Effect of calcium glucoheptonate on proliferation and osteogenesis of osteoblast-like cells in vitro.

Calcium is the key macromineral having a role in skeletal structure and function, muscle contraction, and neurotransmission. Bone remodeling is maintained through a constant balance between calcium resorption and deposition. Calcium deficiency is resolved through calcium supplementation, and among the supplements, water-soluble organic molecules attracted great pharmaceutical interest. Calcium glucoheptonate is a highly water-soluble organic calcium salt having clinical use; however, detailed investigations on its biological effects are limited. We assessed the effects of calcium glucoheptonate on cell viability and proliferation of osteoblast-like MG-63 cells. Calcium uptake and mineralization were evaluated using Alizarin red staining of osteoblast-like MG-63 cells treated with calcium glucoheptonate. Expression of osteogenic markers were monitored by western blotting, immunofluorescence, and qRT-PCR assays. Increased proliferation and calcium uptake were observed in the MG-63 cells treated with calcium glucoheptonate. The treatment also increased the expression of osteopontin and osteogenic genes such as collagen-1, secreted protein acidic and cysteine rich (SPARC), and osteocalcin. Calcium glucoheptonate treatment did not exert any cytotoxicity on colorectal and renal epithelial cells, indicating the safety of the treatment. This is the first report with evidence for its beneficial effect for pharmaceutical use in addressing calcium deficiency conditions.

Caffeic acid derivatives isolated from Galinsoga parviflora herb protected human dermal fibroblasts from UVA-radiation.

BACKGROUND: Among solar radiation, ultraviolet light is the most harmful for the skin, because of intracellular reactive oxygen species formation, leading to oxidative stress, cell damage and apoptosis. Crucial role in skin protection against oxidative stress play antioxidant enzymes regulated by Nrf2 transcription factor. Some plant-derived polyphenols are known to protect skin fibroblasts against UV through induction of Nrf2-dependent antioxidant genes expression. PURPOSE: We previously found out that water extracts from Galinsoga sp. herb protected human dermal fibroblasts against UVA-induced oxidative stress and apoptosis. However, which compounds were responsible for such protective action remained unclear. Here, we investigated photoprotective potential and mechanism of action of two main isolated compounds, 2,3,5(2,4,5)-tricaffeoylaltraric acid and 2,4(3,5)-dicaffeoylglucaric acid, on human dermal fibroblasts (NHDF). STUDY DESIGN/METHODS: NHDF cells were pretreated with tested compounds (6.25-50 µM) and irradiated with UVA (25 J/cm2). Intracellular ROS and GSH level, cell viability, cell membrane integrity and apoptosis were measured. HO-1 protein expression and Nrf2 transcription factor activation were also assessed. RESULTS: Cells pretreated with tested compounds prior to UVA showed inhibition of intracellular ROS formation and increase of GSH level. Significant increase of cell viability was also observed, as well as decrease of LDH release and a the rate of apoptotic cells in comparison to untreated cells. Furthermore, tested compounds increased HO-1 expression and activated the Nrf2 transcription factor in NHDF cells. CONCLUSION: Present study demonstrated that caffeic acid derivatives present in Galinsoga parviflora herb, in particular tricaffeoylaltraric acid may protect dermal fibroblasts against UVA-induced oxidative stress through activation of intracellular antioxidative system. Such caffeic acid derivatives are bioactive compounds which might prevent UV-induced photoageing and photocarcinogenesis.

High-Throughput "FP-Tag" Assay for the Identification of Glycosyltransferase Inhibitors.

Bacterial capsular polysaccharides are important virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing 3-deoxy-ß-d- manno-oct-2-ulosonic acid (ß-Kdo). The ß-Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of glycosyltransferases that shares little homology with other such enzymes, thereby representing an attractive antivirulence target. Here, we report the development of a fluorescence polarization-based, high-throughput screening assay (FP-tag) for ß-Kdo glycosyltransferases, and use it to identify a class of marine natural products as lead inhibitors. This "FP-tag" assay should be readily adaptable to high-throughput screens of other glycosyltransferases.

Octulosonic acid derivatives from Roman chamomile (Chamaemelum nobile) with activities against inflammation and metabolic disorder.

Six new octulosonic acid derivatives (1-6) were isolated from the flower heads of Roman chamomile (Chamaemelum nobile). Their structures were elucidated by means of spectroscopic interpretation. The biological activity of the isolated compounds was evaluated toward multiple targets related to inflammation and metabolic disorder such as NAG-1, NF-κB, iNOS, ROS, PPARα, PPARγ, and LXR. Similar to the action of NSAIDs, all the six compounds (1-6) increased NAG-1 activity 2-3-fold. They also decreased cellular oxidative stress by inhibiting ROS generation. Compounds 3, 5, and 6 activated PPARγ 1.6-2.1-fold, while PPARα was activated 1.4-fold by compounds 5 and 6 only. None of the compounds showed significant activity against iNOS or NF-κB. This is the first report of biological activity of octulosonic acid derivatives toward multiple pathways related to inflammation and metabolic disorder. The reported anti-inflammatory, hypoglycemic, antiedemic, and antioxidant activities of Roman chamomile could be partly explained as due to the presence of these constituents.

Nanoscale amphiphilic macromolecules with variable lipophilicity and stereochemistry modulate inhibition of oxidized low-density lipoprotein uptake.

Amphiphilic macromolecules (AMs) based on carbohydrate domains functionalized with poly(ethylene glycol) can inhibit the uptake of oxidized low density lipoprotein (oxLDL) and counteract foam cell formation, a key characteristic of early atherogenesis. To investigate the influence of lipophilicity and stereochemistry on the AMs' physicochemical and biological properties, mucic acid-based AMs bearing four aliphatic chains (2a) and tartaric acid-based AMs bearing two (2b and 2l) and four aliphatic chains (2g and 2k) were synthesized and evaluated. Solution aggregation studies suggested that both the number of hydrophobic arms and the length of the hydrophobic domain impact AM micelle sizes, whereas stereochemistry impacts micelle stability. 2l, the meso analogue of 2b, elicited the highest reported oxLDL uptake inhibition values (89%), highlighting the crucial effect of stereochemistry on biological properties. This study suggests that stereochemistry plays a critical role in modulating oxLDL uptake and must be considered when designing biomaterials for potential cardiovascular therapies.

Coarse grained molecular dynamics of engineered macromolecules for the inhibition of oxidized low-density lipoprotein uptake by macrophage scavenger receptors.

Atherosclerosis is a condition resulting from the accumulation of oxidized low-density lipoproteins (oxLDLs) in arterial walls. Previously developed macromolecules consisting of alkyl chains and polyethylene glycol (PEG) on a mucic acid backbone, termed nanolipoblockers (NLBs) are hypothesized to mitigate the uptake of oxLDL by macrophage scavenger receptors. In this work, we developed a coarse grained model to characterize the interactions between NLBs with a segment of human scavenger receptor A (SR-A), a key receptor domain that regulates cholesterol uptake and foam cell conversion of macrophages, and studied NLB ability to block oxLDL uptake in PBMC macrophages. We focused on four different NLB configurations with variable molecular charge, charge location, and degree of NLB micellization. Kinetic studies showed that three of the four NLBs form micelles within 300 ns and of sizes comparable to literature results. In the presence of SR-A, micelle-forming NLBs interacted with the receptor primarily in an aggregated state rather than as single unimers. The model showed that incorporation of an anionic charge near the NLB mucic acid head resulted in enhanced interaction with the proposed binding pocket of SR-A compared to uncharged NLBs. By contrast, NLBs with an anionic charge located at the PEG tail showed no interaction increase as NLB aggregates were predominately observed to interact away from the oxLDL binding site. Additionally, using two different methods to assess the number of contacts that each NLB type formed with SR-A, we found that the rank order of contacts coincided with our experimental flow cytometry results evaluating the ability of the different NLBs to block the uptake of oxLDL.

Analysis of two L-Galactono-1,4-lactone-responsive genes with complementary expression during the development of Arabidopsis thaliana.

Unraveling the role of genes annotated as protein of unknown function is of importance in progression of plant science. l-Galactono-1,4-lactone (l-GalL) is the terminal precursor for ascorbic acid (AsA) biosynthesis in Arabidopsis thaliana, and a previous study showed two DUF (domains of unknown function) 642 family genes (At1g80240 and At5g25460, designated as DGR1 and DGR2, respectively) to be sensitive to it. In this work, leaves from wild-type Arabidopsis were fed with d-glucose, l-galactose, l-GalL and AsA, and the expression level of the At1g80240 and At5g25460 genes showed a specific response to l-GalL, but not to the other supplements despite the increases of the tissue AsA contents. Analysis of promoter-ß-glucuronidase (GUS) transgenic plants showed the two genes to be complementarily expressed at the root apex and in the rest of the root excluding the apex, respectively, in both young and old seedlings, and to be expressed at the leaf primordia. The GUS activity under the control of the At5g25460 promoter was high in the cotyledon and leaf veins of young seedlings. These findings were consistent with the results of quantitative real-time PCR. Interestingly, the T-DNA insertion mutant of At5g25460 (SALK_125079) displayed shorter roots and smaller rosettes than Col-0; however, no phenotypic difference was observed between the T-DNA insertion mutant of At1g80240 and the wild type. This is the first report on the expression and functional analysis of these two DUF642 family genes, with the results revealing the contribution of DGR genes to the development of Arabidopsis.

In vivo wound healing activity of the methanolic extract and its isolated constituent, gulonic acid gamma-lactone, obtained from Grewia tiliaefolia.

Grewia tiliaefolia is a subtropical tree, its stem bark is widely used in traditional Indian medicines to heal chronic wounds, gastric ulcers, burning sensation, itching and other allergic ailments. Bioassay-directed fractionation and chromatography of the methanolic extract of G. tiliaefolia stem bark has resulted in the isolation of gulonic acid gamma-lactone. The methanolic extract and the isolated constituent were studied for their potency on three different cutaneous wound models, VIZ., excision, incision and dead space wounds in Wistar rats. In the excision wound model, healing was assessed by the rate of wound contraction and period of epithelisation. In the incision wound model, the degree of healing was analysed by determining the skin breaking strength. In the dead space wound model, the parameters used to confirm the healing process were weight of granulation tissue, its tensile strength, hydroxyproline content and histological studies. The extract as well as the constituent demonstrated wound healing activity. Topical application of gulonic acid gamma-lactone (0.2% w/w ointment) caused faster epithelialisation with 94.02% wound contraction on day 16 post-wounding, while in control animals the duration of healing was extended up to 22 days with 79.53% wound contraction. The tensile strength of the incision wound was significantly increased (561.12 +/- 5.18 g) compared to the control (327.63 +/- 6.37 g). In the dead space wound model, a significant increase in weight, tensile strength and hydroxyproline content of the granuloma tissue was observed following oral administration of gulonic acid gamma-lactone (60 mg/kg). Histology of the granuloma tissue showed increased collagenation and the absence of monocytes. The wound healing effect was compared with that of the standard skin ointment nitrofurazone. The results of this investigation provide supportive scientific evidence for the medicinal use of G. tiliaefolia for healing of cutaneous wound.

[Constituents relating to anti-oxidative and alpha-glucosidase inhibitory activities in Yacon aerial part extract].

Hot water extract of the aerial part of Yacon (Smallanthus sonchifolia, Compositae) showed potent free radical-scavenging activity and inhibitory effects on lipid peroxidation in rat brain homogenate. The most potent antioxidative activity focused on the 50% MeOH-eluted fraction on DIAION HP-20 column chromatography. The structure of the major component in the fraction was identified as 2,3,5-tricaffeoylaltraric acid (TCAA) based on spectroscopic evidence. The antioxidative activity of TCAA is superior to that of natural antioxidants such as (+/-)-catechin, alpha-tocopherol, and ellagic acid, and TCAA also showed selective maltase-inhibitory activity (IC(50) 49 microg/ml). As the hypoglycemic activity of Yacon extract was described in a previous report, the present results showing that the aerial part of Yacon has strong antioxidative activity may encourage its potential use as a food supplement to prevent type II diabetes.

Changes in intracellular and apoplastic peroxidase activity, ascorbate redox status, and root elongation induced by enhanced ascorbate content in Allium cepa L.

Onions (Allium cepa L.) treated with external ascorbic acid or with the immediate precursor of its synthesis L-galactono-gamma-lactone show a stimulated elongation rate of the roots and an increase in the number of new radicles appearing at the bulb base. Treatment with both molecules resulted in an enhanced accumulation of ascorbate and dehydroascorbate along the root axis, but the distribution of these redox forms was not uniform along the root, as detected in intracellular (symplastic) and extracellular (apoplastic) compartments. Thus, those radicular zones metabolically more active, such as the meristem and the elongation zone, accumulated the highest amount of both redox forms of ascorbate. On the other hand, ascorbate and L-galactono-gamma-lactone also stimulated cytosolic glucose-6-phosphate dehydrogenase activity and inhibited peroxidase activity as deduced from in vivo and in vitro experiments. Differences were also found when comparing apoplastic and symplastic activities. These results are compatible with the idea of an ascorbate-mediated stimulation of root growth by inhibiting cell wall stiffening and increasing root metabolism.

Long-distance transport of L-ascorbic acid in potato.

BACKGROUND: Following on from recent advances in plant AsA biosynthesis there is increasing interest in elucidating the factors contributing to the L-ascorbic acid (AsA) content of edible crops. One main objective is to establish whether in sink organs such as fruits and tubers, AsA is synthesised in situ from imported photoassimilates or synthesised in source tissues and translocated via the phloem. In the current work we test the hypothesis that long-distance transport is involved in AsA accumulation within the potato tuber, the most significant source of AsA in the European diet. RESULTS: Using the EDTA exudation technique we confirm the presence of AsA in the phloem of potato plants and demonstrate a correlation between changes in the AsA content of source leaves and that of phloem exudates. Comparison of carboxyflourescein and AgNO3 staining is suggestive of symplastic unloading of AsA in developing tubers. This hypothesis was further supported by the changes in AsA distribution during tuber development which closely resembled those of imported photoassimilates. Manipulation of leaf AsA content by supply of precursors to source leaves resulted in increased AsA content of developing tubers. CONCLUSION: Our data provide strong support to the hypothesis that long-distance transport of AsA occurs in potato. We also show that phloem AsA content and AsA accumulation in sink organs can be directly increased via manipulation of AsA content in the foliage. We are now attempting to establish the quantitative contribution of imported AsA to overall AsA accumulation in developing potato tubers via transgenic approaches.

[Comparison of the efficacy of two different iron supplements for anemia prevention in piglets]. / Vergelijkend onderzoek naar de werkzaamheid van twee verschillende ijzersupplementen voor anemiepreventie in biggen.

In a randomized, confirmatory study performed between July and October 2000 the efficacy of two iron products in preventing iron deficiency anaemia was compared. A total of 102 newborn piglets from ten litters were treated intramuscularly with 200 mg iron as iron dextran per ml, or 200 mg iron as gleptoferron per ml. For true comparison, piglets within a litter of a sow were subdivided into pairs on the basis of birth weight (one pair of the two heaviest piglets, et cetera). Within a pair, treatment with the iron supplements was randomly allocated. One group of piglets was injected at an age of 1 day (experiment 1) and the other group of piglets was injected at an age of 3 days (experiment 2). The piglets were weighed and blood samples were taken at an age of 18 days (experiment 1) or at an age of 19 days (experiment 2). Average daily weight gain and haemoglobin concentrations of both treatment groups were compared. Both products were very effective in preventing anaemia. No significant differences could be found between the two formulations. It can be concluded that iron-dextran and gleptoferron can be used with similar effect for anaemia prevention in piglets.

Novel bismuth compounds have in vitro activity against Helicobacter pylori.

The increasing antimicrobial resistance in Helicobacter pylori has led to the search for new therapeutic agents. In this in vitro study, novel compounds combining bismuth with sialic acid inhibitors were investigated for bactericidal activity using time-kill methodology. The activity of these compounds was compared to bismuth subcitrate against a type strain and a clinical isolate of H. pylori. The compounds tested showed cidal activity which was related to the bismuth component of each drug. These compounds may offer a potential advantage over current bismuth preparations with the sialic acid inhibitor moiety interfering with adhesion of H. pylori to gastric epithelium.

Effect of diet on growth and plasma ascorbic acid in chicks.

Six experiments were conducted to study the effect of diet on growth and plasma ascorbic acid in chickens. D-Glucuronolactone failed to improve growth with either a crude yeast-fish meal diet or a purified diet based on casein and gelatin. With the purified diet, D-glucuronic acid and L-gulonolactone also failed to improve growth and did not influence plasma ascorbic acid levels. Dietary ascorbic acid improved growth of chicks with a purified diet in most cases, but not with a corn-soybean diet. Meat meal and fish meal caused slight increases in plasma ascorbic acid, whereas soybean meal, safflower meal, and cottonseed meal caused greater increases when used in a purified diet. Gulonolactone oxidase activity in the kidney was not different between chicks fed the purified or the corn-soybean diets, but was reduced by 0.1% dietary ascorbic acid. The mechanism for the increase in plasma ascorbic acid with the addition of soybean meal and other plant protein sources to the diet is not known.

Effect of calcium glucarate on beta-glucuronidase activity and glucarate content of certain vegetables and fruits.

Glucarate is normally present in tissues and body fluids and is in equilibrium with D-glucaro-1,4-lactone, a natural inhibitor of beta-glucuronidase activity. Dietary calcium glucarate, a sustained-release from of glucarate, elevates the blood level of D-glucaro-1,4-lactone which suppresses blood and tissue beta-glucuronidase activity. A single dose of CaG (4.5 mmole/kg body weight) inhibited beta-glucuronidase activity in serum and liver, lung, and intestinal microsomes by 57, 44, 37, and 39%, respectively. A chronic administration of calcium glucarate (4% in diet) also decreased beta-glucuronidase activity in intestinal and liver microsomes. Maximal inhibition of beta-glucuronidase activity in serum was observed from 12 noon to 2:00 PM. In contrast, maximum inhibition of beta-glucuronidase activity in intestinal and liver microsomes occurred during mornings, although a secondary depression in intestinal microsomes also occurred around 4 PM. A 4% calcium glucarate supplemented diet also inhibited beta-glucuronidase activity by 70% and 54%, of the bacterial flora obtained from proximal (small intestine) and distal (colon) segments of intestine, respectively. Due to the potential effect of dietary glucarate on net glucuronidation and on other metabolic pathways, glucaric acid levels in various foods were determined. The glucaric acid content varied from a low of 1.12-1.73 mg/100 g for broccoli and potatoes to a high of 4.53 mg/100 g for oranges.

Ascorbic acid as a factor controlling "in vivo" its biosynthetic pathway.

The capacity of ascorbic acid biosynthesis in potato tuber tissue is closely correlated with the ascorbic acid content of the cells: the lower the endogenous content of ascorbic acid, the greater its biosynthesis. At the highest level of ascorbic acid found in the cells, the biosynthetic capacity is virtually zero. In these conditions, adding glucose (the first precursor of ascorbic acid) has no effect whatsoever, whereas adding galactono-gamma-lactone (the last precursor) induces a high rate of ascorbic acid synthesis. It is suggested that AA biosynthesis is subject to a regulatory mechanism "in vivo" which controls an initial step in the biosynthetic pathway. The last step in this pathway, catalyzed by galactone oxidase, is never blocked and, moreover, its activity is greater than that of the preceding steps.

Chemopreventative activity of dietary glucarate on azoxymethane-induced altered hepatic foci in rats.

Previous studies have shown that dietary calcium glucarate, an inhibitor of beta-glucuronidase, is a potent inhibitor of promotion of diethylnitrosamine-induced altered hepatic foci, 7,12-dimethylbenzanthracene-induced mammary tumorigenesis and benzo(a)pyrene-induced lung carcinogenesis. The present study was undertaken to test the chemopreventative activity of calcium glucarate on azoxymethane-induced hepatocarcinogenesis in female Fischer 344 rats. A series of experiments were carried out over 36 weeks to evaluate the effects of calcium glucarate on the initiation and promotion phases separately and also in combination with each other. A calcium gluconate group was included and used as a negative calcium control. Histopathologic evaluation of H&E stained liver sections of all animals in this study showed that a statistically significant inhibition of hepatocarcinogenesis only occurred when dietary calcium glucarate supplementation was provided throughout the combined initiation and promotion phases. This inhibitory effect approximately equaled the summation of that obtained when calcium glucarate was fed only during initiation phase and only during promotion phase.

Effects of the experimental chemopreventative agent, glucarate, on intestinal carcinogenesis in rats.

Dietary calcium glucarate was previously shown to protect effectively against chemically-induced mammary, lung, liver and skin carcinogenesis in rodents, whereas the negative dietary calcium control, calcium gluconate, had no effect. In the present study the chemopreventative activity of dietary calcium glucarate was evaluated in the azoxymethane intestinal carcinogenesis model using the Fischer strain rat. The protocol limited the duration of azoxymethane treatment to 3 weeks to permit the evaluation of the separate effects of glucarate on the initiation and promotion phases. Control rats, treated with azoxymethane and maintained on a low fat chow diet throughout the 32-week experiment had an intestinal adenocarcinoma incidence of 55%, with an equal incidence of 27.7% in the small and large intestines. There was no significant difference between this control group and a negative calcium control group fed 128 mmol/kg chow of calcium as calcium gluconate. In contrast to these two control groups, supplementation of the diet of azoxymethane-treated rats with 128 mmol/kg diet of calcium glucarate during both the initiation and promotion phases significantly inhibited the overall induction of adenocarcinomas in the intestine, the incidence in the entire intestine and in the small and large intestines being 11.8, 5.8 and 5.8%, respectively. When fed only during the initiation phase, the inhibition again was statistically significant, the corresponding values being 11.8%, 5.8 and 5.8%. When calcium glucarate was fed during the promotion phase, a statistically significant inhibition of adenocarcinoma induction was observed only in the colon where the incidence was 5.5%. Weight gain was similar in all groups. These and related data indicate that dietary glucarate exerts a significant inhibitory effect on azoxymethane-induced intestinal and in particular colon carcinogenesis in the rat, decreasing their incidence and size and reducing their metastic potential.

Immunopharmacological activities of 2-keto-3-deoxyoctonic acid-(alpha 2----6)-linked 4-O-phosphono-D-glucosamine derivatives carrying N- and 3-O-acyl substituents.

The immunopharmacological activities of 2-keto-3-deoxyoctonic acid (KDO)-(alpha 2----6)-linked lipid A-subunit analogs, 4-O-phosphono-D-glucosamine derivatives carrying N- and 3-O-acyl substituents, were compared with those of the corresponding analogs without KDO, GLA-27, GLA-47, and GLA-60. Among the analogs tested, GLA-60, a 4-O-phosphono-D-glucosamine carrying N-3-hydroxytetradecanoyl and 3-O-3-tetradecanoyloxytetradecanoyl groups, exhibited the most intensive activities in terms of mitogenicity, adjuvanticity, and mediator (tumor necrosis factor and colony-stimulating factor) induction. Binding (alpha 2----6) of KDO to GLA-60 failed to enhance the activities. Similarly, the activities of GLA-27 and GLA-47 were also decreased by introduction of KDO to the O-6 of the analogs. This indicates that the strengths of the activities of the subunit analogs depend on the kinds of N- and 3-O-linked acyl substituents and not on the presence of the KDO linked to the O-6.