Adenylate kinase as a virulence factor of Pseudomonas aeruginosa

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.     

Disruption of transforming growth factor beta-regulated laminin receptor function by expression of antisense laminin, a chain RNA in human colon cancer cells

Transforming growth factor beta1 (TGFbeta) simultaneously induces the expression of fibronectin, fibronectin receptor, laminin, and laminin receptor (alpha6beta1 integrin) in the human colon cancer cell line Moser (Int J Cancer, 57:742, 1994). Induction of fibronectin and induction of fibronectin receptor by TGFB are tightly coupled, and disrupting fibronectin induction disrupts the induction of fibronectin receptor and cellular adhesion to fibronectin (J Cellular Physiol, 170:138, 1997). We recently demonstrated the efficacy of using antisense chain-specific laminin RNA expression vectors to disrupt the induction by TGFP of the multichain laminin molecule (J Cellular Physiol, 178:296, 1999). We now show in this report that Moser cells used alpha6 and beta1 integrins to adhere to laminin, and, as is the fibronectin and fibronectin receptor system, disrupting the induction by TGFbeta of the ligand laminin by the expression of antisense laminin A chain RNA disrupted the induction of 125I-laminin binding and cellular adhesion to laminin. Disrupting laminin induction also blocked the induction of alpha6 and beta1 integrin laminin receptor by TGFbeta. We conclude that disrupting the induction of the ligand laminin by TGFbeta disrupts TGFbeta-regulated laminin receptor function by suppressing the induction of alpha6 and beta1 integrins. Therefore, targeted disruption of the ligand laminin may be an effective means in disrupting the function of both the ligand and its receptor in cells that utilize the laminin and laminin receptor system in malignant cell behavior.     

The ubiquitin ligase XBAT32 regulates lateral root development in Arabidopsis

Ubiquitin-mediated protein modification plays a key role in many cellular signal transduction pathways. The Arabidopsis gene XBAT32 encodes a protein containing an ankyrin repeat domain at the N-terminal half and a RING finger motif. The XBAT32 protein is capable of ubiquitinating itself. Mutation in XBAT32 causes a number of phenotypes including severe defects in lateral root production and in the expression of the cell division marker CYCB1;1::GUS . The XBAT32 gene is expressed abundantly in the vascular system of the primary root, but not in newly formed lateral root primordia. Treatment with auxin increases the expression of XBAT32 in the primary root and partially rescues the lateral root defect in xbat32 - 1 mutant plants. Thus, XBAT32 is a novel ubiquitin ligase required for lateral root initiation.     

Crystallization and preliminary X-ray structural studies of hemoglobin A2 and hemoglobin E,isolated from the blood samples of beta-thalassemic patients

Hemoglobin A(2) (alpha(2)delta(2)), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is a failure of beta-chain production, HbA(2) acts as the predominant oxygen delivery mechanism. Hemoglobin E, is another common abnormal hemoglobin, caused by splice site mutation in exon 1 of beta globin gene, when combines with beta-thalassemia, causes severe microcytic anemia. The purification, crystallization, and preliminary structural studies of HbA(2) and HbE are reported here. HbA(2) and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from beta-thalassemia minor and E beta-thalassemia. X-ray diffraction data of HbA(2) and HbE were collected upto 2.1 and 1.73 A, respectively. HbA(2) crystallized in space group P2(1) with unit cell parameters a=54.33 A, b=83.73 A, c=62.87 A, and beta=99.80 degrees whereas HbE crystallized in space group P2(1)2(1)2(1) with unit cell parameters a=60.89 A, b=95.81 A, and c=99.08 A. Asymmetric unit in each case contains one Hb tetramer in R(2) state.     

Effect of ascorbic acid on stimulatory status of activated mouse peritoneal phagocytes

Mouse peritoneal macrophages (MPM) when elicited by the antioxidant ascorbic acid have been found to be significantly stimulatory, exhibiting marked alteration at the cellular and enzyme levels. Alterations recorded were as follows--cellular yield per mouse, their protein content, lysosomal acid hydrolase levels and capability to phagocyte, all were significantly enhanced. The new stimulant was observed to produce no synergistic action on MPM when thioglycollate, BCG or endotoxin along with the same stimulated the latter. Levels of antioxidants like ascorbic acid and glutathione were found to be enhanced in elicited macrophages whereas superoxide dismutase levels varied when the three above stimulators were administered. However, the ascorbic acid elicited cells showed an increase in glutathione levels and a decrease in SOD levels but no change in total intracellular ascorbic acid levels. Further, though ascorbic acid interaction enhanced the phagocytic capability of MPM as compared to resident cells, no significant boosting of phagocytic process could be observed when each of three stimulators coupled with ascorbic acid was used for macrophage elicitation.     

Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower

A number of factors that are known to influence genetic transformation were evaluated to optimizeAgrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized intoAgrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the syntheticcryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae.     

Potentiality of a new compound for in vitro differentiation between halophilic and non-halophilic vibrios

Sensitivity of 21 halophilic vibrios and 16 clinical isolates of non-halophilic vibrios was determined against a new possible antivibrio agent, a pyrimidine analogue, 4, 6-dimethylpyrimidine -2-thiol (4,6-DMPT). It appeared to be a vibriocidal agent, having a mean MIC and MBC of 32 microg/ml for halophilic strains and 64 microg/ml for non-halophilic strains and an LD50 of 300 mg/Kg body weight of mice. Thus, 4,6-DMPT may help an in vitro distinction between halophilic and non-halophilic vibrios. Sensitivity of these strains was also studied with respect to pteridine, crystal violet and Tween 80 hydrolysis as further markers distinguishing between these 2 groups which could also be differentiated by their growth on TCBS or/and CLED media.     

Redox proteins in mammalian cell death: an evolutionarily conserved function in mitochondria and prokaryotes

Mammalian cell mitochondria are believed to have prokaryotic ancestry. Mitochondria are not only the powerhouse of energy generation within the eukaryotic cell but they also play a major role in inducing apoptotic cell death through release of redox proteins such as cytochrome c and the apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity. Recent evidence indicates that some present day prokaryotes release redox proteins that induce apoptosis in mammalian cells through stabilization of the tumour suppressor protein p53. p53 interacts with mitochondria either directly or through activation of the genes for pro-apoptotic proteins such as Bax or NOXA or genes that encode redox enzymes responsible for the production of reactive oxygen species (ROS). The analogy between the ancient ancestors of present day bacteria, the mitochondria, and the present day bacteria with regard to their ability to release redox proteins for triggering mammalian cell death is an interesting example of functional conservation during the hundreds of millions of years of evolution. It is possible that the ancestors of the present day prokaryotes released redox proteins to kill the ancestors of the eukaryotes. During evolution of the mitochondria from prokaryotes as obligate endosymbionts, the mitochondria maintained the same functions to programme their own host cell death.     

Novel Pathway for Conversion of Chlorohydroxyquinol to Maleylacetate in Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and β-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.     

Indian Plant Germplasm on the Global Platter: An Analysis

Food security is a global concern amongst scientists, researchers and policy makers. No country is self-sufficient to address food security issues independently as almost all countries are inter-dependent for availability of plant genetic resources (PGR) in their national crop improvement programmes. Consultative Group of International Agricultural Research (CGIAR; in short CG) centres play an important role in conserving and distributing PGR through their genebanks. CG genebanks assembled the germplasm through collecting missions and acquisition the same from national genebanks of other countries. Using the Genesys Global Portal on Plant Genetic Resources, the World Information and Early Warning System (WIEWS) on Plant Genetic Resources for Food and Agriculture and other relevant databases, we analysed the conservation status of Indian-origin PGR accessions (both cultivated and wild forms possessed by India) in CG genebanks and other national genebanks, including the United States Department of Agriculture (USDA) genebanks, which can be considered as an indicator of Indian contribution to the global germplasm collection. A total of 28,027,770 accessions are being conserved world-wide by 446 organizations represented in Genesys; of these, 3.78% (100,607) are Indian-origin accessions. Similarly, 62,920 Indian-origin accessions (8.73%) have been conserved in CG genebanks which are accessible to the global research community for utilization in their respective crop improvement programmes. A total of 60 genebanks including 11 CG genebanks have deposited 824,625 accessions of PGR in the Svalbard Global Seed Vault (SGSV) as safety duplicates; the average number of accessions deposited by each genebank is 13,744, and amongst them there are 66,339 Indian-origin accessions. In principle, India has contributed 4.85 times the number of germplasm accessions to SGSV, in comparison to the mean value (13,744) of any individual genebank including CG genebanks. More importantly, about 50% of the Indian-origin accessions deposited in SGSV are traditional varieties or landraces with defined traits which form the backbone of any crop gene pool. This paper is also attempting to correlate the global data on Indian-origin germplasm with the national germplasm export profile. The analysis from this paper is discussed with the perspective of possible implications in the access and benefit sharing regime of both the International Treaty on Plant Genetic Resources for Food and Agriculture and the newly enforced Nagoya Protocol under the Convention on Biological Diversity.