A yeast artificial chromosome contig containing the complete Duchenne muscular dystrophy gene.

A contig of 36 overlapping yeast artificial chromosome (YAC) clones has been constructed for the complete Duchenne muscular dystrophy (DMD) gene in Xp21. The YACs were isolated from a human 48,XXXX YAC library using the DMD cDNA and brain promoter fragments as hybridization probes. The YAC clones were characterized for exon content using HindIII or EcoRI digests, hybridization of individual DMD cDNA probes, and polymerase chain reaction (PCR) amplification of specific exons near the 5' end of the gene. For comparison to the known long-range restriction map of the DMD gene, YAC clones were digested with SfiI and hybridized with DMD cDNA probes. The combined analysis of the exon content and the SfiI map allowed an approximately 3.2-Mb YAC contig to be constructed. The complete 2.4-Mb DMD gene could be represented in a minimum set of 7 overlapping YAC clones.     

Construction of yeast artificial chromosome libraries by pulsed-field gel electrophoresis

Yeast artificial chromosome (YAC) libraries have been constructed from a variety of organisms using different approaches. This protocol outlines in detail the construction of YAC libraries with large inserts using size fractionation of partially digested DNA by pulsed-field gel electrophoresis.     

High-dose gamma-irradiation enhance expression of transgene under control of immediate-early CMV promoter in stably transfected tumor cells

Gamma-irradiation is a usual method to inactivate whole-cellular anticancer vaccines consisting viable tumor cells. To evaluate the effect of gamma-irradiation to transgene expression in tumor cells we constructed several stably transfected clones of human and mouse cell lines expressing transgenic GM-CSF or GFP under control of IE-CMV promoter. Irradiation of those cells with different doses (ranged from 20 to 100 Gr) of gamma-radiation caused loss of proliferation capacity with survival of the cells population clearly depended on irradiation dose. Cell-cycle staining reveals accumulation of the cells with G2/M DNA content and almost loss of cells in S-phase. Substantial proportion of irradiated cells shows beta-galactosidase activity and morphological changes associated with cell senescence. An irradiated cell shows no changes in the level of mitochondrial dehydrogenase activity regardless irradiation dose exposed. Irradiated cells retain their ability to express transgene. Moreover, amount of the secreted GM-CSF as well as MFI in GFP-expressing cells significantly increases after gamma-irradiation up to 10 fold for cells exposed with 100 Gr. Enhancing of the transgene expression in both human and mouse cells positively correlates with total dose of gamma-irradiation gained by the cells and demonstrates gradual nature. Overall, our results supports using of 100 Gr of gamma-irradiation as the optimal dose for whole-cell anticancer vaccine inactivation.     

Fosmidomycin analogues as inhibitors of monoterpenoid indole alkaloid production in Catharanthus roseus cells

Substituted 3-[2-(diethoxyphosphoryl)propyl]oxazolo[4,5-b]pyridine-2(3H)-ones were obtained by functionalization at 6-position with various substituents (aryl, vinyl, carbonyl chains) via reactions catalysed with palladium. We found that these new fosmidomycin analogues inhibited the accumulation of ajmalicine, a marker of monoterpenoid indole alkaloids production in plant cells. Some of them have greater inhibitory effect than fosmidomycin and fully inhibit alkaloid accumulation at the concentration of 100 microM.     

Extracorporeal photochemotherapy reduces the severity of Lewis rat experimental allergic encephalomyelitis through a modulation of the function of peripheral blood mononuclear cells

Extra corporeal photochemotherapy (ECP) is an immunomodulating procedure used in several nonneurological diseases which, similarly to multiple sclerosis, are likely to be due to T-cell-mediated autoimmunity and it is probable that ECP can modulate the normal activity of peripheral blood mononuclear cells (PBMC). Using the Lewis rat experimental allergic encephalomyelitis (EAE) model of human multiple sclerosis (MS) we examined the effect of extracorporeal UV-A irradiation on psoralen-activated PBMC. In our experiment the comparison between the two groups of animals (ECP or sham-treatment) evidenced that the ECP treatment reduced the severity of EAE on clinical grounds and this result was confirmed by the pathological examination. The changes in the titers of anti-myelin antigen antibodies typical of EAE were also modulated by the procedure. Ex vivo examination evidenced a significant reduction in tumor-necrosis factor-alpha (TNF-alpha) released by PBMC after lipopolysaccharides (LPS) stimulation in culture. We conclude that ECP modifies the normal activity of PBMC during the course of EAE and it is possible that one of the anti-inflammatory mechanisms of action of ECP is correlated to a down-regulation of T-helper 1 lymphocytes activity.     

Synthesis and biological evaluation with plant cells of new fosmidomycin analogues containing a benzoxazolone or oxazolopyridinone ring

Fosmidomycin, 3-(N-formyl-N-hydroxyamido) propylphosphonic acid sodium salt, is an efficient inhibitor of 1-deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway notably present in Plasmodium species. We have synthesized a new series of analogues of fosmidomycin, containing a benzoxazolone, benzoxazolethione or oxazolopyridinone ring. As the MEP pathway is involved in the biosynthesis of all isoprenoids, accumulation of ajmalicine in Catharanthus roseus cells was chosen as a marker of monoterpenoid indole alkaloid (MIA) production. None of the twelve studied phosphonic esters 3 and phosphonic acids 4 affected periwinkle cell growth, but some of them (3c, 3e, 3g and 3h) showed a significant inhibition of ajmalicine accumulation: 45-85% at 125 microM. Surprisingly, this effect disappeared by conversion of 3c and 3g into the corresponding acids 4c and 4g, respectively.     

One-pot synthesis of functionalized 4,5-dihydroisoxazole derivatives via nitrile oxides and biological evaluation with plant cells

1,3 Dipolar cycloadditions of nitrile oxides generated in situ in the presence of a variety of olefins provided 4,5-dihydroisoxazoles. The whole procedure could be performed in a practical and efficient one-pot operation. The products are of excellent purity (95%) and are isolated in 60-83% yields. Some of them enhanced the accumulation of indole alkaloids in periwinkle cell cultures.     

Chromatographic detection of lignin-carbohydrate complexes in annual plants by derivatization in ionic liquid

The opportunity for detecting the presence and the amount of lignin-carbohydrate complexes (LCCs) in renewable feedstocks is a major issue for the complete utilization of biomass. Indeed, LCCs are known to shield cellulose from enzymatic hydrolysis, reducing the efficiency of the digestion processes needed for the production of biobased products. This study is focused on the chromatographic characterization of lignocellulose from agricultural residues (rice husk, wheat straw) and herbaceous energy crops ( Arundo donax , Miscanthus sinesis ) and their fractionation products (hemicellulose, cellulose, and lignin). Exploiting alternative chemical derivatizations on the aforementioned samples, it was possible to discern the connectivity among the various lignocellulosic components. The complete acetylation and benzoylation of the milled native substrates in ionic liquid media, and the systematic comparison between their GPC-UV chromatograms collected at different wavelengths has revealed itself as a straightforward technique in the detection of LCCs. This novel approach proved an extensive connectivity between the lignin and the hemicellulosic for all the analyzed specimens, whereas the cellulosic fraction was conceived as a substantially unbound moiety, accounting for the sample composition at higher molecular weights. Moreover, the collected lignin fractions were extensively characterized by means of (31)P NMR and 2D-HSQC techniques.