Pyrrolo- and pyridoazepine alkaloids as chemical markers in Stemona species

Broad-based phytochemical investigations on 31 Stemona species and geographical provenances led to an overview concerning characteristic accumulation trends and the distribution of different Stemona alkaloids. Two major metabolic differences suggested a taxonomic segregation of the complex Stemona tuberosa group from the other species, and was supported by morphological characters. Whereas most of the Stemona species were characterised by protostemonine type alkaloids, the S. tuberosa group clearly deviated by accumulation trends towards tuberostemonine or croomine derived alkaloids belonging to two different skeletal types. Also of chemotaxonomic relevance was the structural divergence of protostemonine type alkaloids into pyrrolo- or pyridoazepine derivatives represented by stemofoline or oxystemokerrine, respectively, as major constituents. Their common occurrence in different provenances of S. curtisii, also deviating from the other species by various chromosome numbers, deserves special taxonomic attention. Species specific chemical markers were given by the unique accumulation of didehydrostemofoline (=asparagamine A) in S. collinsae and stemokerrine in S. kerrii. In contrast to previous reports, no bisdehydro derivatives with an aromatic pyrrole ring were detected supporting the hypothesis that these alkaloids are artifacts. A new stereoisomer of tuberostemonine was isolated and identified by spectroscopic methods.     

Temperature as a predictive tool for plantar triaxial loading

Diabetic foot ulcers are caused by moderate repetitive plantar stresses in the presence of peripheral neuropathy. In severe cases, the development of these foot ulcers can lead to lower extremity amputations. Plantar pressure measurements have been considered a capable predictor of ulceration sites in the past, but some investigations have pointed out inconsistencies when solely relying on this method. The other component of ground reaction forces/stresses, shear, has been understudied due to a lack of adequate equipment. Recent articles reported the potential clinical significance of shear in diabetic ulcer etiology. With the lack of adequate tools, plantar temperature has been used as an alternative method for determining plantar triaxial loading and/or shear. However, this method has not been previously validated. The purpose of this study was to analyze the potential association between exercise-induced plantar temperature increase and plantar stresses. Thirteen healthy individuals walked on a treadmill for 10 minutes at 3.2 km/h. Pre and post-exercise temperature profiles were obtained with a thermal camera. Plantar triaxial stresses were quantified with a custom-built stress plate. A statistically significant correlation was observed between peak shear stress (PSS) and temperature increase (r=0.78), but not between peak resultant stress (PRS) and temperature increase (r=0.46). Plantar temperature increase could predict the location of PSS and PRS in 23% and 39% of the subjects, respectively. Only a moderate linear relationship was established between triaxial plantar stresses and walking-induced temperature increase. Future research will investigate the value of nonlinear models in predicting plantar loading through foot temperature.     

Genetic characteristics of Kodar snow sheep using SNP markers

For the first time nuclear DNA polymorphisms were studied in Kodar snow sheep (Ovis nivicola kodarensis, KOD). KOD is a representative of a small isolated population of Asian snow sheep (Ovis nivicola Eschscholtz, 1829) inhabiting the Kodar Ridge (Irkutsk oblast, Transbaikal krai). We compared KOD with the geographically closest Yakut subspecies (Ovis nivicola lydekkeri). Genome-wide study of single-nucleotide polymorphisms (SNPs) was performed using the Illumina OvineSNP50 BeadChip (Illumina, United States). The final set of markers for analysis included 1030 SNPs. We found that Kodar snow sheep had almost 10 times lower level of genetic diversity evaluated by multilocus heterozygosity—MLH (0.027 for KOD vs 0.215–0.270 for individuals of Yakut subspecies) and standardized MLH—stMLH (0.116 against 0.910–1.147). The results of multidimensional scaling (MDS), Nei distances calculations (D_N) and STRUCTURE analysis showed a clear genetic differentiation of Kodar snow sheep from Yakut subspecies. Our data is the first step to understanding the demographic history of the original Kodar population of snow sheep.     

The effect of multiple binding modes on empirical modeling of ligand docking to proteins.

A popular approach to the computational modeling of ligand/receptor interactions is to use an empirical free energy like model with adjustable parameters. Parameters are learned from one set of complexes, then used to predict another set. To improve these empirical methods requires an independent way to study their inherent errors. We introduce a toy model of ligand/receptor binding as a workbench for testing such errors. We study the errors incurred from the two state binding assumption--the assumption that a ligand is either bound in one orientation, or unbound. We find that the two state assumption can cause large errors in free energy predictions, but it does not affect rank order predictions significantly. We show that fitting parameters using data from high affinity ligands can reduce two state errors; so can using more physical models that do not use the two state assumption. We also find that when using two state models to predict free energies, errors are more severe on high affinity ligands than low affinity ligands. And we show that two state errors can be diagnosed by systematically adding new binding modes when predicting free energies: if predictions worsen as the modes are added, then the two state assumption in the fitting step may be at fault.     

Secretion of interferon-tau by bovine embryos in long-term culture: comparison of in vivo derived, in vitro produced, nuclear transfer and demi-embryos.

Interferon-tau (IFNtau) is the pregnancy recognition signal of bovine embryos, inhibiting luteolysis. We studied trophoblastic growth and IFNtau secretion of embryos with different developmental potential, i.e., in vivo derived and in vitro produced embryos, cloned embryos and demi-embryos, to evaluate if the ability of secreting IFNtau might be responsible for differences in pregnancy rates after transfer of these categories of embryos to recipients. Day 8 embryos of excellent quality were individually placed in microdrops of buffalo rat liver cell-conditioned medium and maintained for up to 23 days. Embryos were observed on Days 11, 15, 19 and 23, the mean diameter (2r) of attached and spherical embryos was measured, and their trophoblastic area was calculated as r2pi or 4r2pi, respectively. Simultaneously, medium was changed and the IFNtau levels of conditioned media were determined using a bioassay of antiviral activity. Trophoblastic area was smaller (P < 0.05) in demi-embryos than in all other groups, which exhibited similar trophoblastic growth until Day 19. However, on Day 23 trophoblastic area of in vivo derived embryos was more than twice (P < 0.05) as large as those of in vitro produced and nuclear transfer (NT) embryos. IFNtau levels increased only slowly with time in culture of demi-embryos. By contrast, the level of IFNtau doubled from Day 11 to Day 15 in conditioned media from all other groups of embryos. The linear increase in IFNtau production of vivo and in vitro derived embryos continued until the end of the culture period, whereas conditioned media from NT embryos contained significantly (P < 0.05) less IFNtau activity on Days 19 and 23 than those of the former two groups. Our results demonstrate different capabilities of secreting IFNtau for in vivo derived and in vitro produced embryos vs. NT and demi-embryos, which may--at least part--be responsible for the differences in pregnancy rates after transfer to recipients.     

Cardiac specific expression of the green fluorescent protein during early murine embryonic development

We demonstrate the establishment of transgenic mice, where the expression of the green fluorescent protein (GFP) is under control of the human cardiac α-actin promoter. These mice display cardiac specific GFP expression already during early embryonic development. Prominent GFP fluorescence was observed at the earliest stage of the murine heart anlage (E8). Cardiomyocytes of different developmental stages proved GFP positive, but the intensity varied between cells. We further show that contractions of single GFP positive cardiomyocytes can be monitored within the intact embryo. At later stages of embryonic development, the skeletal musculature was also GFP positive, in line with the known expression pattern of cardiac α-actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.