High-performance liquid chromatography method for the determination and quantitation of arabinosylguanine triphosphate and fludarabine triphosphate in human cells

A gradient anion-exchange high-performance liquid chromatographic assay was developed for the simultaneous determination and quantitation of the cytotoxic triphosphates of arabinosylguanine (ara-GTP) and fludarabine (F-ara-ATP). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy using authentic standards. To test this assay in a more complex biological matrix, perchloric acid extracts of circulating human leukemia cells spiked with known concentrations of ara-GTP and F-ara-ATP were examined. Finally, to assess the clinical utility of our method, perchloric acid extracts of circulating human leukemia cells isolated from patients treated with fludarabine and nelarabine were analyzed. The range of quantitation was 0.0125–10 nmol for the ara- and native NTPs in cellular extracts. This assay should be helpful in establishing the mechanistic rationales for drug scheduling and combinations of nelarabine and fludarabine, and for correlating the therapeutic efficacy and levels of the cytotoxic triphosphates in target cells.     

Nitrogen uptake by size-fractionated plankton in permanently well-mixed temperate coastal waters

Nitrogen uptake by net- (15–200 µm), nano- (1–15µm) and picoplankton (<1 µm) was measured overseasonal cycles at two stations with different patterns of biologicaland chemical cycles in the Morlaix Bay (western English Channel).Though assimilable dissolved N nutrient pool at both stationswas nitrate-dominated, characteristics of biomass and N uptakeby netplankton differed from conventional patterns in two respects.In the first, biomass (26–30%) and N uptake (36–43%)were less important than those of nanoplankton. In the second,the netplankton did not show any marked preference for nitrateover ammonium (nitrate to ammonium uptake ratios of 0.98 and1.08). In contrast, nanoplankton had a preference for ammoniumover nitrate (ammonium to nitrate uptake ratios of 2 and 1.2).N uptake by picoplankton was only 8% of total N uptake at bothstations and was supported mainly by regenerated N (66% ammoniumand 17% urea), with nitrate uptake detectable in only one instanceand nitrite uptake in none. Substrate-dependent uptake of ammoniumin all fractions and a higher ammonium uptake in the nanoplanktonfraction in summer at both stations when ambient ammonium concentrationswere high indicated that while nitrate may satisfy a part ofN requirements, availability of ammonium and its flux throughnanoplankton determine the magnitude of total N uptake in thesewaters. Most of the N uptake in picoplankton appears to be autotrophic,suggesting that a substantial part of heterotrophic uptake,if any, could be localized in the fractions >1 µm,and mediated by free-living and particle-bound bacteria.     

Salt stress enhanced antioxidant response in callus of three halophytes (Salsola baryosma, Trianthema triquetra, Zygophyllum simplex) of Thar Desert

Effects of salinity on growth, protein content, proline, catalase and antioxidant enzyme activity in callus of three halophytes of the Thar Desert; Salsola baryosma, Trianthema triquetra and Zygophyllum simplex were evaluated. Callus tissues were cultured on Murashige and Skoog’s medium containing different concentrations of NaCl (50, 100 and 200 mM). Increase in dry weight and soluble proteins were observed in the callus exposed to lower salinity (50 and 100 mM NaCl) in all the three species, whereas on the medium containing 200 mM NaCl, significant decrease in these two growth parameters was recorded. Under the salinity stress maximum proline accumulation was found in S. baryosma with parallel increase in soluble sugars. Among the three species, T. triquetra callus showed maximum CAT activity with 50 and 100 mM NaCl treatment, whereas the enzyme activity decreased at 200 mM NaCl treatment in all three species. The antioxidant potential steadily elevated under salt treatment in all the above three species using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant potential (FRAP) assay. Whereas, superoxide dismutase (SOD) quenching were recorded maximum at low (50 and 100 mM) concentrations in all the three species. However, T. triquetra callus showed maximum total phenolic content (TPC) 15 mg GAE g^?1 with the elevated concentration of NaCl up to 200 mM, and S. baryosma callus showed lower TPC as compared to both species. A significant correlation between antioxidant capacity and TPC was observed indicating that phenolic compounds are the major contributors to the antioxidant potential in these halophyte species. FRAP and DPPH activity of Z. simplex showed maximum correlation (R = 0.992), as compared to other two species. We can conclude that all the three species exhibit a protection mechanism by sustaining growth parameters and antioxidant capacity. Due to high antioxidant property of all these species, the plant extracts may be included in nutraceutical formulations.     

Hyperventilation-induced airway injury and vascular leakage in dogs: effects of alpha 1-adrenergic agonists

Freed, Arthur N., Varsha Taskar, Brian Schofield, andChiharu Omori. Hyperventilation-induced airway injury and vascular leakage in dogs: effects of₁-adrenergic agonists.J. Appl. Physiol. 83(6):1884-1889, 1997.₁-Adrenergic agonistsinhibit hyperventilation-induced bronchoconstriction (HIB) in dogs. Wetested the hypothesis that -agonists inhibit HIB byreducing bronchovascular leakage and edema that theoretically couldcause airway obstruction. Peripheral airways were isolated by using abronchoscope; pretreated with either methoxamine (Mx), norepinephrine(NE), or saline aerosol; and then exposed to a 2,000 ml/min dry-airchallenge (DAC) for 2 min. Colloidal carbon was injected before DAC andused to quantify bronchovascular permeability. Mx-, NE-, andvehicle-treated airways were prepared for morphometric analysis within1 h after DAC. Light microscopy revealed that the 2-min DAC producedminimal bronchovascular leakage and little epithelial damage. However, pretreatment with either Mx or NE significantly enhanced dryair-induced bronchovascular hyperpermeability and mucosal injury. Theincreased damage associated with these₁-agonists implicates aprotective role for the bronchial circulation. The factthat ₁-agonists inhibit HIBsuggests that neither dry air-induced leakage nor injury directlycontributes to the development of airway obstruction. In addition,our data suggest that-agonists attenuate HIB in part byaugmenting hyperventilation-induced bronchovascular leakage and byreplacing airway water lost during a DAC.

     

Analysis of variance components in gene expression data

MOTIVATION: A microarray experiment is a multi-step process, and each step is a potential source of variation. There are two major sources of variation: biological variation and technical variation. This study presents a variance-components approach to investigating animal-to-animal, between-array, within-array and day-to-day variations for two data sets. The first data set involved estimation of technical variances for pooled control and pooled treated RNA samples. The variance components included between-array, and two nested within-array variances: between-section (the upper- and lower-sections of the array are replicates) and within-section (two adjacent spots of the same gene are printed within each section). The second experiment was conducted on four different weeks. Each week there were reference and test samples with a dye-flip replicate in two hybridization days. The variance components included week-to-week, animal-to-animal and between-array and within-array variances. RESULTS: We applied the linear mixed-effects model to quantify different sources of variation. In the first data set, we found that the between-array variance is greater than the between-section variance, which, in turn, is greater than the within-section variance. In the second data set, for the reference samples, the week-to-week variance is larger than the between-array variance, which, in turn, is slightly larger than the within-array variance. For the test samples, the week-to-week variance has the largest variation. The animal-to-animal variance is slightly larger than the between-array and within-array variances. However, in a gene-by-gene analysis, the animal-to-animal variance is smaller than the between-array variance in four out of five housekeeping genes. In summary, the largest variation observed is the week-to-week effect. Another important source of variability is the animal-to-animal variation. Finally, we describe the use of variance-component estimates to determine optimal numbers of animals, arrays per animal and sections per array in planning microarray experiments.     

Recombinant Modified Vaccinia Virus Ankara Expressing Glycoprotein E2 of Chikungunya Virus Protects AG129 Mice against Lethal Challenge

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections.     

Opossum peptide that can neutralize rattlesnake venom is expressed in Escherichia coli

An eleven amino acid ribosomal peptide was shown to completely neutralize Western Diamondback Rattlesnake (Crotalus atrox) venom in mice when a lethal dose of the venom was pre‐incubated with the peptide prior to intravenous injection. We have expressed the peptide as a concatenated chain of peptides and cleaved them apart from an immobilized metal affinity column using a protease. After ultrafiltration steps, the mixture was shown to partially neutralize rattlesnake venom in mice. Preliminary experiments are described here that suggest a potential life‐saving therapy could be developed. To date, no recombinant therapies targeting cytotoxic envenomation have been reported. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:81–86, 2017