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PubMed2005

Legume nodule senescence: roles for redox and hormone signalling in the orchestration of the natural aging process.

Research on legume nodule development has contributed greatly to our current understanding of plant-microbe interactions. However, the factors that orchestrate root nodule senescence have received relatively little attention. Accumulating evidence suggests that redox signals contribute to the establishment of symbiosis and senescence. Although degenerative in nature, nodule senescence is an active process programmed in development in which reactive oxygen species (ROS), antioxidants, hormones and proteinases have key roles. Nodules have high levels of the redox buffers, ascorbate and glutathione, which are important in the nodulation process and in senescence. These metabolites decline with N-fixation as the nodule ages but the resultant decrease in redox buffering capacity does not necessarily lead to enhanced ROS or oxidative stress. We propose models by which ROS and antioxidants interact with hormones such as abscisic acid in the orchestration of nodule senescence.

Antioxidants↗
PubMed2002

Nodule ultrastructure and initial growth of Anadenanthera peregrina (L.) Speg. var. falcata (Benth.) altschul plants infected with rhizobia.

The anatomy and ultrastructure of root nodules of Anadenanthera peregrina var. falcata (Leguminosae-Mimosoideae) were analysed, as was plant growth. To ensure that nodules developed, seedlings were inoculated with a mixture of six strains of rhizobia. Nodules were produced that differed in appearance-and probably also effectiveness-but their structure was similar and they showed characteristics typical of indeterminate nodules, such as persistent meristematic tissue and a gradient of cells at different stages of development. Many starch grains were present in inner cortex cells and interstitial cells of infected tissue. Infected cells were densely packed with bacteroids, which contained many poly-beta-hydroxybutyrate granules. The high incidence of these granules, together with high levels of starch accumulation in interstitial cells, suggested low N2-fixation efficiency of the rhizobia isolates used for inoculation. In the symbiosomes of early-senescent infected cells, reticulum-like structures, small vesicles and a fibrillar material were observed; these may be related to bacteroid degradation. In the cytoplasm of late-senescent infected cells, many vesicles and membrane-like structures were observed, probably associated with membrane degradation of bacteroids and peribacteroids. The total biomass of plants inoculated with rhizobia was low and their xylopodia and shoots had low levels of N compared with noninoculated plants fertilized with ammonium nitrate. However, inoculated plants did not show N-deficiency symptoms and grew better than non-inoculated plants without N fertilization. These growth results, together with ultrastructural observations of nodules, suggest that nitrogen fixation of rhizobia isolates associated with Anadenanthera peregrina var. falcata roots is poor.

Biomass↗
PubMed2006

A retrospective cohort study of Southeast Asian patients with large congenital melanocytic nevi and the risk of melanoma development.

BACKGROUND: The lifetime risk of developing melanoma in Caucasian patients with large congenital melanocytic nevi (LCMN) is estimated to be between 4.5% and 10%. Cohort studies of LCMN and the risk of melanoma development in an Asian population are not available. OBJECTIVE: We sought to determine the risk of melanoma development in a retrospective cohort of patients presenting with LCMN to a dermatology tertiary referral center in Singapore from January 1989 to December 2004. METHODS: Patients with congenital melanocytic nevi (CMN) that covered at least 5% of the body surface area were included in the study. Data were obtained from electronic records and photographic documentation. A search for malignancy was done using the National Cancer Registry database. RESULTS: In all, 39 patients (23 male and 16 female) met the study criteria of LCMN; 15 of 39 patients also met the criteria of having a giant CMN (ie, a CMN that is predicted to be at least 20-cm diameter in adulthood). There were 29 Chinese, 6 Malay, 1 Indian, and 3 Caucasian patients. Their ages ranged from 23 months to 60 years (mean 18.8 years). They presented at a mean age of 26 months and were followed up for an average of 16.9 years. The size of the LCMN ranged from 5% to 40% of body surface area, with a mean of 12.2%. The most common sites were the back (54%), lower limb (28%), and abdomen (26%). Satellite lesions were present in 22 patients. Magnetic resonance imaging of the head or thoracolumbar spine was performed in 7 patients with LCMN on the scalp/face or back, respectively; all produced normal findings. Only one patient was treated: he had carbon-dioxide laser ablation and Q-switched neodymium:yttrium-aluminum-garnet laser treatment of a small part of his LCMN. Skin biopsies were done in 5 patients who had developed nodules; histology showed no evidence of malignancy. No patients had developed any form of malignancy. LIMITATIONS: The addition of 3 adult patients born before the start of the cancer registry may have led to survivor bias. The small sample size did not allow a precise estimate of the risk of melanoma development in our study population. CONCLUSION: The risk of melanoma development in LCMN within a predominantly Southeast Asian cohort appears to be very low. Prophylactic complete excision of LCMN is ideal, but seldom achievable. Hence, patient education, regular melanoma surveillance, and biopsy of suspicious lesions are very important.

Adolescent↗
PubMed2002

Evidence of perchlorate (ClO4-) reduction in plant tissues (poplar tree) using radio-labeled 36ClO4-.

Phytoremediation of perchlorate (ClO4) by poplar trees Populus deltoidex nigrawas investigated using small cuttings growing in hydroponic Hoagland solution and plant tissue cultures, consisting of spherical photosynthetic cell aggregates (i.e. nodules) developing in Murashige and Skoog culture medium. Both plants and nodules were grown under a 16 h/8 h photoperiod cycle and under sterile conditions. Degradation experiments, performed by the incubation of pregrown plants and nodules in the presence of 36Cl radio-labeled ClO4- (25 mg L(-1)), showed a reduction of the initial ClO4- concentration in the solution of about 50% after 30 d of incubation. Analysis of the distribution of radioactivity in different plant fractions indicated that 27.4% of the total was translocated to the leaves, while 66.9% remained in the solution. Very little radioactivity (less than 3.0%) was detected in the other parts of the plants. 32.0% of the radioactivity recovered in the solution was shown to consist of 36Cl- and 68.0% of nontransformed 36ClO4-. The radioactivity recovered in the leaf extracts was distributed as chloride (36Cl-) (1.6% of the total), chlorite (36ClO2-) (2.4%), chlorate (36ClO3-) (4.8%), nontransformed 36ClO4- (21.6%), and an unidentified organic compound (1.4%). The radioactivity recovered in the solution containing submerged nodules consisted of 36Cl- (6.4% of the total), 36ClO3- (1.3%), and nontransformed 36ClO4- (51.5%). Radioactivity detected in the nodule extracts was distributed as 36Cl- (2.0% of the total), 36ClO2- (5.2%), 36ClO3- (6.4%), 36ClO4- (22.7%), and an unidentified organic compound (0.5%). These results provide evidence of perchlorate reduction inside poplar tree tissues. 36ClO4- is partially reduced to 36ClO3-, 36ClO2-, and 36Cl-.

Biodegradation, Environmental↗
PubMed1988

Developmental regulation of nodule-specific genes in alfalfa root nodules.

We have cloned alfalfa nodule-specific cDNAs that code for leghemoglobin (Lb), glutamine synthetase (GS), and three unidentified nodulins. Hybrid-select translation of nodule RNA followed by 2-D gel electrophoresis showed that the Lb-specific cDNA corresponded to at least four Lb species of 12 kDa. One of the unidentified cDNA clones (N-32/34) corresponded to at least five polypeptides of 32-34 kDa; a second unidentified cDNA clone (N-14) corresponded to an individual polypeptide of 14 kDa. The in vitro translation product(s) of the RNA hybrid selected by the third unidentified cDNA clone (N-22) formed a single band at 22 kDa on a one-dimensional gel. Northern and dot blot analyses of RNA isolated from wild-type nodules and from defective nodules elicited by a variety of Rhizobium meliloti mutants showed that 1) RNAs corresponding to the Lb, nodule-specific GS, and three unidentified nodulins were coordinately expressed during the course of nodule development, and 2) all five nodulins were expressed in Fix- nodules that contained infection threads and bacteroids but were not expressed in nodules that lacked infection threads and intracellular rhizobia.

Base Sequence↗
PubMed2002

Construction of a Sinorhizobium meliloti strain carrying a stable and non-transmissible chromosomal single copy of the green fluorescent protein GFP-P64L/S65T.

A single copy of the green fluorescent protein (GFP)-encoding gene gfp-P64L/S65T under the control of the constitutive nptII promoter was introduced in a neutral region of the Sinorhizobium meliloti chromosome, between the genes recA and alaS. Within the same chromosomal region downstream of gfp-P64L/S65T a tetracycline (Tc) resistant cassette was also inserted. Both markers were very stable during at least 40 bacterial generations without any selective pressure. Similarly, the gfp-Tc cassette was stable and functional in all rhizobia that were recovered from alfalfa nodules. The GFP-associated fluorescence derived from the (single copy) chromosomal gfp-P64L/S65T allowed detection of rhizobia during the colonisation of the root, infection thread formation, and nodule development. The gfp-Tc rhizobia showed indistinguishable phenotypes for nodulation, competitiveness, and nitrogen-fixation from the parental strain. The labelling system described here can be used for the stable fluorescent tagging of S. meliloti strains allowing their detection in biologically complex soil environments.

Chromosomes, Bacterial↗
PubMed2006

Cloning and identification of opa22, a new gene involved in nodule formation by Mesorhizobium huakuii.

Using Tn5-sacB insertion mutagenesis, 3000 mutants were obtained from Mesorhizobium huakuii 7653R. Eight nodulation-defective mutants were screened by plant nodulation experiments. The DNA sequences of the contiguous region from the Tn5 insertion site were determined by thermal asymmetric interlaced PCR. A new gene was cloned and designated opa22, as judged from its structural and functional homology. Sequence analysis indicated that opa22 was composed of 774 nucleotides and encoded a protein of 257 amino acids. RPS-BLAST analysis of the Opa22 protein showed a sequence similarity (88.9%) to the opacity protein and related to surface antigens of the bacterial outer membrane, which can mediate various pathogen-host cell interactions and promote invasion. Our results from root hair curling experiments suggested that expression of the opa22 gene might occur at the stage of infection thread formation and nodule development. The complement stain HK24 was able to restore the nodule forming ability of the mutant.

Alphaproteobacteria↗
PubMed1985

Dermal responses to Ostertagia ostertagi in Ostertagia ostertagi- and Cooperia punctata-inoculated calves.

Calves harboring patent Ostertagia ostertagi or Cooperia punctata were given intradermal injections of O ostertagi 3rd-stage larval antigen. The initial injections were followed 30 days later by a 2nd series of injections. Skin thickness was measured at injection sites for 72 hours after injection. Selected injection sites including saline solution control sites were biopsied at 30 minutes, at 3, 24, 48, and 72 hours, and at 30 days after injection. After the 1st series of injections, there was a clear distinction in dermal reactions between O ostertagi-inoculated calves and C punctata-inoculated calves; after 24 hours, reactions were not seen in the C punctata-inoculated calves. Marked dermal reactions occurred in the O ostertagi-inoculated calves. The reactions at 30 minutes and 3 hours were characterized by slight-to-extensive infiltration of neutrophils and dermal edema. The 24-hour cellular reaction was principally due to neutrophil and eosinophil infiltration with edema and necrosis. Reactions at 48 to 72 hours were due to eosinophils and perivascular accumulations of macrophages and lymphocytes. Necrosis, neutrophils, and edema were present in foci where fragments of nematodes were located. On reinjection, a clear distinction in dermal reactions between calves was not seen based on the type of nematode infection. Thirty days after dermal inoculation, large nodules developed at the site of the initial antigen injection. The nodules were characterized by marked intradermal proliferation of lymphocytes in a follicular pattern with occasional macrophages and rare multinucleated giant cells.

Animals↗
PubMed1984

Lymphangiosarcoma in chronic lymphedema that followed dissection of the groin in surgical management of a malignant melanoma.

A 41-year-old white man who had a malignant melanoma in the left lumbar area was treated by wide excision and dissection of the ipsilateral inguinal lymph nodes. Progression and irreversible lymphedema of the left lower extremity developed. Fourteen years after the surgery, numerous purplish plaques and nodules developed on the left leg and thigh. Histologic examination by conventional and electron microscopy yielded the diagnosis of angiosarcoma. The patient died 18 months after the diagnosis was made.

Chronic Disease↗
PubMed2003

Dual genetic pathways controlling nodule number in Medicago truncatula.

We report the isolation and characterization of a new Medicago truncatula hyper-nodulation mutant, designated sunn (super numeric nodules). Similar to the previously described ethylene-insensitive mutant sickle, sunn exhibits a 10-fold increase in the number of nodules within the primary nodulation zone. Despite this general similarity, these two mutants are readily distinguished based on anatomical, genetic, physiological, and molecular criteria. In contrast to sickle, where insensitivity to ethylene is thought to be causal to the hyper-nodulation phenotype (R.V. Penmetsa, D.R. Cook [1997] Science 275: 527-530), nodulation in sunn is normally sensitive to ethylene. Nevertheless, sunn exhibits seedling root growth that is insensitive to ethylene, although other aspects of the ethylene triple response are normal; these observations suggest that hormonal responses might condition the sunn phenotype in a manner distinct from sickle. The two mutants also differ in the anatomy of the nodulation zone: Successful infection and nodule development in sunn occur predominantly opposite xylem poles, similar to wild type. In sickle, however, both infection and nodulation occur randomly throughout the circumference of the developing root. Genetic analysis indicates that sunn and sickle correspond to separate and unlinked loci, whereas the sunn/skl double mutant exhibits a novel and additive super-nodulation phenotype. Taken together, these results suggest a working hypothesis wherein sunn and sickle define distinct genetic pathways, with skl regulating the number and distribution of successful infection events, and sunn regulating nodule organogenesis.

Ethylenes↗
PubMed1994

Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2-acetylaminofluorene (2AAF)-induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N-hydroxy-2AAF sulfotransferase activity was found to decrease 80-90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV activity. In the livers of rats administered only 2AAF (group 1), a general pattern of overall downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF-mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF-induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action.

2-Acetylaminofluorene↗
PubMed1987

Characterization of a slow-growing, transplantable rat mammary tumor (MCR-83): a model for endocrine-related cell kinetic studies.

Out of 24 primary mammary tumors, arising in rats of the WAG/Rij Wistar strain after low dose irradiation, with or without prolonged treatment with estrogen, a slow-growing, well differentiated adenocarcinoma (MCR-83) was selected. This tumor, induced by radiation alone, is independent of estrogen pellets for growth after transplantation into adult female rats, but nontransplantable into males or ovariectomized females. Measurements of tumor growth and contents of both estrogen and progesterone receptors on three successive passages are not indicative of a rapid progression in growth rate or to hormone independency. Ovariectomy and treatment with tamoxifen give a pronounced inhibition of tumor growth, whereas neither methotrexate nor cyclophosphamide is effective. Growth rate is significantly increased when rats are given 17 beta-estradiol. Flow cytometric DNA analysis as well as in situ S-phase cell detection with anti-bromodeoxyuridine antibodies show a 3-fold increase in S-phase fraction cells within 4 days after the onset of estrogen treatment. No spontaneous metastases have been found so far, but lung nodules develop after i.v. inoculation of tumor cells. From one of these nodules a fast-growing, hormone independent subline (MCR-86) has been derived, showing both lymphatic and hematogenous dissemination upon s.c. transplantation. By showing several features of hormone responsive human disease in its early stage of progression the MCR-83 tumor system may be a clinically relevant model for studies on endocrine regulation of tumor growth and its therapeutic manipulation.

Adenocarcinoma↗
PubMed1977

Heme Synthesis in Soybean Root Nodules: I. On the Role of Bacteroid delta-Aminolevulinic Acid Synthase and delta-Aminolevulinic Acid Dehydrase in the Synthesis of the Heme of Leghemoglobin.

During nodulation of soybean (Glycine max) by Rhizobium japonicum, variations in the activities of two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (ALAS) and delta-aminolevulinic acid dehydrase (ALAD) are described. delta-Aminolevulinic acid synthase activity is found in the bacteroid fraction of nodules, but is not detected in the plant fraction. Bacteroid ALAS activity parallels heme accumulation during nodule development. delta-Aminolevulinic acid dehydrase activity is found in both bacteroid and plant cytosol fractions. Bacteroid ALAD activity is constant or increases during nodulation while plant ALAD activity falls.Bacteroid ALAD activity is found in effective, not in inefficient nodules. Plant ALAD activity is found in both effective and inefficient nodules. Plant ALAD activity falls during development of both types of root nodules.These results support the contention that it is the bacteroid ALAS and ALAD activities, not those of the plant, that are directly involved in formation of leghemoglobin heme, suggesting that the bacteroid may be solely responsible for formation of leghemoglobin heme in the nodule symbiosis.

Journal Article↗
PubMed1986

Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules.

The critical problem of oxygen toxicity for nitrogen-fixing organisms may be related to damage caused by oxygen radicals and peroxides. An enzymatic mechanism is described for removal of peroxides in root nodules of soybean (Glycine max). The system utilizes ascorbate as an antioxidant and glutathione as a reductant to regenerate ascorbate. The enzymes involved are ascorbate peroxidase (ascorbate:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1), and glutathione reductase (NADPH:oxidized-glutathione oxidoreductase, EC 1.6.4.2). The reactions are essentially the same as those involving scavenging of H(2)O(2) in chloroplasts. Glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) was not detected. During the course of early nodule development, ascorbate peroxidase and dehydroascorbate reductase activities and total glutathione contents of nodule extracts increased strikingly and were positively correlated with acetylene reduction rates and nodule hemoglobin contents. The evidence indicates an important role of glutathione, ascorbate, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase as components of a peroxide-scavenging mechanism in soybean root nodules.

Journal Article↗
PubMed1995

A mutant Bradyrhizobium japonicum delta-aminolevulinic acid dehydratase with an altered metal requirement functions in situ for tetrapyrrole synthesis in soybean root nodules.

The tetrapyrrole synthesis enzyme delta-aminolevulinic acid (ALA) dehydratase requires Mg2+ for catalytic activity in photosynthetic organisms and in Bradyrhizobium japonicum, a bacterium that can reside symbiotically within plant cells of soybean root nodules or as a free-living organism. ALA dehydratase from animals and other non-photosynthetic organisms is a Zn(2+)-dependent enzyme. A modified B. japonicum ALA dehydratase, ALAD*, was constructed by site-directed mutagenesis of hemB in which three proximal amino acids conserved in plant dehydratases were changed to cysteine residues as is found in the Zn(2+)-dependent enzyme of animals. These substitutions resulted in an enzyme that required Zn2+ rather than Mg2+ for catalytic activity, and therefore a region of the ALA dehydratase from B. japonicum, and probably from plants, was identified that is involved in Mg2+ dependence. In addition, the data show that a change in only a few residues is sufficient to change a Mg(2+)-dependent ALA dehydratase to a Zn(2+)-dependent one. B. japonicum strains were constructed that contained a single copy of either hemB or the altered gene hemB* integrated into the genome of a hemB- mutant. Cultures of the hemB* strain KPZn3 had Zn(2+)-dependent ALA dehydratase activity that functioned in vivo as discerned by its heme prototrophy and expression of wild type levels of cellular hemes. Strain KPZn3 elicited root nodules on soybean that contained viable bacteria and exhibited traits of normally developed nodules, and the symbiotic bacteria expressed nearly wild type levels of cellular hemes. We conclude that the Zn(2+)-dependent ALAD* can function and support bacterial tetrapyrrole synthesis within the plant milieu of root nodules.

Amino Acid Sequence↗
PubMedDate not supplied

Subcutaneous nodules at the injection site of low-molecular-weight heparin: a mimic of metastatic disease at CT.

PURPOSE: To describe the appearance and prevalence of subcutaneous nodules in the anterior abdominal wall seen at CT in cancer patients receiving subcutaneous injections of low-molecular-weight heparin (LMWH). METHODS: CT examinations were reviewed in 426 patients receiving subcutaneous abdominal wall injections of LMWH for the presence of nodules. Nodules were evaluated for contour, maximal diameter, CT attenuation, and presence of hazy changes or air in the surrounding fat. RESULTS: Fourteen (3%) of the 426 patients had nodules attributable to subcutaneous injections of LMWH. Findings included poorly defined borders (100%), adjacent hazy soft tissue changes (100%), adjacent air (57%), and a mean CT attenuation of -4.4 HU (range: -50-40 HU). Three (0.7%) of 426 patients had nodules caused by metastatic disease. One (33%) nodule had poorly defined borders, two (67%) had surrounding hazy changes, one (33%) had air in adjacent tissue, and there was a mean CT attenuation of 44 HU (range: 14-140 HU). One (0.2%) patient had a subcutaneous nodule of uncertain etiology. CONCLUSION: Patients receiving subcutaneous injections of LMWH may develop nodules at the injection sites. Such nodules can resemble metastatic tumor deposits at CT, and careful correlation with clinical history and growth trends of metastatic deposits elsewhere in the patient is needed to avoid misdiagnosis.

Abdominal Muscles↗