Volatiles from Pseudomonas palleroniana Strain B-BH16-1 Suppress Aflatoxin Production and Growth of Aspergillus flavus on Coix lacryma-jobi during Storage.

Semen coicis is not only a traditional Chinese medicine (TCM), but also a typical food in China, with significant medical and healthcare value. Because semen coicis is rich in starch and oil, it can be easily contaminated with Aspergillus flavus and its aflatoxins (AFs). Preventing and controlling the contamination of semen coicis with Aspergillus flavus and its aflatoxins is vital to ensuring its safety as a drug and as a food. In this study, the endosphere bacteria Pseudomonas palleroniana strain B-BH16-1 produced volatiles that strongly inhibited the mycelial growth and spore formation activity of A. flavus. Gas chromatography-mass spectrometry profiling revealed three volatiles emitted from B-BH16-1, of which 1-undecene was the most abundant. We obtained authentic reference standards for these three volatiles; these significantly reduced mycelial growth and sporulation in Aspergillus, with dimethyl disulfide showing the most robust inhibitory activity. Strain B-BH16-1 was able to completely inhibit the biosynthesis of aflatoxins in semen coicis samples during storage by emitting volatile bioactive components. The microscope revealed severely damaged mycelia and a complete lack of sporulation. This newly identified plant endophyte bacterium was able to strongly inhibit the sporulation and growth of Aspergillus and the synthesis of associated mycotoxins, thus not only providing valuable information regarding an efficient potential strategy for the prevention of A. flavus contamination in TCM and food, but potentially also serving as a reference in the control of toxic fungi.

Genome Resource for Acinetobacter schindleri H4-3-C1: An Endophyte of Pseudostellaria heterophylla with Degradation Activity to Toxins Produced by Fungal Pathogens.

Acinetobacter schindleri is an endophyte of Pseudostellaria heterophylla, a traditional Chinese herbal plant. It has high degradation activity to toxins produced by fungal pathogen Fusarium graminearum. Here, we deployed PacBio single-molecule real-time long-read sequencing technology to generate a complete genome assembly for the Acinetobacter schindleri H4-3-C1 strain and obtained 1.59 Gb of clean reads. These reads were assembled to a single circular DNA chromosome with a length of 3,265,024 bp, and no plasmid was found in the genome. Totals of 3,193 coding sequences, 91 transfer RNA, 21 ribosomal RNA, and 75 small RNAs were identified in the genome. This high-quality genome assembly and gene annotation resource will facilitate the excavation of the zearalenone degradation gene and provide valuable resources for preventing and controlling toxigenic fungal diseases of P. heterophylla. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First report of Arcopilus aureus causing leaf black spot disease of Pseudostellaria heterophylla in China.

Pseudostellaria heterophylla (family Caryophyllaceae) is a perennial herbaceous plant. Its tuberous roots are highly valued in traditional Chinese medicine. It is mainly cultivated in a geo-authentic production zone located in the Guizhou, Anhui, Shandong, and Fujian provinces of China (Zhao et al. 2016). The herb is widely used for treating lung diseases and as a spleen tonic (Pang et al. 2011). A severe leaf black spot disease was observed on P. heterophylla in China, from 2018 to 2020. Plants displayed water-soaking symptoms in the early stage of infection, then the watery areas turned brown-red and a black mold appeared on the lesions. At a later stage, the leaf spots showed concentric rings surrounded by a yellow halo, and the initial infection site became dry and necrotic (Supplementary Figure S1). Nine infected plants were collected from three cultivation fields in Shibing County (N 27°4'21", E 108°8'0"), Guizhou province, on April 13th, 2019. The fungus was consistently isolated from symptomatic leaves on potato dextrose agar (PDA) medium according to the method described by Larran et al (2002). A total of 22 isolates were obtained, including 7 isolates of Arcopilus and 15 isolates of Trichoderma. The growth rates of isolate MJ2-2b on PDA and oatmeal agar (OA) medium were 3 to 5 mm/day at 25 °C (Supplementary Figure S2A and S2B). Mycelium of isolate MJ2-2b was dense, yellowish-brown on PDA, while it was sparse, bright-red on OA. Also, the mycelium secreted brownish-red pigment on both PDA and OA. Ascomata when mature were water drop and limoniform. Lateral hairs were brown, erect or flexuous, tapering towards the tips. Ascospores when mature were greyish-white to grey, limoniform, or fusiform to pyriform (Supplementary Figure S2C and S2D). Further, the beta-tubulin gene (Tub2) of the fungus was amplified by using primer pairs T1 and TUB4Rd as described by Wang et al (2016) and subjected to sequencing. NCBI nucleotide BLAST results showed that sequences from seven isolates had a 99.86% identity with A. aureus (strain ChL-C, GenBank accession No. MG889987.1) (Supplementary Figure S2F). Molecular phylogenetic analysis by maximum likelihood method using MEGA 7 confirmed that the fungal isolate clustered with A. aureus. Hence, the causal agent was identified as A. aureus based on morphological and molecular characteristics. The sequence was deposited in GenBank (accession No. MW531453). Pathogenicity tests were conducted on 15-day old tissue-cultured seedlings according to Ghanbary et al (2018) (Supplementary Figure S3). Leaves of 16 seedlings were inoculated with 1×1 mm 5-day-old PDA-grown mycelial plugsof the fungal isolate. The experiment was repeated 3 times. After 10 days, the inoculated leaves showed the same symptoms observed on plants in the field. The associated fungal pathogen was consistently re-isolated from the inoculated seedlings and identified by Tub2 gene sequencing. At present, there are no reports of A. aureus causing disease of plants. To the best of our knowledge, this is the first report of leaf black spot disease on P. heterophylla caused by A. aureus in China.

[Effect of Gastrodiae elata-Phallus impudicus sequential planting pattern on soil microbial community structure].

Gastrodia elata is a heterotrophic plant that needed to be symbiotic with Armillaria. The obstacle of continuous cropping in G. elata is serious during the G. elata cultivation, and the mechanism of obstacle in G. elata continuous cropping had not been solved. The planting of G. elata-Phallus impudicus is a new sequential planting pattern adopted in Guizhou province, but the effect of the cultivation on soil microbial community structure is still unclear. In this study, we collected four soil samples for the research including the soil without planted G. elata as control(CK), rhizosphere soil samples tightly adhering to the G. elata surface(GE), rhizosphere soil samples tightly adhering to Armillaria which was symbiotic with G. elata(AGE), the rhizosphere soil of P. impudicus planting after G. elata cultivation(PI). In order to explore the mechanism, the research study on the soil of G. elata-P. impudicus by using ITS and 16 S rDNA high-throughput sequencing technologies to detect soil microbial community structure including fungi and bacteria in the soil of CK, AGE, GE and PI. OTU clustering and PCA analysis of soil samples showed that the soil microbial diversity was relatively similar in AGE and GE. And the soil microbial in PI and CK clustered together. The results showed that AGE and GE had similar soil microbial diversity, as well as PI and CK. Compared with CK, the soil microbial diversity and abundance in AGE and GE were significantly increased. But the microbial diversity and abundance decreased in PI compared with AGE and GE. The annotation indicated that the abundance of Basidiomycota, Acidobacteria and Chloroflexi decreased, and that of Ascomycota, Zygomycota and Proteobacteria increased in AGE and GE compared with CK. In contrast to AGE and GE, PI was the opposite. The abundance of Basidiomycota, Acidobacteria and Chloroflexi increased in PI compared with AGE and GE. The abundance of microorganisms in the soil of PI and CK was similar. In addition, the co-culture of Armillaria and P. impudicus indicated that P. impudicus had obvious antagonistic effects on the growth of Armillaria. Therefore, it is speculated that the mechanism of G. elata-P. impudicus planting pattern related to the change of soil microbial. And we supposed that P. impudicus might inhibit the growth of Armillaria and change the soil microbial community structure and the abundance of soil microbial. And the soil microbial community structure was restored to a state close to that of uncultivated G. elata. Thus, the structure of soil microbial community planting G. elata could be restored by P. impudicus planting.

[Dissection of seed development of Gastrodia elata at different temperatures].

The study is aimed to create seed materials and dissect the molecular mechanism of sexual propagation of Gastrodia elata. In this research, thirteen characteristics of flowers, flower stem, fruits, seeds and embryo of G.elata f. glauca and G.elata f. elata after bolting at room temperature(RT) and constant temperature(CT, 22 ℃) were determined. It was found that the constant temperature condition could prolong the bolting duration of G.elata and increased the number of flowers, while the variety of G.elata only affected the bolting duration, but had no effect on the number of flowers, and the G.elata f. elata was more likely to bolting than the G.elata f. glauca. The variety of G.elata was the main factor affecting the time of dehiscent fruit of G.elata, the temperature was the main factor affecting the fruits number and fruits diameter, and the constant temperature was more conducive to the fruits shape of G.elata than the room temperature. There was no significant difference in seed phenotype of G.elata varieties, but the seed embryo of G.elata seeds cultivated at constant temperature was fuller than that of G.elata cultivated at room temperature, and temperature had less influence on the seed phenotype of G.elata. But it was interesting to find that temperature and varieties had greater influence on the seed embryo of G.elata, constant temperature cultivation was more conducive to the formation of seed embryo of G.elata, and more the seed embryo of G.elata f. elata was easier to form than the seed embryo of G.elata f. glauca. However, the development of seeds and embryos of G.elata was significantly affected, and the development of seeds and embryos of G.elata f. glauca was more sensitive to temperature than G.elata f. elata. The research suggested that it is advisable for G.elata to produce seed materials by bolting at constant temperature(22 ℃).

Lowering Nitrogen and Increasing Potassium Application Level Can Improve the Yield and Quality of Panax notoginseng.

Excessive nitrogen (N) application and potassium (K) supplement deficiency is a common problem in Panax notoginseng cultivation. However, synergistic effects of lowering N and increasing K application on yield and quality of P. notoginseng have not been reported. Field experiments in two locations with different N and K combined application were conducted to study the effects on yield and quality. Then, the saponin accumulation mechanisms were explored by pot and hydroponic culture with 2- or 3-year-old seedlings. The investigation showed that 70% of P. notoginseng cultivation fields reached abundant levels of total nitrogen (TN) but had deficient levels of total potassium (TK), which may be detrimental to balance the N/K uptake of P. notoginseng. Moreover, the average biomass was 18.9 g, and P. notoginseng saponin (PNS) content was 6.95%; both were influenced by the N/K values of P. notoginseng. The field experiments indicated that compared to the conventional N and K application (N:K = 2:1), lowering N and increasing K application (N:K = 1:2) decreased root rot rate by 36.4-46.1% and increased survival rate, root biomass, and yield, as well as PNS content by 17.9-18.3, 5.7-32.9, 27.8-57.1, and 5-10%, respectively. The mechanism of lowering N and increasing K application on the PNS content improving was due to the decreasing of N/K value, which promoted photosynthesis, sugar accumulation, and the expression of saponin biosynthesis genes. Therefore, lowering N and increasing K application to the ratio of 1:2 would have great potential to improve the synergistic effect on yield and quality of P. notoginseng cultivation.

[Quality assessment of soil fertility of planting Chrysanthemum morifolium areas of Macheng city based on minimum data set].

A minimal data set( MDS) for soil fertility evaluation of Chrysanthemum plantation areas of Macheng city was established by principal component analysis( PCA) combined with Norm values of soil fertility indices and correlation coefficients among indices. A radar map was used to visually reflect the fertility level of individual indicators. Then,the comprehensive index model was used to calculate the soil fertility quality index( SFQI),and the values of SFQI was used to cluster,and the results showed that MDS was composed of five indicators: organic matter( OM),total phosphate( TP),available phosphorus( Av P),available magnesium( Av Mg) and available ferrum( Av Fe). Radar maps showed that the fertility of available phosphorus( Av P) and available copper( Av Cu) was mostly different in the two town,and the fertility of available ferrum( Av Fe) is smallest different. Except for the effective manganese( Av Mn) fertility level of Huangtugang town was higher than that of Futianhe town,the rest were lower than that of Futianhe town. Through analysis,the sensitivity of SFQI value calculated by taking the contribution rate of MDS index in the principal component of the whole data set( TDS) as the weight was the highest,MDS could better replace TDS. The value of SFQI-MDS ranged from 0. 353 to 0. 833,with an average value of 0. 604 and a coefficient of variation of 22%. The results of SFQI-MDS clustering showed that soil fertility could be divided into four categories: grade Ⅰ( 0. 727-0. 833) was superior,accounting for 25. 0%,grade Ⅱ( 0. 615-0. 681)was good,accounting for 29. 2%,mainly distributed in Futianhe Town,grade Ⅲ( 0. 494-0. 589) was medium,accounting for29. 1%,and grade Ⅳ( 0. 353-0. 419) was poor,accounting for 16. 7%,mainly distributed in Huangtugang town. Soil fertility of Futianhe town was better than that of Huangtugang town. It is suggested that boron fertilizer and potassium fertilizer should be supplemented to Chrysanthemum morifolium in production practice,and the amount of phosphate fertilizer,magnesium fertilizer and nitrogen fertilizer should be increased appropriately. At the same time,the amount of organic fertilizer should be increased to enhance soil fertility and improve soil physical and chemical properties.

Soil-Plant Metal Relations in Panax notoginseng: An Ecosystem Health Risk Assessment.

This study features a survey of the content of heavy metals (Pb, Cd, Cr, As, Hg and Cu) in root and cultivation soils of Panax notoginseng (P. notoginseng), carried out in China's Yunnan Province. The average contents of Pb, Cd, Cr, As, Hg, and Cu in the soil were 61.6, 0.4, 102.4, 57.1, 0.3, and 35.1 mg·kg-1, respectively. The heavy metals' pollution indexes can be ranked as follows: As > Cd > Hg > Cu > Cr > Pb. The proportion of soil samples at slight, middle, strong, very strong, and extremely strong levels of potential environmental risk had values of 5.41%, 21.62%, 35.14%, 10.81%, and 27.03%, respectively. The potential environment risk index (RI) showed that 29.73% out of the total sample sites were above the level of strong and extremely strong. The ranges of Pb, Cd, Cr, As, Hg, and Cu content in tuber were 0.04-3.26, 0.04-0.33, 0.22-5.4, 0.10-1.8, 0.00-0.02, and 5.0-20.9 mg·kg-1, respectively. In combination with P. notoginseng consumption data, the estimated heavy metal daily intakes (EDIs) were 0.08-0.23, 0.006-0.019, 0.17-0.52, 0.04-0.12, 0.001-0.002, and 0.59-1.77 µg·kg-1·bw/day. All target hazard quotients (THQs) of individual elements and hazard indexes (HI) were less than one. The present study indicates that most of the P. notoginseng cultivation soil in the province of Yunnan presented slight and moderate ecological risk. Thus, more attention should be given to the heavy metals As, Cd, and Hg when selecting planting areas for the cultivation of P. notoginseng. Health risks associated with the intake of a single element or consumption of the combined metals through P. notoginseng are absent.

[Comparison of agronomic traits of Panax notoginseng between traditional cultivated fields and new cultivated fields].

OBJECTIVE: In order to provide scientific basis for introduction breeding and production regionalization of Panax notoginseng, the environment of producing area, agronomic traits and medicinal material output were investigated. METHOD: Using field survey sampling at harvest time, agronomic traits indicators of leaf segment, stem segment and roots segment etc. of commodity P. notoginseng, longitude and latitude, elevation, soil type, landform of producing area were measured and observed. RESULT: The P. notogiseng cultivation was expanding from traditional area like Wenshang to new areas as Honghe, Kunming, Qujing at large scale. Comparing with traditional cultivated fields, the elevation of new fields, which are red soil of moderate or low mountain slopes and gentle hills between 1 800-2 130 m, increases markedly. The agronomic traits of new cultivated fields such as plant height, stem diameter, the ground and underground biomass were better than those of traditional cultivated fields in varying degree. Furthermore, the root weight, taproot weight, taproot length and other agronomic index augmented more than 20%. Comparison among different cultivated fields showed P. notoginseng of Honghe Shiping Niujie, Kunming Shilin Guishan, Honghe Jiangshui Guanting etc. had better agronomic traits, the plant were thick and tall, the taproot and the top of reed were large, the number of root was more. Inversely, P. notoginseng of Kunming Guandu Xiaoshao, Guangxi Jingxi Lutong, Wenshang Yanshan Jiangna were thin and small. Cluster analysis showed that cultivated fields of P. notoginseng which was across clustered by traditional and new cultivated fields can be divided into three groups, including a high-yielding region insist of three new cultivated fields Honghe Shiping Niujie, Kunming Shilin Guishan, Honghe Jiangshui Guanting and one traditional cultivated field Wenshan Yanshan Jiangna. Correlation analysis indicated that the size and weight of taproot and top of reed were significantly positive correlated with plant height, stem diameter, leaf size, leaf weight and stem weight. Regression analysis pointed out that stem diameter, leaf width, leaf length/width, leaf weight were the main factors affecting the dry weight of taproot, and the main factors influencing the dry weight of top of reed were plant height, petiole, leaf length, leaf width and other agronomic traits. CONCLUSION: From agronomic traits, P. notoginseng in new cultivated fields were more robust than that in traditional fields, but besides the length of taproot, the difference of rest agronomic traits didn't reach the significant level. It is suggested that, in the west and the north of traditional area, the red soil land of moderate or low mountain slopes and gentle hills where the elevation is between 1 800-2 130 m is suitable for cultivating P. notoginseng. Honghe Shiping Niujie, Honghe Jianshui Guanting, Kuming Shilin Gongshan can be used as vital development area for commodity P. notoginseng.

[Double mulching application for Panax notoginseng growing seedlings].

OBJECTIVE: In order to improve the irrigation for Panax notginseng growing seedlings, different mulching ways were carried out to investigate the effects of double mulching. METHOD: Field experiment was applied to study soil moisture, soil temperature and bulk density of different mulching ways while the germination rate and seedlings growth also were investigated. RESULT: Compared with the traditional single mulching with pine leaves or straw, double mulching using plastic film combined with pine leaves or straw could reduce 2/3 volumes of irrigation at the early seedling time Double mulching treatments didn't need to irrigate for 40 days from seeding to germination, and kept soil moisture and temperature steady at whole seedling time about 30% and 9.0-16.6 degrees C, respectively. The steady soil moisture and temperature benefited to resist late spring cold and germinate earlier while kept germination regularly, higher rate and seedlings quality. In contrast, single mulching using pine leaves or straw had poor soil moisture and temperature preserving, needed to irrigate every 12-day, meanwhile dropped the germination and booming time 14 days and 24-26 days, respectively, reduced germination rate about 11.3%-8.7%. However, single pine leaves mulching was better than straw mulching. In addition, though better effects of soil moisture and temperature preserving as well as earlier and higher rate of germination with single plastic films mulching had, some disadvantages had also been observed, such as daily soil temperature changed greatly, seedling bed soil hardened easily, more moss and weeds resulted difficulty in later management. CONCLUSION: To the purpose of saving water and labor as well as getting higher germination rate and seedlings quality, double mulching using plastic films combined pine leaves at the early time and single mulching removing plastic films at the later time is suggested to apply in the growing seedlings of P. notoginseng.

[Different proportion of potassium chloride and potassium sulphate application on cultivation of Panax notoginseng].

In order to make sure whether Panax notoginseng is sensitive to chloridion and guide fertilization in planting of P. notoginseng, the effects of the different proportion of potassium chloride (KCl) and potassium sulfate (K2SO4) on the yield, quality of P. notoginseng were studied. The results showed that K fertilizer significantly improved the growth of P. notoginseng and increased the biomass per plant or per pot and the content of N, P, K and the content of saponin. In cases of conditions such as potassium, and the effects of K2SO4 on increasing the petiole length, leaf size, rhizome length, root length, and content and accumulation of Ginsenoside Rg1 were better than those of KCl. While compared with K2SO4, KCl was more conducive to augmenting height, root width, the biomass of shoot, rhizome, root and the content of Ginsenoside Rb1 and Rd. There was not remarkable difference in agronomic characters, biomass and the content of N, P, K among KCl, K2SO4 and the combination of KCl and K2SO4. However, the content of saponin of the treatment with combination of KCl and K2SO4 was significant higher than that of single KCl or K2SO4 treatments. K fertilizer significantly increased yield and the content of saponins. And P. notoginseng was not sensitive to chloridion. KCl increased the yield and the content of saponins of P. notoginseng as well as K2SO4, and the combination treatment was superior to single treatment. It is recommended that the KCl should be adopted in production, to reduce the cost of potash fertilizer.

[Effects of balanced fertilization and soil amendment on growth and yield of sanqi in continuous cropping].

OBJECTIVE: To explore the solution of continuous cropping obstacle of Panax notoginseng. METHOD: The effects of balanced fertilization and soil amendment on the emergence rate, survival rate, growth and yield of P. notoginseng were studied with a pot culture experiment in 3-year-interval continuous cropping soil. RESULT: The result showed that the content of total N, total P, available N, available P and other available components in continuous cropping soil were higher than those in fresh soil while available K and the rate of K/N were declined, and available Cu and some other micro elements were lack. The way of balanced fertilization "low N + fused calcium-magnesium-phosphate fertilizer + high K + lime + micro elements" would significantly enhance the rate of survival, biomass and yield. It also promoted the growth of P. notoginseng in continuous cropping. The bagasse could relieve the continuous cropping obstacle obviously, the survival rate was improved for 31.6% and the yield of medicinal materials was 19.5%. The fly ash had also some effect in relieving the continuous cropping obstacle. CONCLUSION: The overall results suggested that the adequate fertilization plan is the applying lime, reducing N, applying calcium-magnesium-phosphate fertilizer, improving K and supplying mircoelement as well as applying bagasse to resolve the problem of continuous cropping obstacle of P. notoginseng.

[Status and prospective on nutritional physiology and fertilization of Panax notoginseng].

Sanqi, Panax notoginseng, is a famous traditional Chinese herb, which has more than 400 years cultivation history in China. This paper has reviewed the studies on the suitable growing soil conditions for Sanqi, mineral nutrition effects on Sanqi, and the effects of nutrients uptake on the yield and quality of Sanqi by applying fertilizer. Thereafter, research needed to be put further efforts in the future has raised for discussion, and outlined the following topics for further research like the mechanism of mineral nutrition, methods of nutrients deficiency diagnosis on site, suitable cultivated soil grading special fertizer development, disease resistance, and fertilization recommended by soil testing.

[Inhibition of cell proliferation and C-myc cancer protein expression in human colon adenocarcinoma cell line HT29 with VIP-131I-ASON].

A 15-mer phosphorothioate antisense oligonucleotide (ASON) complementary to the translation start region of the C-myc oncogene mRNA was labeled with 131I or 125I and the labelled compound was linked to the vasoactive intestinal peptide (VIP) to be bound covalently to a polylysine chain so as to deliver oligonucleotide into tumor cells. The effect of the VIP as carrier on cell uptake of ASON in tissue culture was evaluated in a human colon adenocarcinoma HT29 cell line. The efficacy of VIP-131-ASON on cell growth was evaluated using the MTT assay. Expression of c-myc-encoded protein was measured by flow cytometry. Sense and nosense control Oligonucleotides with VIP carrier were used as control. The results showed that VIP competed effectively with VIP-125I-ASON to bind the HT29 cells. Cell uptake was increased 3-4 fold using the VIP carrier compared to the same dosage of naked DNA. HT29 cells treated with VIP-131I-ASON complexes exhibited 4-fold lower proliferation than those treated with 13I-ASON and 6-fold lower proliferation than those treated with radioiodinated Sense and nosense DNA. Cancer protein expression of HT29 cells treated with VIP-131I-ASON was decreased 2-fold compare with that in 131I-ASON treated cell. The use of a VIP carrier greatly increased 131I-ASON cellular uptake and inhibition of cell proliferation and C-myc cancer protein expressing in HT29 cell by radioiodinated antisense Oligonucleotides.

[Preparation and biodistribution of VIP-125I-ASON].

OBJECTIVE: To prepare VIP-125I-ASON and investigate the possibility of using it as an agent for diagnostic imaging and therapy of colon carcinoma. METHODS: The iodination of a 15-base single-stranded antisense oligonucleotide (ASON) complementary to C-myc oncogene mRNA was carried out in the presence of TICl3. The radiolabeled oligonucleotide was complexed with a VIP-polylysine conjugate under certain condition. 3-5 microCi VIP-125I-ASON was injected into the tail vein of the BALB/c nude mice bearing transplanted HT29 colon carcinoma; the nude mice were killed at specific intervals after injection, and the biodistrbution of VIP-125I-ASON in the organs were calculated. RESULTS: The biodistributed experiment showed that the 125I-ASON was excreted by kidney mostly and by liver and spleen in part. The results of studies after the injection of VIP-125I-ASON differed from those of unconjugated 125I-ASON. The conjugation of VIP to the ASON resulted in a decrease in the plasma clearance of the radiopharmaceutical, which may be due to the reduction in the renal clearance of the ASON. The highest uptake of tumor tissue (5.89% ID/g at 2 h) was significantly higher than that in nude mice given unconjugated ASON (P < 0.05). Tumor to blood ratios and tumor to muscle ratios were optimal at 4 h. CONCLUSION: VIP-125I-ASON has desirable stability and higher uptake in tumor. It may provide a new sensitive mean for diagnostic antisense imaging and radiotherapy of tumor in the future.

Antitumor effects of radioiodinated antisense oligonuclide mediated by VIP receptor.

A 15-mer phosphorothioate antisense oligonuclide (ASON) complementary to the translation start region of the C-myc oncogene mRNA was radioiodinated to enhance its antitumor activity, and vasoactive intestinal peptide bound covalently polylysine (VIP-polylysine) was used as a carrier to deliver the oligonucleotide into VIP receptor-positive tumor cells. The antitumor activity of radioiodinated ASON conjugated to VIP-polylysine(VIP-131I-ASON) was investigated in athymic mice bearing HT29 tumor xenografts in comparison with unconjugated radioiodinated ASON(131I-ASON), unlabelled ASON (VIP-ASON) and scrambled oligonucleotide (VIP-131I-MON) conjugated to VIP-polylysine. Conjugation 125I-ASON to VIP-polylysine resulted in a 5.6-fold decrease in the plasma clearance and a 3.4-fold increase in tumor uptake of the radiopharmaceutical. Athymic mice bearing HT29 tumor xenografts were treated with 4 weekly doses of VIP-131I-ASON and the antitumor effects were assessed by use of the slope of the tumor growth curve. VIP-131I-ASON exhibited strong antitumor effects against HT29 xenografts, decreasing tumor growth rate 9.67-, 7.90-fold more effectively than 131I-ASON and VIP-ASON at equivalent doses of ASON. Conversely, 131I-ASON, VIP-ASON or VIP-131I-MON caused no significant effect compared with the normal saline. These data indicated that use of a VIP-polylysine carrier greatly increased HT29 tumor uptake of ASON and treatment with the VIP-131I-ASON complexes resulted in tumor growth delay in human colon cancer xenograft.