Flavonoid production in transgenic hop (Humulus lupulus L.) altered by PAP1/MYB75 from Arabidopsis thaliana L.

Hop is an important source of secondary metabolites, such as flavonoids. Some of these are pharmacologically active. Nevertheless, the concentration of some classes as flavonoids in wild-type plants is rather low. To enhance the production in hop, it would be interesting to modify the regulation of genes in the flavonoid biosynthetic pathway. For this purpose, the regulatory factor PAP1/AtMYB75 from Arabidopsis thaliana L. was introduced into hop plants cv. Tettnanger by Agrobacterium-mediated genetic transformation. Twenty kanamycin-resistant transgenic plants were obtained. It was shown that PAP1/AtMYB75 was stably incorporated and expressed in the hop genome. In comparison to the wild-type plants, the color of female flowers and cones of transgenic plants was reddish to pink. Chemical analysis revealed higher levels of anthocyanins, rutin, isoquercitin, kaempferol-glucoside, kaempferol-glucoside-malonate, desmethylxanthohumol, xanthohumol, α-acids and ß-acids in transgenic plants compared to wild-type plants.

First Report of Hop stunt viroid Infecting Hop in Slovenia.

Hop (Humulus lupulus), of the Cannabaceae family, is a dioecious perennial climbing plant that is native to Asia, North America, and Europe and is commercially grown in many countries for its use in brewing and the pharmaceutical industry. Slovenia has a more than 100-year-old hop-growing tradition and it is an important national agricultural business, with 90% of production exported to foreign markets. Since 2007, symptoms similar to Hop stunt viroid (HSVd) infection have been observed in several hop gardens with cvs. Celeia, Bobek, and Aurora in the Savinja Valley and Koroska Region. Symptoms include stunting, leaf curl, small cone formation, and dry root rot. In the first year of finding the disease, the incidence varied from 1 to 30% and increased rapidly (by as much as 10%) each subsequent year, predominantly along plant rows. For molecular identification of the pathogen, RNA was extracted from leaves and cones of symptomatic and asymptomatic plants from two different hop gardens with cv. Celeia using Tri Reagent (T9424; Sigma-Aldrich, St Louis, MO). Reverse transcription-PCR was carried out using two pairs of specific HSVd primers, HSVdI/HSVdII and HSVdeI/HSVdeII (3,4). Both primer pairs gave a single PCR product from tissue from symptomatic plants, with expected lengths of ~300 bp, but no amplicons were produced using samples from asymptomatic plants. PCR products from HSVdI/HSVdII were subjected to direct sequencing and HSVdeI/HSVdeII products were cloned in PCR Script SK (+) (Stratagene, La Jolla, CA) vector and sequenced. Five sequences (EMBL Accession Nos. HE575344, HE575345, HE575346, HE575347, and HE575348) were obtained, which revealed 96 to 99% sequence identity with various HSVd variants (grapevine, citrus, and cucumber) reported in GenBank of the National Centre for Biotechnology Information (NCBI). HSVd belonging to the Hostuviroid genus, Pospiviroidae family, has been previously reported in hop in Japan, South Korea, North America, and China (1,2). To our knowledge, this is the first report of the detection of HSVd on hop in Europe. Strict phytosanitary measures have been taken to prevent further spread and to eradicate HSVd infections. References: (1) K. C. Eastwell and T. Sano. Hop Stunt. Page 48 in: Compendium of Hop Diseases and Pests. W. F. Mahaffee et al., eds. The American Phytopathological Society, St. Paul, MN, 2009. (2) L. Guo et al. Plant Pathol. 57:764, 2008. (3) J. Matousek et al. Plant Soil Environ. 49:168, 2003. (4) J. Matousek et al. J. Virol. Methods 122:153, 2004.

Antitumor activity of apoptotic nuclease TBN1 from L. esculentum.

Nuclease from tomato (TBN1) was produced by in planta biotechnology purified and tested for its anticarcinogenic properties. The nuclease was cytostatic after its intratumoral administration to nude mice bearing human melanoma or prostate carcinoma or after tumor targeting by TBN1 administration intravenously as conjugate with polyethylene glycol (PEG). Inhibitory effects of TBN1 on tumor growth were comparable to effects of bovine seminal RNase (BS-RNase), but the inhibition was reached at about ten times lower protein concentration. Simultaneously, TBN1 exhibited a lower degree of embryotoxicity compared to BS-RNAse and other nucleases. TBN1 showed significant stability in vivo, because it was readily detected after its administration intratumorally or intravenously by the fluorescence methods. Intravenous administration of TBN1-PEG caused significant inhibition of tumor proliferation without obvious degenerative changes, while direct administration of TBN1 into melanoma tumors led to rapid tumor tissue degeneration. The fact can be essential for the mode of TBN1 biological action that mature nuclease is a small (36 kDa) thermostable glycoprotein that has ability to destroy human 28S, 18S, 7S and 5.8S RNA, circular RNAs, double-stranded RNA in vitro and shows DNase and 3'nucleotidase activities.

Antitumor and biological effects of black pine (pinus nigra) pollen nuclease.

The antitumor effect of black pine (Pinus nigra) pollen nuclease (PN) tested in vitro was negligible in comparison with bovine seminal ribonuclease (BS-RNase). However, in the experiments in vivo a significant decrease of the human melanoma tumor size was observed in the mice treated with this nuclease and also with the animal RNases and DNase I. In nude mice injected intratumoraly with PN (10 microg/dose) the tumor size decreased from 100% in the control mice to 46% in treated mice whereas in counterparts treated with BS-RNase and DNase I the tumor growth was reduced a little more, however after ten times higher doses (100 and 80 microg per dose). Certain aspermatogenic and embryotoxic activity as an expression of side effects of PN and comparative enzymes also appeared, but it was lower compared to the effect of bovine seminal ribonuclease. Immunogenicity of PN was significantly weaker in comparison with BS-RNase. The antibodies against black pine nuclease produced in the injected mice did not inactivate the biological effects of this plant nuclease in vivo. In conclusion PN nuclease proved in vivo higher antitumor activity against human melanoma tumors growing in athymic mice in comparison with animal bovine seminal ribonuclease and DNase I.

Propagation and some physiological effects of Citrus bark cracking viroid and Apple fruit crinkle viroid in multiple infected hop (Humulus lupulus L.).

The hop metabolome important for the brewing industry and for medical purposes is endangered worldwide due to multiple viroid infections affecting hop physiology. Combinatorial biolistic hop inoculation with Citrus bark cracking viroid (CBCVd), Apple fruit crinkle viroid (AFCVd), Hop latent viroid, and Hop stunt viroid (HSVd) showed a low CBCVd compatibility with HSVd, while all other viroid combinations were highly compatible. Unlike to other viroids, single CBCVd propagation showed a significant excess of (-) over (+) strands in hop, tomato, and Nicotiana benthamiana, but not in citruses. Inoculation of hop with all viroids led to multiple infections with unstable viroid levels in individual plants in the pre- and post-dormancy periods, and to high plant mortality and morphological disorders. Hop isolates of CBCVd and AFCVd were highly stable, only minor quasispecies were detected. CBCVd caused a strong suppression of some crucial mRNAs related to the hop prenylflavonoid biosynthesis pathway, while AFCVd-caused effects were moderate. According to mRNA degradome analysis, this suppression was not caused by a direct viroid-specific small RNA-mediated degradation. CBCVd infection led to a strong induction of two hop transcription factors from WRKY family and to a disbalance of WRKY/WDR1 complexes important for activation of lupulin genes.

Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects.

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease (RNase A), is known to display special biological activities namely cytotoxicity for human tumor cells. Because some plant ribonucleases have a similar mass weight and structure as the animal ribonuclease, effects of a commercial product of Mung bean (Phaseolus aureus) nuclease (PhA) were studied on proliferation of ML-2 human tumor cells, as well as it's aspermatogenic, embryotoxic, immunogenic, and immunosuppressive activity, and therapeutic efficiency in athymic mice bearing human melanoma tumor. Concerning the antiproliferative activity, PhA nuclease was almost non-effective in vitro on ML-2 cells and also immunosuppressive activity on human lymphocyte in mixed culture was very low compared to that of BS RNase. However, significant antitumor activity was detected on human melanoma tumor after intratumoral or intraperitoneal administration into the mice. Furthermore conjugate of PhA nuclease with polyethylene glycol (PEG) injected seven times at the dose of 10 microg intraperitoneally showed identical antitumor activity as that of bovine seminal ribonuclease (BS RNase) injected by the same way at ten times higher dose. Both PhA and BS RNases exerted strong aspermatogenic effect on the width of spermatogenic layers while RNase A administration at ten times higher concentration was ineffective. PhA nuclease when compared by means of antibody cross reaction with RNase A, BS RNase and wheat leaf neutral RNase (WLN-RNase) was found to be immunologically similar to RNase A and WLN-RNase, meanwhile BS RNase showed much higher antigenicity in comparison with them.

Some biological actions of PEG-conjugated RNase A oligomers.

Previously we have shown that monomeric RNase A has no significant biological activity, whereas its oligomers (dimer to tetramer) prepared by lyophilizing from 50% acetic acid solutions, show remarkable aspermatogenic and antitumor activities. Furthermore, conjugates prepared by chemical binding of native RNase A to polyethylene glycol (PEG) have shown a significant aspermatogenic and antitumor activities. In this work we show that the chemical conjugation of PEG to the RNase A C-dimer, and to the two RNase A trimers (NC-trimer and C- trimer) decreases the aspermatogenic activity of the oligomers while increasing their inhibitory activity on the growth of the human UB900518 amelanotic melanoma transplanted in athymic nude mice. Moreover, the PEG-conjugated RNaseA oligomers are devoid, like the free oligomers, of any embryotoxic activity.

Complete nucleotide sequence and molecular probing of potato virus S genome.

Complete genomes of three isolates of Potato virus S (PVS) were cloned and sequenced. The PVS ORF-1 was characterized for the first time. It encodes a putative replication protein (RPT) that shares the highest homology (about 52%) with that of Blueberry scorch virus (BlScV). ORF-1 motifs, characteristic for carlaviruses were found for methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp). The complete sequence of PVS genome enabled to develop an immunocapture RT-PCR probing of the PVS genome. Using this system, the sequence variability of 11 genome zones was examined for 34 PVS isolates including 15 PVS-CS variants that caused a systemic infection in Chenopodium quinoa. A broad variability between PVS isolates and diverse sequence variants was found. cDNA fragments covering the coat protein (CP) leader and CP-coding region (approx. 420 bp) were pooled for PVS-O and Chenopodium-systemic PVS isolates (PVS-CS) and corresponding cDNA libraries were screened for sequence variants. Both cDNA pools differred mainly in the 5'-end of the CP gene. Methionine at the position 17 in combination with serine at the position 34 were frequently associated with the CS character of PVS. In general, hydrophobic and polar amino acids were characteristic for the positions 17 and 34, respectively in PVS-CS isolates. Genome probing and evolutionary distances suggested that the PVS-CS isolates analyzed were close to the ordinary European isolates of ordinary strain of PVS (PVS-O) but distant to the original Andean strain of PVS (PVS-A).

Molecular characterization and genome organization of 7SL RNA genes from hop (Humulus lupulus L.).

A wide spectrum of hop 7SL RNA-encoding sequences was detected by temperature gradient-gel electrophoresis. Four hop 7SL RNA genes were cloned and characterized. A new subvariant of the upstream sequence element (USE) 5'TCCCACATGG 3' and two distinct variants of TATA signal were found at positions characteristic for RNA polymerase III-driven transcription in plants. In addition, a more distant conserved sequence element 5' CATGTATAAACTTTCTGC 3' was present in all cloned genes, about 160 bp upstream of the 7SL RNA coding sequence. Consensus secondary structures calculated for hop 7SL RNAs revealed characteristic features, although some structure differences from formerly published models were predicted. Specific in-vitro transcription of plant 7SL RNA genes was observed in a heterologous system (HeLa extract). This in-vitro transcription assay showed significant differences among individual clones in transcription rates, suggesting the requirement of complexity of 7SL RNA sequence for its efficient transcription in HeLa extract. Southern blot analysis of hop DNA revealed 12 7SL-specific signals corresponding to HindIII fragments ranging from 0.45 to 7.8 kb. Several 7SL RNA-encoding sequences and various intergenic spacers were amplified from the individual HindIII fragments of about 1.3 and 2.8 kb. These facts suggest that at least some of the hop 7SL RNA genes are organized in genomic clusters.

Inhibition of potato spindle tuber viroid (PSTVd) infection with DNA oligonucleotides.

The effects of DNA oligonucleotides complementary to potato spindle tuber viroid (PSTVd) on viroid infection were investigated. The oligonucleotides were used to form hybrids with PSTVd in the infection mixture. A 75% reduction of viroid infection was found when an oligonucleotide complementary to nucleotides 79-110 of PSTVd was hybridized with PSTVd at a molar ratio of approximately 5000:1, respectively. A total inhibition of PSTVd infection was observed using an oligonucleotide complementary to nucleotides 42-78 at the same molar excess of DNA over PSTVd, although a 200-fold molar excess was found to be sufficient for the complete blocking of infection by PSTVd. This oligonucleotide caused a significant reduction (about 83%) of viroid infection even if the hybridization was done at a low (30 degrees C) temperature. Shorter oligonucleotides containing 22 and 15 bases corresponding to position 42-62 and 63-78, respectively, exhibited a significant effect only at a high (80 degrees C) initial temperature of molecular hybridization. Heteroduplexes formed between PSTVd RNA and antisense DNA were found to be less stable in a crude nuclease extract from tomato leaves as compared with PSTVd RNA alone. RNase H was demonstrated to cleave the molecular hybrids in vitro.

Immunosuppressive activity of bovine seminal ribonuclease and its mode of action.

Two preparations of dimeric BS RNase-native and recombinant proteins caused identical immunosuppressive effects on MLC-stimulated human lymphocytes. The monomers of RNase A and BS RNase were ten times less active. The inhibitory effect on MLC-stimmulation was followed by 90% inhibition of cell-mediated lympholysis (CML) caused by BS RNase (10 micrograms/ml). This effect indicated that BS RNase suppressed the recognition phase of the cytotoxic reaction, resulting in inhibition of generation of cytotoxic effector cells. BS RNase exerted a similar effect on generation of cytotoxic LAK cells. Cytotoxic activity of LAK cells or CTLs against K562 target cells was abrogated only when BS RNase was added at the beginning of the sensitizing phase, but the cytotoxicity of effector cells in the destruction phase was not influenced. The effect of RNase A on the generation of cytotoxic cells was much less pronounced. To get more information about the site of action, the effect of BS RNase on early lymphocyte stimulation by PHA was investigated by using fluorescein cell probes. BS RNase (100 micrograms/ml) prevented a shift in fluorescein emission occurring within one hour of activation using fluorescein diacetate as a marker for changes in the cytoplasmic matrix. On the contrary, it did not block the shift in fluorescence emission when tested with diphenylhexatrien as a marker for changes in membrane fluidity. Furthermore the effect of BS RNase on expression of membrane antigens expressed on activated human lymphocytes was estimated. BS RNase significantly inhibited the expression of CD25, CD38 and CD71 antigens on PHA-, Con A- and MLC-stimulated human T and B lymphocytes. No substantial change in expression of these antigens was observed on IL-2-stimulated cells, but DNA synthesis was totally abrogated. These results indicate that the mode of action of BS RNase on activated T and B lymphocytes is based mainly on the suppressed expression of receptors for interleukin-2-alpha-chain and transferrin.

Bovine seminal ribonuclease selectively kills human multidrug-resistant neuroblastoma cells via induction of apoptosis.

Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with specific antitumor activity. The cytotoxic action of this agent was examined in human neuroblastoma (NB) cell lines (SK-N-SH and UKF-NB-4) possessing the multidrug resistance (MDR) phenotype and NB cell lines (IMR-32, UKF-NB-1, UKF-NB-2 and UKF-NB-3) without MDR. Although MDR cells expressed large amounts of mdr-1 mRNA, contained functional P-glycoprotein and had 20- to 105-fold lower sensitivities to doxorubicin and vincristine than cells with non-MDR phenotypes, BS-RNase was equally toxic to all NB cells at concentrations employed (0.2 to 100 microg/ml). BS-RNase showed high selectivity for NB cells and was non-toxic to normal fibroblasts and epithelial cells. Ultrastructural investigation and annexin V assay showed that BS-RNase is a powerful inductor of apoptosis. The antitumoral effects of BS-RNase were also demonstrated in vivo using established subcutaneous xenografts in athymic (nude) mice of the MDR-1-bearing UKF-NB-4 cell line. Intratumoral injections (12.5 mg/kg) of BS-RNase over four weeks resulted in complete tumor regression and absence of tumor regrowth over a two-week observation period after cessation of treatment. The results show that BS-RNase selectively kills NB cells by inducing apoptosis and that this agent is active against mdr-1 expressing cells both in vitro and in vivo. BS-RNase fulfills important criteria for a candidate antitumor agent in NB patients with advanced disease.

Biolistic inoculation of plants with viroid nucleic acids.

Parameters for biolistic transfer of viroid nucleic acids using a Helios Gene Gun device were assayed. The main achievement of this method is high efficiency of inoculation with linear monomeric viroid cDNAs and RNAs. This greatly facilitates the study of mutated sequence variants, viroid libraries and mixed populations. The lower limits for efficient inoculation of monomeric cDNA fragments with the sequence of potato spindle tuber viroid (PSTVd) and native PSTVd RNA as detected 21 days p.i. are in the range of 50 ng and 200 pg per tomato plant, respectively. At a higher dose, i.e. 2 ng of native RNA per plant, biolistic transfer causes drastic stunting compared to conventional mechanical inoculation, which points to higher PSTVd titers after the biolistic transfer. Infection is readily achieved with exact length monomeric RNA transcripts having 5'-triphosphate and 3'-OH termini in amounts ranging from 2 to 20 ng per plant, suggesting no need for any supplementary modifications of ends or RNA circularization. The biolistic transfer is efficient for viroid "thermomutants", which exhibit low or no infectivity with conventional mechanical inoculation with Carborundum. The biolistic inoculation is also efficient for two other members of the Pospiviroidae family, hop stunt and hop latent viroid.

Polymer-conjugated bovine pancreatic and seminal ribonucleases inhibit growth of human tumors in nude mice.

The hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for RNase A or BS-RNase modification to prevent their degradation in bloodstream or fast elimination. Two PHPMA chains (classic and star-like) were synthesized and their conjugates with both enzymes were tested on the CD-1 nude mice bearing various human tumors. These RNase conjugates injected intravenously or intraperitoneally into the mice bearing melanoma, neuroblastoma or ovarian tumor caused significant reduction of transplanted tumors following ten daily doses of 2.5 and/or 1 mg/kg, respectively, while free RNase A or BS-RNase injected in doses of 10 mg/kg exerted only negligible antitumor activity. Histological examination confirmed potent cytotoxic effect of RNase A conjugates in ovarian tumor. Despite the antitumor activity observed in vivo, the in vitro cytotoxic activity of RNase A conjugates was not pronounced and did not differ from that caused by the free RNase A. The in vitro experiments with 125I-labeled preparations demonstrated that polymer conjugates were internalized by tumor cells very poorly in contrast to the dose-dependent internalization of the wild enzyme preparation. Surprisingly, mice injected with EL-4 leukemic cells, which were preincubated for 4 h with BS-RNase conjugates, exerted significantly prolonged survival compared with the control non-treated mice. It may be supposed that both BS-RNase and RNase A conjugates with PHPMA act after administration in vivo by a mechanism different from that or those occurring under in vitro conditions because in vivo they exert an antitumor action, whereas in vitro, they are ineffective. The experiments proved that RNase A, when conjugated to PHPMA, produced identical aspermatogenic and antitumor effects as BS-RNase conjugated to this polymer and that this preparation may be regarded as a potential anticancer drug.

Bovine seminal ribonuclease inhibits in vivo growth of human neuroblastoma cells.

Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with specific antitumor activities. It is selectively toxic for neuroblastoma (NB) cells in vitro with no significant effects on the viability of normal human cells. We evaluated the antitumoral effects of BS-RNase on human NB xenografts from UKF-NB-3 cells in athymic (nude) mice. The efficacy of direct intraneoplastic, subcutaneous and systemic delivery of BS-RNase was explored. Systemic administration of BS-RNase (12.5 mg/kg/day intraperitoneally, for 20 days in the course of four weeks) suppressed tumor growth but was not able to induce any cures. Subcutaneous injections (12.5 mg/kg/day for 20 days in the course of four weeks) and intratumoral BS-RNase treatment using the same schedule resulted in complete tumor regression. During 30 days following cessation of treatment no tumor regrowth was observed and animals were free of tumors. Toxic effects of BS-RNase (e.g., on bone marrow and inner organs) were not apparent. This data indicates that BS-RNase fulfills important criteria for a candidate antitumor agent specific for NB.

Bovine seminal ribonuclease exerts selective cytotoxicity toward neuroblastoma cells both sensitive and resistant to chemotherapeutic drugs.

BACKGROUND: Bovine seminal ribonuclease (BS-RNase) exerts selective cytotoxicity toward different types of tumor cells. In the present study, we tested the effects of BS-RNase on cultured neuroblastoma (NB) cells resistant to chemotherapeutic agents. The selectivity of the antitumoral activity of BS-RNase was evaluated using cultures of CD34+ hematopoietic stem cells. MATERIALS AND METHODS: Human NB cell lines including IMR-32, UKF-NB-2 and UKF-NB-3 were selected for resistance against vincristine, doxorubicin or cisplatin by exposure to increasing concentrations of the respective drug. The cytotoxicity of the drugs to NB cells was evaluated using a clonogenic assay in a methylcellulose medium. Peripheral blood progenitor cells were obtained from adult healthy donors by positive selection using specific anti-CD34+ antibodies. The toxicity of BS-RNase to CD34+ cells was assessed in the direct clonogenic assay using methylcellulose medium or in ex vivo expansion culture supplemented with hematopoietic growth factors. RESULTS: In the clonogenic assay it was shown that BS-RNase completely inhibits growth of both parental NB cells and their sublines resistant to chemotherapeutic drugs at concentrations (up to 50 micrograms/ml) which have no significant influence on the growth of colony-forming units, granulocyte macrophage and erythroid burst-forming units. Moreover, BS-RNase had no effect on the ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors. CONCLUSIONS: BS-RNase is a highly efficient agent against NB cells resistant to chemotherapeutic drugs. The lack of toxicity to hematopoietic progenitor cells suggests that BS-RNase is also likely to have tolerable hematopoietic toxicity.

Selective activity of BS-RNase against anaplastic thyroid cancer.

BACKGROUND: Anaplastic thyroid carcinoma is an aggressive solid tumor that fails to adequately respond to any known chemotherapeutic regimen. The development of effective chemotherapy agents would provide the best chance for long-term survival of patients. MATERIALS AND METHODS: The cytotoxic effects of bovine seminal ribonuclease (BS-RNase) against thyroid carcinoma cell lines with different degrees of differentiation in comparison to non-malignant cells, including human foreskin fibroblasts (HFF) and retinal pigment epithelial cells (RPE), were tested using the MTT dye reduction assay. Induction of apoptosis was demonstrated by annexin V assay and expression of proteins related to apoptosis was investigated by flow cytometry. The antitumoral in vivo effects of BS-RNase were assessed on established xenografts of anaplastic thyroid carcinoma cell line 8505C in nude mice using subcutaneous injections of BS-RNase (12.5 mg/kg once a day, on 20 consecutive days). RESULTS: All the tumor cell lines exhibited marked sensitivity against BS-RNase in comparison to HFF and RPE cells. The greatest growth inhibition was seen in the 8505C line, while IC50 values for papillary (B-CPAP) and poorly-differentiated thyroid carcinoma cells were about 6-fold higher. The cytotoxic action of BS-RNase was associated with induction of apoptosis. Expressions of Fas and Fas-ligand were not influenced by BS-RNase completely, while the down-regulation of Bcl-2 in treated cells was observed. In vivo treatment induced significant tumor regression after the course of 20 consecutive days. No apparent toxic effects of BS-RNase toward non-malignant cells were observed during the in vivo treatment. After cessation of therapy (day 20) tumor volume continued to decrease and the tumor was no longer detectable after 30 days of treatment induction in all animals. CONCLUSION: BS-RNase may have beneficial effects for treatment of aggressive anaplastic thyroid cancer.

Aerosol deposition in furnace atomization.

A novel approach to sample deposition in furnace atomization is suggested, which obviates the need for skilled application of microvolumes by syringe. The analyte in aerosol form is deposited under controlled conditions on the internal surface areas of graphite furnaces. Precision approaching that of flame atomization systems is achieved and at the same time, concentrational sensitivity may be increased simply by extending the deposition time. The amount of analyte deposited in the furnace is restricted only by the sample volume available and the matrix concentration. A single standard can be used to construct a calibration curve by simply varying the aerosol deposition time.

Atomic-fluorescence spectroscopy of lead.

The fluorescence spectrum of lead excited with a high-intensity hollow-cathode lamp has been investigated and the probable mechanism of fluorescence transitions is suggested. It is confirmed experimentally that the most intense fluorescence line at 405.78 nm is mostly due to direct-line fluorescence. The premixed air-hydrogen flame, the separated air-acetylene flame, and the oxy-hydrogen flame diluted with argon have been used, the last mentioned giving a detection limit of 0.02 ppm with the line at 405.78 nm.