Vascular proinflammatory responses by aldosterone are mediated via c-Src trafficking to cholesterol-rich microdomains: role of PDGFR

Aims We demonstrated c-Src activation as a novel non-genomic signalling pathway for aldosterone in vascular smooth muscle cells (VSMCs). Here, we investigated molecular mechanisms and biological responses of this phenomenon, focusing on the role of lipid rafts/caveolae and platelet-derived growth factor receptor (PDGFR) in c-Src-regulated proinflammatory responses by aldosterone. Methods and results Studies were performed in cultured VSMCs from Wistar-Kyoto (WKY) rats and caveolin-1 knockout (Cav 1(-/-)) and wild-type mice. Aldosterone stimulation increased c-Src phosphorylation and trafficking to lipid rafts/caveolae. Cholesterol depletion with methyl-beta-cyclodextrin abrogated aldosterone-induced phosphorylation of c-Src and its target, Pyk2. Aldosterone effects were recovered by cholesterol reload. Aldosterone-induced c-Src and cortactin phosphorylation was reduced in caveolin-1-silenced and Cav 1(-/-) VSMCs. PDGFR is phosphorylated by aldosterone within cholesterol-rich fractions of VSMCs. AG1296, a PDGFR inhibitor, prevented c-Src phosphorylation and translocation to cholesterol-rich fractions. Aldosterone induced an increase in adhesion molecule protein content and promoted monocyte adhesion to VSMCs, responses that were inhibited an by cholesterol depletion, caveolin-1 deficiency, AG1296 and PP2, a c-Src inhibitor. Mineralocorticoid receptor (MR) content in flotillin-2-rich fractions and co-immunoprecipitation with c-Src and PDGFR increased upon aldosterone stimulation, indicating MR-lipid raft/signalling association. Conclusion We demonstrate that aldosterone-mediated c-Src trafficking/activation and proinflammatory signalling involve lipid rafts/caveolae via PDGFR.

GABA(A) receptor blockade in dorsomedial and ventromedial nuclei of the hypothalamus evokes panic-like elaborated defensive behaviour followed by innate fear-induced antinociception

Dysfunction in the hypothalamic GABAergic system has been implicated in panic syndrome in humans. Furthermore, several studies have implicated the hypothalamus in the elaboration of pain modulation. Panic-prone states are able to be experimentally induced in laboratory animals to study this phenomenon. The aim of the present work was to investigate the involvement of medial hypothalamic nuclei in the organization of panic-like behaviour and the innate fear-induced oscillations of nociceptive thresholds. The blockade of GABA(A) receptors in the neuronal substrates of the ventromedial. or dorsomedial hypothalamus was followed by elaborated defensive panic-like reactions. Moreover, innate fear-induced antinociception was consistently elicited after the escape behaviour. The escape responses organized by the dorsomedial and ventromedial hypothalamic nuclei were characteristically more elaborated, and a remarkable exploratory behaviour was recorded during GABA(A) receptor blockade in the medial hypothalamus. The motor characteristic of the elaborated defensive escape behaviour and the patterns of defensive alertness and defensive immobility induced by microinjection of the bicuculline either into the dorsomedial. or into the ventromedial hypothalamus were very similar. This was followed by the same pattern of innate fear-induced antinociceptive response that lasted approximately 40 min after the elaborated defensive escape reaction in both cases. These findings suggest that dysfunction of the GABA-mediated neuronal system in the medial hypothalamus causes panic-like responses in laboratory animals, and that the elaborated escape behaviour organized in both dorsomedial and ventromedial hypothalamic nuclei are followed by significant innate-fear-induced antinociception. Our findings indicate that the GABA(A) receptor of dorsomedial and ventromedial hypothalamic nuclei are critically involved in the modulation of panic-like behaviour. (C) 2009 Elsevier B.V. All rights reserved.

THE INFLUENCE OF VIBRISSAL SOMATOSENSORY PROCESSING IN RAT SUPERIOR COLLICULUS ON PREY CAPTURE

The lateral part of intermediate layer of superior colliculus (SCI) is a critical substrate for successful predation by rats. Hunting-evoked expression of the activity marker Fos is concentrated in SCI while prey capture in rats with NMDA lesions in SCI is impaired. Particularly affected are rapid orienting and stereotyped sequences of actions associated with predation of fast moving prey. Such deficits are consistent with the view that the deep layers of SC are important for sensory guidance of movement. Although much of the relevant evidence involves visual control of movement, less is known about movement guidance by somatosensory input from vibrissae. Indeed, our impression is that prey contact with whiskers is a likely stimulus to trigger predation. Moreover, SCI receives whisker and orofacial somatosensory information directly from trigeminal complex, and indirectly from zona incerta, parvicelular reticular formation and somatosensory barrel cortex. To better understand sensory guidance of predation by vibrissal information we investigated prey capture by rats after whisker removal and the role of superior colliculus (SC) by comparing Fos expression after hunting with and without whiskers. Rats were allowed to hunt cockroaches, after which their whiskers were removed. Two days later they were allowed to hunt cockroaches again. Without whiskers the rats were less able to retain the cockroaches after capture and less able to pursue them in the event of the cockroach escaping. The predatory behaviour of rats with re-grown whiskers returned to normal. In parallel, Fos expression in SCI induced by predation was significantly reduced in whiskerless animals. We conclude that whiskers contribute to the efficiency of rat prey capture and that the loss of vibrissal input to SCI, as reflected by reduced Fos expression, could play a critical role in predatory deficits of whiskerless rats. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Modulation of arterial pressure by P2 purinoceptors in the paraventricular nucleus of the hypothalamus of awake rats

In the present study we evaluated the role of purinergic mechanisms in the PVN on the tonic modulation of the autonomic function to the cardiovascular system as well on the cardiovascular responses to peripheral chemoreflex activation in awake rats Guide-cannulae were bilaterally Implanted in the direction of the PVN of male Wistar rats Femoral artery and vein were catheterized one day before the experiments Chemoreflex was activated with KCN (30 mu g/0 05 ml iv) before and after microinjections of P2 receptors antagonist into the PVN Microinjection of PPADS a non selective P2X antagonist Into the PVN (n = 6) produced a significant increase in the baseline MAP (99 +/- 2 vs 112 +/- 3 mmHg) and HR (332 +/- 8 vs 375 +/- 8 bpm) but had no effect on the pressor and bradycardic responses to chemoreflex activation Intravenous injection of vasopres in receptors antagonist after microinjection of PPADS into the PVN produced no effect on the increased baseline MAP Simultaneous microinjection of PPADS and KYN into the PVN (n=6) had no effect in the baseline MAP HR or in the pressor and bradycardic responses to chemoreflex activation We conclude that P2 purinoceptors in the PVN are involved in the modulation of baseline autonomic function to the cardiovascular system but not in the cardiovascular responses to chemoreflex activation in awake rats (C) 2010 Elsevier B V All rights reserved

Activation of lateral parabrachial afferent pathways and endocrine responses during sodium appetite regulation

Modulation of salt appetite involves interactions between the circumventricular organs (CVOs) receptive areas and inhibitory hindbrain serotonergic circuits. Recent studies provide support to the idea that the serotonin action in the lateral parabrachial nucleus (LPBN) plays an important inhibitory role in the modulation of sodium appetite. The aim of the present work was to identify the specific groups of neurons projecting to the LPBN that are activated in the course of sodium appetite regulation, and to analyze the associated endocrine response, specifically oxytocin (OT) and atrial natriuretic peptide (ANP) plasma release, since both hormones have been implicated in the regulatory response to fluid reestablishment. For this purpose we combined the detection of a retrograde transported dye, Fluorogold (FG) injected into the LPBN with the analysis of the Fos immunocytochemistry brain pattern after sodium intake induced by sodium depletion. We analyzed the Fos-FG immunoreactivity after sodium ingestion induced by peritoneal dialysis (PD). We also determined OT and ANP plasma concentration by radioimmunoassay (RIE) before and after sodium intake stimulated by PD. The present study identifies specific groups of neurons along the paraventricular nucleus, central extended amygdala, insular cortex, dorsal raphe nucleus, nucleus of the solitary tract and the CVOs that are activated during the modulation of sodium appetite and have direct connections with the LPBN. It also shows that OT and ANP are released during the course of sodium satiety and fluid reestablishment. The result of this brain network activity may enable appropriate responses that re-establish the body fluid balance after induced sodium consumption. (C) 2009 Elsevier Inc. All rights reserved.

Hypothalamic oxytocin neurons modulate hypophagic effect induced by adrenalectomy

Glucocorticoids have major effects on food intake, as demonstrated by the decrease of food intake following adrenalectomy (ADX); however, the mechanisms leading to these effects are not well understood. Oxytocin (OT) has been shown to reduce food intake. We evaluated the effects of glucocorticoids on OT neuron activation and OT mRNA expression in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei induced by feeding. We also evaluated the effect of pretreatment with OT-receptor antagonist ([d(CH2)5,Tyr(Me)2,Orn8]-vasotocin, OVT) on food intake in ADX rats. Fos/OT neurons in the posterior parvocellular subdivision of the PVN were increased after refeeding, with a higher number in the ADX group, compared with sham and ADX+corticosterone (B) groups, with no difference in the medial parvocellular and magnocellular subdivisions of the PVN. ADX increased OT mRNA expression in the PVN both in fasting and refeeding condition, compared with sham and ADX+B groups. In the SON, refeeding increased the number of Fos/OT neurons, with a higher number in the ADX+B group. In fasted condition, OT mRNA expression in the SON was increased in ADX and ADX+B, compared with sham group. Pretreatment with OVT reversed the ADX-induced hypophagia, with no difference between sham and ADX+B animals. The present results show that glucocorticoid withdrawal induces a higher activation of PVN OT neurons in response to feeding, and an increase of OT mRNA expression in the PVN and OT-receptor antagonist reverses the anorexigenic effect induced by ADX These data indicate that PVN OT neurons might mediate the hypophagic effect induced by adrenalectomy. (C) 2009 Elsevier Inc. All rights reserved.

HYPOTHALAMIC COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT AND CORTICOTROPHIN RELEASING FACTOR NEURONS ARE STIMULATED BY EXTRACELLULAR VOLUME AND OSMOTIC CHANGES

Several studies suggest that hypothalamic cocaine- and amphetamine-regulated transcript (CART) may interact with the hypothalamic-pituitary-adrenal (HPA) axis in the control of neuroendocrine function and may also participate in cardiovascular regulation. Therefore, this study aimed to evaluate, in experimental models of isotonic (I-EVE) and hypertonic (H-EVE) extracellular volume expansion and water deprivation (WD), the activation of CART- and corticotrophin releasing factor (CRF)-immunoreactive neurons, as well as the relative expression of CART and CRF mRNAs in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Both H-EVE (0.30M NaCl, 2mL/100g of body weight, in 1 minute) and 24 hours of WD significantly increased plasma sodium concentrations, producing, respectively, either an increase or a decrease in extracellular volume. I-EVE (0.15M NaCl, 2mL/100g of body weight, in 1 minute) evoked a significant increase in the circulating volume accompanied by unaltered plasma concentrations of sodium. CART-expressing neurons of both magnocellular and parvocellular hypothalamic divisions were activated to produce Fos in response to H-EVE but not in response to I-EVE. Furthermore, increased expression of CART mRNA was found in the PVN of H-EVE but not I-EVE rats. These data show for the first time that EVE not only activates hypothalamic CRF neurons but also increases CRF mRNA expression in the PVN. In contrast, WD increases the number of CART-immunoreactive neurons activated to produce Fos in the PVN and SON but does not change the number of neurons double labeled for Fos and CRF or expression of CRF mRNA in the PVN. These findings provided new insights into the participation of CART in diverse processes within the PVN and SON, including its possible involvement in activation of the HPA axis and cardiovascular regulation in response to changes in extracellular volume and osmolality. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Lateral Parabrachial Afferent Areas and Serotonin Mechanisms Activated by Volume Expansion

Recent evidence has shown that the serotonergic mechanism of the lateral parabrachial nucleus (LPBN) participates in the regulation of renal and hormonal responses to isotonic blood volume expansion (BVE). We investigated the BVE-induced Fos activation along forebrain and hindbrain nuclei and particularly within the serotonergic clusters of the raphe system that directly project to the LPBN. We also examined whether there are changes in the concentration of serotonin (5HT) within the raphe nucleus in response to the same stimulus. With this purpose, we analyzed the cells doubly labeled for Fos and Fluorogold (FG) following BVE (NaCl 0.15 M, 2 ml/100 g b.w., 1 min) 7 days after FG injection into the LPBN. Compared with the control group, blood volume-expanded rats showed a significant greater number of Fos-FG double-labeled cells along the nucleus of the solitary tract, locus coeruleus, hypothalamic paraventricular nucleus, central extended amygdala complex, and dorsal raphe nucleus (DRN) cells. Our study also showed an increase in the number of serotonergic DRN neurons activated in response to isotonic BVE. We also observed decreased levels of 5HT and its metabolite 5-hydroxyindoleacetic acid (measured by high-pressure liquid chromatography) within the raphe nucleus 15 min after BVE. Given our previous evidence on the role of the serotonergic system in the LPBN after BVE, the present morphofunctional findings suggest the existence of a key pathway (DRN-LPBN) that may control BVE response through the modulation of 5HT release. (c) 2008 Wiley-Liss, Inc.

Water deprivation increases Fos expression in hypothalamic corticotropin-releasing factor neurons induced by right atrial distension in awake rats

Atrial mechanoreceptors, sensitive to stretch, contribute in regulating heart rate and intravascular volume. The information from those receptors reaches the nucleus tractus solitarius and then the paraventricular nucleus (PVN), known to have a crucial role in the regulation of cardiovascular function. Neurons in the PVN synthesize CRF, AVP, and oxytocin (OT). Stimulation of atrial mechanoreceptors was performed in awake rats implanted with a balloon at the junction of the superior vena cava and right atrium. Plasma ACTH, AVP, and OT concentrations and Fos, CRF, AVP, and OT immunolabeling in the PVN were determined after balloon inflation in hydrated and water-deprived rats. The distension of the balloon increased the plasma ACTH concentrations, which were higher in water-deprived than in hydrated rats (P < 0.05). In addition, the distension in the water-deprived group decreased plasma AVP concentrations (P < 0.05), compared with the respective control group. The distension increased the number of Fos- and double-labeled Fos/CRF neurons in the parvocellular PVN, which was higher in the water-deprived than in the hydrated group (P < 0.01). There was no difference in the Fos expression in magnocellular PVN neurons after distension in hydrated and water-deprived groups, compared with respective controls. In conclusion, parvocellular CRF neurons showed an increase of Fos expression induced by stimulation of right atrial mechanoreceptors, suggesting that CRF participates in the cardiovascular reflex adjustments elicited by volume loading. Activation of CRF neurons in the PVN by cardiovascular reflex is affected by osmotic stimulation.

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-alpha-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POW mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 mu g/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POW neurons in the hypothalamus and an increased mRNA expression of these neuropeptides. (C) 2008 Elsevier Inc. All rights reserved.

An Experimental Model of Meningoencephalomyelitis by Rocio Flavivirus in Balb/C Mice: Inflammatory Response, Cytokine Production, and Histopathology

Rocio virus (ROCV) is a flavivirus, probably transmitted by Culex mosquitoes and maintained in nature as a zoonosis of wild birds. Rocio virus caused a human epidemic of severe encephalitis that lasted from 1973 to 1980 in the Ribeira valley, in the southeastern coast of Brazil. After this outbreak, serologic evidence of ROCV circulation has been reported and public health authorities are concerned about a return of ROCV outbreaks in Brazil. We show here a study on the pathogenesis and the physiopathology of ROCV disease in the central nervous system of a Balb/C young adult mice experimental model. The animals were intraperitoneally infected by ROCV and followed from 0 to 9 days after infection, when all of them died. Nervous tissue samples were collected from infected animals for immunohistochemistry and molecular biology analysis. We observed the virus in the central nervous system, the inflammatory changes induced by Th1 and Th2 cytokines, and the final irreversible damage of nervous tissues by neuronal degeneration and apoptosis. These findings can help to better understand the pathogenesis and physiopathology of the human meningoencephalomyelitis by ROCV and other flaviviruses.

Effects of Caffeoylquinic Acid Derivatives and C-Flavonoid from Lychnophora ericoides on in vitro Inflammatory Mediator Production

In this study we aimed at evaluating the effect of the major polar constituents of the medicinal plant Lychnophora ericoides on the production of inflammatory mediators produced by LPS-stimulated U-937 cells. The 6,8-di-C-beta-glucosylapigenin (vicenin-2) presented no effect on tumor necrosis factor (TNF)-alpha production, but inhibited, in a dose-dependent manner, the production of prostaglandin (PG) E(2) without altering the expression of cyclooxygenase (COX) -2 protein. 3,5-Dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, at lower concentrations, had small but significant effects on reducing PG E, levels; at higher doses these compounds stimulated PGE(2) and also TNF-alpha production by the cells. All the caffeoylquinic acid derivatives, in a dose-dependent fashion, were able to inhibit monocyte chemoattractant protein-3 synthesis/release, with 4,5-DCQ being the most potent at the highest tested concentration. These results add important information on the effects of plant natural polyphenols, namely vicenin-2 and caffeoylquinic acid derivatives, on the production of inflammatory mediators by cultured cells.

C-Peptide Levels and Insulin Independence Following Autologous Nonmyeloablative Hematopoietic Stem Cell Transplantation in Newly Diagnosed Type 1 Diabetes Mellitus

Context In 2007, the effects of the autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT) in 15 patients with type 1 diabetes mellitus (DM) were reported. Most patients became insulin free with normal levels of glycated hemoglobin A(1c) (HbA(1c)) during a mean 18.8-month follow-up. To investigate if this effect was due to preservation of beta-cell mass, continued monitoring was performed of C-peptide levels after stem cell transplantation in the 15 original and 8 additional patients. Objective To determine C-peptide levels after autologous nonmyeloablative HSCT in patients with newly diagnosed type 1 DM during a longer follow-up. Design, Setting, and Participants A prospective phase 1/2 study of 23 patients with type 1 DM(aged 13-31 years) diagnosed in the previous 6 weeks by clinical findings with hyperglycemia and confirmed by measurement of serum levels of anti glutamic acid decarboxylase antibodies. Enrollment was November 2003-April 2008, with follow-up until December 2008 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirao Preto, Ribeirao Preto, Brazil. Hematopoietic stem cells were mobilized via the 2007 protocol. Main Outcome Measures C-peptide levels measured during the mixed-meal tolerance test, before, and at different times following HSCT. Secondary end points included morbidity and mortality from transplantation, temporal changes in exogenous insulin requirements, and serum levels of HbA1c. Results During a 7- to 58-month follow-up (mean, 29.8 months; median, 30 months), 20 patients without previous ketoacidosis and not receiving corticosteroids during the preparative regimen became insulin free. Twelve patients maintained this status for a mean 31 months (range, 14-52 months) and 8 patients relapsed and resumed insulin use at low dose (0.1-0.3 IU/kg). In the continuous insulin-independent group, HbA(1c) levels were less than 7.0% and mean (SE) area under the curve (AUC) of C-peptide levels increased significantly from 225.0 (75.2) ng/mL per 2 hours pretransplantation to 785.4 (90.3) ng/mL per 2 hours at 24 months posttransplantation (P<.001) and to 728.1 (144.4) ng/mL per 2 hours at 36 months (P=.001). In the transient insulin-independent group, mean (SE) AUC of C-peptide levels also increased from 148.9 (75.2) ng/mL per 2 hours pretransplantation to 546.8 (96.9) ng/mL per 2 hours at 36 months (P=.001), which was sustained at 48 months. In this group, 2 patients regained insulin independence after treatment with sitagliptin, which was associated with increase in C-peptide levels. Two patients developed bilateral nosocomial pneumonia, 3 patients developed late endocrine dysfunction, and 9 patients developed oligospermia. There was no mortality. Conclusion After a mean follow-up of 29.8 months following autologous nonmyeloablative HSCT in patients with newly diagnosed type 1 DM, C-peptide levels increased significantly and the majority of patients achieved insulin independence with good glycemic control. Trial Registration clinicaltrials.gov Identifier: NCT00315133

Chemokines expression during Leptospira interrogans serovar Copenhageni infection in resistant BALB/c and susceptible C3H/HeJ mice

The role of innate immune responses in protection against leptospirosis remains unclear. We examined the expression of the chemokines CCL2/JE (MCP-1), CCL3/MIP-1 alpha (MIP-1 alpha) and CXCL1/KC (IL-8) regarding resistance and susceptibility to leptospirosis in experimental mice models BALB/c and C3H/HeJ, respectively. A virulent strain of Leptospira interrogans serovar Copenhageni was used in this study. Twenty-five animals of each mouse strain of C3H/HeJ and BALB/c, were infected intraperitoneally with 106 cells. Five un-infected animals of each strain were kept as control. Mortality of C3H/HeJ mouse was observed while BALB/c mice were asymptomatic. The presence of leptospire DNA in tissues of infected animals was demonstrated by PCR. Chemokines were measured in serum, spleen, liver, kidney and lung of both strains of animals using immunoenzymatic assay (ELISA). Elevations in the levels of chemokines MCP-1 and IL-8 occurred in all organs and sera of C3H/HeJ and BALB/c infected mice. The levels of MIP-1 alpha were lower when compared to MCP-1 and IL-8 in all analyzed organs, with a slight increase in liver and kidney. Our results indicate that the expression of inflammatory mediators can vary greatly, depending on the tissue and mouse strains. It is possible that the resistance to Leptospira can be partially correlated to the increase of MIP-1 alpha observed in BALB/c mice, while an increasing and a sustained expression of MCP-1 and IL-8 in the lungs of C3H/HeJ mice can be correlated to the severity and progression of leptospirosis. (C) 2009 Elsevier Ltd. All rights reserved.

FURTHER INSIGHTS TOWARD VITAMIN C DETERMINATION AND STABILITY. PROPOSAL OF A NEW QUANTIFICATION METHOD

This work aimed at an evaluation of the classical iodine method for quantification of vitamin C (L-ascorbic acid) in fruit juices, as well as at a search into the stability of this so popular vitamin under different conditions of pH, temperature and light exposition, in addition to a proposal of a new quantification method. Our results point to the persistent reversibility of the blue color of the starch-triiodide complex at the end point when using the classical iodine titration, and the overestimation of the true vitamin concentration in fruit juices. A new quantification method is proposed in order to overcome this problem. Surprising conclusions were obtained regarding the controversial stability of L-ascorbic acid toward atmospheric oxygen, at low pH, even in fruit juice and at room temperature, showing that the major problem concerned with aging of fruit juices is proliferation of microorganisms rather than expontaneous oxidation of L-ascorbic acid.