Dynamic cultivation of human mesenchymal stem cells in a rotating bed bioreactor system based on the Z®RP platform

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three‐dimensional functional tissue‐like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three‐dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stem cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram®. Furthermore, hydroxyapatite coated Sponceram® was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP‐expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram® compared to hydroxyapatite coated Sponceram®. With this study, we present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Targeting Myofibroblasts in Model Systems of Fibrosis by an Artificial α-Smooth Muscle-Actin Promoter Hybrid

Myofibroblasts are the main cell types producing extracellular matrix proteins in a variety of fibrotic diseases. Therefore, they are useful targets for studies of intracellular communication and gene therapeutical approaches in scarring diseases. An artificial promoter containing the −702 bp regulatory sequence of the α-smooth muscle actin (SMA) gene linked to the first intron enhancer sequence of the β-actin gene and the β-globin intron-exon junction was constructed and tested for myofibroblast-dependent gene expression using the green fluorescent protein as a reporter. Reporter expression revealed myofibroblast-specific function in hepatic and renal myofibroblasts, in vitro. In addition, differentiation-dependent activation of the SMA-β-actin promoter hybrid was shown after induction of myofibroblastic features in mesangial cells by stretching treatment. Furthermore, wound healing experiments with SMA-β-actin promoter reporter mice demonstrated myofibroblast-specific action, in vivo. In conclusion, the −702 bp regulatory region of the SMA promoter linked to enhancing β-actin and β-globin sequences benefits from its small size and is suggested as a promising tool to target myofibroblasts as the crucial cell type in various scarring processes.     

Lipid‐Bilayer Permeation of Drug‐Like Compounds

Lipid‐bilayer permeation is determinant for the disposition of xenobiotics in the body. It controls the pharmacokinetic behavior of drugs and is, in many cases, a prerequisite for intracellular targeting. Permeation of in vivo barriers is in general predicted from lipophilicity and related parameters. This article goes beyond the empirical correlations, and elucidates the processes and their interplay determining bilayer permeation. A flip‐flop model for bilayer permeation, which considers the partitioning rate constants beside the translocation rate constants, is compared with the diffusion model based on Fick's first law. According to the flip‐flop model, the ratios of aqueous volumes to barrier area can determine whether partitioning or translocation is rate‐limiting. The flip‐flop model allows permeation of anions and cations, and expands our understanding of pH‐dependent permeation kinetics. Some experimental evidences for ion‐controlled permeation at pH 7 are also included in this work.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

THE EXPRESSION OF APELIN AND ITS RECEPTOR APJ DURING DIFFERENT PHYSIOLOGICAL STAGES IN THE BOVINE OVARY

Recent studies implicate that apelin and its receptor APJ may have important role for the modulation of angiogenesis. The aim of this study was to further characterise the regulation of apelin/APJ system in bovine ovary. Experiment 1: corpora lutea (CL) were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrous cycle and of gravidity (month <3, 3-5, 6-7 and >8). Experiment 2: Follicles during maturation were divided into granulosa cells (GC) and theca interna (TI) and were examined separately. Classification of follicles occurred by follicle size and oestradiol-17β (E2) concentration in the follicular fluid (FF) (<0.5 ng/ml, 0.5-5 ng/ml; 5-40 ng/ml; 40-180 ng/ml; >180 ng/ml). Real-time RT-PCR (qPCR) was applied to investigate mRNA expression of examined factors. In general, the expression level of apelin during the oestrous cycle was significantly higher compared to the one during pregnancy. Apelin mRNA levels were always high during the cycle with a tendency of decrease after CL regression. The APJ mRNA in the CL was significantly up regulated on days 5-7 and 8-12 followed by a decrease on days 13-16, and further on days >18. The expression of APJ does not show any significant regulation in the CL throughout pregnancy. The expression of apelin and APJ was not statistically regulated in GC, but was significantly up regulated in follicles with an E2 concentration of more than 5 ng/ml and showed an increase according to growth and maturation of follicles. In conclusion, our data suggest that apelin/APJ system is involved in the mechanism regulating angiogenesis during follicle maturation as well as during CL formation and function in the bovine ovary.     

Cell physiology and protein secretion of Bacillus licheniformis compared to Bacillus subtilis

The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can secrete numerous proteins into the surrounding medium enabling them to use high-molecular-weight substances, which are abundant in soils, as nutrient sources. The availability of complete genome sequences allows for the prediction of the proteins containing signals for secretion into the extracellular milieu and also of the proteins which form the secretion machinery needed for protein translocation through the cytoplasmic membrane. To confirm the predicted subcellular localization of proteins, proteomics is the best choice. The extracellular proteomes of B. subtilis and B. licheniformis have been analyzed under different growth conditions allowing comparisons of the extracellular proteomes and conclusions regarding similarities and differences of the protein secretion mechanisms between the two species.     

Comparison of exhaled breath condensate pH using two commercially available devices in healthy controls,asthma and COPD patients

Background

Analysis of exhaled breath condensate (EBC) is a non-invasive method for studying the acidity (pH) of airway secretions in patients with inflammatory lung diseases.

Aim

To assess the reproducibility of EBC pH for two commercially available devices (portable RTube and non-portable ECoScreen) in healthy controls, patients with asthma or COPD, and subjects suffering from an acute cold with lower-airway symptoms. In addition, we assessed the repeatability in healthy controls.

Methods

EBC was collected from 40 subjects (n = 10 in each of the above groups) using RTube and ECoScreen. EBC was collected from controls on two separate occasions within 5 days. pH in EBC was assessed after degasification with argon for 20 min.

Results

In controls, pH-measurements in EBC collected by RTube or ECoScreen showed no significant difference between devices (p = 0.754) or between days (repeatability coefficient RTube: 0.47; ECoScreen: 0.42) of collection. A comparison between EBC pH collected by the two devices in asthma, COPD and cold patients also showed good reproducibility. No differences in pH values were observed between controls (mean pH 8.27; RTube) and patients with COPD (pH 7.97) or asthma (pH 8.20), but lower values were found using both devices in patients with a cold (pH 7.56; RTube, p < 0.01; ECoScreen, p < 0.05).

Conclusion

We conclude that pH measurements in EBC collected by RTube and ECoScreen are repeatable and reproducible in healthy controls, and are reproducible and comparable in healthy controls, COPD and asthma patients, and subjects with a common cold.     

Hybrid maize breeding with doubled haploids: V. Selection strategies for testcross performance with variable sizes of crosses and S1 families

In hybrid maize (Zea mays L.) breeding, doubled haploids (DH) are increasingly replacing inbreds developed by recurrent selfing. Doubled haploids may be developed directly from S₀ plants in the parental cross or via S₁ families. In both these breeding schemes, we examined 2 two-stage selecting strategies, i.e., considering or ignoring cross and family structure while selection among and within parental crosses and S₁ families. We examined the optimum allocation of resources to maximize the selection gain ΔG and the probability P(q) of identifying the q% best genotypes. Our specific objectives were to (1) determine the optimum number and size of crosses and S₁ families, as well as the optimum number of test environments and (2) identify the superior selection strategy. Selection was based on the evaluation of testcross progenies of (1) DH lines in both stages (DHTC) and (2) S₁ families in the first stage and of DH lines within S₁ families in the second stage (S₁TC-DHTC) with uniform and variable sizes of crosses and S₁ families. We developed and employed simulation programs for selection with variable sizes of crosses and S₁ families within crosses. The breeding schemes and selection strategies showed similar relative efficiency for both optimization criteria ΔG and P (0.1%). As compared with DHTC, S₁TC-DHTC had larger ΔG and P (0.1%), but a higher standard deviation of ΔG. The superiority of S₁TC-DHTC was increased when the selection was done among all DH lines ignoring their cross and family structure and using variable sizes of crosses and S₁ families. In DHTC, the best selection strategy was to ignore cross structures and use uniform size of crosses.