Simultaneous determination of intact cisplatin and its metabolite monohydrated cisplatin in human plasma

Cisplatin is a cytotoxic platinum compound, used in the treatment of several solid tumors. Cisplatin and to a greater extent its hydrolysis product monohydrated cisplatin are responsible for side-effects like nephrotoxicity. A sensitive, accurate and precise method was developed to simultaneously determine cisplatin and monohydrated cisplatin in plasma. The compounds were separated by high-performance liquid chromatography and quantified by off-line furnace atomic absorption spectrophotometry. The linear ranges for cisplatin and monohydrated cisplatin in deproteinized plasma were 60-600 and 87.5-700 nM, respectively. From plasma, the mean recovery of cisplatin was 83.2% and that of monohydrated cisplatin 79.1%. The lower limits of quantification of cisplatin and monohydrated cisplatin in deproteinized plasma were 60 and 87.5 nM, respectively. Over the whole calibration range, the within- and between-day accuracy of intact cisplatin ranged from 100.7 to 111.4 and 94.8-102.0%, respectively. The within- and between-day accuracy of monohydrated cisplatin ranged from 107.1 to 113.3 and 101.4-104.9%, respectively. The within-day and between-day precision of cisplatin ranged from 3.4 to 11.5 and 7.3-10.3%, respectively. For monohydrated cisplatin, the within-day and between-day precision ranged from 3.7 to 6.2 and 5.6-7.9%, respectively. Currently, the developed assay has been implemented in pharmacokinetic studies of patients treated with cisplatin alone or in combination with other drugs.     

Clavulanic acid degradation in Streptomyces clavuligerus fed-batch cultivations

Clavulanic acid (CA) is an important antibiotic that is produced by Streptomyces clavuligerus. CA is unstable and product degradation has turned out to have a major impact on product titers in fed-batch cultivations. Three different types of experiments have been used to elucidate CA degradation under fed-batch cultivation conditions. First, the influence of individual medium compounds was examined. Second, degradation was monitored during the exponential growth phase in batch cultivations. Third, CA degradation was studied in the supernatant of samples taken during a fed-batch. In addition, data from six fed-batch cultivations were studied to derive information about CA degradation during the production phase. These cultivations were based on a mineral medium, containing glycerol, glutamate, ammonium, and phosphate as the main nutrients. The ammonium concentration had a large influence on the degradation rate constant. In addition, either changes in the substrate availability or high concentrations of ammonium or glycerol cause a major increase in the degradation rate constant. Finally, a linear and a fuzzy logic model were made to predict CA degradation rates in these fed-batches.     

Phage display selects for amylases with improved low pH starch-binding

Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display we selected alpha-amylase variants, which have the ability to bind starch substrate at industrially preferred low pH conditions. First we displayed active alpha-amylase on the surface of phage fd. Secondly we developed a selection system that is based on the ability of alpha-amylase displaying phages to bind to cross-linked starch. This system was used to probe the involvement of specific beta-strands in substrate interaction. Finally, a saturated library of alpha-amylase mutants with one or more amino acid residues changed in their Cbeta4 starch-binding domain was subjected to phage display selection. Mutant molecules with good starch-binding and hydrolytic capacity could be isolated from the phage library by repeated binding and elution of phage particles at lowered pH value. Apart from the wild type alpha-amylase a specific subset of variants, with only changes in three out of the seven possible positions, was selected. All selected variants could hydrolyse starch and heptamaltose at low pH. Interestingly, variants were found with a starch hydrolysis ratio at pH 4.5/7.5 that is improved relative to the wild type alpha-amylase. These data demonstrate that useful alpha-amylase mutants can be selected via surface display on the basis of their binding properties to starch at lowered pH values.     

Similarities and differences in the propagation of slow waves and peristaltic waves

The relationship between slow waves and peristaltic reflexes has not been well analyzed. In this study, we have recorded the electrical activity of slow waves together with that generated by spontaneous peristaltic contractions at 240 extracellular sites simultaneously. Recordings were made from five isolated tubular and six sheet segments of feline duodenum superfused in vitro. In all preparations, slow waves propagated as broad wave fronts along the longitudinal axis of the preparation in either the aborad or the orad direction. Electrical potentials recorded during peristalsis (peristaltic waves) also propagated as broad wave fronts in either directions. Peristaltic waves often spontaneously stopped conducting (46%), in contrast to slow waves that never did. Peristaltic waves propagated at a lower velocity than the slow waves (0.98 +/- 0.25 and 1.29 +/- 0.28 cm/s, respectively; P < 0.001; n = 24) and in a direction independent of the preceding slow wave direction (64% in the same direction, 46% in the opposite direction). In conclusion, slow waves and peristaltic waves in the isolated feline duodenum seem to constitute two separate electrical events that may drive two different mechanisms of contraction in the small intestine.     

Regulation of mitochondrial contact sites in neonatal,juvenile and diabetic hearts

Mitochondrial contact sites (MiCS) are dynamic structures involved in high capacity transport of energy from mitochondria into the cytosole. Previous studies revealed that in normal conditions the actual number of MiCS is in correlation with the energy requirements of the heart, particularly with those for its contractile work. Although the detailed mechanisms of signalling between the processes of energy utilisation and MiCS formation in the heart are not yet elucidated, it is known that intracellular Ca²⁺ transients are intimately involved in this crosstalk. The present study is devoted to investigation of Ca²⁺-linked MiCS formation in healthy adult hearts and in hearts with modified Ca²⁺-handling such as in developing, in juvenile and diabetic myocardium. Experiments were performed on hearts of healthy rats on the 22nd embryonal day, 1st, 4th, 7th and 14th postnatal days as well as on adult hearts. Diabetic hearts were investigated on the 8th day after streptozotocin injection (45 mg.kg^–1 i.v.) to adult rats. Intracellular Ca²⁺ movements were affected by modulation of Ca²⁺ concentration in perfusion solution (1.6 or 2.2 mmol.l^–1) in isolated, Langendorff-perfused hearts, by calcium paradox (CaP) or by replacing of Ca²⁺ by Cd²⁺ ions. Elevation of extracellular Ca²⁺ was reflected by 30.1, 10.4 and 24.1% increase in intracellular free Ca²⁺ concentration in healthy adult, diabetic and 14-day old hearts respectively. In developing hearts the amount of MiCS was culminating on the 4th postnatal day. In adult hearts, elevated calcium in the perfusion solution, CaP as well as diabetes led to a significant increase in the amounts of MiCS formed (58.1, 77.2 and 86.5% respectively; p < 0.05). Diabetic and 14-day old hearts naturally exhibited amounts of MiCS comparable to those obtained by Ca²⁺-stimulation of MiCS formation in adult healthy hearts. In contrast to healthy controls, perfusion of diabetic and 14-day old hearts with elevated Ca²⁺ as well as induction of CaP exerted little influence on MiCS formation (4.4 and 8.2% for elevated Ca²⁺; 2.9 and 10.7% for CaP; p > 0.05). A replacement of Ca²⁺ by Cd²⁺ ions lowered the amount of MiCS in healthy adult and diabetic hearts (61 and 52.2%; p < 0.05). In conclusion, during development, the formation of MiCS may be influenced by both, permanent stimulation by Ca²⁺-signalling and the availability of mCPK. In healthy adult hearts the amount of MiCS is modulated by intracellular Ca²⁺ transients in response to changes in extracellular Ca²⁺ concentration. In diabetic hearts the modulation of MiCS formation is naturally attenuated, apparently as a consequence of persisting alterations in Ca²⁺-handling.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Nucleofection as an efficient nonviral transfection method for human monocytic cells

Despite some progress in the field of gene transfer into hard-to-transfect cells, so far an efficient nonviral method for monocytes has not been available. A comparison of plasmid DNA with capped and polyadenylated mRNA for enhanced green fluorescent protein gene delivery into the commonly used monocytic cell lines U937 and THP-1 suggested that limited DNA trafficking may be the underlying cause of poor transfection results. As Nucleofector technology delivers DNA (or mRNA) straight into the nucleus, we obtained nucleofection efficiencies of up to 80% without significant cell toxicity. Moreover, as the DNA quickly reaches the nucleus, nucleofected cells were ready for analysis after only 2–6 h. The technique is suitable not only for monocytes but also for other hard-to-transfect cells.     

Improved recovery of DNA from polyacrylamide gels after in situ DNA footprinting

Methods used to date for the isolation of DNA from polyacrylamide gels are elution based, time-consuming and with low yield in DNA. This paper describes an improved system employing polyacrylamide gels made of a meltable matrix. The new system was successfully applied to in situ DNA footprinting following gel retardation assays.     

Quantification of mesna and total mesna in kidney tissue by high-performance liquid chromatography with electrochemical detection

A sensitive and selective assay for the determination of mesna and total mesna in tissue was developed and validated. After a simple homogenization, extraction and deproteinization step, mesna could be measured immediately by HPLC with an electrochemical detector provided with a sensitive wall-jet gold electrode. Total mesna (i.e., free mesna and mesna present in mesna disulfides and mixed mesna disulfides) could be measured after pre-column reduction with sodium borohydride to free mesna. The lower limit of quantification of mesna and total mesna was for both compounds 10 nmol/g. The assays for mesna and total mesna in tissue were linear over the ranges of 10-3000 and 10-10000 nmol/g, respectively. The within-day and between-day precisions of both methods were better than 9%. The within-day and between-day accuracy of the mesna assay ranged from 103.7 to 113.6%, whereas the accuracies of the total mesna assay ranged from 97.8 to 106.7%. Mesna in an EDTA containing tissue homogenate or in deproteinized tissue homogenate stored at -80 degrees C was stable for at least 12 weeks. Total mesna was stable under all conditions measured. The developed assays will be applied for the determination of the distribution of mesna and total mesna in tissues of the rat after administration of mesna or BNP7787.