The preparation of pyrogen-free foetal calf serum for use in cell and tissue cultured

A method is described for the preparation of foetal calf serum from blood drawn by heart puncture from calf foetuses obtained from a local municipal abattoir. This method differs from that previously described in that the Seitz Supra EK filter pads used in preliminary purification steps have been abandoned. These pads contain significant amounts of pyrogenic bacterial lipopolysaccharides and attempts to depyrogenate them with sodium hypochlorite, hydrogen peroxide and 1% foetal calf serum were unsuccessful. The modified method uses pyrogen-free glass fibre and cellulose fibre filters and provides an entirely satisfactory serum as proved by negative Limulus amoebocyte lysate tests and the excellent growth of cells.     

Characterization of mouse thrombospondin 2 sequence and expression during cell growth and development.

Thrombospondin (TSP) is an extracellular matrix glycoprotein whose expression has been associated with a variety of cellular processes including growth and embryogenesis. The recent discovery of the existence of a second mouse TSP gene necessitates careful examination of the discrete biochemical and functional properties associated with each molecule. In this report, the primary structures of human TSP, mouse TSP1 (mTSP1), mouse TSP2 (mTSP2), and chicken TSP are compared; and the expression of mTSP1 and mTSP2 during embryogenesis and growth factor-mediated cell proliferation is examined. The cloning and sequencing of the entire coding regions of mTSP1 and mTSP2 revealed considerable conservation of residues critical for TSP structure and function; these data suggest that TSP2 is capable of trimer formation and many of the same cell-surface and ligand interactions that mediate TSP function. Comparison of the various TSP sequences also allowed the assignment based on sequence homology of previously reported human TSP as TSP1 and chicken TSP as TSP2. mTSP2, like mTSP1, was shown to be a primary response gene when quiescent Swiss 3T3 cells were stimulated with serum, platelet-derived growth factor BB, basic fibroblast growth factor, or interleukin-1 beta. Interestingly, TSP1 and TSP2 exhibited markedly different tissue- and stage-specific patterns of mRNA expression during mouse embryogenesis, implying that the two TSP molecules possess discrete functional properties important for development. Additionally, the TSP genes (Thbs1 and Thbs2) were mapped to single loci on mouse chromosomes 2 and 17, respectively.     

Studies on the genetic control of antibody affinity: the independent control of antibody levels and affinity in Biozzi mice.

The levels (Abt) and relative affinity (KR) of antibody produced to protein antigens injected in saline have been measured in the 22nd generation of the Biozzi high (Ab/H) and low (Ab/L) antibody-producing mice. No significant differences in affinity were observed between the two lines of mice (p 0.10) but the levels of antibody (Abt) differed significantly (p 0.0025) when immunized with antigen in saline; however, both Ab/H and Ab/L mice were able to mount a high affinity response to protein antigens injected in Freund's complete adjuvant. These results substantiate earlier observations that in mice, antibody affinity (KR) and antibody level (Abt) are under independent genetic control.     

Apigenin induces apoptosis via downregulation of S‐phase kinase‐associated protein 2‐mediated induction of p27Kip1 in primary effusion lymphoma cells

Objective: The mechanisms that regulate mitogenic and antiapoptotic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we assessed the effect of apigenin (4′,5,7‐trihydroxyflavone), a flavonoid on a panel of PEL cell lines. Materials and methods: We studied the effect of apigenin on four PEL cell lines. Apoptosis was measured by annexin V/PI dual staining and DNA laddering. Protein expression was measured by immunoblotting. Results: Apigenin induced apoptosis in PEL cell lines in a dose dependent manner. Such effects of apigenin appeared to result from suppression of constitutively active kinase AKT resulting in down‐regulation of SKP2, hypo‐phosphorylation of Rb and accumulation of p27Kip1. Apigenin treatment of PEL cells caused dephosphorylation of p‐Bad protein leading to down regulation of the anti‐apoptotic protein, Bcl‐2 and an increase in Bax/Bcl2 ratio. Apigenin treatment also triggered Bax conformational change and subsequently translocation from cytosole to mitochondria causing loss of mitochondrial membrane potential with subsequent release of cytochrome c. Released cytochrome c onto the cytosole activated caspase‐9 and caspase‐3, followed by polyadenosin‐5′‐diphosphate‐ribose polymerase (PARP) cleavage. Finally, treatment of PEL cells with apigenin down‐regulated the expression of inhibitor of apoptosis protein (IAPs). Conclusions: Altogether, these data suggest a novel function for apigenin, acting as a suppressor of AKT/PKB pathway in PEL cells, and raise the possibility that this agent may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.     

The role of mitochondria-rich cells in the chloride current conductance across toad skin

In early studies of salt transport across frog and toad skin, it was assumed that chloride movement is extracellular. However, later studies suggested that chloride movement is largely transcellular. Chloride transport across toad skin is greatly diminished in skins of salt-acclimated toads (Bufo viridis) and was correlated with the number of mitochondria-rich (m.r.) cells in the epithelium. The activated chloride conductance could be recovered upon in vitro incubation with theophylline. It was found that the short-circuit current (Isc) and the chloride conductance (Gcl) in toad skin could be separated experimentally by selective use of synthetic oxytocin (Syntocinon) or theophylline, and by substituting impermeable anions for chloride. With the use of the vibrating probe we demonstrated directly that chloride-dependent peak currents are localized only over m.r. cells, under hyperpolarized (V = -100 mV) conditions. It is concluded that the m.r. cells form the principal site for passive chloride movement across amphibian skin. This cellular pathway is regulated through a cyclic AMP-mediated process. It is suggested that the spatial separation of the sodium and chloride channels is essential to maintain the granulosum cells which are engaged in sodium transport hyperpolarized, and thus providing the driving force for the sodium entry into the cells.     

Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by down-regulating parathyroid hormone receptors.

The effects of the monokines tumor necrosis factor alpha (TNF) and interleukin 1 (IL 1) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adenylate cyclase, with maximal inhibition of PTH response (40% for TNF, 24% for IL 1) occurring at 10(-8) M of either monokine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonreceptor-mediated enzyme activation by cholera toxin and forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radioiodinated agonist mono-[125I]-[Nle8,18, Tyr34]bPTH-(1-34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1-34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertussis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibition of PTH response. In time course studies, brief (1 hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time- and protein synthesis-dependent down-regulation of PTH receptors linked to adenylate cyclase.     

Effect of high intensity interval training on 2,3-diphosphoglycerate at rest and after maximal exercise

The purpose of this study was to examine the effect of intense interval training on erythrocyte 2,3-diphosphoglycerate (2,3-DPG) levels at rest and after maximal exercise. Eight normal men, mean +/- SE = 24.2 +/- 4.3 years, trained 4 days X week-1 for a period of 8 weeks. Each training session consisted of eight maximal 30-s rides on a cycle ergometer, with 4 min active rest between rides . Prior to and after training the subjects performed a maximal 45-s ride on an isokinetic cycle ergometer at 90 rev X min-1 and a graded leg exercise test ( GLET ) to exhaustion on a cycle ergometer. Blood samples were obtained from an antecubital vein before, during and after the GLET only. Training elicited significant increases in the amount of work done during the 45-s ride (P less than 0.05), and also in maximal oxygen uptake (VO2 max: Pre = 4.01 +/- 0.13; Post = 4.29 +/- 0.07 1 X min-1; P less than 0.05) during exercise and total recovery VO2 (Pre = 19.14 +/- 0.09; Post = 21.45 +/- 0.10 1 X 30 min-1; P less than 0.05) after the GLET . After training blood lactate was higher, base excess lower and pH lower during and following the GLET (P less than 0.05 for all variables).(ABSTRACT TRUNCATED AT 250 WORDS)