Norma Sterin-Speziale

The Expression of Sphingosine Kinase-1 in Head and Neck Carcinoma

Sphingosine kinase-1 (SPHK1) modulates the proliferation, apoptosis and differentiation of keratinocytes through the regulation of ceramide and sphingosine-1-phosphate levels. However, studies on the expression of SPHK1 in human head and neck squamous cell carcinoma (HNSCC) specimens are lacking. Therefore, the aim of the present work was to evaluate SPHK1 expression in human primary HNSCCs and to correlate the results with clinical and anatomopathological parameters. We investigated the expression of this protein by immunohistochemistry performed in tissue microarrays of HNSCC and in an independent cohort of 37 paraffin-embedded specimens. SPHK1 expression was further validated by real-time PCR performed on laser capture-microdissected tissue samples. The positive rate of SPHK1 protein in the cancerous tissues was significantly higher (74%) than that in the nontumor oral tissues (23%), and malignant tissues showed stronger immunoreactivity for SPHK1 than normal matching samples. These results were confirmed by real-time PCR quantification of SPHK1 mRNA. Interestingly, the positive expression of SPHK1 was associated with shorter patient survival time (Kaplan-Meier survival curves) and with the loss of p21 expression. Taken together, these results demonstrate that SPHK1 is upregulated in HNSCC and provide clues of the role SPHK1 might play in tumor progression.

Cell proliferation and morphometric changes in the rat kidney during postnatal development.

We have investigated the temporal maturation of the rat kidney during the postnatal developmental period. As a result, we observed the following: an active process of cortical cell proliferation and differentiation occurs as late as day 20. The medulla is the most immature zone at birth and displays the greatest morphological changes during this period. At birth, no distinction exists between inner and outer medulla, and the outer and inner strip of the outer medulla can be distinguished as late as day 30. Remodeling of the ECM surrounding collecting ducts occurs in the medulla twice, stopping at day 11 and it occurs in the papilla three times, stopping at day 20. The increase of kidney size is temporally different for each kidney zone. The cortex and the papilla acquire the morphological appearance of the adult kidney before the medulla does. Consequently, the medulla remains at the highest degree of immaturation among the kidney zones for a relatively long postnatal period.

Compartmental study of rat renal phospholipid metabolism

Phospholipid content and metabolism were studied in rat renal papillary, medullary and cortical slices. The highest concentration of phospholipids was found in cortex and the lowest in papilla samples (ratio cortex/medulla, 1.3; cortex/papilla, 3.7). The profile of the various phospholipids was different depending on the zone. The most important difference was the relative concentrations of sphingomyelin (CerPCho) and phosphatidylinositol (PtdIns) with ratios for PtdIns/CerPCho of 5.0, 3.3 and 2.5 in papilla, medulla, and cortex, respectively. In the three zones, PtdIns showed the highest specific activity for [2-14C]glycerol and [1-14C]arachidonic acid incorporation. By contrast, a higher amount of [1-14C]palmitic acid was incorporated into phosphatidylcholine than into any other phospholipid. The various radioactive precursors were only poorly incorporated into phosphatidylethanolamine. No radioactivity was associated with phosphatidylserine. The papilla possesses the most active phospholipid metabolism of all the pathways studied.

HYDROGEN PEROXIDE METABOLISM AND OXIDATIVE STRESS IN CORTICAL, MEDULLARY AND PAPILLARY ZONES OF RAT KIDNEY

The cortical, medullary and papillary regions of rat kidney were evaluated for a series of parameters related to hydrogen peroxide metabolism and oxidative stress. The rates of oxygen uptake, prostaglandin synthesis and malondialdehyde production by kidney slices were: 47, 0.003 and 0.051 mumol/h g wet wt., respectively, in cortex, 32, 0.023 and 0.035 in medulla and 22, 0.034 and 0.007 in papilla. The activities of superoxide dismutase, catalase and glutathione peroxidase were: 144 +/- 16 U/g wet wt., 880 +/- 100 pmol/g wet wt. and 177 +/- 16 U/g wet wt. in cortex; 97 +/- 9 U/g wet wt., 550 +/- 50 pmol/g wet wt. and 142 +/- 18 U/g wet wt. in medulla; and 23 +/- 2 U/g wet wt., 90 +/- 9 pmol/g wet wt. and 147 +/- 5 U/g wet wt. in papilla. Hydrogen peroxide steady-state concentrations were 0.09 +/- 0.01, 0.07 +/- 0.01 and 0.08 +/- 0.01 microM whereas alpha-tocopherol content was 21 +/- 2, 23 +/- 1 and 34 +/- 3 mumol/g wet wt. and hydroperoxide-initiated chemiluminescence was 22 +/- 2, 33 +/- 2 and 14 +/- 1 cpm. 10(-3)/mg prot for cortex, medulla and papilla, respectively. After 60 min ischemia-30 min reperfusion hydroperoxide-initiated chemiluminescence and hydrogen peroxide steady-state concentration increased by 30% and 60% in cortex and 80% and 60% in medulla, whereas alpha-tocopherol content decreased by 30%, 50% and 2% in cortex, medulla and papilla, respectively. The reperfusion/control ratio of hydroperoxide-initiated chemiluminescence and hydrogen peroxide steady-state concentrations in cortex and medulla indicate the occurrence of oxidative stress after ischemia-reperfusion. The lower sensitivity to oxidative stress found in papilla could be explained by the relatively high relationship of alpha-tocopherol content to hydrogen peroxide production rate in this sub-organ.

Time course of hexachlorobenzene-induced alterations of lipid metabolism and their relation to porphyria

A great deal of information concerning the effects of hexachlorobenzene on the haem metabolic pathway has been obtained but little is known about the effects of the drug on lipid metabolism. Consequently, the time course of phospholipid metabolism alteration caused by this xenobiotic was evaluated as related to changes in porphyrin metabolism with the aim to understand better the interregulation of both metabolisms. Female Wistar rats were treated with HCB (1 g/kg) over a 1-8 week period. Individual phospholipid content, [32P] incorporation, total lipid content, lipid peroxidation, uroporphyrinogen decarboxylase activity, its inhibitor generation and porphyrin content, were the parameters measured in the liver of treated rats. Phospholipid metabolism-with the exception of sphingomyelin-presents a biphasic behaviour, in both the endogenous contents and de novo synthesis. The turning point between both phases is the time at which levels of porphyrin and conjugated dienes increase, the latter compounds being involved in oxidative processes. On the other hand, sphingomyelin decreases continuously during the 8 weeks of treatment. It was also found that the malondialdehyde content increased during the early stages. The time sequence for haem metabolism parameters showed that the accumulation of porphyrins occurs after the decrease in uroporphyrinogen decarboxylase activity and the enzyme inhibitor formation, which are early events (first and second weeks). Porphyrins could not by themselves exacerbate uroporphyrinogen decarboxylase impairment or inhibitor generation. This study shows that hexachlorobenzene alters simultaneously phospholipid and porphyrin metabolisms from the early stages, and generates an oxidative environment that favours porphyrinogens and lipid oxidation at later stages. So, this oxidative environment links the alterations on both metabolisms.

Potentiation of omega-3 fatty acid antidepressant-like effects with low non-antidepressant doses of fluoxetine and mirtazapine.

Despite the advances in psychopharmacology, the treatment of depressive disorders is still not satisfactory. Side effects and resistance to antidepressant drugs are the greatest complications during treatment. Based on recent evidence, omega-3 fatty acids may influence vulnerability and outcome in depressive disorders. The aim of this study was to further characterize the omega-3 antidepressant-like effect in rats in terms of its behavioral features in the depression model forced swimming test either alone or in combination with antidepressants fluoxetine or mirtazapine. Ultimately, we prompted to determine the lowest dose at which omega-3 fatty acids and antidepressant drugs may still represent a pharmacological advantage when employed in combined treatments. Chronic diet supplementation with omega-3 fatty acids produced concentration-dependent antidepressant-like effects in the forced swimming test displaying a behavioral profile similar to fluoxetine but different from mirtazapine. Fluoxetine or mirtazapine at antidepressant doses (10 and 20 mg/kg/day, respectively) rendered additive effects in combination with omega-3 fatty acid supplementation (720 mg/kg/day). Beneficial effects of combined treatment were also observed at sub-effective doses (1 mg/kg/day) of fluoxetine or mirtazapine, since in combination with omega-3 fatty acids (720 mg/kg/day), antidepressants potentiated omega-3 antidepressant-like effects. The antidepressant-like effects occurred in the absence of changes in brain phospholipid classes. The therapeutic approach of combining omega-3 fatty acids with low ineffective doses of antidepressants might represent benefits in the treatment of depression, especially in patients with depression resistant to conventional treatments and even may contribute to patient compliance by decreasing the magnitude of some antidepressant dose-dependent side effects.

Beneficial activity of Enterococcus faecalis CECT7121 in the anti-lymphoma protective response

Aims:  To study the anti‐tumour effects of Enterococcus faecalis CECT7121 on LBC cells, an aggressive murine T‐cell lymphoma that kills the host in 18 days when is intraperitoneally (i.p.) administrated.

Bradykinin stimulates phosphoinositide turnover and phospholipase C but not phospholipase D and NADPH oxidase in human neutrophils.

In response to formyl‐Met‐Leu‐Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2 ‐) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP‐stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2 ‐ production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2 ‐ production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK‐induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2‐PLC measured as the release of [3H]IP3. On the contrary, a PC‐PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA‐derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2 ‐production by fMLP‐activated PMN in a time‐dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO‐ formation. These data show that BK plays an important role in inflammation by modulating the PMN function.

Enhancement of phosphatidylcholine biosynthesis by angiotensin-(1–7) in the rat renal cortex

In the present paper, we investigated the effect of angiotensin-(1-7) (Ang-(1-7)) on phospholipid biosynthesis in the rat renal cortex. A significant increase in phosphatidylcholine (PC) labeling was observed when cortical slices, prelabeled with [32P]orthophosphate, were incubated for 30 min in the presence of Ang-(1-7) (1 pM to 100 nM). Neither the phospholipase C inhibitors, neomycin or db-cAMP nor the protein kinase C inhibitors, chelerythrine or H7, modified the stimulatory effect induced by 0.1 nM Ang-(1-7). The enhancement of PC biosynthesis caused by 0.1 nM Ang-(1-7) was unmodified by either losartan, an AT(1) receptor antagonist, or (1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazol[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate) (PD 123319), an AT(2) receptor antagonist, but was partially blocked by [D-Ala(7)]Ang-(1-7), an Ang-(1-7) specific antagonist. However, losartan potentiated the effect of 100 nM Ang-(1-7) on PC biosynthesis. Losartan by itself increased the de novo synthesis of PC. These results suggest that the Ang-(1-7)-mediated increase in PC biosynthesis is independent of AT(1) and AT(2) receptor activation but mediated by a specific Ang-(1-7) receptor. This mechanism is independent of phospholipase C and PKC activation.

The rate-limiting enzyme in phosphatidylcholine synthesis is associated with nuclear speckles under stress conditions

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity.