Deletion of yeast CWP genes enhances cell permeability to genotoxic agents

We have previously reported the development of a novel genotoxic testing system based on the transcriptional response of the yeast RNR3-lacZ reporter gene to DNA damage. This system appears to be more sensitive than other similar tests in microorganisms, and is comparable with the Ames test. In an effort to further enhance detection sensitivity, we examined the effects of altering major cell wall components on cell permeability and subsequent RNR3-lacZ sensitivity to genotoxic agents. Although inactivation of single CWP genes encoding cell wall mannoproteins had little effect, the simultaneous inactivation of both CWP1 and CWP2 had profound effects on the cell wall structure and permeability. Consequently, the RNR3-lacZ detection sensitivity is markedly enhanced, especially to high molecular weight compounds such as 4-nitroquinoline-N-oxide (> sevenfold) and phleomycin (> 13-fold). In contrast, deletion of genes encoding representative membrane components or membrane transporters had minor effects on cell permeability. We conclude that the yeast cell wall mannoproteins constitute the major barrier to environmental genotoxic agents and that their removal will significantly enhance the sensitivity of RNR-lacZ as well as other yeast-based genotoxic tests.

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction, Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

Gene transfer into conchospores of Porphyra haitanensis (Bangiales, Rhodophyta) by glass bead agitation

Genetic transformation by electroporation of protoplasts is a standard procedure for many plants. However, for the genus Porphyra, the method is not effective because of low viability of protoplasts and is a time-consuming and expensive procedure. Based on the life history of Porphyra, a spore-targeted strategy of genetic transformation was developed, that is, using fresh conchospores of Porphyra haitanensis Chang & Zheng transformed by agitation with glass beads. A SV40 promoter-driven lacZ reporter gene was expressed in conchospores 48 h after the agitation. More transformants were obtained by increasing the agitation time from 10 to 25 s. The maximum number of transformants was more than six out of 1 million agitated conchospores. Transfer of a SV40 promoter-driven egfp gene into conchospores resulted in significant green GFP fluorescence. The expression of lacZ and egfp revealed that this strategy of spore-targeted transformation using glass bead agitation is feasible in P. haitanensis and that the SV40 promoter is effective for monitoring expression of foreign genes in this red algal species.

Cloning and characterization of beta-carotene ketolase gene promoter in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, astaxanthin, under various environmental stresses. beta-carotene ketolase (BKT) plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, an approximate 700 bp 5'-flanking region of the bkt gene containing a putative promoter was cloned through walking upstream. The results of the sequence analysis showed that this bkt 5'-flanking region might have cis-acting elements such as sterol regulatory element (SRE-1)-like motifs, the C-repeat/dehydration responsive element (DRE) and al-3 proximal element (APE)-like motifs, except for typical TATA and CCAAT boxes. The results of the P-galactosidase assay and the transient expression of lacZ driven by a series of sequential deletions revealed that a minimal promoter-like region might exist from -630 to -408 bp, and the highest promoter activity was observed to span the positions from -630 to -308 bp. The results of the site-directed mutagenesis of a C-repeat/DRE and two APE-like motifs in a promoter-like region (-630 to -308 bp) suggested that two APE-like motifs might be essential for transcriptional control of the bkt gene.

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by COILS. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of Delta escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

Genetic transformation in Kappaphycus alvarezii using micro-particle bombardment: a potential strategy for germplasm improvement

This study investigated the delivery of a SV40 promoter driving lacZ gene into cells of Kappaphycus alvarezii using particle bombardment. Thallus pieces 0.5-0.8 mm in diameter and 1 cm in length were prepared as gene recipients. Bombardment parameters of 450 psi (rupture pressures) x 6 cm (particle travel distances), 650 psi x 6 cm, 1,100 psi x 6 cm and 1,100 psi x 9 cm were used. A significant increase in transformation efficiency from about 33% under the rupture pressure of 450 psi to 87% at 650 psi was observed in transformed thalli. Most of the positive cells appeared in epidermal cells bombarded at 450 psi, whereas positive signals were seen in both epidermal and medullary cells at 650 psi. No positive transient expression was detected at a bombardment of 1,100 psi, or in negative or blank controls. For the conditions tested, the best parameter was obtained at 650 psi at a distance of 6 cm. Thus, the strategy of taking vegetative thalli as recipients, using particle bombardment, and combining this with micro-propagation, together with developing an in vivo selectable marker, is a viable way to produce stable transformants, to eliminate chimeric expression, and to achieve transgenic breeding in K. alvarezii.

裙带菜遗传转化模型的建立

该文从转化受体、转化方法、报告基因、再生途径、筛选方法等方面对裙带菜的遗传转化进行了研究,首先,分离并建立了裙带菜雌雄配子体的无性繁殖系,进行了裙带菜的不同再生途径的研究.进行裙带菜配子体孤雌生殖、孤雄生殖和雌雄受精的再生途径实验,结果没有得到孤雄生殖的裙带菜,孤雌生殖的效率低、时间长且发育不正确,雌雄受精的再生途径一个月左右90%以上的雌配子体都正常萌发成幼孢子体.确定在裙带菜遗传转化模型中采用雌雄受精的再生途径.该文初步建立了以雌雄配子体为受体,以基因枪法为转化方法,以SV40启动子为驱动元件,以雌雄受精为再生途径,以氯霉素、潮霉素或basta为筛选压力的裙带菜遗传转化模型.

迟缓爱德华氏菌III型分泌系统(T3SS)输送器蛋白及分子伴侣的研究

迟缓爱德华氏菌（Edwardsiella tarda）是重要的革兰氏阴性致病菌，可以感染包括人类在内的多种动物。由迟缓爱德华氏菌引发的爱德华氏菌病已经在许多水产养殖动物中被发现，每年给淡水和海水水产养殖业带来巨大的损失。目前为止对于迟缓爱德华氏菌病的防治以化学治疗为主，疫苗的研究还在进行中。III型分泌系统（T3SS）是E. tarda重要的致病因子，虽然T3SS基因簇的结构及部分基因的功能得到了初步研究，但其作用机制还未得到阐明。本研究着重开展了迟缓爱德华氏菌T3SS输送器蛋白EseC的分子伴侣的鉴定及功能研究，并对输送器蛋白及其免疫功能进行了初步探讨，希望进一步地了解T3SS在E. tarda致病中的功能及其在疫苗研制中的作用。 一、迟缓爱德华氏菌III型分泌系统（T3SS）输送器蛋白EseC分子伴侣的鉴定和功能研究 以前的工作表明，EseB、EseC和EseD蛋白是E. tarda T3SS输送器蛋白的组成成分，在分泌到细菌细胞外后可以组成输送器装置。分子伴侣对于输送器蛋白的稳定和分泌具有重要的作用，EscC已经被鉴定为EseB和EseD的分子伴侣，而EseC的分子伴侣还没有得到鉴定。在本实验中，我们以EseC作为研究对象，主要开展了EseC分子伴侣鉴定的研究。 生物信息学分析表明，在E. tarda T3SS基因簇上的escA基因与eseC相邻，其编码的蛋白形成一个大的螺旋结构，为分子量较小（17.5kD）的酸性蛋白（pI 4.79），并与已鉴定的分子伴侣具有序列的同源性，这些符合细菌T3SS分子伴侣的特征。研究发现，EscA蛋白分布在细菌的细胞质和细胞膜上。在escA基因缺失后，大大降低了EseC分泌到细菌细胞外的量，同时EseC蛋白在细菌细胞质中的积聚量也减少，当escA基因缺失突变株得到escA基因互补后，EseC的分泌和在细胞质内的积聚恢复到了野生型菌株水平。氯霉素阻断蛋白质合成的实验发现，当细菌不表达EscA的情况下，EseC蛋白逐渐降解，说明了EscA可以影响EseC在胞质中的稳定。蛋白体外结合试验和免疫共沉淀实验发现，EseC和EscA在体外可以结合，在细菌细胞质中也可以相互结合，表明EseC和EscA可以相互作用。上述结果表明，EscA是EseC的分子伴侣。 在确定了EscA是EseC的分子伴侣之后，我们进一步确定EscA对EseC表达的影响，以及两者相互作用的结构域。通过检测转录水平和翻译水平的EseC-LacZ融合蛋白表达情况，发现在EscA缺失的情况下，EseC的转录水平没有变化，而翻译水平下降，表明EscA对EseC的影响在转录后水平。通过构建含有部分结构域缺失的escA或eseC的体外共表达体系，并进行Western blot分析，确定了EseC的31-137氨基酸序列为与EscA结合的区域，而在EscA中并没有找到与EseC结合的区域。EseC的31-137氨基酸片段缺失后，EseC的分泌和在E. tarda细菌细胞中的积聚下降，其下降幅度与escA突变株相当，进一步表明EseC的31-137氨基酸为与EscA相互作用的区域。最后人工感染实验表明，分子伴侣EscA及其与EseC的相互作用对E. tarda的致病力有影响。 二、迟缓爱德华氏菌T3SS输送器蛋白的研究 一些研究表明，T3SS在细菌与宿主相互作用的过程中表达，在体外诱导的条件下也可表达。为了确定E. tarda T3SS体外诱导表达的条件，我们检测了不同培养温度、pH条件下，E. tarda T3SS输送器蛋白表达的情况。研究表明，37°C条件下，E. tarda生长快，T3SS的输送器蛋白表达较低；28°C条件下，T3SS的输送器蛋白表达最高，而在20°C条件下，没有检测到T3SS输送器蛋白的表达。在28°C和37°C的培养条件下，中性和碱性相对酸性来说适合细菌的生长和T3SS输送器蛋白的表达。我们分析了E. tarda野生型和输送器蛋白突变株中的输送器蛋白的细胞分布，并据此推测输送器形成的机制。单一输送器蛋白的缺失不影响其它两个输送器蛋白的积聚，而输送器蛋白的分泌之间存在一定的相互影响。 通过检测输送器蛋白突变株ΔeseB, ΔeseC, ΔeseD生长、泳动、自凝聚和溶血能力的变化，发现在输送器蛋白基因缺失后，体外培养的E. tarda的生长速度变慢，泳动、自凝聚和溶血能力也变弱，说明了输送器蛋白在细菌的生长和功能行使中的重要作用。 为了检测输送器蛋白的免疫保护效果，我们克隆了eseD基因，将其在表达菌株BL21(DE3)中进行表达，并将重组表达的EseD蛋白经Ni-NTA树脂进行纯化。以EseD纯化蛋白作为蛋白抗原对大菱鲆进行注射，EseD蛋白表现出了对鱼类的免疫原性，其抗体效价在第7周达到了最高，为1：5120。攻毒实验表明该蛋白对于保护大菱鲆免疫E. tarda的感染具有帮助作用，在105cfu攻毒浓度下大菱鲆的相对存活率(RPS)为62.5%。结果说明EseD蛋白可以作为蛋白抗原疫苗的候选，并能够在保护鱼类免疫爱德华氏菌病中发挥作用。

转基因海带配子体的制备与高效增殖

海带具有很高的营养价值和经济社会价值。自20世纪90年代以来，本实验室在借鉴高等植物基因工程原理和方法的基础上，根据海带自身特点，建立了海带遗传转化体系（海带孢子体表达系统），它的基本原理是利用基因枪法转化海带配子体，经孤雌或受精途径再生幼孢子体后，用氯霉素筛选幼孢子体获得转基因海带，然后进行海上安全栽培和转基因产品的检测与提取。目前该表达系统已成功实现报告基因（β-半乳糖苷酶基因，lacZ）和功能基因（乙肝表面抗原基因，HBsAg）的稳定表达。 由于海带孢子体表达系统需经孢子体再生和海上栽培等阶段，周期较长，而且转基因安全性问题也在一定程度上制约其研究与应用。因此，我们在海带孢子体表达系统的基础上又建立和优化了海带配子体表达系统，并成功实现了报告基因（绿色荧光蛋白基因，GFP）的瞬间表达和功能基因（瑞替普酶基因，rt-PA）的稳定表达。虽然海带配子体表达系统能避免转基因安全性问题，周期较短，但在表达量和生物量积累方面，与孢子体表达系统相比还有较大差距。 本文首先在海带配子体表达系统中成功实现了人酸性成纤维细胞生长因子基因（hafgf）和鲎素基因（tac）的稳定表达，制备了转基因海带配子体，然后将光生物反应器培养技术应用于转基因海带配子体的高效增殖，以期解决阻碍海带配子体表达系统发展的量的问题，并为转基因海带配子体的大规模培养提供试验依据和技术支持。 本文的研究结果为： 1、人酸性成纤维细胞生长因子基因和鲎素基因可以稳定整合到海带配子体基因组中，实现转基因产物的表达。 2、根据转基因海带配子体的生长特点，研制开发了一套培养体积为300 ml的鼓泡式光生物反应器，它具有操作简便、成本低廉、适合海带配子体生长等特点。随后将培养体系扩大到2.5 L，并研究了光对转基因海带配子体生长的影响，试验结果显示，转基因海带配子体在光强为30 μE m-2 s-1时即可达到光饱和生长，最优光周期为14:10 LD，而且蓝光可促进转基因海带配子体的生长。 3、在前期研究工作的基础上，为改善反应器内的传质条件，我们又设计研制了2.5 L气升式光生物反应器用于转基因海带配子体的高效增殖。研究发现，气升式光生物反应器较鼓泡式光生物反应器能明显地改善反应器内的传质状态，实现转基因海带配子体更高密度的培养（生物量可达到1,990 mg L-1），是一套高密度悬浮培养转基因海带配子体的有效装置和设备。

蓝藻与绿藻类胡萝卜素合成酶基因的比较基因组学及代谢调控研究

类胡萝卜素在生物体内具有重要的生理功能，其中虾青素是一类高附加值值的类胡萝卜素。本文通过比较基因组学和实验手段，探索了类胡萝卜素相关基因的转录调控，为类胡萝卜素的代谢工程奠定的了基础。 1、对已测序的18种蓝藻的类胡萝卜素合成基因进行了比较基因组学研究，发现除了Gloeobacter violaceus PCC 7421的类胡萝卜素合成途径是细菌型外，其余的蓝藻类胡萝卜合成途径均属于植物型。序列比对发现蓝藻中参与合成途径上游的酶基因在进化中保守性较高。研究还发现，一些类胡萝卜素合成酶在结构和功能上存在趋同或趋异进化，揭示它们进化上的多样性。 2、 通过比较基因组学分析，发现在集胞藻Synechocystis sp.PCC6803等几种蓝藻中，没有典型的番茄红素环化酶基因的同源基因，而存在着与绿硫细菌中的γ-carotene环化酶基因相似的基因,例如集胞藻中的sll0147和sll0659。但是研究结果显示sll0147和sll0659的突变对番茄红素环化过程影响不明显，提示在这些蓝藻中可能有其他基因参与环化过程，而sll0659基因可能参与了细胞的分裂过程。 3、 从经济绿藻雨生红球藻中克隆了参与类胡萝卜素合成途径的八氢番茄红素合成酶基因（psy）和八氢番茄红素脱氢酶基因（pds）的cDNA和基因组序列。通过基因组步移的方法克隆了这两个基因的5’侧翼序列，以本实验室建立的lacZ为报告基因的瞬间表达体系研究了这两个基因上游区域的启动子活性。 4、 通过ABA、 N饥饿和高光诱导雨生红球藻积累虾青素，利用半定量RT-PCR方法分析了在相同环境因子诱导下雨生红球藻中类胡萝卜素合成基因的表达调控模式，结果显示在虾青素积累过程中，除了番茄红素环化酶基因变化不明显，其他一些基因均存在明显的转录上调。 本研究首次利用比较基因组学的方法分析了类胡萝卜素基因的进化，发现大部分蓝藻的类胡萝卜合成途径属于植物型，一些类胡萝卜合成酶存在结构和功能的多样性。同时分离了雨生红球藻中类胡萝卜素相关基因八氢番茄红素合成酶基因（psy）和八氢番茄红素脱氢酶基因（pds）的5’上游侧翼序列，验证了它们的启动子功能，研究了雨生红球藻虾青素合成酶基因在相同环境因子诱导下的表达调控模式。本文将基础研究与实验验证相结合，为下一步研究类胡萝卜素合成的代谢工程提供了线索。

雨生红球藻中虾青素合成相关酶基因的转录调控序列分析

虾青素是一种具有极强抗氧化活性的类胡萝卜素，具有广泛的应用价值。雨生红球藻是一种单细胞绿藻，在逆境胁迫条件下能够大量合成并迅速积累虾青素，其积累量最高可达细胞干重的4%，从而成为目前首选的天然虾青素合成工具。但是，虾青素的大量积累总是发生在不适于生物量积累的环境胁迫条件下，虾青素积累与生物量积累之间成为一对矛盾，制约着虾青素大量生产。 异戊烯焦磷酸异构酶（IPI）、β-胡萝卜素酮化酶（BKT）和β-胡萝卜素氧化酶（CRTO）是虾青素合成过程中的相关酶。已有研究结果表明，这些酶基因的表达调控至少是部分发生在转录水平上的，这就为我们从转录水平上研究虾青素生物合成关键酶基因的调控机制提供了重要线索。 本文研究结果如下： 1. 利用基因组步移的方法克隆了两条异戊烯焦磷酸异构酶基因（ipi）的5’上游侧翼序列（1.8kb和2.5kb），预示着ipi的转录由不止一个启动子调控。 2. 利用上述方法克隆了两条β-胡萝卜素氧化酶基因（crtO）5'上游侧翼序列（1kb和2kb）。以lacZ为报告基因的瞬间表达实验结果表明，长度为320bp(-682/-363)的crtO 5'上游侧翼序列具有很强的启动转录活性，提示这段序列包含了启动子的结构。 3. 利用凝胶阻滞的方法研究了雨生红球藻中bkt强启动转录活性区域，即 309bp(-617/-309)启动子区域的转录因子结合位点，并发现在-396/-338的59bp区域内存在特异的核蛋白结合位点。通过序列分析，发现此59bp区域并不包含TATA或者CAAT-box，而是存在对光、缺氧、p-香豆酸及激素反应的G-box。 4. 根据国外已经获得的ipi和crtO全长cDNA序列，利用长距离PCR法从红球藻基因组中扩增到基因组序列。发现ipi和crtO均包含6个外显子和5个内含子，内含子的剪切位点基本符合GU-AG规律。并通过似然比检验（Likelihood ratio test）的方法发现两基因在进化过程中存在着正选择现象。 这些工作为下一步继续寻找与上述特定DNA调控区域特异结合的反式作用因子（蛋白质因子）奠定了基础。

利用海带配子体营养增殖体系实现外源基因表达

1993年以来，本实验室在借鉴高等植物基因工程原理和方法的基础上，利用海带世代交替生活史，建立了海带遗传转化体系,并申请国家专利PCT/CN03/00534（Trends in Biotechnology, 2005, 23,264-268.）。海带遗传转化体系是建立在基因枪转化海带配子体，经孤雌或受精途径再生幼孢子体后用氯霉素筛选以及海上安全栽培等一系列技术平台上的新型体系。目前该体系已经成功实现报告基因（β-半乳糖甘酶基因，LacZ）和目的基因（乙肝表面抗原基因，HBsAg）在孢子体阶段的稳定表达。由于应用该体系表达目的基因须经过孢子体再生以及海上栽培等阶段，周期较长，因此本论文拟通过以下5方面工作初步建立以配子体营养增殖为基础的新型海带表达体系，并尝试在配子体阶段实现转化、筛选、增殖及产物检测等操作。1)确立针对配子体的选择标记。2)利用光生物反应器增殖配子体。3)拓展新的报告基因种类。4)克隆内源高效启动子。5)利用该体系表达rt-PA基因（编码价格昂贵的特效溶栓药，瑞替普酶）。本论文研究结果为1)升高温度能够显著增强草丁膦的筛选效果，根据敏感性实验统计结果确定了草丁膦作为海带配子体遗传转化选择压力的筛选方案为40mg/l连续处理7d×3次。2)利用室内光生物反应器营养增殖海带配子体技术，经草丁膦筛选，实现了SV40启动子驱动草丁膦抗性基因bar在配子体细胞中的稳定表达。3)采用调整配子体营养生长状态的方法减低背景荧光，从而首次观察到增强型绿色荧光蛋白基因（egfp）在海带配子体细胞中的瞬间表达。4)克隆了海带肌动蛋白基因（actin）的基因组序列，这是褐藻肌动蛋白基因组序列克隆的首次报道，通过染色体步移获得海带actin基因上游约450bp的片段。5)在上述工作基础上，与中国药科大学合作构建了瑞替普酶基因（rt-PA）的海带表达载体，并实现该基因在海带配子体细胞中的稳定表达，产物具有正确的生物学活性，平均表达量达到0.159μg/mg可溶蛋白。这是该基因在植物中表达的首次报道。研究结果提示在光生物反应器营养增殖体系基础上构建海带配子体表达系统的方案是可行的。

BMP signaling in the development of the mouse esophagus and forestomach.

The stratification and differentiation of the epidermis are known to involve the precise control of multiple signaling pathways. By contrast, little is known about the development of the mouse esophagus and forestomach, which are composed of a stratified squamous epithelium. Based on prior work in the skin, we hypothesized that bone morphogenetic protein (BMP) signaling is a central player. To test this hypothesis, we first used a BMP reporter mouse line harboring a BRE-lacZ allele, along with in situ hybridization to localize transcripts for BMP signaling components, including various antagonists. We then exploited a Shh-Cre allele that drives recombination in the embryonic foregut epithelium to generate gain- or loss-of-function models for the Bmpr1a (Alk3) receptor. In gain-of-function (Shh-Cre;Rosa26(CAG-loxpstoploxp-caBmprIa)) embryos, high levels of ectopic BMP signaling stall the transition from simple columnar to multilayered undifferentiated epithelium in the esophagus and forestomach. In loss-of-function experiments, conditional deletion of the BMP receptor in Shh-Cre;Bmpr1a(flox/flox) embryos allows the formation of a multilayered squamous epithelium but this fails to differentiate, as shown by the absence of expression of the suprabasal markers loricrin and involucrin. Together, these findings suggest multiple roles for BMP signaling in the developing esophagus and forestomach.

Recombinant Adeno-Associated Virus-Mediated Gene Therapy for Treatment of Familial Hypercholesterolemia in Rabbits

Familial hypercholesterolemia (FH) is a genetic disorder characterized by abnormally high concentrations of low-density lipoprotein-cholesterol (LDLcholesterol) in the blood that can contribute to heart disease. FH can result from a defect in the gene for the LDL receptor (LDL-R). FH patients lacking functional LDL-R may benefit from viral-mediated transfer of a functional copy of the open reading frame (ORF) of the LDL-R. Since a recombinant adeno-associated virus (rAAV) is not immunogenic and can be mass-produced, it shows promise for gene therapy applications. AAV6 and AAV8 have been shown to specifically transduce hepatocytes in several species, which normally remove the majority of LDL-cholesterol from the blood via LDL-R-mediated endocytosis. Because of the potential of rAAV to treat FH by delivery of a correct LDL-R ORF to hepatocytes, the liver specificity of these two AAV serotypes was evaluated. Additionally, rabbits were chosen as the animal model for this study because a specific strain of rabbits, Watanabe heritable hyperlipidemic (WHHL), adequately mimics the pathology of FH in humans. Exposure of rabbit liver to rAAV with the marker LacZ and subsequent inspection of liver tissue showed that AAV8 transduced rabbit liver more efficiently than AAV6. To assess the feasibility of producing a rAAV capable of transferring the LDL-R ORF to rabbit hepatocytes in vivo, rAAV8-LDL-R was mass-produced by a baculovirus system in suspension grown insect cells.

Polycyclic aromatic hydrocarbons (PAHS) (I): Measures of prevention and control

Measures of prevention and control against polycyclic aromatic hydrocarbons (PAHs) focus on an official food control, a code of best practice to reduce PAHs levels by controlling industry and in the development of a chemopreventive strategy. Regulation (EU) 835/2011 establishes maximum levels of PAHs for each food group. In addition, Regulations (EU) 333/2007 and 836/2011 set up the methods of sampling and analysis for its official control. Scientific studies prove that the chemopreventive strategy is effective against these genotoxic compounds effects. Most chemopreventive compounds studied with proven protective effects against PAHs are found in fruit and vegetables.