Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1–60 CTCs), while all samples contained additional CE-positive cells (range 1–41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

Kinetic proofreading is a reaction scheme with a structure more complicated than that of Michaelis kinetics, which leads to a proofreading for errors in the recognition of a correct substrate by an enzyme. We have measured the stoichiometry between ATP hydrolysis and tRNAIle charging, using the enzyme isoleucyl-tRNA synthetase [L-iso-leucine:tRNAIe ligase (AMP-forming), EC 6.1.1.5] and the amino acids isoleucine (correct) and valine (incorrect). The enzymatic deacylation of charged tRNA, which would normally prevent meaningful stoichiometry studies, was eliminated by the use of transfer factor Tu'GTP, (which binds strongly to charged tRNA) in the reaction mixture. For isoleucine, 1.5 ATP molecules are hydrolyzed per tRNA charged, but for valine, 270. These stoichiometry ratios are fundamental to kinetic proofreading, for the energy coupling is essential and proofreading is obtained only by departing from 1:1 stoichiometry between energy coupling and product formation. Within the known reaction pathway, these ratios demonstrate that kinetic proofreading induces a reduction in errors by a factor of 1/180. An overall error rate of about 10-4 for tRNA charging is obtained by a kinetic proofreading using a fundamental discrimination level of about 10-2, and is compatible with the low in vivo error rate of protein synthesis.Many biochemical reactions, particularly those associated with protein synthesis or DNA replication, exhibit high specificity in the selection between similar substrates. The overall error rate in selecting between two similar amino acids (1) in protein synthesis is believed to be about 3 in 104. The normal error rate in DNA synthesis (without post-replication repair) is about 1 in 108 or 109. These low error rates are biologically essential.The elementary description of specificity in biochemical reactions is based on discrimination in a Michaelis complex. [2]When C and D are sufficiently similar, AG will not be large, and fo may be smaller than would be biologically optimal. "Kinetic proofreading" (2) is a method of using twice (or more) the same Michaelis kinetic ability to distinguish between C and D, resulting in an error rate as small as fo2 (or higher power) instead of fo for a given AG. The essential features of kinetic proofreading are contained in the reaction schemeThe first reaction step reversibly forms the usual Michaelis complex. The second step is enzymatically coupled to an energy source, typically the hydrolysis of a nucleoside triphosphate, and is strongly enough driven to be essentially irreversible. (Cc)* is a high energy intermediate, which can decompose in two ways: to free enzyme plus product or to free enzyme and nonproduct C'. C' can be either the original substrate C or a chemically modified form thereof.This scheme is capable of proofreading. For equal concentrations of C and D, the discrimination against D in the initial Michaelis complex and in the formation of (Dc)* will be fo. If the kinetic constants are appropriate (and they can be made so without enhancing...

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Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features in their cofactor binding sites. Then we identify a ligand for this site and use NMR-based binding site mapping (NMR SOLVE) to determine where to extend a combinatorial library, such that diversity elements are directed into the adjacent substrate site. The cofactor mimic is reused in the library in a manner that parallels the reuse of cofactor domains in the oxidoreductase gene family. A library designed in this manner yielded specific inhibitors for multiple oxidoreductases.

Two types of oligonucleotides were synthesized with linker groups attached at the 5'-end. Both were repeating dimers of deoxyribocytidine and deoxyriboadenosine. A 20-mer was prepared with a thiol-containing linker, masked as a disulfide, and a 50-mer was prepared with a vicinal diol-containing linker. A tetraiodoacetylated poly(ethylene glycol) (PEG) derivative was synthesized and reacted with the thiol-containing 20-mer to provide an oligonucleotide PEG conjugate of precisely four oligonucleotides on each PEG carrier. The vicinal diol on the 50-mer was oxidized to an aldehyde and conjugated to keyhole limpet hemocyanin (KLH) to provide an oligonucleotide-KLH conjugate by reductive alkylation. The conjugates were annealed with complementary (TG)n strands. While the double-stranded oligonucleotide-KLH conjugate is an immunogen, eliciting the synthesis of antibodies against oligonucleotides, the PEG conjugate has the biological property of specifically suppressing (tolerizing) B cells which make antibodies against the immunizing oligonucleotide.

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A series of new substituted arylmethyl phenyl ethers has been prepared. These compounds were tested as inhibitors of 5-lipoxygenase (5-LO) in rat neutrophils, in vitro antagonists of leukotriene-induced contraction of guinea pig (GP) lung parenchymal strips, and inhibitors of slow reacting substance of anaphylaxis (SRS-A) mediated bronchospasm in the GP in vivo. Most representatives of this new class of potential antiallergic/antiinflammatory agents showed potent inhibition of 5-LO activity in rat PMNs. The most potent compound, 2-[[3-(1-hydroxyhexyl)phenoxy]-methyl]quinoline (33), had an I50 of 0.12 microM in the rat PMN 5-LO assay and an I50 of 3.6 microM in the leukotriene-induced contraction of GP lung parenchymal strips, and it also showed 91% inhibition of SRS-A-mediated bronchospasm in the GP in vivo at 10 mg/kg, administered intraduodenally. Some of the compounds in this series were also leukotriene antagonists in vitro, and several of them showed in vivo activity against SRS-A-mediated bronchospasm in the GP.