Light-induced tyrosine phosphorylation of BIT in the rat suprachiasmatic nucleus

Circadian changes of protein tyrosine phosphorylation in the hypothalamic suprachiasmatic nucleus have been studied using rats maintained under 12-h light/ 12-h dark cycles as well as constant dark conditions. We found that tyrosine phosphorylation of BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs), a transmembrane glycoprotein of 90-95 kDa, was higher in the light period than in the dark period and was increased after light exposure in the dark period. Similar changes in tyrosine phosphorylation were observed under constant dark conditions, but its amplitude was weaker than that in 12-h light/12-h dark cycles. As the tyrosine-phosphorylated form of BIT is able to bind to the Src homology 2 domain of a protein tyrosine phosphatase, SHP-2, we examined association of these proteins in suprachiasmatic nucleus extracts and found that SHP-2 was coprecipitated with BIT in parallel with its tyrosine phosphorylation. These results suggest that tyrosine phosphorylation of BIT might be involved in light-induced entrainment of the circadian clock.     

Multiple oscillators in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the pacemaker that controls circadian rhythms of a variety of physiological functions. Data strongly indicate the majority of the SCN neurons express self-sustaining oscillations that can be detected as rhythms in the spontaneous firing of individual neurons. The period of single SCN neurons in a dissociated cell culture is dispersed in a wide range (from 20h to 28h in rats), but that of the locomotor rhythm is close to 24h, suggesting individual oscillators are coupled to generate an averaged circadian period in the nucleus. Electrical coupling via gap junctions, glial regulation, calcium spikes, ephaptic interactions, extracellular ion flux, and diffusible substances have been discussed as possible mechanisms that mediate the interneuronal rhythm synchrony. Recently, GABA (γ-aminobutyric acid), a major neurotransmitter in the SCN, was reported to regulate cellular communication and to synchronize rhythms through GABA_A receptors. At present, subsequent intracellular processes that are able to reset the genetic loop of oscillations are unknown. There may be diverse mechanisms for integrating the multiple circadian oscillators in the SCN. This article reviews the knowledge about the various circadian oscillations intrinsic to the SCN, with particular focus on the intercellular signaling of coupled oscillators. (Chronobiology International, 18(3), 371-387, 2001)     

Different patterns of circadian oscillation in the suprachiasmatic nucleus of hamster,mouse, and rat

Although spontaneous neural firing in the mammalian suprachiasmatic nucleus is accepted to peak once during mid-subjective day, dual activity peaks have been reported in horizontal brain slices taken from hamsters. These two peaks were interpreted as new evidence for the theory of dual circadian oscillators and raised the expectation that such activity would be found in other circadian model systems. We examined hamster, mouse, and rat slices in both coronal and horizontal planes and found a second peak of activity only in hamster horizontal preparations. This raises interesting questions about the relative circadian physiology of these important experimental animals.Abbreviations CT circadian time - SCN suprachiasmatic nucleus P.W. Burgoon and P.T. Lindberg contributed equally to this work.     

Chromosomal abnormalities and hormonal disorders of primary amenorrhea patients in Egypt

BACKGROUND:

Primary amenorrhea is defined as the absence of menstruation and secondary sexual characteristics in phenotypic women aged 14 years or older. Hormonal disorders are main causes of primary amenorrhea. Common hormonal cause of primary amenorrhea includes pituitary dysfunction and absent ovarian function. The aim of this study was to estimate the incidence and types of chromosomal abnormalities in patients with primary amenorrhea in Egypt.

MATERIALS AND METHODS:

Chromosomal analysis and hormonal assay were carried out on 223 patients with primary amenorrhea that were referred from different parts of Egypt to Cytogenetic laboratory of Genetic Unit, Children Hospital Mansoura University, from July 2008 to December 2010. FISH technique was carried out in some of cases to more evaluation.

RESULTS:

The frequency of chromosomal abnormalities was 46 (20.63%) in primary amenorrhea patients. The chromosomal abnormalities can be classified into four main types. (1) The numerical abnormalities of the X chromosome were detected in 23 (50 %). (2) Structural abnormalities of the X chromosome were detected in 11 (23.91%). (3) Mosaicism of X chromosome was found in 10 (21.74%). (4) Male karyotype 46, XY was presented in 2 (4.35%).

CONCLUSION:

The present study showed that karyotype and FISH are necessary to detect the causes of primary amenorrhea. This study also revealed the incidence of chromosomal abnormalities in women with primary amenorrhea in Egypt is similar to that reported in previous literatures.     

Spatial and temporal regulation of mitogen-activated protein kinase phosphorylation in the mouse suprachiasmatic nucleus

Circadian and photic regulation of mitogen-activated protein kinase (MAPK) has been shown to associate closely with the function of the circadian clock in vertebrate clock tissues such as the mouse suprachiasmatic nucleus (SCN). Here we show that, in the central region of the mouse SCN, MAPK exhibited circadian and daily rhythms in phosphorylation with a peak at (subjective) night, and this activation was sustained for at least 8 h. In contrast, in the dorsomedial region of the SCN, MAPK showed an overt rhythm in phosphorylation with a transient peak at early subjective day, which was antiphase to that in the central region. Noticeably, the phospho-MAPK-immunoreactive cells observed in the dorsomedial region were distributed from the rostral to the caudal end of the SCN, whereas those observed in the central region were localized within the middle SCN along the rostral-caudal axis. Furthermore, a 15-min light pulse given at subjective night transiently evoked MAPK phosphorylation throughout the ventrolateral region of the SCN peaking within 15 min after the light onset, whereas nighttime-phosphorylated MAPK signals in the central-middle SCN become undetectable within 60 min after the light onset. Thus, the mode of circadian and photic regulation of MAPK phosphorylation varies remarkably among the three subregions within the SCN, suggesting divergent and cell type-specific roles of MAPK in the clock system of the mouse SCN.     

Biogeography and phylogeny of the NOR5/OM60 clade of Gammaproteobacteria

The phylogeny, abundance, and biogeography of the NOR5/OM60 clade was investigated. This clade includes “Congregibacter litoralis” strain KT71, the first cultured representative of marine aerobic anoxygenic phototrophic Gammaproteobacteria. More than 500 16S rRNA sequences affiliated with this clade were retrieved from public databases. By comparative sequence analysis, 13 subclades could be identified, some of which are currently restricted to discrete habitat types. Almost all sequences in the largest subclade NOR5-1 and related subclade NOR5-4 originated from marine surface water samples. Overall, most of the NOR5/OM60 sequences were retrieved from marine coastal settings, whereas there were fewer from open-ocean surface waters, deep-sea sediment, freshwater, saline lakes and soil.     

哺乳动物昼夜节律组构中的下丘脑视交叉上核和松果腺

哺乳动物下丘脑视交叉上核（SCN）是昼夜节律最主要的起搏器,控制着机体的生理和行为的节律。它具有自身内在的节律性,同时也受光照周期信号和一些内源性化学物质的调节。检查腺分泌裉黑素（MEL）受SCN的调控,MEL通过作用于SCN上高亲和性MEL受体,启动第二、第三信使系统,调整SCN的昼夜节律活动。这种调整具有时间敏感性。     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.     

Expression of a GABAB - Receptor in Olfactory Sensory Neurons of Sensilla trichodea on the Male Antenna of the Moth Heliothis virescens

In the olfactory pathway of Drosophila, a GABA_B receptor mediated presynaptic gain control mechanism at the first synapse between olfactory sensory neurons (OSNs) and projection neurons has been suggested to play a critical role in setting the sensitivity and detection range of the sensory system. To approach the question if such a mechanism may be realized in the pheromone recognition system of male moths in this study attempts were made to explore if moth''s pheromone-responsive cells express a GABA_B- receptor. Employing a combination of genome analysis, RT-PCR experiments and screening of an antennal cDNA library we have identified a cDNA which encodes the GABA_B-R1 receptor of Heliothis virescens. Moreover, based on the HvirGABA_B-R1 sequence we could predict a GABA_B-R1 protein from genome sequences of the silkmoth Bombyx mori. To assess whether HvirGABA_B-R1 is expressed in OSNs of male antenna we performed whole-mount in situ hybridization (WM-ISH) experiments. Several HvirGABA_B-R1 positive cells were visualized under long sensilla trichodea, known to contain pheromone-responsive OSNs. In parallel it was shown that cells under long trichoid hairs were labelled with pheromone receptor specific probes. In addition, the HvirGABA_B-R1 specific probe also labelled several cells under shorter olfactory sensilla, but never stained cells under mechanosensory/gustatory sensilla chaetica. Together, the results indicate that a GABA_B receptor is expressed in pheromone-responsive OSNs of H. virescens and suggest a presynaptic gain control mechanism in the axon terminals of these cells.     

Expression of a glucocorticoid receptor (DlGR1) in several tissues of the teleost fish Dicentrarchus labrax

Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization) and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported. Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of DlGR1 localization are discussed.     

Expression and localization of intestinal 15 kDa protein in the rat

Rat intestinal 15 kDa protein (I-15P) is highly homologous to porcine gastrotropin. We studied the occurrence, distribution and subcellular localization of I-15P in the entire rat body, using the immunocytochemistry to localize protein andin situ hybridization to localize mRNA. Both techniques demonstrated the expression of I-15P in the enterocytes of ileum, luteal cells of ovary and a subpopulation of steroid-endocrine cells of adrenal gland. Immuno-electron microscopy further demonstrated that I-15P is localized in both the cytoplasmic and nuclear matrix regions of these cells. The present results suggest roles of I-15P not only in the transport of bile salts but also in the metabolisms of certain steroid hormones.     

Neuropeptide changes in the suprachiasmatic nucleus are associated with the development of hypertension

ABSTRACT

Human postmortem studies as well as experimental animal studies indicate profound changes in neuropeptide expression in the suprachiasmatic nucleus (SCN) in several pathological conditions including hypertension. In addition, animal experimental observations show that the SCN peptides, vasopressin (AVP) and vasoactive intestinal peptide (VIP) are essential for adequate rhythmicity. These data prompted us to investigate whether changes in these neuronal populations could be the cause or consequence of hypertension. Changes in blood pressure and levels of neuropeptide expression in the SCN were determined during development of hypertension in spontaneously hypertensive rats (SHR), in 2K1C reno-vascular induced hypertensive animals and their respective controls. During the pre-hypertensive stage (5 weeks of age), the VIP and AVP content was higher and the somatostatin (SOM) content was lower in the SHR SCN. At the onset of hypertension (12 weeks of age), when blood pressure levels had just reached about 140 mmHg, AVP and SOM content in the SCN was not different anymore in SHRs compared to control, but VIP was still higher. After 16 weeks, the AVP content was decreased, but SOM was increased and the overall level of VIP in the SCN was still higher in SHRs compared to controls. None of the aforementioned changes in the SCN was observed after induction of hypertension in the 2K1C model. However, while VIP was increased in the NTS projecting medial region of the SCN in SHR animals only after the establishment of hypertension, VIP was decreased in the same region in the 2K1C induced hypertensive rats. Consequently, the present findings confirm previous studies in human and rat indicating that changes in the SCN are strongly associated with the development of hypertension. In addition, the changes in peptide content in the 2K1C animals indicate that the SCN is also able to respond to increases in blood pressure.     

Differential effects of chronic treatment with haloperidol and clozapine on the level of preprosomatostatin mRNA in the striatum,nucleus accumbens,and frontal cortex of the rat

1. The goal of this work was to determine the effects of typical and atypical neuroleptics on the level of preprosomatostatin messenger RNA (mRNA) in regions of the rat brain innervated by dopaminergic neurons. 2. Quantitative in situ hybridization histochemistry was used to measure the levels of mRNA encoding preprosomatostatin in neurons of the striatum, the nucleus accumbens, and the medial and lateral agranular areas of the frontal cortex in adult rats treated with either haloperidol or clozapine. 3. In untreated animals, the density of neurons containing preprosomatostatin mRNA was higher in the nucleus accumbens than in the striatum and frontal cortex. The intensity of labeling per neuron, however, was higher in the striatum than in the two other areas examined, suggesting that the expression of preprosomatostatin mRNA is differentially regulated in these brain regions. Chronic administration of haloperidol (1 mg/kg for 28 days) induced a significant decrease in the labeling for preprosomatostatin mRNA in neurons of the nucleus accumbens, frontal cortex, and medial but not lateral striatum. Treatment with clozapine (20 mg/kg for 28 days) increased the levels of preprosomatostatin mRNA in the nucleus accumbens but not in the striatum or the frontal cortex. 4. These results support a role for dopamine in the regulation of central somatostatinergic neurons. The differences in the effects of haloperidol, a neuroleptic which induces extrapyramidal side effects, and clozapine, which does not, suggest that somatostatinergic neurons may play an important role in the regulation of motor behavior.     

Postnatal development of the suprachiasmatic hypothalamic nucleus of the rat

Summary Light and electron microscopy of newborn, four day, one, two, three and five week old rats revealed principally a progressive increase in the diversity and number of synaptic contacts in the suprachiasmatic nucleus (SCN). The major increase in synaptic diversity occurred between four days and one week of age. Correlation between this finding and the adult synaptic morphology of SCN (Güldner, 1976) on the one hand, and the ontogeny of circadian rhythms on the other were made. This suggested that the retinal afferents arriving on day four form asymmetrical contacts with dendrites. While increase in synaptic number was progressive, it was most marked between three and five weeks of age. By five weeks, most features of the adult SCN were present. No significant morphological effects were evident as a result of neonatal retinal lesions.Supported in part by grants NS-12265, NS-12267, HD04583 and HD-08658 from the National Institutes of Health, USPHS. The electron microscopic facilities of the California Regional Primate Center, supported by NIH grant RR-00169, were utilized. The technical assistance of Mrs. Viviana Wong is gratefully acknowledged. A preliminary report of a portion of this data was given at the Society for Neuroscience, November, 1974 in St. Louis, Missouri.     

The role of Clock in the plasticity of circadian entrainment

The mammalian circadian clock lying in suprachiasmatic nucleus (SCN) is synchronized to about 24 h by the environmental light-dark cycle (LD). The circadian clock exhibits limits of entrainment above and below 24 h, beyond which it will not entrain. Little is known about the mechanisms regulating the limits of entrainment. In this study, we show that wild-type mice entrain to only an LD 24 h cycle, whereas Clock mutant mice can entrain to an LD 24, 28, and 32 h except for LD 20 h and LD 36 h cycle. Under an LD 28 h cycle, Clock mutant mice showed a clear rhythm in Per2 mRNA expression in the SCN and behavior. Light response was also increased. This is the first report to show that the Clock mutation makes it possible to adapt the circadian oscillator to a long period cycle and indicates that the clock gene may have an important role for the limits of entrainment of the SCN to LD cycle.     

Microscopical techniques reveal the in situ microbial association inside Aplysina aerophoba, Nardo 1886 (Porifera, Demospongiae, Verongida) almost exclusively consists of cyanobacteria

Sponges (Porifera) are aquatic, sessile filter feeders. As such they are permanently exposed to bacteria in the seawater. Molecular data recovered from sponges by PCR shows a high diversity in bacterial DNA. Hence, sponges are considered to live in close association with a diverse and abundant bacterial community. To recover the spatial distribution of bacteria in sponges we retrieved histological sections of Aplysina aerophoba fixed in situ. By combining signals from fluorescence in situ hybridization (FISH), light microscopy and scanning electron microscopy we revealed a detailed histological picture of the spatial organization of the sponge microbial association within the sponges. Our histological results confirm a high abundance of cyanobacteria inside A. aerophoba while other living bacteria are almost absent. This detailed insight into sponge microbiology could only be achieved by the combination of careful sample preparation and different microscopical and histological methods. It also shows the need to confirm molecular datasets in situ and with a high spatial resolution.     

水稻金属耐受蛋白基因OsMTP2生物信息学及表达分析

为了解水稻(Oryza sativa)响应重金属污染及对重金属积累的机制,利用生物信息学手段对水稻金属耐受蛋白(metal tolerance protein, MTP) OsMTP2的结构特征进行预测,并分析OsMTP2基因的表达特征。结果表明,该蛋白由415个氨基酸组成,具有很强的疏水性。mRNA原位杂交结果表明,OsMTP2在叶片输导组织中有较强的表达,但在根、花药中没有检测到明显的杂交信号。这说明水稻金属耐受蛋白基因OsMTP2主要在输导组织中表达,并在金属离子运输中发挥重要作用。     

Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus:In situ hybridization histochemistry and immunocytochemistry

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.     

Circadian modulation in photic induction of Fos-like immunoreactivity in the suprachiasmatic nucleus cells of diurnal chipmunk,Eutamias asiaticus

Photic induction of immediate early genes including c-fos in the suprachiasmatic nucleus (SCN) has been well demonstrated in the nocturnal rodents. On the other hand, in diurnal rodents, no data is available whether the light can induce c-fos or Fos in the SCN. We therefore examined whether 60 min light exposure induces Fos-like immunoreactivity (Fos-lir) in the SCN cells of diurnal chipmunks and whether the induction is phase dependent, comparing with the results in nocturnal hamsters. We also examined an effect of light on the locomotor activity rhythm under continuous darkness. Fos-lir was induced in the chipmunk SCN. The induction was clearly phase dependent. The light during the subjective night induced strong expression of Fos-lir. This phase dependency is similar to that in hamsters. However, unlike in hamsters, the Fos-lir was induced in some SCN cells of chipmunks exposed to light during the subjective day. In the locomotor rhythm, on the other hand, the light pulse failed to induce the phase shift at phases at which the Fos-lir was induced. These results suggest that the photic induction of Fos-lir in the diurnal chipmunks is gated by a circadian oscillator as well as in the nocturnal hamsters. However, the functional role of Fos protein may be different in the diurnal rodents from in the nocturnal rodents.