Whole-genome resequencing of 292 pigeonpea accessions identifies genomic regions associated with domestication and agronomic traits.

Pigeonpea (Cajanus cajan), a tropical grain legume with low input requirements, is expected to continue to have an important role in supplying food and nutritional security in developing countries in Asia, Africa and the tropical Americas. From whole-genome resequencing of 292 Cajanus accessions encompassing breeding lines, landraces and wild species, we characterize genome-wide variation. On the basis of a scan for selective sweeps, we find several genomic regions that were likely targets of domestication and breeding. Using genome-wide association analysis, we identify associations between several candidate genes and agronomically important traits. Candidate genes for these traits in pigeonpea have sequence similarity to genes functionally characterized in other plants for flowering time control, seed development and pod dehiscence. Our findings will allow acceleration of genetic gains for key traits to improve yield and sustainability in pigeonpea.

Gene expression defines natural changes in mammalian lifespan.

Mammals differ more than 100-fold in maximum lifespan, which can be altered in either direction during evolution, but the molecular basis for natural changes in longevity is not understood. Divergent evolution of mammals also led to extensive changes in gene expression within and between lineages. To understand the relationship between lifespan and variation in gene expression, we carried out RNA-seq-based gene expression analyses of liver, kidney, and brain of 33 diverse species of mammals. Our analysis uncovered parallel evolution of gene expression and lifespan, as well as the associated life-history traits, and identified the processes and pathways involved. These findings provide direct insights into how nature reversibly adjusts lifespan and other traits during adaptive radiation of lineages.

Complete genome sequence of Brucella abortus A13334, a new strain isolated from the fetal gastric fluid of dairy cattle.

Brucella abortus is a major pathogen that infects livestock and humans. A new strain of B. abortus (A13334) was isolated from the fetal gastric fluid of a dairy cow, with the aim of using it to compare genetic properties, analyze virulence factor, and survey the epidemiological relationship to other Brucella species. Here, we report the complete and annotated genome sequence of B. abortus A13334.

Complete genome sequence of Brucella canis strain HSK A52141, isolated from the blood of an infected dog.

Brucella canis infection can be clinically inapparent in dogs, and when infection goes unnoticed, there is a chance for dog-to-human transmission. A new strain of B. canis was isolated from the blood of an infected dog in order to analyze the pathogenic mechanism, compare genetic properties, and develop new genetic tools for early diagnosis of canine brucellosis. Herein, we report the complete genome sequence of the strain B. canis HSK A52141. This is the second complete genome sequence and biological annotation available for a member of B. canis.

Human astrocytic bradykinin B(2) receptor modulates zymosan-induced cytokine expression in 1321N1 cells.

Bradykinin is an important modulator of the neurons and glial cells of the nervous system. Bradykinin secreted from neurons affects astrocytic functions such as neurovascular coupling and astrocytic cytokine production. In human astrocytes, however, the detailed mechanism of bradykinin-mediated modulation of astrocytic functions has not yet been fully elucidated. Here, we report the functional expression of the bradykinin B(2) receptor and its modulation of zymosan-induced cytokine expression in human astrocytoma 1321N1 cells. Bradykinin increased cytosolic [Ca(2+)] in a concentration-dependent manner, whereas [des-Arg(10)] kallidin (an agonist of the B(1) receptor) did not have this effect. Bradykinin also triggered intracellular InsP(3) production. Pretreating the cells with HOE140 (icatibant acetate, a B(2) receptor antagonist) inhibited the bradykinin-induced increase in cytosolic [Ca(2+)] and InsP(3) production. In contrast, [des-Arg(10)]HOE140 (a B(1) receptor antagonist) did not show any inhibitory effect. Bradykinin increased the zymosan-induced expression of TNF-alpha, and interleukin 1beta (IL-1beta) but did not affect the expression of interleukin 6 (IL-6) or interleukin 10 (IL-10). Interestingly, a cyclooxygenase-2 specific inhibitor blocked the bradykinin-induced effect. In contrast to the result in human glioma cells, bradykinin inhibits the zymosan-induced expression of TNF-alpha and IL-1beta in rat astrocytes, which shows a species-dependent manner. These data suggest that bradykinin B(2) receptors are expressed in human astrocytoma cells and that they modulate the expression pattern of inflammatory cytokines.

Pineal function: impact of microarray analysis.

Microarray analysis has provided a new understanding of pineal function by identifying genes that are highly expressed in this tissue relative to other tissues and also by identifying over 600 genes that are expressed on a 24-h schedule. This effort has highlighted surprising similarity to the retina and has provided reason to explore new avenues of study including intracellular signaling, signal transduction, transcriptional cascades, thyroid/retinoic acid hormone signaling, metal biology, RNA splicing, and the role the pineal gland plays in the immune/inflammation response. The new foundation that microarray analysis has provided will broadly support future research on pineal function.

Thyroid hormone and adrenergic signaling interact to control pineal expression of the dopamine receptor D4 gene (Drd4).

Dopamine plays diverse and important roles in vertebrate biology, impacting behavior and physiology through actions mediated by specific G-protein-coupled receptors, one of which is the dopamine receptor D4 (Drd4). Here we present studies on the >100-fold daily rhythm in rat pineal Drd4 expression. Our studies indicate that Drd4 is the dominant dopamine receptor gene expressed in the pineal gland. The gene is expressed in pinealocytes at levels which are approximately 100-fold greater than in other tissues, except the retina, in which transcript levels are similar. Pineal Drd4 expression is circadian in nature and under photoneural control. Whereas most rhythmically expressed genes in the pineal are controlled by adrenergic/cAMP signaling, Drd4 expression also requires thyroid hormone. This advance raises the questions of whether Drd4 expression is regulated by this mechanism in other systems and whether thyroid hormone controls expression of other genes in the pineal gland.

Muscleblind-like 2: circadian expression in the mammalian pineal gland is controlled by an adrenergic-cAMP mechanism.

Muscleblind-like 2 (Mbnl2) is a zinc finger protein first identified in Drosophila. It appears to be essential for photoreceptor development and to be involved in RNA splicing. Here we report that Mbnl2 is strongly expressed in the rat pineal gland. The abundance of pineal Mbnl2 transcripts follows a marked circadian rhythm with peak levels approximately sevenfold higher at night than day levels. Mbnl2 protein exhibits a similar rhythm. In vitro studies indicate that the abundance of Mbnl2 transcripts and protein are controlled by an adrenergic/cAMP mechanism.

Night/day changes in pineal expression of >600 genes: central role of adrenergic/cAMP signaling.

The pineal gland plays an essential role in vertebrate chronobiology by converting time into a hormonal signal, melatonin, which is always elevated at night. Here we have analyzed the rodent pineal transcriptome using Affymetrix GeneChip(R) technology to obtain a more complete description of pineal cell biology. The effort revealed that 604 genes (1,268 probe sets) with Entrez Gene identifiers are differentially expressed greater than 2-fold between midnight and mid-day (false discovery rate <0.20). Expression is greater at night in approximately 70%. These findings were supported by the results of radiochemical in situ hybridization histology and quantitative real time-PCR studies. We also found that the regulatory mechanism controlling the night/day changes in the expression of most genes involves norepinephrine-cyclic AMP signaling. Comparison of the pineal gene expression profile with that in other tissues identified 334 genes (496 probe sets) that are expressed greater than 8-fold higher in the pineal gland relative to other tissues. Of these genes, 17% are expressed at similar levels in the retina, consistent with a common evolutionary origin of these tissues. Functional categorization of the highly expressed and/or night/day differentially expressed genes identified clusters that are markers of specialized functions, including the immune/inflammation response, melatonin synthesis, photodetection, thyroid hormone signaling, and diverse aspects of cellular signaling and cell biology. These studies produce a paradigm shift in our understanding of the 24-h dynamics of the pineal gland from one focused on melatonin synthesis to one including many cellular processes.

Developmental and daily expression of the Pax4 and Pax6 homeobox genes in the rat retina: localization of Pax4 in photoreceptor cells.

Pax4 is a homeobox gene encoding Pax4, a transcription factor that is essential for embryonic development of the endocrine pancreas. In the pancreas, Pax4 counters the effects of the related transcription factor, Pax6, which is known to be essential for eye morphogenesis. In this study, we have discovered that Pax4 is strongly expressed in retinal photoreceptors of the rat. Pax4 expression is not detectable in the foetal eye; however, postnatal Pax4 transcript levels rapidly increase. In contrast, Pax6 exhibits an inverse developmental pattern of expression being more strongly expressed in the foetal eye. Histological analysis revealed that Pax4 mRNA is exclusively expressed in the retinal photoreceptors, whereas Pax6 mRNA and protein are present in the inner nuclear layer and in the ganglion cell layer of the mature retina. In the adult retina, Pax4 transcripts exhibit a diurnal rhythm with maximal levels occurring during the light period, whereas retinal Pax6 transcript levels do not change throughout the day. The daily changes in Pax4 expression may contribute to daily changes in function in the differentiated retinal photoreceptor.

Developmental and diurnal dynamics of Pax4 expression in the mammalian pineal gland: nocturnal down-regulation is mediated by adrenergic-cyclic adenosine 3',5'-monophosphate signaling.

Pax4 is a homeobox gene that is known to be involved in embryonic development of the endocrine pancreas. In this tissue, Pax4 counters the effects of the related protein, Pax6. Pax6 is essential for development of the pineal gland. In this study we report that Pax4 is strongly expressed in the pineal gland and retina of the rat. Pineal Pax4 transcripts are low in the fetus and increase postnatally; Pax6 exhibits an inverse pattern of expression, being more strongly expressed in the fetus. In the adult the abundance of Pax4 mRNA exhibits a diurnal rhythm in the pineal gland with maximal levels occurring late during the light period. Sympathetic denervation of the pineal gland by superior cervical ganglionectomy prevents the nocturnal decrease in pineal Pax4 mRNA. At night the pineal gland is adrenergically stimulated by release of norepinephrine from the sympathetic innervation; here, we found that treatment with adrenergic agonists suppresses pineal Pax4 expression in vivo and in vitro. This suppression appears to be mediated by cAMP, a second messenger of norepinephrine in the pineal gland, based on the observation that treatment with a cAMP mimic reduces pineal Pax4 mRNA levels. These findings suggest that the nocturnal decrease in pineal Pax4 mRNA is controlled by the sympathetic neural pathway that controls pineal function acting via an adrenergic-cAMP mechanism. The daily changes in Pax4 expression may influence gene expression in the pineal gland.

Localization and regulation of dopamine receptor D4 expression in the adult and developing rat retina.

Levels of dopamine and melatonin exhibit diurnal rhythms in the rat retina. Dopamine is high during daytime adapting the retina to light, whereas melatonin is high during nighttime participating in the adaptation of the retina to low light intensities. Dopamine inhibits the synthesis of melatonin in the photoreceptors via Drd4 receptors located on the cell membrane of these cells. In this study, we show by semiquantitative in situ hybridization a prominent day/night variation in Drd4 expression in the retina of the Sprague-Dawley rat with a peak during the nighttime. Drd4 expression is seen in all retinal layers but the nocturnal increase is confined to the photoreceptors. Retinal Drd4 expression is not affected by removal of the sympathetic input to the eye, but triiodothyronine treatment induces Drd4 expression in the photoreceptors. In a developmental series, we show that the expression of Drd4 is restricted to postnatal stages with a peak at postnatal day 12. The high Drd4 expression in the rat retinal photoreceptors during the night supports physiological and pharmacologic evidence that the Drd4 receptor is involved in the dopaminergic inhibition of melatonin synthesis upon light stimulation. The sharp increase of Drd4 expression at a specific postnatal time suggests that dopamine is involved in retinal development.

Daily rhythm in pineal phosphodiesterase (PDE) activity reflects adrenergic/3',5'-cyclic adenosine 5'-monophosphate induction of the PDE4B2 variant.

The pineal gland is a photoneuroendocrine transducer that influences circadian and circannual dynamics of many physiological functions via the daily rhythm in melatonin production and release. Melatonin synthesis is stimulated at night by a photoneural system through which pineal adenylate cyclase is adrenergically activated, resulting in an elevation of cAMP. cAMP enhances melatonin synthesis through actions on several elements of the biosynthetic pathway. cAMP degradation also appears to increase at night due to an increase in phosphodiesterase (PDE) activity, which peaks in the middle of the night. Here, it was found that this nocturnal increase in PDE activity results from an increase in the abundance of PDE4B2 mRNA (approximately 5-fold; doubling time, approximately 2 h). The resulting level is notably higher (>6-fold) than in all other tissues examined, none of which exhibit a robust daily rhythm. The increase in PDE4B2 mRNA is followed by increases in PDE4B2 protein and PDE4 enzyme activity. Results from in vivo and in vitro studies indicate that these changes are due to activation of adrenergic receptors and a cAMP-dependent protein kinase A mechanism. Inhibition of PDE4 activity during the late phase of adrenergic stimulation enhances cAMP and melatonin levels. The evidence that PDE4B2 plays a negative feedback role in adrenergic/cAMP signaling in the pineal gland provides the first proof that cAMP control of PDE4B2 is a physiologically relevant control mechanism in cAMP signaling.

Rhythmic serotonin N-acetyltransferase mRNA degradation is essential for the maintenance of its circadian oscillation.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) is the key enzyme in melatonin synthesis regulated by circadian rhythm. To date, our understanding of the oscillatory mechanism of melatonin has been limited to autoregulatory transcriptional and posttranslational regulations of AANAT mRNA. In this study, we identify three proteins from pineal glands that associate with cis-acting elements within species-specific AANAT 3' untranslated regions to mediate mRNA degradation. These proteins include heterogeneous nuclear ribonucleoprotein R (hnRNP R), hnRNP Q, and hnRNP L. Their RNA-destabilizing function was determined by RNA interference and overexpression approaches. Expression patterns of these factors in pineal glands display robust circadian rhythm. The enhanced levels detected after midnight correlate with an abrupt decline in AANAT mRNA level. A mathematical model for the AANAT mRNA profile and its experimental evidence with rat pinealocytes indicates that rhythmic AANAT mRNA degradation mediated by hnRNP R, hnRNP Q, and hnRNP L is a key process in the regulation of its circadian oscillation.

Methionine adenosyltransferase:adrenergic-cAMP mechanism regulates a daily rhythm in pineal expression.

(S)-adenosylmethionine (SAM) is a critical element of melatonin synthesis as the methyl donor in the last step of the pathway, the O-methylation of N-acetyl 5-hydroxytryptamine by hydroxyindole-O-methyltransferase. The activity of the enzyme that synthesizes SAM, methionine adenosyltransferase (MAT), increases 2.5-fold at night in the pineal gland. In this study, we found that pineal MAT2A mRNA and the protein it encodes, MAT II, also increase at night, suggesting that the increase in MAT activity is caused by an increase in MAT II gene products. The night levels of MAT2A mRNA in the pineal gland were severalfold higher than in other neural and non-neural tissues examined, consistent with the requirement for SAM in melatonin synthesis. Related studies indicate that the nocturnal increase in MAT2A mRNA is caused by activation of a well described neural pathway that mediates photoneural-circadian regulation of the pineal gland. MAT2A mRNA and MAT II protein were increased in organ culture by treatment with norepinephrine (NE), the sympathetic neurotransmitter that stimulates the pineal gland at night. NE is known to markedly elevate pineal cAMP, and here it was found that cAMP agonists elevate MAT2A mRNA levels by increasing MAT2A mRNA synthesis and that drugs that block cAMP activation of cAMP dependent protein kinase block effects of NE. Therefore, the NE-cAMP dependent increase in pineal MAT activity seems to reflect an increase in MAT II protein, which occurs in response to cAMP-->protein kinase-dependent increased MAT2A expression. The existence of this MAT regulatory system underscores the importance that MAT plays in melatonin biogenesis. These studies also point to the possibility that SAM production in other tissues might be regulated through cAMP.