Purification and characterization of erythroid differentiation factor (EDF) isolated from human leukemia cell line THP-1

We isolated a protein, from a cell line of human origin, which exhibits extensive differentiation inducing activity toward Friend leukemia cells. The protein, called Erythroid Differentiation Factor (EDF), was found in a 4 day culture of THP-1 cells performed in the presence of 4 beta-phorbol 12-myristate 13-acetate(PMA). EDF is a homodimer of a molecular weight of 25,000, with an NH2-terminal sequence identical to that of the beta A-chain of porcine Inhibin. It was suggested that a single protein species is responsible for the activities of both EDF and FRP, a FSH releasing protein isolated from porcine ovarian follicular fluid.     

Antigenic and catalytic disparity in the distribution of cytochrome P-450-dependent 25-hydroxyvitamin D3-1α- and 24-hydroxylases

Summary Chick 25-hydroxyvitamin D₃-1-hydroxylase, a cytochrome P-450 monooxygenase with a molecular weight of 57 kDa, can be isolated as described by Mandel et al. (1990b). Under normal physiological circumstances, it occurs exclusively in kidney mitochondria. An isozyme of the 1-hydroxylase, known as the 24-hydroxylase, which uses the same substrate to yield an isomeric product, is also a cytochrome P-450 monooxygenase, has a molecular weight of 55 kDa, and likewise occurs in kidney mitochondria. The amino-terminal sequences of the first 10 residues of the two isozymes are 100% homologous. Monoclonal antibodies of the IgM class raised against the 1-hydroxylase, which quantitatively discriminate against other P-450 cytochromes of mitochondrial or microsomal origin, recognize and interact with the 24-hydroxylase as an antigen. In the present study we show that the intestine, which is the only non-renal tissue with demonstrable 24-hydroxylase activity, gives a positive peroxidase-antiperoxidase immunohistochemical reaction using the monoclonal antibodies against the 1-hydroxylase. The reactions revealed that the antigen in the kidney is restricted to the cortical proximal tubular cells while in the intestine, the antigen is localized in the enterocytes of the villi. In kidney medullary or intestinal crypt cells, or in liver, heart and lung tissues where 1-hydroxylase or 24-hydroxylase activity could not be detected using cell or tissue homogenates, the immunohistochemical reactions were also negative. Since it has been reported that chick embryonic intestine possesses 1-hydroxylase activity that is absent in the mature intestine, our results would suggest that the mature intestinal 24-hydroxylase represents a modified 1-hydroxylase as a consequence of developmentally imposed requirements regulating calcium homeostatic activity in this tissue. The difference in the molecular weights of the two enzymes would indicate either genomic processing prior to the translation of their respective mRNAs, or a post-translational processing of the larger 1-hydroxylase to the smaller 24-hydroxylase. The abbreviations used are: 25-OH-D₃, 25-hydroxyvitamin D₃; 1,25-(OH)₂D₃, 1,25-dihydroxyvitamin D₃; 24,25-(OH)₂D₃, 24,25-dihydroxyvitamin D₃, NADP, nicotinamide adenine dinucleotide phosphate     

理解运动行为抑制调控的新视角：乙酰胆碱门控氯离子通道受体

Acetylcholine, the first identified neurotransmitter, plays crucial roles in various brain functions. One well-known case is its involvement as an activating neurotransmitter in the regulation of locomotion. However, its inhibitory regulatory role, particularly in locomotion, remains poorly understood. In a study conducted by Polat et al., the authors investigated the inhibitory role of acetylcholine in locomotion in C. elegans. In this organism, the acetylcholine-gated chloride channel receptor consists of four subunits. The authors thoroughly examined the loss-of-function of each subunit in movement regulation. Interestingly, the mutant worms were still capable of performing various movements such as forward, backward crawling, and turning, suggesting that the overall movement was not significantly affected. However, quantitative behavior analysis revealed subtle yet significant differences in the timing and postures of the movement in these mutants. Furthermore, the authors employed optogenetics to stimulate a specific neuron involved in backward crawling and demonstrated that the loss-of-function of the receptors in individual neurons affects the transitioning between locomotion modes. This work provides evidence for the inhibitory regulatory role of acetylcholine in locomotion. The loss-of-function of acetylcholine-gated chloride channel receptors likely disrupts the balance of neuronal and circuit physiology, thereby affecting the regulation of locomotion. Moreover, this study highlights the powerful role of quantitative behavior analysis in discovering and understanding more sophisticated functions of neural circuits.     

Carcinogenesis in accessory sex organs of rats induced by 3,2′-dimethyl-4-aminobiphenyl: response to androgen deprivation

 It is generally accepted that early human prostate cancers reveal higher androgen dependency than do advanced ones. In the present study, we examined whether the animal model of prostate cancer has already lost androgen dependency at the early stages of carcinogenesis. At experimental week 46, androgen deprivation was induced in rats and the incidences of atypical hyperplasia and cancer were examined in the ventral, dorsolateral prostate, coagulating glands, and seminal vesicles. Androgen deprivation significantly lowered the incidence of atypical hyperplasia in all four organs. As for the incidence of cancer, no significant differences were observed in the coagulating glands and seminal vesicles. Regarding atypical hyperplasia, androgen deprivation significantly decreased the proliferative cell nuclear antigen labeling index in the coagulating gland and seminal vesicles. The presence of cancer was also decreased in the coagulating gland but not in the seminal vesicles. With control group specimens, more intense staining of androgen receptor was observed in atypical hyperplasias than in cancers. Compared with the atypical hyperplasias, the cancers revealed low androgen dependency at the early stages of carcinogenesis. The cancers in the seminal vesicles also revealed higher androgen independency than did those in the coagulating gland. Accepted: 6 May 1997     

Arabidopsis Raf‐like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling

Given their sessile nature, land plants must use various mechanisms to manage dehydration under water‐deficit conditions. Osmostress‐induced activation of the SNF1‐related protein kinase 2 (SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors [PYR (pyrabactin resistance)/PYL (pyrabactin resistance‐like)/RCAR (regulatory component of ABA receptors) proteins] and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) of the B3 clade of the mitogen‐activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2‐mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress‐induced subclass III SnRK2 activity. Our findings identify a specific type of B3‐MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.     

铁皮石斛花总RNA提取方法的比较研究

为筛选铁皮石斛(Dendrobiumofficinale)花总RNA提取方法,对8种提取方法进行了比较研究,包括改良CTAB-LiCl法(M1)、改良CTAB-异丙醇法(M2)、改良SDS-LiCl法(M3)、改良SDS-异丙醇法(M4)、多糖多酚植物RNA提取试剂盒法(M5)、柱式植物RNAout 2.0试剂盒法(M6)、RNAprep Pure多糖多酚植物总RNA提取试剂盒法(M7)和Biospin多糖多酚植物总RNA提取试剂盒法(M8)。结果表明,以M4和M5提取的总RNA带型清晰,完整性好,A260 nm/A280 nm为1.8～2.0,A260 nm/A230 nm大于2.0,RNA产率分别为(159.45±1.45)和(170.84±3.53)μg/g。利用M4、M5提取霍山石斛、金钗石斛、鼓槌石斛和美花石斛花的总RNA,样品的完整性、浓度和纯度均符合质量要求。以M4、M5提取的铁皮石斛总RNA为模板,扩增Actin基因片段,扩增产物大小与预期一致且条带单一。这说明M4、M5方法操作简便,结果重复性好,能够较好地提取石斛属植物花的总RNA。     

猪流行性腹泻病毒S蛋白胞内区线性B细胞表位最小基序鉴定

[背景] S蛋白是猪流行性腹泻病毒（Porcine Epidemic Diarrhea Virus,PEDV）的主要结构蛋白和免疫原性蛋白,在前期的研究中,本课题组在S蛋白的胞内区鉴定到2个包含线性B细胞表位的短肽。[目的] 鉴定PEDV S蛋白胞内区线性B细胞表位的最小基序。[方法] 原核表达2个短肽的每次后移1个氨基酸的系列8肽,以兔抗S蛋白血清为一抗,通过Western Blot筛选阳性反应8肽,鉴定S蛋白胞内区线性B细胞表位的最小基序。[结果] S蛋白胞内区的2个包含线性B细胞表位的短肽共享一个表位,该表位的最小基序为¹³⁷¹QPYE¹³⁷⁴。同源性分析显示该B细胞表位基序为保守性表位。[结论] 确定了S蛋白胞内区线性B细胞表位的最小基序为¹³⁷¹QPYE¹³⁷⁴;S蛋白抗原表位的鉴定有助于提高对其结构和功能的理解。     

限制性内切酶Mlu I蛋白及其硒代衍生物的制备

【背景】限制性内切酶Mlu I是一种常用的工具酶,在分子生物学领域发挥着重要的作用,其三维结构尚未被解析。【目的】在大肠杆菌中克隆表达、纯化重组Mlu I蛋白及其硒代蛋白,并进行结晶条件的研究。【方法】构建重组表达载体pET28b-Mlu I,在大肠杆菌BL21(DE3)pLysS中诱导表达,利用亲和层析和凝胶过滤层析纯化重组Mlu I蛋白和硒代Mlu I蛋白。对蛋白进行质谱检测、圆二色谱检测以及酶活检测,利用坐滴法进行结晶条件的筛选。【结果】构建了重组表达载体pET28b-Mlu I并纯化获得达到结晶纯度的蛋白,通过质谱检测确定硒代Mlu I蛋白中的8个甲硫氨酸全部被取代,结合酶活测试及圆二色谱检测确定了硒代对Mlu I蛋白的活性、结构无明显影响。采用坐滴法进行初步的晶体生长研究,重组蛋白目前已在1种条件下获得针状晶体并进行初步衍射,获得分辨率在0.32 nm左右的衍射数据。【结论】Mlu I蛋白及硒代Mlu I蛋白纯化体系的构建和结晶条件的研究,可为下一步解析Mlu I三维结构、作用机制的探讨及定向改造奠定基础。     

江西省2010年柯萨奇病毒A组24型变异株的全基因组序列分析

急性出血性结膜炎(Acute hemorrhagic conjunctivitis,AHC)是目前人类最常见的眼病之一,柯萨奇病毒A组24型变异株(Coxsackievirus A24 variant,CV-A24v)是近年来报道引起该病的主要病原体。本研究选取10株来自江西省2010年AHC暴发疫情的CV-A24v,采用特异性引物扩增并测定其全基因组序列。对该10条CV-A24v的全基因组序列进行系统发育分析以及重组分析,计算本研究测定的江西10条以及GenBank中所有22条CV-A24v的全基因组序列的氨基酸置换熵值,并预测其正向选择位点。结果表明,在江西10条CV-A24v基因组序列中未检测到重组。基于全基因组序列构建的最大似然树表明江西10株CV-A24v属于GIV基因型,且分处于两条传播链。对上述32条CV-A24v序列的氨基酸置换熵值计算,共得到25个易突变位点(熵值>0.6),易突变概率最高的区段为2A区。基于Datamonkey中FUBAR和FEL模型分析,发现位于结构蛋白VP2区的234位氨基酸为两种模型共同获得的CV-A24v的正向选择位点。本研究分析了江西10株CV-A24v的全基因组序列特征,为CV-A24v引起的AHC防控工作提供了基础资料。     

益智不同组织多糖含量及其生物合成途径分析

为了解益智（Alpinia oxyphylla）多糖生物合成途径关键酶功能,对其茎、叶、果实中的多糖含量及其单糖组成进行了研究,并采用Real-Time qPCR分析了益智多糖生物合成关键酶基因的表达模式。结果表明,益智多糖含量依次为果实 > 叶 > 茎,主要由葡萄糖、木糖、甘露糖、半乳糖和阿拉伯糖组成;利用益智转录组数据共获得47 690条unigenes,其中31 892条在NR、Swiss-Prot、KEGG、COG、KOG、GO和Pfam数据库获得注释,其中208个unigenes参与益智多糖的生物合成,涉及15个酶。表达分析表明,所筛选的18个基因在茎、叶、果实中均有表达,14个基因在果实中的表达量最高,以糖基转移酶基因和UDP-葡萄糖焦磷酸化酶基因的表达量最高,且其表达模式与不同组织中葡萄糖含量的变化一致。