Effect of serum step-down on protein metabolism and proliferation kinetics of NHIK 3025 cells

Human NHIK 3025 cells growing exponentially in 30% or 3% serum had population doubling times of 19.1 and 27.6 hours, respectively. These values were equal to the calculated protein doubling times (17.6 and 26.5 hours, respectively), showing that the cells were in balanced growth at both serum concentrations. Stepdown from 30% to 3% serum reduced the rate of protein synthesis within 1–2 hours, from 5.7% hour to 4.3% hour, while the rate of protein degradation was unchanged (1.7%/hour). In cells synchronized by mitotic selection from an exponentially growing population, the median cell cycle durations in 30% and 3% serum were 17.2 and 23.6 hours, respectively, which were also in good agreement with the protein doubling times. The median G₁ durations were 7.1 and 9.6 hours, respectively. Thus the duration of G₁ relative to the total cell cycle duration was the same in the two cases. Complete removal of serum for a period of 3 hours resulted in a 3-hour prolongation of the cell cycle regardless of the time after mitotic selection at which the serum was removed. For synchronized cells, the rate of entry into both the S phase and into the subsequent cell cycle were reduced in 3% serum as compared to 30% serum, the former rate being significantly greater than the latter at both serum concentrations. Our results thus indicate that these cells are continuously dependent upon serum throughout the entire cell cycle.     

Isolation and characterization of lambda phages carrying the basic replicon of the resistance plasmid R1

Summary Specialized transducing lambda phages, oriR1, harboring DNA from the resistance plasmid R1drd-19 and its copy mutant pKN103 were isolated. From measurements of CCC-DNA content it is concluded that upon infection the phages can establish themselves as self-replicating plasmids in recA hosts lysogenic for lambda. It is thought that this bypassing of lambda immunity is due to the presence of the R1 origin of replication. The plasmids are sensitive to the incompatibility expressed by plasmid R1. This has been shown mainly by transduction of oriR1 into recipients containing R1 plasmids or plasmid pBR322 carrying the basic replicon. We were able to demonstrate that a copy mutant of plasmid R1 was insensitive to copA ⁺, but sensitive to the conserted action of Pst1 fragments F₁ and F₂. This mutant was previously assumed to be of the dominant type. Physical mapping of the oriR1 derivatives verified that they carry the basic replicon of plasmid R1. The plasmids are not stably maintained, but are lost in a frequency of 1%–2% per cell generation, which is consistent with their lack of the R1par region.     

Summer water relations of the desert phreatophyte Prosopis glandulosa in the Sonoran Desert of southern California

Summary Two shoot populations of the rhizomatous, patchforming herb Solidago canadensis were studied throughout a developmental cycle in two abandoned pasture sites in southern Ontario. The shoot cohorts that emerged in spring dominated the two populations; subsequent recruitment was very low. Shoot mortality was highest in June and was concentrated in the smallest size classes. Both populations showed a pronounced bimodal size structure for most of the growth cycle. Relative growth rate of shoots declined as the growing season progressed and tended to be lowest in the smallest size classes. Inflorescence production depended on shoot size. The calculated relationship between log mean weight and log density of shoots was not constant during the growth cycle and the calculated maximum biomass values do not transgress the ultimate thinning line suggested from previously published data.Address for proofs and present address: Department of Geography, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX. England     

ATP-levels of germinating seeds in relation to vigor

Determination of seed vigor was attempted by comparing ATP-levels of deteriorating seed to germination percentage and production of dry matter. Immediately after imbibition of any seed lot investigated, a production of ATP took place. This ATP-accumulation invariably reached a plateau after 6 h of imbibition. Two well germinating seed lots of rape, one of cauliflower and one of sugar beet, were artificially aged by means of elevated storage temperature and humidity. Every second week through 16 weeks of deterioration the levels of ATP, ADP and AMP after 7 h of imbibition were compared with the germination percentage. While ADP- and AMP-contents of germinating seed displayed no change (when imbibed 7 h) during the period of artificial aging, seed deterioration was reflected in the ATP-levels long before loss of viability could be detected by the conventional germination test.
When ATP-levels per seed were related to germination percentage throughout the aging, all four seed lots displayed similar patterns although the absolute figures differed. In contrast to the conventional "per seed' basis, however, ATP per gram seed not only displayed similar deterioration patterns, but the absolute values were also of the same magnitude.     

Lysosomal pool of free-amino acids

Free (non-protein) amino acids were measured in whole rat liver and in unmodified lysosomes which were prepared from rat liver by the technique of free-flow electrophoresis. Significant intralysosomal pools of threonine, serine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and arginine were found. No efflux occurred from rat liver lysosomes in isotonic buffered sucrose at 0°C, but all amino acids showed various degrees of efflux at 20⁰ and 37⁰.     

Tubulin synthesis and heat shock-induced cell synchrony in Tetrahymena

This paper offers the suggestion that heat shock inhibition of tubulin synthesis accounts for the molecular mechanism by which periodic heat shocks induce cell synchrony in Tetrahymena. Each heat shock (34 °C) represses tubulin synthesis and blocks the division cycle at the point when the oral structure, rich in microtubules, would normally begin to assemble. Recovery (at 28 °C) from each heat shock is characterized by parallel derepression of tubulin synthesis and of oral development. Changes in protein synthesis patterns are complex when the temperature is shifted up and down between 28 and 34 °C and further experimental support is required in support of the hypothesis here forwarded.     

Potentiation of the in vitro cytotoxic response to syngeneic lymphoma cells by soluble products of tuberculin-sensitive lymphoid cells stimulated with PPD

Spleen cells from rats immunized with the syngeneic (C58NT)D Gross virus induced lymphoma have previously been shown to differentiate into cytotoxic effector cells following restimulation with tumor cells in vitro. Previous work has also demonstrated that the addition of PPD-primed syngeneic spleen cells and PPD to cultures of (C58NT)D-primed spleen cells will potentiate the in vitro cytotoxic response to tumor antigens. In the studies presented here, the potentiating effect was found to be mediated by a soluble factor(s) released by nonadherent cells from BCG-primed rats. The release of this immunopotentiating factor(IPF) required the presence of PPD and varied with the concentration of PPD added. IPF was produced by BCG-primed spleen, lymph node, and thymus cells. Maximal production of IPF in PPD-stimulated cultures was obtained after 6–12 hr of incubation. Supernatants obtained after 30 hr of incubation lacked apparent IPF activity when tested initially, but activity was recovered after mild heat treatment. Recovery of IPF activity after heat exposure is best explained by the presence of a heat-labile inhibitor. IPF itself is stable to heat treatment to 56 °C for 40 min. IPF was also shown to be capable of enhancing immune responses to histocompatibility antigens in vitro.     

Role of Heme in Synthesis and Membrane Binding of Succinic Dehydrogenase in Bacillus subtilis

A 5-aminolevulinic acid-requiring mutant of Bacillus subtilis was isolated. When the mutant is shifted from medium containing 5-aminolevulinic acid to medium lacking this growth factor, the bacteria continued to grow at undiminished rate for about three generations. The membranes from these bacteria contained severely reduced amounts of cytochrome. The mutant was used to study the role of heme synthesis on synthesis and membrane binding of succinic dehydrogenase (SDH). The amount of SDH in whole-cell lysates in the soluble cytoplasmic fraction and in membranes was determined by one-dimensional (rocket) immunoelectrophoresis with an SDH-specific antiserum. After heme synthesis was blocked, the relative amount of SDH in the membrane decreased, whereas increasing amounts of SDH antigen were found in the cytoplasm. When heme synthesis was resumed on readdition of 5-aminolevulinic acid, the amount of membrane-bound SDH antigen increased at a much faster rate than net synthesis. During a 3-h growth period without 5-aminolevulinic acid, there was little change in the pattern of membrane proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled membranes, as compared to membranes from control cultures. However, both the 65,000-dalton and the 28,000-dalton polypeptides of the SDH complex (L. Hederstedt, E. Holmgren, and L. Rutberg, J. Bacteriol. 138:370-376, 1979) were present in decreasing amounts in membranes from 5-aminolevulinic acid-starved bacteria. From these results we suggest that SDH in B. subtilis is synthesized as a soluble protein and becomes membrane bound only when it attaches to a site in the membrane, (part of) which is a cytochrome of b type.     

Circadian rhythms of sensitivity to insecticides in Musca domestica (Diptera, Muscidae)

Circadian rhythms of LD₅₀ values to DDT, dieldrin and malathion, topically applied, were determined for houseflies reared under LD 14:10 with dawn at 06.00 hr. There was a marked increase in susceptibility at 05.00 hr in each case. With dawn at 18.00 hr., DDT LD₅₀ values were lowest at 17.00 hr indicating independence of the flies' biological clocks from clock time of day. Flies reared under LD 18:6 and 10:14 also had circadian rhythms of sensitivity to DDT. Mean daily LD₅₀ values were inversely related to photophase length. The ratios of mean daily LD₅₀ to pre-dawn values were greatest for the longer photophases. Flies reared under LD 14:10 until the pupal stage, then DD until testing showed a normal circadian rhythm. Flies reared in total darkness (DD) showed no diel variations in susceptibility. W.H.O. standard strain flies were used for all the experiments. A fully susceptible (Cooper) and a DDT resistant (DEH-DOV) strain also showed significant circadian rhythms of sensitivity to DDT.

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Zusammenfassung Circadianrhythmen der LD 50-Werte gegenüber DDT, Dieldrin und Malathion-topical angewandt wurden bei Stubenfliegen ermittelt, die bei 14:10 h-Tag mit Tagesanbruch um 6.00 Uhr gezüchtet wurden. In allen Fällen war die Empfindlichkeit um 5.00 Uhr wesentlich erhöht. Bei Tagesanbruch um 18.00 Uhr waren die niedrigsten LD 50-Werte um 17.00 Uhr. Dies weist auf die Unabhängigkeit der biologischen Uhr der Fliegen von der Tageszeit hin. Fliegen, die bei 18:6 oder bei 10:14 LD gezüchtet wurden, zeigten ebenfalls einen Circadianrhythmus hinsichtlich der Empfindlichkeit gegenüber DDT. Die mittleren LD 50-Werte waren umgekehrt proportional zur Länge der Photophase. Das Verhältnis der mittleren täglichen LD 50-Werte zu den Vortagesanbruchwerten war am grössten bei längerer Photophase. Fliegen, die bei 14:10 LD bis zum Puppenstadium und anschliessend bei DD bis zur Testung gehalten wurden, zeigten einen normalen circadianen Rhythmus. Bei Züchtung in völliger Dunkelheit zeigten sie keine Tagesschwankungen in der Empfindlichkeit. Für alle Versuche wurde ein WHO-Standardstamm benutzt. Zwei andere Stämme, einer voll empfindlich (Cooper), der andere resistent (DEH-DOV) zeigten ebenfalls signifikante Circadianrhythmen in der DDT-Empfindlichkeir.