Cell growth and tRNA-lys4 synthesis in mouse 3T3 cells

The RPC-5 chromatographic profiles of lys-tRNA were analyzed during the growth of 3T3 cells in culture. An inverse relationship was seen between tRNA₂^lys and tRNA₄^lys which was markedly influenced by medium changes. This interchange of tRNA₂^lys and tRNA₄^lys could be controlled by altering the levels of serum in the medium, or more precisely by altering the serum to cell ratio. A different change in lys-tRNA distribution was seen when the cells reached confluency. The amounts of tRNA₂^lys, tRNA₃^lys and tRNA₄^lys all decreased with a corresponding increase in either tRNA₅^lys or tRNA₆^lys. An identical change in lys-tRNA could be produced by shifting sparse cells into a medium containing 10% calf plasma instead of 10% serum. Both tRNA^lys profiles and cell growth were returned to normal when the cells were returned to medium with 10% fetal calf serum (FCS) or 10% calf plasma and fibroblast growth factor (FGF). A third alteration in tRNA^lys profiles was seen by the addition of cAMP to the cultures. A decrease in tRNA₅^lys and a corresponding increase in tRNA₆^lys was seen upon the addition of 10^?3 M db-cAMP and was accentuated by the simultaneous addition of 10^?3 M methyl isobutylxanthine.These data are consistent with an ordered sequence of tRNA^lys modification involving tRNA₂^lys, tRNA₃^lys, tRNA₄^lys, tRNA_5B^lys and tRNA₆^lys. Several of the factors which control proliferation appear to control the activity of different tRNA-modifying enzymes in this tRNA^lys pathway thereby controlling the levels of tRNA₄^lys, a tRNA previously shown to correlate directly with the proliferative rate of cells.     

Cell growth and tRNA4 synthesis in mouse 3T3 cells

The RPC-5 chromatographic profiles of lys-tRNA were analyzed during the growth of 3T3 cells in culture. An inverse relationship was seen between tRNA₂^lys and tRNA₄^lys which was markedly influenced by medium changes. This interchange of tRNA₂^lys and tRNA₄^lys could be controlled by altering the levels of serum in the medium, or more precisely by altering the serum to cell ratio. A different change in lys-tRNA distribution was seen when the cells reached confluency. The amounts of tRNA₂^lys, tRNA₃^lys and tRNA₄^lys all decreased with a corresponding increase in either tRNA₅^lys or tRNA₆^lys. An identical change in lys-tRNA could be produced by shifting sparse cells into a medium containing 10% calf plasma instead of 10% serum. Both tRNA^lys profiles and cell growth were returned to normal when the cells were returned to medium with 10% fetal calf serum (FCS) or 10% calf plasma and fibroblast growth factor (FGF). A third alteration in tRNA^lys profiles was seen by the addition of cAMP to the cultures. A decrease in tRNA₅^lys and a corresponding increase in tRNA₆^lys was seen upon the addition of 10⁻³ M db-cAMP and was accentuated by the simultaneous addition of 10⁻³ M methyl isobutylxanthine.These data are consistent with an ordered sequence of tRNA^lys modification involving tRNA₂^lys, tRNA₃^lys, tRNA₄^lys, tRNA_5B^lys and tRNA₆^lys. Several of the factors which control proliferation appear to control the activity of different tRNA-modifying enzymes in this tRNA^lys pathway thereby controlling the levels of tRNA₄^lys, a tRNA previously shown to correlate directly with the proliferative rate of cells.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

Fall in oxygen tension of culture medium stimulates the adenosinergic signalling of a human T cell line

We examined the short-course expression of various parameters involved in the adenosinergic signalling of a human T cell line during in vitro decrease of the medium culture oxygen tension mimicking in vivo hypoxia. Fall of 92 mmHg in oxygen tension of culture medium induced in CEM, a CD4+ human T cell line, a continuous production of hypoxia-inducing factor-1α with a plateau value at 9 h, a rapid increase in adenosine production peaking at 3 h and a decrease in adenosine deaminase peaking at 6 h. The adenosine A_2A receptor (A_2AR) protein level of CEM cells was enhanced with a peak at 6 h. Intracellular 3′,5′-cyclic adenosine monophosphate accumulated in CEM cells with a maximal level at 9 h. These results show that a human-cultured T cells line can upregulate its own adenosine production and A_2AR expression during exposure to acute hypoxia. Hypoxia-increased stimulation of the adenosinergic signalling of T cells may have immunosuppressive properties and, consequently, A_2AR agonists may have therapeutic relevance.     

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation

Lipopolysaccharide (LPS) activates innate immune responses through TLR4·MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4·MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV_A, a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV_A activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV_A activation. Computational studies suggested that unique ionic interactions exist between lipid IV_A and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4′-phosphate on lipid IV_A interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys³⁶⁷ and Arg⁴³⁴) at the dimerization interface. When replaced with their negatively charged human counterparts Glu³⁶⁹ and Gln⁴³⁶, mouse TLR4 was no longer responsive to lipid IV_A. In contrast, human TLR4 gained lipid IV_A responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV_A species specificity. Thus, using lipid IV_A as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.     

Evaluation of high salinity adaptation for lipid bio-accumulation in the green microalga Chlorella vulgaris

Aiming at the reutilizing wastewater for algal growth and biomass production, a saline water rejected from reverse osmosis (RO) facility (salinity 67.59 g L⁻¹) was used to cultivate the pre-adapted green microalga Chlorella vulgaris. The inoculum was prepared by growing cells in modified BG-11 medium, and adaptation was performed by applying a gradual increase in salinity (56.0 g L⁻¹ NaCl and 125 ppm FeSO₄·7H₂O) to the culture in 200 L photobioreactor. Experiments using the adapted alga were performed using original-rejected water (ORW) and treated rejected water (TRW) comparing with the recommended growth medium (BG-11). The initial salinity of ORW was chemically reduced to 39.1 g L⁻¹ to obtain TRW. Vertical photobioreactors (15 L) was used for indoor growth experiments. Growth in BG-11 resulted in 1.23 g L⁻¹, while the next adaptation growth reached 2.14 g L⁻¹ of dry biomass. The dry weights of re-cultivated Chlorella after adaptation were 1.49 and 2.19 g L⁻¹ from ORW and TRW; respectively. The cellular oil content was only 12% when cells grown under control conditions verses to 14.3 and 15.42% with original and treated water, respectively. Induction of stress affected the fatty acid methyl esters (FAMEs) profile and the properties of the resulting biodiesel. The present results indicated that induction of stress by high salinity improves the quality of FAMEs that can be used as a promising biodiesel fuel.     

A medium and simplified procedure for growing single cells from Solanum species

A nutrient medium that allows rapid growth of calli from individual cells of several Solanum species has been developed. The medium is based upon that of Kao and Michayluk (Planta, 126 (1975) 105–110). Modifications that improve plating efficiencies at low density include omission of pyruvate, malate, citrate and fumarate and increasing the phosphate level from 1.25 to 5 mM. The inclusion of 0.1–0.2% bovine serum albumin was essential for growth at low density. At a plating density of 80 protoplasts/ml, plating efficiencies of 1.5—2.0% for Solanum tuberosum L. and S. cardiophyllum Lindl. are often obtained. Single cells of these species were mechanically isolated after 48 h of culture at 800 or 8000 protoplasts/ml and plated singly on fresh medium. The single cells divided and formed rapidly-growing celli with plating efficiencies of 37–75%. Plants have been regenerated from these calli.     

Changes in Rhodopseudomonas sphaeroides isoaccepting phenylalanyl-tRNA species during transitions from chemoheterotrophic to photoheterotrophic growth

A new iso-accepting tRNA^phe from extracts of chemoheterotrophic and photoheterotrophic cells of Rhodopseudomonas sphaeroides has been identified by both BDEAE cellulose and RPC-5 chromatography. Rechromatography of each of the tRNA^phe species in either the acylated or deacylated state shows that they migrate as single homogeneous peaks.In steady-state chemoheterotrophic cultures of R. sphaeroides tRNA _I–II ^phe account for 25–30% of the total phenylalanine accepting activity while in steadystate photoheterotrophic cultures tRNA _I–II ^phe account for no more than 10% of the total phenylalanine accepting activity.During the transition from chemoheterotrophic to photoheterotrophic growth conditions the levels of tRNA _I–II ^phe fall in an exponential manner during the first half of the intracytoplasmic membrane induction period. tRNA _I ^phe then remains at a level 10% that of its steady-state chemoheterotrophic level as long as photoheterotrophic growth conditions remain. tRNA _II ^phe , after dropping to 10% of its former chemoheterotrophic level then returns to a level 50% that of its chemoheterotrophic level as long as photoheterotrophic growth conditions remain.Abbreviations BDEAE benzoylated diethyl amino ethyl - RPC reversed phase chromatography - TCA tricholroacetic acid - ICM intracytoplasmic membrane Submitted by WDS in partial fullfilment of requirements for the M.S. degree     

Identification of Gibberellins A4, A9, and A24 from Phaeosphaeria sp. L487 Cultured in a Chemically Defined Medium

Gibberellins A₄, A₉, and A₂₄ were isolated and identified from the new gibberellin-producing fungus, Phaeosphaeria sp. L487 (Loculoascomycetes), cultivated in a chemically defined medium; their yields from the culture filtrate were ca. 1. 7, 0. 3, and 0.4 μg/ml, respectively. Gibberellins A₄ and A₉ significantly stimulated the hypocotyl growth of Chinese cabbage seedlings at a very low concentration of less than 0.01 μg/ml.     

Growth characteristics and toxin production in batch cultures of Anabaena flos-aquae: effects of culture media and duration

The effects of media and culture duration on growth, macromolecular composition and toxicity of an anatoxin- a-producing freshwater cyanobacterium Anabaena flos-aquae (UTEX 2383) were evaluated. The four media A₃M₇, CB, MA and B-12 influenced growth in terms of cell number, chlorophyll-a content and specific growth rate. A₃M₇ medium supported the best growth. The macromolecular composition of cultured cells, viz. total carbohydrate, protein and lipid content varied with media and culture duration reaching maximum concentration at various growth periods. The differences were significant due to interaction of the culture medium and duration. Toxicity of cells grown in different media was compared by Artemia salina bioassay and mouse units. The cells grown in A₃M₇ medium showed highest toxicity and the optimum culture duration was 5 weeks. In terms of both growth characteristics and toxicity the media can be ranked as A₃M₇, MA, CB and B-12 in decreasing order.     

A developmental study on the capacity of rabbit granulosa cells to respond to trophic hormones and secrete progesterone in vitro

To determine when undifferentiated rabbit granulosa cells first develop the capacity to secrete progesterone, pieces of intact ovaries from neonatal rabbits (newborn—30 days old) and pure granulosa cells from 150–1200 μm follicles at 60–600 days old were cultured in vitro for 6–10 days with human chorionic gonadotropin (HCG), Pergonal (LH/FSH), dibutyryl cyslic AMP (Bu₂CAMP), prostaglandin E₂, estradiol-17β, and as controls. The culture medium was collected every 2 days, and progesterone, estrone, and estradiol-17β were measured by radioimmunoassay.None of the neonatal ovaries or granulosa cell cultures secreted estrone or estradiol-17β spontaneously or in response to stimulation by gonadotropins, Bu₂CAMP, or prostaglandin E₂.Control cultures of newborn and 7-day-old ovaries did not secrete progesterone, but ovaries from 17- and 30-day-old rabbits did. Gonadotropins and Bu₂CAMP induced progesterone secretion in 7-day-old ovaries and stimulated its production 5-10-fold in ovaries at 17 and 30 days old, but prostaglandin E₂ and estradiol-17β were without effect.Granulosa cells from all antral follicles (200–1200 μm) secreted progesterone spontaneously, and its production was stimulated 100–1000-fold with gonadotropins and Bu₂CAMP, but not with estradiol-17β or prostaglandin E₂. In contrast, granulosa cells from 100–150 μm preantral follicles from 200-day-old animals did not secrete progesterone under these culture conditions.These results demonstrate that rabbit granulosa cells differentiate the capacity to secrete progesterone at the time the primary follicle develops an antrum, and suggest the differentiation process involves the acquisition of the capacity to respond to gonadotropins perhaps by the synthesis or unmasking of gonadotropin receptors.     

Plant regeneration from stem cortex protoplasts of Brassica napus

Cell layer strips composed of the epidermis and 7–9 layers of subepidermal cells were isolated from the 3–4 terminal internodes of Brassica napus cv Westar plants at the early flowering stage. The strips were precultured for one day in modified liquid MS [11] medium and subsequently incubated for 17–18 h in a 0.4 M mannitol solution containing 1% Macerozyme and 1% Cellulase Onozuka R-10. Protoplast yield was 2–2.8×10⁶ per 1.0g of tissue. Protoplasts were cultured at 1×10⁵/ml in three different media: S₁ [13], B [12] and L[8]. The first cell divisions occurred after 2–8 days of culture at frequencies of 20–54%. The highest growth rate of colonies was obtained in L medium containing 0.4 M sucrose and 2% Ficoll. After 4 weeks, green calli, 1–2 mm in diameter were transferred onto B₅ [2] medium with 3 mgl^-1 zeatin, 1% sucrose, 0.1 M mannitol and 0.5% agarose for shoot regeneration. Up to 20% of the calli regenerated shoots which subsequently were rooted and established in soil in the greenhouse.     

Characteristics of arachidonic acid metabolism of human endothelial cells in culture

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E₂ and thromboxane A₂ in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum.After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E₂ and thromboxane A₂ production showed no variations during the study.     

The physiological state of suspension cultured cells affects the expression of the β-glucuronidase gene following transformation of tall fescue (Festuca arundinacea) protoplasts

Both the stage of the growth cycle and the age of the cell culture used to isolate protoplasts had a pronounced effect on both transient and stable expression of the GUS gene. A level of GUS gene transient expression of 9000 pmol 4MU/μg protein/h and a frequency of GUS gene stable expression of 5.72% were obtained with protoplasts isolated from suspension cultures 10–20 weeks after initiation and 3–4 days after subculturing when an optimized transformation protocol and a rice actin 1 promoter-uidA gene construct were used. The effect of the cell growth cycle on GUS gene transient expression was closely correlated with the growth rate and the rate of protein synthesis in cell cultures whereas prolonged subculturing of the cells resulted in a gradual decline in both transient and stable expression. The length of time cells were digested in cell wall digestion enzyme and the osmolarity of the transformation medium were found to critically affect both the level of transient and stable GUS gene expression. The composition and osmolarity of the protoplast culture medium was less critical for transient GUS gene expression although the osmolarity of the medium was shown to have a significant effect on stable expression of the GUS gene.     

Effect of darkness on isoperoxidases in tobacco tissue cultures

In order to study the effect of light on the tobacco tissue culture WR-132, 5 passages (10 days' growth per passage) of these cells were grown in darkness, and 3 passages were separately grown in intense light (16000 lx). All other growth conditions were the same. The resulting isoperoxidase patterns present in these cells and in their growth media were analyzed at 2-day intervals during this period and then compared with the isoperoxidase patterns of cells grown under dim light conditions (10 lx). A new cathodic isoperoxidase (C_n) appeared in the medium within 2 days after the cells were placed in the dark. C_n was present in all media of WR-132 cell cultures analyzed throughout the 5 passages grown in darkness. The fifth passage in darkness produced total cessation of growth (apparent death). C_n increased and new anodic isoperoxidases A_a, A_b, A_d and A_e appeared in the media as the cells approached death in darkness.     

Decrease in the density of DNA topological turns during in vitro aging of Syrian hamster cells

Primary cultures of Syrian hamster embryonal fibroblasts were grown until the culture flask bottom was completely covered, then passed into a fresh medium with the dilution 1:2 and grown again until complete sealing of the bottom glass surface was achieved. The period required to attain cellular confluency and the density of topological turns (‘titratable superhelical turns’) in nuclear chromosomal superhelical DNA loops were measured in the course of cultivation. Embryonal cells of Syrian hamsters were found to undergo up to twelve culture doublings (passages) in a medium containing 10% bovine serum. The period required to reach confluency increased from 1–2 days for early passage cells, to 4–8 days for late passage cells, which were often able to reach confluency only after change of growth medium. The density of topological turns in nuclear DNA loops was found to decrease, from a range of −0.076 to −0.083 turns per 10 base pairs (bp) in cells of 1–3 passages, to −0.062 turns per 10 bp in cells of 11–12 passages. This decrease in density of DNA-topological turns may play a part in switching the activity of the genes responsible for cellular proliferation and differentiation in the course of aging of primary cell culture.     

Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture

The effects of vitamin K on the morphology and the growth of mouse neuroblastoma (P₂), mouse melanoma (B-16) and rat glioma (C-6) cells in culture were studied. Vitamin K₃ inhibited the growth (due to cell death and partial or complete inhibition of cell division) of all three cell types without causing any morphological differentiation. Vitamin K₃ was more effective than vitamin K₁. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma or glioma cells. Glioma cells did not grow in hormone-supplemented serum-free medium; however, both neuroblastoma and melanoma cells grew to a level 70–80% of that found in serum-supplemented medium. Neuroblastoma cells and melanoma cells cultured in serum-free medium exhibited a 2–3 fold higher sensitivity to vitamin K₃ than those cultured in serum-supplemented medium. This suggests that serum factors attenuate the growth inhibitory effect of vitamin K₃ on tumor cells in culture, probably by reducing the availability of this vitamin to the cells. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma cells even when they were treated in serum-free medium. The fact that micromolar concentrations of vitamin K₃ inhibit the growth of tumor cells in culture suggests that this vitamin may be a potentially useful anticancer agent.     

Intracellular peptidoglycan hydrolases of the bacterium Xanthomonas campestris XL-1

A system of intracellular peptidoglycan hydrolases of Xanthomonas campestris XL-1 comprises about 10 enzymes of different localization and substrate specificity. Seven enzymes (A₁-A₇) are localized in cytosol, one enzyme (A₈) in periplasm, and two enzymes (A₉, A₁₀) were found in the fraction of cell walls and membranes. While the culture is entering the logarithmic growth stage from the stationary stage, a change occurs in the activity of the cytosolic enzymes: A₁ significantly increases, and A₅ and A₆ decrease. The spectrum of cytosolic enzymes also depends on the growth medium composition. The enzyme A₇ present in cells secreting extracellular enzymes (medium 5/5) was not found in non-secreting cells (LB medium). Unlike extracellular enzymes, intracellular peptidoglycan hydrolases are primarily acidic proteins. The data indicate that the system of intracellular peptidoglycan hydrolases of X. campestris is under complex and strict regulation.     

A 2′←5′-Adenylate Trimer With a Positively Charged 2′(3′)-Terminal 8-(4-Aminobunl)Aminoadenosine Residue: Synthesis,Conformation and RNase L Binding

Abstract

An analogue of the 2-5A core trimer containing an 8-(4-aminobutyl)-aminoadenosine (1; A) residue at the 2′(3′)-terminus [2; (2′,5′)A₂A^?] was synthesized. The conformation of (2′,5′)A₂A^? was studied by ₁H, ¹³C-NMR, and CD spectroscopy. The (2′,5′)A₂A^? exhibits very low binding ability to the RNase L of mouse L cells, but slightly enhanced resistance to digestion by SVPD compared to the parent trimer.     

Identification of the Balbiani ring 2 chromomere and determination of the content and compaction of its DNA

Highly purified tRNAs from Drosophila melanogaster were iodinated with ¹²⁵I and hybridized to squashes of polytene chromosomes of Drosophila salivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA ₂ ^Arg , 42A, 84F_1,2; tRNA ₂ ^Asp , 29DE; tRNA ₃ ^Gly , 22BC, 35BC, 57BC; tRNA ₂ ^Lys , 42A, 42E; tRNA ₅ ^Lys , 84AB, 87B; tRNA ₂ ^Met , 48B_5–7, 72F_1–2, 83F-84A; tRNA ₃ ^Met , 46A_1–2, 61D_1–2, 70F_1–2; tRNA ₄ ^Ser , 12DE, 23E; tRNA ₇ ^Ser , 12DE, 23E; tRNA _3a ^Val , 64D; tRNA _3b ^Val , 84D_3–4, 92B_1–9; tRNA ₄ ^Val , 56D_3–7, 70BC.