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The pea late nodulin gene PsNOD6 is homologous to the early nodulin genes PsENOD3/14 and is expressed after the leghaemoglobin genes.

The pea late nodulin gene PsNOD6 has been cloned and sequenced. PsNOD6 is homologous to the pea early nodulin genes PsNOD3 and PsENOD14. In situ hybridization experiments showed that, like the PsENOD3 and PsENOD14 genes, the PsNOD6 gene is only expressed in the infected cell type. The PsNOD6 gene is first expressed at the transition of the pre-fixation zone II into the interzone II-III (the amyloplast-rich zone preceding the fixation zone III), whereas the early nodulin genes PsENOD3 and PsENOD14 are already induced in the pre-fixation zone II. Thus these nodulin genes encoding homologous proteins are induced at consecutive stages of nodule development. The expression of the late nodulin genes encoding leghaemoglobin precedes the expression of the late nodulin gene PsNOD6. Therefore these late nodulin genes have to be regulated by different mechanisms despite the fact they are expressed in the same cell type. This conclusion is consistent with the fact that PsNOD6 lacks one of the conserved regions occurring in the promoters of all other late nodulin genes studied.

Amino Acid Sequence↗

The isolation and characterization of a cDNA clone encoding Lupinus angustifolius root nodule glutamine synthetase.

Glutamine synthetase, purified from Lupinus angustifolius legume nodules, was carboxymethylated and succinylated prior to chemical or enzymatic cleavage. Peptides were purified and sequenced. An oligonucleotide probe was constructed for the sequence MPGQW. This probe was used to identify a glutamine synthetase cDNA clone, pGS5, from a lupin nodule cDNA library constructed in pBR322. pGS5 was sequenced (1043 bp) and computer-assisted homology searching revealed a high degree of conservation between this lupin partial cDNA clone and other plant glutamine synthetases at both the amino acid (greater than 90%) and nucleotide (greater than 80%) level. Northern and Southern analyses using pGS5 supported the conclusion that a multigene glutamine synthetase family exists in lupin which is differentially expressed in both an organ-specific and temporal manner. Western and Northern blot analyses indicated the accumulation of a glutamine synthetase specific mRNA species during nodule development corresponded to the appearance of a novel glutamine synthetase polypeptide between 8 and 10 days after rhizobial inoculation.

Adenosine Triphosphate↗

Nucleosomal structure and histone H1 subfractional composition of pea (Pisum sativum) root nodules, radicles and callus chromatin.

Higher-order packaging of DNA in chromatin structures could be an essential step in the complex chain of events leading to activation/repression of eukaryotic gene expression. With the goal to investigate this aspect of transcriptional regulation of plant genes involved in symbiotic interactions between legumes and rhizobia we analyze here the molecular parameters of chromatin structure in functioning root nodules, callus and radicles of pea. Morphological intactness and the typical nucleosomal organization are preserved in purified nuclei isolated from all three sources. The calculated values of nucleosomal repeat changed from 185 +/- 5 bp in the nuclei of radicles to 168 +/- 5 bp and 195 +/- 6 bp in nodules and callus respectively. The observed changes are due to alterations in linker DNA lengths. The core histones are identical in all cases, but the subfractional composition of H1 linker histone is subjected to quantitative alterations. The most pronounced is the several-fold increase in content of the lowest-molecular-weight subfraction H1-6 which takes place during nodule development.

Cell Nucleus↗

Characterization of the pea ENOD12B gene and expression analyses of the two ENOD12 genes in nodule, stem and flower tissue.

The ENOD12 gene family in pea consists of two different members. The cDNA clone, pPsENOD12, represents the PsENOD12A gene. The second ENOD12 gene, PsENOD12B, was selected from a genomic library using pPsENOD12 as a probe and this gene was sequenced and characterized. The coding regions of the two genes are strikingly similar. Both encode proteins having a signal peptide sequence and a region with pentapeptide units rich in prolines. ENOD12A has a series of rather conserved repeating pentapeptide units, whereas in ENOD12B the number of pentapeptide units is less and these are less conserved. From the amino acid sequence it is obvious that the PsENOD12 genes encode proline-rich proteins which are closely related to proteins that have been identified as components of soybean cell walls (SbPRPs). Previously, Northern blot analyses had shown that ENOD12 genes are expressed in a tissue-specific manner. A high expression level is found in Rhizobium-infected roots and in nodules, whereas expression in flower and stem is lower. This raised the question of which gene is expressed where and when. The availability of the sequences of both ENOD12 genes allowed us to analyse the expression of the two genes separately. Specific oligonucleotides were used to copy the ENOD12 mRNAs and to amplify the cDNAs in a polymerase chain reaction. It was demonstrated that in all the tissues containing ENOD12 mRNA, both genes PsENOD12A and PsENOD12B are transcribed and that the relative amounts of PsENOD12A and PsENOD12B mRNA within each tissue are more or less equal. Moreover, the expression pattern during infection and nodule development is the same for the two genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase.

We have isolated an alfalfa leaf cDNA clone that encodes aspartate aminotransferase (AAT, EC 2.6.1.1) by direct complementation of an Escherichia coli aspartate auxotroph with a plasmid cDNA library. DNA sequence analysis of the recombinant plasmid, pMU1, revealed that a 1514 bp cDNA was inserted in the correct orientation and in-frame with the start of the lacZ coding sequence in the vector, pUC18. The resulting fusion protein is predicted to be 424 amino acids in length with a molecular weight of 46387 Daltons. The cDNA-encoded protein has a characteristic pyridoxal phosphate attachment site motif and has substantial amino acid sequence homology to both animal and bacterial AATs. Plasmid pMU1 encodes an AAT with a Km for aspartate of 3.3 mM, a Km for 2-oxoglutarate of 0.28 mM, and a pH optimum between 8.0 and 8.5. Several lines of evidence including Western blot analysis, the isoelectric point of the encoded protein, and the effect of pH on the activity of the fusion protein, suggest that the cDNA encodes the isozyme AAT-1 rather than AAT-2. Northern blot analysis showed that the aat-1 clone hybridized to a 1.6 kb transcript present in alfalfa leaves, roots and nodules. The relative concentrations of aat-1 mRNA in these tissues were 1:2:5, respectively. Thus, transcription of aat-1 appears to be induced during nodule development. Southern blot analysis suggested that AAT-1 in alfalfa is encoded by either a single-copy gene or a small, multigene family.

Amino Acid Sequence↗

Inhibition of experimental pulmonary metastasis in mice by beta-cyclodextrin-benzaldehyde.

The effect of beta-cyclodextrin-benzaldehyde (CDBA) on experimental pulmonary metastasis in C3H/He mice was examined. In an in vitro assay, the growth of RCT(+) cells was inhibited by 1200 micrograms/ml CDBA using unrenewed media, and by 600 micrograms/ml CDBA in that using daily renewed media. When mice were treated daily with CDBA, 3 weeks later the number of lung nodules developing after i.v. injection of 1 X 10(6) RCT(+) cells was significantly decreased in a dose-dependent manner, i.e., 73.8%, 85.6%, and 95.7% inhibition was observed following 0.5, 5, and 25 mg CDBA/mouse per day p.o. administration, respectively. The same mice showed almost as much natural killer (NK) activity as normal mice. Therefore, experiments were designed to evaluate the effect of CDBA on the NK activity of tumor-free mice whose immunity had been suppressed by 5-fluorouracil (5FU). Injections of 5FU only suppressed this activity to about 50% of normal mice, but the combined treatment with CDBA negated the suppressive effect of 5FU on NK activity. The results suggested that the inhibition of experimental pulmonary metastasis might be induced by the possible combined effects of CDBA; that is, the direct inhibition of tumors and the augmentation of NK cell activity.

Animals↗

Combined immunostimulation (Propionibacterium avidum KP 40) and anticoagulation (heparin) prevents metastatic lung and liver colonization in mice.

The antineoplastic activity of Propionibacterium avidum KP-40 and its enhancement by anticoagulation with heparin was studied. In Balb/c mice syngeneic sarcoma L-1 exclusively caused tumor colonization of the lungs. After neuraminidase treatment the organotropism of this tumor was changed, with tumor nodules developing in lung and liver. After single systemic application of Propionibacterium avidum KP-40 the number of lung and liver colonies decreased evidently. Combination of this immunomodulating therapy with temporary anticoagulation resulted in further reduction of tumor colonies in lung and liver.

Adjuvants, Immunologic↗

Multiple neuroendocrine carcinomas (so-called Merkel cell tumours) of the skin. Report on two cases with unique clinical course.

Two neuroendocrine carcinomas of the skin (so-called Merkel cell tumours) are presented. In both cases multiple tumour nodules developed within the course of the disease. The light and electron microscopic observations correspond with the findings reported in other neuroendocrine carcinomas. As a variable morphological and clinical pattern for these tumours seems to exist we consider our two cases with their unique clinical picture to be an obviously infrequent variant of this tumour disease, we propose for it the term "multiple neuroendocrine carcinoma syndrome".

Adenocarcinoma↗

The soft-tissue tumours induced in Syrian hamsters by herpes simplex virus type 1 and a chemical promoter.

Adult male Syrian hamsters were inoculated subcutaneously with herpes simplex virus type 1 (HSV-1, 10(6) PFU) or ultraviolet-inactivated HSV-1. One week later 12-O-tetradecanoylphorbol 13-acetate (TPA, 2 x 20 nmol weekly) was topically applied to the dorsal skin at the site of virus inoculation for 6 months. Control animals received HSV-1 only or topical treatment with TPA in acetone or acetone alone. Small tumour nodules developed in the HSV-1 group close to the site of virus inoculation 10-15 months after the beginning of the experiment. The neoplasms were classified as angiolipomas, chondromyxomas, a hibernoma, and an unclassified tumor resembling a Kaposi sarcoma in humans. The topical TPA treatment alone induced melanocytic hyperplasia and sebaceous gland hyperplasia. The soft-tissue tumours differed markedly from the structure of the soft-tissue sarcomas induced in Syrian hamsters by viruses of the papova and polyoma groups. Since the spontaneous incidence of benign soft-tissue tumours in our close hamster colony is extremely low, we concluded that mutagenic HSV-1 effects on hamster mesenchymal cell DNA may be involved in the process of formation of the observed benign neoplasms.

Animals↗

Unusual histochemical pattern in preneoplastic hepatic foci characterized by hyperactivity of several enzymes.

In a stop-experiment using the hepatocarcinogen N-nitrosomorpholine, as well as glycogenotic and related lesions, hepatocellular foci with a different histochemical pattern were identified. The outstanding features of these hepatic foci, which may progress to hepatocellular adenoma, were increased activities of mitochondrial glycerol-3-phosphate dehydrogenase (mG3PD), glycogen synthase, pyruvate kinase and glucose-6-phosphatase detected by enzyme histochemistry. Since no decrease in activity of any of the enzymes examined were seen in these foci, compared with normal liver, the term enzymatically hyperactive focus (EHF) is proposed for this type of lesion. Only at the stage of overtly nodular growth did these lesions exhibit some of the characteristic changes seen in nodules developing from glycogenotic foci, namely elevated activities of glucose-6-phosphate dehydrogenase, gamma-glutamyl transferase and glutathione-S-transferase P as well as decreased activities of adenosine-triphosphatase, glucose-6-phosphatase and adenylate cyclase. Some of these enzymes have been used widely in morphometric studies as markers for preneoplastic and neoplastic lesions. The inability to detect early EHF may lead to an underestimation of preneoplastic liver lesions in quantitative studies. Although there are apparent differences in the histochemical patterns of glycogen storing foci and early EHF, these differences tend to disappear during progression to overtly neoplastic lesions. In studies comparing the phenotypic alterations in different types of preneoplastic hepatic lesions, the recognition of EHF may contribute to the distinction of obligatory from facultative phenomena during transformation.

Adenoma↗

[Persistent inflammatory reaction at the injection site of Il-2 with lymphoma-like inflammatory infiltrates].

Interleukin-2 (Il-2) is widely used for treatment of carcinomas, leukemia, and melanoma. Adverse drug effects of Il-2 include various systemic and generalized cutaneous drug reactions. In the following we report on a persistent inflammatory reaction at the injection site of Il-2. A patient received chemoimmuno-therapy for treatment of metastasizing melanoma, including subcutaneous application of recombinant interleukin-2 (Proleukin((R))). Within a few days reddish nodules developed at the injection sites that persisted after cessation of Il-2 injections for two years until final lethal outcome. Histologic examination revealed a lobular panniculitis expressing atypical lymphocytes and multinucleated histiocytes.

Aged↗

[Buruli ulcer. A mycobacterial skin disease].

Buruli ulcer is a chronic ulcerative skin disease caused by Mycobacterium ulcerans. It is the third most common mycobacterial disease in immunocompetent people and affects mainly children living in humid areas of the tropical rain forest. The mode of transmission is unknown. The microorganisms penetrate the skin via microinjuries. A few weeks after infection, a subcutaneous nodule develops, followed by necrosis of the subcutaneous fat and finally by a large dermal ulceration. Typical is the lack of an acute inflammatory response, likely due to an immunosuppressive toxin produced by M. ulcerans called mycolactone. The lesions mostly affect the limbs. Constitutional symptoms are normally absent. The only effective treatment consists of wide excision, often followed by skin grafts. Conservative measures are rarely successful. Buruli ulcer is characterized by low mortality and high morbidity. Early recognition and treatment are decisive for the complete cure and prevention of debilitating deformities.

Adipose Tissue↗

Characterization of soybean genomic features by analysis of its expressed sequence tags.

We analyzed 314,254 soybean expressed sequence tags (ESTs), including 29,540 from our laboratory and 284,714 from GenBank. These ESTs were assembled into 56,147 unigenes. About 76.92% of the unigenes were homologous to genes from Arabidopsis thaliana ( Arabidopsis). The putative products of these unigenes were annotated according to their homology with the categorized proteins of Arabidopsis. Genes corresponding to cell growth and/or maintenance, enzymes and cell communication belonged to the slow-evolving class, whereas genes related to transcription regulation, cell, binding and death appeared to be fast-evolving. Soybean unigenes with no match to genes within the Arabidopsis genome were identified as soybean-specific genes. These genes were mainly involved in nodule development and the synthesis of seed storage proteins. In addition, we also identified 61 genes regulated by salicylic acid, 1,322 transcription factor genes and 326 disease resistance-like genes from soybean unigenes. SSR analysis showed that the soybean genome was more complex than the Arabidopsis and the Medicago truncatula genomes. GC content in soybean unigene sequences is similar to that in Arabidopsis and M. truncatula. Furthermore, the combined analysis of the EST database and the BAC-contig sequences revealed that the total gene number in the soybean genome is about 63,501.

Chromosomes, Artificial, Bacterial↗

The Actinorhizal Symbiosis.

The term "actinorhiza" refers both to the filamentous bacteria Frankia, an actinomycete, and to the root location of nitrogen-fixing nodules. Actinorhizal plants are classified into four subclasses, eight families, and 25 genera comprising more than 220 species. Although ontogenically related to lateral roots, actinorhizal nodules are characterized by differentially expressed genes, supporting the idea of the uniqueness of this new organ. Two pathways for root infection have been described for compatible Frankia interactions: root hair infection or intercellular penetration. Molecular phylogeny groupings of host plants correlate with morphologic and anatomic features of actinorhizal nodules. Four clades of actinorhizal plants have been defined, whereas Frankia bacteria are classified into three major phylogenetic groups. Although the phylogenies of the symbionts are not fully congruent, a close relationship exists between plant and bacterial groups. A model for actinorhizal specificity is proposed that includes different levels or degrees of specificity of host-symbiont interactions, from fully compatible to incompatible. Intermediate, compatible, but delayed or limited interactions are also discussed. Actinorhizal plants undergo feedback regulation of symbiosis involving at least two different and consecutive signals that lead to a mechanism controlling root nodulation. These signals mediate the opening or closing of the window of susceptibility for infection and inhibit infection and nodule development in the growing root, independently of infection mechanism. The requirement for at least two molecular recognition steps in the development of actinorhizal symbioses is discussed.

Journal Article↗

Nodulin 22 from Phaseolus vulgaris protects Escherichia coli cells from oxidative stress.

Plant genes that are induced during the formation and function of a root nodule are called nodulin genes. Cloning and functional analysis of nodule-specific gene products are of valuable help in establishing the role and requirements of the host plant for the specificity and effectiveness of the symbiosis. A cDNA clone (nod22) was isolated from Phaseolus vulgaris L. (common bean) cDNA library derived from Rhizobium-infected roots. Nodulin 22 (Nod22) transcripts are accumulated from early to late stages in root nodule development. RT-PCR in situ studies indicated that Nod22 transcripts are highly accumulated in cortical, vascular bundle and infected cells. The deduced Nod22 protein contains a highly hydrophobic N-terminus, with signal peptide characteristics, and a C-terminal extension with high identity to the alpha-crystallin domains found in alpha-crystallin lens chaperone, and other small heat-shock proteins. These domains have not been previously described in other known nodulins, but have been observed in small heat-shock proteins found in plant tissues exposed to elevated temperature and oxidative stress. Nod22, when it is over-expressed in Escherichia coli, cells confers protection against oxidative stress suggesting its possible role in plant host protection from oxidative toxicity during the Rhizobium-legume symbiosis.

Amino Acid Sequence↗

Localization of H(+)-ATPases in soybean root nodules.

The localization of H(+)-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H(+)-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H(+)-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids.

Microscopy, Electron↗

Subcutaneous nodules in Pseudomonas sepsis.

Approximately 60 nontender, nonfluctuant, red, hot subcutaneous nodules developed on the trunk, extremities, and face due to Pseudomonas aeruginosa septicemia in a 56-year-old woman with stage III ovarian adenocarcinoma. Two years later, these lesions appeared atrophic with central scarring. Complete eradication with systemic antibiotics and without incision and drainage was accomplished.

Adenocarcinoma↗