Joseph A. Vetro

Yeast methionine aminopeptidase type 1 is ribosome‐associated and requires its N‐terminal zinc finger domain for normal function in vivo

Methionine aminopeptidase type 1 (MetAP1) cotranslationally removes N‐terminal methionine from nascent polypeptides, when the second residue in the primary structure is small and uncharged. Eukaryotic MetAP1 has an N‐terminal zinc finger domain not found in prokaryotic MetAPs. We hypothesized that the zinc finger domain mediates the association of MetAP1 with the ribosomes and have reported genetic evidence that it is important for the normal function of MetAP1 in vivo. In this study, the intracellular role of the zinc finger domain in yeast MetAP1 function was examined. Wild‐type MetAP1 expressed in a yeast map1 null strain removed 100% of N‐terminal methionine from a reporter protein, while zinc finger mutants removed only 31–35%. Ribosome profiles of map1 null expressing wild‐type MetAP1 or one of three zinc finger mutants were compared. Wild‐type MetAP1 was found to be an 80S translational complex‐associated protein that primarily associates with the 60S subunit. Deletion of the zinc finger domain did not significantly alter the ribosome profile distribution of MetAP1. In contrast, single point mutations in the first or second zinc finger motif disrupted association of MetAP1 with the 60S subunit and the 80S translational complex. Together, these results indicate that the zinc finger domain is essential for the normal processing function of MetAP1 in vivo and suggest that it may be important for the proper functional alignment of MetAP1 on the ribosomes. J. Cell. Biochem. 85: 678–688, 2002.

Polymer micelles as drug carriers

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Effects of the PPARγ agonist troglitazone on endothelial cells in vivo and in vitro: Differences between human and mouse☆

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists and PPARgamma/alpha dual agonists have been or are being developed for clinical use in the treatment of type 2 diabetes mellitus and hyperlipidemias. A common tumor finding in rodent carcinogenicity studies for these agonists is hemangioma/hemangiosarcoma in mice but not in rats. We hypothesized that increased endothelial cell proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice, and we investigated the effects on endothelial cells utilizing troglitazone, the first clinically used PPARgamma agonist, in vivo and in vitro. Troglitazone (400 and 800 mg/kg/day) induced hemangiosarcomas in mice in a 2-year bioassay. We showed that troglitazone increased endothelial cell proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses after 4 weeks of treatment. Troglitazone was cytotoxic both to human dermal microvascular endothelial cells (HMEC1) and mouse mammary fat pad microvascular endothelial cells (MFP MVEC) at high concentrations. However, MFP MVEC were more resistant to the cytotoxic effects of troglitazone based on the much lower LC(50) in HMEC1 (17.4 muM) compared to MFP MVEC (92.2 muM). Troglitazone increased the proliferation and survival of MFP MVEC but not HMEC1 in growth factor reduced conditions. Our data demonstrate that troglitazone may induce hemangiosarcomas in mice, at least in part, through enhancement of survival and proliferation of microvascular endothelial cells. Such an effect does not occur with human cells, suggesting that human may react differently to exposure to PPAR agonists compared with mice.

Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae

Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged. In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strains reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2. Replacement of the conserved His174 with alanine within the C‐terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro. Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth. Deletion mutants further revealed that the N‐terminal region of MetAP2 (residues 2–57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth. Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N‐terminal region to interfere with wild‐type MetAP2 function.

Preliminary evidence that the novel host-derived immunostimulant EP67 can act as a mucosal adjuvant

EP67 is a complement component 5a (C5a)-derived peptide agonist of the C5a receptor (CD88) that selectively activates DCs over neutrophils. Systemic administration of EP67 covalently attached to peptides, proteins, or attenuated pathogens generates TH1-biased immunogen-specific humoral and cellular immune responses with little inflammation. Furthermore, intranasal administration of EP67 alone increases the proportion of activated APCs in the airways. As such, we hypothesized that EP67 can act as a mucosal adjuvant. Intranasal immunization with an EP67-conjugated CTL peptide vaccine against protective MCMV epitopes M84 and pp89 increased protection of naïve female BALB/c mice against primary respiratory infection with salivary gland-derived MCMV and generated higher proportions of epitope responsive and long-lived memory precursor effector cells (MPEC) in the lungs and spleen compared to an inactive, scrambled EP67-conjugated CTL peptide vaccine and vehicle alone. Thus, EP67 may be an effective adjuvant for mucosal vaccines and warrants further study.

Encapsulation of an EP67-Conjugated CTL Peptide Vaccine in Nanoscale Biodegradable Particles Increases the Efficacy of Respiratory Immunization and Affects the Magnitude and Memory Subsets of Vaccine-Generated Mucosal and Systemic CD8+ T Cells in a Diameter-Dependent Manner

The diameter of biodegradable particles used to coencapsulate immunostimulants and subunit vaccines affects the magnitude of memory CD8+ T cells generated by systemic immunization. Possible effects on the magnitude of CD8+ T cells generated by mucosal immunization or memory subsets that potentially correlate more strongly with protection against certain pathogens, however, are unknown. In this study, we conjugated our novel host-derived mucosal immunostimulant, EP67, to the protective MCMV CTL epitope, pp89, through a lysosomal protease-labile double arginine linker (pp89-RR-EP67) and encapsulated in PLGA 50:50 micro- or nanoparticles. We then compared total magnitude, effector/central memory (CD127/KRLG1/CD62L), and IFN-γ/TNF-α/IL-2 secreting subsets of pp89-specific CD8+ T cells as well as protection of naive female BALB/c mice against primary respiratory infection with MCMV 21 days after respiratory immunization. We found that decreasing the diameter of encapsulating particle from ∼5.4 μm to ∼350 nm (i) increased the magnitude of pp89-specific CD8+ T cells in the lungs and spleen; (ii) partially changed CD127/KLRG1 effector memory subsets in the lungs but not the spleen; (iii) changed CD127/KRLG1/CD62L effector/central memory subsets in the spleen; (iv) changed pp89-responsive IFN-γ/TNF-α/IL-2 secreting subsets in the lungs and spleen; (v) did not affect the extent to which encapsulation increased efficacy against primary MCMV respiratory infection over unencapsulated pp89-RR-EP67. Thus, although not observed under our current experimental conditions with MCMV, varying the diameter of nanoscale biodegradable particles may increase the efficacy of mucosal immunization with coencapsulated immunostimulant/subunit vaccines against certain pathogens by selectively increasing memory subset(s) of CD8+ T cells that correlate the strongest with protection.

Delivery, detection and development in nanomedicine

The Fourth International Nanomedicine and Drug Delivery Symposium (NanoDDS’06; www.nanodds.org/) was held in Omaha, NE, USA, from October 8 to 10, 2006, to ‘provide an overview of the latest advances in all aspects of nanomedicine and drug delivery’ and attracted nearly 160 registered participants from 16 countries, 24 leading experts as guest speakers and over 80 poster presentations. The Symposium was chaired by Professor Alexander (Sasha) Kabanov from the University of Nebraska Medical Center (UNMC; NE, USA), bringing the conference full circle since it was first organized in Omaha by Dr Kabanov and Dr Kataoka as a mini-symposium on ‘Nanomedicine and Drug Delivery’ at UNMC in January 2003 (www.unmc.edu/pharmacy/nanomedicine/).

Complexation of Chol-DsiRNA in place of Chol-siRNA greatly increases the duration of mRNA suppression by polyplexes of PLL(30)-PEG(5K) in primary murine syngeneic breast tumors after i.v. administration

&NA; RNA interference has tremendous potential for cancer therapy but is limited by the insufficient potency of RNAi molecules after i.v. administration. We previously found that complexation with PLL(30)‐PEG(5K) greatly increases the potency of 3′‐cholesterol‐modified siRNA [Chol‐siRNA] in primary murine syngeneic 4T1 breast tumors after i.v. administration but mRNA suppression decreases 24 h after the final dose. We hypothesized that complexation of cholesterol‐modified Dicer‐substrate siRNA (Chol‐DsiRNA) in place of Chol‐siRNA can increase the potency and duration of suppression by polyplexes of PLL(30)‐PEG(5K) in solid tumors. We found that replacing Chol‐siRNA with Chol‐DsiRNA increased polyplex loading and nuclease protection, suppressed stably expressed luciferase to the same extent in primary murine 4T1‐Luc breast tumors under the current dosage regimen, but maintained suppression ˜72 h after the final dose. The kinetics of suppression in 4T1‐Luc over 72 h, however, were similar between DsiLuc and siLuc after electroporation and between polyplexes of Chol‐DsiLuc and Chol‐siLuc after transfection, suggesting that Chol‐DsiRNA polyplexes increase the duration of mRNA suppression through differences in polyplex activities in vivo. Thus, replacing Chol‐siRNA with Chol‐DsiRNA may significantly increase the duration of mRNA suppression by polyplexes of PLL(30)‐PEG(5K) and possibly other PEGylated polycationic polymers in primary tumors and metastases after i.v. administration. Graphical abstract Figure. No caption available.

New Generation of Adjuvants for Protection Against Disease and to Combat Bioterrorism

This chapter highlights the characteristics of traditional and non-traditional adjuvants, the advantages and disadvantages of their use in vaccines. It has so served to introduce the concept of molecular and host-derived adjuvants to improve immune outcomes while minimizing toxicities. A particular emphasis was placed on the generation of conformationally-restricted, response-selective agonists of the complement component C5a. These all appear to have therapeutic, manufacturing, and commercial potential. However, the development and use of such molecular adjuvants for vaccines represent only one approach put forward to meet the modern-day threats posed by antibiotic-resistant strains of bacteria and bioterrorism. It is clear that a well-organized and concerted worldwide effort that utilizes multiple approaches to develop novel adjuvant and vaccine designs will be required to overcome these growing threats to human and animal populations.