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. METHODS AND RESULTS: In vitro studies have shown that LBC cell proliferation was inhibited by Ent. faecalis CECT7121 stimulus in a dose-dependent manner, inducing apoptosis. The production of ceramide was involved in the latter effect. To undertake in vivo studies, syngeneic BALB/c mice pre-treated i.p. with Ent. faecalis CECT7121 (2.5 × 10(8 ) CFU) were challenged i.p. with LBC cells (1.0 × 10(6) cells) the day after. On day 30 post-inoculation of LBC cells, 70% of Ent. faecalis CECT7121 pre-treated mice survived, whereas no survivals were recorded in the control group. A group of surviving mice was re-challenged with LBC cells, and 89% of them survived. Upon stimulation with irradiated LBC cells, spleen cell proliferation, high IFNγ, IL-12 and IL-10 levels were observed in surviving animals. CONCLUSIONS: Enterococcus faecalis CECT7121 affected multiple factors of the tumour establishment by the following methods: down-regulating the LBC cell proliferation and inducing apoptosis in these cells; and enhancing the immune response that protects animals from lymphoma challenge and re-challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrate that Ent. faecalis CECT7121 has potential as a probiotic that could facilitate the development of novel complements to therapeutic strategies against oncological diseases.

High glucose concentrations stimulate renal papillary phosphatidylcholine biosynthesis.

The effects of a high glucose concentration (HGC) on renal phosphatidylcholine (PtdCho) biosynthesis were studied. In control rats, HGC increased papillary PtdCho biosynthesis. In chronic diabetic rats, an increase above that induced by diabetes was observed. Such glucose-responsive phospholipid pools were shown to be transient in adult control rats, while in acute diabetic and aged control and chronic diabetic rats they seem to be of slow breakdown or permanent. Deoxyglucose evokes the HGC effect only in the presence of 5 mM glucose. Neomycin, which blocks phospholipase C action, corrected the HGC effect in control and chronic diabetic rats, but not the increase due to diabetes. CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity was increased by both in vivo and simulated diabetes. Therefore, transient high extracellular glucose levels promote a reversible increase in papillary (32)P-PtdCho, while diabetes causes an irreversible increase resulting in PtdCho accumulation, possibly related to papillary necrosis.

Heme metabolism and lipid peroxidation in rat kidney hexachlorobenzene-induced porphyria: A compartmentalized study of biochemical pathogenic mechanisms.

In the present study, the effects of hexachlorobenzene (HCB) on lipid peroxidation and heme metabolism in the different constitutive suborgans of the kidney were determined. For this purpose, conjugated diene and malondialdehyde levels, as lipid peroxidation parameters, and porphyrin accumulation, uroporphyrinogen decarboxylase activity, and its inhibitor formation, as measures of heme metabolism, were determined in renal cortex, medulla, and papilla. Adult Wistar rats were treated with HCB during 1, 2, 3, or 4 weeks. A significant increase in cortical conjugated dienes was observed from the 1st week of treatment. The malondialdehyde levels rose by 47, 34, and 28% after 2, 3, and 4 weeks of intoxication, respectively. The porphyrin content showed a tenfold increase after 4 weeks of treatment, and the uroporphyrinogen decarboxylase activity was reduced by 26 and 58% with respect to control values after 3 and 4 weeks of treatment, respectively. The results demonstrate a direct correlation between the oxidative environment and the effect elicited by the drug on heme metabolism in the renal cortex. In contrast, in papilla and medulla, where the antioxidant systems were higher, HCB showed no porphyrinogenic effect.

Changes in rat papilla microsomal membrane fluidity during development.

During maturation, rat renal papillary microsomes suffer a rearrangement in their fatty acid phospholipid composition. The most significant changes in total phospholipids are the increase in their content of the 20:4 and a decrease in the levels of 14:0, 16:0, 18:1, 22:6 and 20:3 fatty acids. The changes in total phospholipid fatty acid content are a reflection of the variations in the individual phospholipid composition. During this period, microsomal cholesterol, phospholipid, and protein concentrations present no variations. Steady state fluorescence anisotropy obtained by using TMA-DPH (see text) as a fluorescence probe denoted higher values for 70- versus 10-day-old microsomes. Using DPH as a probe, steady state fluorescence anisotropy was determined in whole microsomes, as well as in total lipid and phospholipid vesicles, from both 10- and 70-day-old papillary cells. No differences were detected in phospholipid and total lipid vesicles between days 10 and 70. On the other hand, 10-day-old microsomes appeared to be less fluid than adult microsomes. The results indicate that these structural changes in kidney membranes during development might affect protein-lipid interaction and, therefore, the activity of many membrane enzymes.

Polyphosphoinositide synthesis in human neutrophils. Effects of a low metabolic energy state.

Phosphatidylinositol (PtdIns) synthesis and polyphosphoinositide (PPI) formation were measured as the incorporation of [32P]orthophosphate ([32P]Pi) or [3H]inositol into non-stimulated intact human neutrophil membrane phospholipids. The rate of PtdIns "de novo" synthesis appeared to be a slow mechanism when compared to the rapid incorporation of [32P]Pi into PPIs. Of the "de novo" synthesized [3H]PtdIns, 70% was further phosphorylated to PPI. Nevertheless, this PPI pool represented less than 0.01% of the total nmols of PPIs formed evaluated as [32P]Pi labeling, indicating that PPI formation mainly involves a no "de novo" synthesized phosphatidylinositol pool. When evaluated at short incubation times, oscillations in the formation of PPIs were detected. A rapid phase was characterized after 30 s of incubation with [32P]Pi Phosphorylation levels returned to an equilibrium state within a minute, and the second phase peaked at 5 min., returning to equilibrium at 15 min. The fluctuant kinetics though not the equilibrium level of PPI formation, could be abolished by neomycin. On the other hand, a selective inhibition of the rapid phase of PPI synthesis occurred in the presence of the tyrosine kinase inhibitor genistein. When the incorporations of [gamma-32P]-adenosine triphosphate (ATP) or [32P]Pi into human neutrophil particulate fraction membranes were evaluated, PPIs synthesis showed fluctuations independently of the precursor used. Noticeably, [32P]from [32P]Pi was incorporated more efficiently into PPIs than that from [gamma-32P]ATP, when evaluated in parallel using equal specific activities for both radiolabeled precursors and under non-ATP synthesizing conditions. Moreover, the incorporation of [32P]Pi into particulate fraction PPIs was not abolished by high concentrations of non-radiolabeled ATP, and metabolically inhibited PMNs showed high rates of PPI synthesis. These data suggest that PPI formation is not necessarily a futile cycle in PMNs.

Fatty acid profiles of rat renal phospholipids during development.

The fatty acid content of rat renal phospholipids was examined during development. An increase in the arachidonic acid content of a particular fraction of phosphatidylcholine (PC2), phosphatidylethanolamine and phosphatidylinositol between 10 and 20 days of age could be observed in the papilla, and these levels were maintained into adulthood, while in the 20:4 content of phosphatidylserine changes were found between 30-day-old rats and adults. In the other fraction of phosphatidylcholine (PC1), saturated fatty acids such as 16:0 and 14:0 decreased, while no changes occurred in the stearic acid (18:0) content. The pattern found for the medulla did not differ significantly from that of the papilla. In contrast, the cortex content of arachidonic acid at 10 days of age was higher than that for papilla and medulla. Levels increased between 10 and 20 days of age, returning to the original values by 30 days with no further variations in the adult. These changes in arachidonic acid content with age in addition to the differences found between the three kidney zones might explain some causes of incomplete renal function in newborns.

Endogenous prostaglandins regulate rat renal phospholipid 'de novo' synthesis.

Rat renal papilla is the zone of the kidney enjoying the most active phospholipid metabolism and also the highest prostaglandin production. We studied the phospholipid biosynthesis and the relationship between phospholipid de novo synthesis and prostaglandin biosynthesis in rat renal papilla. Indomethacin inhibited the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. Exogenous PGF2alpha and PGD2 restored biosynthetic activity in the presence of indomethacin and also increased the activity of the enzymes involved in the Kennedy pathway. The decrease in phospholipid biosynthesis maintained a linear relationship with the decrease in prostaglandin biosynthesis. Moreover, esculetin, which stimulates prostaglandin synthesis, brought about a significant increase in 32P incorporation to the three phospholipids studied. The evidence presented in this paper indicates that renal PGF2alpha and PGD2 modulate phospholipid de novo synthesis in rat renal papilla.

Ontogenic development of membrane lipids in the chick optic lobe.

The developmental profiles of the lipid composition and their de novo synthesis and remodelling in the optic lobe of the chicken were studied. The 32P incorporation to phospholipids showed an active de novo synthesis mainly of phosphatidylinositol and of a particular fraction of phosphatidylcholine during the early stages of the embryo development, concomitantly with the beginning of synaptogenesis. This de novo synthesis of phospholipids strongly increased at hatching. On the other hand, phosphatidylinositol presented an active lipid exchange (acylation-deacylation) in the early stages of embryogenesis, indicating a strong incorporation of 14C-arachidonic acid during this period, followed by a fast drop in specific activity. Two different fractions of phosphatidylcholine were isolated by high-performance thin-layer chromatography with a different profile of fatty acid composition, disclosing their different physicochemical behavior, metabolic activities and evolution during embryogenesis. 32P incorporation into phosphatidylethanolamine remained very low during the earliest stages of embryogenesis, showing an increase when the process of synaptogenesis began, until hatching, when radioactivity reached a plateau. 14C-arachidonic acid incorporation into phosphatidylethanolamine was minimal. Furthermore, the phosphatidylethanolamine pool was progressively enriched in its ethanolamine plasmalogen throughout the development. Chromatographic analysis of lipid extracts showed the presence of cerebroside traces after 16 days of embryo incubation. At hatching, a remarkable increase in non-hydroxylated cerebrosides was observed concurrently with the appearance of hydroxylated ones. These glycosphingolipids, as well as the sulfatides, were markedly increased in the lipid extracts of optic lobes of adult animals, indicating the progressive development and maturity of the myelin sheath.

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.

Is the increase in renal papillary phospholipid biosynthesis a protective mechanism against injury?

Mercuric chloride (HgCl2) is a well-known renal toxic that causes acute renal failure. The effect of HgCl2 treatment and the protection by thyroxine were studied in rat renal papilla (P), outer medullary inner stripe (OMIS), outer medullary outer stripe (OMOS) and cortical phospholipids (PhLs). HgCl2 brought about an increase in the total phospholipid content in P and OMIS but a drop in OMOS and cortex. Only phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) accounted for such changes. Thyroxine, injected on HgCl2-treated rats, partially reversed the effect of the toxic metal in P and OMIS while completely reversed the PtdCho drop in OMOS and cortex. However, the hormone failed to recover the sphingomyelin increase in P, the PtdEtn shortage in OMIS, OMOS and partially reversed the drop in the cortex. When thyroxine was injected without toxic treatment, no effect was observed in the phospholipid content of any kidney zone. Results obtained by using 32P as a precursor to study the PhL de novo synthesis were consistent with those of the phospholipid content. Thus a radioactivity increase--associated with PtdCho and PtdEtn--was observed in the kidney zones where said endogenous PhLs had risen. But in OMOS and cortex, where PtdCho and PtdEtn had dropped, they were also accompanied by a decrease in radioactivity. The thyroxine-induced recovery phase also paralleled the phospholipid content results with those of the de novo synthesis. We suggest that the decrease in the renal phospholipid de novo synthesis may constitute one biochemical explanation of the selective renal toxic effect exerted by HgCl2 and that the increase observed in the renal phospholipid metabolism--induced by the toxic treatment in OMIS and P--may represent a protective mechanism of these zones against toxic injury. Moreover, recovery promoted by thyroxine treatment in OMOS and cortex was accompanied by the reversion of the corresponding PtdCho decrease induced by HgCl2.

Renal phospholipid metabolism in streptozotocin-induced diabetic rats.

The effect of acute and chronic streptozotocin-induced diabetes on phospholipid metabolism in papillary, medullary and cortical slices was studied. No changes were observed in either the phospholipid content or composition in the papilla, medulla and cortex from acute and chronic diabetic rats. With reference to (U-14C)glycerol incorporation in the papilla from chronic diabetic rats, it increased in PtdCho, PtdIns and PtdEtn. In the medulla, the incorporation to PtdCho decreased in the acute diabetic state, while in the chronic diabetic state the incorporation to PtdCho and PtdIns decreased. No changes were observed in cortical phospholipids from diabetic rats. As regards 23P-sodium orthophosphate incorporation to phospholipids, while it increased in PtdCho, PtdIns and PtdEtn in the papilla from acute and chronic diabetic rats, in the medulla from both groups, the incorporation in PtdCho and PtdIns decreased, and in the cortex from acute diabetic rats it decreased only in PtdIns. Even though no changes were detected in phospholipid composition, alterations in phospholipid metabolism were induced by experimental diabetes.

Compartmental study of rat renal prostaglandin synthesis during development.

The biosynthesis of prostaglandins (PGs) from the endogenous and exogenous precursor, arachidonic acid (AA), in renal papilla, medulla and cortex from neonatal to adult rats was investigated. Rat renal papilla and medulla incubated in the presence of [1-14C]AA released radioactive PGE2, PGF2 alpha and PGD2 which increased with age. No radioactive prostaglandins were found in the supernatants of renal cortex at any age studied. The amount of total prostaglandins released from the endogenous precursor also increased from 10 to 70 days of age, PGD2 being the prostaglandin that showed the most important rise. In the cortex, only PGE2 release increased with age. Cyclooxygenase (COX) activity was measured in papillary, medullary and cortical homogenates by using [1-14C]AA as substrate. Papillary and medullary COX activity increased after 10 days of age and continued to rise up to day 30 thereafter remaining unaltered until adulthood. Cortical COX activity was very low and decreased with age. These findings indicate the low capacity of the neonatal rat kidney to synthesize PGs.

Biochemical and morphological changes in the developing kidney.

We have studied microsomal phospholipid, cholesterol and protein concentration in rat renal papilla, medulla and cortex during postnatal development, and the relationship between these membranes biochemical parameters and morphological changes. We also determined DNA concentration in each kidney zone. No changes were observed either in papillary microsomal phospholipids, proteins and cholesterol or in DNA concentration from 10-to 70-day-old rats. Medullary microsomal proteins and cholesterol did not change but a significant increase was observed in the microsomal phospholipid concentration during development; in this case, medullary DNA was significantly lower at 70 than at 10 days. In contrast, all biochemical parameters in renal cortex were significantly higher during development except for DNA concentration which suffered a great decrease. These biochemical findings demonstrate that the developmental pattern is different in each zone of the kidney and confirm the fact that the papilla, in newborn rats, is almost fully developed whereas the renal cortex and medulla are immature.

Short- and long-term treatment with indometacin causes renal phospholipid alteration: a possible explanation for indomethacin nephrotoxicity.

Phospholipid content was studied in kidneys from rats treated with indometacin. Short-term treatment was performed by using low (1 and 5 mg/kg/day) and high (10 and 50 mg/kg/day) doses of indometacin. Long-term treatment was achieved by using only low doses of indometacin. Short-term treatment at low doses did not result in any change in the phospholipid content. In rats administered higher concentrations, indometacin caused a marked increase in all papillary phospholipid contents, but no effect was observed in the medulla, and an increase in sphingomyelin and phosphatidylethanolamine was observed in the cortex. Long-term treatment with administration of 1 mg/kg/day of indometacin led to an increase in all papillary phospholipids from the 2nd week of treatment. Medullary phospholipids also increased, but no changes were observed in cortical phospholipids. These results show that indometacin causes phospholipid accumulation in rat kidney and that the papilla is the most sensitive renal tissue.

Specific and functional insulin receptors in rat renal papilla.

To investigate a possible action of insulin on the rat kidney papilla, the binding of 125I-insulin to papilla microsomes was examined. This binding was specific to insulin in that it was displaced by increasing concentrations of unlabelled porcine insulin and to a lesser extent by porcine proinsulin and IGF-I, but not by IGF-II and bGH. Scatchard plot of the binding data was curvilinear consistent with either two classes of receptors with different affinities or a single class of receptors that showed negative cooperativity. A small fraction of 125I-insulin (maximum 2%) was degraded during incubation, but with a Km two order of magnitude higher than the constant of affinity for binding. Insulin stimulates the incorporation of phosphate to phosphatidylcholine in a dose-dependent manner, reaching a maximum with 10 nM insulin. This data showed both the presence of specific insulin receptors in the kidney papilla and an insulin action through the synthesis of phospholipids by insulin.

Increase of phosphatidylinositol arachidonic acid incorporation induced by mepacrine.

In view of the fact that mepacrine (Mp) is usually used as an inhibitor of the endogenous phospholipase A2, and since this enzyme produces the release of arachidonic acid (AA) from membrane phospholipids, we studied the effect of different concentrations of Mp on the mobilization of [1-14C]AA in rat renomedullary phospholipids. During the acylation period, 0.1 mM Mp did not produce any significant change in the incorporation of [1-14C]AA into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and only a slight increase in phosphatidylinositol (PI). Higher concentrations of Mp (0.5 to 1.0 mM) produced a decrease of radioactivity in PE and PC with an increase in PI. Using prelabeled slices, a dose-dependent decrease in the 14C-radioactivity in PE and PC was observed, with a parallel increase in PI. This effect of Mp persisted even in the presence of a physiological activator of phospholipase A2, bradykinin (BK). No change in the net amount of phospholipids was observed at any of the Mp concentrations used. The results of this study show that Mp, at concentrations generally used to inhibit phospholipase A2, produced a transfer of arachidonic acid from PE and PC to PI, rather than a blockade in the release of AA from membrane phospholipids.

Sow (Sus scrofa) follicular fluid: prostaglandin content and effect on the motility of isolated oviducts.

The present study provides information regarding the effects of the sow follicular fluid (FF) on the motility of isolated segments of swine and rabbit oviducts. In addition, the concentration of prostaglandins (PGs) F2 alpha, E2 and E1 in the follicular fluid of sow ovaries isolated at different stages of the sex cycle as well as the generation of the same PGs by walls of ovarian follicles in early and late proestrus, in estrus, in metestrus and in diestrus, were explored. The stimulatory contractile effect of proestrous FF in isolated segments of sow fimbria was antagonized by polyphloretin phosphate (PPP), a PG receptor blocker and by indomethacin, an inhibitor of PG synthesis. The positive inotropism evoked by the FF was mimiked by bradykinin and the influences of both interventions were similarly antagonized by PPP. It appears plausible that the inotropic effect of the preovulatory FF on the sow fimbria could be not only by PGs already present in the fluid, but also by the stimulation of the synthesis of tubal PGs by follicular fluid bradykinin. The FF also stimulated the ampullary tubal segments isolated from proestrous sows whereas the same volume of FF depressed significantly the isometric developed tension of rabbit ampulla. The total concentration of the three PGs in the FF from late proestrous follicles was significantly greater than that of the same PGs in the other two stages of the sex cycle (early proestrus and diestrus), whereas the concentration of each PG (PGE2, PGF2 alpha or PGE1), did not differ within any of the stages of the cycle. Furthermore, the total amount of the three PGs produced by the walls of follicles from late proestrous ovaries was also significantly greater than that generated by ovarian follicles from early proestrus, estrus, metestrus and diestrus. In summary the results document that the concentration of each one of the PGs measured (E2, E1 or F2 alpha) attained maximal values at the time of ovulation. The results regarding the effects of FF on the inotropic activity of fimbrial and ampullary segments of sow oviducts also suggest that the fluid might play a physiological role, favouring the capture and transfer of ova into the oviducts at the moment of ovulation.