Genes essential for nod factor production and nodulation are located on a symbiotic amplicon (AMPRtrCFN299pc60) in Rhizobium tropici

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.     

Jasmonic acid stimulates the expression of nod genes in Rhizobium

Jasmonates and salicylic acid are considered to be signal molecules that induce a variety of plant genes involved in wound or defence response, as well as affecting nos promoter activity. In this paper we examined whether these chemicals could also affect nod genes from isogenic rhizobia strains. Isogenic strains contain the Rhizobium leguminosarum nodA promoter fused to the lacZ gene of Escherichia coli and differ only in the source of the regulatory nodD gene. Naringenin, jasmonic acid and methyl jasmonate induced expression of nod genes in strain RBL1284 and salicylic acid showed no activity alone or when used in combination with other compounds; addition of naringenin + jasmonic acid produced a synergistic effect. Results obtained with strain RBL5284 were similar to those for RBL1284 albeit the combination of naringenin with the other compounds markedly inhibited nod gene expression. Whereas RBL5283 responded to naringenin with a strong induction, jasmonic acid, methyl jasmonate or salicylic acid showed no significant responses. The inhibitory effect of salicylic acid on nod gene expression indicates that the induction mechanism of jasmonic acid, methyl jasmonate, N-propyldihydrojasmonate and naringenin is probably different from that of salicylic acid.     

Rhizobium tropici teu genes involved in specific uptake of Phaseolus vulgaris bean-exudate compounds

Rhizobium tropici nodulates and fixes nitrogen in bean. In the R. tropici strain CFN299 we identified and characterized teu genes (tropici exudate uptake) induced by bean root exudates, localized by insertion of a promoter-less Tn5-gusA1 transposon. teu genes are present on a plasmid of around 185 kb that is conserved in all R. tropici strains. Proteins encoded by teu genes show similarity to ABC transporters, specifically to ribose transport proteins. No induction of the teu genes was obtained by treatment with root exudates from any of several other plants tested, with the exception of Macroptilium atropurpureum, which is also a host plant for R. tropici. It appears that the inducing compound is characteristic of bean and closely related legumes. It is present in root exudates, but not in seeds. This compound is removed, presumably by metabolism, from the exudates by the majority of bean-nodulating rhizobia (such as R. etli, R. leguminosarum bv. phaseoli and R. giardinii). The principal inducing compound has not been identified, but some induction was obtained using trigonelline. The CFN299 strain seems to have an additional uptake system, as no phenotype is observed in two different mutants. R. tropici strain CIAT899, on the other hand, must have only one uptake system, since a mutant bearing an insertion in the teu genes could not remove the compound from the exudates as efficiently as the wild type, and it showed diminished nodulation competitiveness.     

Declining prevalence of cervicovaginal human papillomavirus infection with age is independent of other risk factors

The purpose of this study was to determine whether the addition of small quantities of minor lecithin components (phosphatidylinositol, phosphatidic acid, lysophosphatidylethanolamine, and cholesterol) and Pluronic F68 to lecithin could improve the stability of lecithin-stabilized perfluorocarbon emulsions. Attempts were made to correlate emulsion stability with interfacial properties (tension and charge). Dynamic interfacial tension was determined using a Teflon Wilhelmy plate method [reported previously (1)]. Emulsions were prepared by microfluidization. Microelectrophoresis was used to measure emulsion droplet charge, and photon correlation spectroscopy and Coulter analysis were used to determine emulsion stability as a function of droplet size. Thermal kinetic accelerated stability testing was conducted. Various droplet size parameters were used to compare emulsion stabilities, and an overall stability ranking, based on these parameters, was obtained for each emulsion. Small quantities of additives altered emulsion stability and these data were correlated with interfacial properties and initial droplet diameters. The addition of cholesterol to lecithin resulted in the most stable perfluorocarbon emulsion.     

Troglitazone action is independent of adipose tissue

We have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL- and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor gamma mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue.     

绿矾硫酸化焙烧-水浸工艺从三元正极废料中同步回收有价金属

针对湿法回收强酸浸出工艺高昂的过程成本和超额的环境负荷问题,通过添加绿矾硫酸化焙烧—水浸工艺实现无酸耗提取废旧三元正极材料中的有价金属,考察了焙烧条件和浸出因素对锂、镍、钴和锰浸出行为的影响。结果表明,在焙烧温度600 ℃、绿矾添加量10.5g/g、焙烧时间180 min、浸出温度45 ℃、液固比5 mL/g、浸出时间120 min的最佳工艺条件下,锂、镍、钴和锰的浸出率分别达到99.64%、96.17%、95.49%和98.17%。     

Agonist-mediated downregulation of G alpha i via the alpha 2-adrenergic receptor is targeted by receptor-Gi interaction and is independent of receptor signaling and regulation

One mechanism of long-term agonist-promoted desensitization of alpha2AR function is downregulation of the cellular levels of the alpha subunit of the inhibitory G protein, Gi. In transfected CHO cells expressing the human alpha2AAR, a 40.1 +/- 3.3% downregulation of Galphai2 protein occurred after 24 h of exposure of the cells to epinephrine, which was not accompanied by a decrease in Galphai2 mRNA. The essential step that targets Gi for degradation by agonist occupancy of the receptor was explored using mutated alpha2AAR lacking specific structural or functional elements. These consisted of 5HT1A receptor and beta2AR sequences substituted at residues 113-149 of the second intracellular loop and 218-235 and 355-371 of the N- and C-terminal regions of the third intracellular loop (altered Gi and Gs coupling), deletion of Ser296-299 (absent GRK phosphorylation), and substitution of Cys442 (absent palmitoylation and receptor downregulation). Of these mutants, only those with diminished Gi coupling displayed a loss of agonist-promoted Gi downregulation, thus excluding Gs coupling and receptor downregulation, palmitoylation, and phosphorylation as necessary events. Furthermore, coupling-impaired receptors consisting of mutations in the second or third loops ablated Gi downregulation, suggesting that a discreet structural motif of the receptor is unlikely to represent a key element in the process. While pertussis toxin ablated Gi downregulation, blocking downstream intracellular consequences of alpha2AAR activation or mimicking these pathways by heterologous means failed to implicate cAMP/adenylyl cyclase, phospholipase C, phospholipase D, or MAP kinase pathways in alpha2AAR-mediated Gi downregulation. Taken together, agonist-promoted Gi downregulation requires physical alpha2AAR-Gi interaction which targets Gi for degradation in a manner that is independent of alpha2AAR trafficking, regulation, or second messengers.     

Autoxidation of ubiquinol-6 is independent of superoxide dismutase

Ubiquinone (Q) is an essential, lipid soluble, redox component of the mitochondrial respiratory chain. Much evidence suggests that ubiquinol (QH2) functions as an effective antioxidant in a number of membrane and biological systems by preventing peroxidative damage to lipids. It has been proposed that superoxide dismutase (SOD) may protect QH2 form autoxidation by acting either directly as a superoxide-semiquinone oxidoreductase or indirectly by scavenging superoxide. In this study, such an interaction between QH2 and SOD was tested by monitoring the fluorescence of cis-parinaric acid (cPN) incorporated phosphatidylcholine (PC) liposomes. Q6H2 was found to prevent both fluorescence decay and generation of lipid peroxides (LOOH) when peroxidation was initiated by the lipid-soluble azo initiator DAMP, dimethyl 2,2'-azobis (2-methylpropionate), while Q6 or SOD alone had no inhibitory effect. Addition of either SOD or catalase to Q6H2-containing liposomes had little effect on the rate of peroxidation even when incubated in 100% O2. Hence, the autoxidation of QH2 is a competing reaction that reduces the effectiveness of QH2 as an antioxidant and was not slowed by either SOD or catalase. The in vivo interaction of SOD and QH2 was also tested by employing yeast mutant strains harboring deletions in either CuZnSOD and/or MnSOD. The sod mutant yeast strains contained the same percent Q6H2 per cell as wild-type cells. These results indicate that the autoxidation of QH2 is independent of SOD.     

Survival of long-lived plasma cells is independent of antigen

Recent studies have shown that persistent specific antibody titer is provided by long-lived plasma cells (PC) which constitute a new kind of 'memory-providing cells'. In the present study, we examine the role of antigen for the long-term survival of PC and the maintenance of specific serum antibody titers. Using a novel cytometric technology, to identify and isolate antigen-specific PC, we analyzed long-lived PC of BALB/c mice, during their development (between day 1 and 10) after secondary immunization with ovalbumin (OVA) and in the phase of the established immune reaction. Most if not all OVA-specific PC were generated within a few days after immunization. Within approximately 3 weeks, they matured, as indicated by down-regulation of expression of MHC class II. These PC are long lived and located in spleen and bone marrow. Upon adoptive transfer, OVA-specific PC from bone marrow, but not memory B cells, conferred specific and long-lasting antibody titers to antigen-free IgH syngeneic recipients. In response to antigenic challenge, new OVA-specific antibody-secreting cells were generated from transferred memory B cells. Antibody secretion by long-lived PC was not affected. Our results confirm that persistent antibody titers are provided by long-lived PC, independent of memory B cells and demonstrate that this humoral memory is inert to antigen.     

Staphylococcus aureus induces platelet aggregation via a fibrinogen-dependent mechanism which is independent of principal platelet glycoprotein IIb/IIIa fibrinogen-binding domains

Platelet aggregation by bacteria is felt to play an important role in the pathogenesis of infective endocarditis. However, the mechanisms involved in bacterium-induced platelet aggregation are not well-defined. In the present study, we examined the mechanisms by which Staphylococcus aureus causes rabbit platelet aggregation in vitro. In normal plasma, the kinetics of S. aureus-induced platelet aggregation were rapid and biphasic. The onset and magnitude of aggregation phase 1 varied with the bacterium-platelet ratio, with maximal aggregation observed at a ratio of 5:1. The onset of aggregation phase 2 was delayed in the presence of apyrase (an ADP hydrolase), suggesting that this later aggregation phase may be triggered by secreted ADP. The onset of aggregation phase 2 was delayed in the presence of prostaglandin I2-treated platelets, and this phase was absent when paraformaldehyde-fixed platelets were used, implicating platelet activation in this process. Platelet aggregation phase 2 was dependent on S. aureus viability and an intact bacterial cell wall, and it was mitigated by antibody directed against staphylococcal clumping factor (a fibrinogen-binding protein) and by the cyclooxygenase inhibitor indomethacin. Similarly, aggregation phase 2 was either delayed or absent in three distinct transposon-induced S. aureus mutants with reduced capacities to bind fibrinogen in vitro. In addition, a synthetic pentadecapeptide, corresponding to the staphylococcal binding domain in the C terminus of the fibrinogen delta-chain, blocked aggregation phase 2. However, phase 2 of aggregation was not inhibited by two synthetic peptides (alone or in combination) analogous to the two principal fibrinogen-binding domains on the platelet glycoprotein (GP) IIb/IIIa integrin receptor: (i) a recognition site on the IIIa molecule for the Arg-Gly-Asp (RGD) sequence of the fibrinogen alpha-chain and (ii) a recognition site on the IIb molecule for a dodecapeptide sequence of the fibrinogen delta-chain. This differs from ADP-induced platelet aggregation, which relies on an intact platelet GP IIb/IIIa receptor with an accessible RGD sequence and dodecapeptide recognition site for fibrinogen. Furthermore, a monoclonal antibody directed against the RGD recognition site on rabbit platelet GP IIb/IIIa receptors failed to inhibit rabbit platelet aggregation by S. aureus. Collectively, these data suggest that S. aureus-induced platelet aggregation requires bacterial binding to fibrinogen but is not principally dependent upon the two major fibrinogen-binding domains on the platelet GP IIb/IIIa integrin receptor, the RGD and dodecapeptide recognition sites.     

NolL of Rhizobium sp. strain NGR234 is required for O-acetyltransferase activity

Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium Rhizobium sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible nolL gene, which is preceded by a nod box, results in the synthesis of NodNGR factors that lack the 3-O- or 4-O-acetate groups. Interestingly, the nodulation capacity of the mutant NGROmeganolL is not impaired, whereas introduction of the nod box::nolL construct into the related strain Rhizobium fredii USDA257 extends the host range of this bacterium to Calopogonium caeruleum, Leucaena leucocephala, and Lotus halophilus. Nod factors produced by a USDA257(pnolL) transconjugant were also acetylated. The nod box::nolL construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the nodD1 gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not.     

Expression of c-met is a strong independent prognostic factor in breast carcinoma

BACKGROUND: The c-met protooncogene encodes the met protein, the receptor for scatter factor/hepatocyte growth factor, a growth factor that modulates the motility and stable interaction of the epithelial cells. This study assesses the expression of met receptor in breast carcinoma and its prognostic value with respect to survival. METHODS: Immunofluorescence was used to evaluate 91 archival breast carcinoma specimens using a polyclonal antibody to the cytoplasmic domain of the receptor. Cases were scored by two pathologists on a percentage basis and then converted to binary scores (positive or negative) on the basis of a bimodal distribution. RESULTS: Strong expression of met was found in 20 invasive ductal breast tumor specimens (22%). The 5-year survival of patients whose tumors showed decreased met expression was 89%, in contrast to a 52% 5-year survival rate in patients whose tumors expressed met (P = 0.008). This trend also was observed in patients without lymph node metastases at presentation, in whom met negative patients had a 95% 5-year survival compared with only 62% for met positive patients (P = 0.006) Multivariate analysis using the Cox proportional hazards model showed met expression to be an independent predictor of survival, with a predictive value nearly equivalent to that associated with lymph node status. CONCLUSIONS: The authors conclude that expression of met in patients with invasive ductal carcinoma of the breast is a strong, independent predictor of decreased survival and may be a useful prognostic marker with which to identify a subset of patients with more aggressive disease.     

Ascitic interleukin-12 is an independent prognostic factor in ovarian cancer

PURPOSE: The clinical impact of endogenous cytokines supplied with deterministic properties in the generation of either T helper (Th)1 -type or Th2-type immune response was investigated in patients with ovarian cancer. Whereas interleukin (IL)- 12 initiates the differentiation of naive Th0 cells toward Th1 phenotype, IL-4 and IL-10 mediate the development of Th2-type immunity. PATIENTS AND METHODS: Cytokines were determined before treatment by means of enzyme-linked immunosorbent assay (ELISA) in ascites fluid and serum of 76 patients with ovarian cancer. Cytokine levels were compared with each other and with standard clinicopathologic parameters. A stepwise logistic regression was calculated to rule out interdependence in the associations of the various variables. Survival analyses were performed with the Kaplan-Meier method and differences in survival were examined according to Mantel and Breslow. Cox proportional hazards analysis was used to identify independent prognostic factors. RESULTS: Whereas IL-10 and IL-12 were detectable in all ascites-fluid samples, IL-4 was measurable in only 43% of the specimens. With the exception of neopterin, macrophage colony-stimulating factor (M-CSF), and IL-4, determined cytokine levels were significantly elevated in ascites fluid compared with serum (P < .01). In univariate analyses, high ascitic-fluid concentrations of either neopterin, tumor necrosis factor-alpha (TNF-alpha), or IL-12 were associated with poor disease-free (P < .005) and overall (P < .01) survival. Multivariate Cox regression analysis showed ascitic-fluid IL-12 levels to be the only immunologic variable that retained independent prognostic significance (P < .03 for disease-free and P < .01 for overall survival), together with residual disease, Fédération Internationale de Gynécologie et d'Obstétrique (FIGO)-stage, and patient age. CONCLUSION: In ovarian cancer, high ascitic-fluid IL-12 levels, which may indicate a local Th1-generated immune response, are associated with disease progression.     

CD44 is an independent prognostic factor in early-stage cervical cancer

Leptin and neuropeptide Y (NPY) are involved in the regulation of food intake and body weight. Both hormones act through specific receptors in the central nervous system. The objective of this study was to investigate the relation of leptin and NPY in human plasma and cerebrospinal fluid (CSF). Leptin and NPY in CSF and in serum/plasma were measured by radioimmunoassays in 35 patients. Leptin concentrations in serum were 100-200 fold higher than in CSF. There was a significant correlation between leptin levels in CSF and in serum (r=0.88, P<0.0001). Female patients had significantly higher leptin serum concentrations than males (16.6+/-10.9 microg/l vs. 6.5+/-7.3 microg/l, P=0.002). In contrast, NPY levels were only twofold higher in CSF than in plasma. There was no relation between leptin and NPY in CSF and serum/plasma, respectively. The ratio of CSF and peripheral leptin levels did not correlate with the respective albumin ratio, indicating that leptin did not merely leak into the CSF via a defective blood-CSF barrier. It is concluded that leptin uptake from the circulation into CSF is a regulated process. The NPY concentration in CSF is not directly related to leptin CSF levels.     

Perioperative risk factors in esophageal cancer: a retrospective study of independent variables

The postoperative outcome of 127 patients with an esophageal carcinoma was investigated retrospectively, to identify independent factors for the perioperative mortality (hospital mortality). Chi-square single factor analysis was employed to test 35 independent variables, in a second analysis stepwise logistic regression was used to determine the factors correlating with the morality. In 41 patients the esophagus was resected by a thoraco-abdominal approach, in 86 by blunt dissection. The hospital mortality was 17.3%, the 30-day mortality 12.6%. Single factor analysis revealed a significant correlation with the variable smoking (p < 0.003), post-operative morbidity (p < 0.008), CEA (p < 0.02), time of operation (p < 0.02) and surgical procedures (p < 0.02). The influence of the surgeon's experience was significant. In the stepwise logistic regression the duration of operation (p < 0.0039), the surgical procedures (p < 0.016), and the units of blood (p < 0.03) were correlated with mortality. The logistic regression showed a significant increase of mortality for the thoracoabdominal approach with increasing duration of the operation. The estimation of survival time according to Kaplan and Meier revealed a significant correlation with the stage of the tumor, but not with the surgical procedure.     

Standard delay eyeblink classical conditioning is independent of awareness.

P. F. Lovibond and D. R. Shanks (see record 2002-00340-001) suggested that all forms of classical conditioning depend on awareness of the stimulus contingencies. This article considers the available data for eyeblink classical conditioning, including data from 2 studies (R. E. Clark, J. R. Manns, & L. R. Squire, 2001; J. R. Manns, R. E. Clark, & L. R. Squire, 2001) that were completed too recently to have been considered in their review. In addition, in response to questions raised by P. F. Lovibond and D. R. Shanks, 2 new analyses of data are presented from studies published previously. The available data from humans and experimental animals provide strong evidence that delay eyeblink classical conditioning (but not trace eyeblink classical conditioning) can be acquired and retained independently of the forebrain and independently of awareness. This conclusion applies to standard conditioning paradigms; for example, to single-cue delay conditioning when a tone is used as the conditioned stimulus (CS) and to differential delay conditioning when the positive and negative conditioned stimuli (CS+ and CS-) are a tone and white noise. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cardiosympathetic neuropathy is independent of cardioparasympathetic neuropathy in patients with non-insulin-dependent diabetes mellitus

The relationship between cardiosympathetic neuropathy and cardioparasympathetic neuropathy was investigated in 103 patients with non-insulin-dependent diabetes mellitus. Cardioparasympathetic nerve function was assessed by comparing electrocardiographically the expiratory and inspiratory R-R interval ratios, during a period of deep breathing, and the coefficients of variation of the R-R intervals. Cardiosympathetic nerve function was assessed by determining the uptake of [123I]metaiodobenzyl guanidine into the myocardium. The results indicate that there is no significant correlation between cardioparasympathetic and cardiosympathetic nerve function.