Difference in the Cyclic Adenosine 3′,5′-Monophosphate Levels in Normal and Transformed Cells

CYCLIC 3′5′-adenosine monophosphate (cyclic AMP) regulates many physiological phenomena^1,2. Cellular morphology changes when the dibutyryl derivative of cyclic AMP is added in vitro to the nutrient media of cultured mammalian cells^3–6. Dibutyryl cyclic AMP has also been shown to restore controlled growth to transformed cells³, change the cell's surface architecture^3,7 and induce axon formation⁸ with an accompanied increase in acetylcholinesterase activity⁹ in neuroblastoma cells growing in culture. These effects suggest that the cyclic AMP moiety may have some basic regulatory action on cell growth and cell specialization.     

Effect of different oxidation states of chromium in causing chromosome alterations in cultured CHO cells

Four chromium salts with different oxidation states were tested for their influence in causing chromosome aberrations and sister-chromatid exchange in Chinese hamster ovary cellsin vitro. Cell cultures were treated with CrO₃, K₂Cr₂O₇, CrCl₂ and Cr(NO₃)₃.9H₂O at concentrations of 10^–7, 10^–6, 10^–5 and 10^–4 M for the aberration assay, and 10^–8, 10^–7, 10^–6 and 10^–5 M for the sister-chromatid exchange assay. It was noticed that Cr (VI) compounds-CrO₃ and K₂Cr₂O₇-considerably enhanced the frequencies of aberrations and sister-chromatid exchanges compared to the control cultures. CrCl₂ and Cr(NO₃)₃.9H₂O–Cr (II) and Cr (III) respectively-caused a slight increase in sister-chromatid exchange rates, but the frequencies of aberrations were almost unchanged compared to the controls. These investigations indicate a definite link between the metals and changes produced in the mammalian chromosomes, reaffirming the evidence of carcinogenic potential of Cr (VI) observed by other investigators.Abbreviations BrdU 5-bromo-2-deoxyuridine - CHO Chinese hamster ovary - SCE sister-chromatid exchange     

Choline and acetylcholine: novel cationic osmolytes in Lactobacillus plantarum

The aim of this work was to study the physiological response of Lactobacillus plantarum subjected to osmotic stress in the presence of three structurally related compatible solutes. Either betaine, choline or acetylcholine was accumulated by osmotically stressed cells when provided in the chemically defined medium. Choline and acetylcholine were accumulated to maximum concentrations of 139 and 222 μmol g (dry weight) of cells⁻¹ respectively and were not converted to betaine. Addition of 0.5 mM choline or 0.5 mM acetylcholine to the medium increased the growth rates of cells in media with various amounts of added sodium chloride. Both choline and acetylcholine are positively charged compounds; therefore, it was presumed that charged intracellular solutes could counterbalance the excess of positive charge. Intracellular inorganic ion levels (K⁺, SO²⁻ ₄, PO³⁻ ₄ and Cl⁻) of cells cultured under conditions of osmotic stress remained similar in the presence of either betaine, choline or acetylcholine. However, cells cultured in the presence of choline or acetylcholine accumulated an additional quantity of approximately 125 or 200 μmol.glutamate (dry weight) cells⁻¹ respectively, as compared to cells grown in the presence of betaine. Hence glutamate appears to be the counterion for choline and acetylcholine. This is the first study demonstrating accumulation of choline and acetylcholine in lactic acid bacteria subjected to osmotic stress. Received: 5 February 1997 / Received revision: 15 April 1997 / Accepted: 19 April 1997     

Mtu1 defects are correlated with reduced osteogenic differentiation

Accumulating evidence has revealed that mitochondria dynamics and function regulation is essential for the successful mesenchymal stem cell (MSC) differentiation. In the present study, the researchers reported for the first time that Mtu1 defects are correlated with reduced osteogenic differentiation. Using in vitro cultured bone marrow MSCs and stromal cell line MS5, we demonstrated that depressed Mtu1 expression was associated with reduced 2-thiouridine modification of the U34 of mitochondrial tRNA^Gln, tRNA^Glu, and tRNA^Lys, which led to respiratory deficiencies and reduced mitochondrial ATP production, and finally suppressed osteogenic differentiation. As expected, these Mtu1-deficient mice exhibited obvious osteopenia. Therefore, our findings in this study provide new insights into the pathophysiology of osteopenia.Subject terms: Cell biology, Post-translational modifications, Mesenchymal stem cells     

Arginine supplementation and exposure time affects polyamine and glucose metabolism in primary liver cells isolated from Atlantic salmon

Arginine has been demonstrated to enhance glucose and lipid oxidation in mammals through activation of polyamine turnover. We aimed to investigate how arginine affects energy utilization through polyamine metabolism and whether this effect is time dependent. Primary liver cells were isolated from Atlantic salmon (2.2 kg body weight) fed diets containing 25.5 (low arginine, LA) or 36.1 (high arginine, HA) g arginine/kg dry matter for 12 weeks, to investigate the effect of long-term arginine supplementation. The cells were cultured for 24 h in L-15 medium to which either alpha-difluoromethylornithine (DFMO) or N ¹,N ¹¹-diethylnorspermine (DENSPM) was added. Analysis of the medium by nuclear magnetic resonance revealed significant differences between the two dietary groups as well as between cells exposed to DFMO and DENSPM, with decreased glucose, fumarate and lactate concentrations in media of the HA cells. Liver cells from fish fed the HA diet had higher spermidine/spermine-N1-acetyltransferase protein abundance and lower adenosine triphosphate concentration as compared to the LA-fed fish, while gene expression was not affected by either diet or treatment. Primary liver cells isolated from salmon fed a commercial diet and cultured in L-15 media with or without arginine supplementation (1.82 or 3.63 mM) for 48 h, representing short-term effect of arginine supplementation, showed differential expression of genes for apoptosis and polyamine synthesis due to arginine supplementation or inhibition by DFMO. Overall, arginine concentration and exposure time affected energy metabolism and gene regulation more than inhibition or activation of key enzymes of polyamine metabolism, suggesting a polyamine-independent influence of arginine on cellular energy metabolism and survival.     

Deletion of Thioredoxin Reductase and Effects of Selenite and Selenate Toxicity in Caenorhabditis elegans

Thioredoxin reductase-1 (TRXR-1) is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se) to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0–2 mM Se as selenite results in inverse, sigmoidal response curves with an LC₅₀ of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and ⁷⁵Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC₅₀ 0.23 mM Se) were identical to selenite, but selenate was 1/4^th as toxic (LC₅₀ 0.95 mM Se) as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.     

Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells

We present a fully defined culture system (adapted Essential8^TM [E8^TM] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8^TM medium was modified by adding (1) l ‐proline, (2) l ‐ornithine, (3) N^ω‐hydroxy‐nor‐l ‐arginine acetate, or by (4) lowering the arginine concentration. Reduction of arginine conversion was best obtained by adding 5 mM l ‐ornithine, followed by 3.5 mM l ‐proline and by lowering the arginine concentration in the medium to 99.5 μM. No major changes in pluripotency and cell amount could be observed for the adapted E8^TM media with ornithine and proline. However, our subsequent ion mobility assisted data‐independent acquisition (high‐definition MS) proteome analysis cautions for ongoing changes in the proteome when aiming at longer term suppression of arginine conversion.     

Synthesis of Cell Coat in Normal and Transformed Cells

THE surface of transformed cells has been a focus of considerable attention recently because some of the properties which distinguish these cells from their precursors, such as decreased cell adhesiveness, altered cell orientation and loss of contact and density dependent inhibition^1–3, may relate to changes on their surface. A common feature of vertebrate cells is the cell coat, a glycoprotein structure surrounding the plasma membrane⁴. Electron microscopy has revealed that transformed cells have a thicker coat than normal cells⁵ and we have now found that coat synthesis in cells transformed by an oncogenic DNA virus and in cells transformed by a chemical carcinogen occurs faster than in normal controls whereas only in the virus-transformed cells is the coat significantly thicker.     

Inhibition of growth of mammalian cell cultures by extracts of arginine-utilizing mycoplasmas

Summary Mechanisms of the inhibition of growth of mammalian cell cultures caused by mycoplasmal infection were investigated by using cell-free extracts of 14 species of mycoplasmas. In four mammalian cell lines tested, the growth of two cell lines, FM3A and MDCK, was inhibited by the extracts of arginine-utilizing mycoplasmas, whereas that of the other two cell lines, Vero and LLC-MK2, was not inhibited by extracts of either arginine- or glucose-utilizing mycoplasmas. These results suggest that there are two types of cell cultures, one susceptible and the other insusceptible to arginine-utilizing mycoplasmas. In a series of experiments using FM3A cells, it was found that the growth inhibition caused by the extracts of arginine-utilizing mycoplasmas was due to removal of arginine from the medium by the action of arginine deiminase present in the extracts and that none of the metabolic products of arginine had any effect on the growth. A highly positive correlation (r=0.96,P<0.01) was observed between the activity of arginine deiminase and the growth-inhibiting activity of extracts of arginine-utilizing mycoplasmas.     

Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells

Summary. The present study was designed to evaluate the relevance of arginine transport in nitric oxide (NO) synthesis in vascular smooth muscle cells. For this purpose, NO synthesis and arginine transport (system B^0,+ and y⁺) were evaluated in cells treated with IL-1β or angiotensin II (Ang II). In addition, the effects of 5 mM lysine and glutamine, competitive inhibitors of systems y⁺ and B^0,+ respectively, were examined. L-arginine transport was estimated with ³H-labelled arginine and NO was determined with the Griess reagent. These studies were done in control conditions, arginine-starved cells, and in cells incubated in media containing 10 mM arginine. Our data indicate that induction of NO biosynthesis by IL-1β depends on external arginine when cells are arginine-depleted for 24 hours. The concentration of arginine producing half maximal activation of NO synthesis in arginine-depleted cells ([arginine]_i < 10 μM) was 41.1 ± 18 μM. By contrast, in normal culture conditions, NO synthesis occurred independently of arginine transport. Neither 5 mM lysine or glutamine which abolished arginine transport through systems y⁺ and B^0,+, respectively, reduced nitrite release in cells incubated in normal media. This suggests that the relevance of arginine uptake to NO synthesis depends on the status of intracellular arginine pools. Intracellular arginine concentrations were not affected by the stimulation of NO production using IL-1β or its inhibition using Ang II, but were markedly reduced by arginine starvation for 48 h. Aspartate levels were also reduced by arginine-depletion, but were not affected in cells incubated with 10 mM arginine. By contrast, glutamate levels were reduced in arginine-starved cells and were increased in cells incubated in arginine-supplemented medium. Ornithine levels were markedly increased by arginine supplementation. Altogether, these findings indicate that NO synthesis is normally independent of membrane transport. However in arginine-depleted cells, membrane transport is essential for NO synthesis. It is concluded that arginine transport is required for the long-term maintenance of intracellular arginine pools. Received February 7, 1999; Accepted June 21, 1999     

Arginine metabolism in Chlamydomonas reinhardi. Conditional expression of arginine-requiring mutants

Summary In Chlamydomonas reinhardi, the arginine-requiring mutants directly derived from the wild-type strain are not able to grow on media containing the routinely used concentration of ammonia. The biochemical basis of this phenomenon has been investigated.The results show that the uptake of arginine is severely depressed by NH ₊ ⁴ and that arginine which succeeds in entering the cells under these conditions is immediately broken down by the first enzyme of the catabolic pathway, arginine deiminase.Accordingly, growth is inhibited as the result of non-availability of exogenous arginine for protein synthesis.It is suggested that similar interactions could be responsible for the lack of other types of amino acid-requiring mutants in Chlamydomonas.Chargé de Recherches du Fonds National Belge de la Recherche Scientifique.     

Hepatic Adaptation Compensates Inactivation of Intestinal Arginine Biosynthesis in Suckling Mice

Suckling mammals, including mice, differ from adults in the abundant expression of enzymes that synthesize arginine from citrulline in their enterocytes. To investigate the importance of the small-intestinal arginine synthesis for whole-body arginine production in suckling mice, we floxed exon 13 of the argininosuccinate synthetase (Ass) gene, which codes for a key enzyme in arginine biosynthesis, and specifically and completely ablated Ass in enterocytes by crossing Ass ^fl and Villin-Cre mice. Unexpectedly, Ass ^fl/fl /VilCre ^tg/- mice showed no developmental impairments. Amino-acid fluxes across the intestine, liver, and kidneys were calculated after determining the blood flow in the portal vein, and hepatic and renal arteries (86%, 14%, and 33%, respectively, of the transhepatic blood flow in 14-day-old mice). Relative to control mice, citrulline production in the splanchnic region of Ass ^fl/fl /VilCre ^tg/- mice doubled, while arginine production was abolished. Furthermore, the net production of arginine and most other amino acids in the liver of suckling control mice declined to naught or even changed to consumption in Ass ^fl/fl /VilCre ^tg/- mice, and had, thus, become remarkably similar to that of post-weaning wild-type mice, which no longer express arginine-biosynthesizing enzymes in their small intestine. The adaptive changes in liver function were accompanied by an increased expression of genes involved in arginine metabolism (Asl, Got1, Gpt2, Glud1, Arg1, and Arg2) and transport (Slc25a13, Slc25a15, and Slc3a2), whereas no such changes were found in the intestine. Our findings suggest that the genetic premature deletion of arginine synthesis in enterocytes causes a premature induction of the post-weaning pattern of amino-acid metabolism in the liver.     

Inverse Relationship of Polyoma Tumour Specific Cell Surface Antigen to H-2 Histocompatibility Antigens

NEOPLASTIC transformation is known to be associated with changes in the strength of normal cellular antigens, but the effect can be either an increase or a decrease. In the former category are Forssman antigens in guinea-pig hepatoma¹ and SV40 transformed cells²; HL-A antigens in leukaemic cells³; and “G” antigen in human tumour cells⁴. On the other hand, the intensity of the expression of mouse H-2 histocompatibility antigens is decreased in TL(+) leukaemia⁵ and methylcholanthrene (MCA)-induced tumours⁶. We set out to tell whether the expression of histocompatibility antigens was also affected by transformation with an oncogenic virus and have found that in tumours induced by polyoma virus, the quantity of H-2 antigens varied inversely with the amount of tumour-specific cell surface antigen.     

Chromosome Replication in Toluenized <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cells

BIOCHEMICAL studies of chromosome replication have been hampered by the unavailability of an adequate in vitro system with the basic features of in vivo DNA replication. The criteria for such a system are: (1) semiconservative replication; (2) normal biological activity of newly synthesized DNA; (3) normal advancement of the original replication fork; (4) rate of DNA replication equivalent to in vivo; and (5) expected phenotypic behaviour of temperature-sensitive dna mutants. Systems in Escherichia coli, a membrane-DNA fraction¹, an agar-embedded cell lysate² and toluene-treated cells³ have met two or three of the requirements. Several laboratories have also reported the expected behaviour of ts-dna E. coli mutants in toluenized cells^3–5.     

Blunted diurnal firing in lateral habenula projections to dorsal raphe nucleus and delayed photoentrainment in stress-susceptible mice

Daily rhythms are disrupted in patients with mood disorders. The lateral habenula (LHb) and dorsal raphe nucleus (DRN) contribute to circadian timekeeping and regulate mood. Thus, pathophysiology in these nuclei may be responsible for aberrations in daily rhythms during mood disorders. Using the 15-day chronic social defeat stress (CSDS) paradigm and in vitro slice electrophysiology, we measured the effects of stress on diurnal rhythms in firing of LHb cells projecting to the DRN (cells^LHb→DRN) and unlabeled DRN cells. We also performed optogenetic experiments to investigate if increased firing in cells^LHb→DRN during exposure to a weak 7-day social defeat stress (SDS) paradigm induces stress-susceptibility. Last, we investigated whether exposure to CSDS affected the ability of mice to photoentrain to a new light–dark (LD) cycle. The cells^LHb→DRN and unlabeled DRN cells of stress-susceptible mice express greater blunted diurnal firing compared to stress-näive (control) and stress-resilient mice. Daytime optogenetic activation of cells^LHb→DRN during SDS induces stress-susceptibility which shows the direct correlation between increased activity in this circuit and putative mood disorders. Finally, we found that stress-susceptible mice are slower, while stress-resilient mice are faster, at photoentraining to a new LD cycle. Our findings suggest that exposure to strong stressors induces blunted daily rhythms in firing in cells^LHb→DRN, DRN cells and decreases the initial rate of photoentrainment in susceptible-mice. In contrast, resilient-mice may undergo homeostatic adaptations that maintain daily rhythms in firing in cells^LHb→DRN and also show rapid photoentrainment to a new LD cycle.

Daily rhythms are disrupted in patients suffering from mood disorders, and it is known that the lateral habenula and dorsal raphe nucleus contribute to circadian timekeeping and regulate mood. This study shows that stress-susceptible mice have blunted and inverted diurnal firing rhythms in lateral habenula cells that project to the dorsal raphe nucleus, and have a slow rate of photoentrainment to a new light cycle.     

Behavior of Mycoplasma hominis in a Human Diploid Cell Culture System

The behavior of Mycoplasma hominis in normal human embryonic lung fibroblast (HAIN-55) cell cultures was investigated. Multiplication patterns of cell-associated mycoplasmas and of extracellular mycoplasmas in the HAIN-55 cultures depended upon the size of the inoculum. This relationship did not vary with the number of days the cells had been cultured, nor with the number of HAIN-55 cell passages. The maximum mycoplasmal growth was obtained with inoculum sizes of 10⁵ to 10⁶ colony-forming units (CFU)/ml. The recovery of mycoplasmas decreased rapidly with inoculum size beyond 10⁷ CFU/ml, and growth of the HAIN-55 cells was inhibited. Growth of the cells was also inhibited by the addition of the cytoplasmic fraction of Mycoplasma hominis.     

In vitro propagation of taro,with spermine,arginine, and ornithine

In vitro growth and multiplication of taro [Colocasia esculenta var. antiquorum cv. Keladi Birah] was improved considerably, when primary shoot apices were cultured on two modifications of Linsmaier and Skoog [1965] medium, containing 5.5 mg 1^–1 naphthaleneacetic acid and 0.2 mg 1^–1 kinetin or 1.85 mg 1^–1 naphthaleneacetic acid and 2 mg 1^–1 kinetin and supplemented with 10^–4 or 10^–3 mol·1^–1 of polyamine spermine or either of the precursors of polyamine putrescine—arginine and ornithine. Plantlets were regenerated directly from primary shoot apices, axillary buds and protocorm-like bodies [PLB]. Frequency of plantlet regeneration, rate of development and growth in height of main plantlets were enhanced by the addition of arginine and ornithine to the media. Secondary plantlet formation from axillary buds and PLB were promoted by spermine and arginine respectively.     

Extracellular and intracellular chemical environments in relation to embryogenesisin vitro

Summary Tissue culture isolated from the root ofDaucus carota readily produced embryos when they were grown on a medium (M_s) containing relatively large amounts of NH₄ ⁺ and nitrate. Embryos were never formed on a second medium (M_w) which contained only nitrate in a low concentration. The cells of M_s-cultures always had a high level of NH₄ ⁺ while it was scarcely detected in M_w-cultures. Addition of either nitrate or glutamate to M_w resulted in the formation of embryos and also in the accumulation of a detectable amount of NH₄ ⁺ in the cells of the tissues. Although the occurrence of NH₄ ⁺ in the medium is not necessary for embryo formationin vitro it appears that a certain level of intracellular NH₄ ⁺ is a prerequisite for this process. Since there is a positive correlation between embryo formation and the contents in the cultures of both soluble and insoluble organic nitrogen, it is probable that NH₄ ⁺ is important for embryo formation because it is an essential substrate for the synthesis of organic nitrogen compounds such as amino acids and proteins. The ability to synthesize organic nitrogen from NH₄ ⁺ decreased conspicuously in the tissues which were cultured on M_s for long periods of time and had lost the capacity to form embryos. A certain level of K⁺ in culture media is necessary for optimal production of embryos on media with NH₄ ⁺, while Na⁺ has no effect on embryo formation.The authors are indebted to Mr. E.Mahn, Miss S.Semenoff, and Mrs. E.Bock for their technical assistances.