Effect of air, anesthetic gas mixture, saline, or 2% lignocaine used for tracheal tube cuff inflation on coughing and laryngotracheal morbidity after tracheal extubation.

BACKGROUND AND AIMS: Coughing and sore throat postoperatively are common clinical problems during general anesthesia which can be avoided by various methods including topicalization of airway with local anesthetics, endotracheal tube cuff (ETT) inflation with local anesthetics, use of intravenous drugs such as dexamethasone, maintaining ETT cuff pressure, intubation by an experienced anethesiologist, etc. The aims of the study were to compare postextubation coughing response, mean number of cuff deflations required intraoperatively, and postoperative airway morbidity in terms of sore throat (2 h and 18-24 h), hoarseness of voice, and dysphagia following inflation of ETT cuff with air, anesthetic gas mixture, saline, and 2% lignocaine during general anesthesia. MATERIAL AND METHODS: One hundred and four patients were randomized into 1 of 4 groups depending on whether air, anesthetic gas mixture, saline, or 2% lignocaine was used to inflate the cuff of ETT using computer-generated randomization table. RESULTS: There was no significant difference in the postextubation cough response among the four groups. The mean number of times the ETT cuff was deflated was significantly in favor of liquid media comapred to gaseous media (P < 0.001). The incidence of sore throat at 2 h and at 18-24 h, hoarseness of voice, and dysphagia were comparable in all groups. CONCLUSION: ETT cuff inflation with air, anesthetic gas mixture, 2% lignocaine, and saline are comparable in terms of incidence of postextubation cough and postoperative airway morbidity symptoms such as sore throat, hoarseness of voice, and dysphagia.

Isolation and functional characterization of bacterial endophytes from Carica papaya fruits.

AIMS: To isolate and characterize the endophytes from papaya fruits and to determine the fermentative potential of the strains. METHODS AND RESULTS: Endophytes provide potential sources for novel natural products for the use in agriculture and nutrition. There is very limited information on isolation and characterization of bacterial endophytes from papaya. We describe isolation and characterization of eighteen endophytes of papaya fruit from four economically important papaya varieties viz 'Red lady', 'Solo', 'Coorg Honey' and 'Bangalore'. The phylogenetic analysis based on the 16S rRNA sequence revealed that isolated endophytes are genetically distinct and cluster as discrete clades in the dendrogram. The Bacillus species is a predominant bacterial endophyte across papaya varieties. The seeds and the endocarp of papaya fruits harbour Kocuria, Acinetobacter and Enterobacter species. The Staphylococcus species were detected in the fruit mesocarp of two papaya varieties used in the study. The endophytes isolated from papaya fruits were capable of producing extracellular enzymes like amylase, cellulase, pectinase and xylanase. Three isolates, Bacillus (PE-LR-1 and PE-LR-3) and Kocuria (PE-LR-2), were selected for fruit fermentation, and antioxidant potential of the fermented product was evaluated. PE-LR-3 fermented product has the free radical scavenging activity of 61·2% and a microbial cocktail of PE-LR-3 with Saccharomyces cerevisiae MTCC 2918 enhances the antioxidant potential to 75·7%. CONCLUSION: These findings suggest that different parts of papaya fruits harbour an array of bacterial endophytes that could be important agents in attributing the high nutritive status to the fruit and can serve as potent microbial cocktails for developing value-added fermented products of this important fruit. SIGNIFICANCE AND IMPACT OF STUDY: This study describes isolation of a bacterial endophyte from papaya fruit that is capable of improving the antioxidant potential of raw papaya after fermentation.

A novel Arabidopsis thaliana protein protects tumor cells from tumor necrosis factor-induced apoptosis.

Recently we have cloned and characterized a novel, oxidative stress-induced Arabidopsis thaliana gene (oxy5), and showed that expression of oxy5 protects bacterial cells from death caused by oxidative stress. As oxidative stress is one pathway of TNF cytotoxicity, we investigated whether the encoded protein could also protect human tumor cells from TNF killing. We stably transfected the oxy5 gene into TNF-sensitive HeLa D98 cells (D98/O.5), and found that all examined transfectants were highly TNF-resistant in the absence of the protein synthesis inhibitor cycloheximide. The acquired TNF resistance of these clones was accompanied by a sharp decrease in the intracellular formation of reactive oxygen species, suggesting the activation of antioxidant enzymes like superoxide dismutases (SODs). Indeed, D98/O.5 clones showed an increased manganous superoxide dismutase (MnSOD) mRNA and protein expression in the absence or presence of TNF stimulation, whereas the expression of the Cu/ZnSOD was not affected. Furthermore, the elevated MnSOD expression in the D98/O.5 clones correlated well with an increased antioxidative activity, which was specifically due to MnSOD as measured by the suppression of xanthine oxidase. Our results demonstrate a novel role for a plant-derived protein in resistance to TNF cytotoxicity, and that the Arabidopsis thaliana protein Oxy5 can exert its protective function across evolutionary boundaries through activation of antioxidant enzymes like MnSOD.

Oxy5, a novel protein from Arabidopsis thaliana, protects mammalian cells from oxidative stress.

The use of molecular oxygen in various cellular processes results in the generation of toxic intracellular by-products termed reactive oxygen species (ROS). In Escherichia coli the oxyR gene product is a transcriptional regulator of the oxyR regulon that is induced in response to hydrogen peroxide-induced oxidative stress (OS). We have previously shown that an annexin-like protein from Arabidopsis thaliana, termed Oxy5, can replace the obligatory role of OxyR in E. coli. Here, we have investigated as to whether oxy5 can function across evolutionary boundaries to protect mammalian cells from OS. Overexpression of the oxyR gene in mammalian tumor cell lines protects them from hydrogen peroxide-induced cell death, and these cells are also highly resistant to the superoxide ion producing compound paraquat. Oxy5 appears to be involved in the detection of calcium flux, as it binds to Ca2+ ions during hydrogen peroxide stress. Moreover, overexpression of Oxy5 leads to lowered protein kinase C activity. Thus, Oxy5 probably functions to sense and initiate protective responses to OS. In addition, Oxy5-overexpressing cells exhibit a reduction in endogenous superoxide ion levels, which concomitantly results in a dramatic decrease in their tumorigenic potential. Taken together, the results demonstrate an antioxidant role for plant oxy5 gene in mammalian cells, which can be potentially utilized in gene therapy programs aimed at reducing the deleterious effects of ROS.

Localization by immunogold labelling of HIV-1 structural proteins on Lowicryl embedded HIV-1 infected cell ultrathin sections.

Using several HIV-1-specific antibodies and the immunogold labelling technique, we have detected and localized distinct viral proteins on ultrathin sections of HIV-1 infected cells embedded in the Lowicryl K4M resin. Monoclonal antibodies (MAbs) against p24, p17 and gp160/gp41 showed a preferential labelling on viral formations still attached to the cell membrane (budding process) or free in the extracellular space. The anti-p24 and the anti-p17 MAb yielded a gold labelling not only on mature but also on immature virions where the gold particles were associated with the ring-like electron-dense material. The three HIV-1-specific antibodies against p24, p17 and p55 yielded a cross-reaction with HIV-2 in agreement with the conservation of the internal antigenic determinants of both viruses. In all instances, there was no specific immunogold labelling over the cells, suggesting that once the virus structural proteins were synthesized, they were promptly utilized for virus assembly at the plasma membrane level.

[Epitope specificity and protective activity of monoclonal antibodies to Venezuelan equine encephalomyelitis virus]. / Epitopnaia spetsifichnost' i protektivnaia aktivnost' monoklonal'nykh antitel k virusu venesuél'skogo éntsefalomielita loshadei.

Epitope specificity and protective activity of 5 types of monoclonal antibodies (MAB) to strain TM-14 of Venezuelan equine encephalomyelitis (VEE) virus were studied. The competing VEE A4 and VEE B5 MAB were shown to recognize the conformation-dependent epitope of glycoprotein E2 third site and to possess an extremely high protective activity. VEE C6 MAB were active in all the studied biological tests (hemagglutination inhibition, neutralization tests, passive defence) and were directed to glycoprotein E2 site six. Competitive radioimmunoassay demonstrated that VEE A5 MAB also interacted with E2-6 site and by the sum of characteristics detected a new epitope of this site. VEE G1 MAB specific to glycoprotein E1 were characterized by protective, but not neutralizing activity, and competed with MAB to sites E2-2 and E2-6. This means that VEE G1 MAB detect a heretofore not described epitope E1 overlapping with two E2 sites.

Structural and functional analyses of a saturated acyl ACP thioesterase, type B from immature seed tissue of Jatropha curcas.

The distinguishing structural and functional domains of plant acyl-acyl carrier protein (ACP) thioesterases and their complex interaction with the ACP-linked fatty acid substrate complex have remained elusive. E. coli based heterologous expression and characterisation of many plant thioesterases reported so far have not been extended and linked to in silico modelling studies to explain the diversity in plant thioesterase substrate specificities. In this study, a thioesterase cDNA isolated from immature seed tissues of Jatropha curcas was found to be type B and specific to stearoyl acyl ACP when expressed in E. coli K27fadD88, a lipid utilisation mutant. Homology modelling and molecular docking of a selected region of the isolated JcFatB protein predicted that it had high affinity towards both stearate (18:0) and palmitate (16:0). Structural analysis of the sequence confirmed the presence of a transit peptide that is processed in multiple steps. The enzyme is localised in the chloroplasts and has an N-terminal inner chloroplast transmembrane domain characteristic of type B plant thioesterases. Docking of ligands with JcFatB and its comparison with a modelled Jatropha thioesterase type A provided further evidence for native substrate preferences of Jatropha thioesterases. This study provides essential clues to develop future methods for large-scale bacterial production of free fatty acids and for design of strategies to modulate the seed oil composition in this important non-edible, seed oil plant.

Synthesis and in vitro antitumor activities of novel 4-anilinoquinazoline derivatives.

A series of 6, 7-dialkoxy-4-anilinoquinazolines were designed, synthesized by substituting different heterocycles on 6-position and a variety of anilines on 4-position of the quinazoline. These novel quinazoline compounds were screened for their cytotoxic effect on epidermal growth factor receptor overexpressing skin epidermoid carcinoma cell line (A431), by using nonoverexpressing tumor cells as negative control (breast adeno carcinoma cell line MCF-7). 2-Butyl-4-chloro-1-{3-[7-methoxy-4-(3-(trifluoromethyl)phenylamino)quinazolin-6-yloxy]-propyl}-1H-imidazole-5-carboxaldehyde (30) and 2-butyl-4-chloro-1-{3-[4-(3-iodophenyl amino)-7-methoxyquinazolin-6-yloxy]propyl}-1H-imidazole-5-carboxaldehyde (33) were found to be more potent against A431 cell line (IC(50) 3.5 and 3 microM) and their activities are comparable to gefitinib. Insilico docking experiments with human EGFR Tyrosine kinase domain (PDB id-2gs2) indicated that 33 docks at the same position as that of gefitinib involving Val702, Ala719, Ser696, and Lys721.

Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis).

Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level.

Hevein, a lectin-like protein from Hevea brasiliensis (rubber tree) is involved in the coagulation of latex.

Hevein, a lectin-like protein is the major protein of vacuolar structures called lutoids in the latex of rubber trees. We have shown both by in planta and ex planta studies that hevein is involved in the coagulation of latex by bringing together rubber particles. This polyvalent bridging between hevein and rubber particles is mediated by N-acetyl-D-glucosamine and involves a receptor glycoprotein of 22 kDa, which is localized on the surface of the rubber particles. The proposed role of hevein helps us to understand the mechanism of coagulation of latex and assigns a physiological intracellular function to one of the smallest lectins.

Accumulation of reactive oxygen species and oxidation of cytokinin in germinating soybean seeds.

Seed germination is an important developmental switch when quiescent seed cells initiate oxidative phosphorylation for further development and differentiation. During early imbibition of soybean seeds (Glycine max L. cv. Weber), a superoxide dismutase (SOD) activity peak was observed, in embryonic axes, after 6 h imbibition. Peroxidase activities, including catalase, were significantly increased after 12 h inhibition and during germination phase III. Catalase was the most efficient enzyme in catabolizing H2O2 in embryonic axes. When stored at 42 degrees C and 100% relative humidity, seeds were stressed and lost their viability in a time-dependent manner. A significant increase in the Cu, Zn-superoxide-dismutase activity, and to a lesser extent, Mn superoxide dismutase activity was observed during germination in low-viability (stressed) seeds as compared to high-viability (unstressed) seeds. Northern blot analysis confirmed that superoxide dismutase induction resulted from an accumulation of its transcripts in response to the production of O2-. The induction of catalase did not occur in low-viability seeds, resulting in dramatic accumulation of H2O2. Using capillary electrophoresis, HPLC and NMR we found that the endogenous cytokinin, zeatin riboside, was present in large quantities in the high-viability seeds, but it was oxidized into adenine in the low-viability seeds. In vitro superoxide anion could also oxidize the cytokinin. Zeatin riboside, but not adenine, was found to act as a scavenger of superoxide anions and may help to maintain seed viability by detoxifying reactive oxygen species. Germination of stressed seeds was partially restored by the addition of exogenous cytokinin (zeatin riboside). Protection against oxidative stress by cytokinin seemed to be a general phenomenon, as Escherichia coli cells were also protected against superoxide stress in the presence of cytokinin.

Wound-induced accumulation of mRNA containing a hevein sequence in laticifers of rubber tree (Hevea brasiliensis).

Hevein is a chitin-binding protein that is present in laticifers of the rubber tree (Hevea brasiliensis). A cDNA clone (HEV1) encoding hevein was isolated by using the polymerase chain reaction with mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea latex cDNA library as a template. HEV1 is 1018 base pairs long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187-amino acid polypeptide. This polypeptide has two striking features. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino termini of wound-inducible proteins in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems, and latex but not in roots.

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene.

The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.

Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress.

Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.

Laticifer-specific gene expression in Hevea brasiliensis (rubber tree).

Natural rubber, cis-1,4-polyisoprene, is obtained from a colloidal fluid called latex, which represents the cytoplasmic content of the laticifers of the rubber tree (Hevea brasiliensis). We have developed a method of extracting translatable mRNA from freshly tapped latex. Analysis of in vitro translation products of latex mRNA showed that the encoded polypeptides are very different from those of leaf mRNA and these differences are visible in the protein profiles of latex and leaf as well. Northern blot analysis demonstrated that laticifer RNA is 20- to 100-fold enriched in transcripts encoding enzymes involved in rubber biosynthesis. Plant defense genes encoding chitinases, pathogenesis-related protein, phenylalanine ammonia-lyase, chalcone synthase, chalcone isomerase, cinnamyl alcohol dehydrogenase, and 5-enolpyruvylshikimate-3-phosphate synthase show a 10- to 50-fold higher expression in laticifers than in leaves, indicating the probable response of rubber trees to tapping and ethylene treatment. Photosynthetic genes encoding ribulose-bisphosphate carboxylase small subunit and chlorophyll a/b-binding protein are not expressed at a detectable level in laticifers. In contrast, genes encoding two hydrolytic enzymes, cellulase and polygalacturonase, are more highly expressed in laticifers than in leaves. Transcripts for the cytoplasmic form of glutamine synthase are preferentially expressed in laticifers, whereas those for the chloroplastic form of the same enzyme are present mainly in leaves. Control experiments demonstrated that beta-ATPase, actin, and ubiquitin are equally expressed in laticifers and leaves. Therefore, the differences in specific transcript abundance between laticifers and leaves are due to differential expression of the genes for these transcripts in the laticifers.

Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Hevea brasiliensis.

Hevea brasiliensis is the major producer of natural rubber which is cis-1,4-polyisoprene. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is involved in the biosynthesis of rubber and other plant products. We have used a hamster HMGR cDNA clone as a heterologous hybridization probe to isolate and characterize cDNA and genomic clones of HMGR from H. brasiliensis. Sequence analysis revealed that these clones fall into two different classes, HMGR1 and HMGR2. Comparison of the two classes shows 86% nucleotide sequence homology and 95% amino acid homology. The carboxy-termini of Hevea HMGRs are highly homologous to those of hamster, yeast and Arabidopsis HMGR. The amino-terminus of Hevea HMGR contains two potential membrane-spanning domains as in Arabidopsis HMGR while seven such domains are found in the HMGRs of other organisms. The apparent molecular mass of Hevea HMGR was estimated in western blot analysis to be 59 kDa. Northern blot analysis indicated that the HMGR1 transcript of 2.4 kb is more highly-expressed in laticifer than in leaf. Genomic Southern analysis using 3'-end cDNA probes indicates the presence of at least two HMGR genes in Hevea.

Distribution patterns of 104 kDa stress-associated protein in rice.

A 104 kDa protein (SAP 104) accumulates in rice seedlings in response to several abiotic stress conditions and immunological homologues of rice SAP 104 have been detected in several monocot and dicot species, as also Neurospora crassa, a fungus. In this report, we show that the amino acid sequence of a tryptic peptide generated from purified SAP 104 bears significant homology with an ATP-binding domain of the HSP 100 family proteins of Arabidopsis thaliana and Glycine max. It is further shown that differential uninduced and induced (by high-temperature stress) levels of this protein are accumulated in various organs of the mature rice plant grown under field conditions. Significant uninduced levels of this protein were in particular found in developing and mature rice grains. Seeds/grains of several other plant genera (i.e. Triticum aestivum, Zea mays, Brassica juncea) were also found to contain high uninduced levels of SAP 104. Importantly, the levels of uninduced SAP 104 in rice grains were found to decline during the seed germination phase: after two days of germination, this protein was undetectable in tissues representing pooled sample of seeds and just-emerged seedlings. Tissue print-immunoblotting analysis has indicated that in seeds high levels of this protein are specifically present in the embryo portion.