Production of proteinase A by Saccharomyces cerevisiae in a cell-recycling fermentation system: experiments and computer simulations

Overproduction of proteinase A by recombinant Saccharomyces cerevisiae was investigated by cultivations in a cell-recycling bioreactor. Membrane filtration was used to separate cells from the broth. Recycling ratios and dilution rates were varied and the effect on enzyme production was studied both experimentally and by computer simulations. Experiments and simulations showed that cell mass and product concentration were enhanced by high ratios of recycling. Additional simulations showed that the proteinase A concentration decreased drastically at high dilution rates and the optimal volumetric productivities were at high dilution rates just below washout and at high ratios of recycling. Cell-recycling fermentation gave much higher volumetric productivities and stable product concentrations in contrast to simple continuous fermentation.     

Dynamic simulations of the molecular conformations of wild type and mutant xanthan polymers suggest that conformational differences may contribute to observed differences in viscosity

Xanthan gum is an exopolysaccharide secreted by the bacterium Xanthamonas campestris whose ability to make solutions viscous at low concentrations and over a pH and temperature range have generated much interest in both academic and industrial environments. Mutant Xanthamonas strains have been derived that produce xanthan gums with an altered or variant subunit chemical structure and different measured viscosities when compared with the wild type (wt) form of the polymer. Two variant gums were targeted as potentially interesting in this study, these being the nonacetylated tetramer (natet) and the acetylated tetramer (atet), which both lack a side-chain terminal mannose residue and in one case (natet) lacks an acetate group on an internal mannose residue. Solutions of these tetrameric gums possess viscosities higher (natet) and lower (atet) than the wt gum, and therefore we have attempted to determine whether these molecules possess unique conformational preferences when compared with the wt and with each other. In this manner we can initiate an understanding of how a polysaccharide's conformation contributes to its solution properties. The GEGOP software permits a sampling of the static and dynamic equilibrium states of carbohydrate molecules, and this software was employed to calculate equilibrium states of representative oligosaccharides with chemical structures representative of xanthan-like molecules. Energy minimization techniques revealed similar local minima for all three molecules. Some of these minima are comprised of elongate backbone conformations (A type) in which side chains fold onto backbone surfaces. Other minima with A backbones possessed side chains in less intimate backbone contact especially when calculations were performed with a low dielectric constant. This phenomenon was particularly pronounced in the wt molecule where an increased number of negatively charged side-chain residues experience charge repulsion resulting in reduced side-chain-backbone contact. Metropolis Monte Carlo (MMC) dynamic simulations performed with an elevated temperature factor (1000 K) allowed a better qualitative representation of conformational space than 300 K simulations. Employing a nonhierarchical cluster analysis method (population density profile: PDP) coupled with a classification scheme, it was possible to partition resulting MMC data sets into conformational families. This analysis revealed that in simulations performed with different dielectric constant values (10, 25, and infinity) all molecules possessed primarily A-type backbones. Less elongate, more open helical backbone forms (B, C, D, J, and Flat-a) did occur during the simulations but were populated to a lesser extent. In the natet molecule significantly open helical backbones existed (E, F, G, H, and I) that did not occur in the lower viscosity wt and atet molecules. PDP clustering methods and subsequent conformational classification applied to the first residue (mannose) of the side chain permitted a determination of side-chain orientation. Comparison of all three molecules indicated a larger population of side-chain conformational families in less direct backbone contact for the wt molecule than either of the variant molecules (natet/atet) suggesting that the side chains in the wt are more flexible. Thus, a major conformational difference between the high viscosity natet and the lower viscosities of the wt/atet is the increased amount of open helical backbone in the natet. In addition, the significant difference between the higher viscosity wt and the lower viscosity atet is the increase side-chain flexibility in the wt. We hypothesize that conformational differences of this kind could form a partial explanation of the observed differences in viscosity between these xanthan-like polymers.     

Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.     

Correlation of the leakage current and charge pumping in silicon oninsulator gate-controlled diodes

The leakage and charge pumping currents were measured in gate-controlled MOS p-i-n diodes fabricated on thin SIMOX substrates. The efficiencies of the techniques as well as their complementary features are analyzed for various experimental conditions. The interface properties of device-grade SIMOX wafers are characterized and shown to be compatible with VLSI requirements. Special interface coupling effects, which occur only in fully depleted SOI devices and modify the conventional signature of charge pumping and leakage current, are thoroughly investigated     

An efficient technique for detecting catastrophic convolutional codes

Catastrophic convolutional codes (CC) cause an infinite number of decoded data bit errors when decoding a finite number of code symbols. A CC displays a catastrophic error propagation if the generating polynomials have a common factor. An efficient algorithm for polynomial factorization in GF(2^m) is used for detecting catastrophic CC for any rate n/m and constraint length k. A general formula is derived to calculate the number of catastrophic codes in any (m, n, k) CC.     

Component-relevancy and characterization results in multistatesystems

Two new versions of component relevancy for multistate structure functions are introduced. They are compared with some existing component-relevance conditions and their general properties are investigated. Based on the two relevance conditions, two component-importance measures for multistate systems are defined; they are most appropriate for comparing components when a certain type of system improvement is considered. Series and parallel structures are characterized within the L-superadditive and L-subadditive structure functions by imposing the two new relevance conditions     

Comment and discussion on digital processing of PD pulses

Some of the more salient aspects of the digital processing technology of PD signals are examined. Most of the efforts in this field are concentrated on the application of digital analyzers for pulse height analysis, pattern recognition and identification of the physical phenomena. It is demonstrated that errors in the signal processing unit can lead to dominant mistakes in the interpretation of the test results