Effect of exogenous hormones on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L.

Inclusion of IAA in the vase water had little effect on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L. while kinetin delayed leaf yellowing at 10^-4M (continuous treatment). Chlorophyll was effectively retained by 10^-7M gibberellic acid (GA) in the vase water or by a 20h pulse at 5°C with 10^-5/10^-4M GA. After 16h of ¹⁴C-GA, uptake at 20°C relatively high levels of ¹⁴C were found in leaves and low levels in stems and flowers. After this treatment about half of the ¹⁴C-GA, in leaves was metabolized into unknown compounds. Corrigendum. Owing to an error in the proofreading process, the article was published incorrectly. The article as it should have been published is presented here.     

Synthesis of 5-Fluoro-1-(3-O-benzoyl-4, 6-dideoxy- β-L-GLYCERO-hex-3-enopyranos-2-ulosyl)uracil

Abstract

Synthesis of the title compound, an unsaturated ketohexo-pyranosyl nucleoside of 5-fluorouracil is reported. It was prepared by oxidation of the corresponding dibenzoylhexopyranosyl nucleoside with pyridinium dichromate/molecular sieves system.     

An Enhanced Heterologous Virus-Like Particle for Human Papillomavirus Type 16 Tumour Immunotherapy

Cervical cancer is caused by high-risk, cancer-causing human papillomaviruses (HPV) and is the second highest cause of cancer deaths in women globally. The majority of cervical cancers express well-characterized HPV oncogenes, which are potential targets for immunotherapeutic vaccination. Here we develop a rabbit haemorrhagic disease virus (RHDV) virus-like particle (VLP)-based vaccine designed for immunotherapy against HPV16 positive tumours. An RHDV-VLP, modified to contain the universal helper T cell epitope PADRE and decorated with an MHC I-restricted peptide (aa 48–57) from the HPV16 E6, was tested for its immunotherapeutic efficacy against the TC-1 HPV16 E6 and E7-expressing tumour in mice. The E6-RHDV-VLP-PADRE was administered therapeutically for the treatment of a pre-existing TC-1 tumour and was delivered with antibodies either to deplete regulatory T cells (anti-CD25) or to block T cell suppression mediated through CTLA-4. As a result, the tumour burden was reduced by around 50% and the median survival time of mice to the humane endpoint was almost doubled the compared to controls. The incorporation of PADRE into the RHDV-VLP was necessary for an E6-specific enhancement of the anti-tumour response and the co-administration of the immune modifying antibodies contributed to the overall efficacy of the immunotherapy. The E6-RHDV-VLP-PADRE shows immunotherapeutic efficacy, prolonging survival for HPV tumour-bearing mice. This was enhanced by the systemic administration of immune-modifying antibodies that are commercially available for use in humans. There is potential to further modify these particles for even greater efficacy in the path to development of an immunotherapeutic treatment for HPV precancerous and cancer stages.     

Efficient routes to epimerically-pure side-chain derivatives of lanosterol

A technically simple route is described to individual epimers of side-chain derivatives of lanosterol (3beta-hydroxy-5alpha-lanosta-8,24-diene). Epimerically pure 24,25-epoxy-, 24,25-dihydroxy- and 24-bromo-25-hydroxy-lanosterol have been prepared in good yield from commercial (50-60%) lanosterol. Hypophosphorous acid was used as a catalyst for the cohalogenation of the Delta24(25) bond and also for the efficient conversion of 24,25-epoxy- and 24-bromo-25-hydroxylanosterol to epimerically pure 24(R) or 24(S)-24,25-dihydroxylanosterols.     

A low-toxicity method for the separation of lanosterol and dihydrolanosterol from commercial mixtures

We describe an inexpensive, low-toxicity and high-yielding method for the production of pure lanosterol and dihydrolanosterol from the commercially available mixture. Optimum conditions are presented for the one-pot production of the intermediate 24,25 vicinal diol of lanosterol acetate (via either epoxidation or hydroxyhalogenation) which is readily separated from the unreacted dihydrolanosterol acetate. The lanosterol diol can then be converted to pure (>97%) lanosterol. Hypophosphorous acid was used for both the conversion of the epoxide to the diol, and as a catalyst for the hydroxyhalogenation by N-halosuccinimides of the olefinic bond.     

Platelet-associated NAD(P)H oxidase contributes to the thrombogenic phenotype induced by hypercholesterolemia

Elevated cholesterol levels promote proinflammatory and prothrombogenic responses in venules and impaired endothelium-dependent arteriolar dilation. Although NAD(P)H oxidase-derived superoxide has been implicated in the altered vascular responses to hypercholesterolemia, it remains unclear whether this oxidative pathway mediates the associated arteriolar dysfunction and platelet adhesion in venules. Platelet and leukocyte adhesion in cremasteric postcapillary venules and arteriolar dilation responses to acetylcholine were monitored in wild-type (WT), Cu,Zn-superoxide dismutase transgenic (SOD-TgN), and NAD(P)H oxidase-knockout (gp91(phox-/-)) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. HC elicited increased platelet and leukocyte adhesion in WT mice versus ND. Cytosolic subunits of NAD(P)H oxidase (p47phox and p67phox) were expressed in platelets. This was not altered by hypercholesterolemia; however, platelets and leukocytes from HC mice exhibited elevated generation of reactive oxygen species compared to ND mice. Hypercholesterolemia-induced leukocyte recruitment was attenuated in SOD-TgN-HC and gp91(phox-/-)-HC mice. Recruitment of platelets derived from WT-HC mice in venules of SOD-TgN-HC or gp91(phox-/-)-HC recipients was comparable to ND levels. Adhesion of SOD-TgN-HC platelets paralleled the leukocyte response and was attenuated in SOD-TgN-HC recipients, but not in WT-HC recipients. However, gp91(phox-/-)-HC platelets exhibited low levels of adhesion comparable to those of WT-ND in both hypercholesterolemic gp91(phox-/-) and WT recipients. Arteriolar dysfunction was evident in WT-HC mice, compared to WT-ND. Overexpression of SOD or, to a lesser extent, gp91(phox) deficiency restored arteriolar vasorelaxation responses toward WT-ND levels. These findings reveal a novel role for platelet-associated NAD(P)H oxidase in producing the thrombogenic phenotype in hypercholesterolemia and demonstrate that NAD(P)H oxidase-derived superoxide mediates the HC-induced arteriolar dysfunction.     

ATM protein kinase mediates full activation of Akt and regulates glucose transporter 4 translocation by insulin in muscle cells

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. Patients with A-T also have high incidences of type 2 diabetes mellitus. The gene mutated in this disease, ATM (A-T, mutated), encodes a protein kinase. Previous studies have demonstrated that cytoplasmic ATM is an insulin-responsive protein and a major upstream activator of Akt following insulin treatment. To further investigate the function of ATM in insulin signal transduction, insulin resistance was induced in rats by feeding them a high-fat diet. Muscle tissue of rats with insulin resistance had both dramatically reduced ATM levels and substantially decreased Akt phosphorylation at Ser473 in comparison to that of regular chow-fed controls. The decreased ATM expression suggests that ATM is involved in the development of insulin resistance through down-regulation of Akt activity. The role of ATM in activation of Akt was further confirmed in mouse embryonic fibroblast (MEF) A29 (ATM+/+) and A38 (ATM-/-) cells. In addition, insulin-mediated Akt phosphorylation in mouse L6 muscle cells was greatly reduced by KU-55933, a specific inhibitor of ATM. A 2-deoxyglucose incorporation assay showed that this inhibitor also caused a significant reduction in insulin-mediated glucose uptake in L6 cells. An immunofluorescence experiment demonstrated that in L6 cells transfected with wild-type (WT) ATM, insulin caused a dramatic increase of the cell surface glucose transporter 4 (GLUT4), while in cells transfected with kinase-dead (KD) ATM, translocation of GLUT4 to the cell surface in response to insulin was markedly inhibited.     

Coating and selective deposition of nanofilm on silicone rubber for cell adhesion and growth

A recently developed method for surface modification, layer-by-layer (LbL) assembly, has been applied to silicone, and its ability to encourage endothelial cell growth and control cell growth patterns has been examined. The surfaces studied consisted of a precursor, with alternating cationic polyethyleneimine (PEI) and anionic sodium polystyrene sulfonate (PSS) layers followed by alternating gelatin and poly-d-lysine (PDL) layers. Film growth increased linearly with the number of layers. Each PSS/PEI bilayer was 3 nm thick, and each gelatin/PDL bilayer was 5 nm thick. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. The contact angle was primarily dictated by the outermost layer. Of the coatings studied, gelatin was the most hydrophilic. A film of (PSS/PEI)₄/(gelatin/PDL)₄/ gelatin was highly favorable for cell adhesion and growth, in contrast to films of (PSS/PEI)₈ or (PSS/PEI)₈/PSS. Cell growth patterns were successfully controlled by selective deposition of microspheres on silicone rubber, using microcontact printing with a silicone stamp. Cell adhesion was confined to the region of microsphere deposition. These results demonstrate that the LbL self-assembly technique provides a general approach to coat and selectively deposit films with nanometer thickness on silicone rubber. Furthermore, they show that this method is a viable technique for controlling cellular adhesion and growth.     

The Effect of Short-Term H2S Fumigation on Nitrate Reductase Activity in Spinach Leaves

Short-term exposure of spinach plants to 250 ppb H₂S at a photonfluence rate of 35µmol m^–2s^–1 (within the400–700 nm range) in the ambient air did not affect invitro nitrate reductase activity (NRA) in the leaves. Likewise,H₂S exposure did not significantly affect in vivo NRA measuredunder anaerobic conditions. In vivo NRA of untreated plantswas apparently inhibited in the presence of oxygen. However,shortterm H₂S exposure increased in vivo "aerobic" NRA up tofive fold of that of untreated plants. H₂S induced increaseof in vivo "aerobic" NRA depended on the sulfide concentration.After 24 hours of exposure maximal increase (two to five fold)of in vivo NRA "aerobic" was observed at 220 ppb H₂S. It isproposed that H₂S inhibited NADH oxidizing enzymes, which resultedin an increase in NADH supply to nitrate reductase (NR) in thepresence of oxygen. It was unlikely that the increase in invivo "aerobic" NRA in sulfide exposed plants was due to an alteredcompetition between mitochondrial respiration and NR since leafrespiration was not affected by an exposure to 250 ppb H₂S (Received February 12, 1986; Accepted June 27, 1986)