Gypsy moth cell lines divergent in viral susceptibility

Summary A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis virus. This research was funded in part through a reimbursable agreement with the U.S. Forest Service.     

Physical conditions of propagation media and their influence on the rooting of cuttings

Summary The air content in three types of propagation media, Jiffy-7 and Jiffy-9 which are Sphagnum peat and Grodan which is rockwool, were investigated when they were held at moisture tensions of 0,6 and 12 cm measured from the base of the media. At 0 cm tension the air content (vol. %) was highest in Jiffy-9 and lowest in Jiffy-7. At 12 cm tension the air content was higher in Grodan than in Jiffy-9 and Jiffy-7. Oxygen diffusion coefficients (ODC) and oxygen diffusion rates (ODR) were measured at the different air contents. At air contents below 20 vol. % ODC was about the same for Jiffy-9 and Grodan but at air contents above 20 vol.% it was larger for Jiffy-9 than for Grodan. The oxygen diffusion rate was measured at 0, 4 and 8 cm moisture tension. At all tensions it was approximately 20% higher in Jiffy-9 than in Grodan and Jiffy-7. The ODR in Jiffy-7 and Grodan were affected equally at the same tension, although Grodan contained more air. Report no 253     

Spectrophotometric titrations of micellar Schiff''s bases prepared from pyridoxal 5′-phosphate

Electron absorption and equilibrium of the Schiffs bases prepared between pyridoxal 5′-phosphate (PLP) and dodecylamine (DODA) or some other shorter chain amines have been studied in nonionic and cationic micellar solutions with various pH of the bulk solution. In the presence of the nonionic (Triton X-100) micelles the Schiffs bases formed between PLP and DODA were embedded into the micelles because the absorption occured at 335 nm, indicative of the nonpolar milieu. This absorption was constant at pH 5–10. At pH 3–5, the tautomeric form absorbing at 415 nm appeared. This resembles the titration of glycogen phosphorylate or that of Schiffs bases in methanol. Short chain amines absorbed at 415 nm, which is typical of Schiffs bases in aqueous solutions. Tryptophan also absorbed first at 415 nm but the absorption changed to 325 nm with a half-time of ～20 min. This was interpreted as being due to formation of the cyclic structure catalysed by micelles. The pH-dependent equilibrium constant of the reaction between PLP and DODA in Triton X-100 solution had a maximum at pH9, the value being 3500 M^?1, about ten times greater than the value of ethylamine at the same pH. Spectral properties of PLP-DODA imines in the cationic micelles (cetyltrimethylammonium bromide) resembled those in the nonionic micelles, except that at low pH the absorption peak in the 415 nm region did not appear. The equilibrium constant of PLP-DODA had maximum at pH 9, the value being as high as 118000 M^?1. Different properties of nonionic and cationic micelles and the design of micellar model systems of PLP enzymes are discussed.     

Ultracentrifugation in polyethylene tubing: An efficient method to concentrate protein solutions on the microliter scale

An ultracentrifugation procedure is described to concentrate protein solutions on the microliter scale. Protein solutions were centrifuged in U-shaped lengths of polyethylene tubing at 160 000 × g for 15 h and thereafter fractionated by cutting the tubing. The method can be performed in a conventional ultracentrifuge and needs no special equipment.     

Prostaglandin D2, a potential antineoplastic agent

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D₂ (PGD₂) was found most active. PGD₂ exerted a dose dependent inhibition of L1210 cell growth over 3.6 μ$\text{M}$. At 14.3 μ$\text{M}$ growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC₅₀ value of PGD₂ on L1210 cell growth was calculated to be 6.9 μ$\text{M}$ (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD₂ was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA₂ showed a comparable, and PGE₂ a less but significant growth inhibitory activity, while PGB₂, PGF_2α and PGI₂ had no such effects on cell proliferation at 14.3 μ$\text{M}$ concentration. These results suggest a potential antineoplastic activity of PGD₂.     

Estimating the maintenance energy and biomass concentration of Saccharomyces cerevisiae by continuous calorimetry

Continuous calorimetry has been applied to monitoring the heat evolution of Saccharomyces cerevisiae grown on d-glucose. The heat evolution, together with the energy and carbon balances, was used to evaluate the energetic efficiency of biomass, by-product biosynthesis, fermentative heat evolution as well as the maintenance energy of S. cerevisiae in ‘aerobic fermentation’ and ‘aerobic respiration’. In aerobic fermentation, under catabolite repression, the fraction of substrate energy converted to heat evolution, maintenance requirement, and biomass decreased with the increase of d-glucose concentration. The fraction of substrate energy converted to ethanol is the highest value and it could contribute up to 70% of the total substrate energy. In aerobic respiration, 43% of the total substrate energy was evolved as heat. While 50% of the total substrate energy was converted into biomass, only 7% of the total substrate energy was used for maintenance functions. The maintenance energy coefficient of S. cerevisiae was determined to be 0.427 MJ kg^?1 cell h^?1 (0.102 kcal g^?1 cell h^?1). For the first time, heat evolution together with yield-maintenance energy was used to predict biomass concentration during the fed-batch cultivation of S. cerevisiae.     

Ion flow through membranes and the resting potential of cells

Summary A knowledge of the relationship between ion flow, both passive and active, ionic concentration, and membrane potential is essential to the understanding of cellular function. The problem has been analyzed on the basis of elementary physical and biophysical principles, providing a theoretical model of current flow and resting potential of cells, including those in epithelia. The model assumes that the permeability of the ion channets is not voltage dependent, but applies to gated channels when the gates are open. Two sources of nonlinearity of the current-voltage relationship are included in the analysis: ionic depletion and accumulation at the channels' mouths, and channel saturation at higher concentrations. The predictions of the model have been quantitative, validated by comparison with experiment, which has been limited to the only two cases in which adequate data was found. Application of the theory to the scala media of the mammalian cochlea has explained the source of its high positive potential and provided estimates of the Na⁺ and K⁺ permeabilities of the membranes of its marginel cess. This analysis provides a theoretically sound alternative to the widely used Goldman equation, the limited validity of which was emphasized by Goldman (D.E. Goldman, 1943,J. Gen. Physiol.27:37–60), as well as its derivatives, including the Goldman-Hodgkin-Katz equation for resting potentials.     

Template-directed synthesis of oligonucleotides under eutectic conditions

Summary One of the most important sets of model prebiotic experiments consists of reactions that synthesize complementary oligonucleotides from preformed templates under nonenzymatic conditions. Most of these experiments are conducted at 4°C using 0.01–0.1 M concentrations of activated nucleotide monomer and template (monomer equivalent). In an attempt to extend the conditions under which this type of reaction can occur, we have concentrated the reactants by freezing at –18°C, which is close to the NaCl–H₂O eutectic at –21°C.The results from this set of experiments suggest that successful syntheses can occur with poly(C) concentrations as low at 5×10^–4 M and 2MeImpG concentrations at 10^–3 M. It was also anticipated that this mechanism might allow the previously unsuccessful poly(A)-directed synthesis of oligo(U)s to occur. However, no template effect was seen with the poly(A) and ImpU system. The failure of these conditions to allow template-directed synthesis of oligo(U)s supports the previously proposed idea that pyrimidines may not have been part of the earliest genetic material.Because of the low concentrations of monomer and template that would be expected from prebiotic syntheses, this lower temperature could be considered a more plausible geologic setting for template-directed synthesis than the standard reaction conditions.     

Synergistic Regulation of Cytosolic Ca2+ Concentration in Mouse Astrocytes by NK1 Tachykinin and Adenosine Agonists

The effects on cytosolic Ca2+ concentration of 2-chloroadenosine and [L-Pro9]-substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L-Pro9]-substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2-chloroadenosine alone occurred. When simultaneously applied, [L-Pro9]-substance P and 2-chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihydropyridines, La3+, and Co2+, excluding the implication of voltage-operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L-Pro9]-substance P and 2-chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L-Pro9]-substance P in potentiating the 2-chloroadenosine-evoked response. Like 2-chloroadenosine, pinacidil, which hyperpolarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L-Pro9]-substance P. Conversely, depolarization with 50 mM KCl canceled the effects of either pinacidil or 2-chloroadenosine applied with [L-Pro9]-substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2-chloroadenosine.