ABCG2/BCRP transport mechanism revealed through kinetically excited targeted molecular dynamics simulations.

ABCG2/BCRP is an ABC transporter that plays an important role in tissue protection by exporting endogenous substrates and xenobiotics. ABCG2 is of major interest due to its involvement in multidrug resistance (MDR), and understanding its complex efflux mechanism is essential to preventing MDR and drug-drug interactions (DDI). ABCG2 export is characterized by two major conformational transitions between inward- and outward-facing states, the structures of which have been resolved. Yet, the entire transport cycle has not been characterized to date. Our study bridges the gap between the two extreme conformations by studying connecting pathways. We developed an innovative approach to enhance molecular dynamics simulations, 'kinetically excited targeted molecular dynamics', and successfully simulated the transitions between inward- and outward-facing states in both directions and the transport of the endogenous substrate estrone 3-sulfate. We discovered an additional pocket between the two substrate-binding cavities and found that the presence of the substrate in the first cavity is essential to couple the movements between the nucleotide-binding and transmembrane domains. Our study shed new light on the complex efflux mechanism, and we provided transition pathways that can help to identify novel substrates and inhibitors of ABCG2 and probe new drug candidates for MDR and DDI.

Juxtapositions between the somatostatinergic and growth hormone-releasing hormone (GHRH) neurons in the human hypothalamus.

Somatostatin is a 14-28 amino acid peptide that is located not only in the gastrointestinal system but also in multiple sites of the human brain. The inhibitory effect of somatostatin on the growth hormone (GH) secretion of the pituitary gland is a well-established phenomenon. There is a general consensus that somatostatin is released into the hypophysial portal blood and modulates GH secretion by hormonal action. In the present study, we explored the possibility that in addition to the hormonal route, somatostatin may also influence GH secretion via influencing the growth hormone-releasing hormone (GHRH) secretion by direct contacts that may be functional synapses. Since the verification of these putative synapses by electron microscopy is virtually impossible in humans due to the long post mortem time, in order to reveal the putative somatostatinergic-GHRH juxtapositions, light microscopic double-label immunohistochemistry was utilized. By examining the slides with high magnification, we observed that the vast majority of the GHRH perikarya received contacting somatostatinergic axonal varicosities in the arcuate nucleus. In contrast, GHRH axonal varicosities rarely contacted somatostatinergic perikarya. The morphology and the abundance of somatostatin to GHRH juxtapositions indicate that these associations are functional synapses, and they represent, at least partially, the morphological substrate of the somatostatin-influenced GHRH secretion. Thus, in addition to influencing the GH secretion directly via the hypophysial portal system, somatostatin may also modulate GH release from the anterior pituitary by regulating the hypothalamic GHRH secretion via direct contacts. The rare GHRH to somatostatin juxtapositions indicate that the negative feedback effect of GH targets the somatostatinergic system directly and not via the GHRH system.

Intimate associations between the endogenous opiate systems and the growth hormone-releasing hormone system in the human hypothalamus.

Although it is a general consensus that opioids modulate growth, the mechanism of this phenomenon is largely unknown. Since endogenous opiates use the same receptor family as morphine, these peptides may be one of the key regulators of growth in humans by impacting growth hormone (GH) secretion, either directly, or indirectly, via growth hormone-releasing hormone (GHRH) release. However, the exact mechanism of this regulation has not been elucidated yet. In the present study we identified close juxtapositions between the enkephalinergic/endorphinergic/dynorphinergic axonal varicosities and GHRH-immunoreactive (IR) perikarya in the human hypothalamus. Due to the long post mortem period electron microscopy could not be utilized to detect the presence of synapses between the enkephalinergic/endorphinergic/dynorphinergic and GHRH neurons. Therefore, we used light microscopic double-label immunocytochemistry to identify putative juxtapositions between these systems. Our findings revealed that the majority of the GHRH-IR perikarya formed intimate associations with enkephalinergic axonal varicosities in the infundibular nucleus/median eminence, while endorphinergic-GHRH juxtapositions were much less frequent. In contrast, no significant dynorphinergic-GHRH associations were detected. The density of the abutting enkephalinergic fibers on the surface of the GHRH perikarya suggests that these juxtapositions may be functional synapses and may represent the morphological substrate of the impact of enkephalin on growth. The small number of GHRH neurons innervated by the endorphin and dynorphin systems indicates significant differences between the regulatory roles of endogenous opiates on growth in humans.

Catecholaminergic system innervates galanin-immunoreactive neurons in the human diencephalon.

Galanin released into the hypophysial portal circulation in the hypothalamus may function as a hypophysiotropic factor regulating the anterior pituitary function or it may function as a neurotransmitter/neuromodulator acting at synaptic sites regulating neuronal activity of many neurons in the brain. Catecholamines (adrenaline, noradrenaline, and dopamine) primarily regulate anterior pituitary functions indirectly via innervating hypophysiotropic neurons. The aim of the present studies was to explore with double-label immunocytochemistry if, as in rodents, catecholamines interact with galanin in the human diencephalon. Due to the long post-mortem period and subsequent lack of optimal preservation of the cell membranes in the brain, electron microscopy could not be employed to show the presence of catecholaminergic-immunoreactive synapses on galanin-immunoreactive neurons. Therefore, we used light microscopic immunocytochemistry and high-magnification microscopy with oil immersion to identify putative juxtapositions between catecholamines and galanin-utilizing antisera against key enzymes of catecholamine synthesis (tyrosine hydroxylase (TH), representing all three catecholamines; dopamine-beta-hydroxylase (DBH), representing noradrenaline; and phenylethanolamine-N-methyltransferase (PNMT), representing adrenaline) and galanin. Our studies show that among the three catecholamines, dopamine is the most abundant and the vast majority of catecholaminergic contacts on galanin-immunoreactive neurons is dopaminergic. The number of DBH-immunoreactive contacts is less and the number of PNMT-immunopositive contacts is negligible. Among the hypothalamic regions, the periventricular region above the infundibulum (infundibular or arcuate nucleus) contained the largest number of contacts. These en passant-type intimate associations between catecholamine- and galanin-immunoreactive neuronal elements may be functional synapses and may provide the morphological basis for the catecholamine-mediated galanin release.

Glycosaminoglycans and neuroprotection.

Glycosaminoglycans (GAGs) are basic building blocks of the ground substance of the extracellular matrix and present at the cellular level as an important component of the glycocalyx covering the cell membrane. In addition to the general role of GAGs in maintaining the integrity of the cell and extracellular matrix by retaining water, certain GAGs exhibit anticoagulant and neuroprotective properties and serve as cell-surface receptors for various molecules. Although heparin, a highly sulfated GAG, has been used as a drug for more than 70 years due to its anticoagulant attributes, the neuroprotective properties of GAGs came into focus only in recent years. The discovery of some of the roles GAGs play in the pathomechanism of numerous neurodegenerative disorders as well as shedding light on the neuroprotective properties of these compounds in animal studies raised the possibility that GAGs may provide an entirely new avenue in the treatment of neurodegenerative diseases. Indeed, some GAGs were successfully used to improve the cognitive function of patients with various neurodegenerative conditions (Ban et al. (1991, 1992); Conti et al. (1989a, b); Passeri and Cucinotta, (1989); Santini (1989). Although the mechanism by which the GAGs exhibit neuroprotective properties is not entirely clear, there is a general consensus that the major factors of the neuroprotective attributes of GAGs include the impact of GAGs on amyloidogenesis and the regulatory action of GAGs in the apoptotic pathway.

Distribution and morphology of the catecholaminergic neural elements in the human hypothalamus.

Previous studies have demonstrated that catecholaminergic, tyrosine hydroxylase (TH)-immunoreactive (IR) perikarya and fibers are widely distributed in the human hypothalamus. Since TH is the key and rate-limiting enzyme for catecholaminergic synthesis, these IR neurons may represent dopaminergic, noradrenergic or adrenergic neural elements. However, the distribution and morphology of these neurotransmitter systems in the human hypothalamus is not entirely known. Since the different catecholaminergic systems can be detected by identifying the neurons containing the specific key enzymes of catecholaminergic synthesis, in the present study we mapped the catecholaminergic elements in the human hypothalamus using immunohistochemistry against the catecholaminergic enzymes, TH, dopamine beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT). Only a few, PNMT-IR, adrenergic neuronal elements were found mainly in the infundibulum and the periventricular zone. DBH-IR structures were more widely distributed in the human hypothalamus occupying chiefly the infundibulum/infundibular nucleus, periventricular area, supraoptic and paraventricular nuclei. Dopaminergic elements were detected by utilizing double label immunohistochemistry. First, the DBH-IR elements were visualized; then the TH-IR structures, that lack DBH, were detected with a different chromogen. In our study, we conclude that all of the catecholaminergic perikarya and the majority of the catecholaminergic fibers represent dopaminergic neurons in the human hypothalamus. Due to the extremely small number of PNMT-IR, adrenergic structures in the human hypothalamus, the DBH-IR fibers represent almost exclusively noradrenergic neuronal processes. These findings suggest that the juxtapositions between the TH-IR and numerous peptidergic systems revealed by previous reports indicate mostly dopaminergic synapses.

Intimate associations between the neuropeptide Y system and the galanin-immunoreactive neurons in the human diencephalon.

Galanin and neuropeptide Y (NPY) are among the most abundant neuropeptides in the hypothalamus. The role of NPY and galanin in the regulation of the secretory activity of the anterior pituitary has been well established. In addition, the two peptides interact with a number of neurons synthesizing the releasing and inhibiting hormones and a large number of other neuropeptides. The aim of the present studies was to explore if, as in rodents, NPY innervates galanin-immunoreactive (IR) neurons in the human diencephalon. Due to the long post mortem period and subsequent lack of optimal preservation of the cell membranes in the brain, electron microscopy could not be employed to show the presence of NPY-IR synapses on galanin-IR neurons. Therefore, we used light microscopic double label immunocytochemistry and high magnification microscopy with oil immersion to identify putative juxtapositions between NPY and galanin. Our studies show that similarly to rats, numerous NPY-IR nerve terminals surrounded galanin-IR neurons in the human hypothalamus. Among the hypothalamic regions, the infundibulum (infundibular or arcuate nucleus) contained the largest number of galanin-IR neurons heavily surrounded with NPY-IR nerve terminals. These en passant-type intimate associations between NPY-IR and galanin-IR neuronal elements may be functional synapses and may provide the morphological basis for the NPY-mediated galanin release. Consequently, NPY-galanin communication may mediate effects of NPY on neuronal systems innervated by galanin, and therefore may play a pivotal role in the regulation of reproduction, growth, energy and metabolism.

Catecholaminergic input to the oxytocin neurosecretory system in the human hypothalamus.

Previous studies revealed that oxytocin release is increased by various forms of stress. Hypertonic saline injection, immobilization, and several other stressors elevated the blood level of oxytocin in rats. However, the mechanism of the stress-induced oxytocin release in human is not elucidated yet. Although numerous studies indicate that catecholamines play a pivotal role in modulating the release of oxytocin, there is a lack of data regarding the morphological substrate of this phenomenon. In order to reveal putative juxtapositions between tyrosine hydroxylase-immunoreactive (TH-IR) catecholaminergic and the oxytocinergic systems in the human hypothalamus, we utilized double-label immunohistochemistry in the present study. Numerous TH-IR axon varicosities abutted on oxytocin-IR neurons in the supraoptic and paraventricular nuclei, forming synapse-like associations. Close examination of these juxtapositions with high magnification failed to reveal any gaps between the contacting elements. In summary, the intimate associations between the TH-IR and oxytocin-IR elements may be functional synapses and may represent the morphological substrate of stress-influenced oxytocin release. The finding that several oxytocin-IR perikarya did not receive apparent TH innervation suggests that additional mechanisms may play significant roles in the oxytocin modulation by stressors.

Associations between the human growth hormone-releasing hormone- and neuropeptide-Y-immunoreactive systems in the human diencephalon: a possible morphological substrate of the impact of stress on growth.

Previous studies revealed that stress is a pivotal factor in the regulation of growth. Psychological harassment may result in psychosocial dwarfism with delayed puberty, short stature and depression. Growth hormone (GH) secretion is suppressed by stress, possibly via the attenuation of growth hormone-releasing hormone (GHRH) secretion. However, the morphological substrate of this phenomenon has not been elucidated yet. Since neuropeptide Y (NPY) levels in the plasma is increased by administration of various stressors, the common consensus is that NPY plays a crucial role in the stress response. In the present study, we examined the putative juxtapositions between the NPY- and GHRH-immunoreactive (IR) systems in the human hypothalamus using double-label immunohistochemistry. Our findings revealed that the majority of the GHRH-IR perikarya formed intimate associations with NPY-IR fiber varicosities. The majority of these juxtapositions were found in the infundibular nucleus/median eminence where NPY-IR fiber varicosities often covered a significant surface area of the GHRH neurons. Since the juxtapositions between the GHRH-IR perikarya and NPY-IR fiber varicosities may be functional synapses, they may represent the morphological substrate of stress-suppressed GH secretion. The large number of contacting elements indicates that NPY plays a pivotal role in GH release, and may be considered as a major factor in the attenuation of growth by stress in humans.

Neuroprotective properties of glycosaminoglycans: potential treatment for neurodegenerative disorders.

Previous studies suggest that proteoglycans and glycosaminoglycans (GAGs) may play an important role in the pathogenesis and/or alleviation of neurodegenerative disorders, including Alzheimer's disease (AD). Proteoglycans increase the formation of neurofibrillary tangles, and stimulate the aggregation of beta-amyloid (Abeta). This effect, on the other hand, is believed to be competitively inhibited by certain GAGs. Over the past few years, we have examined the neuroprotective properties of Neuroparin (C3), a low-molecular-weight GAG (approx. 2.1 kDa), in animal models of lesions characteristic of AD. Neuroparin is composed of 4-10 oligosaccharides, and it is derived from heparin involving depolymerization of heparin by gamma irradiation. In our experiments, Neuroparin protected against cholinergic lesions induced by intracerebroventricular injection of a specific cholinotoxin, AF64A, in rats. Administration of Neuroparin attenuated AF64A-stimulated, low-affinity nerve growth factor receptor-immunoreactive axonal varicosities in the rat septum, and increased arborization of hippocampal CA1 neurons. Neuroparin also reduced the septal caspase 3 immunoreactivity induced by AF64A treatment. Moreover, Neuroparin reduced tau 2 immunoreactivity in the rat hippocampus, stimulated by intra-amygdaloid injection of Abeta(25-35). These findings are in good agreement with our previous data indicating a neuroprotective role of GAGs. These results, plus others, all suggest that Neuroparin may possess neuroprotective properties against many of the characteristic neural lesions in AD. Since our pharmacokinetic studies revealed that Neuroparin is capable of crossing the blood-brain barrier, Neuroparin may, conceivably, open an entirely new avenue in the treatment of neurodegenerative disorders. Phase I studies have been completed, and have proven to be extremely supportive in that regard.

Neutron detection on the Foton-M2 satellite by a track etch detector stack.

In the frame of a European Space Agency (ESA) project called 'Biology and Physics in Space', a returning satellite, Foton-M2, was orbiting a container, the BIOPAN-5, loaded with biological experiments and facilities for radiation dosimetry (RADO) in the open space. One of the RADO experiments was dedicated to the detection of the primary cosmic rays and secondary neutrons by a track etch detector stack. The system was calibrated at high-energy particle accelerators and neutron generators. The developed detectors were investigated by an image analyser, and from the track parameters the linear energy transfer spectra and the absorbed dose were determined (26 microGy/d). Also, the neutron flux was estimated below 5 MeV and found to be 2.4 cm(-2) s(-1) directly from the space. The construction of the stack allowed to investigate the neutrons also from the direction of the carrying satellite, where the flux was found somewhat higher.

Vascular anomalies in a case of situs inversus.

Situs inversus is a developmental condition in which the thoracic and abdominal organs fail to negotiate their normal migration patterns and the result is a mirror-image arrangement of these viscera. The literature provides evidence that individuals with this condition have a higher incidence of other congenital malformations (e.g. heart anomalies). Here we describe the dissection of a 71 year-old female cadaver with situs inversus, in which we discovered multiple anomalous vessels associated with the coeliac trunk directed toward the liver. In addition, we identified the inferior vena cava on the left side and a persistent supracardinal vein on the right, constituting a double inferior vena cava. Finally, we identified multiple abnormal venous channels associated with the sub-renal inferior vena cava. These vascular patterns are indeed a rare finding and have surgical implications but may indicate a higher incidence of vascular anomalies in cases of situs inversus.

Morphological substrate of the catecholaminergic input of the vasopressin neuronal system in humans.

It has been postulated that the stress response is associated with water balance via regulating vasopressin release. Nausea, surgical stress and insulin-induced hypoglycaemia were shown to stimulate vasopressin secretion in humans. Increased vasopressin release in turn induces water resorption through the kidneys. Although the mechanism of the stress-mediated vasopressin release is not entirely understood, it is generally accepted that catecholamines play a crucial role in influencing water balance by modulating the secretion of vasopressin. However, the morphological substrate of this modulation has not yet been established. The present study utilised double-label immunohistochemistry to reveal putative juxtapositions between tyrosine hydroxylase (TH)-immunoreactive (IR) catecholaminergic system and the vasopressin systems in the human hypothalamus. In the paraventricular and supraoptic nuclei, numerous vasopressin-IR neurones received TH-IR axon varicosities. Analysis of these juxtapositions with high magnification combined with oil immersion did not reveal any gaps between the contacted elements. In conclusion, the intimate associations between the TH-IR and vasopressin-IR elements may be functional synapses and may represent the morphological basis of vasopressin release modulated by stressors. Because certain vasopressin-IR perikarya receive no detectable TH innervations, it is possible that additional mechanisms may participate in the stress-influenced vasopressin release.

Three-dimensional representation of the neurotransmitter systems of the human hypothalamus: inputs of the gonadotrophin hormone-releasing hormone neuronal system.

The gonadotrophin-releasing hormone (GnRH) represents the final common pathway of a neuronal network that integrates multiple external and internal factors to control fertility. Among the many inputs GnRH neurones receive, oestrogens play the most important role. In females, oestrogen, in addition to the negative feedback, also exhibits a positive feedback influence upon the activity and output of GnRH neurones to generate the preovulatory luteinising hormone surge and ovulation. Until recently, the belief has been that the GnRH neurones do not contain oestrogen receptors and that the action of oestrogen upon GnRH neurones is indirect, involving several, oestrogen-sensitive neurotransmitter and neuromodulator systems that trans-synaptically regulate the activity of the GnRH neurones. Although this concept still holds for humans, recent studies indicate that oestrogen receptor-beta is expressed in GnRH neurones of the rat. This review provides three dimensional stereoscopic images of GnRH-immunoreactive (IR) and some peptidergic (neuropeptide Y-, substance P-, beta-endorphin-, leu-enkaphalin-, corticotrophin hormone-releasing- and galanin-IR) and catecholaminergic neurones and the communication of these potential oestrogen-sensitive neuronal systems with GnRH neurones in the human hypothalamus. Because the post-mortem human tissue does not allow the electron microscopic identification of synapses on GnRH neurones, the data presented here are based on light microscopic immunocytochemical experiments using high magnification with oil immersion, semithin sections or confocal microscopy.

Low intracerebroventricular doses of cholinotoxin AF64A do not affect the morphology of gonadotropin hormone-releasing hormone (GnRH)-immunoreactive fibers in the rat septum.

Ethylcholine aziridinium (AF64A) induces cholinergic lesion in animal models of AD. Although higher concentrations of AF64A are known to induce nonspecific, cholinergic, and non-cholinergic lesions, low concentrations are believed to be selectively cholinotoxic. However, morphological evidence of this phenomenon has not been demonstrated yet. The present study demonstrates that while AF64A damaged septal cholinergic fibers, periventricular GnRH-immunoreactive fibers remained intact, confirming the highly selective cholinotoxicity of AF64A at appropriate concentrations.

Bi-directional associations between galanin and luteinizing hormone-releasing hormone neuronal systems in the human diencephalon.

Evidence suggests that galanin plays an important role in the regulation of reproduction in the rat. Galanin is colocalized with luteinizing hormone (LH)-releasing hormone (LHRH) in a subset of LHRH neurons in female rats and galanin-immunoreactive (galanin-IR) nerve terminals innervate LHRH neurons. Recent studies indicate that galanin may control gonadal functions in rats at two different levels: (i) via direct modulation of pituitary LH secretion and/or (ii) indirectly via the regulation of the hypothalamic LHRH release. However, the morphological substrate of any similar modulation is not known in human. In the present series of experiments we first mapped the galanin-IR and LHRH-IR neural elements in human brain, utilizing single label immunohistochemistry. Then, following the superimposition of the maps of these systems, the overlapping sites were identified with double labeling immunocytochemistry and examined in order to verify the putative juxtapositions between galanin-IR and LHRH-IR structures. LHRH and galanin immunoreactivity were detected mainly in the medial basal hypothalamus, in the medial preoptic area and along the diagonal band of Broca. Careful examination of the IR elements in the overlapping areas revealed close, bi-directional contacts between galanin-IR and LHRH-IR structures, which have been verified in semithin plastic sections. These galanin-LHRH and LHRH-galanin juxtapositions were most numerous in the medial preoptic area and in the infundibulum/median eminence of the human diencephalon. In conclusion, the present study is the first to reveal bi-directional juxtapositions between galanin- and LHRH-IR neural elements in the human diencephalon. These galanin-LHRH and LHRH-galanin contacts may be functional synapses, and they may be the morphological substrate of the galanin-controlled gonadal functions in humans.

Glycosaminoglycan C3 protects against AF64A-induced cholinotoxicity in a dose-dependent and time-dependent manner.

Several studies revealed that proteoglycans (PGs) and glycosaminoglycans (GAGs) play a pivotal role in the pathogenesis of Alzheimer's disease (AD). PGs have affinity to amyloid beta (Abeta) and protect it against proteolysis, and the consequent aggregation is the cause of neurotoxicity. This effect is believed to be attenuated by GAGs. Moreover, a low-molecular-weight GAG C3 derived from unfractionated heparin has been reported to protect against Abeta-induced tau-2 immunoreactivity and cholinergic damage induced by a cholinotoxin, AF64A, in rat. However, the optimal dose and the timeframe of administration of C3 are still unknown. In our studies, we revealed the concentration-dependent and time-dependent effects of C3 on AF64A-induced cholinergic lesion in rat. C3 was administered orally in 5, 10, and 25 mg/kg/day concentration, 7 days before and/or 7 days after intracerebroventricular (i.c.v.) AF64A administration. Our results have shown that 25 mg/kg/day C3 effectively protects against AF64A-generated cholinotoxicity if administered both 7 days before and 7 days after the AF64A injection. In contrast to these findings, administration of 5 or 10 mg/kg/day C3 or 25 mg/kg/day C3, given 7 days before or 7 days after stereotaxic AF64A injection, did not show cholinoprotective effects. In conclusion, the time-dependent effects of C3 on AF64A-induced cholinergic lesion suggest that C3 may act via the processes of both neuroprotection and neurorepair. Moreover, the effects of C3 depend largely on the administered dose of this low-molecular-weight GAG. The present findings also indicate that C3, administered in the effective concentration and timeframe, may play a pivotal role in the treatment of AD.

Close anatomical associations between beta-endorphin and luteinizing hormone-releasing hormone neuronal systems in the human diencephalon.

Endogenous opiates, such as beta-endorphin, inhibit the release of luteinizing hormone (LH) release in the pituitary gland of several species including rat, pig, sheep, and human. Although it is generally believed that beta-endorphin influences gonadal functions via the regulation of hypothalamic LH-releasing hormone (LHRH) release, the morphological substrate underlying this regulation in humans remains elusive. In the present series of experiments the beta-endorphin-immunoreactive (IR) and LHRH-IR neural elements, utilizing single label immunohistochemistry, were mapped. Following the superimposition of the maps of these systems, the overlapping sites were identified and examined in order to verify the putative juxtapositions between the beta-endorphin-IR and LHRH-IR structures. LHRH-IR elements were detected mainly in the medial basal hypothalamus, in the medial preoptic area and along the diagonal band of Broca. Beta-endorphin-IR perikarya were observed in the infundibular region/median eminence, whereas beta-endorphin-IR axon varicosities were detected periventricularly in the preoptic and tuberal regions, in the medial basal hypothalamus and around the mamillary bodies. Careful examination of the immunoreactive elements in the overlapping areas revealed close contacts between beta-endorphin-IR and LHRH-IR structures, which have been verified in semithin plastic sections. These putative beta-endorphin-LHRH juxtapositions were most numerous in the medial preoptic area and in the infundibulum/median eminence of the human diencephalon. In conclusion, the present paper is the first study that revealed close juxtapositions between the beta-endorphin-IR and LHRH-IR neural elements in the human diencephalon. These beta-endorphin-LHRH contacts may be functional synapses, and they may be the morphological substrate of the beta-endorphin control on gonadal functions in man.

Protective effect of the heparin-derived oligosaccharide C3, on AF64A-induced cholinergic lesion in rats.

Alzheimer's disease (AD) literature indicates that glycosaminoglycans (GAGs) may prevent proteoglycan-induced amyloid-beta (Abeta) aggregation, decrease Abeta-induced tau-2 immunoreactivity, and increase the axonal growth and arborization of hippocampal neurons. However, there is no information about the impact of GAGs on cholinergic lesions. Here, AF64A was administered stereotaxically into the lateral ventricles of rats, at doses that are selective for cholinotoxicity (1 and 2 nmol). The heparin-derived oligosaccharide (HDO), C3 (25mg/kg), was administered orally, once daily for 7 days before, and 7 days after AF64A administration. Choline acetyltransferase (ChAT) immunohistochemistry revealed that C3 administration reduced AF64A-induced cholinergic damage in the septum and cingulum bundle. Quantitative neuronal cell counts showed that C3 attenuated, by 60%, the decrease in cell number in the medial septum. Enzyme analysis showed that C3 also significantly restored ChAT (30%) and acetylcholinesterase (AChE) enzyme activity (45%), which had been diminished by AF64A. Our data suggest that, in addition to its effects of anti-Abeta aggregation, anti-Abeta-induced tau-2 immunoreactivity, and neurotrophic effects, C3 also effectively reduces AF64A-induced cholinergic damage; hence it may have potential therapeutic value in AD patients.

Oral and subcutaneous administration of the glycosaminoglycan C3 attenuates Abeta(25-35)-induced abnormal tau protein immunoreactivity in rat brain.

High molecular weight glycosaminoglycans (GAG) and proteoglycans (PG) affect pathological changes of the brain in Alzheimer's disease (AD). PG stimulate the processing and aggregation of amyloid-beta (Abeta), protect the protein from proteolysis, and increase the formation of neurofibrillary tangles by inducing the hyperphosphorylation of tau protein. These effects may be competitively inhibited by GAG. We have studied the effects of orally (by gavage) and subcutaneously (s.c.) administered low molecular weight heparin, C3 (4-10 oligosaccharides; MW = 2.1 kDa; USP value = 12 U/mg), on abnormal tau-2 protein immunoreactivity in the rat hippocampus following a single, unilateral intra-amygdaloid administration of Abeta(25-35). Oral administration of C3 (25 mg/kg; once daily) was initiated 3 days prior to Abeta(25-35) administration, and was continued daily for an additional 14 days. S.c. administration of C3 (2.5 mg/kg, twice daily), was started 3 days prior to, and was continued for 32 days after, Abeta(25-35) administration. Animal brains were subsequently processed for tau-2, ChAT-immunoreactivity, choline acetyltransferase (ChAT) activity and acetylcholinesterase (AChE) activity. Both oral and s.c. administration of C3 attenuated Abeta(25-35) induced appearance of tau-2-immunoreactive (IR) perikarya in the ipsilateral hippocampus (P < 0.05). Hippocampal cholinergic enzyme activity in C3 treated animals was not significantly different from control animals. The present findings suggest that C3 might be used successfully to prevent abnormal tau protein formation in chronic neurologic diseases, such as AD. Moreover, our data demonstrate that the mechanism of this effect does not appear to influence the cholinergic system of the brain.