Biomedical subjects

Michael L Shuler

Publications and source records attributed to Michael L Shuler.

At least 19 recordsLinked to original sources

PubMed2007

A genomically/chemically complete module for synthesis of lipid membrane in a minimal cell.

A minimal cell is a hypothetical cell defined by the essential functions required for life. We have developed a module for the synthesis of membrane precursors for a mathematical minimal cell model. This module describes, with chemical and genomic detail the production of the constituents required to build a cell membrane and identifies the corresponding essential genes. Membranes allow selective nutrient passage, harmful substance exclusion, and energy generation. Bacterial membrane components range from lipids to fatty acids with embedded proteins and are structurally similar to eukaryotic cell membranes. Membranes are dynamic structures and experimental analyses show great variations in bacterial membrane composition. The flexibility of the model is such that different membrane compositions could be obtained in response to simulated changes in culture conditions. The model's predictions are in close agreement with the observed biological trends. The model's predictions correspond well with the experimental values of total lipid content in cells grown in chemostat culture, but less well with data from batch growth. Cell shape and size results agree especially well for data for growth rate relative to maximum growth rate larger than 0.5; and DNA, RNA, and protein predictions are consistent with experimental observations. A better understanding of the simplest bacterial membrane should lead to insights on the more complex behavior of membranes of higher species as well as identification of potential targets for antimicrobials.

Biotechnology↗

PubMed2006

Cloning, expression and enantioselective hydrolytic catalysis of a microsomal epoxide hydrolase from a marine fish, Mugil cephalus.

The cDNA of a marine fish microsomal epoxide hydrolase (mEH) gene from Mugil cephalus was cloned by rapid amplification of cDNA ends (RACE) techniques. The homology model for the mEH of M. cephalus showed a characteristic structure of alpha/beta-hydrolase-fold main domain with a lid domain over the active site. The characteristic catalytic triad, consisting of Asp(238), His(444), and Glu(417), was highly conserved. The cloned mEH gene was expressed in Escherichia coli and the recombinant mEH exhibited (R)-preferred hydrolysis activity toward racemic styrene oxide. We obtained enantiopure (S)-styrene oxide with a high enantiopurity of more than 99% enantiomeric excess and yield of 15.4% by batch kinetic resolution of 20 mM racemic styrene oxide.

Amino Acid Sequence↗

PubMed2006

Effect of the plant peptide regulator, phytosulfokine-alpha, on the growth and Taxol production from Taxus sp. suspension cultures.

Phytosulfokine-alpha (PSK-alpha) is a small plant peptide (5 amino acids) that displays characteristics typically associated with animal peptide hormones. PSK-alpha was originally isolated based on its mitogenic activity with plant cultures; it has been reported to increase production of tropane alkaloids from Atropa belladonna, although its general influence on secondary metabolite production is unknown. The studies reported in this article were initiated to evaluate the effects of PSK-alpha supplementation on production of Taxol (paclitaxel) from plant cell cultures of Taxus sp. particularly when methyl jasmonate (MeJA) is added as an elicitor of secondary metabolism. The response to PSK-alpha supplementation was cell line dependent. Taxus cuspidata P93AF showed no statistically significant response to PSK-alpha supplementation while Taxus canadensis C93AD and T. cuspidata PO93X displayed a concentration-dependent response (up to 100 nM PSK-alpha added in first 24 h of culture) with a decrease in initial growth rate, an increase in cell density (dry weight/fresh weight), and increased Taxol production. More remarkably with T. canadensis (C93AD), a very strong synergistic response of PSK-alpha (100 nM) and methyl jasmonate (MeJA, 100 microM) elicitation was observed, resulting in Taxol level of 35.3 +/- 2.1 mg/L or 1.83 +/- 0.02 mg Taxol/g dry cell weight achieved at day 21, a level of approximately 10-fold higher than for either treatment by itself. Although the level of Taxol production achieved is not remarkable, this synergistic treatment was able to partially revive taxane production in cultures that have lost productivity due to extended time (over 10 years) in continuous subculture.

Acetates↗

PubMed2006

Antibody-based surface plasmon resonance detection of intact viral pathogen.

Surface plasmon resonance (SPR) technique was used to directly detect an intact form of insect pathogen: the baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). An SPR sensor chip with three bio-functional layers was used to detect the intact AcMNPV: amine-reactive crosslinker with a disulfide bond that chemisorbs to gold film, Protein A, and a mouse IgG monoclonal antibody raised against a surface protein of the target viral pathogen. A two-channel (reference & test) micro-fluidic SPR system is used for reliable measurement. Bio-specific response to the AcMNPV is compared with the response for tobacco mosaic virus (TMV) as control. Successive exposure of the sensor chip to both viruses verifies a specific response to AcMNPV. This serves as a prerequisite to the development of a new type of viral pathogen detection sensors.

Antibodies, Viral↗

PubMed2005

A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis.

We have developed a prototype three-channel microfluidic chip that is capable of generating a linear concentration gradient within a microfluidic channel and is useful in the study of bacterial chemotaxis. The linear chemical gradient is established by diffusing a chemical through a porous membrane located in the side wall of the channel and can be established without through-flow in the channel where cells reside. As a result, movement of the cells in the center channel is caused solely by the cells chemotactic response and not by variations in fluid flow. The advantages of this microfluidic chemical linear gradient generator are (i) its ability to produce a static chemical gradient, (ii) its rapid implementation, and (iii) its potential for highly parallel sample processing. Using this device, wildtype Escherichia coli strain RP437 was observed to move towards an attractant (e.g., l-asparate) and away from a repellent (e.g., glycerol) while derivatives of RP437 that were incapable of motility or chemotaxis showed no bias of the bacteria's distribution. Additionally, the degree of chemotaxis could be easily quantified using this assay in conjunction with fluorescence imaging techniques, allowing for estimation of the chemotactic partition coefficient (CPC) and the chemotactic migration coefficient (CMC). Finally, using this approach we demonstrate that E. coli deficient in autoinducer-2-mediated quorum sensing respond to the chemoattractant l-aspartate in a manner that is indistinguishable from wildtype cells suggesting that chemotaxis is insulated from this mode of cell-cell communication.

Cell Communication↗

PubMed2005

Portable in situ fluorescence cytometry of microscale cell-based assays.

A portable fluorescence cytometric system has been developed for characterizing chemical concentration and cellular status in microscale cell culture analog (microCCA) devices. Based on discrete optical components, the system provides a modular platform for real-time image measurements applicable to a variety of cell-based microassays. As a feasibility study, we investigated the real-time dynamics of daunorubicin uptake with cultured mouse L-cells in a microCCA compartment. Time course results measured by the portable fluorescence cytometric system confirmed that in the microCCA devices daunorubicin accumulation is proportional to the liquid turnover rate.

Animals↗

PubMed2005

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy.

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 microm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 microm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 microm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).

Biofilms↗

PubMed2005

Behavior of lead in a model microbial predator-prey system.

Predation at the microbial level can affect the fate of toxic trace metals. Metals associated with bacterial prey can be released into the dissolved phase following digestion by a predator, and/or metals can remain in the predator and be transferred potentially to the next level of the food chain. Toxic metal ions in the aqueous phase also are expected to modify the growth and predation rate of a microbial predator. A model predator-prey system was developed to test the effects of Pb on cells and to help elucidate the fate of Pb in this type of interaction. Established methods that have been shown to be suitable for distinguishing dissolved, prey-bound, predator-bound, and ingested Pb were used to establish the pathway of Pb over time. Growth parameters were measured using batch reactors for the protozoan predator Tetrahymena thermophila and the bacterial prey Pseudomonas putida without Pb and at several concentrations of Pb. The effect of prey density on predation and Pb phase distribution also was investigated. Results demonstrate that some kinetic parameters related to prey consumption and growth of T. thermophila are altered by Pb. Upon addition of predator to prey cells in equilibrium with dissolved Pb, dissolved and prey-bound Pb become associated with the predator through ingestion and adsorption. Ingested Pb is excreted later as a bound metal associated with T. thermophila waste matter. A preliminary mathematical model was developed to describe predator-prey dynamics and their influence on the behavior and fate of Pb. Growth data were used to obtain model parameters, and model simulations for Pb fractionation are compared to experimental observations.

Animals↗

PubMed2004

Robust control of initiation of prokaryotic chromosome replication: essential considerations for a minimal cell.

A genomically and chemically detailed mathematical model of a "minimal cell" would be useful to understand better the "design logic" of cellular regulation. A "minimal cell" will be a prokaryote with the minimum number of genes necessary for growth and replication in an ideal environment (i.e., preformed precursors, constant temperature, etc.). The Cornell single-cell model of Escherichia coli serves as the basic framework upon which a minimal cell model can be constructed. A critical issue for any cell model is to describe a mechanism for control of initiation of chromosome replication. There is strong evidence that the essence of chromosome replication control is highly conserved in eubacteria and even extends to the archae. A generalized mechanism is possible based on binding of the protein DnaA-ATP to the origin of replication (oriC) as a primary control. Other features, such as regulatory inactivation of DnaA (RIDA) by conversion of DnaA-ATP to DnaA-ADP and titration of DnaA by binding to other DnaA boxes on the chromosome, have emerged as critical elements in obtaining a functional system to control initiation of chromosome synthesis. We describe a biologically realistic model of chromosome replication initiation control embedded in a complete whole-cell model that explicitly links the external environment to the mechanism of replication control. The base model is deterministic and then modified to include stochastic variation in the components for replication control. The stochastic model allows evaluation of the model's robustness, employing a low standard deviation of interinitiation time as a measure of robustness. Four factors were examined: DnaA synthesis rate; DnaA-ATP binding sites at oriC; the binding rate of DnaA-ATP to the nonfunctional DnaA boxes; and the effect of changing the number of nonfunctional binding sites. The observed DnaA synthesis rate (2000 molecules/cell) and the number of DnaA binding sites per origin (30) are close to the values predicted by the model to provide good control (low variance of interinitiation time), with a reasonable expenditure of cell resources. At relatively high binding rates for DnaA-ATP to the DnaA boxes (10(16) M(-1) s(-1)), increasing the number of DnaA binding sites to about 300, improved control (but little further improvement was seen by extension to 1000 boxes); however, at a low binding rate (10(10) M(-1) s(-1)), an increase in DnaA boxes had an adverse effect on control. The combination of all four factors is probably necessary to obtain a robust control system. Although this mechanism of replication initiation control is highly conserved, it is not clear if simpler control in a minimal cell might exist based on experimental observations with Mycoplasma. This issue is discussed in this investigation.

Bacterial Proteins↗

PubMed2004

An endothelial and astrocyte co-culture model of the blood-brain barrier utilizing an ultra-thin, nanofabricated silicon nitride membrane.

The endothelial cells comprising brain capillaries have extremely tight intercellular junctions which form an essentially impermeable barrier to passive transport of water soluble molecules between the blood and brain. Several in vitro models of the blood-brain barrier (BBB) have been studied, most utilizing commercially available polymer membranes affixed to plastic inserts. There is mounting evidence that direct contact between endothelial cells and astrocytes, another cell type found to have intimate interaction with the brain side of BBB capillaries, is at least partially responsible for the development of the tight intercellular junctions between BBB endothelial cells. However, the membranes commonly used for BBB in vitro models are lacking certain attributes that would permit a high degree of direct contact between astrocytes and endothelial cells cultured on opposing sides. This work is based on the hypothesis that co-culturing endothelial and astrocyte cells on opposite sides of an ultra-thin, highly porous membrane will allow for increased direct interaction between the two cell types and therefore result in a better model of the BBB. We used standard nanofabrication techniques to make membranes from low-stress silicon nitride that are at least an order of magnitude thinner and at least two times more porous than commercial membrane inserts. An experimental survey of pore sizes for the silicon nitride membranes suggested pores approximately 400 nm in diameter are adequate for restricting astrocyte cell bodies to the seeded side while allowing astrocyte processes to pass through the pores and interact with endothelial cells on the opposite side. The inclusion of a spun-on, cross-linked collagen membrane allowed for astrocyte attachment and culture on the membranes for over two weeks. Astrocytes and endothelial cells displayed markers specific to their cell types when grown on the silicon nitride membranes. The transendothelial electrical resistances, a measure of barrier tightness, of endothelial and astrocyte co-cultures on the silicon nitride membranes were comparable to the commercial membranes, but neither system showed synergy between the two cell types in forming a tighter barrier. This lack of synergy may have been due to the loss of ability of commercially available primary bovine brain microvascular endothelial cells to respond to astrocyte differentiating signals.

Animals↗

PubMed2004

Lipid-gel and poly(dimethylsiloxane) film to mimic bioaccumulation in adipocytes.

Bioaccumulation is an increasingly important consideration in validation studies of the safety and efficacy of potential drugs. Although an "adipocyte" cell line model has been proven successful to mimic the accumulation of naphthalene in adipocytes, the prolonged incubation time limits its use in high-throughput studies and reduces reproducibility. In this investigation, naphthalene and naphthol accumulation and uptake kinetics of thin poly(dimethylsiloxane) (PDMS) film and lipid nanospheres suspended in a crosslinked gelatin gel (lipid-gel) were compared with those of adipocytes. Unlike the PDMS film, the lipid-gel can mimic the kinetics and extent of naphthalene accumulation in the adipocytes reasonably well. However, the lipid-gel accumulated about twice as much naphthol as the adipocytes, suggesting that hydrophobicity/hydrophilicity of the metabolite may be an important factor in the accuracy of accumulation studies with the lipid-gel. Nonetheless, the lipid-gel system shows promise as an inexpensive, convenient, and reproducible fat mimic for bioaccumulation studies.

3T3-L1 Cells↗

PubMed2004

A self-priming microfluidic diaphragm pump capable of recirculation fabricated by combining soft lithography and traditional machining.

Fluid transport is crucial in the development of microanalytical devices. While there are many micropump designs available, most are incapable of sustaining recirculation of fluid at microL/min to mL/min levels. We have designed and fabricated a positive displacement micropump by combining soft lithography with traditional bulk machining. The micropump is actuated through pneumatic pressure. The pump is self-priming and is suitable for recirculating fluid through a microfluidic device containing mammalian cell culture. By custom designing the volume of the pumping chamber, tight control of the output flow rate can be obtained by changing the actuation frequency. It can also be fabricated easily on plastic substrates without access to expensive microfabrication equipment.

Cell Culture Techniques↗

PubMed2004

Mobilization of adsorbed copper and lead from naturally aged soil by bacterial extracellular polymers.

Sorption of pollutants is a dominant phase transfer process affecting the fate and transport of metals through the subsurface. The movement of contaminants is retarded by sorption to the stationary subsurface porous media and can seriously hinder remediation efforts. Research has shown that the binding of adsorbed metals becomes more pronounced the longer the contaminant is in the subsurface and the release rates of aged metal contaminants have not received the research attention given to freshly added metals in laboratory studies. Metal release rates are also influenced by the presence of dissolved ligands that compete with mineral soil surfaces by providing binding sites. Dissolved organic matter such as bacterial extracellular polymers are common in natural soil solutions and the metal binding properties of bacterial polymers are well established. Therefore, binding of metals to dissolved biopolymers may result in mobilization of an adsorbed metal. This is important for cases where the metals are assumed to be relatively immobile such as in the case of land applied biosolids. In addition, naturally occurring adherent bacteria commonly produce extracellular polymers and thus may modify the bioavailability of meal contaminants at the point of their attachment. In this study samples from three sites, one a land applied sludge test site, were used to investigate the ability of bacterial extracellular polymers to release metals from soils with long-term exposures. The presence of ?200mg/L bacterial extracellular polymer was found to increase the short-term (less than 350h) release of Cu and Pb by a factor of 2-4-fold.

Adsorption↗

PubMed2004

Predicting the rate and extent of cadmium and copper desorption from soils in the presence of bacterial extracellular polymer.

The movement of cationic transition metals through the subsurface is strongly retarded by sorption to the porous media. However, dissolved organic ligands can compete with soil surfaces by providing binding sites for metals in solution. An extracellular polymer produced by a bacterium isolated from soil was used in this study to observe and model the influence of a naturally occurring ligand on the release of adsorbed metals from two test soils. Experimental results show that the presence of dissolved extracellular polymer enhanced the rate and extent of desorptive release of soil-bound cadmium and copper. A kinetic model that uses a gamma distribution of rate constants to account for the physical and chemical heterogeneity of the soil matrix was employed to describe the release of cadmium and copper in batch experiments. Model parameters describing soil, metal and extracellular polymer interactions were obtained through separate experiments. With these parameters the model successfully predicted the influence of dissolved polymer on the rate and extent of release of cadmium and copper from soil in independent batch experiments. These results suggest that the presence of natural metal-binding ligands such as bacterial extracellular polymers can act to increase the driving force for desorption by lowering the aqueous concentration of free unbound metals in solution.

Adsorption↗

PubMed2004

Development of a model microbial predator-prey system suitable for studies of the behavior of toxic trace metals.

Interactions between microbial predators and their prey can significantly influence the behavior of toxic trace metals. Ingested bacterial prey-bound metals can either accumulate within a predator or be excreted and potentially reintroduced into the dissolved phase. A defined predator-prey system suitable for developing a more fundamental understanding of metal behavior in simple microbial food chains was designed and tested by using lead (Pb) as a representative cationic transition metal. Desired features of this system were the ability to define the chemical speciation of dissolved metals as well as to distinguish between prey- and predator-bound metals. Pseudomonas putida and the ciliate protozoan Tetrahymena thermophila were selected as representative bacterial prey and predator species, respectively. In addition, the use of fluorescent microspheres was evaluated as an experimental surrogate for bacterial prey. Filtration techniques for size-selective separation were developed so that the distribution of Pb between cells of T. thermophila, cells of P. putida or microspheres, and the dissolved phase could be assessed. Filtration units were selected based on their ability to perform separations with minimal metal loss at circumneutral pH. Five-micron polycarbonate filter membranes successfully separated T. thermophila from P. putida with good cell retention and low metal loss. Centrifuge filters successfully separated dissolved and particle-bound metal (<5,000 nominal molecular wt limit). Exemplary experimental results are presented and show that predation on Pb-exposed cells of P. putida or microspheres increases uptake of Pb by T. thermophila.

Animals↗

PubMed2003

Effect of silkworm hemolymph on N-linked glycosylation in two Trichoplusia ni insect cell lines.

A recombinant N-linked glycoprotein, secreted human placental alkaline phosphatase (SEAP), was produced in two Trichoplusia ni insect cell lines using the baculovirus expression vector. Silkworm hemolymph (SH) was added to TNMFH + 10% fetal bovine serum (FBS) medium to a concentration of 2.5% or 5%, and SEAP production and glycosylation in the presence of SH were compared with controls devoid of hemolymph. Growing Tn-4s cells in 5% SH-supplemented medium required progressive adaptation of the cells to SH, and adapted cells had a SEAP specific yield decreased by 2.5-fold compared with control cells not exposed to SH. Although SEAP produced in the control possessed little complex glycosylation (<1%), SEAP produced by SH-adapted cells in the presence of 5% SH possessed 8.7% sialylated structures, as well as unusual, asialylated, agalactosylated structures with a high degree of polymerization (DP). On the basis of enzymatic and mass-spectrometric analyses, we propose that these structures are glucosylated, high-mannose oligosaccharides. SEAP was also produced by Tn-4s cells without adaptation to SH when SH was added just prior to baculovirus infection, but SEAP specific yield was adversely affected (approximately fourfold reduction compared with control devoid of hemolymph), and glycosylation of SEAP produced under these conditions was characterized by large amounts of high-mannose and high-DP structures and an absence of complex structures. Similarly, Tn5B1-4 cells that were not adapted to SH had a SEAP specific yield reduced by approximately fivefold in SH-containing medium; however, these cells were able to produce 13.5% sialylated SEAP in the presence of 2.5% SH, whereas complex structures were not produced in the absence of SH. We propose that SH improves glycosylation either directly or indirectly by decreasing SEAP specific yield.

Alkaline Phosphatase↗

PubMed2003

Structural characterization of plant-derived hepatitis B surface antigen employed in oral immunization studies.

Several subunit vaccine antigens have been successfully expressed in plants and recently the hepatitis B surface antigen (HBsAg), expressed in potatoes, was shown to be orally immunogenic in animal studies. However, to date, a detailed analysis of the plant-derived antigen is lacking. Herein, we comprehensively characterize the structure and post-translational processing of HBsAg from potato tuber and two plant cell suspension cultures. The HBsAg was found to accumulate intracellularly as tubular structures, with a complex size distribution, differing substantially from the virus-like particle (VLP) preparations of the current commercial vaccines. Extensive disulfide-bond cross-linking, which is important for immunogenicity, was evident and 21-37% of total HBsAg protein displayed epitopes which correlate with vaccine potency. The significance of these results with regard to the production of cost-effective orally delivered vaccines is discussed.

Administration, Oral↗

PubMed2002

Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed.

Antigens↗