The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2-3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.

六种重组别藻蓝蛋白的抗氧化活性研究

藻胆蛋白（ phycobiliprotein ）是某些藻类中一类重要的捕光色素蛋白，由脱辅基蛋白（ apoprotein ）和四吡咯结构的藻胆素（ phycocyanobilin ）共价结合组成，具有抗氧化和抗肿瘤等多种生物活性。本实验室利用两种载体pMAL-p2X和pET28a对APC及其亚基分别进行重组表达，产生了MBP-APC、MBP-αAPC（Mα）、MBP-βAPC（Mβ）、6×His-αAPC（Hα）、6×His-βAPC（Hβ）和6×His-APC（HAPC）六种重组别藻蓝蛋白。本研究将表达产物和天然APC一起进行抗氧化活性研究，以期筛选出分子量更小、活性更强的组分。 本研究通过以下模型比较了六种重组别藻蓝蛋白和天然别藻蓝蛋白在体外不同抗氧化模型中的活性氧清除作用： 1. 通过比较对体外纯化学体系产生的羟自由基的清除作用，发现MBP系列的重组别藻蓝蛋白组分对体外化学体系产生的羟自由基均有一定的清除能力，清除活性大小依次为：MαAPC、rAPC、MβAPC、HβAPC、HαAPC、HAPC、Native APC。 2. 通过比较对体外纯化学体系产生的氢过氧自由基的清除作用，发现带有MBP标签的重组别藻蓝蛋白组分除MβAPC 有很低的抗氧化活性外，MαAPC和rAPC均无明显的抗氧化活性；而带有His标签的HβAPC和HAPC均高于天然APC，且随着浓度的提高，清除能力随之提高，呈现良好的量效关系。 3. 通过比较对体外纯化学体系产生的超氧阴离子自由基的清除作用，发现除MβAPC和MαAPC外，不同浓度的天然及重组别藻蓝蛋白组分对超氧阴离子自由基均有一定程度的清除作用，且随着浓度的增加清除率随之增加。其中，rAPC和HβAPC对超氧阴离子自由基的清除效果最强，IC50值分别达到36.98和42.27μg/mL。 4. 通过比较对O2-损伤红细胞膜的保护作用发现，天然及6种重组别藻蓝蛋白及其亚基对O2-损伤红细胞膜的作用均无显著的抑制作用。 5. 通过比较对•OH诱导的脂质过氧化的保护作用发现，，在100~500μg/mL的剂量范围内， rAPC和HβAPC有显著的抑制脂质过氧化的作用，IC50值分别为230.50和217.35μg/mL；HAPC 和Native APC有弱抑制作用；而MαAPC、MβAPC和HαAPC则无明显抑制脂质过氧化的作用。 我们又对其中各组分在不同体系中的抗氧化效果进行比较后发现：不同的抗氧化体系中，各种重组别藻蓝蛋白组分的抗氧化活性虽然各不相同，但带有His标签的重组别藻蓝蛋白组分明显高于带有MBP标签的重组别藻蓝蛋白组分；其中，HβAPC对体外羟自由基、氢过氧自由基、超氧阴离子自由基以及生物互作产生的•OH均有较强的清除作用，有望开发成为新一代抗氧化剂。 本研究筛选出了具有高效抗氧化活性的新型重组别藻蓝蛋白HβAPC（海普克），分子量小，且抗氧化活性比雷普克显著，这为进一步探讨雷普克和海普克的抗肿瘤作用机理提供了资料。

紫球藻藻红蛋白和藻蓝蛋白共价交联物的构建及其与天然藻胆体特性的比较研究

用蔗糖密度梯度超速离心的方法从单细胞红藻---紫球藻（Porphyridium cruentum）中分离纯化了完整的藻胆体。光谱分析表明，藻胆体在可见光区域有四个吸收峰（545nm, 565nm, 618nm 和 650nm）和一个吸收肩（498nm）；荧光发射峰分别在680nm和595nm，二者的比值（λ_(680nm)~(em)/λ_(595nm)~(em)）可高达10以上，说明分离的藻胆体非常完整。非变性电泳结果显示，分离的藻胆体至少有三种颜色的条带，分别为：B－PE，R－PC和b－PE，APC可能含量很少而未见到。SDS－PAGE电泳鉴定至少有三条明显的带，为PE的α和β的重叠带，γ亚基和RPC的亚基。用羟基磷灰石层析和Sephadex G-200层析的方法分离到了高纯度的B－藻红蛋白、b-藻红蛋白和较纯的R－藻蓝蛋白。光谱检验发现，分离得到的藻胆蛋白均为天然态，其中藻红蛋白的荧光发射峰在580nm左右，藻蓝蛋白的荧光发射峰在640nm左右。SDS－PAGE电泳分析，B-藻红蛋白有两条带，分子量大约在20kD，31kD。小分子量的条带极宽，是因为α和β亚基的分子量相近，所以有重叠；大分子量的条带是γ亚基。相对应的是b-藻红蛋白只有20kD的条带，无γ亚基的条带。藻蓝蛋白有两条明显的条带，大约为16kD和20kD。用戊二醛共价交连的方法得了紫球藻B-藻红蛋白和R－藻蓝蛋白和共价结合体。吸收光谱表明，交联体和对照最明显的区别是紫外光区278nm的峰高增加并且蓝移到268nm；交联体的荧光发射光谱与对照相比，498nm激发产生的峰值在588nm（游离的PE的发射峰）和650nm（交联体中PC的发射峰），而对照只有590nm的发射峰，并且交联体在588nm的峰高明显低于对照。非变性电泳鉴定，发现有与对照不同的新的条带出现。这一切证明藻胆蛋白能量传递模型共价交联成功。人工构建的交联物和天然的藻胆体相比，对低离子强度和低温（4 ℃）更为稳定，但能量传递效率相对较低。

坛紫菜中藻胆蛋白和多糖的化学结构特征研究

本文对北移坛紫菜（Porphyra haitanensis）中的藻胆蛋白和多糖进行了深入的研究，并发现其化学结构特征与南方坛紫菜存在一定差异，其主要结果如下：坛紫菜的水溶性色素粗提物经过耱酸铵沉淀和羟基磷灰石（HA）柱层析后，分离到三种藻胆蛋白，即藻蓝蛋白（RPC）、藻红蛋白（RPE）和变藻蓝蛋白（APC）。在中性介质中，其吸收光谱和荧光发射光谱与文献报告基本一致，但在酸性（PH_3）或碱性（PH_(12)）介质中，吸收光谱有明显改变，原有的荧光性质也消失。RPC和APC只分离到一种聚集体，但RPE有两种不同的聚体，用Sephadex G-100凝胶过滤方法，测得藻胆蛋白的分子量分别为：RPC 117，000，APC 122，000，小分子RPE 34，000，大分子RPE 232，000。 而且RPE的两种聚集体的吸收光谱、荧光光谱以及等电点等物理性质都有所差异。对RPE的氨基酸分析结果表明，以天冬氨酸、丙氨酸和谷氨酸为主，且酸性氨基酸的含量大于碱性基酸的含量。用分级法对南北方两种坛紫菜的热水和冷水提取多糖进行了层析分级，用~(13)C-NMR和红外光谱研究了各级分的化学结构特征，南北方坛紫菜琼胶分别主要由用1.0M和0.5M NaCl从DEAE-Sephadex A50层析柱洗脱下的带电荷的琼胶分子组成，~(13)C-NMR谱图表明坛紫菜主要由琼胶糖结构及其生物前体（硫酸基结合在L-半乳糖的第6位碳上）构成，且甲基化琼胶糖在南方坛紫菜中的含量明显比北移坛紫菜高，这是坛紫菜由南方移植北方后的明显差异，这种差异对于进一步研究坛紫菜的生理学特点是有意义的。

Molecular genetic analysis of DNA alterations in Irish colorectal cancer

Colorectal cancer is the most common cause of death due to malignancy in nonsmokers in the western world. In 1995 there were 1,757 cases of colon cancer in Ireland. Most colon cancer is sporadic, however ten percent of cases occur where there is a previous family history of the disease. In an attempt to understand the tumorigenic pathway in Irish colon cancer patients, a number of genes associated with colorectal cancer development were analysed in Irish sporadic and HNPCC colon cancer patients. The hereditary forms of colon cancer include Familial adenomatous polyposis coli (FAP) and Hereditary Non-Polyposis Colon Cancer (HNPCC). Genetic analysis of the gene responsible for FAP, (the APC gene) has been previously performed on Irish families, however the genetic analysis of HNPCC families is limited. In an attempt to determine the mutation spectrum in Irish HNPCC pedigrees, the hMSH2 and hMLHl mismatch repair genes were screened in 18 Irish HNPCC families. Using SSCP analysis followed by DNA sequencing, five mutations were identified, four novel and a previously reported mutation. In families where a mutation was detected, younger asyptomatic members were screened for the presence of the predisposing mutation (where possible). Detection of mutations is particularly important for the identification of at risk individuals as the early diagnosis of cancer can vastly improve the prognosis. The sensitive and efficient detection of multiple different mutations and polymorphisms in DNA is of prime importance for genetic diagnosis and the identification of disease genes. A novel mutation detection technique has recently been developed in our laboratory. In order to assess the efficacy and application of the methodology in the analysis of cancer associated genes, a protocol for the analysis of the K-ras gene was developed and optimised. Matched normal and tumour DNA from twenty sporadic colon cancer patients was analysed for K-ras mutations using the Glycosylase Mediated Polymorphism Detection technique. Five mutations of the K-ras gene were detected using this technology. Sequencing analysis verified the presence of the mutations and SSCP analysis of the same samples did not identify any additional mutations. The GMPD technology proved to be highly sensitive, accurate and efficient in the identification of K-ras gene mutations. In order to investigate the role of the replication error phenomenon in Irish colon cancer, 3 polyA tract repeat loci were analysed. The repeat loci included a 10 bp intragenic repeat of the TGF-β-RII gene. TGF-β-RII is involved in the TGF-β epithelial cell growth pathway and mutation of the gene is thought to play a role in cell proliferation and tumorigenesis. Due to the presence of a repeat sequence within the gene, TGFB-RII defects are associated with tumours that display the replication error phenomenon. Analysis of the TGF-β-RII 10 bp repeat failed to identify mutations in any colon cancer patients. Analysis of the Bat26 and Bat 40 polyA repeat sequences in the sporadic and HNPCC families revealed that instability is associated with HNPCC tumours harbouring mismatch repair defects and with 20 % of sporadic colon cancer tumours. No correlation between K-ras gene mutations and the RER+ phenotype was detected in sporadic colon cancer tumours.

Characterisation of the microbiota of traditional fermented beverages and screening these and other populations for novel antimicrobial producers and gene clusters

To screen for novel ribosomally synthesised antimicrobials, in-silico genome mining was performed on all publically available fully sequenced bacterial genomes. 49 novel type 1 lantibiotic clusters were identified from a number of species, genera and phyla not usually associated with lantibiotic production, and indicates high prevalence. A crucial step towards the commercialisation of fermented beverages is the characterisation of the microbial content. To achieve this goal, we applied next-generation sequencing techniques to analyse the bacterial and yeast populations of the organic, symbiotically-fermented beverages kefir, water kefir and kombucha. A number of minor components were revealed, many of which had not previously been associated with these beverages. The dominant microorganism in each of the water kefir grains and fermentates was Zymomonas, an ethanol-producing bacterium that had not previously been detected on such a scale. These studies represent the most accurate description of these populations to date, and should aid in future starter design and in determining which species are responsible for specific attributes of the beverages. Finally, high-throughput robotics was applied to screen for the presence of antimicrobial producers associated with these beverages. This revealed a low frequency of bacteriocin production amongst the bacterial isolates, with only lactococcins A, B and LcnN of lactococcin M being identified. However, a proteinaceous antimicrobial produced by the yeast Dekkera bruxellensis, isolated from kombucha, was found to be active against Lactobacillus bulgaricus. This peptide was patially purified.

A cross-site, comparative effectiveness study of an integrated HIV and substance use treatment program.

Co-occurrence of HIV and substance abuse is associated with poor outcomes for HIV-related health and substance use. Integration of substance use and medical care holds promise for HIV patients, yet few integrated treatment models have been reported. Most of the reported models lack data on treatment outcomes in diverse settings. This study examined the substance use outcomes of an integrated treatment model for patients with both HIV and substance use at three different clinics. Sites differed by type and degree of integration, with one integrated academic medical center, one co-located academic medical center, and one co-located community health center. Participants (n=286) received integrated substance use and HIV treatment for 12 months and were interviewed at 6-month intervals. We used linear generalized estimating equation regression analysis to examine changes in Addiction Severity Index (ASI) alcohol and drug severity scores. To test whether our treatment was differentially effective across sites, we compared a full model including site by time point interaction terms to a reduced model including only site fixed effects. Alcohol severity scores decreased significantly at 6 and 12 months. Drug severity scores decreased significantly at 12 months. Once baseline severity variation was incorporated into the model, there was no evidence of variation in alcohol or drug score changes by site. Substance use outcomes did not differ by age, gender, income, or race. This integrated treatment model offers an option for treating diverse patients with HIV and substance use in a variety of clinic settings. Studies with control groups are needed to confirm these findings.

Pairing QuantiFERON gold in-tube with opt-out HIV testing in a tuberculosis contact investigation in the Southeastern United States.

Knowing one's HIV status is particularly important in the setting of recent tuberculosis (TB) exposure. Blood tests for assessment of tuberculosis infection, such as the QuantiFERON Gold in-tube test (QFT; Cellestis Limited, Carnegie, Victoria, Australia), offer the possibility of simultaneous screening for TB and HIV with a single blood draw. We performed a cross-sectional analysis of all contacts to a highly infectious TB case in a large meatpacking factory. Twenty-two percent were foreign-born and 73% were black. Contacts were tested with both tuberculin skin testing (TST) and QFT. HIV testing was offered on an opt-out basis. Persons with TST >or=10 mm, positive QFT, and/or positive HIV test were offered latent TB treatment. Three hundred twenty-six contacts were screened: TST results were available for 266 people and an additional 24 reported a prior positive TST for a total of 290 persons with any TST result (89.0%). Adequate QFT specimens were obtained for 312 (95.7%) of persons. Thirty-two persons had QFT results but did not return for TST reading. Twenty-two percent met the criteria for latent TB infection. Eighty-eight percent accepted HIV testing. Two (0.7%) were HIV seropositive; both individuals were already aware of their HIV status, but one had stopped care a year previously. None of the HIV-seropositive persons had latent TB, but all were offered latent TB treatment per standard guidelines. This demonstrates that opt-out HIV testing combined with QFT in a large TB contact investigation was feasible and useful. HIV testing was also widely accepted. Pairing QFT with opt-out HIV testing should be strongly considered when possible.

The impact of shame on health-related quality of life among HIV-positive adults with a history of childhood sexual abuse.

Childhood sexual abuse is prevalent among people living with HIV, and the experience of shame is a common consequence of childhood sexual abuse and HIV infection. This study examined the role of shame in health-related quality of life among HIV-positive adults who have experienced childhood sexual abuse. Data from 247 HIV-infected adults with a history of childhood sexual abuse were analyzed. Hierarchical linear regression was conducted to assess the impact of shame regarding both sexual abuse and HIV infection, while controlling for demographic, clinical, and psychosocial factors. In bivariate analyses, shame regarding sexual abuse and HIV infection were each negatively associated with health-related quality of life and its components (physical well-being, function and global well-being, emotional and social well-being, and cognitive functioning). After controlling for demographic, clinical, and psychosocial factors, HIV-related, but not sexual abuse-related, shame remained a significant predictor of reduced health-related quality of life, explaining up to 10% of the variance in multivariable models for overall health-related quality of life, emotional, function and global, and social well-being and cognitive functioning over and above that of other variables entered into the model. Additionally, HIV symptoms, perceived stress, and perceived availability of social support were associated with health-related quality of life in multivariable models. Shame is an important and modifiable predictor of health-related quality of life in HIV-positive populations, and medical and mental health providers serving HIV-infected populations should be aware of the importance of shame and its impact on the well-being of their patients.

Emi2-mediated inhibition of E2-substrate ubiquitin transfer by the anaphase-promoting complex/cyclosome through a D-box-independent mechanism.

Vertebrate eggs are arrested at Metaphase II by Emi2, the meiotic anaphase-promoting complex/cyclosome (APC/C) inhibitor. Although the importance of Emi2 during oocyte maturation has been widely recognized and its regulation extensively studied, its mechanism of action remained elusive. Many APC/C inhibitors have been reported to act as pseudosubstrates, inhibiting the APC/C by preventing substrate binding. Here we show that a previously identified zinc-binding region is critical for the function of Emi2, whereas the D-box is largely dispensable. We further demonstrate that instead of acting through a "pseudosubstrate" mechanism as previously hypothesized, Emi2 can inhibit Cdc20-dependent activation of the APC/C substoichiometrically, blocking ubiquitin transfer from the ubiquitin-charged E2 to the substrate. These findings provide a novel mechanism of APC/C inhibition wherein the final step of ubiquitin transfer is targeted and raise the interesting possibility that APC/C is inhibited by Emi2 in a catalytic manner.

Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A.

Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.

Patient-derived endothelial progenitor cells improve vascular graft patency in a rodent model.

Late outgrowth endothelial progenitor cells (EPCs) derived from the peripheral blood of patients with significant coronary artery disease were sodded into the lumens of small diameter expanded polytetrafluoroethylene (ePTFE) vascular grafts. Grafts (1mm inner diameter) were denucleated and sodded either with native EPCs or with EPCs transfected with an adenoviral vector containing the gene for human thrombomodulin (EPC+AdTM). EPC+AdTM was shown to increase the in vitro rate of graft activated protein C (APC) production 4-fold over grafts sodded with untransfected EPCs (p<0.05). Unsodded control and EPC-sodded and EPC+AdTM-sodded grafts were implanted bilaterally into the femoral arteries of athymic rats for 7 or 28 days. Unsodded control grafts, both with and without denucleation treatment, each exhibited 7 day patency rates of 25%. Unsodded grafts showed extensive thrombosis and were not tested for patency over 28 days. In contrast, grafts sodded with untransfected EPCs or EPC+AdTM both had 7 day patency rates of 88-89% and 28 day patency rates of 75-88%. Intimal hyperplasia was observed near both the proximal and distal anastomoses in all sodded graft conditions but did not appear to be the primary occlusive failure event. This in vivo study suggests autologous EPCs derived from the peripheral blood of patients with coronary artery disease may improve the performance of synthetic vascular grafts, although no differences were observed between untransfected EPCs and TM transfected EPCs.

Current status and perspectives of accelerated carbonation processes on municipal waste combustion residues

The increasing volumes of municipal solid waste produced worldwide are encouraging the development of processes to reduce the environmental impact of this waste stream. Combustion technology can facilitate volume reduction of up to 90%, with the inorganic contaminants being captured in furnace bottom ash, and fly ash/APC residues. The disposal or reuse of these residues is however governed by the potential release of constituent contaminants into the environment. Accelerated carbonation has been shown to have a potential for improving the chemical stability and leaching behaviour of both bottom ash and fly ash/APC residues. However, the efficacy of carbonation depends on whether the method of gas application is direct or indirect. Also important are the mineralogy, chemistry and physical properties of the fresh ash, the carbonation reaction conditions such as temperature, contact time, CO2 partial pressure and relative humidity. This paper reviews the main issues pertaining to the application of accelerated carbonation to municipal waste combustion residues to elucidate the potential benefits on the stabilization of such residues and for reducing CO2 emissions. In particular, the modification of ash properties that occur upon carbonation and the CO2 sequestration potential possible under different conditions are discussed. Although accelerated carbonation is a developing technology, it could be introduced in new incinerator facilities as a "finishing step" for both ash treatment and reduction of CO2 emissions.

Epidermal Langerhans Cells are Dispensable for Humoral and Cell-Mediated Immunity Elicited by Gene Gun Immunization.

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity.