The Effects of Six-Gram D-Aspartic Acid Supplementation on the Testosterone, Cortisol, and Hematological Responses of Male Boxers Subjected to 11 Days of Nocturnal Exposure to Normobaric Hypoxia.

The aim of this study was to evaluate the effects of D-aspartic acid (DAA) supplementation during a simulated altitude protocol on the hormonal and hematological responses in athletes. We hypothesized that DAA supplementation would contribute to an increase in the luteinizing hormone (LH), free, and testosterone and a greater increase in hematological variables. Sixteen male boxers participated; they were randomly assigned to an experimental group (DAA) or a control group (C) and underwent 14 days of supplementation, 6 g/day of DAA. Both DAA and C participants were exposed to normobaric hypoxia (FiO2 = 15.5%; 2500 m) for 10-12 h a day over a period of 11 days. The results showed that DAA had no significant effect on resting, LH, or the testosterone/cortisol ratio during the training camp. Hypoxic exposure significantly (p < 0.05) increased red blood cell and reticulocyte counts as well as hemoglobin and hematocrit concentrations in both groups, but DAA had no significant effect on these changes. In conclusion, we found that DAA supplementation at a dose of 6 g/day for 14 days does not affect the testosterone, cortisol, or hematological responses of athletes during.

Molecular clocks in ancient proteins: Do they reflect the age at death even after millennia?

Age at death estimation in cases of human skeletal finds is an important task in forensic medicine as well as in anthropology. In forensic medicine, methods based on "molecular clocks" in dental tissues and bone play an increasing role. The question, whether these methods are applicable also in cases with post-depositional intervals far beyond the forensically relevant period, was investigated for two "protein clocks", the accumulation of D-aspartic acid (D-Asp) and the accumulation of pentosidine (Pen) in dentine. Eight teeth of skeletons from different burial sites in Austria and with post-depositional intervals between c. 1216 and c. 8775 years were analysed. The results of age at death estimation based on D-Asp and Pen in dentine were compared to that derived from a classical morphological examination. Age at death estimation based on D-Asp resulted consistently in false high values. This finding can be explained by a post-mortem accumulation of D-Asp that may be enhanced by protein degradation. In contrast, the Pen-based age estimates fitted well with the morphological age diagnoses. The described effect of post-mortem protein degradation is negligible in forensically relevant time horizons, but not for post-depositional intervals of thousands of years. That means that the "D-Asp clock" loses its functionality with increasing post-depositional intervals, whereas Pen seems to be very stable. The "Pen-clock" may have the potential to become an interesting supplement to the existing repertoire of methods even in cases with extremely long post-depositional intervals. Further investigations have to test this hypothesis.

New insights on the influence of free d-aspartate metabolism in the mammalian brain during prenatal and postnatal life.

Free d-aspartate is abundant in the mammalian embryonic brain. However, following the postnatal onset of the catabolic d-aspartate oxidase (DDO) activity, cerebral d-aspartate levels drastically decrease, remaining constantly low throughout life. d-Aspartate stimulates both glutamatergic NMDA receptors (NMDARs) and metabotropic Glu5 receptors. In rodents, short-term d-aspartate exposure increases spine density and synaptic plasticity, and improves cognition. Conversely, persistently high d-Asp levels produce NMDAR-dependent neurotoxic effects, leading to precocious neuroinflammation and cell death. These pieces of evidence highlight the dichotomous impact of d-aspartate signaling on NMDAR-dependent processes and, in turn, unveil a neuroprotective role for DDO in preventing the detrimental effects of excessive d-aspartate stimulation during aging. Here, we will focus on the in vivo influence of altered d-aspartate metabolism on the modulation of glutamatergic functions and its involvement in translational studies. Finally, preliminary data on the role of embryonic d-aspartate in the mouse brain will also be reviewed.

Short-Term d-Aspartic Acid Supplementation Does Not Affect Serum Biomarkers Associated With the Hypothalamic-Pituitary-Gonadal Axis in Male Climbers.

D-aspartic acid (DAA) is promoted as a testosterone (T) enhancing supplement by mechanisms involving the hypothalamic-pituitary-gonadal (HPG) axis. Here, we investigated the short-term effects of DAA on serum biomarkers of the HPG-axis in male climbers. Using a single-blinded, placebo-controlled design, 16 climbers were randomly assigned to either a DAA (3 g/day) or placebo (3 g/day) supplement for 2 weeks. The reverse treatment commenced after a 2-week washout, with all conditions administered in a balanced manner. The subjects maintained their normal weekly training across this study. Serum samples taken before and after each treatment were analyzed for T, luteinizing hormone, sex hormone binding globulin, and cortisol (C), and free T was calculated (cFT). The DAA supplement did not significantly affect serum T, cFT, and luteinizing hormone levels. Only a main effect of time on sex hormone binding globulin (6.8% increase) and C (13.6% decrease) emerged (p < .03). Significant negative associations were identified between pretest values and changes (%) in T, cFT, luteinizing hormone, and C levels with DAA and/or placebo, but these relationships did not differ between treatments (p > .46). Additional measures of physical function and serum hematology also failed to respond to DAA. In summary, a daily dose of DAA during a short training period did not influence T and selected indicators of the HPG-axis in male climbers. Other parameters linked to athletic performance and health status were also unaffected. Our findings support evidence showing that DAA (including DAA-blended supplements) at either recommended or higher dosages does not afford any ergogenic benefits for athletic males.

Therapeutic effects of D-aspartate in a mouse model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. EAE is mainly mediated by adaptive and innate immune responses that leads to an inflammatory demyelization and axonal damage. The aim of the present research was to examine the therapeutic efficacy of D-aspartic acid (D-Asp) on a mouse EAE model. EAE induction was performed in female C57BL/6 mice by myelin 40 oligodendrocyte glycoprotein (35-55) in a complete Freund's adjuvant emulsion, and D-Asp was used to test its efficiency in the reduction of EAE. During the course of study, clinical evaluation was assessed, and on Day 21, post-immunization blood samples were taken from the heart of mice for the evaluation of interleukin 6 and other chemical molecules. The mice were sacrificed, and their brain and cerebellum were removed for histological analysis. Our findings indicated that D-Asp had beneficial effects on EAE by attenuation in the severity and delay in the onset of the disease. Histological analysis showed that treatment with D-Asp can reduce inflammation. Moreover, in D-Asp-treated mice, the serum level of interleukin 6 was significantly lower than that in control animals, whereas the total antioxidant capacity was significantly higher. The data indicates that D-Asp possess neuroprotective property to prevent the onset of the multiple sclerosis.

The effects of d-aspartic acid supplementation in resistance-trained men over a three month training period: A randomised controlled trial.

CONTEXT: Research on d-aspartic acid (DAA) has demonstrated increases in total testosterone levels in untrained men, however research in resistance-trained men demonstrated no changes, and reductions in testosterone levels. The long-term consequences of DAA in a resistance trained population are currently unknown. OBJECTIVE: To evaluate the effectiveness of DAA to alter basal testosterone levels over 3 months of resistance training in resistance-trained men. DESIGN: Randomised, double-blind, placebo controlled trial in healthy resistance-trained men, aged 18-36, had been performing regular resistance training exercise for at least 3 d.w-1 for the previous 2 years. Randomised participants were 22 men (d-aspartic acid n = 11; placebo n = 11) (age, 23.8±4.9 y, training age, 3.2±1.5 y). INTERVENTION: D-aspartic acid (6 g.d-1, DAA) versus equal-weight, visually-matched placebo (PLA). All participants performed 12 weeks of supervised, periodised resistance training (4 d.w-1), with a program focusing on all muscle groups. MEASURES: Basal hormones, total testosterone (TT), free testosterone (FT), estradiol (E2), sex-hormone-binding globulin (SHBG) and albumin (ALB); isometric strength; calf muscle cross-sectional area (CSA); calf muscle thickness; quadriceps muscle CSA; quadriceps muscle thickness; evoked V-wave and H-reflexes, were assessed at weeks zero (T1), after six weeks (T2) and after 12 weeks (T3). RESULTS: No change in basal TT or FT were observed after the intervention. DAA supplementation (n = 10) led to a 16%, 95% CI [-27%, -5%] reduction in E2 from T1-T3 (p<0.01). The placebo group (n = 9) demonstrated improvements in spinal responsiveness (gastrocnemius) at the level of the alpha motoneuron. Both groups exhibited increases in isometric strength of the plantar flexors by 17%, 95% CI [7%, 28%] (p<0.05) as well as similar increases in hypertrophy in the quadriceps and calf muscles. CONCLUSIONS: The results of this paper indicate that DAA supplementation is ineffective at changing testosterone levels, or positively affecting training outcomes. Reductions in estradiol and the blunting of peripheral excitability appear unrelated to improvements from resistance training. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12617000041358.

Supplementation of sperm media with zinc, D-aspartate and co-enzyme Q10 protects bull sperm against exogenous oxidative stress and improves their ability to support embryo development.

High levels of reactive oxygen species in the semen of infertile patients or spontaneously generated during in vitro sperm handling may impair sperm quality, fertilization and embryo developmental competence. We recently reported that zinc, d-aspartate and co-enzyme Q10, contained in the dietary supplement Genadis® (Merck Serono), have protective effects on human and bull sperm motility, lipid peroxidation and DNA fragmentation in vitro; furthermore, in bovine, treated spermatozoa had an improved ability to support embryo development. However, only a few studies have investigated the protective role of antioxidants during in vitro sperm handling in the presence of an exogenous oxidative stress. Herein, to simulate such conditions in an animal model, we induced exogenous oxidative stress on spermatozoa through the xanthine-xanthine oxidase system and investigated its effects on sperm function and subsequent embryo developmental competence in the presence of zinc, d-Asp and CoQ10 protection. The main results showed that exogenous oxidative stress decreased sperm motility, increased sperm DNA fragmentation, and reduced fertilization and blastocyst rates and quality. Pre-treatment with zinc, d-aspartate and co-enzyme Q10 before exogenous oxidative stress was able to prevent these effects. Supplementation of sperm culture media with zinc, d-aspartate and co-enzyme Q10 could protect sperm from oxidative stress damage during in vitro handling in assisted reproductive technologies.

Evaluation of enantiomeric purity of magnesium-L-aspartate dihydrate.

Magnesium supplementation in form of organic magnesium salts is a very popular practice. We examined the enantiomeric purity of "Magnesium aspartate dihydrate" monographed in the European Pharmacopeia. A chiral capillary zone electrophoresis using (2-hydroxypropyl)-ß-cyclodextrin coupled to laser induced fluorescence detection and a HPLC-fluorescence method with chiral derivatization using o-phthaldialdehyde and N-acetyl-L-cysteine as an orthogonal method were developed and validated. Two batch samples of this substance and three drug products containing the salt were analyzed by means of both methods. The concentration of the D-enantiomer of aspartic acid ranged from 0.03 to 0.12%. Simulations of the synthesis revealed that the d-aspartic acid content is elevated if the dissolution of L-aspartic acid was performed at acidic pH values.

Treatment with zinc, d-aspartate, and coenzyme Q10 protects bull sperm against damage and improves their ability to support embryo development.

Reactive oxygen species (ROS) are physiologically generated during mitochondrial respiration and are involved in several signaling mechanisms. However, under pathological conditions, the concentration of ROS may exceed the antioxidant scavenging systems and subsequently lead to cell damage. High ROS levels have been proven to be detrimental to spermatozoa and furthermore compromise sperm function through lipid peroxidation, protein damage, and DNA strand breakage. Although the oral administration of antioxidants has been demonstrated to improve the semen quality in subfertile men, it is still a matter of debate if it can positively influence fertilization outcome and embryo developmental competence. Studies carried out in suitable animal models could resolve these fundamental questions. Hence, the main aims of the present study were to evaluate: (1) the effects of zinc, d-aspartate, and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on bull sperm motility and DNA fragmentation; and (2) whether treated spermatozoa have a superior competence in fertilization and in supporting the development of healthy embryos. Our data indicate that this treatment prevents the loss of sperm motility and the rise in sperm DNA fragmentation over time. Moreover, blastocyst rate was found to be significantly higher in oocytes fertilized by treated spermatozoa, and these blastocysts harbored a significantly lower percentage of apoptotic cells.

D-aspartic acid supplementation combined with 28 days of heavy resistance training has no effect on body composition, muscle strength, and serum hormones associated with the hypothalamo-pituitary-gonadal axis in resistance-trained men.

It was hypothesized that D-aspartic acid (D-ASP) supplementation would not increase endogenous testosterone levels or improve muscular performance associated with resistance training. Therefore, body composition, muscle strength, and serum hormone levels associated with the hypothalamo-pituitary-gonadal axis were studied after 28 days of resistance training and D-ASP supplementation. Resistance-trained men resistance trained 4 times/wk for 28 days while orally ingesting either 3 g of placebo or 3 g of D-ASP. Data were analyzed with 2 × 2 analysis of variance (P < .05). Before and after resistance training and supplementation, body composition and muscle strength, serum gonadal hormones, and serum D-ASP and d-aspartate oxidase (DDO) were determined. Body composition and muscle strength were significantly increased in both groups in response to resistance training (P < .05) but not different from one another (P > .05). Total and free testosterone, luteinizing hormone, gonadotropin-releasing hormone, and estradiol were unchanged with resistance training and D-ASP supplementation (P > .05). For serum D-ASP and DDO, D-ASP resulted in a slight increase compared with baseline levels (P > .05). For the D-ASP group, the levels of serum DDO were significantly increased compared with placebo (P < .05). The gonadal hormones were unaffected by 28 days of D-ASP supplementation and not associated with the observed increases in muscle strength and mass. Therefore, at the dose provided, D-ASP supplementation is ineffective in up-regulating the activity of the hypothalamo-pituitary-gonadal axis and has no anabolic or ergogenic effects in skeletal muscle.

The effects of 15 Hz trans-spinal magnetic stimulation on locomotor control in mice with chronic contusive spinal cord injury.

The effects of repetitive trans-spinal magnetic stimulation (rTSMS), combined with acrobatic exercise on functional locomotor recovery in chronic spinal-contused mice were tested. The exposure to magnetic stimulation was initiated 3 weeks after injury, when the animals entered chronic stage. The rTSMS was applied for a total of 4 weeks over a 9-week duration trial. Seventeen mice with the spinal cord contusion injured at level T13 were separated into two groups. While one group consisting of 10 animals was exposed to rTSMS (15 Hz), the other seven animals served as controls. Functional recovery measured with Basso mouse scale and horizontal ladder scale showed significantly better functional recovery in rTSMS-treated animals. The progress in recovery continued even after cessation of magnetic stimulation. In vitro experiments revealed that the release of glutamate analog, radioactive D-aspartate from the segments of the spinal cord exposed to rTSMS was significantly elevated. In conclusion, the exposure to rTSMS, applied to injured spinal cord during chronic post-surgery stage remarkably improves the functional recovery. This recovery may be correlated by magnetically induced elevation in the release of major excitatory neurotransmitter, glutamate from injured tissue.

Interaction of PGHS-2 and glutamatergic mechanisms controlling the ovine fetal hypothalamus-pituitary-adrenal axis.

Prostaglandins, generated within the fetal brain, are integral components of the mechanism controlling the fetal hypothalamus-pituitary-adrenal (HPA) axis. Previous studies in this laboratory demonstrated that prostaglandin G/H synthase isozyme 2 (PGHS-2) inhibition reduces the fetal HPA axis response to cerebral hypoperfusion, blocks the preparturient rise in fetal plasma ACTH concentration, and delays parturition. We also discovered that blockade of N-methyl-d-aspartate (NMDA) receptors reduces the fetal ACTH response to cerebral hypoperfusion. The present study was designed to test the hypothesis that PGHS-2 action and the downstream effect of HPA axis stimulation are stimulated by NMDA-mediated glutamatergic neurotransmission. Chronically catheterized late-gestation fetal sheep (n = 8) were injected with NMDA (1 mg iv). All responded with increases in fetal plasma ACTH and cortisol concentrations. Pretreatment with resveratrol (100 mg iv, n = 5), a specific inhibitor of PGHS-1, did not alter the magnitude of the HPA axis response to NMDA. Pretreatment with nimesulide (10 mg iv, n = 6), a specific inhibitor of PGHS-2, significantly reduced the HPA axis response to NMDA. To further explore this interaction, we injected NMDA in six chronically catheterized fetal sheep that were chronically infused with nimesulide (n = 6) at a rate of 1 mg/day into the lateral cerebral ventricle for 5-7 days. In this group, there was no significant ACTH response to NMDA. Finally, we tested whether the HPA axis response to prostaglandin E(2) (PGE(2)) is mediated by NMDA receptors. Seven chronically catheterized late-gestation fetal sheep were injected with 100 ng of PGE(2), which significantly increased fetal plasma ACTH and cortisol concentrations. Pretreatment with ketamine (10 mg iv), an NMDA antagonist, did not alter the ACTH or cortisol response to PGE(2). We conclude that generation of prostanoids via the action of PGHS-2 in the fetal brain augments the fetal HPA axis response to NMDA-mediated glutamatergic stimulation.

Inhibition of brown adipose tissue thermogenesis by neurons in the ventrolateral medulla and in the nucleus tractus solitarius.

Neurons in the ventrolateral medulla (VLM) and in the nucleus tractus solitarius (NTS) play important roles in the regulation of cardiovascular and other autonomic functions. In the present study, we demonstrate an inhibition of brown adipose tissue (BAT) thermogenesis evoked by activation of neurons in the VLM, as well as by neurons in the intermediate NTS, of chloralose/urethane-anesthetized, artificially ventilated rats. Activation of neurons in either rostral VLM or caudal VLM with N-methyl-d-aspartate (12 nmol) reversed the cold-evoked increase in BAT sympathetic nerve activity (SNA), BAT temperature, and end-expired CO(2). Disinhibition of neurons in either VLM or NTS with the GABA(A) receptor antagonist, bicuculline (30 pmol), reversed the increases in BAT SNA, BAT temperature, and end-expired CO(2) that were elicited 1) by cold defense; 2) during the febrile model of nanoinjection of prostaglandin E(2) into the medial preoptic area; 3) by activation of neurons in the dorsomedial hypothalamus or in the rostral raphe pallidus (rRPa); or 4) by the micro-opioid receptor agonist fentanyl. Combined, but not separate, inhibitions of neurons in the VLM and in the NTS, with the GABA(A) receptor agonist, muscimol (120 pmol/site), produced increases in BAT SNA, BAT temperature, and expired CO(2), which were reversed by nanoinjection of glycine (30 nmol) into the rRPa. These findings suggest that VLM and NTS contain neurons whose activation inhibits BAT thermogenesis, that these neurons receive GABAergic inputs that are active under these experimental conditions, and that neurons in both sites contribute to the tonic inhibition of sympathetic premotor neuronal activity in the rRPa that maintains a low level of BAT thermogenesis in normothermic conditions.

Protective effects of the pyrolyzates derived from bamboo against neuronal damage and hematoaggregation.

ETHNOPHARMACOLOGICAL RELEVANCE: Bamboo species are thought to be originally from Central China, but are now found in many temperate and semi-tropical regions around the world. Although the extracts from bamboo may have antioxidant activities and anti-inflammatory effects, their exact biological activities have not been elucidated. AIM OF THE STUDY: Two biological activities of bamboo-derived pyrolyzates were investigated; the protective effects against N-methyl-d-aspartate (NMDA)-induced cell death in primary cultured cortical neuron and the anti-plasmin effects determined by using fibrin and fibrinogen degradation products (FDPs) assay. RESULTS: Treatment of neuronal cells with pyrolyzates of Phyllostachys pubescens, Phyllostachys nigra and Phyllostachys bambusoides resulted in restored cell viability when compared to untreated cells in an NMDA-induced neuronal cell death assay. In addition, cortical neurons treated with Phyllostachys pubescens and Phyllostachys nigra showed a reduction of apoptosis following exposure to NMDA, as determined by Hoechst 33342 staining. In addition, Phyllostachys nigra pyrolyzates also exhibited anti-plasmin action in a FDP assay. It is of interest to note that pyrolyzates exhibited activities of NMDA-receptor antagonist and antifebrin (ogen), since a combination of NMDA receptor antagonists, glucocorticosteroids, GABAergic drugs and heparin are useful for treatment in delayed postischemic injury. CONCLUSION: Our results indicate that the pyrolyzates derived from bamboo may have anti-apoptotic effects, and can be useful as a supplement for ischemic injury treatment.

DL-Aspartic acid administration improves semen quality in rabbit bucks.

Recently, D-aspartic acid (d-Asp) has been suggested as being involved in mechanisms regulating reproduction activity in animals and human. In this study we analyzed the effects of DL-Asp oral administration on sperm production in the rabbit. Bucks from 60, bred in a genetic centre and used for semen production, were divided in 2 subgroups of 6 individuals. The treated group was fed with a concentrate containing DL-Asp which assured a daily administration of 1.3g dl-Asp/head; the control group was fed with the same concentrate without DL-Asp. The treatment was carried out for 2wk and animals were monitored weekly, from 1wk before the start of the treatment to 3wk after the end of the treatment. Through the experimental period there were no significant variations in semen volume between the two groups. A significant increase in both sperm concentration and kinetic parameters, i.e., the overall percentage of motile spermatozoa, the average path velocity, the percentage of progressively motile spermatozoa, etc., was found in the supplemented group. L-Asp values in blood serum and seminal plasma did not vary through the experimental period. D-Asp concentration in blood serum increased more than 4-fold than baseline (P<0.01) at the end of the treatment and was maintained at higher than baseline values for up to 3wk after the end of the treatment. D-Asp concentration in seminal plasma was higher than in blood serum before the start of the treatment (13.7+/-1.6nM vs 3.5+/-3.3nM; P<0.01) which suggests an elective storage of D-Asp in the male genital tract. Baseline values of d-Asp concentration in seminal plasma significantly increased following treatment and were back to initial values 1wk after the end of the treatment. In conclusion, DL-Asp administration improved sperm quality in bucks and the high D-Asp content in seminal plasma suggests a primary role for this D-amino acid in regulatory mechanisms of reproductive activity.

D-aspartate prevents corticostriatal long-term depression and attenuates schizophrenia-like symptoms induced by amphetamine and MK-801.

Since their discovery in the mammalian CNS, D-aspartate and D-serine have aroused a strong interest with regard to their role as putative neuromodulatory molecules. Whereas the functional role of D-serine as an endogenous coagonist of NMDA receptors (NMDARs) has been elucidated, the biological significance of D-aspartate in the brain is still mostly unclear. In the present study, we demonstrated that nonphysiological high levels of D-aspartate (1) increased in vivo NMDAR activity, (2) attenuated prepulse inhibition deficits induced by amphetamine and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate], (3) produced striatal adaptations of glutamate synapses resembling those observed after chronic haloperidol treatment, and (4) enhanced hippocampal NMDAR-dependent memory. This evidence was obtained using two different experimental strategies that produced an abnormal increase of endogenous D-aspartate levels in the mouse: a genetic approach based on the targeted deletion of the D-aspartate oxidase gene and a pharmacological approach based on oral administration of D-aspartate. This work provides in vivo evidence of a neuromodulatory role exerted by D-aspartate on NMDAR signaling and raises the intriguing hypothesis that also this D-amino acid, like D-serine, could be used as a therapeutic agent in the treatment of schizophrenia-related symptoms.

Behavioural alterations in male mice lacking the gene for D-aspartate oxidase.

D-serine and D-aspartate are important regulators of mammalian physiology. D-aspartate is found in nervous and endocrine tissue, specifically in hypothalamic supraoptic and paraventricular nuclei, pituitary, and adrenal medullary cells. Endogenous D-aspartate is selectively degraded by D-aspartate oxidase. We previously reported that adult male mice lacking the gene for D-aspartate oxidase (Ddo(-/-) mice) display elevated concentrations of D-aspartate in several neuronal and neuroendocrine tissues as well as impaired sexual performance and altered autogrooming behaviour. In the present study, we analyzed behaviours relevant to affect, cognition, and motor control in Ddo(-/-) mice. Ddo(-/-) mice display deficits in sensorimotor gating and motor coordination as well as reduced immobility in the forced swim test. Basal corticosterone concentrations are elevated. The Ddo(-/-) mice have D-aspartate immunoreactive cells in the cerebellum and adrenal glands that are not observed in the wild-type mice. However, no differences in anxiety-like behaviour are detected in open field or light-dark preference tests. Also, Ddo(-/-) mice do not differ from wild-type mice in either passive avoidance or spontaneous alternation tasks. Although many of these behavioural deficits may be due to the lack of Ddo during development, our results are consistent with the widespread distribution of D-aspartate and the hypothesis that endogenous D-aspartate serves diverse behavioural functions.

Mini-alphaB-crystallin: a functional element of alphaB-crystallin with chaperone-like activity.

Alpha-crystallin is a member of the family of small heat-shock proteins (sHSP) and is composed of two subunits, alphaA-crystallin and alphaB-crystallin, which exhibit molecular chaperone-like properties. In a previous study, we found that residues 70-88 in alphaA-crystallin can function like a molecular chaperone by preventing the aggregation and precipitation of denaturing substrate proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we show that the complementary sequence in alphaB-crystallin, residues 73-92 (DRFSVNLDVKHFSPEELKVK), is the functional chaperone site of alphaB-crystallin. Like the mini-alphaA-crystallin chaperone, the mini-alphaB-crystallin chaperone interacts with 1,1'-bi(4-anilino) naphthalene-5,5'-disulphonic acid (bis-ANS) and also possesses significant beta-sheet and random coil structure. Deletion of four residues (DRFS) from the N-terminus or deletion of C-terminus LKVK residues from the 73-92 peptide abolishes the chaperone-like activity against denaturing alcohol dehydrogenase. However, removal of DRFS or HFSPEELKVK is necessary to completely abolish the antiaggregation property of the peptide in insulin reduction assay. Substitution of Asp at a site corresponding to D80 in alphaB-crystallin with d-Asp or beta-Asp results in a significant loss of chaperone-like activity. Kynurenine modification of His in the peptide abolishes the antiaggregation property of the mini-chaperone. These data suggest that the 73-92 region in alphaB-crystallin is one of the substrate binding sites during chaperone activity.

Synthesis and evaluation of water-soluble polymeric bone-targeted drug delivery systems.

Four polymeric bone-targeting conjugates were synthesized based on poly(ethylene glycol) (PEG, two conjugates) and poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA, two conjugates). The well-known bone-targeting compounds, alendronate and aspartic acid peptide, were used as bone-targeting moieties. Fluorescein isothiocyanate (FITC) was attached to the conjugates as a model drug for detection purposes. The bone-targeting potential of these conjugates was tested in vitro with hydroxyapatite (HA) and in mice. The data obtained indicated that these novel delivery systems could specifically accumulate in the bone tissue.

Location of putative glutamatergic neurons projecting to the medial preoptic area of the rat hypothalamus.

The medial preoptic area is a key structure in the neural control of reproduction. Considerable evidence has accumulated indicating that glutamatergic innervation of the area plays an important role in this control. Sources of the glutamatergic input are unknown. Present investigations were aimed at studying this question. [3H]D-aspartate, which is selectively taken up by high-affinity uptake sites at presynaptic endings that use glutamate or aspartate as a transmitter, and is transported back to the cell body, was injected into the medial preoptic area. The neurons retrogradely labelled with [3H]D-aspartate were detected autoradiographically. Labelled cells were found in several telencephalic and diencephalic structures, but not in the brainstem. Within the telencephalon, labelled neurons were detected in the lateral septum, bed nucleus of the stria terminalis and amygdala. Diencephalic structures included the medial preoptic area itself, hypothalamic paraventricular, suprachiasmatic, ventromedial, arcuate, ventral premammillary, supramammillary and thalamic paraventricular nuclei. All of them are known to project to this area. The findings provide the first neuromorphological data on the location of putative glutamatergic neurons projecting to the medial preoptic area. Furthermore, they indicate that local putative glutamatergic neurons as well as several telencephalic and diencephalic structures contribute to the glutamatergic innervation of the area.