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.     

Reduction of liver manganese concentration in response to the ingestion of excess zinc: identification using metallomic analyses

To date, minerals of interest have been analyzed individually to understand mineral dynamics and metabolism. Our recent development of metallomic analyses enabled us to evaluate minerals in an unbiased and global manner. Here, we evaluated the effects of ingestion of excess zinc to plasma and tissue concentrations of minerals in growing rats. A total of 26 minerals were simultaneously evaluated by metallomic analyses using inductively coupled plasma-mass spectrometry (ICP-MS) in semi-quantification mode; the concentrations of several minerals exhibited consistent changes in response to the concentrations of dietary zinc. Manganese concentrations in plasma and femur increased, while concentrations in the liver and pancreas decreased with increasing dietary zinc concentrations. Because the interaction between zinc and manganese is not known, we further focused our analysis on liver manganese. Quantitative analyses also indicated that the hepatic concentration of manganese decreased in response to the ingestion of diets containing excess zinc, a result that is partly explained by the decreased expression of hepatic Zip8, a manganese transporter. The present study reveals mineral interaction by using metallomic analyses and proposes a possible mechanism that underlies this novel interaction.     

Brain-Derived Neurotrophic Factor from Bone Marrow-Derived Cells Promotes Post-Injury Repair of Peripheral Nerve

Brain-derived neurotrophic factor (BDNF) stimulates peripheral nerve regeneration. However, the origin of BNDF and its precise effect on nerve repair have not been clarified. In this study, we examined the role of BDNF from bone marrow-derived cells (BMDCs) in post-injury nerve repair. Control and heterozygote BDNF knockout mice (BDNF+/−) received a left sciatic nerve crush using a cerebral blood clip. Especially, for the evaluation of BDNF from BMDCs, studies with bone marrow transplantation (BMT) were performed before the injury. We evaluated nerve function using a rotarod test, sciatic function index (SFI), and motor nerve conduction velocity (MNCV) simultaneously with histological nerve analyses by immunohistochemistry before and after the nerve injury until 8 weeks. BDNF production was examined by immunohistochemistry and mRNA analyses. After the nerve crush, the controls showed severe nerve dysfunction evaluated at 1 week. However, nerve function was gradually restored and reached normal levels by 8 weeks. By immunohistochemistry, BDNF expression was very faint before injury, but was dramatically increased after injury at 1 week in the distal segment from the crush site. BDNF expression was mainly co-localized with CD45 in BMDCs, which was further confirmed by the appearance of GFP-positive cells in the BMT study. Variant analysis of BDNF mRNA also confirmed this finding. BDNF+/− mice showed a loss of function with delayed histological recovery and BDNF+/+→BDNF+/− BMT mice showed complete recovery both functionally and histologically. These results suggested that the attenuated recovery of the BDNF+/− mice was rescued by the transplantation of BMCs and that BDNF from BMDCs has an essential role in nerve repair.     

Facile synthesis and evaluation of C-functionalized benzyl-1-oxa-4,7,10-triazacyclododecane-N,N',N″-triacetic acid as chelating agent for ¹¹¹In-labeled polypeptides

A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N,N′,N″-triacetic acid (ODTA), has been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetraacetic acid (DOTA). However, neither synthetic procedure for a C-functionalized ODTA (C-ODTA) nor its chelating ability with a trace amount of radioactive indium-111 (¹¹¹In) has been elucidated. We herein present a facile synthetic procedure for C-ODTA, and estimated its ability as a chelating agent for radiolabeling peptides and proteins with ¹¹¹In. The synthetic procedure involves the synthesis of a linear precursor using a para-substituted phenylalanine derivative as a starting material. The following intramolecular cyclization reaction was best performed (>73% yield) when Boc-protected linear compound and the condensation reagent, HATU, were simultaneously added to the reaction vessel at the same flow rate. The cyclic compound was then reduced with BH₃ and alkylated with tert-butyl bromoacetate. The synthetic procedure was straightforward and some optimization would be required. However, most of the intermediate compounds were obtained easily in good yields, suggesting that the present synthetic procedure would be useful to synthesize C-ODTA derivatives. The intramolecular cyclization reaction might also be applicable to synthesize polyazamacrocycles of different ring sizes and cyclic peptides. In ¹¹¹In radiolabeling reactions, C-ODTA provided ¹¹¹In chelates in higher radiochemical yields at low ligand concentrations when compared with C-DOTA. The ¹¹¹In-labeled C-ODTA remained unchanged in the presence of apo-transferrin. The biodistribution studies also showed that the ¹¹¹In-labeled compound was mainly excreted into urine as intact. These findings indicate that C-ODTA would be useful to prepare ¹¹¹In-labeled peptides of high specific activities in high radiochemical yields.     

Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs

Mesenchymal stromal cells are crucial components of secondary lymphoid organs (SLOs). Organogenesis of SLOs involves specialized stromal cells, designated lymphoid tissue organizer (LTo) in the embryonic anlagen; in the adult, several distinct stromal lineages construct elaborate tissue architecture and regulate lymphocyte compartmentalization. The relationship between the LTo and adult stromal cells, however, remains unclear, as does the precise number of stromal cell types that constitute mature SLOs are unclear. From mouse lymph nodes, we established a VCAM-1(+)ICAM-1(+)MAdCAM-1(+) reticular cell line that can produce CXCL13 upon LTbetaR stimulation and support primary B cell adhesion and migration in vitro. A similar stromal population sharing many characteristics with the LTo, designated marginal reticular cells (MRCs), was found in the outer follicular region immediately underneath the subcapsular sinus of lymph nodes. Moreover, MRCs were commonly observed at particular sites in various SLOs even in Rag2(-/-) mice, but were not found in ectopic lymphoid tissues, suggesting that MRCs are a developmentally determined element. These findings lead to a comprehensive view of the stromal composition and architecture of SLOs.