Novel regioselective formation of S- and N-hydroxyl-alkyls of 5-(3-chlorobenzo[b]thien-2-yl)-3-mercapto-4H-1,2,4-triazole and a facile synthesis of triazolo-thiazoles and thiazolo-triazoles. Role of catalyst and microwave

Regioselective alkylation of 5-(3-chlorobenzo[b]thien-2-yl)-4H-1,2,4-triazole (1) with hydroxy alkylating agents 2, 3, 13, and the 2,3-O-isopropylidene-1-O-(p-tolylsulfonyl)-glycerol (10) afforded the corresponding S-alkylated derivatives 6, 7, 11, and 14 under both conventional and microwave irradiation conditions; bentonite as a solid support gave better results, with no change in regioselectivity. A facile intramolecular dehydrative ring closure of 6, 7, 11, and 14 using K(2)CO(3) in DMF afforded the corresponding fused triazolo-thiazines and thiazolo-triazole 17-19. The isopropylidenes and acetyl derivatives of the products were prepared.     

Role of transthyretin in thyroxine transfer from cerebrospinal fluid to brain and choroid plexus

The transport of 125I-labeled thyroxine (T4) from the cerebrospinal fluid (CSF) into brain and choroid plexus (CP) was measured in anesthetized rabbit [0.5 mg/kg medetomidine (Domitor) and 10 mg/kg pentobarbitonal sodium (Sagatal) iv] using the ventriculocisternal (V-C) perfusion technique. 125I-labeled T4 contained in artificial CSF was continually perfused into the lateral ventricles for up to 4 h and recovered from the cisterna magna. The %recovery of 125I-labeled T4 from the aCSF was 47.2+/-5.6% (n=10), indicating removal of 125I-labeled T4 from the CSF. The recovery increased to 53.2+/-6.3% (n=4) and 57.8+/-14.8% (n=3), in the presence of 100 and 200 microM unlabeled-T4, respectively (P<0.05), indicating a saturable component to T4 removal from CSF. There was a large accumulation of 125I-labeled T4 in the CP, and this was reduced by 80% in the presence of 200 microM unlabeled T4, showing saturation. In the presence of the thyroid-binding protein transthyretin (TTR), more 125I-labeled T4 was recovered from CSF, indicating that the binding protein acted to retain T4 in CSF. However, 125I-labeled T4 uptake into the ependymal region (ER) of the frontal cortex also increased by 13 times compared with control conditions. Elevation was also seen in the hippocampus (HC) and brain stem. Uptake was significantly inhibited by the presence of endocytosis inhibitors nocodazole and monensin by >50%. These data suggest that the distribution of T4 from CSF into brain and CP is carrier mediated, TTR dependent, and via RME. These results support a role for TTR in the distribution of T4 from CSF into brain sites around the ventricular system, indicating those areas involved in neurogenesis (ER and HC).     

Use of bioluminescence imaging to monitor Campylobacter survival in chicken litter

Aim: The aim of this study was to develop a novel approach for characterizing the growth and persistence of Campylobacter in different poultry‐rearing environments. Specifically, we constructed bioluminescent Campylobacter strains and used them to monitor the survival of these pathogens in litter (bedding) material. Methods and Results: We inserted shuttle plasmids carrying the luminescence genes (luxCDABE) into C. jejuni and C. coli to construct bioluminescent strains of these pathogens. The strains were spiked into microcosms containing samples of litter‐washings and dry litter collected from different enclosures that housed broiler chickens. Our results show that C. jejuni and C. coli survived for at least 20 days in reused (old) litter while the growth of these pathogens was inhibited in clean (new) litter. Furthermore, our results suggest that the availability of nutrients and the condition of the litter (reused vs new) are important factors in the persistence of these pathogens. Conclusions: Reused litter can potentially predispose chickens to Campylobacter contamination and maintaining clean litter might reduce the incidences of colonization with these pathogens. Significance and Impact of the Study: Bioluminescence provided a simple, sensitive, and rapid approach for analysing the growth dynamics of Campylobacter. Using this technology, we highlighted the potential role of litter material in maintaining these pathogens in the chicken environment.     

Derivation and characterisation of hESC lines from supernumerary embryos, experience from Odense, Denmark

The derivation and characterisation of human embryonic stem cells provides a source of pluripotent stem cells with potential for clinical applications. Utilising locally sourced embryos from two IVF clinics, we derived and characterised five new cell lines for use in a non-clinical setting. Analysis of clinical data showed that the majority of embryos (94.5%) failed to reach the blastocyst stage of development and of all embryos, regardless of developmental status, 248 embryos were needed to create one stem cell line. From the number of embryos (69) which developed to the blastocyst stage 8.7% developed into cell lines. Using outgrowth of the whole blastocyst, we derived five new, unreported cell lines in Odense, Denmark between 2005 and 2006. Characterisation was carried out using RT-PCR, staining, karyotyping, EB formation and teratoma formation. The KMEB hESC lines will, in the future, be made available through the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk/).     

Aphids secrete watery saliva into plant tissues from the onset of stylet penetration

Aphid feeding requires the secretion of two types of saliva: gelling saliva (from the principal gland) that forms an intercellular sheath for the penetrating stylet, and watery saliva [from accessory salivary glands (ASGs)] that facilitates intracellular penetration and phloem feeding. Plant viruses can be used as salivary markers to investigate key steps in aphid feeding, and penetration can be monitored electrically using the electrical penetration graph (EPG) approach. We conducted a series of EPG‐controlled transmission experiments using Cucurbit aphid‐borne yellows virus [CABYV; Polerovirus spec. (Luteoviridae)], which is retained in the ASGs, as a marker for watery saliva secretions. The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), was used as a vector and melon seedlings, Cucumis melo L. (Cucurbitaceae), as host plants. Viruliferous aphids were interrupted at various stages during stylet penetration, i.e., during intercellular penetration prior to intracellular puncture and following a potential drop within the first probe. Viruliferous aphids and leaf disc samples obtained from the stylet penetration site were used to detect CABYV by quantitative real‐time RT‐PCR. Approximately half of the inoculated leaf discs were found to be infected with CABYV after very brief (12.9 ± 1.9 s) intercellular stylet probes and before intracellular stylet puncture. The number of virus particles ejected during such probes was similar to the number ejected by aphids during longer probes including a single intracellular puncture. Our results therefore suggest that watery saliva is secreted by aphids from the onset of stylet penetration.     

β cell membrane remodelling and procoagulant events occur in inflammation‐driven insulin impairment: a GLP‐1 receptor dependent and independent control

Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell‐derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon‐like peptide (GLP)‐1 analogue, is known to promote insulin secretion and β‐cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin‐m5f β‐cell function, TF activity mediated by MPs and their modulation by 1 μM liraglutide were examined in a cell cross‐talk model. Methyl‐β‐cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N‐éthylmaleimide‐sensitive‐factor Attachment protein Receptor (SNARE)‐dependent exocytosis. Cytokines induced a two‐fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two‐fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD‐treated cells showed similar patterns. Cells pre‐treated by saturating concentration of the GLP‐1r antagonist exendin (9‐39), showed a partial abolishment of the liraglutide‐driven insulin secretion and liraglutide‐decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP‐1r‐dependent and ‐independent pathways. Our results confirm an integrative β‐cell response to GLP‐1 that targets receptor‐mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation‐driven procoagulant events.     

Diterpenoids from Euphorbia paralias.

Two segatane diterpenoids containing a bicyclic [4,3,1] ring system were isolated, together with 11 known diterpenoids, four segetanes, five jatrophanes and two paralianes, from the aerial parts of Euphorbia paralias. The structures of the new compounds were established by spectroscopic means, including by 2D NMR and CD spectroscopic analyses. The antifeedant and antiviral activities of the isolated compounds are also described.     

Uptake of methionine sulfoximine by some N2 fixing bacteria, and its effect on ammonium transport

The N2 fixing bacteria Klebsiella pneumoniae, Azospirillum brasilense, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum, but not Azotobacter vinelandii accumulate the glutamine analogue methionine sulfoximine in the cell. In the accumulating cells methionine sulfoximine inhibits ammonium transport. Accumulation and inhibition are prevented by glutamine.