Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 ?-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa’s staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa’s staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase. The transduction did not affect the expression of Fas molecule on cells.     

Serum and insulin inhibit cell death induced by cycloheximide in the human breast cancer cell line MCF-7

Summary Continuous exposure of cells to cycloheximide (CHM) terminates in cell death. This may result from CHM’s inhibition of protein synthesis. In the present study we investigated the effect of serum and insulin on cell death induced by CHM in the human breast cancer cell line MCF-7, and correlated this effect to the inhibition of protein synthesis. Cell death was evaluated by measuring either dead cells by the trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHM (0.1 to 50 μg/ml) was shown to induce cell death in a time- and concentration-dependent manner. Including either fetal bovine serum or insulin in the culture medium inhibited this cell death in a concentration-dependent manner. Protein synthesis as measured by [³H]leucine incorporation was inhibited by the increasing concentration of CHM, However, fetal bovine serum and insulin did not alter the protein synthesis inhibition rate induced by CHM. These results indicate that inhibition of protein synthesis is not enough for cell death to proceed. Insulin or factors present in serum may stabilize some crucial cell proteins (key enzymes, cytoskeletal or membrane components) which are vital for cell life.     

Upregulation of FIk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells (NSCs) constitute the cellular basis for embryonic brain development and neurogenesis.The processis regulated by NSC niche including neighbor cells such as vascular and glial cells.Since both vascular and glial cellssecrete vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF),we assessed the effect ofVEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonicstem cells.VEGF alone did not have any significant effect.When bFGF was added,however,VEGF stimulated NSCproliferation in a dose-dependent manner,and this stimulation was inhibited by ZM323881,a VEGF receptor (Flk-1)-specific inhibitor.Interestingly,ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF,suggestingthat VEGF autocrine plays a role in the proliferation of NSCs.The stimulatory effect of VEGF on NSC proliferationdepends on bFGF,which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphoryla-tion of ERK1/2.Collectively,this study may provide insight into the mechanisms by which mieroenvironmental nichesignals regulate NSCs.     

Kv1.3 channel blockade enhances the phagocytic function of RAW264.7 macrophages

This study aimed to comprehend the largely unknown role of voltage-gated potassium channel 1.3(Kv1.3) in the phagocytic function of macrophages. We found that blocking of the Kv1.3 channel with 100 pmol L-1 Stichodactyla helianthus neurotoxin(Sh K) enhanced the phagocytic capacities of both resting and lipopolysaccharide(LPS)-stimulated RAW264.7 macrophages in the chicken erythrocyte system. In the fluorescein isothiocyanate(FITC)-labeled Escherichia coli k-12 system, Sh K increased the phagocytic capacities of resting RAW264.7 cells, but not of the LPS-stimulated cells, as LPS alone stimulated almost saturated phagocytosis of the macrophages. Sh K increased the nitric oxide(NO) production in LPS-activated cells, but not in resting RAW264.7 cells. There was no effect of Sh K alone on the cytokine secretions in resting RAW264.7 cells, but it suppressed IL-1β secretion in LPS-stimulated RAW264.7 cells. At a concentration of 100 pmol L-1, Sh K did not affect the viability of the tested cells. Kv1.3 was expressed in RAW264.7 cells; this expression was downregulated by LPS, but significantly upregulated by disrupting caveolin-dependent endocytosis with filipin III. In addition, cytochalasin D, an inhibitor of actin polymerization, did not affect the Kv1.3 expression. Thus, blocking of the Kv1.3 channel enhances the phagocytic capacity and NO production of this cell line. Our results suggest that Kv1.3 channel serves as a negative regulator of phagocytosis in macrophages and can therefore be a potential target in the treatment of macrophage dysfunction.     

Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells （NSCs） constitute the cellular basis for embryonic brain development and neurogenesis. The process is regulated by NSC niche including neighbor cells such as vascular and glial cells. Since both vascular and glial cells secrete vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）, we assessed the effect of VEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonic stem cells. VEGF alone did not have any significant effect. When bFGF was added, however, VEGF stimulated NSC proliferation in a dose-dependent manner, and this stimulation was inhibited by ZM323881, a VEGF receptor （Flk-1）- specific inhibitor. Interestingly, ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF, suggesting that VEGF autocrine plays a role in the proliferation of NSCs. The stimulatory effect of VEGF on NSC proliferation depends on bFGF, which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphorylation of ERK1/2. Collectively, this study may provide insight into the mechanisms by which microenvironmental niche signals regulate NSCs.     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDR1 ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDR1 ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzed in vitro and in vivo ; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDR1 ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effe     

Interaction between a Nanovirus-like Component and the Tobacco Curly Shoot Virus／Satellite Complex

The biological role of DNA1, a nanovirus-like component shown to be associated with the begomovirus/satellite complex, has not yet been identified. Here, we demonstrated that DNA1 of Tobacco curly shoot virus isolate Y35 (TbCSV-Y35) attenuated leaf-curling symptoms induced by TbCSV-Y35 or TbCSV-Y35 plus Y35 DNAβ in the early stage of symptom development and induced leaf cluster at a later stage of symptom development in Nicotiana benthamiana plants. The leaf disc assay demonstrated that TbCSV-Y35 DNA1 replicated autonomously. Southern blot analysis revealed that TbCSV-Y35 DNA1 reduced viral DNA accumulation. Viral DNA accumulation was not reduced when plants were co-inoculated with TbCSV-Y35 DNAβ, but the TbCSV-Y35 DNAβ level was dramatically reduced in the presence of TbCSV-Y35 DNA1. To determine whether the interaction between TbCSV/satellite complex and DNA1 had isolate specificity, DNA1 of TbCSV isolate Y132 was cloned and sequenced. It was found to have 75% nucleotide sequence identity with TbCSV-Y35 DNA1. Infectivity tests showed that TbCSV-Y132 DNA1 had no effect on the symptoms induced by TbCSV-Y35 or TbCSV-Y35 and Y35 DNAβ in N. benthamiana plants, although Y 132 DNA1 could replicate in these plants.     

刺激大鼠穹窿下器官诱发饮水和脑内c—fos表达的胆碱能机制

实验以饮水行为脑内c-fos表达为指标,,观察刺激大鼠穹窿下器官（SFO）的效应,结果显示,刺激SFO能诱发明显的饮水行为,与此同时,前脑8个部位（终板血管器官,正中视前核,室旁核,视上核,下丘脑外侧区,穹窿周核背侧区,丘脑联合核和无名质）和后脑3个部位（最后区,孤束核和壁旁外侧核）的Fos蛋白表达明显增强,免疫组化双重染色结果显示,刺激SFO能诱导视上核和室旁核中部分神经元呈Fos蛋白和加压素共同表达。脑室注射阿托品能部分阻断刺激SFO诱发的饮水行为,脑内上述各部位所诱导的Fos蛋白表达也明显减弱,以上结果提示,M胆碱能机制参与 刺激SFO诱发的饮水行为和脑内Fos蛋白的表达。     

Effect of progesterone on the activation of neurones of the supraoptic nucleus during parturition

Parturition is driven by a pulsatile pattern of oxytocin secretion, resulting from burst firing activity of supraoptic oxytocin neurones and reflected by induction of Fos expression. Rats were injected with progesterone on day 20 of pregnancy to investigate the role of the decreasing progesterone:ratio oestrogen ratio, which precedes delivery, in the activation of supraoptic neurones. Progesterone delayed the onset of birth by 28 h compared with vehicle (control) and prolonged the duration of delivery, which was overcome by pulsatile injections of oxytocin, indicating that the slow delivery may reflect impaired oxytocin secretion. Parturient rats pretreated with progesterone had fewer Fos immunoreactive nuclei in the supraoptic nucleus than did parturient rats pretreated with vehicle. The number of Fos immunoreactive nuclei was not restored after oxytocin injection, indicating that appropriate activation of oxytocin neurones is impaired by progesterone and also that there is a lack of stimulatory afferent drive. Fos expression increased in the nucleus of the tractus solitarius during parturition in rats pretreated with either vehicle or progesterone, but not in rats that had been pretreated with progesterone and induced with oxytocin, indicating that this input was inhibited. Endogenous opioids inhibit oxytocin neurones in late pregnancy and the opioid antagonist, naloxone, increases Fos expression in supraoptic nuclei by preventing inhibition. However, progesterone attenuated naloxone-induced Fos expression in the supraoptic nucleus in late pregnancy and naloxone administered during parturition did not accelerate the duration of births delayed by progesterone administration, indicating that progesterone does not act by hyperactivation of endogenous opioid tone. RU486, a progesterone receptor antagonist, enhanced supraoptic neurone Fos expression in late pregnancy, indicating progesterone receptor-mediated actions. Thus, progesterone withdrawal is necessary for appropriate activation of supraoptic and tractus solitarius neurones during parturition.     

Effect on vasopressin release of microinjection of cholinergic agonists into the rat supraoptic nucleus.

It is likely that central cholinergic pathways to the paraventricular and supraoptic nuclei participate in the control of vasopressin release. We have shown previously that this is due, in part, to activation of muscarinic, but not nicotinic, receptors in the paraventricular nucleus. There is, however, reason to believe that this cholinergic effect in the supraoptic nucleus may be the result of activation of nicotinic receptors. To test this possibility, we have studied in conscious unrestrained rats the effect of microinjection of muscarinic and nicotinic agonists into the supraoptic nucleus on vasopressin release, mean arterial blood pressure, and heart rate. Under ether anesthesia, a stainless steel guide cannula was placed in the supraoptic nucleus 5-7 days before the experiment, and femoral, arterial, and venous catheters were implanted 1 day before the experiment. Microinjection of nicotine into the supraoptic nucleus at doses of 1 and 10 micrograms resulted in transient increases in the plasma vasopressin concentration that were 7-fold and 11-fold greater, respectively, than control values at 3 min. There were also small transient increases in mean arterial blood pressure, but heart rate was unchanged. The microinjection of 2 and 20 ng of oxotremorine, a muscarinic agonist, into the supraoptic nucleus had no effect on the plasma vasopressin concentration, mean arterial blood pressure, or heart rate. These doses of oxotremorine were previously shown to have potent stimulatory effects on vasopressin release when microinjected into the paraventricular nucleus. These findings suggest that the central cholinergic stimulation of vasopressin release is due, in part, to activation of muscarinic receptors in the paraventricular nucleus and nicotinic receptors in the supraoptic nucleus.     

Alpha 2-adrenergic receptors in brown adipose tissue of infant rats--I. Identification and characteristics of binding sites in isolated plasma membranes

1. Alpha 2-Adrenoceptor antagonists [3H]yohimbine and [3H]RX 781094 and the partial alpha 2-agonist [3H]clonidine exhibited specific binding to plasma membrane fragments isolated from interscapular brown fat of 7-day-old rats. 2. Competition studies with epinephrine, yohimbine and practolol revealed that [3H]norepinephrine, the principal in vivo agonist acting upon brown adipocytes, can readily bind to alpha 2-adrenoceptors in brown fat of infant rats. 3. The presence of alpha 2-adrenoceptor subclass in brown fat of infant rats may play a role in the sympathetic regulation of this rapidly proliferating tissue.     

Xylazine activates oxytocinergic but not vasopressinergic hypothalamic neurons under normal and hyperosmotic conditions in rats

Role of central alpha2-adrenoceptors in the regulation of hypothalamic magnocellular cells was studied under hyperosmotic challenge elicited by hypertonic saline (HS). Rats pretreated with receptor agonist, xylazine (XYL), were injected intraperitoneally with different (low: 0.375, moderate: 0.75, high: 1.5 M) HS 30 min later. The activity of the paraventricular (PVN) and supraoptic (SON) vasopressin and oxytocin perikarya was established by Fos-dual-immunohistochemistry 60 min after HS administration. Results showed that 1/XYL is a potent stimulus for oxytocin but not vasopressin magnocellular cells under basal and weak hyperosmotic conditions 2/highHS completely overlaps the effect of XYL. In addition, XYL partially suppressed Fos expression in the parvocellular PVN cells activated by highHS. The data suggest that alpha2-adrenoceptors may play an important role in the regulation of oxytocinergic PVN and SON neurons under basal and weak hyperosmotic conditions and that alpha2-adrenoceptors may also participate in the control of PVN parvocellular cells under intense osmotic challenge.     

Different effects of chronic Na+, Cl−, and K+ depletion on brain vasopressin mRNA and plasma vasopressin in young rats

1. We studied the effects of selective chronic dietary sodium, chloride, or potassium depletion in young rats on vasopressin mRNA levels in the supraoptic and paraventricular nuclei, an index of vasopressin formation, and in plasma vasopressin levels, an index of vasopressin release. 2. All diets significantly increased plasma renin activity, contracted the extracellular fluid volume, and decreased serum osmolarity. 3. In the supraoptic nucleus, vasopressin mRNA levels were significantly decreased in the low-sodium group but were not significantly affected by chloride depletion. 4. There were no significant changes in vasopressin mRNA in the paraventricular nucleus after sodium or chloride dietary depletion. 5. After 2 weeks of potassium depletion, vasopressin mRNA levels were decreased in the supraoptic nucleus. When potassium depletion was prolonged for 3 weeks, vasopressin mRNA levels increased in both supraoptic and paraventricular nuclei. 6. Plasma vasopressin levels were high in animals subjected to dietary chloride depletion or to 3 weeks of potassium depletion. Dietary sodium depletion or 2 weeks of dietary potassium depletion did not significantly affect plasma vasopressin. 7. Our results show that chronic sodium, chloride, or potassium depletion differentially affect brain vasopressin mRNA and vasopressin release in young rats. 8. The effect of these diets may be mediated through changes in the extracellular fluid volume, serum osmolarity, and/or renin angiotensin system.     

Role of the locus ceruleus in baroreceptor regulation of supraoptic vasopressin neurons in the rat

The goal of this study was to identify the source of baroreceptor-related noradrenergic innervation of the diagonal band of Broca (DBB). Male Sprague-Dawley rats underwent sinoaortic denervation (SAD, n = 13) or sham SAD surgery (n = 13). We examined Fos expression produced by baroreceptor activation and dopamine-beta-hydroxylase immunofluorescence in hindbrain regions that contain noradrenergic neurons. Baroreceptors were stimulated by increasing blood pressure >40 mmHg with phenylephrine (10 microgram. kg(-1). min(-1) iv) in sham SAD and SAD rats. Controls were infused with 0.9% saline. Only the locus ceruleus (LC) demonstrated a baroreceptor-dependent increase in Fos immunoreactivity in dopamine-beta-hydroxylase-positive neurons. In a second experiment, normal rats received rhodamine-labeled microsphere injections in the DBB (n = 12) before phenylephrine or vehicle infusion. In these experiments, only the LC consistently contained Fos-positive cells after phenylephrine infusion that were retrogradely labeled from the DBB. Finally, we lesioned the LC with ibotenic acid and obtained extracellular recordings from identified vasopressin neurons in the supraoptic nucleus. LC lesions significantly reduced the number of vasopressin neurons that were inhibited by acute baroreceptor stimulation. Together, these results suggest that noradrenergic neurons in the LC participate in the baroreflex activation of the DBB and may thus be important in the baroreflex inhibition of vasopressin-releasing neurons in the supraoptic nucleus.     

Differential effects of water and saline intake on water deprivation-induced c-Fos staining in the rat

We studied c-Fos staining in adult male rats after 48 h of water deprivation and after 46 h of water deprivation with 2 h of access to water or physiological saline. Controls were allowed ad libitum access to water and physiological saline. For immunocytochemistry, anesthetized rats were perfused with a commercially available antibody for c-Fos. Dehydration significantly increased plasma vasopressin (AVP), osmolality, plasma renin activity (PRA), hematocrit, and sodium concentration and decreased urinary volume. Fos staining was significantly increased in the median preoptic nucleus, organum vasculosum of the lamina terminalis, supraoptic nucleus (SON), and magnocellular and parvocellular paraventricular nucleus (PVN), as well as the area postrema, nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVL). Rehydration with water significantly decreased AVP levels and Fos staining in the SON, PVN, and RVL and significantly increased Fos expression in the perinuclear zone of the SON, NTS, and parabrachial nucleus. Rehydration with water was associated with decreased urinary sodium concentration and hypotonicity, and hematocrit and PRA were comparable to levels seen after dehydration. After rehydration with saline, plasma osmolality, hematocrit, and PRA were not different from control, but plasma AVP and urinary sodium concentration were increased. In the SON, Fos staining was significantly increased, with a great percentage of the Fos cells also stained for oxytocin compared with water deprivation. Changes in Fos staining were also observed in the NTS, RVL, parabrachial nucleus, and PVN. Rehydration with water or saline produces differential effects on plasma AVP, Fos staining, and sodium concentration.