Intra-colonic administration of the TLR7 agonist R-848 induces an acute local and systemic inflammation in mice

Imidazoquinoline compounds, such as resiquimod (R-848), are well known topically active immune modifiers that bind to toll-like receptor 7 (TLR7). The aim of this study was to characterize the R-848 induced inflammatory response in mice and to validate the response using methyl-prednisolone and anti-TNF antibody.Intra-colonic application of R-848 to BALB/c mice induced a systemic transient elevation of TNF, CXCL1, IL-6, and IL-12p40 and a colonic elevation of cytokines/chemokines and iNOS, without infiltration of immune cells or epithelial destruction. Treatment with methyl-prednisolone or anti-TNF antibody attenuated the systemic (TNF, IL-6, IL-12p40, and CXCL1) and local (colonic TNF and iNOS mRNA expression) response induced by R-848.In summary, intra-colonic administration of R-848 induces an acute systemic and local inflammatory response, which can be attenuated by steroids or anti-TNF antibody. We suggest that the R-848 inflammatory model can be useful in future validation of new drugs for gastrointestinal inflammatory conditions.     

Serotonin localization in Phallusia mammillata larvae and effects of 5-HT antagonists during larval development

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an important role in a wide range of non-neural processes. Using immunofluorescence with an antiserotonin antibody, 5-HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5-HT on development, we treated embryos with two different 5-HT receptor subtype antagonists. Treatment at the gastrula stage with 10 microM ondansetron, an antagonist of the 5-HT(3) receptor, induced anterior truncation and a short tail. At 10 microM, ritanserin, a 5-HT(2B) receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5-HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5-HT is suggested.     

Ericoid mycorrhizal fungi: some new perspectives on old acquaintances

Many ericaceous species colonize as pioneer plants substrates ranging from arid sandy soils to moist mor humus, in association with their mycorrhizal fungi. Thanks to the symbiosis with ericoid mycorrhizal fungi, ericaceous plants are also able to grow in highly polluted environments, where metal ions can reach toxic levels in the soil substrate. For a long time this mycorrhizal type has been regarded as an example of a highly specific interaction between plants and fungi. More recent studies have been challenging this view because some ericoid mycorrhizal endophytes seem also able to colonise plants from very distant taxa. A molecular approach has allowed the investigation of genetic diversity and molecular ecology of ericoid mycorrhizal fungi, and has revealed that ericaceous plants can be very promiscuous, with multiple occupancy of their thin roots. The molecular analysis of sterile morphotypes involved in this symbiosis has also led to deeper understanding of the species diversity of ericoid fungi. Genetic polymorphism of ericoid fungi is wider than previously thought, and often increased by the presence of Group I introns in the nuclear small subunit rDNA.     

High-yield method for isolation and culture of endothelial cells from rat coronary blood vessels suitable for analysis of intracellular calcium and nitric oxide biosynthetic pathways

We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This method makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.     

Enzymatic conversions of polar lipids. Principles, problems and solutions

This text provides a brief overview of the principles of enzymatic lipid conversion and some recent advances in the enzymatic conversion of glycerophospholipids and galactolipids. Lipases and phospholipases are used to exchange fatty acids or the polar group in the lipids. The reactions can be carried out either as hydrolysis–esterification sequences or as one-step transferase reactions. The scope and limitations of the different methods are discussed.     

Statistical modelling of insect behavioural responses in relation to the chemical composition of test extracts

Abstract. The use of generalized linear models (GLM) for relating changes in insect behaviour to changes in the chemical composition of a plant extract is presented and applied to data from an experimental study of the olfactory response of Cydia pomonella L. (Lepidoptera: Tortricidae) to apple volatiles. The volatiles were collected from healthy apples, artificially damaged apples or apples infested with C. pomonella larvae (either instar I, IV or V). These treatments produced a blend of 23 major components and the chemical composition of the blends differed substantially amongst the treatments.
A statistically significant relationship was found between the concentration of hexyl hexanoate and 2-methylbutyl acetate in each extract and the number of moths moving upwind. Statistically significant models were developed which suggested that a relationship exists between the concentration of Z , E -α-farnesene, hexyl hexanoate and 2-methylbutyl acetate and the number and duration of movements made by the moths.
Subsequently Y-tube assays were carried out to validate the predictions made with respect to the orientation of mated female C. pomonella . The results of these assays confirm hexyl hexanoate as an attractant. There were indications that 2-methylbutyl acetate acted as a repellent although differences were not statistically significant. Previous bioassays have shown that C. pomonella displays a statistically significant negative linear dose–response to α-farnesene ( Hern & Dorn, 1999 ).
The statistical methods employed are very flexible and fairly easy to implement, offering the potential to screen plant extracts for bioactive compounds with a minimum of biological constraints. Their general applicability has yet to be demonstrated and as such these analyses only offer evidence of statistical relationships; the results must be validated by additional bioassays before conclusions can be drawn.     

Cloning, expression and chromosomal location of NKX6B to 10q26, a region frequently deleted in brain tumors

Nkx6-2 (former Gtx) is a murine-homeobox-containing gene localized distally on Chromosome (Chr) 7. Analysis of the expression pattern, together with DNA binding assays, suggests that this gene product might be important for differentiated oligodendrocyte function and in the regulation of myelin gene expression. We now report on the cloning and characterization of the human homolog (NKX6B). DNA sequence analysis of an 11-kb genomic fragment revealed that the complete human gene spans 1.2 kb and is composed of three exons. NKX6B is predicted to encode a polypeptide of 277 amino acids with 97% identity to mouse Nkx6-2. Northern blot experiments showed that NKX6B expression is tightly controlled in a tissue-specific fashion with the highest site of expression being the brain. Finally, using STS content mapping and RH analyis, we demonstrated that NKX6B maps to the 10q26, a region where frequent loss of heterozygosity has been observed in various malignant brain tumors. These results may implicate NKX6B as a candidate tumor suppressor gene for brain tumors, particularly for oligodendrogliomas. Received: 14 June 2000 / Accepted: 21 September 2000     

Synthesis and preliminary biological characterization of a new potential I-Radioligand for dopamine and serotonin receptors

The synthesis and a preliminary biological characterization of a new class of N-benzyl-aminoalcohols which have serotonin (5-HT₂) and dopamine (D₂) receptor affinity is described. In vitro competition binding studies were conducted with the new molecules and ³H-spiperone on crude membrane preparation from rat striatum and frontal cortex. One of these compounds, 3-benzylamino-1-(4-fluoro-2-iodophenyl)-propan-1-ol (6f), whose IC₅₀ values are in the micromolar range for both the D₂ and 5-HT₂ receptors, was prepared in iodine-125 labelled form (6i) by nucleophilic substitution of the bromine atom of 3-benzylamino-1-(2-bromo-4-fluorophenyl)-propan-1-ol (6d). In the in vivo studies, conducted on rats, the radiolabelled molecule 6i shows a good capacity to cross the blood–brain barrier (BBB) with a mean value of first pass cerebral extraction (E) of ca. 50% when the regional cerebral blood flow, measured with microsphere technique, is in the experimental animal's physiologic range (0.8–1 mL/min/g). A preliminary in vitro autoradiographic distribution on coronal rat brain slices of the radioiodinated molecule showed that it was preferentially localized in the striatum and in the cerebral regions rich in dopamine- and serotonin receptors, even if a high non-specific binding was observed.     

Cloning, sequencing and mutational analysis of the cytochrome c552 gene (cycB) from Bradyrhizobium japonicum strain 110

We report the cloning and nucleotide sequence analysis of the cytochrome c552 gene (cycB) of Bradyrhizobium japonicum strain 110. The gene was identified with help of an oligonucleotide that was designed on the basis of the amino acid sequence determined for purified cytochrome c552 of B. japonicum strain CC705. The cycB gene product has an N-terminal 23-amino acid signal peptide that is missing in the mature cytochrome c552 protein. A B. japonicum cycB insertion mutant was constructed which had no observable phenotypic defects in denitrification and symbiotic nitrogen fixation. Thus, the function of c552 remains unknown.