Enzyme stabilization by glass-derived silicates in glass-exposed aqueous solutions.

OBJECTIVES: To analyze the solutes leaching from glass containers into aqueous solutions, and to show that these solutes have enzyme activity stabilizing effects in very dilute solutions. METHODS: Enzyme assays with acetylcholine esterase were used to analyze serially succussed and diluted (SSD) solutions prepared in glass and plastic containers. Aqueous SSD preparations starting with various solutes, or water alone, were prepared under several conditions, and tested for their solute content and their ability to affect enzyme stability in dilute solution. RESULTS: We confirm that water acts to dissolve constituents from glass vials, and show that the solutes derived from the glass have effects on enzymes in the resultant solutions. Enzyme assays demonstrated that enzyme stability in purified and deionized water was enhanced in SSD solutions that were prepared in glass containers, but not those prepared in plastic. The increased enzyme stability could be mimicked in a dose-dependent manner by the addition of silicates to the purified, deionized water that enzymes were dissolved in. Elemental analyses of SSD water preparations made in glass vials showed that boron, silicon, and sodium were present at micromolar concentrations. CONCLUSIONS: These results show that silicates and other solutes are present at micromolar levels in all glass-exposed solutions, whether pharmaceutical or homeopathic in nature. Even though silicates are known to have biological activity at higher concentrations, the silicate concentrations we measured in homeopathic preparations were too low to account for any purported in vivo efficacy, but could potentially influence in vitro biological assays reporting homeopathic effects.

Mutational analysis of aspartoacylase: implications for Canavan disease.

Mutations that result in near undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-l-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.

Laboratory research in homeopathy: con.

Alternative medical approaches to human diseases such as cancer are becoming increasingly popular, but reports on their success rates have been highly variable. Homeopathy is an alternative medical practice often applied to less critical human diseases but one that has also been applied sporadically to the treatment of cancer. Animal studies on the use of homeopathy to treat experimental cancer are few and the evidence provided to date is far from conclusive. The debate presented here concerns the utility of animal studies on cancer treatment with homeopathic preparations. As part of a Point-Counterpoint feature, this review and its companion piece in this issue by Khuda-Bukhsh (Integr Cancer Ther. 2006;5:320-332) are composed of a thesis section, a response section in reaction to the companion thesis, and a rebuttal section to address issues raised in the companion response.

Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators.

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N-acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH-SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2'-O-dibutyryl cAMP) and protein kinase C (PMA; phorbol-12-myristate-13-acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l-[(3)H]glutamine, whereas PMA-induced differentiation reduced both. Exogenously applied NAA caused dose dependent increases in intracellular NAA levels, and NAAG biosynthesis from l-[(3)H]glutamine, suggesting precursor-product and mass-action relationships between NAA and NAAG. Incorporation of l-[(3)H]aspartate into NAA and NAAG occurred sequentially, appearing in NAA by 1 h, but not in NAAG until between 6 and 24 h. Synthesis of NAAG from l-[(3)H]aspartate was increased by dbcAMP and decreased by PMA at 24 h. The effects of PMA on l-[(3)H]aspartate incorporation into NAA were temporally biphasic. Using short incubation times (1 and 6 h), PMA increased l-[(3)H]aspartate incorporation into NAA, but with longer incubation (24 h), incorporation was significantly reduced. These results suggest that, while the neuronal production of NAA and NAAG are biochemically related, significant differences exist in the regulatory mechanisms controlling their biosynthesis.

Progress toward acetate supplementation therapy for Canavan disease: glyceryl triacetate administration increases acetate, but not N-acetylaspartate, levels in brain.

Canavan disease (CD) is a fatal genetic neurodegenerative disorder caused by mutations in the gene for aspartoacylase, an enzyme that hydrolyzes N-acetylaspartate (NAA) into L-aspartate and acetate. Because aspartoacylase is localized in oligodendrocytes, and NAA-derived acetate is incorporated into myelin lipids, we hypothesize that an acetate deficiency in oligodendrocytes is responsible for the pathology in CD, and we propose acetate supplementation as a possible therapy. In our preclinical efforts toward this goal, we studied the effectiveness of orally administered glyceryl triacetate (GTA) and calcium acetate for increasing acetate levels in the murine brain. The concentrations of brain acetate and NAA were determined simultaneously after intragastric administration of GTA. We found that the acetate levels in brain were increased in a dose- and time-dependent manner, with a 17-fold increase observed at 1 to 2 h in 20- to 21-day-old mice at a dose of 5.8 g/kg GTA. NAA levels in the brain were not significantly increased under these conditions. Studies using mice at varying stages of development showed that the dose of GTA required to maintain similarly elevated acetate levels in the brain increased with age. Also, GTA was significantly more effective as an acetate source than calcium acetate. Chronic administration of GTA up to 25 days of age did not result in any overt pathology in the mice. Based on these results and the current Food and Drug Administration-approved use of GTA as a food additive, we propose that it is a potential candidate for use in acetate supplementation therapy for CD.

Rapid sodium cyanide depletion in cell culture media: outgassing of hydrogen cyanide at physiological pH.

During the course of in vitro studies on cyanide exposure with SH-SY5Y human neuroblastoma cells, we found that sodium cyanide (NaCN) up to a concentration of 10 mM had no significant toxic effect under our culture conditions. Further investigation of this apparent cyanide resistance revealed that the sodium cyanide was being rapidly depleted from the cell culture medium. Cyanide was interacting with constituents of the cell culture medium and was somehow being detoxified or removed from solution. The reaction of cyanide with cell culture media in 96-well culture plates reduced cyanide concentrations rapidly (80-90% in 2 h at 37 degrees C). Running the same reaction in capped tubes significantly reduced cyanide loss from solution. Incubation of cyanide with individual constituents of the cell culture medium in solution showed that glucose, phenol red, and amino acids all acted to detoxify or remove cyanide from solution. When amino acids or buffers were incubated with sodium cyanide in aqueous solution at pH 7.4, hydrogen cyanide (HCN) was found to degas from the solutions. We compared HCN outgassing over a range of pH values. As expected, HCN remained very soluble at high pH, but as the pH was reduced to 7.0, the rate of HCN formation and outgassing increased dramatically. Acid-base reactions involving cyanide and proton donors, such as amino acids and other cell culture media constituents, at physiological pH result in rapid HCN outgassing from solution at 37 degrees C. These results indicate that previous in vitro cyanide toxicity studies done in standard culture media with prolonged incubation times using gas-exchanging culture containers might have to be reevaluated in light of the fact that the effective cyanide concentrations in the culture media were significantly lower than reported.

Defective N-acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in Canavan's disease.

Canavan's disease (CD) is a fatal, hereditary disorder of CNS development that has been linked to mutations in the gene for the enzyme aspartoacylase (ASPA) (EC 3.5.1.15). ASPA acts to hydrolyze N-acetylaspartate (NAA) into l-aspartate and acetate, but the connection between ASPA deficiency and the failure of proper CNS development is unclear. We hypothesize that one function of ASPA is to provide acetate for the increased lipid synthesis that occurs during postnatal CNS myelination. The gene encoding ASPA has been inactivated in the mouse model of CD, and here we show significant decreases in the synthesis of six classes of myelin-associated lipids, as well as reduced acetate levels, in the brains of these mice at the time of peak postnatal CNS myelination. Analysis of the lipid content of white matter from a human CD patient showed decreased cerebroside and sulfatide relative to normal white matter. These results demonstrate that myelin lipid synthesis is significantly compromised in CD and provide direct evidence that defective myelin synthesis, resulting from a deficiency of NAA-derived acetate, is involved in the pathogenesis of CD.

SH-SY5Y neuroblastoma cells: a model system for studying biosynthesis of NAAG.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide that is thought to modulate neurotransmitter release through pre-synaptic mGluR3 receptors. Despite years of research into NAAG biochemistry, almost nothing is known about NAAG biosynthesis. To date, NAAG biosynthesis has only been demonstrated conclusively in explanted animal neural tissues, including frog retina, rat dorsal root ganglia and crayfish nerve cord, but not in human cells or tissues. We show here that a human neuroblastoma cell line, SH-SY5Y, provides a good model system for the study of NAAG biosynthesis. Radiolabled NAAG synthesis occurred using both L-[3H]glutamic acid and L-[3H]glutamine as precursors, with glutamine being the preferred substrate. Differentiation of SH-SY5Y cells with retinoic acid resulted in decreased radiolabel incorporation into NAAG, whereas differentiation with nerve growth factor did not affect radiolabel incorporation.

Immunohistochemical localization of aspartoacylase in the rat central nervous system.

Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cell-specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (approximately 37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotin-peroxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.

Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain.

Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (approximately 670 kDa), which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, approximately 0.5 mm) and acetyl CoA (approximately 0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed.

Developmental increase of aspartoacylase in oligodendrocytes parallels CNS myelination.

Canavan disease, an autosomal-recessive neurogenetic disorder, is caused by mutations in aspartoacylase, an enzyme that deacetylates N-acetylaspartate to generate free acetate in the brain. Earlier studies have shown that aspartoacylase is primarily restricted to myelin synthesizing cells (oligodendroglia) in the CNS. These findings have led us to investigate the developmental expression of aspartoacylase gene in the rat brain in an attempt to shed more light on the role of this enzyme in myelination. In situ hybridization using a 35S riboprobe based on murine aspartoacylase cDNA was used in this study. The probe hybridized mostly to the white matter tracts with different densities depending on the age of the animal and region of the brain examined. Little or no hybridization signals were detected in the 1-day-old rats, whereas the signal was clearly detectable in most of the white matter regions of the CNS in the 11-day-old rats. The signal density markedly increased at postnatal day 17, the peak of myelination. Thereafter, the hybridization signals decreased somewhat but still could be observed in the adult animals. Thus, the developmental expression pattern of aspartoacylase gene in the postnatal brain closely parallels myelination in the CNS. In the CNS, the hybridization signal of ASPA appeared to be restricted primarily to oligodendrocytes, the primary myelin synthesizing cell type in the CNS. However, the signal was not detectable in rat sciatic nerve (Schwann cells) of the peripheral nervous system. These findings indicate that the role of N-acetylaspartate in myelin synthesis is restricted to the CNS. Furthermore, they provide additional support for the acetate deficiency hypothesis of Canavan disease and also make a stronger case for acetate supplementation as an immediate and inexpensive therapy for Canavan disease.

Aspartoacylase is restricted primarily to myelin synthesizing cells in the CNS: therapeutic implications for Canavan disease.

Canavan disease is a devastating neurodegenerative childhood disease caused by mutations in aspartoacylase, an enzyme that deacetylates N-acetylaspartate to generate free acetate in the brain. Localization of aspartoacylase in different cell types in the rat brain was examined in an attempt to understand the pathogenesis of Canavan disease. In situ hybridization histochemistry with a riboprobe based on murine aspartoacylase cDNA was used in this study. The hybridization signal was detectable primarily in the myelin-synthesizing cells, namely oligodendroglia. These findings provide strong additional support for insufficient myelin synthesis as the pathogenic basis of Canavan disease and make a compelling case for acetate supplementation as a simple and noninvasive therapy for this fatal disease with no treatment.

A radiometric assay for aspartoacylase activity in cultured oligodendrocytes.

Recent studies have shown that aspartoacylase (ASPA), the defective enzyme in Canavan disease, is detectable in the brain only in the oligodendrocytes. Studying the regulation of ASPA is central to the understanding the pathogenesis of Canavan disease and to the development of therapeutic strategies. Toward this goal, we have developed a sensitive method for the assay of ASPA in cultured oligodendrocytes. The method involves: (a) chemical synthesis of [14C]N-acetylaspartate (NAA) from L-[14C]Asp; (b) use of [14C]NAA as substrate in the assay; and (c) separation and quantitation of the product L-[14C]Asp using a TLC system. This method can detect as low as 10pmol of product and has been optimized for cultured oligodendrocytes. Thus, this method promises to be a valuable tool for understanding the biochemical mechanisms involved in the cell-specific expression and regulation of ASPA in oligodendrocytes.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC ). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN --> RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the K(m) for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

cAmp regulation of arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87): a new cell line (1E7) provides evidence of intracellular AANAT activation.

Arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase, AANAT, EC ) is the penultimate enzyme in melatonin synthesis. As described here, a cell line (1E7) expressing human AANAT (hAANAT) has been developed to study the human enzyme. 1E7 hAANAT is detectable in immunoblots as a 23-kDa band and is immunocytochemically visualized in the cytoplasm. The specific concentration of hAANAT in homogenates is comparable to that of the night rat pineal gland. Kinetics of AANAT extracted from 1E7 cells are the same as those of bacterially expressed hAANAT; both preparations of hAANAT are equally sensitive to the inhibitor CoA-S-N-acetyltryptamine. Studies of cAMP regulation indicate that treatment with forskolin, dibutyryl cAMP, isobutylmethylxanthine, or isoproterenol activate cellular hAANAT within intact 1E7 cells approximately 8-fold without markedly increasing the abundance of AANAT protein or the activity of AANAT in broken cell preparations; and, that forskolin, isobutylmethylxanthine and isoproterenol elevate cyclic AMP production. These observations extend our understanding of cAMP regulation of AANAT activity, because it is currently thought that this only involves changes in the steady-state levels of AANAT protein. This previously unrecognized switching mechanism could function physiologically to control melatonin production without changing AANAT protein levels.

Murine aspartoacylase: cloning, expression and comparison with the human enzyme.

Canavan disease is caused by mutations in aspartoacylase, the enzyme that degrades N-acetylaspartate (NAA) into acetate and aspartate. Murine aspartoacylase (mASPA) was cloned using sequence information from mouse expressed sequence tags homologous to the human cDNA. The open reading frame was cloned into a thioredoxin fusion vector, overexpressed in bacteria, and the protein was purified using affinity chromatography to near homogeneity. Recombinant human ASPA (hASPA) was prepared by a similar method. Both recombinant enzymes were highly specific to NAA, with about 10% of the NAA activity toward N-acetylasparagine. More interestingly, the product of N-acetylasparagine was aspartate but not asparagine, indicating that ASPA catalyzed deacetylation as well as hydrolysis of the beta acid amide. Our success in preparing the recombinant ASPA in high purity should permit multiple lines of investigations to understand the pathogenic mechanisms of Canavan disease and the functional roles of NAA.

Selective metabolism of kynurenine in the spleen in the absence of indoleamine 2,3-dioxygenase induction.

The kynurenine pathway of L-tryptophan degradation is differentially regulated dependent on the level of immune system activation. During inflammation and disease, activity of the hepatocellular enzyme tryptophan 2,3-dioxygenase (TDO) decreases and a second enzyme, indoleamine 2,3-dioxygenase (IDO), is induced in extrahepatic sites. Substantial formation of a metabolise downstream of this step, quinolinic acid (Quin), subsequently occurs only in select regions of the lymphoid tissues, such as spleen, in a temporally restricted manner. The goal of this study was to determine the localization of Quin in unstimulated mice under conditions where rate-limiting control of the pathway by both TDO and IDO was by-passed. Supplementation of drinking water with L-kynurenine, a pathway intermediate that lies between tryptophan and Quin, resulted in a dose-dependent increase in Quin immunoreactivity in the follicles and discontinuous regions of the marginal zones of the spleen. Strongly immunoreactive cells in the periarteriole lymphoid sheaths adopted a highly reactive morphology despite the lack of immunostimulation and IDO induction. In contrast, a patchy to diffuse pallor of staining was observed in the liver parenchyma with 1 and 10 mM L-kynurenine ingestion, respectively. These data show that selective tryptophan metabolism can occur in discrete subcompartments of the lymphoid tissues beyond the level of IDO. In vivo manipulation of Quin synthesis in the absence of IDO induction may serve as a model for studying regulation and function of the kynurenine pathway activation in the immune system.

Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function.

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish larvae indicated that zfAANAT-2 mRNA abundance exhibits a dramatic 24-h rhythm in a 14-h light, 10-h dark cycle, with high levels at night. This rhythm persists in constant darkness, indicating that the zfAANAT-2 mRNA rhythm is driven by a circadian clock at this stage. The techniques described in this report were also used to determine that zfAANAT-2 expression is altered in two well characterized genetic mutants, mindbomb and floating head. The observations described here suggest that zfAANAT-2 mRNA may be a useful marker to study development of the pineal gland and of circadian clock mechanisms in zebrafish.

The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 A resolution with a bisubstrate analog.

Serotonin N-acetyltransferase, a member of the GNAT acetyltransferase superfamily, is the penultimate enzyme in the conversion of serotonin to melatonin, the circadian neurohormone. Comparison of the structures of the substrate-free enzyme and the complex with a bisubstrate analog, coenzyme A-S-acetyltryptamine, demonstrates that acetyl coenzyme A (AcCoA) binding is accompanied by a large conformational change that in turn leads to the formation of the serotonin-binding site. The structure of the complex also provides insight into how the enzyme may facilitate acetyl transfer. A water-filled channel leading from the active site to the surface provides a pathway for proton removal following amine deprotonation. Furthermore, structural and mutagenesis results indicate an important role for Tyr-168 in catalysis.

Natural melatonin 'knockdown' in C57BL/6J mice: rare mechanism truncates serotonin N-acetyltransferase.

Pineal melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin) is severely compromised in most inbred strains of mice, in many cases because serotonin is not acetylated by serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT). We have found that in the C57BL/6J strain, AANAT mRNA encodes a severely truncated AANAT protein, because a pseudo-exon containing a stop codon is spliced in. This is the first identification of a natural mutation which knocks down melatonin synthesis. The decrease in melatonin signaling may have been a selective factor in the development of laboratory strains of mice because melatonin can inhibit reproduction and modify circadian rhythmicity.