C NMR study on peracetylated derivatives of cyclomaltoheptaose (β-cyclodextrin, β-CD) and its methylated derivative

Peracetylated samples of cyclomaltoheptaose (β-cyclodextrin, β-CD) and its methylated derivative were studied by ¹³C NMR. The acetyl carbonyl carbon signal in peracetylated β-CD was resolved into a triplet, and the three peaks were assigned by long-range C---H COSY and INAPT techniques. The individual carbonyl peak was found to be indicative of the location of the acetyl group on the 2, 3, and 6 position in the glucose residues. An acetylated derivative of a partly methylated β-CD was also subjected to ¹³C NMR analysis to determine the distribution of acetyl and, subsequently, methyl groups on the glucose residues.     

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.     

Synergistic inhibitory activity of α- and β-LFA-1 peptides on LFA-1/ICAM-1 interaction

Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the α-subunit and the I-like domain of the β-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.     

Chiral separation of vinpocetine using cyclodextrin-modified micellar electrokinetic chromatography

A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) technique has been developed for enantioseparation of vinpocetine using an inexpensive 2-hydroxypropyl-β-CD (HP-β-CD) as the chiral selector (CS). The best chiral separation was achieved using 40 mM HP-β-CD as the CS in 50 mM phosphate buffer (pH 7.0) consisting of 40 mM sodium dodecyl sulfate (SDS) at a separation temperature and separation voltage of 25°C and 25 kV, respectively. To the author's best knowledge, this is the first CD-MEKC study able to successfully separate the four stereoisomer of vinpocetine in separation time of 9.5 min and resolution of 1.04-3.87.     

Expression, purification, and structural study of the EC4 domain of E-cadherin

The objective of this work was to produce unlabeled and 15N-labeled EC4 domain protein from E-cadherin for studying its structure and binding properties to other EC domains as well as to E-cadherin peptides. The EC4 domain of E-cadherin was expressed in Escherichia coli from the vector pASK-IBA6 and localized in the periplasmic space of E. coli. This protein contains a Streptag sequence at the N-terminus, and thus was purified using a Strep-Tactin affinity column. However, at high concentrations the 15N-labeled EC4 protein showed an unstable conformation. Conditions for stabilizing the conformation of this protein were evaluated using CD spectroscopy. The CD results showed that this protein has high conformational stability in Tris buffer at pH 6.0 in the presence of 10 mM calcium chloride.     

Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice

Anti‐angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain–hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion‐associated domain in MUC4 and other proteins (AMOP) domain at the C‐terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C‐terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)‐basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF‐stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase‐dependent pathway. ISM binds to αvβ5 integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmen‐tal vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.     

Differential phosphorylation of NG2 proteoglycan by ERK and PKCalpha helps balance cell proliferation and migration

Two distinct Thr phosphorylation events within the cytoplasmic domain of the NG2 proteoglycan help regulate the cellular balance between proliferation and motility. Protein kinase Calpha mediates the phosphorylation of NG2 at Thr2256, resulting in enhanced cell motility. Extracellular signal-regulated kinase phosphorylates NG2 at Thr2314, stimulating cell proliferation. The effects of NG2 phosphorylation on proliferation and motility are dependent on beta1-integrin activation. Differential cell surface localization of the two distinctly phosphorylated forms of NG2 may be the mechanism by which the NG2-beta1-integrin interaction promotes proliferation in one case and motility in the other. NG2 phosphorylated at Thr2314 colocalizes with beta1-integrin on microprotrusions from the apical cell surface. In contrast, NG2 phosphorylated at Thr2256 colocalizes with beta1-integrin on lamellipodia at the leading edges of cells. Thus, phosphorylation and the resulting site of NG2-integrin localization may determine the specific downstream effects of integrin signaling.     

GWAS of follicular lymphoma reveals allelic heterogeneity at 6p21.32 and suggests shared genetic susceptibility with diffuse large B-cell lymphoma

Non-Hodgkin lymphoma (NHL) represents a diverse group of hematological malignancies, of which follicular lymphoma (FL) is a prevalent subtype. A previous genome-wide association study has established a marker, rs10484561 in the human leukocyte antigen (HLA) class II region on 6p21.32 associated with increased FL risk. Here, in a three-stage genome-wide association study, starting with a genome-wide scan of 379 FL cases and 791 controls followed by validation in 1,049 cases and 5,790 controls, we identified a second independent FL-associated locus on 6p21.32, rs2647012 (OR(combined) = 0.64, P(combined) = 2 × 10(-21)) located 962 bp away from rs10484561 (r(2)<0.1 in controls). After mutual adjustment, the associations at the two SNPs remained genome-wide significant (rs2647012:OR(adjusted) = 0.70, P(adjusted) = 4 × 10(-12); rs10484561:OR(adjusted) = 1.64, P(adjusted) = 5 × 10(-15)). Haplotype and coalescence analyses indicated that rs2647012 arose on an evolutionarily distinct haplotype from that of rs10484561 and tags a novel allele with an opposite (protective) effect on FL risk. Moreover, in a follow-up analysis of the top 6 FL-associated SNPs in 4,449 cases of other NHL subtypes, rs10484561 was associated with risk of diffuse large B-cell lymphoma (OR(combined) = 1.36, P(combined) = 1.4 × 10(-7)). Our results reveal the presence of allelic heterogeneity within the HLA class II region influencing FL susceptibility and indicate a possible shared genetic etiology with diffuse large B-cell lymphoma. These findings suggest that the HLA class II region plays a complex yet important role in NHL.     

Disulfide bond formation promotes the cis- and trans-dimerization of the E-cadherin-derived first repeat

Cadherin is a cell adhesion molecule crucial for epithelial and endothelial cell monolayer integrity. The previously solved x-ray crystallographic structure of the E-CAD12 cis-dimer displayed an unpaired Cys(9), which protruded away from the Cys(9) on the other protomer. To investigate the possible biological function of Cys(9) within the first repeat (the E-cadherin-derived N-terminal repeat), E-CAD1 was overexpressed and secreted into the periplasmic space of Escherichia coli cells. Recombinant E-CAD1 produced a mixed monomer and dimer in an equilibrium fashion. The dimer was linked by a disulfide through Cys(9) pairing. Analysis by high pressure liquid chromatography and electron microscopy suggested the existence of oligomeric complexes. Mutation at Trp(2) appears to indicate that these oligomeric complexes trans-dimerize. Interestingly, mutation of Cys(9) affected not only the cis-dimerization, but also the trans-oligomerization of E-CAD1. Accordingly, it is plausible that, under oxidative stress, the homophilic interactions of E-cadherin through E-CAD1 may be promoted and stabilized by this disulfide bond.     

NG2 proteoglycan promotes endothelial cell motility and angiogenesis via engagement of galectin-3 and alpha3beta1 integrin

The NG2 proteoglycan is expressed by microvascular pericytes in newly formed blood vessels. We have used in vitro and in vivo models to investigate the role of NG2 in cross-talk between pericytes and endothelial cells (EC). Binding of soluble NG2 to the EC surface induces cell motility and multicellular network formation in vitro and stimulates corneal angiogenesis in vivo. Biochemical data demonstrate the involvement of both galectin-3 and alpha3beta1 integrin in the EC response to NG2 and show that NG2, galectin-3, and alpha3beta1 form a complex on the cell surface. Transmembrane signaling via alpha3beta1 is responsible for EC motility and morphogenesis in this system. Galectin-3-dependent oligomerization may potentiate NG2-mediated activation of alpha3beta1. In conjunction with recent studies demonstrating the early involvement of pericytes in angiogenesis, these data suggest that pericyte-derived NG2 is an important factor in promoting EC migration and morphogenesis during the early stages of neovascularization.