Structure-Function Relationship of Substituted Bromomethylcoumarins in Nucleoside Specificity of RNA Alkylation

Selective alkylation of RNA nucleotides is an important field of RNA biochemistry, e.g. in applications of fluorescent labeling or in structural probing experiments, yet detailed structure-function studies of labeling agents are rare. Here, bromomethylcoumarins as reactive compounds for fluorescent labeling of RNA are developed as an attractive scaffold on which electronic properties can be modulated by varying the substituents. Six different 4-bromomethyl-coumarins of various substitution patterns were tested for nucleotide specificity of RNA alkylation using tRNA from Escherichia coli as substrate. Using semi-quantitative LC-MS/MS analysis, reactions at mildly acidic and slightly alkaline pH were compared. For all tested compounds, coumarin conjugates with 4-thiouridine, pseudouridine, guanosine, and uridine were identified, with the latter largely dominating. This data set shows that selectivity of ribonucleotide alkylation depends on the substitution pattern of the reactive dye, and even more strongly on the modulation of the reaction conditions. The latter should be therefore carefully optimized when striving to achieve selectivity. Interestingly, the highest selectivity for labeling of a modified nucleoside, namely of 4-thiouridine, was achieved with a compound whose selectivity was somewhat less dependent on reaction conditions than the other compounds. In summary, bromomethylcoumarin derivatives are a highly interesting class of compounds, since their selectivity for 4-thiouridine can be efficiently tuned by variation of substitution pattern and reaction conditions.     

The Role of CHI3L1 (Chitinase-3-Like-1) in the Pathogenesis of Infections in Burns in a Mouse Model

In severe burn injury the unique setting of a depleted, dysfunctional immune system along with a loss of barrier function commonly results in opportunistic infections that eventually proof fatal. Unfortunately, the dynamic sequence of bacterial contamination, colonization and eventually septic invasion with bacteria such as Pseudomonas species is still poorly understood although a limiting factor in clinical decision making. Increasing evidence supports the notion that inhibition of bacterial translocation into the wound site may be an effective alternative to prevent infection. In this context we investigated the role of the mammalian Chitinase-3-Like-1 (CHI3L1) non-enyzmatic protein predominately expressed on epithelial as well as innate immune cells as a potential bacterial-translocation-mediating factor. We show a strong trend that a modulation of chitinase expression is likely to be effective in reducing mortality rates in a mouse model of burn injury with superinfection with the opportunistic PA14 Pseudomonas strain, thus demonstrating possible clinical leverage.     

Stimulation of liver tryptophan pyrrolase during heat exposure.

Exposure of rats to heat (39 +/- 1 degree C) stimulated liver tryptophan pyrrolase 2-fold between 3 and 48 h. Plasma corticosterone increased 2-fold after 1 h of heat exposure and decreased to a low value of 50% by 16 h. The effect of heat exposure on the enzyme was obtained in adrenalectomized animals. Stimulation by cortisol and tryptophan of the enzyme was also obtained in heat exposure, and the effects seemed to be additive. The concentration of tryptophan in the liver remained unchanged, and that in the plasma decreased to about 50% at 8 h exposure to heat and reverted to normal by 46 h. Simultaneous administration of noradrenaline to heat-exposed rats had no effect, whereas that of thyroxine partly prevented the stimulation of the enzyme activity. Hypothyroid conditions obtained by thyroidectomy or treatment with propylthiouracil significantly stimulated the enzyme activity. Cycloheximide treatment of heat-exposed rats did not prevent the stimulation of the enzyme activity. The results indicate that the effect of heat exposure on liver tryptophan pyrrolase is obtained, due to the accompanying hypothyroid condition, by increasing the activity of the existing protein by a mechanism possibly different from those known at present.     

An exploration of microbial and associated functional diversity in the OMZ and non-OMZ areas in the Bay of Bengal

Depletion of oxygen in certain marine areas creates oxygen minimum zones (OMZs), which can alter the species composition and abundance. We have carried out high-throughput 16S rRNA gene amplicon profiling from the Bay of Bengal (BOB) OMZ and non-OMZ areas. Typically, a total of 35 families of micro-organisms were identified as biomarkers for OMZ and non-OMZ regions in the BOB. Our analysis has identified families Pseudoalteromonadaceae, OM60 and Synechococcaceae to be abundant in oxygenated water, whereas organisms belonging to families Pelagibacteraceae and Caulobacteraceae, which are involved in sulphur and nitrogen metabolism, were prominent in the OMZ areas. Predictive functional analysis for these identified bacteria clearly that suggested an abundance of microbes with assimilatory sulphur-reducing genes (cysl and csH) in the non-OMZ, while bacteria involved in dissimilatory sulphate reduction (known to carry aprA and aprB genes) were enriched in the OMZ areas. Comparative analysis with OMZ areas from Peru and Chile revealed that OMZ areas in the BOB are characterized by specific and distinctive bacterial diversity. Overall, the current analysis provides valuable documentation about the bacterial populations and their characteristics, which can generate pointers for their functional significance in the BOB.     

Isolation,and Virulence Profiles,of Aeromonas hydrophila Implicated in an Outbreak of Food Poisoning in Sweden

A case of food poisoning outbreak involving Aeromonas hydrophila is reported in this study. A group of 27 people consumed a typical Swedish food “landgång” which is a type of smörgåsbord containing shrimps with mayonnaise, liver paté, ham, sausage, and legume salad which was purchased from a food store. Twenty-two of the 27 persons became ill within 20-34 hr of consumption of the food and reported the symptoms ranging from severe acute diarrhea, abdominal pain, headache, fever and vomiting. One person also fainted. The symptoms lasted for a couple of days. Of the remaining 5 healthy persons who consumed the left-over food the next day, 2 became ill with similar symptoms. The bacteriological examination of left-over food samples resulted in the isolation of A. hydrophila from shrimps with mayonnaise, smoked sausage, liver paté and boiled ham. The total number of A. hydrophila in these foods were log 10⁶ to log >10⁷ organisms per gram of food sample. A. hydrophila was however, not isolated from legume/mayonnaise salad samples. All the food samples tested showed low numbers of other expected food contaminating organisms such as coliforms at 37 C and 44 C, fecal streptococci, Staphylococcus aureus, fungi and yeast etc., while Bacillus cereus, Clostridium perfringens and Salmonella spp. were not detected in the food samples. Investigations of the virulence profiles of the A. hydrophila isolates showed their capacity to produce β-hemolysin, cytotoxins, cytotonic toxins, enterotoxins, and adhesion to and invasion of human intestinal (Henle 407) cells in culture.     

A convenient and validated enantiomer separation of chiral aliphatic amines as nitrobenzoxadiazole derivatives on polysaccharide‐derived chiral stationary phases under simultaneous ultraviolet and fluorescence detection

A convenient method using a fluorogenic agent, 4‐chloro‐7‐nitro‐1,2,3‐benzoxadiazole (NBD‐Cl), was developed for enantiomer separation of chiral aliphatic amines including amino alcohols by normal high‐performance liquid chromatography. The enantiomer separation of chiral aliphatic amines as NBD derivatives was performed on six covalently bonded and four coated‐type polysaccharide‐derived chiral stationary phases (CSPs) under simultaneous ultraviolet (UV) and fluorescence detection (FLD). Among the covalently bonded CSPs, Chiralpak IE showed the best enantiomer separation for most analytes. The other CSPs also showed good enantioselectivity except for Chiralpak IB. On the other hand, Chiralpak AD‐H and Amylose‐1 generally exhibited better enantiomer separation of NBD derivatized chiral amines among the coated CSPs. The developed analytical technique was also applied to determine the optical purity of commercially available (R)‐ and (S)‐leucinol; the impurity was found to be 0.06%. The developed method was validated and proved to be an accurate, precise, sensitive, and selective method suitable for separation of chiral aliphatic amines as NBD derivatives under simultaneous UV and FLD.     

The Essential Oil Compositions of Ocimum basilicum from Three Different Regions: Nepal,Tajikistan, and Yemen

The aerial parts of Ocimum basilicum L. were collected from four different geographical locations, Sindhuli and Biratnagar (Nepal), Chormaghzak village (Tajikistan), and Sana'a (Yemen). The essential oils were obtained by hydrodistillation and analyzed by gas chromatography/mass spectrometry. A cluster analysis of 179 essential oil compositions revealed six major chemotypes: Linalool, eugenol, estragole, methyl eugenol, 1,8‐cineole, and geraniol. All four of the basil oils in this study were of the linalool‐rich variety. Some of the basil oils were screened for bioactivity including antimicrobial, cytotoxicity in human cancer cells, brine shrimp lethality, nematicidal, larvicidal, insecticidal, and antioxidant. The basil oils in this study were not notably antibacterial, cytotoxic, antioxidant, nor nematicidal, but were active in the brine shrimp lethality test, and did show larvicidal and insecticidal activities.     

Elevated CO<Subscript>2</Subscript> influences host plant defense response in chickpea against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Global atmospheric concentration of CO₂ is likely to increase from 350 to 750 ppm over the next 100 years. The present studies were undertaken to understand the effects of elevated CO₂ on enzymatic activity and secondary metabolites in chickpea in relation to expression of resistance to pod borer, Helicoverpa armigera. Fifteen-day-old chickpea plants [ICCL 86111—resistant and JG 11—commercial cultivar] grown in the greenhouse were transferred to open-top chambers (OTC) and kept under 350, 550 and 750 ppm of CO₂. Twenty neonates of H. armigera were released on each plant at 7 days after shifting the pots to the OTCs. Un-infested plants were maintained as controls. After 7 days of infestation, the activities of defensive enzymes [peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL)] and amounts of total phenols and condensed tannins increased with an increase in CO₂ concentration in chickpea. The nitrogen balance index was greater in plants kept at 350 ppm CO₂ than in plants kept under ambient conditions. The H. armigera-infested plants had higher H₂O₂ content; amounts of oxalic and malic acids were greater at 750 ppm CO₂ than at 350 ppm CO₂. Plant damage was greater at 350 ppm than at 550 and 750 ppm CO₂. This information will be useful for understanding effects of increased levels of CO₂ on expression of resistance to insect pests to develop strategies to mitigate the effects of climate change.     

Immunochemical relationship between glucoamylases I and II ofAspergillus niger

Rabbit antisera were prepared against the purified glucoamylases I and II ofAspergillus niger. Relationships between the two enzyme forms were investigated by using the antisera in immunodiffusion and immunoinhibition experiments. Both the forms of glucoamylase gave a single continuous precipitin band demonstrating very close structural resemblance. They gave almost identical immunoprecipitation patterns and had the same equivalence points indicating that the two forms ofA. niger gluoamylases were immunologically identical. The enzyme treated with periodate was immunologically identical with the controls and had slightly less enzyme activity but showed greatly reduced stability on storage at 4‡ C.     

Biological studies onTrichogrammatoidea armigera Nagaraja a new dimorphic egg parasite ofHeliothis armigera (Hübner) in India

A new species ofTrichogrammatoidea which is being described byH. Nagaraja asT. armigera, has been reared from eggs ofHeliothis armigera onPolianthes tuberosa and from those of an unidentified Lepidopteron onCajanus cajan. In the laboratory the parasite was successfully bred onCorcyra cephalonica, Achaea janata, Gnorimoschema operculella andPlutella xylostella; it did not show any perceptible preference for any of these hosts. It also parasitised eggs ofSpodoptera litura, but although development proceeded to the adult stage, adults failed to emerge, suggesting unsuitability of this host. The males ofT. armigera are found to exhibit dimorphism — one form being alate and the other typically apterous. The apterous males were almost exclusively produced only by fertilised females and in the progeny of virgin females these forms were extremely rare (1 apterous male: 1,500 winged males). Among the progeny of a single mated female, an apterous male developed invariably in association with a female, but this rule did not apply when a singleCorcyra egg was parasitised by more than one parental female. However, in no case did an apterous male alone emerge from a single host egg. The biology ofT. armigera has been studied at 25°C±1°C. and R.H. 75%, usingC. cephalonica eggs. The parasite completed its life-cycle in 7–9 days — the egg, larval and pupal periods occupying, 1, 2–3 and 4–5 days, respectively. When fed honey, the average longevity of females was 7 (max. 11) days, of alate males 6 (max. 10) days and of apterous males 1 (max. 2) day. The maximum fecundity was 118 while the average was about 63. From 2–26 (average 9) eggs were parasitised per day. The sex-ratio was 62% females: 38% males (35% alate and 3% apterous). An alate male during its lifetime inseminated upto 10 (Av. 9) females while an apterous one inseminated up to 4 (Av. 3) females.