isolation-quantification-botulinum-neurotoxin-from-complex-matrices

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

BioSentinel Inc., Madison, WI

The success of liver grafts is currently limited by the length of time organs are cold preserved before transplant. Novel insights to improve viability of cold-stored organs may emerge from studies with animals that naturally experience low body temperatures (T(b)) for extended periods. In this study, we tested whether livers from hibernating ground squirrels tolerate cold ischemia-warm reperfusion (cold I/R) for longer times and with better quality than livers from rats or summer squirrels. Hibernators were used when torpid (T(b) < 10 degrees C) or aroused (T(b) = 37 degrees C). Livers were stored at 4 degrees C in University of Wisconsin solution for 0-72 h and then reperfused with 37 degrees C buffer in vitro. Lactate dehydrogenase (LDH) release after 60 min was increased 37-fold in rat livers after 72 h cold I/R but only 10-fold in summer livers and approximately three- to sixfold in torpid and aroused hibernator livers, despite twofold higher total LDH content in livers from hibernators compared with rats or summer squirrels. Reperfusion for up to 240 min had the least effect on LDH release in livers from hibernators and the greatest effect in rats. Compared with rats or summer squirrels, livers from hibernators after 72 h cold I/R showed better maintenance of mitochondrial respiration, bile production, and sinusoidal lining cell viability, as well as lower vascular resistance and Kupffer cell phagocytosis. These results demonstrate that the hibernation phenotype in ground squirrels confers superior resistance to liver cold I/R injury compared with rats and summer squirrels. Because hibernation-induced protection is not dependent on animals being in the torpid state, the mechanisms responsible for this effect may provide new strategies for liver preservation in humans.

Natural resistance to liver cold ischemia-reperfusion injury associated with the hibernation phenotype.

Prolactin regulates a variety of physiological processes, including mammary gland growth and differentiation, and recent findings support an important role in breast cancer development and progression. However, little is known about the trafficking of its receptor, a member of the cytokine receptor superfamily. In the present study, we examined the effect of ligand on the endogenous "long" isoform of the prolactin receptor in breast cancer cells. We found that prolactin caused rapid and prolonged down-regulation of this receptor. The prolactin-induced increase in degradation was blocked by inhibitors of both proteasomes and lysosomes. However, the ubiquitin-conjugating system was not required for internalization. Prolactin also resulted in the concomitant appearance of a cell-associated prolactin receptor fragment containing the extracellular domain. This latter process required proteasomal, but not metalloprotease, activity, distinguishing it from ectodomain "shedding" of other membrane receptors, which are secreted as binding proteins. The prolactin receptor fragment was labeled by surface biotinylation and independent of protein synthesis. Together, these data indicated that prolactin binding initiates limited proteasomal cleavage of its receptor, generating a cell-associated fragment containing the extracellular domain. Our findings described a new potential mediator of prolactin action and a novel mechanism whereby proteasomes modulate cellular processes.

Proteasomes mediate prolactin-induced receptor down-regulation and fragment generation in breast cancer cells.

Despite the growing body of evidence supporting prolactin (PRL) actions in human breast cancer, little is known regarding PRL regulation of its own receptor in these cells. Ligand-initiated endocytosis is a key process in the regulation of receptor availability and signaling cascades that may lead to oncogenic actions. Although exposure to exogenous PRL accelerates degradation of the long isoform of the PRL receptor (lPRLR), neither the signals initiated by PRL that lead to lPRLR internalization and subsequent down-regulation, nor the relationship to downstream pathways are understood in breast cancer cells. In this study, we showed that PRL-induced down-regulation of the lPRLR was reduced by inhibition of src family kinases (SFKs), but not Janus kinase 2, in MCF-7 cells. Inhibition of SFKs also resulted in accumulation of a PRL-induced PRLR fragment containing the extracellular domain, which appeared to be generated from newly synthesized PRLR. lPRLR was constitutively associated with SFKs in lipid rafts. PRL-induced SFK activation led to recruitment of the guanosine triphosphatase, dynamin-2, to an internalization complex, resulting in endocytosis. Inhibition of endocytosis by small interfering RNA-mediated knockdown of dynamin-2 blocked PRL-induced down-regulation of lPRLR, confirming that internalization is essential for this process. Endocytosis also was required for optimal phosphorylation of ERK1/2 and Akt, but not for Janus kinase 2 or signal transducer and activator of transcription 5, indicating that internalization selectively modulates signaling cascades. Together, these data indicate that SFKs are key mediators of ligand-initiated lPRLR internalization, down-regulation, and signal transduction in breast cancer cells, and underscore the importance of target cell context in receptor trafficking and signal transduction.

SRC family kinases accelerate prolactin receptor internalization, modulating trafficking and signaling in breast cancer cells.

Normal mammary development requires coordinated interactions of numerous factors, including prolactin (PRL) and insulin-like growth factor I (IGF-I), both of which have also been implicated in breast cancer pathogenesis and progression. We previously reported that PRL and IGF-I synergize in breast cancer cells to activate ERK1/2 and AKT, leading to increased proliferation, survival, and invasion. Intriguingly, PRL co-treatment with IGF-I augments IGF-I receptor (IGF-IR) phosphorylation 2-fold higher than IGF-I alone. Here, we showed the importance of the tyrosine phosphatase SHP-2 in this cross-talk using pharmacological inhibition and small interfering RNA. SHP-2 recruitment to IGF-IR was significantly attenuated by PRL co-treatment. Src family kinase activity was required for IGF-IR association with SHP-2, ligand-induced IGF-IR internalization, and PRL-enhanced IGF-IR phosphorylation. Inhibition of internalization, via knockdown of the GTPase, dynamin-2, prevented not only IGF-IR dephosphorylation, but also PRL-enhanced IGF-IR phosphorylation. Consistently, PRL diminished IGF-I-induced IGF-IR internalization, which may result from reduced SHP-2 association with IGF-IR, because we demonstrated an essential role for SHP-2 in IGF-IR internalization. Together, these findings describe a novel mechanism of cross-talk between PRL and IGF-I in breast cancer cells, with implications for our understanding of tumor progression and potential therapeutic strategies.

Prolactin enhances insulin-like growth factor I receptor phosphorylation by decreasing its association with the tyrosine phosphatase SHP-2 in MCF-7 breast cancer cells.

Methods 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 4h and BoNT/E in 20h, 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.

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters.

The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.

Calpain-mediated proteolysis of paxillin negatively regulates focal adhesion dynamics and cell migration.

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.

In vitro detection and quantification of botulinum neurotoxin type e activity in avian blood.

Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-μl samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices.

Timothy M. Piazza

BioSentinel Inc., Madison, WI

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays