Immunological Characterization of Honey Proteins and Identification of MRJP 1 as an IgE-Binding Protein

We encountered a fourth case of honey allergy in Japan. We characterized and identified the IgE-binding proteins in honey using the serum of a honey-allergenic patient. Immunoblot analysis revealed that IgE in the patient serum specifically bound to four proteins in each honey sample. At least three of these IgE-binding proteins were N-linked glycoproteins. To identify the 60-kDa IgE-binding protein in dandelion honey, the N-terminal sequences of the fragmented protein were analyzed, revealing the protein to be major royal jelly protein 1 (MRJP 1). Three IgE-binding proteins removed of N-linked oligosaccharide showed a large reduction in IgE-binding activity as compared with the intact protein. This suggests that the carbohydrates in the IgE-binding proteins are a major epitope for patient IgE.     

Raf-1蛋白激酶在细胞信号转导研究中的新进展

重点讨论Raf-1蛋白激酶的特征、激活方式及其与其他蛋白质相互作用等方面的研究进展.Raf-1蛋白激酶是酪氨酸激酶相关信号转导途径中的重要信号分子之一,可直接下传与Ras蛋白相关的细胞增殖信号.近年来发现,Raf-1蛋白激酶还可与其他信号分子作用或相互调节,参与多种细胞生物学过程的信号转导与调控.     

核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

目的 构建谷胱甘肽转硫酶（GST）与EGFP相融合的新型蛋白质示踪载体--pGST-EGFP,以用于蛋白质细胞亚定位信号序列的深入分析.方法 以质粒pEGFP-N1为骨架,融合从pGEX-2TK载体中扩增的GST编码序列,构建成pGST-EGFP融合表达质粒;再插入人工合成的已知核定位蛋白SV40的核定位序列（NLS）,构建成pGST-EGFP-SV40 NLS作为阳性对照;另外,构建小分子量蛋白TNNI2在pGST-EGFP的融合表达质粒.将对照pEGFP-N1和各重组质粒分别用脂质体介导,瞬时转染HeLa细胞,荧光显微镜下观察蛋白的核定位情况.结果 单独表达的EGFP呈全细胞分布,而GST-EGFP融合蛋白只存在于细胞浆;SV40 NLS能将GST-EGFP融合蛋白带进细胞核.虽然TNNI2-EGFP融合蛋白的细胞亚定位呈现核内丰度更高的特点,但TNNI2-GST-EGFP融合蛋白仅限定于胞浆分布,提示TNNI2不能主动定位到细胞核中.结论 成功构建了蛋白质细胞亚定位示踪载体--pGST-EGFP.作为核定位信号分析系统,其对小分子蛋白细胞亚定位的示踪效果优于传统的pEGFP载体,更适用于科研工作中小分子量蛋白质核定位信号序列的研究.     

小鼠P311蛋白与类赖氨酰氧化酶-1相互作用的鉴定

P311是利用抑制差减杂交技术从小鼠肺组织中筛选到的一个肺泡发育上游调节基因.它高水平表达于肺泡发育的相关阶段,而在慢性阻塞性肺病(chronic obstructive pulmonary disease,COPD)患者肺组织中表达水平大大下降.为深入探讨P311蛋白的作用机制,构建pGBKT7-P311诱饵表达载体,利用酵母双杂交技术,从小鼠肺组织cDNA文库中筛选出P311相互作用蛋白类赖氨酰氧化酶-1(lysyl oxidase-like 1,Loxl-1).并通过体内外免疫共沉淀及双分子荧光互补实验进行验证.为进一步研究P311蛋白的生物学功能提供新的思路。     

HIV-1 Vpu蛋白的原核表达、鉴定和纯化

目的：克隆、表达、纯化人免疫缺陷病毒Ⅰ型（HIV-1）Vpu蛋白,为其功能及免疫学研究奠定基础。方法：PCR扩增Vpu基因,纯化、酶切后克隆到原核表达载体pET32a中,转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,IPTG诱导蛋白表达,免疫印迹鉴定目的蛋白,亲和层析纯化蛋白。结果：构建了HIV-1Vpu蛋白的原核表达载体Vpu-pET32a,并在大肠杆菌中高效表达,目的蛋白呈可溶性形式存在,免疫印迹检测显示为目的蛋白,经Ni—NTAAgarose纯化获得了高纯度的目的蛋白。结论：在原核表达系统中表达了可溶性HIV-1Vpu蛋白,为进一步进行HIV-1Vpu蛋白的免疫原性和功能研究奠定了基础。     

应用蛋白组学方法筛选和鉴定乙型肝炎病毒PreS1结合蛋白

前S1蛋白（PreS1）在乙型肝炎病毒与宿主的相互作用中起至关重要的作用．为筛选乙型肝炎病毒PreS1结合蛋白,进一步探讨其在病毒感染过程中的作用,原核表达、纯化了PreS1-谷胱甘肽-S-转移酶（glutathione-S-transferase,GST）融合蛋白,利用此蛋白与HepG2细胞裂解液进行Pull-down实验,其产物进行双向凝胶电泳分离. 结果发现2个PreS1特异结合蛋白,经质谱鉴定为分子伴侣蛋白——葡萄糖调节蛋白78(GRP78)和葡萄糖调节蛋白75(GRP75)．通过免疫共沉淀和Western印迹分析证实,PreS1与GRP75之间存在相互作用．实验结果表明,GRP75为新发现乙型肝炎病毒PreS1特异结合蛋白,其与PreS1结合后的生理功能以及在HBV感染过程中的作用值得深入研究．     

丙型肝炎病毒蛋白作用于细胞信号转导途径的研究进展

细胞信号转导异常往往与人类疾病的发生、发展密切相关。一些病毒致病和感染机制即为病毒抗原蛋白作用宿主细胞信号转导途径,导致宿主细胞内信号转导发生紊乱。丙型肝炎病毒（HCV）是引发慢性丙型肝炎,导致肝硬化和肝细胞癌发生的主要病原体,但目前HCV的致病机制与宿主内持续感染机制尚不清楚。HCV致病机制可能与HCV表达的蛋白质干扰宿主细胞信号转导途径而导致异常的细胞信号转导有关。研究HCV蛋白对宿主细胞信号转导途径的影响不仅有助于阐明其致病机制,还能为新药设计和寻找新的治疗方法提供新思路和新靶点。本文主要综述了近年来国内外有关HCV蛋白作用细胞信号转导途径的研究进展。     

Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situ hybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.     

Identification of an interleukin-1 beta binding protein in human plasma

A covalent cross-linking technique was used to bind iodinated interleukin-1 (IL1) alpha and beta to plasma proteins. One specific IL1 beta binding protein was observed, that when cross-linked to ¹²⁵I-ILl beta migrated to approximately 60 kDa on SDS-PAGE. The protein did not bind IL1 alpha. The 43 -kDa protein was partially purified using a wheat germ agglutinin affinity column. The isolated factor again specifically bound IL1 beta, and appeared to consist of single chain glycoprotein. The protein was heat stable and had a rapid association time with IL1 beta. This protein may be an important carrier molecule for IL1 beta in vivo.     

Identification of a CArG Box-Dependent Enhancer within the Cysteine-Rich Protein 1 Gene That Directs Expression in Arterial but Not Venous or Visceral Smooth Muscle Cells

Smooth muscle cells (SMCs) are heterogeneous with respect to their contractile, synthetic, and proliferative properties, though the regulatory factors responsible for their phenotypic diversity remain largely unknown. To further our understanding of smooth muscle gene regulation, we characterized the cis-regulatory elements of the murine cysteine-rich protein 1 gene (CRP1/Csrp1). CRP1 is expressed in all muscle cell types during embryogenesis and predominates in vascular and visceral SMCs in the adult. We identified a 5-kb enhancer within the CRP1 gene that is sufficient to drive expression in arterial but not venous or visceral SMCs in transgenic mice. This enhancer also exhibits region-specific activity in the outflow tract of the heart and the somites. Within the 5-kb CRP1 enhancer, we found a single CArG box that binds serum response factor (SRF), and by mutational analysis, demonstrate that the activity of the enhancer is dependent on this CArG element. Our findings provide further evidence for the existence of distinct regulatory programs within SMCs and suggest a role for SRF in the activation of the CRP1 gene.     

Hormone-sensitive lipase in brown adipose tissue: Identification and effect of cold exposure

Hormone-sensitive lipase (HSL) in brown adipose tissue from mice was identified through immunoprecipitation with a polyclonal antibody (anti-HSL) towards rat white fat HSL and Western blotting. An 82 kDa polypeptide, slightly smaller than the rat white fat HSL 84 kDa subunit, was detected and its identity as HSL verified by inhibition properties. The HSL concentration per g tissue was several-fold higher in the mouse brown adipose tissue than in the rat white adipose tissue, but the specific activities per mg protein were similar. Cold-exposure (4°C of the mice for 24 h approximately doubled the HSL concentration but this increase parallelled the overall protein increase and did not reflect a specific effect on the HSL.     

GST pull-down验证流感病毒PB1-F2蛋白与热休克蛋白40的相互作用

为体外验证流感病毒PB1-F2与热休克蛋白Hsp40相互作用,通过两个方向的GST pull-down试验验证PB1-F2与Hsp40的相互作用。构建GST-多肽融合蛋白原核表达载体pGEX-6P-1-PB1-F2和pGEX-6P-1-Hsp40,并在大肠杆菌(E.co-li)BL21中诱导表达;构建真核表达载体pLEGFP-Hsp40及pCAGGS-PB1-F2,并分别转染293T细胞使其表达Hsp40及PB1-F2融合蛋白,然后进行GST pull-down试验验证二者的相互作用。成功地构建了两种蛋白的各种表达载体,经表达、纯化获得了可溶性的GST-多肽融合蛋白,GST pull-down试验正反两方向都证实了PB1-F2与Hsp40的相互作用,初步证实了流感病毒PB1-F2在体外能与Hsp40发生相互作用。     

兔抗人热激蛋白70样蛋白1多克隆抗体的制备与初步鉴定

目的：制备兔抗人热激蛋白70样蛋白1（HSP70L1）的多克隆抗体并进行初步鉴定。方法：在大肠杆菌中重组表达融合蛋白GST-HSP70L1和His-HSP70L1并纯化;将GST-HSP70L1融合蛋白用于免疫新西兰大耳白兔获得多克隆抗体,用His-HSP70L1对抗血清进行分离纯化,得到抗HSP70L1多克隆抗体,用Western印迹、免疫沉淀对其进行初步鉴定。结果：获得了高表达的GST-HSP70L1和His-HSP70L1重组融合蛋白;纯化获得抗HSP70L1抗体,此抗体可用于Western印迹和免疫沉淀实验。结论：获得了兔抗人HSP70L1的多克隆抗体,为进一步研究HSP70L1的生物功能提供了有用的工具。     

Not just the sum of its parts: Geographic variation and nonadditive effects of pyrazines in the chemical defence of an aposematic moth

Chemical defences often vary within and between populations both in quantity and quality, which is puzzling if prey survival is dependent on the strength of the defence. We investigated the within- and between-population variability in chemical defence of the wood tiger moth (Arctia plantaginis). The major components of its defences, SBMP (2-sec-butyl-3-methoxypyrazine) and IBMP (2-isobutyl-3-methoxypyrazine), are volatiles that deter bird attacks. We hypothesized that (1) variation in the chemical defences of male wood tiger moths reflects the local predation pressure; (2) observed differences in quantity and quality of defence among populations have a genetic basis; and (3) increasing concentrations of SBMP and IBMP will elicit greater aversive reactions in predators, with the two pyrazines having an additive effect on predators' avoidance. We found that (1) the chemical defence of wild moths partly reflects local predator selection: high predation pressure populations (Scotland and Georgia) had stronger chemical defences, but not lower variance, than the low-predation populations (Estonia and Finland). (2) Based on the common garden results, both genetic and environmental components seem to influence the strength of chemical defence in moth populations; and (3) IBMP alone did not provide protection against bird predators but worked against bird attacks only when combined with SBMP, and while SBMP was more effective at higher concentrations, IBMP was not. Altogether this suggests that, when it comes to pyrazine concentration, more is not always better, highlighting the importance of testing the efficacy of chemical defence and its components with relevant predators, as extrapolating from chemical data may be less than straightforward.     

Stronger Suppression of Plasma Cholesterol and Enhancement of the Fecal Excretion of Steroids by a Buckwheat Protein Product than by a Soy Protein Isolate in Rats Fed on a Cholesterol-free Diet

We investigated the effects of a buckwheat protein product (BWP), soy protein isolate (SPI) and casein on the plasma cholesterol level and fecal steroid excretion in rats fed on a cholesterol-free diet. The consumption of BWP suppressed plasma cholesterol by enhancing the fecal excretion of both neutral and acidic steroids. These effects of BWP were stronger than those of SPI.     

转录因子X-box结合蛋白1相互作用蛋白的筛选和鉴定

X-box 结合蛋白 1 是一种重要的转录因子,参与体内多项信号转导过程. 为进一步研究 XBP1 的生物学功能,运用酵母双杂交技术在肝细胞文库中筛选 XBP1 的结合蛋白. 首先运用 PCR 技术扩增获得 XBP1 的编码序列,克隆至 pGEM-T 载体,经测序鉴定后,亚克隆至诱饵载体 pGBKT7 中,转化酵母 AH109(a type). 免疫印迹检测诱饵质粒 pGBKT7-XBP1 在AH109 酵母中的表达之后,含有诱饵质粒的酵母 AH109 与含有肝细胞 cDNA 文库质粒 pACT2 的酵母 Y187(αtype)配合,配合后的二倍体酵母生长在含有 X-α-gal 的营养缺陷型培养基上 (SD/-Trp-Leu-His-Ade) 进行选择和筛选,经测序和序列比对确定阳性克隆的开放读码框 ORF,得到 7 种不同的蛋白质. 为了进一步验证这些筛选蛋白质与 XBP1 的相互作用,克隆其中一种蛋白质 MT1E,并运用 GST pulldown 和免疫共沉淀技术成功检测了 MT1E 和 XBP1 的相互作用(体外 / 体内),结果提示,MT1E 可能是 XBP1 的一个新的调节蛋白. 通过酵母双杂交技术筛选得到的 7 种蛋白质分别与肝细胞基础代谢、蛋白质的合成与运输、细胞的增殖与凋亡密切相关. 上述结果有助于揭示 XBP1 的生物学功能,为进一步探讨 XBP1 的表达和调控机制提供新线索.     

Selective Expression of Huntingtin-associated Protein 1 in ��-Cells of the Rat Pancreatic Islets

Huntingtin-associated protein-1 (HAP1) was initially identified as a binding partner of huntingtin, the Huntington''s disease protein. Based on its preferred distribution among neurons and endocrine cells, HAP1 has been suggested to play roles in vesicular transportation in neurons and hormonal secretion of endocrine cells. Given that HAP1 is selectively expressed in the islets of rat pancreas, in this study, we analyzed the expression pattern of HAP1 in the islets. In rats injected intraperitoneally with streptozotocin, which can selectively destroy β-cells of the pancreatic islets, the number of HAP1 immunoreactive cells was dramatically decreased and was accompanied by a parallel decrease in the number of insulin-immunoreactive cells. Immunofluorescent double staining of pancreas sections showed that, in rat islets, HAP1 is selectively expressed in the insulin-immunoreactive β-cells but not in the glucagon-immunoreactive α-cells and somatostatin immunoreactive δ-cells. In isolated rat pancreatic islets, ∼80% of cells expressed both HAP1 and insulin. Expression of HAP1 in the INS-1 rat insulinoma cell line was also demonstrated by immunofluorescent staining. Western blotting further revealed that HAP1 in both the isolated rat pancreatic islets and the INS-1 cells also has two isoforms, HAP1A and HAP1B, which are the same as those in the hypothalamus. These results demonstrated that HAP1 is selectively expressed in β-cells of rat pancreatic islets, suggesting the involvement of HAP1 in the regulation of cellular trafficking and secretion of insulin. (J Histochem Cytochem 58:255–263, 2010)     

Identification of the Protease-Binding Domain in the N-Terminal Region of Influenza Virus A Matrix Protein M1

A region responsible for protease binding by influenza virus A matrix protein M1 was identified. Trypsin binding was observed with the N-proximal 9-kDa fragment obtained by cleaving M1 with formic acid. The binding was inhibited by monoclonal antibodies (mAb) to region 46–70 of M1 and by an antiserum to region 21–45, whereas mAb to the middle and C-terminal regions had no effect. Thus, the protease-binding domain was mapped to the N-terminal part of M1.     

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day^1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).Although circadian rhythms in running-wheel activity are typically linked to the SCN clock^6-8, circadian oscillators in many other regions of the brain and body^9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN^15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators^17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.