An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.     

Determination of triamterene and its main metabolite hydroxytriamterene sulfate in human urine by capillary electrophoresis using ultraviolet absorbance and laser-induced fluorescence detection

Two capillary electrophoresis methods have been developed for the direct determination of triamterene and its main metabolite hydroxytriamterene sulfate in human urine. Analytes were detected using conventional UV detection as well as laser-induced fluorescence (LIF) detection with an HeCd-laser operating at a wavelength of 325 nm. The results of both detection techniques were compared. Indeed, the limit of quantification was eightfold lower using LIF detection (50 ng/ml) in comparison to UV detection (400 ng/ml). As no interference due to endogenous urine compounds was observed, direct urine analysis was feasible. Analysis was very simple and fast-one run could be performed within less than 10 min (CE-UV method) and 2.5 min (CE-LIF method), respectively. Both assays were fully validated and applied to urine samples from a human volunteer. The results of the application of the CE-LIF method to human urine samples are presented in this publication.     

Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9

A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.     

Monitoring the effects of drug treatment in rat models of disease by serum protein analysis

In this review we list from literature investigations on rat serum proteins using electrophoretic techniques in connection with drug testing. From our own research work, we provide annotated two-dimensional maps of rat serum proteins under control and experimental conditions. Emphasis is on species-specific components and on the effects of acute and chronic inflammation. We discuss our project of structural proteomics on rat serum as a minimally invasive approach to pharmacological investigation, and we outline a typical experimental plan for drug testing according to the above guidelines. We then report in detail on the results of our trials of anti-inflammatory drugs on adjuvant arthritis, an animal model of disease resembling in many aspects human rheumatoid arthritis. We demonstrate a correlation between biochemical parameters and therapeutic findings and outline the advantages of the chosen methodological approach, which proved also sensitive in revealing "side effects" of the test drugs. In an appendix we describe our experimental protocol when performing two-dimensional electrophoresis of rat serum.     

Assessment of the performance of a fed-batch cultivation from the preculture quality using an electronic nose

An electronic nose, a gas-phase multisensor system, was used to monitor precultivations of a recombinant tryptophan-producing Escherichia coli strain. The electronic nose signals showed a high correlation toward the main stages of the precultivations, namely, exponential growth, oxygen-limited growth, and glucose depletion. Principal component analysis (PCA) of the electronic nose signals was performed and shown to be useful for monitoring preculture progression. More importantly, PCA also allowed a qualitative assessment of the preculture performance during subsequent fed-batch cultivations. The electronic nose signals from the precultures showed, furthermore, a high correlation to the time of phosphate limitation and the tryptophan yield coefficient of the subsequent fed-batch cultivations, which allowed an accurate prediction of these process variables using partial least squares (PLS). The results demonstrate on data from 12 cultivations how the electronic nose can be a useful tool for the assessment of inoculum quality, thereby providing means of reducing batch-to-batch variation and increasing the productivity of bioprocesses.     

Pharmacological comparison of a recombinant CB1 cannabinoid receptor with its G(alpha 16) fusion product

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB(1) agonist-mediated signal transduction via an engineered G(alpha 16) system is different than that of the G(i/o) coupling normally preferred by the CB(1) receptor, we transfected the human recombinant CB(1) receptor (hCB(1)) or a fusion protein comprising the hCB(1) receptor and G(alpha 16) (hCB(1)-G(alpha 16)) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB(1)-G(alpha 16) fusion protein was found to be similar to that for hCB(1): HU 210 > CP 55,940 > or = SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [(35)S]GTP gamma S binding or inhibited forskolin-stimulated cAMP, presumably by coupling to G(i/o), in cells expressing hCB(1) but not hCB(1)-G(alpha 16). However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCB(1)-G(alpha 16) but not hCB(1). These data demonstrate that ligand affinities for the hCB(1) receptor are not affected by fusion to the G(alpha 16) subunit. Furthermore, there is essentially no difference in the function of the hCB(1) receptor when coupled to G(i/o) or G (alpha 16).     

Monitoring of clinical and laboratory data in two cases of imported Lassa fever

During 2000, four cases of fatal Lassa fever were imported from Africa to Europe. In two patients, consecutive serum samples were available for monitoring of virus load and cytokine levels in addition to standard laboratory data. Both patients had non-specific early clinical symptoms including high fever. Patient 1 developed multi-organ failure and died of hemorrhagic shock on day 15 of illness, while patient 2 died of respiratory failure due to aspiration without hemorrhage on day 16. Ribavirin was administered to both patients beginning only on day 11. High serum aspartate aminotransferase and lactate dehydrogenase (LDH) levels were remarkable in both patients. Patient 1 had an initial virus load of 10(6) S RNA copies/ml as measured by real-time RT-PCR. Viremia increased steadily and reached a plateau of approximately 10(8)-10(9) copies/ml 4 days before death, while IFN-gamma and TNF-alpha rose to extremely high levels only shortly before death. In contrast, in patient 2 the virus load decreased from 10(7) to 10(6) copies/ml during the late stage of illness which was paralleled by a decrease in the IFN-gamma and TNF-alpha levels. The IL-10 level increased when specific IgM and IgG appeared. These data suggest that a high virus load and high levels of pro-inflammatory cytokines in the late stage of Lassa fever play an important role in the pathogenesis of hemorrhage, multi-organ failure, and shock in Lassa fever.     

Statistical process control for large scale microarray experiments

MOTIVATION: Maintaining and controlling data quality is a key problem in large scale microarray studies. In particular systematic changes in experimental conditions across multiple chips can seriously affect quality and even lead to false biological conclusions. Traditionally the influence of these effects can be minimized only by expensive repeated measurements, because a detailed understanding of all process relevant parameters seems impossible. RESULTS: We introduce a novel method for microarray process control that estimates quality based solely on the distribution of the actual measurements without requiring repeated experiments. A robust version of principle component analysis detects single outlier microarrays and thereby enables the use of techniques from multivariate statistical process control. In particular, the T(2) control chart reliably tracks undesired changes in process relevant parameters. This can be used to improve the microarray process itself, limits necessary repetitions to only affected samples and therefore maintains quality in a cost effective way. We prove the power of the approach on 3 large sets of DNA methylation microarray data.     

Metabolomics: quantification of intracellular metabolite dynamics

The rational improvement of microbial strains for the production of primary and secondary metabolites ('metabolic engineering') requires a quantitative understanding of microbial metabolism. A process by which this information can be derived from dynamic fermentation experiments is presented. By applying a substrate pulse to a substrate-limited, steady state culture, cellular metabolism is shifted away from its metabolic steady state. With the aid of a rapid sampling and quenching routine it is possible to take 4-5 samples per second during this process, thus capturing the metabolic response to this stimulus. Over 30 metabolites, nucleotides and cofactors from Escherichia coli metabolism can be extracted and analysed using a range of different techniques, for example enzymatic assays, HPLC and LC-MS methods. Using different substrates as limiting and pulse-substrates (glucose, glycerol), different metabolic pathways and substrate uptake systems are investigated. The resulting plots of intracellular metabolite concentrations against time serve as a data basis for modelling microbial metabolic networks.