High-level expression of the <Emphasis Type="Italic">Penicillium notatum</Emphasis> glucose oxidase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using codon optimization

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml⁻¹ (2.5 g protein l⁻¹) in a 3 l fermentor—410% higher than GOD-w (148 U ml⁻¹), and thus is a low-cost alternative for the bread baking industry.     

Semi-synthesis of chondroitin sulfate-E from chondroitin sulfate-A

Chondroitin sulfate-E (chondroitin-4, 6-disulfate) was prepared from chondroitin sulfate-A (chondroitin-4 - sulfate) by regioselective sulfonation, performed using trimethylamine sulfur trioxide in formamide under argon. The structure of semi-synthetic chondroitin sulfate-E was analyzed by PAGE, (1)H NMR, (13)C NMR, 2D NMR and disaccharide analysis and compared with natural chondroitin sulfate-E. Both semi-synthetic and natural chondroitin sulfate-E were each biotinylated and immobilized on BIAcore SA biochips and their interactions with fibroblast growth factors displayed very similar binding kinetics and binding affinities. The current semi-synthesis offers an economical approach for the preparation of the rare chondroitin sulfate-E from the readily available chondroitin sulfate-A.     

Time‐Varying Latent Effect Model for Longitudinal Data with Informative Observation Times

Summary In analysis of longitudinal data, it is not uncommon that observation times of repeated measurements are subject‐specific and correlated with underlying longitudinal outcomes. Taking account of the dependence between observation times and longitudinal outcomes is critical under these situations to assure the validity of statistical inference. In this article, we propose a flexible joint model for longitudinal data analysis in the presence of informative observation times. In particular, the new procedure considers the shared random‐effect model and assumes a time‐varying coefficient for the latent variable, allowing a flexible way of modeling longitudinal outcomes while adjusting their association with observation times. Estimating equations are developed for parameter estimation. We show that the resulting estimators are consistent and asymptotically normal, with variance–covariance matrix that has a closed form and can be consistently estimated by the usual plug‐in method. One additional advantage of the procedure is that it provides a unified framework to test whether the effect of the latent variable is zero, constant, or time‐varying. Simulation studies show that the proposed approach is appropriate for practical use. An application to a bladder cancer data is also given to illustrate the methodology.     

Elucidating essential role of conserved carboxysomal protein CcmN reveals common feature of bacterial microcompartment assembly

Bacterial microcompartments are organelles composed of a protein shell that surrounds functionally related proteins. Bioinformatic analysis of sequenced genomes indicates that homologs to shell protein genes are widespread among bacteria and suggests that the shell proteins are capable of encapsulating diverse enzymes. The carboxysome is a bacterial microcompartment that enhances CO(2) fixation in cyanobacteria and some chemoautotrophs by sequestering ribulose-1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase in the microcompartment shell. Here, we report the in vitro and in vivo characterization of CcmN, a protein of previously unknown function that is absolutely conserved in β-carboxysomal gene clusters. We show that CcmN localizes to the carboxysome and is essential for carboxysome biogenesis. CcmN has two functionally distinct regions separated by a poorly conserved linker. The N-terminal portion of the protein is important for interaction with CcmM and, by extension, ribulose-1,5-bisphosphate carboxylase/oxygenase and the carbonic anhydrase CcaA, whereas the C-terminal peptide is essential for interaction with the carboxysome shell. Deletion of the peptide abolishes carboxysome formation, indicating that its interaction with the shell is an essential step in microcompartment formation. Peptides with similar length and sequence properties to those in CcmN can be bioinformatically detected in a large number of diverse proteins proposed to be encapsulated in functionally distinct microcompartments, suggesting that this peptide and its interaction with its cognate shell proteins are common features of microcompartment assembly.     

Hepatocyte-specific deletion of Janus kinase 2 (JAK2) protects against diet-induced steatohepatitis and glucose intolerance

Non-alcoholic fatty liver disease (NAFLD) is becoming the leading cause of chronic liver disease and is now considered to be the hepatic manifestation of the metabolic syndrome. However, the role of steatosis per se and the precise factors required in the progression to steatohepatitis or insulin resistance remain elusive. The JAK-STAT pathway is critical in mediating signaling of a wide variety of cytokines and growth factors. Mice with hepatocyte-specific deletion of Janus kinase 2 (L-JAK2 KO mice) develop spontaneous steatosis as early as 2 weeks of age. In this study, we investigated the metabolic consequences of jak2 deletion in response to diet-induced metabolic stress. To our surprise, despite the profound hepatosteatosis, deletion of hepatic jak2 did not sensitize the liver to accelerated inflammatory injury on a prolonged high fat diet (HFD). This was accompanied by complete protection against HFD-induced whole-body insulin resistance and glucose intolerance. Improved glucose-stimulated insulin secretion and an increase in β-cell mass were also present in these mice. Moreover, L-JAK2 KO mice had progressively reduced adiposity in association with blunted hepatic growth hormone signaling. These mice also exhibited increased resting energy expenditure on both chow and high fat diet. In conclusion, our findings indicate a key role of hepatic JAK2 in metabolism such that its absence completely arrests steatohepatitis development and confers protection against diet-induced systemic insulin resistance and glucose intolerance.     

Expression, purification and characterization of recombinant human angiogenin in Pichia pastoris

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.     

Treatment with chicken-egg-white or whole-egg extracts maintains and enhances the survival and differentiation of spleen cells

The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells would be widely useful in cellular biology research. In this study, we compared the different abilities of spleen cells to survive and differentiate in vivo after permeabilization by five different types of egg extracts. Five types of egg extracts were prepared. The spleen cells from male GFP-transgenic mice were permeabilized by the extracts for 30 min, cultured for 12 days, and then transfused into irradiated female mice. At varying days after transplantation, the percentage of GFP-expressing surviving spleen cells was detected in the peripheral blood by flow cytometry. At 120 days after transplantation, bone marrow cells from the female mice were analyzed for the presence of cells containing the Y chromosome. Surviving GFP-positive spleen cells that had been permeabilized with either chicken-egg-white or whole-egg extracts could be detected in the female mice after transplantation. A lower percentage of GFP-positive cells was also detected after permeabilization by the other extracts tested, and no GFP-positive cells were found in the female mouse transfused with spleen cells permeabilized with Hank’s Buffered Salt Solution (HBSS) as a control. At 120 days after transplantation, the percentage of cells containing a Y chromosome in the bone marrow positively correlated with the percentage of GFP-positive cells in the peripheral blood. After permeabilization by chicken-egg-white or whole-egg extracts, spleen cells demonstrated significantly enhanced survival and differentiation functions compared with the spleen cells treated with the other egg extracts tested. These results show that chicken-egg-white and whole-egg extracts have roles in maintaining and enhancing the survival and differentiation of spleen cells. Therefore, these two types of extracts may be of future use in maintaining the function of stem cells.     

Quantitative comparison of errors in 15N transverse relaxation rates measured using various CPMG phasing schemes

Nitrogen-15 Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation experiment are widely used to characterize protein backbone dynamics and chemical exchange parameters. Although an accurate value of the transverse relaxation rate, R(2), is needed for accurate characterization of dynamics, the uncertainty in the R(2) value depends on the experimental settings and the details of the data analysis itself. Here, we present an analysis of the impact of CPMG pulse phase alternation on the accuracy of the (15)N CPMG R(2). Our simulations show that R(2) can be obtained accurately for a relatively wide spectral width, either using the conventional phase cycle or using phase alternation when the r.f. pulse power is accurately calibrated. However, when the r.f. pulse is miscalibrated, the conventional CPMG experiment exhibits more significant uncertainties in R(2) caused by the off-resonance effect than does the phase alternation experiment. Our experiments show that this effect becomes manifest under the circumstance that the systematic error exceeds that arising from experimental noise. Furthermore, our results provide the means to estimate practical parameter settings that yield accurate values of (15)N transverse relaxation rates in the both CPMG experiments.     

Coarse-grained event tree analysis for quantifying Hodgkin-Huxley neuronal network dynamics

We present an event tree analysis of studying the dynamics of the Hodgkin-Huxley (HH) neuronal networks. Our study relies on a coarse-grained projection to event trees and to the event chains that comprise these trees by using a statistical collection of spatial-temporal sequences of relevant physiological observables (such as sequences of spiking multiple neurons). This projection can retain information about network dynamics that covers multiple features, swiftly and robustly. We demonstrate that for even small differences in inputs, some dynamical regimes of HH networks contain sufficiently higher order statistics as reflected in event chains within the event tree analysis. Therefore, this analysis is effective in discriminating small differences in inputs. Moreover, we use event trees to analyze the results computed from an efficient library-based numerical method proposed in our previous work, where a pre-computed high resolution data library of typical neuronal trajectories during the interval of an action potential (spike) allows us to avoid resolving the spikes in detail. In this way, we can evolve the HH networks using time steps one order of magnitude larger than the typical time steps used for resolving the trajectories without the library, while achieving comparable statistical accuracy in terms of average firing rate and power spectra of voltage traces. Our numerical simulation results show that the library method is efficient in the sense that the results generated by using this numerical method with much larger time steps contain sufficiently high order statistical structure of firing events that are similar to the ones obtained using a regular HH solver. We use our event tree analysis to demonstrate these statistical similarities.