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.     

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.     

Proteoglycan synthesis in organ cultures from regions of bovine tendon subjected to different mechanical forces.

Synthesis of proteoglycans by morphologically and chemically distinct regions of bovine flexor tendon was investigated in explant cultures. Proximal regions of the flexor tendon which experience only tensile forces and have low contents of proteoglycans initially exhibited relatively low rates of proteoglycan synthesis but high rates of collagen synthesis. The predominant proteoglycan produced by all proximal explants was of small hydrodynamic size and appeared similar to that extracted from proximal tissue. In contrast, explants derived from the distal tendon region, which experiences frictional and compressive forces in addition to tensile forces, and has a high content of proteoglycans, showed relatively high initial rates of proteoglycan synthesis and lower rates of collagen synthesis. These distal explants produced primarily large proteoglycans on the first day in culture. Turnover of newly synthesized proteoglycans was not detectable in proximal tissue, and was low in distal tissue. Loss of unlabelled proteoglycan from proximal and distal explants was not detected during the 12 days of culture. These observations suggest that the increase in specific types of proteoglycans in regions of tendon subjected to frictional and compressive forces is the result of elevated synthesis rates in this tissue. Two alterations in proteoglycan synthesis occurred during the 12-day culture period. (1) The rate of proteoglycan synthesis by all explants increased with time in culture. (2) The proportion of small proteoglycans synthesized by distal explants increased from 32% of the total proteoglycan produced on day 1, to 80% of that produced on day 12. Explants from proximal tendon continued to produce only small proteoglycans throughout the 12 days in culture. This switch in proteoglycan phenotype, resulting in decreased synthesis of large proteoglycans by the distal tissue, may be due to a lack of compressive forces on the cultured explants.     

Detection of BrdUrd incorporation in mammalian chromosomes by a BrdUrd antibody

Summary A bromodeoxyuridine antibody staining technique (BAT) was applied for the analysis of human chromosomes of different chromosomal band resolution. For this purpose lymphocyte cultures were synchronized and labeled with bromodeoxyuridine during the second half of the S-phase. Generally BAT was found comparable to GTG banding though some prominent GTG bands and the constitutive heterochromatin exhibit less intense staining with this technique.