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PubMed2000

Mass spectrometry applied to the analysis of estrogens in the environment.

Environmental analytical chemistry has recently changed focus from analysis of non-polar, persistent contaminants (e.g. polychlorinated biphenyls and dioxins) to more polar and labile compounds that interfere with biological processes. For example, natural and synthetic estrogens and their metabolites have been detected in sewage treatment plant effluents at nanogram/liter concentrations that are similar to those at which both total sex reversal and intersex (containing both testes and ova) is induced in fish exposed to these compounds in laboratory experiments. The development of techniques for the analysis of natural and synthetic estrogens in biological fluids (i.e. serum and urine) has been a priority in the biomedical field. However, the recent recognition that estrogen hormones are contaminants in the environment that may contribute to endocrine disruption has focused attention on the need for highly sensitive and specific techniques that are applicable for trace analysis in complex environmental matrices. Three optimized mass spectrometric protocols have been developed for the determination and quantitation of steroid hormones in environmental matrices using gas chromatography/tandem mass spectrometry (GC/MS/MS), liquid chromatography/mass spectrometry selected ion monitoring, (LC/MS - SIM) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The advantages and disadvantages of each method are presented.

Chromatography, Liquid↗
PubMed2000

Modeling of dose-response-time data: four examples of estimating the turnover parameters and generating kinetic functions from response profiles.

The most common approach to in vivo pharmacokinetic and pharmacodynamic modeling involves sequential analysis of the plasma concentration versus time and then response versus time data, such that the plasma kinetic model provides an independent function, driving the dynamics. However, response versus time data, even in the absence of measured drug concentrations, inherently contain useful information about the turnover characteristics of response (turnover rate, half-life of response), the drug's biophase kinetics (F, half-life) as well as the pharmacodynamic characteristics (potency, intrinsic activity). Previous analyses have assumed linear kinetics, linear dynamics, no time lag between kinetics and dynamics (single-valued response), and time constant parameters. However, this report demonstrates that the drug effect can be indirect (antinociception, cortisol/adrenocorticotropin (ACTH), body temperature), display nonlinear kinetics, display feedback mechanisms (nonstationarity, cortiso/ACTH) and exhibit hysteresis with the drug levels in the biophase (antinociception, body temperature). It is also demonstrated that crucial determinants of the success of modeling dose-response-time data are the dose selection, multiple dosing, and to some extent different input rates and routes. This report exemplifies the possibility of assigning kinetic forcing functions in pharmacodynamic modeling in both preclinical and clinical studies for the purpose of characterizing (discrimination between turnover and drug-specific parameters) response data and optimizing subsequent clinical protocols, and for identification of inter-individual differences.

Animals↗
PubMed2000

Health-related quality of life following bilateral intrastriatal transplantation in Parkinson's disease.

Intrastriatal transplantation of embryonic dopaminergic tissue is a new, experimental approach for the treatment of Parkinson's disease (PD). Clinical trials have shown longterm graft survival and therapeutically valuable improvements with decreased L-dopa dose and time spent in the "off"-phase, and reduced rigidity and hypokinesia. We have measured health-related quality of life (HRQoL) using the Nottingham Health Profile (NHP) in five patients subjected to bilateral transplantation in the caudate and putamen to explore the influence of intrastriatal grafts on HRQoL and the value of such measures in trials of restorative therapies. The results demonstrate improved HRQoL following transplantation, with individual patients showing striking improvements within different dimensions of the NHP as well as the NHP distress index (NHPD). The most pronounced improvements after grafting were observed for physical mobility along with emotional reactions and energy. These results indicate that intrastriatal transplantation of embryonic dopaminergic tissue can give rise to improvements within most areas of HRQoL, and that HRQoL measurements provide important information additional to that obtained by traditional, symptom-oriented assessment protocols. However, the optimal approach to HRQoL measurement in PD remains to be determined.

Activities of Daily Living↗
PubMed2003

Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells.

OBJECTIVE: To compare the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (BMSC) and adipose tissue-derived stromal cells (ATSC), because the availability of an unlimited cell source replacing human chondrocytes could be strongly beneficial for cell therapy, tissue engineering, in vitro drug screening, and development of new therapeutic options to enhance the regenerative capacity of human cartilage. METHODS: Quantitative gene expression of common cartilage and cell interaction molecules was analyzed using complementary DNA array technology and reverse transcription-polymerase chain reaction during optimization of cell differentiation, in order to achieve a molecular phenotype similar to that of chondrocytes in cartilage. RESULTS: The multilineage potential of BMSC and ATSC was similar according to cell morphology and histology, but minor differences in marker gene expression occurred in diverse differentiation pathways. Although chondrogenic differentiation of BMSC and ATSC was indistinguishable in monolayer and remained partial, only BMSC responded (with improved chondrogenesis) to a shift to high-density 3-dimensional cell culture, and reached a gene expression profile highly homologous to that of osteoarthritic (OA) cartilage. CONCLUSION: Hypertrophy of chondrocytes and high matrix-remodeling activity in differentiated BMSC spheroids and in OA cartilage may be the basis for the strong similarities in gene expression profiles between these samples. Differentiated stem cell spheroids represent an attractive tool for use in drug development and identification of drug targets in OA cartilage-like tissue outside the human body. However, optimization of differentiation protocols to achieve the phenotype of healthy chondrocytes is desired for cell therapy and tissue engineering approaches.

Adipose Tissue↗
PubMed2006

Kinetics of the interaction of desAABB-fibrin monomer with immobilized fibrinogen.

The soluble and stable fibrin monomer-fibrinogen complex (SF) is well known to be present in the circulating blood of healthy individuals and of patients with thrombotic diseases. However, its physiological role is not yet fully understood. To deepen our knowledge about this complex, a method for the quantitative analysis of interaction between soluble fibrin monomers and surface-immobilized fibrinogen has been established by means of resonant mirror (IAsys) and surface plasmon resonance (BIAcore) biosensors. The protocols have been optimized and validated by choosing appropriate immobilization procedures with regeneration steps and suitable fibrin concentrations. The highly specific binding of fibrin monomers to immobilized fibrin(ogen), or vice versa, was characterized by an affinity constant of approximately 10(-8)M, which accords better with the direct dissociation of fibrin triads (KD approximately 10(-8) -10(-9) M) (J. R. Shainoff and B. N. Dardik, Annals of the New York Academy of Science, 1983, Vol. 27, pp. 254-268) than with earlier estimations of the KD for the fibrin-fibrinogen complex (KD approximately 10(-6) M) (J. L. Usero, C. Izquierdo, F. J. Burguillo, M. G. Roig, A. del Arco, and M. A. Herraez, International Journal of Biochemistry, 1981, Vol. 13, pp. 1191-1196).

Biopolymers↗
PubMed2001

Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae.

An integrated approach is used to develop a rapid sampling strategy for the quantitative analysis of in vivo kinetic behavior based on measured concentrations of intracellular metabolites in Saccharomyces cerevisiae. Emphasis is laid on small sample sizes during sampling and analysis. Subsecond residence times are accomplished by minimizing the dead volume of the sterile sampling system and by maximizing flow rates through application of vacuum to the sampling tubes in addition to the overpressure in the fermenter. A specially designed sample tube adapter facilitates sampling intervals of 4 to 5 s for various test tube types. Statistical analysis of the results obtained from enzymatic and liquid chromatography mass spectrometry (LC-MSMS) analysis of the metabolite concentrations was used to optimize the sampling protocol. The most notable improvement is reached through the introduction of vacuum drying of the cell extract. The presented system is capable of reliably dealing with fermenter samples as small as 1-g with a variation of less than 3%, and is thus ideally suited for intracellular measurements on small, lab-scale fermenters.

Biomass↗
PubMed2004

Theoretical and experimental analysis of analyte transport in a fiber-optic, protein C immuno-biosensor.

Protein C (PC) is an important anticoagulant in human blood plasma, and early diagnosis of PC deficiency is critical for preventing dangerous thromboembolic complications. A fiber-optic PC immuno-biosensor has been under development in our research group for real-time PC-deficiency diagnosis. The sensor has demonstrated a good sensitivity and specificity for quantifying PC in buffered solutions. However, for plasma samples, with a limited sample reaction time, the sensor produced only 30% of the signal intensity of PC in buffer. The high plasma viscosity (1.9 cP) was speculated as the major reason for signal intensity reduction. In this investigation, the sensing performance of the fiber-optic PC biosensor is systematically characterized in terms of physical and chemical properties of the sample media. Theoretical and experimental analyses indicate that the reduced diffusion rate of PC molecules in viscous samples caused the sensing system to be more mass-transfer-limited. Convective flow of sample/reagent solutions during immunoreactions can increase the rate of the analyte mass transport from the bulk solution to the sensor surface, with reaction kinetics changing from mass-transfer-limited to reaction-limited as flow velocity increases. It was shown that PC sensor performance was significantly improved for plasma samples with convection. The effect of the flow velocity and incubation times for samples and reagents on the sensor performance was also systematically analyzed to optimize the assay protocol for PC sensing. Currently, a 6-cm-long immuno-biosensor is capable of quantifying PC in plasma (1 mL) in the heterozygous PC deficiency range (0.5 to 2.5 microg/mL) within 5 minutes, at an average signal-to-noise ratio of 50.

Biosensing Techniques↗
PubMed2007

A simplified bioprocess for human alpha-fetoprotein production from inclusion bodies.

A simple and effective Escherichia coli (E. coli) bioprocess is demonstrated for the preparation of recombinant human alpha-fetoprotein (rhAFP), a pharmaceutically promising protein that has important immunomodulatory functions. The new rhAFP process employs only unit operations that are easy to scale and validate, and reduces the complexity embedded in existing inclusion body processing methods. A key requirement in the establishment of this process was the attainment of high purity rhAFP prior to protein refolding because (i) rhAFP binds easily to hydrophobic contaminants once refolded, and (ii) rhAFP aggregates during renaturation, in a contaminant- dependent way. In this work, direct protein extraction from cell suspension was coupled with a DNA precipitation-centrifugation step prior to purification using two simple chromatographic steps. Refolding was conducted using a single-step, redox-optimized dilution refolding protocol, with refolding success determined by reversed phase HPLC analysis, ELISA, and circular dichroism spectroscopy. Quantitation of DNA and protein contaminant loads after each unit operation showed that contaminant levels were reduced to levels comparable to traditional flowsheets. Protein microchemical modification due to carbamylation in this urea-based process was identified and minimized, yielding a final refolded and purified product that was significantly purified from carbamylated variants. Importantly, this work conclusively demonstrates, for the first time, that a chemical extraction process can substitute the more complex traditional inclusion body processing flowsheet, without compromising product purity and yield. This highly intensified and simplified process is expected to be of general utility for the preparation of other therapeutic candidates expressed as inclusion bodies.

Escherichia coli↗
PubMed1989

Characterizing aquatic bacteria according to population, cell size, and apparent DNA content by flow cytometry.

Flow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high-intensity UV excitation, condensing the laser beam onto a small area, using blemish-free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high-transmittance optical filters, collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 micron 3 as calibrated by Coulter impedance. Plastic spheres down to 0.014 micron 3, 0.3 micron in diameter, were resolved. Aquatic bacteria 0.05 micron 3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA-dependent because DNase treatment eliminated most fluorescence. These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA-free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.

Bacteria, Anaerobic↗
PubMed1990

Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells.

A new method for clonal growth of Dictyostelium axenic amoebae has been developed. Cells are plated in growth medium containing 1% ultra-low gelling temperature agarose. Cells grow normally in the agarose and form colonies up to several millimeters in diameter. When the colonies have grown to a sufficient size, they begin multicellular development. Pseudoplasmodia are formed, migrate to the surface of the agar, and then undergo fruiting body formation. Cells can be removed from the soft agarose colonies with a toothpick or by picking spores from the fruiting bodies. This method should be useful for drug, auxotrophic, and temperature selections where clonal maintenance of axenic colonies is important. This method has been used in combination with a selection for resistance to G418 to isolate independent colonies following DNA-mediated transformation. Several parameters in the calcium phosphate and electroporation transformation protocols have been optimized and the transformation frequency quantified. Independent transformed colonies are obtained at a frequency of 1 in 10(4) to 1 in 10(5) cells when integrating plasmids are introduced using calcium phosphate coprecipitation. The frequency is about tenfold higher when extrachromosomal shuttle vectors are introduced into cells.

Agar↗
PubMed2004

General method for the modification of different BAC types and the rapid generation of BAC transgenic mice.

Most genome projects have relied on the sequencing of bacterial artificial chromosomes (BACs), which encompass 100-300 kb of genomic DNA. As a consequence, several thousand BAC clones are now mapped to the human and mouse genome. It is therefore possible to identify in silico a BAC clone that carries a particular gene and obtain it commercially. Given the large size of BACs, most if not all regulatory sequences of a gene are present and can be used to direct faithful and tissue-specific expression of heterologous genes in vitro in cell cultures and in vivo in BAC-transgenic mice. We describe here an optimized and comprehensive protocol to select, modify, and purify BACs in order to generate BAC-transgenic mice. Importantly, this protocol includes a method to generate, within 2 days, complex plasmid cassettes required to modify BACs, and to efficiently modify different types of BACs selected from the two major BAC libraries available. Altogether, using a combination of genomic database analysis, overlap PCR cloning, and BAC recombination in bacteria, our approach allows for the rapid and reliable generation of "pseudo knockin" mice. genesis 38:39-50, 2004.

Animals↗
PubMed2001

Detection of apoB-100 R3500Q mutation by competitive allele-specific polymerase chain reaction.

The apolipoprotein B-100 mutation R3500Q is one of the most common inherited defects causing abnormality of the lipid metabolism. We describe a one-step, single-tube PCR technique for detection of the mutation based on competition between allele-specific primers. Three oligonucleotides are used: two allele-specific primers differing in their 3' nucleotide (for the wild-type and the mutant allele) together with a common primer, resulting in simultaneous amplification of both alleles. This provided internal control of successful amplification and is expected to result in increased specificity. The allele-specific primers differ also in length, allowing us to distinguish both alleles by their size in a single electrophoretic run. For optimization of the protocol, DNAs genotyped before by oligonucleotide ligation assay were used. The individual genotypes obtained by CAS-PCR coincided fully with the ones from a referent OLA test: seven heterozygous individuals were found, 4 of them among 150 unrelated hypercholesterolemic individuals studied and other three in the pedigrees of heterozygous carriers. On the overall 160 genotypes were determined, neither false-positive (0 out of 153 non-carriers) nor false-negative (0 out of 7 carriers) results were obtained. No homozygous mutant genotypes were identified in this sample.

Alleles↗
PubMedDate not supplied

Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus expressing the 10A1 envelope protein.

BACKGROUND: Introduction of recombinant genes in the genome of primary lymphocytes by virtue of a replication-deficient retrovirus can be used in immunological studies and for cell-based gene therapy. METHODS: Packaging cells GP+E86 producing replication-deficient retrovirus incorporating the genes of enhanced green fluorescent protein (eGFP), C2gamma or C2xi, were generated by calcium phosphate-mediated transfection. Clones with the highest titres of retrovirus vector were isolated from them and their supernatants were used for transduction of PT67 cells. Primary mouse lymphocytes and T-cell hybridoma MD.45 were transduced by centrifugation with retroviral stock. The retroviral content of packaging cell supernatants was determined by dot blotting and hybridization with a DNA probe. RESULTS: PT67 cells produced approximately 50 times more retrovirus vector than the original GP+E86 clones. When retroviral stocks of PT67 and GP+E86 cells were used at 1/50 dilution and undiluted, respectively (to normalize them for retroviral RNA content), the transduction efficiency of mouse T-cell hybridoma was 40% and 5%, respectively. Centrifugation of target cells with retroviral stock at 2000 g for 60 min increased the percentage of transduced cells two- to three-fold. Within a population of cells isolated from the draining lymph nodes of an immunized mouse and reactivated with an antigen, up to 60% of CD4(+) T cells and up to 80% of B cells could be transduced with a transgene in replication-deficient retrovirus packaged by PT67 cells using the optimized gene transfer protocol. CONCLUSIONS: This protocol allows for the generation of packaging cells producing high titres of retrovirus vector. The 10A1 envelope protein is superior to the ecotropic one for the transduction of mouse lymphocytes.

Animals↗
PubMed2004

Biodistribution of the RD114/mammalian type D retrovirus receptor, RDR.

BACKGROUND: The limited expression of viral receptors on target cells is a recognized barrier to therapeutic gene transfer. Previous analysis of receptor expression has been performed using indirect methods due to a lack of receptor-specific antibodies. METHODS: In this report we have used anti-RDR antiserum to provide direct histochemical and flow cytometric analysis of the expression of RDR, which is the cognate receptor for RD114-pseudotyped vectors as well as being a neutral amino acid transporter. RESULTS: RDR was present on a range of normal tissues with relevance to gene therapy including: colon, testis, ovary, bone marrow and skeletal muscle. It was also highly expressed on immature cells present in the squamous epithelia of skin, cervix, nasal mucosa, bronchus and tonsil. Of relevance to possible germline gene transfer, we demonstrated a lack of RDR expression on male or female germ cells. RDR expression on mature hemopoietic cell subsets showed up to 5-fold variability between individuals within each lineage-with some individuals expressing low levels of RDR across all blood lineages. Both myeloid and monocytic lineages contained the highest fraction of cells expressing RDR, whereas lymphoid lineages showed the lowest. Coexpression of CD34 and RDR ranged from 2.04 to 0.44% in G-CSF-mobilized peripheral blood samples. CONCLUSIONS: As a means to optimize gene transfer protocols, biodistribution studies such as these are fundamental to enable targeting of the virus receptor most abundantly expressed on relevant populations. The inter-individual variation of receptor expression seen here also raises the possible requirement for tailor-made gene therapy protocols.

3T3 Cells↗
PubMedDate not supplied

Dynamic signal intensity changes in liver with superparamagnetic MR contrast agents.

The dynamic effects of three different superparamagnetic magnetic resonance (MR) contrast agents on liver signal were evaluated with an echo-planar imaging technique. The contrast agents were (a) USPIO (ultrasmall superparamagnetic iron oxide), which has a long blood half-life and was developed for MR imaging of lymph nodes and bone marrow; (b) AG (arabinogalactan)-USPIO, an asialoglycoprotein receptor--directed iron oxide with hepatocyte uptake; and (c) AMI-25, a conventional reticuloendothelial iron oxide agent. Dynamic liver signal intensity (SI) curves reflect different uptake mechanisms for the different agents. Receptor blocking experiments indicate that intracellular redistribution or clustering of the AG-USPIO (known to occur from electron microscopy studies) does not contribute to the decrease in liver SI. Monitoring liver SI over time with echo-planar imaging may provide a better understanding of the kinetics of the growing number of MR contrast agents and allow optimization of imaging protocols to exploit peak enhancement.

Animals↗
PubMedDate not supplied

Target definition and trajectory optimization for interactive MR-guided biopsies of brain tumors in an open configuration MRI system.

We present an imaging strategy for planning and guiding brain biopsies in an open configuration MR system. Preprocedure imaging was performed in a 1.5-T MR system and was designed to provide, in a clinically efficient manner, high resolution anatomical and functional/physiologic information for precise definition and tissue characterization of the target, aiming at optimization of the biopsy trajectory for planning a safe and accurate procedure. The interventions were performed in a .5-T open bore magnet, and imaging was optimized to provide the imaging quality and temporal resolution necessary for performing the procedure interactively in near real time. Brain biopsies of 21 patients were performed in a 10-month period. Segmentation and surface rendering analysis of the lesions and vascular structures and dynamic MR perfusion and cortical activation studies provided an efficient and comprehensive way to appreciate the relationship of the target to surrounding vital structures, improved tissue characterization and definition of the tumor margins, and demonstrated the location of essential cortex, allowing appropriate placement of the burr hole and choice of optimal trajectory. Interactive protocols provided good visualization of the target and the interventional devices and offered the operator real-time feedback and control of the procedure. No complications were encountered. Advanced methods of image acquisition and processing for accurate planning of interventional brain procedures and interactive imaging with MR guidance render feasible the performance of safe and accurate neurointerventional procedures.

Adult↗
PubMed2005

Magnetic resonance imaging artifacts caused by aneurysm clips and shunt valves: dependence on field strength (1.5 and 3 T) and imaging parameters.

PURPOSE: To evaluate artifact sizes at 3 T compared to at 1.5 T, and to evaluate the influence of scanning parameters with respect to artifact size on a 3-T magnetic resonance imaging (MRI) system. MATERIALS AND METHODS: Two aneurysm clips and five shunt valves were imaged in a water phantom at 1.5 and 3 T. At 3 T the influence of bandwidth (spin echo (SE) images) and echo time (gradient echo (GRE) images) on artifact size (area and extension in two orthogonal directions) was investigated. RESULTS: Artifact sizes increased substantially (typically 5-10 mm) at 3 T, compared to at 1.5 T, for implants entirely made of metallic materials, whereas the increase was the size less prominent (0-5 mm) for implants only partly containing metal. Artifact areas could be altered by changing the bandwidth or the echo time to about the same extent as it was affected by the increased field strength. CONCLUSION: Artifact sizes increase at 3 T, compared to at 1.5 T, depending on the type and composition of the implant, but can be substantially reduced by altering the imaging parameters. Optimization of imaging protocols to minimize artifacts is therefore important at higher field strengths.

Artifacts↗