Genomic

Conventional approaches for therapeutic targeting of viral pathogens have consistently faced obstacles arising from the development of resistant strains and a lack of broad-spectrum application. Influenza represents a particularly problematic therapeutic challenge since high viral mutation rates have often confounded many conventional antivirals. Newly emerging or engineered strains of influenza represent an even greater threat as typified by recent interest in avian subtypes of influenza. Based on the limitations associated with targeting virally-encoded molecules, we have taken an orthogonal approach of targeting host pathways in a manner that prevents viral propagation or spares the host from virus-mediated pathogenicity. To this end, we report herein the application of an improved technology for target discovery, Random Homozygous Gene Perturbation (RHGP), to identify host-oriented targets that are well-tolerated in normal cells but that prevent influenza-mediated killing of host cells. Improvements in RHGP facilitated a thorough screening of the entire genome, both for overexpression or loss of expression, to identify targets that render host cells resistant to influenza infection. We identify a set of host-oriented targets that prevent influenza killing of host cells and validate these targets using multiple approaches. These studies provide further support for a new paradigm to combat viral disease and demonstrate the power of RHGP to identify novel targets and mechanisms.

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We describe the vaccination of Panamanian monkeys (Aotus sp.) with two recombinant blood stage antigens that each contain a portion of the N-terminal region of the SERA (serine repeat antigen) protein of the malaria parasite Plasmodium falciparum. We immunized with either a 262-amino-acid SERA fragment (SERA I) that contains amino acids 24 to 285 of the 989-amino-acid protein or a 483-amino-acid SERA fragment (SERA N) that contains amino acids 24 to 506 as part of a fusion protein with human gamma interferon. The recombinant proteins were shown to stimulate protective immunity when administered with complete and incomplete Freund adjuvant. Four of six immunized monkeys challenged by intravenous inoculation with blood stage P. falciparum developed parasitemias that were reduced by at least 1,000-fold. Two of six immunized monkeys developed parasitemias which were comparable to the lowest parasitemia in one of four controls and were 50- to 1,000-fold lower than in the other three controls.

Cyclohexanone oxygenase, from Acinetobacter NCIB 9871, has been incubated with (2S,6S)-[2,6-2H2]-and (27?) -[ 2-2H, ]cyclohexanone. The resulting labeled c-caprolactone (2-oxepanone) samples were degraded to 1-pentanol, which was esterified by using (-)-camphanyl chloride. Analysis of the camphanates by deuterium NMR spectroscopy, using Eu(dpm)3, showed that the conversion of ketone to lactone had in each case proceeded with complete retention of configuration at the migrating carbon center. A similar degradation of (27?)-[2-2H1]cyclohexanone itself showed that reduction of [2-2H,]cyclohex-2-enone by Beauveria bassiana ATCC 7159 is also completely stereoselective. A method has been developed for assessing the enantioselectivity of enzymes toward racemic substrate mixtures. (2R)-2-[methyl-2H3]-and (2S)-2-[methyl-13C] methylcyclohexanone were synthesized and mixed in equal amounts, and the resulting mixture (a virtual racemate) was incubated with cyclohexanone oxygenase. The course of the reaction was followed by both 13C and 2H NMR spectroscopy, showing that the initial rate of oxidation of the 2S enantiomer was nearly twice that of the 2R enantiomer. (27?)-and (2S)-2-methylcyclohexanone were both converted by cyclohexanone oxygenase to 6-methyl-e-caprolactone (7-methyl-2-oxepanone).Advantages of the virtual racemate/multinuclear NMR technique over existing methodology are described.While the Baeyer-Villiger reaction3 has been a standard tool of organic chemistry for the better part of a century, only in recent years has it become clear that nature makes widespread use of this reaction in biodegradative pathways.4 Studies in several f Dedicated to Dr. Ulrich Weiss on the occasion of his 75th birthday.