Objective: Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL). In patients with primary APS, expression of tissue factor (TF) on the surface of monocytes is increased, which may contribute to thrombosis in these patients. However, the intracellular mechanisms involved in aPL‐mediated up‐regulation of TF on monocytic cells are not understood. This study was undertaken to investigate the intracellular signals induced by aPL that mediate TF activation in monocytes from APS patients.Methods: We analyzed, both in vivo and in vitro, aPL interactions with proteins that have signaling functions, including mitogen‐activated protein kinases (MAP kinases) and NF‐κB/Rel proteins.Results: In vivo studies demonstrated significantly higher levels of both TF messenger RNA and TF protein in monocytes from APS patients compared with controls. At the molecular level, increased proteolysis of IκBα and activation of NF‐κB were observed. Constitutive activation of both p38 and ERK‐1 MAP kinases was also found. Treatment of normal monocytes with aPL activated ERK‐1 and p38 MAP kinases, as well as the IκB/NF‐κB pathway, in a dose‐dependent manner. NF‐κB activation and IκBα degradation induced by aPL were inhibited by the NF‐κB inhibitor SN50 and the p38 MAP kinase inhibitor SB203580, thus suggesting crosstalk between these pathways. However, the MEK‐1/ERK inhibitor PD98059 did not affect aPL‐induced NF‐κB binding activity. TF expression induced by aPL was significantly inhibited by combined treatment with the 3 inhibitors.Conclusion: Our results suggest that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK‐1/ERK proteins, and the p38 MAP kinase–dependent nuclear translocation and activation of NF‐κB/Rel proteins.

Hypocotyls of kidney beans (Phaseolus vulgaris L.) accumulated ascorbate after preincubation with a number of possible precursors, mainlyl-galactono-γ-lactone (l-GL) andl-gulono-γ-lactone. The increase in the intracellular ascorbate concentration was parallel to the high stimulation of thel-GL dehydrogenase (l-GLD) activity measured in vitro using l-GL as a substrate and cytochromec as an electron acceptor. Cell fractionation using a continuous linear Percoll gradient demonstrated that l-GLD is associated with mitochondria; therefore, pure mitochondria were isolated and subjected to detergent treatment to separate soluble from membrane-linked proteins. l-GLD activity was mainly associated with the detergent phase, suggesting that a membrane-intrinsic protein is responsible for the ascorbic acid biosynthetic activity. Subfractionation of mitochondria demonstrated that l-GLD is located at the inner membrane.

Cytochrome b₅ reductases (CYB5R) are required for the elongation and desaturation of fatty acids, cholesterol synthesis and mono-oxygenation of cytochrome P450 enzymes, all of which are associated with protection against metabolic disorders. However, the physiological role of CYB5R in the context of metabolism, healthspan and aging remains ill-defined. We generated CYB5R-overexpressing flies (CYB5R-OE) and created a transgenic mouse line overexpressing CYB5R3 (CYB5R3-Tg) in the C57BL/6J background to investigate the function of this class of enzymes as regulators of metabolism and age-associated pathologies. Gender- and/or stage-specific induction of CYB5R, and pharmacological activation of CYB5R with tetrahydroindenoindole extended fly lifespan. Increased expression of CYB5R3 was associated with significant improvements in several metabolic parameters that resulted in modest lifespan extension in mice. Diethylnitrosamine-induced liver carcinogenesis was reduced in CYB5R3-Tg mice. Accumulation of high levels of long-chain polyunsaturated fatty acids, improvement in mitochondrial function, decrease in oxidative damage and inhibition of chronic pro-inflammatory pathways occurred in the transgenic animals. These results indicate that CYB5R represents a new target in the study of genes that regulate lipid metabolism and healthspan.

We previously demonstrated that extracellular calcium regulates vitamin D receptor (VDR) expression by parathyroid cells. Since the calcimimetic R-568 potentiates the effects of calcium on the calcium-sensing receptor, it was hypothesized that administration of R-568 may result in increased VDR expression in parathyroid tissue. In vitro studies of the effect of R-568 on VDR mRNA and protein were conducted in cultures of whole rat parathyroid glands and human hyperplastic parathyroid glands. In vivo studies in Wistar rats examined the effect of R-568 and calcitriol alone and in combination. Incubation of rat parathyroid glands in vitro with R-568 (0.001–1 μM) resulted in a dose-dependent decrease in parathyroid hormone (PTH) secretion and an increase in VDR expression (mean ± SE). Incubation in 1 mM calcium + 0.001 μM R-568 elicited an increase in VDR mRNA (306 ± 46%) similar to the maximum increase detected with 1.5 mM calcium (330 ± 42%). In vivo, VDR mRNA was increased after administration of R-568 (168 ± 9%, P < 0.001 vs. control) or calcitriol (198 ± 16%, P < 0.001 vs. control). Treatment with R-568 also increased VDR protein in normal rat parathyroid glands and in human parathyroid glands with diffuse, but not nodular, hyperplasia. In conclusion, the present study shows that the calcimimetic R-568 exerts a stimulatory effect on VDR expression in the parathyroid glands of study models and provides additional evidence for the use of calcimimetics in the treatment of secondary hyperparathyroidism.

Low extracellular calcium (Ca) stimulates parathyroid hormone (PTH) secretion and also increases the renal synthesis of calcitriol (CTR), which is known to decrease PTH production. This study began with the hypothesis that the parathyroid cell response to CTR may be modulated by extracellular Ca concentration through an effect on parathyroid cell vitamin D receptor (VDR). In the present study, rat parathyroid glands were incubated in low (0.6 mM) and high (1.5 mM) Ca concentration. The parathyroid VDRmRNA was higher in 1.5 than 0.6 mM Ca. Furthermore, this effect was not observed in incubated slices of kidney cortex and medulla, tissues which also possess both Ca and vitamin D receptors. Experiments were also performed to evaluate the effect of Ca on VDR expression in vivo. Male Wistar rats received intraperitoneal injections of CaCl₂ or a single intramuscular injection of EDTA to obtain 6 h of hypercalcemic (ionized Ca, 1.4 to 1.6 mM) or hypocalcemic (ionized Ca, 0.85 to 0.95 mM) clamp; a third group of rats was used as control. A small dose of CTR was administered to hypercalcemic rats to match the serum CTR levels of hypocalcemic rats. Parathyroid gland VDRmRNA and VDR protein were increased in hypercalcemic rats as compared with hypocalcemic rats. Increasing doses of CTR upregulated VDRmRNA and VDR only in hypercalcemic rats. Additional experiments showed that the decrease in VDR in hypocalcemic rats prevented the inhibitory effect of CTR on PTHmRNA. In conclusion, our study shows that extracellular Ca regulates VDR expression by parathyroid cells independently of CTR and that by this mechanism hypocalcemia may prevent the feedback of CTR on the parathyroids.