Intracellular flux analysis in hybridomas using mass balances and in vitro (13)C nmr

Intracellular fluxes are important in defining cellular physiology and its changes in response to environmental variations. Stoichiometric balances combined with extra cellular metabolite measurements were applied to the estimation of intracellular fluxes and the study of energy metabolism in the hybridoma cell line ATCC CRL 1606. Redundant measurements allowed the evaluation of the consistency of the stoichiometry, measurements, and pseudo-steady-state assumption leading to refinement of the assumed biochemistry and identification of measurement errors. To validate the flux estimates, two batch experiments were performed with glucose labeled in the 1 position with (13)C. The distribution of (13)C in secreted lactate was measured via nuclear magnetic resonance spectroscopy (NMR) and compared to that predicted from the estimated intracellular fluxes. There was good agreement between the measured and estimated isotope distributions, demonstrating the validity of the flux estimates obtained from stoichiometric balances. (c) 1995 John Wiley & Sons, Inc.     

BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells

Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H₂O₂ treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H₂O₂-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H₂O₂ but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H₂O₂ exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.     

Effects of anandamide on gestation in pregnant rats

The present study was to test whether the recently described endogenous ligand for the cannabinoid receptor; arachidonyl-ethanolamide (anandamide, ANA), may produce similar effects on pregnancy as the main psychoactive component of marihuana: Δ⁹-tetrahydrocannabinol (THC) in rats. ANA, THC (0.02 mg/kg i.p./day, respectively) or vehicle were injected daily over the third week of pregnancy. The pregnant rats were either killed on day 21 of pregnancy or followed up to delivery. Results show a significant increase in the duration of pregnancy after both THC and ANA treatment. Both drugs caused an increase in the frequency of stillbirths. The mothers' hormone contents in tissues and sera were measured. Decreased LH content was observed in the serum of treated animals. No changes in FSH content were observed either in the pituitary or in the sera. Pituitary prolactin (PRL) levels was lower in ANA treated animals as compared both to controls or THC treated subjects. The serum PRL content decreased in all experimental groups. Decrease in serum progesterone was more prominent in treated rats. Serum levels of prostaglandins (PGF 1 and PGF 2) were significantly decreased after THC and ANA treatment. We conclude that ANA has the same tendency to change reproductory parameters in pregnant rats as THC, although in some cases the effects of ANA were slightly different from that of THC. Both endogenous and exogenous cannabinoids inhibit PG synthesis in pregnant rats and this maybe responsible for the delay constitute the mechanism in the onset of labour.     

1α,25-dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclea envelope of osteoblasts

1α,25-Dihydroxyvitamin D₃ (1α, 25-(OH)₂D₃) has been shown to increase cytosolic calcium and inositol trophosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1α, (OH)₂D₃-induced changes in the calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei wa assessed. 1α,25 (OH)₂D₃, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1β,25-(OH)₂D₃, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1α,25-(OH)₂D₃, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium with 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effects of ATP on nuclear calcium was not additive with 1α, 25-(OH)₂D₃, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1α,25-(OH)₂D₃ amd ATP rapidly increase inositol triphosphate levels in isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid nuclear calcium. Thus, the 1α,25-(OH)₂D₃ and ATP effects of nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblastlike cells. © Wiley-Liss, Inc.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

SAR in rats exposed in 2,450-MHz circularly polarized waveguides

Average specific absorption rates (SARs) for live rats exposed in 2,450-MHz circularly polarized waveguides were estimated from the total system loss determined from measurements using five power meters, and a correction factor representing actual SAR/apparent SAR. The actual SAR was measured by twin-well calorimetry and the apparent SAR by power meters. Values were obtained for carcasses of various body masses for five orientations. The average SAR with free movement in the cages changed less than threefold as the rats grew from 200 to 700 g. The ratio of peak to average SAR in the body was less than 3. These results indicate relatively constant energy disposition in rats exposed in the circularly polarized waveguide.     

Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.