scispace - formally typeset
Search or ask a question
Author

Adamina Vocero-Akbani

Other affiliations: Howard Hughes Medical Institute
Bio: Adamina Vocero-Akbani is an academic researcher from Washington University in St. Louis. The author has contributed to research in topics: Fusion protein & Transduction (genetics). The author has an hindex of 5, co-authored 5 publications receiving 3801 citations. Previous affiliations of Adamina Vocero-Akbani include Howard Hughes Medical Institute.

Papers
More filters
Journal ArticleDOI
03 Sep 1999-Science
TL;DR: It is shown that intraperitoneal injection of the 120-kilodalton beta-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically active fusion protein to all tissues in mice, including the brain.
Abstract: Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical properties of the proteins. Here it is shown that intraperitoneal injection of the 120-kilodalton β-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically active fusion protein to all tissues in mice, including the brain. These results open new possibilities for direct delivery of proteins into patients in the context of protein therapy, as well as for epigenetic experimentation with model organisms.

2,640 citations

Journal ArticleDOI
TL;DR: Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27 Kip1 induces cell migration and promotes cell migration in mice.
Abstract: Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27 Kip1 induces cell migration

1,014 citations

Journal ArticleDOI
TL;DR: Observations demonstrate that TGF-beta signaling mediates a G(1) arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Ctk2 and one for Cdk4/6.
Abstract: Transforming growth factor β (TGF-β)-mediated G1 arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. We report here that TGF-β-treated human HepG2 hepatocellular carcinoma cells arrest in G1, but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-β-treated cells failed to induce p15INK4b, down-regulate CDC25A, or increase levels of p21CIP1, p27KIP1, and p57KIP2. However, TGF-β treatment resulted in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr160 phosphorylation on Cdk2. Whole-cell lysates from TGF-β-treated cells showed inhibition of Cdk2 Thr160 Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, was not altered. Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7. Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regulated in response to TGF-β treatment. Taken together, these observations demonstrate that TGF-β signaling mediates a G1 arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.

102 citations

Book ChapterDOI
TL;DR: It is concluded that the methodology generates highly efficient transducible proteins that are biologically active and have broad potential in the manipulation of biological experimental systems, such as apoptotic induction, cell cycle progression, and differentiation, and in the delivery of pharmacologically relevant proteins.
Abstract: Currently, delivery of expression vectors, proteins, and/or pharmacologically important peptidyl mimetics to target cells is problematic because of the low percentage of cells targeted, overexpression, size constraints, and bioavailability Concentration-dependent transduction of full-length proteins and domains directly into cells would serve to alleviate these problems Previous researchers have demonstrated the ability of proteins linked to the human immunodeficiency virus (HIV) Tat transduction domain to transduce into cells; but because of inefficiencies, this methodological potential has not significantly progressed since 1988 We describe, in this chapter, a significant increase in transduction efficiency of proteins and ease of use by (1) generation of a Tat protein transduction domain in-frame bacterial expression vector, pTAT-HA, and (2) development of a purification protocol yielding denatured proteins We have transduced full-length Tat fusion proteins ranging in size from 15 to 115 kDa into approximately 100% of all target cells examined, including peripheral blood lymphocytes, all cells present in whole blood, bone marrow stem cells, diploid fibroblasts, fibrosarcoma cells, and keratinocytes Transduction occurs in a concentration-dependent manner, achieving maximum intracellular concentrations in less than 10 min We conclude that our methodology generates highly efficient transducible proteins that are biologically active and have broad potential in the manipulation of biological experimental systems, such as apoptotic induction, cell cycle progression, and differentiation, and in the delivery of pharmacologically relevant proteins

81 citations

Book ChapterDOI
TL;DR: Transduction of full-length Tat fusion proteins directly into ∼100% of primary or transformed cells has broad implications for manipulating intracellular processes in both experimental in vitro tissue culture systems and animal models.
Abstract: Publisher Summary This chapter describes the methodology to generate and transduce full-length Tat fusion proteins that can be applied to a broad spectrum of proteins independent of size or function. Bacterially expressed N'-terminal in-frame Tat fusion proteins are isolated from bacteria by sonication in 8 M urea. The use of 8 M urea achieves two goals. First, the majority of recombinant proteins in bacteria are present in inclusion bodies as denatured insoluble proteins, especially full-length proteins. Sonication in urea solubilizes this material, thus allowing for its isolation. Second, denatured Tat fusion proteins have an enhanced potential to elicit biological responses. The denatured Tat fusion proteins are made soluble in an aqueous buffer and added directly to the medium of cells in tissue culture. To date, this strategy is used to generate and transduce more than 60 full-length proteins and domains from 15 to 120 kDa from a variety of classes, suggesting that many proteins may be transduced into cells. Transduction of full-length Tat fusion proteins directly into ∼100% of primary or transformed cells has broad implications for manipulating intracellular processes in both experimental in vitro tissue culture systems and animal models.

58 citations


Cited by
More filters
Journal ArticleDOI
03 Sep 1999-Science
TL;DR: It is shown that intraperitoneal injection of the 120-kilodalton beta-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically active fusion protein to all tissues in mice, including the brain.
Abstract: Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical properties of the proteins. Here it is shown that intraperitoneal injection of the 120-kilodalton β-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically active fusion protein to all tissues in mice, including the brain. These results open new possibilities for direct delivery of proteins into patients in the context of protein therapy, as well as for epigenetic experimentation with model organisms.

2,640 citations

Journal ArticleDOI
13 Oct 2000-Cell
TL;DR: The author would like to thank S. H. Roan for all her help and members of the Massague laboratory for insightful discussions.

2,303 citations

Journal ArticleDOI
TL;DR: Two nonviral gene delivery systems using either biodegradable poly(D,Llactide-co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells.
Abstract: The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,Llactide-co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (~200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less

2,189 citations

Journal ArticleDOI
TL;DR: A cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles is developed, which efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10–30 pg of super paramagnetic iron per cell.
Abstract: The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells

1,788 citations

Journal ArticleDOI
TL;DR: Overall, a transporter has been developed that is superior to Tat(49-57), protease resistant, and more readily and economically prepared and suggest that the guanidinium groups of Tat( 49-57) play a greater role in facilitating cellular uptake than either charge or backbone structure.
Abstract: Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat(49-57) (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat(49-57) were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alanine-substituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat(49-57) play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Tat(49-57) in cellular uptake. In contrast, a 9-mer of l-arginine (R9) was 20-fold more efficient than Tat(49-57) at cellular uptake as determined by Michaelis-Menton kinetic analysis. The d-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat(49-57) play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this analysis, we designed and synthesized a class of polyguanidine peptoid derivatives. Remarkably, the subset of peptoid analogues containing a six-methylene spacer between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat(49-57) and even to r9. Overall, a transporter has been developed that is superior to Tat(49-57), protease resistant, and more readily and economically prepared.

1,710 citations