Role of Vitamin A in Mammary Gland Development and Lactation.

Vitamin A (all-trans-retinol), its active derivatives retinal and retinoic acid, and their synthetic analogues constitute the group of retinoids. It is obtained from diet either as preformed vitamin A or as carotenoids. Retinal plays a biological role in vision, but most of the effects of vitamin A are exerted by retinoic acid, which binds to nuclear receptors and regulates gene transcription. Vitamin A deficiency is an important nutritional problem, particularly in the developing world. Retinol and carotenoids from diet during pregnancy and lactation influence their concentration in breast milk, which is important in the long term, not only for the offspring, but also for maternal health. In this study, we review the role of vitamin A in mammary gland metabolism, where retinoid signaling is required not only for morphogenesis and development of the gland and for adequate milk production, but also during the weaning process, when epithelial cell death is coupled with tissue remodeling.

Time course of memory formation for an appetitive, one-trial, water-reward task in day-old chicks.

Researchers studying learning and memory in the day-old chick have developed a one-trial taste avoidance learning task, in which a chick pecks a bead coated with an aversive-tasting liquid such as methylanthranilate. The current study examined, in two experiments, whether chicks could demonstrate learning of an appetitive version of the one-trial task, and how long this memory might last. The results demonstrate that chicks show significantly decreased latencies to peck a test bead similar to that pecked at training coated with water, and that this memory lasted for at least 6 h after training. These results demonstrate that this appetitive task produces significant learning.

Vitamin A Deficiency and the Lung.

Vitamin A (all-trans-retinol) is a fat-soluble micronutrient which together with its natural derivatives and synthetic analogues constitutes the group of retinoids. They are involved in a wide range of physiological processes such as embryonic development, vision, immunity and cellular differentiation and proliferation. Retinoic acid (RA) is the main active form of vitamin A and multiple genes respond to RA signalling through transcriptional and non-transcriptional mechanisms. Vitamin A deficiency (VAD) is a remarkable public health problem. An adequate vitamin A intake is required in early lung development, alveolar formation, tissue maintenance and regeneration. In fact, chronic VAD has been associated with histopathological changes in the pulmonary epithelial lining that disrupt the normal lung physiology predisposing to severe tissue dysfunction and respiratory diseases. In addition, there are important alterations of the structure and composition of extracellular matrix with thickening of the alveolar basement membrane and ectopic deposition of collagen I. In this review, we show our recent findings on the modification of cell-junction proteins in VAD lungs, summarize up-to-date information related to the effects of chronic VAD in the impairment of lung physiology and pulmonary disease which represent a major global health problem and provide an overview of possible pathways involved.

Amelioration of scopolamine-induced amnesia by phosphatidylserine and curcumin in the day-old chick.

In the one-trial taste-avoidance task in day-old chicks, acetylcholine receptor activation has been shown to be important for memory formation. Injection of scopolamine produces amnesia, which appears to be very similar in type to that of Alzheimer's disease, which is correlated with low levels of acetylcholine in the brain. Traditional pharmacological treatments of Alzheimer's disease, such as cholinesterase inhibitors and glutamate receptor blockers, improve memory and delay the onset of impairments in memory compared with placebo controls. These agents also ameliorate scopolamine-induced amnesia in the day-old chick trained on the one-trial taste-avoidance task. The present experiments examined the ability of two less traditional treatments for Alzheimer's disease, phosphatidylserine and curcumin, to ameliorate scopolamine-induced amnesia in day-old chicks. The results showed that 37.9 mmol/l phosphatidylserine and 2.7 mmol/l curcumin significantly improved retention in chicks administered scopolamine, whereas lower doses were not effective. Scopolamine did not produce state-dependent learning, indicating that this paradigm in day-old chicks might be a useful one to study the effects of possible Alzheimer's treatments. In addition, chicks administered curcumin or phosphatidylserine showed little avoidance of a bead associated with water reward, indicating that these drugs did not produce response inhibition. The current results extend the findings that some nontraditional memory enhancers can ameliorate memory impairment and support the hypothesis that these treatments might be of benefit in the treatment of Alzheimer's disease.

Discrimination of shape and size sues by day-old chicks in two one-trial learning tasks.

The ability of day-old chicks (Gallus gallus domesticus) to discriminate between the shape and size of beads was investigated in two one-trial tasks, taste avoidance and sickness-conditioned learning. Previous studies determined that color is a critical classification cue for conditioned stimuli in these tasks. In taste avoidance learning, a chick pecks a bead coated with a bitter substance. In sickness-conditioned learning, chicks peck a dry bead and are injected 30min later with lithium chloride. Chicks could discriminate beads of different sizes, but not different shapes, when trained in the taste avoidance task, whereas in the sickness-conditioned learning task, chicks could discriminate shape, but not size. These results suggest that chicks use a number of classificatory cues to remember an avoidance response, and, in the absence of color cues, chicks rely on different cues for different learning tasks.

Memantine improves observational learning in day-old chicks.

Evidence of observational learning (social learning) is present in many species. One such task is the one-trial taste-avoidance task, in which Actor chicks peck a bead coated with an aversant substance. Observer chicks learn to avoid beads that are similar in appearance to the one presented to the Actors. It has been firmly established that active learning of the one-trial taste-avoidance task is dependent on a constrained level of glutamate receptor activation. The current study examined the effects of memantine, a noncompetitive N-methyl-D-aspartate receptor antagonist, on the learning by Observers. Memantine produced an inverted U-shaped dose-dependent response curve; 1.0 mmol/l memantine produced significant improvement. These results demonstrate that memantine influences memory formation for observational learning in the day-old chick and support the hypothesis that memantine can improve memories by altering levels of glutamate during memory formation.

Vitamin a deficiency and alterations in the extracellular matrix.

Vitamin A or retinol which is the natural precursor of several biologically active metabolites can be considered the most multifunctional vitamin in mammals. Its deficiency is currently, along with protein malnutrition, the most serious and common nutritional disorder worldwide. It is necessary for normal embryonic development and postnatal tissue homeostasis, and exerts important effects on cell proliferation, differentiation and apoptosis. These actions are produced mainly by regulating the expression of a variety of proteins through transcriptional and non-transcriptional mechanisms. Extracellular matrix proteins are among those whose synthesis is known to be modulated by vitamin A. Retinoic acid, the main biologically active form of vitamin A, influences the expression of collagens, laminins, entactin, fibronectin, elastin and proteoglycans, which are the major components of the extracellular matrix. Consequently, the structure and macromolecular composition of this extracellular compartment is profoundly altered as a result of vitamin A deficiency. As cell behavior, differentiation and apoptosis, and tissue mechanics are influenced by the extracellular matrix, its modifications potentially compromise organ function and may lead to disease. This review focuses on the effects of lack of vitamin A in the extracellular matrix of several organs and discusses possible molecular mechanisms and pathologic implications.

Gambling with our health: smoke-free policy would not reduce tribal casino patronage.

BACKGROUND: Tribal sovereignty exempts tribal casinos from statewide smoking bans. PURPOSE: To conduct a tribally-led assessment to identify the characteristics of casino patrons at Lake of the Torches Resort Casino in Lac du Flambeau WI and their preferences for a smoke-free casino. METHODS: A survey was administered from April to August 2011 to a stratified random sample of 957 members of the casino players club to assess their preferences for a smoke-free casino. These members were categorized into three groups: those who reported being likely to (1) visit more; (2) visit less; or (3) visit the same if the casino prohibited smoking. They were characterized by age, education, sex, race/ethnicity, annual income, players club level, and reasons for visiting the casino. Statistical analyses were conducted on weighted data in October to December 2011. Weighted logistic regression was calculated to control for potential confounding of patron characteristics. RESULTS: Of the 957 surveyed patrons, 520 (54%) patrons were likely to visit more; 173 (18%) patrons to visit less; and 264 (28%) patrons were indifferent to the smoke-free status. Patrons more likely to prefer a smoke-free casino tended to be white, elderly, middle class and above, and visit the casino restaurants. Patrons within the lower tiers of the players club, almost half of the players club members, also showed a higher preference for a smoke-free casino. CONCLUSIONS: This tribal casino would likely realize increased patronage associated with smoke-free status while also contributing to improved health for casino workers and patrons.

Vitamin A deficiency disturbs collagen IV and laminin composition and decreases matrix metalloproteinase concentrations in rat lung. Partial reversibility by retinoic acid.

Vitamin A is essential for lung development and pulmonary cell differentiation. Its deficiency leads to altered lung structure and function and to basement membrane architecture and composition disturbances. Previously, we showed that lack of retinoids thickens the alveolar basement membrane and increases collagen IV, which are reversed by retinoic acid, the main biologically active vitamin A form. This study analyzed how vitamin A deficiency affects the subunit composition of collagen IV and laminin of lung basement membranes and pulmonary matrix metalloproteinase content, plus the recovering effect of all-trans-retinoic acid. Male weanling pups were fed a retinol-adequate/-deficient diet until 60 days old. A subgroup of vitamin-A-deficient pups received daily intraperitoneal all-trans-retinoic acid injections for 10 days. Collagen IV and laminin chain composition were modified in vitamin-A-deficient rats. The protein and mRNA contents of chains α1(IV), α3(IV) and α4(IV) increased; those of chains α2(IV) and α5(IV) remained unchanged; and the protein and mRNA contents of laminin chains α5, ß1 and γ1 decreased. The mRNA of laminin chains α2 and α4 also decreased. Matrix metalloproteinases 2 and 9 decreased, but the tissue inhibitors of metalloproteinases 1 and 2 did not change. Treating vitamin-A-deficient rats with retinoic acid reversed all alterations, but laminin chains α2, α4 and α5 and matrix metalloproteinase 2 remained low. In conclusion, vitamin A deficiency alters the subunit composition of collagen IV and laminin and the lung's proteolytic potential, which are partly reverted by retinoic acid. These alterations could contribute to impaired lung function and predispose to pulmonary disease.

Memantine ameliorates scopolamine-induced amnesia in chicks trained on taste-avoidance learning.

In day-old chicks trained on the one-trial taste-avoidance task, activation of NMDA receptors by glutamate is particularly important in the initial stages of memory consolidation. In addition, acetylcholine receptor activation has been shown to be a necessary component of memory formation for this task because injection of scopolamine produces amnesia. Memantine, a non-competitive NMDA receptor antagonist, improves memory formation under certain impairing circumstances, despite inhibiting the activation of NMDA receptors. The present experiments tested the hypothesis that memantine can ameliorate scopolamine-induced amnesia in day-old chicks (Gallus gallus domesticus) trained on the one-trial taste-avoidance task. Three experiments assessed the effects of scopolamine, memantine, and glutamate in this task. The results of Experiment 1 demonstrated that 50.0mM scopolamine produces significant amnesia. In Experiment 2, 1.0mM memantine reversed the scopolamine-induced amnesia, while other doses were ineffective. In Experiment 3, injection of 50.0mM glutamate in combination with scopolamine reversed the memantine amelioration. These results indicate a relationship between glutamate and acetylcholine in memory formation in the day-old chick.

Vitamin A deficiency increases protein catabolism and induces urea cycle enzymes in rats.

Chronic vitamin A deficiency induces a substantial delay in the rates of weight and height gain in both humans and experimental animals. This effect has been associated with an impaired nutrient metabolism and loss of body protein. Therefore, we analyzed the effect of vitamin A deficiency on endogenous proteolysis and nitrogen metabolism and its reversibility with all-trans retinoic acid (RA). Male weanling rats, housed in pairs, were pair-fed a vitamin A-deficient (VAD) or control diet until they were 60 d old. A group of deficient rats were further treated with daily intraperitoneal injections of all-trans RA for 10 d. Final body and tissue (i.e. liver and heart) weights were significantly lower and tissue:body weight ratios were similar in VAD rats and in controls. Conversely, the epididymal white fat:body weight ratio and the plasma concentrations of alanine aminotransferase and adiponectin were significantly higher in VAD rats, which also had hepatic macrovesicular lipid accumulations. Plasma and gastrocnemius muscle 3-methylhistidine, urine nitrogen, and plasma and urine urea concentrations were all significantly higher in the VAD group. The expression of the genes encoding urea cycle enzymes and their activities increased in VAD livers. These changes were partially reverted by all-trans RA. We propose that fuel partitioning in vitamin A deficiency may shift from fatty acids to protein catabolism as an energy source. Our results emphasize the importance of vitamin A on the energy balance control system and they provide an explanation for the role of vitamin A in protein turnover, development, and growth.

Memantine improves memory for taste-avoidance learning in day-old chicks exposed to isolation stress.

Activation of NMDA receptors by glutamate is particularly important in the initial stages of memory consolidation. Memantine, a noncompetitive NMDA receptor antagonist, ameliorates memory impairment under certain circumstances, despite blocking the activation of NMDA receptors. The present experiments tested the hypothesis that memantine can improve memory deficits induced by isolation stress in day-old chicks (Gallus gallus domesticus) trained in a one-trial taste-avoidance task. Three experiments assessed the effects of memantine at different concentrations and in combination with isolation stress. The results of Experiment 1 indicate that, under normal, non-stressed conditions, memory in control animals is strong and 15.0 mM memantine impairs memory, similar to that seen in many studies of the effects of NMDA receptor antagonists on learning. However, the results of Experiments 2 and 3 showed that, when chicks were exposed to isolation stress during the pre-training period, memory formation for saline-injected control animals was impaired and 5.0 mM memantine significantly improved memory in an inverted U-shaped dose response function. The current results extend the findings that memantine can ameliorate memory impairment and supports the hypothesis that memantine, despite its action to reduce NMDA receptor activity, can facilitate normalized memory acquisition.

Vitamin A deficiency alters rat lung alveolar basement membrane: reversibility by retinoic acid.

Vitamin A is essential for lung development and pulmonary cell differentiation and its deficiency results in alterations of lung structure and function. Basement membranes (BMs) are also involved in those processes, and retinoic acid, the main biologically active form of vitamin A, influences the expression of extracellular matrix macromolecules. Therefore, we have analyzed the ultrastructure and collagen content of lung alveolar BM in growing rats deficient in vitamin A and the recovering effect of all-trans retinoic acid. Male weanling pups were fed a retinol-adequate or -deficient diet until they were 60 days old. A group of vitamin A-deficient pups were recovered by daily intraperitoneal injections of all-trans retinoic acid for 10 days. Alveolar BM in vitamin A-deficient rats doubled its thickness and contained irregularly scattered collagen fibrils. Immunocytochemistry revealed that these fibrils were composed of collagen I. Total content of both collagen I protein and its mRNA was greater in vitamin-deficient lungs. In agreement with the greater size of the BM the amount of collagen IV was also increased. Proinflammatory cytokines, IL-1alpha, IL-1beta and TNF-alpha, did not change, but myeloperoxidase and TGF-beta1 were increased. Treatment of vitamin A-deficient rats with retinoic acid reversed all the alterations, but the BM thickness recovered only partially. Retinoic acid recovering activity occurred in the presence of increasing oxidative stress. In conclusion, vitamin A deficiency results in alterations of the structure and composition of the alveolar BM which are probably mediated by TGF-beta1 and reverted by retinoic acid. These alterations could contribute to the impairment of lung function and predispose to pulmonary disease.

Vitamin E deficiency induces liver nuclear factor-kappaB DNA-binding activity and changes in related genes.

The biological functions of vitamin E have been classically attributed to its property as a potent inhibitor of lipid peroxidation in cellular membranes. However, in 1991, Azzi's group first described that alpha-tocopherol inhibits smooth muscle cell proliferation in a protein kinase C (PKC)-dependent way, demonstrating a non-antioxidant cell signalling function for vitamin E. More recently, the capacity of alpha-tocopherol to modulate gene expression with the implication of different transcription factors, beyond its antioxidant properties, has also been established. This study was to determine the effect of vitamin E-deficiency on liver nuclear factor-kappa B (NF-kappaB) DNA-binding activity and the response of target antioxidant-defense genes and cell cycle modulators. Rats were fed either control diet or vitamin-E free diet until 60 or 90 days after birth. Vitamin E-deficiency enhanced liver DNA-binding activity of NF-kappaB [electrophoretic mobility-shift assay, (EMSA)] and up-regulated transcription of gamma-glutamylcysteine synthetase (gamma-GCSM; gamma-GCSC), cyclin D1 and cyclin E. We also showed down-regulation of p21(Waf1/Cip1) transcription. Western-blot analysis demonstrated that gamma-glutamylcysteine synthetase catalytic subunit (gamma-GCSC) and cyclin D1 showed a similar pattern to that found in the RT-PCR analysis. Moreover, chromatin immunoprecipitation (ChIP) assay demonstrated that NF-kappaB directly regulates transcription of gamma-GCS (both subunits) and cyclin D1 through the binding of NF-kappaB to the corresponding gene promoters, which was enhanced in vitamin E-deficiency. These findings show that vitamin E-deficiency induces significant molecular regulatory properties in liver cells with an altered expression of both antioxidant-defense genes and genes that control the cell cycle and demonstrate that liver NF-kappaB activation is involved in this response. Our results emphasize the importance of maintaining an adequate vitamin E consumption not only to prevent liver oxidative damage but also in modulating signal transduction.

Weaning induces NOS-2 expression through NF-kappaB modulation in the lactating mammary gland: importance of GSH.

At the end of lactation the mammary gland undergoes involution, a process characterized by apoptosis of secretory cells and tissue remodelling. To gain insight into this process, we analysed the gene expression profile by oligonucleotide microarrays during lactation and after forced weaning. Up-regulation of inflammatory mediators and acute-phase response genes during weaning was found. Expression of IkappaBalpha (inhibitory kappaBalpha), a protein known to modulate NF-kappaB (nuclear factor-kappaB) nuclear translocation, was significantly up-regulated. On the other hand, there was a time-dependent degradation of IkappaBalpha protein levels in response to weaning, suggesting a role for NF-kappaB. Furthermore, we have demonstrated, using chromatin immunoprecipitation assays, binding of NF-kappaB to the NOS-2 (inducible nitric oxide synthase) promoter at the early onset of events triggered during weaning. The three isoforms of NOS are constitutively present in the lactating mammary gland; however, while NOS-2 mRNA and protein levels and, consequently, NO production are increased during weaning, NOS-3 protein levels are diminished. Western blot analyses have demonstrated that protein nitration is increased in the mammary gland during weaning, but this is limited to a few specific tyrosine-nitrated proteins. Interestingly, inhibition of GSH synthesis at the peak of lactation partially mimics these findings, highlighting the role of NO production and GSH depletion during involution.

Vitamin A deficiency alters the structure and collagen IV composition of rat renal basement membranes.

Retinoids can modulate the expression of extracellular matrix (ECM) proteins with variable results depending on other contributing factors. Because changes in these proteins may alter the composition and impair the function of specialized ECM structures such as basement membranes (BMs), we studied the effects of vitamin A deficiency on renal BMs during the growing period. Newborn male rats were fed a vitamin A-deficient (VAD) diet for 50 d. The ultrastructure of renal BMs was analyzed by electron microscopy. Total collagen IV, the different alpha(IV) chains, matrix degrading metalloproteinases (MMP), and tissue inhibitors of metalloproteinases (TIMP) were quantified by immunocytochemistry and/or Western blotting. Tumor necrosis factor-alpha and interleukin-1beta were measured by ELISA. Semiquantitative RT-PCR was used for determining the steady-state levels for each alpha(IV) chain mRNA. VAD renal BMs showed an irregular thickening, particularly tubular BM. The total collagen IV content was increased, but there was a differential expression of the collagen IV chains. The protein amounts for alpha1(IV), alpha4(IV), and alpha5(IV) were similarly increased, whereas alpha2(IV) and alpha3(IV) were decreased. The levels of mRNA for each collagen IV chain changed in parallel with those of the corresponding protein. Both MMP2 and MMP9 were diminished, but no change was detected in TIMP1 or TIMP2. Our data indicate that nutritional VAD leads to alterations in the structure of renal BMs and to quantitative and qualitative variations in its collagen IV composition. These changes may be a factor predisposing to or resulting in kidney malfunction and renal disease.

Inhibition of liver trans-sulphuration pathway by propargylglycine mimics gene expression changes found in the mammary gland of weaned lactating rats: role of glutathione.

In the lactating mammary gland, weaning produces mitochondrial cytochrome c release and nuclear DNA fragmentation, as determined by gel electrophoresis. This is followed by a significant decrease in lactation. Weaning for 2 h produces an early induction of the tumour suppressor/transcription factor p53, whereas the oncoprotein c-Jun and c-Jun N-terminal kinase are elevated after 24 h of weaning when compared with controls. The expression of p21(cip1) and p27(kip1), cyclin-dependent kinase inhibitors, was significantly higher in weaned rats when compared with control lactating rats. All the changes mentioned above also happen in the lactating mammary gland when propargylglycine, an inhibitor of the liver trans-sulphuration pathway, is administered. This effect is partially reversed by N -acetylcysteine administration. The administration of buthionine sulphoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, to lactating rats produces a decrease in GSH levels and changes in protein concentrations and gene transcripts similar to those in rats with impaired trans-sulphuration pathway. These data suggest that the inter-tissue flux of GSH is an important mechanism of L-cysteine delivery to the lactating mammary gland and emphasize the importance of this physiological event in maintaining the gene expression required to sustain lactation.

In vivo studies of altered expression patterns of p53 and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis.

Several clinical trials have revealed that individuals who were given beta-carotene and vitamin A did not have a reduced risk of cancer compared to those given placebo; rather, vitamin A could actually have caused an adverse effect in the lungs of smokers [Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S. & Hammar, S. N. Engl. J. Med (1996) 334, 1150-1155; Hennekens, C.H., Buring, J.E., Manson, J.E., Stampfer, M., Rosner, B., Cook, N.R., Belanger, C., LaMotte, F., Gaziano, J.M., Ridker, P.M., Willet, W. & Peto, R. (1996) N. Engl. J. Med. 334, 1145-1149]. Using differential display techniques, an initial survey using rats showed that liver RNA expression of c-H-Ras was decreased and p53 increased in rats with chronic vitamin A deficiency. These findings prompted us to evaluate the expression of c-Jun, p53 and p21WAF1/CIF1 (by RT-PCR) in liver and lung of rats. This study showed that c-Jun levels were lower and that p53 and p21WAF1/CIF1 levels were higher in chronic vitamin A deficiency. Vitamin A supplementation increased expression of c-Jun, while decreasing the expression of p53 and p21WAF1/CIF1. Western-blot analysis demonstrated that c-Jun and p53 showed a similar pattern to that found in the RT-PCR analyses. Binding of retinoic acid receptors (RAR) to the c-Jun promoter was decreased in chronic vitamin A deficiency when compared to control hepatocytes, but contrasting results were found with acute vitamin A supplementated cells. DNA fragmentation and cytochrome c release from mitochondria were analyzed and no changes were found. In lung, an increase in the expression of c-Jun produced a significant increase in cyclin D1 expression. These results may explain, at least in part, the conflicting results found in patients supplemented with vitamin A and illustrate that the changes are not restricted to lung. Furthermore, these results suggest that pharmacological vitamin A supplementation may increase the risk of adverse effects including the risk of oncogenesis.