Recent advances in gene‐enhanced bone tissue engineering

The loss of bone tissue represents a critical clinical condition that is frequently faced by surgeons. Substantial progress has been made in the area of bone research, providing insight into the biology of bone under physiological and pathological conditions, as well as tools for the stimulation of bone regeneration. The present review discusses recent advances in the field of gene‐enhanced bone tissue engineering. Gene transfer strategies have emerged as highly effective tissue engineering approaches for supporting the repair of the musculoskeletal system. By contrast to treatment with recombinant proteins, genetically engineered cells can release growth factors at the site of injury over extended periods of time. Of particular interest are the expedited technologies that can be applied during a single surgical procedure in a cost‐effective manner, allowing translation from bench to bedside. Several promising methods based on the intra‐operative genetic manipulation of autologous cells or tissue fragments have been developed in preclinical studies. Moreover, gene therapy for bone regeneration has entered the clinical stage with clinical trials for the repair of alveolar bone. Current trends in gene‐enhanced bone engineering are also discussed with respect to the movement of the field towards expedited, translational approaches. It is possible that gene‐enhanced bone tissue engineering will become a clinical reality within the next few years.     

Membrane transport as controlling pacemaker of glycolysis in Saccharomyces carlsbergensis

The frequency of anaerobic NADH oscillations in Saccharomyces carlsbergensis following addition of fructose is 1.5–2.0 times slower than that following addition of glucose. Since catabolism of the two sugars differs in the sugar phosphorylation pathway only, the reason for the observed alternation of the frequency must be sought among these reactions. It has been found that:

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1. The equilibrium of hexose phosphates, as catalized by phosphoglucose isomerase, is nearly independent of the added hexose.     

Collective Cell Migration in Embryogenesis Follows the Laws of Wetting

Collective cell migration is a fundamental process during embryogenesis and its initial occurrence, called epiboly, is an excellent in vivo model to study the physical processes involved in collective cell movements that are key to understanding organ formation, cancer invasion, and wound healing. In zebrafish, epiboly starts with a cluster of cells at one pole of the spherical embryo. These cells are actively spreading in a continuous movement toward its other pole until they fully cover the yolk. Inspired by the physics of wetting, we determine the contact angle between the cells and the yolk during epiboly. By choosing a wetting approach, the relevant scale for this investigation is the tissue level, which is in contrast to other recent work. Similar to the case of a liquid drop on a surface, one observes three interfaces that carry mechanical tension. Assuming that interfacial force balance holds during the quasi-static spreading process, we employ the physics of wetting to predict the temporal change of the contact angle. Although the experimental values vary dramatically, the model allows us to rescale all measured contact-angle dynamics onto a single master curve explaining the collective cell movement. Thus, we describe the fundamental and complex developmental mechanism at the onset of embryogenesis by only three main parameters: the offset tension strength, α, that gives the strength of interfacial tension compared to other force-generating mechanisms; the tension ratio, δ, between the different interfaces; and the rate of tension variation, λ, which determines the timescale of the whole process.     

An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system.

We have expanded the use of the Semliki Forest virus (SFV) by infecting chromaffin cells with synaptic proteins at high efficiency. Using the SFV gene expression system, up to 40% of cultured bovine chromaffin cells express the protein of interest within 12-48 h after infection. In order to learn about the basic physiological properties of infected cells, we performed membrane capacitance measurements using the whole-cell patch-clamp technique and monitored catecholamine release with amperometry. We found that chromaffin cells infected with green fluorescent protein (GFP) were comparable to control cells in intracellular calcium concentrations ([Ca2+]i), leak currents and cell sizes. In response to depolarization, calcium currents were elicited and the cells secreted catecholamine. Comparison of the calcium current amplitude and the size of the readily releasable pool of vesicles revealed a small decrease in these parameters compared to control cells. The refilling kinetics after pool depletion, however, were not altered. Overexpressed munc13-1 translocates to the plasma membrane in response to phorbol esters, an effect that is also observed in fibroblasts transfected with conventional methods. Thus, the use of the SFV gene expression system to infect chromaffin cells represents a major improvement in infection efficiency compared to other methods. It opens up new opportunities to introduce synaptic proteins into chromaffin cells and study their role in secretion.     

Dominant-negative alleles of 14-3-3 proteins cause defects in actin organization and vesicle targeting in the yeast Saccharomyces cerevisiae

14-3-3 Proteins are thought to function as adapters in signaling complexes [1,2], thereby participating in cellular processes including vesicle trafficking and exocytosis [3,4]. To delineate further the function of 14-3-3 proteins during vesicle trafficking, we generated dominant-negative alleles of the two 14-3-3 homologues, Bmh1p and Bmh2p, in budding yeast and analyzed their phenotype in respect to exocytosis. Cells overexpressing the carboxy-terminal region of Bmh2p failed to polarize vesicular transport although bulk exocytosis remained unaffected and showed a disrupted actin cytoskeleton. Our data suggest that 14-3-3 proteins may act primarily on the actin cytoskeleton to regulate vesicle targeting.     

Glycine receptors: recent insights into their structural organization and functional diversity

Strychnine-sensitive glycine receptors (GlyRs) are known to mediate synaptic inhibition in spinal cord, brainstem and other regions of the CNS. During the past 5 years, considerable progress has been made in delineating structural determinants of ligand binding and channel activation in recombinant GlyRs. Furthermore, immunohistochemical and gene inactivation studies have disclosed distinct distributions and functions of differentially expressed GlyR subtypes in retina, hippocampus and the dorsal horn of the spinal cord. Accordingly, GlyRs regulate not only the excitability of motor and sensory neurones, but are also essential for the processing of photoreceptor signals, neuronal development and inflammatory pain sensitization. Hence, these receptors constitute promising targets for the development of clinically useful compounds.