The induction of multinuclearity in agitated and in aging cultures of Acanthamoeba sp. Neff

Partially synchronized secondary cultures of chicken embryo fibroblasts were exposed to Rous sarcoma virus (RSV) at various times in the cell cycle. The initiation of normal virus production could be inhibited by treatment with excess thymidine or with cytosine arabinoside without any effect on mitosis. This result is consistent with the hypothesis that the provirus of RSV is DNA, although the virion of RSV contains only RNA. It was, also, found that treatment which prevented or interferred with the first mitosis after exposure to virus prevented the initiation of normal virus production. Later mitoses did not appear to be necessary. A corollary of this finding is that virus production is synchronized by mitosis and that the length of the latent period depends upon when in the cell cycle a cell is exposed to virus.     

Amino Acid Requirements of Aeromonas

Four of the 12 cultures of Aeromonas hydrophila, 5 of the 10 A. shigelloides, and 9 of the 10 A. salmonicida that were studied required arginine and lysine, among other amino acids, for their growth.     

Isolation and characterization of a cycloheximide-resistant mutant of Acanthamoeba castellanii Neff.

A cycloheximide-resistant mutant was isolated from the amoeba Acanthamoeba castellanii Neff. Drug resistance was found to be due to a ribosomal modification.     

Lysosomal Hydrolases in Acanthamoeba sp.

SYNOPSIS. Homogenates of axenic Acanthamoeba sp. had ribonuclease, phosphatase, proteinase, α-glucosidase, β- N -acetylglucosaminidase, and β-glucuronidase activities; all had acid pH optima. After isopycnic density-gradient centrifugation all showed the same relatively broad and unimodal distribution pattern, with a peak at a density of 1.17. This differs from the distribution of mitochondrial malate dehydrogenase and of peroxisomal urate oxidase and catalase. The findings are believed to reflect the occurrence of lysosomes in Acanthamoeba.     

Amino Acid Requirements and Proteolytic Activity of Streptococcus sanguis

The growth response of Streptococcus sanguis groups 1:A and 1:B in a complete chemically defined medium was not influenced by the oxygen concentration of the growth atmosphere. All of the cultures required cysteine and arginine; tyrosine and branched-chain amino acids were frequently required. Proteolysis of casein, mucin, and the anionic proteins of germfree rat saliva by S. sanguis was demonstrated. Hydrolytic activity toward casein was found in the soluble contents of the cells and in the cellular debris after disruption of the cells, with the soluble fractions exhibiting greater proteolytic activity toward casein. The soluble fractions from S. sanguis did not hydrolyze mucin, but this substrate was hydrolyzed by the cell debris fraction. When the amino acid requirements and proteolytic activity of S. sanguis and S. mutans were compared, these two oral streptococcal species exhibited distinct and characteristic differences.     

Amino Acid Uptake and Metabolism of Legionella pneumophila Hosted by Acanthamoeba castellanii

Legionella pneumophila survives and replicates within a Legionella-containing vacuole (LCV) of amoebae and macrophages. Less is known about the carbon metabolism of the bacteria within the LCV. We have now analyzed the transfer and usage of amino acids from the natural host organism Acanthamoeba castellanii to Legionella pneumophila under in vivo (LCV) conditions. For this purpose, A. castellanii was ¹³C-labeled by incubation in buffer containing [U-¹³C₆]glucose. Subsequently, these ¹³C-prelabeled amoebae were infected with L. pneumophila wild type or some mutants defective in putative key enzymes or regulators of carbon metabolism. ¹³C-Isotopologue compositions of amino acids from bacterial and amoebal proteins were then determined by mass spectrometry. In a comparative approach, the profiles documented the efficient uptake of Acanthamoeba amino acids into the LCV and further into L. pneumophila where they served as precursors for bacterial protein biosynthesis. More specifically, A. castellanii synthesized from exogenous [U-¹³C₆]glucose unique isotopologue mixtures of several amino acids including Phe and Tyr, which were also observed in the same amino acids from LCV-grown L. pneumophila. Minor but significant differences were only detected in the isotopologue profiles of Ala, Asp, and Glu from the amoebal or bacterial protein fractions, respectively, indicating partial de novo synthesis of these amino acids by L. pneumophila. The similar isotopologue patterns in amino acids from L. pneumophila wild type and the mutants under study reflected the robustness of amino acid usage in the LCV of A. castellannii.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.     

Cultivation of Acanthamoeba castellanii, Neff Strain, at High Hydrostatic Pressures

SYNOPSIS. Log-phase cultures of Acanthamoeba castellanii , Neff strain, have been maintained at elevated hydrostatic pressures over periods of several days and the population has been recounted at the end of the experimental period. A pressure of 2,000 psi (136 atm) depressed growth of the population, but was quickly reversed on release. A pressure of 4,000 psi (272 atm) severely depressed population growth, and any increase was slight and short-lasting at 5,000 psi (340 atm). Growth of the population was resumed only after an interval of 1 or more days after release.     

Mitochondrial Genetic Codes Evolve to Match Amino Acid Requirements of Proteins

Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular—and contrary to the predictions of the mutation-drift hypothesis—the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins.     

Amino acid sequence of Acanthamoeba actin

By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene.     

Correlation Between Pigment Production and Amino Acid Requirements in Bacillus subtilis

Several Bacillus subtilis W-23 auxotrophs were unable to produce wild-type pigment normally on minimal agars supplemented sufficiently for growth. This offers a reliable means for scoring genotypes.     

Carbohydrate and Amino Acid Fermentation in the Free-Living Primitive Protozoon Hexamita sp.

Hexamita sp. is an amitochondriate free-living diplomonad which inhabits O₂-limited environments, such as the deep waters and sediments of lakes and marine basins. ¹³C nuclear magnetic resonance spectroscopy reveals ethanol, lactate, acetate, and alanine as products of glucose fermentation under microaerobic conditions (23 to 34 μM O₂). Propionic acid and butyric acid were also detected and are believed to be the result of fermentation of alternative substrates. Production of organic acids was greatest under microaerobic conditions (15 μM O₂) and decreased under anaerobic (<0.25 μM O₂) and aerobic (200 to 250 μM O₂) conditions. Microaerobic incubation resulted in the production of high levels of oxidized end products (70% acetate) compared to that produced under anoxic conditions (20% acetate). In addition, data suggest that Hexamita cells contain the arginine dihydrolase pathway, generating energy from the catabolism of arginine to citrulline, ornithine, NH₄⁺, and CO₂. The rate of arginine catabolism was higher under anoxic conditions than under microaerobic conditions. Hexamita cells were able to grow in the absence of a carbohydrate source, albeit with a lower growth rate and yield.     

Eigenschaften des Elektronentransportsystems von Mitochondrien des Protisten Acanthamoeba castellanii Neff. II. Reinigung und Eigenschaften der NADH-Akzeptor-Oxydoreduktase des Elektronentransportsystems

ZUSAMMENFASSUNG. Es wird die Reinigung einer mitochondrialen NADH-Akzeptor-Oxydoreduktase (EC. 1.6.99.3) über folgende Stufen beschrieben: fraktionierte Ammonsulfatfällung, Chromatographie an DEAE-Cellulose und Molekularsiebchromatographie an Sephadex G 100. Das gewonnene Enzympräparat erwies sich als elektrophoretisch rein. Die Michaeliskonstante für NADH als einziges Substrat liegt bei 6,9 × 10⁻⁵ M NADH. Ein ebenfalls isoliertes extramitochondriales Enzym underscheidet sich vom intramitochondrialen in drei voneinander unabhängigen Eigenschaften: Substratspezifität, elektrophoretiche Mobilität, Molekulargewicht. Das isolierte intramitochondriale Enzym verhält sich gegenüber Atmungsketteninhibitoren ähnlich wie isolierte Mitochondrien.
SYNOPSIS. Mitochondrial NADH reductase (EC. 1.6.99.3) was isolated from Acanthamoeba castellanii by the following steps: fractionation by (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, and chromatography on Sephadex G 100 columns. Purity of the enzyme preparation was demonstrated by electrophoresis. With NADH as the sole substrate, the K _m= 6.9 × 10⁻⁵ M NADH. Extramitochondrial enzyme, also isolated from the ameba, differed from the mitochondrial one in substrate specificity, electrophoretic mobility, and molecular weight. The mitochondrial NADH reductase and isolated mitochondria were affected in the same way by respiratory chain inhibtors.     

Phagocytosis of erythrocytes by Acanthamoeba sp

Phagocytic recognition by the unicellular soil organism Acanthamoeba sp. (Neff strain) was examined with fresh or modified erythrocytes. Several parameters were studied of the interaction of glutaraldehyde-treated red cells with amoebae attached to glass. Attachment and ingestion steps of particle uptake were found to have differing temperature dependence. Particle-phagocyte interaction required the addition of Na⁺ or Ca²⁺ and was inhibited by high osmolarity or ionic strength. These features are similar to those previously described for mammalian macrophages. A quantitative spectrophotometric technique was adapted to the measurement of erythrocyte uptake after lysis of noningested red cells. Rates of uptake of six species of red cells spanned a 100-fold range. While untreated sheep red cells were taken up at very low rates, ingestion of red cells treated with aldehyde, tannic acid, polylysine, carbodiimide, ferrous sulfate or salt-free sucrose was appreciably increased. Some but not all of these modified red cells were previously found to interact with macrophages and insect hemocytes. Thus Acanthamoeba displays phagocytic recognition of untreated and modified erythrocytes. The results also indicate that the particle vocabulary ingested by the amoebae overlaps in part with that of certain metazoan phagocytes.     

Influence of Incubation Atmosphere on Growth and Amino Acid Requirements of Streptococcus mutans

The growth response of Streptococcus mutans representing antigenic type a or d in a chemically defined medium was influenced by the oxygen concentration of the growth atmosphere. Under controlled aerobic (1.5% O(2)) conditions these cultures attained a greater density than when the atmosphere contained 0.006% O(2) or less. The growth of S. mutans strains representing antigenic types b or c in the defined medium was independent of the oxygen concentration of the growth environment. Under the conditions used in this study, none of the strains tested could utilize ammonium ion as a sole source of nitrogen for growth. The requirement for certain amino acids and inhibition by other amino acids varied with antigenic type and relative oxygen concentration of the growth environment. Under conditions where the atmospheric oxygen was reduced to 0.0006% O(2) or less, the amino acid requirements of the cultures became either more numerous or more stringent. S. mutans strains of type c generally required the least number of amino acids, whereas cultures of type d had more numerous requirements. Nearly every culture tested under the anaerobic atmosphere was inhibited by one of the branched-chain amino acids, leucine, valine, or isoleucine. Methionine and lysine were also found to be inhibitory, particularly toward the type c strains.