Trophic factors are proteins that support and protect subpopulations of cells. A number have been reported to act on dopaminergic neurons in vitro and in vivo, making them potential therapeutic candidates for Parkinson's disease. All of these candidate factors protect dopaminergic neurons if given prior to, or with, selective neurotoxins. Fewer trophic factors, primarily glial-derived neurotrophic factor (GDNF) and its relative, neurturin (NRTN; also known as NTN), have been shown to restore function in damaged dopamine neurons after the acute effects of neurotoxins have subsided. A major barrier to clinical translation has been delivery. GDNF delivered by intracerebroventricular injection in patients was ineffective, probably because GDNF did not reach the target, the putamen, and intraputaminal infusion was ineffective, probably because of limited distribution within the putamen. A randomized clinical trial with gene therapy for NRTN is underway, in an attempt to overcome these problems with targeting and distribution. Other strategies are available to induce trophic effects in the CNS, but have not yet been the focus of human research. To date, clinical trials have focused on restoration of function (i.e., improvement of parkinsonism). Protection (i.e., slowing or halting disease progression and functional decline) might be a more robust effect of trophic agents. Laboratory research points to their effectiveness in protecting neurons and even restoring dopaminergic function after a monophasic neurotoxic insult. Utility for such compounds in patients with Parkinson's disease and ongoing loss of dopaminergic neurons remains to be proven.

The ubiquitin-proteasome pathway is particularly important for the regulated degradation of various proteins which control a vast array of biological processes. Therefore, proteasome inhibitors are promising candidates for anti-tumoral or anti-inflammatory drugs. N-Acetyl-Leu-Leu-Norleucinal (Ac-LLN-al, also termed calpain inhibitor I) was one of the first proteasome inhibitors discovered and has been widely used to study the 20S proteasome core particle (CP) function in vivo, despite its lack of specificity. Vinyl sulfones, like Ac-PRLN-vs, show covalent binding of the beta-carbon atom of the vinyl sulfone group to the Thr1Ogamma only of subunit beta2. However, vinyl sulfones have similar limitations as peptide aldehydes as they have been reported also to bind and block intracellular cysteine proteases. A more specific proteasome inhibitor is the natural product lactacystin, which can be isolated from Streptomyces. It was found that this compound forms an ester bond only to the Thr1Ogamma of the chymotrypsin-like active subunit beta5 due to specific P1 interactions. In contrast to most other proteasome inhibitors, the natural alpha',beta'-epoxyketone peptide epoxomicin binds specifically to the small class of N-terminal nucleophilic (Ntn) hydrolases (CPs belong to this protease family) with the formation of a morpholino adduct. All previously described proteasome inhibitors bind covalently to the proteolytic active sites. However, as the proteasome is involved in a variety of biological important functions, it is of particular interest to block the CP only for limited time in order to reduce cytotoxic effects. Recently, the binding mode of the natural specific proteasome inhibitor TMC-95 obtained from Apiospora montagnei was investigated. The crystal structure revealed that the TMC-95 blocks the active sites of the CP noncovalently in the low nanomolar range. This review summarizes the current structural knowledge of inhibitory compounds bound to the CP, showing the proteasome as a potential target for drug development in medical research.

Glial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two structurally related, potent survival factors for sympathetic, sensory and central nervous system neurons. GDNF mediates its actions through a multicomponent receptor system composed of a ligand-binding glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) and the transmembrane protein tyrosine kinase Ret. In contrast, the mechanism by which the NTN signal is transmitted is not well understood. Here we describe the identification and tissue distribution of a GPI-linked protein (designated NTNR-alpha) that is structurally related to GDNFR-alpha. We further demonstrate that NTNR-alpha binds NTN (K[d] approximately 10 pM) but not GDNF with high affinity; that GDNFR-alpha binds to GDNF but not NTN with high affinity; and that cellular responses to NTN require the presence of NTNR-alpha. Finally, we show that NTN, in the presence of NTNR-alpha, induces tyrosine-phosphorylation of Ret, and that NTN, NTNR-alpha and Ret form a physical complex on the cell surface. These findings identify Ret and NTNR-alpha as signalling and ligand-binding components, respectively, of a receptor for NTN and define a novel family of receptors for neurotrophic and differentiation factors composed of a shared transmembrane protein tyrosine kinase and a ligand-specific GPI-linked protein.

1. Measurements of the unloaded sliding speed of and isometric force exerted on single thin filaments in in vitro motility assays were made to evaluate the role of regulatory proteins in the control of unloaded thin filament sliding speed and isometric force production. 2. Regulated actin filaments were reconstituted from rabbit F-actin, native bovine cardiac tropomyosin (nTm), and either native bovine cardiac troponin (nTn), troponin containing a TnC mutant, CBMII, in which the sole regulatory site in cardiac TnC (site II) is inactivated (CBMII-Tn), or troponin containing a point mutation in TnT (I79N, where isoleucine at position 79 is replaced with asparagine) associated with familial hypertrophic cardiomyopathy (FHC). 3. Addition of regulatory proteins to the thin filament increases both the unloaded sliding speed and the isometric force exerted by myosin heads on the thin filaments. 4. Variation of thin filament activation by varying [Ca2+] or the fraction of CBMII/TnC bound to the thin filament at pCa 5, had little effect on the unloaded filament sliding speed until the fraction of the thin filament containing calcium bound to TnC was less than 0.15. These results suggest that [Ca2+] primarily affects the number of attached and cycling crossbridges. 5. The presence of the FHC TnT mutant increased the thin filament sliding speed but reduced the isometric force that heavy meromyosin exerted on regulated thin filaments. These latter results, together with the increased sliding speed and isometric force seen in the presence of regulatory proteins, suggest that thin filament regulatory proteins exert significant allosteric effects on the interaction of crossbridges with the thin filament.

Hirschsprung disease (HSCR), or congenital intestinal aganglionosis, is a relatively common disorder of neural crest migration. It has a strong genetic basis, although simple Mendelian inheritance is rarely observed. Hirschsprung disease is associated with several other anomalies and syndromes, and animal models for these conditions exist. Mutations in the RET gene are responsible for approximately half of familial cases and a smaller fraction of sporadic cases. Mutations in genes that encode RET ligands (GDNF and NTN); components of another signaling pathway (EDNRB, EDN3, ECE-1); and the transcription factor, SOX10, have been identified in HSCR patients. A subset of these mutations is associated with anomalies of pigmentation and/or hearing loss. For almost every HSCR gene, incomplete penetrance of the HSCR phenotype has been observed, probably due to genetic modifier loci. Thus, HSCR has become a model of a complex polygenic disorder in which the interplay of different genes is currently being elucidated.

Both glial cell line-derived neurotrophic factor (GDNF) and its recently discovered congener, neurturin (NTN), have been shown to exert neuroprotective effects on lesioned nigral dopamine (DA) neurons when administered at the level of the substantia nigra. In the present study, we have explored the relative in vivo potency of these two neurotrophic factors using two alternative routes of administration, into the striatum or the lateral ventricle, which may be more relevant in a clinical setting. In rats subjected to an intrastriatal (IS) 6-hydroxydopamine (6-OHDA) lesion, GDNF and NTN were injected every third day for 3 weeks starting on the day after the 6-OHDA injection. GDNF provided almost complete (90-92%) protection of the lesioned nigral DA neurons after both IS and intracerebroventricular (ICV) administration. NTN, by contrast, was only partially effective after IS injection (72% sparing) and totally ineffective after ICV injection. Although the trophic factor injections protected the nigral neurons from lesion-induced cell death, the level of expression of the phenotypic marker, tyrosine hydroxylase (TH), was markedly reduced in the rescued cell bodies. The extent of 6-OHDA-induced DA denervation in the striatum was unaffected by both types of treatment; consistent with this observation, the high rate of amphetamine-induced turning seen in the lesioned control animals was unaltered by either GDNF or NTN treatment. In the GDNF-treated animals, and to a lesser extent also after IS NTN treatment, prominent axonal sprouting was observed within the globus pallidus, at the level where the lesioned nigrostriatal axons are known to end at the time of onset of the neurotrophic factor treatment. The results show that GDNF is highly effective as a neuroprotective and axon growth-stimulating agent in the IS 6-OHDA lesion model after both IS and ICV administration. The lower efficacy of NTN after IS, and particularly ICV, administration may be explained by the poor solubility and diffusion properties at neutral pH.

Neurturin (NTN) is a potent survival factor for midbrain dopaminergic neurons. CERE-120, an adeno-associated virus type 2 (AAV2) vector encoding human NTN (AAV2-NTN), is currently being developed as a potential therapy for Parkinson's disease. This study examined the bioactivity and safety/tolerability of AAV2-NTN in the aged monkey model of nigrostriatal dopamine insufficiency. Aged rhesus monkeys received unilateral injections of AAV2-NTN into the caudate and putamen, with each animal therefore serving as its own control. Robust expression of NTN within the nigrostriatal system was observed 8 months postadministration. (18)F-fluorodopa imaging using positron emission tomography revealed statistically significant increases in (18)F-fluorodopa uptake in the injected striatum compared with the uninjected side at 4 and 8 months. In addition, at 8 months postadministration, a significant enhancement in tyrosine hydroxylase immunoreactive fibers and an increase in the number of tyrosine hydroxylase immunoreactive cells was observed in the AAV2-NTN injected striatum compared with the uninjected side. Robust activation of phosphorylated extracellular signal-regulated kinase immunoreactivity in the substantia nigra was also observed. Histopathological analyses revealed no adverse effects of AAV2-NTN in the brain. Collectively, these results are consistent with the neurotrophic effects of NTN on the dopaminergic nigrostriatal system and extend the growing body of evidence supporting the concept that AAV2-NTN may have therapeutic benefit for Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.

Previously it was shown that the TNF superfamily member TWEAK (TNFSF12) acts through its receptor, Fn14, to promote proinflammatory responses in kidney cells, including the production of MCP-1, RANTES, IP-10 and KC. In addition, the TWEAK/Fn14 pathway promotes mesangial cell proliferation, vascular cell activation, and renal cell death. To study the relevance of the TWEAK/Fn14 pathway in the pathogenesis of antibody-induced nephritis using the mouse model of nephrotoxic serum nephritis (NTN), we induced NTN by passive transfer of rabbit anti-glomerular antibodies into Fn14 knockout (KO) and wild type (WT) mice. Severe proteinuria as well as renal histopathology were induced in WT but not in Fn14 KO mice. Similarly, a pharmacologic approach of anti-TWEAK mAb administration into WT mice in the NTN model significantly ameliorated proteinuria and improved kidney histology. Anti-TWEAK treatment did not affect the generation of mouse anti-rabbit antibodies; however, within the kidney there was a significant decrease in glomerular immunoglobulin deposition, as well as macrophage infiltrates and tubulointerstitial fibrosis. The mechanism of action is most likely due to reductions in downstream targets of TWEAK/Fn14 signaling, including reduced renal expression of MCP-1, VCAM-1, IP-10, RANTES as well as Fn14 itself, and other molecular pathways associated with fibrosis in anti-TWEAK treated mice. Thus, TWEAK/Fn14 interactions are instrumental in the pathogenesis of nephritis in the NTN model, apparently mediating a cascade of pathologic events locally in the kidney rather than by impacting the systemic immune response. Disrupting TWEAK/Fn14 interactions may be an innovative kidney-protective approach for the treatment of lupus nephritis and other antibody-induced renal diseases.

Polycystin-1 (PC1), the PKD1 gene product, plays a critical role in renal tubule diameter control and disruption of its function causes cyst formation in human autosomal dominant polycystic kidney disease. Recent evidence shows that PC1 undergoes cleavage at the juxtamembrane G protein-coupled receptor proteolytic site (GPS), a process likely to be essential for its biological activity. Here we further characterized the proteolytic cleavage of PC1 at the GPS domain. We determined the actual cleavage site to be between leucine and threonine of the tripeptide HLT(3049) of human PC1. Cleavage occurs in the early intracellular secretory pathway and requires initial N-glycan attachment but not its subsequent trimming. We provide evidence that the cleavage occurs via a cis-autoproteolytic mechanism involving an ester intermediate as shown for Ntn hydrolases and EMR2.

Neurturin (NTN) and glial cell line-derived neurotrophic factor (GDNF) are the first two members of the GDNF family (GF) of neurotrophic factors. These two proteins are potent survival factors for several populations of central and peripheral neurons in mature and developing rodents. The receptor for these factors is a multicomponent complex that includes the RET (rearranged during transfection) tyrosine kinase receptor and one of two glycosyl phosphatidylinositol (GPI)-linked ligand-binding components called GDNF family receptor alphas (GFRalpha-1 and GFRalpha-2). We have used in situ hybridization to study the mRNA expression of NTN, GDNF, RET, GFRalpha-1, and GFRalpha-2 in the central nervous system (CNS) of adult mice. GF receptors are expressed in several areas in which neuronal populations known to respond to NTN and GDNF are located, including the ventral horn of the spinal cord and the compacta region of the substantia nigra. In addition, we have demonstrated receptor expression in other areas of the brain including the thalamus and hypothalamus. Neurons in these areas express GF receptors, and therefore, may respond to NTN or GDNF. NTN and GDNF are expressed in targets of neurons that express GF receptors. The pattern of GF factor and receptor expression in the adult brain suggests a role for these factors in maintaining neuronal circuits in the mature CNS.

During the development of nephrotoxic nephritis (NTN) in the mouse, we find that a variety of chemokines and chemokine receptors are induced: CCR1 (RANTES, MIP-1alpha), CCR2 (MCP-1), CCR5 (RANTES, MIP-1alpha, MIP-1beta), CXCR2 (MIP-2), and CXCR3 (IP-10). Their timing of expression indicated that CXCR2 and CCR1 are probably important in the neutrophil-dependent heterologous phase of the disease, whereas CCR1, CCR2, CCR5, and CXCR3 accompany the subsequent mononuclear cell infiltration characteristic of autologous disease. We therefore assessed the role of CCR1 in NTN using CCR1(-/-) mice. We found that neutrophil accumulation in CCR1(-/-) mice was comparable to that in wild-type animals but that renal recruitment of CD4(+) and CD8(+) T cells and macrophages increased significantly. Moreover, CCR1(-/-) mice developed more severe glomerulonephritis than did controls, with greater proteinuria and blood urea nitrogen, as well as a higher frequency of crescent formation. In addition, CCR1(-/-) mice showed enhanced Th1 immune responses, including titers of antigen-specific IgG2a antibody, delayed-type hypersensitivity responses, and production of IFN-gamma and TNF-alpha. Lastly, using recombinant proteins and transfected cells that overexpressed CCR1, we demonstrated that MIP-1alpha, but not RANTES, bound CCR1 and induced cell chemotaxis. Thus, rather than simply promoting leukocyte recruitment during NTN, CCR1 expression profoundly alters the effector phase of glomerulonephritis. Therapeutic targeting of chemokine receptors may, on occasion, exacerbate underlying disease.

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor (GDNF). Both factors promote the survival of a variety of neurons, and GDNF is required for the development of the enteric nervous system and kidney. GDNF signals through a receptor complex consisting of the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)-linked receptor termed GDNFR-alpha. Here we report the cloning of a new GPI-linked receptor termed NTNR-alpha that is homologous with GDNFR-alpha and is widely expressed in the nervous system and other tissues. By using microinjection to introduce expression plasmids into neurons, we show that coexpression of NTNR-alpha with Ret confers a survival response to neurturin but not GDNF, and that coexpression of GDNFR-alpha with Ret confers a survival response to GDNF but not neurturin. Our findings indicate that GDNF and neurturin promote neuronal survival by signalling through similar multicomponent receptors that consist of a common receptor tyrosine kinase and a member of a GPI-linked family of receptors that determines ligand specificity.

Neurturin (NTN) is a neurotrophic factor that shares homology with glial cell line-derived neurotrophic factor (GDNF). Recently, a receptor complex has been identified for GDNF that includes the Ret tyrosine kinase receptor and a glycosylphosphatidylinositol-linked protein termed "GDNFRalpha." However, differences in the phenotype of Ret and GDNF knockout animals suggest that Ret has at least one additional ligand. In this report, we demonstrate that NTN induces Ret phosphorylation in primary cultures of rat superior cervical ganglion (SCG) neurons. NTN also caused Ret phosphorylation in fibroblasts that were transfected stably with Ret and GDNFRalpha but not in cells expressing Ret alone. A glycosylphosphatidylinositol-linked protein also was important for NTN and GDNF signaling in SCG neurons; phosphatidylinositol-specific phospholipase C treatment of SCG cultures reduced the ability of NTN to phosphorylate Ret and the ability of NTN or GDNF to activate the mitogen-activated protein kinase pathway. NTN and GDNF also caused sustained activation of Ret and the mitogen-activated protein kinase pathway in SCG neurons. Finally, both NTN and GDNF activated the phosphatidylinositol 3-kinase pathway in SCG neurons, which may be important for the ability of NTN and GDNF to promote neuronal survival. These data indicate that NTN is a physiologically relevant ligand for the Ret receptor and suggest that NTN may have a critical role in the development of many neuronal populations.

Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN-hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family.

Convection-enhanced delivery (CED) distributes macromolecules in the brain in a homogeneous, targeted fashion in clinically useful volumes. However, the binding of growth factors to heparin-binding sites in the extracellular matrix may limit the volume of distribution (V(d)). To overcome this limitation, we examined the effects of heparin coinfusion on V(d) of glial-derived neurotrophic factor (GDNF), neurturin (NTN), artemin, and a nonspecifically bound protein, albumin. Heparin coinfusion significantly enhanced the V(d) of GDNF and GDNF-homologous trophic factors, probably by binding and blocking heparin-binding sites in the extracellular matrix. Furthermore, coinfusion of heparin with NTN enhanced striatal dopamine metabolism, compared to trophic factor administered alone. The negligible benefit of GDNF in recent clinical trials of Parkinson's disease may result from limited tissue distribution. Heparin coinfusion during CED targeting the striatum may alleviate this important limitation. This study demonstrates the influence of receptor binding on the distribution of trophic factors in the CNS.

Growth-arrest specific gene 6 (Gas6) is a vitamin K-dependent growth factor for mesangial and epithelial cells. To investigate whether Gas6 is essential for progressive glomerular injury, we constructed Gas6(-/-) mice and examined the role of Gas6 in accelerated nephrotoxic nephritis (NTN), a model of progressive glomerulonephritis. We found less mortality and proteinuria in Gas6(-/-) mice than in wild-type mice following injection of nephrotoxic serum. Glomerular cell proliferation, glomerular sclerosis, crescent formation, and deposition of fibrin/fibrinogen in glomeruli were also reduced in Gas6(-/-) mice. Furthermore, administering Gas6(-/-) mice recombinant wild-type Gas6, but not Gas6 lacking a previously characterized N-terminal gamma-carboxyl group, induced massive proteinuria, glomerular cell proliferation, and glomerulosclerosis, comparable to responses seen in wild-type mice. These data indicate that Gas6 induces glomerular cell proliferation in NTN and suggest that this factor contributes to glomerular injury and the progression of chronic nephritis.

Bacterial bile salt hydrolases catalyze the degradation of conjugated bile acids in the mammalian gut. The crystal structures of conjugated bile acid hydrolase (CBAH) from Clostridium perfringens as apoenzyme and in complex with taurodeoxycholate that was hydrolyzed to the reaction products taurine and deoxycholate are described here at 2.1 and 1.7 A resolution, respectively. The crystal structures reveal close relationship between CBAH and penicillin V acylase from Bacillus sphaericus. This similarity together with the N-terminal cysteine classifies CBAH as a member of the N-terminal nucleophile (Ntn) hydrolase superfamily. Both crystal structures show an identical homotetrameric organization with dihedral (D(2) or 222) point group symmetry. The structure analysis of C. perfringens CBAH identifies critical residues in catalysis, substrate recognition, and tetramer formation which may serve in further biochemical characterization of bile acid hydrolases.

Hirschsprung disease (HSCR) is a frequent neurocristopathy characterized by the absence of submucosal and myenteric plexuses in a variable length of the gastrointestinal tract. Pedigrees and segregation analyses suggested the involvement of one or several dominant genes with low penetrance in HSCR. Considering that RET and glial cell line-derived neurotrophic factor (GDNF) mutations have been reported in the disease, we regarded the other RET ligand, neurturin (NTN), as an attractive candidate gene, especially as it shares large homologies with GDNF. Here, we report on the finding of a heterozygous missense NTN mutation in a large non-consanguineous family including four children affected with a severe aganglionosis phenotype extending up to the small intestine. Interestingly, it appears that the NTN mutation reported here is not sufficient to cause HSCR, and this multiplex family also segregates a RET mutation. This cascade of independent and additive genetic events fits well with the multigenic pattern of inheritance expected in HSCR, and further support the role of RET ligands in development of the enteric nervous system.

Cloning strategies were used to identify a gene termed glial cell line-derived neurotrophic factor receptor-beta (GDNFR-beta) related to GDNFR-alpha. In situ hybridization was then used to map cellular expression of the GDNF-related trophic factor neurturin (NTN) and GDNFR-beta mRNA in developing and adult mice, and comparisons with GDNFR-alpha and RET were made. Neurturin is expressed in postnatal cerebral cortex, striatum, several brainstem areas, and the pineal gland. GDNFR-beta mRNA was more widely expressed in the developing and adult CNS, including cerebral cortex, cerebellum, thalamus, zona incerta, hypothalamus, brainstem, and spinal cord, and in subpopulations of sensory neurons and developing peripheral nerves. NTN colocalized with RET and GDNFR-alpha in ureteric buds of the developing kidney. The circular muscle layer of the developing intestines, smooth muscle of the urether, and developing bronchiolae also expressed NTN. GDNFR-beta was found in myenteric but not submucosal intestinal plexuses. In developing salivary glands NTN had an epithelial expression, whereas GDNFR-beta was expressed in surrounding tissue. Neurturin and GDNFR-beta were present in developing sensory organs. In the gonads, NTN appeared to be expressed in Sertoli cells and in the epithelium of the oviduct, whereas GDNFR-beta was expressed by the germ cell line. Our findings suggest multiple roles for NTN and GDNFR-beta in the developing and adult organism. Although NTN and GDNFR-beta expression patterns are sometimes complementary, this is not always the case, suggesting multiple modi operandi of GDNF and NTN in relation to RET and the two binding proteins, GDNFR-alpha and GDNFR-beta.

BACKGROUND

Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), which is usually obtained by chemical deacylation of cephalosporin C (CPC). The chemical production of 7-ACA includes, however, several expensive steps and requires thorough treatment of chemical wastes. Therefore, an enzymatic conversion of CPC to 7-ACA by cephalosporin acylase is of great interest. The biggest obstacle preventing this in industrial production is that cephalosporin acylase uses glutaryl-7ACA as a primary substrate and has low substrate specificity for CPC.

RESULTS

We have solved the first crystal structure of a cephalosporin acylase from Pseudomonas diminuta at 2.0 A resolution. The overall structure looks like a bowl with two "knobs" consisting of helix- and strand-rich regions, respectively. The active site is mostly formed by the distinctive structural motif of the N-terminal (Ntn) hydrolase superfamily. Superposition of the 61 residue active-site pocket onto that of penicillin G acylase shows an rmsd in Calpha positions of 1.38 A. This indicates structural similarity in the active site between these two enzymes, but their overall structures are elsewhere quite different.

CONCLUSIONS

The substrate binding pocket of the P. diminuta cephalosporin acylase provides detailed insight into the ten key residues responsible for the specificity of the cephalosporin C side chain in four classes of cephalosporin acylases, and it thereby forms a basis for the design of an enzyme with an improved conversion rate of CPC to 7-ACA. The structure also provides structural evidence that four of the five different classes of cephalosporin acylases can be grouped into one family of the Ntn hydrolase superfamily.

OBJECTIVE

To determine if the contribution of the sympathetic nervous system to blood pressure could be evidenced by low-frequency oscillations of systolic blood pressure (LF(SBP)), reflecting vascular sympathetic modulation, or by the decrease in blood pressure after autonomic blockade.

METHODS

We studied multiple system atrophy (MSA) patients, in whom supine hypertension is maintained by residual sympathetic tone ('positive controls'); pure autonomic failure (PAF) patients, in whom supine hypertension is largely independent of sympathetic tone ('negative controls'); essential hypertensive patients (HTN) and normotensive subjects (NTN).

RESULTS

Supine systolic blood pressure (SBP) was 204 +/- 8, 185 +/- 6, 177 +/- 9 and 130 +/- 4 mmHg in MSA, PAF, HTN and NTN, respectively. LF(SBP) was higher in MSA and HTN (5.7 +/- 1.5 and 5.8 +/- 1.4 mmHg(2) compared to NTN and PAF (3.3 +/- 0.5 and 1.1 +/- 0.5 mmHg(2). Trimethaphan 2-4 mg/min induced complete autonomic blockade and lowered SBP below 125 mmHg in all NTN and all but one MSA (to 111 +/- 3 and 97 +/- 9 mmHg). SBP remained elevated in PAF (164 +/- 7 mmHg). Responses in HTN were variable; SBP decreased below 125 mmHg in three and remained elevated in four patients. The decrease in LF(SBP) correlated with the reduction in SBP, with a steeper slope in MSA and HTN compared to NTN (29.0 +/- 5.5, 8.4 +/- 1.6 and 3.6 +/- 1.2 mmHg/mmH (2), respectively).

CONCLUSIONS

Ganglionic blockade, alone or coupled to LF(SBP), discriminated between human models of sympathetic-dependent (MSA) and independent (PAF) hypertension. This approach may aid in assessing the contribution of the sympathetic nervous system in essential hypertension, in which sympathetic dependence is variably expressed.

The netrin family of secreted proteins provides migrational cues in the developing central nervous system. Recently, netrins have also been shown to regulate diverse processes beyond their functions in the brain, incluing the ochrestration of inflammatory events. Particularly netrin-1 has been implicated in dampening hypoxia-induced inflammation. Here, we hypothesized an anti-inflammatory role of endogenous netrin-1 in acute kidney injury (AKI). As homozygous deletion of netrin-1 is lethal, we studied mice with partial netrin-1 deletion (Ntn-1(+/-) mice) as a genetic model. In fact, Ntn-1(+/-) mice showed attenuated Ntn-1 levels at baseline and following ischemic AKI. Functional studies of AKI induced by 30 min of renal ischemia and reperfusion revealed enhanced kidney dysfunction in Ntn-1(+/-) mice as assessed by measurements of glomerular filtration, urine flow rate, urine electrolytes, serum creatinine and creatinine clearance. Consistent with these findings, histological studies indicated a more severe degree kidney injury. Similarly, elevations of renal and systemic inflammatory markers were enhanced in mice with partial netrin-1 deficiency. Finally, treatment of Ntn-1(+/-) mice with exogenous netrin-1 restored a normal phenotype during AKI. Taking together, these studies implicate endogenous netrin-1 in attenuating renal inflammation during AKI.

Neurturin (NTN) and glial cell line-derived neurotrophic factor (GDNF), two members of the GDNF family of growth factors, exert very similar biological activities in different systems, including the substantia nigra. Our goal in the present work was to compare their function and define whether nonoverlapping biological activities on midbrain dopaminergic neurons exist. We first found that NTN and GDNF are differentially regulated during postnatal development. NTN mRNA progressively decreased in the ventral mesencephalon and progressively increased in the striatum, coincident with a decrease in GDNF mRNA expression. This finding suggested distinct physiological roles for each factor in the nigrostriatal system. We therefore examined their function in ventral mesencephalon cultures and found that NTN promoted survival comparable with GDNF, but only GDNF induced sprouting and hypertrophy of developing dopaminergic neurons. We subsequently examined the ability of NTN to prevent the 6-hydroxydopamine-induced degeneration of adult dopaminergic neurons in vivo. Fibroblasts genetically engineered to deliver high levels of GDNF or NTN were grafted supranigrally. NTN was found to be as potent as GDNF at preventing the death of nigral dopaminergic neurons, but only GDNF induced tyrosine hydroxylase staining, sprouting, or hypertrophy of dopaminergic neurons. In conclusion, our results show selective survival-promoting effects of NTN over wider survival, neuritogenic, and hypertrophic effects of GDNF on dopaminergic neurons in vitro and in vivo. Such differences are likely to underlie unique roles for each factor in postnatal development and may ultimately be exploited in the treatment of Parkinson's disease.