Fluorescence decay curves under pulsed excitation: distinction between free and bound NAD(P)H on isolated living cells

Under pulsed excitation the decay curve of the intrinsic cellular fluorescence has been recorded using an original equipment. Cell by cell experiments are feasible so that the NAD(P)H bound/free ratio and the relaxation time characteristic of bound NAD(P)H can be used in order to build up histograms characterizing the cell population.     

Spatial and temporal expression of histone demethylase,Kdm2a,during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.     

Effect of C-reactive protein on platelet-activating factor-induced platelet aggregation and membrane stabilization

C-reactive protein (CRP) is an acute phase reactant which humoral concentration rises drastically following tissue injury or inflammation. CRP of all species binds to phosphorylcholine residues. In the present studies CRP was found to inhibit platelet-activating factor-induced platelet aggregation, and to stabilize platelet membranes against the lytic effect of lysophosphatidylcholine. Inhibition of platelet aggregation by CRP is accompanied by an inhibition of arachidonic acid release from both phosphatidylcholine and phosphatidylinositol. This suggests that phospholipases are inhibited. Hydrolysis of multilamellar dipalmitoylphosphatidylcholine liposomes by purified phospholipase A2, was also inhibited by CRP. These results suggest that CRP can stabilize membranes from the detergent-like effects of lysolipids and from potentially toxic materials such as platelet-activating factor. By inhibition of phospholipases, production of inflammatory mediators would be blocked. CRP might thus act as an early protective recognition mechanism in acute inflammatory states.     

Plasma immunoreactive N-terminal parathyroid hormone and cyclic AMP and cyclic GMP urinary levels in man after I. M. administration of two different doses of porcine calcitonin

The effects of acute porcine calcitonin (pCT) administration were studied in 11 healthy volunteers with no metabolic disease. Each subject was given, intramuscularly, 1 MRC unit of pCT in glycine vehicle, 160 units of pCT in gelatine vehicle, and placebo, according to a crossover design. The following parameters were studied: blood calcium, phosphorus and immunoreactive parathyroid hormone (iPTH); urine calcium, phosphorus, cyclic AMP and GMP. Both the pCT preparations produced, at the same time after administration, a hypocalcemic effect (P less than 0.01) which was not dose related, without any modification of urinary calcium excretion, implying that both doses are able to inhibit completely bone destruction. Despite the blood calcium decrease, no significant modifications in plasma iPTH levels were observed. pCT administration did not modify the urinary excretion of cyclic AMP, while it increased the urinary levels of cyclic GMP, particularly at the higher dose employed. Blood phosphorus decrease and urinary phosphate excretion increase were observed only after the administration of 160 MRC units of pCT. These observations suggest that the effects on urinary cyclic GMP and on blood and urine phosphorus are not mediated by PTH but could be the result of a direct action of calcitonin seen only when high doses are employed. In conclusion, one MRC unit of pCT is sufficient to inhibit bone resorption.     

New method for the detection of reactive oxygen species in anti-tumoural activity of adriamycin: A comparison between hypoxic and normoxic cells

Tumour hypoxia plays a role in chemoresistance in several human tumours. However, how hyperbaric oxygen leads to chemotherapeutic gain is unclear. This study investigates the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of adriamycin (ADR) on CCRF-CEM cells under hypoxic (2% O₂) and normoxic (21% O₂) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. At 24 h, ADR, probably via semiquinone radical, enhances ROS levels in normoxic cells compared to hypoxic cells. Long-term studies show that ROS are also generated by a second mechanism related to cell functions perturbation. ADR arrests the cell cycle progression both under hypoxia and normoxia, indicating that oxygen and ROS does not influence the DNA damaging activity of ADR. The findings reveal that moderate improvement of ADR cytotoxicity results from higher ROS formation in normoxic cells, leading to elevated induction of cell death.     

Essential role for IkappaB kinase beta in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation

T cell receptor (TCR) signaling to IkappaB kinase (IKK)/NF-kappaB is controlled by PKCtheta-dependent activation of the Carma1, Bcl10, and Malt1 (CBM) complex. Antigen-induced phosphorylation of Bcl10 has been reported, but its physiological function is unknown. Here we show that the putative downstream kinase IKKbeta is required for initial CBM complex formation. Further, upon engagement of IKKbeta/Malt1/Bcl10 with Carma1, IKKbeta phosphorylates Bcl10 in the C terminus and thereby interferes with Bcl10/Malt1 association and Bcl10-mediated IKKgamma ubiquitination. Mutation of the IKKbeta phosphorylation sites on Bcl10 enhances expression of NF-kappaB target genes IL-2 and TNFalpha after activation of primary T cells. Thus, our data provide evidence that IKKbeta serves a dual role upstream of its classical substrates, the IkappaB proteins. While being essential for triggering initial CBM complex formation, IKKbeta-dependent phosphorylation of Bcl10 exhibits a negative regulatory role in T cell activation.