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Describes a device for preparing cell spreads. Steps involved in preparing a suitable cell suspension; Features of the apparatus used in the process; Instrument's use of centrifugal force to speed up the sedimentation of the cells.

It was shown previously that cell elongation in the roots of certain calcifuge grasses was inhibited by concentrations of bicarbonate and malate which promoted cell elongation in relate calcicole species. The present paper reports experiments in which [2-[sup14]C]-acetate was supplied to excised roots of a calcifuge Deschampsia flexuosa and a calcicole species Arrhenatherum elatius. Time course studies of the pattern of [sup14]C incorporation into the organic and amino acids of th roots indicates an active tricarboxylic acid cycle in both species but differing in that in Deschampsia the respiratory malate pool was more completely compartmented from other malate pool in the cells, whereas in Arrhenatherum a progressive mixing of the malate pools occurred Treatment of roots with a pulse of [2-[sup14]C]-acetate followed by exposure to 10 m-equiv./I HCO[sup-,sub3] for 3 hours resulted in a substantial modification in the pattern of [sup14[C incorporation from acetate in Deschampsia but not in Arrhenatherum. The results are discussed in relation to the differential effects of bicarbonate on root growth in the two species. [ABSTRACT FROM AUTHOR]

We have further characterized the polynucleotide ligase purified from cultures of the human heteroploid line EUE [Spadari, Ciarrocchi and Falaschi, Eur. J. Biochem. 22, 75 (1971)]. The 350-fold purified enzyme gives positive response with four different assays; the rates with the different substrates are different, but the variations are identical to those observed with the purified ligase induced by T4-phage. The enzyme can reconstitute the transforming activity of Bacillus subtilis DNA inactivated by pancreatic DNAase. The human cell enzyme is unable to use a hybrid substrate where an interrupted polydeoxynucleotide is annealed to a polyribonucleotide, whereas in the same conditions the T4 enzyme gives appreciable activity. The partial dependence of the purified enzyme on proteins present in a boiled crude extract, reported in the previous paper, seems due to an aspecific protective effect of the soluble proteins of cell extracts. The enzyme does not show any appreciable sequence specificity in the phosphodiester bond it can form. The purified enzyme can be fractionated into two molecular forms, one having a molecular weight of 190000, the other 95000; fresh extracts of EUE cells contain almost exclusively the high molecular-weight form; ageing of the extract or purification lead to the appearance of the second peak, without variations in the total activity. This could correspond to the conversion of a dimer structure into a monomer. The “dimer” has a pH optimum close to 8.1, whereas the “monomer” has its optimum at 7.5; this explains the bimodal pH curve previously described in the purified enzyme. [ABSTRACT FROM AUTHOR]

After administration of 14C-labelled 2-deoxy-D-glucose or ³H-labelled nucleosides to cells in tissue culture the following activated intermediates of the deoxysugar have been isolated and characterized: the 6-phosphate derivative, the 1-phosphate derivative, the 1,6-bisphosphate derivative. UDP-2-deoxy-D-glucose, GDP-2-deoxy-D-glucose and 2-deoxy-D-gluconic acid 6-phosphate. After extraction with acid and organic solvents. 2-deoxy-D-glucose could be recovered by acid hydrolysis from the cells. Labelled 2-deoxy-D-galactose was not found either bound to UDP or to the cells, indicating that no significant epimerisation of 2-deoxy-D-glucose at the UDP level occurred in these cells. [ABSTRACT FROM AUTHOR]

The results presented in this paper establish relationships between structural morphological and functional properties of cytoplasmic membrane vesicles isolated from an Escherichia coli unsaturated fatty acid auxotroph. The membranes were isolated from cells grown in the presence of either oleic, linoleic, linolenic or elaidic acids. High-angle X-ray diffraction studies show that the order-disorder transitions induced by temperature variations and associated with the paraffin chains of the lipids are a function of the fatty acid composition of the membranes. In some cases "cocrystallization" of various lipid species takes place within a single type of ordered domains. In other cases there is segregation of various lipid species into more than one distinct type of ordered domain. The various order-disorder transitions observed induce morphological changes in the hydrophobic core of the membranes which can be detected by freeze-etch electron microscopy. A random distribution of particles on the fracture faces is observed when the paraffin chains of the lipids are disordered. Upon ordering of the paraffin chains, particles are excluded from the ordered domains and as a consequence, smooth areas and areas with densely packed particles are observed. The ratio of the smooth surface to particulated surface is proportional to the amount of ordered paraffin chains present. Moreover, the size of the smooth domains is a function of the fatty acid composition of the membranes. Discontinuities in the rate of D-lactate-dependent proline uptake as a function of temperature correlate with the order-disorder transitions observed. The high energies of activation at low temperatures are attributed to decreased mobility of the carrier proteins upon aggregation. In contrast, phosphoenolpyruvate-dependent vectorial phosphorylation does not respond to the ordering of the paraffin chains. [ABSTRACT FROM AUTHOR]

This paper describes further confirmation of the successful application of the macrophage electrophoretic mobility (MEM) test to the detection of malignant disease. Results from fifty-one patients and eighteen controls are presented. The apparatus and technique used for the measurement of macrophage elctrophoretic mobility differed from those previously used in other investigations. Further information is presented on the nature and behaviour of macrophage slowing factor(s), and experiments concerning the possible use of cells other than guinea-pig macrophages as indicators of slowing factor production are also described. [ABSTRACT FROM AUTHOR]

This section presents technical abstracts on police science. In the article, Multiple Thickness Cell Assemblies--Application to Ultraviolet Spectrophotometry, D. D. Tunnicliff has devised multiple cell assemblies with cells of varying thicknesses to take advantage of varying cell length rather than concentrations in ultraviolet absorption spectrophotometry. The article, Plastic Vessel for pH Measurements of Small Samples, by Kenneth M. Richter, furnished dimensions and sketches of an adapter shaped to the Beckman standard electrodes for samples as small as 0.25 ml. A shoulder in one well of the adapter serves to prevent damage to the calomel electrode. The paper, Control of Fine Chemicals and Pharmaceuticals--Report for Analysts by W. B. Fortune, was presented at the Symposium on Analysis of Fine Chemicals and Pharmaceuticals at the American Chemical Society meeting in Atlantic City, New Jersey in September, 1956. The paper points out the increasingly difficult problems of identification and analysis of the many new drugs and chemicals being produced. Classical methods are inadequate, and new methods are hardly keeping pace with development and production of new products. Of particular interest to the Forensic Chemist or Criminalist are the comments about the need of applying confirmatory tests and not relying on single or inadequate identification tests.

The article discusses the study regarding the distribution of the Bacillariophyceae algae living on the sediments of some lakes of the English Lake District. The author mentions that the study measures the seasonal change in total diatom population and in certain genera by obtaining the summation of the counts of cell numbers of all species and other individual genera. The study confirms the reports of earlier papers that the benthic epipelic diatom community goes through distinct seasonal changes including low winter growth, high spring growth, and moderate autumn growth.

Ficoll, a polymer of sucrose, and bovine serum albumin have been compared as media for preparation of density gradients for separation of lymphoid cells. Direct vapour pressure osmometer measurements demonstrated that Ficoll at high concentrations has a marked effect on the osmolarity of the medium in which it is dissolved. Albumin, prepared in the manner we have described in earlier papers of this series, has no effect, and density gradients prepared with this albumin do not contain gradients of osmolarity. Antibody forming calls give basically similar density distributions after separation in both media, apart from a slight overall shift to the dense end due to shrinkage in Ficoll. Heterogeneous, multiple peak distribution profiles, or relatively simple, homogeneous profiles may be obtained in both media, depending on the source of antibody-forming cells, the stage of differentiation of the population, and the degree of resolution of the gradient. Antibody-forming cell density heterogeneity is a property of the cell population, rather than an artifact of the separation system. Different animal species have a wide variation in the osmolarity of their sera. Albumin-gradients of osmolarity which were not iso-osmotic for the mouse caused a general shift in the density of mouse cell populations but no marked change in the relative distribution of cells. However, a change from iso-osmotic conditions did affect the ability of cells to reband on a second gradient. The use of gradients iso-osmotic for the particular species under study is recommended as a general procedure. [ABSTRACT FROM AUTHOR]

The present paper deals with some of the quantitative aspects of immunoglobulin formation in vitro. The results of this study show that isolated spleen cells synthesize about 1.25 μg IgG per 108 lymphoid cells during the first 6 hours of incubation. The initial rate of synthesis was not maintained throughout the 48 hours of incubation, but decreased markedly after 6 hours. Comparison of the intensity of the autoradiographic line with the amount of radioactive immunoglobulin in the precipitation line revealed that a very distinct line (gradation + + +) corresponds to about 0.0125 μg IgG. The limit of visibility (+) is reached at approximately 0.0002/μg radioactive IgG. [ABSTRACT FROM AUTHOR]

The Mie theory of scattering is used to provide new information on how changes in particle volume, with no change in dry weight, should influence light scattering for various scattering angles and particle sizes. Many biological cells (e.g., algal cells, erythrocytes) and large subcellular structures (e.g., chloroplasts, mitochondria) in suspension undergo this type of reversible volume change, a change which is related to changes in the rates of cellular processes. A previous study examined the effects of such volume changes on total scattering. In this paper scattering at 10 degrees is found to follow total scattering closely, but scattering at 45 degrees , 90 degrees , 135 degrees , and 170 degrees behaves differently. Small volume changes can cause very large observable changes in large angle scattering if the sample particles are uniform in size; however, the natural particle size heterogeneity of most samples would mask this effect. For heterogeneous samples of most particle size ranges, particle shrink-age is found to increase large angle scattering.

1. Nuclei were prepared from frozen rat liver by a modification of the technique of Behrens, and were studied with regard to the content of free amino acids and nucleic acid. 2. Under rigorously controlled conditions, preparations of nuclei are obtained by the Behrens' method which form a gel in the presence of 5 or 10 per cent NaCl or of water plus a small amount of dilute alkali; whereas when conditions are less rigorously controlled, nuclei are obtained which form no such gel. The property of forming gels with alkali is probably characteristic of all cell nuclei which have not undergone autolysis. 3. Nuclei prepared by the Behrens' technique contain the enzymes arginase, catalase, and esterase in very appreciable concentrations. 4. The free amino acids of the isolated cell nuclei, as well as of other liver cell fractions, have been investigated using the technique of paper chromatography. 5. The chromatographic patterns of the free amino acids of whole cells, ground cytoplasm, and isolated cell nuclei were very similar or identical. A feature of interest in these chromatograms was the faintness or absence of the spots due to a number of the essential amino acids, as compared to the intensities of the spots due to glycine, alanine, and glutamic acid. Glutathione was present in the isolated nuclei as well as in the whole cells. 6. Chromatograms made from hydrolysates of nuclei showed high concentrations of the essential amino acids and were similar to chromatograms of hydrolysates of typical proteins.

An RNA ligase which is dependent on ATP and Mg2+ for maximal activity with 5'-32P-labeled poly(I) · poly(C) as substrate has been purified from Escherichia coli infected with phage T4. Ligase activity was demonstrated by the transfer of alkaline phosphatase sensitive 5'-32P termini into an alkaline phosphatase resistant form. The formation of phosphodiester bond was verified by hydrolysis of the reaction product and determining the transfer of 32p from the 5' position to the 2'(3') position by electrophoresis. An increase in sedimentation rate of poly(I) · poly(C) was also observed after reaction with RNA ligase. The enzyme also showed an ATP-PP1 exchange activity. The activity on the synthetic polyribonueleotides poly(A), poly(U), poly(A) · poly(U) and poly(I) · poly(C) was examined and an increased efficiency of the substrate was obtained when [5'-32P]poly(U) formed a complex with poly(A). [5'-32P]Poly(A) was a good substrate both with and without the complementary strand. With 5'-32P-labeled poly(I) · poly(C) as substrate RNA-ligase activity was detected in mammalian cells such as KB, HeLa and L-cells. The activity seems to increase after infection with encephalomyelocarditis (EMC) virus or poliovirus, but not aider infection with adenovirus type 2. The RNA ligase activity was confined to the cytoplasmic fraction of the cells. [ABSTRACT FROM AUTHOR]

HeLa cells, prelabeled with [³H[leucine for several generations, were pulse-labeled with [14c]-leucine. The 14C/³H ratio of the leucine in the medium, in the total amino acid pool and bound to transfer RNA was determined.It was found that the 14C/³H ratio of the leucyl-tRNA was identical with that of the extracellular medium while that of the amino acid pool was much lower. These results confirm previous reports that selection of amino acids for protein synthesis takes place before the amino acids are released intracellularly.Further, it is concluded that amino acids resulting from intracellular proteolysis are not reutilized directly under the conditions of our experiments.A possible model of intracellular amino acid pathways is discussed. [ABSTRACT FROM AUTHOR]

Lymph node cells from immunized rabbits were cultured with varying concentrations of antigen in preheated (56°, 30 minutes) autologous serum which had been collected before immunization. [3H]Thymidine was present for the last 6 hours of the 24-hour culture period and the radioactive labelling of acid-precipitable material was then determined. Changes in labelling due to variations of culture conditions were interpreted according to whether they were specific for control or antigen-treated cultures or non-specific. Cell concentration and serum concentration were predominantly non-specific variables influencing the labelling in control and antigen-treated cultures to a proportionate extent. However, at serum concentrations below 5 per cent labelling was disproportionately inhibited in antigen-treated cultures; there were further minor disproportionate inhibitions at higher serum concentrations. Labelling was inhibited by increasing the concentration of serum from 25 to 50 per cent, mainly due to a non-diffusible competitive inhibitory activity. Isotope-dilution analysis of the effects of serum on labelling over a wide range of serum concentrations indicated that the relationship was a complex one with at least three step-wise stimulations of the maximum labelling rate being produced by increasing the serum concentrations from 0 to 25 per cent. Labelling in antigentreated cultures containing post-immunization serum was less than labelling in cultures containing an equal volume of preimmunization serum, but labelling in control cultures was enhanced by post-immunization serum. These results are shown to be compatible with the proposals (i) that labelling in control cultures reflects the response of cells to low concentrations of endogenous antigens, and (ii) that preimmunization serum and post-immunization serum contain ‘natural’ and ‘acquired’ antibodies respectively, which normally buffer cell-borne receptor sites against reaction with endogenous and exogenous antigens. [ABSTRACT FROM AUTHOR]

Immediately after plucking the hairs from a mouse, with its skin in the resting phase of the hair growth cycle, both the labeling index and incorporation levels of ³HTdR are lowered. This depression persists until stimulated cells enter S, usually about 10 hours after plucking. These observations are confirmed using a combination of liquid scintillation and autoradiographic procedures. The dpm per punch of fixed skin, the labeling index of the basal layer, the grains per labeled basal layer cell and the proportion of labeled DNA are all reduced following plucking. The dpm per punch of fresh skin is concomitantly enlarged by plucking through an increase in the fixative-soluble pool, which may be due to the induction of vascular changes. Anesthesia apparently reduces slightly the incorporation levels into fixed unplucked skin. The vascular changes and anesthesia depression of incorporation cannot account for the depression in incorporation into fixed tissue which must be due to a more direct effect on DNA synthesis. The data could be explained on the basis of a reduction in the rate of synthesis combined with a block to entry into S or to progression through S. Using the autoradiographic and liquid scintillation techniques presented in this paper and published previously, a number of calculations were made regarding the proliferative population size in the epidermis and hair follicle. The cycle time for basal layer cells in unplucked skin is estimated to be 141 hours with an upper limit on the growth fraction of 0.92. [ABSTRACT FROM AUTHOR]

A procedure to isolate Escherichia coli mutants defective in ribonucleotide reduction is described. Using a parental strain defective in dCTP deaminase (paxA) and thus unable to synthesize dUMP from deoxycytidine nucleotides, we selected for cells unable to reduce UDP at a rate sufficient to satisfy the cell's need for thymidine nucleotides. This was accomplished by selecting for cells resistant to 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine (trimethoprim) in the presence of thymidine and screening these cells for a thymidine requirement that could be satisfied by deoxyuridine. Genetic analysis as well as enzyme assays of four such mutants chosen for study indicated that three had a defective thymidylate synthetase. One, LD195, which had normal thymidylate synthetase activity, was found to have a conditional deoxyuridine requirement that is only expressed under favourable growth conditions. Since restoration of a functional dCTP deaminase to derivatives of LD195 eliminated the deoxyuridine requirement, it was concluded that a combination of two mutations is responsible for the expression of this phenotype. Analysis of nucleotide pools of LD195 grown exponentially under permissive conditions revealed that both the dTMP and dTTP pools were 6-fold lower than in the parent indicating a deficiency in the production of a thymidine nucleotide precursor. Enzyme assays of crude extracts of LD195 indicated that this deficiency was due to a defective ribonucleosidediphosphate reductase. This conclusion is proven by an extensive enzymatic analysis presented in an accompanying paper. [ABSTRACT FROM AUTHOR]

In this paper the metabolism of amino sugars in the retina and in the pigment epithelial cells has Been studied. Extracts obtained from the retina and from the pigment epithelial cells are capable, in the presence of suitable cofactors, of converting [1.14C]glucosamine into UDP-N-acetylhexosamines. Chromatographic investigations with the use of Dowex resin column have permitted the identitication of the principal intermediaries of amino sugar metabolism: GlcNAc, GIcN-6-P, Glc-NAc-6-P, UDP-GlcNAc, UDP-GalNAc. Both the retina and the pigment epithelial cells are, further, capable of synthesizing those amounts of acetyl coenzyme A necessary for the formation of acetylated amino sugars. The metabolism of amino sugars in the retina would appear to be much more active than in the pigment epithelial cells; this would indicate that the greater part of the synthesis of mucopolysaccharides of the interstitial matrix takes place in the retina. In the course of this research, furthermore, the presence of three compounds has been ascertained, the identification of which is still in progress. [ABSTRACT FROM AUTHOR]

Using the labeled CO2 trapping method, tryptophan hydroxylase activity was detected in the nucleated thrombocytes of the domestic fowl, but not in the mammalian platelets. This enzyme activity was around a 5-hydroxytryptophan production of 60 pmol×mg protein-1××h-1. Certain properties such as optimal pH (7.0), Km value (12 μM), requirement for cofactor (2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydroptetidine) and inhibition by p-chlorophenylalanine indicated a close resemblance to tryptophan hydroxylase from brain tissue. Solubilization of this enzyme was to some extent successful by digitonin treatment of the cells. [ABSTRACT FROM AUTHOR]

This work shows that Pseudomonas fluorescens can synthesize two different inducible enzymes which possess β-ketoadipate: succinyl-CoA transferase activity. Transferase I is operative in the activation of free β-ketoadipic acid and is specifically induced in cells grown either with this acid, or with an aromatic precursor (benzoate) oxidized through the β-ketoadipate pathway. Transferase II is specifically induced m cells grown with saturated dicarboxylic adds of chain length C6 through C10, and its physiological function is that of an adipate :succinyl-CoA transferase; its ability to activate β-ketoadipic acid reflects non-specificity with regard to the CoA acceptor. [ABSTRACT FROM AUTHOR]

This article describes a technique for growing algae and flagellata cultures. There are five biological groups of microorganisms that may be grown in soil-water cultures. Equipment needed include hard glass, tubes, and larger containers such as glass cylinders or milk bottles. Organic substances supplying food materials are placed at the bottom of the vessel and covered with soil in a bid to prevent excessive putrefaction. An appropriate source of nitrogen and carbon compounds are casein, gelatine and similar substances. An advantage of this technique is that the organisms to be cultivated are chosen and separated from other forms at the outset. The cells to be used for inoculation should be passed through a sterile fluid to avoid contamination of the cultures with small algae.

Lymph node cells from Black Hooded (BH) rats immunized with Freund's complete adjuvant (CFA) are only poorly cytotoxic to target cells treated with target cell specific antibody. Normal spleen cells are highly cytotoxic to antibody coated target cells. When 106 lymph node cells, from rats immunized with Chang cells in CFA, are mixed with 107 spleen cells from unimmunized rats, the mixture is 8.6 times more cytotoxic than the sum of the cytotoxicities of the two lymphoid cell populations alone. Immune lymph node cells were no more cytotoxic in the presence of 5 per cent fresh guinea-pig serum than in the presence of heat inactivated guinea-pig serum. Immune spleen cells are prevented from damaging target cells by antagonists of protein synthesis such as Puromycin. Puromycin, however, does not alter the cytotoxic effect of either immune or nonimmune spleen cells on antibody coated target cells. From these experiments it is tentatively suggested that lymphoid cells from immunized animals mediate target cell damage by a reaction involving firstly immunologically specific responder cells which synthesise antibody and secondly non-specific effector cells which damage the antibody coated target cells. Evidence is presented which suggests that stable soluble factors may not be responsible for mediating target cell damage in this system. [ABSTRACT FROM AUTHOR]

A number of serum protein fractions were tested for their ability to suppress the immune response of rats injected with washed sheep cells. Significant depression of the immune response was produced by human serum proteins, and by bovine α-globulin fractions. The mechanism of this effect is at present not clear.

The article focuses on the physiological prerequisites required for the transition of autotrophic plants from water to terrestrial life. It is stated that the presence of cells with central vacuoles seems to be a prerequisite for invasion of land by plants. An inner aqueous medium is provided by their cell sap which determines the status of protoplasmic hydration. There is an reemphasis of the fact that the protoplasm is the site of all vital processes. Therefore, it is important to study the hydration of protoplasm.

In mechanically synchronized L-cells, the activity of thymidine kinase and dCMP deaminase shows a distinct periodicity during the mitotic cycle. The rise in enzyme activities occurs at about the onset of DNA synthesis, 6 hours after mitosis, and reaches a maximum 16 hours after mitosis. The maximum rate of thymidine incorporation into DNA is found at 10 hours. The increase of enzyme activity is 6 fold for thymidine kinase and 5 fold for dCMP deaminase. A rapid fall of both enzyme activities is observed between 18 and 22 hours, coinciding with the second synchronous mitosis. The increase in enzyme activities is probably due to de novo synthesis, as judged by the effect of actinomycin D and puromycin. It. is suggested that during the presynthetic phase of the cell cycle, messenger RNAs are synthesized for these enzymes. The enzymes in turn are required for the following DNA synthesis. [ABSTRACT FROM AUTHOR]

A procedure is described which can be employed to isolate the thermosensitive mutants of Escherichia coli affected in protein synthesis. The technique involves the enrichment of cells unable to propagate MS2 phage at 42 °C and the subsequent screening for thermosensitive growth and protein synthesis. Two of the mutants isolated in this way, QL-152 and QL-199, were studied in some detail, The synthesis of glutamyl-tRNA by QL-152 supernatant enzyme appeared to be thermosensitive. In the case of QL-199 a 65% reduction of the glutaminyl-tRNA synthesis was measured. Moreover QL-199 cells contain an elongation factor EF-Tu which is more thermolabile than wild-type EF-Tu. [ABSTRACT FROM AUTHOR]

A method for the separation of isoenzymes by counter-current distribution in aqueous biphasic systems is described. The phase systems used contained dextran and trimethylamino-poly(ethylene glycol). The method has been tested with three isoenzyme systems, namely, lactic dehydrogenase, matate dehydrogenase and enolase. Separation of isoenzymes in extracts of tissue or cells as well as in a mixture of commercial isoenzyme preparations were successfully achieved by this technique. [ABSTRACT FROM AUTHOR]

Minicells and filaments of Escherichia coli strain P678-54 provide a convenient means of preparing cell-envelope fractions that are, respectively, greatly enriched or reduced in material derived from the cell poles. The inner (cytoplasmic) membrane, outer membrane, and sacculus were prepared both from minicells and from filaments purified from a single culture. One protein component and phosphatidylglycerol were found to be enriched in minicell inner membrane. [ABSTRACT FROM AUTHOR]

Low-molecular-weight RNA in the 4--5-S range is present after all incubation times, but without a distinct 4-S peak while 5-S ENA increases in amount with time of incubation. An additional low-molecular-weight RNA possessing a molecular weight of about 0.77 × 105 was detected in preparations of nucleolar RNA labelled for at least 3 h. RNA from chromosomes I--III contains, besides HRNA and low-molecular-weight RNA, RNAs with molecular weights 1.55 × 106 ("30-S"), 0.96 × 106 (23-S) and/or 0.72 × 106 (18-S) representing (pre)-rRNA species. "30-S" and 18-S RNA are also present in the nuclear sap while a distinct 23-S peak is absent. Although in whole larvae mature rRNAs can only be found after 1-h labelling, clear rRNA peaks in cytoplasmic RNA from salivary glands are not found earlier than after a 6-h incubation. An RNA of molecular weight 0.65 × 105 first observed in nucleolar RNA preparations and accumulated in cytoplasm is suggested to be (pre)-rRNA formed during processing by an endonucleolytic activity.

A quantitative in vitro method for phytohaemagglutinin (PHA) induced lymphocyte transformation is described. This method has the following advantages. Cultures require as few as 105 lymphocytes and are performed in microtrays, over a short incubation period (48 hr). A short pulse time of 4 hr with [³H] thymidine is utilized. This enables high levels of [³H]thymidine to be maintained throughout the labelling period, ensures maximum incorporation of thymidine into cellular DNA, and diminishes cellular damage by internal irradiation. The extraction process has been simplified by using a communal washing procedure after drying the cultures on glass-fibre filter discs. The procedure is both quicker and more reproducible (coefficient of variation 6%) than extraction and drying on filtration manifolds (coefficient of variation 23%). The effect of adjusting variables such as the number of cells, incubation time, concentration of [³H]thymidine, concentration of PHA, specific activity of [³H]- thymidine and duration of [³H]thymidine pulse has been studied in order to approach optimal labelling conditions for the assay system. The reproducibility of the method has been investigated by repeated testing of normal individuals on a day-to-day basis and over extended periods. The mean coefficient of variation for samples repeated daily and over longer time intervals (weeks or months between samples) was 15%. [ABSTRACT FROM AUTHOR]

Immunoglobulin molecules on the surfaced mouse thymus cells have been shown by immunofluorescence. Ninety-ave to 100 per cent of thymus lymphocytes were found to bind polyvalent rabbit and-mouse imrnunoglobulin, although the density of the fluorescence was much less than that on the surface of B lymphocytes derived from the spleen. Two purified antisera, an anti-IgM and an anti-kappa chain strum, also stained the cells. The resynthesis of these immunoglobulin molecules in vitro was demonstrated after they had been removed with pronase. [ABSTRACT FROM AUTHOR]

Guinea-pigs made tolerant to dinitrochlorobenzene (by prior intracardiac injection of dinitrobenzene sulphonate) failed to give positive skin reactions after contact sensitization, but nevertheless had peritoneal cells which reacted with the hapten in macrophage migration inhibition (MMI) tests. This reactivity was blocked in vitro by the addition of serum from tolerant animals but not by serum from hyper-sensitive animals. Cells from hypersensitive guinea-pigs were anomalous, in that their reaction with hapten in MMI was not blocked by tolerant serum. Hypersensitive serum, though not active by itself in MMI, was able to prevent blocking by tolerant serum when the two sera were mixed. This was interpreted as an `unblocking' phenomenon and suggested that hypersensitive cells were insusceptible to blocking because they themselves produced an unblocking substance (antibody ?), although preliminary efforts to demonstrate this directly were not successful. Hypersensitive serum had an analogous activity in vivo, since when passively transferred to otherwise tolerant animals it enabled them to produce typical skin reactions; that is, it broke tolerance. Tolerance or non-reactivity in vivo in the situation investigated thus appears to be an enhancement-like process, characterized by the presence of reactive lymphoid cells and a blocking factor (antigen-antibody complex?) detectable in the serum of the tolerant animals. [ABSTRACT FROM AUTHOR]

The article offers information on the research living cells grown in tube. A research was conducted at the National Institutes of Health in the United States, of devising an artificial circulatory network that allows them to grow living animal cells in the laboratory to density that resembles natural body tissue. Scientists involved to the research include Richard A. Knazek, Peter O. Kohler, and William R. Kidwell. With the research many types of human and animal cells can be grown in the test tube.

The effects of nucleotide analogues on DNA synthesis ere studied in nucleotide permeable Escherichia coli cells. 2′,3′,Dideoxyribosylthymine 5′-triphosphate inhibited replicative DNA synthesis far more strongly than endonuclease-induced DNA repair synthesis. In ϕX174-infected cells this analogue caused the formation of short pieces (7 S) of ϕX replicative form DNA in which newly synthesized DNA was found covalently linked to preformed primer. Deoxyuridine triphosphate led to even shorter pieces (3 S) of ϕX replicative form DNA. Nicotinamide mononucleotide, an inhibitor of E. coli DNA ligase, promoted accumulation of short (10 S) E. coil DNA pieces. Newly synthesized 5′-triphosphate ends were not detected in these nucleotide-permeable cells during the early stages of ϕX replicative form replication, nor was evidence obtained that nuoleoside monophosphates are immediate precursors of DNA. [ABSTRACT FROM AUTHOR]

Two DNA specific exonucleases, DNase III and DNase IV, are present in mammalian cell nuclei. The activity of the purified enzymes with ultraviolet-irradiated polydeoxynucleotides and DNA as substrates has been investigated. DNase III shows a very poor ability to release pyrimidine dimers, although excision can be demonstrated by using high concentrations of enzyme. In contrast, DNase IV liberates dinners in an effective fashion in vitro, and is a likely candidate to serve in this function also in vivo. The pyrimidine dimers are released by DNase IV as parts of oligonucleotides containing 5-8 residues. Neither of the two exonucleases is markedly inhibited by caffeine. [ABSTRACT FROM AUTHOR]

In a system of synchronously dividing mouse fibroblasts (L cells), the activity of several enzymes was determined in relation to initiation of DNA synthesis. Synchrony of DNA synthesis and cell division was obtained by selective mechanical detachment of cells in mitosis, thereby avoiding the use of blocking agents. The activity of DNA polymerase under assay conditions permitting terminal or replicative activity to occur shows characteristic fluctuations throughout the life cycle of the cell: whereas the terminal enzyme activity exhibits a peak at about 12 h, the replicative form of the enzyme has its maximum 16 h after mitosis. DNA synthesis starts at about 9 h and reaches its peak at 16 h after mitosis. The two enzyme fractions examined (particulate and supernatant) prefer native DNA as primer. The supernatant enzyme exhibits exclusively terminal activity with heat-denatured DNA as primer. The degradation of TTP by TTPase is subject to characteristic fluctuations in the mitotic cycle, whereby a decrease of TTPase activity of the particulate enzyme fraction in the S-phase (phase of DNA synthesis) may contribute to a preservation of the TTP pool, thus acting as an additional regulatory mechanism. DNase activity remains nearly constant throughout the mitotic cycle. [ABSTRACT FROM AUTHOR]

Alterations in the phosphorylation of [³H]uridine and in RNA synthesis resulting from restrictions in the availability of glucose as the only source of energy were studied in rat thymus cells in vitro and compared to the influence of prednisolone injected in vivo on both parameters. The following results were obtained: 1. In the absence of glucose, an impairment in the phosphorylation of [³H]uridine is observed after 15-30 rain of incubation. A corresponding decrease; in the formation of RNA is not clearly established before 60 min of incubation. 2. Fluctuations in the concentration of glucose in the incubation medium between 1.0 mg and 0.1 mg of glucose/ml produce only modest changes in the capacity of rat thymus cells to synthesize RNA. 3. Phosphorylation of [³H]uridine and RNA synthesis appear to be inhibited in a synchronous rather than a sequential fashion after the administration of prednisolone. 4. The phosphorylation of [³H]uridine is also inhibited in rat thymus cells under the influence of prednisolone when RNA synthesis is completely blocked by actimycin D or by incubation of thymus cells at 0°. 5. on the other hand, thymus nuclei from prednisolone-treated rats which were depleted of their capacity to phosphorylate [³H]uridine by repeated treatment with a buffer solution containing Triton X-100, still displayed an impairment in their capacity to utilize [³H]ATP for RNA synthesis as compared to corresponding nuclear preparations from normal animals. 6. The steroid-mediated decrease in the phosphorylation of [³H]uridine does not reflect a direct, steroid-effect on the kinases but appears to be caused by changes in the structural environment to which these enzymes are normally bound. Our results appear to indicate that prednisolone exerts its catabolic or antianabolic effect on rat thymus cells by at least two distinct mechanisms, one of which involves a decrease in precursor phosphorylation while the other leads to an impairment of RNA synthesis. [ABSTRACT FROM AUTHOR]

The labeling kinetics of rRNA in exponentially growing Tetrahymena pyriformis were analyzed quantitatively in order to determine pool-sizes and turnover rates of nuclear 17 and 25 S rRNA. The mathematical analysis of the system indicates that, during the steady state of growth, there is an inverse proportionality between pool-size and turnover rate of nuclear rRNA. Thus, from the labeling kinetics of rRNA and the growth rate constant (4.6 × 10-3 min-1), the pool-size of macronuclear 17 S rRNA was estimated at 0.5% of that of cytoplasmic 17 S rRNA corresponding to a turnover rate constant of 0.92 min-1 The analogues figures for 25 S rRNA were estimated at 1.6% and 0.29 min-1. Since the RNA content of macronuclei is about 2% of the cellular RNA content, it is concluded that during growth, most nuclear RNA is found in precursors of ribosomes and that macronuclei contain few, if any, finished ribosomes. Apart from its nsc on rRNA, the method described here may be generally useful in determining pool-sizes and turnover rates of other precursors, such as free ribosomal proteins. [ABSTRACT FROM AUTHOR]

A yeast strain, enriched and isolated from soil using phenol as major carbon source, was identified as Trichosporon cutaneum. After growth on phenol washed cell suspensions of the organism oxidized phenol and some phenol derivatives without delay, others after varying lag periods. Cell-free preparations from the yeast strain were capable of hydroxylating various phenols as judged by a substrate- and NADPH-dependent oxygen uptake. Certain properties and the cofactor requirement of this hydroxylating system were investigated. Diphenols were hydroxylated at rates considerably higher than monophenols. Mono-halogen substituted phenols were hydroxylated at rates comparable to that of phenol hydroxylation. The relative hydroxylating activities with respect to different phenol derivatives were markedly changed after 3-5 weeks' storage, thus indicating the presence of several hydroxylating enzymes. The cell-free preparations also contained the enzyme catechol 1,2-oxygenase. Results of studies with intact cells and cell-free preparations indicated that not only phenol but also resorcinol, quinol and catechol were further hydroxylated prior to ring fission. Catechol when supplied as substrate of catechol 1,2-oxygenase preparations, was oxidized by the "ortho type" of ring fission. The enzyme involved has broad substrate specificity. [ABSTRACT FROM AUTHOR]

Fatty acid synthesis in B. subtilis and E. coli is compared. Cell free fatty acid synthesizing systems from both organisms require malonyl-CoA, NADPH and a "priming" substrate. In the case of E. coli the priming substrate is acetyl-CoA and the products of fatty acid synthesis are saturated and unsaturated straight-chain fatty acids. B. subtilis synthesizes branched-chain fatty acids predominantly and uses branched-chain acyl-CoA esters as priming substrates. The fundamental difference between the fatty acid synthesizing systems of these two organisms lies in the specificity of the initial acyl-CoA:acyl cagier protein transacylase and the role this enzyme plays in dictating the nature of the fatty acids formed. 1. The utilisation of various acyl-CoA primers by a B. subtilis cell-free fatty acid synthesizing system was studied. This system was unable to utilize acetyl-CoA. However, if acetyl acyl carrier protein was used as the priming substrate, the initial acyl-CoA:acyl carrier protein transacylase was by-passed and acetate was rapidly incorporated info straight-chain fatty acids. 2. Similar experiments using a cell-free system from E. coli indicated that the substrate specificity of the E. coli acyl-CoA:acyl carrier protein transacylase was much less critical. The branched-chain CoA esters and intermediates were, as a whole, poorly utilized by the E. coli system. [ABSTRACT FROM AUTHOR]

The flux rates of Pi from blood plasma into the extracellular and intracellular space of mouse liver have been measured in vivo with the [32P]Pi-tracer technique. The specific radioactivities of plasma Pi and total liver Pi have been measured 0.1-4 min after rejection of carrier-free [32P]Pi. The results have been treated with the analogue computer technique. The main conclusions are: 1. The flux rate of Pi from blood plasma into the extracellular liver space amounts to 1.14 µmoles and that from the extracellular into the intracellular liver space to 0.39 µmoles per g fresh liver per min. 2. The influx of Pi through the liver cell membrane takes place without transient transformation into another phosphate compound rapidly exchangeable with large intracellular phosphate pools. 3. The specific radioactivity of intracellular Pi is somewhat higher than that of [γ-32P]ATP, thus indicating the absence of intracellular Pi-compartmentation with isotope kinetic relevance. [ABSTRACT FROM AUTHOR]

In cultured human cells (Chang, liver) labeled thymidine is incorporated during short labeling periods into a cytoplasmic DNA, presumably located in the mitochondria, with a buoyant density of 1.688 g/ml (nuclear DNA main band 1.700 g/ml). This DNA is apparently metabolically labile and becomes completely delabeled after a chasing period of approximately three cell generations. During such a chase period an increasing amount of radioactive label appears in closed circular duplex DNA (buoyant density 1.706 g/ml). Pulse ([Me-³H]thymidine)-chase-pulse ([2-14C]thymidine) experiments suggest, that the "light cytoplasmic" DNA is a precursor of the closed circular duplex DNA. [ABSTRACT FROM AUTHOR]