Initiation of protein synthesis in cell-free extracts of Tetrahymena pyriformis.
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ABSTRACT:
SUBMITTER: Smith KE
PROVIDER: S-EPMC1163041 | biostudies-other | 1981 May
REPOSITORIES: biostudies-other
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Project description:1. We have examined methods necessary for preparing post-mitochondrial supernatants from Tetrahymena pyriformis strain HSM that are capable of efficient cell-free protein synthesis. 2. The requirements for optimum synthesis in these extracts are described. 3. Data relating to the kinetics of protein synthesis and the initiation capacity of these supernatants are presented.
| S-EPMC1162811 | biostudies-other
Project description:The behavior of ciliates has been studied for many years through environmental biology and the ethology of microorganisms, and recent hydrodynamic studies of microswimmers have greatly advanced our understanding of the behavioral dynamics at the single-cell level. However, the association between single-cell dynamics captured by microscopic observation and pattern dynamics obtained by macroscopic observation is not always obvious. Hence, to bridge the gap between the two, there is a need for experimental results on swarming dynamics at the mesoscopic scale. In this study, we investigated the spatial population dynamics of the ciliate, Tetrahymena pyriformis, based on quantitative data analysis. We combined the image processing of 3D micrographs and machine learning to obtain the positional data of individual cells of T. pyriformis and examined their statistical properties based on spatio-temporal data. According to the 3D spatial distribution of cells and their temporal evolution, cells accumulated both on the solid wall at the bottom surface and underneath the air-liquid interface at the top. Furthermore, we quantitatively clarified the difference in accumulation levels between the bulk and the interface by creating a simple behavioral model that incorporated quantitative accumulation coefficients in its solution. The accumulation coefficients can be compared under different conditions and between different species.
| S-EPMC8622496 | biostudies-literature
Project description:Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates.
| S-EPMC7109869 | biostudies-literature
Project description:A soluble dolichyl phosphate phosphatase from Tetrahymena pyriformis was purified about 68-fold. The enzyme appeared to be specific for dolichyl phosphate and existed in two interrelated forms, one of mol.wt. about 500000 and the other of mol.wt. about 63000. The enzyme was strongly inhibited by 5 mM-Mn2+ and was strongly stimulated by Mg2+. Tetrahymena in the exponential growth phase contained more of this enzymic activity than cells in stationary or lag phase. The dolichyl phosphate phosphatase may be loosely bound to mitochondrial membranes. Two roles proposed for this enzyme are (1) that of releasing dolichol from its phosphorylated biosynthetic form for its use in the cell as unesterified dolichol or dolichyl ester and/or (2) that of regulation of synthesis of glycoproteins or some other glycosylated compound.
| S-EPMC1163075 | biostudies-other
Project description:In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.
| S-EPMC4013096 | biostudies-literature
Project description:Toxicity evaluation is an extremely important process during drug development. It is usually initiated by experiments on animals, which is time-consuming and costly. To speed up such a process, a quantitative structure-activity relationship (QSAR) study was performed to develop a computational model for correlating the structures of 581 aromatic compounds with their aquatic toxicity to tetrahymena pyriformis. A set of 68 molecular descriptors derived solely from the structures of the aromatic compounds were calculated based on Gaussian 03, HyperChem 7.5, and TSAR V3.3. A comprehensive feature selection method, minimum Redundancy Maximum Relevance (mRMR)-genetic algorithm (GA)-support vector regression (SVR) method, was applied to select the best descriptor subset in QSAR analysis. The SVR method was employed to model the toxicity potency from a training set of 500 compounds. Five-fold cross-validation method was used to optimize the parameters of SVR model. The new SVR model was tested on an independent dataset of 81 compounds. Both high internal consistent and external predictive rates were obtained, indicating the SVR model is very promising to become an effective tool for fast detecting the toxicity.
| S-EPMC5564774 | biostudies-literature
Project description:Snake venoms possess lethal activities against different organisms, ranging from bacteria to higher vertebrates. Several venoms were shown to be active against protozoa, however, data about the anti-protozoan activity of cobra and viper venoms are very scarce. We tested the effects of venoms from several snake species on the ciliate Tetrahymena pyriformis. The venoms tested induced T. pyriformis immobilization, followed by death, the most pronounced effect being observed for cobra Naja sumatrana venom. The active polypeptides were isolated from this venom by a combination of gel-filtration, ion exchange and reversed-phase HPLC and analyzed by mass spectrometry. It was found that these were cytotoxins of the three-finger toxin family. The cytotoxins from several cobra species were tested and manifested toxicity for infusorians. Light microscopy revealed that, because of the cytotoxin action, the infusorians' morphology was changed greatly, from teardrop-like to an almost spherical shape, this alteration being accompanied by a leakage of cell contents. Fluorescence microscopy showed that the fluorescently labelled cytotoxin 2 from cobra N. oxiana was localized mainly at the membrane of killed infusorians, indicating that cytotoxins may kill T. pyriformis by causing membrane rupture. This work is the first evidence of the antiprotozoal activity of cobra venom cytotoxins, as demonstrated by the example of the ciliate T. pyriformis.
| S-EPMC7290292 | biostudies-literature
Project description:The Protozoan, Tetrahymena pyriformis, is capable of phosphorylating dolichol in the presence of CTP. Other nucleotides (ATP, UTP and GTP) were ineffective. The enzyme was activated independently by the bivalent cations Mg2+, Mn2+ and Ca2+. The Ca2+ stimulation of the enzyme activity was calmodulin-dependent. A substantial increase in the enzyme activity was seen in the presence of UTP. The apparent Km values for CTP and dolichol, as calculated from the Lineweaver-Burk plot, were 3 mM and 70 microM respectively. Although the presence of detergent was essential, at higher concentrations there was a decrease in the enzyme activity. The enzyme had two pH optima (pH 7.4 and 9.0) and at both the activity was Ca2+-calmodulin-dependent.
| S-EPMC1152507 | biostudies-other
Project description:1. Mitochondria of the obligately aerobic ciliate protozoon, Tetrahymena pyriformis strain ST, are unusual in that they possess a cytochrome oxidase system that does not react with reduced mammalian cytochrome c; the presence of cytochromes a(603)+a(3) is masked in the alpha-band region of spectra by the broad absorption band of cytochrome a(620). 2. Other haemoproteins present include cytochromes b(560), b(556), c(553) and c(549). 3. The reaction of reduced cytochrome a(3) with CO is reversed by flash photolysis, and in the presence of O(2) the subsequent oxidation of this cytochrome is followed by that of cytochrome a(603). 4. Cytochromes a(620) and b(560) also react with CO and with KCN; the latter cytochrome corresponds with that designated cytochrome o by other workers. 5. The contribution of cytochrome a(603) to difference spectra is revealed by making use of the fact that it does not react with KCN. 6. Cytochrome a(620) is unstable, and its alpha-absorption band is lost from spectra of mitochondria which have been aged or treated with ultrasound, detergents or organic solvents. 7. Possible pathways of electron transport via the several different terminal oxidases in Tetrahymena mitochondria are proposed.
| S-EPMC1174005 | biostudies-other
Project description:This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours.
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