Reconstitution of a bacterial/plant polyamine biosynthesis pathway in Saccharomyces cerevisiae.

Polyamine synthesis in most organisms is initiated by the decarboxylation of ornithine to form putrescine via ornithine decarboxylase (ODC). Plants, some bacteria and some fungi and protozoa generate putrescine from arginine, via arginine decarboxylase (ADC) and agmatine ureohydrolase (AUH) or agmatine iminohydrolase. A polyamine-requiring strain of Saccharomyces cerevisiae with a mutation in the gene encoding ODC was transformed with plasmids bearing genes encoding Escherichia coli ADC and AUH. Transformants regained the ability to grow in the absence of exogenous polyamines and contained enzyme activities consistent with the presence of both prokaryotic enzymes. Similar results were obtained when a plasmid containing a gene encoding oat (Avena sativa L.) ADC was substituted for the E. coli gene. These data demonstrate the successful complementation of a yeast biosynthetic polyamine synthesis defect by genes encoding an alternative pathway found in bacteria; they also show that plant ADC can substitute for the bacterial enzyme in this pathway. The recombinant yeast provides a tool for the study of the functional properties of these enzymes and for discovery of compounds that specifically inhibit this pathway.

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae.

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.

Arginine decarboxylase (polyamine synthesis) mutants of Arabidopsis thaliana exhibit altered root growth.

Putrescine and polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase; the other with ornithine decarboxylase. The authors developed an in vivo screening strategy to identify mutants with low levels of arginine decarboxylase activity. The screen requires both a primary screen of the M2 generation and a secondary screen of the M3 generation. The method used was to screen 15,000 EMS-mutagenized M2 seedlings for low levels of arginine decarboxylase (ADC) activity and identified seven mutants that fall into two complementation groups. These mutants have from 20% to 50% of wild-type enzyme activity. Morphological alterations common among the mutants include increased levels of lateral root branching. The authors obtained a double mutant combining the alleles with the lowest activities from the two complementation groups; this has lower ADC enzyme activity and putrescine levels than either of the single mutants. The double mutant has highly kinked roots that form a tight cluster; it also has narrower leaves, sepals, and petals than either single mutant or wild-type, and delayed flowering. These results suggest there may be more than one ADC gene in Arabidopsis, and that ADC and polyamine levels play roles in root meristem function and in lateral growth of leaf-homolog organs.

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

Regulation of Arabidopsis thaliana (L.) Heynh Arginine decarboxylase by potassium deficiency stress.

Arginine decarboxylase (ARGdc) is the first enzyme in one of the two pathways to putrescine in plants. ARGdc enzyme activity has been shown to be induced by many environmental factors, including potassium deficiency stress. We investigated the mechanism for induction of ARGdc activity during potassium deficiency stress in Arabidopsis thaliana (L.) Heynh. We show that A. thaliana responds to potassium deficiency stress by increasing ARGdc activity by up to 10-fold over unstressed plants with a corresponding increase in putrescine levels of up to 20-fold. Spermidine and spermine levels do not increase proportionately. Northern analysis showed no increase in ARGdc mRNA levels correlated with the increase in ARGdc enzyme activity. Western analysis revealed that there was no difference between ARGdc protein levels in stressed plants compared with controls. The increase in ARGdc enzyme activity due to potassium deficiency stress does not appear to involve changes in mRNA or protein abundance.

Lung cancer in West Sweden 1976-1985. A study of trends and survival with special reference to surgical treatment.

We reviewed 3 285 consecutive cases of lung cancer diagnosed in West Sweden during the period 1976-1985. Data were collected from the regional cancer registry, the Swedish National Population Registry, and medical records. During the study period, the annual female/male ratio increased from 0.29 to 0.42. In females, there was an increase primarily in the incidence of tobacco-related morphologic tumour types (i.e. squamous and small cell lung cancers). In males, a moderate increase of adenocarcinomas was seen, although squamous cell cancer remained the most common tumour type. The overall 5-year survival rate was 8.3%. In 641 patients (20%) a surgical tumour resection was carried out. The 5-year survival rate following resection was 38%, and the probability of 10-year survival was estimated at 25%. In a multifactorial model including gender, age, histology, pTNM stage and extent of resection, pTNM stage and, to a lesser degree, age were statistically significant independent predictors of postoperative survival. The five-year survival was 57% in stage 1, 21-27% in stage II and IIIa, and 10% in stage IIIb. Of all resected patients, 4.2% died within two months after resection. In males, early postoperative mortality was predicted by preoperative bicycle ergometry. The prognosis in non-resected patients was poor, with only 2% surviving 5 years or longer. In conclusion, the results indicate that some progress has been made with regard to surgical management of lung cancer, but they also point to the fact that the vast majority of patients are not amenable to curative treatments, and that the overall survival in lung cancer has improved only marginally during the last decades.

Arginine decarboxylase of oats is activated by enzymatic cleavage into two polypeptides.

Oat arginine decarboxylase is synthesized as a 66-kDa proenzyme, but the soluble enzyme is found in oats as a complex of 42- and 24-kDa polypeptide fragments, both derived from the 66-kDa precursor. We report here that this proteolytic cleavage is the result of a processing enzyme, distinct from arginine decarboxylase itself, that leads to activation of the arginine decarboxylase. The proteolysis is resistant to a standard set of protease inhibitors, but is inhibited by high concentrations of Zn2+, as is the activation of arginine decarboxylase. Agmatine, putrescine, spermidine, and spermine, as well as the arginine decarboxylase inhibitor difluoromethylarginine, each had no effect on the reaction. Oat arginine decarboxylase is thus similar to some other amino acid decarboxylases in requiring a proteolytic cleavage for activation; however, it differs in that the other examples are auto-catalytic self-processing.

Arginine decarboxylase of oats is clipped from a precursor into two polypeptides found in the soluble enzyme.

We have examined soluble oat (Avena sativa) arginine decarboxylase by probing its structure with polyclonal antibodies that separately recognize amino-terminal and carboxyl-terminal antigens and with a monoclonal antibody that immunoprecipitates enzyme activity. These experiments indicated that oat arginine decarboxylase is clipped from a 66,000-D precursor polypeptide into 42,000- and 24,000-D produce polypeptides. Both of these are found in the enzyme and may be held together by disulfide bonds. A full-length precursor protein could not be detected in plants but could be produced by expression of the cDNA in Escherichia coli. Analysis of the expression of the cDNA in E. coli, with antibodies and using pulse labeling with [(35)S]methionine, indicated that the bulk of the expressed protein was the full-length 66,000-D form. Small amounts of 42,000- and 24,000-D polypeptides could also be detected. A reconstruction experiment, adding a radioactively labeled full-length protein isolated from E. coli to powdered oat leaves, supported the idea that the protein extraction method used for western blots was not likely to result in artifactual proteolytic degradation.

Rates of corolla growth in tobacco determined with the plastochron index.

The growth of vegetative and reproductive shoots of Nicotiana tabacum L. cv. Xanthi is analyzed with the plastochron index to estimate the relationship between corolla growth and time. The plastochron of leaves 9 through 20 declines steadily at each successive node. The flower plastochron increases steadily during the growth of an individual cyme, with the most distal flower to open having the longest plastochron. Variation in the flower plastochron is the result of variation in the rate of flower initiation, not the growth rate of individual flowers. The corolla has an extended phase of approximately constant relative growth in length (between 0.2 · d(-1) and 0.3 · d(-1)) until a peak of growth (0.5 · d(-1)) 2-3 d before anthesis. Corollas also have periodic peaks and troughs of growth that are low in amplitude (0.1 · d(-1)), but persist throughout most of corolla development. The pattern of corolla expansion contrasts strongly with earlier reports of the pattern of tobacco leaf growth.

Developmentally regulated antigen associated with calcium crystals in tobacco anthers.

We have found and characterized an antigen associated with crystal-containing cells in the stomium and connective tissue of the anthers of Nicotiana tabacum L. (tobacco). The antigen, defined by the monoclonal antibody NtF-8B1, localizes to subcellular regions surrounding the crystals. At the light-microscope level, the antigen is detectable just after the first appearance of crystals in the connective tissue of the anther, and at approximately the same time as the appearance of crystals in the stomium. The antigen is not detectable on a Western blot, and gave inconclusive results on a test of periodate sensitivity. It is not the crystals themselves, nor is the presence of the crystals required for antibody recognition. The antigen is sensitive to heat and protease treatment, indicating that it is a protein. The antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Chemical tests indicate that the druse crystals in the stomium are calcium oxalate.

Analysis of a cDNA encoding arginine decarboxylase from oat reveals similarity to the Escherichia coli arginine decarboxylase and evidence of protein processing.

Arginine decarboxylase is the first enzyme in one of the two pathways of putrescine synthesis in plants. We purified arginine decarboxylase from oat leaves, obtained N-terminal amino acid sequence, and then used this information to isolate a cDNA encoding oat arginine decarboxylase. Comparison of the derived amino acid sequence with that of the arginine decarboxylase gene from Escherichia coli reveals several regions of sequence similarity which may play a role in enzyme function. The open reading frame (ORF) in the oat cDNA encodes a 66 kDa protein, but the arginine decarboxylase polypeptide that we purified has an apparent molecular weight of 24 kDa and is encoded in the carboxyl-terminal region of the ORF. A portion of the cDNA encoding this region was expressed in E. coli, and a polyclonal antibody was developed against the expressed polypeptide. The antibody detects 34 kDa and 24 kDa polypeptides on Western blots of oat leaf samples. Maturation of arginine decarboxylase in oats appears to include processing of a precursor protein.

Alternative pathways of tobacco placental development: time of commitment and analysis of a mutant.

Previous work from our lab identified mutants, Mgr3 and Mgr9, of tobacco (Nicotiana tabacum) that produced unusual elongated green outgrowths from placentae in vivo. Similarly appearing stigmatoid growths were described developing from some in vitro cultures of excised placentae of tobacco (Hicks and McHughen, 1974, 1977). Here we report a developmental analysis and comparison of the unusual stigmatoid outgrowths seen in in vitro cultures of wild-type and mutant placentae, as well as the green outgrowths seen in vivo in the mutants. The growths produced by wild-type and mutant placental cultures in vitro, and the growths produced by the mutants in vivo, are identified as abnormal stigmas and styles. Wild-type in vitro placental cultures also produce outgrowths identified as homologs of whole carpels. Carpel fusion is not required for differentiation of stigma, style, transmitting tract, vascular traces, ovary, and ovules in these structures. The type and extent of stigmatoid growth production depends upon the age of the explant at excision and culture initiation. Before ovule primordium initiation, few growths are seen in culture; for a short window of time afterward, the primordia are competent to give rise to stigmatoid and carpelloid growths when cultured. After commitment to ovule development occurs, the primordia produce only ovules when cultured. The behavior of the mutant placental cultures is dimorphic. Explants from early stages behave similarly to wild-type when cultured, but differences between wild-type and the mutant behaviors in culture arise at the time when the stigmatoid growths begin to appear in vivo in the mutants. These results imply that ovule primordia pass through stages of distinct sequential restrictions of fate, first to growth as gynoecia, and then second to growth as ovules. The mutant strains described here perturb the commitment to differentiation as ovules.

Polyamine levels in petunia genotypes with normal and abnormal floral morphologies.

We characterized the polyamine pathway in Petunia hybrida genotypes that were either wild type or that had been identified as having altered floral morphology. Analysis of four normal morphology lines revealed two patterns of endogenous levels of putrescine and arginine decarboxylase: two with higher levels of putrescine, two with lower levels of putrescine. Analysis of F1 and backcross progeny between high putrescine and low putrescine strains is consistent with their differences being due to a dominant allele for low putrescine content and arginine decarboxylase activity. Four Petunia mutants with floral morphology changes were also screened. One of these mutants, alf, showed high levels of putrescine and high levels of arginine decarboxylase late in development; these high levels were found whether the alf line was present in either of the two types of normal morphology genetic backgrounds that had been characterized.

Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis.

Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium.

Biochemical differentiation in the tobacco flower probed with monoclonal antibodies.

We have isolated a series of monoclonal antibodies that react to antigens in flowers of Nicotiana tabacum L. (tobacco) displaying specificity or preferentiality in their cell and tissue distributions. We immunized mice with extracts from tobacco flowers and then screened the hybridomas by enzyme-linked immunosorbent assay (ELISA) against extracts from leaves, sepals, petals, stamens and pistils; twenty five were chosen from the total screened. The antigens detected by about half of the antibodies were periodate-sensitive, implying that the epitopes were carbohydrate. Competition ELISA assays were used to determine if any antibodies were reacting to the same epitopes. Western blot analysis showed that while some antibodies reacted to specific bands, the bulk either failed to react or reacted to multiple bands, consistent with a glyco-conjugate nature for many of the antigens. Analysis of the spatial pattern of antigen distribution within tobacco flowers by immunolocalization showed that some antibodies recognized epitopes that were limited to very specific cells and tissues. We used the immunolocalization technique to analyze a mutant with stigmoid anthers: an antibody recognizing a pistil transmitting-tract antigen also reacted to cells in stigmoid anthers. Our results with this antibody set imply that biochemical differentiation within the tobacco flower includes cell-and tissue-specific glyco-moeities, and also that similarities, at the biochemical level, exist between a normal floral organ and the abnormal organ in a phenotype with a developmental switch.

Chemotherapy for adenocarcinoma of the lung (WHO III): A randomized study of vindesine versus lomustine, cyclophosphamide, and methotrexate versus all four drugs.

Two hundred seventy-nine patients with previously untreated nonresectable adenocarcinoma of the lung (ACL) entered a prospective randomized trial, comparing vindesine (VDS) to a combination of lomustine (CCNU), cyclophosphamide (CTX), and methotrexate (MTX), and to a regimen including all four drugs. Response assessment was possible in 218 patients, while 259 were evaluable for survival. Response rates were similar (22%, 23%, and 27%, respectively) as were median durations of response (15 weeks overall) and survival (29 weeks overall). Patients with dose-limiting toxicity had significantly higher response rate and longer survival than patients without toxicity. The major toxicity was peripheral neuropathy with VDS treatment and myelosuppression with the other two regimens. The VDS single-agent activity in ACL was confirmed, but addition of VDS to the three-drug regimen did not increase activity. Future studies of VDS in combination with other active agents, and comparison to a matched control group on supportive care, are indicated.

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase.

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.