a b s t r a c tMethods that do not require animal sacrifice to detect botulinum neurotoxins (BoNTs) are critical for BoNT antagonist discovery and the advancement of quantitative assays for biodefense and pharmaceutical applications. Here we describe the development and optimization of fluorogenic reporters that detect the proteolytic activity of BoNT/A, B, D, E, F, and G serotypes in real time with femtomolar to picomolar sensitivity. Notably, the reporters can detect femtomolar concentrations of BoNT/A in 4 h and BoNT/E in 20 h, sensitivity that equals that of animal-based methods. The reporters can be used to determine the specific activity of BoNT preparations with intra-and inter-assay coefficients of variation of approximately 10%. Finally, we find that the greater sensitivity of our reporters compared with those used in other commercially available assays makes the former attractive candidates for high-throughput screening of BoNT antagonists.Ó 2011 Elsevier Inc. All rights reserved.Botulinum neurotoxins (BoNTs), 1 produced by the bacteria of the genus Clostridium, are the most lethal substances known. The zinc-dependent endopeptidases act by entering neurons and cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and, thus, compromise the protein machinery responsible for neurotransmitter release [1][2][3][4][5][6][7][8]. Failure to promptly treat a victim of BoNT poisoning can result in flaccid paralysis, respiratory failure, or death [9]. The combination of extreme potency and a lack of medical treatments other than antitoxin administration and intensive care has made BoNT a biodefense priority requiring the discovery and development of assays to quantify toxins and to identify antagonists to counteract intoxication [10][11][12][13]. Despite their lethality, BoNTs are widely used for cosmetic and pharmaceutical applications due in part to their exquisite specificity for the neuromuscular junction. BoNTs provide relief of muscle tension and pain by inhibiting neurons that cause excessive muscle contractions. Therapeutic preparations of BoNT/A and B serotypes are Food and Drug Administration (FDA) approved for treating glabellar lines, strabismus, cervical dystonia, blepharospasm, cranial nerve VII disorders, and primary axillary hyperhidrosis. Dozens of ''off-label'' BoNT clinical applications have also been documented [14][15][16][17].The mouse bioassay, or lethality test, has been the standard for testing BoNT-containing samples for the past 30 years [18][19][20]. Government agencies use this method for testing food and serum samples for the presence of BoNT, whereas the pharmaceutical industry uses it for quality control and to quantify ''for human use'' BoNT preparations. The test is carried out by injecting mice intraperitoneally with approximately 0.5 ml of sample per mouse and recording the number of deaths over a 1-to 7-day period. The assay is very sensitive, with a detection limit of 5-10 pg for BoNT/A [21,22]. Results for the mouse bioassay are reported in...

A peptide with high intrinsic activity for specifically stimulating the secretion of immunoreactive growth hormone (GH; somatotropin) has been characterized and reproduced by total synthesis. This peptide, human pancreatic growth hormone-releasing factor, 44-amino-acid form (hpGRF1-44-NH2), was isolated from a tumor localized in the pancreas of a patient with acromegaly. We report here the effect of this growth hormone-releasing factor (GRF) on GH release and the GH mRNA levels in monolayer cultures of rat pituitary. The cytoplasmic dot hybridization technique was used to examine the effect of GRF on GH mRNA levels. Incubation of rat pituitary cultures with GRF for 72 hr resulted in a 2-to 2.5-fold increase in GH mRNA levels, and the maximal levels of stimulation were achieved at GRF concentrations as low as 1 fM. GRF did not stimulate prolactin release, nor did it affect specific prolactin mRNA levels in the pituitary cultures. We conclude that GRF is a potent and specific GH secretagogue that also affects specifically GH mRNA levels in normal pituitary cells.The hypothalamus fulfills a dual function in the regulation of growth hormone (GH; somatotropin) release. An inhibitory factor, somatostatin, has been characterized since 1972, whereas the stimulatory factor [growth hormone-releasing factor (GRF) or somatocrinin] has eluded investigators until recently. In order to circumvent the problem of low abundance of GRF in the hypothalamus, extrahypothalamic sources of GRF production were sought. A solid tumor of the pancreas obtained at surgery from a patient who had produced the clinical syndrome of acromegaly was used to isolate and characterize the primary structure of three peptides possessing high intrinsic GH-releasing activity called hpGRF-40, hpGRF-44, and hpGRF-37, in which hp designates human pancreatic (1). Based upon their common NH2 terminal sequence and lower specific activities in vitro, hpGRF-40 and -37 are most probably proteolytic degradation products of hpGRF-44. Most recently the isolation and structural characterization of bovine (2), porcine (3), and murine (4) hypothalamic GRF have been reported. Both bovine and porcine GRF have 44 amino acid residues and share extensive structural homology with the 44-amino-acid form of hpGRF designated hpGRF1-44 NH2-The experiments reported here were conducted with the synthetic replicate of hpGRFl-44-NH2 (henceforth referred to as GRF or somatocrinin). GRF already has been shown to stimulate GH-release in vivo (5) and in vitro (6) without affecting release of other pituitary hormones. We report here that GRF also stimulates GH mRNA sequences in rat pituitary cells. A portion of these findings has been presented in preliminary form (7).

Molecular cloning has established the primary structures of two precursors of the human pancreas growth hormone-releasing factor (hpGRF-44), somatocrinin. Both polypeptides contain the sequence of hpGRF-44 flanked by basic processing sites. Furthermore, the precursors include a putative signal sequence and a carboxyl-terminal amidation signal for hpGRF-44. The two forms of mRNA code for pre-pro-GRF-107 and pre-pro-GRF-108. Pre-pro-GRF-108 differs from pre-pro-GRF-107 by the insertion of a serine in the carboxyl-terminal portion of the precursor. In vitro translation of tumor poly(A)+ RNA followed by immunoprecipitation with GRF-specific antiserum and gel electrophoresis showed the molecular weight of preprosomatocrinin to be approximately 13,000, which is in good agreement with the molecular weight deduced from the sequences of the cDNA clones.A 44-amino acid peptide (hpGRF-44, somatocrinint) with high intrinsic growth hormone-releasing activity in vitro and in vivo was recently isolated from a human pancreatic tumor and characterized (1, 2). Preliminary biological (3), immunological (4), and physico-chemical (unpublished data) evidence indicates that the tumor-derived hpGRF-44 is identical to hypothalamic growth hormone-releasing factor. The availability of tumor material, highly enriched in the growth hormone-releasing factor compared with hypothalamic tissue, has permitted the molecular cloning and structural characterization of the mRNA encoding hpGRF-44. MATERIALS AND METHODSPoly(A)+ RNA was obtained from 15 g of tumor tissue (stored in liquid nitrogen or at -80°C) by using the guanidine thiocyanate procedure (5) and chromatography on oligo(dT)-cellulose (6). Double-stranded cDNA was synthesized by using a modification of a published method (ref. 7; unpublished data). A cDNA library was constructed by homopolymer tailing of the cDNA with dGTP, hybridizing it to EcoRV-cut pBR322 that had been tailed with dCTP and transforming Escherichia coli RR1 with the chimeric plasmid (8). The solid-phase phosphotriester method (9) was used for the synthesis of two mixed oligodeoxynucleotide probes (see Fig. 1), which were labeled at their 5' ends with [y-32P]ATP (10). The tetradecamer probe was used for colony screening (11) as follows: hybridizations were carried out in 0.75 M NaCl/0.075 M sodium citrate/0. 1% NaDodSO4/ 0.2% bovine serum albumin/0.2% polyvinylpyrrolidone/0.2% Ficoll/partially hydrolyzed yeast RNA (100 ug/ml) at 300C for 2 hr using 1 pmol of labeled probe per ml. The filters were quickly rinsed in 0.3 M NaCl/0.03 M sodium citrate at room temperature. After a high-stringency wash in 0.6 M NaCl/0.06 M sodium citrate for 60 min at 40'C, the filters were exposed to x-ray film overnight.DNA sequence analysis, Southern and RNA blot analyses, nick-translation, primer extension, and NaDodSO4/polyacrylamide and denaturing agarose gel electrophoresis were carried out according to standard procedures (10,(12)(13)(14)(15)(16)(17)(18). Antibodies to hpGRF were produced in rabbits following immunization with...

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Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by Förster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n ‫؍‬ 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity.