RhNGF slow unfolding is not due to proline isomerization: possibility of a cystine knot loop-threading mechanism.

The unfolding of recombinant human beta-NGF (NGF) in guanidine hydrochloride (GdnHCl) was found to be time dependent with the denaturation midpoint moving to lower GdnHCl concentration over time. Dissociation and extensive unfolding of the NGF dimer occurred rapidly in 5 M GdnHCl, but further unfolding of the molecule occurred over many days at 25 degrees C. Fluorescence spectroscopy, size-exclusion and reversed-phase HPLC, ultra-centrifugation, and proton NMR spectroscopy were used to ascertain that the slow unfolding step was between two denatured monomeric states of NGF (M1 and M2). Proton NMR showed the monomer formed at early times in GdnHCl (M1) had little beta-sheet structure, but retained residual structure in the tryptophan indole and high-field methyl regions of the spectrum. This residual structure was lost after prolonged incubation in GdnHCl giving a more fully unfolded monomer, M2. From kinetic unfolding experiments in 5 M GdnHCl it was determined that the conversion of M1 to M2 had an activation energy of 26.5 kcal/mol, a half-life of 23 h at 25 degrees C, and the rate of formation of M2 was dependent on the GdnHCl concentration between 5 and 7.1 M GdnHCl. These properties of the slow unfolding step are inconsistent with a proline isomerization mechanism. The rate of formation of the slow folding monomer M2 increases with truncation of five and nine amino acids from the NGF N-terminus. A model for the slow unfolding reaction is proposed where the N-terminus threads through the cystine knot to form M2, a loop-threading reaction, increasing the conformational freedom of the denatured state.     

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at 相似文献   

Characterization of the Responses of Purkinje Cells to Neurotrophin Treatment

Abstract: The ability of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron-specific enolase-immunopositive cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, ∼200%, following BDNF, NT-4/5, or NT-3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT-4/5 (46%), whereas NT-3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT-3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT-4/5, and NT-3, but not NGF, induced the rapid expression of the immediate early gene c- fos . Immunocytochemical double-labeling with antibodies to c-fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c-fos. After 4 days in vitro, both BDNF and NT-3 induced the formation of c-fos protein in calbindin-immunopositive neurons, whereas NT-4/5 did not. The latter results suggest that although BDNF and NT-4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT-4/5 are not identical.     

Immunohistochemical distribution of NGF, BDNF, NT-3, and NT-4 in adult rhesus monkey brains.

Immunohistochemical distribution and cellular localization of neurotrophins was investigated in adult monkey brains using antisera against nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Western blot analysis showed that each antibody specifically recognized appropriate bands of approximately 14.7 kDa, 14.2 kDa, 13.6 kDa, and 14.5 kDa, for NGF, BDNF, NT-3, and NT-4, respectively. These positions coincided with the molecular masses of the neurotrophins studied. Furthermore, sections exposed to primary antiserum preadsorbed with full-length NGF, BDNF, NT-3, and NT-4 exhibited no detectable immunoreactivity, demonstrating specificities of the antibodies against the tissues prepared from rhesus monkeys. The study provided a systematic report on the distribution of NGF, BDNF, NT-3, and NT-4 in the monkey brain. Varying intensity of immunostaining was observed in the somata and processes of a wide variety of neurons and glial cells in the cerebrum, cerebellum, hippocampus, and other regions of the brain. Neurons in some regions such as the cerebral cortex and the hippocampus, which stained for neurotrophins, also expressed neurotrophic factor mRNA. In some other brain regions, there was discrepancy of protein distribution and mRNA expression reported previously, indicating a retrograde or anterograde action mode of neurotrophins. Results of this study provide a morphological basis for the elucidation of the roles of NGF, BDNF, NT-3, and NT-4 in adult primate brains.     

Probing the structure-function relationship of nerve growth factor

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED₅₀ for biological activation is approximately 100-fold lower than theK _d for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (K _d=10^–9 M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF: NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.     

Human nerve growth factor: Lack of immunocrossreactivity with mouse nerve growth factor

Human β-nerve growth factor (hNGF) was purified from term human placenta. The biological potency of hNGF in the chick dorsal root ganglion assay did not differ significantly from that of mouse NGF (mNGF). Molecular weight determinations of mNGF and hMGF were also similar. No immunological crossreactivity was noted between hNGF, at a concentration of 100 μg/ml, and mNGF in a radioimmunoassay for mNGF using 6 different antisera to mNGF. hNGF shares several properties with mNGF but is immunological distinct. The results of studies in man using antisera to mNGF should be interpreted with caution.     

Decrease in the levels of NGF and BDNF in brains of mice fed a tryptophan-deficient diet

The roles of dietary tryptophan (Trp) were evaluated in regulation of production of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 in the various brain regions in ddY mice. Feeding the mice a Trp-deficient diet for 2 weeks significantly decreased in the hippocampal level of NGF but not those of BDNF and NT-3, as compared with feeding an adequate Trp diet. The mice fed excess Trp did not have different levels of any of these neurotrophins than in the mice fed an adequate Trp diet. The levels of BDNF in the cerebral cortex were also significantly lower in the mice fed on a Trp-deficient diet, while the levels of NGF and NT-3 in the region were not modulated upon feeding of the diet. The dietary Trp level had no significant effect on the levels of NGF, BDNF, or NT-3 in the entorhinal cortex nor septum of the mice. These results demonstrate that the brain levels of NGF and BDNF are dependent on the dietary content of tryptophan.     

Neurotrophin-4/5, brain-derived neurotrophic factor,and neurotrophin-3 promote survival of cultured vestibular ganglion neurons and protect them against neurotoxicity of ototoxins

The ability of neurotrophin-4/5 (NT-4/5), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) to promote survival of postnatal rat vestibular ganglion neurons (VGNs) was examined in dissociated cell cultures. Of the four neurotrophins, NT-4/5 and BDNF were equally effective but more potent than NT-3 in promoting the survival of VGNs. In contrast, NGF showed no detectable effects. As expected, TrkB-IgG (a fusion protein of extracellular domain of TrkB and Fc domain of human immunoglobulin G) specifically inhibited the survival-promoting effects by NT-4/5 or BDNF and TrkC-IgG fusion protein completely blocked that of NT-3. Immunohistochemistry with TrkB, TrkA, and p75 antisera revealed that VGNs made TrkB and p75 proteins, but not TrkA protein. Ototoxic therapeutic drugs such as cisplatin and gentamicin often induce degeneration of hair cells and ganglion neurons in both auditory and vestibular systems that leads to impairment of hearing and balance. When cisplatin and gentamicin were added to the dissociated VGN culture in which the hair cells were absent, additional cell death of VGNs was induced, suggesting that the two ototoxins may have a direct neurotoxic effect on ganglion neurons in addition to their known toxicity on hair cells. However, if the cultures were co-treated with neurotrophins, NT-4/5, BDNF, and NT-3, but not NGF, prevented or reduced the neurotoxicity of the two ototoxins. Thus, the three neurotrophins are survival factors for VGNs and are implicated in the therapeutic prevention of VGN loss caused by injury and ototoxins. © 1995 John Wiley & Sons, Inc.     

Neurotrophin-4/5 (NT-4/5) increases adult rat retinal ganglion cell survival and neurite outgrowth in vitro

Retinal ganglion cell (RGC) survival and neurite outgrowth were investigated in retinal explants from adult rats. Neutrotrophin-4/5 (NT-4/5) caused dose-dependent increases in neurite outgrowth with one-half maximal effects at approximately 0.5 ng/ml and maximal effects at 5 ng/ml. In explants treated for 7 days, the actions of NT-4/5 were similar to those of brain-derived neurotrophic factor (BDNF); with either neurotrophin, nearly twice as many RGCs survived and there was a two- to threefold increase in the number of neurites formed by RGCs. Combinations of saturating concentrations of NT-4/5 and BDNF did not enhance these in vitro effects, implying that both neurotrophins share a common signaling pathway. In contrast, nerve growth factor (NGF), neurotrophin-3 (NT-3), or ciliary nuerotrophic factor (CNTF) appeared to exert minimal influences on RGC survival or neurite outgrowth. 1994 John Wiley & Sons, Inc.     

性别及日龄对转人神经生长因子基因小鼠唾液中蛋白分泌的影响

神经生长因子(Nerve growth factor,NGF)是一种能促进神经元发育、分化、再生的蛋白。为高效生产药效更佳的人源NGF (hNGF)药物,最近,笔者实验室构建出唾液腺特异表达hNGF的转基因小鼠,并从该转基因小鼠唾液中纯化获得具有高生物学活性的h NGF蛋白。为了选择性别和日龄最适宜的转hNGF基因小鼠用于收集纯化hNGF蛋白,文中比较了28日龄(性成熟前)雄性、雌性,63日龄(性成熟后)雄性、雌性转hNGF基因小鼠,共4组转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液鼠源NGF (mNGF)蛋白量和唾液h NGF蛋白量等指标。结果显示,63日龄的转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液mNGF蛋白量和唾液hNGF蛋白量显著高于28日龄同一性别的转hNGF基因小鼠,且63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF蛋白量显著高于同一日龄的雌性转hNGF基因小鼠;在4组小鼠中,63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF含量最高,比28日龄雌性转hNGF基因小鼠高出约46倍,最适宜用于收集唾液并从中纯化hNGF。     

Dimeric structure and conformational stability of brain-derived neurotrophic factor and neurotrophin-3.

We have examined the molecular structure of the related neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) by physical methods, including gel filtration, velocity sedimentation, sedimentation equilibrium, urea gel electrophoresis, fluorescence spectroscopy, and far-ultraviolet circular dichroism. The results of these studies indicate that at physiologically relevant concentrations both recombinant proteins exist as tightly associated dimers. The dimers are stable even in 8 M solutions of urea. In solutions of guanidine hydrochloride, BDNF and NT-3 undergo slow unfolding between 3 and 5 M concentration of denaturant. Circular dichroism spectroscopy revealed approximately 70% beta-sheet and 20% beta-turn content in the native structure of both neurotrophic factors. In this respect, BDNF and NT-3 resemble other polypeptide growth factors whose receptors are also integral protein-tyrosine kinases.     

Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae

Nerve growth factor (NGF) is a trophic agent that is essential for the development and survival of sympathetic and sensory nerves. A chemically-synthesized DNA fragment encoding human NGF (hNGF) and a cDNA encoding mouse NGF (mNGF) were engineered for expression in the yeast, Saccharomyces cerevisiae. Expression and secretion of hNGF and mNGF was attempted under the direction of the yeast PGK promoter and with various leader sequences. Among the leader sequences tested, that of the yeast -factor successfully directed secretion of both hNGF and mNGF that were correctly processed. The content of the recombinant NGF (reNGF) in the culture supernatant was estimated to be 1 g/ml. The yeast-produced reNGF was able to bind to NGF receptors in rat pheochromocytoma (PC12) cells as efficiently as the standard mNGF, and partially purified reNGF could induce neurite outgrowth of PC12 cells. Thus, we have demonstrated that biologically active human and mouse reNGF can be produced in yeast cells. Correspondence to: M. Nishizawa     

Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons

Regulation of calbindin and calretinin expression by brain-derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1-10 ng/ml) resulted in a concentration-dependent decrease in the number of calretinin-positive neurons and a concentration-dependent increase in the number of calbindin-immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up-regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin- and calbindin-containing neurons. Among other members of the neurotrophin family, neurotrophin-4 (NT-4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT-4, incubation of cortical neurons with neurotrophin-3 (NT-3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT-4, NT-3 did not affect calbindin expression. Double-labeling experiments evidenced that calretinin- and calbindin-containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT-4 exert opposite effects according to the neurochemical phenotype of the target cell.     

Intermediates in the inactivation and unfolding of dimeric arginine kinase induced by GdnHCl

Equilibrium studies of guanidine hydrochloride (GdnHCl)-induced unfolding of dimeric arginine kinase (AK) from sea cucumber have been performed by monitoring by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphthalene-8sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking. The unfolding is a multiphasic process involving at least two dimeric intermediates. The first intermediate, I1, which exists at 0-0.4 M GdnHCl, is a compact inactive dimer lacking partial global structure, while the second dimeric intermediate, I2, formed at 0.5-2.0 M GdnHCl, possesses characteristics similar to the globular folding intermediates described in the literature. The whole unfolding process can be described as follows: (1) inactivation and the appearance of the dimeric intermediate I1; (2) sudden unwinding of I1 to another dimeric intermediate, I2; (3) dissociation of dimeric intermediate I2 to monomers U. The refolding processes initiated by rapid dilution in renaturation buffers indicate that denaturation at low GdnHCl concentrations (below 0.4 M GdnHCl) is reversible and that there seems to be an energy barrier between the two intermediates (0.4-0.5 M GdnHCl), which makes it difficult for AK denatured at high GdnHCl concentrations (above 0.5 M) to reconstitute and regain its catalytic activity completely.     

Expression of neurotrophins and their receptors in peripheral lung cells of mice

Neurotrophins play an essential role in nerve systems. Recent reports indicated that neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)] have numerous effects on non-neural cells, especially on immune cells. However, whether lung cells express neurotrophins and/or their receptors (TrkA for NGF, TrkB for BDNF and NT-4/5, and TrkC for NT-3) has never been systematically investigated. We investigated constitutive expression of neurotrophin family and their Trk receptor family in alveolar macrophages and other peripheral lung cells of mice. New findings were: (1) RT-PCR for neurotrophins and their receptors detected NT-3 and NT-4/5 in alveolar macrophages, BDNF, NT-4/5, trkA, the truncated form of trkB, and trkC in lung homogenate, but no trks in alveolar macrophages, (2) immunohistochemistry for neurotrophin receptors detected TrkA in capillary cells, the truncated form of TrkB, and TrkC in interstitial macrophages, (3) immunoelectron microscopy for TrkC revealed expression of TrkC on the surface of interstitial macrophages, and (4) in situ hybridization for neurotrophins detected BDNF in interstitial macrophages and alveolar type I cells, NT-3 in alveolar macrophages, and NT-4/5 in alveolar and interstitial macrophages. These findings indicate that a previously unknown signal trafficking occurs through neurotrophins in peripheral lung.     

Dissociation and aggregation of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride

The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.     

Unfolding pathway of the dimeric and tetrameric forms of phosphofructokinase-2 from Escherichia coli

Escherichia coli phosphofructokinase-2 (Pfk-2) is an oligomeric enzyme characterized by two kinds of interfaces: a monomer-monomer interface, critical for enzymatic activity, and a dimer-dimer interface formed upon tetramerization due to allosteric binding of MgATP. In this work, Pfk-2 was denatured by guanidine hydrochloride (GdnHCl) and the impact of ligand binding on the unfolding pathway of the dimeric and the tertrameric forms of the enzyme was examined. The unligated dimeric form unfolds and dissociates from 0.15 to 0.8 M GdnHCl without the accumulation of native monomers, as indicated by circular dichroism and size exclusion chromatography measurements. However, a monomeric intermediate with an expanded volume and residual secondary structure accumulates above 0.8 M GdnHCl. The dimeric fructose-6-P-enzyme complex shows a shift in the simultaneous dissociation and unfolding process to elevated GdnHCl concentrations (from 0.8 to 1.4 M) together with the expulsion of the ligand detected by intrinsic fluorescence measurements. The unfolding pathway of the tetrameric MgATP-enzyme complex shows the accumulation of a tetrameric intermediate with altered fluorescence properties at about 0.4 M GdnHCl. Above this concentration a sharp transition from tetramers to monomers, without the accumulation of either compact dimers or monomers, was detected by light scattering measurements. Indeed, the most populated species was a partially unfolded monomer about 0.7 M GdnHCl. On the basis of these results, we suggest that the subunit contacts are critical for the maintenance of the overall structure of Pfk-2 and for the binding of ligands, explaining the reported importance of the dimeric state for enzymatic activity.     

Efficacy of Brain-Derived Neurotrophic Factor and Neurotrophin-3 on Neurochemical and Behavioral Deficits Associated with Partial Nigrostriatal Dopamine Lesions

Abstract: Brain-derived neurotrophic factor (BDNF) promotes the survival of dopamine (DA) neurons, enhances expression of DA neuron characteristics, and protects these cells from 6-hydroxydopamine (6-OHDA) toxicity in vitro. We tested the ability of BDNF or neurotrophin-3 (NT-3) to exert similar protective effects in vivo during chronic delivery of 6-OHDA to the rat neostriatum. Chronic infusions of BDNF or NT-3 (12 µg/day) above the substantia nigra were started 6 days before and continued during an 8-day chronic intrastriatal infusion of 6-OHDA. In control and neurotrophin-treated animals, 6-OHDA treatment selectively depleted 50–60% of nigrostriatal DA nerve terminals but produced little if any loss of pars compacta DA cell bodies. This partial DA lesion resulted in three rotations per minute toward the lesioned hemisphere after treatment with the DA release-inducing drug d-amphetamine. Compared with supranigral infusions of vehicle, BDNF and NT-3 decreased the number of these ipsiversive rotations by 70 and 48% and increased by 20- and 10-fold, respectively, the number of contraversive rotations observed after amphetamine injection. When challenged with the DA receptor agonist apomorphine, BDNF- and NT-3-treated animals also exhibited a seven- and 3.5-fold increase in the number of contraversive rotations relative to the vehicle group, respectively. Compared with vehicle, BDNF increased striatal levels of homovanillic acid (HVA; 86%), 3,4-dihydroxyphenylacetic acid (DOPAC; 42%), and 5-hydroxyindoleacetic acid (5-HIAA; 32%) and the HVA/DA (43%) and 5-HIAA/serotonin (34%) ratios in the DA-denervated striatum. NT-3 augmented only striatal 5-HIAA levels (24%). Neither factor altered the 6-OHDA-induced decrease in striatal DA levels or high-affinity DA uptake and thus did not protect against the destruction of DA terminals and did not alter striatal D₁ or D₂ ligand binding. Choline, GABA, and glutamate uptake in the striatum were not altered by the lesion or neurotrophin treatment. Thus, BDNF and to a lesser extent NT-3 reverse rotational behavioral deficits and augment striatal DA and 5-HT metabolism in a partial DA lesion model.     

Monocytes of allergics and non-allergics produce, store and release the neurotrophins NGF, BDNF and NT-3

INTRODUCTION: Recent studies have shown that neurotrophins (NTs) are involved in inflammatory processes. Elevated plasma levels of NTs were found allergic diseases with the highest levels in allergic asthma. However, the exact cellular sources involved in the regulation and release of neurotrophins in allergic inflammation are still not well defined. OBJECTIVE: The aim of this study was to assess whether monocytes of allergic and non-allergic subjects produce, store and release the neurotrophins NGF, BDNF and NT-3. METHODS: Monocytes of allergic and non-allergic donors were purified by immunomagnetic selection. APAAP-staining for the presence of NTs and their receptors was performed. RT-PCR and Western blot evaluated the production and storage of NTs. Monocytes were incubated and supernatants were collected for measurement of neurotrophic factors after stimulation with lipopolysaccharide (LPS) as inflammatory stimulus. The neurotrophin content in lysates and cell culture supernatants was determined by ELISA. RESULTS: Human monocytes express the neurotrophins NGF, BDNF and NT-3 but also their specific receptors TrkA, TrkB and TrkC. RT-PCR amplification of isolated mRNA demonstrated expression of the examined neurotrophins. Proteins were detectable by Western blot. NTs were found in the monocyte lysates and supernatants at different levels in allergic and non-allergic donors. Cell stimulation with LPS leads to release of NGF and NT3. CONCLUSIONS: Monocytes, produce, store and release NGF, BDNF and NT-3. They are a possible source of elevated neurotrophin levels found in allergy and asthma.     

NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.

To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF. Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity--high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have paralleled as well as reciprocal roles in vivo.