Epitope-defined monoclonal antibodies against multiplexin collagens demonstrate that type XV and XVIII collagens are expressed in specialized basement membranes

Type XV and type XVIII collagens are classified as part of multiplexin collagen superfamily and their C-terminal parts, endostatin and restin, respectively, have been shown to be anti-angiogenic in vivo and in vitro. The alpha1(XV) and alpha1(XVIII) collagen chains are reported to be localized mainly in the basement membrane zone, but their distributions in blood vessels and nonvascular tissues have yet to be thoroughly clarified. In the present study, we raised monoclonal antibodies against synthetic peptides of human alpha1(XV) and alpha1(XVIII) chains and used them for extensive investigation of the distribution of these chains. We came to the conclusion that nonvascular BMs contain mainly one of two types: subepithelial basement membranes that contained type XVIII in general, or skeletal and cardiac muscles that harbored mainly type XV. But basement membranes surrounding smooth muscle cells in vascular tissues contained one or both of them, depending on their locations. Interestingly, continuous capillaries contained both type XV and type XVIII collagens in their basement membranes; however, fenestrated or specialized capillaries such as glomeruli, liver sinusoids, lung alveoli, and splenic sinusoids expressed only type XVIII in their basement membranes, lacking type XV. This observation could imply that different functions of basement membranes in various tissues and organs use different mechanisms for the endogenous control of angiogenesis.     

Glycodelin and beta-lactoglobulin, lipocalins with a high structural similarity, differ in ligand binding properties.

Human glycodelin, a lipocalin with a high amino acid similarity to beta-lactoglobulins, appears as various glycoforms with different biological activities in endometrium (glycodelin-A) and seminal plasma (glycodelin-S). We found that the structures of these glycodelins and beta-lactoglobulin are similar. Despite this structural similarity, unlike beta-lactoglobulin, glycodelin-A binds neither retinoic acid nor retinol. It was impossible to detect any endogenous retinoids or steroids in any of the two purified glycodelins. Both their glycoforms share similar thermodynamic parameters of reversible denaturation suggesting that native folding of glycodelin-A and glycodelin-S is not influenced by the differences in glycosylation or by ligand binding.     

Drosophila type XV/XVIII collagen, Mp, is involved in Wingless distribution

Multiplexin (Mp) is the Drosophila orthologue of vertebrate collagens XV and XVIII. Like them, Mp is widely distributed in the basement membranes of the developing embryos, including those of neuroblasts in the central and peripheral nervous systems, visceral muscles of the gut, and contractile cardioblasts. Here we report the identification of mutant larvae bearing piggyBac transposon insertions that exhibit decrease Mp production associated with abdominal cuticular and wing margin defects, malformation of sensory organs and impaired sensitivity to physical stimuli. Additional findings include the abnormal ultrastructure of fatbody associated with abnormal collagen IV deposition, and reduced Wingless deposition. Collectively, these findings are consistent with the notion that Mp is required for the proper formation and/or maintenance of basement membrane, and that Mp may be involved in establishing the Wingless signaling gradients in the Drosophila embryo.     

Coordination polymers derived from a bis-pyridyl-bis-amide ligand: Supramolecular structural diversities and anion binding properties

Six new coordination polymers namely [{Cu(μ-L1)(CH₃COO)₂}]_∝1a, [{Cu(μ-L1)₂(CH₃COO)₂]_∝1b, [{Cu(μ-L1)₂(H₂O)₂}(NO₃)₂]_∝2, [{Cu(μ-L1)₂(H₂O)₂}(ClO₄)₂]_∝3, [{Cu(μ-L1)(H₂O)₂(μ-SO₄)}·3H₂O]_∝4a and [{Cu(μ-L1)₂SO₄}·X]_∝4b (L1 = N,N′-bis-(3-pyridyl)terephthalamide) have been synthesized. Single crystal structures of five coordination polymers namely 1a, 2-4b and the free ligand L1 are discussed in the context of the effect of conformation dependent ligating topology of the ligands, hydrogen bonding backbone, counter anions on the resultant supramolecular structures observed in these coordination polymers. It was revealed from the single crystal X-ray structure analysis that conformation dependent ligating topology of the bis-amide ligand L1, counter anion’s ligating strength dependent metal: ligand ratio, hydrogen bonding ability of the ligand as well as counter anions are responsible for the formation of 1D zigzag, 1D looped chain, 2D corrugated sheet in 1a, 2-3, 4a−4b, respectively. By following in situ coordination polymer crystallization technique, anion binding and separation studies have also been performed; nitrate anion has been separated as neat coordination polymer crystals from a complex mixture of anions.     

Astrasieversianins IX,XI and XV,cycloartane derived saponins from Astragalus sieversianus

Sixteen triterpenoid saponins, astrasieversianins I–XVI, have been isolated from the methanol extract of the roots of Astragalus sieversianus. By heterogeneous acidic hydrolysis, all glycosides produced only one common aglycone which was identified as the known natural product astramembrangenin or cycloastragenol. On the basis of spectral analysis and chemical reactions, the structures of two new triterpenoid glycosides, astrasieversianin IX and XI, were assigned as the 3-0-[α-l-rhamnopyranosyl(1 → 2)]-(3'-O-acetyl)-β-d-xylopyranosyl-6-O-β-d-xylopyranoside and the 3-O-[α-l-rhamnopyranosyl(1 → 2)]-(4'-O-acetyl)-β-d-xylopyranosyl-6-β-d-xylopyranoside of cycloastragenol. Astrasieversianin XV was identified as the 20,24-epimer (20R,24S) of cyclosieversiside G (20S,24R).     

Lytic activity and structural differences of amphipathic peptides derived from trialysin

Trialysin is a pore-forming protein found in the saliva of Triatoma infestans (Hemiptera, Reduviidae), the insect vector of Chagas' disease. The protein is active against a broad range of cell types from bacteria to eukaryotic cells. Recognizing that the N-terminus of trialysin harbors the lytic motif [Amino, R., Martins, R. M., Procopio, J., Hirata, I. Y., Juliano, M. A., and Schenkman, S. (2002) J. Biol. Chem. 277, 6207-6213], we designed a set of peptides scanning this region to investigate the structural basis of its biological function. Peptides encompassing residues 1-32 (P6), 1-27 (P7), and 6-32 (P5) efficiently induced lysis of the protozoan parasite Trypanosoma cruzi and Escherichia coli in the 0.4-9.0 microM range, while much higher concentrations were required to cause hemolysis. Other more internal peptides, including peptide P2 (residues 21-47) and others up to residue 52, were less effective. P6 turned out to be the most active of all. P7 has a significantly higher activity than P5 against E. coli, while P5 has a hemolytic activity comparable to that of P6. CD spectroscopy showed that all tested peptides acquire a comparable helical content in solvent mixtures or in detergent micelles. The solution structure of P2 and P5-P7 was determined in a 30% trifluoroethanol/water mixture by nuclear magnetic resonance. All peptides exhibit a structure characterized by a central helical fold, and except for P2, which does not show a continuous hydrophobic surface, they are amphipathic. The structural models show that P5 and P7 extend their structural similarities with the most active peptide, P6, in either the C-terminus or the N-terminus. Amino acid substitutions in the N-terminus of P6 improved hemolysis but did not change the activity against T. cruzi. These results suggest that while amphipathicity is essential for the lytic activity, the selectivity of the active peptides for specific organisms appears to be associated with the structural features of their N- and C-termini.     

GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity

We investigated the influence of a glycosylphosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic domains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its ectodomain released with the enzyme PI-phospholipase C (PI-PLC), were 9S trimers. Compared to wt-HA, the GPI-HA ectodomain underwent additional terminal oligosaccharide modifications; some of these occurred near the receptor binding pocket and completely inhibited the ability of GPI-HA to bind erythrocytes. Growth of GPI-HA-expressing cells in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) abrogated the differences in carbohydrate modification and restored the ability of GPI-HA to bind erythrocytes. The ectodomain of GPI-HA produced from cells grown in the presence or absence of dMM underwent characteristic low pH-induced conformational changes (it released its fusion peptides and became hydrophobic and proteinase sensitive) but at 0.2 and 0.4 pH units higher than wt-HA, respectively. These results demonstrate that although GPI-HA forms a stable trimer with characteristics of the wt, its structure is altered such that its receptor binding activity is abolished. Our results show that transmembrane and GPI-anchored forms of the same ectodomain can exhibit functionally important differences in structure at a great distance from the bilayer.     

Respiratory tissue properties derived from flow transfer function in healthy humans

Tomalak, W., R. Peslin, and C. Duvivier. Respiratorytissue properties derived from flow transfer function in healthy humans. J. Appl. Physiol. 82(4):1098-1106, 1997.Assuming homogeneity of alveolar pressure, therelationship between airway flow and flow at the chest during forcedoscillation at the airway opening [flow transfer function(FTF)] is related to lung and chest wall tissue impedance (Zti):FTF = 1 + Zti/Zg, where Zg is alveolar gas impedance, which isinversely proportional to thoracic gas volume. By using a flow-typebody plethysmograph to obtain flow rate at body surface, FTF has beenmeasured at oscillation frequencies (f_os) of 10, 20, 30 and 40 Hz in eight healthy subjects during both quiet and deepbreathing. The data were corrected for the flow shunted through upperairway walls and analyzed in terms of tissue resistance (Rti) andeffective elastance (Eti,eff) by using plethysmographically measuredthoracic gas volume values. In most subjects, Rti was seen to decreasewith increasingf_os and Eti,effto vary curvilinearly withf_os²,which is suggestive of mechanical inhomogeneity. Rti presented a weakvolume dependence during breathing, variable in sign according tof_os and amongsubjects. In contrast, Eti,eff usually exhibited a U-shaped patternwith a minimum located a little above or below functional residualcapacity and a steep increase with decreasing or increasing volume(30-80 hPa/l²) on eitherside. These variations are in excess of those expected from the sigmoidshape of the static pressure-volume curve and may reflect the effect ofrespiratory muscle activity. We conclude that FTF measurement is aninteresting tool to study Rti and Eti,eff and that these parametershave probably different physiological determinants.

     

Matricryptins derived from non fibrillar collagens,MMP-2 and SPARC are involved in the control of angiogenesis

The name matricryptin was proposed by Davis et al. (2000) for enzymatic fragments of extracellular matrix containing exposed matricryptic sites. The exposure of these sites occurred after structural or conformational modifications. Matricryptins derived from non fibrillar collagens (IV, VIII, XV and XVIII) and from matrix metalloproteinase-2 inhibit angiogenesis and tumor growth. Proteolysis of SPARC releases several peptides which exert opposite effects on angiogenesis. Matricryptins derived from glycosaminoglycans also participate in the control of angiogenesis.     

Identification of novel short peptides derived from the alpha 4, alpha 5, and alpha 6 fibrils of type IV collagen with anti-angiogenic properties

Angiogenesis, or neovascularization, is tightly controlled by positive and negative regulators, many of which reside in the extracellular matrix. We have now identified eight novel 19- to 20-residue peptides derived from the alpha4, alpha5, and alpha6 fibrils of type IV collagen, which we have designated tetrastatins, pentastatins, and hexastatins, respectively. We have shown that these endogenous peptides suppress the proliferation and migration of HUVECs in vitro. By performing clustering analyses of the sequences using sequence similarity criteria and of the experimental results using a hierarchical algorithm, we report that the clusters identified by the experimental results coincide with the sequence-based clusters, indicating a tight relationship between peptide sequence and anti-angiogenic potency. These peptides may have potential as anti-angiogenic therapeutic agents.     

Distinctive binding and structural properties of piscine transthyretin

The thyroid hormone binding protein transthyretin (TTR) forms a macromolecular complex with the retinol-specific carrier retinol binding protein (RBP) in the blood of higher vertebrates. Piscine TTR is shown here to exhibit high binding affinity for L-thyroxine and negligible affinity for RBP. The 1.56 A resolution X-ray structure of sea bream TTR, compared with that of human TTR, reveals a high degree of conservation of the thyroid hormone binding sites. In contrast, some amino acid differences in discrete regions of sea bream TTR appear to be responsible for the lack of protein-protein recognition, providing evidence for the crucial role played by a limited number of residues in the interaction between RBP and TTR. Overall, this study makes it possible to draw conclusions on evolutionary relationships for RBPs and TTRs of phylogenetically distant vertebrates.     

Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue

The biologic characteristics of mesenchymal stem cells (MSCs) isolated from two distinct tissues, bone marrow and adipose tissue were evaluated in these studies. MSCs derived from human and non-human primate (rhesus monkey) tissue sources were compared. The data indicate that MSCs isolated from rhesus bone marrow (rBMSCs) and human adipose tissue (hASCs) had more similar biologic properties than MSCs of rhesus adipose tissue (rASCs) and human bone marrow MSCs (hBMSCs). Analyses of in vitro growth kinetics revealed shorter doubling time for rBMSCs and hASCs. rBMSCs and hASCs underwent significantly more population doublings than the other MSCs. MSCs from all sources showed a marked decrease in telomerase activity over extended culture; however, they maintained their mean telomere length. All of the MSCs expressed embryonic stem cell markers, Oct-4, Rex-1, and Sox-2 for at least 10 passages. Early populations of MSCs types showed similar multilineage differentiation capability. However, only the rBMSCs and hASCs retain greater differentiation efficiency at higher passages. Overall in vitro characterization of MSCs from these two species and tissue sources revealed a high level of common biologic properties. However, the results demonstrate clear biologic distinctions, as well.     

Isolation, properties and tissue distribution of rat glutathione transferase E

Uptake of Li+ induced by the addition of proline to a cell suspension of Escherichia coli was detected using an Li+-selective electrode. This Li+ uptake was inhibited by L-azetidine 2-carboxylic acid, a competitive inhibitor of the proline transport system. Thus, direct evidence for Li+-proline cotransport via the proline transport system was obtained. Kinetic parameters of the Li+ uptake were determined.     

Comparative structural studies of two natural isoforms of ammodytoxin,phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity

Ammodytoxin A (AtxA) and its natural isoform AtxC from the venom of Vipera ammodytes ammodytes belong to group IIA-secreted phospholipases A₂ which catalyze the hydrolysis of glycerophospholipids and exhibit strong neurotoxic and anticoagulant effects. The two isoforms, which differ in sequence by only two amino acid residues (Phe124 > Ile and Lys128 > Glu), display significant differences in toxicity and anticoagulant properties and act on protein targets including neurotoxic proteic receptors and coagulation factor Xa with significantly different strengths of binding.In order to characterize the structural basis of these functional differences, we have determined the crystal structures of the two isoforms. Comparison of the structures shows that the mutation Lys128 > Glu in AtxC could perturb interactions with FXa, resulting in lower anticoagulant activity, since the side chain of Glu128 is partly buried, making a stabilizing hydrogen bond with the main-chain nitrogen atom of residue Thr35. This interaction leads to a displacement of the main polypeptide chain at positions 127 and 128 (identified by mutagenesis as important for interaction with FXa), and a different orientation of the side chain of unmutated Lys127. The mutation Phe124 > Ile in AtxC induces no significant conformational changes, suggesting that the differences in toxicity of the two isoforms are due essentially to differences in surface complementarity in the interaction of the toxin with the neurotoxic protein receptor. The crystal structures also reveal a novel dimeric quaternary association involving significant hydrophobic interactions between the N-terminal α-helices of two molecules of ammodytoxin related by crystallographic symmetry. Interactions at the dimer interface include important contributions from Met7, which is unique to ammodytoxin. Equilibrium sedimentation experiments are consistent with the crystallographic model.Competition experiments using SPR technology show complete inhibition of AtxA binding to FXa by calmodulin (CaM). The crystal structure shows that the C-terminal region, important for binding to FXa and CaM, is fully exposed and accessible for interaction with proteic receptors in both the monomeric and dimeric forms of ammodytoxin described here.