Psen1 knock-in mice into which a pathogenic mutation (P117L) had been introduced to generate a new model that exhibits early deposition of wild-type human Aβ by crossbreeding the App^NL-F line with the Psen1^P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new third-generation mouse model showed more cored plaque pathology and neuroinflammation than App^NL-G-F mice and will help accelerate the development of disease-modifying therapies to treat preclinical AD.     

Pathophysiological concentrations of amyloid beta proteins directly inhibit rat brain and recombinant human type II phosphatidylinositol 4-kinase activity

We previously found that pathophysiological concentrations (< or = 10 nm) of an amyloid beta protein (Abeta25-35) reduced the plasma membrane phosphatidylinositol monophosphate level in cultured rat hippocampal neurons with a decrease in phosphatidylinositol 4-monophosphate-dependent Cl- -ATPase activity. As this suggested an inhibitory effect of Abeta25-35 on plasma membrane phosphatidylinositol 4-kinase (PI4K) activity, in vitro effects of Abetas on PI4K activity was examined using rat brain subcellular fractions and recombinant human type II PI4K (PI4KII). Abeta25-35 (10 nm) inhibited PI4KII activity, but neither PI 3-kinase (PI3K) nor type III PI4K (PI4KIII) activity, in microsomal fractions, while 100 nm Abeta25-35 inhibited PI3K activity in mitochondrial fractions. In plasma membrane-rich fractions, Abetas (> 0.5 nm) dose-dependently inhibited PI4KII activity, the maximal inhibition to 77-87% of control being reached around 10 nm of Abetas without significant changes in apparent Km values for ATP and PI, suggesting non-competitive inhibition by Abetas. The inhibition by 10 nm Abeta25-35 was reversible. In recombinant human PI4KIIalpha, inhibition profiles of Abetas were similar to those in rat brain plasma membranes. Therefore, pathophysiological concentrations of Abetas directly and reversibly inhibited plasma membrane PI4KII activity, suggesting that plasma membrane PI4KII is a target of Abetas in the pathogenesis of Alzheimer's disease.     

Mapping of unit boundaries of a protein: exhaustive search for permissive sites for duplication by complementation analysis of random fragment libraries of tryptophan synthase alpha subunit

To identify peptide units that make up a single-domain protein, we searched for possible combinations of N and C-fragments that exhibit functional complementation, and attempted an exhaustive evaluation of peptide unit boundaries. The tryptophan synthase alpha subunit was used as a model enzyme, which has a single TIM (beta8/alpha8) barrel domain. Libraries comprising randomly digested N and C-fragments were constructed, and clones expressing enzymatic activity were selected by the ability to confer growth of the Escherichia coli trpA mutant on a medium lacking tryptophan. More than 50 clones were obtained, and two cleavable positions were found on the loops after extra-helix 2' and helix 5. Half of the clones harbored N and C-fragments having an overlap between two fragments. The remaining clones harbored one fragment made by the fusion of N and C-fragments with insertional sequence duplication. Mapping the frequency of occurrence of fragment overlap and insertional duplication showed significant peaks at two loops, which coincide with the cleavable sites. These results suggest that the boundaries of unit fragments are located at these positions, and that bisection, fragment overlap and insertion are all possible at the unit boundaries.     

BRCA1 phosphorylation by Aurora-A in the regulation of G2 to M transition

Aurora-A/BTAK/STK15 localizes to the centrosome in the G(2)-M phase, and its kinase activity regulates the G(2) to M transition of the cell cycle. Previous studies have shown that the BRCA1 breast cancer tumor suppressor also localizes to the centrosome and that BRCA1 inactivation results in loss of the G(2)-M checkpoint. We demonstrate here that Aurora-A physically binds to and phosphorylates BRCA1. Biochemical analysis showed that BRCA1 amino acids 1314-1863 binds to Aurora-A. Site-directed mutagenesis indicated that Ser(308) of BRCA1 is phosphorylated by Aurora-A in vitro. Anti-phospho-specific antibodies against Ser(308) of BRCA1 demonstrated that Ser(308) is phosphorylated in vivo. Phosphorylation of Ser(308) increased in the early M phase when Aurora-A activity also increases; these effects could be abolished by ionizing radiation. Consistent with these observations, acute loss of Aurora-A by small interfering RNA resulted in reduced phosphorylation of BRCA1 Ser(308), and transient infection of adenovirus Aurora-A increased Ser(308) phosphorylation. Mutation of a single phosphorylation site of BRCA1 (S308N), when expressed in BRCA1-deficient mouse embryo fibroblasts, decreased the number of cells in the M phase to a degree similar to that with wild type BRCA1-mediated G(2) arrest induced by DNA damage. We propose that BRCA1 phosphorylation by Aurora-A plays a role in G(2) to M transition of cell cycle.