Biomedical subjects

Wei-Shou Hu

Publications and source records attributed to Wei-Shou Hu.

At least 19 recordsLinked to original sources

PubMed2005

A kinetic model of quantitative real-time polymerase chain reaction.

Real-time polymerase chain reaction (PCR) is one of the most sensitive and accurate methods for quantifying transcript levels especially for those expressed at low abundance. The selective amplification of target DNA over multiple cycles allows its initial concentration to be determined. The amplification rate is a complex interplay of the operating conditions, initial reactant concentrations, and reaction rate constants. Experimentally, the compounded effect of all factors is quantified in terms of an effective efficiency, which is estimated by curve fitting to the amplification data. We present a comprehensive model of PCR to study the effect of various reactant concentrations on the amplification efficiency. The model is used to calculate the kinetic progression of the target DNA concentration with cycle number under conditions when different species are stoichiometrically or kinetically limiting. The reaction efficiency remains constant for the initial cycles. As the primer concentration becomes limiting, the efficiency is marked by a gradual decrease. This is in contrast to a steep decline under nucleotide limiting conditions. Under some conditions, commonly used experimentally, increasing primer concentration has the adverse effect of reducing the final amplified template concentration. This phenomenon seen at times experimentally is explained by the simulation results under rate limiting enzyme concentrations. Primer dimer formation is shown to significantly affect the reaction rates, effective efficiency, and the estimated initial concentrations. This model, by describing the interplay of the many operating variables, will be a useful tool in designing PCR conditions and evaluating its results.

Computer Simulation↗

PubMed2005

EST sequencing for gene discovery in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are one of the most important cell lines in biological research, and are the most widely used host for industrial production of recombinant therapeutic proteins. Despite their extensive applications, little sequence information is available for molecular based research. To facilitate gene discovery and genetic engineering, two cDNA libraries were constructed from three CHO cell lines grown under various conditions. The average insert size for both libraries is approximately 800-850 bp, and each library has comparable redundancy levels of 36%-38% for the sequences isolated. Random sequencing of 4,608 ESTs yielded 2,602 unique assemblies, 76% of which were annotated as orthologs of sequences in the GenBank database. A high abundance of mitochondrial genome transcripts facilitated the assembly of the complete mitochondrial genome by PCR walking. Comparative analysis of sequences from both mitochondrial and nuclear genomes with orthologous genes from other species shows that CHO sequences are generally most similar to mouse; however, examples with highest similarity to rat or human are common. A cDNA microarray, including all 4,608 ESTs, was constructed. The microarray results reveal a high level of consistency between transcript abundance in the libraries and fluorescence intensities. Inclusion of redundant clones in the microarray, additionally, allows small changes in abundant mRNAs to be discerned with a high degree of confidence. The information and tools generated provide access to genomic technology for this important cell line.

Amino Acid Sequence↗

PubMed2005

Large-scale gene expression analysis of cholesterol dependence in NS0 cells.

NS0, a nonsecreting mouse myeloma cell, is a major host line used for recombinant antibody production. These cells have a cholesterol-dependent phenotype and rely on an exogenous supply of cholesterol for their survival and growth. To better understand the physiology underlying cholesterol dependence, we compared NS0 cells, cultivated under standard cholesterol-dependent growth conditions (NS0), to cells adapted to cholesterol-independent conditions (NS0 revertant, NS0_r). Large-scale transcriptional analyses were done using the Affymetrix GeneChip array, MG-U74Av2. The transcripts expressed differentially across the two cell lines were identified. Additionally, proteomic tools were employed to analyze cell lysates from these two cell lines. Cellular proteins from both NS0 and NS0_r were subjected to 2D gel electrophoresis. MALDI-TOF mass spectrometry was performed to determine the identity of the differentially expressed spots. We examined the expression level of mouse genes directly involved in cholesterol biosynthesis, lipid metabolism, and central energy metabolism. Most of these genes were downregulated in the revertant cell type, NS0_r, compared to NS0. Overall, a large number of genes are expressed differentially, indicating that the reversal of cholesterol dependency has a profound effect on cell physiology. It is probable that a single gene mutation, activation, or inactivation is responsible for cholesterol auxotrophy. However, the wide-ranging changes in gene expression point to the distinct possibility of a regulatory event affecting the reversibility of auxotrophy, either directly or indirectly.

Animals↗

PubMed2005

An analysis of the use of genomic DNA as a universal reference in two channel DNA microarrays.

BACKGROUND: DNA microarray is an invaluable tool for gene expression explorations. In the two-dye microarray, fluorescence intensities of two samples, each labeled with a different dye, are compared after hybridization. To compare a large number of samples, the 'reference design' is widely used, in which all RNA samples are hybridized to a common reference. Genomic DNA is an attractive candidate for use as a universal reference, especially for bacterial systems with a low percentage of non-coding sequences. However, genomic DNA, comprising of both the sense and anti-sense strands, is unlike the single stranded cDNA usually used in microarray hybridizations. The presence of the antisense strand in the 'reference' leads to reactions between complementary labeled strands in solution and may cause the assay result to deviate from true values. RESULTS: We have developed a mathematical model to predict the validity of using genomic DNA as a reference in the microarray assay. The model predicts that the assay can accurately estimate relative concentrations for a wide range of initial cDNA concentrations. Experimental results of DNA microarray assay using genomic DNA as a reference correlated well to those obtained by a direct hybridization between two cDNA samples. The model predicts that the initial concentrations of labeled genomic DNA strands and immobilized strands, and the hybridization time do not significantly affect the assay performance. At low values of the rate constant for hybridization between immobilized and mobile strands, the assay performance varies with the hybridization time and initial cDNA concentrations. For the case where a microarray with immobilized single strands is used, results from hybridizations using genomic DNA as a reference will correspond to true ratios under all conditions. CONCLUSION: Simulation using the mathematical model, and the experimental study presented here show the potential utility of microarray assays using genomic DNA as a reference. We conclude that the use of genomic DNA as reference DNA should greatly facilitate comparative transcriptome analysis.

DNA↗

PubMed2005

Growth cones turn and migrate up an immobilized gradient of the laminin IKVAV peptide.

Growth cone navigation is guided by extrinsic environmental proteins, called guidance cues. Many in vitro studies have characterized growth cone turning up and down gradients of soluble guidance cues. Although previous studies have shown that axonal elongation rates can be regulated by gradients of surface-bound molecules, there are no convincing demonstrations of growth cones turning to migrate up a surface-bound gradient of an adhesive ligand or guidance cue. In order to test this mode of axonal guidance, we used a photo-immobilization technique to create grids and gradients of an adhesive laminin peptide on polystyrene culture dish surfaces. Chick embryo dorsal root ganglia (DRGs) were placed on peptide grid patterns containing surface-bound gradients of the IKVAV-containing peptide. DRG growth cones followed a path of surface-bound peptide to the middle of a perpendicularly oriented gradient with a 25% concentration difference across 30 microm. The majority of growth cones turned and migrated up the gradient, turning until they were oriented directly up the gradient. Growth cones slowed their migration when they encountered the gradient, but growth cone velocity returned to the previous rate after turning up or down the gradient. This resembles in vivo situations where growth cones slow at a choice point before changing the direction of axonal extension. Thus, these results support the hypothesis that mechanisms of axonal guidance include growth cone orientation by gradients of surface-bound adhesive molecules and guidance cues.

Animals↗

PubMed2005

Characterization of a hollow fiber bioartificial liver device.

A three-compartment bioartificial liver (BAL) has been developed for potential treatment of fulminant hepatic failure. It has been shown previously that viability and liver-specific functions were maintained in laboratory-scale bioreactors of such design. In this study, the performance of hepatocytes in a clinical-scale bioartificial liver was verified by sustained specific production rates of albumin and urea, along with oxygen consumption rates for up to 56 h and liver-specific gene expression for up to 72 h. In addition, transmission of porcine endogenous retrovirus and other type C retroviral particles across the hollow fibers was not detected under both normal and extreme operating fluxes. These results demonstrate that the clinical-scale BAL performs at a level similar to the laboratory scale and that it offers a viral barrier against porcine retroviruses.

Albumins↗

PubMed2004

A diffusion-reaction model for DNA microarray assays.

DNA microarrays are extensively used for the quantification of the degree of differential mRNA expression. The assay involves hybridization of mobile DNA strands with immobilized complementary DNA strands to form duplexes. The overall duplex formation rate depends on the rate of transport of strands in solution to the corresponding spot on the surface, and the rate of the hybridization reaction. We present a theoretical model that incorporates both kinetics of the reversible hybridization reaction and diffusional transport of the labeled strands, and analyze DNA microarray hybridization using this model. Simulations are carried out in a geometrically realistic domain for labeled DNA concentrations corresponding to rare and abundant transcripts for typical assay conditions. The rate of strand diffusion in solution is shown to strongly affect the overall hybridization rate. We compute the minimum inter-spot spacing for replicate spots to enhance sensitivity. We also determine the hybridization time for which reliable estimates of the relative mRNA abundance of two species can be obtained using total fluorescence intensities. An analytical solution for the concentration distribution of mobile strands at intermediate hybridization times provides a convenient tool to calculate the mobile strand concentration profiles. This model provides a framework for the process analysis of all microarray assays currently used for genomic transcriptional analysis.

Computer-Aided Design↗

PubMed2004

Large scale gene expression profiling of metabolic shift of mammalian cells in culture.

The metabolic state of hybridoma cells in continuous culture varies with the cultivation condition from which the culture is initiated. At a metabolically shifted state, cells have markedly reduced glucose and other nutrient consumption and lactate production as compared to cells in batch culture or in continuous culture without a metabolic shift. Taking a combined genomics and proteomics approach, we investigated the molecular mechanism of metabolic shift. Cells from continuous cultures at two different steady states with a glucose consumption to lactate production molar ratio (DeltaL/DeltaG) of 0.08 and 1.4 were studied. Affymetrix GeneChips as well as cDNA microarrays were employed to identify differentially expressed mRNA transcripts, and the differentially expressed proteins were identified using the 2D gel electrophoresis-mass spectrometry approach. The decrease in glucose metabolism upon metabolic shift is accompanied by a decrease in gene expression of a number of genes involved in its metabolism. However, the number of genes differentially expressed and the extent of differential expression upon metabolic shift are relatively moderate. The change in the expression of metabolic genes at the transcriptional level was confirmed by real time PCR. The results suggest that metabolic shift is a combined effect of both biochemical events at reaction level and gene expression at transcription and translation level. This approach of integrating transcriptional profiling, proteomic techniques and biochemical analysis provides a more global view of the metabolism of mammalian cells in culture.

Animals↗

PubMed2004

Non-linear reduction for kinetic models of metabolic reaction networks.

Kinetic models of metabolic networks are essential for predicting and optimizing the transient behavior of cells in culture. However, such models are inherently high dimensional and stiff due to the large number of species and reactions involved and to kinetic rate constants of widely different orders of magnitude. In this paper we address the problem of deriving non-stiff, reduced-order non-linear models of the dominant dynamics of metabolic networks with fast and slow reactions. We present a method, based on singular perturbation analysis, which allows the systematic identification of quasi-steady-state conditions for the fast reactions, and the derivation of explicit non-linear models of the slow dynamics independent of the fast reaction rate expressions. The method is successfully applied to detailed models of metabolism in human erythrocytes and Saccharomyces cerevisiae.

Carbon↗

PubMed2004

A Boolean algorithm for reconstructing the structure of regulatory networks.

Advances in transcriptional analysis offer great opportunities to delineate the structure and hierarchy of regulatory networks in biochemical systems. We present an approach based on Boolean analysis to reconstruct a set of parsimonious networks from gene disruption and over expression data. Our algorithms, Causal Predictor (CP) and Relaxed Causal Predictor (RCP) distinguish the direct and indirect causality relations from the non-causal interactions, thus significantly reducing the number of miss-predicted edges. The algorithms also yield substantially fewer plausible networks. This greatly reduces the number of experiments required to deduce a unique network from the plausible network structures. Computational simulations are presented to substantiate these results. The algorithms are also applied to reconstruct the entire network of galactose utilization pathway in Saccharomyces cerevisiae. These algorithms will greatly facilitate the elucidation of regulatory networks using large scale gene expression profile data.

Algorithms↗

PubMed2003

Unveiling steady-state multiplicity in hybridoma cultures: the cybernetic approach.

Mammalian cells grown in suspension produce waste metabolites such as lactate, alanine, and ammonia, which reduce the yield of cell mass and the desired product on the nutrients supplied. Previous studies (Cruz et al., 1999; Europa et al., 2000; Follstad et al., 1999) have shown that the cells can be made to alter their metabolism by starving them on their nutrients in continuous cultures at low dilution rates or starting the culture as a fed-batch. This leads to multiple steady states in continuous reactors, with some states being more favorable than others. Mathematical models that take into account the metabolic regulation that leads to these multiple steady states are invaluable tools for bioreactor control. In this article we present a cybernetic modeling strategy in which Metabolic Flux Analysis (MFA) is used to guide the cybernetic formulation. The hybridoma model presented as a result of this strategy considers the partially substitutable, partially complementary nature of glucose and glutamine. The choice of competitions within the network is guided by MFA and the model is successful in explaining the three multiple steady states observed. The cybernetic model though identified for the hybridoma experiments of Hu and others (Europa et al., 2000) seem generally applicable to mammalian systems as it captures the pathways that are common to mammalian cells grown in suspension. The model presented here could be used for start-up strategies for continuous reactors and model-based feedback control for maintaining high productivity of the reactor.

Animals↗

PubMed2002

Structural polarity and functional bile canaliculi in rat hepatocyte spheroids.

Primary hepatocytes self-assemble into spheroids that possess tight junctions and microvilli-lined channels. We hypothesized that polarity develops gradually and that the channels structurally and functionally resemble bile canaliculi. Immunofluorescence labeling of apical and basolateral proteins demonstrated reorganization of the membrane proteins into a polarized distribution during spheroid culture. By means of fluorescent dextran diffusion and confocal microscopy, an extensive network of channels was revealed in the interior of the spheroids. These channels connected over several planes and opened to pores on the surface. To examine the content of apical proteins in the channel membranes, the bile canalicular enzyme dipeptidyl peptidase IV (DPPIV) was localized using a fluorogenic substrate, Ala-Pro-cresyl violet. The results show that DPPIV activity is heterogeneously distributed in spheroids and localized in part to channels. Bile acid excretion was then investigated to demonstrate functional polarity. A fluorescent bile acid analogue, fluorescein isothiocyanate-labeled glycocholate, was taken up into the spheroids and excreted into bile canalicular channels. Due to the structural polarity of spheroids and their ability to excrete bile into channels, they are a unique three-dimensional model of in vitro liver tissue self-assembly. (Videoanimations of some results are available at http://hugroup.cems.umn.edu/research_movies).

Animals↗

PubMed2002

Differential gene expression analysis during porcine hepatocyte spheroid formation.

Primary porcine hepatocytes cultured in suspension self-assemble into multicellular aggregates or spheroids that display enhanced liver-specific functional capability and remain viable for an extended period of time in vitro. The molecular events underlying the process of spheroid formation were explored by differential gene expression analysis. Critical time points in spheroid formation were first identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of stress-related gene expression levels at different stages of spheroid formation. Suppression subtractive hybridization was used to identify transcripts up- or down-regulated at different stages of spheroid formation. Subsequently, three sets of reciprocal subtractions, comparing freshly isolated hepatocytes, spheroid-forming hepatocytes, and mature spheroids were carried out, and differentially expressed transcripts were isolated, cloned, sequenced, and annotated. A total of 65 genes and 14 novel transcripts were identified as differentially expressed, and very high sequence conservation between pig and human transcripts was observed. The resultant expressed sequence tags (ESTs) revealed a rapid decrease in the transcript levels of a subset of liver-specific genes, cytochrome P450s, and enzymes involved in heme biosynthesis, as well as up-regulation of genes involved in calcium-dependent vesicle trafficking and a number of acute-phase proteins in mature spheroids. Previous morphological and functional data on hepatocyte spheroid formation support cellular polarization of the hepatocyte into apical and basolateral domains in spheroids. This is important for the re-emergence of differentiated functions in vitro and is reflected by differences in gene expression patterns.

Animals↗

PubMed2002

An auxin surge following fertilization in carrots: a mechanism for regulating plant totipotency.

All plants exhibit the property of cellular totipotency, whereby individual cells can regenerate into an entire organism. However little is known about the underlying mechanisms regulating totipotency. Using a preparative microtechnique, we report an 80-fold surge in the concentration of free auxin that is correlated with the initial stages of zygotic embryogenesis in carrots. The concentration of free IAA increases from a basal level of ca. 25 ng/g FW in unfertilized ovules to ca. 2.000 ng/g FW in the late globular and early heart stages, then back to the basal level in the torpedo stage. This initial increase in IAA levels is diagnostic of the activity of the tryptophan-mediated pathway for IAA biosynthesis, while the maintenance of the basal levels is attributed to the tryptophan-independent pathway for IAA biosynthesis. Our observations are consistent with the hypothesis that the sequential activation of alternative IAA biosynthetic pathways is a critical mechanism for regulating carrot (Daucus carota L. cv. Danvers 126) embryogenesis and other instances of plant totipotency.

Daucus carota↗

PubMed2002

Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.

We have derived from normal human, mouse, and rat postnatal bone marrow primitive, multipotent adult progenitor cells (MAPCs) that can differentiate into most mesodermal cells and neuroectodermal cells in vitro and into all embryonic lineages in vivo. Here, we show that MAPCs can also differentiate into hepatocyte-like cells in vitro. Human, mouse, and rat MAPCs, cultured on Matrigel with FGF-4 and HGF, differentiated into epithelioid cells that expressed hepatocyte nuclear factor-3beta (HNF-3beta), GATA4, cytokeratin 19 (CK19), transthyretin, and alpha-fetoprotein by day 7, and expressed CK18, HNF-4, and HNF-1alpha on days 14-28. Virtually all human, as well as a majority of rodent cells stained positive for albumin and CK18 on day 21; 5% (rodent) to 25% (human) cells were binucleated by day 21. These cells also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital-inducible cytochrome p450, could take up LDL, and stored glycogen. MAPCs, which can be expanded in vitro and maintained in an undifferentiated state for more than 100 population doublings, can thus differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. MAPCs may therefore be an ideal cell for in vivo therapies for liver disorders or for use in bioartificial liver devices.

Adult↗

PubMed2000

Heterogeneous distribution of lysine 6-aminotransferase during cephamycin C biosynthesis in Streptomyces clavuligerus demonstrated using green fluorescent protein as a reporter.

The cellular distribution of the cephamycin biosynthetic enzyme lysine 6-aminotransferase (LAT) has been studied in Streptomyces clavuligerus hyphae by confocal microscopy using the S65T mutant of green fluorescent protein (GFP) as a reporter. LAT mediates the first committed step in the biosynthesis of the secondary metabolite cephamycin C by S. clavuligerus. The enzymic activity of LAT varies with time during the growth of S. clavuligerus in liquid medium. To investigate if this temporal variation occurs uniformly amongst all hyphae, S. clavuligerus was transformed with a plasmid containing the LAT-encoding gene translationally fused to the GFP-encoding gene. The LAT-GFP fusion product displayed fluorescence spectral characteristics of GFP, and showed similar temporal characteristics of LAT activity compared to the wild-type strain of S. clavuligerus. The transformed strain exhibited a heterogeneous distribution of fluorescence in mycelia grown in liquid cultures. This distribution varied significantly as the batch progressed: only a fraction of the mycelia fluoresced in the early growth phase, whereas nearly all hyphae fluoresced by the late growth phase. Thereafter, a non-uniform distribution of fluorescence was again observed in the declining growth phase. A large fraction of the non-fluorescent cells in the declining growth phase were found to be non-viable. Observations of S. clavuligerus colonies grown on solid agar also showed variation of LAT-GFP expression at different stages of growth. These observations in the solid phase can be explained in terms of nutrient deprivation and signalling molecules. The results suggest that physiological differentiation of S. clavuligerus mycelia leading to cephamycin C biosynthesis is both temporally and spatially distributed. The findings also revealed that the observed heterogeneity was independent of the position of individual cell compartments within the hypha. The potential of GFP as a reporter for the quantitative study of cephamycin biosynthesis at the cellular level has also been demonstrated.

Amino Acid Sequence↗

PubMedDate not supplied

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production.

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.

Adenoviridae↗

PubMedDate not supplied

Transcriptional response of Escherichia coli to temperature shift.

Temperature shift is often practiced in the cultivation of Escherichia coli to reduce undesired metabolite formation and to maximize synthesis of correctly folded heterologous protein. As the culture temperature is decreased below the optimal 37 degrees C, growth rate decreases and many physiological changes occur. In this study, we investigated the gene expression dynamics of E. coli on switching its cultivation temperature from 37 to 33 and 28 degrees C using whole genome DNA microarrays. Approximately 9% of the genome altered expression level on temperature shift. Overall, the alteration of transcription upon the downshift of temperature is rapid and globally distributed over a wide range of gene classes. The general trends of transcriptional changes at 28 and 33 degrees C were similar. The largest functional class among the differentially expressed genes was energy metabolism. About 12% of genes in energy metabolism show a decrease in their level of expression, and approximately 6% show an increase. Consistent with the decrease in the glucose uptake rate, many genes involved in glycolysis and the PTS sugar transport systems show decreased expression. Genes encoding enzymes related to amino acid biosynthesis and transport also have reduced expression levels. Such decrease in expression probably reflects the reduced growth rate and the accompanying reduction in energy and amino acid demand at lower temperatures. However, nearly all genes encoding enzymes in the TCA cycle have increased expression levels, which may well be compensating the reduction of the activity of TCA cycle enzymes at lower temperatures. Temperature shift also results in shift of the cytochromes from the high affinity cytochrome o system to the low affinity cytochrome d system. There is no evidence that protein processing genes are selectively altered to create favorable conditions for heterologous protein synthesis. Our results indicate that the beneficial effect of temperature shift in many biotechnological processes is likely to be attributed to the general effect of reduced growth and metabolism.

Adaptation, Physiological↗