Micropropagation and Cryoconservation of the Endangered Plant Species Artemisia laciniata (Asteraceae).

BACKGROUND: Artemisia laciniata, mainly distributed in Siberia and Central Asia, is classified as critically endangered in Europe. OBJECTIVES: This study developed a protocol for its micropropagation and cryopreservation. MATERIALS AND METHODS: In vitro cultures from fresh seed and in vivo shoots were initiated. Micropropagation and cryopreservation protocols were developed. Bacteria detected after cryopreservation were investigated using 16S rRNA analysis. Genome size measurements of regenerated plants after cryopreservation using flow cytometry and carbon isotope measurements to evaluate stress status were also carried out. RESULTS: A. laciniata from both starting materials could be successfully propagated on MS medium with 0.5 uM BAP. Material initiated from in vivo shoots yielded lower regeneration percentages (16%) after cryopreservation than material generated from seed (57 and 63%) using the droplet-vitrification method and PVS3. Bacteria occurring after cryopreservation belonged to the genera Sphingomonas, Staphylococcus, Curtobacterium and Gordonia. There was no significant difference in the genome size and stress status between non-cryopreserved and cryopreserved plants. CONCLUSION: A. laciniata could be readily micropropagated and cryopreserved. No negative effects of cryopreservation on plant water use efficiency or on genetic stability were found.

Interploidal hybridization and mating patterns in the Sphagnum subsecundum complex.

Polyploidization is thought to result in instant sympatric speciation, but several cases of hybrid zones between one of the parental species and its polyploid derivative have been documented. Previous work showed that diploid Sphagnum lescurii is an allopolyploid derived from the haploids S. lescurii (maternal progenitor) and S. subsecundum (paternal progenitor). Here, we report the results from analyses of a population where allodiploid and haploid S. lescurii co-occur and produce sporophytes. We tested (i) whether haploids and diploids form hybrid triploid sporophytes; (ii) how hybrid and nonhybrid sporophytes compare in fitness; (iii) whether hybrid sporophytes form viable spores; (iv) the ploidy of any viable gametophyte offspring from hybrid sporophytes; (v) the relative viability of sporelings derived from hybrid and nonhybrid sporophytes; and (vi) if interploidal hybridization results in introgression between the allopolyploid and its haploid progenitor. We found that triploid hybrid sporophytes do occur and are larger than nonhybrid sporophytes, but exhibit very low germination percentages and produce sporelings that develop more slowly than those from nonhybrid sporophytes. All sporophytes attached to haploid gametophytes were triploid and were sired by diploid males, but all sporophytes attached to diploid gametophytes were tetraploid. This asymmetric pattern of interploidal hybridization is related to an absence of haploid male gametophytes in the population. Surprisingly, all sporelings from triploid sporophytes were triploid, yet were genetically variable, suggesting some form of aberrant meiosis that warrants further study. There was limited (but some) evidence of introgression between allodiploid and haploid S. lescurii.

Three-genome mosses: complex double allopolyploid origins for triploid gametophytes in Sphagnum.

This paper documents the occurrence of allotriploidy (having three differentiated genomes) in gametophytes of two Southern Hemisphere Sphagnum species (S. australe, S. falcatulum). The pattern of microsatellite alleles indicates that both species are composed of a complex of allodiploid and allotriploid gametophytes, with the latter resulting from two allopolyploidization events. No haploid (n = x) gametophytes were found for either species. The ploidal levels suggested by the pattern of microsatellite alleles were confirmed by flow cytometry and Feulgen DNA image densitometry. For both S. australe and S. falcatulum, the respective allodiploid plants (or their ancestors) are one of the parent species of the allotriploid plants. This is the first report of triploidy in Sphagnum gametophytes occurring in nature and also the first report of the presence of three differentiated genomes in any bryophyte. It is also the first report of intersectional allopolyploidy in Sphagnum, with S. australe appearing to have parental species from Sphagnum sections Rigida and Sphagnum, and S. falcatulum having parental species from Sphagnum sections Cuspidata and Subsecunda. In both species, the allotriploid cytotypes were the most prevalent cytotype on the South Island of New Zealand. The pattern of microsatellite alleles shows the presence of two genetically distinct populations of allodiploid S. australe, possibly indicating multiple origins of polyploidy for that allodiploid cytotype. Morphological evidence is also highly indicative of recurrent polyploidy in the allotriploid cytotype of S. falcatulum. Allopolyploidy has clearly played a major evolutionary role in these two Southern Hemisphere taxa. This study, in conjunction with other recent research, indicates that allopolyploidy is a common, if not the predominant, form of polyploidy in Sphagnum.

Comparative analysis of cancer-associated antigen CA-195, CA 19-9 and carcinoembryonic antigen in diagnosis, follow-up and monitoring of response to chemotherapy in patients with gastrointestinal cancer.

To establish further the clinical significance of the CA-195 tandem immunoradiometric assay in gastro-intestinal malignancies, the sera of a total of 222 subjects have been analysed and compared with assays of the "classical gastrointestinal tumour markers", CA19-9 and carcinoembryonic antigen (CEA). CA-195 elevations above normal (greater than 10 U/ml) were noted in 51/72 (70.8%) colorectal, 15/15 (100%) pancreatic, and in 6/12 (50%) gastric cancer patients. Whereas CA19-9 was increased (greater than 37 U/ml) in 65%, 93%, and 42% of cases, only 54% colorectal, 45% pancreatic, and 42% gastric cancer patients had pathologically elevated serum CEA levels (greater than 5 ng/ml). No abnormal increase of both CA-195 and CA19-9 was found in healthy volunteers, whereas 3/20 (smoking) individuals had CEA levels slightly above normal. With a 29% false-positive rate noted among 103 patients with benign gastrointestinal disorders, the specificity of CA-195 was superior to that of CA19-9 (58%) and comparable with that of CEA (31%). A significant correlation between CA-195 levels and the clinical/pathological stage of disease was noted in colorectal (P less than 0.01) and pancreatic cancer patients (P less than 0.007). Preliminary results of serial measurements of CA-195 in colorectal cancer suggest that this new marker protein, which has no cross-reactivity with CEA, may be useful as a non-invasive test for postoperative surveillance of patients to detect disease recurrence, and serve to complement (though certainly not replace) standard clinical measurements of response to chemotherapy.

Comparative analysis of CA72-4, CA195 and carcinoembryonic antigen in patients with gastrointestinal malignancies.

To establish further the clinical significance of the novel quantitative immunoradiometric assay system CA72-4 in patients with gastric and other digestive tract malignancies, the sera of a total of 208 subjects have been analysed and levels of carcinoembryonic antigen (CEA) and another promising new tumour marker, CA195, have been compared. Twenty patients had gastric (GC), 60 colorectal (CC), and 14 pancreatic carcinomas (CC); 94 patients had benign disorders, and 20 were healthy volunteers. CA72-4 elevations above normal (greater than 4 U/ml) were observed in 6 (30%) GC, 17 (28%) CC, and 8 (57%) PC patients. CA195 appeared more sensitive and was increased (greater than 10 U/ml) in 35% GC, 70% CC, and in 100% PC patients; CEA levels above normal (greater than 5 ng/ml) were noted in 35%, 45%, and 66% of patients respectively. CA72-4 had a rather high specificity and was increased in only 6/94 (6%) patients with benign diseases, whereas CA195 had a false positive rate of 23%, and CEA of 33%. Among 20 healthy donors, none had elevated levels of CA72-4 or CA195, but marginal elevations of CEA were noted in 3 smokers. Despite some advantage of the new tumour marker CA72-4 in terms of specificity, its value as a serodiagnostic test in gastrointestinal cancer patients seems inferior to that of CA195 and CEA.

A study of various strategies to enhance the cytotoxic activity of 5-fluorouracil/leucovorin in human colorectal cancer cell lines.

Several different strategies to improve the in vitro cytocidal effect of 5-fluorouracil/leucovorin (5FU/LV), including modulation of dosage and schedule and combination with other cytotoxic agents or biochemical modulators, were examined in the COLO 320DM and Ht-29 cell lines by means of the Bactec system. Modest enhancement of 5FU activity by coadministration of LV was observed in both human colon cancer cell lines. Neither increased concentrations of LV nor prolonged drug exposure or preincubation with LV were found to enhance significantly the growth inhibitory activity of combined 5FU/LV. The only parameter that was found to affect the killing potential of the combination was the concentration of 5-FU, suggesting that lower doses of the antimetabolite would be more effective (COLO 320DM: P less than 0.003; Ht-29 P less than 0.02). The addition of either cisplatin, hyaluronidase or dipyridamole to 5-FU/LV yielded synergistic growth inhibition in 3/6, 2/6 and 2/6 human colon cancer cell lines, respectively. Strictly additive effects were noted for the combination with BCNU as well as concurrent exposure of the cells to 42 degrees C hyperthermia. Whether or not certain combined 5FU/LV drug regimens will result in an improved therapeutic index, however, remains to be determined in properly designed clinical trials.

In vitro evaluation of the anticancer drug modulatory effect of hyaluronidase in human gastrointestinal cell lines.

In an attempt to establish whether the combination of anticancer drugs with hyaluronidase would result in enhanced cytotoxicity, we have tested a range of 6 continuous cell lines against 4 different chemotherapeutic drugs with or without the addition of various concentrations of the enzyme. Measurement of cytotoxic drug effects has been performed using the Bactec system, a new semiautomated radiometric technique. In only 15 of a total of 144 experiments (11%) was a significant hyaluronidase-mediated potentiation of the single agents' activity seen. In the large majority of experiments, the antiproliferative effect of the combined treatment was classified as additive or subadditive, while in 23% it was antagonistic. Evaluation of the drug modulatory mechanism of hyaluronidase suggested that the combined drug-hyaluronidase effects were independent of the nature of the drug, the exposure mode and the concentration of the enzyme employed. Among the various tumor cell lines tested there was a marked heterogeneity in the sensitivity to the combined effect (P less than 0.0001). In summary, we have not been able to confirm the promising results of early reports of in vitro and in vivo enhancement of the cytotoxicity of antitumor agents by hyaluronidase. Our data emphasize the need for further controlled clinical studies in order to prove or disprove this new therapeutic approach.

Application of an optical immersion-gel in a flow cytometer with horizontally oriented objective.

In certain flow cytometry systems, it is desirable to use immersion optics to obtain optimum fluorescence yield. This is important when propidium iodide and other DNA fluorochromes are used that have weaker fluorescence emission compared to DAPI, when a lamp is used instead of a laser and when the DNA concentrations are low. Our Partec PA II with a horizontally oriented objective and a vertically oriented flow chamber precludes using a liquid immersion medium. The problem was solved using an optical gel with appropriate characteristics. This gel is commercially available and commonly used for connecting glass fiber cables, but has never been used for microscopy before. Compared to the manufacturer's objective (40 x, aperture 0.8), the fluorescence yield was improved approximately four-fold using the optical gel and a 40 x glycerol objective (aperture 1.25). This innovation widens the applicability of flow cytometers with horizontally oriented objectives and vertical flow chambers. We expect it to facilitate the use of propidium iodide as a DNA stain, especially when interspecific genome size comparisons are to be done and base ratio dependent bias must be avoided.

Patterns, sources and ecological implications of clonal diversity in apomictic Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae).

Sources and implications of genetic diversity in agamic complexes are still under debate. Population studies (amplified fragment length polymorphisms, microsatellites) and karyological methods (Feulgen DNA image densitometry and flow cytometry) were employed for characterization of genetic diversity and ploidy levels of 10 populations of Ranunculus carpaticola in central Slovakia. Whereas two diploid populations showed high levels of genetic diversity, as expected for sexual reproduction, eight populations are hexaploid and harbour lower degrees of genotypic variation, but maintain high levels of heterozygosity at many loci, as is typical for apomicts. Polyploid populations consist either of a single AFLP genotype or of one dominant and a few deviating genotypes. genotype/genodive and character incompatibility analyses suggest that genotypic variation within apomictic populations is caused by mutations, but in one population probably also by recombination. This local facultative sexuality may have a great impact on regional genotypic diversity. Two microsatellite loci discriminated genotypes separated by the accumulation of few mutations ('clone mates') within each AFLP clone. Genetic diversity is partitioned mainly among apomictic populations and is not geographically structured, which may be due to facultative sexuality and/or multiple colonizations of sites by different clones. Habitat differentiation and a tendency to inhabit artificial meadows is more pronounced in apomictic than in sexual populations. We hypothesize that maintenance of genetic diversity and superior colonizing abilities of apomicts in temporally and spatially heterogeneous environments are important for their distributional success.

Genome size variation in Arachis hypogaea and A. monticola re-evaluated.

Genome size variation within species is a frequently reported, but still a controversial problem. In the present study, we re-evaluated recently published Feulgen densitometric data on genome size and its infraspecific variation in Arachis hypogaea, and also conducted measurements in one accession of its wild relative A. monticola. The methods applied were propidium iodide flow cytometry and Feulgen densitometry using Pisum sativum as an internal standard. The 2C DNA contents previously published cannot be confirmed, but values obtained in this study are about half as large. Additionally, we could not reproduce the previously reported 1.15-fold variation within A. hypogaea; our data indicate genome size stability between respective accessions of this species. Based on 8.84 pg (2C) for Pisum sativum the DNA amounts (2C) were: 5.914 pg in A. hypogaea, and 5.979 pg in A. monticola.

Genome size in Arachis duranensis: a critical study.

Arachis duranensis is a diploid wild relative of the tetraploid cultivated peanut Arachis hypogaea. The literature indicates two 2C genomic DNA mean values (genome size) for A. duranensis, 4.92 and 5.64 pg, and intraspecific variation of up to 11% negatively correlated with altitude above sea level of the collection sites has been reported. Our recent investigations of Arachis species have shown that unrecognized technical problems with peanut material may have influenced previous genome-size data and rendered them open to critical comments. In the present study, 20 accessions of A. duranensis were investigated by means of DNA flow cytometry (propidium iodide staining) and several of these also by Feulgen DNA image analysis. Pisum sativum was used as the internal standard (2C = 8.84 pg). 2C values in A. duranensis were about half those described previously and varied between 2.49 and 2.87 pg (flow cytometry). This variation was statistically significant and reproducible. There was a negative correlation of genome size with latitude and altitude above sea level of the collection sites. Such a correlation had been already found in one of the previous studies. However, the incongruences between the absolute DNA content values obtained in the present investigation and those in the literature point to the importance of carrying out methodological studies on best practice in DNA-content determinations in plants.

Search for improved culture conditions for clonogenic growth of human colorectal cancer cells in vitro.

In order to analyze and define potentially better growth conditions for colonic stem cell proliferation, we chose four established human colorectal cancer cell lines that differed in biologic cell properties. We studied variables of standard cloning conditions including culture medium, serum supplement, solidifying agent, addition of specific growth factors and use of capillaries as an alternative culture vessel. While modulation of serum concentration as well as use of various standard formulations of culture base media did not result in a reproducible increase of plating efficiencies (PEs), a significant increase in colony formation (when compared to the conventional assay procedure) was achieved; by use of 0.3% agarose or boiled agar as semisolid matrix and by culturing of cells in enriched 'GMF medium'. Specific growth factors, such as EGF or glucagon resulted in "occasionally better" in vitro growth. This suggests a retention of the ability of cells in culture to respond to physiologic regulators of growth. To verify and extend these initial results obtained with continuous cell lines, growth enhancing modifications of the original cloning technique were subsequently applied to in vitro growth of 15 human colorectal cancer specimens obtained directly from patients. Specimens that grew 30 or more colonies under standard plating conditions displayed a more than two-fold increase in PEs which was reproducible for the two specific variables mentioned above, but the overall success rate of the assay could not be improved. In addition to the possibility that several deficient basic requirements for achieving optimal environmental conditions for colonic stem cell growth have not been defined, we believe a major reason for failing to improve the number of drug-assayable specimens is related to an inherent interneoplastic diversity in terms of growth requirements of human colorectal malignancies.

Application of a new radiometric system for identification of potentially useful drug combinations for treatment of human gastrointestinal adenocarcinoma.

As an alternative to empirical clinical evaluation of combined drug effects in human tumors, and in an attempt to establish whether the combination of mitoxantrone (DHAD) with other standard drugs would be of any benefit to the patient with advanced gastrointestinal cancer, we have examined the results of simultaneous single-agent testing in vitro in a panel of 8 human colorectal (HCC) and 5 gastric cancer (HGC) cell lines. Cytotoxic drug effects were measured by the use of a new semiautomated radiometric technique (Bactec system), and were quantitated with attention to potentially clinically relevant plasma concentrations. Among several different drug combinations tested, maximal synergistic cell kill was found for DHAD + 5-fluorouracil; continuous incubation of the cells at 1/100 of the peak plasma concentration achievable in humans yielded in vitro responses in 8/8 HCC and 5/5 HGC cell lines. With regard to our results of single-agent testing, our finding of a significant level of in vitro arabinoside-C activity, using prolonged exposure at an in vitro dose corresponding to a clinical high-dose regimen, may provide a rational basis for (re)evaluation of the compound in gastrointestinal cancer patients.

[In vitro evaluation of the chemosensitivity of malignant gastrointestinal tumors by stem cell assay]. / In-vitro-Evaluierung der Chemosensitivität maligner gastrointestinaler Tumoren mittels des Stammzell-Assays.

The Human Tumor Stem Cell Assay, originally described by Hamburger and Salmon, was shown to be a useful in-vitro technique for predicting response or lack of response in individual patients' tumors. In the present study 34 GI-tumors were assayed for evaluation of in-vitro growth characteristics and sensitivity-patterns to standard chemotherapeutic drugs as well as to recombinant interferon alpha-2(rIF). Sufficient growth for evaluation of anticancer drug activity (greater than 30 colonies/control plate) was obtained in 56% of specimens: 2/9 colorectal, 0/3 stomach, 0/3 pancreatic tumors and 1/4 hepatomas revealed a 50% (or more) decrease of TCFUs, that was considered the minimum for in-vitro efficacy. Our results suggest a very limited overall activity of rIF in gastrointestinal malignancies. Only 1 pancreatic cancer (of 18 evaluable specimens) showed a significant decrease of colony formation (70%), when 100 U of interferon/ml were added to the culture system.

Effect of recombinant interferon-alpha2C on reticuloendothelial function in patients with thrombocytosis.

The aim of the study was to investigate the influence of recombinant interferon-alpha 2C (rIFN-alpha 2C) on the in vivo Fc-dependent phagocytic activity of the reticuloendothelial (RE) system. Fourteen patients with excessive thrombocytosis due to myeloproliferative disorders were studied before and 3 months after initiation of therapy. RE function was determined by measuring the clearance of autologous red blood cells (RBC) labeled with 51Cr and sensitized with anti-D antibody. Eleven of the 14 patients responded to rIFN-alpha 2 treatment (platelets, less than 440 X 10(9)/liter). Rather in contrast to a shortening of platelet half-life and an increase (trendwise) in platelet-bound IgG, rIFN-alpha 2 caused a significant impairment of RE function. Although this finding could in part be accounted for by the treatment-related decrease in splenic volume, statistical analysis revealed a direct influence of rIFN-alpha 2 on RBC clearance (p less than 0.01). Our study results might be explained by an interferon (IFN)-induced, intensified expression of Fc receptors on platelet (and leukocyte) surfaces, possibly enhancing unspecific binding of IgG to their cellular membranes. The subsequent increased platelet uptake may lead to an overloading of the RE system causing impaired reactions to additional stimuli such as IgG-coated RBC.

In vitro phase II trial of recombinant interferon alpha-2 in gastrointestinal cancer.

The antitumor effects of recombinant interferon alpha-2 (rIF) on clonogenic tumor cells were investigated in 29 cases of gastrointestinal cancer. An in vitro response (greater than or equal to 50% inhibition of tumor colony-forming units) was observed in 17% of the tumors, including 2 of 8 pancreatic, 2 of 6 gastric, and 1 of 10 colon cancer specimens. The relative efficacy of rIF in tissue cultures of pancreatic and gastric tumors was further substantiated by the resistance against simultaneously tested single conventional cytostatic drugs. Preliminary results of comparative studies of cloned interferon alpha-2 and human purified leukocyte interferon (hlIF) in 2 human colon cancer cell lines and 11 fresh tumor specimens suggest similar trends in terms of colony inhibition in individual assays. However, the interpatient differences indicate an overall superiority of the natural preparation (P less than 0.02).

[Prognosis of malignant tumors of the exocrine pancreas: effect of clinical and pathologico-anatomic variables on patient survival]. / Prognose maligner Tumore des exokrinen Pankreas: der Einfluss klinischer und pathologisch-anatomischer Variabler auf die Uberlebenszeit der Patienten.

Survival patterns of 112 patients with histologically ascertained pancreatic cancer were analysed retrospectively in an attempt to determine the relationship to various clinical and pathologic-anatomic prognostic factors. Stage of disease, localisation of the primary tumor, as well as histologic grade were found to influence the patient's survival significantly. A limited anatomic involvement with tumor, localisation within the head of the pancreas, and high-grade differentiation were associated with an increased median survival. An evaluation of non-anatomic prognostic variables suggested a relative survival advantage for females, younger age groups, patients with favourable initial performance status and short symptom duration. There was no obvious relationship between survival and qualitative or quantitative indication of weight loss. In order to permit a critical evaluation of the value of any treatment program in pancreatic cancer, our observations reemphasize the need to define characteristics of patients under study.