New Method for the Large-Scale Preparation of Diptheria Toxoid: Purification of Toxin

Diptheria toxin of high purity was prepared in batch cultures of 1 to 40 liters by procedures capable of processing 200-liter batches without modification. The procedure incorporates preliminary purification of the growth medium and, after deep fermentation or surface culture of Corynebacterium diphtheriae, both concentration and partial purification of the toxin by membrane ultrafiltration. Final purification is achieved by Sephadex G-100 gel filtration. Purities of 2,000 to 2,500 flocculation units per mg of protein nitrogen (260 to 410 flocculation units per unit of absorbance at 280 nm) were routinely obtained with only a 10% loss of toxin. The toxin appeared pure on immunoelectrophoresis and ultracentrifugation, and only minor amounts of lower-molecular-weight impurities were revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Significant advantages of the procedure are its rapidity and reproducibility and the fact that all stages are performed at 4 C in neutral isotonic buffer.     

The occurrence of two types of deoxyribonucleic acid polymerase activity in the main classes of nuclei obtained from the brains of infant and adult rats

1. The influence of exogenous or activated DNA template on the DNA polymerase activity in the different types of intact nuclei from rat brain tissue was determined. The different amounts or physical state of the DNA template did not produce significant differences in the relative distribution of the DNA polymerase activity between the separate groups of nuclei. 2. The DNA polymerase activities, fractionated by sucrose gradient centrifugation into enzyme A and enzyme B, were found to be present in the extracts of all types of rat brain nuclei. The distribution of these two activities in the ;particulate' and ;soluble' fractions of the separate groups of nuclei from 10-day-old and adult rats was studied. The findings are related to the DNA-synthetic activity in vivo of the intact nuclei and the possible biological functions of the DNA polymerase activities are discussed.     

Hexose phosphate synthase in trimethylamine-grown bacterium 2B2, a facultative methylotroph (Short Communication)

Hexose phosphate synthase and hexulose phosphate isomerase activities were found in trimethylamine-grown bacterium 2B2, a facultative methylotroph. The activities were separated by column chromatography of cell extracts on DEAE-cellulose. Hexulose phosphate isomerase activity was measured spectrophotometrically by using the product of the hexose phosphate synthase reaction as substrate.     

Defective transport in S-adenosylmethionine synthetase mutants of Escherichia coli

The transport of several metabolites is decreased in mutant strains of Escherichia coli (Met K, E4 and E40), which contain decreased levels of S-adenosylmethionine synthetase. The rates and extents of uptake for lysine, leucine, methionine, and α-methylglucoside in both whole cells and membrane vesicles isolated from these mutants are 2- to 10-fold lower than in corresponding preparations from wild-type cells, although proline uptake is normal. The addition of S-adenosylmethionine to cultures of strain E40 can partially restore the rate and extent of lysine uptake. Lysine transport is lower in mutant vesicles in the presence of either d-lactate, succinate, α-hydroxylbutyrate, or NADH even though these substrates are oxidized at rates comparable to those in wild-type vesicles. This suggests that the defect is not related to the ability of vesicles to oxidize electron donors, but is very likely related to the ability of mutant vesicles to couple respiration to lysine transport. In addition, temperature-induced efflux of α-methylglucoside phosphate and dinitrophenol-induced efflux of lysine are similar in both the mutant and wild-type membranes, indicating that the barrier properties of the membrane and the activity of the lysine carrier are normal.     

A Simplified Resectioning Technique

A simple and rapid technique is outlined for 20-40 mUm sectioning of epoxy embedded tissue, removal of areas of interest from the thick sections, resectiening of the selected areas for ultrastructural use. Use of the embeddmg mixture desaibed by Kushida in 1971- makes possible thick sectioning without fracturing. Mounting on glass slides with immersion oil for light microscopic observation facilitates later demouutng for isolation of selected areas. Identification of specific areas of interest is aided by an osmium intensifier stain, p-phenylenediamine. The method is useful for both localizing specific tissue areas and for systematic sampling at precise intervals.     

Reactivity of Two Gonococcal Antigens in an Automated Microhemagglutination Procedure

In studies of several hundred sera, a passive-hemagglutination technique with soluble antigen of sonically treated gonococci as the sensitizing material for tanned erythrocytes and Neisseria sicca sonically treated material as an absorbent detected gonococcal antibodies in 77% of males and 88% of females infected with uncomplicated gonorrhea. However, 6% of the sera from individuals in celibate religious orders and 18% of the sera from a group of females having cervical cultures negative for gonococci were also reactive with this procedure. Erythrocytes sensitized with an alkaline extract of gonococci reacted with 23% of the sera from infected males, 49% of the sera from infected females, and 2% of the sera from celibate females.     

A spectrophotometric study of the secondary structure of ribonucleic acid isolated from the smaller and larger ribosomal subparticles of rabbit reticulocytes

The spectrum of RNA from the smaller and larger subparticles of rabbit reticulocyte ribosomes was studied as a function of pH, ionic strength, urea concentration and temperature. It was inferred that both RNA species form short double-helical segments of not more than about 10 base-pairs in length. Not more than about 70% of the base residues may be located in double-helical segments. RNA from the larger subparticle is richer in guanine and cytosine residues and its secondary structure is the more stable. These conclusions are based on the use of double-helical RNA from virus-like particles and of unfractionated Escherichia coli tRNA as model systems.