Neurturin (NTN), a potent neurotrophic factor acting specifically on dopaminergic neurons, is comprised of 102 amino acids as a mature protein. We artificially synthesized a gene for mature human NTN (hNTN) using codons preferred by the yeast Pichia pastoris. This synthesized gene, fused in frame with sequences encoding the alpha-factor signal peptide gene from Saccharomyces cerevisiae was cloned into P. pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-hNTN was then transformed into the yeast and stable multicopy recombinant P. pastoris strains were selected by G418 resistance. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hNTN, a 16kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using CM-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Bioactivity of the recombinant hNTN was confirmed by the ability of the protein to stimulate growth of nerve fibers from the dorsal root ganglia of chick embryos in vitro.

Experimental nephrotoxic serum nephritis (NTN) is a model for T-cell-mediated human rapid progressive glomerulonephritis. T-cell receptor stimulation involves intracellular signaling events that ultimately lead to the activation of transcription factors, such as NF-κB. We explored the involvement of the NF-κB components IKK-2 and NEMO in NTN, by using cell-specific knockouts of IKK-2 and NEMO in CD4+ T lymphocytes. Our results demonstrate that although the course of disease was not grossly altered in CD4xIKK2Δ and CD4xNEMOΔ animals, renal regulatory T cells were significantly reduced and T helper (Th)1 and Th17 cells significantly increased in both knockout mouse groups. The expression of the renal cytokines and chemokines IL-1β, CCL-2, and CCL-20 was also significantly altered in both knockout mice. Lymphocyte transcriptome analysis confirmed the increased expression of Th17-related cytokines in spleen CD4+ T cells. Moreover, our array data demonstrate an interrupted canonical NF-κB pathway and an increased expression of noncanonical NF-κB pathway-related genes in nephritic CD4xNEMOΔ mice, highlighting different downstream effects of deletion of IKK-2 or NEMO in T lymphocytes. We propose that better understanding of the role of IKK-2 and NEMO in nephritis is essential for the clinical application of kinase inhibitors in patients with glomerulonephritis.-Guo, L., Huang, J., Chen, M., Piotrowski, E., Song, N., Zahner, G., Paust, H.-J., Alawi, M., Geffers, R., Thaiss, F. T-lymphocyte-specific knockout of IKK-2 or NEMO induces Th17 cells in an experimental nephrotoxic nephritis mouse model.

The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes l-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an αβ-βα sandwich fold homodimer that cleaves its own peptide backbone at the G167-T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167-T168 peptide bond reaches only ≤50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure-activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member.

We have identified a novel L-asparaginase, abASNase3, from Aquabacterium sp. A7-Y. abASNase3 is composed of 306 amino acids and exhibits 34 % sequence homology to human asparaginase (hASNase3). Further analysis revealed that abASNase3 belongs to the N-terminal nucleophile (Ntn) family of hydrolases. Previous reports about the Ntn hydrolase family and the results of our study suggest that abASNase3 must form two subunits by self-cleavage between Gly189 and Thr190 to attain catalytic activity. The two subunits remained tightly associated to build a single functional unit. The optimum pH for abASNase3 was found to be 8.0 in Tris-HCl buffer and the enzyme was found to be stable over a broad pH range from pH 6.0 to 12.0. The optimum temperature for abASNase3 was found to be approximately 40 °C, and the enzyme was stable below 65 °C. abASNase3 showed high substrate specificity toward L-asparagine and had no or only slight activity toward D-asparagine, L-glutamine and D-glutamine. abASNase3 was significantly activated by Mg(2+) and was substantially inhibited by Ni(2+), Cu(2+), Mn(2+) and Co(2+). The Michaelis-Menten constant and turnover number of abASNase3 for L-asparagine were estimated to be 3.37 × 10(-2) M and 8.72 × 10(-3) s(-1), respectively. Our results indicate that abASNase3 is a novel L-asparaginase in the Ntn family of hydrolases.

A novel bacterial strain that was capable of growing on the beta-tripeptide H-betahVal-betahAla-betahLeu-OH as the sole carbon and nitrogen source was isolated from an enrichment culture. On the basis of physiological characterization, partial 16S rRNA sequencing, and fatty acid analysis, strain 3-2W4 was identified as a member of the family Sphingomonadaceae. Growth on the beta-tripeptide and the beta-dipeptide H-betahAla-betahLeu-OH was observed, and emerging metabolites were characterized. Small amounts of a persisting metabolite, the N-acetylated beta-dipeptide, were identified in both media. According to dissolved organic carbon measurements, 74 to 80% of the available carbon was dissimilated. The beta-peptide-degrading enzyme was purified from the crude cell extract of cells from strain 3-2W4 grown on complex medium. The enzyme was composed of two subunits, and the N-terminal sequences of both were determined. With this information, it was possible to identify the complete nucleotide sequence and to deduce the primary structure of the gene bapA. The gene encoded a beta-peptidyl aminopeptidase (BapA) of 402 amino acids that was synthesized as preprotein with a signal sequence of 29 amino acids. The enzyme was cleaved into two subunits (residues 30 to 278 and 279 to 402). It belonged to the N-terminal nucleophile (Ntn) hydrolase superfamily.

Siderophore biosynthesis by Pseudomonas aeruginosa enhances virulence and represents an attractive drug target. PvdQ functions in the type-1 pyoverdine biosynthetic pathway by removing a myristoyl anchor from a pyoverdine precursor, allowing eventual release from the periplasm. A circularly permuted version of PvdQ bypasses the self-processing step of this Ntn-hydrolase and retains the activity, selectivity, and structure of wild-type PvdQ, as revealed by a 1.8 Å resolution X-ray crystal structure. A 2.55 Å resolution structure of the inactive S1A/N269D-cpPvdQ mutant in complex with the pyoverdine precursor PVDIq reveals a specific binding pocket for the d-Tyr of this modified peptide substrate. To our knowledge, this structure is the first of a pyoverdine precursor peptide bound to a biosynthetic enzyme. Details of the observed binding interactions have implications for control of pyoverdine biosynthesis and inform future drug design efforts.

Conformational transitions and functional stability of the bile salt hydrolase (BSH; cholylglycine EC: 3.5.1.24) from Bifidobacterium longum (BlBSH) cloned and expressed in E. coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and CD spectroscopy. Thermal and Gdn-HCl-mediated denaturation of BlBSH is a multistep process of inactivation and unfolding. The inactivation and unfolding of the enzyme was found to be irreversible. Enzyme activity seems sensitive to even minor conformational changes at the active site. Thermal denaturation as such did not result in any insoluble protein aggregates. However, on treating with 0.25 - 1 M Gdn-HCl the enzyme showed increasing aggregation at temperatures of 40 - 55 degrees C indicating more complex structural changes taking place in the presence of chemical denaturants. The enzyme secondary structure was still intact at acidic pH (pH 1 - 3). The perturbation in the tertiary structure at the acidic pH was detected through freshly formed solvent exposed hydrophobic patches on the enzyme. These changes could be due to the formation of an acid-induced molten globule-like state.

HpxW from the ubiquitous pathogen Klebsiella pneumoniae is involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.

The nonspecific cell-mediated immunocompetence of 51 patients with gestational trophoblastic disease (GTD), including 16 patients with hydatid mole (HM), 24 with nonmetastatic trophoblastic neoplasia (NTN) and 15 with metastatic TN (MTN), was studied with the use of both in vitro and in vivo parameters of cell-mediated immunity (CMI) such as lymphocyte blastogenic response to phytohemagglutinin (PHA), subpopulation constitution, and delayed cutaneous hypersensitivity responses to 2,4-dinitrochlorobenzene (DNCB) and to purified protein derivative of tuberculin (PPD). The pretreatment cell-mediated immune status of patients with HM developing no malignant sequelae was shown to be essentially similar, in terms of both in vitro PHA and in vivo DNCB reactivities, to that of normal women and of patients with benign gynecological diseases. In patients with TN, however, there was a significant depression in the blastogenic lymphocyte response to PHA before evacuation of the mole, which was persistently demonstrated after uterine evacuation and more marked throughout the course of disease in patients with MTN, than in those with NTN, with a tendency to return to normal in remission. Moreover, patients with TN had a significant depletion of T lymphocytes as determined by rosette-forming cell procedures before treatment, which was most evident in patients with MTN. Plasma from the MTN patients was also shown to have an inhibitory effect on PHA responsiveness of lymphocytes from normal women. There was an increased incidence of impaired reactivity to DNCB in patients with TN (higher in MTN than in NTN), compared with HM and benign diseases, while no such difference in incidence was observed in response to PPD. On the basis of these findings, a preliminary characterization of altered immunocompetence in patients with TN and its mechanism are discussed.

Glial cell line-derived neurotrophic factor (GDNF) was initially discovered as a neurotrophic factor that enhances survival of midbrain dopaminergic neurons. Findings in 1994 and 1995 extended the spectrum of biological activities of GDNF to different populations of neurons (motor and sensory). Recent findings revealed that GDNF, Neurturin (NTN), Persephin (PSP), and Artemin (ARTN) are members of the GDNF protein family and are structurally related to transforming growth factor protein family. They are survival factors for peripheral and central neurons, for oligodendrocytes and can promote morphogenesis of kidney in vitro.

National networks detect multi-state trends in element deposition using direct measurement methods. Biomonitoring techniques have been used to examine deposition in local areas and around point sources. We sought to determine the efficacy of a moss bag technique to detect element deposition trends on a mid-range (state) scale, and to compare these results with those of the National Acid Deposition Program/National Trends Network (NADP/NTN, 1999). We sampled heavy metals, sulfur, and nitrogen deposition (21 elements) using mesh bags containing Sphagnum russowii at nine sites, over a 375 km transect crossing southern Wisconsin (upper Midwest, USA). We found statistically significant trends of decreasing deposition in a northwesterly direction for 13 elements: Al, B, Ca, Cd, Co, Cu, Cr, Fe, Mg, Mn, Ni, S, and Zn. Six of these have moderate to large changes in concentration (14-37%). The trends for Ca, Mg, and S are consistent with regional deposition patterns in 1998 isopleth maps from the NADP/NTN (1999) which are derived from a sampling array far less dense than the transect sites. This national network indicates that Ca and Mg increase to the southeast, beyond Wisconsin borders. The fact that the present study demonstrates strong correlations between both of these elements (Ca and Mg) and Al, B, Cr, Cu, Fe, Mn, Ni, and Zn (mean r for all correlations = 0.75, p < 0.02) implies that these correlated elements also increase to the southeast in neighboring states.

Injury to the recurrent laryngeal nerve (RLN) leads to the loss of ipsilateral laryngeal fold movement, with dysphonia, and occasionally dysphagia. Functional movement of the vocal folds is never restored due to misrouting of regenerating axons to agonist and antagonist laryngeal muscles. Changes of neurotrophic factor expression within denervated muscles occur after nerve injury and may influence nerve regeneration, axon guidance and muscle reinnervation. This study investigates the expression of certain neurotrophic factors in the laryngeal muscles during the course of axonal regeneration using RT-PCR. The timing of neurotrophic factor expression was correlated to the reinnervation of the laryngeal muscles by motor axons. Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF) and Netrin-1 (NTN-1) increased their expression levels in laryngeal muscles after nerve section and during regeneration of RLN. The upregulation of trophic factors returned to control levels following regeneration of RLN. The expression levels of the neurotrophic factors were correlated with the innervation of regenerating axons into the denervated muscles. The results suggest that certain neurotrophic factor expression is strongly correlated to the reinnervation pattern of the regenerating RLN. These factors may be involved in guidance and neuromuscular junction formation during nerve regeneration. In the future, their manipulation may enhance the selective reinnervation of the larynx.

A facile method to produce high-quality ZnO nanostructures; either tetrapod (TP), nanotetraneedle (NTN) or multipod (MP) with a high degree of homogeneity for advanced field emission (FE) applications is presented. Among these nanostructures, NTN has been successfully employed to demonstrate enhanced current densities (2.6 mA cm(-2)), turn-on field (1.5 V microm(-1)) and field-enhancement factors (6930) over conventional multiwalled carbon nanotubes (MWCNTs), TP, MP and ZnO-spheroids. A comparative study of FE from various ZnO nanostructures, morphologies and site densities has lead to the conclusion that diameter of the tip is one of the vital parameters in enhancing the overall FE properties.

Ice-core samples from Upper Fremont Glacier (UFG), Wyoming, were used as proxy records for the chemical composition of atmospheric deposition. Results of analysis of the ice-core samples for stable isotopes of nitrogen (δ15N, ) and sulfur (δ34S, ), as well as and deposition rates from the late-1940s thru the early-1990s, were used to enhance and extend existing National Atmospheric Deposition Program/National Trends Network (NADP/NTN) data in western Wyoming. The most enriched δ34S value in the UFG ice-core samples coincided with snow deposited during the 1980 eruption of Mt. St. Helens, Washington. The remaining δ34S values were similar to the isotopic composition of coal from southern Wyoming. The δ15N values in ice-core samples representing a similar period of snow deposition were negative, ranging from -5.9 to -3.2 ‰ and all fall within the δ15N values expected from vehicle emissions. Ice-core nitrate and sulfate deposition data reflect the sharply increasing U.S. emissions data from 1950 to the mid-1970s.

Buckypapers (BPs) are free standing thin sheets made of carbon nanotubes, such as single-walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs) or their mixtures. In this research, through in situ electrical resistance measurements, we studied the electrical conductance changes of carbon nanotube networks (NTNs) in various BP samples from complete immersion to evaporation using different chemical solvents. BP samples demonstrated a 20-30% decrease in conductance upon the immersion and almost full recovery after the drying process. We found that by pre-stressing, BP samples demonstrated highly reproducible patterns of conductance changes corresponding to solvent quantity. This feature can be potentially used for sensor applications to simultaneously detect both the occurrence and the amount of organic solvent leakage.

Leptospirosis caused by Leptospira is a zoonotic disease of global importance but it is considered as an emerging or re-emerging infectious disease in many areas in the world. Until now, the mechanisms about pathogenesis and transmission of Leptospira remains poorly understood. As eukaryotic and prokaryotic proteins can be denatured in adverse environments and chaperone-protease/peptidase complexes degrade these harmful proteins, we speculate that infection may also cause leptospiral protein denaturation, and the HslU and HslV proteins of L. interrogans may compose a complex to degrade denatured proteins that enhances leptospiral survival in hosts. Here we show that leptospiral HslUV is an ATP-dependent chaperone-peptidase complex containing ATPase associated with various cellular activity (AAA+) and N-terminal nucleophile (Ntn) hydrolase superfamily domains, respectively, which hydrolyzed casein and chymotrypsin-like substrates, and this hydrolysis was blocked by threonine protease inhibitors. The infection of J774A.1 macrophages caused the increase of leptospiral denatured protein aggresomes, but more aggresomes accumulated in hslUV gene-deleted mutant. The abundant denatured leptospiral proteins are involved in ribosomal structure, flagellar assembly, two-component signaling systems and transmembrane transport. Compared to the wild-type strain, infection of cells in vitro with the mutant resulted in a higher number of dead leptospires, less leptospiral colony-forming units and lower growth ability, but also displayed a lower half lethal dose, attenuated histopathological injury and decreased leptospiral loading in lungs, liver, kidneys, peripheral blood and urine in hamsters. Therefore, our findings confirmed that HslUV AAA+ chaperone-Ntn peptidase complex of L. interrogans contributes to leptospiral survival in hosts and transmission of leptospirosis.

The 20S proteasome is a key player in eukaryotic and archaeal protein degradation, but its progenitor in eubacteria is unknown. Recently, the ancestral β-subunit protein (Anbu) was predicted to be the evolutionary precursor of the proteasome. We crystallized Anbu from Hyphomicrobium sp. strain MC1 in four different space groups and solved the structures by SAD-phasing and Patterson search calculation techniques. Our data reveal that Anbu adopts the classical fold of Ntn-hydrolases, but its oligomeric state differs from that of barrel-shaped proteases. In contrast to their typical architecture, the Anbu protomer is a tightly interacting dimer that can assemble into a helical superstructure. Although Anbu features a catalytic triad of Thr1Oγ, Asp17Oδ1 and Lys32Nε, it is unable to hydrolyze standard protease substrates. The lack of activity might be caused by the incapacity of Thr1NH2 to function as a Brønsted acid during substrate cleavage due to its missing activation via hydrogen bonding. Altogether, we demonstrate that the topology of the proteasomal fold is conserved in Anbu, but whether it acts as a protease still needs to be clarified.

BACKGROUND

Glial cell line-derived neurotrophic factor (GDNF) and a related family member, neurturin (NTN), as well as their cognate receptors (GDNF receptors, GFRalpha-1 and GFRalpha-2, respectively) are involved in nervous system development and murine hair cycle control. To date, their expression in human scalp skin is still unknown.

METHODS

The expression pattern of these proteins was examined in human scalp skin by immunofluorescence and immunoalkaline phosphatase staining methods as well as RT-PCR (GDNF). A total of 50 normal human scalp skin biopsy specimens were examined (healthy females, 53-57 years).

RESULTS

The expression of GDNF protein was strong in the epidermis and sebaceous and sweat glands. In the epidermis, GDNF protein expression was seen in all layers except the stratum corneum. It was strong in the basal layer and decreased gradually towards the granular layer. The results of RT-PCR analysis revealed that GDNF protein is synthesised in the epidermis. The expression of NTN, GFRalpha-1, and GFRalpha-2 proteins was strong in the papillary dermis and sebaceous and sweat glands. In the epidermis, NTN protein expression was absent. The expression of GFRalpha-1 and GFRalpha-2 proteins was moderate in the epidermis. The expression of c-Ret protein was consistently strong in the epidermis and sebaceous and sweat glands. These proteins were strongly expressed in both epithelial and mesenchymal compartments of human anagen VI scalp hair follicles.

CONCLUSIONS

Our investigation reports, for the first time, the expression patterns of GDNF, NTN, GFRalpha-1, GFRalpha-2, and c-Ret proteins in human scalp skin. The expression of these proteins in the skin suggests their possible roles in skin homeostasis. The clinical ramifications of these observations mandate further investigations. Adly MA, Assaf HA, Hussein MR, Paus R. Analysis of the expression pattern of glial cell line-derived neurotrophic factor, neurturin, their cognate receptors GFRalpha-1 and GFRalpha-2, and a common signal transduction element c-Ret in the human scalp skin.

The centrifugal blood pump with a magnetically suspended impeller has shown its superiority as compared to other artificial heart pumps. However, there is still insufficient understanding of fluid mechanics related issues in the clearance gap. The design nature of the pump requires sufficient washout in the clearance between the impeller and the stationary pump housing inner surface. In this study, numerical simulations were carried out to investigate the flow fields in the gap of the Kyoto-NTN centrifugal blood pump. The flow patterns in the gap region of the blood pump were presented and regions of high and low velocity were identified. It was found that the radial velocity of the blood in the gap was closely related to the pressure distribution at the exit of the impeller, both the highest pressure gradient and the highest radial velocity in the gap occurred at an angular position of 170 degrees . The mass flow rate in the gap was estimated to be 25.2% of the pump outflow, which is close to the measurement results of a five times enlarged test pump. The wall shear stresses on the gap surface were found to be over 21 Pa and below 300 Pa, which is correspondingly higher than the threshold of thrombi formation and is lower than the shearing threshold of red blood cells. Comparison of the 1 : 1 simulation model with the measurement results on a five times enlarged test pump indicates that there are some differences in the resulting radial velocity distributions in the gap and thus the washout mechanism. Two symmetrical high washout regions at both the cutwater and splitter plate were observed in the simulation instead of a single washout region at the splitter plate found in the experimental study. This may be due to the scaling effect of the enlarged test pump; also the medium used in the experiment is different from the simulation.

Neurturin (NTN) belongs to the glial cell-line derived neurotrophic factor (GDNF) family of growth factors. Both NTN and GDNF have been shown to potently prevent the degeneration of dopaminergic neuron in vitro and in vivo. The GDNF family receptor alpha 2 (GFR alpha-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFR alpha-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFR alpha-2b and GFR alpha-2c). All three isoforms are expressed in all mammalian tissues examined, including human fetal brain. However, the expression levels of these isoforms have yet to be quantified. In this report, we have developed a real time polymerase chain reaction (PCR) detection method using SYBR Green I to detect the expression levels of the three splice variants (GFR alpha-2a, GFR alpha-2b and GFR alpha-2c). Of the three isoforms, GFR alpha-2a was found to be the most abundant receptor expressed in the whole murine brain. The real time PCR detection method using SYBR Green I developed in this report can be used to unambiguously quantitate expression levels of the GFR alpha-2 isoforms and can be extended to the quantitation of other alternatively spliced isoforms.

An accelerated model of nephrotoxic serum nephritis (NTN) was induced in two groups of Sprague-Dawley rats on the following regimens: (a) a diet deficient in essential fatty acids that contained 20% coconut oil (rats A); (b) a diet supplemented with essential fatty acids that contained 20% safflower oil (rats B). Animals from both groups developed a severe proteinuria reaching its maximum 4 days after the injection of nephrotoxic serum (NTS). Kidney tissue was studied by light and immunofluorescent microscopy 2 to 21 days after NTS injection. The glomeruli of rats A exhibited much more fibrinoid deposition than did those of rats B. A comparable glomerular infiltration by mononuclear cells was observed in both groups of animals between the 2nd and 5th day following the injection of NTS. The number of intrinsic glomerular cells incorporating 3H-thymidine in vivo, however, was significantly lower in rats A than it was in rats B. The outgrowth capacity of glomerular cells in vitro was significantly lower in glomerular explants from rats A than it was in glomerular explants from rats B. These findings demonstrate that, in this model of rat NTN, a diet deficient in essential fatty acids has no major effects on the course of the disease during the first 3 weeks following NTS injection. They further show that the proliferation of intrinsic glomerular cells can be modulated by altering prostaglandin metabolism.

We examined the mRNA expression of neurturin (NTN) and its receptor GFRalpha2 in rat substantia nigra (SN) and striatum by northern analysis at ages ranging from postnatal day (PND) 2 to adult. NTN mRNA expression is developmentally regulated in striatum with a peak at PND10, but its expression in striatum is low, and less than that of SN. In SN, there is no developmental regulation. GFRalpha2 was expressed most highly during the first two postnatal weeks. Like NTN, GFRalpha2 mRNA was also more abundant in SN, at both PND2 and 14. Our results show that NTN expression is relatively low in the striatum, the target of dopamine (DA) neurons, and there is no apparent pattern of developmental regulation in SN. Thus these studies are not strongly supportive of a role for NTN in regulating natural cell death (NCD) in DA neurons, either as a target-derived or as a local paracrine factor.