Upregulated Guanine Deaminase Is Involved in Hyperpigmentation of Seborrheic Keratosis via Uric Acid Release.

Seborrheic keratosis, which is a benign tumor composed of epidermal keratinocytes, develops common in the elderly. Uric acid generated by upregulated guanine deaminase (GDA) has been identified to cause UV-induced keratinocyte senescence in seborrheic keratosis. Seborrheic keratosis is also frequently pigmented. Growing evidences indicate that hyperuricemia is a risk factor of acanthosis nigricans, an acquired skin hyperpigmentation. The objective of this study was to investigate role of GDA and its metabolic end product, uric acid, in hyperpigmentation of patients with seborrheic keratosis using their lesional and non-lesional skin specimen sets and cultured primary human epidermal keratinocytes with or without GDA overexpression or uric acid treatment. GDA-overexpressing keratinocytes or their conditioned media containing uric acid increased expression levels of MITF and tyrosinase in melanocytes. Uric acid released from keratinocytes was facilitated by ABCG2 transporter with the help of PDZK1 interaction. Released uric acid was taken by URAT1 transporter in melanocytes, stimulating melanogenesis through p38 MAPK activation. Overall, GDA upregulation in seborrheic keratosis plays a role in melanogenesis via its metabolic end product uric acid, suggesting that seborrheic keratosis as an example of hyperpigmentation associated with photoaging.

Structure guided mutagenesis reveals the substrate determinants of guanine deaminase.

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.

GDAP1 mutations are frequent among Brazilian patients with autosomal recessive axonal Charcot-Marie-Tooth disease.

Mutations in ganglioside-induced differentiation-associated-protein 1 (GDAP1) are associated with several subtypes of Charcot-Marie-Tooth (CMT) disease, including autosomal recessive and demyelinating (CMT4A); autosomal recessive and axonal (AR-CMT2K); autosomal dominant and axonal (CMT2K); and an intermediate and recessive form (CMTRIA). To date, at least 103 mutations in this gene have been described, but the relative frequency of GDAP1 mutations in the Brazilian CMT population is unknown. In this study, we investigated the frequency of GDAP1 mutations in a cohort of 100 unrelated Brazilian CMT patients. We identified five variants in unrelated axonal CMT patients, among which two were novel and probably pathogenic (N64S, P119T) one was novel and was classified as VUS (K207L) and two were known pathogenic variants (R125* and Q163*). The prevalence rate of GDAP1 among the axonal CMT cases was 7,14% (5/70), all of them of recessive inheritance, thus suggesting that the prevalence was higher than what is observed in most countries. All patients exhibited severe early-onset CMT that was rapidly progressive. Additionally, this study widens the mutational spectrum of GDAP1-related CMT through identification of two novel likely pathogenic variants.

Guanine Deaminase in Human Epidermal Keratinocytes Contributes to Skin Pigmentation.

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.

Guanine Deaminase Stimulates Ultraviolet-induced Keratinocyte Senescence in Seborrhoeic Keratosis via Guanine Metabolites.

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.

A Novel Short Isoform of Cytosolic PSD-95 Interactor (Cypin) Regulates Neuronal Development.

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.

Overexpression of cypin alters dendrite morphology, single neuron activity, and network properties via distinct mechanisms.

OBJECTIVE: This study investigates the effect that overexpression of cytosolic PSD-95 interactor (cypin), a regulator of synaptic PSD-95 protein localization and a core regulator of dendrite branching, exerts on the electrical activity of rat hippocampal neurons and networks. APPROACH: We cultured rat hippocampal neurons and used lipid-mediated transfection and lentiviral gene transfer to achieve high levels of cypin or cypin mutant (cypinΔPDZ; PSD-95 non-binding) expression cellularly and network-wide, respectively. MAIN RESULTS: Our analysis revealed that although overexpression of cypin and cypinΔPDZ increase dendrite numbers and decrease spine density, cypin and cypinΔPDZ distinctly regulate neuronal activity. At the single cell level, cypin promotes decreases in bursting activity while cypinΔPDZ reduces sEPSC frequency and further decreases bursting compared to cypin. At the network level, by using the Fano factor as a measure of spike count variability, cypin overexpression results in an increase in variability of spike count, and this effect is abolished when cypin cannot bind PSD-95. This variability is also dependent on baseline activity levels and on mean spike rate over time. Finally, our spike sorting data show that overexpression of cypin results in a more complex distribution of spike waveforms and that binding to PSD-95 is essential for this complexity. SIGNIFICANCE: Our data suggest that dendrite morphology does not play a major role in cypin action on electrical activity.

Analysis of co-expression networks for circular RNAs and mRNAs reveals that circular RNAs hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 are candidate oncogenes in gastric cancer.

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.

Arxula adeninivorans recombinant guanine deaminase and its application in the production of food with low purine content.

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.

The role of PSD-95 and cypin in morphological changes in dendrites following sublethal NMDA exposure.

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.

BDNF-promoted increases in proximal dendrites occur via CREB-dependent transcriptional regulation of cypin.

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.

Transcriptional regulation of the gene cluster encoding allantoinase and guanine deaminase in Klebsiella pneumoniae.

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.

Global gene expression profiling of somatic motor neuron populations with different vulnerability identify molecules and pathways of degeneration and protection.

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.

The hunt for 8-oxoguanine deaminase.

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.

Guanine deaminase functions as dihydropterin deaminase in the biosynthesis of aurodrosopterin, a minor red eye pigment of Drosophila.

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.

Phylogenetic analysis and molecular evolution of guanine deaminases: from guanine to dendrites.

Guanine deaminase (GDA; guanase) is a ubiquitous enzyme that catalyzes the first step of purine metabolism by hydrolytic deamination of guanine, resulting in the production of xanthine. This hydrolase subfamily member plays an essential role in maintaining homeostasis of cellular triphosphate nucleotides for energy, signal transduction pathways, and nitrogen sources. In mammals, GDA protein levels can play a role in neuronal development by regulating dendritic arborization. We previously demonstrated that the most abundant alternative splice form of GDA in mammals, termed cypin (cytosolic PSD-95 interactor), interacts with postsynaptic density proteins, regulates microtubule polymerization, and increases dendrite number. Since purine metabolism and dendrite development were previously thought to be independent cellular processes, this multifunctional protein serves as a new target for the treatment of cognitive disorders characterized by aberrant neuronal morphology and purine metabolism. Although the enzymatic activity of GDA has been conserved during evolution from prokaryotes to higher eukaryotes, a detailed evolutionary assessment of the principal domains in GDA proteins has not yet been put forward. In this study, we perform a complete evolutionary analysis of the full-length sequences and the principal domains in guanine deaminases. Furthermore, we reconstruct the molecular phylogeny of guanine deaminases with neighbor-joining, maximum-likelihood, and UPGMA methods of phylogenetic inference. This study can act as a model whereby a universal housekeeping enzyme may be adapted to act also as a key regulator of a developmental process.

Purine utilization by Klebsiella oxytoca M5al: genes for ring-oxidizing and -opening enzymes.

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.

Structural characterization of the zinc binding domain in cytosolic PSD-95 interactor (cypin): Role of zinc binding in guanine deamination and dendrite branching.

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.

GUD1 (YDL238c) encodes Saccharomyces cerevisiae guanine deaminase, an enzyme expressed during post-diauxic growth.

Purine salvage is a complex pathway allowing a correct balance between adenylic and guanylic derivatives. In this paper, we show that GUD1 (YDL238c) encodes guanine deaminase, a catabolic enzyme producing xanthine and ammonia from guanine. Importantly, Gud1p activity was higher during post-diauxic growth, suggesting that a decrease of the guanylic nucleotide pool could be required when cells shift from proliferation to quiescence.

Expression of axon guidance molecules and their related genes during development and sexual differentiation of the olfactory bulb in rats.

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.