Evidence for the presence of a receptor for the cytolethal distending toxin (CLDT) of Campylobacter jejuni on CHO and HeLa cell membranes and development of a receptor-based enzyme-linked immunosorbent assay for detection of CLDT

Abstract Using ligand blotting, it was found that partially purified cytolethal distending toxin prepared from and enterotoxigenic strain of Campylobacter jejuni , bound to two peptides of molecular masses of approximately 59 kDa and 45 kDa and to a single peptide of 59 kDa in protein blots prepared from HeLa and CHO cell membranes, respectively. In contrast, labile toxin of Escherichia coli and cholera toxin bound to a single peptide of the same molecular mass (15 kDa) on protein blots prepared from both CHO and HeLa cell crude membranes resolved by gel electrophoresis. This banding pattern was identical using SDS-solubilized membrane, with or without heat treatment, but no band was obtained when reduced (treatment with 2-mercaptoethanol) samples were used for the gel electrophoresis. The differences between receptors of cytolethal distending toxin and cholera toxin/labile toxin were exploited to develop a receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin which involved the consecutive addition of either solubilized CHO or HeLa membranes, antigen and antibody. This enzyme-linked immunosorbent assay consistently detected crude cytolethal distending toxin diluted up to 16-fold. The receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin developed in this study is a suitable alternative assay which can be performed easily in laboratories with minimal facilities and, more importantly, the results are available within a few hours as compared to times of up to 5 days in the conventional tissue culture detection of cytolethal distending toxin.     

Optimized protocol for the pilot-scale preparation of fungal cellulase

Summary A 25-l scale protocol is devised for the optimal secretion and recovery of fungal cellulase. Using a selected higher yieldingTrichoderma viride SMC strain, a protocol consisted of: a) an optimized production medium rich in microcrystalline cellulose (MCC), fortified with 1% (w/v) ammonium sulphate, 0.5% (w/v) soybean flour, 0.1% (v/v) Tween-80 and other trace nutrients; b) optimized physical parameters of production, such as an inoculum containing a homogeneous suspension of 6×10⁷ conidia per 1,28±1°C, pH 4.0±0.5, 300±20 rpm, 11000±1000 l/h aeration, and 170–220 h duration; c) optimal recovery through a filter press (450 l/h rate of filtration) followed by precipitation with 2.5–3.0 volumes of acetone (15°C and basket centrifugation (27°C, 1700 rpm)); and d) vacuum drying (35°C, 4–6 h). This afforded 70% recovery of cellulase in the form of white fluffy powder containing 20000±2000 carboxy methyl cellulase and 1000±50 units filter paperase per g activities, with raw material cost of US$ 8–10 per million carboxy methyl cellulase units. During storage for 18 months at 4°C, ambient temperature and 37°C, the cellulase preparation was found to retain 100, 75 and 60% of its initial activity, respectively.     

Synthesis and Biological Activity of a Bis-Substituted 3′-Deoxyadenosine Analog of 2–5A

Abstract

The 5′-monophosphate, p5′(3′dA)2′p5′A2′5′(3′dA), was synthesized and found to bind to the 2–5A-dependent endonuclease of mouse L cells only two-three times less effectively than the parent p5′A2′p5′A2′p5′A. When evaluated for its ability to activate the 2–5A-dependent endonuclease, ppp5′(3′dA)2′p5′A2′p5′(3′dA) was found to be fifty times more effective than ppp5′A2′p5′(3′dA)2′p5′A and ten times less effective than 2–5A as an endonuclease activator     

Recent Progress in Oligoribonucleotide Synthesis

Abstract

The usefulness of the p-nitrophenylethylsulfonyl (NPES) group for 2′-OH protection in oligoribonucleotide synthesis is further investigated. The difficulties of uridine protection are discussed and the p-cyanophenylethyl (CPE) group introduced as a versatile new 0⁴-blocking group.     

Molecularly imprinted polyaniline molecular receptor–based chemical sensor for the electrochemical determination of melamine

Molecularly imprinted polymer‐modified glassy carbon electrode (GCE)‐based electrochemical sensor is prepared using the electropolymerization of aniline in the presence of melamine (MA) as a template. In this work, the advantages of molecularly imprinted conducting polymers (MICPs) and electroanalytical methods were combined to obtain an electronic device with better performances. The sensor performance was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) with the linear range of 0.6‐16 × 10^?9M, quantification limit of 14.9 × 10^?10M, and detection limit of 4.47 × 10^?10M (S/N = 3). The selectivity of the sensor was tested in the presence of acetoguanamine (AGA), diaminomethylatrazine (DMT), casein, histidine, and glycine interfering molecules taken at the triple concentration with MA that demonstrated too small current response compared with that of the analyte indicating high specificity of the sensor towards the template. The sensor was successfully applied to determine MA in infant formula samples with significant recovery greater than 96% and relative standard deviation (RSD) less than 4.8%. Moreover, the good repeatability, recyclability, and stability make this sensor device promising for the real‐time monitoring of MA in different food stuffs.     

Increasing productivity of Spirulina platensis in photobioreactors using artificial neural network modeling

Although production of microalgae in open ponds is conventionally practiced due to its economy, exposure of the algae to uncontrollable elements impedes achievement of quality and it is desirable to develop closed reactor cultivation methods for the production of high value products. Nevertheless, there are several constraints which affect growth of in closed reactors, some of which this study aims to address for the production of Spirulina. Periodic introduction of fresh medium resulted in increased trichome numbers and improved algal growth compared to growth in medium that was older than 4 weeks in 20 L polycarbonate bottles. Mixing of the cultures by bubbling air and use of draft tube reduced the damage to the growing cells and permitted increased growth. However, there was better growth in inclined cylindrical reactors mixed with bubbling air. The oxygen production rates were very similar irrespective differences in the maintained cultures densities. The uniformity in oxygen production rate suggested a tendency towards homeostasis in Spirulina cultures. The frequency of biomass harvest on the productivity of Spirulina showed that maintenance of moderate culture density between 0.16 and 0.32 g/L resulted in about 14% more productivity than maintaining the cell density between 0.16 and 0.53 g/L or 48% more than by daily harvest above 0.16 g/L. An artificial neural network based predictive model was developed, and the variables useful for predicting biomass output were identified. The model could predict the growth of Spirulina up to 3 days in advance with a coefficient of determination >0.94.