G. W. Kidder

The Biochemistry of Ciliates in Pure Culture

Publisher Summary During the past years, the field of microbiology has taken its place as an important branch of investigation contributing to the knowledge of biochemistry. It seems clear that Tetrahymena and ciliates, in general, possess the enzyme systems that are somewhat comparable to those of higher animals. From a consideration of the requirements of Tetrahymena, it can be seen that successful assay procedures should be possible for the amino acids, such as, histidine, leucine, lysine, isoleucine, methionine, phenylalanine, threonine, and tryptophan. Tetrahymena is singularly suited for investigations of many phases of cellular metabolism. Much valuable information can be attained in a short time regarding animal nutrition by the use of Tetrahymena. Applications to higher animals, of information gained in this way, regarding interactions of basic materials and proportional relations of constituents of foods should prove useful. The response of Tetrahymena to many compounds of potential value in connection with chemotherapy makes it possible to use this organism as a tool.

A COMPARATIVE STUDY OF THE USE OF MICROORGANISMS IN THE SCREENING OF POTENTIAL ANTITUMOR AGENTS

The selection of compounds of possible interest as antitumor agents presents a major problem to those concerned with the chemotherapy of neoplastic disease in man. The ideal method for the selection of such compounds must not only be simple, rapid, inexpensive, and adaptable to large-scale operation, but should detect the maximum number of antitumor agents with a rate of “false positives” sufficiently low not to invalidate the use of the method as a screening procedure. The members of the Subcommittee on Microbiology, Screening Panel, Cancer Chemotherapy National Service Center (CCNSC), National Cancer Institute, Public Health Service, Bethesda, Md., have been engaged in the comparative study of several microbiological assay systems to determine the extent of the usefulness of such methods for the detection of potential antitumor agents. The application of bacterial, fungal, and protozoan bioassay methods to this problem is not a new approach; however, the majority of earlier studies have been based upon the use of a single bacterial (or other) bioassay system for this purp0se.l It should not be expected that any single in vitro system will detect all compounds exhibiting antitumor activity in any one or more diverse in vivo experimental tumor systems; this requires no elaboration. I n the expectation that any in vitro microbiological screening procedure must be based upon multiple bioassay systems covering a variety of diverse metabolic pathways, these collaborative studies were organized to include sixteen microbial

Partial purification and properties of a nucleoside hydrolase from Crithidia

Abstract An enzyme catalyzing the hydrolysis of nucleosides was found to occur in Crithidia fasciculata and was partially purified (30- to 40-fold) by treatment with either streptomycin sulfate or MnCl2, ammonium sulfate fractionation, acidification and neutralization, passage through Sephadex G-200, and isoelectric focusing. The specific activity of these preparations was about 6 μmnoles of uridine hydrolyzed per mg protein per min. Specificity for the puriue or pyrimidine base was very broad; uridine gave the maximum rate of hydrolysis. Deoxyribosides were not hydrolyzed. The enzyme is relatively stable to heat and to acidification and can be stored frozen. Hydrolysis of uridine is inhibited by borate ions and by adenosine, inosine, and guanosine, but not by cytidine or xanthosine.

Adenine deaminase of a eukaryotic animal cell, Crithidia fasciculata☆

Abstract Adenine deaminase (adenine aminohydrolase, EC 3.5.4.2) has been found to occur in Crithidia fasciculata with a specific activity higher than that of the same enzymes of bacteria and yeasts. It is remarkable for its stability to heat, exhibiting no appreciable loss of activity after 60 min at 55 °C. It occurs in the soluble portion of cell extracts but can be released into the suspending medium by osmotic and/or cold shock.

Amino acid antagonisms in Tetrahymena.

Abstract The response of Tetrahymena pyriformis W to each of the amino acids essential for its growth was tested in media of increased total amino acid concentration. Inhibition occurred in the utilization of all the required amino acids except lysine. Specific interrelationships were found to occur between certain amino acids when tested by increasing the concentration of only one amino acid at a time. Synergistic effects occur when two or more specifically antagonistic amino acids are added to the medium together. Supplying amino acids in the form of peptides had only a small effect on the inhibitions produced by other amino acids.

Deazapurines as growth inhibitors.

Summary 1. Deazaadenine (7-amino-1-imidazo( b )pyridine) inhibits the of Tetrahymena pyriformis W. This inhibition is antagonized by ? The half-maximal inhibition index is 6. 2. In glucose-containing medium, not only is there growth inhibitory but complete cytolysis occurs as the concentration of the inhibitory creases. Cytolysis can be prevented (in the lower concentrations of inhibitor) by increases in the adenine content of the medium. 3. Deazaguanine (5-amino-7-hydroxy-1-imidazo( b )pyridine) is inhibitory to Tetrahymena , but the solubility of the compound is so that effective concentrations high enough for half-maximal inhibitory cannot be obtained.

Thiosemicarbazide toxicity in mice.

Summary 1. Fatal convulsions follow the administration of doses of 20 mg of thiosemicarbazide (TSC)/kg or more to mice. 2. Semicarbazide is approximately 1/20 as toxic as thiosemicarbazide, while aminoguanidine produces no toxic symptoms at a dose of 650 mg/kg. 3. The toxicity of thiosemicarbazide is completely reversed by stoichiometric doses of pyridoxamine administered either orally, intraperitoneally or subcutaneously. The administration of pyridoxamine is effective either preceding or following the dosage with TSC at appropriate intervals. 4. Dietary B6NH2 deficiency does not influence the toxicity of TSC. 5. Pyruvic acid, α-ketoglutaric acid, γ-aminobutyric acid, glutamic acid, glucose and calcium chloride did not reverse the toxicity of TSC nor enhance the activity of suboptimal doses of pyridoxamine.

The purine phosphoribosyltransferases of Crithidia fasciculata.

The purine phosphoribosyltransferases of Crithidia fasciculata were identified and some of their properties described. The organism possesses three separate enzymes for the production of AMP, IMP, and GMP. The evidence for this comes from the observed differences in elution patterns from gel filtration columns, differences in heat sensitivity, and especially the clear separation of hypoxanthine phosphoribosyltransferase from guanine phosphoribosyltransferase by affinity chromatography on GMP-agarose. APRTase is activated most efficiently by Zn++, whereas HPRTase and GPRTase are activated most effectively by Co++. In no case did the product mononucleotides produce strong inhibition of the transferase activities.

Multiple inhibition by 6-methylpurine☆

Abstract Inhibition of the growth of Tetrahymena pyriformis by 1-deaza-6-methyl-purine is not readily reversed by adenine. This purine analog also causes the death and eventual lysis of the cells. While it does not have a lethal effect, 6-methylpurine (6MeP) is a more potent inhibitor of growth, and is reversed by high concentrations of adenine. Low concentrations are ineffective. Synergistic reversing effects on inhibition by 6MeP are exhibited by combinations of small amounts of adenine (10–15 μg/ml) with acetate. Metabolites such as sterols and phospholipids produced biosynthetically from acetate are capable of replacing the mixture of acetate plus adenine (10–15 μg/ml). Peptides, but not amino acids, give additional release of inhibition in the presence of lipid materials. It is suggested that 6MeP interferes specifically with the production of acyl or amino acyl compounds, probably by affecting the synthesis or utilization of ATP, as well as by interfering with adenine utilization for nucleic acid synthesis.

GROWTH STUDIES ON CILIATES VI. DIAGNOSIS, STERILIZATION AND GROWTH CHARACTERISTICS OF PERISPIRA OVUM

The growth characteristics of a number of species of ciliates have been reported recently in the literature. Most of these papers deal with saprozoic forms (Phelps, 1935, 1936, etc.) or with bacteria-feeders (Johnson, 1933; Kidder and Stuart, 1939b). The report which is to follow deals with observations of a quantitative nature made on the holotrichous ciliate, Perispira ovum, and represents the first attempt at an anlaysis of the growth characteristics of an obligate carnivore in bac teria-free medium, although Brown (1940) has published a brief ac count of some of the growth characteristics of Leucophrys patula, a facultative carnivore. Due to the fact that Perispira is not a common genus and its previous descriptions have been contradictory (Stein, 1859; Levander, 1894; KahI, 1926), a rather complete description of its organization will be given. This description will serve to establish the identity of our experimental organism and should eliminate con fusion in future investigations, regarding its specific designation. We agree with the views expressed by Taylor and Furgason (1938) and Furgason (1940) that the space devoted to a purely morphological de scription is warranted as a means of standardization of experimental material.