Extract of Ginkgo biloba leaves reverses yohimbine-induced spatial working memory deficit in rats

Extract of Ginkgo biloba is used to alleviate age-related decline in cognitive function, which may be associated with the loss of catecholamines in the prefrontal cortex. The purpose of this study was to verify whether alpha-2 adrenergic activity is involved in the facilitative effects of extract of Ginkgo biloba on prefrontal cognitive function. Male Wistar rats were trained to reach criterion in the delayed alternation task (0, 25, and 50-s delay intervals). A pilot study found that 3 or 4 mg/kg of yohimbine (intraperitoneal) reduced the choice accuracy of the delayed alternation task in a dose and delay-dependent manner, without influencing motor ability or perseverative behaviour. Acute oral pre-treatment with doses of 50, 100, or 200 mg/kg (but not 25 mg/kg) of extract of Ginkgo biloba prevented the reduction in choice accuracy induced by 4 mg/kg yohimbine. These data suggest that the prefrontal cognition-enhancing effects of extract of Ginkgo biloba are related to its actions on alpha-2-adrenoceptors.

Extracellular carbohydrate polymers from five desert soil algae with different cohesion in the stabilization of fine sand grain

Extracellular polymeric substances (EPS) from four filamentous cyanobacteria Microcoleus vaginatus, Scytonema javanicum, Phormidium tenue and Nostoc sp. and a coccoid single-cell green alga Desmococcus olivaceus that had been separated from desert algal crusts of Tegger desert of China, were investigated for their chemical composition, structure,and physical properties. The EPS contained 7.5-50.3% protein (in polymers ranging from 14 to more than 200 kD, SDS-PAGE) and 16.2-46.5% carbohydrate (110-460 kD, GFC). 6-12 kinds of monosaccharides, including 2-O-methyl rhamnose, 2-O-methyl glucose, and N-acetyl glucosamine were found. The main carbohydrate chains from M. vaginatus and S. javanicum consisted mainly of equal proportion of Man, Gal and Glc, that from P. tenue consisted mainly of arabinose, glucose and rhamnose. Arabinose was present in pyranose form, mainly alpha-L 1 --> 3 linked, with branches on C4 of almost half of the units. Glucose was responsible for the terminal units, in addition of having some units as beta1 --> 3 and some as beta1 --> 4 linked. Rhamnose was mainly 1 --> 3 linked with branches on C2 on half of the units. The carbohydrate polymer from D. olivaceus was composed mainly of beta1 --> 4 linked xylose, galactose and glucose. The galactose part was present both in beta-pyranose and -furanose forms. Arabinose in alpha-L-furanose form was mainly present as 1 --> 2 and 1 --> 2, 5 linked units, rhamnose only as alpha 1 --> 3 and xylose as beta 1 --> 4. The backbone of the polysaccharide from Nostoc sp. was composed of beta-1 --> 4 linked xylose, galactose and glucose. Most of the glucose was branched on position C6, terminal glucose and 2-O-methyl glucose units are also present. The relationship between structure, physical properties and potential biological function is discussed. (C) 2003 Elsevier Ltd. All rights reserved.

猕猴胚胎干细胞无饲养层、无血清培养及诱导分化成O2A 胶质前体细胞的研究

灵长类胚胎干细胞（ES 细胞）不仅能为研究生殖发育生物学基础理论提供良好的 模型，而且可为细胞替代治疗提供大量的供体细胞，因此具有重要的研究价值。当前 灵长类ES 细胞研究还有很多问题需要解决，如分离建立更多的胚胎干细胞系，优化培 养体系，提高ES 细胞定向分化为特定细胞的比例，研究ES 细胞自我更新和分化的机 制等。本文一方面概括了灵长类ES 细胞的研究进展，另一方面并对制备抗体，免疫外 科手术法分离灵长类胚胎内细胞团，建立猕猴ES 细胞的无饲养层、无血清培养体系和 诱导猕猴ES 细胞分化成高纯度的O2A 神经胶质前体细胞进行了研究。主要结论如下： 1）分别以猕猴脾脏淋巴细胞和人外周血单个核细胞作为免疫原，免疫日本大耳白兔， 得到免疫血清。在补体介导的细胞毒作用下，兔抗人和兔抗猕猴免疫血清可以裂解人 和猕猴囊胚滋养层细胞，从而分离出内细胞团，用于分离培养人和猕猴胚胎干细胞。2） 猕猴ES 细胞在以层粘连蛋白（laminin）为胞外基质，含转化生长因子beta1（TGFβ1） 的无血清培养基（SFM）中可以稳定的增殖至少22 代，保持不分化，并具有分化成三 个胚层细胞的能力。进一步的研究发现去除TGFβ1 后，猕猴ES 细胞出现分化，整合 素表达降低，推测TGFβ1 可能通过促进猕猴ES 细胞整合素的表达，加强其与胞外基 质的相互作用，从而维持ES 细胞的自我更新。然而猕猴ES 细胞不能在纤粘连蛋白 （fibronectin）和明胶上生长。3）无饲养层、无血清培养体系中长期培养的猕猴ES 细 胞，分化出拟胚体，14 天的拟胚体在血清中分化培养一周后，在含碱性成纤维生长因 子bFGF、表皮生长因子EGF 和胰岛素＋转铁蛋白＋亚硒酸钠ITS 的培养基中培养， 获得97％的O2A 胶质前体细胞，得到的O2A 细胞能够稳定增殖，并且可以自发分化 为II 型星型胶质细胞和少突胶质细胞。本实验的结果有助于猕猴ES 细胞分离建系和培 养系统的优化、推动猕猴ES 细胞自我更新和诱导为神经胶质细胞机制的研究，便于建 立ES 细胞替代治疗的猕猴模型，从而为人类ES 细胞的临床疾病治疗提供参考。

Differential proteomic analysis of neonatal cardiomyocytes in response to beta-adrenergic receptor stimulation

beta-Adrenoceptors(beta-ARs) play a critical role in regulating cardiac functions under both physiological and pathological conditions. To further explore the mechanisms through which beta-ARs perform its actions, proteomic approaches were adopted to study the global protein patterns in cultured neonatal rat cardiomyocytes exposed to isoproterenol (ISO). A modified method, "Mirror Images in One Gel", was used to improve the reproducibility and resolution power of two-dimensional electrophoresis. A 2-DE map with a good reproducibility was obtained in which 1281 70 spots were detected and about 1191 +/- 54 spots were matched, with an average matching rate of 92.9%. Nine proteins with significant changes were identified by using peptide mass fingerprinting(PMF) data obtained via MALDI-MS.

Pregnancy-specific glycoprotein function, conservation and receptor investigation

Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt to replicate previously reported cytokine responses to PSG-treatment of immune cells and subsequently to investigate functionally important amino acids within PSG1. 2). To determine whether candidate receptor, integrin αvβ3, was a binding partner for PSG1 and to investigate whether PSG1 possessed functionality in a leukocyte-endothelial interaction assay. 3). To determine whether proteins generated from recently identified putative PSG genes in the horse shared functional properties with PSGs from other species. Outcomes: 1). Sequential domain deletion of PSG1 as well as mutation of conserved residues within the PSG1 Ndomain did not affect PSG1-induced TGF-β1. The investigated response was subsequently found to be the result of latent TGF-β1 contaminating the recombinant protein. Protein further purified by SEC to remove this showed no induction of TGF-β1. The most N-terminal glycosylation site was demonstrated to have an important role in PSG N domain secretion. PSG1 attenuated LPS-induced IL-6 and TNF-α. Investigations into signalling underpinning this proved inconclusive. 2). Integrin αvβ3 was identified as a novel PSG1 receptor mediating an as yet unknown function. Preliminary investigations into a role for PSGs as inhibitors of leukocyte endothelial interactions showed no effect by PSG1. 3). Horse PSG protein, CEACAM49, was shown to be similarly contaminated by latent TGF-β1 particle and once removed did not demonstrate TGF-β1 release. Interestingly horse PSG did show anti-platelet properties through inhibition of the plateletfibrinogen interaction as previously published for mouse and human PSGs.

Repeated concanavalin A challenge in mice induces an interleukin 10-producing phenotype and liver fibrosis.

Weekly injections of Concanavalin A (Con A) were performed in BALB/c mice to evaluate the pattern of cytokine production and liver injury. High serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), IL-4, and interferon gamma (IFN-gamma) were found in the serum after the first 2 injections of Con A but rapidly decreased from the third injection. Conversely, IL-10 serum levels after repeated Con A challenge increased by 7 times from week 1 to 20. In vivo depletion studies indicated that CD4(+) T cells are essential in IL-10 production. Hepatocyte necrosis was only observed after the first injections of Con A whereas centrilobular inflammatory infiltrates persisted up to 20 weeks. Perisinusoidal liver fibrosis was also increasingly detected in BALB/c mice, whereas no fibrous change was observed in nude mice after 6 weeks of Con A challenge. The number of stellate cells, detected by immunostaining, increased after 20 weeks of Con A injections. Liver cytokine messenger RNA (mRNA) expression after 20 weeks showed expression of transforming growth factor beta1 (TGF-beta1), IL-10, and IL-4 whereas IL-2 was no more expressed. The present study shows that mice repeatedly injected with Con A develop liver fibrosis. The cytokine-release pattern observed after 1 injection of Con A is rapidly shifted towards an immunomodulatory phenotype characterized by the systemic production of large amounts of IL-10.

Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis.

Interleukin (IL)-10, a potent anti-inflammatory cytokine, limits the severity of acute pancreatitis and downregulates transforming growth factor (TGF)-beta release by inflammatory cells on stimulation. Proinflammatory mediators, reactive oxygen species, and TGF-beta can activate pancreatic stellate cells and their synthesis of collagen I and III. This study evaluates the role of endogenous IL-10 in the modulation of the regeneration phase following acute pancreatitis and in the development of pancreatic fibrosis. IL-10 knockout (KO) mice and their C57BL/6 controls were submitted to repeated courses (3/wk, during 6 wk, followed by 1 wk of recovery) of cerulein-induced acute pancreatitis. TGF-beta(1) release was measured on plasma, and its pancreatic expression was assessed by quantitative RT-PCR and immunohistochemistry. Intrapancreatic IL-10 gene expression was assessed by semiquantitative RT-PCR, and intrapancreatic collagen content was assessed by picrosirius staining. Activated stellate cells were detected by immunohistochemistry. S phase intrapancreatic cells were marked using tritiated thymidine labeling. After repeated acute pancreatitis, IL-10 KO mice had more severe histological lesions and fibrosis (intrapancreatic collagen content) than controls. TGF-beta(1) plasma levels, intrapancreatic transcription, and expression by ductal and interstitial cells, as well as the number of activated stellate cells, were significantly higher. IL-10 KO mice disclosed significantly fewer acinar cells in S phase, whereas the opposite was observed for pseudotubular cells. Endogenous IL-10 controls the regeneration phase and limits the severity of fibrosis and glandular atrophy induced by repeated episodes of acute pancreatitis in mice.

Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.

-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.

Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.

No association between TGFB1 polymorphisms and late radiotherapy toxicity: a meta-analysis.

BACKGROUND: Transforming growth factor-beta 1 (TGF-β1) protein may be multifunctional and related to the development of fibrosis, induction of apoptosis, extracellular signaling and inhibition of proliferation in response to radiation-induced DNA damage. Several studies have investigated associations between single nucleotide polymorphisms (SNPs) in the TGFB1 gene and risk of late radiation-induced injury of normal tissue, but the conclusions remain controversial. METHODS: We searched three electronic databases (i.e., MEDLINE, EMBASE and EBSCO) for eligible publications and performed a meta-analysis assessing the association of three commonly studied SNPs in TGFB1 (i.e., rs1800469, rs1800470 and rs1800471) with risk of late radiation-induced injury of normal tissue. RESULTS: We finally included 28 case-only studies from 16 publications on aforementioned SNPs in TGFB1. However, we did not find statistical evidence of any significant association with overall risk of late radiotherapy toxicity in the pooled analysis or in further stratified analysis by cancer type, endpoint, ethnicity and sample size. CONCLUSIONS: This meta-analysis did not find statistical evidence for an association between SNPs in TGFB1 and risk of late radiation-induced injury of normal tissue, but this finding needs further confirmation by a single large study.

Kv1.5 is a major component underlying the A-type potassium current in retinal arteriolar smooth muscle.

Little is known about the molecular characteristics of the voltage-activated K(+) (K(v)) channels that underlie the A-type K(+) current in vascular smooth muscle cells of the systemic circulation. We investigated the molecular identity of the A-type K(+) current in retinal arteriolar myocytes using patch-clamp techniques, RT-PCR, immunohistochemistry, and neutralizing antibody studies. The A-type K(+) current was resistant to the actions of specific inhibitors for K(v)3 and K(v)4 channels but was blocked by the K(v)1 antagonist correolide. No effects were observed with pharmacological agents against K(v)1.1/2/3/6 and 7 channels, but the current was partially blocked by riluzole, a K(v)1.4 and K(v)1.5 inhibitor. The current was not altered by the removal of extracellular K(+) but was abolished by flecainide, indicative of K(v)1.5 rather than K(v)1.4 channels. Transcripts encoding K(v)1.5 and not K(v)1.4 were identified in freshly isolated retinal arterioles. Immunofluorescence labeling confirmed a lack of K(v)1.4 expression and revealed K(v)1.5 to be localized to the plasma membrane of the arteriolar smooth muscle cells. Anti-K(v)1.5 antibody applied intracellularly inhibited the A-type K(+) current, whereas anti-K(v)1.4 antibody had no effect. Co-expression of K(v)1.5 with K(v)beta1 or K(v)beta3 accessory subunits is known to transform K(v)1.5 currents from delayed rectifers into A-type currents. K(v)beta1 mRNA expression was detected in retinal arterioles, but K(v)beta3 was not observed. K(v)beta1 immunofluorescence was detected on the plasma membrane of retinal arteriolar myocytes. The findings of this study suggest that K(v)1.5, most likely co-assembled with K(v)beta1 subunits, comprises a major component underlying the A-type K(+) current in retinal arteriolar smooth muscle cells

Functional interaction of E1AF and Sp1 in glioma invasion.

Transcription factor E1AF is widely known to play critical roles in tumor metastasis via directly binding to the promoters of genes involved in tumor migration and invasion. Here, we report for the first time E1AF as a novel binding partner for ubiquitously expressed Sp1 transcription factor. E1AF forms a complex with Sp1, contributes to Sp1 phosphorylation and transcriptional activity, and functions as a mediator between epidermal growth factor and Sp1 phosphorylation and activity. Sp1 functions as a carrier bringing E1AF to the promoter region, thus activating transcription of glioma-related gene for beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38). Biologically, E1AF functions as a positive invasion regulator in glioma in cooperation with Sp1 partly via up-regulation of GalT V. This report describes a new mechanism of glioma invasion involving a cooperative effort between E1AF and Sp1 transcription factors.

Cell surface beta 1, 4-galactosyltransferase 1 promotes apoptosis by inhibiting epidermal growth factor receptor pathway.

Our previous studies have shown that overexpression of beta1,4-galactosyltransferase1 (beta1,4GT1) leads to increased apoptosis induced by cycloheximide (CHX) in SMMC-7721 human hepatocarcinoma cells. However, the role of beta1,4GT1 in apoptosis remains unclear. Here we demonstrated that cell surface beta1,4GT1 inhibited the autophosphorylation of epidermal growth factor receptor (EGFR) especially at Try 1068. The phosphorylation of protein kinase B (PKB/Akt) and extracellular signal-regulated protein kinase1/2 (ERK1/2), which are downstream molecules of EGFR, were also reduced in cell surface beta1,4GT1-overexpressing cells. Furthermore, the translocations of Bad and Bax that are regulated by PKB/Akt and ERK1/2 were also increased in these cells. As a result, the release of cytochrome c from mitochondria to cytosol was increased and caspase-3 was activated. In contrast, RNAi-mediated knockdown of beta1,4GT1 increased the autophosphorylation of EGFR. These results demonstrated that cell surface beta1,4GT1 may negatively regulate cell survival possibly through inhibiting and modulating EGFR signaling pathway.

The beta-(1--

The stimulatory effects of the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond on the mouse spleen were studied for elucidation of the mechanism of their antitumor activity, and their stimulatory effects were compared with Lentinan. The mouse spleen's weight was increased after the intraperitoneal (i.p.) injection of the oligosaccharides compared with the saline group. In addition, routinely hematoxylin and eosin (HE)-stained spleen sections showed that the injection also changed the spleen's histopathology. RNA samples were isolated from splenocytes of oligosaccharides, Lentinan or saline-injected mice. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot showed that the administration of the oligosaccharides or Lentinan enhanced mouse spleen mRNA production of TNF-alpha but not IL-2. The injection also enhanced Concanavalin A (Con A)-induced mouse splenocytes proliferation, but the in vitro administration of the oligosaccharides did not have the proliferation-enhancing effect. Taken together, these results suggest that the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond have similar stimulatory effects as Lentinan. Additionally, they may exert their antitumor effects through the induction of splenocytes mediated immune responses.

Protein kinase B/Akt activity is involved in renal TGF-beta 1-driven epithelial-mesenchymal transition in vitro and in vivo

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-beta1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3beta phosphorylation. TGF-beta1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and alpha-smooth muscle actin expression, consistent with the fibrotic actions of TGF-beta1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-beta1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3beta, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-beta1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.