Bibliothek

Notizen: We have studied the influence of strain of organism, temperature, and medium on the production of the antileukemic intracellular enzyme L-asparaginase by E. coli B grown in shaken flasks. Five strains of E. coli B exhibited wide differences in their capacities to synthesize the EC-2 form of L-asparaginase active against leukemia. For the most productive strain, when grown in a casein hydrolysate medium, maximal production of L-asparaginase occurred at 25°C. At this temperature, the organism required glycerol, glucose, or other mono-saccharides to synthesize L-asparaginase. Synthesis was stimulated when glycerol was used in place of glucose, but not in its presence. The effect of glycerol on L-asparaginase synthesis was most evident when the cells were grown at 37°C, rather than at 25°C. With 0.25% glucose, cells had a specific activity of 409 I.U./g; with glycerol cells had a specific activity of 553 I.U./g. At 25°C, both cell and L-asparaginase synthesis were increased by the use of 0.25% glycerol resulting in only a slight increase in specific activity of the cells. The addition of zinc, copper, manganese, iron, L-asparagine, L-glutamine, or L-aspartic acid had no effect on L-asparaginase synthesis in the casein hydrolysate medium. L-aspartic acid (10-2 M) enhanced L-asparaginase synthesis in a synthetic medium that lacked these metals or L-asparagine, L-glutamine, or L-aspartic acid; cells grown under these conditions had a specific activity of 90 I.U./g.In the casein hydrolysate medium, cell morphology was correlated with temperature of incubation.

Notizen: Some results of our studios on transformation of steroids by mixed culture fermentation are presented in this paper. Arthrobacter simplex was paired in turn with each of the following: Streptomyces roseochromogenes, Curvularia lunata, Absidia coerulea, and Aspergillus ochraceus. The steroid substrates examined for multiple transformation were 16α-hydroxy-cortexolone, 16α-hydroxy-cortexolone 16,17-acetonide, 9α-fluorohydrocortisone, 9α-fluorohydrocortisone 21-acetate, and 9α-fluorohydrocortisone 21-hemisuccinate. The effects of media, steroid substrate, and microbial interaction in a mixed culture on the induction and repression of steroid transforming enzymes were unique to each case studied. The reaction mechanism of the multiple steroid transformation was also found to vary from one mixed culture system to another. Two different reaction mechanisms were observed, namely, consecutive and parallel. In the former, one of the two enzymatic reactions always preceded the other, while in the latter, two different enzyme reactions occurred simultaneously, thereby giving rise to two different intermediates. Multiple transformation of steroids by a single step mixed culture fermentation has potential economic advantages.

Notizen: This investigation used the glucose oxidase system to simulate oxygen transfer rate in fermentation broths. It was demonstrated that the fungal preparation contained sufficient lactonase activity so that D-glucono-δ-lactone did not accumulate and that the rate of production of gluconic acid was proportional to the oxygen uptake rate. Enzyme concentrations of 1.5-2 g/1 were found adequate to determine oxygen absorption rates in shake flasks while maintaining the dissolved oxygen concentration of low levels. The apparent Michaelis constant for oxygen, Km(O2), was found to be 27% saturation with air; this value along with experimentally determined uptake rates could be used to calculate dissolved oxygen concentration in lieu of using a dissolved oxygen probe. Enzyme concentrations of 5 g/l were sufficient to give linear acid production and low dissolved oxygen concentrations in a bench-scale fermenter with no foaming or enzyme deactivation. The method is considered more valid and easier to employ than previously utilized techniques such as sulfite oxidation. Extension of the system to evaluating aeration effectiveness and scaleup of fermentation equipment is discussed.

Notizen: Growth kinetics of heterogeneous populations of sewage origin were studied in completely mixed reactors of the once-through type at a high concentration of incoming substrate, 3000 mg/l glucose, and in systems employing cell feedback or sludge recycle at an incoming substrate concentration of 1000 mg/1 glucose. The recycle flow rate employed was 25% of the incoming feed flow, and the concentration of cells in the recycle was maintained as closely as possible at 150% of the cell concentration in the reactor. Studies were made at various dilution rates. Throughout these studies, batch experiments using cells grown at the various dilution rates were made to determine ks and μm values. As in previous studios using heterogeneous populations, the relationship between specific growth rates μ and substrate concentration S was represented better by the Monod equation than by any other which was tested. The growth “constants” μm, ks, and Y were found to fall in the same general range as those determined in previous studies in once-through systems operated at 1000 mg/l glucose. It was observed that cell recycle, even at the relatively low concentration factor employed in these studies, greatly enhanced the flocculating and settling characteristics of the cells.

Notizen: A mathematical model for the prediction of product formation in batch processes is presented. The suggested procedure allows production of a prognose at any given moment of a run concerning the product concentration at any future moment of the same experiment. The series of subsequent prognoses thus produce a trend, and it is assumed that an analysis of the trend may provide information, which is useful in the evaluation of different decision alternatives in pilot plant batch experiments.

Notizen: A high intensity light system (HILIS) was designed and constructed to define the environmental parameters affecting production of algae. The HILIS incorporates the basic concepts of an aerobic fermenter for heterotrophic cells with high intensity illumination for photosynthetic studies. Of nine parameters considered, temperature and light intensity studies using Chlorella 71105 have been completed. Total illumination was varied from 25,000 to 300,000 lumens (30 times intensity of sunlight as measured at earth's surface) in 7.7-1, culture. The effect of illumination upon growth was measured as cell concentration and total daily algal production when operating the HILIS as a continuous system at a dilution rate of 0.91 per day.Growth may be expressed as a long function of illumination. A maximum algal concentration of 25.5g/l., dry weight basis, was attained at 300,000 lumens.

Notizen: A description is given of the design and operation of high-power magnetic drives developed to enable shaft seals and glands to be dispensed within deepculture vessels, in tissue homogenizers, and in mixing and filling processes where sterility is essential. The drives operate at speeds of 300 to 2000 rpm in volumes of 300 1. to 10 ml with clearances up to 16 mm between the pole faces of the magnets.Two types of drive are described, one in which the driving and driven magnets form an integral unit on the lid of a vessel: such vessels are used for transporting material. To intiate stirring, it is only necessary to connect a motor directly, or through a cable-drive, to the magnetic-drive assembly. In the other type of unit the driving magnet is attached permanently to the driving motor. Locating pins on the base of the motor and corresponding sockets on the lid of the vessel ensure that when the motor is in position, the driving and driven magnets are located correctly in relation to one another.The design of these drives is based on the use of multipole, ceramic magnets. The advantages of their use in such units, compared with metal magnets, are discussed. Earlier magnetic drives are also discussed and explanations offered for the difficulties formerly met in scaling up.

Notizen: Experiments were performed on a cellulose acetate ultrafiltration membrane (HF-200, ABCOR Inc., Cambridge, Mass.) to test its efficacy in concentrating and purifying a crude enzyme (trypsin) preparation. Studies were also made to determine the influence of inorganic salts, pressure, and temperature on the rate of ultrafiltration for this membrane. The results showed reductions in the rates will be encountered due to the presence of inorganic salts. However, the reduced rates were still sufficiently high to make this method extremely attractive. Operating at filtration pressures above 75 psi at, 20 to 30°C for this membrane does not show any beneficial effect in terms of ultrafiltration rates. However, at 10°C there were continual increases in the filtration rates up to 100 psi. Concentration and purification studies with trypsin yielded a concentration factor of 8.35 and a purification factor 2.35. It was shown concretely that the purification of the enzyme was due to the passage of low molecular weight proteins (below 20,000) through the membrane. Enzyme activity slightly greater than 90% was obtained: 70% was found in the concentrate and 20% in the filtrate. It is concluded that membrane ultrafiltration is an ideal simple, rapid, and economical method for the recovery of biological active substances.

Notizen: A procedure for measuring the rate of heat production from a fermentation has been developed. The method is based on measuring the rate of temperature rise of the fermentation broth resulting from metabolism, when the temperature controller is turned off. The heat accumulation measured in this manner is then corrected for heat losses and gains. A sensitive thermistor is used to follow the temperature rise with time. This procedure is shown to be as accurate as previous methods but much simpler in execution. Using this technique, the rate of heat production during metabolism was found to correlate with the rate of oxygen consumption. Experiments were performed using bacteria (E. coli and B. subtilis), a yeast (C. intermedia), and a mold (A. niger). The substrates investigated included glucose, molasses, and soy bean meal. The proportionality constant for the correlation is independent of the growth rate, slightly dependent on the substrate, and possibly dependent On the type of organism growth. This correlation has considerable potential for predicting heat evolution from the metabolism of microorganisms on simple or complex substrates and providing quantitative parameters necessary for heat removal calculations.

Notizen: A multistage tower laboratory fermentor has been constructed consisting of eight compartments separated by sieve plates. Flow of substrate and air is concurrent from the bottom to the top of the column. It, was hoped that this system could be used to reproduce, simultaneously on a continuous basis, eight distinct phases of a batch growth curve. It was believed that the extent of batch curve simulation would depend upon the character of hydraulic mean residence time of broth in the column and in the individual compartments. The expected relationship did not occur. Rather it was found that growth in the column involved residence time characteristics not only for the fluid but also for the microorganisms, and for the growth limiting substrate. Depending upon the column operation, these could be distinct and different.The purpose of this investigation was to study the residence time distribution (RTD) of the continous (fluid) and dispersed (microorganisms) phases for model systems as well as for a yeast fermentation. Various degrees of flow nonideality, i.e., fluid blackflow and dispersed phase sedimentation, were noticed. The former seems to be due to interaction of the concurrent gas and liquid flow; it is particularly dependent upon void area of the sieve plate holes. Sedimentation is probably a function of plate design as well as cell size and density. It wa concluded that for a particular plate design the gas hold-up wass controlled by superficial air velocity and was the main parameter governing the differences between dispersed and continous phase(Rt1). This conclusion was supported by a computeraided styudy utilizing a mathematical model of fluid flow to fit the growth kinetics and cell distribution observed experimentally throughout the fermentor.Some advantages of foam control in the tower fermentor by surface active compounds are mentioned. Also, suggestions are made for carrying out fermentations that have two liquid phases, such as a hydrocarbon fermentation. The possibility of closely approximating plug-flow conditions in the multistage tower fermentor, a necessary condition for batch growth simulation, is discussed from a practical point of view.

Notizen: A mixed culture of methanol oxidizing bacteria has been cultivated on simple inorganic salts medium supplemented with methanol. Optimal growth occurred at 31°C, pH 6.0-6.3, and a methanol concentration between 1 and 2 ml/1, of medium. The maximum yield was 4.5 g dw/I and the mean generation time 3.2 hr.It was estimated that 41% of methanol carbon was converted into cell-carbon, and that 73% of the inorganic nitrogen was converted to organic nitrogen.

Notizen: The current knowledge concerning the biosynthesis of chloramphenicol is discussed. Cultures of Streptomyces sp. 3022a fed 14C-shikimie acid incorporated the label to the same extent into phenylalanine, tyrosine, and chloramphenicol. Of possible precursors of the phenylpropanoid nucleus of this antibiotic only p-aminophenylalanine and DL-threo-p-amino phenylserine specifically labeled chloramphenicol. On the basis of these results a pathway for the biosynthesis of chloramphenicol is presented. The lack of specific incorporation of 15N-nitrogen from a competitive feeding experiment in which both l5N-nitrate and 14N-DL-serine were fed to growing cultures suggests that both the amido- and the nitro-nitrogen atom present in this antibiotic are derived from a common pool. Studies on the enzyme, DAHP synthetase, show that in streptomyces sp. 3022a it is not subject to feed back inhibition by either phenylalanine, tyrosine, or chloramphenicol.

Notizen: 9α-Hydroxy-19-norandrostenedionc (9α-hydroxy-Δ4-estrene 3, 17-dione) (IV) was prepared by fermentation of 19-norandroslenedione with Corynespora melanis or Norcardia restriclus. When incubated with a growing culture of Arthrobacter simplex or its acetone-dried cells, IV was converted to 9α-hydroxyestronc (VII) and 9-keto-9, 10-secoestrone (VI). 9α-Hydroxyestrone undergoes spontaneous as well as enzymic dehydration to form Δ9(11)-estrone (IX). Both VI and IX have been isolated and identified as such while VII was isolated as its 3-acetate.

Notizen: A protease, excreted by a sporogeneous strain of B. megaterium, growing exponentially in a minimum glucose ammonium medium, was isolated. It is a neutral endopeptidase, stabilized by Ca++, inhibited by o-phenanthroline, but not by di-isopropylfluorophosphate. The specificity, studied on insulin B-chain, glucagon, cytochrome c, and dipeptides substrates, indicated the need for a dipeptide backbone with both substituted amino and carboxyl groups. A requirement was observed for a nonpolar lateral chain in the amino acid whose amino group was involved in the peptide bond (Leu, Phe, Ala, He, Val). Rates of hydrolysis varied also with the amino acid whose carboxyl group was involved (e.g., His 〉 Ser 〉 Ala 〉 Gly). In complex medium, supplemented with Yeast Extract, the biosynthesis of the protease was repressed during growth, but the same enzyme was excreted during sporulation. The repression was apparently of the same nature as that controlling sporulation during and after growth (e.g., repression by a mixture of amino acids or high concentration of glucose). An asporogeneous mutant showed a normal product ion of protease under all conditions, and a low intracellular protease turnover after growth. A mutant unable to produce protease showed a normal sporulation and a high protein turnover. This protease, here termed megapeptidase, seems to be a typical growth enzyme, not related to either the sporulation process or to the protein turnover after growth.

Notizen: A description is given of a needle valve press for disrupting cells and microorganisms. The main features of the design are reproductibility and ease of control of the needle valve settings due to absence of screw threads, case of dismantling and cleaning, and the availability of a wide range of alternative piston and cylinder sizes.

Notizen: The stability of foam formed during fermentation is decisively affect ed by the nature of the nutrient media used. In froth-flotation models, (a) the foam formation time, characteristic of the tendency to foam, and (b) foam subsistence time, characteristic of the stability of foams formed, have been studied. With the utilization of these two parameters, the stability of foam from aqueous solution of several surface active components of nutrient media has been noted as a function of concentrations. Further, but, without attempting completeness, the viscosity enhancing effect of carbohydrate components, and the effect of the subsistence time of their foam, upon the stability of foam have been studied together with the correlation between “standing” time after sterilization and tendency to foam. Taking soy-bean meal as a model, the stability of foam films in function of pi I, at constant concentration, has been studied. It seems that though a proper control of the factors mentioned, nutrient media with a low tendency of foaming can be formulated.

Notizen: Cultivation of Norcardia sp., Mycobacterium phlei, and Candida lipolytica in inorganic salt solution containing n-alkanes C10-C20 as solo carbon and energy source was investigated. Generation times of 0.5-7.0 hr were typical during the exponential growth phase. The final cell concentrations (dry weight) were usually 9-26 g/l with n-alkane mixtures ranging from n-decane through n-eicosane. A linear dependence was found between the production of cell mass and the consumption of n-alkanes. The rest concentration of n-alkanes in the cell mass is in all experiments smaller than 0.5% (w/w). Cell yields were Ysub 60-142% and for Ye 50-97% based on n-alkane utilization. In one case, with the Nocardia NBZ 23, the substrate specifity on hydrocarbons and on a n-alkane mixture C10-C20 was studied. The cell mass recovered from the fermentations contained 47.8-57.7% carbon, 5.6-9.95% nitrogen, 7.2-9.4% hydrogen, 35-62% crude protein, and 6-36% lipid. Cellular protein and lipid synthesized by an organism is influenced by the type of nitrogen source. The amino acid, glucosamine, muramic acid, 2,6-diaminopimelinic acid, and fatty acid distribution in organisms grown on n-alkanes compared with a corresponding fermentation on glucose as sole carbon source were also estimated.

Notizen: Mathematical models which can be used to describe batch growth in fermentations with two liquid phases are developed for systems in which the growth limiting substrate is dissolved the dispersed liquid phase. In the models, the possibilities of growth occurring at the surface of the dispersed phase and in the continuous phase are considered. It is assumed that the composition of the dispersed phase is such that substrate utilization from it causes little of no change, in interfacial area. Three special cases are examined. In the first, it is assumed that all growth occurs at the surface of the dispersed phase. In the second and third, it is assumed that growth occurs both at the interface and in the continuous phase. The second case assumes that substrate equilibrium is continuously established between the two phases while the third assumes substrate consumption in the continuous phase is limited by rate of transport of substrate to that phase. Comparison of the first model with available experimental data shows good agreement between model and data.

Notizen: A number of improvements have been made in a totally-automated antibiotic bioassay machine previously described. The new machine accepts unmeasured, untreated, opaque suspensions of fermentation beers three times faster (120 samples per hour) and supplies printed potencies sooner (in just over two hours). Whereas the original machine employed a self-cleaning filter and used disposable two milliliter beakers, this version involves a batch-dialysis scheme for effecting sample purification, and provides for automated cleaning of incubation chambers.In operation, a measured, portion of thoroughly-mixed fermentation beer is automatically diluted and transferred into one side of an incubation chamber, the two halves of which are separated by a dialysis membrane. The other half is filled with inoculated media. During the two hour incubation at 37°, dialyzable antibiotic limits growth of the inoculum in proportion to its concentration. After incubation, the turbidity of the inoculum is simultaneously read by an online computer and plotted on a strip chart recorded. The computer suplies printed potency values and sample identification on site, while the recording provides the operator with an analog record of turbidity. Fiber optics are employed in the turbidmetric readout, and an electric typewrite provides the printout.

Notizen: An engineering-scale procedure for the recovery of 300-400 g batches of mixed transfer ribonucleic acids is described. Semicontinuous growth of E. coli K-12 MO7 yielded 77 kg of harvested cells in four days. Phenol extraction and ethanol precipitation recovered a crude tRNA material that was further purified by DKAE-cellulose chromatography in runs of 1 × 106 A260 units each on a 6 × 30 in. column using a 240 1, gradient elution. The purified tRNAs were partially concentrated and resolved into three groups.

Notizen: The presence of active small particles, such as bacterial cells, in a liquid will affect the rate of gas-liquid interfacial mass transfer. A theoretical analysis of the situation is presented in this article.

Notizen: Streplomyces griscus var. X-2455 produces an antibiotic complex which is active in vitro against a number of gram-positive and gram-negative bacteria and in mice against systemic infections caused by K, pneumoniae and D, pneumoniac. In view of the favorable chemotherapeutic index and the broad in vitro spectrum of crude concentrates, isolation of the pure antibiotic complex and the individual constituents was undertaken. The antibiotics referred to as Ho 5-2667, Ro 7-7730, and Ho 7-7731 can be differentiated by tle, ultraviolet light absorption spectra, and in vitro antibacterial activities. They all contain iron and may be classified as sideromycins.From antibiotic concentrates an antibacterially inactive substance was isolated and identified as N-acetyltyramine.

Notizen: A brief review of the development of the research on erythorbic acid fermentation was presented. A previously proposed scheme of the acid biosynthesis has been proved to be correct. D-glucono-γ-lactone dehydrogenase was purified to 50 fold and compared with other lactone dehydrogenases. For the purpose of commercial development, screening and mutagenie treatments of strains and studies on fundamental cultural conditions were carried out. Penicillium, but no other genera, was obtained as a producer. The experiments of ultraviolet irradiation and various cultural conditions were successful in elevating the yield of the acid over 40% in jar-fermentor to glucose supplied. The continuous multibed extraction system of anion-exchange resin was developed and a yield of 19.2% of the acid from fermentation broth was obtained.

Notizen: Bromination of the thiazole ring by the enzyme chloroperoxidase, produced by the fungus Caldariomyces fumago, was demonstrated. Both 2-acetoacetamido-4-methylthiazole and 2-acetamidothiazole were brominated on C-5 of the thiazole ring in the presence of chloroperoxidase, bromide, and hydrogen peroxide in 0.06 M Phosphate solution at pH 3.0. No reaction occurred in the absence of enzyme.

Notizen: Red kidney bean (Phascolus vulgaris) cells, derived from roof, callus, were grown in suspension culture in shake flasks and in laboratory fermentors using batch and continuous batch culture techniques. The medium contained casein hydrolysate, sucrose, inorganic salts, vitamins, and growth hormones. In continuous batch culture yields of up to 171 g wet weight, (8.5 g dry weight) per liter were obtained in 7 days. Organic nitrogen was used preferentially. Growth on nitrate was considerably slower than on organic nitrogen sources. Indole acetic and naphthalene acetic acids were not essential for good growth of the cells whereas kinetin and 2, 4-D were. The optimum pH for growth was about p11 4.5. The presence of amylase and peroxidase was detected in culture filtrates. Amylase activity was low in either the presence or the absence of starch in the medium. Peroxidase production could be related directly with growth of the culture. Maximum peroxidase yield, as measured by the guaiacol method and expressed as horse radish peroxidase, was 1.25 × 10-8 M.

Notizen: Batch experiments were run using heterogeneous populations to determine whether a hyperbolic equation of the type suggested by Monod could be used to depict the relation between specific growth rate, μ, and NH3-N concentration when ammonia N was the growth-limiting nutrient. The heterogeneous populations employed were developed from sewage seed grown on glucose at various levels of nitrogen and various dilution rates in completely mixed continuous flow reactors. It was found that the hyperbolic function could be used. Values of μm in the range of 0.4-0.7 hr-1 were observed, and values of Ks, in general, ranged from 1.5 to 4.0 mg/l. Variation in the values of these growth “constants” did not follow any discernible pattern related to past growth history (i.e., COD:N ratio or dilution rate at which the cells were previously grown).

Notizen: Heterogeneous populations of sewage origin were grown continuously at, dilution rates from 1/12 hr-1 to dilute-out (1/1 hr-1) using glucose (1000 mg/l) as carbon source and three concentrations of NH3-N as the nitrogen source (COD:N = 70:1, 40:1, and 25:1). The effects of nitrogen level and growth rate (dilution rate) on substrate removal, biological solids production, cellular carbohydrate and protein, and NH4+-N in the effluent were examined. It was found that the optimum level of nitrogen supplementation for the synthetic nitrogen-deficient waste employed should not be based solely on the desired effluent quality with respect to COD removal but should include due consideration of reactor detention time (or dilution rate) and the allowable (or desirable) level of nitrogen leakage in the effluent.

Notizen: A synthetic waste (with glucose as carbon source) devoid of a source of nitrogen was purified in a laboratory scale pilot plant by a new modification of the activated sludge process. The process makes use of a separate carbon assimilation (oxidative assimilation) phase and an endogenous phase in which ammonia is added to a portion of the settled sludge and non-nitrogenous products stored in the cells in the assimilation phase are converted to protein. It was found that sludge so treated, when recycled to the assimilation tank, could carry out continuous oxidative assimilation of the waste. Various COD:N ratios were studied. At the highest, 70:1, 90% purificaton efficiency was achieved.

Notizen: Continuous saccharification of Solka Floc (cellulose pulp) in single and four-vessel stirred-tank reactor systems has been possible employing enzymes obtained directly from submerged fermentation of Trichoderma viride QM 6a. Studies on the effect of modification of the solid substrate, enzyme stability, substrate concentration, and the influence of reducing sugar concentration on the rate of hydrolysis are reported. While susceptibility of substrate to digestion is not affected by heating alone, it is strikingly increased by heating plus grinding, or by grinding following heating. Batch and steady state continuous saccharification experiments have yielded more than 5% reducing sugar in the effluent with a dilution rate of 0.025 hr-1 at 50°C, at a substrate level of 10%. An average glucose concentration of 3.4% has been obtained in the effluent of a continuous saccharification using 5% substrate at the same dilution rate and temperature.

Notizen: Kinetic studies on fermentation processes were made and a general equation of production rate was newly presented applying the kinetic theory on mierobial cell growth which was reported previously by the authors.l,2 Equations for product concentration in fermentation time courses were derived by developing mathematically the general equation of production rate, and characteristic properties of fermentation processes were clarified. Some examples of fermentations were analyzed kinetically using the new kinetic theory. The calculated values of product, and cell concentrations were in good agreement with the observed values.

Notizen: Amyloglucosidase (EC. 3.2.1.3), partially purified from an Aspergillus species, was chemically attached to DEAE cellulose using the bifunctional reagent 2-amino-4,6-dichloro-s-triazine. The action of the insolubilized enzyme derivative on dilute maltose and dextrin solutions was studied in a packed bed. A second and deeper bed was used to demonstrate the possibility of a continuous process for raising the dextrose; equivalents of “glucose” liquors of high concentration formed by acid hydrolysis of maize starch.

Notizen: Acid soluble extracts obtained at 30 min intervals from cells of C. utilis growing in synchrony in a phased culture (cycle time 51/2 hr) were fractionated on a Dowex-1-formate column. The series of fractionation profiles showed changes in number and amounts of components over the cell cycle. Transient accumulations of numerous components over the complex pool were observed. The significance of the changes are discussed in relation to practical applications and cell metabolism.

Notizen: The participation of polyketides in the biogenesis of natural products has long been bolstered by chemical analogies. Many isotopic tracer studies have validated the acetate-polymalonate route, via presumptive extended poly-β-carbonyl intermediates, to a variety of fungal metabolites. Though implicit as antibiotic precursors, the ephemeral polyketides have not been isolated, nor perhaps with the exception of acetoacetate, can oligoketides become incorporated intact into secondary metabolites. However, a prototypical oligoketides in its stable lactone form, methyltriacetic lactone (3, 6-dimethyl-l-hydroxy-2-pyrone), has been obtained from the tropolone-producing mold P. Stipitatum. A convenient synthesis of this metabolite, by methylation of triacetic lactone followed by partition chromatographic separation of the resultant positional isomers, has been devised. In an experiment with 14C-formate, it was shown that the hypothetical, enzyme-bound polyketide precursor to methyltriacetic lactone is probably involved in stipitatie arid formation, and that the origin of the “extra” methyl or methyl-derived carbons of both substances arises from the identical “C1” pool. Radioactive tracer experiments concerning the biogenesis of pulvilloric acid, a fairly unstable antibiotic substance produced by P. Pulvillorum, showed that its exocyclic carboxyl is formed following initial methyl transfer, whereas the ring system of the molecule is essentially acetate-polymalonate derived. In order to test the hypothesis that methyl-branched C14 polyketide precursors to pulvilloric acid exist and may become integrated into the fatty acid multienzyme complex, presumptive fatty acid congeners to pulvilloric acid such as. 1-methylmyristie, 4-methyllauric, or 2-methyllauric acids were sought. These substances were, however, absent from the mycelial fatty acid spectrum, as well as from the fatty acid moieties of a crystalline glyceridc mixture obtained from the beer. Alternative approaches to the detection or isolation of polyketides are discussed.

Notizen: The research carried out by several scientists has made possible the industrial preparation of β-carotene by fermentation. A fungus, Blakeslea trispora, abundantly synthesizes carotenoids when its two opposite forms are cultivated together in a special fatty medium. When ionones or other natural substances are introduced into the culture, a very obvious increase in the biosynthesis of carotenoids, more specifically of β-carotene, is obtained.Our own work has shown that; (1) several synthetic products chemically related to β-ionone, such as 2,6,6-trimethyl-l-acetyleyelohexene, can advantageously replace either partially or totally the ionones as inductors of the biosysnthesis of β-carotene; (2) various nitrogen-containing substances when added to the culture medium can considerably enhance the biosysnthesis of carotenoids while sometimes very specically orienting it. Their action comes on top of that of the ionones or their substitutes; actually this action is unexplained.Thus certain amides, imides, lactams, hydrazides, or substituted pyradines, and in particular succinimide and isonicotinoylhydrazine, have produced a two or threefold increase in the quantity of β-carotene present in the culture media of Blackeslea trispora.Conversely some heterocyclic substances such as pyridine itself or imidazole totally inhibit the biosysnthesis of β-carotene but induce the production of very important quantities of lycopene.

Notizen: One of the kinteic equations derived previously from a series of sophisticated batch and continuous alcohol fermentations by using a respiration-deficient mutant of baker's yeast is as follows: \documentclass{article}\pagestyle{empty}\begin{document}$$ {{dp} \mathord{\left/ {\vphantom {{dp} {dt}}} \right. \kern-\nulldelimiterspace} {dt}} = v_0 e^{ - k_2 p} \left[{{S \mathord{\left/ {\vphantom {S {\left({K_s ^\prime + S} \right)}}} \right. \kern-\nulldelimiterspace} {\left({K_s ^\prime + S} \right)}}} \right]X $$\end{document} where dp/dt = ethanol production rate, v0 = specific rate of ethanol production at p = 0, k2 = empirical constant, K′s = saturation constant, S = glucose concentration, and X = cell mass concentration. The above equation was confirmed in the previous paper to fit, the brewing of “sake.”The temperature of the specific brewing is not always constant (10 to 18°C). The effect of temperature on v0 was assessed from the Arrhenius plot, assuming that k2 was independent of temperature. Values of dp/dt taken from the “sake” brewing data were rearranged, taking the temperature change into account. These datu, corrected for the temperature, were found to follow quite favorably the kinetic equation mentioned above. So far, a prediction of the ethanol production rate in practice was rectified to the extent of p = 19%.

Notizen: Utilization of n-heptane by a Pseudomonad was studied in pilot-size butch cultures. Optimal pH and temperature were determined by a factorial design and a medium based upon mineral uptake rates was formulated. High cell yields were obtained by volatilizing heptane in the incoming air and thereby achieving good hydrocarbon dispersion. Hydrocarbon carried by effluent gases was recovered and recycled. In cultures where pH is not controlled, decrease in the electrolytic conductivity of the medium was found to be indicative of viable cells and was used in monitoring bacterial propagation. If not checked, increase in salinity in pH controlled cultures was found to affect cell production negatively. Viscosity changes were not very significant. Heptane to aqueous medium ratio was found to affect oxygen supply to the system due to higher dissolved oxygen concentrations associated with hydrocarbons.

Notizen: Candida lipolytica, strain Phaff, was grown on 1.0% 1-hexadecene as sole source of carbon. Several oxidative intermediates were isolated and identified. Based on these intermediates two pathways are proposed for the degradation of the 1-alkene via the methyl group and the double bond. Subterminal oxidation of the 1-alkene was also indicated. Cell yield, lipid content, fatty acid profile and 1, 2-diol concentration are given for various rates of aeration during growth in a fermentor.

Notizen: In the course of submerged cultivation of low-production and industrial production strains of Streptomyces aureofaciens, the activity of enzymes of the tricurboxylic acid cycle was studied. The activities of citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37) were estimated spectrophotometrically in cell-free preparations. In the growth phase, mainly the initial reactions of the cycle were active with both strains. In production-phase, the activities of enzymes in the low-production strain were 2-5 × higher than in the production strain. Benzylthioeyanate, at a concentration of 5 × l0-5M, stimulated chlortetracycline production of both strains with accompanying decrease in activity of the enzymes of the tricarboxylic acid cycle. The role of the tricarboxylic acid cycle in control of chlortetracycline biosynthesis is discussed.

Notizen: Methane biosynthesis within the anaerobic ecosystem results from the diphasic conversion of complex organic substrates through a series of biologically mediated oxidation-reduction reactions which arc pH dependent. Routine parametric analysis of the products emanating from the corresponding acid and methane fermentation phases of the anaerobic digestion process provide a basis for determination of process condition and efficiency. Of these analyses, pH and electrode potential of pE are intensive parameters of considerable significance but for which interpretation is often difficult and misleading. Attention has been focussed on the analogy and distinction between pH and pE determinations and their application to the anaerobic ecosystem. Theoretical and practical limitations are discussed and experimental evidence presented which supports the possible use of pH and pE determinations for process evaluation and control.

Notizen: With a chemostat culture, both the bacterial growth rate and the growth environment can be independently varied between wide limits. Changing the growth rate of Aerobacter aerogenes organisms (in either a glycerol-limited medium or a Mg2+-limited medium) affected the bacterial cell wall content; invariably slow growing organisms were smaller than faster growing ones and had a higher cell wall/biomass ratio. Changing the growth rate also influenced the composition of the walls but in this respect glycerol-limited organisms and Mg2+-limited organisms behaved differently. Thus, whereas increasing the growth rate of glycerol-limited cultures caused the cell wall 2-keto-3-deoxyoctonic acid (KDO) and heptose contents to increase progressively, with Mg2+-limited cultures they decreased. Furthermore, although KDO and heptose are both components of the lipopolysaccharide layer, their ratio varied with growth rate, and with the nature of the growth-limitation, indicating changes in the lipopolysaccharide composition. These results are discussed with particular reference to the influence of environment on cell wall content and composition, and the use of continuous culture for the production of bacterial vaccines.

Notizen: Continuous phased growth produces a culture in which most of the cells in the population are in the same stage of their development. The cell, thereby amplified by the size of the synchronous population, may be examined in the phased culture at any desired growth rate. Changes taking place in the cell after the cell cycle, i.e., post-cycle changes, may be examined by a modification of the procedure. Further systematic applications of the method permit a rational approach to problems of cell growth and metabolism.The phasing technique recognizes the cells as the fundamental unit for experimental investigation, and offers a great potential in the analysis of the cell throughout its cycle, a relatively unexploited field in cell physiology and fermentation. Experiments with yeasts and bacteria illustrate some of the applications and progress made so far.

Notizen: Homogeneous technique facilitates the cultivation of large quantities of cells, reduces the risk of contamination by eliminating many manipulations, and makes practical the control of conditions such as pH and oxygen tension. Although most animal cells will not multiply in free suspension, certain cell lines have lost the requirement of being attached to a solid surface. These cells can be subcultured indefinitely but have some resemblance to cancer cells such as their abnormal karyotype. Certain cell linen developed from human embryonic tissue maintain their diploid character after repeated subculture and would seem to be ideal for the production of vaccines. However, strict regulations exist for viral products for human injection in that only cells taken from normal tissue and subcultured but once may be used.A microcarrier method in which cells adhere to DEAE-Sephadex beads permits a suspension culture which may be termed quasihomogeneous. The attached cells may be retained by sedimentation or by screening as the medium is replaced. Cell debirs from the original tissue is difficult to remove from microcarrier cultures; modifications of the trypsinization technique have alleviated but not solved this problem.Conditions for virus replication can be less critical than those for cell growth in that oxygen tension seems to have little influence on virus production. In cases where rate of virus production increases with specific growth rate of cells, homogeneous culture would have a advantage in maintaining a high cell mogeneous culture would have a valuble advantage in maintaining a high cell growth rate for a longer time. Some virus infections destroy cells, but others cause little change in cellular mteabolism except that virus is continually produced. The latter type can be conducted with a microcarrier in continuous culture with a virus titer exceeding 107 plaque forming units per milliliter for over 50 days with Rubella-infected BHK cells.

Notizen: Frog retinal rod outer segments were detached from dark adapted retinas by (1) agitation in frog Ringer's solution or (2) by crushing between two glass surfaces. The resulting suspensions were further purified by low and high speed centrifugation procedures in Ficoll density gradients. The density of the outer segments in Ficoll solutions was found to be 1.09. The large frog outer segments, unlike bovine outer segments, are not readily separated from nuclei, which were estimated to comprise 2.6-8% of the material, based on DNA analyses. The RNA/DNA ratio was 0.4-0.5, like that of neuronal nuclei. Representative enzymes of glycolysis (lactic dehydrogenase and glyceraldehyde phosphate dehydrogenase), phosphogluconate oxidation (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), the citric acid cycle (malic dehydrogenase) and ATP degradation (ATPase) were assayed. A major part of the malic dehydrogenase activity was probably due to inner segments attached to some of the outer segments. Glyceraldehyde phosphate dehydrogenase and glucose-6-phosphate dehydrogenase (but not lactic dehydrogenase) activities were lower in detached outer segments purified on Ficoll gradients than in samples of outer segment layer microdissected from freeze-dried sections of frog retina, as well as in whole retina. The data suggest that the activities of all the enzymes studied are intrinsically low in rod outer segments.

Notizen: The frog, Rana pipiens, hibernates through the winter with ovaries containing oocytes which, in size and appearance, are ready for ovulation and maturation. From November through April, the normal time of egg laying, ovulation and maturation can be induced by interrupting hibernation and administering gonadotropic hormones. In the studies reported here, it has been found that oocytes taken from hibernating animals in early winter take up amino acids from a saline medium at a relatively rapid rate. Respiratory inhibition produced by such agents as dinitrophenol (DNP) and anaerobiosis, does not completely stop uptake but slows it down markedly.In late winter, amino acid uptake tends to be slower in normal cells and when DNP or cyanide is added, a marked acceleration of the rate of uptake is observed. The uptake in poisoned cells is accumulative, producing internal concentrations higher than that of the medium. At this concentration of DNP, amino acid incorporation is almost completely stopped. Fluoride abolishes the DNP stimulation of amino acid uptake. Removal of sodium ion from the incubating medium has no effect on either uptake or incorporation.These data are interpreted to mean that a capacity for oxidative phosphorylation in oocytes diminishes during winter hibernation. In the spring either an anaerobic capacity comes into being or becomes capable of being switched on. During this time the rate of amino acid uptake, even in the downhill phase, is limited by energy availability and is not dependent on a sodium gradient.

Notizen: The effect of temperature on intracellular ATP levels in cells of the ascitic Hepatoma 129 has been determined using an UV-enzymic method. ATP levels are found to vary inversely with temperature over the range (4°-37°C) studied, and this variation is found to be reversible. An indication of a possible direct correlation of ADP level with temperature was also obtained.A correlation is deduced between rise in intracellular ATP level and decrease in cytoplasmic viscosity, both from the present results, and from similar effects recently reported by Landau ('61) for increase in pressure.The significance of these observations is discussed with reference to the model for cytoplasmic viscosity proposed by Marsland ('56) and the wider biological significance of this effect is explored.

Notizen: Dietary zinc deficiency has been shown to decrease the in vivo incorporation of thymidine into the nuclear DNA of liver parenchymal cells in the rat. A single injection of 100 μg of zinc was sufficient to rapidly reverse the effect of zinc deficiency on the synthesis of DNA.

Notizen: It has been shown by re-cloning of colonies formed in vitro from rat bone marrow cells, that normal granulocyte colonies can originate from single cells. No mixed macrophage (M) and granulocyte (G) colonies were obtained after re-cloning either M or G colonies. The results indicate, that clones of normal granulocytes and macrophages can be obtained in vitro, and that the mixed primary M and G colonies formed after seeding hematopoietic cells from animals presumably originate from a mixture of M and G cells.

Notizen: The cytotoxic effect of high, as well as low, oxygen tension of proliferation and metabolism of Low line cells in culture is reversible even after several days of exposure provided the cells are returned to 95% air + 5% CO2 environment. This suggests that the activity of certain mechanisms within the cells may have been altered or in other ways inhibited by the abnormal environments but are quite rapidly regenerated once the adverse condition is removed. The cells tolerate a low O2 exposure for at least 20 days while continuous exposure to high O2 atmosphere results in degeneration and death after 7-10 days. Both glucose utilization and lactic acid production are elevated in cultures exposed to either low or high O2 tensions, although they are markedly higher in the latter condition. When cell so exposed are returned to an air + 5% CO2 atmosphere, rate of glucose uptake and lactic acid formation soon approaches that found in control cultures.

Notizen: Histologic examination of the spleens in RFM/Un mice killed 6 to 9 days after 350 to 800 R whole-body x-irradiation revealed hemopoietic colonies, the numbers of which decreased exponentially with increasing radiation dose. In such animals, myelocytic colonies were the predominant type on the sixth to the eighth day. However, they decreased in number with time, being fewer than erythropoietic colonies by the ninth day after irradiation. In C57BL mice, erythropoietic colonies were relatively more numerous, markedly predominating on both the eighth and the thirteenth days.RFM/Un mice injected with nonirradiated syngeneic bone marrow cells within 24 hours after 750 R developed colonies, predominantly of erythropoietic and undifferentiated types, the numbers of which were proportional to the numbers of marrow cells injected. The number of colonies formed from exogenous marrow cells increased slightly between the sixth and ninth days after inoculation, possibly because of a greater likelihood of counting them due to an increase in their size.

Notizen: In Tetrahymena pyriformis, mating type I, variety 1, cycloheximide rapidly and completely inhibited incorporation of 14C-L-leucine into protein. Actinomycin D (25 μg per ml) inhibited incorporation of 14C-uracil into cold-TCA-insoluble material, after a 5-10 minute lag. Frequently a subsequent decline in the amount of radioactivity was observed. Protein synthesis continued in actinomycintreated cultures for a variable time after cessation of RNA synthesis.Oral development was affected by cycloheximide virtually immediately, and by actinomycin D after a 10-15 minute lag. Cells affected by either drug before the onset of oral membranelle formation were permanently arrested in the stomatogenic field phase. Cells affected in the early and middle stages of membranelle formation completed development of membranelles, but did not invariably complete cell division. Cycloheximide, when added at the beginning of membranelle formation, brought about arrest or resorption of membranelles after they were completed. Actinomycin did not elicit resorption, but sometimes brought about blockage during cell division. Cells affected by either drug after membranelles were fully formed (and cell division was just beginning) completed oral development, nuclear divisions, and cell division. These results suggest that concurrent RNA and protein synthesis are essential for the initiation but not for the completion of membranelle differentiation. The results also suggest that a specific messenger RNA(s) with a very short half-life is required for the synthesis of proteins involved in the initiation of membranelle differentiation.

Notizen: Growth of KB cells was inhibited by both spermine and spermidine, but the inhibition is reduced in conditioned medium. The amount of spermine required for 50% inhibition of plating varied according to the type of serum used with medium 199 (calf, fetal bovine, and horse; 0.55, 0.9, and 24 μg/ml respectively). The spermine oxidase activity of the three sera was calf 〉 horse 〉 fetal bovine, which is not the same ordering as was obtained for the inhibition. When the concentration of sera in the media was varied, the inhibition decreased as calf and fetal bovine sera concentration increased, whereas, with horse serum, an increase in serum concentration increased the inhibition. The opposite effects of increasing concentrations of the sera on the inhibition suggest that at least two factors are involved in the inhibition. A scheme which involves three factors (spermine oxidase, another enzyme and its activator) is postulated to account for the inhibitions and reversals observed. Spermine oxidase alone cannot account for the action of polyamine on cells.

Notizen: Reduction in alkaline phosphatase activity was observed when HeLa S3 cells were grown in Puck's medium containing high concentrations of human serum. This effect was not seen with the enzyme of Chang liver 8A cells. The induction of increased alkaline phosphatase in HeLa S3 by prednisolone or by osmolality changes was not prevented by serum. The concentration of serum in the culture medium had no influence on acid phosphatase activity.

Notizen: Functionally active 30S ribosomes can be reconstituted in vitro from 16S RNA and a mixture of 30S ribosomal proteins under certain defined conditions. Our previous studies on the specificity and kinetics of reconstitution are summarized and discussed. The reconstitution reaction is first-order with respect to formation of active 30S ribosomes. The rate-limiting reaction is probably unimolecular, and it represents the structural rearrangement of an intermediate. Presumed reconstitution intermediates, or RI particles, have been isolated from reconstitution mixtures incubated at low temperature. It has been concluded that the reconstitution takes place in stepwise fashion: 16S ribosomes. New experimental results that show the highly cooperative nature of the assembly reaction are described; the number of sites, per RNA chain, which can bind ribosomal proteins independently from each other (i.e., without cooperativity) is at most two to three. Finally, another approach to the study of ribosome assembly in vivo is described. It utilizes cold-sensitive E. coli mutants that are defective in ribosome biosynthesis at low temperature and accumulate incomplete “intermediate” ribosomal particles.

Notizen: A cell line was established in vitro from a spontaneous myeloid leukemia of SL strain mice. This cell line was cytologically identified as myeloblast, and its normal differentiation appeared to be completely blocked in mass culture. When the line cells were seeded in soft agar with a conditioned medium from normal cells, either macrophages or neutrophil granulocytes appeared from a single clone. The rate of formation of colonies containing differentiated cells always increased with an increase in the concentration of conditioned medium. The conditioned medium from this line cell was not as effective as was that from normal cells in inducing differentiation.

Notizen: Considerable variability has been found in the yield of cells in batch cultures of Tetrahymena pyriformis grown axenically in 1% tryptone/0.05% yeast extract. This variability has been traced to the photolysis by visible light of the flavin mononucleotide and thiamine components of yeast extract.

Notizen: Colony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7-day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.

Notizen: Maturation of bacterial viruses requires the formation of mature viral nucleic acid in the form found in progeny virus particles, the “packaging” of this viral nucleic acid within a capsule, and the assembly of “packaged” nucleic acid with the accessory structures necessary for infectivity. Recognition that the replicating viral nucleic acid is frequently in a form distinct from that found in mature particles has, in some instances, led to the postulation and formal description of presently unknown processes that must participate in the synthesis of the mature form. In at least some instances, packaging of the mature nucleic acid is clearly integrated with its synthesis.Examples involving the DNA bacteriophages T4, λ, and φ X174 and the RNA bacteriophages are presented to illustrate these points.

Notizen: The development of a virus is programmed by a series of negative and positive controls which determine the timing and the segment on either of the two DNA strands (l or r) to be transcribed into specific messenger RNA's. Bacteriophage λ provides one of the most deeply studied systems for following the development of lysogenic viruses. In the lysogenic repressed state, only 2-4% of the λ genome is expressed. This pc-cI-rex region is transcribed leftward to produce a repressor protein which prevents any further transcription by blocking the oL and oR operators flanking the cI-rex operon (figs. 1, 2). This negative control is relieved by destruction of the repressor, and the result is the “induction” of viral development. The earliest post-induction or postinfection events are the leftward transcription of the pLoL N region from strand l and the rightward transcription mainly of the pRoR-x segment from strand r. The N product acts as a positive control, permitting a leftward transcription beyond gene N and a rightward transcription of genes cII-O-P and also Q. The int-xis system controls the excision of the λ genome, whereas the act of rightward transcription and the products of genes O and P initiate the replication of λ DNA. The product of gene Q, still another positive control, stimulates rightward transcription of the late genes which control the synthesis and assembly of the phage heads and tails as well as cell lysis. Among other types of negative control are the possible competition between the two divergent transcriptions originating in region x, the “antirepressor” effect of the x product, and the interference between the two convergent transcriptions which collide in the central b2 region. The majority of controls are based on protein-DNA interactions and can be modified by mutations. For instance, transcription can be rendered independent of negative repressor control either by constitutive, v, mutations which decrease or abolish the affinity of the o operators for the repressor or by insertion of new promoters-e.g., c17 or ric- on the “downstream” side of the operator. The need for the positive N and Q controls may also be obviated by mutations in the N- or Q-dependent promoter or terminator elements.The specific DNA structure within the controlling sites is not known. However, a remarkable coincidence was observed; namely, the occurrence of pyrimidine-rich clusters in those segments of the individual DNA strands acting as templates for RNA synthesis. This observation, which pertains to all studied DNA's, including those of phages T2, T3, T4, T5, T6, T7, λ, and φ 80, formed the basis for a proposal that implicates pyrimidine-rich clusters in the initiation, control and/or termination of transcription, and also in the determination of the preferred strand and, consequently, the orientation of transcription. General considerations regarding the possible role of the structural singularities, especially those represented by the pyrimidine clusters, in the bipartite structure of the recognition regions in DNA are discussed.

Notizen: These studies have been conducted as a first step toward investigating the molecular mechanism by which DNA-dependent RNA polymerase recognizes a specific region of DNA. The DNA moieties are either bound to or freed from RNA polymerase. Results obtained so far indicate that the DNA moieties remaining as resistant segments after deoxyribonuclease digestion should be regarded as the binding regions on DNA. The results described here were carried out by use of E. coli and lambda DNA.

Notizen: Sarcoplasmic reticulum fragments, prepared from skeletal muscle homogenates, were found to consist of two major types when examined after negative staining. One type possessed 90 Å subunits and was thought to be of mitochondrial origin. The other had 35 Å subunits and ws presumably derived from the sarcoplasmic reticulum. Only the latter type accumulated visible calcium oxalate deposits inside the vesicle when they were exposed to a medium containing ATP, MgCl2, K2C2O4 and CaCl2. The calcium oxalate loaded vesicles were strikingly angular in shape and did not have tails. The calcium oxalate loaded vesicles had identical membrane subunit arrangement to inactive companion membranes and nonincubated controls; this suggested that the membrane subunits were not the critical structural requirement for calcium transport. A method was described whereby the calcium accumulating membranes could be purified 3- to 4-fold on the basis of the best previously used preparation procedures.

Notizen: The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10-5 M FUdR was very low (0.0001-0.0003) whether the drug was administered during early G1, late G1, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10-7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.

Notizen: The effects of cycloheximide on synchronized Tetrahymena pyriformis strain GL-C were investigated at concentrations ranging from 0.01 to 10 μg/ml. The initial inhibition of protein synthesis was nearly total (〉85%) at 1 μg/ml and above, partial (50-80%) at 0.2 to 0.05 μg/ml, and slight (〈30%) at 0.02 μg/ml. Eventual recovery of protein synthesis to a rate approaching that of the controls took place at concentrations of 1 μg/ml and less. When the drug was added before a “transition point” at 55 minutes after the end of the synchronizing treatment (EST), cell division was blocked by 10 μg/ml, and delayed at concentrations of 1 μg/ml or less. The duration of delay was related to the degree of initial inhibition, and to the time required for recovery of protein synthesis; it also depended on the time after EST at which the drug was added. At a given concentration, maximum division delay was observed just prior to the “transition point;” this maximum delay was correlated with resorption of differentiating oral primordia, followed by the appearance of new primordia. The lesser delays observed at earlier times were correlated with temporary blockage of development of primordia in the “stomatogenic field” stage. Resumption of oral primordium development was, in both cases, temporally correlated with a substantial recovery of protein synthesis. After the “transition point,” cell division, and completion of oral development, was delayed slightly at the lower concentrations, and more substantially at 1 and 10 μg/ml, with some division-arrest at the latter concentration. Except for the recovery phenomenon, the developmental responses elicited by cycloheximide were similar to those observed earlier with puromycin.The bearing of these findings on the mechanism of synchronization in Tetrahymena is considered in the Discussion.

Notizen: Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.

Notizen: Theoretical osmotic fragility curves were calculated and drawn by computer using the van't Hoff equation and the isotonic areas and volumes of 1000 individual erythrocytes. We studied the influence on the calculated curves of theoretically altering the fraction of the volume which was osmotically active from 50 to 70%, and of altering the permissible stretch before hemolysis from zero to 10%. With the two assumptions-that the membrane does not stretch before hemolysis, and that the osmotically active fraction of the cell volume is 0.58-it was possible to duplicate the general shape of the standard fragility curve; the exact NaCl concentration, however, at which there was 50% hemolysis was approximately 0.1 gm/100 ml higher than found in vitro. The calculated osmotic fragility curves can be made quantitatively similar to in vitro ones if the following statements are true: the osmotically active volume is 58%, the permissible stretch of the membrane without lysis is 6%, the cell membrane resists a slight osmotic pressure gradient of approximately 0.1 atmospheres, and hemolysis is an all or nothing phenomenon. This set of values for the relevant factors is sufficient but not unique in causing the superposition of the calculated and experimental curves. The frequency distribution of the cells according to the hemolytic salt concentrations (the sodium chloride concentration at which an individual cell just hemolyzes) was skewed positively and was leptokurtic for each of the seven normal subjects studied.

Notizen: The fraction of cell capable of division was determined for (1) population of the human diploid cell strains, WI38 after different numbers of subcultivations in vitro and (2) a single population of WI38 cells at intervals throughout its entire in vitro lifespan. In both cases the percentage of cells capable of division decreased with increasing age in tissue culture. The rate and the magnitude of the decrease is sufficient to account for the limited in vitro lifespan reported by other investigators. Furthermore, the decrease in the fraction of cells capable of division in similar in some respects of senescence among human populations.

Notizen: All tRNA sequences so far known can be folded into a cloverleaf structure. Physical data and chemical reactions allow us to draw conclusions on secondary (cloverleaf) and tertiary structure. N-oxidation of adenosine to adenosine-1-N-oxide can be done with monoperphthalic acid in non-base-paired regions of polynucleotides and can be followed easily by changes in absorption of ultraviolet light. Thus this method can be used to determine the structure of tRNA's. A fingerprint of the N-oxidation product of tRNAyeastPhe reveals that all adenosine residues are protected except the 3′-terminal adenosine and the three adenosine residues in or adjacent to the anticodon. On this basis a conformation of tRNAyeastPhe is proposed. Similar tertiary structures can be constructed for the other tRNA's. In order to connect tertiary structure of a tRNA and recognition by its aminoacylating enzyme, the rate of aminoacylation, as a function of temperature, was measured. Neither changes in the anticodon nor specific changes at the 3′-terminal adenosine abolish aminoacylation. Single crystals of tRNAyeastPhe were obtained from aqueous solutions upon addition of various organic solvents.

Notizen: Assemblies of protein molecules represent a fundamental level of biological organization. The dynamic behavior of these systems-including both the assembly process and functional rearrangements-may be accounted for by the specificity of the protein interactions, which depend on environmental conditions. Analysis of the self-assembly of virus particles has established that the design of an ordered structure can be built into the specific bonding properties of the constituent proteins. Any structure which can change its state of organization is, by definition, polymorphic. The distinctive aspect of polymorphism in protein structures, contrasted with nonliving states of matter, is that the molecular design has been selected to carry out a function and that this function is part of an integrated system. The differences in molecular conformation and arrangement in all polymorphic structures-for example, allosteric enzymes or ice crystals-depend on the intrinsic interaction properties of the molecules themselves. The structures of ice and water illustrate relations between specificity and polymorphism which are relevant to the form and function of protein assemblies.Two types of polymorphism can be distinguished: modal polymorphism, which is externally moderated, as in phase transitions between different crystals forms; and positional polymorphism, which is internally moderated, as in the different disposition of identical molecules within a single crystal lattice. Positional polymorphism, exemplified by the quasi-equivalent bonding of icosahedral virus coat proteins and the different arrangement of myosin and paramyosin at the center and polar portions of the bipolar filaments, results from specific interactions that are not compatible with a strictly equivalent packing of identical molecules. The structural rearrangements in muscle contraction and the switching between the oxy and deoxy forms of hemoglobin represent the formation of different structures in response to altered external conditions. The different structural states of many protein assemblies are characterized by conserved connections which may be regarded as providing the framework for functional rearrangements. The types of polymorphism displayed by hemoglobin, virus, and muscle proteins demonstrate the relevance of the simple view that the function of a protein is determined by the potential structures it can form.

Notizen: Analysis of the equilibrium of the normal biconcave human red cell, in terms of its tension and a pressure inside, suggests a force of attraction between the opposite membranes at the dimple regions. The analogous attraction that causes rouleaux formation is mediated by long-chain molecules. Single cells hanging on edge between polarizer and analyser, almost “crossed,” were photographed at different angles to the axis of the polarizer. Enlarged prints were scanned by a photometer. For single cells the records showed non-significant fluctuations of intensity, but mean values for 32 cells showed a very significant sinusoidal variation with angle, as predicted by theory for birefringence in the cell at the dimple region. For the rim region, the averaged data showed no variation with angle. In cells moderately osmotically swollen, birefringence in the centre of the dimple region was absent, but persisted close to the inside of the membranes. The latter disappeared in cells further swollen to a biconvex shape. The data is interpreted as indicating oriented chains of molecules across the interior of the cell at the dimple region. The behaviour on swelling was what had been seen in a model with nylon fibres oriented between the charged plates of a condenser, in which the variation of attractive force with distance was adequate to explain the equilibrium of the red cell membrane.

Notizen: H3-Uridine microinjected in the giant axons of the squid is incorporated in a TCA insoluble material. There is no difference between stimulated and resting axons as to the amount incorporated.The amount incorporated is increased if the stimulation precedes the microinjection of the tracer.RNA was purified and characterized from the axoplasm, axon sheaths and from a purified membrane preparation obtained from squid retinal nerve.

Notizen: Diploid human fibroblasts in culture (WI 38) were allowed to reach a stationary phase and were then stimulated to reenter DNA synthesis and cell division by addition of serum to the culture medium. The rate of protein synthesis increased during the first hours after addition of serum reaching at three hours a plateau value that continued for at least 24 hours after serum addition. Inhibition of protein synthesis during the early hours after serum addition abolished the stimulation of DNA synthesis occurring 20 to 28 hours later.Increased protein synthesis was preceded by a rapid decrease in the intracellular pool size of most amino acids. These changes were independent of concomitant protein synthesis. They suggest that serum exerts an immediate effect on the function of the cell membrane.

Notizen: Cellular transformation was induced with avian myeloblastosis virus strain BAI-A (standard AMV) and with a strain of AMV containing subgroup B only. Cultures of muscle tissues from either chick embryo or day old chicks were used for this study. Results were similar in C/O and C/A cells. Leukemogenic virus was continuously produced by these transformed cultures.

Notizen: In the presence of 1 mM ATP in the external medium, TA3 mouse ascites tumor cells showed a dramatic loss of potassium and gain of sodium down their respective concentration gradients. The volume changes detected by size discrimination with the Coulter counter have been adequately confirmed by densimetric techniques. Further, some experiments were so designed that net losses of both ions occurred and the cell shrank in response to ATP, a response which was predictable if the volume change was produced by a loss of cell water. We believe that ATP produces a major change in the passive permeability of the membrane to these ions and the effect may be due to a response of a contractile protein in the membrane to ATP.

Notizen: Growth hormone, oxytocin, parathyroid hormone, prolactin and lysine vasopressin strongly stimulate mitotic activity in rat thymocyte populations maintained in vitro. These hormones have no mitotic effect on cells maintained in calcium-free medium. It is concluded that they stimulate mitosis only indirectly by sensitising the mitotically competent segment of a thymocyte population to the action of calcium. The stimulatory action of calcium itself is opposed by low concentrations of the mucopolysaccharide chondroitin sulphate. However, the inhibitory action of chondroitin sulphate can be overcome by growth hormone.A possible common mechanism of action of these hormones on mitotically competent cells is discussed.

Notizen: A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 107 cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r2/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.

Notizen: The progression of rat thynocytes (maintained in vitro) into mitosis is profoundly affected by the level of magnesium in the medium. Increasing the extracellular MgC12 concentration from 0 to 1.0 mM does not affect mitotic activity, but a further increase to 1.2 and 2.5 mM rapidly and strongly stimulates the flow of cells into mitosis. The level of extracellular magnesium also governs the mitogenic effectiveness of calcium ions. The mitogenic action of magnesium stems from an ability to stimulate the initiation of deoxyribonucleic acid synthesis.

Notizen: Colony-forming cells (CFU), which have the general properties of hemopoietic “stem” cells, appear to be augmented in the mouse fetal liver from 12-18 days gestation and then decrease in the newborn. This finding suggests that few, if any, hemopoietic “stem” cells remain in the adult liver, an organ which appears to be unable to function erythropoietically, even at times of severe crises. In the spleen, and active adult as well as embryonic hematopoietic organ, the total number of CFU increases from 18 days gestation until at least 7 days after birth.Spleen and liver CFU augmentation seems to occur in cojunction with an analogous expansion of non-hematopoietic cells. The data suggests, in fact, that while there is an increase in the total number of liver CFU, there is also a dilution of liver CFU in the total cell population at successively later gestational ages.