A modification of the single radial immunodiffusion potency test (SRD) for rabies vaccines

In 1983 a WHO collaborative study showed that the single radial immunodiffusion test provided a reliable system for the assay of the glycoprotein content of inactivated rabies vaccines. We have modified the method used in the collaborative study by using round plastic plates with wells arranged in a circle. Potency estimates were made by the multiple slope ratio method and not by single slope estimation procedures adjusted for a common intercept. We carried out a statistical evaluation of the accuracy and reliability of the precipitation area calculation. We confirmed that for the purpose of the potency estimation the area calculations were sufficiently precise in most instances. By analysing the sources of variation for round and for glass plates and by testing the variability of these methods we found that the round plates were as good as the glass plates. The advantage of the round plates was mainly in the ease of their handling: the agarose solution was poured in the plates without the need for a separate mould; an integral moist chamber with these plates was readily available; the precipitation zones were measured easily using a viewer which gave fourfold enlargements and the method required less antiglycoprotein.     

Structure of Cu2(indomethacin)4 and the reaction with superoxide in aprotic systems

The copper complex of indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole acetate), a common anti-inflammatory drug, was prepared and characterized. Crystal structure determination revealed the dimeric form of the 1:2 complex, namely Cu₂(indomethacin)₄ · L₂, in the unit cell. Suprisingly, the copper-copper distance (263 pm) was very close to metallic copper (256 pm). The two coordination sites in the copper-copper axis can be readily replaced by superoxide. An intriguing similarity to Cu₂(acetate)₄ was seen.Due to the lipophilic nature of the indomethacin ligand, this copper complex reacted with superoxide in aprotic solvents. The superoxide dismutating activity was successfully demonstrated in Me₂SO/water and acetonitrile/water mixtures using the nitro-blue tetrazolium assay and pulse radiolysis. The second-order rate constant of 6 · 10⁹ M^?1 · s^?1 in strictly aqueous systems dropped only slightly to 1.1 · 10⁹ M^?1 · s^?1 when aprotic solvents were used. This is the fastest rate constant ever observed for a copper-dependent dismutation of superoxide. The KO₂-induced lipid peroxidation in both erythrocytes and liver microsomes was suppressed by 70% in the presence of 1 · 10^?10 mol · ml^?1 of Cu₂(indomethacin)₄. The inhibitory action dropped to 25% when Cu₂Zn₂superoxide dismutase was employed. The formation of copper · indomethacin in rat serum after administration of indomethacin was shown in vitro and in vivo.     

A phosphorus-31 nuclear magnetic resonance study of phosphate uptake and storage in cultured Catharanthus roseus and Daucus carota plant cells

High resolution 31P NMR spectra (103.2 MHz) of oxygenated Catharanthus roseus and Daucus carota cells grown in suspension cultures were obtained using a solenoidal perfusion probe. The spectra showed resonances for various phosphorylated metabolites such as ATP, ADP, NAD(P)(H), nucleoside diphosphoglucose, and sugar phosphates. The relative levels of the phosphorylated metabolites remained constant throughout the growth curve. No resonances for storage compounds such as polyphosphates, pyrophosphate, or phytates were observed. Two resolved resonances for Pi indicated an intracellular pH of 7.3 and 5.7 (or below) for the cytoplasm and vacuoles, respectively. The time course of Pi uptake and storage during growth in fresh culture medium was followed by studying the level of vacuolar Pi with 31P NMR (145.7 MHz). Simultaneously, the level of Pi in the culture medium was followed with radioactive 32P. C. roseus quickly takes up all the Pi from the culture medium (maximum rate 1.7 mumol min-1 g-1 (dry weight of cells]. The Pi is first stored in the vacuoles; subsequently, one part of this pool is used to keep a constant cytoplasmic Pi level while another part is apparently accumulated as an NMR invisible Pi store, probably in another cell organelle. In contrast, D. carota does not accumulate Pi in the vacuoles and consequently it takes up Pi from the medium at a much slower rate (0.05 mumol min-1 g-1 (dry weight of cells].     

Biologically addressable monolayer structures formed by templates of sulfur-bearing molecules.

We demonstrate that the combined application of Langmuir-Blodgett and self-assembly techniques allows the fabrication of patterns with contrasting surface properties on gold substrates. The process is monitored using fluorescence microscopy and surface plasmon spectroscopy and microscopy. These structures are suitable for the investigation of biochemical processes at surfaces and in ultrathin films. Two examples of such processes are shown. In the first example, the structures are addressed through the binding of a monoclonal antibody to a peptide. This demonstrates the formation of self-assembled monolayers by cysteine-bearing peptides on gold, and the directed binding of proteins to the structured layers. A high contrast between specific and unspecific binding of proteins is observed by the patterned presentation of antigens. Such films possess considerable potential for the design of multichannel sensor devices. In the second example, a structured phospholipid layer is produced by controlled self-assembly from vesicle solution. The structures created--areas of phospholipid bilayer, surrounded by a matrix of phospholipid monolayer--allow formation of a supported bilayer which is robust and strongly bound to the gold support, with small areas of free-standing bilayer which very closely resemble a phospholipid cell membrane.     

Elimination of X-ray-induced chromosomal aberrations in the progeny of female mice

Female NMRI mice were irradiated with various doses of X-rays and induced chromosome aberrations were scored in MII oocytes (Dosage: 0.222, 0.666, 2 and 6 Gy). After irradiation with 2 Gy, early zygotes were examined in the 2-cell stage; additional dominant lethals were counted and surviving embryos were examined after 13.5 days of pregnancy. 87.2% of the MII oocytes showed structural chromosomal aberrations after irradiation with 2 Gy. Surviving embryos, however, failed to show any increase in the aberration rate. This result points to (almost) complete elimination of genetically damaged oocytes and zygotes already before birth. In addition to the structural aberrations, aneuploidies were induced. Most of them, however, were hypoploidies. Hence, the study confirmed the well-known susceptibility of oocytes around the time of fertilization for induced chromosome loss. Induced hyperploidies, however, were very rare. Evidence for induction of meiotic non-disjunction was weak. In surviving embryos, no increase in numerical aberrations, either hypoploid or hyperploid was discovered. The significance of these data for the prediction of chromosomal damage due to to ionizing radiation in humans is discussed. Recent risk estimates of UNSCEAR and other agencies represent very cautious upper levels.