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PubMed1989

Developmentally Regulated Expression of the Gene Family for Cytosolic Glutamine Synthetase in Pisum sativum.

In Pisum sativum, two classes of genes encode distinct isoforms of cytosolic glutamine synthetase (GS). The first class comprises two nearly identical or "twin" GS genes (GS341 and GS132), while the second comprises a single GS gene (GS299) distinct in both coding and noncoding regions from the "twin" GS genes. Gene-specific analyses were used to monitor the individual contribution of each gene for cytosolic GS during root nodule development and in cotyledons during germination, two contexts where large amounts of ammonia must be assimilated by GS for nitrogen transport. mRNAs corresponding to all three genes for cytosolic GS were shown to accumulate coordinately during a time course of nodule development. All the GS mRNAs also accumulate to wild-type levels in mutant nodules formed by a nifD(-) strain of Rhizobium leguminosarum indicating that induced GS expression in pea root nodules does not depend on the production of ammonia. Distinct patterns of expression for the two classes of GS genes were observed in certain mutant root nodules and most dramatically in cotyledons of germinating seedlings. The different patterns of expression between the two classes of genes for cytosolic GS suggests that their distinct gene products may serve nonoverlapping functions during pea development.

Journal Article↗
PubMed1976

The development and proliferation of the trophoblast from ectopic mouse embryo allografts of increasing gestational age.

The development and proliferation of trophoblast from ectopic mouse embryo allografts and isografts of increasing gestational age in nonimmune and specifically preimmunized hosts was studied. Specific preimmunization of recipients significantly inhibited haemorrhagic nodule development from 3-3 1/2 and 4-4 1/2 day post coitum homozygous blastocysts and from 5-5 1/2 day homozygous egg cylinders. Preimmunization had no effect on the percentage of haemorrhagic nodules which developed from 6-6 1/2 day homozygous egg cylinders. Haemorrhagic nodule development was significantly increased for F1 hybrid allografts compared to homozygous allografts to preimmunized recipients. Coincident with the immune inhibition of haemorrhagic nodule formation was the microscopic finding that in some cases trophoblast cells developed without subsequent proliferation in preimmunized hosts. The results are discussed in relation to the expression of transplantation antigens by the developing conceptus, the continued differentiation of the embryo after transplantation, and the inductive influences of the inner cell mass on trophoblast ontogeny.

Animals↗
PubMed2004

Safety of fine-needle aspiration in patients with small hepatocellular carcinoma.

OBJECTIVE.: This study was to clarify the safety of fine-needle aspiration of small hepatocellular carcinoma (HCC). METHODS.: Ninety consecutive patients with small hepatocellular carcinoma (46 with single nodule, 44 with multiple nodules, all </=3cm) who had a comparative angiography performed within 50 days (median 16 days) after aspiration were included. RESULTS.: The angiographic detection of hepatocellular carcinoma was not influenced by the procedure of aspiration except in one (1.1%) with small arterioportal shunt. The newly developed nodules of hepatocellular carcinoma were not localized in the segment of aspirated nodule located. None of the new nodules was located along the needle tract. Patients with single nodule caused by hepatitis C had a significantly higher incidence of appearance of new nodule than those caused by hepatitis B (38.46% versus 4.35%, p=0.0159). Patients with multiple nodules had a significantly higher incidence of appearance of new nodule than patients with single nodule (60% versus 18.18%, p<0.005). These results indicated that the newly developed nodules were related to the disease process rather than the procedure of aspiration. CONCLUSIONS.: Fine-needle aspiration is a safe procedure in the collection of specimens for the diagnosis of small hepatocellular carcinoma.

Journal Article↗
PubMed1994

Bacterial and plant glycoconjugates at the Rhizobium-legume interface.

Many classes of bacterial and plant glycoconjugate have been shown to be involved in establishing the Rhizobium root nodule symbiosis with peas (Pisum sativum). It was demonstrated, using techniques of molecular genetics, that a group of Rhizobium nodulation genes (nod genes) co-operate to synthesize a lipo-oligosaccharide signal molecule that specifically initiates nodule development on legume hosts. An additional gene function, encoded by nodX, has been found to extend the host range of Rhizobium leguminosarum bv. viciae to include nodulation of a pea mutant, cultivar Afghanistan; the nodX gene product specifies the addition of an acetyl group to the terminal N-acetylglucosamine residue at the reducing end of the pentasaccharide core of this signal molecule. Several other classes of bacterial glycoconjugate have also been shown by genetic analysis to be essential for normal nodule development and function: these include a capsular extracellular polysaccharide; lipopolysaccharide in the outer membrane; and cyclic glucans present in the periplasmic space. Potential functions for these glycoconjugates are discussed in the context of tissue and cell invasion by Rhizobium. Some plant components involved in symbiotic interactions have been identified by the analysis of nodule-specific gene expression (early nodulins). Several of the cDNA clones encoding these early nodulins specify proline-rich proteins that presumably correspond to cell wall glycoproteins or membrane arabinogalactan proteins. Other plant glycoconjugates have been identified using monoclonal antibodies as probes. A plant glycoprotein present in intercellular spaces has been identified as a component of the luminal matrix of infection threads. Because it attaches to the surface of bacteria and is itself susceptible to oxidative cross-linking, this glycoprotein may be involved in limiting the progress of microbial infections. Endocytosis of bacteria into the plant cytoplasm is apparently driven by direct interactions between the bacterial surface and the plasma membrane that is exposed within an unwalled infection droplet; glycoprotein and glycolipid components of the plant membrane glycocalyx have been defined using monoclonal antibodies. Differentiation of endosymbiotic bacteroids is preceded by differentiation of the plant-derived peribacteroid membrane which encloses the symbiosome compartment. Using a monoclonal antibody that identifies a group of plant membrane-associated, inositol-containing glycolipids, we have identified a very early marker for the differentiation of peribacteroid membrane from plasma membrane.

Carbohydrate Sequence↗
PubMed1992

Molecular dissection of structure and function in the lipopolysaccharide of Rhizobium leguminosarum strain 3841 using monoclonal antibodies and genetic analysis.

Following treatment with nitrosoguanidine, mutant derivatives of Rhizobium leguminosarum strain 3841 were isolated which failed to react with AFRC MAC 203. This monoclonal antibody normally recognizes a strain-specific lipopolysaccharide epitope which is developmentally regulated during legume nodule differentiation. Structural modification of lipopolysaccharide (LPS) was analysed by examining reactivity with a range of monoclonal antibodies with different epitope specificities, and also by analysis of LPS mobility changes after electrophoresis on polyacrylamide gels. One class of these LPS-defective mutants induced normal nitrogen-fixing (Fix+) nodules on peas (Pisum sativum), while another two classes of Fix- mutants were also identified, suggesting that a component of the LPS antigen that is part of the MAC 203 epitope is essential for normal nodule development leading to symbiotic nitrogen fixation. When grown under low-oxygen or low-pH culture conditions, one class of Fix- mutants completely lacked LPS-1 (the species that carries O antigen) and a second class showed a modified and truncated form of LPS-1. Mutants with defective LPS structure were also obtained after Tn5 mutagenesis of R. leguminosarum 3841 and all nine Fix- mutants were also found to lack the MAC 203 epitope. Three of these transposon-induced mutants synthesized a truncated form of LPS-1 that was structurally similar to that of the class of the NTG-induced mutants described above. These transposon-induced mutations, and the nitrosoguanidine-induced Fix- mutations, were closely linked and could be suppressed by the same cloned fragment of chromosomal DNA. The data presented here suggest that a precondition for normal nodule development of R. leguminosarum 3841 within pea nodules is the ability to synthesize relatively long-chain LPS-1 macromolecules under the physiological conditions encountered within the nodule. All mutants that lacked the ability to elongate LPS-1 macromolecules also failed to express the MAC 203 epitope.

Antibodies, Bacterial↗
PubMed2006

Structure, expression, and mapping of two nodule-specific genes identified by mining public soybean EST databases.

Numerous nodule-specific genes, which are involved in the root nodule development and function, have been known and are still being discovered. Here, we reported the structure, expression, and genetic map location of two novel nodule-specific genes. First, two EST groups, one obtained from a nodule library and the other from all aboveground tissue libraries, were clustered with regard to in silico expression profiles. We compiled a pool of 103 putative nodule-specific sequence clusters. Then, two representative ESTs were selected for further experimental analyses. According to the full-length cDNA sequences, one was an EST of a novel nodule-specific polygalacturonase gene, GmPGN, and the other was an EST of a new short nodule-specific gene, GmEKN. The results of expression analyses of the GmPGN cDNAs indicated that GmPGN expression was not detectable in any of the soybean tissues except in the nodule tissue and may be regulated via alternative splicing. GmEKN expression was the most strongly detected in the nodule. The predicted GmEKN protein is both glutamic acid- and lysine-rich, and is also highly hydrophilic. Genetic mapping located GmPGN near the known quantitative trait locus conferring resistance to soybean cyst nematode on soybean molecular linkage group (MLG) B1, and GmEKN on MLG A2. These results provide useful information for the use of these genes in research on the orchestration of numerous genes in nodule development and function.

Amino Acid Sequence↗
PubMed2002

Expression of genes encoding late nodulins characterized by a putative signal peptide and conserved cysteine residues is reduced in ineffective pea nodules.

Five nodulin genes, PsN1, PsN6, PsN314, PsN335, and PsN466, with reduced expression in ineffective nodules on the pea (Pisum sativum) mutant E135 (sym13) were characterized. They encode small polypeptides containing a putative signal peptide and conserved cysteine residues and show homology to the nodulins PsENOD3/14 and PsNOD6. For each gene, multiple bands were detected by genomic Southern analysis. Northern analysis showed that all five genes were expressed exclusively in nodules and that their temporal expression patterns were similar to that of the leghemoglobin (Lb) gene during nodule development. Their transcripts were localized predominantly from the interzone II-III to the distal part of nitrogen-fixing zone III in effective nodules, resembling the Lb gene. However, transcripts in ineffective E135 nodules were localized in narrower regions than those in the effective nodules. These results indicate that these nodulins are abundant in pea nodules and that their successive expression during nodule development is associated with nitrogen-fixing activity.

Amino Acid Sequence↗
PubMed1998

Srchi13, a novel early nodulin from Sesbania rostrata, is related to acidic class III chitinases.

On the tropical legume Sesbania rostrata, stem-borne nodules develop after inoculation of adventitious root primordia with the microsymbiont Azorhizobium caulinodans. A cDNA clone, Srchi13, with homology to acidic class III chitinase genes, corresponds to an early nodulin gene with transiently induced expression during nodule ontogeny. Srchi13 transcripts accumulated strongly 2 days after inoculation, decreased from day 7 onward, and disappeared in mature nodules. Induction was dependent on Nod factor-producing bacteria. Srchi13 was expressed around infection pockets, in infection centra, around the developing nodule and its vascular bundles, and in uninfected cells of the central tissue. The specific and transient transcript accumulation together with the lipochitooligosaccharide degradation activity of the recombinant protein hint at a role of Srchi13 in normal nodule ontogeny by limiting the action of Nod factors.

Amino Acid Sequence↗
PubMed1990

Symbiotic pseudorevertants of Rhizobium meliloti ndv mutants.

Nodule development (ndv) mutants of Rhizobium meliloti cannot invade alfalfa to establish a nitrogen-fixing symbiosis and instead induce the formation of small, white, unoccupied nodules on alfalfa roots. Such mutants also fail to produce the unusual cyclic oligosaccharide beta-(1----2)-glucan and show defects in several aspects of vegetative growth and function. Here we show that ndv mutants are severely reduced, although not totally deficient, in the ability to attach to and initiate infection threads on alfalfa seedlings, and we demonstrate that the symbiotic deficiency can be separated from the rest of the mutant phenotype by isolating second-site pseudorevertants. Pseudorevertants selected for restoration of motility, a vegetative property, regained a substantial amount of attachment capability but only slight infection thread initiation and symbiotic ability. Such strains also regained partial tolerance to growth at low osmolarity, even though they did not recover the ability to synthesize periplasmic beta-(1----2)-glucan. Pseudorevertants selected on alfalfa for restoration of symbiosis were unrestored for beta-(1----2)-glucan production or any other vegetative property and regained little or no attachment or infection thread initiation capability. We take these data to indicate that wild-type R. meliloti normally has considerable excess capability for both attachment and infection thread initiation and that the symbiotic block in ndv mutants lies further along the developmental pathway than either of these processes, probably at the level of infection thread extension. Further, the fact that neither type of pseudorevertant recovered the ability to produce periplasmic beta-(1----2)-glucan raises the possibility that this oligosaccharide is not directly required for nodule development.

Bacteriophages↗
PubMed1997

Immunolocalization of PsNLEC-1, a lectin-like glycoprotein expressed in developing pea nodules.

The pea (Pisum sativum) nodule lectin gene PsNlec1 is a member of the legume lectin gene family that is strongly expressed in infected pea nodule tissue. A full-length cDNA sequence of PsNlec1 was expressed in Escherichia coli and a specific antiserum was generated from the purified protein. Immunoblotting of material from isolated symbiosomes revealed that the glycoprotein was present in two antigenic isoforms, PsNLEC-1A and PsNLEC-1B. The N-terminal sequence of isoform A showed homology to an eight-amino acid propeptide sequence previously identified from the cDNA sequence of isoform B. In nodule homogenates the antiserum recognized an additional fast-migrating band, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was associated with a 100,000 g nodule membrane fraction, suggesting an association with cytoplasmic membrane or vesicles. Immunogold localization in pea nodule tissue sections demonstrated that the PsNLEC-1 antigen was present in the symbiosome compartment and also in the vacuole but revealed differences in distribution between infected host cells in different parts of the nodule. These data suggest that PsNLEC-1 is subject to posttranslational modification and that the various antigenic isoforms can be used to monitor membrane and vesicle targeting during symbiosome development.

Amino Acid Sequence↗
PubMed1994

Root nodulation of Sesbania rostrata.

The tropical legume Sesbania rostrata can be nodulated by Azorhizobium caulinodans on both its stem and its root system. Here we investigate in detail the process of root nodulation and show that nodules develop exclusively at the base of secondary roots. Intercellular infection leads to the formation of infection pockets, which then give rise to infection threads. Concomitantly with infection, cortical cells of the secondary roots dedifferentiate, forming a meristem which has an "open-basket" configuration and which surrounds the initial infection site. Bacteria are released from the tips of infection threads into plant cells via "infection droplets," each containing several bacteria. Initially, nodule differentiation is comparable to that of indeterminate nodules, with the youngest meristematic cells being located at the periphery and the nitrogen-fixing cells being located at the nodule center. Because of the peculiar form of the meristem, Sesbania root nodules develop uniformly around a central axis. Nitrogen fixation is detected as early as 3 days following inoculation, while the nodule meristem is still active. Two weeks after inoculation, meristematic activity ceases, and nodules then show the typical histology of determinate nodules. Thus, root nodule organogenesis in S. rostrata appears to be intermediate between indeterminate and determinate types.

Cell Differentiation↗
PubMed1984

Endoscopic study of chronological changes leading to cancer in the esophagus of dogs induced by N-ethyl-N'-nitro-N-nitrosoguanidine.

An endoscopical study of chronological change during the carcinogenetic process of cancer of the esophagus induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) in 16 male beagle dogs was performed to clarity whether or not precancerous lesions exist in the esophagus. Redness was first observed in the mucosa in the lower and/or middle portions of the esophagus. A small nodule developed in the redness, followed by a nodule. The nodule then developed into a flat polyp which finally developed into lesions such as an elevated lesions, protrusions or elevations with depressions. Histologically, esophagitis or acanthosis in the redness and acanthosis were found in most of the small nodules. With regard to the nodules, papillomatosis was observed in half of the lesions, while either acanthosis or atypical epithelial proliferation were found in most of the flat polyps. Almost all the elevated or protruding lesions were found to be carcinoma.

Animals↗
PubMed2000

Postoperative suppressive therapy for thyroid adenomas.

Thyroid adenoma is a common disease. If partial thyroidectomy is performed, postoperative suppression therapy is often given to avoid nodule development in the remaining thyroid. It is unclear whether this treatment is warranted. Patients who underwent a partial thyroidectomy with a histologic diagnosis of follicular thyroid adenoma from January 1985 until February 1998 were studied retrospectively. Patients were analyzed on the basis of postoperative therapy, new thyroid nodule growth, and costs. Seventy-six patients were identified with a recurrence rate of 4 per cent (3/76). Sixty-one per cent (46/76) were treated with postoperative thyroid suppression therapy, and no difference in new nodule development was noted with at least 6 months of follow-up (P = 0.274). No patients required reoperation. A large cost saving was shown for patients who were not treated with levothyroxine. We conclude that postoperative thyroid suppression may not be routinely indicated. A prospective, randomized study would be necessary to answer this question conclusively.

Adenoma↗
PubMed1997

Establishment of an experimental intrapulmonary tumor nodule model.

BACKGROUND: A pulmonary tumor model is necessary to study the biology and therapy of lung cancer. Methods to establish a solitary intrapulmonary nodule are not well defined. Two methods for solitary intrapulmonary tumor nodule development in the Fischer rat are described. METHODS: Methylcholanthrene-induced sarcoma cell suspensions were introduced into lung parenchyma of Fischer rats via limited thoracotomy and lung puncture, or instilled into a distal airway after tracheal puncture and catheterization. Intrapulmonary tumor location, implantation mortality, procedure length, and animal survival were recorded. RESULTS: Single pulmonary nodules developed at the implanted position in 100% (n = 320) and 95% (62/65) of animals after direct injection into the pulmonary parenchyma or via tracheal puncture and instillation. Operative mortality was 2% and 5% via lung or tracheal implantation, respectively. Less than 5 minutes was required for each implantation. Mean survival time was 24 +/- 2 and 26 +/- 6 days after lung or tracheal implantation in animals allowed to survive until tumor-induced death. CONCLUSIONS: These easily performed, reproducible methods of establishing solitary intrapulmonary tumors are useful tools for lung cancer research.

Animals↗
PubMed2003

Endoreduplication mediated by the anaphase-promoting complex activator CCS52A is required for symbiotic cell differentiation in Medicago truncatula nodules.

In Medicago nodules, endoreduplication cycles and ploidy-dependent cell enlargement occur during the differentiation of bacteroid-containing nitrogen-fixing symbiotic cells. These events are accompanied by the expression of ccs52A, a plant ortholog of the yeast and animal cdh1/srw1/fzr genes, acting as a substrate-specific activator of the anaphase-promoting complex (APC) ubiquitin ligase. Because CCS52A is involved in the transition of mitotic cycles to endoreduplication cycles, we investigated the importance of somatic endoploidy and the role of the M. truncatula ccs52A gene in symbiotic cell differentiation. Transcription analysis and ccs52A promoter-driven beta-glucuronidase activity in transgenic plants showed that ccs52A was dispensable for the mitotic cycles and nodule primordium formation, whereas it was induced before nodule differentiation. The CCS52A protein was present in the nucleus of endoreduplication-competent cells, indicating that it may activate APC constitutively during the endoreduplication cycles. Downregulation of ccs52A in transgenic M. truncatula plants drastically affected nodule development, resulting in lower ploidy, reduced cell size, inefficient invasion, and the maturation of symbiotic cells, accompanied by early senescence and finally the death of both the bacterium and plant cells. Thus, ccs52A expression is essential for the formation of large highly polyploid symbiotic cells, and endoreduplication is an integral part of normal nodule development.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1996

Low proliferative activity in macroregenerative nodules: evidence for an alternate hypothesis concerning human hepatocarcinogenesis.

UNLABELLED: Macroregenerative nodules are commonly thought to be hyperplastic lesions, deriving both their large size and premalignant potential from an increased proliferative rate. We have previously suggested an alternate model of macroregenerative nodule development in which neither size nor premalignant potential of macroregenerative nodules would depend on increased proliferation. We tested this hypothesis by examining the proliferative activity in macroregenerative and surrounding cirrhotic nodules. METHODS: Eighteen macroregenerative nodules, including five type I and 13 type II, were immunostained for proliferating cell nuclear antigen (PCNA). Type II macroregenerative nodules included ten with diffuse large (7) or small (3) liver cell dysplasia only and eight containing nodule-in-nodule lesions. Five nodule-in-nodule lesions met the histologic criteria for hepatocellular carcinoma. PCNA labeling indices (PCNA-LIs; percentage positive hepatocyte nuclei/500 randomly counted cells) were determined in macroregenerative nodules and the four largest adjacent cirrhotic nodules. Nodule-in-nodule lesions were assessed separately from the background macroregenerative nodule. RESULTS: 4/5 type I and 12/13 type II macroregenerative nodules (exclusive of NIN lesions) had PCNA-LIs lower than the mean of surrounding cirrhotic nodules. All nodule-in-nodule lesions, whether atypical or overtly malignant, had PCNA-LIs greater than any surrounding nodules. In conclusion, macroregenerative nodules have PCNA-LIs indistinguishable from, and often lower than, surrounding cirrhotic nodules. Increased proliferative activity only occurs with the development of atypia and transition to hepatocellular carcinoma. CONCLUSION: Macroregenerative nodules derive neither their size nor their premalignant potential from on-going rapid proliferation, a finding consistent with our alternate hypothesis of macroregenerative nodule development.

Carcinoma, Hepatocellular↗
PubMed2003

Solitary congenital hypertrophy of the retinal pigment epithelium: clinical features and frequency of enlargement in 330 patients.

OBJECTIVE: To describe the clinical features of solitary congenital hypertrophy of the retinal pigment epithelium (CHRPE) and to determine the frequency of enlargement of this lesion DESIGN: Retrospective, observational, noncomparative case series. PARTICIPANTS: Three hundred thirty consecutive patients with solitary CHRPE. MAIN OUTCOME MEASURES: The 3 main outcome measures included flat lesion enlargement, intralesional lacunae enlargement, and development of an elevated nodule within the lesion. The clinical features at the time of presentation were analyzed for their impact on the main outcomes using a series of Cox proportional hazards regressions. RESULTS: The most common referring diagnosis included choroidal nevus (26%), choroidal melanoma (15%), CHRPE (9%), and unspecified lesion (48%). The median age at diagnosis was 45 years (range, 1-80 years), and there were no patients with familial adenomatous polyposis or related colon cancer, although a history of cancer was noted in 8% of patients, most commonly breast cancer (3%). The lesion most frequently was located inferotemporally (31%) and at the equatorial region (45%). Rarely, it was located in the macula (1%) or peripapillary region (1%). The median largest basal diameter was 4.5 mm, and the lesion was flat in all cases except in 5 (1.5%), in which there was an intralesional lesion nodule. The lesion was pigmented in 88% of cases and nonpigmented in 12%. Lacunae were noted in 43% of the pigmented CHRPE, and the lacunae showed gradual enlargement in 32%. Factors related to lacunae enlargement included number and relative size of lacunae. Flat enlargement of the lesion was documented in 46% of patients with comparative photographic follow-up and in 83% of those followed up for more than 3 years. The median rate of enlargement was 10 micro m per month. The most important factor associated with flat lesion enlargement was relative size of the lacunae within CHRPE. There were no cases of CHRPE in which a nodule developed while the patient was being followed up. Of the 5 lesions that had a nodule, progressive enlargement of the nodule was found in 3. CONCLUSIONS: Congenital hypertrophy of the retinal pigment epithelium generally has been regarded as a benign, stable lesion, but subtle, flat enlargement was noted in most patients (83%) followed up for 3 or more years using meticulous photographic comparison. Flat enlargement of the lesion appeared to be related to percentage of the mass occupied by lacunae.

Adolescent↗
PubMed1986

A plant flavone, luteolin, induces expression of Rhizobium meliloti nodulation genes.

The symbiotic interaction of Rhizobium meliloti and alfalfa results in the formation of nitrogen-fixing root nodules. Rhizobium meliloti nodABC genes are required for the early host responses of cortical cell divisions and root hair curling. The induction of nodABC expression by alfalfa exudates demonstrates host-symbiont signaling at an early stage in nodule development. The inducer molecule for nodABC expression was isolated from plant exudate by constructing a nodABC-lacZ fusion to monitor the inducing activity. From ultraviolet-visible absorption spectra, proton nuclear magnetic resonance, and mass spectrometry, the inducer was determined to be 3',4', 5,7-tetrahydroxyflavone (luteolin). Luteolin is a normal secondary plant metabolite found throughout the plant kingdom that may serve to control nodABC expression during nodule development. This regulatory role for a flavone contrasts with the function of some flavonoids as defense compounds.

Flavonoids↗