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.

Pineal N-acetyltransferase is inactivated by disulfide-containing peptides: insulin is the most potent.

Pineal N-acetyltransferase can be inactivated in broken cell preparations by cystamine through a mechanism of thiol-disulfide exchange. Some, but not all, disulfide-containing peptides can inactivate this enzyme; the most potent inactivator is insulin. These findings suggest that a disulfide-containing peptide with high reactivity toward N-acetyltransferase may participate in the intracellular regulation of this enzyme.

5-hydroxytryptophan elevates serum melatonin.

Daytime administration of 5-hydroxytryptophan to sheep elevated serum melatonin more than sevenfold within 2 hours. This suggests that administration of 5-hydroxytryptophan could be used as the basis of a clinical test of pineal function and that melatonin might mediate some clinical effects of 5-hydroxytryptophan.

Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis.

Pineal serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, or AA-NAT) generates the large circadian rhythm in melatonin, the hormone that coordinates daily and seasonal physiology in some mammals. Complementary DNA encoding ovine AA-NAT was cloned. The abundance of AA-NAT messenger RNA (mRNA) during the day was high in the ovine pineal gland and somewhat lower in retina. AA-NAT mRNA was found unexpectedly in the pituitary gland and in some brain regions. The night-to-day ratio of ovine pineal AA-NAT mRNA is less than 2. In contrast, the ratio exceeds 150 in rats. AA-NAT represents a family within a large superfamily of acetyltransferases.

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.

Temporal and spatial changes of quinolinic acid immunoreactivity in the immune system of lipopolysaccharide-stimulated mice.

Quinolinic acid (Quin), a metabolite of tryptophan, is a neurotoxin that has been implicated in a variety of neuropathologic disorders that have immune components. The goal of this study was to characterize the changes in the cellular localization of Quin immunoreactivity in a paradigm of immune stimulation with lipopolysaccharide (LPS) in vivo to provide a basis for further studies on the physiological role of Quin in the immune system. Intraperitoneal LPS injection significantly increased Quin immunoreactivity (IR) in lymphoid tissues within 24 h. Spatial changes in splenic Quin-IR demonstrated a shift from the periarterial lymphoid sheaths to the follicles before returning to control levels by 72 h post-LPS. The strongly Quin-IR cells were tentatively identified as interdigitating dendritic cells and macrophages. Only minimal Quin-IR was detected in liver and lung, even under conditions of LPS stimulation combined with tryptophan loading. These data emphasize the temporally and spatially specific nature of Quin-IR changes in lymphoid tissues under conditions of immune stimulation and raise the possibility that Quin may have an immunomodulatory function.

Localization of quinolinic acid in the murine AIDS model of retrovirus-induced immunodeficiency: implications for neurotoxicity and dendritic cell immunopathogenesis.

OBJECTIVE AND DESIGN: Using murine AIDS (MAIDS) as a model of retrovirus-induced immunodeficiency, the aims of this study were (1) to determine the cellular source(s) of quinolinic acid (Quin) with regard to its significance as a potential neuroexcitotoxin in AIDS dementia complex, and (2) to characterize the relationship between dendritic cell Quin immunoreactivity and the histopathological changes associated with the progression of disease. METHODS: Mice with MAIDS were sacrificed from 1 to 16 weeks post-infection. Temporal and spatial changes in the in vivo distribution of Quin at the cellular level were determined by carbodiimide-based immunohistochemical methods. RESULTS: Cellular Quin immunoreactivity was chronically elevated in lymphoid tissues of mice with MAIDS. In contrast, no cellular Quin immunoreactivity was visible in the brain parenchyma at any timepoint studied. CONCLUSION: These findings are consistent with the view that select immune cells in the peripheral lymphoid tissues may be the primary source of Quin, which may contribute to neurotoxic complications in retrovirus-induced immunodeficiency syndromes. The predominant Quin immunoreactive cell types changed with the progression of disease. A significant finding was the marked increase in the number of Quin immunoreactive dendritic cells in the early phase of MAIDS, suggesting a relationship between dendritic cells and Quin in retroviral infection.

Identification and characterization of two isozymic forms of arylamine N-acetyltransferase in Syrian hamster skin.

Arylamine N-acetyltransferase (EC 2.3.1.5) activity was examined using skin from Syrian hamster. Two isozymes of arylamine N-acetyltransferase, designated NAT-1 and NAT-2, were detected on anion-exchange high-performance liquid chromatography analysis. Both enzyme activities had indistinguishable molecular masses (30 kDa), but differed significantly in their specificity toward the aromatic amines including serotonin, dopamine, methoxytryptamine, tryptamine, para-phenetidine, para-aminobenzoic acid, and sulphamethazine. Specifically, NAT-2 but not NAT-1 catalyzed acetylation of dopamine to N-acetyldopamine and acetylation of serotonin to form N-acetylserotonin, a direct precursor of melatonin. The two isozymes were also distinguishable based upon their sensitivity toward methotrexate inhibition (50% inhibiting dose for NAT-1 = 380 microM; NAT-2 > 2 mM). The presence of these two activities in the skin raises new questions about the physiologic role of this enzyme in general and in the skin-specific functions in particular.

Ovine pineal alpha 1-adrenoceptors: characterization and evidence for a functional role in the regulation of serum melatonin.

Plasma melatonin in sheep increases to nocturnal levels rapidly (10-20 min) after dark onset. This increase is blocked by iv prazosin (1 mg), but not propranolol (6 mg). Prazosin also blocks the elevation in pineal melatonin content after dark onset, but does not significantly alter the rise in N-acetyltransferase activity or the elevation in pineal N-acetylserotonin content. Since the nocturnal elevation in N-acetyltransferase, a neurally regulated event, was unaltered, this suggests that prazosin does not significantly impair the transmission of neural signals from the eye to the gland, but does act on pineal alpha 1-adrenoceptors to block melatonin production. This is supported by binding studies in ovine pineal membranes using [125I] iodo-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone, which revealed that binding is rapid, reversible, saturable, and stereo-specific. Saturation studies indicated the presence of a single class of binding sites, with an equilibrium binding constant (Kd) of 32 +/- 6 pM and a maximum binding of 139 +/- 19 fmol/mg protein. The relative potencies of several adrenergic agonists and antagonists in competition studies indicated that the receptor belongs to the alpha 1-subclass of adrenoceptors. Together, these data suggest that melatonin synthesis in the sheep pineal gland is controlled in part by an alpha 1-adrenoceptor mechanism at a step beyond N-acetylation.

Reciprocal day/night relationship between serotonin oxidation and N-acetylation products in the rat pineal gland.

Pineal tryptophan, serotonin [5-hydroxytryptamine (5-HT)], N-acetylserotonin, melatonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, 5-methoxytryptophol, and 5-methoxyindoleacetic acid were measured by high pressure liquid chromatography with electrochemical detection. A complete analysis required less than the equivalent of two rat pineal glands. Samples were obtained at eight time points. A reciprocal physiological relationship was found between oxidation and N-acetylation products of 5-HT. 5-HT and the oxidation products 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, 5-methoxytryptophol, and 5-methoxyindoleacetic acid decreased at night, when the N-acetylation products N-acetylserotonin and melatonin increased. These observations are consistent with the hypothesis that circadian changes in the N-acetylation of 5-HT by indoleamine N-acetyltransferase is the major factor controlling circadian changes in the amounts of 5-HT, and 5-HT oxidation and N-acetylation products in the rat pineal gland.

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.

Iodinated melatonin mimics melatonin action and reveals discrete binding sites in fetal brain.

Iodinated melatonin was used to study melatonin sites of action in brain. Iodomelatonin mimicked the effects of melatonin on reproductive development in Djungarian hamster fetuses. 125I-melatonin injected into the dam was recovered from fetal brain. In vitro autoradiographic studies revealed a remarkably discrete distribution of competitive 125I-melatonin-binding sites in the fetal brain, with binding in median eminence/arcuate nucleus area greater than suprachiasmatic nucleus greater than pineal gland much greater than anterior pituitary gland much greater than preoptic area. 125I-melatonin promises to be a useful tool for understanding the sites and mechanism of action of melatonin.

Localization and Synaptic Release of N-acetylaspartylglutamate in the Chick Retina and Optic Tectum.

The neuropeptide, N-acetylaspartylglutamate (NAAG), was identified in the chick retina (1.4 nmol/retina) by HPLC, radioimmunoassay and immunohistochemistry. This acidic dipeptide was found within retinal ganglion cell bodies and their neurites in the optic fibre layer of the retina. Substantial, but less intense, immunoreactivity was detected in many amacrine-like cells in the inner nuclear layer and in multiple bands within the inner plexiform layer. In addition, NAAG immunoreactivity was observed in the optic fibre layer and in the neuropil of the superficial layers of the optic tectum, as well as in many cell bodies in the tectum. Using a newly developed, specific and highly sensitive (3 fmol/50 microl) radioimmunoassay for NAAG, peptide release was detected in isolated retinas upon depolarization with 55 mM extracellular potassium. This assay also permitted detection of peptide release from the optic tectum following stimulation of action potentials in retinal ganglion cell axons of the optic tract. Both of these release processes required the presence of extracellular calcium. Electrically stimulated release from the tectum was reversibly blocked by extracellular cadmium. These findings suggest that NAAG serves an extracellular function following depolarization-induced release from retinal amacrine neurons and from ganglion cell axon endings in the chick optic tectum. These data support the hypothesis that NAAG functions in synaptic communication between neurons in the visual system.

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.

Quinolinic acid immunoreactive cells in the choroid plexus, leptomeninges and brain vasculature of the immune-stimulated gerbil.

Antibodies to quinolinic acid were utilized to study the cellular localization of this endogenous neurotoxin in the gerbil brain subsequent to systemic immune stimulation with pokeweed mitogen. Immunohistochemistry of carbodiimide fixed spleen revealed a dramatic increase in the number of quinolinic acid-positive cells in the red pulp in the immune-stimulated animals. Quinolinic acid immunoreactivity in the brain was observed in cells within the choroid plexus, vasculature and leptomeninges of the stimulated group only. No immunoreactivity was observed in brain parenchyma. These results are supportive of an immune system origin for the increases in quinolinic acid in CSF and brain during immune stimulation in a rodent model system.

Enhanced carbodiimide fixation for immunohistochemistry: application to the comparative distributions of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat brain.

To improve carbodiimide-based immunohistochemistry, carbodiimide-mediated coupling of radiolabeled N-acetylaspartylglutamate (NAAG) to bovine serum albumin was assayed in vitro. Various perfusion protocols, based on assay results, were tested for their ability to improve the immunohistochemical localization of two nervous system-specific molecules, NAAG and N-acetylaspartate (NAA) in the spinal cord, medulla, hippocampus, and cerebral cortex of the rat. Coupling of [3H]-NAAG to BSA in vitro was optimal with 100 mM carbodiimide and 1 mM N-hydroxysuccinimide in water at 37 degrees C. Optimal fixation of tissue was defined as permitting the identification of the NAAG and NAA in neuronal somata, dendritic arborizations, fine axons, and synaptic terminals with minimal diffuse background immunoreactivity. These conditions were obtained at 37 degrees C with 6% carbodiimide, 1 mM N-hydroxysuccinimide, and 5% dimethylsulfoxide perfused transcardially. Strong NAAG and NAA immunoreactivities were co-distributed in the majority of neurons in the spinal cord. Large-diameter spinal sensory afferents were stained for NAAG in the dorsal horn. The dorsal column nuclei were immunoreactive for NAAG and NAA, but only NAA staining was observed in the nucleus of the solitary tract. In cerebral cortex and hippocampus, NAAG and NAA immunoreactivities appeared to be exclusive, with NAAG staining observed in interneurons throughout all cortical layers, and NAA immunoreactivity present in most pyramidal neurons.

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.

N-acetylaspartate as an acetyl source in the nervous system.

To understand the role of N-acetylaspartate (NAA) as an acetyl donor, we investigated the metabolism of NAA in brain and liver slice preparations. The tissue slices were incubated with [14C-acetyl]NAA (SA = 3 microCi/mumol) or [14C]acetate (SA = 3 microCi/mumol) for 2 h. The tissue was homogenized and was extracted using chloroform/methanol (2:1). The aqueous phase was initially analyzed using anion exchange HPLC while the lipid phase was analyzed using a two-dimensional TLC system. Further resolution of the NAA peak from the anion exchange HPLC was performed using a reverse phase HPLC system. The aqueous phase of both the liver and brain samples incubated with [14C-acetyl]NAA revealed similar patterns of three distinct radioactivity peaks corresponding to NAA, acetate and an early eluting unknown molecule. Further resolution of the NAA peak using reverse phase HPLC indicated that it corresponded to NAA and acetyl CoA. There was significant incorporation of radioactivity into various lipid components in both the brain and liver samples. Patterns similar to that observed with NAA were detected in the case of [14C]acetate in both the brain and liver slice preparations. These results demonstrate that NAA metabolism is not restricted to the nervous system, although its biosynthesis is. It is clear that acetyl moiety of NAA is incorporated into lipids and partially hydrolyzed to free acetate in both brain and liver preparations. Further, production of acetyl CoA from NAA indicates that the acetyl group of NAA is incorporated into lipids and perhaps other acetylated molecules via the acetyl CoA route. A working hypothesis on the metabolic role of NAA is presented.

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.