QUANTITATIVE STUDIES ON THE PHOTOLETHAL EFFECTS OF QUARTZ ULTRA-VIOLET RADIATION UPON PARAMECIUM

Paramecia grown under controlled conditions were irradiated at known intensities of light of wave-lengths 2537, 2654, 2804, 3025, and 3130 A. The approximate absorption of the light by the Parmecia was found to be greatest and of the same order of magnitude at the three shortest wave-lengths, considerably less at 3025, and least at 3130 A. Paramecia did not die when irradiated with high dosages of intense light of wave-length 3130 A. At the other wave-lengths 50 per cent vesiculation occurred when between 10¹² and 10¹³ quanta had been absorbed by a Paramecium. This would indicate that a very large number of molecules in a Paramecium are affected before vesiculation occurs.     

Macronuclear DNA organization and transcription in Paramecium primaurelia

Macronuclear DNA was isolated from Paramecium primaurelia, stock 168. Although the macronucleus is polyploid to the extent of 840C, in other respect the DNA appears to be simply organized, having neither satellite sequences nor substantial amounts of intermediately repetitive sequence. The sequence complexity of macronuclear DNA is quite low for a eukaryote cell, being approximately 19 times more complex than the genome of Escherichia coli. In addition, the GC content is low (25%) and the isolated DNA molecules have lengths mostly in the range 0.2–5 μm. In these various respects, the macronuclear DNA of Paramecium is similar to that of other ciliates. A clone of Paramecium cultured under controlled conditions contains polyadenylated RNA sequences which are homologous to 5–8% of the macronuclear DNA. Sequence complexity analysis indicates that the polyadenylated RNA contains two abundance classes of molecules, one present at low frequency and transcribed from approximately 10⁴ genes, the other at 100 times greater concentration and transcribed from about 100 genes. The relevance of these results to the control of gene expression in Paramecium is discussed.     

A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein

Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10–30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd²⁺-ions.     

CELL POPULATION KINETICS OF EXCISED ROOTS OF PISUM SATIVUM

The cell population kinetics of excised, cultured pea roots was studied with the use of tritiated thymidine and colchicine to determine (1) the influence of excision, (2) the influence of sucrose concentration, (3) the average mitotic cycle duration, and (4) the duration of mitosis and the G₁, S, and G₂ periods of interphase.¹ The results indicate that the process of excision causes a drop in the frequency of mitotic figures when performed either at the beginning of the culture period or after 100 hours in culture. This initial decrease in frequency of cell division is independent of sucrose concentration, but the subsequent rise in frequency of division, after 12 hours in culture, is dependent upon sucrose concentration. Two per cent sucrose maintains the shortest mitotic cycle duration. The use of colchicine indicated an average cycle duration of 20 hours, whereas the use of tritiated thymidine produced an average cycle duration of 17 hours.     

Cold-sensitive Ca2+ Influx in Paramecium

The concentration of intracellular calcium, [Ca²⁺] _i , in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca²⁺] _i at the anterior region of the cell. Increase in [Ca²⁺] _i was not observed at any region in Ca²⁺-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K⁺ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca²⁺. The cold-induced inward current was lost upon replacing extracellular Ca²⁺ with equimolar concentration of Co²⁺, Mg²⁺ or Mn²⁺, but it was not affected significantly by replacing with equimolar concentration of Ba²⁺ or Sr²⁺. These results indicate that Paramecium cells have Ca²⁺ channels that are permeable to Ca²⁺, Ba²⁺ and Sr²⁺ in the anterior soma membrane and the channels are opened by cooling. Received: 1 April 1996/Revised: 23 July 1996     

Binding of Ca ions by Paramecium caudatum

Binding of ⁴⁵Ca by live Paramecium caudatum was determined under various external ionic conditions. It was found that calcium uptake was separable into at least two components, a rapid and a slow one. The rapid component was influenced by the presence of certain other ions in a manner which agrees with the law of mass action. It appears that an ion exchange system may be involved in a binding equilibrium established between Paramecium, Ca⁺⁺, and certain other ions. K⁺, Rb⁺, and Ba⁺⁺ in the equilibrium medium are among those ions which inhibit calcium uptake. It is proposed that liberation of Ca⁺⁺ from binding sites on Paramecium by an exchange reaction with competing ions is the first step in the mechanism of ciliary reversal in the response to external application of these ions.     

Defective ion regulation in a class of membrane-excitation mutants in Paramecium

The “paranoiac” mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na⁺, unlike wild-type paramecia, which jerk back and forth in Na⁺ solutions. The paranoiac mutants in Na⁺ solutions also show large losses of cellular K⁺ and large influxes of Na⁺. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na⁺-dependent loss of cellular K⁺ and a much smaller Na⁺ influx. In K⁺-containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K⁺ content.The Na⁺ influx, the K⁺ loss, and the duration of backward swimming are all proportional to the extracellular Na⁺ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na⁺ influxes and the large K⁺ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.     

Concanavalin A as a peripherally acting inhibitor of thyroxin-mediated metamorphosis in amphibians

Stimulated by reports that Concanavalin A (Con A), a plant protein and lectin from jack bean, has an inhibitory effect on thyroid activation induced by thyrotropin, we set out to test whether Con A inhibits thyroid action on hormone-sensitive target tissues in amphibians. We noted that premetamorphic tadpoles injected with 0.15 ml of thyroxin (T₄ 0.24 μM) responded by accelerating metamorphic change as indicated by precocious disappearance of the tail, and appreciable growth of the hind limbs and changes in mouth-part morphology. Tadpoles given an injection of thyroxin immediately followed by an injection of Con A (9.6 μM) showed no such metamorphic changes. In the second series of experiments tail fin discs obtained from premetamorphic tadpoles when placed in cultures supplemented with T₄ (0.24 μM) had completely shrunk within 96 hr. Tail fin discs that were raised in vitro in medium containing Con A as well as thyroxin failed to regress. In the third series of experiments tail discs were initially cultured in medium containing thyroxin and transferred within 48 hr to medium containing Con A. When Con A was added after this 48-hr exposure to thyroxin it was no longer effective in preventing tail fin disc resorption. We conclude tentatively (1) that Con A is a peripheral inhibitor of thyroxin and (2) this it somehow binds to the tissue or interacts with thyroxin rendering it ineffective before the hormone has a chance to act. The significance of finding a peripherally active inhibitor of thyroid hormone for studies of mechanism of action of this hormone on development and differentiation of hormone-sensitive target structures is obvious.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : II. STIMULATION OF CELLULAR OXIDATION AND REVERSIBLE INHIBITION OF CELL DIVISION BY DIHALO AND TRIHALOPHENOLS

The dihalo and trihalophenols, and phenols containing both halo and nitro substituents in the same molecule, produce, in fertilized eggs of Arbacia punctulata, a rise in rate of oxygen consumption and a reversible block to cell division. To define the conditions which affect the degree of this activity, the following factors have been varied: the arrangement of substituents in the molecule, the concentration of reagent, and the time after fertilization at which the reagent is added. The stimulation of oxygen consumption and reversible block to cell division produced by the dihalophenols are qualitatively the same as those previously produced in fertilized Arbacia eggs by certain dinitrophenols. To yield optimum respiratory effect and maximum division block, it usually requires a higher concentration of dihalo than of the corresponding dinitrophenol. For example, with fertilized Arbacia eggs at 20°C. 2,4-dinitrophenol, in optimum concentration of 3 x 10^–5 molar, raises oxygen consumption to 292 per cent of normal (4). The corresponding values for two dihalo analogues are: 2,4-dichlorophenol, 10^–4 molar and 236 per cent; 2,4-dibromophenol, 6 x 10^–5 molar and 282 per cent. The halophenols differ from the nitrophenols in two interesting respects: (a) The monohalophenols produce little or no oxidative stimulation or division block in fertilized Arbacia eggs; p-nitrophenol is very active in both respects. (b) The symmetrical trihalophenols have an appreciable ability to stimulate oxygen consumption and block division; symmetrical trinitrophenol is inactive in both respects (4). The increases in oxygen consumption produced in fertilized Arbacia eggs by 2,4-dichloro and 2,4-dinitrophenol are larger than the percentage increases given by methylene blue and o-cresol indophenol under the same experimental conditions. The dihalo and dinitrophenols produce a reversible block to the cell division of fertilized marine eggs. The oxidation-reduction indicators, in contrast to the dihalo and dinitrophenols, block cell division irreversibly and fertilized eggs of Arbacia do not recover from optimum respiratory stimulating concentrations of these oxidation-reduction dyes. The present experiments with halophenols are in harmony with and lend considerable support to the hypothesis (4) that nitro and similarly substituted phenols derive their biological activity from the presence and properties of the phenolic OH group, as modified by proper substitution in the phenolic benzene ring.     

Simultaneous recording of cytosolic Ca2+ levels inDidinium andParamecium during aDidinium attack onParamecium

Summary The carnivorous ciliateDidinium nasutum captures prey such asParamecium by discharging extrusomes, known as toxicysts, while the attackedParamecium defensively discharges trichocysts. Several authors have suggested that both discharges, the toxicysts ofDidinium and the trichocysts ofParamecium, are evoked by the rise in cytosolic Ca²⁺ level in each cell. However, these putative increases in cytosolic Ca²⁺ levels have not as yet been recorded simultaneously in these cells during aDidinium attack onParamecium. We injected the fluorescent Ca²⁺ indicator Ca-Green 1 dextran into bothDidinium andParamecium, and simultaneously observed the cytosolic Ca²⁺ levels in these cells asDidinium attackedParamecium. When aParamecium came into contact with theDidinium proboscis, theDidinium showed a significant rise in cytosolic Ca²⁺ in the basal portion of the proboscis. One video frame (33 ms) after the onset of the Ca²⁺ rise inDidinium, theParamecium also showed an increase in cytosolic Ca²⁺. This is the first simultaneous recording of changes in the Ca²⁺ level during a predator-prey interaction in ciliates. The possible roles of these Ca²⁺ increases are discussed in relation to the discharge of toxicysts during theDidinium attack and of trichocysts as a defensive behavior ofParamecium.Abbreviations AED aminoethyldextran - P_i inorganic phosphate - FITC fluorescein isothiocyanate     

Rapid Separation and Concentration of Food-Borne Pathogens in Food Samples Prior to Quantification by Viable-Cell Counting and Real-Time PCR

Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low- and high-speed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10¹ to 10³ CFU/g using the RTi-qPCR assay, and amounts as small as 10⁰ to 10¹ CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10¹ to 10² CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.     

Reduction in the latent period of the response to thyroxin by tadpole tail discs fused to discs pretreated with thyroxin.

It has been previously shown that tissue blocks prepared from the tail fins of tadpoles (Rana pipiens) survive in vitro for several weeks and respond to thyroxin by shrinkage after a latent period of four or more days (20°C). It has been postulated that this shrinkage corresponds to that of normal tail tissue during metamorphic climax. The long latent period of thyroid action is presumed to depend upon the time required to form the necessary cellular or biochemical intermediates. If such intermediates are activated by thyroxin in the amphibian tail tissue, it may be possible to shorten the latent period of the thyroxin response of fin tissue by fusing untreated blocks to blocks previously treated with thyroxin. Experiments are reported here in which tail fin blocks obtained from tadpoles previously immersed in thyroxin for 3 days were docked to recipient blocks not exposed to this hormone. Such recipient tissues responded with characteristic resorptive activity within 24 hr, instead of the minimum of 4 days required by control tissues exposed directly to thyroxin. It is inferred that some component other than thyroxin which is active in inducing resorption is transmitted from the treated to the untreated block. Presumably, this is a product normally present late in the latent period of thyroxin action.     

Calcium channel activation and inactivation inParamecium biochemically measured by cyclic GMP production

Summary The Ca-inward current ofParamecium is related to cGMP production by a Ca-dependent guanylate cyclase. Excitation with Ba²⁺ increases cGMP levels about ninefold to 45 pmol/ mg within 15 sec. Inhibition of cGMP hydrolysis reveals a large rate of synthesis of up to 25 pmol cGMP/mg·sec^–1, or about 1.2 ·10⁸ molecules/cell·sec^–1. Because no other factors than the Ca-inward current were found to affect cGMP formation inParamecium, we used it as a quantitative measure of Ca²⁺ channel activity. After a transient stimulation of cGMP formation by 1mm Ba²⁺, an additional increase of Ba²⁺ to 5mm did not result in a renewed elevation of cGMP levels. The extent of desensitization towards a second stimulus was graded with the strength of the first stimulus. Termination of the first stimulus after various time intervals and restimulation after 3 min with 1mm Ba²⁺ revealed a time-dependent inactivation of the Ca²⁺ channel, which could be fitted by a single exponential. The inactivated form of the channel was stable for a few minutes at room temperature. The partial desensitization ofParamecium reduced the maximal response, but did not shift the dose-response curve for Ba²⁺. Veratridine, which activates the Ca²⁺ channel, was also used as a first stimulus. It effectively and transiently inactivated the channel resulting in a complete loss of both a behavioral response ofParamecium and cGMP elevation towards a second stimulus. The time course of reactivation of channel excitability was studied at different temperatures. Half times of recovery were 51 and 7.5 min at 12 and 25°C, respectively. Reactivation curves can be described by a single exponential, indicating a first order reaction. The activation energy was 100 kJ/mol.The extremely high rate of cGMP turnover inParamecium is reminiscent of findings in visual cells. A model for regulation of the voltage-dependent Ca channel ofParamecium is proposed.     

ON THE QUANTA OF LIGHT PRODUCED AND THE MOLECULES OF OXYGEN UTILIZED DURING CYPRIDINA LUMINESCENCE

A study of the oxygen consumed per lumen of luminescence during oxidation of Cypridina luciferin in presence of luciferase, gives 11.4 x 10^–5 gm. oxygen per lumen or 88 molecules per quantum of λ = 0.48µ, the maximum in the Cypridina luminescence spectrum. For reasons given in the text, the actual value is probably somewhat less than this, perhaps of the order of 6.48 x 10^–5 gm. per lumen or 50 molecules of oxygen and 100 molecules of luciferin per quantum. It is quite certain that more than 1 molecule per quantum must react. On the basis of a reaction of the type: luciferin + 1/2 O₂ = oxyluciferin + H₂O + 54 Cal., it is calculated that the total efficiency of the luminescent process, energy in luminescence/heat of reaction, is about 1 per cent; and that a luciferin solution containing 4 per cent of dried Cypridina material should rise in temperature about 0.001°C. during luminescence, and contain luciferin in approximately 0.00002 molecular concentration.     

THE EFFECT OF AUXIN AND GUANINE ON CELL EXPANSION AND CELL DIVISION IN THE GAMETOPHYTE OF THE FERN,ONOCLEA SENSIBILIS

Miller , J. H. (Yale U., New Haven, Conn.) The effect of auxin and guanine on cell expansion and cell division in the gametophyte of the fern, Onoclea sensibilis. Amer. Jour. Bot. 48(9): 816–819. Illus. 1961.—Auxin and guanine promote cell expansion in 0. sensibilis gametophytes. The optimum concentration of auxin for total expansion is 10^−-5 M, but the optimum for elongation is 10^−-6 M. Above this concentration the cells expanded isodiametrically. Guanine is active at higher concentrations than auxin. Increasing concentrations of auxin progressively inhibit red light-induced cell division, while guanine has no effect on cell division. Neither kinetin nor adenine promotes cell expansion or cell division.     

Na+ and K+ uptake and exchange by the amphibian oocyte during the first meiotic division

The uptake and efflux of ²²Na and ⁴²K were studied in denuded Rana pipiens oocytes following progesterone induction of the resumption of meiotic maturation. Coincident with the breakdown of the large nucleus, or germinal vesicle, there is a virtual disappearance of K⁺ permeability of the oocyte plasma membrane. Only about 1–2% of the total [K⁺]_i is exchanged by completion of nuclear breakdown (8–10 hr) and accounts for the finding that there is no detectable change in total [K⁺]_i during the first meiotic division (20–24 hr). In the case of Na⁺, influx, exchange, and efflux kinetics were unchanged during the first meiotic division, with 20 and 35% of the total oocyte Na⁺ exchanging by the completion of nuclear breakdown and first meiotic division, respectively. Removal of Na⁺ from the incubation medium produced and earlier nuclear breakdown, whereas a K-free medium delayed breakdown. There was no effect of 10 μm/ml tetrodotoxin or 10^?5M strophanthidin on the time course of nuclear breakdown. Thus one action of progesterone appears to be a selective turning off of “K channels” in the oocyte plasma membrane. The disappearance of K selectivity of the oocyte plasma membrane coincides with plasma membrane depolarization, as well as nuclear swelling and breakdown.     

Bioassay of a nucleopolyhedrosis virus of the gypsy moth, Porthetria dispar

The pathogenicity of an American isolate of the nucleopolyhedrosis virus of Porthetria dispar was studied. Laboratory data on third-instar larvae showed that mortality was directly related to virus concentration. The computed LD₅₀ was 1,729 PIBs/larva or 72 PIBs/mg larval body weight. The LT₅₀'s for 2.5 × 10⁶, 2.5 × 10⁵, 2.5 × 10⁴, 5 × 10³, and 2.5 × 10³ PIBs/larva were 8.1, 9.9, 11.3, 12.2, and 13.1 days, respectively. Approximately 37 and 60% of the total larval mortality occurred during the third- and fourth-instar, respectively. The periods to pupation and the pupal weights of survivors apparently were not affected by virus concentration. Moth emergence from surviving pupae was not reduced.     

CYTOLOGICAL STUDIES ON THE ANTIMETABOLITE ACTION OF 2,6-DIAMINOPURINE IN VICIA FABA ROOTS

At a concentration of 9.6 x 10^–5 M, 2,6-diaminopurine (DAP) completely inhibited cell enlargement, cell division, and DNA synthesis (determined by microphotometric measurement of Feulgen dye) in Vicia faba roots. Inhibition of cell enlargement was partially reversed by adenine, guanine, xanthine, adenosine, and desoxyadenosine. Guanine and the nucleosides gave the greatest reversal, suggesting that one point of DAP action upon cell enlargement is a disruption of nucleoside or nucleotide metabolism, possibly during pentosenucleic acid synthesis. DAP inhibited cell division by preventing onset of prophase. At the concentrations used it had no significant effect on the rate or appearance of mitoses in progress. Inhibition of entrance into prophase was not directly due to inhibition of DNA synthesis since approximately half of the inhibited nuclei had the doubled (4C) amount of DNA. Adenine competitively reversed DAP inhibition of cell division, giving an inhibition index of about 0.5. Guanine gave a slight reversal while xanthine, hypoxanthine, adenosine, and desoxyadenosine were inactive. A basic need for free adenine for the onset of mitosis was suggested by this reversal pattern. Meristems treated with DAP contained almost no nuclei with intermediate amounts of DNA, indicating that DAP prevented the onset of DNA synthesis while allowing that underway to reach completion. The inhibition of DNA synthesis was reversed by adenine, adenosine, and desoxyadenosine although synthesis appeared to proceed at a slower rate in reversals than in controls. Inhibition of DNA synthesis by DAP is probably through nucleoside or nucleotide metabolism. A small general depression of DNA content of nuclei in the reversal treatments was observed. This deviation from DNA "constancy" cannot be adequately explained at present although it may be a result of direct incorporation of DAP into DNA. The possible purine precursor, 4-amino-5-imidazolecarboxamide gave no reversal of DAP inhibition of cell elongation and cell division and only a slight possible reversal of inhibition of DNA synthesis.     

THE EFFECT OF LIGHT ON THE PERMEABILITY OF PARAMECIUM

1. The permeability of Paramecium to NH₄OH is greater when the cells are exposed to light than it is when they are in darkness. 2. This change can be demonstrated in cells exposed to monochromatic red light, though it is small. It becomes greater as the wave lengths shorten, and is greatest in the near ultra-violet. 3. The permeability increases as the duration of exposure to light is prolonged. 4. These experiments demonstrate the necessity of controlling the illumination when using Paramecium in physiological tests.