Injection Of Nuclei Research Articles

Effect of Long-Term Exposure of Donor Nuclei to the Oocyte Cytoplasm on Production of Cloned Mice Using Serial Nuclear Transfer.

Although animal cloning is becoming increasingly practicable, cloned embryos possess many abnormalities and so there has been a low success rate for producing live animals. This is most likely due to incomplete reprogramming of somatic cell nuclei before they start to develop as the donor nuclei are usually only exposed to the oocyte cytoplasm for 1-2 hours before reconstructed oocytes are activated to avoid oocyte aging. Therefore, in this study, we attempted to extend the exposure period of somatic cell nuclei to the oocyte cytoplasm to determine whether this could enhance reprogramming of donor nuclei. Donor nuclei were transferred into oocytes, following which pseudo-MII spindles (pMIIs) derived from these were extracted and injected into newly collected enucleated oocytes 24 hours after the first nuclear transfer (NT). These serial NT oocytes were then activated and their developmental potential was examined to full term. There was no obvious difference in the pMIIs of reconstructed oocytes at 6 and 24 hours after donor nucleus injection; however, in both of these, the chromosomes were more widely spread inside the spindle than in fresh intact oocytes. Furthermore, a few chromosomes remained in 25% and 47% of these enucleated oocytes at 6 and 24 hours after donor nucleus injection, respectively. When these pMIIs were injected into fresh enucleated oocytes, the developmental rate to blastocyst was significantly lower, but we could still obtain several healthy cloned offspring. Thus, serial NT at intervals of 24 hours using fresh oocytes is possible, but the success rate could not be improved due to loss of chromosomes during the second NT.

Regulatory effects of paraventricular nucleus injections of ghrelin on the hypothalamus-pituitary-thyroid axis via CB1 receptors in male rats

Paraventricular nucleus (PVN) neurons play critical roles in thyrotropin-releasing hormone (TRH) synthesis and the regulation of energetic homeostasis through the hypothalamus-pituitary-thyroid (HPT) axis. Endocannabinoids induce inhibitory effects on TRH-producing neurons via the type 1 cannabinoid receptor [CB1]. The administration of ghrelin modulates thyrotropes and decreases TSH and T4. The aim of present study was to evaluate the interactions of ghrelin on the HPT axis and CB1 receptors in the PVN. In this experimental study, 54 male Wistar rats weighing 320–370 g were divided into 10 groups and stereotaxic surgery was performed on all animals. control group received a saline injection, while the experimental groups received one of the following injections: ghrelin (5 nm/μL), ACPA (CB1 receptor agonist; 1.25, 2.5, or 5 ng/μL), AM251 (CB1 receptor antagonist; 50, 100, or 200 ng/μL), ACPA + ghrelin (5 ng/μL + 5 ng/μL) and AM251 + ghrelin (200 ng/μL + 5 nm/μL) into the PVN once a day for 3 days. Serum TSH, T3, T4, and T3 uptake levels were evaluated by radioimmunoassay, and FT4I and FT3I were calculated. The present study showed that PVN injections of ACPA and ghrelin alone inhibited thyroid gland secretion, and AM251 stimulated this secretion. ACPA and AM251 increased TSH levels, while ghrelin decreased these levels. The coadministration of ACPA with ghrelin increased TSH levels, but AM251 with ghrelin enhanced serum FT4I. Lastly, injections of ACPA and ACPA with ghrelin decreased thyroid hormones and increased TSH levels. Central injections of ghrelin modulate the HPT axis via the CB1 receptor. ACPA, ghrelin, and ACPA with ghrelin play inhibitory roles in the regulation of thyroid hormones in the PVN, while AM251 was able to stimulate thyroid hormone secretion.

Chronic activation of NPFFR2 stimulates the stress-related depressive behaviors through HPA axis modulation

Neuropeptide FF (NPFF) is a morphine-modulating peptide that regulates the analgesic effect of opioids, and also controls food consumption and cardiovascular function through its interaction with two cognate receptors, NPFFR1 and NPFFR2. In the present study, we explore a novel modulatory role for NPFF-NPFFR2 in stress-related depressive behaviors. In a mouse model of chronic mild stress (CMS)-induced depression, the expression of NPFF significantly increased in the hypothalamus, hippocampus, medial prefrontal cortex (mPFC) and amygdala. In addition, transgenic (Tg) mice over-expressing NPFFR2 displayed clear depression and anxiety-like behaviors with hyperactivity in the hypothalamic-pituitary-adrenal (HPA) axis, reduced expression of glucocorticoid receptor (GR) and neurogenesis in the hippocampus. Furthermore, acute treatment of NPFFR2 agonists in wild-type (WT) mice enhanced the activity of the HPA axis, and chronic administration resulted in depressive and anxiety-like behaviors. Chronic stimulation of NPFFR2 also decreased the expression of hippocampal GR and led to persistent activation of the HPA axis. Strikingly, bilateral intra-paraventricular nucleus (PVN) injection of NPFFR2 shRNA predominately inhibits the depressive-like behavior in CMS-exposed mice. Antidepressants, fluoxetine and ketamine, effectively relieved the depressive behaviors of NPFFR2-Tg mice. We speculate that persistent NPFFR2 activation, in particular in the hypothalamus, up-regulates the HPA axis and results in long-lasting increases in circulating corticosterone (CORT), consequently damaging hippocampal function. This novel role of NPFFR2 in regulating the HPA axis and hippocampal function provides a new avenue for combating depression and anxiety-like disorder.

605. Cerebrospinal Fluid for Delivery of AAV Gene Therapy in GM1 Gangliosidosis

GM1 gangliosidosis, a hereditary lysosomal storage disease caused by a deficiency of lysosomal β-galactosidase, is characterized by rapidly progressing and fatal neurologic disease. The most common form of GM1 affects children and is fatal by approximately 4 years of age. Currently, there is no effective treatment for GM1 gangliosidosis beyond palliative or supportive care. There are several naturally occurring models of GM1 gangliosidosis, including a well-characterized feline model that closely reproduces human disease progression and facilitates testing of experimental therapies for clinical application. Adeno-associated viral (AAV) therapy has proven effective in GM1 cats, with a greater than 6 fold increase in lifespan after thalamic and deep cerebellar nuclei injections. Prior to initiation of human clinical trials, CSF delivery was evaluated to circumvent the risk of cerebellar parenchymal injection and to improve cortical biodistribution. AAVrh10 was delivered at a total dose of 1e12 vector genomes/kg body weight via 3 routes: cisterna magna (CM), bilateral intracerebroventricular (ICV), or lumbar cistern (LC). Cats were followed for 4 weeks post injection, after which biodistribution of enzyme and vector were assessed. After LC injection, enzyme was limited to the spinal cord, where it reached a maximum of 0.7 fold normal. As expected, the highest vector concentration was limited to the lumbar spinal cord. This indicates limited cranial flow of vector from the lumbar cistern of injected cats, minimizing the efficacy of lumbar delivery. Delivery by the CM led to 0.4 fold - 1.5 fold normal levels of enzyme activity in the spinal cord and up to 2.1 fold the cerebellum and thalamic regions. Following CM injection, vector distributed well to the caudal region of the cerebrum, all of the cerebellum, and throughout the spinal cord. Enzyme activity after ICV injections showed increases from 0.2 fold - 0.8 fold normal in the spinal cord and 0.1 fold - 0.5 fold in the brain. ICV injections led to vector distribution throughout the cerebrum as well as the spinal cord. CSF delivery via cisterna magna, ICV, or lumbar injection is substantially less invasive than cerebellar injection. Based on the results presented here, CM or ICV injection may prove beneficial in clinical trials. Additionally, combining cisterna magna and ICV injection of vector could further enhance therapeutic effect.View Large Image | Download PowerPoint Slide

Strong magnetic fields and SGRs/AXPs as white dwarf pulsar: a source of ultra-high energy cosmic rays

The origin of highest energy cosmic rays still remains a mystery in Astrophysics. In this work we consider the Soft Gamma Repeaters (SGRs) and Anomalous X-ray Pulsars (AXPs) as possible sources of ultra-high cosmic rays. These stars described as white dwarfs pulsars can achieved large electric potential differences in their surface and accelerate particles up to Lorentz factors γ ∼ 1010. Pulsars offer favorable sites for the injection of electrons and heavy nuclei, and accelerate them to ultrahigh energies. Once accelerated in the pulsar this particles can escape from the magnetosphere and produce the radiation observed. Here, we discuss the possibility of SGRs/AXPs as white dwarf pulsars to be possible sources of ultra-high energetic photons with E ∼ 1021eV.

Transgenic mouse offspring generated by ROSI.

The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes.

Role of the Paraventricular Nucleus of the Hypothalamus in the Sympathoexcitatory Effects of Leptin.

Leptin binds to receptors in multiple hypothalamic nuclei to increase sympathetic nerve activity; however, the neurocircuitry is unclear. Here, using anesthetized male Sprague-Dawley rats, we investigated the role of the paraventricular nucleus of the hypothalamus. Intracerebroventricular injection of leptin slowly increased lumbar sympathetic nerve activity (LSNA), heart rate, mean arterial pressure, and baroreflex control of LSNA and heart rate. Inhibition of the paraventricular nucleus with muscimol completely reversed leptin's effects. Blockade of paraventricular melanocortin 3/4 receptors with SHU9119 or ionotropic glutamate receptors with kynurenate, alone or together, each partially reversed the effects of leptin, implicating increased activation of glutamate and melanocortin 3/4 receptors. Conversely, although blockade of neuropeptide Y Y1 receptors in the paraventricular nucleus increased LSNA, mean arterial pressure, and heart rate, these responses were prevented by intracerebroventricular or arcuate nucleus injections of leptin, suggesting that, at least in part, leptin also increases sympathetic nerve activity by suppression of tonic neuropeptide Y inhibitory inputs from the arcuate nucleus. Injection of the melanocortin 3/4 receptor agonist melanotan-II into the paraventricular nucleus increased LSNA, mean arterial pressure, and heart rate only after blockade of neuropeptide Y Y1 receptors. Therefore, we conclude that leptin increases LSNA in part via increased glutamatergic and α-melanocyte-stimulating hormone drive of paraventricular sympathoexcitatory neurons, the latter of which requires simultaneous withdrawal of tonic neuropeptide Y inhibition.

The fate of ultrahigh energy nuclei in the immediate environment of young fast-rotating pulsars

Young, fast-rotating neutron stars are promising candidate sources for the production of ultrahigh energy cosmic rays (UHECRs). The interest in this model has recently been boosted by the latest chemical composition measurements of cosmic rays, that seem to show the presence of a heavy nuclear component at the highest energies. Neutrons stars, with their metal-rich surfaces, are potentially interesting sources of such nuclei, but some open issues remain: 1) is it possible to extract these nuclei from the star's surface? 2) Do the nuclei survive the severe conditions present in the magnetosphere of the neutron star? 3) What happens to the surviving nuclei once they enter the wind that is launched outside the light cylinder? In this paper we address these issues in a quantitative way, proving that for the most reasonable range of neutron star surface temperatures (T<107 K), a large fraction of heavy nuclei survive photo-disintegration losses. These processes, together with curvature losses and acceleration in the star's electric potential, lead to injection of nuclei with a chemical composition that is mixed, even if only iron is extracted from the surface. We show that under certain conditions the chemical composition injected into the wind region is compatible with that required in previous work based on purely phenomenological arguments (typically ∼50% protons, ∼30% CNO and ∼20% Fe), and provides a reasonable explanation of the mass abundance inferred from ultra high energy data.

Haemodynamic and renal sympathetic responses to V1b vasopressin receptor activation within the paraventricular nucleus.

What is the central question of this study? Does antagonism of V1b receptors prevent the haemodynamic and renal sympathetic nerve responses that occur with application of exogenous vasopressin into the paraventricular nucleus (PVN) of conscious, chronically instrumented rats? What is the main finding and its importance? Microinjection of vasopressin into the PVN increased mean arterial pressure, heart rate and renal sympathetic nerve activity, all of which were inhibited by pre-injection of the PVN with the V1b antagonist, nelivaptan. The administered vasopressin did not enter the peripheral circulation or increase plasma vasopressin. Ganglionic blockade prevented each of the responses, consistent with mediation by enhanced sympathetic output rather than an increase in circulating vasopressin. Vasopressin (VP) participates in regulation of haemodynamics and volume. Besides more classical actions as a circulating hormone, VP may act via release from axons and dendrites within the CNS. The paraventricular nucleus (PVN) possesses vasopressinergic neurons and a dense complement of VP receptors, including the V1b receptor, which has been implicated in several types of stress responses. We tested the hypothesis that antagonism of V1b receptors will prevent VP-induced increases in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA). Studies were performed in conscious male Sprague-Dawley rats chronically instrumented with vascular catheters, renal nerve electrodes and a cannula stereotaxically directed into the PVN. Unilateral microinjection of VP into the PVN significantly increased MAP, HR and RSNA, peaking at 10min. Pre-injection of the PVN with the selective V1b receptor antagonist, nelivaptan, did not alter baseline values but blocked the responses to VP. Ganglionic blockade with chlorisondamine decreased MAP and HR and abolished their increase in response to subsequent PVN application of VP. Injection of VP into the PVN did not alter plasma VP levels. Paraventricular nucleus injection with radiolabelled VP resulted in negligible radiolabelled VP in peripheral blood. These findings support the concept that, in basal conditions, PVN V1b receptor activation (rather than VP release into the periphery) may be implicated in the increases in MAP, HR and RSNA due to increased sympathetic outflow. While the role of V1a and oxytocin receptors cannot be excluded, these data suggest that further studies of the role of V1b receptor activation by endogenous VP during stress to effect neuroexcitation are warranted.

Systematic study of the uncertainties in fitting thecosmic positron data by AMS-02

The operation of AMS-02 opens a new era for the study of cosmic ray physics with unprecedentedly precise data which are comparable with the laboratory measurements. The high precision data allow a quantitative study on the cosmic ray physics and give strict constraints on the nature of cosmic ray sources. However, the intrinsic errors from the theoretical models to interpret the data become dominant over the errors in the data. In the present work we try to give a systematic study on the uncertainties of the models to explain the AMS-02 positron fraction data, which shows the cosmic ray e+e− excesses together with the PAMELA and Fermi-LAT measurements. The excesses can be attributed to contributions from the extra e+e− sources, such as pulsars or the dark matter annihilation. The possible systematic uncertainties of the theoretical models considered include the cosmic ray propagation, the treatment of the low energy data, the solar modulation, the pp interaction models, the nuclei injection spectrum and so on. We find that in general a spectral hardening of the primary electron injection spectrum above ∼50–100 GeV is favored by the data. Furthermore, the present model uncertainties may lead to a factor of ∼2 enlargement in the determination of the parameter regions of the extra source, such as the dark matter mass, annihilation rate and so on.

Process-model simulations of cloud albedo enhancement by aerosols in the Arctic.

A cloud-resolving model is used to simulate the effectiveness of Arctic marine cloud brightening via injection of cloud condensation nuclei (CCN), either through geoengineering or other increased sources of Arctic aerosols. An updated cloud microphysical scheme is employed, with prognostic CCN and cloud particle numbers in both liquid and mixed-phase marine low clouds. Injection of CCN into the marine boundary layer can delay the collapse of the boundary layer and increase low-cloud albedo. Albedo increases are stronger for pure liquid clouds than mixed-phase clouds. Liquid precipitation can be suppressed by CCN injection, whereas ice precipitation (snow) is affected less; thus, the effectiveness of brightening mixed-phase clouds is lower than for liquid-only clouds. CCN injection into a clean regime results in a greater albedo increase than injection into a polluted regime, consistent with current knowledge about aerosol-cloud interactions. Unlike previous studies investigating warm clouds, dynamical changes in circulation owing to precipitation changes are small. According to these results, which are dependent upon the representation of ice nucleation processes in the employed microphysical scheme, Arctic geoengineering is unlikely to be effective as the sole means of altering the global radiation budget but could have substantial local radiative effects.

Neuropeptide Y is crucial for nutritional state-dependent regulation of maternal behavior.

Lactation is indispensable for the survival of mammalian pups. However, any excess of energy expenditure for lactation over energy intake threatens the mother's survival. Here, we report that an orexigenic molecule, neuropeptide Y (NPY), mediates nutritional state-dependent regulation of maternal behavior. After 9h of fasting, dams showed a dramatic decrease in the expression of maternal behavior. Intracerebroventricular or direct dorsal raphe nucleus (DRN) injection of NPY inhibited the expression of maternal behavior in non-fasted dams. In contrast, injection of the NPY Y1 receptor antagonist BIBP-3226 into the DRN, in which the expression of the Y1 receptor was confirmed in serotonergic (5-HT) and GABAergic interneurons, recovered the expression of maternal behavior in fasted dams. When the pups were presented, the increase in the number of c-Fos-positive GABAergic, but not serotonergic, neurons was smaller in fasted than in non-fasted dams. These results suggest that NPY may inhibit pup-induced activation of GABAergic neurons via the Y1 receptor. Injection of a 5-HT1A agonist, GABAA receptor antagonist, or GABAB receptor antagonist into the DRN induced incomplete maternal behavior in non-fasted dams. In contrast, each of a 5-HT2A receptor agonist or a GABAB receptor agonist, but not a GABAA receptor agonist, recovered separate components of maternal behavior in fasted dams. These results suggest that NPY inhibits both 5-HT neuronal activity and its modulation via the GABA receptor in the DRN, resulting in the suppression of maternal behavior under food-restricted conditions.

Nesfatin-1 influences the excitability of glucosensing neurons in the dorsal vagal complex and inhibits food intake.

Nesfatin-1 is a recently discovered metabolic peptide hormone that decreases food intake after lateral, third, or fourth brain ventricle; cisterna magna; or paraventricular nucleus (PVN) injection in ad libitum fed rats. Additional micro-injection studies will improve the understanding of how nesfatin-1 acts on the brain and define specific nuclei responsive to nesfatin-1, which will provide insight on its effects on food intake. We evaluated how nesfatin-1 injection into the dorsal vagal complex (DVC) modulates food intake response in rats during the dark phase. Consistent with previous observations, nesfatin-1-injected rats significantly reduced cumulative food intake over a 5-h period in rats. Chronic administration of nesfatin-1 into the DVC reduced body weight gain over a 10-day period. Because glucosensing neurons in the DVC are involved in glucoprivic feeding and homeostatic control of blood glucose, we examined the effect of nesfatin-1 on the excitability of DVC glucosensing neurons. Nesfatin-1 inhibited most of the glucose-inhibitory (GI) neurons and excited most of the glucose-excitatory (GE) neurons in the DVC. Current-clamp electrophysiology recordings from DVC glucosensing neurons in slice preparation showed that bath applied nesfatin-1(10 nM) increased the firing frequency of GE neurons and inhibited the firing rate of GI-neurons. Nesfatin-1 inhibited 88.9% (16/18) of gastric distension inhibitory (GD-INH) neurons and excited 76.2% (32/42) of gastric distension excitatory (GD-EXC) neurons. Thus, nesfatin-1 may control food intake by modulating the excitability of glucosensing neurons in the DVC.

Central pupillary light reflex circuits in the cat: I. The olivary pretectal nucleus.

The central pathways subserving the feline pupillary light reflex were examined by defining retinal input to the olivary pretectal nucleus (OPt), the midbrain projections of this nucleus, and the premotor neurons within it. Unilateral intravitreal wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) injections revealed differences in the pattern of retinal OPt termination on the two sides. Injections of WGA-HRP into OPt labeled terminals bilaterally in the anteromedian nucleus, and to a lesser extent in the supraoculomotor area, centrally projecting Edinger-Westphal nucleus, and nucleus of the posterior commissure. Labeled terminals, as well as retrogradely labeled multipolar cells, were present in the contralateral OPt, indicating a commissural pathway. Injections of WGA-HRP into the anteromedian nucleus labeled fusiform premotor neurons within the OPt, as well as multipolar cells in the nucleus of the posterior commissure. Connections between retinal terminals and the pretectal premotor neurons were characterized by combining vitreous chamber and anteromedian nucleus injections of WGA-HRP in the same animal. Fusiform-shaped, retrogradely labeled cells fell within the anterogradely labeled retinal terminal field in the OPt. Ultrastructural analysis revealed labeled retinal terminals containing clear spherical vesicles. They contacted labeled pretectal premotor neurons via asymmetric synaptic densities. These results provide an anatomical substrate for the pupillary light reflex in the cat. Pretectal premotor neurons receive direct retinal input via synapses suggestive of an excitatory drive, and project directly to nuclei containing preganglionic motoneurons. These projections are concentrated in the anteromedian nucleus, indicating its involvement in the pupillary light reflex.

Cirrus cloud susceptibility to the injection of ice nuclei in the upper troposphere

AbstractDue to their net warming effect, cirrus clouds play a crucial role in the climate system. A recently proposed climate engineering mechanism (CEM) intends to reduce high cloud cover by seeding cirrus clouds with efficient ice nuclei (IN) and therefore cool climate. Here, the susceptibility of cirrus clouds to the injection of ice nuclei in the upper troposphere is investigated in the extended Community Atmospheric Model version 5 (CAM5). Due to large uncertainties associated with the dominant ice nucleation mechanism in cirrus clouds, different control cases were simulated. In addition to pure homogeneous and heterogeneous nucleation, cases with competition between homogeneous and heterogeneous nucleation and different fractions of mineral dust active as IN were considered. Whereas seeding in the pure heterogeneous case leads to a strong warming due to overseeding, an optimal seeding IN concentration of approximately 18 l−1 was found for the other cases. For the optimal seeding concentration, a reduction in the net cloud forcing (NCF) of up to 2 W m−2 was simulated, corresponding to a strong cooling effect. To optimize the cooling and minimize the amount of seeding material, globally nonuniform seeding strategies were tested, with minimal seeding in the summer hemisphere and in the tropics. With seeding applied to less than half the globe, an even stronger reduction in the NCF was achieved. This suggests that the CEM could work for an atmosphere even with considerable heterogeneous ice nucleation and that the desired cooling could be obtained without seeding the entire globe.

Exogenous Urocortin 1 Alters the Respiratory Exchange Ratio after Administration into the Lateral Septum

Previous reports have suggested that hypothalamic urocortin 1 (Ucn1) exerts inhibitory control on energy metabolism as direct paraventricular nucleus injections dose-dependently decrease the respiratory energy exchange ratio (RER). Other evidence indicates that Ucn1 injections into the lateral septum may alter metabolic function. Consequently, the present study was designed to further characterize the effects of lateral septal Ucn1 signaling on eating and energy metabolism of adult Sprague-Dawley rats. Ucn1 was infused at the onset of the nocturnal cycle at doses of 10 - 100 pmol. In both females and males the peptide elicited a reliable suppression of food intake and significantly lowered RER over a 4 h postinjection period. The decrease in RER is consistent with enhanced lipid oxidation. Overall these findings suggest that, similar to the paraventricular nucleus, the lateral septum is a critical site of action in mediating the effects of Ucn1 on food intake and energy substrate utilization.

FERMI BUBBLE γ-RAYS AS A RESULT OF DIFFUSIVE INJECTION OF GALACTIC COSMIC RAYS

Recently, the {\it{Fermi}} space telescope has discovered two large $\gamma$-ray emission regions, the so-called "Fermi bubbles", that extend up to $\sim 50^\circ$ above and below the Galactic center. The $\gamma$-ray emission from the bubbles are found to follow a hard spectrum with no significant spatial variation in intensity and spectral shape. The origin of the emission is still not clearly understood. Suggested explanations include injection of cosmic-ray nuclei from the Galactic center by high-speed Galactic winds, electron acceleration by multiple shocks and stochastic electron acceleration inside the bubbles. In this letter, it is proposed that the $\gamma$-rays can be the result of diffusive injection of Galactic cosmic-ray protons during their propagation through the Galaxy. Considering that the bubbles are slowly expanding, and cosmic rays undergo much slower diffusion inside the bubbles than in the averaged Galaxy and at the same time suffer losses due to adiabatic expansion and inelastic collisions with the bubble plasma, this model can explain the observed intensity profile, the emission spectrum and the measured luminosity without invoking any additional particle production processes unlike other existing models.

Arcuate nucleus injection of an anti-insulin affibody prevents the sympathetic response to insulin

Accumulating evidence suggests that insulin acts within the hypothalamus to alter sympathetic nerve activity (SNA) and baroreflex function. Although insulin receptors are widely expressed across the hypothalamus, recent evidence suggests that neurons of the arcuate nucleus (ARC) play an important role in the sympathoexcitatory response to insulin. The purpose of the present study was to determine whether circulating insulin acts directly in the ARC to elevate SNA. In anesthetized male Sprague-Dawley rats (275-425 g), the action of insulin was neutralized by microinjection of an anti-insulin affibody (1 ng/40 nl). To verify the efficacy of the affibody, ARC pretreatment with injection of the anti-insulin affibody completely prevented the increase in lumbar SNA produced by ARC injection of insulin. Next, ARC pretreatment with the anti-insulin affibody attenuated the lumbar sympathoexcitatory response to intracerebroventricular injection of insulin. Third, a hyperinsulinemic-euglycemic clamp increased lumbar, but not renal, SNA in animals that received ARC injection of a control affibody. However, this sympathoexcitatory response was absent in animals pretreated with the anti-insulin affibody in the ARC. Injection of the anti-insulin affibody in the adjacent ventromedial hypothalamus did not alter the sympathoexcitatory response to insulin. The ability of the anti-insulin affibody to prevent the sympathetic effects of insulin cannot be attributed to a general inactivation or nonspecific effect on ARC neurons as the affibody did not alter the sympathoexcitatory response to ARC disinhibition by gabazine. Collectively, these findings suggest that circulating insulin acts within the ARC to increase SNA.

26 REPROGRAMMING OF TWO SOMATIC NUCLEI IN THE SAME MOUSE OOPLASM LEADS TO PLURIPOTENT NOT TOTIPOTENT EMBRYOS

The use of cloned embryos to derive pluripotent embryonic stem cells (ESC) has raised expectations among many for its potential to benefit regenerative medicine. However, diploid cloned embryos also have the ability for totipotency and they occasionally develop to term. Thus, a therapeutic application cannot be kept totally separate from reproductive cloning. In contrast to diploidy, tetraploidy is incompatible with full development in mammals; yet, tetraploid cells arise after normal diploid development and contribute to certain organs of the adult body. In this context, we asked if single oocytes are able to reprogram 2 simultaneously transplanted somatic nuclei, and whether resultant embryos may serve as a source for functional pluripotent cell lines. We modified the conventional nuclear cloning method by simultaneously injecting 2 nuclei of cumulus cells into a single ooplasm of the B6C3F1 mouse strain. Following activation of these reconstructed oocytes, the blastocyst rates were lower compared with conventional cloned embryos (1 nucleus injection), but similar to tetraploid fertilized embryos generated by intracytoplasmic sperm injection (ICSI) of 2 sperm heads and prevention of second polar body extrusion using cytochalasin B. Upon transfer of tetraploid cloned blastocysts to pseudopregnant recipients, no fetuses were found at midgestation (Table 1). Tetraploid cloned blastocysts supported the derivation of ESC (Table 1), which maintain a stable tetraploid karyotype over more than 20 passages. These tetraploid ESC possess the major hallmarks of pluripotency, including matching gene expression profiles (MouseRef-8 v2 expression BeadChip, Illumina) compared with those of diploid counterparts (&gt;99% similarity) as well as the ability to differentiate into derivatives of the 3 germ layers when forming teratomas in severe-combined immunodeficient mice. Upon injection into wild-type blastocysts, tetraploid ESC populated the inner cell mass; however, after transfer of these chimeras to pseudopregnant recipients, derivatives of tetraploid ESC were found only in extraembryonic tissues of midgestation fetuses. In sum, we have shown that the reprogramming capacity of mouse oocytes is sufficient for 2 somatic nuclei, and that resultant cloned embryos support pluripotency but not totipotency. Our approach to therapeutic cloning is free of reproductive implications. Table 1.Developmental competence of single (2N) and double nucleus transplanted (4N) cloned mouse embryos compared with fertilized controls

Вплив модуляції Na+, К+-АТФази нейронів довгастого мозку на гемодинамічні ефекти у щурів із генетично детермінованою гіпертензією

The study was conducted in normotensive and spontaneously hypertensive rats anesthetized with urethane (1600 mg/kg of animal weight, intraperitoneally). It has been shown that in normotensive rats, injections of a specific inhibitor of Na+, K(+)-ATPase ouabain (10(-8)-10(-5) mol/l) in the populations of the neurons within nucleus of the solitary tract (NTS), paramedian reticular nucleus (PMn) and lateral reticular nucleus (LRN) were accompanied by the development of the hypertensive responses in a dose-dependent fashion. These data suggest that Na+, K(+)-ATPase of the neuron somatic membranes in the medullary cardiovascular nuclei is involved in neural control of the cardiovascular function, and its inhibition by microinjections of ouabain promotes the development of hypertension. In contrast to normotensive rats, ouabain injected in the medullary nuclei of spontaneously hypertensive animals induced either enhanced hypertensive or hypotensive responses. Biochemical analysis revealed that the activity of Na+, K(+)-ATPase in the microsomal fraction of the medulla oblongata of spontaneously hypertensive rats significantly exceeded its activity in the medulla oblongata of normotensive animals. Possible mechanisms of ouabain effects in spontaneously hypertensive rats have being discussed. Activation of Na+, K(+)-ATPase activity of the cardiovascular neurons with asparkam injections in the medullary nuclei resulted in hypotensive responses in both normotensive and spontaneously hypertensive rats.