Growth and morphology of neuronal cell lines cultured in perfusion

Summary To optimize culture conditions and gain a more reliable culturing system for studies of metabolic properties of neuronal cells, a simplified perfusion chamber was developed. It consists of two parts: a perfusion block and a standard plastic culture dish. To confirm the suitability of this chamber for continuous culturing of anchorage-dependent cells, the growth and morphology of the four neuronal cell lines glioma C6 and glioma 138MG, neuroblastoma C1300, clones N1E115 and N18 were followed for 4 d using both traditional and perfusion techniques. A marked increase in growth and a decrease in the degree of morphological differentiation were obtained with the latter technique compared to the former. This work was supported by grants from the National Swedish Board for Technical Development (Grant 81-5009), the Swedish Work Environmental Foundation (Grant 76-53), and Ollie and Elof Ericssons Foundation for Scientific Research.     

Tubulin synthesis and heat shock-induced cell synchrony in Tetrahymena

This paper offers the suggestion that heat shock inhibition of tubulin synthesis accounts for the molecular mechanism by which periodic heat shocks induce cell synchrony in Tetrahymena. Each heat shock (34 °C) represses tubulin synthesis and blocks the division cycle at the point when the oral structure, rich in microtubules, would normally begin to assemble. Recovery (at 28 °C) from each heat shock is characterized by parallel derepression of tubulin synthesis and of oral development. Changes in protein synthesis patterns are complex when the temperature is shifted up and down between 28 and 34 °C and further experimental support is required in support of the hypothesis here forwarded.     

Ubiquitin‐specific protease‐like 1 (USPL1) is a SUMO isopeptidase with essential,non‐catalytic functions

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity‐based search with the suicide inhibitor haemagglutinin (HA)‐SUMO‐vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin‐specific protease‐like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l—an essential but distant zebrafish homologue of USPL1—also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low‐abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.     

Structural understanding of stabilization patterns in engineered bispecific Ig‐like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

An inside job for siRNAs

Among the three main categories of small silencing RNAs in insects and mammals-siRNAs, miRNAs, and piRNAs-siRNAs were thought to arise primarily from exogenous sources, whereas miRNAs and piRNAs arise from endogenous loci. Recent work in flies and mice reveals several classes of endogenous siRNAs (endo-siRNAs) that contribute to functions previously reserved for miRNAs and piRNAs, including gene regulation and transposon suppression.     

Actin exposure upon tissue injury is a targetable wound site-specific protein marker

BackgroundIdentification of wound-specific markers would represent an important step toward damaged tissue detection and targeted delivery of biologically important materials to injured sites. Such delivery could minimize the amount of therapeutic materials that must be administered and limit potential collateral damage on nearby normal tissues. Yet, biological markers that are specific for injured tissue sites remain elusive.MethodsIn this study, we have developed an immunohistological approach for identification of protein epitopes specifically exposed in wounded tissue sites.ResultsUsing ex-vivo tissue samples in combination with fluorescently-labeled antibodies we show that actin, an intracellular cytoskeletal protein, is specifically exposed upon injury. The targetability of actin in injured sites has been demonstrated in vivo through the specific delivery of anti-actin conjugated particles to the wounded tissue in a lethal rat model of grade IV liver injury.ConclusionsThese results illustrate that identification of injury-specific protein markers and their targetability for specific delivery is feasible.General significanceIdentification of wound-specific targets has important medical applications as it could enable specific delivery of various products, such as expression vectors, therapeutic drugs, hemostatic materials, tissue healing, or scar prevention agents, to internal sites of penetrating or surgical wounds regardless of origin, geometry or location.     

Material properties of films from enzymatically tailored arabinoxylans

Rye arabinoxylan, with an initial arabinose to xylose (Ara/Xyl) ratio of 0.50, was enzymatically modified with alpha-L-arabinofuranosidase. Different enzyme dosages were used to prepare arabinoxylan samples with a gradient of arabinose content varying from Ara/Xyl ratio 0.50 to 0.20. The degree of polymerization of the arabinoxylans was not affected by the enzymatic treatment, as detected with SEC-MALLS. Arabinoxylan samples with an Ara/Xyl ratio of 0.30 and below agglomerated in a water solution as seen by changes in light scattering. All samples, however, formed cohesive films upon drying, without addition of external plasticizers. The film from untreated arabinoxylan was completely amorphous; whereas films of the enzyme-treated arabinoxylans were semicrystalline with an increasing degree of crystallinity with decreasing arabinose content as determined by WAXS. Oxygen permeability measurements of the films showed that decreased arabinose content also resulted in lower oxygen permeability of the films. All films were strong and relatively stiff, but showed variations in strain at break. The moderately debranched film with an Ara/Xyl ratio of 0.37 had highest strain at break among all the films tested, yet was stiff and strong. This material also exhibited yielding and had stress/strain behavior similar to synthetic semicrystalline polymers, with a tendency to strain-induced crystallization. Such a combination of mechanical properties combined with oxygen barrier properties is very attractive for packaging applications.     

Stochastic exits from dormancy give rise to heavy‐tailed distributions of descendants in bacterial populations

Variance in reproductive success is a major determinant of the degree of genetic drift in a population. While many plants and animals exhibit high variance in their number of progeny, far less is known about these distributions for microorganisms. Here, we used a strain barcoding approach to quantify variability in offspring number among replicate bacterial populations and developed a Bayesian method to infer the distribution of descendants from this variability. We applied our approach to measure the offspring distributions for five strains of bacteria from the genus Streptomyces after germination and growth in a homogenous laboratory environment. The distributions of descendants were heavy‐tailed, with a few cells effectively ‘winning the jackpot’ to become a disproportionately large fraction of the population. This extreme variability in reproductive success largely traced back to initial populations of spores stochastically exiting dormancy, which provided early‐germinating spores with an exponential advantage. In simulations with multiple dormancy cycles, heavy‐tailed distributions of descendants decreased the effective population size by many orders of magnitude and led to allele dynamics differing substantially from classical population genetics models with matching effective population size. Collectively, these results demonstrate that extreme variability in reproductive success can occur even in growth conditions that are far more homogeneous than the natural environment. Thus, extreme variability in reproductive success might be an important factor shaping microbial population dynamics with implications for predicting the fate of beneficial mutations, interpreting sequence variability within populations and explaining variability in infection outcomes across patients.