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PubMed2002

Vascular effects of a soy leaves (Glycine max) extract and kaempferol glycosides in isolated rat carotid arteries.

We have recently purified genistin, and six kaempferol glycosides from a soy leaves ( Glycine max L. Merr.) butanol extract. Here we report the vascular effects of the extract and purified genistin and kaempferol glycosides on contractions induced by different constricting agonists in isolated rat carotid arteries. The butanol extract relaxed artery rings preconstricted by 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F 2 alpha (U46619) or [5 Z,9alpha,11alpha,13 E,15 S]-9,11,15-trihydroxyprosta-5,13-dienoic acid (PGF 2 alpha ) in a dose-dependent manner and this effect was independent of the presence of endothelium. The extract also inhibited the concentration-dependent contraction to U46619 with a slight reduction of the maximal response. The extract produced partial relaxation of both phenylephrine-preconstricted endothelium-intact and -denuded rings. In contrast, the extract had no effect on the contractile response to 50 mM extracellular K (+). None of the six kaempferol glycosides affected vessel tension induced by U46619. A mixture of kaempferol glycosides prepared according to their relative composition in the extract had no effect either. However, kaempferol relaxed U46619- and high K (+)-contracted rings to the same extent. Endothelium played no role in kaempferol-induced relaxation. Genistein induced concentration-dependent relaxation and this effect was attenuated in the endothelium-denuded rings. Genistin caused a smaller relaxant effect. The present results indicate that a butanol extract from soy leaves causes endothelium-independent relaxation in rat carotid artery rings. Kaempferol glycosides, accounting for approximately 48 % of the extract in weight, are not the ingredients responsible for the extract-induced relaxation. Genistein and genistin also caused relaxation, however, the dose range is beyond that of the extract causing relaxation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2002

Characterization of the acid stability of glycosidically linked neuraminic acid: use in detecting de-N-acetyl-gangliosides in human melanoma.

The glycosidic linkage of sialic acids is much more sensitive to acid hydrolysis than those of other monosaccharides in vertebrates. The commonest sialic acids in nature are neuraminic acid (Neu)-based and are typically N-acylated at the C5 position. Unsubstituted Neu is thought to occur on native gangliosides of certain tumors and cell lines, and synthetic de-N-acetyl-gangliosides have potent biological properties in vitro. However, claims for their natural existence are based upon monoclonal antibodies and pulse-chase experiments, and there have been no reports of their chemical detection. Here we report that one of these antibodies shows nonspecific cross-reactivity with a polypeptide epitope, further emphasizing the need for definitive chemical proof of unsubstituted Neu on naturally occurring gangliosides. While pursuing this, we found that alpha2-3-linked Neu on chemically de-N-acetylated G(M3) ganglioside resists acid hydrolysis under conditions where the N-acetylated form is completely labile. To ascertain the generality of this finding, we investigated the stability of glycosidically linked alpha- and beta-methyl glycosides of Neu. Using NMR spectroscopy to monitor glycosidic linkage hydrolysis, we find that only 47% of Neualpha2Me is hydrolyzed after 3 h in 10 mm HCl at 80 degrees C, whereas Neu5Acalpha2Me is 95% hydrolyzed after 20 min under the same conditions. Notably, Neubeta2Me is hydrolyzed even slower than Neualpha2Me, indicating that acid resistance is a general property of glycosidically linked Neu. Taking advantage of this, we modified classical purification techniques for de-N-acetyl-ganglioside isolation using acid to first eliminate conventional gangliosides. We also introduce a phospholipase-based approach to remove contaminating phospholipids that previously hindered efforts to study de-N-acetyl-gangliosides. The partially purified sample can then be N-propionylated, allowing acid release and mass spectrometric detection of any originally existing Neu as Neu5Pr. These advances allowed us to detect covalently bound Neu in lipid extracts of a human melanoma tumor, providing the first chemical proof for naturally occurring de-N-acetyl-gangliosides.

Acetylation↗
PubMed2004

Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A.

The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.

Catalysis↗
PubMed2005

Complexes of Thermoactinomyces vulgaris R-47 alpha-amylase 1 and pullulan model oligossacharides provide new insight into the mechanism for recognizing substrates with alpha-(1,6) glycosidic linkages.

Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of alpha-(1,4), alpha-(1,4), and alpha-(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes alpha-(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes alpha-(1,6) glycosidic linkages with a lesser efficiency. X-ray structures of three complexes comprising an inactive mutant TVAI (D356N or D356N/E396Q) and a pullulan model oligosaccharide (P2; [Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]2 or P5; [Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]5) were determined. The complex D356N/P2 is a mimic of the enzyme/product complex in the main catalytic reaction of TVAI, and a structural comparison with Aspergillus oryzaealpha-amylase showed that the (-) subsites of TVAI are responsible for recognizing both starch and pullulan. D356N/E396Q/P2 and D356N/E396Q/P5 provided models of the enzyme/substrate complex recognizing the alpha-(1,6) glycosidic linkage at the hydrolyzing site. They showed that only subsites -1 and -2 at the nonreducing end of TVAI are effective in the hydrolysis of alpha-(1,6) glycosidic linkages, leading to weak interactions between substrates and the enzyme. Domain N of TVAI is a starch-binding domain acting as an anchor in the catalytic reaction of the enzyme. In this study, additional substrates were also found to bind to domain N, suggesting that domain N also functions as a pullulan-binding domain.

Bacterial Proteins↗
PubMed2000

Medicinal foodstuffs. XVIII. Phytoestrogens from the aerial part of Petroselinum crispum MIll. (Parsley) and structures of 6"-acetylapiin and a new monoterpene glycoside, petroside.

In the course of our screening for natural estrogenic compounds from Occidental medicinal herbs, the extracts of several herbs were found to show proliferative activity in MCF-7 (an estrogen-sensitive breast cancer cell line). Among these active herbs, the methanolic extract from the aerial parts of Petroselinum crispum (parsley) showed potent estrogenic activity, which was equal to that of isoflavone glycosides from soybean. Through bioassay-guided separation, we isolated several flavone glycosides and a new flavone glycoside, 6"-acetylapiin, with estrogenic activity together with a new monoterpene glucoside, petroside. The structures of 6"-acetylapiin and petroside were characterized by the chemical and physicochemical evidence. Estrogenic activities of these flavone glycosides were found to be enhanced by removal of their glycoside moieties. The EC50 values (concentration needed to enhance the MCF-7 proliferation 50% compared to non-estrogen treated cell) of their aglycones are as follows, apigenin (1.0 microM), diosmetin (2.9 microM), and kaempferol (0.56 microM). The estrogenic activities of these flavones are nearly equal to those of the isoflavones, daidzein (0.61 microM) and genistein (0.60 microM). The methanolic extract of parsley, apiin, and apigenin restored the uterus weight in ovariectomized mice when orally administered for consecutive 7 days.

Animals↗
PubMed1984

[The determination of the recognition site of an antiserum detecting desialylation on O-glycosidic carbohydrates of hCG and its clinical applications. The relationship between the presence of asialo-hCG and malignant change in trophoblastic disease].

Human chorionic gonadotropin (hCG) is a specific tumor marker glycoprotein hormone for trophoblastic diseases. It contains 4 asparagine-linked and 4 serine-linked carbohydrate units. Recently, variations in the carbohydrate moieties of hCG in chorio-carcinoma have been suggested. However, the immunological method of detecting these malignant transformational changes of carbohydrate units in hCG have not been investigated. We therefore attempted to assess the possibility of establishing a radioimmunoassay system which can detect these transformational changes in serine-O-glycosidically linked carbohydrate units of hCG. HCG-specific hCG beta COOH-terminal portion contains all 4 O-glycosidically linked sugar chains (positions 121, 127, 132 and 138). An antiserum (R141) generated against the enzymatically cleaved, desialylated hCG beta COOH-terminal peptide (residue 123-145) by toepad immunization method was extensively characterized. This antiserum reacts with asialo-hCG better than with native hCG. It does not bind with synthetic COOH-terminal peptides nor monosaccharides such as N-acetyl-D-galactosamine and D-galactose, which are sugar components in O-glycosidic carbohydrate chains. The HF-treated asialo-hCG, in which galactose residues are further removed, still reacts with the antiserum. The antiserum requires both the peptide sequence and N-acetyl-D-galactosamine residues for its binding. Glycophorine and fetuin, which also contain the same O-glycosidic carbohydrate structure, do not react with the antiserum. Beta subunit gains its binding capability to the antiserum only upon desialylation, while native alpha and beta subunits do not react. The antiserum has high specificity toward asialo-hCG in which particularly O-glycosidic carbohydrate units are desialylated. With a specific radioimmunoassay system using this antiserum (R141), urinary hCG preparations from 29 patients with various trophoblastic diseases were analyzed. Asialo-hCG immunoactivity was insignificant in all patients with hydatidiform mole, invasive hydatidiform mole and persisted trophoblastic disease. However, asialo-hCG was found in 2 out of 6 cases of choriocarcinoma. The existence of asialo-hCG may be related to the advancement of disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Asialoglycoproteins↗
PubMed1989

A multivariate approach to saccharide quantitative structure-activity relationships exemplified by two series of 9-hydroxyellipticine glycosides.

Multivariate saccharide quantitative structure-activity relationships (QSARs) have been developed for two series of 9-hydroxyellipticine glycosides. In order to describe the structural variation of the glycoside moieties, thirteen chromatographic variables were used. Eleven D-glycosides and seven L-glycosides were used in separate QSARs to model 9-hydroxyellipticine glycoside antitumour activity against L 1210 leukemia. The multivariate partial least squares (PLS) method was used to establish the QSARs.

Animals↗
PubMed1979

Species restriction of the mitogenicity induced by lanatoside C. Lymphocyte activation by digitalis glycosides is confined to cells from digitalis resistant species.

Activation of Na+, K+-ATPase has previously been suggested to be the triggering signal in mitogen-induced cell activation. Using a digitalis glycoside known to be a potent polyclonal B-cell activator, this hypothesis could be tested since digitalis activates ATPase at different concentrations in various species, depending on the degree of sensitivity to the toxic effects of glycosides. Lanatoside C was found to stimulate lymphocytes from glycoside resistant species such as rat, mouse and hamster. The possible involvement of Na+, K+-ATPase was made less likely by the similarity in dose--response profile in these cells although they have been reported to display varying degrees of glycoside resistance. Furthermore, using lymphocytes from digitalis-sensitive species such as man, guinea-pig or rabbit, no mitogenicity could be recorded, strongly suggesting a lack of correlation between glycoside-induced effects on Na+, K+-ATPase and cell activation.

Adenosine Triphosphatases↗
PubMed1988

Differences in color discrimination between three cardioactive glycosides.

Color discrimination ability of 100 in-patients suffering from congestive heart failure and treated with digitoxin (D), pengitoxin (P), or digoxin (Dg) was determined with the Farnsworth-Munsell 100 Hue test (FM 100) and compared with the color discrimination of 72 in-patients who were not treated with digitalis glycosides (control group C). Parallel to the performance of the FM 100, the glycoside plasma level was measured by radioimmunoassay. The total error score (TES) of the FM 100 was correlated with the glycoside plasma level and the patient's age. In the C as well as in the D or P groups up to 172 errors and in the Dg group up to 586 errors were observed. With the exception of Dg, no differences were observed between the regression lines indicating an age-dependent increase in TES even under D or P treatment. In contrast to the two glycosides, Dg enhances the TES in therapeutically relevant plasma concentrations. The differences between the glycosides are due to differences in their volume of distribution and their plasma protein binding.

Acetyldigoxins↗
PubMed1977

Influence of canrenoate-K and cardiac glycosides on their tissue distribution and elimination.

The combination of cardiac glycosides and canrenoate-potassium (CR-K) produces synergistic effects on hemodynamics. On the other hand, CR-K antagonizes digitalis-induced cardiac arrhythmias. Therefore, it was the purpose of this study to determine interactions between these substances, particularly of their myocardial uptake. The additional administration of CR-K leads to significantly higher concentrations of digoxin and ouabain in heart, liver, adrenal gland and spleen. Contrary to this, additional digoxin reduces the concentration of CR-K in the tissue. Particularly obvious is the reduced concentration in the kidney, adrenal gland, pancreas, brain and spleen. The renal excretion of digoxin and ouabain is reduced by the additional administration of CR-K, while digoxin accelerates the CR-K excretion within the first 60 min after application. Metabolic interference was not detected in the combination of cardiac glycosides and CR-K. The mechanisms for the interactions between cardiac glycosides and CR-K during the distribution phase are discussed. The inhomogenous interference of their myocardial uptake makes a common cardiac receptor for the synergistic effect of cardiac glycosides and CR-K rather unlikely. CR-K does not have a suppressant effect on digitalis-induced arrhythmias due to any diminution of the glycoside uptake by myocardial tissue.

Animals↗
PubMed1996

Quantitative determination of cardiac glycosides in Digitalis lanata leaves by reversed-phase thin-layer chromatography.

An analytical method for the determination of cardiac glycosides in Digitalis lanata leaves by reversed-phase thin-layer chromatography (RP-TLC) was developed. The procedure consisted of extraction of dry leaf powder with 50% methanol and clean-up by Sep-Pak cartridges prior to RP-TLC analysis. RP-TLC was performed on an octadecylsilyl bonded silica gel plate, using a developing solvent of acetonitrile-methanol-0.5 M NaCl (1:1:1) for primary glycosides and acetonitrile-methanol-0.5 M NaCl (12:7:9) for secondary glycosides. The plate was scanned with a reflectance densitometer at 225 nm. The quantitation was carried out by the internal standard method. The present method is reliable and relatively simple for the determination of cardiac glycosides in Digitalis lanata leaves.

Cardiac Glycosides↗
PubMed2001

The role of non-Saccharomyces species in releasing glycosidic bound fraction of grape aroma components--a preliminary study.

AIMS: The purpose of the study was to evaluate the effect of beta-glycosidase activity in wine yeasts in releasing terpene glycosides from grape juice. METHODS AND RESULTS: Glycosidase activity was screened in 160 yeasts by testing their ability to hydrolyse arbutine on agar plates. Only non-Saccharomyces species exhibited beta-glycosidase activity. Enzyme activity, based on hydrolytic activity on p-nitrophenyl-beta-glycoside, was mainly located in the whole cell fraction, with smaller amounts in permeabilized cells being released into the growth medium. The hydrolysis of glycosides was determined by HRGC-MS, confirming the role of yeast in the liberation of monoterpenols, especially linalool and geraniol. CONCLUSION: The results indicate the potential of microbial beta-glycosidases for releasing flavour compounds from glycosidically-bound, non-volatile precursors, with significant implications for wines made from less aromatic grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the role of non-Saccharomyces species in enhancing wine aroma and flavour, suggesting that the future lies with controlled use of mixed cultures in winemaking.

Arbutin↗
PubMed1986

The role of intracellular Ca ions in the therapeutic and toxic effects of cardiac glycosides and catecholamines.

Many inotropic maneuvers act by increasing the intracellular calcium concentration [( Ca2+]i). The present report illustrates this with respect to the positive inotropic effects of cardiac glycosides and catecholamines. It is shown that the increased contractility produced by cardiac glycosides is accompanied by an increase in intracellular Na concentration and, furthermore, that the relationship between contraction and Na is very steep. This steep dependence, which may result from a Na-Ca exchange which exchanges several Na ions per Ca, means that maneuvers that have only small effects on Na will have significant effects on contraction. Cardiac glycosides also produce abnormal pacemaker activity and cardiac arrhythmias. These originate from a transient inward current activated by oscillations of [Ca2+]i, which result from spontaneous oscillatory release of Ca ions from the sarcoplasmic reticulum. The local anesthetic group of antiarrhythmic agents abolishes the transient inward current. Catecholamines also increase systolic [Ca2+]i and, in high enough concentrations, can produce oscillations of [Ca2+]i. This tendency of glycosides and catecholamines to produce arrhythmogenic oscillations of [Ca2+]i is a major limitation to their use. Therefore, inotropic agents that act by means other than increasing [Ca2+]i may be of great efficacy.

Animals↗
PubMed2000

Acute yellow oleander (Thevetia peruviana) poisoning: cardiac arrhythmias, electrolyte disturbances, and serum cardiac glycoside concentrations on presentation to hospital.

OBJECTIVE: To describe the cardiac arrhythmias, electrolyte disturbances, and serum cardiac glycoside levels seen in patients presenting to hospital with acute yellow oleander (Thevetia peruviana) poisoning and to compare these with published reports of digitalis poisoning. DESIGN: Case series. SETTING: Medical wards of Anuradhapura District General Hospital, Sri Lanka, and coronary care unit of the Institute of Cardiology, National Hospital of Sri Lanka, Colombo, the national tertiary referral centre for cardiology. PATIENTS: 351 patients with a history of oleander ingestion. MEASUREMENTS: ECG and blood sample analysis on admission. RESULTS: Most symptomatic patients had conduction defects affecting the sinus node, the atrioventricular (AV) node, or both. Patients showing cardiac arrhythmias that required transfer for specialised management had significantly higher mean serum cardiac glycoside and potassium but not magnesium concentrations. Although there was considerable overlap between groups, those with conduction defects affecting both sinus and AV nodes had significantly higher mean serum cardiac glycoside levels. CONCLUSIONS: Most of these young previously healthy patients had conduction defects affecting the sinus or AV nodes. Relatively few had the atrial or ventricular tachyarrhythmias or ventricular ectopic beats that are typical of digoxin poisoning. Serious yellow oleander induced arrhythmias were associated with higher serum cardiac glycoside concentrations and hyperkalaemia but not with disturbances of magnesium.

Adolescent↗
PubMed1999

Liquid chromatography/electrospray ionization mass spectrometric characterization of flavonol glycosides in tomato extracts and human plasma.

Flavonoids continue to attract wide attention as possible very useful agents for combating free radical pathologies, i.e. the pathological states associated with free radical overproduction. Commonly used methods for the analysis of plant flavonoids include high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). On the other hand, the soft-ionization approach based on electrospray ionization (ESI-MS) permits highly selective analysis of complex matrices. In this work, we examined firstly the ESI-MS behaviour of representative aglycones and glycosides of flavonols, flavones and isoflavones with the aim of suggesting a possible relationship between structure and mass spectra. Using HPLC coupled to a diode array detector (DAD) for on-line UV spectra acquisition, and in parallel to ESI-MS for mass spectra (LC/DAD-ESI-MS), we have developed methodology to observe flavonols directly in tomato puree extract. In this way, it has been possible to detect intact flavonol glycosides in tomato extracts and to characterize a flavonol trisaccharide. For the first time, using LC/ESI-MS, it has been possible to detect intact flavonol glycosides in plasma of healthy volunteers and to provide further evidence on the absorption of flavonoid glycosides after consumption of common vegetables like tomatoes.

Calibration↗
PubMed2006

From linden flower to linden honey. Part 2: Glycosidic precursors of cyclohexa-1,3-diene-1-carboxylic acids.

The presence of two unusual, recently identified terpene acids, i.e., 4-(1-hydroxy-1-methylethyl)cyclohexa-1,3-diene-1-carboxylic acid (1) and 4-(1-methylethenyl)cyclohexa-1,3-diene-1-carboxylic acid (2), was now also confirmed in (Swiss) linden honey, after solid-phase extraction and HPLC purification. NMR Spectroscopy, in combination with UPLC/MS analysis, showed the presence of several glycosides of 1, which accounted for ca. 0.6 weight-% of the honey, as quantified by UPLC-UV. The major 'glycoside' of 1, compound 5, could be isolated and identified by 2D-NMR experiments as the corresponding beta-gentiobiosyl ester (rather than the classical compound with a glycosidic bond between an aglycone OH group and the sugar). The same diglycosides found in linden honey were also detected in linden nectar; also, chestnut and fir honeys contained these glycosides in minor quantities, but not colza, acacia, or dandelion honeys (Table 2).

Carboxylic Acids↗
PubMed2005

Effect of silybin and its glycosides on the expression of cytochromes P450 1A2 and 3A4 in primary cultures of human hepatocytes.

Four beta-glycosides of flavonoligan silybin, i.e. silybin beta-galactoside, silybin beta-glucoside, silybin beta-maltoside, silybin beta-lactoside were synthesized in order to improve silybin water solubility and bioavailability (Kren et al., J Chem Soc, Perkin Trans 1, 2467-2474, 1997). The presented paper deals with the effect of silybin and its synthetic beta-glycosides on the expression of two major cytochrome P450 isoforms, CYP1A2 and CYP3A4. Primary cultures of human hepatocytes were the model of choice. mRNAs were analyzed using Northern blot and P-radiolabelled probes. CYP protein content was determined by immunoblotting using specific antibodies. Silybin and its beta-glycosides do not induce expression of CYP1A2 and CYP3A4. Tested compounds did not affect inducible expression of CYP1A2 and CYP3A4 by dioxin and rifampicin, respectively, as evaluated at the level of mRNAs and proteins. Silybin and its beta-glycosides do not interfere with the expression of CYP1A2 and CYP3A4, are not likely to produce drug-drug interactions in terms of the inducibility of two important cytochromes P450.

Aryl Hydrocarbon Hydroxylases↗
PubMed2007

Novel environmental toxins: steryl glycosides as a potential etiological factor for age-related neurodegenerative diseases.

Amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) is a unique neurodegenerative disease found on the island of Guam. This disease presents as a spectrum of neurological disorders characterized by features of ALS, parkinsonism, dementia, or a combination. The strongest epidemiological link has been to the consumption of the seeds from the cycad plant that purportedly contained a neurotoxin. Mice fed washed cycad flour show signs that mimic ALS-PDC, which include progressive deficits in motor, cognitive, and olfactory functions associated with neuron loss in the spinal cord, nigrostriatal system, cortex, hippocampus, and olfactory bulb. Through a series of chemical extractions of washed cycad flour, we identified steryl glycoside molecules as bioactive molecules that are neurotoxic in culture and in mice. A detailed review of this class of molecule revealed that the molecules are abundant in the environment, particularly in plants and bacteria. Lipid analysis showed that some bacteria that are associated with some forms of neurodegenerative disorders have the capacity to synthesize steryl glycosides. Furthermore, certain steryl glycosides have been found to be a cell stress mediator and may have some immunomodulary effects. We hypothesize that steryl glycosides are putative neurotoxins involved in the etiopathogenesis of several age-related neurodegenerative disorders.

Aging↗