Determination of a new antibacterial agent (Ro 23-9424) by multidimensional high-performance liquid chromatography with ultraviolet detection and direct plasma injection

Analysis of a new antibacterial agent, Ro 23-9424 (I), in plasma has been complicated by the fact that its metabolite, fleroxacin (II), is formed not only in vivo, but also nonenzymatically by the hydrolysis of the ester bond of I. In order to minimize sample preparation time and possible hydrolysis during sample preparation, a high-performance liquid chromatographic procedure was developed which features direct injection of plasma and multidimensional chromatography. The first dimension size-exclusion separation allows plasma proteins to elute with the column void volume. The second dimension reversed-phase column provides a high-resolution separation dependent upon the hydrophobicity of the sample species. With a 5-μl injection, the limit of quantitation of the method is 0.35 μg/ml for I and 0.27 μg/ml for II. The method was used to determine steady state plasma vs. time profiles for I and II from 750 mg i.v. doses of I administered twice daily.     

The low temperature,rapid dissolution of gellan away from root cultures

Summary A buffer system consisting of 50 mM Tris-HCl-TRIZMA base plus 10 mM EDTA was used to rapidly dissolve gellan gels used for maintaining transformed carrot root cultures. The optimum conditions of pH 7.5 in the presence of 10 mM EDTA for dissolving gellan were first worked out on a model test system containing 0.4% gellan, 0.025% MgSO₄·7H₂O, and blue dye. The conditions were then tested on gellan gels (0.2% gellan plus nutrients) containing carrot roots. This gel dissolution system was rapid (18 to 20 min), did not require heating, and could also be efficiently performed at 4 °C. Furthermore, the buffer system used for gel dissolution is a standard one used for plant cell fractionation studies.     

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)₅ and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.     

Leukotriene B4 binding to human neutrophils

[³H] Leukotriene B₄ (LTB₄) binds concentration dependency to intact human polymorophonuclear leukocytes (PMN's). The binding is saturable, reaches equilibrium in 10 min at 4°C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [³H] LTB₄ indicating two discrete populations of binding sites. The high affinity binding sites have a dissociation constant of 0.46 × 10⁻⁹M and B_max of 1.96 × 10⁴ sites per neutrophil; the low affinity binding sites have a dissociation constant of 541 × 10⁻⁹M and a B_max of 45.6 × 10⁴ sites per neutrophil. Competitive binding experiments with structural analogues of LTB₄ demonstrate that the interaction between LTB₄ and the binding site is stereospecific, and correlates with the relative biological activity of the analogs. At 25°C[³H] LTB₄ is rapidly dissociated from the binding site and metabolized to 20-OH and 20-COOH-LTB₄. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [³H] LTB₄ binding, suggesting that LTB₄ is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB₄ in neutrophils are tightly coupled processes.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

Inhibitors of Urokinase and Thrombin in Cultured Neural Cells

Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum-free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate-stable complexes with 125I-urokinase or 125I-thrombin. Rinsed glioblastoma possessed two components that complexed 125I-urokinase. One was type 1 plasminogen activator inhibitor (PAI-1), because the 125I-urokinase-containing complexes were immunoprecipitated with anti-PAI-1 antibodies. The other component formed complexes with 125I-urokinase that were not recognized by antibodies to PAI-1 or protease nexin-1 (PN-1). Its identity is unknown. In addition to these cell-bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with 125I-urokinase; one was PAI-1, and the other was PN-1. The secreted PN-1 also formed complexes with 125I-thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes with either 125I-urokinase or 125I-thrombin. However, human neuroblastoma cells did contain very low levels of PN-1 mRNA and PN-1 protein. Added PN-1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN-1 and blocked the ability of PN-1 to form complexes with 125I-urokinase. Thus, cell-bound PN-1 was a specific thrombin inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosphingolipids of normal and leukemic human leukocytes

Summary We have reviewed the studies on neutral glycosphingolipids and gangliosides of normal and leukemia human leukocytes. In this review, we examine (a) the glycosphingolipid composition of various leukocyte populations, (b) the differences in glycosphingolipids found among subsets of these cells, (c) the possible use of these compounds as markers of differentiation, and (d) the changes in glycosphingolipid composition that occur with leukemogenesis.     

THE INTERACTION OF AUXIN AND ETHYLENE IN THE MAINTENANCE OF LEAF BLADE FORM IN PHASEOLUS VULGARIS L. VAR. PINTO

Auxin treatment results in hyponastic curvature of the primary leaves of Phaseolus vulgaris L. var pinto. Ethylene production by hyponastic leaves is detected within 1 hr after treatment with IAA in concentrations at or above 1 μm. The amount of ethylene detected is proportional to the concentration of auxin applied. Untreated control leaves and leaves treated with 2,3,5-tri-iodobenzoic acid or gibberellic acid did not produce ethylene detectable by our equipment. The hyponastic curvature induced by auxin treatment can be inhibited by exogenous application of ethylene or ethylene-generating compounds, and these treatments produce epinasty in auxin-treated leaves. Treatment with inhibitors of ethylene synthesis or action, such as aminoethoxy-vinylglycine, carbon dioxide, or heat treatment, prolong hyponasty. The planar form, therefore, appears to be affected by both hyponastic auxin effect and an epinastic ethylene effect.