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Valacyclovir, enzymatically hydrolyzed in the body to acyclovir, is a guanine-based nucleoside analog commonly prescribed as an antiviral therapy. Previous reports suggest that guanosine analogs bind to guanine deaminase; however, it is unclear whether they act as inhibitors or substrates. Data from our laboratory suggest that inhibition of guanine deaminase by small molecules attenuates spinal cord injury-induced neuropathic pain. Here, we examine whether the guanosine analogs valacyclovir and acyclovir are deaminated by cypin (cytosolic PSD-95 interactor), the major guanine deaminase in the body, or if they act as cypin inhibitors. Using purified Rattus norvegicus cypin, we use NADH-coupled assay to confirm deamination of valacyclovir and determined Michaelis-Menten constants. Subsequently, we use tryptophan fluorescence quenching assay to calculate dissociation constants for valacyclovir and acyclovir and find that inclusion of the valine motif in valacyclovir increases affinity for cypin compared to acyclovir. To our knowledge, neither K m nor K D values for cypin has been previously reported for either compound. We use Amplex Red assay and demonstrate that both valacyclovir and acyclovir are cypin substrates and that their metabolites are further processed by xanthine oxidase and uricase. Using molecular dynamics simulations, we demonstrate that an alpha helix near the active site is displaced when valacyclovir binds to cypin. Furthermore, we used LC-MS-based assay to directly confirm deamination of valacyclovir by cypin. Taken together, our results demonstrate a novel role for cypin in deamination of valacyclovir and acyclovir and suggest that therapeutics based on purine structures may be inactivated by cypin, decreasing inhibitory efficacy., (© 2024 The Author(s). PROTEINS: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Background: Glucocorticoids are widely known for their immunomodulatory action. Their synthetic analogs are used to treat several autoimmune diseases, including immune thrombocytopenia. However, their efficacy and mechanisms of action in immune thrombocytopenia are not fully understood., Objectives: To investigate the mechanism of glucocorticoid actions on platelet production., Methods: The actions of glucocorticoids on platelet production were studied combining in vivo, ex vivo and in vitro approaches., Results: Dexamethasone reduced bleeding in mice and rapidly increased circulating young platelet counts. In vitro glucocorticoid treatment stimulated proplatelet formation by megakaryocytes and platelet-like particle release. This effect was blocked by glucocorticoid receptor antagonist RU486, indicating a glucocorticoid receptor-dependent mechanism. Genome-wide analysis revealed that dexamethasone regulates the expression of >1000 genes related to numerous cellular functions, including predominant cytoplasm and cytoskeleton reorganization. Dexamethasone and other glucocorticoids induced the expression of Gda (the gene encoding guanine deaminase), which has been reported to have a role in dendrite development. Inhibition of guanine deaminase enzymatic activity blocked dexamethasone stimulation of proplatelet formation, implicating a critical role for this enzyme in glucocorticoid-mediated platelet production., Conclusion: Our findings identify glucocorticoids as new regulators of thrombopoiesis., Competing Interests: Declaration Of Competing Interests There are no competing interests to disclose., (Published by Elsevier Inc.)

Guanine deaminases (GDs) are essential enzymes that regulate the overall nucleobase pool. Since the deamination of guanine to xanthine results in the production of a mutagenic base, these enzymes have evolved to be very specific in nature. Surprisingly, they accept structurally distinct triazine ammeline, an intermediate in the melamine pathway, as one of the moonlighting substrates. Here, by employing NE0047 (a GD from Nitrosomonas europaea), we delineate the nuance in the catalytic mechanism that allows these two distinct substrates to be catalyzed. A combination of enzyme kinetics, X-ray crystallographic, and calorimetric studies reveal that GDs operate via a dual proton shuttle mechanism with two glutamates, E79 and E143, crucial for deamination. Additionally, N66 appears to be central for substrate anchoring and participates in catalysis. The study highlights the importance of closure of the catalytic loop and of maintenance of the hydrophobic core by capping residues like F141 and F48 for the creation of an apt environment for activation of the zinc-assisted catalysis. This study also analyzes evolutionarily distinct GDs and asserts that GDs incorporate subtle variations in the active site architectures while keeping the most critical active site determinants conserved., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Guanine deaminases (GD) are essential enzymes that help in regulating the nucleobase pool. Since the deamination reaction can result in the accumulation of mutagenic bases that can lead to genomic instability, these enzymes are tightly regulated and are nonpromiscuous. Here, we delineate the basis of their substrate fidelity via entailing the reaction mechanism of deamination by employing density functional theory (DFT) calculations on NE0047, a GD from Nitrosomonas europaea . The results show that, unlike pyrimidine deaminases, which require a single glutamic acid as a proton shuttle, GDs involve two amino acids, E79 and E143 (numbering in NE0047), which control its reactivity. The hybrid quantum mechanics/molecular mechanics (QM/MM) calculations have shown that the first Zn-bound proton transfer to the N3 atom of the substrate is mediated by the E79 residue, and the second proton is transferred to the amine nitrogen of substrate via E143. Moreover, cluster models reveal that the crystallographic water molecules near the active site control the reactivity. A comparison with human GD reveals that the proposed catalytic mechanism is generic, and the knowledge generated here can be effectively applied to design selective inhibitors.

Epidermal keratinocytes are considered as the most important neighboring cells that modify melanogenesis. Our previous study used microarray to show that guanine deaminase (GDA) gene expression is highly increased in melasma lesions. Hence, we investigated the role of GDA in skin pigmentation. We examined GDA expression in post-inflammatory hyperpigmentation (PIH) lesions, diagnosed as Riehl's melanosis. We further investigated the possible role of keratinocyte-derived GDA in melanogenesis by quantitative PCR, immunofluorescence staining, small interfering RNA-based GDA knockdown, and adenovirus-mediated GDA overexpression. We found higher GDA positivity in the hyperpigmentary lesional epidermis than in the perilesional epidermis. Both UVB irradiation and stem cell factor (SCF) plus endothelin-1 (ET-1) were used, which are well-known melanogenic stimuli upregulating GDA expression in both keratinocyte culture alone and keratinocyte and melanocyte coculture. GDA knockdown downregulated melanin content, while GDA overexpression promoted melanogenesis in the coculture. When melanocytes were treated with UVB-exposed keratinocyte-conditioned media, the melanin content was increased. Also, GDA knockdown lowered SCF and ET-1 expression levels in keratinocytes. GDA in epidermal keratinocytes may promote melanogenesis by upregulating SCF and ET-1, suggesting its role in skin hyperpigmentary disorders.

DNA damage and oxidative stress play a critical role in photoageing. Seborrhoeic keratosis (SK) affects sunlight-exposed sites in aged individuals. This study examined the mechanism of photoageing in SK. The guanine deaminase gene, which is involved in purine metabolism, was upregulated with uric acid levels and p21 in SK. Guanine deaminase was detectable in keratinocytes. Repeated exposure to ultraviolet (UV) increased levels of guanine deaminase, together with DNA damage, such as γ-H2AX and cyclobutane pyrimidine dimer formation, generation of reactive oxygen species, and keratinocyte senescence, which were reversed by guanine deaminase knockdown. However, guanine deaminase overexpression and H2O2 formed γ-H2AX, but not cyclobutane pyrimidine dimer. Loss-of-function guanine deaminase mutants reduced the metabolic end-product uric acid, which was increased by exposure to exogenous xanthine. Repeated exposure to UV increased levels of uric acid. Exogenous uric acid increased cellular senescence, reactive oxygen species, and γ-H2AX, similar to guanine deaminase. Overall, guanine deaminase upregulation increased UV-induced keratinocyte senescence in SK, via uric acid mediated by reactive oxygen species followed by DNA damage.

Guanine deaminase is a metabolic enzyme, found in all forms of life, which catalyzes the conversion of guanine to xanthine. Despite the availability of several crystal structures, the molecular determinants of substrate orientation and mechanism remain to be elucidated for the amidohydrolase family of guanine deaminase enzymes. Here, we report the crystal structures of Escherichia coli and Saccharomyces cerevisiae guanine deaminase enzymes (EcGuaD and Gud1, respectively), both members of the amidohydrolase superfamily. EcGuaD and Gud1 retain the overall TIM barrel tertiary structure conserved among amidohydrolase enzymes. Both proteins also possess a single zinc cation with trigonal bipyrimidal coordination geometry within their active sites. We also determined a liganded structure of Gud1 bound to the product, xanthine. Analysis of this structure, along with kinetic data of native and site-directed mutants of EcGuaD, identifies several key residues that are responsible for substrate recognition and catalysis. In addition, after a small library of compounds had been screened, two guanine derivatives, 8-azaguanine and 1-methylguanine, were identified as EcGuaD substrates. Interestingly, both EcGuaD and Gud1 also exhibit secondary ammeline deaminase activity. Overall, this work details key structural features of substrate recognition and catalysis of the amidohydrolase family of guanine deaminase enzymes in support of our long-term goal to engineer these enzymes with altered activity and substrate specificity.

As endogenous biomolecules, guanine, guanine-based nucleosides, and nucleotides are essential for cellular DNA/RNA synthesis, energy metabolism, and signal transduction. However, these biomolecules have been found to have a cell-specific antiproliferation effect at higher concentrations, and the mechanism is unclear. In this study, we demonstrate that guanine deaminase (GDA) is a major factor in determining the cell-type selectivity to the antiproliferation effect of guanine-based biomolecules. GDA catalyzes the deamination of guanine to xanthine, which is an essential part of the guanine degradation pathway. GDA deficient cells could not efficiently remove the excess guanine-based biomolecules. These excess molecules disturb the metabolism of adenine-, cytosine-, and thymine-based nucleotides; subsequently inhibit the DNA synthesis and cell growth; and eventually result in the apoptosis/death of GDA deficient cells. The inhibition of DNA synthesis could be relieved by simultaneous addition of adenine- and cytosine-based nucleosides, and the inhibited DNA synthesis could be restarted by post addition of them, which subsequently reduces the antiproliferation effect of guanine-based biomolecules or even totally restores the cell proliferation. These results provide important information for the development of guanine-based drugs or guanine-rich oligonucleotide drugs, as well as for the safety evaluation of food with a high level of guanine-based compounds.

Background: The mammalian guanine deaminase (GDA), called cypin, is important for proper neural development, by regulating dendritic arborization through modulation of microtubule (MT) dynamics. Additionally, cypin can promote MT assembly in vitro. However, it has never been tested whether cypin (or other GDA orthologs) binds to MTs or modulates MT dynamics. Here, we address these questions and characterize Xenopus laevis GDA (Gda) for the first time during embryonic development., Results: We find that exogenously expressed human cypin and Gda both display a cytosolic distribution in primary embryonic cells. Furthermore, while expression of human cypin can promote MT polymerization, Xenopus Gda has no effect. Additionally, we find that the tubulin-binding collapsin response mediator protein (CRMP) homology domain is only partially conserved between cypin and Gda. This likely explains the divergence in function, as we discovered that the cypin region containing the CRMP homology and PDZ-binding domain is necessary for regulating MT dynamics. Finally, we observed that gda is strongly expressed in the kidneys during late embryonic development, although it does not appear to be critical for kidney development., Conclusions: Together, these results suggest that GDA has diverged in function between mammals and amphibians, and that mammalian GDA plays an indirect role in regulating MT dynamics. Developmental Dynamics 248:296-305, 2019. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Cytosolic PSD-95 interactor (cypin), the primary guanine deaminase in the brain, plays key roles in shaping neuronal circuits and regulating neuronal survival. Despite this pervasive role in neuronal function, the ability for cypin activity to affect recovery from acute brain injury is unknown. A key barrier in identifying the role of cypin in neurological recovery is the absence of pharmacological tools to manipulate cypin activity in vivo. Here, we use a small molecule screen to identify two activators and one inhibitor of cypin's guanine deaminase activity. The primary screen identified compounds that change the initial rate of guanine deamination using a colorimetric assay, and secondary screens included the ability of the compounds to protect neurons from NMDA-induced injury and NMDA-induced decreases in frequency and amplitude of miniature excitatory postsynaptic currents. Hippocampal neurons pretreated with activators preserved electrophysiological function and survival after NMDA-induced injury in vitro, while pretreatment with the inhibitor did not. The effects of the activators were abolished when cypin was knocked down. Administering either cypin activator directly into the brain one hour after traumatic brain injury significantly reduced fear conditioning deficits 5 days after injury, while delivering the cypin inhibitor did not improve outcome after TBI. Together, these data demonstrate that cypin activation is a novel approach for improving outcome after TBI and may provide a new pathway for reducing the deficits associated with TBI in patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The guanine deaminase cypin (cytosolic PSD-95 interactor) binds to PSD-95 (postsynaptic density protein 95) and regulates dendrite branching by promoting microtubule polymerization. Here, we identify a novel short isoform of cypin, termed cypinS, which is expressed in mouse and human, but not rat, tissues. Cypin and cypinS mRNA and protein levels peak at P7 and P14 in the mouse brain, suggesting a role for these isoforms during development. Interestingly, although cypinS lacks guanine deaminase activity, overexpression of cypinS increases dendrite branching. This increase occurs further away from soma than do increases resulting from overexpression of cypin. In contrast, overexpression of cypin, but not cypinS, decreases dendritic spine density and maturity. This suggests that changes to spines, but not to dendrites, may be dependent on guanine deaminase activity. Furthermore, overexpression of either cypin or cypinS increases miniature excitatory postsynaptic current (mEPSC) frequency, pointing to a presynaptic role for both isoforms. Interestingly, overexpression of cypinS results in a significantly greater increase in frequency than does overexpression of cypin. Thus, cypin and cypinS play distinct roles in neuronal development.

Accumulating evidence has suggested that circular RNAs (circRNAs) play important roles in oncogenesis and tumor progression. However, our knowledge of circRNAs in gastric cancer (GC) remains limited. To investigate circRNAs involved in GC oncogenesis, we examined differentially-expressed circRNAs and mRNAs in GC tissues and paired noncancerous mucosa tissues using circRNA and mRNA microarrays. Next, we built gene co-expression networks according to the degree of correlation to predict the critical circRNAs in GC. Through bioinformatics analysis, we observed three newly identified circRNAs that are substantially upregulated in GC: hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15. Additionally, hsa_circ_0047905 and hsa_circ_0138960 positively correlated with their parental gene mRNA. Knockdown of hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 in GC cells, resulted in downregulation of parental gene expression. Functional assays suggested that inhibition of these three circular RNAs suppresses GC cell proliferation and invasion in vitro. Those findings suggest that hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 might act as tumor promoters in the pathogenesis of gastric cancer.

Bariatric surgery alleviates obesity and ameliorates glucose tolerance. Using metabolomic and proteomic profiles, we evaluated metabolic changes in serum and liver tissue after duodenal-jejunal bypass (DJB) surgery in rats fed a normal chow diet. We found that the levels of vitamin B 12 in the sera of DJB rates were decreased. In the liver of DJB rats, betaine-homocysteine S-methyltransferase levels were decreased, whereas serine, cystathionine, cysteine, glutathione, cystathionine β-synthase, glutathione S-transferase, and aldehyde dehydrogenase levels were increased. These results suggested that DJB surgery enhanced trans-sulfuration and its consecutive reactions such as detoxification and the scavenging activities of reactive oxygen species. In addition, DJB rats showed higher levels of purine metabolites such as ATP, ADP, AMP, and inosine monophosphate. Decreased guanine deaminase, as well as lower levels of hypoxanthine, indicated that DJB surgery limited the purine degradation process. In particular, the AMP/ATP ratio and phosphorylation of AMP-activated protein kinase increased after DJB surgery, which led to enhanced energy production and increased catabolic pathway activity, such as fatty acid oxidation and glucose transport. This study shows that bariatric surgery altered trans-sulfuration and purine metabolism in the liver. Characterization of these mechanisms increases our understanding of the benefits of bariatric surgery., (Copyright © 2016 the American Physiological Society.)

HPV infection has been identified recently as the causative agent of a subset of squamous cell carcinomas arising in oropharyngeal tonsils. Factors influencing the susceptibility of tonsillar epithelium to HPV-induced oncogenesis are far from being elucidated. A 5-protein signature including cytokeratin (CK)7, anterior gradient (AGR)2, cluster differentiation (CD)63, matrix metalloproteinase (MMP)7, and guanine deaminase (GDA) has recently been found to identify a residual embryonic cell population in the squamocolumnar (SC) junction of the cervix, susceptible to HPV infection, and cancers originating from these cells. The expression of SC junction markers was investigated with immunohistochemistry in normal tonsils and in oropharyngeal carcinomas (OPC) fully characterised for HPV. All markers were constantly expressed in the reticulated epithelial cells of the tonsillar crypts, with variable diffusion and intensity; in OPC, positivity was observed in 36,5%, 29,2%, 39%, 17%, and 25% of cases with respectively AGR2, CK7, GDA, CD63, and MMP7 antibodies. No OPC was positive for all markers; 6 were completely negative. AGR2 and CK7 showed significant association with tumor- and HPV-related parameters. AGR2 expression was associated with tumor origin in the tongue base (p=0.013); CK7 was associated with non-keratinising morphology (p=0.013). p16 tumor cell expression was associated with AGR2 (p=0.021); transcriptionally active HPV infection was associated with AGR2 and CK7 (p=0.024 and 0.043). Expression of SC junction markers in tonsillar crypt cells might be related to the embryological development of tonsillar structures; their partial association with HPV oncogenic infection could help to identify HPV-susceptible cells and related OPC.

Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods., (© 2014 S. Karger AG, Basel.)

Guanine deaminases (GDs) are important enzymes involved in purine metabolism as well as nucleotide anabolism pathways that exhibit a high degree of fidelity. Here, the structural basis of the substrate specificity of GDs was investigated by determining a series of X-ray structures of NE0047 (GD from Nitrosomonas europaea) with nucleobase analogues and nucleosides. The structures demonstrated that the interactions in the GD active site are tailor-made to accommodate only guanine and any substitutions in the purine ring or introduction of a pyrimidine ring results in rearrangement of the bases in a catalytically unfavorable orientation, away from the proton shuttling residue E143. In addition, X-ray structural studies performed on cytidine revealed that although it binds in an optimal conformation, its deamination does not occur because of the inability of the enzyme to orchestrate the closure of the catalytically important C-terminal loop (residues 181-189). Isothermal calorimetry measurements established that these nucleoside moieties also disrupt the sequential mode of ligand binding, thereby abrogating all intersubunit communication. Intriguingly, it was recently discovered that GDs can also serve as endogenous ammeline deaminases, although it is structurally nonhomologous with guanine. To understand the mechanism of dual-substrate specificity, the structure of NE0047 in complex with ammeline was determined to a resolution of 2.7 Å. The structure revealed that ammeline not only fits in the active site in a catalytically favorable orientation but also allows for closure of the C-terminal loop.

Guanase is an important enzyme of the purine salvage pathway of nucleic acid metabolism and its inhibition has beneficial implications in viral, bacterial, and cancer therapy. The work described herein is based on a hypothesis that azepinomycin, a heterocyclic natural product and a purported transition state analog inhibitor of guanase, does not represent the true transition state of the enzyme-catalyzed reaction as closely as does iso-azepinomycin, wherein the 6-hydroxy group of azepinomycin has been translocated to the 5-position. Based on this hypothesis, and assuming that iso-azepinomycin would bind to guanase at the same active site as azepinomycin, several analogs of iso-azepinomycin were designed and successfully synthesized in order to gain a preliminary understanding of the hydrophobic and hydrophilic sites surrounding the guanase binding site of the ligand. Specifically, the analogs were designed to explore the hydrophobic pockets, if any, in the vicinity of N1, N3, and N4 nitrogen atoms as well as O(5) oxygen atom of iso-azepinomycin. Biochemical inhibition studies of these analogs were performed using a mammalian guanase. Our results indicate that (1) increasing the hydrophobicity near O(5) results in a negative effect, (2) translocating the hydrophobicity from N3 to N1 also results in decreased inhibition, (3) increasing the hydrophobicity near N3 or N4 produces significant enhancement of inhibition, (4) increasing the hydrophobicity at either N3 or N4 with a simultaneous increase in hydrophobicity at O(5) considerably diminishes any gain in inhibition made by solely enhancing hydrophobicity at N3 or N4, and (5) finally, increasing the hydrophilic character near N3 has also a deleterious effect on inhibition. The most potent compound in the series has a Ki value of 8.0±1.5μM against rabbit liver guanase., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

NE0047 from Nitrosomonas europaea has been annotated as a zinc-dependent deaminase; however, the substrate specificity is unknown because of the low level of structural similarity and sequence identity compared to other family members. In this study, the function of NE0047 was established as a guanine deaminase (catalytic efficiency of 1.2 × 10(5) M(-1) s(-1)), exhibiting secondary activity towards ammeline. The structure of NE0047 in the presence of the substrate analogue 8-azaguanine was also determined to a resolution of 1.9 Å. NE0047 crystallized as a homodimer in an asymmetric unit. It was found that the extreme nine-amino acid C-terminal loop forms an active site flap; in one monomer, the flap is in the closed conformation and in the other in the open conformation with this loop region exposed to the solvent. Calorimetric data obtained using the full-length version of the enzyme fit to a sequential binding model, thus supporting a cooperative mode of ligand occupancy. In contrast, the mutant form of the enzyme (ΔC) with the deletion of the extreme nine amino acids follows an independent model of ligand occupancy. In addition, the ΔC mutant also does not exhibit any enzyme activity. Therefore, we propose that the progress of the reaction is communicated via changes in the conformation of the C-terminal flap and the closed form of the enzyme is the catalytically active form, while the open form allows for product release. The catalytic mechanism of deamination was also investigated, and we found that the mutagenesis of the highly conserved active site residues Glu79 and Glu143 resulted in a complete loss of activity and concluded that they facilitate the reaction by serving as proton shuttles.

In our long and broad program to explore structure-activity relationships of the natural product azepinomycin and its analogues for inhibition of guanase, an important enzyme of purine salvage pathway of nucleic acid metabolism, it became necessary to investigate if the nucleoside analogues of the heterocycle azepinomycin, which are likely to be formed in vivo, would be more or less potent than the parent heterocycle. To this end, we have resynthesized both azepinomycin (1) and its two diastereomeric nucleoside analogues (2 and 3), employing a modified, more efficient procedure, and have biochemically screened all three compounds against a mammalian guanase. Our results indicate that the natural product is at least 200 times more potent toward inhibition of guanase as compared with its nucleoside analogues, with the observed K(i) of azepinomycin (1) against the rabbit liver guanase=2.5 (±0.6)×10(-6) M, while K(i) of Compound 2=1.19 (±0.02)×10(-4) M and that of Compound 3=1.29 (±0.03)×10(-4) M. It is also to be noted that while IC(50) value of azepinomycin against guanase in cell culture has long been reported, no inhibition studies nor K(i) against a pure mammalian enzyme have ever been documented. In addition, we have, for the first time, determined the absolute stereochemistry of the 6-OH group of 2 and 3 using conformational analysis coupled with 2-D (1)H NMR NOESY., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of "off target effects." However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔT(agg) around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design.

Focal swelling or varicosity formation in dendrites and loss of dendritic spines are the earliest indications of glutamate-induced excitotoxicity. Although it is known that microtubule dynamics play a role in varicosity formation, very little is known about the proteins that directly impact microtubules during focal swelling and dendritic spine loss. Our laboratory has recently reported that the postsynaptic protein PSD-95 and its cytosolic interactor (cypin) regulate the patterning of dendrites in hippocampal neurons. Cypin promotes microtubule assembly, and PSD-95 disrupts microtubule organization. Thus, we hypothesized that cypin and PSD-95 may play a role in altering dendrite morphology and spine number in response to sublethal NMDA-induced excitotoxicity. Using an in vitro model of glutamate-induced toxicity in rat hippocampal cultures, we found that cypin overexpression or PSD-95 knockdown increases the percentage of neurons with varicosities and the number of varicosities along dendrites, decreases the size of varicosities after sublethal NMDA exposure, and protects neurons from NMDA-induced death. In contrast, cypin knockdown or PSD-95 overexpression results in opposite effects. We further show that cypin regulates the density of spines/filopodia: cypin overexpression decreases the number of protrusions per micrometer of dendrite while cypin knockdown results in an opposite effect. Cypin overexpression and PSD-95 knockdown attenuate NMDA-promoted decreases in protrusion density. Thus, we have identified a novel pathway by which the microtubule cytoskeleton is regulated during sublethal changes to dendrites.

Alterations in dendrite branching and morphology are present in many neurodegenerative diseases. These variations disrupt postsynaptic transmission and affect neuronal communication. Thus, it is important to understand the molecular mechanisms that regulate dendritogenesis and how they go awry during disease states. Previously, our laboratory showed that cypin, a mammalian guanine deaminase, increases dendrite number when overexpressed and decreases dendrite number when knocked down in cultured hippocampal neurons. Here, we report that exposure to brain-derived neurotrophic factor (BDNF), an important mediator of dendrite arborization, for 72 h but not for 24 h or less increases cypin mRNA and protein levels in rat hippocampal neurons. BDNF signals through cypin to regulate dendrite number, since knocking down cypin blocks the effects of BDNF. Furthermore, BDNF increases cypin levels via mitogen-activated protein kinase and transcription-dependent signaling pathways. Moreover, the cypin promoter region contains putative conserved cAMP response element (CRE) regions, which we found can be recognized and activated by CRE-binding protein (CREB). In addition, exposure of the neurons to BDNF increased CREB binding to the cypin promoter and, in line with these data, expression of a dominant negative form of CREB blocked BDNF-promoted increases in cypin protein levels and proximal dendrite branches. Together, these studies suggest that BDNF increases neuronal cypin expression by the activation of CREB, increasing cypin transcription leading to increased protein expression, thus identifying a novel pathway by which BDNF shapes the dendrite network.

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k(cat) = 49 s(-1), K(m) = 72 μM, and k(cat)/K(m) = 6.7 × 10(5) M(-1) s(-1). The kinetic constants for the deamination of cytosine are as follows: k(cat) = 45 s(-1), K(m) = 302 μM, and k(cat)/K(m) = 1.5 × 10(5) M(-1) s(-1). Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.

Purines can be used as the sole source of nitrogen by several strains of K. pneumoniae under aerobic conditions. The genes responsible for the assimilation of purine nitrogens are distributed in three separated clusters in the K. pneumoniae genome. Here, we characterize the cluster encompassing genes KPN_01787 to KPN_01791, which is involved in the conversion of allantoin into allantoate and in the deamination of guanine to xanthine. These genes are organized in three transcriptional units, hpxSAB, hpxC, and guaD. Gene hpxS encodes a regulatory protein of the GntR family that mediates regulation of this system by growth on allantoin. Proteins encoded by hpxB and guaD display allantoinase and guanine deaminase activity, respectively. In this cluster, hpxSAB is the most tightly regulated unit. This operon was activated by growth on allantoin as a nitrogen source; however, addition of allantoin to nitrogen excess cultures did not result in hpxSAB induction. Neither guaD nor hpxC was induced by allantoin. Expression of guaD is mainly regulated by nitrogen availability through the action of NtrC. Full induction of hpxSAB by allantoin requires both HpxS and NAC. HpxS may have a dual role, acting as a repressor in the absence of allantoin and as an activator in its presence. HpxS binds to tandem sites, S1 and S2, overlapping the -10 and -35 sequences of the hpxSAB promoter, respectively. The NAC binding site is located between S1 and S2 and partially overlaps S2. In the presence of allantoin, interplay between NAC and HpxS is proposed.

Synthesis and biochemical inhibition studies of a novel transition state analog inhibitor of guanase bearing the ring structure of azepinomycin have been reported. The compound was synthesized in five-steps from a known compound and biochemically screened against the rabbit liver guanase. The compound exhibited competitive inhibition profile with a K(i) of 16.7±0.5μM., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Guanine deaminase (GDA; cypin) is an important metalloenzyme that processes the first step in purine catabolism, converting guanine to xanthine by hydrolytic deamination. In higher eukaryotes, GDA also plays an important role in the development of neuronal morphology by regulating dendritic arborization. In addition to its role in the maturing brain, GDA is thought to be involved in proper liver function since increased levels of GDA activity have been correlated with liver disease and transplant rejection. Although mammalian GDA is an attractive and potential drug target for treatment of both liver diseases and cognitive disorders, prospective novel inhibitors and/or activators of this enzyme have not been actively pursued. In this study, we employed the combination of protein structure analysis and experimental kinetic studies to seek novel potential ligands for human guanine deaminase. Using virtual screening and biochemical analysis, we identified common small molecule compounds that demonstrate a higher binding affinity to GDA than does guanine. In vitro analysis demonstrates that these compounds inhibit guanine deamination, and more surprisingly, affect GDA (cypin)-mediated microtubule assembly. The results in this study provide evidence that an in silico drug discovery strategy coupled with in vitro validation assays can be successfully implemented to discover compounds that may possess therapeutic value for the treatment of diseases and disorders where GDA activity is abnormal., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Different somatic motor neuron subpopulations show a differential vulnerability to degeneration in diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy. Studies in mutant superoxide dismutase 1 over-expressing amyotrophic lateral sclerosis model mice indicate that initiation of disease is intrinsic to motor neurons, while progression is promoted by astrocytes and microglia. Therefore, analysis of the normal transcriptional profile of motor neurons displaying differential vulnerability to degeneration in motor neuron disease could give important clues to the mechanisms of relative vulnerability. Global gene expression profiling of motor neurons isolated by laser capture microdissection from three anatomical nuclei of the normal rat, oculomotor/trochlear (cranial nerve 3/4), hypoglossal (cranial nerve 12) and lateral motor column of the cervical spinal cord, displaying differential vulnerability to degeneration in motor neuron disorders, identified enriched transcripts for each neuronal subpopulation. There were striking differences in the regulation of genes involved in endoplasmatic reticulum and mitochondrial function, ubiquitination, apoptosis regulation, nitrogen metabolism, calcium regulation, transport, growth and RNA processing; cellular pathways that have been implicated in motor neuron diseases. Confirmation of genes of immediate biological interest identified differential localization of insulin-like growth factor II, guanine deaminase, peripherin, early growth response 1, soluble guanylate cyclase 1A3 and placental growth factor protein. Furthermore, the cranial nerve 3/4-restricted genes insulin-like growth factor II and guanine deaminase protected spinal motor neurons from glutamate-induced toxicity (P < 0.001, ANOVA), indicating that our approach can identify factors that protect or make neurons more susceptible to degeneration.

An enzyme from Pseudomonas aeruginosa, Pa0142 (gi|9945972), that is able to catalyze the deamination of 8-oxoguanine (8-oxoG) to uric acid has been identified for the first time. 8-Oxoguanine is formed by the oxidation of guanine residues within DNA by reactive oxygen species, and this lesion results in G:C to T:A transversions. The value of k(cat)/K(m) for the deamination of 8-oxoG by Pa0142 at pH 8.0 and 30 degrees C is 2.0 x 10(4) M(-1) s(-1). This enzyme can also catalyze the deamination of isocystosine and guanine at rates that are approximately an order of magnitude lower. The three-dimensional structure of a homologous enzyme (gi|44264246) from the Sargasso Sea has been determined by X-ray diffraction methods to a resolution of 2.2 A (PDB entry). The enzyme folds as a (beta/alpha)(8) barrel and is a member of the amidohydrolase superfamily with a single zinc in the active site. This enzyme catalyzes the deamination of 8-oxoG with a k(cat)/K(m) value of 2.7 x 10(5) M(-1) s(-1). Computational docking of potential high-energy intermediates for the deamination reaction to the X-ray crystal structure suggests that active-site binding of 8-oxoG is facilitated by hydrogen-bond interactions from a conserved glutamine that follows beta-strand 1 with the carbonyl group at C6, a conserved tyrosine that follows beta-strand 2 with N7, and a conserved cysteine residue that follows beta-strand 4 with the carbonyl group at C8. A bioinformatic analysis of available protein sequences suggests that approximately 200 other bacteria possess an enzyme capable of catalyzing the deamination of 8-oxoG.

Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.

Aim: To assess the diagnosis of the activity of a pathological process in patients with ankylosing spondyloarthritis (AS) and to reveal purine metabolic (PM) changes in relation to the clinical features of AS., Materials and Methods: The serum activities of the PM enzymes: xanthine oxidase (XO), guanine deaminase (GDA), guanosine deaminase (GSDA), purine nucleoside phosphorylase (PNP), guanosine phosphorylase (GP), and adenosine deaminase (ADA) were determined in 55 patients (51 males and 4 females) aged 36.0 +/- 1.4 years). A control group comprised 30 apparently age- and gender-matched healthy individuals, as in the study group., Results: On admission, the patients were found to have increased XO, GDA, PNP, and GD activities and decreased GSDA activity. The higher activity of the process was observed, the greater activities of GDA, XO, PNP, GP and the lower activity of GSDA and ADA were. There were the enzymatic activity differences that depended on the degree of pathological process activity, clinical form, the magnitude of X-ray changes, and the degree of joint functional insufficiency., Conclusion: The findings suggest that there may be PM disturbances in the immunocompetent cells.

Dihydropterin deaminase, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was purified 5850-fold to apparent homogeneity from Drosophila melanogaster. Its molecular mass was estimated to be 48 kDa by gel filtration and SDS-PAGE, indicating that it is a monomer under native conditions. The pI value, temperature, and optimal pH of the enzyme were 5.5, 40 degrees C, and 7.5, respectively. Interestingly the enzyme had much higher activity for guanine than for 7,8-dihydropterin. The specificity constant (k(cat)/K(m)) for guanine (8.6 x 10(6) m(-1).s(-1)) was 860-fold higher than that for 7,8-dihydropterin (1.0 x 10(4) m(-1).s(-1)). The structural gene of the enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis as CG18143, located at region 82A1 on chromosome 3R. The cloned and expressed CG18143 exhibited both 7,8-dihydropterin and guanine deaminase activities. Flies with mutations in CG18143, SUPor-P/Df(3R)A321R1 transheterozygotes, had severely decreased activities in both deaminases compared with the wild type. Among several red eye pigments, the level of aurodrosopterin was specifically decreased in the mutant, and the amount of xanthine and uric acid also decreased considerably to 76 and 59% of the amounts in the wild type, respectively. In conclusion, dihydropterin deaminase encoded by CG18143 plays a role in the biosynthesis of aurodrosopterin by providing one of its precursors, 7,8-dihydrolumazine, from 7,8-dihydropterin. Dihydropterin deaminase also functions as guanine deaminase, an important enzyme for purine metabolism.

The purine contents of alcoholic beverages were determined in order to utilize them in the dietary care of gout and hyperuricemia. In the management of these diseases, restriction of both alcohol and purine intake are important. The method employed in this study is a quantitative determination of purine contents by HPLC. Alcoholic beverages were hydrolyzed to corresponding purine bases, which were then separated by HPLC, and base peaks were identified using an enzymatic peak-shift technique. This method is sufficiently accurate and reproducible to examine the purine contents of various alcoholic beverages that patients consume. Purine contents were as follows: spirits, 0.7-26.4 micromol/L; regular beer, 225.0-580.2 micromol/L; low-malt beer, 193.4-267.9 micromol/L; low-malt and low-purine beer, 13.3 micromol/L; other liquors, 13.1-818.3 micromol/L. Some local and low-alcohol beers were found to contain about 2.5 times more purines than regular beer. As some alcoholic beverages contain considerable amounts of purines, we recommend that excess consumption of these beverages be avoided. These data should be useful in the management of hyperuricemia and gout, not only for patients but also for physicians., ((c) 2009 John Wiley & Sons, Ltd.)

Altering the specificity of an enzyme requires precise positioning of side-chain functional groups that interact with the modified groups of the new substrate. This requires not only sequence changes that introduce the new functional groups but also sequence changes that remodel the structure of the protein backbone so that the functional groups are properly positioned. We describe a computational design method for introducing specific enzyme-substrate interactions by directed remodeling of loops near the active site. Benchmark tests on 8 native protein-ligand complexes show that the method can recover native loop lengths and, often, native loop conformations. We then use the method to redesign a critical loop in human guanine deaminase such that a key side-chain interaction is made with the substrate ammelide. The redesigned enzyme is 100-fold more active on ammelide and 2.5e4-fold less active on guanine than wild-type enzyme: The net change in specificity is 2.5e6-fold. The structure of the designed protein was confirmed by X-ray crystallographic analysis: The remodeled loop adopts a conformation that is within 1-A Calpha RMSD of the computational model.

The enterobacterium Klebsiella oxytoca uses a variety of inorganic and organic nitrogen sources, including purines, nitrogen-rich compounds that are widespread in the biosphere. We have identified a 23-gene cluster that encodes the enzymes for utilizing purines as the sole nitrogen source. Growth and complementation tests with insertion mutants, combined with sequence comparisons, reveal functions for the products of these genes. Here, we report our characterization of 12 genes, one encoding guanine deaminase and the others encoding enzymes for converting (hypo)xanthine to allantoate. Conventionally, xanthine dehydrogenase, a broadly distributed molybdoflavoenzyme, catalyzes sequential hydroxylation reactions to convert hypoxanthine via xanthine to urate. Our results show that these reactions in K. oxytoca are catalyzed by a two-component oxygenase (HpxE-HpxD enzyme) homologous to Rieske nonheme iron aromatic-ring-hydroxylating systems, such as phthalate dioxygenase. Our results also reveal previously undescribed enzymes involved in urate oxidation to allantoin, catalyzed by a flavoprotein monooxygenase (HpxO enzyme), and in allantoin conversion to allantoate, which involves allantoin racemase (HpxA enzyme). The pathway also includes the recently described PuuE allantoinase (HpxB enzyme). The HpxE-HpxD and HpxO enzymes were discovered independently by de la Riva et al. (L. de la Riva, J. Badia, J. Aguilar, R. A. Bender, and L. Baldoma, J. Bacteriol. 190:7892-7903, 2008). Thus, several enzymes in this K. oxytoca purine utilization pathway differ from those in other microorganisms. Isofunctional homologs of these enzymes apparently are encoded by other species, including Acinetobacter, Burkholderia, Pseudomonas, Saccharomyces, and Xanthomonas.

Deaminases are enzymes that catalyze the hydrolysis of amino groups of nucleosides or their bases. Because these enzymes play important roles in nucleotide metabolism, they are relevant targets in anticancer and antibacterial therapies. Mammalian deaminases are commercially available but the use of bacterial whole cells, especially as biocatalysts, is continuously growing because of their economical benefits. Moreover, deaminases are useful for the preparative chemoenzymatic transformation of nucleoside and base analogues into a variety of derivatives. The purine deaminase activities of Arthrobacter oxydans, a gram-positive bacterium utilized widely in bioremediation, were studied. The presence of adenosine, adenine and guanine deaminases was demonstrated and some purine bases and nucleosides were analyzed as substrates. Using A. oxydans whole cells as the biocatalyst, different purine compounds such as the anti-HIV, 2',3'-dideoxyinosine (73%, 2 h) were obtained.

Dendrite morphology regulates how a postsynaptic neuron receives information from presynaptic neurons. The specific patterning of dendrite branches is promoted by extrinsic and intrinsic factors that trigger the activation of functional signaling pathways. However, most of the regulating factors and the biochemical mechanisms involved in regulating dendrite branching are unknown. Our laboratory previously reported that cypin (cytosolic PSD-95 interactor) plays an active role in regulating dendrite branching in hippocampal neurons. Cypin-promoted increases in dendrite number are dependent on guanine deaminase activity. In order to identify the specific structural role of zinc-binding in cypin-mediated dendrite branching and guanine deaminase activity, we employed computational homology modeling techniques to construct a three dimensional structural model of cypin. Analysis of the protein-ion sequestration scaffold of this model identified several histidines and aspartic acid residues responsible for zinc binding. Single substitution mutations in these specific sites completely disrupted the guanine deaminase enzymatic activity and rendered cypin unable to promote dendrite branching in rat hippocampal neurons. The specific zinc ion-binding function of each residue in the protein scaffold was also confirmed by Inductively Coupled Plasma-Optic Emission Spectrometry. Inspection of our structural model confirmed that His82 and His84 coordinate with the zinc ion, together with His240, His279, and Asp330, residues that until now were unknown to play a role in this regard. Furthermore, promotion of dendrite branching by cypin is zinc-dependent., ((c) 2007 Wiley-Liss, Inc.)

The way a dendrite is patterned determines how a neuron will receive information. The Rho GTPases have been reported to play increasingly well defined roles in determining dendritic branch and spine development and morphology. Much is known about how these small GTPases regulate the actin cytoskeleton; however, very little is known about how they alter the microtubule cytoskeleton. Our laboratory previously cloned and characterized cypin, a guanine deaminase that increases dendrite number by binding to tubulin heterodimers and promoting microtubule assembly. In the present study, we show that cypin and RhoA are part of a common pathway that regulates dendrite number. Inhibition of Rho kinase activity does not affect cypin-mediated dendrite branching. Furthermore, cypin does not affect the activity of RhoA, as measured by GTP binding to RhoA. In fact, activated RhoA acts to inhibit cypin protein expression and, by doing so, decreases dendrite number. In addition, this decrease in cypin protein occurs via a translation-dependent mechanism. Together, our data suggest that cypin acts downstream of the small GTPase RhoA to regulate dendrite branching in hippocampal neurons, providing a novel mechanism for RhoA action on microtubule dynamics.

The catalytic mechanism of Bacillus subtilis guanine deaminase (bGD), a Zn metalloenzyme, has been investigated by a combination of quantum mechanical calculations using the multilayered ONIOM method and molecular dynamics simulations. In contrast to a previously proposed catalytic mechanism, which requires the bound guanine to assume a rare tautomeric state, the ONIOM calculations showed that the active-site residues of the enzyme do not affect the tautomeric state of guanine, and consequently the bound guanine is a tautomer that is the most abundant in aqueous solution. Two residues, Glutamate 55 and Aspartate 114, were found to play important roles in proton shuttling in the reaction. The proposed reaction path is initiated by proton transfer from a Zn-bound water to protonate Asp114. This process may be quite complex and rather dynamic in nature, as revealed by the molecular dynamics (MD) simulations, whereby another water may bridge the Zn-bound water and Asp114, which then is eliminated by positioning of guanine in the active site. The binding of guanine stabilizes protonated Asp114 by hydrogen bond formation. Asp114 can then transfer its proton to the N3 of the bound guanine, facilitating the nucleophilic attack on C2 of the guanine by the Zn-bound hydroxide to form a tetrahedral intermediate. This occurs with a rather low barrier. Glu55 then transfers a proton from the Zn-hydroxide to the amino group of the reaction intermediate and, at this point, the C2-N2 bond has lengthened by 0.2 A compared to guanine, making C2-N2 bond cleavage more facile. The C2-N2 bond breaks forming ammonia, with an energy barrier of approximately 8.8 kcal/mol. Ammonia leaves the active site, and xanthine is freed by the cleavage of the Zn-O2 bond, with a barrier approximately 8.4 kcal/mol. Along this reaction path, the highest barrier comes from C2-N2 bond cleavage, while the barrier from the cleavage of the Zn-O2 bond is slightly smaller. The Zn-O2 bond can be broken without the assistance of water during the release of xanthine.

Human guanase is known as a specific enzyme in the liver, kidney, and brain. However, its functional significance remains poorly understood. In addition, interestingly, a different organ distribution between humans and rats was suggested. Here, we performed immunohistochemical staining with anti-human nedasin (neuronal and endocrine discs large/SAP102 associated protein), whose sequence was identical to that of guanase, antibody and histochemical staining for guanase in normal tissues of rat and human liver, kidney, and small intestine, and compared the results. Guanase activity was observed uniformly in the rat hepatocytes, biliary epithelium and vascular endothelium cells, while it was localized to the hepatocytes and biliary epithelium in the human liver. When the histochemical staining for guanase and the immunohistochemical staining for nedasin were compared, the stained regions were different in the rat liver but were almost consistent in all human tissues. Totally consistent staining results were also obtained between rats and humans in the other organization except the liver. Based upon the research reports to date, the experiments on guanase and nedasin in rat organs performed in this study are considered to have important implications in the investigation of their physiological significance.

Guanase is known as an enzyme released from the liver. Recently, cloning and sequencing of the guanase gene were reported. In addition, almost simultaneously, it was reported that an unknown protein that binds to neuronal and endocrine lethal(1)-discs large (NE-dlg), one of the membrane-associated guanylate kinase homologues (MAGUK) family proteins involved in synaptic connection between neurons, was cloned and named nedasin (NE-dlg associated protein), whose sequence was almost identical to that of guanase. We immunostained fresh frozen sections of surgically removed human liver, kidney, and small intestine with anti-nedasin antibody, and simultaneously performed histochemical staining for guanase for comparison. Histochemically, guanase activity was observed in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the mucosal epithelium on the small intestine, and in the proximal tubule on the kidney. Immunohistochemically, a brown discoloration due to DAB oxidation was seen in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the proximal tubule but in the distal tubule a little on the kidney, in the mucosal epithelium on the small intestine. The stained region of the liver and the small intestine were different from that of the kidney. The different staining properties dependent on the organs were considered to be due to different isozymes. The physiological significance of guanase may vary with the isozymes, further studies are considered necessary.

Temporal and spatial assembly of signal transduction machinery determines dendrite branch patterning, a process crucial for proper synaptic transmission. Our laboratory previously cloned and characterized cypin, a protein that decreases PSD-95 family member localization and regulates dendrite number. Cypin contains zinc binding, collapsin response mediator protein (CRMP) homology, and PSD-95, Discs large, zona occludens-1 binding domains. Both the zinc binding and CRMP homology domains are needed for dendrite patterning. In addition, cypin binds tubulin via its CRMP homology domain to promote microtubule assembly. Using a yeast two-hybrid screen of a rat brain cDNA library with cypin lacking the carboxyl terminal eight amino acids as bait, we identified snapin as a cypin binding partner. Here, we show by affinity chromatography and coimmunoprecipitation that the carboxyl-terminal coiled-coil domain (H2) of snapin is required for cypin binding. In addition, snapin binds to cypin's CRMP homology domain, which is where tubulin binds. We also show that snapin competes with tubulin for binding to cypin, resulting in decreased microtubule assembly. Subsequently, overexpression of snapin in primary cultures of hippocampal neurons results in decreased primary dendrites present on these neurons and increased probability of branching. Together, our data suggest that snapin regulates dendrite number in developing neurons by modulating cypin-promoted microtubule assembly.

Synthesis of several pryrazolo[4,3-e][1,2,4]-triazines is described. The absorption spectrum of some 5-substituted derivatives was found to extend to the visible region. These compounds were found to inhibit some enzymes of purine metabolism, like xanthine oxidase or bacterial purine-nucleoside phosphorylase with Ki values in the 10(-3) -10(-5) M range.

Plasma guanine deaminase (guanase; GD) is well established as an indicator of hepatocellular disease, recently being applied in the detection of hepatitis C in donor blood and in the diagnosis of hepatoma. No totally efficient, simple method for the estimation of plasma GD activity is routine since both guanine and 8-azaguanine, the substrates of the enzyme, are scarcely soluble in water. This difficulty in preparing stable substrates of sufficient concentration has resulted in methods that are both troublesome and inaccurate. Here we describe the development of new colorimetric and high-performance liquid chromatography (HPLC) methods utilizing guanosine as a "prosubstrate." After an initial breakdown of the guanosine to guanine using purine nucleoside phosphorylase, the ammonia formed as a result of the breakdown of the guanine by GD was estimated colorimetrically by the Berthelot reaction. As an alternative or a complementary assay, the xanthine also formed was measured using an isocratic HPLC method. These methods are suitable for routine assays for measuring plasma GD over a wide range of activities.

Axon guidance molecules and related proteins such as semaphorin 3A, neuropilin-1, plexin-1, netrin-1, growth-associated protein, olfactory marker protein, cypin and collapsin response mediator proteins guide the development of neural circuits in the olfactory bulb. In this study, transcriptions of these genes were examined in the olfactory bulb of female, male and neonatal testosterone propionate-treated female rats at the ages of 2, 5, 10, 15, 20, 25, 30 and 45 days. The semaphorin 3A, neuropilin-1, growth-associated protein and collapsin response mediator protein 1-5 genes were expressed significantly higher during the early development stages than in adulthood while the opposite is true for the olfactory marker protein. The expression profile of cypin and netrin-1 was relatively constant through development. A late effect of the neonatal testosterone propionate treatment on netrin-1, growth-associated protein, olfactory marker protein, collapsin response mediator proteins 1, 3, 4 and cypin gene expression was observed. The expression profiles of collapsin response mediator proteins and their related genes in the developing olfactory bulb confirmed most studies on the relationship between collapsin response mediator proteins and development in the brain. Sex differences of semaphorin 3A, neuropilin-1 as well as collapsin response mediator protein 3 at the early development stage and the late effect of neonatal testosterone propionate treatment on the expressions of netrin-1, growth-associated marker protein, cypin and collapsin response mediator proteins 1, 3 and 5 genes may indicate a possible role of these molecules on sexual differentiation of the olfactory bulb.

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was preferentially degraded to adenosine with concomitant appearance of inosine and hypoxanthine. In the presence of ATP, AMP was preferentially degraded via IMP. The nucleosides generated in the course of the reactions are further degraded, almost exclusively, via nucleoside phosphorylase using as cofactor the P(i) generated in the reaction mixture. In order to quantify the effect of each one of the enzymes involved in the degradation of NMP, two complementary approaches were followed: (i) the V:(max) and K:(m) values of the enzymes acting in the intermediate steps of the reactions were determined; (ii) these data were introduced into differential equations describing the concentration of the nucleotides and their degradation products as a function of the time of incubation. Factors affecting kinetic parameters of the equation velocity as a function of ATP concentration were introduced when required. The differential equations were solved with the help of Mathematica 3.0. The theoretical method can be used to simulate situations not feasible to be carried out, such as to measure the influence of nM-microM concentrations of ATP on the metabolism of AMP.

Bacillus subtilis can utilize the purine bases adenine, hypoxanthine and xanthine as nitrogen sources. The utilization of guanine as a nitrogen source is reported here. The first step is the deamination of guanine to xanthine catalysed by guanine deaminase (GDEase). To isolate mutants defective in GDEase activity, a collection of mutant strains was screened for strains unable to use guanine as a nitrogen source. The strain BFA1819 (yknA) showed the expected phenotype and no GDEase activity could be detected in this strain. A new name for yknA, namely gde, is proposed. The gde gene encodes a 156 amino acid polypeptide and was preceded by a promoter sequence that is recognized by the sigma(A) form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular mass inducer molecule. The level of GDEase was found to be subjected to global nitrogen control exerted by the GlnA/TnrA-dependent signalling pathway. The two regulatory proteins of this pathway, TnrA and GlnR, indirectly and positively affected gde expression. This is the first instance of a gene whose expression is positively regulated by GlnR. The GDEase amino acid sequence shows no homology with the mammalian enzyme. In agreement with this are the different physiological roles for the two enzymes.

Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a K(m) of 15 microM with guanine and a k(cat) of 3.2 s(-1). The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. coli guanine deaminase locus is 3' from an open reading frame which shows homology to a bacterial purine base permease.

The activities of the enzymes in Echinococcus multilocularis metacestodes involved in purine salvage were studied by HPLC. As in most parasites, this cestode relies entirely on salvage of preformed bases and nucleosides for its purine requirement. Therefore, these enzymes may be targets for drugs in the chemotherapeutic treatment of diseases caused by this parasite. The animals used in this study were gerbils (Meriones unguiculatus). Enzyme activities from sera and hepatic tissue in control and infected animals were similar with the exception of adenine phosphoribosyltransferase which showed an activity 4-fold greater in the serum from control than in serum from infected animals. In the parasite, adenine and hypoxanthine-guanine phosphoribosyltransferases and adenosine deaminase had the highest activities. Therefore, in E. multilocularis metacestodes, this pathway seems to be important for the parasite's metabolism.

Mobilization of the ribose moiety and of the amino group of guanosine may be realized in rat liver extract by the concerted action of purine nucleoside phosphorylase and guanase. Ribose 1-phosphate formed from guanosine through the action of purine nucleoside phosphorylase acts as ribose donor in the synthesis of xanthosine catalyzed by the same enzyme. The presence of guanase, which irreversibly converts guanine to xanthine, affects the overall process of guanosine transformation. As a result of this purine pathway, guanosine is converted into xanthosine, thus overcoming the lack of guanosine deaminase in mammals. Furthermore, in rat liver extract the activated ribose moiety stemming from the catabolism of purine nucleosides can be transferred to uracil and, in the presence of ATP, used for the synthesis of pyrimidine nucleotides; therefore, purine nucleosides can act as ribose donors for the salvage of pyrimidine bases.