Spectroscopic characterization of 2-isopropylmalic acid-aluminum(III) complex

Spectroscopic elucidation of a 2-isopropylmalic acid (2-iPMA)-aluminum(III) complex has been carried out using (1)H, (13)C and (27)Al NMR spectroscopy, diffusion-ordered NMR spectroscopy (DOSY) and electrospray ionization mass spectrometry (ESI-MS). 2-iPMA is secreted by Saccharomyces cerevisiae and can dissolve Al(III) in the culture medium. The (1)H chemical shift perturbation and (1)H DOSY clearly indicated the formation of the 2-iPMA-Al(III) complex. The measurements of (13)C and (27)Al NMR spectroscopy and ESI-MS demonstrated that the major form of a complex is comprised four 2-iPMA and two Al(III) species. This compound is expected to possess strong Al(III)-detoxification capability.     

Signaling involved in pituitary adenylate cyclase-activating polypeptide-stimulated ADNP expression

Activity-dependent neurotrophic protein (ADNP) was discovered as a novel response gene for VIP and has neuroprotective potential. When the VIP paralog, PACAP38 was added to mouse neuron-glia co-cultures, it induced ADNP mRNA expression in a bimodal fashion at subpico- and nanomolar concentrations with greater response at subpicomolar level. The response was attenuated by a PAC1-R antagonist at both concentrations and by a VPAC1-R antagonist at nanomolar concentration only. An IP3/PLC inhibitor attenuated the response at both concentrations of PACAP38, but a MAPK inhibitor had no effect. A PKA inhibitor suppressed the response at nanomolar concentration only. These findings suggest that ADNP expression is mediated through multiple receptors and signaling pathways that are regulated by different concentrations of PACAP.     

Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2

Numb has been implicated in cortical neurogenesis during nervous system development, as a result of its asymmetric partitioning and antagonizing Notch signaling. Recent studies have revealed that Numb functions in clathrin-dependent endocytosis by binding to the AP-2 complex. Numb is also expressed in postmitotic neurons and plays a role in axonal growth. However, the functions of Numb in later stages of neuronal development remain unknown. Here, we report that Numb specifically localizes to dendritic spines in cultured hippocampal neurons and is implicated in dendritic spine morphogenesis, partially through the direct interaction with intersectin, a Cdc42 guanine nucleotide exchange factor (GEF). Intersectin functions as a multidomain adaptor for proteins involved in endocytosis and cytoskeletal regulation. Numb enhanced the GEF activity of intersectin toward Cdc42 in vivo. Expression of Numb or intersectin caused the elongation of spine neck, whereas knockdown of Numb and Numb-like decreased the protrusion density and its length. Furthermore, Numb formed a complex with EphB2 receptor-type tyrosine kinase and NMDA-type glutamate receptors. Knockdown of Numb suppressed the ephrin-B1-induced spine development and maturation. These results highlight a role of Numb for dendritic spine development and synaptic functions with intersectin and EphB2.     

Eukaryotic phylotypes in aquatic moss pillars inhabiting a freshwater lake in East Antarctica, based on 18S rRNA gene analysis

Aquatic mosses of Leptobryum species form unique tower-like pillars of vegetation termed “moss pillars” in Antarctic lakes. Moss pillars have distinct redox-affected sections: oxidative exteriors and reductive interiors. We have proposed that a “pillar” is a community and habitat of functionally interdependent organisms and may represent a mini-biosphere. Batteries of 16S rRNA genotypes, or phylotypes, of eubacteria and cyanobacteria, but no archaea, have been identified in moss pillars. However, detailed identification or phylogenetic analyses of the moss and their associated eukaryotic microbiota have not been performed. This study analyzed near-full-length 18S rRNA gene sequences obtained from two whole moss pillars. In total, 28 PCR clone libraries from two whole moss pillars were constructed, and 96 clones from each library (total 2,688 clones) were randomly selected and sequenced. Molecular phylogenetic analysis revealed that the phylotype belonging to Bryophyta, considered to be derived from moss, was closely related (99.9?%) to the 18S rRNA gene sequence from Leptobryum pyriforme. Unexpectedly, phylotypes belonging to a novel clade of fungi dominated (approximately 27–75?%) the moss pillar libraries. This suggests that fungi may contribute to carbon cycling in the moss pillar as parasites or decomposers. In addition, phylotypes related to ciliates and tardigrades were subdominant in the exterior, while the phylotype of the ameba-like, single-celled eukaryote, Cercomonas (Cercozoa), was detected only in the interior. These features were shared by both moss pillars. The 18S rRNA gene-based profiles demonstrated that redox-related factors may control distribution of some eukaryotic microbes in a whole moss pillar.     

Triiodothyronine induces UCP-1 expression and mitochondrial biogenesis in human adipocytes

Uncoupling protein (UCP)-1 expressed in brown adipose tissue plays an important role in thermogenesis. Recent data suggest that brown-like adipocytes in white adipose tissue (WAT) and skeletal muscle play a crucial role in the regulation of body weight. Understanding of the mechanism underlying the increase in UCP-1 expression level in these organs should, therefore, provide an approach to managing obesity. The thyroid hormone (TH) has profound effects on mitochondrial biogenesis and promotes the mRNA expression of UCP in skeletal muscle and brown adipose tissue. However, the action of TH on the induction of brown-like adipocytes in WAT has not been elucidated. Thus we investigate whether TH could regulate UCP-1 expression in WAT using multipotent cells isolated from human adipose tissue. In this study, triiodothyronine (T(3)) treatment induced UCP-1 expression and mitochondrial biogenesis, accompanied by the induction of the CCAAT/enhancer binding protein, peroxisome proliferator-activated receptor-γ coactivator-1α, and nuclear respiratory factor-1 in differentiated human multipotent adipose-derived stem cells. The effects of T(3) on UCP-1 induction were dependent on TH receptor-β. Moreover, T(3) treatment increased oxygen consumption rate. These findings indicate that T(3) is an active modulator, which induces energy utilization in white adipocytes through the regulation of UCP-1 expression and mitochondrial biogenesis. Our findings provide evidence that T(3) serves as a bipotential mediator of mitochondrial biogenesis.     

RAGE mediates vascular injury and inflammation after global cerebral ischemia

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in a diverse range of pathological conditions. To analyze the roles of RAGE and its decoy receptor, endogenous secretory RAGE (esRAGE), in the global cerebral ischemia, three different mouse cohorts, wild-type, RAGE^−/−, and esRAGE transgenic (Tg) mice were subjected to bilateral common carotid artery occlusion (BCCAO). RT-PCR and immunohistochemical analysis revealed that expression of RAGE was induced in the vascular cells at 12 h, and then in the neurons and glia from 3 to 7 days in the hippocampus after BCCAO. The numbers of surviving neurons in the hippocampal CA1 region were significantly higher in RAGE^−/− and esRAGE Tg mice than those in wild-type mice in the periods between 24 h and 7 days after BCCAO. Lower levels of 3-nitrotyrosine (3-NT) and higher levels of endothelial nitric oxide synthase (eNOS), together with enlarged vascular areas were observed in RAGE^−/− and esRAGE Tg mice at 12 h after BCCAO. In the later periods, expressions of glia-derived inflammatory mediators TNFα and inducible nitric oxide synthase (iNOS) were reduced in RAGE^−/− and esRAGE Tg mice. These results suggest that RAGE may contribute to delayed neuronal death after global cerebral ischemia by enhancing vascular injury and deleterious glia-mediated inflammation.     

Regioselective glucosidation of <Emphasis Type="Italic">trans</Emphasis>-resveratrol in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing glucosyltransferase from <Emphasis Type="Italic">Phytolacca americana</Emphasis>

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.     

Iron overload inhibits calcification and differentiation of ATDC5 cells

There is a little information about the effects of iron overload on cartilage metabolism. In the present study, we examined the effects of excess iron on the differentiation and mineralization of cultured chondrocytes, ATDC5 cells. We used ferric ammonium citrate (FAC) as a ferric ion donor and desferrioxamine (DFO) as a ferric ion chelator. Neither chemical affected the production of proteoglycan, a marker of an early stage of ATDC5 differentiation. In contrast, FAC inhibited the deposition of calcium, a late-stage event in chondrocyte differentiation, by ATDC5 cells in a dose-dependent manner, and DFO accelerated it. Energy dispersive X-ray spectroscopy/scanning electron microscope analysis revealed that the levels of iron and calcium in cells treated with FAC were increased and decreased, respectively. Furthermore, FAC inhibited the expression of matrix metalloproteinase 13 mRNA, another marker of late-stage chondrocyte differentiation. In addition, we found that the heavy and light chains of ferritin were expressed specifically at a late stage of ATDC5 differentiation, and the levels of both proteins were enhanced by the addition of iron. These results suggest that iron overload might give rise to osteopenia and arthritis by inhibiting chondrocyte differentiation and mineralization.     

Screening of an α-amylase inhibitor peptide by photolinker-peptide array

Peptide arrays in which peptides were immobilized on cellulose membranes through photolinkers were synthesized. The peptides were subsequently detached from the arrays by ultraviolet (UV) photolysis for 3 h, and were used to search for functional peptides that inhibit the activity of α-amylase derived from human pancreatic juice. Amino acid replacement with high-molecular-size amino acids, Arg (R), Phe (F), Trp (W), or Tyr (Y), for the first and seventh residues of amylase inhibitor peptide, GHWYYRCW, as previous reported, led to enhancement of the inhibitory effect of the peptide on α-amylase. In particular, one of the resulting peptides, RHWYYRYW, showed a stronger inhibitory effect than acarbose (which is used as a hypoglycemic agent) or inhibitor peptide GHWYYRCW.     

Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2(-/-)) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2(-/-) growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2(-/-) growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2(-/-) growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2(-/-) mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.