Studies of Microthrix parvicella in situ and in laboratory culture: production and use of specific antibodies

Physiological studies on M. parvicella have been conducted to determine the rate of growth of this organism in pure culture. The organism displayed a doubling time of 128 days despite its profuse abundance in a local Wastewater Treatment Plant (WWTW). An extensive survey has been ongoing since February 2000 into the extent of M. parvicella in the WWTW. A suite of monoclonal and polyclonal antibodies has been developed to detect and quantify M. parvicella.

Individual mRNA expression profiles reveal the effects of specific microRNAs

BACKGROUND: MicroRNAs (miRNAs) are oligoribonucleotides with an important role in regulation of gene expression at the level of translation. Despite imperfect target complementarity, they can also significantly reduce mRNA levels. The validity of miRNA target gene predictions is difficult to assess at the protein level. We sought, therefore, to determine whether a general lowering of predicted target gene mRNA expression by endogenous miRNAs was detectable within microarray gene expression profiles. RESULTS: The target gene sets predicted for each miRNA were mapped onto known gene expression data from a range of tissues. Whether considering mean absolute target gene expression, rank sum tests or 'ranked ratios', many miRNAs with significantly reduced target gene expression corresponded to those known to be expressed in the cognate tissue. Expression levels of miRNAs with reduced target mRNA levels were higher than those of miRNAs with no detectable effect on mRNA expression. Analysis of microarray data gathered after artificial perturbation of expression of a specific miRNA confirmed the predicted increase or decrease in influence of the altered miRNA upon mRNA levels. Strongest associations were observed with targets predicted by TargetScan. CONCLUSION: We have demonstrated that the effect of a miRNA on its target mRNAs' levels can be measured within a single gene expression profile. This emphasizes the extent of this mode of regulation in vivo and confirms that many of the predicted miRNA-mRNA interactions are correct. The success of this approach has revealed the vast potential for extracting information about miRNA function from gene expression profiles.

Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology

Paralytic shellfish poisoning (PSP) toxin monitoring in shellfish is currently performed using the internationally accredited AOAC mouse bioassay. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. The feasibility of using a surface plasmon resonance optical biosensor to detect PSP toxins in shellfish tissue below regulatory levels was examined. Three different PSP toxin protein binders were investigated: a sodium channel receptor (SCR) preparation derived from rat brains, a monoclonal antibody (GT13-A) raised to gonyautoxin 2/3, and a rabbit polyclonal antibody (R895) raised to saxitoxin (STX). Inhibition assay formats were used throughout. Immobilization of STX to the biosensor chip surface was achieved via amino-coupling. Specific binding and inhibition of binding to this surface was achieved using all proteins tested. For STX calibration curves, 0 - 1000 ng/mL, IC50 values for each binder were as follows: SCR 8.11 ng/mL; GT13-A 5.77 ng/mL; and R895 1.56 ng/mL. Each binder demonstrated a different cross-reactivity profile against a range of STX analogues. R895 delivered a profile that was most likely to detect the widest range of PSP toxins at or below the internationally adopted regulatory limits.

Production and characterization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues

Previous studies have shown that glycation of insulin occurs in pancreatic beta -cells under conditions of hyperglycaemia and that the site of glycation is the N-terminal Phe(1) of the insulin B-chain. To enable evaluation of glycated insulin in diabetes, specific antibodies were raised in rabbits and guinea-pigs by using two synthetic peptides (A: Phe-Val-Asn-Gln-His-Leu-Cys-Tyr, and B: Phe-Val-Asn-Gln-His-Leu-Tyr-Lys) modified by N-terminal glycation and corresponding closely to the N-terminal sequence of the glycated human insulin B-chain. For immunization, the glycated peptides were conjugated either to keyhole limper haemocyanin or ovalbumin using glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester or 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. Antibody titration curves, obtained using I-125-tyrosylated tracer prepared from glycated peptide A, revealed high-titre antisera in five groups of animals immunized for 8-28 weeks. The highest titres were observed in rabbits and guinea-pigs immunized with peptide B coupled to ovalbumin using glutaraldehyde. Under radioimmunoassay conditions, these antisera exhibited effective dose (median) (ED50) values for glycated insulin of 0.3-15 ng/ml and 0.9-2.5 ng/ml respectively, with negligible cross-reactivity against insulin or other islet peptides. The degree of cross-reaction with glycated proinsulin was approximately 50%. Glycated insulin in plasma of control and hydrocortisone-treated diabetic rats measured using rabbit 3 antiserum (1:10 000 dilution; sensitivity