Neurturin (NTN), a member of glial cell line-derived neurotrophic factor (GDNF) family, is known as an important neurotrophic factor for penis-projecting neurons. We recently demonstrated significant protection from erectile dysfunction (ED) following a replication-defective herpes simplex virus (HSV) vector-mediated GDNF delivery to the injured cavernous nerve. Herein, we applied HSV vector-mediated delivery of NTN to this ED model. Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20 microl of vector stock was administered directly to the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before being killed to assess neuronal survival. Four weeks after nerve injury, rats treated with HSV-NTN exhibited significantly higher ICP/AP compared with untreated or control vector-treated groups. The HSV-NTN group had more FG-positive major pelvic ganglion neurons than the control group following injury. HSV vector-mediated delivery of NTN could be a viable approach for the improvement of ED following cavernous nerve injury.

Huntington's disease (HD) is a devastating neurodegenerative disease characterized by the selective loss of neurons in the striatum and cerebral cortex. This study tested the hypothesis that an adenoassociated viral (AAV2) vector encoding for the trophic factor neurturin (NTN) could provide neuroprotection in the rat 3-nitropropionic acid (3NP) model of HD. Rats received AAV2-NTN (CERE-120), AAV2-eGFP or Vehicle, followed 4 weeks later by the mitochondrial toxin 3NP. 3NP induced motor impairments were observed on the rotarod test, the platform test, and a clinical rating scale in all groups. However, each of these deficits was attenuated by AAV2-NTN (CERE-120). Stereological counts revealed a significant protection of NeuN-ir striatal neurons from 3NP toxicity by AAV2-NTN. These data support the concept that AAV2-NTN might be a valuable treatment for patients with Huntington's disease.

Potato virus Y (PVY) is one of the most important viruses of potato world-wide, several strain groups are recognized. In the past two decades, novel PVY variants have appeared causing necrotic symptoms on potato tubers. Implicated are two groups of recombinant strains: PVY(N)W and PVY(NTN), and NA-PVY(NTN). While the first two are recombinants between PVY-N- and O-strains the latter is a recombinant between an N-strain and an unknown PVY strain or other Potyvirus. Available biological and molecular data on PVY suggest that classification of PVY strains has to be revised. Some drawbacks have been found with recently published primers used in RT-PCR based differentiation of PVY strains as some defined isolates could not be identified correctly. Consequently we developed new primers using both recently available sequences and newly generated complete sequences of PVY strains. The reliability of these newly developed primers and procedures was successfully demonstrated on nearly 100 biologically and serologically characterised PVY isolates.

Penicillin G acylase is an important enzyme in the commercial production of semisynthetic penicillins used to combat bacterial infections. Mutant strains of Providencia rettgeri were generated from wild-type cultures subjected to nutritional selective pressure. One such mutant, Bro1, was able to use 6-bromohexanamide as its sole nitrogen source. Penicillin acylase from the Bro1 strain exhibited an altered substrate specificity consistent with the ability of the mutant to process 6-bromohexanamide. The X-ray structure determination of this enzyme was undertaken to understand its altered specificity and to help in the design of site-directed mutants with desired specificities. In this paper, the structure of the Bro1 penicillin G acylase has been solved at 2.5 A resolution by molecular replacement. The R-factor after refinement is 0.154 and R-free is 0.165. Of the 758 residues in the Bro1 penicillin acylase heterodimer (alpha-subunit, 205; beta-subunit, 553), all but the eight C-terminal residues of the alpha-subunit have been modeled based on a partial Bro1 sequence and the complete wild-type P. rettgeri sequence. A tightly bound calcium ion coordinated by one residue from the alpha-subunit and five residues from the beta-subunit has been identified. This enzyme belongs to the superfamily of Ntn hydrolases and uses Ogamma of Ser beta1 as the characteristic N-terminal nucleophile. A mutation of the wild-type Met alpha140 to Leu in the Bro1 acylase hydrophobic specificity pocket is evident from the electron density and is consistent with the observed specificity change for Bro1 acylase. The electron density for the N-terminal Gln of the alpha-subunit is best modeled by the cyclized pyroglutamate form. Examination of aligned penicillin acylase and cephalosporin acylase primary sequences, in conjunction with the P. rettgeri and Escherichia coli penicillin acylase crystal structures, suggests several mutations that could potentially allow penicillin acylase to accept charged beta-lactam R-groups and to function as a cephalosporin acylase and thus be used in the manufacture of semi-synthetic cephalosporins.

The severity of glomerular injury in the heterologous phase of NTN is dependent on proinflammatory cytokines including TNF alpha and IL-1 beta, and can be enhanced by LPS. We have previously shown that passive immunization against IL-1 beta and TNF partially abrogated the LPS effect in this model. In the present work, we have assessed the effects on glomerular injury of blocking and binding of IL-1 to its receptor by rh IL-1 receptor antagonist (IL-1ra) and by neutralizing IL-1 and TNF with rm soluble IL-1 receptor type1 (sIL-1Rt1) and rh sTNF receptor (sTNFr p55), respectively. Pretreatment with either IL-1ra, sIL-1Rt1, or sTNFr partially abrogated the effects of LPS and reduced albumin excretion from 45 +/- 8, 66 +/- 9, and 101 +/- 17 mg/24 hr at 13 +/- 4 (P < 0.02), 14 +/- 4 (P < 0.001), and 21 +/- 7 mg/24 hr (P < 0.001), respectively. Similarly, these inhibitors reduced the prevalence of glomerular capillary thrombi and the intensity of glomerular neutrophil infiltration. Glomerular thrombosis was reduced from 18 +/- 3%, 28 +/- 5%, and 25 +/- 7% to 3 +/- 2% (P < 0.002), 6 +/- 2% (P < 0.001), and 3 +/- 2 (P < 0.001), respectively, and glomerular neutrophil infiltration was reduced from 46 +/- 3, 54 +/- 2, 59 +/- 8 to 19 +/- 2 (P < 0.001), 25 +/- 2 (P < 0.001), and 28 +/- 2 neutrophils/50 glomeruli in section, respectively. Coadministration of both soluble receptors of IL-1 and TNF caused a further decrease in glomerular injury. The protective effect was also noticed at four hours after induction of nephritis, and even when these inhibitors were administered after the LPS injection and at the same time of induction of nephritis. All three treatments reduced circulating TNF concentration (down to 20%, 34%, and 0%, respectively) but without detectable glomerular TNF gene expression. Glomerular IL-1 beta mRNA levels were also reduced by 41%, 53%, and 67%, respectively, when assessed by densitometric analysis of Northern blots. In contrast, the glomerular expression of IL-1ra was not affected by its exogenous administration but was mildly reduced by sIL-1Rt1 and sTNFr, which demonstrates the potential role for host derived IL-1ra as an endogenous negative feedback mediator in the glomerulus. These results confirm the direct involvement of IL-1 and TNF in LPS-enhanced hNTN and demonstrate the potency of these inhibitors in modulating injury even when administered after LPS and in time of induction of nephritis. They were more specific and effective than passive immunization with polyclonal antibodies, and this demonstrates their potential usefulness in the management of nephritis.

The neurotrophic factor neurturin (NTN) is structurally related to the glial-derived neurotrophic factor (GDNF) and has been shown to prevent the degeneration of dopaminergic neurons both in vitro and in vivo. The preferred receptor for NTN is the GDNF family receptor alpha 2 (GFRalpha-2). To date, three protein-coding alternatively spliced GFRalpha-2 isoforms (GFRalpha-2a, GFRalpha-2b, GFRalpha-2c) have been identified in mammalian tissues. An accurate quantification of the expression levels is necessary when determining the contributions of these isoforms to NTN signaling in tissues. In this report, sequence independent real time RT-PCR is used to determine the expression levels of GFRalpha-2 isoforms at different developmental stages of the murine embryos, and in various adult tissues. In the adult murine brain, GFRalpha-2a was found to be the most abundant, GFRalpha-2c was slightly less and GFRalpha-2b was 10-fold lower. The testis did not appear to express significant levels of GFRalpha-2a, 2b or 2c, compared to the brain. A novel finding in this study is that in some tissues, including the adult brain, the expression levels of GFRalpha-2, as quantified by the amplification of the 3' sequences encoding the putative glycosyl-phosphatidylinositol anchor signal sequence, were significantly higher than the combined levels of GFRalpha-2a, GFRalpha-2b and GFRalpha-2c. This indicates the existence of yet to be identified forms of GFRalpha-2 in some tissues that may be of physiological significance.

Developmentally, semaphorin 3A (sema3A) is an important chemorepellent that guides centrally projecting axons of dorsal root ganglion (DRG) neurons. Sema3A-mediated growth cone collapse can be prevented by cyclic GMP (cGMP) and nerve growth factor (NGF) in embryonic neurons. Sema3A may also play a role in directing regrowth of injured axons in adults, and interactions with neurotrophic factors near the injury site may determine the extent and targeting of both regenerative and aberrant growth. The aim of this study was to determine whether NGF, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) modulate sema3A-mediated growth cone collapse in cultured adult rat DRG neurons. Sema3A caused a significant increase in growth cone collapse, which was completely prevented by prior treatment with NGF, GDNF or NTN. Immunocytochemical experiments showed that sema3A-sensitive neurons were heterogeneous in their expression of neurotrophic factor receptors and responses to neurotrophic factors, raising the possibility of novel, convergent signaling mechanisms between these substances. Increasing cGMP levels caused growth cone collapse, whereas sema3A-mediated collapse was prevented by inhibition of guanylate cyclase or by increasing cyclic AMP levels. In conclusion, sema3A signaling pathways in adult neurons differ to those described in embryonic neurons. Three different neurotrophic factors each completely prevent sema3A-mediated collapse, raising the possibility of novel converging signaling pathways. These studies also show that there is considerable potential for neurotrophic factors to regulate sema3A actions in the adult nervous system. This may provide insights into the mechanisms underling misdirected growth and targeting of sensory fibers within the spinal cord after injury, that is thought to contribute to development of autonomic dysreflexia and neuropathic pain.

The neurorestorative effects of exogenous neurturin (NTN) delivered directly into the putamen via multiport catheters were studied in 10 MPTP-lesioned rhesus monkeys expressing stable parkinsonism. The parkinsonian animals were blindly assigned to receive coded solutions containing either vehicle (n = 5) or NTN (n = 5, 30 microg/day). Both solutions were coinfused with heparin using convection-enhanced delivery for 3 months. The NTN recipients showed a significant and sustained behavioral improvement in their parkinsonian features during the treatment period, an effect not seen in the vehicle-treated animals. At study termination, locomotor activity levels were increased by 50% in the NTN versus vehicle recipients. Also, DOPAC levels were significantly increased by 150% ipsilateral (right) to NTN infusion in the globus pallidus, while HVA levels were elevated bilaterally in the NTN-treated animals by 10% on the left and 67% on the right hemisphere. No significant changes in DA function were seen in the putamen. Volumetric analysis of putamenal NTN labeling showed between-subject variation, with tissue distribution ranging from 214 to 744 mm3, approximately equivalent to 27-93% of area coverage. Our results support the concept that intraparenchymal delivery of NTN protein may be effective for the treatment of PD. More studies are needed to determine strategies that would enhance tissue distribution of exogenous NTN protein, which could contribute to optimize its trophic effects in the parkinsonian brain.

Peptidase family U34 consists of enzymes with unclear catalytic mechanism, for instance, dipeptidase A from Lactobacillus helveticus. Using extensive sequence similarity searches, we infer that U34 family members are homologous to penicillin V acylases (PVA) and thus potentially adopt the N-terminal nucleophile (Ntn) hydrolase fold. Comparative sequence and structural analysis reveals a cysteine as the catalytic nucleophile as well as other conserved residues important for catalysis. The PVA/U34 family is variable in sequence and exhibits great diversity in substrate specificity, to include enzymes such as choloyglycine hydrolases, acid ceramidases, isopenicillin N acyltransferases, and a subgroup of eukaryotic proteins with unclear function.

Glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally homologous, and they seem to produce similar effects in vitro. Tissue distributions of their respective receptors, GFR alpha-1 and GFR alpha-2, reveal overlapping but distinct patterns of expression, which implies that the in vivo actions of GDNF and NTN may be different. In the present study, a direct comparison of the in vivo effects of GDNF and NTN was performed using osmotic minipumps delivering either GDNF or NTN over a 30-day period into rat lateral cerebral ventricles. Amphetamine-induced activity levels were increased in both NTN- and GDNF-treated animals, with higher activity levels achieved by GDNF than NTN. The increase in amphetamine-induced activity levels persisted for 2 weeks and returned to control levels at the end of the third week. NTN-treated rats showed higher dopamine levels in the mediodorsal striatum, relative to the ventrolateral striatum. In contrast, no significant change in the regional distribution of dopamine levels was observed in GDNF treated or control animals. On the other hand, an increase in ventrolateral and mediodorsal striatal dopamine utilization was apparent in GDNF-treated animals, while NTN-treated animals showed increased levels of dopamine utilization only in the ventrolateral striatum. With respect to potential adverse effects, GDNF administration resulted in weight loss and the emergence of allodynia. No weight loss or allodynia was detectable with chronic NTN administration. These results suggest that although GDNF and NTN share structural and functional similarities, they may have differential effects in vivo.

Glial cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) protect retinal ganglion cells (RGCs) from axotomy-induced apoptosis. It is likely that neuroprotection by GDNF or NTN in the adult central nervous system (CNS) involves indirect mechanisms and independent signal transduction events. Extracellular glutamate is a trigger of apoptosis in injured RGCs, and glutamate transporter levels can be upregulated by GDNF. Therefore, GDNF may indirectly protect RGCs by enhancing glutamate uptake in the retina. We studied the upregulation of the glutamate transporters GLAST-1 and GLT-1 by GDNF and NTN, and the intracellular pathways required for GDNF/NTN neuroprotection. GDNF required phosphoinositide-3 kinase (PI3K) and Src activity to upregulate GLAST-1 and GLT-1. NTN required PI3K activity to upregulate GLAST-1 and did not affect GLT-1 levels. PI3K activity was also important for GDNF and NTN neuroprotection following optic nerve transection. However, GDNF also required Src and mitogen-activated protein kinase activity to prevent RGC apoptosis. RNA interference demonstrated that the upregulation of GLAST-1 by GDNF and NTN is required to rescue RGCs. Thus, additional independent signal transduction events, together with the upregulation of GLT-1 by GDNF, differentiate the biological activity of GDNF from NTN. Furthermore, the upregulation of the glial glutamate transporter GLAST-1 by both factors is an indirect neuroprotective mechanism in the CNS.

Immunomodulatory enzyme indoleamine 2, 3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. Via catalysing tryptophan degradation, IDO suppresses adaptive T cell-mediated immunity and plays an important role in various forms of immune tolerance. Its role in T helper type 1 (Th1)-directed, cell-mediated crescentic glomerulonephritis (GN) is still unclear. Therefore, we investigated the activity and role of IDO in crescentic GN using a model of nephrotoxic serum nephritis (NTN), and IDO activity was inhibited by 1-methyl-tryptophan (1-MT) in vivo. Our results showed that activity of IDO, as determined by high performance liquid chromatography analysis of the kynurenine/tryptophan ratio, was increased markedly in the serum and renal tissue of NTN mice, and immunohistochemistry revealed that expression of IDO was up-regulated significantly in glomeruli and renal tubular epithelial cells during NTN. Treatment with 1-MT resulted in significantly exacerbated kidney disease with increased glomerular crescent formation, accumulation of CD4(+)T cells and macrophages in renal tissue, and augmented renal injury compared with phosphate-buffered saline-treated NTN mice, which was associated with enhanced Th1 responses and intrarenal cellular proliferation. These findings suggest that the development of NTN was regulated negatively by increased IDO activity, and IDO might play an important role in the pathogenesis of crescentic GN.

Nitrite, a stable product of nitric oxide (NO), is synthesized in vitro by glomeruli in experimental glomerulonephritis. We have now studied the expression of the gene for inducible NO synthase (iNOS) in accelerated nephrotoxic nephritis (NTN). The purpose of the study was to confirm in vivo induction of iNOS in this model of immune complex disease, and to relate the onset of induction and the level of expression to pathogenic events in the model. Glomeruli from rats with NTN were isolated at 6 h, 24 h and 2, 4 and 7 days and total RNA extracted. RNA (10 micrograms) was reverse transcribed and polymerase chain reaction (PCR) was performed with primers homologous to rat vascular smooth muscle iNOS and rat beta actin. A 222-base PCR product corresponding to iNOS mRNA was present in all experimental animals. iNOS expression was also found in activated macrophages, neutrophils and IL-1-stimulated but not unstimulated mesangial cells. Quantitative competitive PCR was carried out on glomerular samples using a 514-bp mutant of a 735-bp PCR product. iNOS expression was present at low levels in normal glomeruli and was markedly enhanced at 6 h after the induction of glomerulonephritis and peaked at 24 h. Increased iNOS expression persisted to day 7. beta actin mRNA levels were similar in all glomerular specimens. This study demonstrates that there is in vivo induction of iNOS in immune complex glomerulonephritis, corresponding to the generation of nitrite we have previously reported. iNOS gene expression is detectable within 6 h of induction of NTN, indicating the onset of gene transcription is closely related to the initial formation of immune complexes.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are members of the transforming growth factor-beta family and have been shown to elicit neurotrophic effects upon several classes of neurons including dopaminergic neurons, motoneurons, parasympathetic, sympathetic as well as primary sensory neurons. However, there is little information available on their roles in cutaneous innervation. Herein, we have studied the regulation of gdnf, ntn and the GDNF family receptors and examined their role in the development of facial cutaneous innervation in GDNF mutant mice. A dynamic spatial and temporal regulation of gdnf, ntn and their ligand binding receptors within the follicle-sinus complex correlate with development of distinct subclasses of sensory nerve endings. Furthermore, development of NGF-dependent myelinated mechanoreceptors, i.e. reticular and transverse lanceolate endings also require GDNF during ending formation and maintenance. In addition, ligand and receptor association seems to be intricately linked to a local Schwann cell-axon interaction essential for sensory terminal formation. Our results suggests that functionally specified nerve endings depend on different GDNF family members and that in contrast to neurotrophins, this family of neurotrophic factors may be acting at local sites of terminal Schwann cell-axon growth cone interactions and that they collaborate with neurotrophins by supporting the same populations of neurons but at different times in development.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two closely related growth factors reported to selectively act on distinct neuronal populations in the CNS. Both GDNF and NTN signal through a receptor complex consisting of the signal transducing subunit, Ret, and a ligand-specific binding subunit, termed GDNF family receptor (GFR)alpha-1 and GFRalpha-2, respectively. By using RT-PCR, we observed that mRNAs encoding the subunits of both receptor complexes are widely expressed throughout the developing brain, suggesting the presence of targets for these growth factors other than the ones known today. We provide evidence that these targets include glial cells.

miRNA variants (termed isomiRs) have been reported as potential functional molecules that may affect miRNA stability or target selection. The aims of the present study were to comprehensively survey and characterize non-template nucleotides (NTNs) and isomiR repertoires in miRNAs. Over 50 % of the NTNs were located in the 3' ends (also termed 3' additions), followed by the 5' ends and adjacent positions to 5' and 3' ends. The similar distributions of NTNs and isomiR repertoires might be detected between homologous or clustered miRNAs. miRNA might be stably expressed based on the typical analysis, but its isomiRs might be strongly up- or down-regulated. IsomiRs with novel seed sequences were mainly derived from "seed shifting" events in 5' isomiRs, NTNs in 5' ends or in seed sequences. IsomiRs from a miRNA locus or homologous miRNA loci maybe have the same seed sequences, but they would have various enrichment levels and 3' ends. Interestingly, isomiR species with novel seed sequences via NTNs in the seed region were always stably expressed. These novel seed sequences could lead to novel functional roles through driving the potential novel target mRNAs. Integrated predicted target mRNAs and further microarray validation showed that these isomiRs have versatile biological roles. Collectively, multiple isomiR products and miRNA maturation processes provide opportunities to perform versatile roles in the regulatory network, which further enriches and complicates the regulation of biological processes.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and their receptors, GDNF family receptor alpha-1 (GFRalpha-1) and GDNF family receptor alpha-2 (GFRalpha-2), are critically important for kidney and nervous system development. However, their role in skin biology, specifically in hair growth control, is as yet unknown. We have studied expression and function of GDNF, neurturin, GFRalpha-1, and GFRalpha-2 in murine skin during the cyclic transformation of the hair follicle (HF) from its resting state (telogen) to active growth (anagen) and then through regression (catagen) back to telogen. GDNF protein and GFRalpha-1 messenger RNA are prominently expressed in telogen skin, which lacks NTN and GFRalpha-2 transcripts. Early anagen development is accompanied by a significant decline in the skin content of GDNF protein and GFRalpha-1 transcripts. During the anagen-catagen transition, GDNF, GFRalpha-1, NTN, and GFRalpha-2 transcripts reach maximal levels. Compared with wild-type controls, GFRalpha-1 (+/-) and GFRalpha-2 (-/-) knockout mice show a significantly accelerated catagen development. Furthermore, GDNF or NTN administration significantly retards HF regression in organ-cultured mouse skin. This suggests important, previously unrecognized roles for GDNF/GFRalpha-1 and NTN/GFRalpha-2 signaling in skin biology, specifically in the control of apoptosis-driven HF involution, and raises the possibility that GFRalpha-1/GFRalpha-2 agonists/antagonists might become exploitable for the treatment of hair growth disorders that are related to abnormalities in catagen development.

OBJECTIVE

To develop CERE-120 (AAV-NTN) as a novel therapy for Parkinson's disease (PD) that might restore function of degenerating dopamine neurons and prevent further degeneration.

METHODS

A nonclinical program demonstrated that NTN expression can be predictably controlled following CERE-120 administration, provides clear evidence of efficacy in numerous animal models and is safe at dose multiples that far exceed those required for efficacy. Preliminary, open label evidence in PD subjects offers corroborative support for these observations.

CONCLUSIONS

CERE-120 may represent an important, novel therapy for PD, though the clinical data require confirmation with additional clinical tests, including an ongoing multi-center, double-blinded controlled trial.

The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors.

Glutamate synthases play with glutamine synthetase an essential role in nitrogen assimilation processes in microorganisms, plants, and lower animals by catalyzing the net synthesis of one molecule of L-glutamate from L-glutamine and 2-oxoglutarate. They exhibit a modular architecture with a common subunit or region, which is responsible for the L-glutamine-dependent glutamate synthesis from 2-oxoglutarate. Here, a PurF- (Type II- or Ntn-) type amidotransferase domain is coupled to the synthase domain, a (beta/alpha)8 barrel containing FMN and one [3Fe-4S]0,+1 cluster, through a approximately 30 angstroms-long intramolecular tunnel for the transfer of ammonia between the sites. In bacterial and eukaryotic GltS, reducing equivalents are provided by reduced pyridine nucleotides thanks to the stable association with a second subunit or region, which acts as a FAD-dependent NAD(P)H oxidoreductase and is responsible for the formation of the two low potential [4Fe-4S]+1,+2 clusters of the enzyme. In photosynthetic cells, reduced ferredoxin is the physiological reductant. This review focus on the mechanism of cross-activation of the synthase and glutaminase reactions in response to the bound substrates and the redox state of the enzyme cofactors, as well as on recent information on the structure of the alphabeta protomer of the NADPH-dependent enzyme, which sheds light on the intramolecular electron transfer pathway between the flavin cofactors.

Flavobacterium glycosylasparaginase was cloned in an Escherichia coli expression system. Site-directed mutagenesis was performed at residues suggested to be important in the catalytic mechanism based on the crystal structure of the human enzyme and other biochemical studies. In vitro autoproteolysis allowed the mutant enzymes to be activated, including those that were slow to self-cleave. Based on the activity of the mutant enzymes, six catalytically essential amino acids were identified: Trp-11, Asp-66, Thr-152, Thr-170, Arg-180, and Asp-183. Kinetic analysis of each mutant further defined the function of these residues in substrate specificity and reaction rate. Mutagenesis of the N-terminal nucleophile residue Thr-152 confirmed the key function of its side-chain hydroxyl group. Partial activities of mutants T152S/C were in agreement with the general mechanism of N-terminal nucleophile (Ntn)-amidohydrolases. The side-chain hydroxyl of Thr-170 contributes to the reaction rate based on studies of mutants T170S/C/A. Residues Asp-183 and Arg-180 were found to H-bond, respectively, with the charged alpha-amino and alpha-carboxyl group of the substrate (Asn-GlcNAc). Mutants R180Q/L and D183E/N had greatly decreased substrate affinity and reduced reaction rates. Kinetic studies also showed that Trp-11 is involved in regulation of the enzyme reaction rate, contradictory to a previous suggestion that this residue is involved in substrate binding. Asp-66 is a new residue found to be important in enzyme activity. The overall active site structure involving these catalytic residues resembles the glutaminase domain of glucosamine 6-phosphate synthase, another member of the Ntn-amidohydrolase family of enzymes.

Parkinson's disease (PD) is the second most common neurodegenerative disorder marked by cell death in the Substantia nigra (SN). Docosahexaenoic acid (DHA) is the major polyunsaturated fatty acid (PUFA) in the phospholipid fraction of the brain and is required for normal cellular function. Glial cell line derived neurotrophic factor (GDNF) and neurturin (NTN) are very potent trophic factors for PD. The aim of the study was to evaluate the neuroprotective effects of GDNF and NTN by investigating their immunostaining levels after administration of DHA in a model of PD. For this reason we hypothesized that DHA administration of PD might alter GDNF, NTN expression in SN. MPTP neurotoxin that induces dopaminergic neurodegeneration was used to create the experimental Parkinsonism model. Rats were divided into; control, DHA-treated (DHA), MPTP-induced (MPTP), MPTP-induced+DHA-treated (MPTP+DHA) groups. Dopaminergic neuron numbers were clearly decreased in MPTP, but showed an increase in MPTP+DHA group. As a result of this, DHA administration protected dopaminergic neurons as shown by tyrosine hydroxylase immunohistochemistry. In the MPTP+DHA group, GDNF, NTN immunoreactions in dopaminergic neurons were higher than that of the MPTP group. In conclusion, the characterization of GDNF and NTN will certainly help elucidate the mechanism of DHA action, and lead to better strategies for the use of DHA to treat neurodegenerative diseases.

Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the distal gut, resulting in bowel obstruction shortly after birth. Aganglionosis usually affects the distal colon, but may also extensively involve the entire colon and, rarely, the more proximal bowel. Recently, germline mutations of RET, GDNF, and NTN genes have been reported in HSCR. Here we describe the results of mutational analysis of these genes in 15 Japanese child patients with total colonic aganglionosis with small bowel involvement. DNA sequences of all the RET/GDNF/NTN coding regions were determined by the direct dyedeoxy terminator cycle method. Eight different RET mutations were identified in exons 1, 7, 10, 12, 15, and 17 in 10 of the 15 patients. Of these eight mutations, five were found in the tyrosine kinase domain. No GDNF or NTN mutation was found. Compared with typical HSCR, this patient group appeared to exhibit a higher percentage of RET mutations and accumulation of mutations in the tyrosine kinase domain. A homozygous (or hemizygous) RET mutation was found in a male baby with total intestinal aganglionosis, while the heterozygosity of the same mutation resulted in a less severe type of aganglionosis. In familial cases, all heterozygous for the same mutation, aganglionosis was more severe in male than in female siblings. These results also urge us to examine if the RET germline mutation may cause critical alteration of the GDNF/NTN-Ret signal transduction more severely in homo(hemi)zygosity and in male fetuses during organogenesis.

Pseudomonas sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported ntn gene cluster. NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)(+) independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)(+)-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization.