A multi-generation analysis of the stability of transgenic virus resistance in doubled-haploid tobacco lines

Plants can be genetically engineered for virus resistance by transformation with a viral gene. We transformed tobacco with the tomato spotted wilt virus (TSWV) nucleocapsid gene from the Hawaiian L isolate in order to obtain TSWV resistant breeding lines. Doubled-haploid lines were produced from primary transgenic plants that were selected for resistance to the virus. Several of these lines showed very high levels of resistance and were symptomless after inoculation with the Hawaiian L isolate of TSWV. The accumulation of only low levels of full-length transgene RNA and protein observed in these lines is consistent with an RNA-mediated mechanism of resistance. The lines that were highly resistant to the Hawaiian L isolate of TSWV were also found to be highly resistant to several other isolates of TSWV, while lines that were only moderately resistant to the Hawaiian L isolate were often susceptible to the other isolates. The highly resistant lines were advanced over several generations by self-pollination. Although these lines were fully homozygous, several lines lost resistance in later generations, indicating that the resistance was unstable. Selection for resistance in these unstable lines did not prevent the occurrence of susceptible progeny in subsequent generations. Therefore, testing over several generations is required to determine the stability of resistance when breeding crops with transgenic virus resistance.     

Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkins lymphoma (NHL) associated with an aggressive clinical course. Inhibition of the ubiquitin-proteasome pathway modulates survival and proliferation signals in MCL and has shown clinical benefit in this disease. This has provided rationale for exploring combination regimens with B-cell selective immunotherapies such as rituximab. In this study, we examined the effects of combined treatment with bortezomib and rituximab on patient-derived MCL cell lines (Jeko, Mino, SP53) and tumor samples from patients with MCL where we validate reversible proteasome inhibition concurrent with cell cycle arrest and additive induction of apoptosis. When MCL cells were exposed to single agent bortezomib or combination bortezomib/rituximab, caspase dependent and independent apoptosis was observed. Single agent bortezomib or rituximab treatment of Mino and Jeko cell lines and patient samples resulted in decreased levels of nuclear NFκB complex(es) capable of binding p65 consensus oligonucleotides, and this decrease was enhanced by the combination. Constitutive activation of the Akt pathway was also diminished with bortezomib alone or in combination with rituximab. On the basis of in vitro data demonstrating additive apoptosis and enhanced NFκB and phosphorylated Akt depletion in MCL with combination bortezomib plus rituximab, a phase II trial of bortezomib-rituximab in patients with relapsed/refractory MCL is underway.Key words: mantle cell lymphoma, proteasome inhibition, CD20, survival and death pathways, apoptosis     

Elucidating and Minimizing the Loss by Recombinant Vaccinia Virus of Human Immunodeficiency Virus Gene Expression Resulting from Spontaneous Mutations and Positive Selection

While characterizing modified vaccinia virus recombinants (rMVAs) containing human immunodeficiency virus env and gag-pol genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the recombinant gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the recombinant gene to a site between essential, highly conserved MVA genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag-pol genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary recombinant vaccines.Vaccinia virus (VACV), the first recombinant virus shown to induce a protective immune response against an unrelated pathogen (21, 22), is being employed as a vector for veterinary and wildlife vaccines (19). Development of recombinant VACV for human use, however, has been impeded by safety concerns. For this reason, there is interest in modified VACV Ankara (MVA), a highly attenuated smallpox vaccine with an exemplary safety profile even in immunodeficient animals (17, 26, 27). MVA is severely host range restricted and propagates poorly or not at all in most mammalian cells because of a block in virion assembly (29). Initial experiments with recombinant MVA (rMVA) demonstrated its ability to robustly express foreign proteins (29) and induce protective humoral and cell-mediated immunity (30). Currently, rMVA candidate vaccines expressing genes from a wide variety of pathogens are undergoing animal and human testing (13).While developing candidate human immunodeficiency virus (HIV) and other vaccines, we encountered a tendency for mutant rMVA that had lost the ability to express foreign proteins to arise after tissue culture passage (28, 34, 37). This instability may initially go undetected, however, unless individual plaques are isolated and analyzed. Nevertheless, once established in the population, the nonexpressors can rapidly overgrow the original rMVA. These considerations are particularly important for production of large vaccine seed stocks of rMVA. The instability of cloned genes in MVA is surprising, since MVA had already undergone genetic changes during its adaptation through hundreds of passages in chicken embryo fibroblasts (CEFs) and is now quite stable. Indeed, identical 167,000-bp genome sequences have been reported for three independent plaque isolates, accession numbers {"type":"entrez-nucleotide","attrs":{"text":"U94848","term_id":"2772662","term_text":"U94848"}}U94848, {"type":"entrez-nucleotide","attrs":{"text":"AY603355","term_id":"47088326","term_text":"AY603355"}}AY603355, and {"type":"entrez-nucleotide","attrs":{"text":"DQ983236","term_id":"115607417","term_text":"DQ983236"}}DQ983236, and by Antoine et al. (1). Although the cause of the instability of the gene inserts had not been previously investigated, harmful effects of the recombinant protein seem to play a role in the selective advantage of nonexpressing mutants. Thus, reducing the expression level of parainfluenza virus and measles virus transmembrane proteins and deleting part of the cytoplasmic tail of HIV Env improves the stability of rMVAs (28, 34, 37). Reducing expression, however, can also decrease immunogenicity and therefore may be undesirable (36).In view of the importance of understanding and overcoming this pernicious instability problem, we carried out a systematic study of HIV env and gag-pol genes that were unstable in rMVA. We also considered that the analysis would provide basic information regarding the kinds of errors that can occur during replication of the VACV genome, which encodes its own cytoplasmic replication system (20). The most common mutations, which led to loss of recombinant gene expression, were large deletions that extended deep into the MVA flanks and frameshift mutations within consecutive identical nucleotides in the insert. The frequency of viable mutations was minimized by introducing the recombinant gene between two essential, highly conserved MVA genes and by making silent codon alterations to interrupt the homonucleotide runs. In addition, we constructed a panel of recombinant viruses with chimeric and truncated env genes to determine the basis for the selection of nonexpressing mutants and to prevent their expansion during virus propagation. Understanding the causes of the instability and taking preemptive actions should facilitate the development of MVA and other poxviruses as human and veterinary vaccines. In addition, these insights may have application to other DNA expression vectors.     

Expression of genes associated with carbohydrate metabolism in cotton stems and roots

Background  

Cotton (Gossypium hirsutum L) is an important crop worldwide that provides fiber for the textile industry. Cotton is a perennial plant that stores starch in stems and roots to provide carbohydrates for growth in subsequent seasons. Domesticated cotton makes these reserves available to developing seeds which impacts seed yield. The goals of these analyses were to identify genes and physiological pathways that establish cotton stems and roots as physiological sinks and investigate the role these pathways play in cotton development during seed set.     

3-Substituted gem-cyclohexane sulfone based gamma-secretase inhibitors for Alzheimer's disease: conformational analysis and biological activity

Previously, chemistry effort on the gem-cyclohexane series of gamma-secretase inhibitors has focused on the 4-position of the cyclohexane ring. Recently chemistry has been directed towards the 3-position and substitution here has also provided compounds with high gamma-secretase activity.     

4-substituted cyclohexyl sulfones as potent, orally active gamma-secretase inhibitors

The protease gamma-secretase plays a pivotal role in the synthesis of pathogenic amyloid-beta in Alzheimer's disease (AD). Here, we report a further extension to a series of cyclohexyl sulfone-based gamma-secretase inhibitors which has allowed the preparation of highly potent compounds which also demonstrate robust Abeta(40) lowering in vivo (e.g., compound 32, MED 1mg/kg p.o. in APP-YAC mice).