Evaluation of Uridine Metabolism in Human and Animal Spermatozoa

The objective of this study was to elucidate the role of uridine for spermatozoa, since this pyrimidine nucleoside was found in millimolar concentration in human seminal plasma. Here, the degradative activity of uridine-phosphorylase [EC 2.4.2.3] and the salvage activity of uridine kinase [EC 2.7.1.48] were detected in human spermatozoa. HPLC analysis depicted the uptake of exogeneous ¹⁴C-labelled adenine, but not of uridine and of hypoxanthine, into nucleotide pools of boar spermatozoa. On addition of uridine, the computer-assisted semen analysis (CASA) of human cells revealed a reduction of the percentage of motile spermatozoa in contrast to an elevation of some velocity parameters. It is concluded that exogeneous uridine could function as suppressor for early capacitation and as a substrate for phosphorolysis, if ribose is needed, rather than to satisfy a demand for intracellular pyrimidine nucleotides.     

An efficient method for routine epstein-barr virus immortalization of human B lymphocytes

Summary A variety of methods exist for the immortalization of B lymphocytes by Epstein-Barr virus due to the simplicity of such techniques to establish cell lines with stable genomic DNA. Two different methods for immortalizing lymphoblastoid cell lines were compared for differences in techniques and materials, time between initiation and immortalization, and success rate of immortalization. An incubation period in Epstein-Barr virus and the use of conditioned media improved immortalization efficiency from 86 to 98% and decreased the time (usually weeks) from culture initiation to cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in non-insulin-dependent diabetes mellitus.     

Reiteration in the Monodominant Tropical Tree Dicymbe corymbosa (Caesalpiniaceae) and its Potential Adaptive Significance

The tropical monodominant tree Dicymbe corymbosa reiterates via epicormic shoots and roots, resulting in multistemmed trees with complex pseudotrunks and root mounds. In 2 ha of primary forest on the Guiana Shield, we quantified the reiterative structure and aboveground soil development of 307 D. corymbosa individuals ≥ 10 cm dbh and investigated the potential adaptive significance of reiteration in terms of genet persistence and root exploitation of aboveground soil accumulations. We also investigated the incidence of the heart rot fungus Phellinus robustus in D. corymbosa and examined its relationship to the reiteration process. Large trees contained more and larger reiterations, greater trunk, root mound, and organic soil volumes, and a higher incidence of Phellinus than smaller trees. Roots and ectomycorrhizas were abundant in aboveground soils on the trees, occurred at higher densities than those of the surrounding forest floor, and may be important in recycling mineral nutrients. Stem turnover and reiteration were associated with Phellinus heart rot and appeared to be cumulative over time, resulting in persistent, structurally complex trees of indeterminate lifespan. Dicymbe corymbosa provides a rare example of a tree species that exploits both persistence and recruitment niches, as it successfully recruits through mast fruiting.     

Hepatic overexpression of murine Abcb11 increases hepatobiliary lipid secretion and reduces hepatic steatosis

Abcb11 encodes for the liver bile salt export pump, which is rate-limiting for hepatobiliary bile salt secretion. We employed transthyretin-Abcb11 and BAC-Abcb11 transgenes to develop mice overexpressing the bile salt export pump in the liver. The mice manifest increases in bile flow and biliary secretion of bile salts, phosphatidylcholine, and cholesterol. Hepatic gene expression of cholesterol 7alpha-hydroxylase and ileal expression of the apical sodium bile salt transporter are markedly reduced, whereas gene expression of targets of the nuclear bile salt receptor FXR (ileal lipid-binding protein, short heterodimer partner (SHP) is increased. Because these changes in gene expression are associated with an increased overall hydrophobicity of the bile salt pool and a 4-fold increase of the FXR ligand taurodeoxycholate, they reflect bile salt-mediated regulation of FXR and SHP target genes. Despite the increased biliary secretion of bile salts, fecal bile salt excretion is unchanged, suggestive of an enhanced enterohepatic cycling of bile salts. Abcb11 transgenic mice fed a lithogenic (high cholesterol/fat/cholic acid) diet display markedly reduced hepatic steatosis compared with wild-type controls. We conclude that mice overexpressing Abcb11 display an increase in biliary bile salt secretion and taurodeoxycholate content, which is associated with FXR/SHP-mediated changes in hepatic and ileal gene expression. Because these mice are resistant to hepatic lipid accumulation, regulation of Abcb11 may be important for the pathogenesis and treatment of steatohepatitis.     

In situ sensor techniques in modern bioprocess monitoring

New reactor concepts as multi-parallel screening systems or disposable bioreactor systems for decentralized and reproducible production increase the need for new and easy applicable sensor technologies to access data for process control. These sophisticated reactor systems require sensors to work with the lowest sampling volumes or, even better, to measure directly in situ, but in situ sensors are directly incorporated into a reactor or fermenter within the sterility barrier and have therefore to stand the sterilization procedures. Consequently, these in situ sensor technologies should enable the measurement of multi-analytes simultaneously online and in real-time at a low price for the robust sensing element. Current research therefore focuses on the implementation of noninvasive spectroscopic and optical technologies, and tries to employ them through fiber optics attached to disposable sensing connectors. Spectroscopic methods reach from ultraviolet to infrared and further comprising fluorescence and Raman spectroscopy. Also, optic techniques like microscopy are adapted for the direct use in bioreactor systems (Ulber et al. in Anal Bioanal Chem 376:342–348, 2003) as well as various electrochemical methods (Joo and Brown in Chem Rev 108:638–651, 2008). This review shows the variety of modern in situ sensing principles in bioprocess monitoring with emphasis on spectroscopic and optical techniques and the progress in the adaption to latest reactor concepts.     

A systems biology approach to analyse leaf carbohydrate metabolism in Arabidopsis thaliana

Plant carbohydrate metabolism comprises numerous metabolite interconversions, some of which form cycles of metabolite degradation and re-synthesis and are thus referred to as futile cycles. In this study, we present a systems biology approach to analyse any possible regulatory principle that operates such futile cycles based on experimental data for sucrose (Scr) cycling in photosynthetically active leaves of the model plant Arabidopsis thaliana. Kinetic parameters of enzymatic steps in Scr cycling were identified by fitting model simulations to experimental data. A statistical analysis of the kinetic parameters and calculated flux rates allowed for estimation of the variability and supported the predictability of the model. A principal component analysis of the parameter results revealed the identifiability of the model parameters. We investigated the stability properties of Scr cycling and found that feedback inhibition of enzymes catalysing metabolite interconversions at different steps of the cycle have differential influence on stability. Applying this observation to futile cycling of Scr in leaf cells points to the enzyme hexokinase as an important regulator, while the step of Scr degradation by invertases appears subordinate.     

Modeling the spread of Phytophthora

We consider a model for the morphology and growth of the fungus-like plant pathogen Phytophthora using the example of Phytophthora plurivora. Here, we are utilizing a correlated random walk describing the density of tips. This random walk incorporates a delay in branching behavior: newly split tips only start to grow after a short while. First, we question the effect of such a delay on the running fronts, for uniform- as well as non-uniform turning kernels. We find that this delay primarily influences the slope of the front and therewith the way of spatial appropriation, and not its velocity. Our theoretical predictions are confirmed by the growth of Phytophthora in concrete experiments performed in Petri dishes. The second question addressed in this paper, concerns the manner tips are interacting, especially the point why tips stop to grow “behind” the interface of the front, respectively in confrontation experiments at the interface between two colonies. The combination of experimental data about the spatially structured time course of the glucose concentration and simulations of a model taking into account both, tips and glucose, reveals that nutrient depletion is most likely the central mechanism of tip interaction and hyphal growth inhibition. We presume that this is the growing mechanism for our kind of Phytophthora in infected plant tissue. Thus, the pathogen will sap its hosts via energy depletion and tissue destruction in infected areas.