Patrick H. Roseboom

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.

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.

Avian melatonin synthesis: Photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N‐acetyltransferase (arylalkylamine N‐acetyltransferase; AA‐NAT; EC 2.3.1.87). Here we determined that the chicken AA‐NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA‐NAT mRNA was not detected in other tissues. The AA‐NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA‐NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6‐h light pulse late in subjective night in DD. Nocturnal AA‐NAT mRNA levels do not change during a 20‐min exposure to light, whereas this treatment dramatically decreases AA‐NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA‐NAT activity reflect, at least in part, clock‐generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light‐induced decrease in AA‐NAT activity.

New light is shining on the melatonin rhythm enzyme: the first postcloning view.

One of the most interesting molecules in circadian biology is serotonin N-acetyltransferase (arylalkyfamine N-acetyltransferase, AANAT), the enzyme that controls the daily rhythm in pineal melatonin production and blood melatonin. The recent cloning of AANAT cDNA has led to the characterization of the human gene; the realization that AANAT represents a unique gene family; the discovery of circadian rhythms in AANAT mRNA; the determination of the basis of transsynaptic and cellular regulation of expression of the AANAT gene; a new understanding of the relationship of AANAT mRNA and activity; and the surprising finding of strong expression of the AANAT gene in the retina and significant levels in select brain regions, the pituitary gland, and testes. The cloning of AANAT cDNA has not only made it possible to answer longstanding questions in circadian biology, but has also raised stimulating new issues.

Norepinephrine-induced phosphorylation of the transcription factor CREB in isolated rat pinealocytes: an immunocytochemical study

In the present study we investigated whether norepinephrine, which stimulates melatonin biosynthesis in the mammalian pineal organ, causes phosphorylation of the cyclic AMP responsive element binding protein (CREB) in rat pinealocytes. Cells isolated from the pineal organ of adult male rats and cultured on coated coverslips were treated with norepinephrine, β- or α1 agonists for 1, 5, 10, 20, 30, 60 or 300 min and then immunocytochemically analyzed with an antibody against phosphorylated CREB (p-CREB). Treatment with norepinephrine or β-adrenergic agonists resulted in a similar, time-dependent induction of p-CREB immunoreactivity, exclusively found in cell nuclei. The α1 agonist phenylephrine did not induce p-CREB immunoreactivity at low doses (0.1 μM) or when high doses (10 μM) were applied in combination with a β-antagonist (propranolol, 0.1 μM). This indicates that induction of CREB phosphorylation is elicited by β-adrenergic receptor stimulation. The response was first seen after 10 min and reached a maximum after 30 to 60 min when more than 90% of the cells displayed p-CREB immunoreactivity. The intensity of the p-CREB immunoreactivity showed marked cell-to-cell variation, but nearly all immunoreactive cells were identified as pinealocytes by double-labeling with an antibody against the S-antigen, a pinealocyte-specific marker. The results show that norepinephrine stimulation induces p-CREB immunoreactivity by acting upon β-adrenergic receptors in virtually all rat pinealocytes. The findings support the notion that phosphorylation of CREB is a rather rapid and uniform response of pinealocytes to noradrenergic stimulation and thus is an important link between adrenoreceptor activation and subsequent gene expression in the rat pineal organ.

2D-PAGE analysis: Adrenergically regulated pineal protein AIP 37/6 is a phosphorylated isoform of cytosolic malate dehydrogenase

The adrenergic transmitter norepinephrine (NE) dramatically increases the prominence of only two out of the hundreds of [35S]methionine-labeled pineal proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). One of these regulated proteins is AIP 37/6 (37 kDa, pI approximately 6). The labeling of this protein is increased approximately 100-fold by NE. In the study presented here the identity of AIP 37/6 was investigated. The results of microsequencing, immunochemical analysis of 2D-PAGE blots and size exclusion chromatography indicate that AIP 37/6 is an isoform of cytosolic malate dehydrogenase (cMDH; approximately 36.3 kDa; pI approximately 6.5). Associated studies indicate that this isoform is phosphorylated whereas the bulk of cMDH is not. Cotranslational phosphorylation of cMDH is discussed.Washington State continues to grow more racially and ethnically diverse. The state’s population of color (non-white and Latino/Hispanic) increased from 13 percent in the 1990 census to an estimated 22.4 percent in 2004, according to the Office of Financial Management (OFM). Census 2000 reports that 10.4 percent (16,500) of the state’s citizens were immigrants. Of those, more than one in every four (26.4 percent) were recent newcomers to Washington, having arrived between 1995 and 2000. The number of non-English speaking persons 18 and over doubled in the period from 1990 to 2000, from about 117,000 to more than 261,000.