The present study was intended to reveal the antibacterial activity of various cold organic solvent extracts of Abrus precatorius L. (Fabaceae) and Asystasia gangetica (L.) T. Anderson (Acanthaceae) against the selected pathogens. Powders of whole plant parts of A. precatorius and A. gangetica were extracted with petroleum ether, benzene, chloroform and ethanol at ambient temperature. The dried extracts were tested for antibacterial activity by agar disc diffusion method. Among the different extracts of A. precatorius, ethanolic extracts showed maximum zone of inhibition (21 mm) against Bacillus subtilis followed by 13 mm against Staphylococcus aureus. Broadest spectrum of activity was exhibited in petroleum ether extracts of A. precatorius against different bacterial pathogens (5/7 pathogens). In A. gangetica, benzene extracts exhibited broadest spectrum of activity with the maximum level of inhibition (12 mm) against B. subtilis followed by 11 mm in ethanolic extracts against Salmonella typhi. Petroleum ether extracts of A. gangetica did not show any activity against the selected pathogens. The present study results clearly show that the extracts of A. precatorius and A. gangetica had significant and considerable antibacterial activity against various pathogens and further evaluation is necessary to find out the active principle compound responsible for bioactivity. Keywords: Abrus precatorius, antibacterial, Asystasia gangetica, bioactivity, pathogens.

Primary fatty acid amides are a group of bioactive lipids that have been linked with a variety of biological processes such as sleep regulation and modulation of monoaminergic systems. As novel forms of these molecules continue to be discovered, more emphasis will be placed on selective, trace detection. Currently, there is no published experimental determination of collision induced dissociation of PFAMs. A select group of PFAM standards, 12 to 22 length carbon chains, were directly infused into an electrospray ionization source Quadrupole Time of Flight Mass Spectrometer. All standards were monitored in positive mode using the [M + H](+) peak. Mass Hunter Qualitative Analysis software was used to calculate empirical formulas of the product ions. All PFAMs showed losses of 14 m/z indicative of an acyl chain, while the monounsaturated group displayed neutral losses corresponding to H(2)O and NH(3). The resulting spectra were used to propose fragmentation mechanisms. Isotopically labeled PFAMs were used to validate the proposed mechanisms. Patterns of saturated versus unsaturated standards were distinctive, allowing for simple differentiation. This determination will allow for fast, qualitative identification of PFAMs. Additionally, it will provide a method development tool for selection of unique product ions when analyzed in multiple reaction monitoring mode.

A highly sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed and validated for the quantification of glycerophosphoinositol (GroPIns), glycerophosphocholine (GroPCho), glycerol 3-phosphate (GroP), inositol, and choline in the extracellular medium of Saccharomyces cerevisiae. The media samples were pretreated with a single two-phase liquid extraction. Chromatographic separation was achieved on a Waters Xbridge HILIC (150 mm x 4.6 mm, 5 mu m) column under isocratic conditions using a mobile phase composed of acetonitrile/water, 70:30 (v/v) with 10 mM ammonium acetate (pH adjusted to 4.5) at a flow-rate of 0.5 mL/min. Using a triple quadrupole tandem mass spectrometer, samples were detected in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curves were linear (r(2) >= 0.995) over the range of 0.5-150 nM, with the lower limit of quantitation validated at 0.5 nM for all analytes. The intra- and inter-day precision (calculated by coefficient of variation, CV%) ranged from 1.24 to 5.88% and 2.46 to 9.77%, respectively, and intra- and inter-day accuracy (calculated by relative error, RE%) was between -8.42 to 8.22% and -9.35 to 6.62%, respectively, at all quality control levels. The extracellular metabolites were stable throughout various storage stability studies. The fully validated method was successfully applied to determine the extracellular levels of phospholipid-related metabolites in S. cerevisiae. (C) 2012 Elsevier B.V. All rights reserved.

ABSTRACT Glycerophosphodiesters are the products of phospholipase-mediated deacylation of phospholipids. In Saccharomyces cerevisiae , a single gene, GIT1 , encodes a permease responsible for importing glycerophosphodiesters, such as glycerophosphoinositol and glycerophosphocholine, into the cell. In contrast, the Candida albicans genome contains four open reading frames (ORFs) with a high degree of similarity to S. cerevisiae GIT1 ( ScGIT1 ) Here, we report that C. albicans utilizes glycerophosphoinositol (GroPIns) and glycerophosphocholine (GroPCho) as sources of phosphate at both mildly acidic and physiological pHs. Insertional mutagenesis of C. albicans GIT1 ( CaGIT1 ) (orf19.34), the ORF most similar to ScGit1 , abolished the ability of cells to use GroPIns as a phosphate source at acidic pH and to transport [ 3 H]GroPIns at acidic and physiological pHs, while reintegration of a GIT1 allele into the genome restored those functions. Several lines of evidence, including the detection of internal [ 3 H]GroPIns, indicated that GroPIns is transported intact through CaGit1. GroPIns transport was shown to conform to Michaelis-Menten kinetics, with an apparent K m of 28 ± 6 μM. Notably, uptake of label from [ 3 H]GroPCho was found to be roughly 50-fold greater than uptake of label from [ 3 H]GroPIns and roughly 500-fold greater than the equivalent activity in S. cerevisiae. Insertional mutagenesis of CaGIT1 had no effect on the utilization of GroPCho as a phosphate source or on the uptake of label from [ 3 H]GroPCho. Growth under low-phosphate conditions was shown to increase label uptake from both [ 3 H]GroPIns and [ 3 H]GroPCho. Screening of a transcription factor deletion set identified CaPHO4 as required for the utilization of GroPIns, but not GroPCho, as a phosphate source.

This article presents a novel method for small-scale lipidomics of bacterial cells by integrating extraction of glycerophospholipids on a microchip with a nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometer (nanoESI-Q-TOF MS/MS). The standard starting point for typical macroscale lipid analysis is a multiphase liquid-liquid extraction. Working with small populations of cells (1 to about 1000) requires a scaled down process in order to minimize dilution and facilitate the interface with microscale separation methods for sample cleanup and introduction to mass spectrometry. We have developed a microfluidic system that allows for lysis of bacterial cells, capture of lipids, and elution of captured lipids from a solid phase for microscale purification of lipids. The best on-chip extraction efficiency for glycerophospholipids was as high as 83.3% by integrating silica beads as the packing material with methanol as the eluent. A total of 10 successive measurements were evaluated indicating that the microchip packed with fresh silica beads is capable of being reused four times without any loss in the lipid extraction process. The initial screening based on high-resolution tandem mass spectrometry data along with a discovery profiling approach revealed the presence of 173 identified phospholipid species from microfluidic cell extracts. This work demonstrates the potential of incorporating microchip-based lipid extraction into cellular lipidomics research.

Phospholipase B‐mediated cleavage of glycerophospholipids produces both intracellular and extracellular glycerophosphodiesters. Saccharomyces cerevisiae and Candida albicans contain genes encoding phospholipases of this type. In S. cerevisiae, a single gene, ScGIT1, encodes a permease responsible for transporting glycerophosphodiesters, such as glycerophosphoinositol (GroPIns) and glycerophosphocholine (GroPCho) across the cell membrane. In contrast, the C. albicans genome contains four ORFs with a high degree of homology to the ScGIT1. C. albicans transports GroPCho at a rate of roughly 10‐fold greater magnitude as compared to S. cerevisiae. In addition, C. albicans utilizes GroPIns and GroPCho as sole sources of phosphate at the physiologically relevant pH of 7.5. GroPIns transport, but not GroPCho transport is regulated by phosphate in C. albicans. Deletion of CaGIT1 (ORF 19.34) abolishes GroPIns transport activity and the ability to use GroPIns as a phosphate source, but does not affect GroPCho transport.

Singular value decomposition (SVD) analysis on spectrophotometric data obtained from an oxygen atom transfer (OAT) reaction involving a molybdoenzyme model system is reported. Specifically, the rate of solvolysis reaction of a phosphoryl intermediate complex has been compared with independent measurements. The SVD derived reaction rates are consistent with other measurements. This generalized approach is applicable in examining other bioinorganic reactions, and data processing.

Primary fatty acid amides (R-CO-NH2) and N-acylglycines (R-CO-NH-CH2-COOH) are classes of compounds that have only recently been isolated and characterized from biological sources. Key questions remain regarding how these lipid amides are produced and degraded in biological systems. Relative to the fatty acids, little has been done to develop methods to separate and quantify the fatty acid amides and N-acylglycines. We describe reversed phase HPLC methods for the separation of C2-C12 primary fatty acid amides and N-acylglycines and also C12-C22 fatty acid amides. Separation within each class occurs primarily on the basis of simple interactions between the acyl chain and the chromatographic stationary phase, but the polar headgroups on these and related fatty acids and N-acylethanolamides modulate the absolute retention in reversed phase mode. We use these methods to measure the enzyme-mediated, two-step conversion of N-octanoylglycine to octanoamide.

Laser-induced fluorescence is an extremely sensitive method for detection in chemical separations. In addition, it is well-suited to detection in small volumes, and as such is widely used for capillary electrophoresis and microchip-based separations. This review explores the detailed instrumental conditions required for sub-zeptomole, sub-picomolar detection limits. The key to achieving the best sensitivity is to use an excitation and emission volume that is matched to the separation system and that, simultaneously, will keep scattering and luminescence background to a minimum. We discuss how this is accomplished with confocal detection, 90degrees on-capillary detection, and sheath-flow detection. It is shown that each of these methods have their advantages and disadvantages, but that all can be used to produce extremely sensitive detectors for capillary- or microchip-based separations. Analysis of these capabilities allows prediction of the optimal means of achieving ultrasensitive detection on microchips.

Gel electrophoresis is the most widely accepted technique for analysis and separation of DNA fragments. Standard gel electrophoresis is used for fragment sizes up to approximately 50 kb in length. Larger fragments must be separated by some form of pulsed field electrophoresis (1). Capillary gel electrophoresis (2) and ultrathin slab gel electrophoresis (3) are currently being developed to allow for high speed separation of DNA sequencing ladders for sizes less than 1 kb. No matter what form of electrophoresis is used, the separation is highly non-linear and generally has an upper limit to the size of the DNA that can be analyzed. In addition, conventional gel-based separations may take many hours, depending on required resolution and detection method.

The authors have demonstrated flow cytometric detection and sizing of single pieces of fluorescently stained lambda DNA (48.5 kb) and individual Kpn I restriction fragments of lambda DNA at 17.05 kb and 29.95 kb. DNA fragments were stained stoichiometrically with an intercalating dye such that the fluorescence from each fragment was directly proportional to fragment length. Laser powers range from 10 to 100 mW and transit times through the focused laser beam were several milliseconds. Measurements were made using time-resolved single photon counting of the detected fluorescence emission from individual stained DNA fragments. Samples were analyzed at rates of about 50 fragments per second. The measured fluorescence intensities are linearly correlated with DNA fragment length over the range measured. Detection sensitivity and resolution needed for analysis of small pieces of DNA are discussed and a comparison of single photon counting measurements of DNA fragments to measurements using more conventional flow cytometers is made. Applications of this methodology to DNA sizing and DNA fingerprinting are discussed.