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The cdc25A protein phosphatase inhibitor dysidiolide (1) has been synthesized enantioselectively, starting from the enantiomerically pure ketal enone 2 and using a cationic rearrangement as the key step to produce the fully substituted bicyclic core of the natural product. Once the central portion of 1 was established, elaboration of the side chains was accomplished expediently via steps that included (1) vinyl cuprate displacement of an iodide to complete the C-1 side chain, (2) a highly diastereoselective oxazaborolidine-catalyzed (CBS) reduction to form carbinol 11, and (3) photochemical oxidation of 11 to generate the γ-hydroxybutenolide functionality of 1. Additionally, this synthesis proves the absolute stereochemistry of dysidiolide (1).

A series of 1,2,3,4,5,6-hexafluorobicyclo[4.2.0]octa-2,4-dienes was subjected to direct irradiation with ultraviolet light and to triplet sensitization. While all of the dienes cyclized smoothly upon direct irradiation to tricyclo[4.2.0.0 2,5 ]oct-3-enes, their behavior when triplet sensitized was strongly dependent upon the substituents in the 7- and 8-positions. Responses included no reaction, cyclization to tricyclooctene, and fragmentation to hexafluorobenzene plus olefin. Fragmentation occurred only when geminal chlorines were present at the 7-position. This observation is consistent with the view that the four-membered ring opens homolytically at the C 6 C 7 bond, and only if sufficient stabilization is available for a radical center at the 7-position.

We investigated the ability of a novel polyphosphazene polyelectrolyte, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) to enhance antigen-specific immune responses. BALB/c mice were immunized once subcutaneously with either bovine serum albumin (BSA) or influenza virus X:31 antigen alone, or in combination with PCEP, or either of the adjuvants poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) and alum. Both PCEP and PCPP significantly enhanced serum antigen-specific total IgG, IgG1 and IgG2a antibody titers, and these responses were highest in PCEP-immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of X:31 antigen by 25-fold had no effect on antibody responses in mice immunized with PCPP and PCEP, but resulted in reduced titers in those immunized with alum. Analysis of X:31 antigen-specific cytokines revealed that alum and PCPP were associated with a predominantly IL-4 response. In contrast, PCEP was associated with production of both IFNγ and IL-4. We conclude that PCEP is a potent enhancer of antigen-specific Th1 and Th2 immune responses and is a promising adjuvant for vaccine applications.

The advent of modern molecular biology has provided us with a means of producing antigens with unprecedented ease and precision. It is ironic that these new methodologies generate purified antigens that do not generally induce a strong immune response in the absence of an effective adjuvant. The development of improved vaccine adjuvants for use in humans has therefore become a priority area of research. Nevertheless, research on adjuvants has lagged seriously behind the work done on antigens. For decades the only adjuvant widely used in humans has been alum. Saponin and its purified component Quil A, complete Freund’s adjuvant (CFA) and other adjuvants used in research and veterinary applications have toxicities that limit their potential use in human vaccines. New chemically defined preparations such as QS-21, muramyl dipeptide, and monophosphoryl lipid A are being studied.

The messenger RNAs present in HeLa cells late in adenovirus 2 infection have been characterized utilizing three complementary methods of analysis. In each case the mRNAs were defined functionally, by identification of the polypeptides they encode when translated in a reticulocyte cell-free system. The RNAs were examined by (1) electrophoretic fractionation in agarose gels containing methyl-mercury hydroxide to estimate the sizes of the functional RNAs; (2) selection by annealing to viral DNA fragments immobilized on nitrocellulose to define the genomic origin of their constituent sequences; (3) hybrid-arrested translation using viral DNA fragments to locate their protein coding sequence. The results allow the positioning of both the coding and non-coding regions of all the late mRNAs with respect to the DNA. They demonstrate that the mature mRNAs originating in the major late transcription unit fall into five families. Within each family the mRNAs overlap in a staggered fashion, sharing the same 3′ terminus but differing at their 5′ termini. The data also reveal the existence of two new late proteins of 52,000 and 55,000 Mr encoded between map co-ordinates 30 to 34 and permit the positioning of the late RNAs, according to the polypeptides they encode, as follows: 52, 55K · IIIa: III · pVII · V: pVI · II: 100 K/33 K · pVIII: IV. (The colons signify the ends of families; centre dots the junctions within families; the slash an overlap of coding sequences; and the comma, coding sequences which have not been separated in this analysis. A 55 K protein has Mr = 55,000.) Those mRNAs which are either the only transcripts from a specific region (such as those for polypeptides IX, IVa2 and IV) or are the 3′-terminal transcripts within a family (such as those coding for polypeptides IIIa, V, II and pVIII) migrate as discrete species upon electrophoresis in agarose gels containing methyl-mercury hydroxide. However, the 5′-proximal RNAs within a family (such as those encoding polypeptides 52, 55 K, III, pVII, pVI, 100 K and 33 K) exhibit a broad distribution of molecular weights.

copia-specific RNA was isolated from Drosophila melanogaster tissue culture cells by hybridization of cytoplasmic polyadenylylated RNA to copia DNA immobilized on cellulose. The purified RNA was translated in reticulocyte lysates. One major polypeptide of approximately 51,000 daltons was synthesized in addition to several others between 18,000 and 38,000 daltons. The 51,000-dalton polypeptide and several of the others are encoded by mRNAs of about 2000 nucleotides. The approximate locations on the copia element of the coding sequences for the 51,000-dalton polypeptide and several other proteins were determined by hybrid-arrested translation with copia restriction fragments. The relative abundance of copia-specific RNA was determined at various stages of the Drosophila life cycle. The level of copia-specific RNA is modulated during development of the organism, with the highest level occurring during the larval stages.

Extracts of wheat germ are capable of synthesizing the major capsid protein of simian virus 40. Poly(A)-containing RNA from BS-C-1 cells infected with simian virus 40 directed the synthesis of a novel polypeptide that migrates in polyacrylamide gels together with the major capsid polypeptide of simian virus 40, VP-1. The patterns of the major tryptic peptides of purified VP-1 and the novel polypeptide synthesized in vitro were identical after two-dimensional paper electrophoresis. The novel polypeptide was not synthesized in response to poly(A)-rich RNA from uninfected cells or from virus-infected cells treated with cytosine arabinoside. Messenger RNA from infected cells purified by selective hybridization to DNA of simian virus 40 directs the synthesis of a major polypeptide of electrophoretic mobility similar to that of VP-1 of simian virus 40. This approach should prove useful in identifying additional products specified by DNA tumor viruses.

A linked cell-free system has been developed which is capable of transcribing and translating mamalian viral DNA, and its characteristics and requirements are outlined. In this system, simian virus 40 (SV40) DNA Form I (supercoiled) directed the synthesis of discrete polypeptides up to 85,000 daltons in size. One of these products was indistingusihable from authentic major virus capsid protein VPI, as judged by mobility on sodium dodecyl sulfate/polyacrylamide gels, antibody predipitation, and peptide analyses. The cell-free products larger than VPI comprised a number of polypeptides ranging in molecular weight from 50,000 to 85,000. These polypeptides demonstrated no immunological relationship whatsoever to the structural protein VPI. However, two of these products, along with one of approximately 25,000 dlatons, were precipitated with antiserum to SV40 tumor antigen. Linear SV40 DNA generated by the cleavage of Form I DNA with the restriction endonuclease EcoR1 was an efficient template in this system and also directed the synthesis of a polypeptide migrating with VPI on polyacrylamide gels. The potential of this system for defining a functional map of a DNA genome is discussed.

A method is described for mapping regions of eukaryotic viral DNA coding for specific proteins, utilizing a linked transcription-translation cell-free system primed with DNA fragments generated by restriction endonucleases. Three simian virus 40 (SV40) DNA fragments derived from that region of the DNA expressed late in lytic infection were purified. They were: Hpa I-A (0.76-0.175 map units), Bgl I-EcoRI-B (0.672-0), and Hpa II-EcoRI-B (0.735-0). (Fragments are named from the cleaving restriction endonuclease and electrophoretic mobility. End positions on the conventional map are in clockwise order.) These fragments efficiently stimulated the incorporation of [3H]UTP and [35S]methionine into trichloroacetic-acid-insoluble material in the linkec system. The location of the region of DNA coding for the viral structural proteins VPI, VP2 and VP3 was determined from the spectrum of polypeptide synthesis directed by the individual intact fragments and their specific endonucleolytic digests. The polypeptides synthesized in the cell-free system were characterized on urea-sodium dodecyl sulfate polyacrylamide gradient gels and by two-dimensional tryptic peptide analysis. ..

The Epstein-Barr virus (EBV)-specified polypeptides induced upon viral replication in the P3HR-1 cell line have been examined by immunoprecipitation with a high-titer human anti-EBV serum. Twenty-five predominant polypeptides were identified in cell extracts, whereas 18 polypeptides were precipitated from cell-free translation reactions directed by total mRNA. Hybrid selection of mRNA to the BamHI DNA clones of the EBV genome and immunoprecipitation of the corresponding cell-free translation products revealed 98 EBV-specified polypeptides and their coding location along the viral genome. In addition, the viral polypeptides that bind reversibly to DNA-cellulose have been characterized and the deduced map locations of this functional group of EBV-specified polypeptides is presented.

Tobacco mosaic virus (TMV) RNA stimulates amino acid incorporation into protein in cell-free extracts from wheat germ, rye embryo and Escherichia coli . The properties of the wheat germ system are examined and the nature of the viral RNA-induced products studied with the aid of a virus mutant carrying a threonine → methionine replacement in its coat protein. A peptide containing this methionine residue is present in tryptic digests of mutant RNA-directed cell-free products, and is absent from digests of wild type RNA-directed products. The undigested cell-free product contains a very large number of polypeptides with molecular weights from 10,000 to 140,000, but little or no synthesis of correct sized coat protein is observed.

Herein we outline three methods that, when coupled with cell-free protein synthesis, permit the identification of recombinant DNA molecules encoding specific polypeptides. RNAs enriched by fractionation on methylmercury hydroxide agarose gels are used to prepare sequence-specific probes. Recombinant DNA clones thus identified are further characterized as to their encoded polypeptides by either hybrid selection or hybrid arrest of translation.

We describe a simple procedure for isolating specific mRNAs and for mapping them to the regions of the DNA from which they originate. The method involves hybridization of total cytoplasmic RNA to restriction fragments of DNA that have been fractionated in agarose gels and immobilized on nitrocellulose filters. The hybridization-selected RNAs are eluted and translated in a cell-free system in order to identify their encoded polypeptides. Optimal hybridization and filter washing conditions are given for selection of mRNAs from adenovirus 2-infected cells and transformed cells.

Host-range mutants of adenovirus 5 that contain a defect in region E1A (0-4.5 units) fail to replicate in HeLa cells and to transform rodent cells. In HeLa cells, these mutants synthesize only the two RNAs from E1A that share the same 5' and 3' termini but differ in length by the amount of internal sequence removed by splicing. RNA from wild-type virus, selected by hybridization to DNA from region E1A, translates into polypeptides of Mr 51,000 and 48,000 that are highly acidic in isoelectric focusing gels. These acidic Mr 51,000 and Mr 48,000 polypeptides are encoded by the longer and shorter E1A RNAs, respectively. Two of the host-range mutants, H5hr1 and H5hr2, fail to synthesize the Mr 51,000 polypeptide but do produce the Mr 48,000 polypeptide and a novel polypeptide thought to be a truncated portion of the Mr 51,000 polypeptide. H5hr1 and H5hr2 are hypothesized to have termination codons in sequences found only in RNA encoding the Mr 51,000 polypeptide. This prediction is verified for H5hr1 by DNA sequence analysis. The other three host-range mutants (H5hr3-5) synthesize both acidic polypeptides and are predicted to be missense. These results strongly imply that the Mr 51,000 polypeptide, alone or in combination with the Mr 48,000 polypeptide, is needed to regulate expression of adjacent viral genes during the early phase of adenovirus infection.

Rabbit beta globin DNA sequence, excised from plasmid pbetaG1, directs in vitro synthesis of beta-globin in a transcription-translation cell-free system, even after specific elimination of the entire 3'-noncoding region. A DNA restriction fragment carrying this 3' noncoding region and hybridized to globin mRNA cannot arrest the cell-free translation of beta-globin mRNA.

Fine structural investigations of non-viable rye grains indicate recognisible abnormalities in the plasmalemma and mitochondrial membranes of the unimbibed embryo. Once such grains are wetted there is rapid and progressive disorganisation of the tissue. Biochemical studies show a reduced uptake of water, lack of respiratory activity and a failure in nucleic acid and protein synthesis. Whereas total DNA, RNA and protein levels are unchanged on loss of viability, the integrity of DNA and RNA is impaired and ribosomal RNA and soluble protein levels are reduced.