A single amino acid defines cross-species reactivity of tree shrew (Tupaia belangeri) CD1d to human invariant natural killer T (iNKT) cells

P>The non-classical major histocompatibility complex (MHC) class I molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells, which are an important part of the innate immune system. CD1d/iNKT systems are highly conserved in evoluti

Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3' and 5' RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5'-terminal untranslated region (UTR), a 355 bp T-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74-96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1 -k long genomic DNA of carp NKEF-B containing six exons and five introns. Realtime RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity. (C) 2008 Elsevier Ltd. All rights reserved.

Alterations of immune functions induced by 12C6+ion irradiation in mice

Purpose: To estimate the biological risks to the immune system of the type of space radiation, 12C6+, encountered by cosmonauts during long-term travel in space. Materials and methods: The Kun-Ming strain mice were whole-body irradiated by 12C6+ ion with 0, 0.01, 0.05, 0.075, 0.2, 0.3, 0.5, 0.75, 1 or 2 Gy, at a dose rate of 1 Gy/min. At 35 days after irradiation, the thymus and spleen weights were measured, the natural killer (NK) cells activity of spleen was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), and the interferon-gamma (IFN-gamma) levels in serum and thymus were detected with enzyme-linked immunosorbent assays (ELISA). Results: The results showed that the thymus weight, IFN-gamma levels in serum and the activity of splenic NK-cells had significantly increased at a dose of 0.05 Gy. With further dose increase, the weight of spleen continued to increase but the weight of thymus, IFN-gamma level and NK-cells activity declined. Conclusions: These results suggest that the dose of 0.05 Gy irradiation has a stimulatory effect on mouse immunity; this effect declined with increasing dose.

Immunomodulation and antitumor activity of kappa-carrageenan oligosaccharides

The modulation of carrageenan oligosaccharides from Kappaphycus striatum on the immune system in S 180-bearing mice was investigated. The mice inoculated with S180 cell suspension were treated p.o. with carrageenan oligosaccharides (50, 100 and 200 mu g/g) for 14 days. The effects of carrageenan oligosaccharides on transplantable tumors and macrophage phagocytosis, quantitative hemolysis of sheep red blood cells (QHS),. lymphocyte proliferation, the activity of natural killer cells (NK), production of interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) were studied. Carrageenan oligosaccharides could significantly inhibit the growth of transplantable sarcoma S180 and increase macrophage phagocytosis, the form of antibody secreted by spleen cells, spleen lymphocyte proliferation, NK cells activity, serumal IL-2 and TNF-alpha level in S 180-bearing mice. Considering all these results, it is suggested that carrageenan oligosaccharides exert their antitumor effect by promoting the immune system. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

低剂量12C6+辐射对小鼠免疫系统损伤和抗氧化剂氮乙酰半胱氨酸的保护作用研究

实验目的：随着科技的发展，人类活动范围已经逐渐向外太空扩展，对于人类太空探索的最大威胁是太空中的各种粒子辐射。这些辐射包括太阳辐射（质子和电子）和银河辐射（质子占85%，氦离子占14%，重离子占1%）。众所周知，重离子与常规X和γ射线相比有较高的传能线密度（linear energy transfer, LET）和相对生物学效应（relative biological effectiveness, RBE），对机体组织和器官有较强的影响。放射治疗是肿瘤治疗的重要手段之一，由于肿瘤细胞的异质性，其对放、化疗的反应相差悬殊。本研究的目的是： 1评估辐射对健康机体产生的生物学风险； 2研究抗氧化剂氮乙酰半胱氨酸（NAC）对机体辐射损伤的保护作用 3不同肿瘤细胞辐射敏感性的差异。实验方法： 1 X射线或12C6+离子对小鼠进行不同剂量的全身辐射。NAC处理组小鼠在照射前1小时腹腔注射200mg/kg的NAC，对照组注射等体积的生理盐水。照射后不同时间点取样，利用流式细胞仪检测小鼠免疫细胞周期和凋亡情况，单细胞电泳检测淋巴细胞DNA损伤，MTT法（3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide）检测脾脏NK（natural killer，NK）细胞活性，微核法检测淋巴细胞染色体损伤情况，小鼠体内干扰素－γ（Interferon-γ，IFN-γ）由ELISA方法得到，小鼠血清中超氧化物岐化酶(Surperoxide dismutase SOD）由分光光度法测定，并观察胸腺和脾脏指数变化。 2 不同剂量X射线和12C6+离子辐射人肺腺癌细胞H1299和A549，用细胞克隆法检测照射后细胞存活曲线，流式细胞仪检测细胞周期和凋亡，Western-blot 检测A549 细胞P53蛋白表达。 结果： 1小鼠外周血淋巴细胞、胸腺细胞和脾脏淋巴细胞周期随着X射线照射剂量的增大而被阻滞在了G0/G1期，相同剂量的12C6+离子辐射时外周血淋巴细胞周期被阻滞在S期，分次连续X射线照射时，外周血淋巴细胞周期随着累积剂量的增加被阻滞在G2/M期；细胞凋亡比例随着照射剂量的增加而增加。小鼠血清中IFN-γ水平和脾脏中NK细胞活性在重离子照射剂量为0.05Gy时有显著增加，脾脏NK细胞活性随着照射剂量的增加而减弱。 2重离子照射后，小鼠淋巴细胞DNA和染色体的损伤随辐射剂量和照射后时间的延长而加剧。脾脏NK细胞活性在照射后各个时间点减弱，血清中IFN-γ水平和SOD酶活性随着重离子照射剂量的增加而降低。预防性给予NAC，12C6+离子辐射对淋巴细胞DNA和染色体所致损伤，胸腺细胞周期和凋亡，脾脏NK细胞活性，血清中IFN-γ的水平和SOD酶的活性的损伤与盐水组比较均有显著改善。 3 X射线照射对肺腺癌H1299细胞周期和凋亡率未产生明显影响，重离子照射后随着照射剂量的增加细胞周期被阻滞在G2/M期，细胞凋亡率也呈剂量依赖性；X射线和12C6+离子照射A549细胞后，细胞周期均被阻滞在G2/M期，凋亡率剂量依赖性增加。A549细胞P53蛋白的表达水平随着重离子照射剂量的增加而增加。结论： 1重离子辐射造成细胞DNA和染色体损伤随着照射剂量的增加和照射后时间的延长而增加，比X射线辐射损伤复杂和难以修复，产生这种现象的机理为辐射导致活性氧分子簇的产生，细胞因子和与细胞氧化反应有关的酶活性的变化，同时这种损伤对胸腺细胞周期、凋亡和胸腺、脾脏指数以及机体免疫系统都有影响；低剂量重离子辐射（0.05Gy）对小鼠机体的免疫力有刺激作用，机体免疫能力随着照射剂量增加和照射后时间的推移而减弱，不同的免疫器官对辐射的敏感性也不同； 2 200mg/kg 的NAC对辐射所致小鼠免疫系统损伤有很好的保护作用； 3 肺腺癌细胞H1299比同系A549具有较强的辐射敏感性，A549细胞凋亡的增加与P53蛋白表达水平升高有关

The role of cell-surface-bound phosphatases in species competition within natural phytoplankton assemblage: an in situ experiment

Despite it is widely acknowledged that the ability to hydrolyze dissolved organic matter using extracellular phosphatases is diverse in fresh water phytoplankton, the competition within single species related to presence and quantity of cell-surface-bound phosphatases has not been examined in natural conditions yet. Here, we studied phytoplankton species competition in a freshwater reservoir during an in situ experiment. A natural plankton community, with the exclusion of large zooplankton, was enclosed in permeable dialysis bags inside two large containers of different bioavailable phosphate concentrations. Phytoplankton species biomass and the abundance of bacteria were determined in purpose to compare the development of enclosed microbial communities. Total and cell-surface-bound phosphatase activities in the phytoplankton were investigated using the Fluorescently Labelled Enzyme Activity (FLEA) technique that allows for direct microscopic detection of phosphatase-positive cells and, with image cytometry, enables quantification of phosphatase hydrolytic capacity. Production of extracellular phosphatases was not completely inhibited or stopped in the phosphate-enriched environment, phytoplankton cells only showed the activity less often. Under the phosphate-nonenriched conditions, the production of phosphatases was enhanced, but active species did not proliferate amongst phytoplankton assemblage. Further, specific growth rates of the phosphatase-positive species in the non-enriched environment were lower than the same phosphatase-positive species in phosphate-enriched environment. Interestingly, the phosphatase-positive cells of Ankyra ancora increased their size in both treatments equally, although the population in phosphate-enriched environment grew much faster and the cell-specific phosphatase activity was lower. We hypothesize that brand new daughter cells had sufficient phosphorus reserves and therefore did not employ extracellular phosphatases until they matured and needed extra bioavailable phosphorus to support their metabolism before cell division. Based on presented in situ experiment, we propose that the ability to hydrolyze organic polymers and particles with cell-surface-hound phosphatases is advantageous for longer persistence of given population in a phosphate-scarce environment; although phosphatase-positive species cannot dominate the reservoir phytoplankton solely because of specific phosphorus-scavenging strategy.

Kinetic measurements of cell surface E-selectin/carbohydrate ligand interactions

Selectin/ligand interactions initiate the multistep adhesion and signaling cascades in the recruitment of leukocytes from circulation to inflamed tissues and may also play a role in tumor metastasis. Kinetic properties of these interactions are essential determinants governing blood-borne cells' tethering to and rolling on the vessel wall. Extending our recently developed micropipette method, we have measured the kinetic rates of E-selectin/ligand interactions. Red cells coated with an E-selectin construct were allowed to bind HL-60 or Colo-205 cells bearing carbohydrate ligands. Specific adhesions were observed to occur at isolated points, the frequency of which followed a Poisson distribution. These point attachments were formed at the same rate with both the HL-60 and Colo-205 cells (0.14 +/- 0.04 and 0.13 +/- 0.03 mum(2) s(-1) per unit density of E-selectin, respectively) but dissociated from the former at a rate twice as fast as did from the latter (0.92 +/- 0.23 and 0.44 +/- 0.10 s(-1), respectively). The reverse rates agree well with those measured by the flow chamber. The forward rates are orders of magnitude higher than those of Fc gamma receptors interacting with IgG measured under similar conditions, consistent with the rapid kinetics requirement for the function of E-selectin/ligand binding, which is to capture leukocytes on endothelial surfaces from flow.

Stability Of Adhesion Clusters And Cell Reorientation Under Lateral Cyclic Tension

This work is motivated by experimental observations that cells on stretched substrate exhibit different responses to static and dynamic loads. A model of focal adhesion that can consider the mechanics of stress fiber, adhesion bonds, and substrate was developed at the molecular level by treating the focal adhesion as an adhesion cluster. The stability of the cluster under dynamic load was studied by applying cyclic external strain on the substrate. We show that a threshold value of external strain amplitude exists beyond which the adhesion cluster disrupts quickly. In addition, our results show that the adhesion cluster is prone to losing stability under high-frequency loading, because the receptors and ligands cannot get enough contact time to form bonds due to the high-speed deformation of the substrate. At the same time, the viscoelastic stress fiber becomes rigid at high frequency, which leads to significant deformation of the bonds. Furthermore, we find that the stiffness and relaxation time of stress fibers play important roles in the stability of the adhesion cluster. The essence of this work is to connect the dynamics of the adhesion bonds (molecular level) with the cell's behavior during reorientation (cell level) through the mechanics of stress fiber. The predictions of the cluster model are consistent with experimental observations.

Microfluidic effects of transporting signaling components in cell coculture chips

A theoretical model has been developed to investigate the microfluidic transport of the signaling chemicals in the cell coculture chips. Using an epidermal growth factor (EGF)-like growth factor as the sample chemical, the effects of velocities and channel geometry were studied for the continuous-flow microchannel bioreactors. It is found that different perfusion velocities must be applied in the parallel channels to facilitate the communication, i.e., transport of the signaling component, between the coculture channels. Such communication occurs in a unidirectional way because the signaling chemicals can only flow from the high velocity area to the low velocity area. Moreover, the effect of the transport of the signaling component between the coculture channels on the growth of the monolayer cells and the multicellular tumor spheroid (MTS) in the continuous-flow coculture environment were simulated using 3D models. The numerical results demonstrated that the concentration gradients will induce the heterogeneous growth of the cells and the MTSs, which should be taken into account in designing the continuous-flow perfusion bioreactor for the cell coculture research.

Melanoma cell growth inhibition by beta gamma-CAT, which is a novel non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin

In vertebrates, non-lens beta gamma-crystallins are widely expressed in various tissues but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained

Long-term depression in rat CA1-subicular synapses depends on the G-protein coupled mACh receptors

The subiculum, which is the primary target of CA1 pyramidal neurons and sending efferent fibres to many brain regions, serves as a hippocampal interface in the neural information processes between hippocampal formation and neocortex. Long-term depression (LTD) is extensively studied in the hippocampus, but not at the CA1-subicular synaptic transmission. Using whole-cell EPSC recordings in the brain slices of young rats, we demonstrated that the pairing protocols of low frequency stimulation (LFS) at 3 Hz and postsynaptic depolarization of -50 mVelicited a reliable LTD in the subiculum. The LTD did not cause the changes of the paired-pulse ratio of EPSC. Furthermore, it did not depend on either NMDA receptors or voltage-gated calcium channels (VGCCs). Bath application of the G-protein coupled muscarinic acetylcholine receptors (mAChRs) antagonists, atropine or scopolamine, blocked the LTD, suggesting that mAChRs are involved in the LTD. It was also completely blocked by either the Ca2+ chelator BAPTA or the G-protein inhibitor GDP-beta-S in the intracellular solution. This type of LTD in the subiculum may play a particular role in the neural information processing between the hippocampus and neocortex. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Sexual Behavior Modulates Contextual Fear Memory Through Dopamine D1/D5 Receptors

Traumatic events always lead to aversive emotional memory, i.e., fear memory. In contrast, positive events in daily life such as sex experiences seem to reduce aversive memory after aversive events. Thus, we hypothesized that post-traumatic pleasurable ex

Chronic Morphine Treatment Impaired Hippocampal Long-Term Potentiation and Spatial Memory via Accumulation of Extracellular Adenosine Acting on Adenosine A(1) Receptors

Chronic exposure to opiates impairs hippocampal long-term potentiation (LTP) and spatial memory, but the underlying mechanisms remain to be elucidated. Given the well known effects of adenosine, an important neuromodulator, on hippocampal neuronal excitability and synaptic plasticity, we investigated the potential effect of changes in adenosine concentrations on chronic morphine treatment-induced impairment of hippocampal CA1 LTP and spatial memory. We found that chronic treatment in mice with either increasing doses (20-100 mg/kg) of morphine for 7 d or equal daily dose (20 mg/kg) of morphine for 12 d led to a significant increase of hippocampal extracellular adenosine concentrations. Importantly, we found that accumulated adenosine contributed to the inhibition of the hippocampal CA1 LTP and impairment of spatial memory retrieval measured in the Morris water maze. Adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine significantly reversed chronic morphine-induced impairment of hippocampal CA1 LTP and spatial memory. Likewise, adenosine deaminase, which converts adenosine into the inactive metabolite inosine, restored impaired hippocampal CA1 LTP. We further found that adenosine accumulation was attributable to the alteration of adenosine uptake but not adenosine metabolisms. Bidirectional nucleoside transporters (ENT2) appeared to play a key role in the reduction of adenosine uptake. Changes in PKC-alpha/beta activity were correlated with the attenuation of the ENT2 function in the short-term (2 h) but not in the long-term (7 d) period after the termination of morphine treatment. This study reveals a potential mechanism by which chronic exposure to morphine leads to impairment of both hippocampal LTP and spatial memory.

Immunolocalization and expression of vascular endothelial growth factor receptors (VEGFRs) and neuropilins (NRPs) on Keratinocytes in human epidermis

Vascular endothelial growth factor (VEGF) plays an important role in normal and pathological angiogenesis. VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-2, and VEGFR-3) and neuropilins (NRPs, including NRP-1 and NRF-2) are high-affinity receptors for V