17beta-estradiol pretreatment prevents the global ischemic injury-induced decrease of Akt activation and bad phosphorylation in gerbils

Estradiol acts as a neuroprotective factor against ischemic brain injury. This study investigated whether estradiol modulates neuroprotective mechanism through the activation of Akt and its downstream target such as Bad in global ischemic injury. Adult female gerbils were ovariectomized and treated with estradiol prior to ischemic injury. Transient cerebral ischemia was accomplished by bilateral clipping of the common carotid artery for 5 min. Brains were collected on 1, 3, 5 day after injury. In hippocampal CA1 region of non-treated gerbils, most of neuronal cells exhibited pyknotic nuclei and showed the positive reaction of TUNEL staining on 5 day after injury. However, estradiol significantly reduced the neuronal cell death. Potential activation was measured by phosphorylation of Akt at Ser473 and Bad at Ser136 using western blot analysis. The levels of pAkt and pBad were significantly decreased in non-treated gerbils on 1-5 day after injury. However, estradiol prevents the global ischemic injury-induced decrease of pAkt and pBad. Our findings suggest that estradiol prevents cell death due to global ischemic injury and that Akt activation and Bad phosphorylation by estradiol mediated these protective effects.     

Evaluation of the Akt kinase activation in a PTEN deficient canine osteosarcoma cell line

Introduction: Osteosarcoma is a devastating disease in both human and veterinary medicine. The dog has been recognized as an important model system for the study of this disease, with a high incidence of tumor development noted annually. The cure rates in veterinary oncology are between 20 and 30% of patients seen. Akt is a serine/threonine kinase that primarily protects the cell from apoptosis and may prompt proliferation and growth. Akt activation involves sequential phosphorylation of threonine at position 308 (T308) and serine at position 473 (S473). While for the T308 phosphorylation responsible is PDK1 kinase, for S473 phosphorylation no definitive mechanism has yet been identified. The tumor suppressor PTEN, which has been reported to be absent in canine osteosarcoma, negatively regulates Akt. In our study we evaluated the Akt activation sequence in response to serum deprivation. Methods: “Buck,” an immortalized PTEN deficient canine OSA cell line established and characterized in our laboratory was used. Buck cells were grown in the absence of fetal bovine serum for 8, 24, and 48 hours. Activity of Akt was assayed directly by a method described by Kang et al. The phosphorylation status of Akt at T308 and S473, together with serine at position 241 (S241) of PDK1 was assessed by immunoblotting. Results: We noted marked decrease of the Akt kinase activity with the nadir at the 24‐hour time point and subsequent increase of Akt activity at the 48‐hour time point. Decoupling of the two Akt phosphorylation sites at the 24, and 48‐hour time points was observed (p < 0.01). The phosphorylation status of T308 declined steadily throughout the experiment. The phosphorylation status of S473 declined until the 24‐hour time point, and then increased in parallel with the results noted in the kinase activity assay. Phosphorylation of PDK1 S241 was examined and found to decrease at 24 hours, with subsequent increase after 48 hours of serum starvation, in parallel with the phosphorylation pattern of Akt S473 (p < 0.01). Conclusions: We report parallel patterns of PDK1 S241 and Akt S473 phosphorylation in the absence of growth factors. PDK1 activity is not assessed directly, but its phosphorylation status indicates that more PDK1 is in an activated from at the 48‐hour time point than at 24 hours. The increase of Akt activity and S473 phosphorylation at the 48‐hour time point is presumably a response to the apoptogenic conditions of the serum free medium. The same response of PDK1 may indicate the presence of a regulatory autophosphorylation mechanism that members of the AGC kinase family share. Surprisingly, the increased amount of activated PDK1 does not correlate with the phosphorylation pattern of Akt T308, and could be attributed to cytoplasmic sequestration of PDK1.     

Campylobacter jejuni induces an anti-inflammatory response in human intestinal epithelial cells through activation of phosphatidylinositol 3-kinase/Akt pathway

Campylobacter jejuni (C. jejuni) is the most common cause of human acute bacterial gastroenteritis. Poultry is a major reservoir of C. jejuni and considered an important source of human infections, thus, it is important to understand the host response to C. jejuni from chicken origin. In this study, we demonstrated firstly that a chicken isolate SC11 colonized chicks faster than clinical isolate NCTC11168. Using the SC11, we further studied the host responds to C. jejuni in terms of inflammatory response and involvement of cellular signaling pathways. Infection of C. jejuni SC11 was able to activate phosphatidylinositol 3-kinase (PI3K)/Akt pathway and induce pro-inflammatory interleukin-8 (IL-8) as well as anti-inflammatory cytokine IL-10 in human intestinal epithelial cell line Colo 205. The signalling pathways PI3K/Akt and mitogen-activated protein (MAP) kinases ERK and p38 were involved in C. jejuni-induced IL-8 and IL-10 expression. Inhibition of PI3K resulted in augmentation of C. jejuni-induced IL-8 production, concomitant with down-regulation of IL-10 mRNA, indicating an anti-inflammatory response was activated and associated with the activation of P13K/Akt. Similar effect was observed for cytolethal distending toxin (CDT) deficient mutants. Moreover, we demonstrated that heat-killed bacteria were able to induce IL-8 and IL-10 expression to a lower level than live bacteria. We therefore conclude that C. jejuni activate a PI3K/Akt-dependent anti-inflammatory pathway in human intestinal epithelial cells which may benefit the intracellular survival of C. jejuni during infection.     

MicroPET detection of regional brain activation induced by colonic distention in a rat model of visceral hypersensitivity

Using microPET and (18)F-fluorodeoxyglucose ((18)F-FDG) as a tracer, we investigated regional brain activation in a rat model of visceral hypersensitivity induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). TNBS injection into the proximal colon through laparotomy resulted in a significant, sustained decrease in the pain threshold to mechanical distention of the distal colon, indicating a phenomenon referred to as visceral hypersensitivity. When TNBS-induced colonic hypersensitivity was fully developed, all the TNBS-treated rats presented characteristic pain behaviors in response to colonic distention at previously innocuous pressure (0-35 mmHg) that produced no abdominal pain in sham-operated control animals. In microPET study, colonic distention at the normally non-painful pressure produced significant increases in (18)F-FDG uptake in the thalamus and sensory cortex I of TNBS-treated rats. Since the increases in (18)F-FDG uptake in the brain regions were completely abolished by an analgesic dose of morphine (375 microg/kg, s.c.), it is most likely that the regional brain activation detected by microPET is a pain-related central event. The pharmacological and microPET data indicate that colonic distention at the normally non-painful pressure activates specific brain regions in rats with TNBS-induced visceral hypersensitivity, and the microPET protocol described here could provide an objective measure to test visceral analgesic compounds.     

猪血凝性脑脊髓炎病毒感染可激活脑内的小胶质细胞

以猪血凝性脑脊髓炎病毒(PHEV)易感的BALB/c小鼠为实验动物模型,以小胶质细胞表面特异性标记物Iba1蛋白为检测对象,利用荧光定量PCR、Western-blot、免疫组化等方法,分别从基因转录水平、蛋白水平和组织学水平对PHEV感染后不同时间点小鼠脑内小胶质细胞的增殖、活化情况进行了检测分析。结果表明,与对照组相比,试验组小鼠脑组织内的Iba1基因转录水平、蛋白表达量均有升高,且都随着感染时间延长而增大。将采集的小鼠脑组织制作石蜡切片,免疫组化结果证实PHEV的感染激活了小胶质细胞,表现为细胞数量增多,呈聚集样分布;同时细胞形态变化也很明显,胞体变大并呈阿米巴状。上述试验证实PHEV感染小鼠后小胶质细胞发生了明显增殖、活化现象,为后期深入开展PHEV所致神经系统炎性损伤的机制研究奠定了基础。     

Morphology of spontaneous brain tumors in the rat.

In lifespan studies of 2,242 rats of three strains, 32 neoplasms were identified in brain, meninges and pineal gland. They were astrocytoma (16 Wistar, three Sprague-Dawley), ependymoma (four Osborne-Mendel), meningioma (two Osborne-Mendel, one Wistar), pinealoma (two Osborne-Mendel), reticulosis (two Wistar), oligodendroglioma (one Wistar), and gliomatosis (one Wistar).     

Role of gap junctional intercellular communication (GJIC) through p38 and ERK1/2 pathway in the differentiation of rat neuronal stem cells

Gap junctional intercellular communications (GJIC) contributes to neural function in development and differentiation of CNS. In this study, we have investigated the expression of GJIC during the differentiation of neuronal stem cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neuronal stem cell-derived cells from rat brain. During neuronal stem cell differentiation, expressions of Cx43 and 32 were increased for the duration of 72 hr, however the effect were decreased on the 7d. In the neuronal stem cell-derived cells, pretreatments with p38 MAP kinase inhibitor, SB203580, and MEK inhibitor, PD98059, could protect GJIC against TPA-induced inhibition of GJIC. Our data suggest that GJIC plays an important role during neuronal stem cell differentiation, and ERK1/2 and p38 MAP kinase signaling pathway may be closely related functionally to regulate gap junction in rat neuronal stem cell-derived cells.     

Characteristics of ventricular activation and recovery patterns in the rat.

Bipolar or unipolar epicardial and intraseptum electrograms were recorded from the in situ rat hearts with spontaneous sinus rhythm or myocardial electrical stimulation of 400 bpm in order to investigate the sequence and duration of cardiac activation in the rat. The ventricular epicardial isochronous maps in the rat showed the activation sequence comparable to those in humans and dogs, although the activation time (6 to 13 msec) of epicardium was approximately one-third of those of such species. The delay of activation between septum and ventricular surface in the rat was 5 to 8 msec, which was also shorter as compared with those in humans and dogs. However, the conduction velocity of the ventricular epicardium in the rat was approximately 40 cm/sec, mostly equal to that of humans. These findings might be accounted for by the possible presence of the bundle branch-purkinje system, at least functionally, similar to humans. The local QT interval in the septum were relatively longer (69 to 123 msec), compared with that of the ventricular surface (50 to 98 msec), and such durations of ventricular epicardium and septum were considerably less than those of dogs and humans. These results are consistent with the configuration of QRS-T on the limb lead ECG.     

用科学力量战胜疫情

中共中央总书记、国家主席胡锦涛20日考察了军事医学科学院微生物流行病研究所和中国科学院北京基因组研究所,对在防治非典型肺炎斗争中取得重大科技成果的科研人员表示衷心感谢和崇高敬意,勉励科研人员再接再厉、坚定信心,继续发扬爱国奉献、勇攀高峰、为民造福的精神,为战胜疫病、保护人民的身体健康和生命安全作出更大的贡献。在军事医学科学院微生物流行病研究所,胡锦涛考察了三级生物安全实验室、病毒学生物实验室,并不时向科研人员询问科技攻关的进展情况和科研成果运用于临床的前景。在中国科学院北京基因组研究所,胡锦涛考察了测序…     

Rapid brain cooling in intubated pigs through nasal flushing with oxygen: prevention of brain hyperthermia

Local cooling of the brain by the respiratory air is found in many animal species. The mechanism is based on cooling of the nasal vein blood and heat transfer in the cavernous sinus/carotid artery complex and is therefore not active in anaesthetised, intubated animals. The present experiment was made to investigate the effects of oxygen flushing of the nasal cavities in such animals. Nine anaesthetised, intubated male pigs were used. The temperatures in the third ventricle and rectum were measured continuously. Oxygen was infused into the nasal cavities during 10 min periods interrupted by 10 min without flow. The nasal oxygen flow constantly induced a rapid, reversible and flow dependant decrease in brain temperature: 0.25 degree C +/- 0.04, (n = 2) (mean +/- SD, n) at < 4 l/min; 1.35 degrees C +/- 0.78, (n = 20) at 4-6 l/min; and 1.44 degrees C +/- 0.62, (n = 6) at > 6 l/min. The ventricle temperature decreased 0.59 degree C +/- 0.23, (n = 8) when the animals were transferred to spontaneous respiration and the tracheal tube removed. It may be possible to protect the brain in intubated animals and humans from heat-induced damages by establishment of nasal flushing.     

Intragastric gavage with denatonium benzoate acutely induces neuronal activation in the solitary tract nucleus via the vagal afferent pathway

Natural toxic substances have a bitter taste and their ingestion sends signals to the brain leading to aversive oral sensations. In the present study, we investigated chronological changes in c-Fos immunoreactivity in the nucleus tractus solitarius (NTS) to study the bitter taste reaction time of neurons in the NTS. Equal volumes (0.5 mL) of denatonium benzoate (DB), a bitter tastant, or its vehicle (distilled water) were administered to rats intragastrically. The rats were sacrificed at 0, 0.5, 1, 2, 4, 8, or 16 h after treatment. In the vehicle-treated group, the number of c-Fos-positive nuclei started to increase 0.5 h after treatment and peaked 2 h after gavage. In contrast, the number of c-Fos-positive nuclei in the DB-treated group significantly increased 1 h after gavage. Thereafter, the number of c-Fos immunoreactive nuclei decreased over time. The number of c-Fos immunoreactive nuclei in the NTS was also increased in a dose-dependent manner 1 h after gavage. Subdiaphragmatic vagotomy significantly decreased DB-induced neuronal activation in the NTS. These results suggest that intragastric DB increases neuronal c-Fos expression in the NTS 1 h after gavage and this effect is mediated by vagal afferent fibers.     

Higher sensitivity of LEC strain rat in radiation-induced acute intestinal death.

LEC strain rats (LEC rats), which have been known to develop hereditarily spontaneous fulminant hepatitis 4-5 months after birth, were highly sensitive to whole-body X-irradiation as compared to WKAH strain rats (WKAH rats). Radiation-induced acute intestinal death occurred at doses higher than 6.5 Gy in LEC rats, and at doses higher than 12.8 Gy in WKAH rats, respectively. By the probit analysis of survival data, it was shown that the LD50/7 value of LEC rats was estimated to be 7.03 Gy which was significantly lower than that (12.99 Gy) of WKAH rats. Histopathological examinations of small intestines from LEC rats 2 days after irradiation at the dose of 8.5 Gy showed severe epithelial death together with edema, whereas little or no significant changes were noted in intestinal epithelium of 8.5 Gy-irradiated WKAH rats. These results suggest that the radiosensitivity of LEC rats to ionizing radiation appears to be higher than that of other strains of rats.     

Supporting the extensive dairy sheep smallholders of the semi-arid region of Crete through technical intervention

The objective of this field study was to depict the extensive system of dairy sheep farming in the semi-arid environment of the island of Crete and to assess the potential margins of improvement through technical intervention. Forty-three family-run farms keeping a total of 13 870 sheep were surveyed in seven representative areas of the island. Several parameters were dealt with, concerning socio-economy, flock management and productivity. Study areas differed widely regarding feeds supplied per sheep, land cultivated for feeds, grazing land utilized and housing space. A range of parameters were recorded on flock size and their production characteristics such as births, fertility and number of lambs weaned. Milk yield and parameters associated with milk quality, such as somatic cell counts and total microbial flora, were also recorded. Technical intervention was directed towards removal of non-productive animals, programming of matings, balancing of diets, management of grazing lands and health care. Ewe fertility and numbers of lambs weaned per ewe, as well as harvested milk and milk quality (based on somatic cell counts and microbial load of milk) were also significantly improved. Information derived from this study stresses the important role of extension services to small farm sustainability and contributes to our knowledge of the dairy sheep farming systems in countries around the Mediterranean and elsewhere.     

Alterations of rat brain lipids in methyl mercury intoxication.

Lipid alterations due to experimental intoxication by methyl mercury were studied in rat brain. The methyl mercury was administered perorally as a hydroxide bound to bovine liver protein. The lipids were separated by thin layer chromatography and the fatty acids identified by gasliquid chromatography. The lipids studied included free fatty acids, cholesterol and the main phosphatidylesters, phosphatidylcholine and phosphatidylethanolamine.The methyl mercury treatment resulted in an increase in total brain lipids and palmitic acid (16:0) in the free fatty acid fraction. This increase is interpreted as the cellular response to compensate for the loss of membrane proteins known to take place in the brain due to methyl mercury intoxication.     

Liposome-encapsulated oxymorphone prevents hyperalgesia for 7 days in a rat model of neuropathic pain

A delayed‐release formulation of liposome‐encapsulated oxymorphone (LEO) was produced using a novel dehydration–rehydration technique. Preparations were standardized spectrophotometrically against a known concentration of the drug. The purpose of this study was to test the analgesic properties of LEO in a rat model of neuropathic pain. Sprague–Dawley rats were divided into control (non‐neuropathic) and test (neuropathic) groups. Control and test groups were administered one SC injection of (i) vehicle liposomes (negative control treatment); (ii) liposome‐encapsulated morphine, 2.8 mg kg^?1 (positive control treatment); or (iii) LEO, 1.2 mg kg^?1. All treatments were administered after baseline thermal withdrawal latencies (TWL) were determined (9.2 ± 0.39 seconds (mean ± SEM)). Test groups then underwent sciatic ligation to induce neuropathic pain. TWL were determined in all six groups (n = 8) daily for 1 week. In a separate group of age‐matched rats, blood (0.3 mL from the jugular vein) and urine (1–2 mL via metabolism cages) were collected daily for 7 days after administration of LEO (1.2 mg kg^?1). TWL did not change in the control rats given liposome‐encapsulated sucrose or morphine. There was a small increase (p = 0.04) in TWL in control rats given LEO, likely as a result of the relatively higher dose of oxymorphone compared with morphine based on receptor affinity. TWL in test rats given blank liposomes decreased significantly (p < 0.001) by day 4 (7.1 ± 0.5 seconds), with a maximal decrease by day 7 (5.1 ± 0.36 seconds), indicating development of full hyperalgesia. In contrast, rats given liposome‐encapsulated morphine or oxymorphone had no change in TWL at day 4, indicating that these preparations prevented hyperalgesia after a single injection. This treatment effect persisted through day 7. Serum concentrations of oxymorphone after a single injection of LEO peaked at 4 hours (6.8 ± 0.82 ng mL^?1) and were detectable through day 4 (0.98 ± 0.003 ng mL^?1), while urine concentrations of drug were detectable through day 7. This result suggests that oxymorphone metabolites might have been responsible for the protracted analgesic response. The encapsulation efficiency of oxymorphone using this novel technique was approximately 96%. In conclusion, liposome encapsulation of oxymorphone proved to be an efficient mechanism to provide a delayed‐release formulation of this opioid. This single dose of subcutaneously administered liposome‐encapsulated oxymorphone was effective in preventing hyperalgesia for 7 days in this animal model of neuropathic pain.     

CD4(+) T-cell responses against the VP1-unique region in individuals with recent and persistent parvovirus B19 infection

To date cellular immune responses against parvovirus B19 (B19) have not been studied extensively. The aim of this study was to examine the T-cell response against the VP1-unique region as the immunodominant part of the viral structural protein VP1 in individuals with different courses of B19 infection. Therefore, a group of 13 parvovirus-positive probands was separated into subgroups characterized for recent or acute, past or persistent infection by means of the presence of specific immunoglobulin (Ig)M and IgG isotypes and of viral DNA in blood and tissue. Transiently transfected B-cells expressing VP1-unique region were used in ELISpot assays to investigate T-cell responses directed against the VP1-unique region in peripheral blood mononuclear cells (PBMC) of individual donors. Significant numbers of interferon-gamma (IFN-gamma) secreting lymphocytes were detectable in PBMC of all individuals with recent, acute or persistent B19 infection, but not in PBMC of donors with past B19 infection and seronegative individuals. A more detailed analysis of IFN-gamma producing cells by intracellular cytokine staining by flow cytometry revealed, that CD4(+) T cells but not CD8(+) cytotoxic lymphocytes (CTL) were the major subpopulation of IFN-gamma producing cells. These data strongly suggest the need of virus protein production for the maintenance of VP1-unique region-specific CD4(+) T-helper cell responses in B19-infected individuals.     

Crop milk protein is synthesised following activation of the IRS1/Akt/TOR signalling pathway in the domestic pigeon (Columba livia)

1. The experiment was conducted to study whether insulin receptor substance 1 (IRS1) / Protein kinase B (Akt)/target of the rapamycin (TOR) signalling pathway activation stimulates crop milk protein synthesis in the domestic pigeon (Columba livia).

2. Crop milk was collected from ten 1-d-old squabs and analysed for nutrient content. During the non-breeding period and the first day of lactation, blood samples were collected from 5 pairs of breeding pigeons and the levels of prolactin and insulin were determined.

3. Crop samples were collected from 5 pairs of breeders at d 14 and 16 of the incubation period and d 1, 3 and 7 of the lactation period. Crop samples were evaluated for changes in crop weight and thickness and changes in the expression patterns of IRS1/Akt/TOR signalling pathway–related proteins.

4. The results demonstrated that prolactin induces a gradual increase in the relative weight and thickness of the crop, with crops reaching a maximum size at the third day of lactation.

5. Pigeon crop milk contains 64.1% crude protein and 29.7% crude fat based on dry weight.

6. Serum prolactin and insulin levels in the lactation period were significantly higher than those in the non-breeding period. Compared with non-breeding pigeons, the expression of the phosphorylated IRS1 phosphorylated Akt, phosphorylated TOR, phosphorylated ribosomal protein S6 kinase, phosphorylated S6, phosphorylated eukaryotic initiation factor 4E binding protein 1 and eukaryotic initiation factor 4E were significantly up-regulated in the crop of pigeons in the lactation period.

7. In conclusion, prolactin might induce changes in crop tissue and form the physiological structure for crop milk synthesis. Furthermore, the synthesis of crop milk protein is regulated by activation of the IRS1/Akt/TOR signalling pathway.

     

Preferential infection of neuronal and astroglia cells by Akabane virus in primary cultures of fetal bovine brain

Akabane virus is a member of the genus Bunyavirus; it is pathogenic for ruminants and transmitted by arthropod vectors. Infection of adult cattle and sheep causes a transient viremia without obvious clinical signs, while infection of pregnant animals often causes fetal abnormalities including hydranencephaly, poliomyelitis and arthrogryposis. Infectious virus or viral antigens is present in the brain, spinal cord and skeletal muscle of infected fetuses. To understand the interaction between Akabane virus and bovine brain cells, we investigated the viral tropism using primary cultures of fetal bovine brain. The cultured neuronal cells, astroglia cells and microglia cells were distinguished by cell type specific antisera. Akabane virus was found to infect neuronal cells and astroglia cells, which led to degenerative death. No microglia cells were found infected. In some brain cultures, we observed different sensitivities of the cells to two Akabane virus strains: an attenuated strain infected and spread more readily than wild type virus. This difference was not observed in a hamster fibroblast cell line. Both viral and host determinants might be involved in the different susceptibility of brain cells to Akabane virus infection.