Evaluation of some essential oils as botanical fungitoxicants in management of post-harvest rotting of citrus fruits

During screening of some essential oils against Penicillium italicum, the oils of Mentha arvensis, Ocimum canum and Zingiber officinale were found to exhibit absolute fungitoxic activity against the test fungus. The oils were subsequently standardized through physico-chemical and fungitoxic properties. Practical applicability of the essential oils was observed in control of blue mould rot of oranges and lime fruits caused by P. italicum during storage. The Mentha oil-treated oranges and lime fruits showed enhancement of storage life of 6 and 8 days, respectively. The storage life of Ocimum oil-treated oranges and lime fruits was found to be enhanced by 6 days while in the case of Zingiber oil, it was 4 and 8 days enhancement of shelf life of oranges and lime fruits, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Catabolic pathways and biotechnological applications of microbial caffeine degradation

Catabolism of caffeine (1,3,7-trimethylxanthine) in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline (1,3-dimethylxanthine), whereas theobromine (3,7-dimethylxanthine) is the major metabolite in bacteria. In certain bacterial species, caffeine has also been oxidized directly to trimethyl uric acid in a single step. The conversion of caffeine to its metabolites is primarily brought about by N-demethylases (such as caffeine demethylase, theobromine demethylase and heteroxanthinedemethylase), caffeine oxidase and xanthine oxidase that are produced by several caffeine-degrading bacterial species such as Pseudomonas putida and species within the genera Alcaligenes, Rhodococcus and Klebsiella. Development of biodecaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods removal of caffeine, which are costly, toxic and non-specific to caffeine. This review mainly focuses on the biochemistry of microbial caffeine degradation, presenting recent advances and the potential biotechnological application of caffeine-degrading enzymes.     

Probing the dynamics of a His73-heme alkaline transition in a destabilized variant of yeast iso-1-cytochrome c with conformationally gated electron transfer methods

The alkaline transition of cytochrome c involves substitution of the Met80 heme ligand of the native state with a lysine ligand from a surface Ω-loop (residues 70 to 85). The standard mechanism for the alkaline transition involves a rapid deprotonation equilibrium followed by the conformational change. However, recent work implicates multiple ionization equilibria and stable intermediates. In previous work, we showed that the kinetics of formation of a His73-heme alkaline conformer of yeast iso-1-cytochrome c requires ionization of the histidine ligand (pK(HL) ~ 6.5). Furthermore, the forward and backward rate constants, k(f) and k(b), respectively, for the conformational change are modulated by two auxiliary ionizations (pK(H1) ~ 5.5, and pK(H2) ~ 9). A possible candidate for pK(H1) is His26, which has a strongly shifted pK(a) in native cytochrome c. Here, we use the AcH73 iso-1-cytochrome c variant, which contains an H26N mutation, to test this hypothesis. pH jump experiments on the AcH73 variant show no change in k(obs) for the His73-heme alkaline transition from pH 5 to 8, suggesting that pK(H1) has disappeared. However, direct measurement of k(f) and k(b) using conformationally gated electron transfer methods shows that the pH independence of k(obs) results from coincidental compensation between the decrease in k(b) due to pK(H1) and the increase in k(f) due to pK(HL). Thus, His26 is not the source of pK(H1). The data also show that the H26N mutation enhances the dynamics of this conformational transition from pH 5 to 10, likely as a result of destabilization of the protein.     

ITS-RFLP fingerprinting and molecular marker for detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">ciceris</Emphasis>

Genetic diversity of 11 representative isolates of Fusarium oxysporum f.sp. ciceris causing chickpea wilt was determined through internal transcribed spacer (ITS) region of the ribosomal DNA-restriction fragment length polymorphism (ITS-RFLP). ITS1+5.8s+ITS2 regions of the isolates were amplified with a set of primers ITS1 and ITS4 and amplified products were digested with 4 restriction enzymes (AluI, MboI, RsaI, MseI). Six different kinds of ITS-RFLP patterns were obtained. The ITS region of these isolates was sequenced and deposited to NCBI GeneBank. The nucleotide sequence homology of ITS region grouped the isolates into 5 categories. Primers were designed with sequence information using Primer 3 software. F. oxysporum f.sp. ciceris specific markers (FOC F2 and FOC R2) based on ITS region were developed for the first time for detection of the pathogen. The markers produced an amplicon of 292 bp; they were validated against the isolates of the pathogen collected from different locations of India.     

Ticks on snakes: The ecological correlates of ectoparasite infection in free‐ranging snakes in tropical Australia

Parasites profoundly influence the lives of their hosts, yet the dynamics of host–parasite interactions are poorly understood – especially in reptiles. We examined the ecological correlates of parasitism by ixodid ticks in an assemblage of 10 snake species in tropical Australia. In total, we recorded 3803 ticks on 1841 individual snakes of six species (no ticks were found on the other species). Molecular analyses confirmed the tropical reptile tick (Amblyomma fimbriatum: Ixodidae) to be the most common snake tick at our study site, with inter‐ and intraspecific variation in tick prevalence and intensity. Tick attachment sites were random on most snake species, but both male and female ticks congregated on the heads of the colubrid snake Boiga irregularis and the python Simalia amethistina. In these same species, tick loads were higher on snakes captured in woodland than in rainforest. Females of two python species (Aspidites melanocephalus and S. amethistina) had higher tick loads than did males. In B. irregularis, individuals captured in the dry season had higher tick loads than those captured in the wet season. In most parasitized snake species, larger individuals had greater tick loads. Data from snake recaptures confirmed individual tick burdens frequently varied, with little correlation between tick loads on the same snake at successive captures (except for B. irregularis). Finally, tick intensity was not correlated with (and thus, presumably did not influence) the body condition of any snake species in our study. Use of specific types of refuge sites may strongly influence tick loads on snakes in this system.     

Aerobiology of subtropical Eastern Hymalayas (Kurseong), India

Summary Aerobiology studies of subtropical Eastern Hymalayas (Kurseong) were done for two years. A total of 32 airborne pollen types were recorded along with a few fern spores. In all 4961 pollen grains were trapped during 1981–82 and 4697 during 1982–83. The data reveals seasonal variation of pollen incidence. The incidence of tree pollen dominated over grasses and weeds. The trapped tree pollen originated fromAlnus, Betula, Engelhardtia, Quercus, Bucklandia, Acer, Salix, Ilex, Cryptomeria, Pinus, Cupressus, etc. among others, some of which are known to be allergenically significant in Kurseong. The pollen grains of weeds likeUrtica, Plantago, Cannabis, Rumex, Amaranthaceae, Chenopodiaceae etc. are also proved to be allergenically significant in Kurseong. Grass pollen grains established as highly allergenic, occurred in large amount round the year. The seasonal variation of pollen incidence was dependent mainly on variable flowering periods and climatic variations.     

Manganese-excess induces oxidative stress,lowers the pool of antioxidants and elevates activities of key antioxidative enzymes in rice seedlings

Manganese (Mn) is an essential element for plant growth but in excess, specially in acidic soils, it can become phytotoxic. In order to investigate whether oxidative stress is associated with the expression of Mn toxicity during early seedling establishment of rice plants, we examined the changes in the level of reactive oxygen species (ROS), oxidative stress induced an alteration in the level of non-enzymic antioxidants and activities of antioxidative enzymes in rice seedlings grown in sand cultures containing 3 and 6 mM MnCl₂. Mn treatment inhibited growth of rice seedlings, the metal increasingly accumulated in roots and shoots and caused damage to membranes. Mn treated plants showed increased generation of superoxide anion (O₂ ^.−), elevated levels of H₂O₂ and thiobarbituric acid reactive substances (TBARS) and decline in protein thiol. The level of nonprotein thiol, however, increased due to Mn treatment. A decline in contents of reduced ascorbate (AsA) and glutathione (GSH) as well as decline in ratios of their reduced to oxidize forms was observed in Mn-treated seedlings. The activities of antioxidative enzymes superoxide dismutase (SOD) and its isoforms Mn SOD, Cu/Zn SOD, Fe SOD as well as guaiacol peroxidase (GPX) increased in the seedlings due to Mn treatment however, catalase (CAT) activity increased in 10 days old seedlings but it declined by 20 days under Mn treatment. The enzymes of Halliwell-Asada cycle, ascorbate peroxidase (APX) monodehydoascorbate reductase (MDHAR), dehyroascorbate reductase (DHAR) and glutathione reductase (GR) increased significantly in Mn treated seedlings over controls. Results suggest that in rice seedlings excess Mn induces oxidative stress, imbalances the levels of antioxidants and the antioxidative enzymes SOD, GPX, APX and GR appear to play an important role in scavenging ROS and withstanding oxidative stress induced by Mn.     

Effect of asiaticoside on the healing of skin wounds in the carp Cirrhinus mrigala: An immunohistochemical investigation

In the present study effect of asiaticoside, on healing of skin wounds in Cirrhinus mrigala is reported. Skin wound, approx. 2 mm in diameter was excised using sterile disposable biopsy punch. Immediately after infliction of the wound, epidermis from wound edge starts migrating as thin sheet toward wound gap. Fronts of migrating epidermis gradually advance, and results in complete epithelialization of wound. Experiments were conducted for 30 days and fishes were divided into control, sham, vehicle control and asiaticoside treated groups. Immunohistochemical localization of proliferating cell nuclear antigen positive cells indicating cellular proliferation and caspase 3 positive cells reflecting apoptosis was carried out and their density at different post wound intervals in each fish group was analyzed. Significant increase in cellular proliferation as well as decrease in apoptosis in both epidermis and dermis in fish treated with asiaticoside compared to sham and vehicle control fish is observed at different intervals of wound repair. This suggests that in treated group healing of skin wounds in fish is enhanced than in sham and vehicle control groups. Asiaticoside treatment in healing of skin wounds would greatly be beneficial to fish farmers as it could protect fish from invasion of pathogens and check fish mortality.     

The N-terminus of m5C-DNA methyltransferase MspI is involved in its topoisomerase activity.

DNA cytosine methyltransferase MspI (M.MspI) must require a different type of interaction of protein with DNA from other bacterial DNA cytosine methyltransferases (m5C-MTases) to evoke the topoisomerase activity that it possesses in addition to DNA-methylation ability. This may require a different structural organization in the solution phase from the reported consensus structural arrangement for m5C-MTases. Limited proteolysis of M.MspI, however, generates two peptide fragments, a large one (p26) and a small one (p18), consistent with reported m5C-MTase structures. Examination of the amino-acid sequence of M.MspI revealed similarity to human topoisomerase I at the N-terminus. Alignment of the amino-acid sequence of M.MspI also uncovered similarity (residues 245-287) to the active site of human DNA ligase I. To evaluate the role of the N-terminus of M.MspI, 2-hydroxy-5-nitrobenzyl bromide (HNBB) was used to truncate M.MspI between residues 34 and 35. The purified HNBB-truncated protein has a molecular mass of approximately equal 45 kDa, retains DNA binding and methyltransferase activity, but does not possess topoisomerase activity. These findings were substantiated using a purified recombinant MspI protein with the N-terminal 34 amino acids deleted. Changing the N-terminal residues Trp34 and Tyr74 to alanine results in abolition of the topoisomerase I activity while the methyltransferase activity remains intact.     

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes

Bacillus flexus strain SSAI1 isolated from agro-industry waste, Tuem, Goa, India displayed high arsenite resistance as minimal inhibitory concentration was 25 mM in mineral salts medium. This bacterial strain exposed to 10 mM arsenite demonstrated rapid arsenite oxidation and internalization of 7 mM arsenate within 24 h. The Fourier transformed infrared (FTIR) spectroscopy of cells exposed to arsenite revealed important functional groups on the cell surface interacting with arsenite. Furthermore, scanning electron microscopy combined with electron dispersive X-ray spectroscopy (SEM-EDAX) of cells exposed to arsenite revealed clumping of cells with no surface adsorption of arsenite. Transmission electron microscopy coupled with electron dispersive X-ray spectroscopic (TEM-EDAX) analysis of arsenite exposed cells clearly demonstrated ultra-structural changes and intracellular accumulation of arsenic. Whole-genome sequence analysis of this bacterial strain interestingly revealed the presence of large number of metal(loid) resistance genes, including aioAB genes encoding arsenite oxidase responsible for the oxidation of highly toxic arsenite to less toxic arsenate. Enzyme assay further confirmed that arsenite oxidase is a periplasmic enzyme. The genome of strain SSAI1 also carried glpF, aioS and aioE genes conferring resistance to arsenite. Therefore, multi-metal(loid) resistant arsenite oxidizing Bacillus flexus strain SSAI1 has potential to bioremediate arsenite contaminated environmental sites and is the first report of its kind.