VEGF-C and angiopoietins in lymphangiogenesis

Angiogenesis and lymphangiogenesis occur during development as the result of tightly coordinated signalling programs to generate two hierarchically organised vascular systems. All tissues and organs are dependent on a functional blood vasculature for oxygen and nutrients, whereas the lymphatic vasculature functions to collect excess tissue fluid, passing it through lymph nodes for immune surveillance, and returning it to the blood circulation. Effectors that control developmental angiogenesis and lymphangiogenesis are also involved in pathological settings, and therefore potential targets for therapy. Vascular endothelial growth factor (VEGF) and angiopoietin (Ang) growth factors, signalling through endothelial VEGFR and Tie receptors, have been established as key regulators of angiogenic and lymphangiogenic processes in development and disease. In this study, we aimed to obtain a clearer understanding of the vascular effects of stimulation by VEGF-C, Ang1 and Ang2, all known to be involved in lymphangiogenesis. In cell culture models, we found that both intrinsic and microenvironmental regulatory mechanisms are involved in the regulation of endothelial cell phenotypes, and distinct responses to VEGF signalling are induced by specific receptor pathways in different endothelial cell types. Surprisingly, we also found that Ang1 induces sprouting lymphangiogenesis in vivo by a VEGFR-3 dependent mechanism, establishing Ang1 as a novel lymphangiogenic factor. Using inducible transgenic mouse models, we found that VEGF-C-induced lymphatic hyperplasia persisted independently of the growth factor, indicating that short pro-lymphangiogenic therapy could lead to lasting improvements in tissue oedema. While VEGF-C had blood vessel effects in embryos, no angiogenic side effects were observed in adult tissues. Furthermore, inducible transgenic expression of Ang2 during embryonic development confirmed Ang2 as an important regulator of lymphatic remodelling and mural cell contacts. The unexpected similarity of the lymphatic maturation defects caused by excess Ang2 to those observed in Ang2 deficient mice demonstrated that correct doses of Ang2 are crucial for the control of lymphatic development. Unlike Ang1, Ang2 did not induce lymphatic sprouting. Although Ang1 has been shown to be able to substitute for Ang2 during developmental lymphangiogenesis, their lymphatic effects are not identical. These findings further our understanding of the basic mechanisms of angiogenesis and lymphangiogenesis, important for the future development of targeted therapies for vascular diseases such as cancer, inflammation, lymphoedema and ischemia. VEGF-C and Ang1 especially emerged as promising candidates for pro-lymphangiogenic therapy.

1H and 13C NMR spectral studies of C-2 substituted isomeric exo- and endo-5-methyl-bicyclo[3.2.1]octane-6,8-diones

A detailed analysis of the 1H and 13C NMR spectra of C-2 aryl and alkyl/desalkyl substituted isomeric exo- and endo-5-methylbicyclo[3.2.1]octane-6,8-diones is presented. The chemical shift of the C-5 angular methyl, the C-2 alkyl/olefinic (C-10)/C-2 methine protons, the aromatic proton shieldings and the characteristic AMX and ABX spectral pattern of the ketomethylene and bridgehead protons were found to be sensitive to the phenyl ring orientation (anisotropy). These distinctive features could be used for configurational distinction for this class of compounds. With increasing ortho-methoxy substitution on the phenyl ring, considerable deshilelding of the bridgehead proton was observed (ca. 0.6 ppm). Absence of the C-2 alkyl group in the desalkyl isomers resulted in substantial changes in the chemical shifts of different protons. A study of the NMR spectra of the corresponding bicyclic compounds with C-2 methoxy/hydroxy substitution instead of the aryl group revealed that the anisotropy of the phenyl ring and the electronegative oxygen substituents have opposite effects. The 13C NMR spectral assignment of each carbon resonance of C-2 aryl and alkyl/desalkyl substituted isomeric exo- and endo-5-methylbicyclo[3.2.1]octane-6,8-diones and the corresponding C-2 methoxy/hydroxy/chloro and methyl bicyclic compounds are reported. Additional ortho-methoxy substitution on the phenyl ring was found to produce considerable high field shifts of the C-10 and C-1 carbon resonances. A high-field shift was observed for the C-6 and C-8 carbonyl carbons, presumably due to 1,3-dicarbonyl interactions. The chemical shifts of C-1 aromatic, C-10 alkyl and C-2 carbons, which are sensitive to exo/endo isomerism, could be utilized in differentiating a pair of isomers.

Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Evaluation of wood characteristics of tropical post-mid rotation plantation Eucalyptus cloeziana and E. pellita: Part (c) Wood quality and structural properties.

This study provides information about wood quality, structural properties, processing characterists and product suitability of wood harvested from fast-grown hardwood plantations. Wood quality attributes tested included density, extractive content, unit shrinkage, heartwood proportion and sapwood width. Structural properties tested included small clear and full section strength and stiffness, hardness, joint group, visual grade assessment and natural vibration-based grade assessment. The variation between the inner, intermediate and outer heartwood zones and the variation between provenances was also tested. Overall, the wood qualtiy attributes measured for 19 year-old E. cloeziana and 15 year-old E. pellita plantation material fall between those expected from the wood of mature, native forest trees and those found in younger plantation material of the same species.

VEGF-C/VEGFR-3 and PDGF-B/PDGFR-b pathways in embryonic lymphangiogenesis

The circulatory system comprises the blood vascular system and the lymphatic vascular system. These two systems function in parallel. Blood vessels form a closed system that delivers oxygen and nutrients to the tissues and removes waste products from the tissues, while lymphatic vessels are blind-ended tubes that collect extravasated fluid and cells from the tissues and return them back to blood circulation. Development of blood and lymphatic vascular systems occurs in series. Blood vessels are formed via vasculogenesis and angiogenesis whereas lymphatic vessels develop via lymphangiogenesis, after the blood vascular system is already functional. Members of the vascular endothelial growth factor (VEGF) family are regulators of both angiogenesis and lymphangiogenesis, while members of the platelet-derived growth factor (PDGF) family are major mitogens for pericytes and smooth muscle cells and regulate formation of blood vessels. Vascular endothelial growth factor C (VEGF-C) is the major lymphatic growth factor and signaling through its receptor vascular endothelial growth factor receptor 3 (VEGFR-3) is sufficient for lymphangiogenesis in adults. We studied the role of VEGF-C in embryonic lymphangiogenesis and showed that VEGF-C is absolutely required for the formation of lymph sacs from embryonic veins. VEGFR-3 is also required for normal development of the blood vascular system during embryogenesis, as Vegfr3 knockout mice die at mid-gestation due to failure in remodeling of the blood vessels. We showed that sufficient VEGFR-3 signaling in the embryo proper is required for embryonic angiogenesis and in a dosage-sensitive manner for embryonic lymphangiogenesis. Importantly, mice deficient in both VEGFR-3 ligands, Vegfc and Vegfd, developed a normal blood vasculature, suggesting VEGF-C- and VEGF-D- independent functions for VEGFR-3 in the early embryo. Platelet-derived growth factor B (PDGF-B) signals via PDGFR-b and regulates formation of blood vessels by recruiting pericytes and smooth muscle cells around nascent endothelial tubes. We showed that PDGF-B fails to induce lymphangiogenesis when overexpressed in adult mouse skin using adenoviral vectors. However, mouse embryos lacking Pdgfb showed abnormal lymphatic vessels, suggesting that PDGF-B plays a role in lymphatic vessel maturation and separation from blood vessels during embryogenesis. Lymphatic vessels play a key role in immune surveillance, fat absorption and maintenance of fluid homeostasis in the body. However, lymphatic vessels are also involved in various diseases, such as lymphedema and tumor metastasis. These studies elucidate the basic mechanisms of embryonic lymphangiogenesis and add to the knowledge of lymphedema and tumor metastasis treatments by giving novel insights into how lymphatic vessel growth could be induced (in lymphedema) or inhibited (in tumor metastasis).

Precise Values of the "C Component Vicinal Coupling Constants for Calculating Side-Chain Conformations in Amino Acids

From consideration of 'H-lH vicinal coupling constants and '"G'H long-range coupling constants in a series of amino acid derivatives, the precise values of uC component vicinal coupling constants have been calculated for the three minimum energy staggered rotamers for the C(or)H-C(P)H, side-chains of amino acids.

Analyzing hysteresis behavior of capacitance-voltage characteristics of IZO/C₆₀/pentacene/Au diodes with a hole-transport electron-blocking polyterpenol layer by electric-field-induced optical second-harmonic generation measurement

By using electric-field-induced optical second-harmonic generation (EFISHG) measurement, we analyzed hysteresis behavior of capacitance-voltage (C-V) characteristics of IZO/polyterpenol (PT)/C₆₀/pentacene/Au diodes, where PT layer is actively working as a hole-transport electron-blocking layer. The EFISHG measurement verified the presence of interface accumulated charges in the diodes, and showed that a space charge electric field from accumulated excess electrons (holes) that remain at the PT/C₆₀ (C₆₀/pentacene) interface is responsible for the hysteresis loop observed in the C-V characteristics.

Reciprocal and advanced generation hybrids between Corymbia citriodora and C-torelliana: forestry breeding and the risk of gene flow

Corymbia F1 hybrids have high potential for plantation forestry; however, little is known of their reproductive biology and potential for genetic pollution of native Corymbia populations. This study aims to quantify the influence of reproductive isolating barriers on the success of novel reciprocal and advanced generation Corymbia hybrids. Two maternal taxa, Corymbia citriodora subsp. citriodora and Corymbia torelliana, were pollinated using five paternal taxa, C. citriodora subsp. citriodora, C. torelliana, one C. torelliana x C. citriodora subsp. citriodora hybrid and two C. torelliana x C. citriodora subsp. variegata hybrids. Pollen tube, embryo and seed development were assessed. Reciprocal hybridisation between C. citriodora subsp. citriodora and C. torelliana was successful. Advanced generation hybrids were also created when C. citriodora subsp. citriodora or C. torelliana females were backcrossed with F1 hybrid taxa. Prezygotic reproductive isolation was identified via reduced pollen tube numbers in the style and reduced numbers of ovules penetrated by pollen tubes. Reproductive isolation was weakest within the C. citriodora subsp. citriodora maternal taxon, with two hybrid backcrosses producing equivalent capsule and seed yields to the intraspecific cross. High hybridising potential was identified between all Corymbia species and F1 taxa studied. This provides opportunities for advanced generation hybrid breeding, allowing desirable traits to be amplified. It also indicates risks of gene flow between plantation and native Corymbia populations.

Novel intramolecular reductive C-C coupling during borohydride reduction of a bi-enone

Borohydride reduction of the bi-enone (1) gave the structurally and mechanistically interesting compounds (4) and (5a-c) resulting from intramolecular carbon-carbon coupling.

Structure of l-Diphenylmethyl-3-hydroxyazetidinium Chloride,* C 16 H lsN O+.CI -

M r=275.8, monoclinic, P21/a, a= 12.356 (5), b=9.054 (4), c= 14.043 (4) A, t= 100.34 (3) ° , V=1545.5A 3, Z=4, D,,,= 1.14, D x = 1.185 Mg m -3, p(Mo Ka, /l = 0.7107 ]k) = 2.77 mm -1, F(000) = 584.0, T= 293 K, R = 0.053 for 1088 reflections. The four-membered ring is buckled 13.0 ° (0= 167.0°). The azetidinium moiety is linked to the C1- ion through a hydrogen bond [O-H...C1 = 3.166 (5) A].

Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana × C. citriodora

A protocol was developed for short-term preservation and distribution of the plantation eucalypt, Corymbia torelliana × C. citriodora, using alginate-encapsulated shoot tips and nodes as synthetic seeds. Effects of sowing medium, auxin concentration, storage temperature and planting substrate on shoot regrowth or conversion into plantlets were assessed for four different clones. High frequencies of shoot regrowth (76–100%) from encapsulated explants were consistently obtained in hormone-free half- and full-strength Murashige and Skoog (MS) sowing media. Conversion into plantlets from synthetic seeds was achieved on half-strength MS medium by treating shoot tips or nodes with 4.9–78.4 μM IBA prior to encapsulation. Pre-treatment with 19.6 μM IBA provided 62–100% conversion, and 95–100% of plantlets survived after acclimatisation under nursery conditions. Synthetic seeds containing explants pre-treated with IBA were stored for 8 weeks much more effectively at 25°C than at 4°C, with regrowth frequencies of 50–84% at 25°C compared with 0–4% at 4°C. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile planting substrates. Highest frequencies (46–90%) of plantlet formation from pre-converted synthetic seeds were obtained by transferring shoot tip-derived synthetic seeds onto an organic compost substrate. These plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds.