Design and synthesis of reagents for phage display screening of dehalogenases

Bifunctional molecules containing both a biotin and a substrate unit have been designed and synthesized for phage display screening of mutant libraries of haloalkane dehalogenase enzymes. The molecules were assembled using a convergent modular synthetic strategy. One molecule was synthesized to evaluate the concept of covalent capture and a second for screening of phage libraries for enantioselectivity.     

Insulin‐like growth factor axis targeting in cancer and tumour angiogenesis – the missing link

Numerous molecular players in the process of tumour angiogenesis have been shown to offer potential for therapeutic targeting. Initially denoted to be involved in malignant transformation and tumour progression, the insulin‐like growth factor (IGF) signalling axis has been subject to therapeutic interference, albeit with limited clinical success. More recently, IGFs and their receptors have received attention for their contribution to tumour angiogenesis, which offers novel therapeutic opportunities. Here we review the contribution of this signalling axis to tumour angiogenesis, the mechanisms of resistance to therapy and the interplay with other pro‐angiogenic pathways, to offer insight in the renewed interest in the application of IGF axis targeting agents in anti‐cancer combination therapies.     

Photosynthesis,water use efficiency,and δ<Superscript>13</Superscript>C in two rice genotypes with contrasting response to water deficit

The effects of water deficit and re-irrigation were studied in glasshouse-grown rice plants (cvs. Cimarrón and Fonaiap 2000) which differ in their susceptibility to water deficit. Relative water content decreased from >90 to 67–69 % and recovered to pre-stress values within 24 h after re-irrigation. The irradiance-saturated rate of photosynthesis (P _sat), transpiration rate (E), and stomatal conductance (g _s) decreased with water deficit. E and g _s decreased similarly in both cultivars, but P _sat was more strongly inhibited in Cimarrón than in Fonaiap 2000. Water deficit increased water use efficiency (WUE_T) over 2-fold in Fonaiap 2000 and by 1.5-fold in Cimarrón. The ratio of intercellular to ambient CO₂ concentration (C _i/C _a) decreased in Fonaiap 2000 during mild stress but increased at severe stress. Contrarily, Cimarrón did not change C _i/C _a with water deficit. After re-irrigation Fonaiap 2000 recovered P _sat to ca. 80 % of control values 24 h after re-irrigation, whereas Cimarrón recovered to 60 % of control values 48 h after re-irrigation. E and g _s recovered to a lesser extent (50 %) than P _sat, after 48 h of re-irrigation in both cultivars. Total aboveground and green (live) biomass were unaffected by water deficit in Fonaiap 2000 but were reduced by 21 and 40 % in Cimarrón, respectively. Dead biomass increased in stressed plants of both cultivars but to a larger extent in Cimarrón than in Fonaiap 2000. Water deficit increased δ¹³C in Fonaiap 2000, whereas Cimarrón was unaffected by water deficit showing lower values than those of Fonaiap 2000. δ¹³C was highly and linearly correlated to the ratio C _i/C _a. WUE_T was also significantly correlated to δ¹³C.     

New acylated triterpenoid saponins from Maesa lanceolata

Ten new acylated triterpenoid saponins were isolated from the leaves of Maesa lanceolata. For their structure elucidation extensive use was made of homo- and heteronuclear 2D NMR techniques such as COSY, NOESY, HSQC and HMBC. All saponins identified contained the same tetraglycosidic side chain, but the triterpenoid moiety showed a variable esterification pattern. Monoester, diester and triester derivatives were present. Maesasaponin I was a 21-monoester derivative, i.e. ?3 beta-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl- (1-->3)]-[beta-D-galactopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl+ ++?-21 beta-angeloyloxy-13 beta, 28-oxidoolean-16 alpha, 22 alpha, 28 alpha-triol. Maesasaponins III, IV3, V3 and VI2 had an additional acetyl, propanoyl, n-butanoyl and angeloyl substituent, respectively, in position 22. Maesasaponins II, IV2, V2, VI3 and VII1 were characterised as the 16-acetyl derivatives of maesasaponins I, III, IV3, V3 and VI2, respectively. Structures of saponins previously reported in M. lanceolata had to be revised.     

Selective requirement for CD40-CD154 in drug-induced type 1 versus type 2 responses to trinitrophenyl-ovalbumin

CD154 is transiently expressed by activated T cells and interacts with CD40 on B cells, dendritic cells, macrophages, and monocytes. This costimulatory receptor-ligand couple seems decisive in Ag-driven immune responses but may be differentially involved in type 1 vs type 2 responses. We studied the importance of CD40-CD154 in both responses using the reporter Ag popliteal lymph node assay in which selectively acting drugs generate clearly polarized type 1 (streptozotocin) or type 2 (D-penicillamine, diphenylhydantoin) responses to a constant coinjected Ag in the same mouse strain. Treatment of mice with anti-CD154 reduced characteristic immunological parameters in type 2 responses (B and CD4(+) T cell proliferation, IgG1 and IgE Abs, and IL-4 secretion) and only slightly affected the type 1 response (small decrease in IFN-gamma production, influx of CD11c(+) and F4/80(+) cells, and prevention of architectural disruption of the lymph node, but no effect on IgG2a Ab and TNF-alpha secretion or B and CD4(+) T cell proliferation). The findings indicate that the CD40-CD154 costimulatory interaction is a prerequisite in drug-induced type 2 responses and is only marginally involved in type 1 responses. The observed expression patterns of CD80 and CD86 on different APC (B cells in type 2 and dendritic cells in type 1) may be responsible for this discrepancy.     

Characterisation of new oligoglycosidic compounds in two Chinese medicinal herbs

A series of caffeic acid derivatives (3,5-dicaffeoyl-quinic acid, 3,4-dicaffeoyl-quinic acid, and 4,5-dicaffeoyl-quinic acid), and the new compound beta,3,4-trihydroxyphenethyl-O-[beta-apiofuranosyl-(1-->4)-alpha- rhamnopyranosyl-(1-->3)]-(4-O-caffeoyl)-beta-glucopyranoside (wedelosin), as well as three known flavonoid glycosides (quercetin 3-O-beta-glucoside, kaempferol 3-O-beta-apiosyl-(1-2)-beta-glucoside, and astragalin or kaempferol 3-O-beta-glucoside) were isolated from the Chinese medicinal herb Wedelia chinensis. Wedelosin showed an inhibitory activity on both the classical and the alternative activation pathway of the complement system. Another Chinese medicinal herb, Kyllinga brevifolia, yielded two known flavonoid glycosides [kaempferol 3-O-beta-apiosyl-(1-2)-beta-glucoside and isorhamnetin 3-O-beta-apiosyl-(1-2)-beta-glucoside], and a new quercetin triglycoside [quercetin 3-O-beta-apiofuranosyl-(1-->2)-beta-glucopyranoside 7-O-alpha-rhamnopyranoside]. The latter compound showed a moderate anti-viral activity.     

New 2-substituted 1,2,3,4-tetrahydrobenzofuro[3,2-c]pyridine having highly active and potent central alpha 2-antagonistic activity as potential antidepressants

The synthesis and biological activity of a series of benzofuro[3,2-c]pyridines and a benzothieno[3,2-c]pyridine are described. These compounds exhibit high affinity for the alpha 2-adrenoceptor, with high selectivity versus the alpha 1-receptor. Compound 1 also shows potent in vivo central activity and has been selected for further biological and clinical evaluation.     

Antigen 84, an effector of pleiomorphism in Mycobacterium smegmatis

While in most rod-shaped bacteria, morphology is based on MreB-like proteins that form an actin-like cytoskeletal scaffold for cell wall biosynthesis, the factors that determine the more flexible rod-like shape in actinobacteria such as Mycobacterium species are unknown. Here we show that a Mycobacterium smegmatis protein homologous to eubacterial DivIVA-like proteins, including M. tuberculosis antigen 84 (Ag84), localized symmetrically to centers of peptidoglycan biosynthesis at the poles and septa. Controlled gene disruption experiments indicated that the gene encoding Ag84, wag31, was essential; when overexpressed, cells became longer and wider, with Ag84 asymmetrically distributed at one pole. Many became grossly enlarged, bowling-pin-shaped cells having up to 80-fold-increased volume. In these cells, Ag84 accumulated predominantly at a bulbous pole that was apparently generated by uncontrolled cell wall expansion. In some cells, Ag84 was associated with exceptional sites of cell wall expansion (buds) that evolved into branches. M. bovis BCG Ag84 was able to form oligomers in vitro, perhaps reflecting its superstructure in vivo. These data suggested a role for Ag84 in cell division and modulating cell shape in pleiomorphic actinobacteria.     

Methotrexate-induced mucositis in mucin 2-deficient mice

The mucin Muc2 or Mycin2 (Muc2), which is the main structural component of the protective mucus layer, has shown to be upregulated during chemotherapy-induced mucositis. As Muc2 has shown to have protective capacities, upregulation of Muc2 may be a counter reaction of the intestine protecting against mucositis. Therefore, increasing Muc2 protein levels could be a therapeutic target in mucositis prevention or reduction. Our aim was to determine the role of Muc2 in chemotherapy-induced mucositis. Mucositis was induced in Muc2 knockout (Muc2(-/-)) and wild type (Muc2(+/+)) mice by injecting methotrexate (MTX). Animals were weighed and sacrificed on Days 2-6 after MTX treatment and jejunal segments were analyzed. Before MTX treatment, the small intestine of Muc2(+/+) and Muc2(-/-) mice were similar with respect to epithelial morphology and proliferation. Moreover, sucrase-isomaltase and trefoil factor-3 protein expression levels were comparable between Muc2(+/+) and Muc2(-/-) mice. Up to Day 3 after MTX treatment, percentages of weight-loss did not differ. Thereafter, Muc2(+/+) mice showed a trend towards regaining weight, whereas Muc2(-/-) mice continued to lose weight. Surprisingly, MTX-induced intestinal damage of Muc2(-/-) and Muc2(+/+) mice was comparable. Prior to MTX-injection, tumor necrosis factor-alpha and interleukin-10 mRNAs were upregulated in Muc2(-/-) mice, probably due to continuous exposure of the intestine to luminal antigens. Muc2 deficiency does not lead to an increase in chemotherapy-induced mucositis. A possible explanation is the mechanism by which Muc2 deficiency may trigger the immune system to release interleukin-10, an anti-inflammatory cytokine before MTX-treatment.