Use of the nuclease inhibitor aurintricarboxylic acid (ATA) for improved non-viral intratumoral in vivo gene transfer by jet-injection

BACKGROUND: Stability, integrity and retention of the DNA within the targeted tissue is decisive for efficient gene transfer using naked DNA. Pre-clinical and clinical studies require reproducible transfection rates by preventing rapid degradation of naked DNA in the transduced tissue. Tumor tissues contain nuclease activity, which can affect DNA stability if naked DNA is used. Therefore, inhibition of nuclease-mediated DNA degradation by the nuclease inhibitor aurintricarboxylic acid (ATA) might lead to improved gene transfer efficiency in tumor tissues. METHODS: For both, DNA-degradation analysis and in vivo gene transfer experiments, the beta-galactosidase (LacZ)-expressing pCMVbeta and the cytosine deaminase (CD)-expressing pCMV-CD plasmid were used. Influence of the nuclease inhibitor ATA was determined in tumors, in which naked pCMVbeta or pCMV-CD DNA and ATA was co-administered by jet-injection. The nuclease activity and inhibition by ATA was analyzed using the DNase Alert detection system. The influence of ATA on LacZ expression was determined by specific ELISA and its effect on the therapeutic efficacy of CD gene transfer on tumor growth was determined in vivo. RESULTS: The screening of different human mammary and colon carcinoma models revealed strong nuclease activity rapidly degrading naked plasmid DNA. Co-administration of ATA with pCMVbeta or pCMV-CD for in vivo jet-injection of tumors prevented DNA from nuclease degradation associated with either increased LacZ gene expression or improved reduction in tumor growth. CONCLUSIONS: Tumor-associated nuclease activity is a notable hurdle in gene transfer of naked DNA and therefore inhibition of nucleolytic degradation of plasmid DNA facilitates intratumoral gene expression.     

Needleless in vivo gene transfer into muscles by jet injection in combination with electroporation

Previously, we have established an in vivo electroporation method for gene transfer into muscle by injection of DNA with a needle followed by electric pulse delivery using needle-type electrodes and proved that this method is effective for the systemic delivery of cytokines. To perform the needleless gene delivery, we combined jet injection of DNA with electroporation using plate-type electrodes. For delivery of beta-galactosidase- and enhanced green fluorescent protein (EGFP)-expressing plasmids into muscles, there was no significant difference between the previous needle-mediated method and the newly developed jet-injection method. When pCAGGS-IL-5 was introduced into tibialis anterior, quadricipital and back sural muscles by this new method, the serum IL-5 levels reached 3.4 +/- 0.9, 5.7 +/- 1.7 and 8.4 +/- 2.7 ng/ml at day 5, respectively. Although the peak values of IL-5 achieved by the jet-injection method in these muscles were lower than that of the highest value achieved by needle-mediated gene delivery into anterior tibial muscle, this new method could deliver plasmid into relatively large muscles with better efficiency than the needle-mediated method. Thus the jet-injection method provides a useful means of gene delivery into large muscles, which is essential for future use in human gene therapy.     

Population Growth of Pratylenchus penetrans on Winter Cover Crops Grown in the Pacific Northwest

Population growth of Pratylenchus penetrans on 13 fall and winter cover crops was studied in the greenhouse and field. All crops except oat cv. Saia supported population growth of P. penetrans in greenhouse experiments, although the response of P. penetrans to oat cv. Saia varied considerably between experiments. The mean ratio of the final population density/initial population density (Pf/Pi) after 16 weeks for P. penetrans added to a greenhouse soil mix was 0.09, whereas Pf/Pi values after 10 weeks for two experiments with naturally infested soil were 0.95 and 2.3. Although P. penetrans increased on sudangrass cv. Trudan 8 and sudangrass × sorghum hybrid cv. SS 222, subsequent incorporation of sudangrass vegetation into soil reduced P. penetrans populations to preplant levels. Field experiments were inconclusive but suggested that oat cv. Saia or rye cv. Wheeler may be better choices for winter cover than weed-contaminated fallow or other crops on P. penetrans-infested sites in the Pacific Northwest.     

Mapping the Naked Neck (NA) and Polydactyly (PO) mutants of the chicken with microsatellite molecular markers

The bulked segregant analysis methodology has been used to map, with microsatellite markers, two morphological mutations in the chicken: polydactyly (PO) and naked neck (NA). These autosomal mutations show partial dominance for NA, and dominance with incomplete penetrance for PO. They were mapped previously to different linkage groups of the classical map, PO to the linkage group IV and NA being linked to the erythrocyte antigen CPPP. An informative family of 70 offspring was produced by mating a sire, heterozygous for each of the mutations, to 7 dams homozygous recessive for each locus. Three DNA pools were prepared, pool PO included 20 chicks exhibiting at least one extra-toe, pool NA included 20 non-polydactyly chicks showing the typical phenotype associated with heterozygosity for the naked neck mutation, and pool NP included 20 chicks exhibiting neither of the mutant phenotypes. Typings were done on an ABI-373 automatic sequencer with 147 microsatellite markers covering most of the genome. An unbalanced distribution of sire marker alleles were detected between pool PO, and pools NA and NP, for two markers of chromosome 2p, MCW0082 and MCW0247. A linkage analysis taking into account the incomplete penetrance of polydactyly (80%) was performed with additional markers of this region and showed that the closest marker to the PO locus was MCW0071 (5 cM, lod score = 9). MCW0071 lies within the engrailed gene EN2 in the chicken. In the mouse, the homologous gene maps on chromosome 5, close to the hemimelic extra-toes mutation Hx. In the case of the NA locus, markers of chromosome 3 were selected because CPPP was mapped on this chromosome. Analysis of individual typings showed a linkage of 5.7 cM (lod score = 13) between the NA locus and ADL0237 in the distal region of chromosome 3q. These results contribute to connecting the former classical map to the molecular genetic map of the chicken, and open the way to the identification of the molecular nature of two developmental mutations of the chicken that are known to occur in many breeds of chickens.     

Oat plant effects on net nitrogen mineralization

Living plants have been reported to stimulate, inhibit, or have no effect on net nitrogen mineralization in soil. A series of experiments were conducted to evaluate the influence of living oat plants Avena sativa on net N mineralization. Oat plants were grown in plastic cylinders containing soil, and net N mineralization was assessed by determining the N balance in these microcosms. Measured N inputs included N contained in the oat seeds and N₂ fixation. N losses by NH₃ volatilization and denitrification were also measured. We observed that in some soils net N mineralization was stimulated by as much as 81%, but in other soils there was no effect of living oat plants on net N mineralization. N mineralization responses are related to past cropping histories of the soils.     

A measurement system to detect small plant movements

A measurement system based on a photodiode array technique is presented, together with some results from recordings of phototropic responses of Avena sativa L. (cv. Seger I) coleoptiles.
A commercial photodiode array (RL-256G) was used, controlled by a likewise commercial control board RC-301. Electronic circuits were built to connect the control board to the interface card IO-801 of an Apple IIe microcomputer. The circuits constitute a fast temporary memory, which mainly contains two shift registers and two timers. Programs, which control the measurements, have been written and are presented.
Detailed recordings of blue light induced phototropic movements are presented: resolution and other features of the equipment are discussed.     

Uptake and Distribution of Stable Strontium in 26 Cultivars of Three Crop Species: Oats,Wheat, and Barley for Their Potential Use in Phytoremediation

The main objective of this study was to investigate the accumulation and distribution of strontium (Sr) in 26 cultivars of wheat (Triticum aestivum L.), husk oat (Avena sativa L) and naked oat (Avena nuda), and barley (Hordeum vulgare L.) for their potential use in phytoremediation.Sr levels had no effect on the accumulation of shoot biomass at tillering or at maturity. Mean shoot Sr concentration of naked oat and barley at tillering was significantly (P < 0.05) higher than that of wheat; Neimengkeyimai-1, a naked oat cultivar, had the highest Sr concentrations. At maturity, of four naked oat cultivars, Neimengkeyimai-1 had the highest Sr content at all measured Sr levels. Leaves had the highest Sr concentrations, followed by roots and straw, and then grain with the lowest. Mean enrichment coefficients from soil to shoots ranged from 0.521 to 1.343; the percentage of stable Sr removed from the soil to the shoots at harvest time was more than 1.4% after 120 days. Neimengkeyimai-1 could be used as a model for further research to find more effective cultivars; and naked oat plants could be selected for phytoremediation to clean up contaminated soil.     

炭疽病对山西晋北饲用燕麦地上部碳氮磷化学计量学特征的影响

C、N、P化学计量学特征有助于了解植物生长过程中的碳同化能力和养分元素的利用效率,并诊断限制性元素,但病害对植物化学计量特征影响的研究尚缺乏报道。本研究以山西晋北罹患炭疽病的饲用燕麦为对象,测定了不同发病程度植株地上部C、N、P含量及化学计量值,并初步探讨了其与病害发生持续时间、品种和种植模式之间的关系。结果表明: 随着发病程度增加,C、N、P含量呈下降趋势,其中重度发病植株的C、N、P含量分别较健康植株显著降低了5.4%、20.0%、24.2%;C∶N和C∶P呈升高趋势,其中重度发病植株的C∶N、C∶P较健康植株显著增加了18.5%和22.2%。不同采样点的发病程度对C含量均未造成影响,表明3种饲用燕麦品种具有一定的耐病保产能力;但N、P的化学计量值存在地点间变异,采样点病害持续时间越长,N、P的损失越严重。与单播燕麦相比,炭疽病降低了混播燕麦对P的利用优势。总之,炭疽病对晋北饲用燕麦碳同化能力造成一定程度的负面影响,加重了植株地上部受N、P养分限制的程度,但显著提高了N、P的利用效率。生产实践中需采取必要的防治措施,减轻病害对饲用燕麦经济收益和生态功能的不利影响。