Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.     

Isolation of an autonomously replicating sequence (ARS) from satellite DNA of Drosophila melanogaster

Summary Autonomously replicating sequences (ARSs) were cloned from the 1.688 satellite DNA of D. melanogaster using YIp5, consisting of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^– yeast strain as the recipient. Three out of six clones contained an ARS and the average frequncy of the occurrence of ARS was thus calculated to be approximately one per 14 kbp of the satellite DNA. A 500 bp ARS fragment (BgHS500) was obtained from one of the resultant clones (pYDS57). BgHS500 does not hybridize with the major repeating unit (370 bp) but it does with the minor unique sequence of the satellite. The sequence of BgHS500 was determined and found to be rich in AT and to contain the sequence, 5AAAACATAAAA3, a sequence common to yeast ARSs. However, a smaller fragment (150 bp) isolated from BgHS500 and containing the 11 bp sequence did not exhibit the characteristics of an ARS. The average copy number in the transformants of pBgHS500, a recombinant molecule of BgHS500 and YIp5, ranged from 0.05–0.5, while that of the parent plasmid, pYDS57, was about 2–10. On the basis of these results, it is postulated that the sequence 5AAAACATAAAA3 may possibly consiitute the core of ARSs and certain other sequences may also be necessary to insure that the ARS consistently undergoes at least one complete replication in each cell cycle. The role of ARSs in the genome of D. melanogaster is discussed.     

Increase of (Ca2++Mg2+)-ATPase activity of renal basolateral membrane by parathyroid hormone via cyclic AMP-dependent membrane phosphorylation

Studies were made on the mechanism of the effect of parathyroid hormone (PTH) on the activity of (Ca2++Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme from the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.3 X 10(-7) M, Vmax = 200 nmol Pi/mg/min). At 1 X 10(-7) M free Ca2+, both PTH (10(-7)-10(-6) M) and cAMP (10(-6)-10(-4) M) stimulated (Ca2++Mg2+)-ATPase activity dose-dependent and their stimulatory effects were inhibited completely by 5 microM H-8, an inhibitor of cAMP-dependent protein kinase. PTH (10(-7) M) also caused 40% increase in 32P incorporation into the BLM and 5 microM H-8 inhibited this increase too. PTH (10(-7) M) was found to stimulate phosphorylation of a protein of Mr 9000 by cAMP dependent protein kinase and 5 microM H-8 was found to block this stimulation also. From these results, it is proposed that PTH stimulates (Ca2++Mg2+)-ATPase activity by enhancing its affinity for free Ca2+ via cAMP-dependent phosphorylation of a BLM protein of Mr 9000.     

Pork peptone stimulates cholecystokinin secretion from enteroendocrine cells and suppresses appetite in rats

We found that soybean beta-conglycinin peptone (BconP) suppresses food intake through cholecystokinin (CCK) release from enteroendocrine cells in association with binding of the peptone to rat small intestinal brush border membrane (BBM). The aim of the present study was to find new appetite suppressing peptides. Peptones from chicken, pork, beef, beef liver, and egg white were examined for activities to bind with rat BBM, CCK-release from enteroendocrine cell line STC-1, and induce satiety in rats. Chicken and pork peptone (ChickP and PorkP) bound to BBM with highest ability as evaluated with a surface plasmon biosensor. PorkP and ChickP released CCK in higher amounts than BconP from STC-1 cells dose-dependently, with highest stimulation by PorkP. An orogastric preload of PorkP, but not ChickP, suppressed food intake similarly to BconP, dose-dependently. These results suggest that PorkP interacts directly with the small intestinal CCK cells to release CCK, and that it suppresses appetite in rats.     

The Xdsg protein in presumptive primordial germ cells (pPGCs) is essential to their differentiation into PGCs in Xenopus

In order to know the role of the Xdsg gene in presumptive PGCs (pPGCs) of Xenopus, we attempted to inhibit the translation of Xdsg mRNA in pPGCs by injecting antisense morpholino oligo (asMO), together with Fluorescein Dextran-Lysine (FDL), into single germ plasm-bearing cells of 32-cell embryos. Among three types of asMOs complementary to different parts of the 5'-untranslated region of Xdsg mRNA tested, only one asMO, designated as Xdsg-3, inhibited the translation of the mRNA in FDL-labeled pPGCs, resulting in the absence of labeled PGCs in experimental tadpoles. On the other hand, two other asMOs, Xdsg-1 and -2, did not inhibit the translation, so that a similar number of labeled PGCs found in FDL-injected but asMO-uninjected control tadpoles were observed in experimental tadpoles derived from asMO-injected embryos. Surprisingly, use of Xdsg-3 asMO resulted in the disappearance of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) from FDL-labeled pPGCs by inhibiting the translation of XVLG1 mRNA. However, the effect of Xdsg-3 asMO on the translation of Xdsg and XVLG1 mRNAs and PGC formation could be canceled by the coinjection with Xdsg mRNA. Consequently, the Xdsg protein in pPGCs may play an important role in the formation of PGCs by regulating the production of XVLG1 protein.     

Application of menthol to the skin of whole trunk in mice induces autonomic and behavioral heat-gain responses

When ambient temperature is decreased in mammals, autonomic and behavioral heat-gain responses occur to maintain their core temperatures. However, what molecules in cutaneous sensory nerve endings mediate cooling-induced responses is unclear. Recently, transient receptor potential melastatin-8 (TRPM8) has been identified in cell bodies of sensory neurons as low-temperature and menthol-activated cation channel. We hypothesized that TRPM8 mediates cooling-induced autonomic and behavioral heat-gain responses. To activate TRPM8 specifically, we applied 1-10% menthol to the skin of whole trunk in mice instead of cooling and measured core temperatures and autonomic and behavioral heat-gain responses. Solvent of menthol (100% ethanol) was used as control. Significant elevation of core temperatures was observed between 20 and 120 min after menthol application. Pretreatment with diclofenac sodium, an antipyretic drug, did not affect this hyperthermia, indicating that the menthol-induced hyperthermia is not fever. Menthol application induced a rise in oxygen consumption, shivering-like muscle activity, tail skin vasoconstriction (autonomic responses), and heat-seeking behavior. All of them are typical heat-gain responses. These results support the hypothesis that TRPM8 mediates cooling-induced autonomic and behavioral heat-gain responses.     

Impaired NO-mediated vasodilation with increased superoxide but robust EDHF function in right ventricular arterial microvessels of pulmonary hypertensive rats

Pulmonary hypertension (PH) causes right ventricular (RV) hypertrophy and, according to the extent of pressure overload, eventual heart failure. We tested the hypothesis that the mechanical stress in PH-RV impairs the vasoreactivity of the RV coronary microvessels of different sizes with increased superoxide levels. Five-week-old male Sprague-Dawley rats were injected with monocrotaline (n=126) to induce PH or with saline as controls (n=114). After 3 wk, coronary arterioles (diameter = 30-100 microm) and small arteries (diameter = 100-200 microm) in the RV were visualized using intravital videomicroscopy. We evaluated ACh-induced vasodilation alone, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), in the presence of tetraethylammonium (TEA) or catalase with or without L-NAME, and in the presence of SOD. The degree of suppression in vasodilation by L-NAME and TEA was used as indexes of the contributions of endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), respectively. In PH rats, ACh-induced vasodilation was significantly attenuated in both arterioles and small arteries, especially in arterioles. This decreased vasodilation was largely attributable to reduced NO-mediated vasoreactivity, whereas the EDHF-mediated vasodilation was relatively robust. The suppressive effect on arteriolar vasodilation by catalase was similar to TEA in both groups. Superoxide, as measured by lucigenin chemiluminescence, was significantly elevated in the RV tissues in PH. SOD significantly ameliorated the impairment of ACh-induced vasodilation in PH. Robust EDHF function will play a protective role in preserving coronary microvascular homeostasis in the event of NO dysfunction with increased superoxide levels.     

Photoreactions of Tyr8- and Gln50-mutated BLUF domains of the PixD protein of Thermosynechococcus elongatus BP-1: photoconversion at low temperature without Tyr8

We studied the photoreaction of a blue-light sensor PixD protein of Thermosynechococcus elongatus that has the blue-light-using flavin (BLUF) domain. The Tyr8 and Gln50 residues of the protein were modified to phenylalanine, alanine, or asparagine (Y8F, Y8A, Q50N, and Q50A) by site-directed mutagenesis. The following results were obtained. (1) At room temperature, blue-light illumination induced the red shift of the absorption bands of flavin in the wild-type (WT) protein but not in the Y8F, Y8A, Q50A, and Q50N mutant proteins, as reported [Okajima, K., et al. (2006) J. Mol. Biol. 363, 10-18]. (2) At 80 K, neither the Q50N nor the Q50A mutant protein accumulated the red-shifted form. (3) At 80 K, the Y8F protein photoaccumulated the red-shifted forms to an extent that was half that in the WT protein at a 43-fold slower rate, and the Y8A protein to the one-fourth the extent at a 137-fold slower rate. (4) The red-shifted form in the Y8F protein was stable below 240 K and became unstable above 240 K in the dark. (5) The illumination of the Y8F protein at 150 K accumulated the red-shifted form at the beginning, and the prolonged illumination accumulated the flavin anions by the secondary photoreaction. (6) The results indicate that Tyr8 is not indispensable for the accumulation of the red-shifted form at least at 80 K. (7) Photoconversion mechanisms in the WT and Tyr8-mutated proteins are discussed in relation to the schemes with and without the electron transfer between Tyr8 and flavin in the first step of the photoconversion.