Simultaneous quantitative analysis of main components in linderae reflexae radix with one single marker

Establish a quantitative analysis of multi-components by the single marker (QAMS) method for quality evaluation and validate its feasibilities by the simultaneous quantitative assay of four main components in Linderae Reflexae Radix. Four main components of pinostrobin, pinosylvin, pinocembrin, and 3,5-dihydroxy-2-(1-p-mentheneyl)-trans-stilbene were selected as analytes to evaluate the quality by RP-HPLC coupled with a UV-detector. The method was evaluated by a comparison of the quantitative results between the external standard method and QAMS with a different HPLC system. The results showed that no significant differences were found in the quantitative results of the four contents of Linderae Reflexae Radix determined by the external standard method and QAMS (RSD <3%). The contents of four analytes (pinosylvin, pinocembrin, pinostrobin, and Reflexanbene I) in Linderae Reflexae Radix were determined by the single marker of pinosylvin. This fingerprint was the spectra determined by Shimadzu LC-20AT and Waters e2695 HPLC that were equipped with three different columns.     

Wall slip of mayonnaises in viscometers

The shear flow of mayonnaise is generally characterized by an apparent yield stress, shear thinning in steady flow, stress overshoots upon inception of flow and other time-dependent effects. These observations are usually understood to be the result of structural rearrangement within the material. Additionally and separately, the possibility that emulsions may exhibit apparent wall slip on a microscopic scale at a solid-liquid boundary has been reported by some researchers. Thus, observed rheological behavior is likely to be the result of the interplay between these two phenomena. In the present work, it is demonstrated that when measurements are sought to be made on mayonnaise using rotational viscometers visible wall slip occurs, rendering such instruments ineffective for the purpose of making viscosity measurements even at shear rates as low as 10^–3s^–1. The factors that influence the onset and extent of slip are investigated with the help of parallel plate viscometers, and it is concluded that the observed “yielding” of mayonnaise is actually an artifact of the onset of macroscopic slip. Slip effects are also found in capillary flow but are ameliorated with increasing shear rate. To circumvent these problems, it is proposed that extensional viscometry be employed for determining the flow behavior of mayonnaises. Received: 18 August 1997 Accepted: 1 April 1998     

The putative use of alpha-1-acid glycoprotein as a non-invasive marker of fibrosis

The acute phase response to injury or infection results in alterations in the expression of the plasma proteins produced by the liver. Many of these biomolecules are glycosylated with oligosaccharide chains covalently attached to the polypeptide backbone and the extent and composition of this glycosylation can be altered in a disease-dependent manner. Of particular interest is the observation that the acute phase glycoprotein, alpha-1-acid glycoprotein (AGP) has altered glycosylation in several physiological and pathological conditions. It is posited that changes induced in liver diseases may reflect disease severity and may therefore act as a non-invasive marker of fibrosis. This study has investigated the glycosylation of AGP in the plasma of people with varying degrees of cirrhosis and fibrosis. Hyperfucosylation was observed in all disease samples in comparison to normal plasma and was significantly increased in cirrhosis. Both sialic acid and N-acetylgalactosamine (GalNAc) were negatively associated with fibrosis. Two samples were found to express GalNAc, which as a constituent of the glycosylation of serum proteins is rare. In conclusion, fucose, sialic acid and other aspects of the glycosylation of AGP are influenced by the degree of fibrosis and as such may prove a valuable prognostic indicator of the development of cirrhosis.     

A secretome-based methodology may provide a better characterization of the virulence of Listeria monocytogenes: preliminary results

Four strains of Listeria monocytogenes with different levels of virulence were studied. Two strains were consistently evaluated as virulent (strain 3077) and of low virulence (strain 3993), whereas the other two strains (3006 and 3049) originated conflicting results in what the evaluation tests were concerned: both were shown to exhibit low virulence when evaluated by in vitro assays, but virulent when the analyses were performed under in vivo conditions.To clarify the virulence potential of the selected strains, a proteomic approach was used after incubating L. monocytogenes cultures under conditions favoring the expression of virulence factors (minimal medium, at 37 °C). Bacterial proteins present in the liquid culture media were precipitated from late exponential phase cultures, fractionated by SDS-PAGE and identified by MALDI-TOF-MS.Three virulence factors differentially expressed were detected: protein p60, listeriolysin O (LLO) and internalin C (InlC). Clustering analysis of the four L. monocytogenes strains based on their secretome profiles allowed their categorization in two groups: the virulent group, composed by strains 3077 and 3049, and the low virulence group, containing strains 3993 and 3006. The results presented in this work suggest that the virulent potential of a particular L. monocytogenes strain may be predicted from the levels of both listeriolysin O (LLO) and internalin C (InlC) present in its secretome when the bacterium is grown under conditions favoring the expression of virulence factors. Following validation of this proposal through the analysis of a large array of strains, this methodology exhibits a great potential to be developed into an accurate and rapid method to characterize L. monocytogenes strain virulence.     

Assessment of Genetic Stability in Traditional Ginger Cultivated in Manipur,India Based on Molecular and Chemical Markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyze the genetic stability of ten local cultivars collected from nine districts of Manipur, India with the released ginger variety Nadia. A total of 15 RAPD and 8 ISSR primers resulted in 107 and 53 distinct and reproducible bands, respectively. A lack of polymorphism revealed the genetic stability among the local cultivars. Unlike molecular markers, analysis of essential oil composition by gas chromatography–mass spectrometry (GC-MS) revealed variation among 11 clones. Among eight major constituents obtained by GC-MS technique, cinnamyl acetate was found only in IBSD/Z-41d cultivars, whereas, in IBSD/Z-41o no trace of trans-geraniol was observed. Moreover, concentration of 6-gingerol determined by high-performance liquid chromatographic (HPLC) method shows that IBSD/Z-41r contains the highest and IBSD/Z-41i contains the lowest gingerol percentage.     

Real-time monitoring of cell migration,phagocytosis and cell surface receptor dynamics using a novel,live-cell opto-microfluidic technique

We report an opto-microfluidic method for continuous and non-interfering monitoring of cell movement and dynamic molecular processes in living cells enabled by the microfluidic “Lab-in-a-Trench” (LiaT) platform. To demonstrate real-time monitoring of heterogeneous cell–cell interactions, cell tracking and agent-induced cell activation dynamics, we observe phagocytosis of Escherichia coli by murine macrophages, migration of active macrophages and LPS-induced CD86 expression in macrophages. The visualization of phagocytosis is facilitated through the loading of green fluorescent protein (GFP) expressing E. coli to the array of cell capture modules before the introduction of macrophages. Simple migration tracking of active macrophages is enabled by a spatio-temporal control of the environment conditions within the LiaT platform. Furthermore, we report an interference-free monitoring of non-modified, endogenous changes in protein expression on the surface of living cells using traditional, antibody immuno-reagents. Throughout the experiment, murine macrophages were captured in the LiaT device and exposed to sub-background levels of fluorescently labeled anti-CD86 antibody. Upon lipopolysaccharide (LPS) stimulation, CD86 changes were visualized in real-time by time-lapse microscopy. This novel opto-microfluidic effect is controlled by the equilibrium of convective–diffusive replenishment of fluorescently labeled antibodies and antibody affinity. Overall, our non-interfering analysis method allows the studying of active cellular processes and endogenous protein dynamics in live cells in a simple and cost-efficient manner.     

Analysis of normal and modified nucleosides in urine by capillary electrophoresisa)

Summary Modified nucleosides excreted in urine have been studied as potential diagnostic markers for cancer and AIDS, and as indicators for the whole-body turnover of RNA. Until now, reversed-phase (RP) HPLC and, to some extent, immunoassays are the preferred analytical methods for urinary nucleosides. A new capillary electrophoretic method for the analysis of normal and modified nucleosides in urine has been developed and optimized in our laboratory. The separation of nucleosides extracted from normal human urine on phenyl boronic acid affinity chromatography columns was performed in uncoated 565 mm (500 mm to detection window) × 50 μm i.d. capillary tubing using a 300 mM SDS—25 mM borate—50 mM phosphate buffer (pH 6.7), a 45-s load, a voltage of 7.5 kV (41 μA) and UV detection at 260 and 210 nm. The average recovery of the nucleosides was 91 %. The calibration curves were linear over all physiological and pathophysiological concentration ranges and the limits of detection were at micromolar levels. Reproducibility of migration times were better than 1 % (coefficient of variation,CV), and the reproducibilities of the determined concentrations were better than 5 % for standards and 6–15 % for extracted urine. The developed method was used to quantify 15 normal and modified nucleosides in 25 normal urines to establish reference ranges. The analysis time was less than 45 min. Dedicated to Professor E. Bayer on the occasion of his 70th birthday. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.