Combinational biosynthesis of a fluorescent cyanobacterial holo-alpha-allophycocyanin in Escherichia coli

Allophycocyanin ( APC) is a phycobiliprotein with various biological and pharmacological properties. An expression vector containing five essential genes in charge of biosynthesis of cyanobacterial APC holo-alpha subunit ( holo- ApcA) was constructed, resulting in over- expression of a fluorescent holo- ApcA in E. coli. After being cultured for 16 h, the dry cell density reached 22.5 gl(-1), and the expression of holo- HT- ApcA was up to 1 gl(-1) broth. The recombinant protein showed similar spectral features to native APC.

Molecular cloning and characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) gene homologue from Zhikong scallop Chlamys farreri

LPS-induced TNF-alpha factor (LITAF) is a novel transcriptional factor that was first discovered in LPS-stimulated human macrophage cell line THP-1. LITAF can bind to TNF-a promoter to regulate its expression. The first scallop LITAF (named as CfLITAF) was cloned from Zhikong scallop Chlamys farreri by Expressed Sequence Tag (EST) and Polymerase Chain Reaction (PCR) techniques. The cDNA of CfLITAF was of 1240 bp and consisted of a 5' untranslated region (UTR) of 112 bp, a 3' UTR of 678 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 16.08 kDa and theoretical isoelectric point of 6.77. A typical conserved LITAF-domain was identified in CfLITAF by SMART analysis. Homology analysis of the deduced amino acid sequence of CfLITAF with other known sequences by using the BLAST program revealed that CfLITAF was homologous to the LITAF from human and rat (Identity = 46%), cattle, horse, mouse and chicken (Identity = 48%), western clawed frog (Identity = 42%), and zebrafish (Identity = 50%). The mRNA expression of CfLITAF in different tissues including haemocytes, muscle, mantle, heart, gill and gonad, and the temporal expression in haemocytes challenged by LPS or peptidoglycan (PGN) were measured by Real-time RT-PCR. CfLITAF mRNA transcripts could be detected in all tissues examined and be up-regulated in haemocytes after LPS challenge. No significant changes were observed after PGN stimulation. All these data indicated the existence of LITAF in scallop and also provided clue on the presence of TNF-alpha-like molecules in invertebrates. (C) 2007 Elsevier Ltd. All rights reserved.

海带根中alpha-葡萄糖苷酶抑制剂的提取及其酶抑制作用动力学研究

alpha-葡萄糖苷酶是一类能够催化碳水化合物alpha-1,4-糖苷键水解的酶，是动物体内许多代谢途径中的关键酶，具有多方面的靶向药理活性。研究表明，alpha-葡萄糖苷酶与糖尿病，艾滋病，恶性肿瘤、溶酶体贮积症等疾病的发生机制有重要关系，已经成为治疗这些疾病的重要靶点。 海带（Laminaria japonica） 属褐藻门、海带属植物。自二十世纪四十年代从日本引入中国以来，已成为中国沿海重要的海洋经济作物。海带藻体叶片部分大都用来提炼碘、甘露醇和藻酸盐，但其假根部分一直被当作化工工业和食品工业的废料，而未得到有效的利用。在中国的沿海一带，海带根作为一种抗糖尿病的民间药物，有很长的历史。因此从海带根中提取分离降血糖成分并研究其作用机制将具有重要的意义。 本论文第一部分进行了海带根中具有alpha-葡萄糖苷酶抑制作用的活性组分的筛选。首先我们按照海带根中所含成分的极性不同进行了提取分离，即将海带根乙醇提取物依次用石油醚、乙酸乙酯、正丁醇萃取。结果乙醇粗体物、石油醚部位、乙酸乙酯部位、正丁醇部位与水部位的酶抑制IC50分别为1589µg/L、970µg/L、 380µg/L、1616µg/L、1106µg/L。乙酸乙酯部位的酶抑制活性最强。 本论文第二部分对海带根乙酸乙酯部位的提取物的体内降糖活性进行了研究，结果表明乙酸乙酯部位可以降低糖尿病大鼠餐后血糖，与阴性对照差异极显著（P<0.01）。小鼠服药后表现出的生命体征与伺服拜糖平后表现出的生命体征类似。 在以上工作基础上我们以alpha-葡萄糖苷酶的抑制活性为导向，用天然产物化学的方法对活性成分进行追踪，通过光谱、波谱与理化性质鉴定出了具有显著alpha-葡萄糖苷酶抑制活性的化合物：丁基-异丁基-邻苯酸二甲酯，并对其进行了酶抑制动力学研究。研究表明丁基-异丁基-邻苯酸二甲酯对alpha-葡萄糖苷酶表现为非竞争性抑制类型，IC50达到13.6μg/mL，且Km=2.39 g/L 本研究工作为进一步研究海带根降血糖成分的作用机理提供了研究基础。

Preparation, characterization and antifungal properties of 2-(alpha-arylamino phosphonate)-chitosan

In this paper, 20 kinds of different 2-(alpha-arylamino phosphonate)-chitosan (2-alpha-AAPCS) were prepared by different Schiff bases of chitosan (CS) reacted with di-alkyl phosphite in benzene solution. The structures of the derivatives (2-alpha-AAPCS) were characterized by FT-IR spectroscopy and elemental analysis. In addition, the antifungal activities of the derivatives against four kinds of fungi were evaluated in the experiment. The results indicated that all the prepared 2-alpha-AAPCS had a significant inhibiting effect on the investigated fungi when the derivatives concentration ranged from 50 to 500 mu g mL(-1). Furthermore, the antifungal activities of the derivatives increased with increasing the molecular weight and concentration. And the antifungal activities of the derivatives were affected by their dimensional effect and charge density. Besides, the rule and mechanism of the antifungal activities of them were discussed in this paper. (C) 2009 Elsevier B.V. All rights reserved.

2-(Benzotriazol-1-yl)-2-(alpha-hydroxybenzyl)-N-phenylthioacrylamide

In the title compound, C21H16N4OS, the dihedral angles between the planes of the benzotriazole and N-phenyl rings and the plane of the atoms that link these two rings are 79.56 (6) and 59.02 (5) degrees, respectively, while that between the two benzene rings is 64.12 (6) degrees. There are some inter- and intramolecular interactions in the crystal structure.

Hypoxia-inducible factor 1 alpha cDNA cloning and its mRNA and protein tissue specific expression in domestic yak (Bos grunniens) from Qinghai-Tibetan plateau

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated a-subunit and a constitutively expressed beta-subunit. How animals living on Qinghai-Tibetan plateau adapt to the extreme hypoxia environment is known indistinctly. In this study, the Qinghai yak which has been living at 3000-5000 m attitude for at least two millions of years was selected as the model of high hypoxia-tolerant adaptation species. The HIF-1 alpha ORFs (open reading frames) encoding for two isoforms of HIF-1 alpha have been cloned from the brain of the domestic yak. Its expression of HIF-1 alpha was analyzed at both mRNA and protein levels in various tissues. Both its HIF-1 alpha mRNA and protein are tissue specific expression. Its HIF-1 alpha protein's high expression in the brain, lung, and kidney showed us that HIF-1 alpha protein may play an important role in the adaptation to hypoxia environment. (c) 2006 Elsevier Inc. All rights reserved.

Cloning of hypoxia-inducible factor 1 alpha cDNA from a high hypoxia tolerant mammal - plateau pika (Ochotona curzoniae)

Hypoxia-inducible factor I is a transcription factor composed of HIF-1alpha and HIF-1beta. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species livin only at 3000-5000m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1alpha cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1alpha cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822 bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1alpha and pSHIF-1alpha. The HIF-1alpha mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1alpha mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1alpha may play an important role in the pika's adaptation to hypoxia, especially in brain and kidney, and pika HIF-1alpha function pattern may be different from that of mouse HIF-1alpha. Further-more, for the high ratio of HIF-1alpha homology among the animals, the HIF-1alpha gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa. (C) 2004 Elsevier Inc. All rights reserved.

去甲肾上腺素参与吗啡神经适应性改变的中枢机制

That relapse still exists even after prolonged withdrawal is a difficult issue in the medical cure of drug addiction. Neuro-adaptation induced by prolonged exposure to addictive drugs is the neural mechanisms of both compulsive drug seeking and relapse.Neuro-adaptation caused by addictive drugs increases the individuals’ response to drugs and on the other hand, it reduces the response to natural reward in withdrawn individuals.There must be common neural mechanisms between the co-existing phenomena, and there must also be unique neural mechanisms in the drugs.To reveal the neuro-adaptation arising in the process from random, controllable drug-use to uncontrollable compulsive drug seeking is of great significance both theoretically and practically.Based on the above hypothesis, in order to reveal the function of alpha adrenergic receptor in compulsive drug-seeking motivation during the process of drug addiction, using sensitization of morphine-induced psychomotor activity as behavioral model, through the method of behavioral pharmacology, the neural mechanisms of alpha adrenergic receptor’s involvement in the process of addiction has been studied.The adjustment function caused by alpha receptors in medial prefrontal cortex and nucleus accumbens to morphine-induced psychomotor activity has been compared in the period of first use of drugs and in repetitive-use period. Furthermore, the effect on novelty seeking caused by alpha-receptors in relevant brain areas has also been compared. Major results are as follow: 1 After prolonged morphine exposure, rats’ response to morphine-induced psychomotor activity is strengthened and response to novel object induced reward weakened. 2 Injection of prazosin in medial prefrontal cortex will block morphine-induced psychomotor activity of naïve rats, however, it will not block that of morphine-withdrawn rats, but it will block the novelty seeking behavior of morphine-withdrawn rats. 3 Injection of clonidine in medial prefrontal cortex will block morphine-induced psychomotor effect of both naïve rats and morphine-withdrawn rats, and will block the novelty seeking behavior of morphine-withdrawn rats. 4 Injection of prazosin in nucleus accumbens will not affect the morphine-induced psychomotor effect of either naïve rats or morphine-withdrawn rats, nor will it affect the novelty seeking behavior of morphine-withdrawn rats. 5 Injection of clonidine in nucleus accumbens will block morphine-induced psychomotor effect of naïve rats, however, it will not block that of morphine-withdrawn rats, nor will it affect the novelty seeking behavior of morphine-withdrawn rats. These results show: 1 The weakening of the function of alpha1 receptors in medial prefrontal cortex and alpha2 receptors in nucleus accumbens caused by repetitive exposure to morphine is probably the cause of compulsive drug-seeking activity. 2 Blocking alpha1 receptors in medial prefrontal cortex accelerates the loss of interest in natural reward after morphine withdrawal. 3 Blocking alpha2 receptors in medial prefrontal cortex not only restrains drug-seeking motivation, but also blocks the individual’s seeking motivation for novelty stimulus, which suggests that, while selecting medicine for curing addiction, it should be considered to reduce the influence on natural reward as much as possible and to avoid major side-effect.