Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

14-3-3 proteins in pineal photoneuroendocrine transduction: how many roles?

Recent studies suggest that a common theme links the diverse elements of pineal photoneuroendocrine transduction--regulation via binding to 14-3-3 proteins. The elements include photoreception, neurotransmission, signal transduction and the synthesis of melatonin from tryptophan. We review general aspects of 14-3-3 proteins and their biological function as binding partners, and also focus on their roles in pineal photoneuroendocrine transduction.

Genetic, temporal and developmental differences between melatonin rhythm generating systems in the teleost fish pineal organ and retina.

Complete melatonin rhythm generating systems, including photodetector, circadian clock and melatonin synthesis machinery, are located within individual photoreceptor cells in two sites in Teleost fish: the pineal organ and retina. In both, light regulates daily variations in melatonin secretion by controlling the activity of arylalkylamine N-acetyltransferase (AANAT). However, in each species examined to date, marked differences exist between the two organs which may involve the genes encoding the photopigments, genes encoding AANAT, the times of day at which AANAT activity and melatonin production peak and the developmental schedule. We review the fish pineal and retinal melatonin rhythm generating systems and consider the evolutional pressures and other factors which led to these differences.

14-3-3 Proteins and photoneuroendocrine transduction: role in controlling the daily rhythm in melatonin.

This paper describes the role 14-3-3 proteins play in vertebrate photoneuroendocrine transduction. 14-3-3 proteins form a complex with arylalkylamine N-acetyltransferase (AANAT), the enzyme which turns melatonin production on during the day and off at night. Complex formation is triggered at night by cAMP-dependent phosphorylation of the enzyme, and results in activation and protection against proteolysis. This enhances melatonin production >10-fold. Light exposure results in dephosphorylation of the enzyme and disassociation from 14-3-3, leading to destruction and a rapid drop in melatonin production and release and circulating levels.

Characterization of the Saccharomyces cerevisiae homolog of the melatonin rhythm enzyme arylalkylamine N-acetyltransferase (EC 2.3.1.87).

Arylalkylamine N-acetyltransferase (AANAT, serotonin N-acetyltransferase, EC ) plays a unique transduction role in vertebrate physiology by converting information about day and night into a hormonal signal: melatonin. Only vertebrate members of the AANAT family have been functionally characterized. Here a putative AANAT from Saccharomyces cerevisiae (scAANAT) was studied to determine whether it possessed the catalytic activity of the vertebrate enzyme. scAANAT is 47% similar to ovine AANAT, but lacks the regulatory N- and C-terminal flanking regions conserved in all vertebrate AANATs. It was found to have enzyme activity generally typical for AANAT family members, although the substrate preference pattern was somewhat broader, the specific activity was lower, and the pH optimum was higher. Deletion of scAANAT reduced arylalkylamine acetylation by S. cerevisiae extracts, indicating that scAANAT contributes significantly to this process. The scAANAT sequence conformed to the three-dimensional structure of ovine AANAT catalytic core; however, an important structural element (loop 1) was found to be shorter and to lack a proline involved in substrate binding. These differences could explain the lower specific activity of scAANAT, because of the importance of loop 1 in catalysis. Data base analysis revealed the presence of putative AANATs in other fungi but not in the nearly complete genomes of Drosophila melanogaster or Caenorhabditis elegans. These studies indicate that the catalytic and kinetic characteristics of fungal and vertebrate enzymes can be considered to be generally similar, although some differences exist that appear to be linked to changes in one structural element. Perhaps the most striking difference is that fungal AANATs lack the regulatory domains of the vertebrate enzyme, which appear to be essential for the regulatory role the enzyme plays in photochemical transduction.

Breast cancer screening for primary care trainees: comparison of two teaching methods.

BACKGROUND: This study compared the efficacies of two methods of teaching breast cancer screening to primary care trainees. METHODS: Fifty-one nurse-practitioner students were assigned by class section and 47 medical residents by practice site to receive a lecture-demonstration class or individual/small-group instruction from a standardized patient. Prior to instruction and one year later, participants took a written test to assess knowledge and standardized patients evaluated their skills. RESULTS: Overall, the participants improved their breast cancer screening skills. CONCLUSION: The standardized patient teaching method was of greater benefit to the nurse-practitioner students.

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.

Breast cancer screening education: comparing outcome skills of nurse practitioner students and medical residents.

BACKGROUND: Nurse practitioner students, along with all primary care trainees, need breast cancer screening education. The purpose of the study was to compare the performances of nurse practitioner students and medical residents before and after receiving training. METHODS AND RESULTS: In a pretest/posttest design, 51 nurse practitioner students and 47 medical residents received training either from a standardized patient or from a lecture/demonstration class. Before training and 1 year after, participants took the written test and had their skills evaluated by a standardized patient. There were no significant differences between the nurse practitioner students and the medical residents in the mean scores on the written pretest or on the written posttest with both groups improving their scores. The nurse practitioner students had significantly higher scores on the practicum posttest (P <.05). CONCLUSIONS: Nurse practitioner students perform well in learning breast cancer screening. More than one method of teaching is effective.

Regulation of arylalkylamine N-acetyltransferase-2 (AANAT2, EC 2.3.1.87) in the fish pineal organ: evidence for a role of proteasomal proteolysis.

In fish, individual photoreceptor cells in the pineal organ and retina contain complete melatonin rhythm generating systems. In the pike and seabream, this includes a photodetector, circadian clock, and melatonin synthesis machinery; the trout lacks a functional clock. The melatonin rhythm is due in part to a nocturnal increase in the activity of the arylalkylamine N-acetyltransferase (AANAT) which is inhibited by light. Two AANATs have been identified in fish: AANAT1, more closely related to AANATs found in higher vertebrates, is specifically expressed in the retina; AANAT2 is specifically expressed in the pineal organ. We show that there is a physiological day/night rhythm in pineal AANAT2 protein in the pike, and that light exposure at midnight decreases the abundance of AANAT2 protein and activity. In culture, this decrease is blocked by inhibitors of the proteasomal degradation pathway. If glands are maintained under light at night, treatment with these inhibitors increases AANAT2 activity and protein. Organ culture studies with the trout and seabream also indicate that the light-induced decrease of AANAT2 activity is prevented when proteasomal proteolysis is blocked. A cAMP-dependent pathway protects AANAT2 protein from degradation. These results provide a clue to understanding how light regulates the daily rhythm in melatonin secretion in fish photoreceptor cells and provides evidence that proteasomal proteolysis is a conserved element in the regulation of AANAT in vertebrates.

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.

Evaluation of Spartan semi-empirical molecular modeling software for calculations of molecules on surfaces: CO adsorption on Ni(111).

This paper reports the use of the PM3(tm) semi-empirical method in the Spartan molecular modeling software to optimize geometries and calculate vibrational frequencies for increasingly complex transition metal- and carbon monoxide (CO)-containing systems, culminating in calculations of CO adsorbed on a Ni(111) surface. Mononuclear and dinuclear transition metal carbonyl molecular species were used to establish the level of accuracy that could be expected for vibrational frequencies to provide a context for the results from the adsorbed molecule calculations. One to four CO molecules adsorbed on the (111) face of a 22-atom-nickel crystal were then modeled, and the accuracy of the adsorption geometry and vibrational frequency was evaluated. The calculated CO stretching vibrational frequencies were within 8% larger than the gas phase experimental values for the molecular species and were approximately 10% larger than the range of experimental values for CO on the nickel surface. The geometry optimization predicted that the CO molecules on the Ni(111) surface occupy three-fold hollow sites with no preference for sites over Ni atoms, in agreement with recent structural data and other theoretical calculations. The software was less successful in calculating the CO bond angle to the surface and the distance of the CO molecules from the surface, but the calculation did produce a reasonable distance between CO molecules on the surface. In general, the PM3(tm) method in Spartan shows promise for predicting adsorption sites and vibrational frequencies of molecules on metal surfaces.

Mechanism of glucocorticoid-mediated reversal of inhibition of Cl(-)/HCO(-)(3) exchange during chronic ileitis.

In the normal ileum, coupled NaCl absorption occurs via the dual operation of Na(+)/H(+) and Cl(-)/HCO(-)(3) exchange on the brush-border membrane (BBM) of villus cells. In a rabbit model of chronic small intestinal inflammation we determined the cellular mechanism of inhibition of NaCl absorption and the effect of steroids on this inhibition. Cl(-)/HCO(-)(3) but not Na(+)/H(+) exchange was reduced in the BBM of villus cells during chronic ileitis. Cl(-)/HCO(-)(3) exchange was inhibited secondary to a decrease in the affinity for Cl(-) rather than an alteration in the maximal rate of uptake of Cl(-) (V(max)). Methylprednisolone (MP) stimulated Cl(-)/HCO(-)(3) exchange in the normal ileum by increasing the V(max) of Cl(-) uptake rather than altering affinity for Cl(-). MP reversed the inhibition of Cl(-)/HCO(-)(3) exchange in rabbits with chronic ileitis. However, MP alleviated the Cl(-)/HCO(-)(3) exchange inhibition by restoring the affinity for Cl(-) rather than altering the V(max) of Cl(-) uptake. These data suggest that glucocorticoids mediate the alleviation of Cl(-)/HCO(-)(3) exchange inhibition in chronically inflamed ileum by reversing the same mechanism that was responsible for inhibition of this transporter rather than exerting a direct effect on the transporter itself, as was the case in normal ileum.

An analysis of printed breast cancer information for African American women.

BACKGROUND: The 1995-1998 Delta Project was designed to increase breast cancer screening among disadvantaged African American women with limited literacy skills by educating their health care professionals about breast health. The research team intended to provide onsite training and appropriate educational materials; however, they found no suitable materials. This article presents the results of an assessment of available materials and defines the need for suitable materials. METHODS: Nineteen organizations that develop cancer-related publications submitted materials intended for African American audiences. Sixty-one documents were examined for readability and cultural sensitivity. The Flesch Reading Ease (FRE), Flesch-Kincaid (F-K), and Cultural Sensitivity Assessment Tools (CSAT) were used in testing. RESULTS: The mean FRE score of 65 yielded a F-K mean grade level of 7.5 (desired level: 3.5). Using CSAT, 16 documents (26%) were eliminated because they had no visuals. Twenty-two publications (37%) were culturally sensitive for all audiences and 19 (31%) were for white audiences. Four (6%) pieces specifically addressed African American women. CONCLUSIONS: Printed educational materials on breast cancer do not adequately provide information to undereducated, economically disadvantaged African American women.

Melatonin synthesis: arylalkylamine N-acetyltransferases in trout retina and pineal organ are different.

Serotonin N-acetyltransferase (AANAT) is the first enzyme in the conversion of serotonin to melatonin. Changes in AANAT activity determine the daily rhythm in melatonin secretion. Two AANAT genes have been identified in the pike, pAANAT-1 and pAANAT-2, expressed in the retina and in the pineal, respectively. The genes preferentially expressed in these tissues encode proteins with distinctly different kinetic characteristics. Like the pike, trout retina primarily expresses the AANAT-1 gene and trout pineal primarily expresses the AANAT-2 gene. Here we show that the kinetic characteristics of AANAT in these tissues differ as in pike. These differences include optimal temperature for activity (pineal: 12 degrees C; retina: 25 degrees C) and relative affinity for indoleethylamines compared to phenylethylamines. In addition, retinal AANAT exhibited substrate inhibition, which was not seen with pineal AANAT. The kinetic differences between AANAT-1 and AANAT-2 appear to be defining characteristics of these gene subfamilies, and are not species specific.

Use of a kinetic energy orifice as a probe of metastable dissociation in Fourier transform ion cyclotron resonance mass spectrometry.

Although Fourier transform ion cyclotron resonance mass spectrometry is a powerful tool in the qualitative observation of gas phase reactions, ion detection is on the millisecond time scale, orders of magnitude longer than typically found when using a sector instrument. Observations of short-lived species such as chemically activated adduct ions can be accomplished using selective ion excitation as a probe of intermediate lifetime. Whereas ion elimination has been shown to be effective in monitoring ion lifetimes on the microsecond time scale, problems associated with detecting ions produced with high kinetic energies limits the technique. Use of a kinetic energy orifice as an ion skimmer effectively eliminates ions near the center of the ion cell at relatively low kinetic energies. By modifying a single section cell to include a kinetic energy orifice, the lifetimes of chemically activated adduct ions have been investigated.

Genetic variability in plasma melatonin in sheep is due to pineal weight, not to variations in enzyme activities.

This study was conducted to determine the origin of the high variability in the mean nocturnal plasma melatonin concentration (MC) in sheep. Two extreme groups of 25 lambs each [low (L) and high (H)] were obtained by calculating their genetic value on the basis of the MC of their parents. The MC of lambs was significantly higher in the H group than in the L group (L: 189.7 +/- 24.4 vs. H: 344.1 +/- 33.0 pg/ml, P < 0.001). Within each group, 13 lambs were slaughtered during the day (D) and 12 lambs during the night (N). Pineal weight was significantly higher in the H group than in the L group (L: 83.5 +/- 6.7 vs. H: 119.1 +/- 9.2 mg, P < 0.01) but did not differ between D and N. The amount of melatonin released in vitro per milligram of pineal gland, the arylalkylamine N-acetyltransferase (AANAT) activity, the AANAT protein content, and the level of AANAT mRNA differed significantly between D and N but not with genetic group. Hydroxyindole O-methyltransferase activity did not differ significantly between D and N or between genetic groups. Therefore, the genetic difference in MC between the two groups of lambs was attributed to a difference in pineal size, not in enzymatic activity of the pinealocytes.

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.

Ambient sound levels in a state psychiatric hospital.

High environmental and psychosocial stress contribute to the onset and relapse of major psychiatric disorders. High sound levels in general hospitals are common and may be indirectly associated with negative physical effects because of increased physiological stress on the body. Excessive sound also interferes with cognitive functioning, especially affecting prefrontal cortical processes, but no information about sound levels in psychiatric hospitals was available. This study critically examines literature on sound stress and reports findings from an exploratory study of sound levels in a tertiary care psychiatric hospital. An overall mean sound level of 75.68 dB was found, with peak sound levels as high as 85 to 90 dB, in the range that causes hearing loss. These levels, higher than sound levels on medical, surgical, and intensive care units, suggest the need for more attention to the effect that environmental sound has on the behavior of patients hospitalized with acute psychiatric symptoms.

Two arylalkylamine N-acetyltransferase genes mediate melatonin synthesis in fish.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the first enzyme in the conversion of serotonin to melatonin. Large changes in AANAT activity play an important role in the daily rhythms in melatonin production. Although a single AANAT gene has been found in mammals and the chicken, we have now identified two AANAT genes in fish. These genes are designated AANAT-1 and AANAT-2; all known AANATs belong to the AANAT-1 subfamily. Pike AANAT-1 is nearly exclusively expressed in the retina and AANAT-2 in the pineal gland. The abundance of each mRNA changes on a circadian basis, with retinal AANAT-1 mRNA peaking in late afternoon and pineal AANAT-2 mRNA peaking 6 h later. The pike AANAT-1 and AANAT-2 enzymes (66% identical amino acids) exhibit marked differences in their affinity for serotonin, relative affinity for indoleethylamines versus phenylethylamines and temperature-activity relationships. Two AANAT genes also exist in another fish, the trout. The evolution of two AANATs may represent a strategy to optimally meet tissue-related requirements for synthesis of melatonin: pineal melatonin serves an endocrine role and retinal melatonin plays a paracrine role.

Ovine arylalkylamine N-acetyltransferase in the pineal and pituitary glands: differences in function and regulation.

The enzyme arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) has been conventionally linked with the biosynthesis of melatonin within the pineal gland and retina. This study establishes that AANAT messenger RNA (mRNA) and functional enzyme occurs within the pars tuberalis (PT) and to a lesser degree within the pars distalis (PD) of the sheep pituitary gland; expression in these tissues is approximately 1/15th (PT) and 1/300th (PD) of that in the ovine pineal gland. AANAT mRNA in the PT appears to be expressed in the same cells as the Mel1a receptor. No evidence was obtained to indicate that either PT or PD cells have the ability to synthesize melatonin, suggesting that this enzyme plays a different functional role in the pituitary. We also found that cAMP regulation of the abundance of AANAT mRNA differs between the PT and pineal gland. Forskolin (10 microM) has no effect on pineal AANAT mRNA levels, yet represses expression in the PT. This suppressive influence could be mediated by ICER (inducible cAMP response early repressor), which is induced by forskolin in both tissues. Although it appears that the specific function and regulation of AANAT in the pituitary gland differ from that in the pineal gland, it seems likely that AANAT may play a role in the broader area of signal transduction through the biotransformation of amines.