Vaccine protection against simian immunodeficiency virus by recombinant strains of herpes simplex virus

An effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.     

Characterization of Ricinus communis phloem profilin,RcPRO1

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.     

Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity

We tested the role of the “spring-loaded” conformational change in the fusion mechanism of the influenza hemagglutinin (HA) by assessing the effects of 10 point mutants in the region of high coiled-coil propensity, HA2 54–81. The mutants included proline substitutions at HA2 55, 71, and 80, as well as a double proline substitution at residues 55 and 71. Mutants were expressed in COS or 293T cells and assayed for cell surface expression and structural features as well as for their ability to change conformation and induce fusion at low pH. We found the following: Specific mutations affected the precise carbohydrate structure and folding of the HA trimer. All of the mutants, however, formed trimers that could be expressed at the cell surface in a form that could be proteolytically cleaved from the precursor, HA0, to the fusion-permissive form, HA1-S-S-HA2. All mutants reacted with an antibody against the major antigenic site and bound red blood cells. Seven out of ten mutants displayed a wild-type (wt) or moderately elevated pH dependence for the conformational change. V55P displayed a substantial reduction (~60– 80%) in the initial rate of lipid mixing. The other single mutants displayed efficient fusion with the same pH dependence as wt-HA. The double proline mutant V55P/ S71P displayed no fusion activity despite being well expressed at the cell surface as a proteolytically cleaved trimer that could bind red blood cells and change conformation at low pH. The impairment in fusion for both V55P and V55P/S71P was at the level of outer leaflet lipid mixing. We interpret our results in support of the hypothesis that the spring-loaded conformational change is required for fusion. An alternate model is discussed.     

Identification of immunologically related proteins in sieve-tube exudate collected from monocotyledonous and dicotyledonous plants

The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein 2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the enucleate sieve-tube system of higher plants. Received: 12 February 1998 / Accepted: 16 March 1998     

Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf)

Blackspot, caused by the Ascomycete fungus Diplocarpon rosae, is the most widespread and pernicious disease of cultivated roses. While some species of rose possess resistance to D. rosae, none of the modern-day rose cultivars are fully resistant to the pathogen. In the current study, Biolistic gene delivery was used to introduce a rice gene, encoding a basic (Class I), chitinase into embryogenic callus of the blackspot-susceptible rose (Rosa hybrida L.) cv. Glad Tidings. The plasmid used for transformation carried the neomycin phosphotransferase (nptII) gene facilitating the selection and regeneration of transgenic plants on medium containing 250 mg/l kanamycin. Southern analysis confirmed integration of 2–6 copies of the chitinase gene into the rose genome; gene expression was confirmed by enzyme assay. Bioassays demonstrated that expression of the chitinase transgene reduced the severity of blackspot development by 13–43%. This degree of resistance to the pathogen correlated with the level of chitinase expression in the transgenic rose plants. The introduction of disease defence genes into rose provides a method of producing blackspot-resistant rose cultivars sought by breeders and growers.     

Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin

Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.     

KRAS Mutation Analysis by PCR: A Comparison of Two Methods

Background

KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.

Methods

DNA was extracted by Maxwell (Promega) from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer’s instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies). The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.

Results

Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT) for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT) for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.

Conclusion

There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.