The role of enzymatic activities of antiviral proteins from plants for action against plant pathogens.

Antiviral proteins (AVPs) from plants possess multiple activities, such as N-glycosidase, RNase, DNase enzymatic activity, and induce pathogenesis-related proteins, salicylic acid, superoxide dismutase, peroxidase, and catalase. The N-glycosidase activity releases the adenine residues from sarcin/ricin (S/R) loop of large subunit of ribosomes and interfere the host protein synthesis process and this activity has been attributed for antiviral activity in plant. It has been shown that AVP binds directly to viral genome-linked protein of plant viruses and interfere with protein synthesis of virus. AVPs also possess the RNase and DNase like activity and may be targeting nucleic acid of viruses directly. Recently, the antifungal, antibacterial, and antiinsect properties of AVPs have also been demonstrated. Gene encoding for AVPs has been used for the development of transgenic resistant crops to a broad range of plant pathogens and insect pests. However, the cytotoxicity has been observed in transgenic crops using AVP gene in some cases which can be a limiting factor for its application in agriculture. In this review, we have reviewed various aspects of AVPs particularly their characteristics, possible mode of action and application.

Role of virtual bronchoscopy in children with a vegetable foreign body in the tracheobronchial tree.

OBJECTIVES: Multidetector computed tomography virtual bronchoscopy is a non-invasive diagnostic tool which provides a three-dimensional view of the tracheobronchial airway. This study aimed to evaluate the usefulness of virtual bronchoscopy in cases of vegetable foreign body aspiration in children. METHODS: The medical records of patients with a history of foreign body aspiration from August 2006 to August 2010 were reviewed. Data were collected regarding their clinical presentation and chest X-ray, virtual bronchoscopy and rigid bronchoscopy findings. Cases of metallic and other non-vegetable foreign bodies were excluded from the analysis. Patients with multidetector computed tomography virtual bronchoscopy showing features of vegetable foreign body were included in the analysis. For each patient, virtual bronchoscopy findings were reviewed and compared with those of rigid bronchoscopy. RESULTS: A total of 60 patients; all children ranging from 1 month to 8 years of age, were included. The mean age at presentation was 2.01 years. Rigid bronchoscopy confirmed the results of multidetector computed tomography virtual bronchoscopy (i.e. presence of foreign body, site of lodgement, and size and shape) in 59 patients. In the remaining case, a vegetable foreign body identified by virtual bronchoscopy was revealed by rigid bronchoscopy to be a thick mucus plug. Thus, the positive predictive value of virtual bronchoscopy was 98.3 per cent. CONCLUSION: Multidetector computed tomography virtual bronchoscopy is a sensitive and specific diagnostic tool for identifying radiolucent vegetable foreign bodies in the tracheobronchial tree. It can also provide a useful pre-operative road map for rigid bronchoscopy. Patients suspected of having an airway foreign body or chronic unexplained respiratory symptoms should undergo multidetector computed tomography virtual bronchoscopy to rule out a vegetable foreign body in the tracheobronchial tree and avoid general anaesthesia and invasive rigid bronchoscopy.

Synthesis of mesoporous silica nanoparticles and drug loading of poorly water soluble drug cyclosporin A.

Mesoporous silica nanoparticles (MSNs) are introduced as chemically and thermally stable nanomaterials with well-defined and controllable morphology and porosity. It is shown that these particles possess external and internal surfaces that can be selectively functionalized with multiple organic and inorganic groups. Silica nano-particles were synthesized by chemical methods from tetraethylorthosilicate (TEOS), methanol (CH3OH) and deionised water in the presence of sodium hydroxide as catalyst at 80°C temperature. The nature and morphology of particles was investigated by scanning electron microscopy (SEM), N2 adsorption/desorption method using BET instrument and X-ray diffraction (XRD). Silica nanoparticles are applicable to a wide range of therapeutic entities from small molecule to peptides and proteins including hydrophobic and hydrophilic entities. Drug loading does not require chemical modification of the molecule; there are no changes in the drug structure or activity after loading and subsequent release of the drug. Thus, well suited to solve formulation problems associated with hydrophobic drugs such as peptide and protein drugs like cyclosporine A. Silica nanoparticles improved the solubility of poorly water soluble drugs and enhanced the absorption and bioavailability of these compounds.

Ribonuclease, deoxyribonuclease, and antiviral activity of Escherichia coli-expressed Bougainvillea xbuttiana antiviral protein 1.

A full-length cDNA encoding ribosome-inactivating/antiviral protein from the leaves of Bougainvillea xbuttiana was recently isolated. The coding region of cDNA was cloned and expressed in Escherichia coli, and the protein product was designated as BBAP1 (Bougainvillea xbuttiana antiviral protein 1). BBAP1 showed ribonuclease activity against Torula yeast RNA. It also exhibited depurination activity against supercoiled pBlueScript SK+ plasmid DNA in a concentration dependent manner, and was found to convert nicked circular DNA into linear form only at higher concentration. On bioassay, BBAP1 exhibited antiviral activity against sunnhemp rosette virus infecting Cyamopsis tetragonoloba leaves in which 95% inhibition of local lesion formation was observed.

Genetic and epigenetic regulation of stem cell homeostasis in plants.

Plants generate new organs through the activity of small populations of stem cells present in specialized niches called meristems. Stem cell homeostasis is attained by dynamic regulatory networks involving transcriptional regulators, hormones, and other intercellular signals that specify cell fate and convey positional information to the apical stem cells and the organizing center located immediately below. The balance between stem cell maintenance within the shoot apical meristem (SAM) and differentiation of cells that are displaced from the niche to form new organs involves the epigenetic silencing of stem cell regulatory genes. Recent advances have identified highly conserved chromatin remodeling factors as epigenetic regulators of stem cell fate that confer plasticity in plant development and ensure the stable inheritance of repressed expression states during organogenesis. These advances reveal that common mechanisms contribute to stem cell homeostasis in plants and animals.

Diabetic retinopathy is a poor predictor of type of nephropathy in proteinuric type 2 diabetic patients.

BACKGROUND: Both diabetic nephropathy (DN) and nondiabetic nephropathy (NDN) are reported to occur in patients with type 2 diabetes mellitus (DM). The precise diagnosis of the type of nephropathy has obvious clinical and prognostic implication. The aim of the study was to evaluate the histologic spectrum of nephropathy in proteinuric type 2 diabetic patients and to find the correlation between type of nephropathy and diabetic retinopathy (DR). METHODS: Twenty eight proteinuric type 2 diabetic patients were included in the study. Five patients (ADPKD 3 and chronic pyelonephritis 2) were excluded from biopsy. Percutaneous renal biopsy was carried in remaining 23 patients. RESULTS: There was a preponderance of male (75%) and majority of the patients were in the age group of 30-78 years. Duration of diabetes ranged between 4 months to 25 years with mean +/- SD of 10.53 +/- 7.62 years. The presenting features were nephrotic syndrome 14 (60.9%), non-nephrotic proteinuria 9 (39.1%) and impaired renal function in 19 (82.6%) patients. Renal biopsy in 23 cases revealed; isolated diabetic nephropathy 13 (56.2%), NDN7 (13.43%) and 3 (13%) patients had NDN superimposed on diabetic nephropathy. Membranous nephropathy (2), focal segmental glomerulosclerosis (2), mesangiocapillary glomerulonephritis (1) were the nondiabetic glomerular disease in our type 2 diabetic patients. Chronic pyelonephritis and ischemic interstial nephropathy was the predominant tubulointerstial lesion in this study. Diabetic retinopathy (DR) observed in 12 (75%) patients with biopsy proven DN and absent in 4 (25%) patients with DN. The distribution of renal lesions in patients with DR (n = 15) showed DN in 9 (60%), NDN 3 (20%) and remaining 3 patients had combined lesions. Renal biopsy in 8 patients without DR showed typical DN in 4 (50%) and NDN in 4 (50%) patients. CONCLUSION: This study demonstrates presence of both glomerular and tubulointerstitial lesions unrelated to diabetes (NDN) in proteinuric type 2 diabetic patients. Further presence or absence of DR was a poor predictor of diabetic nephropathy because DN was noted in 50% of patients without DR and 40% of patients with DR had non-diabetic nephropathy either alone or in combination with DN.

Purification, characterization and cloning of antiviral/ribosome inactivating protein from Amaranthus tricolor leaves.

An antiviral protein (AVP), imparting high level of resistance against sunnhemp rosette virus (SRV) was purified from the dried leaves of Amaranthus tricolor. The purified protein (AAP-27) exhibited approximately 98% inhibition of local lesion formation at a concentration range of approximately 30 microg ml(-1). The protein was found to be highly basic glycoprotein monomer (pI approximately 9.8) of Mr 27 kDa, with neutral sugar content of 4%. The purified protein exhibited N-glycosidase and RNase activities. We have also isolated full-length cDNA clone, encoding this protein designated as A. tricolor antiviral protein-1 (AAP-1). Two primers, one designed on the basis of N-terminal sequence of the purified protein and the other from the conserved active peptides of other AVPs/RIPs were used for PCR amplification of double stranded cDNA, isolated from the leaves of A. tricolor. The amplified fragment was used as a probe for library screening. The isolated full-length cDNA consisted of 1058 nucleotides with an open reading frame encoding a polypeptide of 297 amino acids. The deduced amino acid sequence of AAP-1 has a putative active domain conserved in other AVPs/RIPs and shows varying homology to the RIPs from other plant species.

An antiviral protein having deoxyribonuclease and ribonuclease activity from leaves of the post-flowering stage of Celosia cristata.

An antiviral protein named CCP-27 was purified from the leaves of Celosia cristata at the post-flowering stage by anion-exchange, cation-exchange, and gel-filtration chromatography. It exhibited resistance against sunnhemp rosette virus in its test host Cyamopsis tetragonoloba. It also exhibited deoxyribonuclease activity against supercoiled pBlueScript SK+ plasmid DNA. It was found to nick supercoiled DNA into nicked circular form at lower protein concentration followed by nicked to linear form conversion at higher protein concentration. CCP-27 also possesses strong ribonuclease activity against Torula yeast rRNA.

Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.

RNase and DNase activities of antiviral proteins from leaves of Bougainvillea xbuttiana.

Antiviral proteins (AVPs) purified from the leaves of Bougainvillea xbuttiana cv Mahara exhibited RNase activity against viral RNA of the tobamoviruses, Tobacco mosaic virus (TMV) and Sunnhemp rosette virus (SRV). They caused complete degradation of viral RNAs in a concentration-dependent manner. RNase activity gel assay ruled out the possibility of the presence of contaminating nucleases. AVPs also showed DNase activity, as indicated by conversion of supercoiled form of plasmid DNA into relaxed and linear forms. The implications of these activities in controlling plant viruses are discussed.

Possible mechanism of action of antiviral proteins from the leaves of Chenopodium album L.

Antiviral proteins (AVPs) named CAP-I and CAP-II purified from the leaves of Chenopodium album cv Pusa Bathua-1 induced systemic resistance against tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) in both hypersensitive as well as systemic hosts. An increased accumulation of two polypeptides (approximately 17 kDa and approximately 26 kDa) was observed in untreated upper leaves of Cyamopsis tetragonoloba plants whose basal leaves were treated with CAP-I/CAP-II. Both AVPs exhibited ribosomal RNA N-glycosidase activity on 28S rRNA of tobacco leaves and also caused in vitro degradation of TMV RNA. It is suggested that the CAP-I and -II are multi-functional and may be acting at multiple levels to ensure maximum possible inhibition of viral infection.

A systemic resistance inducing antiviral protein with N-glycosidase activity from Bougainvillea xbuttiana leaves.

An antiviral protein from Bougainvillea xbuttiana leaves induced systemic resistance in host plants N. glutinosa and Cyamopsis tetragonoloba against TMV and SRV, respectively which was reversed by actinomycin D, when applied immediately or shortly after antiviral protein treatment. When the inhibitor was applied to the host plant leaves post inoculation, it was effective if applied upto 4 h after virus infection. It also delayed the expression of symptoms in systemic hosts of TMV. The inhibitor showed characteristic N-glycosidase activity on 25S rRNA of tobacco ribosomes, suggesting that it could also be interfering with virus multiplication through ribosome-inactivation process.

Purification and properties of antiviral proteins from the leaves of Bougainvillea xbuttiana.

A non-phytotoxic, resistance inducing, proteinaceous antiviral principle was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration from the leaves of Bougainvillea xbuttiana. It imparted resistance against tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) in their respective test hosts viz. Nicotiana glutinosa, N. tabacum var. Samsun NN, and Cyamopsis tetragonoloba, respectively. The purified principle eluted as a single peak upon gel filtration, but exhibited two polypeptides on SDS-PAGE with Mr 28,000 and 24,000. The two polypeptides were found to be highly basic, rich in lysine with pI around 10.0 and 10.5, respectively. Since this principle effected local lesion inhibition in both treated and untreated top leaves of test host, it might be acting in the initial stages of virus infection as a systemic inducer.

Cloning of acyl-acyl carrier protein (ACP) thioesterase gene from Brassica juncea.

Acyl-acyl carrier protein (ACP) thioesterase is the chain-length-determining enzyme in de novo biosynthesis of plant fatty acids. For cloning the gene encoding acyl-ACP thioesterase from Brassica juncea, genomic DNA was used as a template to amplify a 0.7 kb thioesterase fragment in a PCR with the primers designed from the known sequences available in the GenBank. This 0.7 kb fragment was used as a probe to study the expression of the gene in developing seeds and also to screen a genomic library of B. juncea constructed in lambdaEMBL-3 to get the full length of the gene. A 4.0 kb BamHI fragment containing the full gene was finally cloned in a plasmid vector from a recombinant phage clone lambda5.12 after a series of screening, sub-cloning and Southern hybridization.

Effect of combined nitrogen on the expression of nitrate reductase and nitrite reductase in Azorhizobium caulinodans.

In aerobically grown Azorhizobium caulinodans strain IRBG 46, in vivo expression of nitrate reductase (NR) and nitrite reductase (NiR) requires the presence of either nitrate or nitrite. On the contrary mere microaerobic conditions are sufficient for the expression of NR and NiR, however, addition of nitrate to the growth medium enhanced the activities of the enzymes. Optimum concentration of nitrate for maximum expression of NR and NiR activities was different in aerobic and microaerobic conditions. Nitrite was released into the medium both in aerobic and microaerobic conditions beyond a particular concentration of nitrate in the medium. Dissimilatory nitrate reduction was affected to a lesser extent by ammonium compared to assimilatory nitrate reduction.

Effect of nitrate on nitrogen fixation and nitrate assimilation in Azorhizobium-Sesbania rostrata symbiotic system.

Root nodule formation was inhibited by 30% and 50% respectively at low concentration of 1 mM and 2 mM nitrate, while stem nodule formation was enhanced by 50% only at 1 mM nitrate. The nodule specific nitrogenase activity decreased with the increasing concentration of nitrate. At 1 mM nitrate nitrogenase activity per plant stem nodule was not affected, but it was less than 50% in the root nodules as compared to control. Increasing concentration of nitrate increased in vivo activity of nitrate reductase (NR) significantly in stem, root nodules and leaves. Nodule cytosolic NR utilized both NADH and succinate as electron donor, but not reduced MV. However bacteroidal NR utilised reduced MV as reductant more efficiently than succinate.

Expression of Rhizobium meliloti symbiotic promoters in Azorhizobium caulinodans.

Plasmids containing Rhizobium meliloti symbiotic promoters P1 (promoter of nifHDK) and P2 (promoter of fixABCX) when mobilized into the cells of Azorhizobium caulinodans strain IRBG 46 showed strong expression of these promoters under free-living microaerobic as well as symbiotic conditions. Under free-living conditions microaerobiosis (3% or less O2) was found to be sufficient to activate these promoters; expression being higher at 1% than at 3% O2 concentration. Under symbiotic conditions the expression was much more stronger-with bacteroids in stem nodules showing higher expression than those in root nodules. Under both the conditions expression of the promoters in the native R. meliloti strain Rm102F34 was lower than that in the A. caulinodans strain IRBG 46. The results suggest a functional homology of these promoters in the heterologous background of A. caulinodans.

Integration ofhup cosmid pHU52 into the chromosomal DNA ofCicer-Rhizobium using Tn5 as an homologous sequence.

Cosmid pHU52, which carrieshup genes ofBradyrhizobium japonicum, has been integrated into theCicer-Rhizobium G36-84 genome via Tn5-mediated homologous recombination. Tn5 was inserted into both the cosmid pHU52 and the chromosome ofCicer-Rhizobium to provide a region of DNA homology, without affecting the expression of necessary genes. An incompatible plasmid, pPH1JI, was used to select those few cells that had undergone recombination. The integration of the cosmid was demonstrated by Southern blot analysis. Chromosomal integration of thehup genes maximized stability and minimized the potential for their horizontal transfer to other bacterial species. The integratedhup genes were found to expressex planta as well in nodules. The method described illustrates how a given gene can be stably integrated into the chromosome.