MiR-126-3p suppresses the growth, migration and invasion of NSCLC via targeting CCR1.

OBJECTIVE: MicroRNA (miRNA) plays vital roles in the development of different cancers. In the current work, we explored the function of miR-126-3p in the growth and metastasis of non-small-cell lung cancer (NSCLC) cell in vitro and in vivo. PATIENTS AND METHODS: The expressions of miR-126-3p in NSCLC cell lines were assessed using the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation, wound healing and transwell invasion were applied to reveal the role of miR-126b-3p on NSCLC cell growth, migration and invasion. The expressions of epithelial-mesenchymal transition (EMT) associated markers (E-cadherin and N-cadherin) were assessed by immunofluorescence staining. The Xenograft model and lung metastasis model were applied to explore the impact of miR-126-3p on the growth and aggressiveness of NSCLC cell in vivo. RESULTS: MiR-126-3p was significantly down-regulated in NSCLC cell lines and tissues. The up-regulation of miR-126-3p inhibited the growth, colony formation, migration and invasion of NSCLC cell. Furthermore, the xenograft model indicated that miR-126-3p suppressed NSCLC cell growth and lung metastasis by targeting chemokine (C-C motif) receptor 1 (CCR1). In addition, we demonstrated that the over-expression of CCR1 rescued the inhibitory effects of miR-126-3p on NSCLC cells growth, migration and invasion. Finally, knocked-down of CCR1 was able to mimic the inhibitory effects of miR-126-3p on the progression of NSCLC cell. CONCLUSIONS: These findings indicate that miR-126-3p plays an important role in the growth, migration and invasion of NSCLC by targeting CCR1.

Towards an open grapevine information system.

Viticulture, like other fields of agriculture, is currently facing important challenges that will be addressed only through sustained, dedicated and coordinated research. Although the methods used in biology have evolved tremendously in recent years and now involve the routine production of large data sets of varied nature, in many domains of study, including grapevine research, there is a need to improve the findability, accessibility, interoperability and reusability (FAIR-ness) of these data. Considering the heterogeneous nature of the data produced, the transnational nature of the scientific community and the experience gained elsewhere, we have formed an open working group, in the framework of the International Grapevine Genome Program (www.vitaceae.org), to construct a coordinated federation of information systems holding grapevine data distributed around the world, providing an integrated set of interfaces supporting advanced data modeling, rich semantic integration and the next generation of data mining tools. To achieve this goal, it will be critical to develop, implement and adopt appropriate standards for data annotation and formatting. The development of this system, the GrapeIS, linking genotypes to phenotypes, and scientific research to agronomical and oeneological data, should provide new insights into grape biology, and allow the development of new varieties to meet the challenges of biotic and abiotic stress, environmental change, and consumer demand.

First Report of Hop stunt viroid in Apricot in China.

Hop stunt viroid (HSVd), a member of the family Pospiviroidae, was first described as the causal agent of hop stunt disease in Japan. It has since been found in a wide range of hosts including herbaceous and woody hosts (e.g., hop, cucumber, grapevine, citrus, plum, peach, pear, apricot, almond, and pomegranate). It was also detected and characterized in apricot where infection appears to be latent (1). The viroid occurs frequently in apricot. In southeastern Spain, the presence of HSVd was found to infect 81% of apricot trees (2). Apricots originated in China and are extensively cultivated, but HSVd infection in this host has not been reported. In September 2005, a single symptomatic apricot tree, 'Yin Bai', one of the most popular and widely grown cultivars in China, was discovered at the Institute of Fruit Science in Changping District in Beijing, Peoples Republic of China. Observed symptoms included a number of yellow spots with an irregular border that scattered in an irregular manner over the leaf surface. Total RNA was extracted and used for return-polyacrylamide gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR) (4). Results of both assays were positive for HSVd. A 297-bp full-length DNA fragment was amplified by RT-PCR using primers R1 (5'-GCTGGATTCTGAGAAGAGTT-3') complementary to HSVd residues 87-106 for the RT reaction, followed by R2 (5'-AACCCGGGGCTCCTTTCTCA-3') complementary to HSVd residues 67-84 and forward primer F3 (5'-AACCCGGGGCAACTCTTCTC-3') residues 79-96 for PCR. The primers are located in the strictly conserved central region of the conserved HSVd group and contain the unique endonuclease restriction site SmaI. The amplified products were cloned into pGEM-T (Promega, Madison, WI) and selected for further analysis on the basis of the results of restriction digests. Six individual clones were sequenced and three different sequences were obtained. Nucleic acid sequence (GenBank Accession No. DQ362901) obtained from one clone was 99.3% (nucleotide changes T206→C, C233→T) identical to HSVd.apr8 (GenBank Accession No. Y09349) (3). Sequence (GenBank Accession No. DQ362904) obtained from three clones was 99.7% (nucleotide change C233→T) and a third sequence (GenBank Accession No. DQ362905) obtained from two clones was 99.3% (nucleotide changes G107→A, C233→T) identical to HSVd.apr8. Further investigation is necessary to determine whether the symptoms observed are associated with the viroid infection. To our knowledge, this is the first report of HSVd isolated from apricot in China. References: (1) N. Astruc et al. Eur. J. Plant Pathol. 102:837, 1996. (2) M. C. Cañzres et al. Acta Hortic. 472:581, 1998. (3) S. A. Kofalvi et al. J. Gen. Virol. 78:3177, 1997. (4) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995.

High level expression of a recombinant acid phytase gene in Pichia pastoris.

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.

Characterization of X-Disease Phytoplasmas in Chokecherry from North Dakota by PCR-RFLP and Sequence Analysis of the rRNA Gene Region.

Genetic variation of X-disease phytoplasma strains from chokecherry (ChX) in North Dakota and nearby sites, and their relatedness with three standard strains of the X-disease phytoplasma group, eastern X-disease (CX), western X-disease (WX), and goldenrod yellows (GR1) phyto-plasmas, were studied. Primer pairs were developed to amplify the 23S ribosomal RNA (rRNA) gene and the 16S/23S spacer region. The rRNA genes (16S rRNA, 23S rRNA, and two ribosomal protein [rp] genes) and the 16S/23S spacer region were amplified by polymerase chain reactions. The restriction fragment length polymorphism (RFLP) patterns of 16S rRNA, 23S rRNA, and rp genes, generated by digestion with four restriction enzymes (AluI, HpaII, MseI, and RsaI), showed no difference among 43 ChX phytoplasma isolates. Sequencing of the 441-bp 16S/23S spacer region revealed variation at four positions among 12 ChX phytoplasma strains. A tRNAIle and other conserved sequences were identified in the spacer region. Among X-disease subgroups, RFLP analysis indicated that ChX is similar to WX, closely related to CX, and easily distinguished from GR1. Sequencing indicated that ChX is closer to CX than to WX. Together, the analyses indicated that ChX phytoplasmas are genetically different from the standard strains of other X-disease phytoplasma subgroups.

Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation.

The effects of timentin on shoot regeneration of tobacco (Nicotiana tabaccum) and Siberian elm (Ulmus pumila L.) and its use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were determined. Timentin is a mixture of ticarcillin and clavulanic acid, and at concentrations of 200-500 mg/l with ratios of ticarcillin:clavulanic acid of 50:1 and 100:1, it had little effect on shoot regeneration of tobacco or Siberian elm. Timentin was as effective in suppressing A. tumefaciens as carbenicillin and cefatoxime at concentrations commonly used in transformation. The disarmed A. tumefaciens strain LBA4404 in infected tobacco leaf tissues was visually undetectable after three subcultures on medium with 500 mg/l of timentin and 250 mg/l carbenicillin. Timentin was stable in solid agar medium and remained effective for at least 70 days, but was unstable when stored as a mixed stock solution or as separate ticarcillin and clavulanic acid stock solutions at -20°C or -80°C freezer for 4 weeks. Timentin may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.

The use of cell culture for subchromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.

Barley yellow dwarf virus (BYDV) resistance has been transferred to wheat from a group 7 chromosome of Thinopyrum (Agropyron) intermedium. The source of the resistance gene was the L1 disomic addition line, which carries the 7Ai-1 chromosome. The resistance locus is on the long arm of this chromosome. BYDV resistant recombinant lines were identified after three or more generations of selection against a group 7 Th. intermedium short arm marker (red coleoptile) and selection for the presence of BYDV resistance. One recombinant line produced by ph. mutant induced homoeologous pairing and 14 recombinant lines induced by cell culture have been identified. Resistance in seven of the cell culture induced recombinants has been inherited via pollen according to Mendelian segregation ratios for up to eight generations. Meiotic analysis of heterozygotes indicates that the alien chromatin in the cell culture induced recombinants is small enough to allow regular meiotic behaviour. The ph-induced recombinant was less regular in meiosis. A probe, pEleAcc2, originally isolated from Th. elongatum and that hybridizes to dispersed repeated DNA sequences, was utilised to detect Th. intermedium chromatin, which confers resistance to BYDV, in wheat backgrounds. Quantification of these hybridization signals indicated that the translocations involved a portion of alien chromatin that was smaller than the complete long arm of 7Ai-1. Restriction fragment length polymorphism analysis confirmed the loss of the short arm of 7Ai-1 and indicated the retention of segments of the long arm of 7Ai-1. Two 7Ai-1L DNA markers always assorted with the BYDV resistance. A third 7Ai-IL DNA marker was also present in seven of eight recombinants. In all recombinants except TC7, the 7Ai-1L markers replaced the 7DL markers. None of the wheat group 7 markers was missing from TC7. It is concluded that all the resistant lines are the result of recombination with wheat chromosome 7D, except line TC7, which is the result of recombination with an unidentified nongroup 7 chromosome.

[Pharmacological studies on the effects of huanglian decoction on experimental gastric lesions in rats and antiemetic in pigeons].

The results showed that Huanglion decoction has protective effect on ethanol-,HCl- and aspirin-induced gastric hemorrhagic lesions in rats and antemetic effect on CuSO4-induced vomiting in pigeons. A dose of 27g/(kg.d) po applied in mice showed no toxic action. This dose is 400 times that of clinical application.

[Experimental studies on Uncaria sinensis (Oliv.) Havil and Achyranthes bidentata Blume and their compacibility].

Pharmacological studies were conducted on Uncaria sinensis and Achyranthes bidentata both separately and combined. Comparison was made on the hypotensive effect on normal and renal-type hypertensive rats as well anti-spasmodic and sedative effects in mice. The results showed that Uncaria sinensis and Achyranthes bidentata have obvious synergic action in compatibility.

[Pharmacognostic studies on the Chinese drug guanzhong produced in East China].

By on-the-spot investigation we have checked up on 8 kinds of botanical origin of the Chinese traditional drug Guanzhong produced in East China and have given keys for their identification. The major tissue features of basal petioles and the external characteristics of the crude drugs have also been given.

Shoot regeneration from petioles and leaves of Vitis X labruscana 'Catawba'.

Shoot regeneration and normal plants were obtained from leaf and petiole explants derived from in vitro grown shoots of Vitis X labruscana 'Catawba'. Regeneration was induced in the presence of both 6-benzylaminopurine and indole-3-butyric acid; combinations of 2,4-dichlorophenoxyacetic acid or 2-naphthoxyacetic acid with 6-benzylaminopurine did not permit regeneration from leaf explants. Up to 15% of leaf and 70% of petiole explants regenerated shoots on media with 5.0-10.0 µM BA and 0.1-0.5 µM IBA. Incubation in the dark was required to obtain regeneration. About 50% of shoots developed normally following transfer to light. An average of one shoot regenerated from leaf explants and 3.3 shoots regenerated per petiole explant. Regeneration from petioles and leaves was always from the basipetal end. The interaction of 6-benzylaminopurine with indole-3-butyric acid was also examined.

Low dose cytosine arabinoside: partial remission of acute myeloid leukaemia without evidence of differentiation induction.

Thirteen patients with acute myeloid leukaemia aged from 19 to 81 were treated with low dose cytosine arabinoside (ARA-C) in a dose of 10-15 mg/m2 twice daily subcutaneously. Three complete remissions were obtained. Partial responses were observed in a further two patients. To analyse the action of low dose ARA-C freshly isolated leukaemic cells and cells from the cloned promyelocytic leukaemia cell line (HL60) were cultured in vitro in the presence of cytosine arabinoside. Minimal evidence of differentiation induction was observed when compared with the cytotoxic effects of the drug. These results suggest that ARA-C does not exert its anti-leukaemic effects by halting proliferation through differentiation induction. Rather, it appeared that the capacity of this agent to kill cells in S-phase produced a progressive depletion of the cycling leukaemic cells. This resulted in a corresponding steady decline in the total leukaemic cell population.

Mitoxantrone treatment in advanced breast cancer.

Half of our patients presenting with breast cancer will eventually require cytotoxic chemotherapy. In searching for an effective drug with low toxicity, many new drugs have been investigated. Seventeen patients with advanced breast cancer were treated with mitoxantrone, an analogue of adriamycin. The response rate was 44% (CR 6% + PR 38%). Side effects were fewer than those normally anticipated for most cytotoxic chemotherapy in that only two patients suffered hair loss and three nausea and vomiting. There was, however, significant bone marrow depression with granulocytopenia and two patients died of septicaemia following treatment. Cardiac toxicity occurred in two patients: of these, both had prior treatment with adriamycin, and one had prior radiotherapy to the chest wall. Both of these patients died.

Lectin-binding affinities of human breast tumors.

Paraffin-embedded sections from a variety of breast lesions were stained with a number of lectins labeled with fluorescein isothiocyanate and examined by fluorescence microscopy: 95 non-neoplastic tissues and 69 malignant tumors were examined. Lectin binding was demonstrated in all malignant tumors, the fluorescence being confined to the plasma membrane of the tumor cells. Normal and hyperplastic tissues either failed to stain or showed a grossly diminished level of fluorescence. The significance of the staining reaction is discussed.