常氧下高原鼠兔HIF-1α mRNA的表达

低氧诱导因子-1(HIF-1)是一种由低氧诱导因子-1α(HIF-1α)和芳香烃核受体转录因子(ARNT)组成的二聚体转录因子，在生物体氧平衡调节中起关键作用。高原鼠兔(Ochotona curzoniae)是生活在青藏高原海拔3000 m以上区域，有着极强的低氧、低温耐受能力的特有自然种群。它通过高的基础代谢率和高的氧利用率来适应高原低温、缺氧环境。将高原鼠兔带到北京，常氧室温条件下适应7 d。 RT-PCR扩增高原鼠兔HIF-1αcDNA片断，~（32）p标记作为特异性探针，Northern杂交检测了常氧条件下高原鼠兔HIF-1α的组织特异性表达。结果显示，HIF-1α mRNA在高原鼠兔多种组织中均有表达，并且表现出明显的组织差异性。其中脑的表达量最大，肾其次，心、脾和肝的表达相对较少。

高原鼠兔低氧诱导因子-1α的初步研究

高寒和缺氧是高原地区的两个重要的限制生态因子，高原土著动物在长期的适应进化过程中必将形成自己独特的低温、低氧适应策略。高原鼠兔（Ochotona curzoniae）是生活在青藏高原海拔以上地区的特有物种，具有极强的低温、低氧耐受能力。它主要通过高的基础代谢率和增加非颤抖性产热以及高的氧利用率来适应高寒、缺氧环境[1-4] 。低氧诱导因子-l（Hypoxia-Induced FacHIF一l）是 1992年Semanza等在低氧的肝癌细胞株Hep3B中发现的一种特异性结合于红细胞生成素（EPO）增强子寡核昔酸序列的转录因子[5]，它HIF-la和HIF-lβ/ARNT两个亚单位组成［6〕。HIF-l。受低氧诱导，属功能性亚基，HIF-1侧ARNT在细胞中构建型表达，不受低氧诱导[7]。为进一步揭示原鼠兔低氧适应的分子机理，我们正在克隆高原鼠兔HIF-1。基因的CDNA全长，并试图对其在低氧适应中的作用进行研究，本文报道了高原鼠兔低氧诱导因子-la编码区部分片段的克隆。

Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using X-RACE technology and named AtKP1 (for Arabidopsis kinesin protein 1). This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coli and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.

Resumos...

Análise proteômica de raízes de feijão (Phaseolus vulgaris) inoculadas com Rhizobium tropici. Teste para comprovar a eficiência do método de extração e determinação de ácido fítico em sementes de soja utilizado na Embrapa Soja. Auditoria de comunicação: avaliando os veículos de comunicação interna da Embrapa Soja. Utilização da técnica de espectrofotometria do infravermelho próximo (NIR) para análise discriminante dos ácidos graxos oléico e linoléico de genótipos de girassol. Análise da disponibilidade hídrica para a cultura da soja nas safras 2004/05 e 2009/10 em Londrina, PR . Calibração de psicrômetros para avaliações de potencial hídrico foliar. Avaliação de ácidos graxos da soja: grão inteiro, casca, cotilédones e hipocótilo. Variabilidade temporal da produtividade da soja após conversão do preparo convencional para o sistema plantio direto. COMUT na biblioteca da Embrapa Soja. EM DIA: Boletim interno compartilhando informações. Sistema Web para estimativas de perdas por seca na cultura da soja. Portais Web desenvolvidos no laboratório de Bioinformática da Embrapa Soja. Validação de um método para detecção e quantificação de soja cultivance® tolerante a herbicidas imidazolinonas por PCR . Índice de vegetação por diferença normalizada (NDVI) de cultivares de soja sob três níveis de disponibilidade hídrica no solo. Avaliação do fluxo de seiva em cultivares de soja em três níveis de disponibilidade hídrica no solo. A importância do controle de qualidade dos inoculantes. Comportamento exploratório de ninfas recém eclodidas de Edessa meditabunda (F.) (Heteroptera: Pentatomidae) sobre a superfície dos córions. Produção de brotos de soja da cultivar BRS 216. Teste de aceitabilidade de brotos de soja da cultivar BRS 216. Análise de lignina com diferentes massas de tegumento de soja utilizando método gravimétrico. Desenvolvimento e caracterização físico-química de biscoitos com farinha de soja orgânica de cultivares especiais para a alimentação humana. Análise sensorial de biscoitos com farinha de soja orgânica de cultivares especiais para a alimentação humana. Avaliação de genótipos de soja de diferentes grupos de maturação e resistência aos percevejos. Métodos químicos para extração de boro no solo. Abordagem computacional para a identificação de elementos cis-regulatórios no genoma da soja. Quebra de dormência em sementes de girassol silvestre utilizando ácido giberélico. Teor relativo de água em cultivares de soja sob três níveis de disponibilidade hídrica no solo. Soybean gene express: plataforma para análise de expressão diferencial em bibliotecas subtrativas de CDNA. Biologia e exigências térmicas do ácaro vermelho Tetranychus gigas. Características biológicas de Telenomus remus em diferentes hospedeiros após serem criados em de ovos de Anticarsia gemmatalis e Spodoptera frugiperda por uma geração. Enfoque sobre o plano de saúde dos empregados da Embrapa no contexto do pacote de benefícios sociais oferecidos pela empresa. Dinâmica populacional do ácaro verde Mononychellus planki em cultivares de soja. Envelhecimento, trabalho e tempo livre: elaborando projetos de vida. Teor de isoflavonas em vinte cultivares de soja semeadas em Londrina e Ponta Grossa. Utilização de Pseudomonas fluorescens no controle biológico de Macrophomina phaseolina. Algoritmo computacional para determinar o perfil mínimo de marcadores moleculares que discriminam um conjunto de cultivares. Qualidade física do solo em um sistema de integração lavoura-pecuária com diferentes pressões de pastejo. Diversidade de estirpes do gênero Burkholderia em solos do cerrado brasileiro baseado no sequenciamento do gene ribossomal 16S. Estudo da diversidade genética de isolados de Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei.

Cloning and structure analysis of hydrogenase gene from Chlamydomonas reinhardtii SE

The cDNA of Chlamydomonas reinhardtii SE encoding hydrogenase (HydA2) was obtained from the total RNA of C reinhardtii SE by RT-PCR. The DNA of hydrogenase was amplified by PCR from the genomic DNA of C reinhardtii SE. The cDNA and DNA of hydrogenase were sequenced, respectively. The structure of hydrogenase gene was analyzed by biology software. The open reading frame predicts that the hydrogenase is composed of 3584 bp encoding 505 amino acids in length with a predicted M.W. of 53.69 kDa. Ten exons (including 1518 bp) and nine introns (including 2066 bp) have been found in the hydrogenase, and there were two potential N-glycosylate sites, eight protein kinase C phosphorylation site, eight casein kinase H phosphorylation site and one sulphorylation in the sequence. The theory pI was 6.15. Total number of negatively charged residues (Asp + Glu) and positively charged residues (Arg + Lys) were 55 and 61, respectively. (c) 2005 Elsevier Ltd. All rights reserved.

Taking advantage of an old concept, "illegitimate transcription", for a proposed novel method of genetic diagnosis of McArdle disease

McArdle disease is a metabolic disorder caused by pathogenic mutations in the PYGM gene. Timely diagnosis can sometimes be difficult with direct genomic analysis, which requires additional studies of cDNA from muscle transcripts. Although the "nonsense-mediated mRNA decay" (NMD) eliminates tissue-specific aberrant transcripts, there is some residual transcription of tissue-specific genes in virtually all cells, such as peripheral blood mononuclear cells (PBMCs).We studied a subset of the main types of PYGM mutations (deletions, missense, nonsense, silent, or splicing mutations) in cDNA from easily accessible cells (PBMCs) in 12 McArdle patients.Analysis of cDNA from PBMCs allowed detection of all mutations. Importantly, the effects of mutations with unknown pathogenicity (silent and splicing mutations) were characterized in PBMCs. Because the NMD mechanism does not seem to operate in nonspecific cells, PBMCs were more suitable than muscle biopsies for detecting the pathogenicity of some PYGM mutations, notably the silent mutation c.645G>A (p.K215=), whose effect in the splicing of intron 6 was unnoticed in previous muscle transcriptomic studies.We propose considering the use of PBMCs for detecting mutations that are thought to cause McArdle disease, particularly for studying their actual pathogenicity.

Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L.

J. A. Gallagher, A. J. Cairns and C. J. Pollock (2004). Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L. Journal of Experimental Botany, 55 (397) pp.557-569 Sponsorship: BBSRC RAE2008

Construction of a Festuca pratensis BAC library for map-based cloning in IFestulolium substitution lines

Iain S. Donnison, Donal M. O Sullivan, Ann Thomas, Peter Canter, Beverley Moore, Ian Armstead, Howard Thomas, Keith J. Edwards and Ian P. King (2005). Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines. Theoretical and Applied Genetics, 110 (5) pp.846-851 Sponsorship: BBSRC;BBSRC RAE2008

The identification and characterisation of Rab4 interacting proteins

Rab4 is a member of the Rab superfamily of small GTPases. It is localized to the early sorting endosome and plays a role in regulating the transport from this compartment to the recycling and degradative pathways. In order to further our understanding of the role Rab4 plays in endocytosis, a yeast two-hybrid screen was performed to identify putative Rab4 effectors. A constitutively active mutant of Rab4, Rab4Q67L, when used as bait to screen a HeLa cDNA library, identified a novel 80kDa protein that interacted with Rab4-GTP. This protein was called Rab Coupling Protein (RCP). RCP interacts preferentially with the GTP-bound form of Rab4. Subsequent work demonstrated that RCP also interacts with Rab11, and that this interaction is not nucleotide-depenedent. RCP is predominantly membrane-bound and localised to the perinuclear recycling compartment. Expression of a truncation mutant of RCP, that contains the Rab binding domain, in HeLa cells, results in the formation of an extensive tubular network that can be labelled with transferrin. These tubules are derived from the recycling compartment since they are inaccessible to transferrin when the ligand is internalised at 18oC. The truncation mutant-induced morphology can be rescued by overexpression of active Rab11, but not active Rab4. This suggests that RCP functions between Rab4 and Rab11 in the receptor recycling pathway, and may act as a ‘molecular bridge’ between these two sequentially acting small GTPases. Quantitative assays demonstrated that overexpression of the truncation mutant results in a dramatic inhibition in the rate of receptor recycling. Database analysis revealed that RCP belongs to a family of Rab interacting proteins, each characterised by a carboxy-terminal coiled-coil domain and an amino-terminal phospholipid-binding domain. KIAA0941, an RCP homologue, interacts with Rab11, but not with Rab4. Overexpression of its Rab binding domain also results in a tubular network, however, this tubulation cannot be rescued by active Rab11.

Zinc finger nuclease gene repair as a treatment for cystinosis

Cystinosis is a multi-system autosomal recessive disorder caused by mutations and/or deletions in both alleles of CTNS, a gene encoding for the low pH dependent lysosomal cystine exporter cystinosin. Cystinosis occurs in approximately 1:200,000 newborns worldwide and is characterised by an accumulation of cystine in the lysosomes. The most severe form of the disorder is nephropathic cystinosis presenting Fanconi syndrome and leads without treatment to an end-stage renal failure before the age of ten. The only treatment available so far is cysteamine therapy, which delays disease progression by five years, but does not provide a cure for cystinosis patients. Current gene and cell based therapeutic approaches have not yet provided a suitable alternative. A potentially approach for a long-term treatment could be to generate autologous gene–modified stem cells by repairing the gene. Zinc Finger Nucleases (ZFNs) serve as a tool to increase HDR up to a 200,000-fold by introducing a double-stranded break (DSB). Thus, simple mutations in the CTNS gene could be corrected by introduction of a double-stranded break using ZFNs to boost the process of HDR with a suitable donor DNA sequence. A permanent repair of the most common lesion CTNS, a 57 kb deletion, could be achieved by ZFN-mediated HDR using a minigene CTNS promoter/cDNA construct. The thesis describes the design and testing of seven zinc finger nuclease pairs for their cleavage activity in vitro and in cellulo.. A highly sensitive assay to detect even low levels of ZFN-mediated HDR was also developed. Finally, to further investigate the role of autophagy in tissue injury in cystinotic cells an assay to monitor autophagy levels in the cells was successfully developed. This assay provides the opportunity to demonstrate functional restoration of CTNS after successful ZFN-HDR in cystinotic cells.

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.

Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools

Cystic Fibrosis (CF) is an autosomal recessive monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene with the ΔF508 mutation accounting for approximately 70% of all CF cases worldwide. This thesis investigates whether existing zinc finger nucleases designed in this lab and CRISPR/gRNAs designed in this thesis can mediate efficient homology-directed repair (HDR) with appropriate donor repair plasmids to correct CF-causing mutations in a CF cell line. Firstly, the most common mutation, ΔF508, was corrected using a pair of existing ZFNs, which cleave in intron 9, and the donor repair plasmid pITR-donor-XC, which contains the correct CTT sequence and two unique restriction sites. HDR was initially determined to be <1% but further analysis by next generation sequencing (NGS) revealed HDR occurred at a level of 2%. This relatively low level of repair was determined to be a consequence of distance from the cut site to the mutation and so rather than designing a new pair of ZFNs, the position of the existing intron 9 ZFNs was exploited and attempts made to correct >80% of CF-causing mutations. The ZFN cut site was used as the site for HDR of a mini-gene construct comprising exons 10-24 from CFTR cDNA (with appropriate splice acceptor and poly A sites) to allow production of full length corrected CFTR mRNA. Finally, the ability to cleave closer to the mutation and mediate repair of CFTR using the latest gene editing tool CRISPR/Cas9 was explored. Two CRISPR gRNAs were tested; CRISPR ex10 was shown to cleave at an efficiency of 15% and CRISPR in9 cleaved at 3%. Both CRISPR gRNAs mediated HDR with appropriate donor plasmids at a rate of ~1% as determined by NGS. This is the first evidence of CRISPR induced HDR in CF cell lines.

The relevance of bacterial isoprenoid biosynthetic pathways for host-microbe interactions

This thesis was undertaken to investigate the relevance of two bacterial isoprenoid biosynthetic pathways (Mevalonate (MVAL) and 2-C-methyl-D-erythritol 4-phosphate (MEP)) for host-microbe interactions. We determined a significant reduction in microbial diversity in the murine gut microbiota (by next generation sequencing) following oral administration of a common anti-cholesterol drug Rosuvastatin (RSV) that targets mammalian and bacterial HMG-CoA reductase (HMG-R) for inhibition of MVAL formation. In tandem we identified significant hepatic and intestinal off-target alterations to the murine metabolome indicating alterations in inflammation, bile acid profiles and antimicrobial peptide synthesis with implications on community structure of the gastrointestinal microbiota in statin-treated animals. However we found no effect on local Short Chain Fatty Acid biosynthesis (metabolic health marker in our model). We demonstrated direct inhibition of bacterial growth in-vitro by RSV which correlated with reductions in bacterial MVAL formation. However this was only at high doses of RSV. Our observations demonstrate a significant RSV-associated impact on the gut microbiota prompting similar human analysis. Successful deletion of another MVAL pathway enzyme (HMG-CoA synthase (mvaS)) involved in Listeria monocytogenes EGDe isoprenoid biosynthesis determined that the enzyme is non-essential for normal growth and in-vivo pathogenesis of this pathogen. We highlight potential evidence for alternative means of synthesis of the HMG-CoA substrate that could render mvaS activity redundant under our test conditions. Finally, we showed by global gene expression analysis (Massive Analysis of cDNA Ends (MACE RNA-seq) a significant role for the penultimate MEP pathway metabolite (E)-4-hydroxy-3-methyl-2-but-2-enyl pyrophosphate (HMBPP) in significant up regulation of genes of immunity and antigen presentation in THP-1 cells at nanomolar levels. We infected THP-1 cells with wild type or HMBPP under/over-producing L. monoctyogenes EGDe mutants and determined subtle effects of HMBPP upon overall host responses to Listeria infection. Overall our findings provide greater insights regarding bacterial isoprenoid biosynthetic pathways for host-microbe/microbe-host dialogue.

Gait and behavior in an IL1β-mediated model of rat knee arthritis and effects of an IL1 antagonist.

Interleukin-1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over-expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse-associated pathology. IL1β over-expression was induced in the right knees of 30 Wistar rats via intra-articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra-articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β-infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra-articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over-expression is associated limb hypersensitivity and gait compensation. Intra-articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.

Intracoronary adenovirus-mediated delivery and overexpression of the beta(2)-adrenergic receptor in the heart : prospects for molecular ventricular assistance.

BACKGROUND: Genetic modulation of ventricular function may offer a novel therapeutic strategy for patients with congestive heart failure. Myocardial overexpression of beta(2)-adrenergic receptors (beta(2)ARs) has been shown to enhance contractility in transgenic mice and reverse signaling abnormalities found in failing cardiomyocytes in culture. In this study, we sought to determine the feasibility and in vivo consequences of delivering an adenovirus containing the human beta(2)AR cDNA to ventricular myocardium via catheter-mediated subselective intracoronary delivery. METHODS AND RESULTS: Rabbits underwent percutaneous subselective catheterization of either the left or right coronary artery and infusion of adenoviral vectors containing either a marker transgene (Adeno-betaGal) or the beta(2)AR (Adeno-beta(2)AR). Ventricular function was assessed before catheterization and 3 to 6 days after gene delivery. Both left circumflex- and right coronary artery-mediated delivery of Adeno-beta(2)AR resulted in approximately 10-fold overexpression in a chamber-specific manner. Delivery of Adeno-betaGal did not alter in vivo left ventricular (LV) systolic function, whereas overexpression of beta(2)ARs in the LV improved global LV contractility, as measured by dP/dt(max), at baseline and in response to isoproterenol at both 3 and 6 days after gene delivery. CONCLUSIONS: Percutaneous adenovirus-mediated intracoronary delivery of a potentially therapeutic transgene is feasible, and acute global LV function can be enhanced by LV-specific overexpression of the beta(2)AR. Thus, genetic modulation to enhance the function of the heart may represent a novel therapeutic strategy for congestive heart failure and can be viewed as molecular ventricular assistance.