Depletion of Brain Glutathione in Preweanling Mice by L-Buthionine Sulfoximine

Previous studies indicated that DL-buthionine sulfoximine (DL-BSO), an agent that inhibits the biosynthesis of GSH in liver and other peripheral organs, fails to suppress levels of GSH in the CNS. In the current study, preweanling mice responded to repeated injections of L-BSO with marked declines (79.6-86.5%) of GSH content in brain and spinal cord. In adult mice, the same treatment schedule produced only modest declines (17.8-29.2%) of GSH content in brain and a 55.9% decline in spinal cord. Pretreatment of preweanling mice with L-BSO represents a tool for studying the role of GSH in the CNS.     

Phenotypic and cytotoxic characteristics of the immune cells of the human placenta

Despite some functional impairment of the newborn's T-cell immune system, most infants survive the intrauterine and perinatal period without succumbing to infection or maternal lymphocyte engraftment. The placenta may play a crucial role in protecting the infant from microbial and histocompatibility antigens. Accordingly, we studied phenotypic and functional capacities of placental cells. Placentas were obtained from uncomplicated pregnancies. Matched cord blood and maternal peripheral blood were also obtained in many instances. Fresh minced placental tissue was washed and digested with collagenase and DNase and mononuclear cells were obtained by density gradient centrifugation. The average yield was 10(6) cells/g of tissue with greater than 80% viability. Chromosome analysis of five placental preparations indicated that these cells were of fetal rather than maternal origin. The isolated placental cells consisted of trophoblasts, lymphocytes (74 +/- 3%), monocytes (16 +/- 3%), and granulocytes (8 +/- 2%). E-rosette forming cells (T cells) made up 65 +/- 2% and surface membrane immunoglobulin positive cells made up 8 +/- 1% of the placental mononuclear cells. Fluorescent activated analysis of the mononuclear cells indicated less Leu 4-positive cells (Pan-T) 43 +/- 3%, and less Leu 3-positive (T-helper cells) (25 +/- 2%), than cord and maternal cell preparations. Leu-2, DR, and B1 positive cells were similar to those in cord and maternal blood. Leu 7 and especially Leu 11 positive cells, markers for natural killer cells, were abundant in placental cells, making up 4 +/- 0.7% and 20 +/- 3%, respectively. The Leu 7/Leu 11 ratio of the placental cells was different from either the maternal or cord blood cells. Natural killer activity of placental cells against a K562 natural killer target was low, despite the abundance of cells with NK markers. The K562 activity was low in the placental cells, similar to the low NK activity of maternal and cord cells. Molt 4f killer activity was near normal. Lectin-dependent cytotoxicity using an EL-4 cell target plus PHA was low in placentas, compared to normal, maternal, or cord cell cytotoxicity. Matched samples indicated that LDCC activity was mother greater than cord greater than placenta. Antibody-dependent cytotoxicity (Raji target) of placental cells showed low activity, and again the paired studies indicated that normal controls greater than maternal greater than cord greater than placenta cytotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)     

A stimulating effect of guanyl nucleotides on the rat-liver soluble cyclic GMP high-affinity phosphodiesterase activity

The high affinity (low Km) cyclic GMP phosphodiesterase (PDE) is activated by GTP, while the cyclic AMP PDE is not. GTP and its hydrolysis-resistant analogue, guanylylimidodiphosphate (GppNHp), display a half-maximal stimulating effect at almost the same concentration (5 X 10(-6) M). The GTP stimulating effect is not observed when the socalled cyclic GMP low affinity (high Km) PDE is operative. GTP cooperates with the increase of the substrate concentration on removing the IBMX inhibitory effect. The isolation through a classical chromatographic procedure on a DEAE-cellulose column, of a PDE fraction specific for cyclic GMP, results in the loss of the GTP stimulating effect.     

Cardiovascular adaptations to 10days of cycle exercise

Mier, Constance M., Michael J. Turner, Ali A. Ehsani, andRobert J. Spina. Cardiovascular adaptations to 10 days of cycleexercise. J. Appl. Physiol. 83(6):1900-1906, 1997.We hypothesized that 10 days of training wouldenhance cardiac output (CO) and stroke volume (SV) during peak exerciseand increase the inotropic response to -adrenergic stimulation. Tensubjects [age 26 ± 2 (SE) yr] trained on a cycleergometer for 10 days. At peak exercise, training increasedO₂ uptake, CO, and SV(P < 0.001). Left ventricular (LV)size and function at rest were assessed with two-dimensional echocardiography before (baseline) and after atropine injection (1.0 mg) and during four graded doses of dobutamine. LV end-diastolic diameter increased with training (P < 0.02), whereas LV wall thickness was unchanged. LV contractileperformance was assessed by relating fractional shortening (FS) to theestimated end-systolic wall stress(_ES). Training increased theslope of the FS-_ES relationship (P < 0.05), indicating enhancedsystolic function. The increase in slope correlated with increases inCO (r = 0.71,P < 0.05) and SV(r = 0.70,P < 0.05). The increase in bloodvolume also correlated with increases in CO(r = 0.80, P < 0.01) and SV (r = 0.85, P < 0.004). These datashow that 10 days of training enhance the inotropic response to-adrenergic stimulation, associated with increases in CO and SVduring peak exercise.

     

Inorganic phosphate enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin via a p53‐dependent pathway

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (Pi) inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53‐wild type U2OS cells (and not of p53‐null Saos and p53‐mutant MG63 cells) by slowing‐down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin‐induced cytotoxicity in U2OS, and not in Saos and MG63 cells, by apoptosis induction, as revealed by a marked increase of sub‐G1 population, Bcl‐2 downregulation, caspase‐3 activation, and PARP cleavage. Remarkably, Pi/doxorubicin combination‐induced cytotoxicity was accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine‐alpha. Moreover, the doxorubicin‐induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53‐dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy. J. Cell. Physiol. 228: 198–206, 2013. © 2012 Wiley Periodicals, Inc.     

Urocortin 1 administered into the hypothalamic supraoptic nucleus inhibits food intake in freely fed and food-deprived rats

Peptides of the corticotropin-releasing hormone/Urocortin (CRH/Ucn) family are known to suppress appetite primarily via CRH₂ receptors. In the rat hypothalamic supraoptic nucleus (SON), synthesis of both Ucn1 and CRH₂ receptors has been reported, yet little is known about the effects of Ucn1 in the SON on feeding behaviour. We first established the dose-related effects of Ucn1 injected into the SON on the feeding response in both freely fed and 24-h food-deprived rats. A conditioned taste avoidance paradigm was performed to investigate possible generalised effects of local Ucn1 treatment. Administration of Ucn1 into the SON at doses equal to or higher than 0.5 μg significantly decreased food intake in both freely fed and food-deprived rats. The Ucn1-mediated suppression of food intake was delayed in freely fed as compared to food-deprived animals. Conditioning for taste aversion to saccharine appeared at 0.5 and 1 μg of Ucn1. Both the early and the delayed onset of anorexia observed after intra-SON injection of Ucn1 under fasting and fed conditions, respectively, suggest the possible involvement of different CRH receptor subtypes in the two conditions, while the conditioned taste aversion seems to be responsible for the initial latency to eat the first meal in these animals.