The effect of tyrphostin AG-556 on intimal thickening in a mouse model of arterial injury

BACKGROUND: Inflammation has been shown to play an important role in promoting the response to arterial injury and proinflammatory cytokines, such as tumor necrosis factor (TNF) alpha, are candidate mediators. AG-556 is a tyrosine kinase inhibitor proven to be effective in a model of multiple sclerosis-like syndrome in mice due to its immunomodulating effect. In the current study, we investigated the effect of the tyrphostin AG-556 on neointimal thickening and cytokine profile in a model of arterial injury in the mouse. METHODS: Injury was induced by external cuff placement on the left femoral artery of wild-type C57BL/6 mice. AG-556 dissolved in DMSO was injected intraperitoneally daily to the injured mice in a dosage of 2 mg/mouse. Control mice received DMSO injections. Histological analysis was carried out to assess neointimal formation. Splenocytes were cultured in the absence and presence of a mitogen for evaluation of thymidine incorporation and cytokine production. RESULTS: AG-556 treatment significantly attenuated intimal thickening (43,000+/-17,000 microm2; n=11) when compared to DMSO administration (286,000+/-127,000 microm2; n=10; P<0.05). Basal interferon-gamma production by splenocytes from AG-556-treated mice was increased by approximately 20-fold in comparison with levels in DMSO-treated animals, whereas Con-A induced secretion of the cytokine was similar between both groups. Levels of TNF-alpha, IL-4 and IL-10 in the culture supernatant from treated and non-treated animals did not differ significantly. CONCLUSION: The tyrosine kinase inhibitor AG-556 may have a role in the reduction of intimal thickening. The effect could be mediated via an immune modulating effect involving a significant increase in the smooth muscle cell inhibitory cytokine IFN-gamma.     

Biotransformation by plant cells immobilized in cross-linked polyacrylamide-hydrazide

Plant cells were entrapped by mixing suspended MENTHA cells with linear, water soluble polyacrylamide-hydrazide chains followed by the stoichiometric addition of glyoxal as the cross linking agent (PAAH-G entrapment). In parallel, some cells were entrapped in calcium-alginate beads, as previously described. The capability of both immobilized cell systems to reduce monoterpenes was compared with freely suspended MENTHA cells. Entrapment by either alginate or PAAH-G did not impair cell vitality, as observed by fluorescein diacetate staining. Biotransformation of (-) menthone to (+) neomenthol by M-cells and of (+) pulegone to (+) isomenthone by P-cells indicated that the transformation efficiency of the cells entrapped in PAAH-G is as high as that of freely suspended cells. Moreover, the distribution of both precursor and product in the medium versus their content in the cells (or cells contained in gel-beads) showed that less monoterpenes were retained in cells entrapped in PAAH-G, as compared to the freely suspended cells. Thus prolonged incubation (e.g. 24 hr), which usually results in appreciable loss of monoterpenes from the chloroform extract of freely-suspended-cells, caused considerably less loss from the PAAH-G entrapped-cells. In a preliminary test it was shown that PAAH-G entrapped cells were capable to perform three, consecutive, batch-type monoterpene biotransformations, without significant decrease of transformation capability. The capability to immobilize living plant cells within this synthetic chemically crosslinked gel system, combined with the favourable beads/ free-medium ratio of monoterpene distribution, point towards a potential development of a continuous biotransformation process carried out by plant-cells entrapped in this system.     

Biotransformation of monoterpenes by Mentha cell lines

Previous results indicated that all tested cell suspension lines derived from various MENTHA chemotypes were capable to biotransform (-) menthone to (+)-neomentol but only some of these lines converted (+)-pulegone to (+)-isomenthone. In order to quest whether only the natural secondary metabolites or also other compounds with similarities to pulegone are biohydrogenated by MENTHA cell suspensions, we incubated such suspensions with 5 unsaturated alpha-beta; ketones. No conversion was detected when mesityl oxide, trans-6-tert. butyl pulegone or 3-isopropylidine-9-methyl-decalone-2 were incubated with MENTHA cells, while saturation of the alpha-beta double bond of 2-isopropylidine cyclohexanone and of trans-6-methyl pulegone was observed in suspensions of those cell lines which are capable of pulegone transformation. Suspensions of MENTHA cell lines which were incapable to hydrogenate pulegone did not biotransform the two latter pulegone analogues.     

V-region mutation in vitro,in vivo,and in silico reveal the importance of the enzymatic properties of AID and the sequence environment

The somatic hypermutation of Ig variable regions requires the activity of activation-induced cytidine deaminase (AID) which has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) motif hot spots in in vivo and in vitro assays. We compared mutation profiles of in vitro assays for the 3′ flanking intron of VhJ558-Jh4 region to previously reported in vivo profiles for the same region in the Msh2^−/−Ung^−/− mice that lack base excision and mismatch repair. We found that the in vitro and in vivo mutation profiles were highly correlated for the top (nontranscribed) strand, while for the bottom (transcribed) strand the correlation is far lower. We used an in silico model of AID activity to elucidate the relative importance of motif targeting in vivo. We found that the mutation process entails substantial complexity beyond motif targeting, a large part of which is captured in vitro. To elucidate the contribution of the sequence environment to the observed differences between the top and bottom strands, we analyzed intermutational distances. The bottom strand shows an approximately exponential distribution of distances in vivo and in vitro, as expected from a null model. However, the top strand deviates strongly from this distribution in that mutations approximately 50 nucleotides apart are greatly reduced, again both in vivo and in vitro, illustrating an important strand asymmetry. While we have confirmed that AID targeting of hot and cold spots is a key part of the mutation process, our results suggest that the sequence environment plays an equally important role.     

The Nerium oleander aphid Aphis nerii is tolerant to a local isolate of Aphid lethal paralysis virus (ALPV)

In a survey that was conducted during the year 2011, a local strain of Aphid lethal paralysis virus (ALPV) was identified and isolated from a wild population of Aphis nerii aphids living on Nerium oleander plants located in northern Israel. The new strain was tentatively named (ALPV-An). RNA extracted from the viral particles allowed the amplification and determination of the complete genome sequence. The virus genome is comprised of 9835 nucleotides. In a BLAST search analysis, the ALPV-An sequence showed 89 % nucleotide sequence identity with the whole genome of a South African ALPV and 96 and 94 % amino acid sequence identity with the ORF1 and ORF2 of that strain, respectively. In preliminary experiments, spray-applied, purified ALPV virions were highly pathogenic to the green peach aphid Myzus persicae; 95 % mortality was recorded 4 days post-infection. These preliminary results demonstrate the potential of ALPV for use as a biologic agent for some aphid control. Surprisingly, no visible ALPV pathogenic effects, such as morphological changes or paralysis, were observed in the A. nerii aphids infected with ALPV-An. The absence of clear ALPV symptoms in A. nerii led to the formulation of two hypotheses, which were partially examined in this study. The first hypothesis suggest that A. nerii is resistant or tolerant of ALPV, while the second hypothesis propose that ALPV-An may be a mild strain of ALPV. Currently, our results is in favor with the first hypothesis since ALPV-An is cryptic in A. nerii aphids and can be lethal for M. persicae aphids.