Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

Many biopharmaceuticals are now produced as secreted glycoproteins from mammalian cell culture. The glycosylation profile of these proteins is essential to ensure structural stability and biological and clinical activity. However, the ability to control the glycosylation is limited by our understanding of the parameters that affect the heterogeneity of added glycan structures. It is clear that the glycosylation process is affected by a number of factors including the 3-dimensional structure of the protein, the enzyme repertoire of the host cell, the transit time in the Golgi and the availability of intracellular sugar-nucleotide donors. From a process development perspective there are many culture parameters that can be controlled to enable a consistent glycosylation profile to emerge from each batch culture. A further, but more difficult goal is to control the culture conditions to enable the enrichment of specific glycoforms identified with desirable biological activities. The purpose of this paper is to discuss the cellular metabolism associated with protein glycosylation and review the attempts to manipulate, control or engineer this metabolism to allow the expression of human glycosylation profiles in producer lines such as genetically engineered Chinese hamster ovary (CHO) cells.     

Vibration induced osteogenic commitment of mesenchymal stem cells is enhanced by cytoskeletal remodeling but not fluid shear

Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell's sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04 Pa to 5 Pa. Vibrations were applied at magnitudes of 0.15g, 1g, and 2g using frequencies of both 100 Hz and 30 Hz. After 14 d and under low fluid shear conditions associated with 100 Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30 Hz vibrations enhanced mineralization only in the 2g group. Over 3 d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott–Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity.     

The feasibility of modal testing for measurement of the dynamic characteristics of goat vertebral motion segments

Structural vibration testing might be a promising method to study the mechanical properties of spinal motion segments as an alternative to imaging and spinal manipulation techniques. Structural vibration testing is a non-destructive measurement technique that measures the response of a system to an applied vibration as a function of frequency, and allows determination of modal parameters such as resonance frequencies (ratio between stiffness and mass), vibration modes (pattern of motion) and damping. The objective of this study was to determine if structural vibration testing can reveal the resonance frequencies that correspond to the mode shapes flexion-extension, lateroflexion and axial rotation of lumbar motion segments, and to establish whether resonance frequencies can discriminate specific structural alterations of the motion segment. Therefore, a shaker was used to vibrate the upper vertebra of 16 goat lumbar motion segments, while the response was obtained from accelerometers on the transverse and spinous processes and the anterior side of the upper vertebra. Measurements were performed in three conditions: intact, after dissection of the ligaments and after puncturing the annulus fibrosus. The results showed clear resonance peaks for flexion-extension, lateral bending and axial rotation for all segments. Dissection of the ligaments did not affect the resonance frequencies, but puncturing the annulus reduced the resonance frequency of axial rotation. These results indicate that vibration testing can be utilised to assess the modal parameters of lumbar motion segments, and might eventually be used to study the mechanical properties of spinal motion segments in vivo.     

External mechanical control of the timing of bend initiation in sea urchin sperm flagella

The movement parameters of a sea urchin sperm flagellum can be manipulated mechanically by applying various modes of periodic vibrations to the sperm head held by suction in the tip of a micropipette. The beat frequency of the flagellum readily synchronizes with the frequency of the externally imposed lateral vibration, and the plane of flagellar bending waves adapts itself to the plane of the pipette vibration (Gibbons et al., J. Cell Biol. 101:270a, 1985; Nature 325: 351-352, 1987). In this study, we observed the particular effects of external asymmetric forces on flagellar beating parameters by vibrating the micropipette holding the sperm head in a transverse sawtooth-like motion composed of a rapid effective stroke and a slower recovery stroke, while keeping the vibration frequency constant. The results demonstrate that the timing of bend initiation within the flagellar beat cycle can be controlled mechanically by changing the time point within the vibration cycle at which the micropipette changes its direction of motion. A switch in the sidedness of the asymmetric movement of the micropipette produces dramatic changes in the profiles of bend growth in the basal 5 microns of the flagellum but has almost no effect on the asymmetry or other parameters of bending in the mid- and distal regions of the flagellum. Our results suggest that elastic strain within the basal region of the flagellar structure may play a more significant role in the process of bend initiation than has been realized heretofore.     

Biological significance of circulating polyamines in oncology.

Malignant cell proliferation is associated with an increase intracellular polyamine metabolism which itself appears to be in equilibrium with the extracellular circulating polyamine compartments. Erythrocyte polyamine contents may be used clinically as an index of cell proliferation, but the exact biological roles of circulating polyamines, considered as physio(patho)logical parameters involved in the homeostatic(dys)regulation of cell proliferation, remain obscure. It is known that circulating polyamines help promote malignant cell proliferation and metastatic dissemination, but their ultimate targets are not yet completely understood. Either produced by actively proliferating normal or cancer cells, or absorbed from the gastro-intestinal tract (food and colonic microfloral population), circulating polyamines could favour in vivo malignant cell proliferation. 1) Since these organic polycations are more rapidly internalized by cancer cells than by normal ones, do they join and facilitate the malignant intracellular polyamine metabolism? 2) Does binding of polyamines to specific acceptor sites at the surface of cancer cells, thereby modulating endocytosis of biological factors present in the extracellular spaces, modify the homeostatic control of cell proliferation and differentiation? 3) Do modifications of blood polyamine compartmentalization, observed in cancerous organisms, responsible for new enzyme and/or immune capacities, contribute to tumor progression? Answering the above-mentioned questions would lead to new therapeutic approaches in human oncology.     

Fibronectin quantification without antibodies: a bioassay for the detection of the gelatin-captured macromolecule

We have developed a new cell-adhesion-bioassay (CAA) for the quantitative determination of fibronectin in biological fluids. The assay is based on two particular properties of fibronectin: it specifically binds to gelatin with high affinity and simultaneously it can anchor to different surface molecules of a cell. First fibronectin, derived from very different biological fluids, is purified in situ, within the wells of the microtiter plates applied for the assay, using solid surface bound gelatin. After capturing the macromolecule, it is quantified based on its cell adhesive properties. In contrast to ELISA the CAA does not require specific antibodies, and as the Jurkat cells used as indicator cells, seem to recognize fibronectin from different species equally; species specificity of the reagent plays smaller, perhaps negligible, role in the determination of the amount of the macromolecule. The CAA method may not replace fibronectin specific ELISA-s, but using its principle, improved applications, for example a capture EIA for determining fibronectin can easily be envisioned and CAA may serve as a viable alternative for EIA-s when specific antibodies are not available or when relative measurement of not only the soluble but cell surface associated fibronectin is necessary.     

Crystallographic evaluation of internal motion of human alpha-lactalbumin refined by full-matrix least-squares method

The low temperature form of human alpha-lactalbumin (HAL) was crystallized from a 2H2O solution and its structure was refined to the R value of 0.119 at 1.15 A resolution by the full-matrix least-squares method. Average estimated standard deviations of atomic parameters for non-hydrogen atoms were 0.038 A for coordinates and 0.044 A2 for anisotropic temperature factors (Uij). The magnitude of equivalent isotropic temperature factors (Ueqv) was highly correlated with the distance from the molecular centroid and fitted to a quadratic equation as a function of atomic coordinates. The atomic thermal motion was rather isotropic in the core region and the anisotropy increased towards the molecular surface. The statistical analysis revealed the out-of-plane motion of main-chain oxygen atoms, indicating that peptide groups are in rotational vibration around a Calpha.Calpha axis. The TLS model, which describes the rigid-body motion in terms of translation, libration, and screw motions, was adopted for the evaluation of the molecular motion and the TLS parameters were determined by the least-squares fit to Uij. The reproduced Ueqvcal from the TLS parameters was in fair agreement with observed Ueqv, but differences were found in regions of residues, 5-22, 44-48, 70-75, and 121-123, where Ueqv was larger than Ueqvcal because of large local motions. To evaluate the internal motion of HAL, the contribution of the rigid-body motion was determined to be 42.4 % of Ueqv in magnitude, which was the highest estimation to satisfy the condition that the Uijint tensors of the internal motion have positive eigen values. The internal motion represented with atomic thermal ellipsoids clearly showed local motions different from those observed in chicken-type lysozymes which have a backbone structure very similar to HAL. The result indicates that the internal motion is closely related to biological function of proteins.     

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.     

Gewinnung und Charakterisierung von Prolidase aus,Escherichia coli B., I. Gewinnung des Enzyms

An enzyme being able to hydrolize the imido linkage at the N-terminal end of proline is isolated from E. coli B. This fact corresponds to the specifity of hydrolization of the animal prolidase. Enzyme synthesis within the cells of E. coli B is carried out independently from growth. Changed environmental factors may influence the formation rate of enzyme in a restricted way. A relatively high enzyme biosynthesis can be reached by cultivating the strain E. coli B at a temperature of 37°C as well as an initial pH-value of the medium of 7.0 in submerged culture (400–500 rpm) By variation of the medium composition enzyme synthesis does not change considerably, however, biomass yield can be increased about 100% If mechanical cell desintegration is optimized by means of ultrasonic or vibration homogenisator the cell components may be easily released with a higher proline specific activity as animal prolidase.     

Dynamics of mammalian NER proteins

Despite detailed knowledge on the genetic network and biochemical properties of most of the nucleotide excision repair (NER) proteins, cell biological analysis has only recently made it possible to investigate the temporal and spatial organization of NER. In contrast to several other DNA damage response mechanisms that occur in specific subnuclear structures, NER is not confined to nuclear foci, which has severely hampered the analysis of its arrangement in time and space. In this review the recently developed tools to study the dynamic molecular transactions between the NER factors and the chromatin template are summarized. First, different procedures to inflict DNA damage in a part of the cell nucleus are discussed. In addition, technologies to measure protein dynamics of NER factors tagged with the green fluorescent protein (GFP) will be reviewed. Most of the dynamic parameters of GFP-tagged NER factors are deduced from different variants of 'fluorescence recovery after photobleaching' (FRAP) experiments and FRAP analysis procedures will be briefly evaluated. The combination of local damage induction, genetic tagging of repair factors with GFP and microscopy innovations have provided the basis for the determination of NER kinetics within living mammalian cells. These new cell biological approaches have disclosed a highly dynamic arrangement of NER factors that assemble in an orderly fashion on damaged DNA. The spatio-temporal analysis tools developed for the study of NER and the kinetic model derived from these studies can serve as a paradigm for the understanding of other chromatin-associated processes.     

A Computer-Assisted 3D Model for Analyzing the Aggregation of Tumorigenic Cells Reveals Specialized Behaviors and Unique Cell Types that Facilitate Aggregate Coalescence

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named “facilitators” and “probes.” A third cell type, the “dervish”, is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.     

Characteristics and Stimulation Potential with BMP-2 and BMP-7 of Tenocyte-Like Cells Isolated from the Rotator Cuff of Female Donors

Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient’s age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml) in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and –III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff.     

Perception of biological motion in schizophrenia and healthy individuals: a behavioral and FMRI study

Background

Anomalous visual perception is a common feature of schizophrenia plausibly associated with impaired social cognition that, in turn, could affect social behavior. Past research suggests impairment in biological motion perception in schizophrenia. Behavioral and functional magnetic resonance imaging (fMRI) experiments were conducted to verify the existence of this impairment, to clarify its perceptual basis, and to identify accompanying neural concomitants of those deficits.

Methodology/Findings

In Experiment 1, we measured ability to detect biological motion portrayed by point-light animations embedded within masking noise. Experiment 2 measured discrimination accuracy for pairs of point-light biological motion sequences differing in the degree of perturbation of the kinematics portrayed in those sequences. Experiment 3 measured BOLD signals using event-related fMRI during a biological motion categorization task.Compared to healthy individuals, schizophrenia patients performed significantly worse on both the detection (Experiment 1) and discrimination (Experiment 2) tasks. Consistent with the behavioral results, the fMRI study revealed that healthy individuals exhibited strong activation to biological motion, but not to scrambled motion in the posterior portion of the superior temporal sulcus (STSp). Interestingly, strong STSp activation was also observed for scrambled or partially scrambled motion when the healthy participants perceived it as normal biological motion. On the other hand, STSp activation in schizophrenia patients was not selective to biological or scrambled motion.

Conclusion

Schizophrenia is accompanied by difficulties discriminating biological from non-biological motion, and associated with those difficulties are altered patterns of neural responses within brain area STSp. The perceptual deficits exhibited by schizophrenia patients may be an exaggerated manifestation of neural events within STSp associated with perceptual errors made by healthy observers on these same tasks. The present findings fit within the context of theories of delusion involving perceptual and cognitive processes.     

Bifurcation analysis applied to a model of motion integration with a multistable stimulus

A computational study into the motion perception dynamics of a multistable psychophysics stimulus is presented. A diagonally drifting grating viewed through a square aperture is perceived as moving in the actual grating direction or in line with the aperture edges (horizontally or vertically). The different percepts are the product of interplay between ambiguous contour cues and specific terminator cues. We present a dynamical model of motion integration that performs direction selection for such a stimulus and link the different percepts to coexisting steady states of the underlying equations. We apply the powerful tools of bifurcation analysis and numerical continuation to study changes to the model’s solution structure under the variation of parameters. Indeed, we apply these tools in a systematic way, taking into account biological and mathematical constraints, in order to fix model parameters. A region of parameter space is identified for which the model reproduces the qualitative behaviour observed in experiments. The temporal dynamics of motion integration are studied within this region; specifically, the effect of varying the stimulus gain is studied, which allows for qualitative predictions to be made.     

Characteristics of Motor Resonance Predict the Pattern of Flash-Lag Effects for Biological Motion

Background

When a moving stimulus and a briefly flashed static stimulus are physically aligned in space the static stimulus is perceived as lagging behind the moving stimulus. This vastly replicated phenomenon is known as the Flash-Lag Effect (FLE). For the first time we employed biological motion as the moving stimulus, which is important for two reasons. Firstly, biological motion is processed by visual as well as somatosensory brain areas, which makes it a prime candidate for elucidating the interplay between the two systems with respect to the FLE. Secondly, discussions about the mechanisms of the FLE tend to recur to evolutionary arguments, while most studies employ highly artificial stimuli with constant velocities.

Methodology/Principal Finding

Since biological motion is ecologically valid it follows complex patterns with changing velocity. We therefore compared biological to symbolic motion with the same acceleration profile. Our results with 16 observers revealed a qualitatively different pattern for biological compared to symbolic motion and this pattern was predicted by the characteristics of motor resonance: The amount of anticipatory processing of perceived actions based on the induced perspective and agency modulated the FLE.

Conclusions/Significance

Our study provides first evidence for an FLE with non-linear motion in general and with biological motion in particular. Our results suggest that predictive coding within the sensorimotor system alone cannot explain the FLE. Our findings are compatible with visual prediction (Nijhawan, 2008) which assumes that extrapolated motion representations within the visual system generate the FLE. These representations are modulated by sudden visual input (e.g. offset signals) or by input from other systems (e.g. sensorimotor) that can boost or attenuate overshooting representations in accordance with biased neural competition (Desimone & Duncan, 1995).