Endothelial and lipoprotein lipases in human and mouse placenta

Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin-Sepharose affinity chromatography, the EL protein eluted as a single peak without detectable phospholipid or triglyceride (TG) lipase activity. The major portion of LPL protein eluted slightly after EL. This peak also had no lipase activity and most likely contained monomeric LPL. Fractions eluting at a higher NaCl concentration contained small amounts of LPL protein (most likely dimeric LPL) and had substantial TG lipase activity. In situ hybridization studies showed EL mRNA expression in syncytiotrophoblasts and endothelial cells and LPL mRNA in syncytiotrophoblasts. In contrast, immunohistochemistry showed EL and LPL protein associated with both cell types. In mouse placentas, lack of LPL expression resulted in increased EL mRNA expression. These results suggest that the cellular expression of EL and LPL in human placenta is different. Nevertheless, the two lipases might have overlapping functions in the mouse placenta. Our data also suggest that the major portions of both proteins are stored in an inactive form in human term placenta.     

One-step affinity purification protocol for human telomerase.

Human telomerase is a ribonucleoprotein (RNP) enzyme, comprising protein components and an RNA template that catalyses telomere elongation through the addition of TTAGGG repeats. Telomerase function has been implicated in aging and cancer cell immortalization. We report a rapid and efficient one-step purification protocol to obtain highly active telomerase from human cells. The purification is based on affinity chromatography of nuclear extracts with antisense oligonucleotides complementary to the template region of the human telomerase RNA component. Bound telomerase is eluted with a displacement oligonucleotide under mild conditions. The resulting affinity-purified telomerase is active in PCR-amplified telomerase assays. The purified telomerase complex has a molecular mass of approximately 550 kDa compared to the approximately 1000 kDa determined for the telomerase RNP in unfractionated nuclear extracts. The purification protocol provides a rapid and efficient tool for functional and structural studies of human telomerase.     

Molecular cloning of the chicken myelomonocytic growth factor (cMGF) reveals relationship to interleukin 6 and granulocyte colony stimulating factor.

Normal as well as retrovirally transformed avian myeloid precursor cells require the colony stimulating factor cMGF for their survival, proliferation and colony formation in vitro. cMGF has been shown to be a glycoprotein which is active in the picomolar concentration range. Co-expression of kinase type oncogenes in v-myb or v-myc transformed myeloid cells induces cMGF expression and confers factor independence via an autocrine mechanism. Here we describe the molecular cloning of cMGF from a myeloblast cDNA library and show that it is a 201 amino acid residue secretory protein which is modified by signal peptide cleavage and glycosylation during translocation into the lumen of membrane vesicles. A bacterially expressed trpE-cMGF fusion protein induces proliferation of E26 transformed myeloblasts in a cMGF bioassay suggesting that glycosylation is not absolutely necessary for biological activity. Sequence comparison reveals that cMGF is distantly related to G-CSF and IL-6.     

Regeneration of fertile plants from protoplasts of different Arabidopsis thaliana genotypes

Summary A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of Arabidopsis thaliana is reported. Protoplasts were isolated from leaves of 21-to 28-day-old Arabidopsis plants grown in a controlled environment. Sustained divisions were achieved when protoplasts were embedded in beads formed by 1.4% sodium alginate in the presence of 50mM CaCl₂ in 0.4 mannitol, which was then exchanged againts modified B5 medium. About 0.4%–0.6% of the protoplasts developed into colonies of which 80%–90% formed shoots and subsequently regenerated to fertile plants. Seeds harvested from more than 200 independently regenerated plants were sown and germination frequencies of more than 95% were obtained. Furthermore, the F₁ plants did not show any evidence of somaclonal variation on visual inspection. This protocol was originally developed for Arabidopsis thaliana Columbia; however it was shown to be applicable also for the genotypes Wassilewskija, Landsberg erecta and Estland though with differing efficiencies.Abbreviations FDA fluorescein diacetate - CM culture medium - SRM shoot regeneration medium - SEM shoot elongation medium - RM rooting medium - PE plating effciency - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - Kin kinetin - 2-iP 2-isopentenyladenine - GA₃ gibberetic acid     

Friction in Total Hip Joint Prosthesis Measured In Vivo during Walking

Friction-induced moments and subsequent cup loosening can be the reason for total hip joint replacement failure. The aim of this study was to measure the in vivo contact forces and friction moments during walking. Instrumented hip implants with Al₂O₃ ceramic head and an XPE inlay were used. In vivo measurements were taken 3 months post operatively in 8 subjects. The coefficient of friction was calculated in 3D throughout the whole gait cycle, and average values of the friction-induced power dissipation in the joint were determined. On average, peak contact forces of 248% of the bodyweight and peak friction moments of 0.26% bodyweight times meter were determined. However, contact forces and friction moments varied greatly between individuals. The friction moment increased during the extension phase of the joint. The average coefficient of friction also increased during this period, from 0.04 (0.03 to 0.06) at contralateral toe off to 0.06 (0.04 to 0.08) at contralateral heel strike. During the flexion phase, the coefficient of friction increased further to 0.14 (0.09 to 0.23) at toe off. The average friction-induced power throughout the whole gait cycle was 2.3 W (1.4 W to 3.8 W). Although more parameters than only the synovia determine the friction, the wide ranges of friction coefficients and power dissipation indicate that the lubricating properties of synovia are individually very different. However, such differences may also exist in natural joints and may influence the progression of arthrosis. Furthermore, subjects with very high power dissipation may be at risk of thermally induced implant loosening. The large increase of the friction coefficient during each step could be caused by the synovia being squeezed out under load.     

Postoperative Changes in In Vivo Measured Friction in Total Hip Joint Prosthesis during Walking

Loosening of the artificial cup and inlay is the most common reasons for total hip replacement failures. Polyethylene wear and aseptic loosening are frequent reasons. Furthermore, over the past few decades, the population of patients receiving total hip replacements has become younger and more active. Hence, a higher level of activity may include an increased risk of implant loosening as a result of friction-induced wear. In this study, an instrumented hip implant was used to measure the contact forces and friction moments in vivo during walking. Subsequently, the three-dimensional coefficient of friction in vivo was calculated over the whole gait cycle. Measurements were collected from ten subjects at several time points between three and twelve months postoperative. No significant change in the average resultant contact force was observed between three and twelve months postoperative. In contrast, a significant decrease of up to 47% was observed in the friction moment. The coefficient of friction also decreased over postoperative time on average. These changes may be caused by ‘running-in’ effects of the gliding components or by the improved lubricating properties of the synovia. Because the walking velocity and contact forces were found to be nearly constant during the observed period, the decrease in friction moment suggests an increase in fluid viscosity. The peak values of the contact force individually varied by 32%-44%. The friction moment individually differed much more, by 110%-129% at three and up to 451% at twelve months postoperative. The maximum coefficient of friction showed the highest individual variability, about 100% at three and up to 914% at twelve months after surgery. These individual variations in the friction parameters were most likely due to different ‘running-in’ effects that were influenced by the individual activity levels and synovia properties.     

Reassembly of somatic cells and testicular organogenesis in vitro

Testicular organogenesis in vitro requires an environment allowing a reassembly of testicular cell types. Previous in vitro studies using male murine germ cells cultured in a defined three-dimensional environment demonstrated tubulogenesis and differentiation into spermatozoa. Combining scaffolds as artificial culture substrates with testicular cell culture, we analysed the colonization of collagen sponges by rat testicular cells focusing on cell survival and reassembly of tubule-like-structures in vitro. Isolated testicular cells obtained from juvenile Sprague Dawley and eGFP transgenic rats were cultured on collagen sponges (DMEM high glucose + Glutamax, 35 °C, 5% CO₂ with or without gonadotropins). Live cell imaging revealed the colonization of cells across the entire scaffold for up to 35 days. After two days, histology showed cell clusters attached to the collagen fibres and displaying signs of tubulogenesis. Clusters consisted mainly of Sertoli and peritubular cells which surrounded some undifferentiated spermatogonia. Flow cytometry confirmed lack of differentiation as no haploid cells were detected. Leydig cell activity was detected by a rise of testosterone after gonadotropin stimulation. Our approach provides a novel method which is in particular suitable to follow the somatic testicular cells in vitro an issue of growing importance for the analysis of germ line independent failure of spermatogenesis.     

Cell cultivation under different gravitational loads using a novel random positioning incubator

Important in biotechnology is the establishment of cell culture methods that reflect the in vivo situation accurately. One approach for reaching this goal is through 3D cell cultivation that mimics tissue or organ structures and functions. We present here a newly designed and constructed random positioning incubator (RPI) that enables 3D cell culture in simulated microgravity (0 g). In addition to growing cells in a weightlessness‐like environment, our RPI enables long‐duration cell cultivation under various gravitational loads, ranging from close to 0 g to almost 1 g. This allows the study of the mechanotransductional process of cells involved in the conversion of physical forces to an appropriate biochemical response. Gravity is a type of physical force with profound developmental implications in cellular systems as it modulates the resulting signaling cascades as a consequence of mechanical loading. The experiments presented here were conducted on mouse skeletal myoblasts and human lymphocytes, two types of cells that have been shown in the past to be particularly sensitive to changes in gravity. Our novel RPI will expand the horizon at which mechanobiological experiments are conducted. The scientific data gathered may not only improve the sustainment of human life in space, but also lead to the design of alternative countermeasures against diseases related to impaired mechanosensation and downstream signaling processes on earth. Biotechnol. Bioeng. 2014;111: 1180–1190. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Gaussian-weighted RMSD superposition of proteins: a structural comparison for flexible proteins and predicted protein structures

Many proteins contain flexible structures such as loops and hinged domains. A simple root mean square deviation (RMSD) alignment of two different conformations of the same protein can be skewed by the difference between the mobile regions. To overcome this problem, we have developed a novel method to overlay two protein conformations by their atomic coordinates using a Gaussian-weighted RMSD (wRMSD) fit. The algorithm is based on the Kabsch least-squares method and determines an optimal transformation between two molecules by calculating the minimal weighted deviation between the two coordinate sets. Unlike other techniques that choose subsets of residues to overlay, all atoms are included in the wRMSD overlay. Atoms that barely move between the two conformations will have a greater weighting than those that have a large displacement. Our superposition tool has produced successful alignments when applied to proteins for which two conformations are known. The transformation calculation is heavily weighted by the coordinates of the static region of the two conformations, highlighting the range of flexibility in the overlaid structures. Lastly, we show how wRMSD fits can be used to evaluate predicted protein structures. Comparing a predicted fold to its experimentally determined target structure is another case of comparing two protein conformations of the same sequence, and the degree of alignment directly reflects the quality of the prediction.     

Systems biology of virus entry in mammalian cells

In this article, we define systems biology of virus entry in mammalian cells as the discipline that combines several approaches to comprehensively understand the collective physical behaviour of virus entry routes, and to understand the coordinated operation of the functional modules and molecular machineries that lead to this physical behaviour. Clearly, these are extremely ambitious aims, but recent developments in different life science disciplines slowly allow us to set them as realistic, although very distant, goals. Besides classical approaches to obtain high-resolution information of the molecules, particles and machines involved, we require approaches that can monitor collective behaviour of many molecules, particles and machines simultaneously, in order to reveal design principles of the systems as a whole. Here we will discuss approaches that fall in the latter category, namely time-lapse imaging and single-particle tracking (SPT) combined with computational analysis and modelling, and genome-wide RNA interference approaches to reveal the host components required for virus entry. These techniques should in the future allow us to assign host genes to the systems' functions and characteristics, and allow emergence-driven, in silico assembly of networks that include interactions with increasing hierarchy (molecules-multiprotein complexes-vesicles and organelles), and kinetics and subcellular spatiality, in order to allow realistic simulations of virus entry in real time.