Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein.

Genetic and embryological studies in the mouse demonstrated functional differences between parental chromosomes during development. This is due to imprinted genes whose expression is dependent on their parental origin. In a recent systematic screen for imprinted genes, we detected Peg3 (paternally expressed gene 3). Peg3 is not expressed in parthenogenones. In interspecific hybrids, only the paternal copy of the gene is expressed in the embryos, individual tissues examined in d9.5-13.5 embryos, neonates and adults. Peg3 mRNA is a 9 kb transcript encoding an unusual zinc finger protein with eleven widely spaced C2H2 type motifs and two groups of amino acid repeats. Peg3 is expressed in early somites, branchial arches and other mesodermal tissues, as well as in the hypothalamus. Peg3 maps to the proximal region of chromosome 7. Consistent with our findings, maternal duplication of the proximal chromosome 7 causes neonatal lethality. This region is syntenic with human chromosome 19q13.1-13.3 (refs 10,11), where the genes for myotonic dystrophy and a putative tumour suppressor gene are located.

Expression and imprinting status of human PEG8/IGF2AS, a paternally expressed antisense transcript from the IGF2 locus, in Wilms' tumors.

A large imprinted gene cluster in human chromosome 11p15.5 has been implicated in Beckwith-Wiedemann syndrome and Wilms' tumor. We have identified a paternally expressed imprinted gene, PEG8/IGF2AS, in this locus. It is transcribed in the opposite direction to the IGF2 transcripts and some genomic regions are shared with the IGF2 gene, as in the case of the mouse imprinted Igf2as gene reported previously by T. Moore et al. As to the relationship between these genomic regions, the human and mouse genes are very similar but there is no homology in their middle parts. Interestingly, PEG8/IGF2AS and IGF2 were found to be overexpressed in Wilms' tumor samples, at levels over ten and a hundred times higher than that in normal kidney tissues neighboring the tumors, respectively. These findings indicate that PEG8/IGF2AS is a good marker of Wilms' tumor and also suggest the possibility of PEG8/IGF2AS being one of the candidate Wilms' tumor genes.

Cilostazol, a selective cAMP phosphodiesterase inhibitor, dilates retinal arterioles and increases retinal and choroidal blood flow in rats.

The effects of cilostazol, a selective cyclic AMP (cAMP) phosphodiesterase inhibitor, on retinal and choroidal blood flow and retinal arteriole diameter were examined in anesthetized rats. The retinal and choroidal blood flow was measured using laser Doppler flowmetry, and the diameter of the retinal arterioles was measured using digital video microscopy. Cilostazol was administered by two routes; systemically via the intravenous route, and directly into the retinal vessels via the intra-arterial route. When administered intravenously, 1 mg/kg of cilostazol produced a biphasic blood flow response, composed of an initial decrease which was dependent on a depressor response of mean arterial pressure, and a subsequent slight but significant increase which was independent of changes in mean arterial pressure. When administered intra-arterially over a 2-min period, 40-55 and 400-440 microg of cilostazol both produced an increase in the blood flow in a dose-dependent manner, while a depressor effect was observed only at the dose of 400-440 microg. The diameter of the retinal arterioles was increased after the intra-arterial injection of cilostazol (400 microg). It is concluded that intra-arterially administered cilostazol induces vasodilation of the retinal arterioles of rats, which results in an increase in blood supply to the retina, independent of changes in mean arterial pressure.

Central effects of (5RS, 1'SR)-5-benzyl-3-(3'-morpholino-1'-phenylpropyl)-1,3-oxazolidin-2 -one monofumarate on the function of the bladder and periurethral skeletal muscle in anesthetized rats.

The effects of a newly developed drug for incontinence, (5RS, 1'SR)-5-benzyl-3-(3'-morpholino-1'-phenylpropyl)-1,3-oxazolidin-2- one monofumarate (NC-1800), on bladder and periurethral skeletal muscle functions were tested in urethane-anesthetized rats. When the bladder pressure was low, i.v. administration of NC-1800 at doses of 4 to 16 mg/kg induced dose-dependent increases of vesical pressure associated with increases in pelvic efferent nerve activity. When the bladder was expanded, the same administration of NC-1800 induced dose-dependent inhibitions of both vesical micturition contractions and rhythmic pelvic burst discharges. Hypogastric efferent nerve activity was not affected. The periurethral electromyogram (EMG) activity was excited when the bladder was contracted, and EMG activity was inhibited when the bladder was relaxed by NC-1800. Pelvic ganglionic transmission, neuromuscular transmission of both bladder and urethra, and muscle contractility itself of bladder and urethra were not affected by NC-1800. These results suggest that NC-1800 modulates the functions of the bladder and urethra by influencing pelvic and pudendal nerve activity via the central nervous system.

Effect of stimulation of nicotinic cholinergic receptors on cortical cerebral blood flow and changes in the effect during aging in anesthetized rats.

The effect of intravenous injection of nicotine on cortical cerebral blood flow (CBF) was examined in urethane anesthetized rats. Nicotine (3-30 microg/kg) increased cortical CBF, independent of mean arterial pressure. This response was attenuated to about a half of the control one after lesioning the nucleus basalis of Meynert (NBM) bilaterally. The response was not significantly influenced after blocking the muscarinic receptors, but was abolished after blocking the nicotinic receptors in the parenchyma of the brain. It is concluded that the nicotine-induced cortical vasodilation was mediated by activation of the nicotinic receptors in the NBM and also in the cortex of the brain. The threshold dose of nicotine for increasing cortical CBF was shifted in aged rats of 23-26 months, and the nicotine-induced increase in cortical CBF was much reduced in aged rats of 32-36 months. Activation of nicotinic receptors in the brain may be of therapeutic value in aged subjects in facilitating the cholinergic neural vasodilative system.

The role of the spinal cord as a reflex center for the somatically induced reflex responses of splenic sympathetic and natural killer cell activity in anesthetized rats.

Somatic afferent regulation of splenic natural killer (NK) cell activity by hindpaw pinching has been proven, in anesthetized rats, to be a reflex response whose reflex center is in the brain and efferent arc is a splenic sympathetic nerve. Using central nervous system (CNS)-intact and acutely spinalized anesthetized rats, the present study aimed to examine the possibility of whether afferent stimulation (pinching) of the skin over the abdominal segments could influence cytotoxic activity of splenic NK cells and splenic sympathetic nerve activity at the spinal segmental level. In CNS-intact rats, pinching stimulation of the skin of the abdomen with surgical clamps for 30 min did not significantly change cytotoxic activity of splenic NK cells although splenic sympathetic nerve activity increased slightly. In acutely spinalized rats the same stimulation reduced cytotoxic activity of splenic NK cells and was accompanied by an intense reflex increase in splenic sympathetic nerve activity. It is concluded that the spinal cord is capable of producing propriospinally the reflex suppression of cytotoxic activity of splenic NK cells via reflex activation of the splenic sympathetic efferent nerve following stimulation to the abdominal segments whose afferent information enters the spinal cord at the same segments or segments overlapping the splenic sympathetic outflow. A possible mechanism of inhibition of this spinal reflex by inhibitory descending pathways is discussed.

Effects of age on cholinergic vasodilation of cortical cerebral blood vessels in rats.

The present study examined the age-related changes in the cholinergic vasodilative system originating in the nucleus basalis of Meynert (NBM) and projecting to the cerebral cortex using Wistar rats of three different ages; young adult (4-7 months), old (24-25 months), and very old (32-42 months) rats. The vasodilative responses in frontal and parietal cortices, measured by laser Doppler flowmetry, induced by electrical stimulation of NBM without blood pressure response were well maintained in old rats, but declined significantly in very old rats. Extracellular acethylcholine (ACh) release in both cortices collected by a microdialysis technique showed both basal levels and response to NBM stimulation to be well maintained in both old and very old rats. The vasodilative cerebral blood flow response elicited by stimulation of the muscarinic ACh receptors, using their agonist, arecoline, was also well maintained in old and very old rats. Considering the present data and our previous finding that the cerebral cortical vasodilative response to activation of the nicotinic ACh receptors using their agonist, nicotine, was markedly reduced in very old rats (Neurosci. Lett., 228 (1997) 203), it was concluded that the age-related decline of nicotinic ACh receptor activity was a cause of the decline of the vasodilative responses elicited by NBM stimulation in very old rats. This result suggests that a reduction of the cholinergic vasodilative system in very old rats due to decreased activity of the nicotinic ACh receptor may cause insufficient blood flow in the cortex when the cortical neurons require.

Effects of nicotine on blood flow and delayed neuronal death following intermittent transient ischemia in rat hippocampus.

A cholinergic neural vasodilative response in the cerebral cortex and hippocampus, independent of metabolic vasodilation, was recently demonstrated by activating the nicotinic acetylcholine receptors (nAChRs) via activation of cholinergic neurons originating in the nucleus basalis of Meynert and septal complex in the basal forebrain and projecting to the cortex and hippocampus (see reviews by Sato A and Sato Y: Neurosci Res 14: 242--274, 1992; Sato A and Sato Y: Alzheimer Dis Assoc Disord 9: 28--38, 1995). In the present study, we aimed to examine whether an increase in regional blood flow in the hippocampus (Hpc-BF) following stimulation of the nAChRs by i.v. injection of nicotine could improve the delayed death of the hippocampal neurons following transient ischemia in rats. Hpc-BF was measured by using a laser Doppler flowmeter. During intermittent (every 2 min) transient occlusion for a total of 6 min of bilateral carotid arteries besides permanent ligation of bilateral vertebral arteries, Hpc-BF decreased to about 16% of the preocclusion level, and 5 or 7 d later, after the occlusion, delayed neuronal death occurred in approximately 70% of the CA1 hippocampal neurons. Hpc-BF was increased dose-dependently by injection of nicotine (30--100 microg/kg, i.v.), independent of mean arterial pressure. Nicotine (30--100 microg/kg) administered 5 min before occlusion slightly but significantly attenuated the occlusion-induced decrease in Hpc-BF. The delayed death of the CA1 hippocampal neurons occurring after transient occlusion was attenuated by pretreatment with nicotine (30--100 microg/kg) to approximately 50% of the total neurons. The results indicate that nAChR stimulation-induced increases in Hpc-BF can protect against ischemia-induced delayed death of hippocampal neurons.

Effect of acupuncture-like stimulation on cortical cerebral blood flow in anesthetized rats.

The effect of acupuncture-like stimulation of various areas (cheek, forepaw, upper arm, chest, back, lower leg, hindpaw, perineum) on cortical cerebral blood flow (CBF) was examined in anesthetized rats. An acupuncture needle (diameter, 340 microm) was inserted into the skin and underlying muscles at a depth of about 5 mm and twisted to the right and left once a second for 1 min. CBF of the cortex was measured using a laser Doppler flowmeter. Stimulation of the cheek, forepaw, upper arm and hindpaw produced significant increases in CBF, but stimulation of the chest, back, lower leg and perineum did not produce significant responses. Stimulation of the cheek, forepaw, and hindpaw produced an increase in mean arterial pressure (MAP), while stimulation of the back produced a decrease in MAP. Stimulation of the upper arm, chest, lower leg and perineum did not produce a significant MAP response. After spinal transection at the 1st to 2nd thoracic level, the blood pressure response to stimulation of the cheek and forepaw was suppressed, whereas an increase in CBF still took place. The increase in CBF induced by forepaw stimulation was abolished by severance of the somatic nerves at the brachial plexus. Forepaw stimulation enhanced the activity of the radial, ulnar and median nerves. Furthermore, in the present study, passing of an electric current through acupuncture needles showed that excitation of group III (Adelta) and group IV (C) afferent fibers in the somatic nerve was capable of producing an increase in CBF, whereas excitation of group I (Aalpha) and group II (Abeta) fibers was ineffective. The increase in CBF induced by forepaw stimulation was almost abolished by intravenous administration of muscarinic and nicotinic cholinergic blocking agents (atropine 5 mg/kg and mecamylamine 20 mg/kg), and by bilateral lesions in the nucleus basalis of Meynert. Acupuncture-like stimulation of a forepaw increased acetylcholine release in the cerebral cortex. We concluded that the increase in CBF, independent of systemic blood pressure, elicited by acupuncture stimulation is a reflex response in which the afferent nerve pathway is composed of somatic group III and IV afferent nerves, and efferent nerve pathway includes intrinsic cholinergic vasodilators originating in the nucleus basalis of Meynert.

Peg5/Neuronatin is an imprinted gene located on sub-distal chromosome 2 in the mouse.

We have established a systematic screen for imprinted genes using a subtraction-hybridization method with day 8.5 fertilized and parthenogenetic embryos. Two novel imprinted genes, Peg1/Mest and Peg3, were identified previously by this method, along with the two known imprinted genes, Igf2 and Snrpn. Recently three additional candidate imprinted genes, Peg5-7 , were detected and Peg5 is analyzed further in this study. The cDNA sequence of Peg5 is identical to Neuronatin, a gene recently reported to be expressed mainly in the brain. Two novel spliced forms were detected with some additional sequence in the middle of the known Neuronatin sequences. All alternatively spliced forms of Peg5 were expressed only from the paternal allele, confirmed using DNA polymorphism in a subinterspecific cross. Peg5/Neuronatin maps to sub-distal Chr 2, proximal to the previously established imprinted region where imprinted genes cause abnormal shape and behavior in neonates.