Analysis of DNA-bound advanced glycation end-products by LC and mass spectrometry

Sugars and sugar degradation products readily react in vitro with guanine derivatives, resulting in the formation of DNA-bound advanced glycation end-products (DNA-AGEs). The two diastereomers of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG(A,B)) and the cyclic adduct of methylglyoxal and 2'-deoxyguanosine (mdG) (N(2)-7-bis(1-hydroxy-2-oxopropyl)-2'-deoxyguanosine have also been detected in cultured cells and/or in vivo. LC-MS/MS methods have been developed to analyze sensitively DNA adducts in vitro and in vivo. In this paper, the chemical structures of possible DNA-AGEs and the application of LC-MS/MS to measure DNA-AGEs are reviewed.     

High Prevalence of Antibodies against the Bacterium Treponema pallidum in Senegalese Guinea Baboons (Papio papio)

The bacterium Treponema pallidum is known to cause syphilis (ssp. pallidum), yaws (ssp. pertenue), and endemic syphilis (ssp. endemicum) in humans. Nonhuman primates have also been reported to be infected with the bacterium with equally versatile clinical manifestations, from severe skin ulcerations to asymptomatic. At present all simian strains are closely related to human yaws-causing strains, an important consideration for yaws eradication. We tested clinically healthy Guinea baboons (Papio papio) at Parc National Niokolo Koba in south eastern Senegal for the presence of anti-T. pallidum antibodies. Since T. pallidum infection in this species was identified 50 years ago, and there has been no attempt to treat non-human primates for infection, it was hypothesized that a large number of West African baboons are still infected with simian strains of the yaws-bacterium. All animals were without clinical signs of treponematoses, but 18 of 20 (90%) baboons tested positive for antibodies against T. pallidum based on treponemal tests. Yet, Guinea baboons seem to develop no clinical symptoms, though it must be assumed that infection is chronic or comparable to the latent stage in human yaws infection. The non-active character is supported by the low anti-T. pallidum serum titers in Guinea baboons (median = 1:2,560) versus serum titers that are found in genital-ulcerated olive baboons with active infection in Tanzania (range of medians among the groups of initial, moderate, and severe infected animals = 1:15,360 to 1:2.097e+7). Our findings provide evidence for simian infection with T. pallidum in wild Senegalese baboons. Potentially, Guinea baboons in West Africa serve as a natural reservoir for human infection, as the West African simian strain has been shown to cause sustainable yaws infection when inoculated into humans. The present study pinpoints an area where further research is needed to support the currently on-going second WHO led yaws eradication campaign with its goal to eradicate yaws by 2020.     

Susceptibility of four inbred mouse strains to a low-pathogenic isolate of <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis>

EUMORPHIA (European Union Mouse Research for Public Health and Industrial Application) is a research program involved in developing new approaches in phenotyping, mutagenesis, and informatics to improve characterization of mouse models for understanding human physiology and disease. Secondary screen experiments include the development of assays to identify mice with altered susceptibility or resistance to infections. In this context we developed a new model and established a standard operating procedure for the experimental infection of mice with Yersinia (Y.) enterocolitica. In contrast with previous studies that dealt with high-pathogenic Y. enterocolitica, we used the low-pathogenic Y. enterocolitica strain E40 to analyze differences in the immune response of four strains of inbred mice (BALB/c, C3H/HeN, 129P2, C57BL/6) after oral infection. The determination of colony-forming units in Peyer’s patches and histologic analysis supported the observations that BALB/c are less able to ameliorate the infection within 21 days. The immune defense of C57BL/6 mice against Yersinia was the most effective resulting in a nearly complete elimination of bacteria after 21 days. C3H/HeN and 129P2 were intermediate. Analysis of serum immunoglobulins (Ig) by Luminex showed a significant increase of IgG2b levels 21 days after infection in all four inbred strains. The other immunoglobulins remained nearly constant. Our infection model discriminates between the efficiency of an infection at an early time point (3 days) and immunity at a later time point (21 days). It is furthermore an appropriate model to characterize genetic differences in resistance and immunity of inbred and mutant mouse lines.     

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.     

Sm2, a paralog of the Trichoderma cerato-platanin elicitor Sm1, is also highly important for plant protection conferred by the fungal-root interaction of Trichoderma with maize

Background

The proteins Sm1 and Sm2 from the biocontrol fungus Trichoderma virens belong to the cerato-platanin protein family. Members of this family are small, secreted proteins that are abundantly produced by filamentous fungi with all types of life-styles. Some species of the fungal genus Trichoderma are considered as biocontrol fungi because they are mycoparasites and are also able to directly interact with plants, thereby stimulating plant defense responses. It was previously shown that the cerato-platanin protein Sm1 from T. virens - and to a lesser extent its homologue Epl1 from Trichoderma atroviride - induce plant defense responses. The plant protection potential of other members of the cerato-platanin protein family in Trichoderma, however, has not yet been investigated.

Results

In order to analyze the function of the cerato-platanin protein Sm2, sm1 and sm2 knockout strains were generated and characterized. The effect of the lack of Sm1 and Sm2 in T. virens on inducing systemic resistance in maize seedlings, challenged with the plant pathogen Cochliobolus heterostrophus, was tested. These plant experiments were also performed with T. atroviride epl1 and epl2 knockout strains. In our plant-pathogen system T. virens was a more effective plant protectant than T. atroviride and the results with both Trichoderma species showed concordantly that the level of plant protection was more strongly reduced in plants treated with the sm2/epl2 knockout strains than with sm1/epl1 knockout strains.

Conclusions

Although the cerato-platanin genes sm1/epl1 are more abundantly expressed than sm2/epl2 during fungal growth, Sm2/Epl2 are, interestingly, more important than Sm1/Epl1 for the promotion of plant protection conferred by Trichoderma in the maize-C. heterostrophus pathosystem.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0333-0) contains supplementary material, which is available to authorized users.     

Long-term perfusion of the isolated rat liver maintenance of its functional state by use of a fluorocarbon emulsion

In order to establish a long-term perfusion system a fluorocarbon emulsion was developed and employed for the perfusion of isolated rat liver up to 20 h. Its suitability for maintaining some specific organ functions was compared with that of a commonly used red cell-containing medium. All livers perfused with the fluorocarbon medium released phosphoglucose isomerase, glutamate-oxaloacetate transaminase and glutamate dehydrogenase almost linearly at a low basal rate, glutamate dehydrogenase release beginning after 5 h perfusion. In contrast to that, a certain percentage of the livers perfused with the red cell-containing medium showed an exponential enzyme release which was over two standard deviations above the mean of the livers perfused with fluorocarbon medium, the values being 25% for phosphoglucose isomerase, 38% for glutamate-oxaloacetate transmiinase and 87% for glutamate dehydrogenase after 10 h of perfusion. In each case the exponential release of phosphoglucose isomerase signaled the functional impairment of the preparation.Thus, defining those livers as “intact” only if their phosphoglucose isomerase release was within two standard deviations of the means of the fluorocarbon-perfused livers, the following liver functions were examined in fluorocarbon-perfused and, for comparison, in “intact” cell-perfused livers during a 10-h period: Metabolite state, galactose elimination from the perfusate, induction of tyrosine aminotransferase by dexamethasone, and gluconeogenesis from lactate and bile production. It was found that the fluorocarbon medium provided at least the same or an even better hepatic function than did the red cell-containing medium. However, while in red cell-perfused livers functional impairment always occurred at various percentages under the conditions mentioned above, this was never observed with the fluorocarbon medium.Electron microscopic examination of the livers perfused with the fluorocarbon medium showed no disturbance of the mitochondrial matrix and cristae after a 10 h perfusion. While within a large number of liver cells the ergastoplasm was seen in normal appearance, in other liver cells the cisternae of rough endoplasmic reticulum were vacuolated.Some important physicochemical data of the fluorocarbon medium such as O₂ capacity, viscosity and particle size are reported, and the technique and the problems of its preparation are described. The advantages of the fluorocarbon medium for long as well as short term perfusion experiments are discussed.     

Characterization of human apolipoprotein B100 oligosaccharides in LDL subfractions derived from normal and hyperlipidemic plasma: deficiency of alpha-N-acetylneuraminyllactosyl-ceramide in light and small dense LDL particles

The carbohydrate composition of apolipoprotein (apo) B100, particularly its degree of sialylation, may contribute to the atherogenic properties of low-density lipoprotein (LDL). We analyzed LDL apoB100 glycans derived from normolipidemic, hypercholesterolemic, and hypertriglyceridemic diabetic subjects. Using exoglycosidase carbohydrate sequencing and matrix-assisted laser desorption/ionization mass spectrometry to analyze fluorescently labeled oligosaccharides, we report evidence for several carbohydrates not previously identified on apoB100, including truncated complex biantennary N-glycans and hybrid N-glycans. The distribution and diversity of the apoB100 glycans isolated from all individuals was highly conserved. The N-glycan composition of apoB100 derived from five LDL subpopulations (LDL1, d = 1.018-1.023; LDL2, d = 1.023-1.030; LDL3, d = 1.030-1.040; LDL4, d = 1.040-1.051; LDL5, d = 1.051-1.065 g/ml) did not vary in normolipidemic or hypercholesterolemic subjects. Furthermore, we found no evidence for "desialylated" apoB100 glycans in any of the samples analyzed. Analysis of the most abundant LDL ganglioside, alpha-N-acetylneuraminyllactosyl-ceramide, revealed a deficiency in small dense LDL and in the most buoyant subpopulation. These data provide a novel explanation for the apparent deficiency of sialic acid in small dense LDL and indicate that the global apoB100 N-glycan composition is invariable in the patient groups studied.