Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity

In order to enhance the enzymatic detoxification rate of organophosphorus (OP) nerve agents we have searched for more active variants of recombinant mammalian paraoxonase (PON1). We have previously identified three key positions in PON1 that affect OP hydrolysis: Leu69, Val346 and His115, that significantly enhance the hydrolysis of cyclosarin (GF), soman, chlorpyrifos-oxon (ChPo), O-isopropyl-O-(p-nitrophenyl)methylphosphonate (IMP-pNP) and diisopropyl fluorophosphate (DFP). GC/FPD analysis compared to residual AChE inhibition assay displayed stereoselective hydrolysis of GF, soman and IMP-pNP, indicating that wild type PON1 and its variant V346A are more active toward the less toxic P(+) optical isomer. In order to obtain new PON1 variants with reversed stereoselectivity, displaying augmented activity toward the more toxic isomer P(-) of nerve agents, we synthesized new asymmetric fluorogenic OPs (Flu-OPs). Six Flu-OPs were prepared containing either ethyl (E), cyclohexyl (C) or pinacolyl (P) alkyl radicals attached to methyl-phosphonyl (MP) moiety analogous to the structure of VX, GF and soman, respectively. The fluorescent moieties are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy-9,9-dimethyl-9H-acridin-2-one (DDAO). The kinetics of AChE and BChE inhibition by these new Flu-OPs display k(i) values 8.5x10(4) to 8.5x10(7) and 5x10(4) to 2x10(6)M(-1)min(-1), respectively. EMP-MeCyC and EMP-DDAO are the most active inhibitors of AChE whereas CMP-MeCyC and CMP-DDAO are better inhibitors of BChE than AChE, indicating accommodation of bulky cyclohexyl group inside the active site of BChE. PMP-MeCyC and PMP-DDAO are the least active inhibitors of both AChE and BChE. CMP-MeCyC and CMP-DDAO were significantly detoxified only by the five-site mutations PON1 variant L69V/S138L/S193P/N287D/V346A. Degradation kinetics of Flu-OPs measured by increase in absorbance of the released fluorogenic group was fit by a two exponential function, indicating faster hydrolysis of the less toxic optical isomer. Interestingly, wt PON1 caused only 50% degradation of racemic EMP-MeCyC, CMP-MeCyC and CMP-DDAO indicating complete hydrolysis of P(+) isomer. This remarkable stereoselectivity was used for the enzymatic separation of the P(-) isomer of CMP-MeCyC. The bimolecular rate constant k(i) for human AChE inhibition by the isolated P(-) isomer of CMP-MeCyC is five-fold larger than that of its P(+) isomer. The marked preference of wt PON1 toward P(+) stereo-isomer of CMP-MeCyC and CMP-DDAO renders their P(-) stereo-isomers suitable for the selection of new OP hydrolase variants with reversed stereoselectivity.     

Collection of large-scale expanded lymphocyte cultures for adoptive immunotherapy using a COBE Spectra apheresis machine

Adoptive cell transfer represents an important mode of cancer immunotherapy and posttransplant associated viral infections. This technique involves massive volume reduction, as the procedure starts with large-scale ex vivo expansion of lymphocyte cultures (volume up to 60 L), which are harvested at the end of the in vitro process and terminates in a small volume to be infused into the patient. Toward this end, we develop a novel efficient process based on the COBE Spectra apheresis machine usually used for apheresis process. As the COBE Spectra cell separator is easy to use and often available at medical centers, this novel technique is applicable to many transplant and cell processing centers. Our results show a high recovery yield (98%+/-15%) and viability (ranged 79% to 99%) of the large-scale expanded lymphocytes. It preserves sterility of the product and is therefore suitable for immunotherapy treatments.     

Related F-box proteins control cell death in Caenorhabditis elegans and human lymphoma

Cell death is a common metazoan cell fate, and its inactivation is central to human malignancy. In Caenorhabditis elegans, apoptotic cell death occurs via the activation of the caspase CED-3 following binding of the EGL-1/BH3-only protein to the antiapoptotic CED-9/BCL2 protein. Here we report a major alternative mechanism for caspase activation in vivo involving the F-box protein DRE-1. DRE-1 functions in parallel to EGL-1, requires CED-9 for activity, and binds to CED-9, suggesting that DRE-1 promotes apoptosis by inactivating CED-9. FBXO10, a human protein related to DRE-1, binds BCL2 and promotes its degradation, thereby initiating cell death. Moreover, some human diffuse large B-cell lymphomas have inactivating mutations in FBXO10 or express FBXO10 at low levels. Our results suggest that DRE-1/FBXO10 is a conserved regulator of apoptosis.     

Surgical feasibility of ipsilateral multifocal non-small cell lung cancer in different lobes: excellent survival in node-negative subgroup

Objective: The management of ipsilateral multifocal non-small cell lung cancer (NSCLC) in different lobes is still controversial. We analyzed our surgical results and the prognostic factors with the findings of other studies and evaluated the surgical feasibility. Methods: Between January 1, 1983 and December 31, 2001, 1408 patients underwent operation for primary NSCLC, including 20 patients who received complete resections for multifocal NSCLC of the same histological type in ipsilateral different lobes. Results: The 1-, 2- and 5-year survival rate of the 20 patients were 60.0, 39.3 and 28.1%, respectively. There were no statistically significant differences in T-status, gender, pathological type, and stage. An excellent 5-year survival rate of 66.7% (median, 101 months) in the group without node involvement was found (N0 vs. N1+2, P=0.0872). Conclusion: Our data suggest that surgical resection is mandatory in patients with ipsilateral multifocal NSCLC when there is no evidence of node metastasis.     

In vivo imaging of insulin receptors in monkeys using 18F-labeled insulin and positron emission tomography.

We previously described a prosthetic group methodology for incorporating 18F into peptides and showed that 18F-labeled insulin (18F-insulin) binds to insulin receptors on human cells (IM-9 lymphoblastoid cells) with affinity equal to that of native insulin (1). We now report studies using 18F-insulin with positron emission tomography to study binding to insulin receptors in vivo. Positron emission tomography scans were performed in six rhesus monkeys injected with 0.3-1.4 mCi of 18F-insulin (approximately 0.1 nmol, SA 4-11 Ci/mumol). Integrity of the tracer in blood, determined by immunoprecipitation, was 94% of control for the first 5 min and decreased to 31% by 30 min. Specific, saturable uptake of 18F was observed in the liver and kidney. Coinjection of unlabeled insulin (200 U, approximately 1 nmol) with the 18F-insulin reduced liver and increased kidney uptake of the labeled insulin. Liver radioactivity was decreased by administration of unlabeled insulin at 3 min, but not 5 min, after administration of the tracer, while some kidney radioactivity could be displaced 5 min after injection. Clearance of 18F was predominantly in bile and urine. 18F-insulin is a suitable analogue for studying insulin receptor-ligand interactions in vivo, especially in the liver and kidney.     

Effect measure modification and confounding of severe head injury mortality by age and multiple organ injury severity

PURPOSE: The study aim is to assess confounding and effect measure modification of the relationship between head injury severity (measured using the Glasgow Coma Scale [GCS]) and mortality by age and multiple organ injury (measured using the Injury Severity Score [ISS]). Head injury-related mortality is affected by head injury severity, as well as age and multiple organ injury. However, little is known about the effect modification of these relationships. METHODS: Stratifications and multiple logistic regression analyses examined the association of mortality with GCS score less than 9, controlled for ISS of 16 and higher or age of 55 years and older. Receiver operating curve (ROC) analyses were used to explore the relationship of GCS cutoff values and prediction of death by the ISS and age categories mentioned. RESULTS: Both age and ISS modified head injury mortality in a similar direction: there was a negative interaction between age and ISS and more severe head injury. Lower GCS values (indicating more severe head injury) related more strongly to mortality in younger persons. Lower GCS values related more strongly to mortality when extracranial injuries were less severe. These data indicate effect modification. Data indicate negative confounding of the association between GCS and mortality by age and positive confounding by ISS. Multivariate analyses indicate that only younger age modifies the effect of more severe head injury with statistical significance when both age and multiple organ injury are considered. ROC analyses conducted with stratified and logistic regression analyses indicate that GCS is a better predictor of death in those with younger age. CONCLUSIONS: The relationship of head injury severity and mortality is modified and confounded by age and ISS. GCS is a better predictor of death in younger patients.