Protein transduction domains (PTDs), both naturally occurring and synthetic, have been increasingly utilized to deliver biologically active agents to a variety of cell types in vitro and in vivo. We report that in addition to previously characterized arginine-rich PTDs, including TAT, lysine homopolymers were able to mediate transduction of a wide variety of cell types, as measured by flow cytometric and enzymatic assays. The efficiency of PTD-mediated transduction was influenced by the cell type tested, although polylysine homopolymers demonstrate levels of internalization that consistently exceeded those of TAT and arginine homopolymers. Transduction of arginine/lysine-rich PTDs occurred at 4°C and following depletion of cellular ATP pools, albeit generally at reduced levels. Although transduction was reduced in Chinese hamster ovary mutant lines deficient in either heparan sulfate or glycosaminoglycan synthesis, uptake was restored to wild-type levels by incubating target cells with dextran sulfate. The enhancement of transduction by dextran sulfate suggests that electrostatic interactions play an important first step in the process by which PTDs and their cargoes traverse the plasma membrane.

We have previously demonstrated that adenoviral gene transfer of the NF-B inhibitor I B to human islets results in protection from interleukin (IL)-1␤-mediated dysfunction and apoptosis. Here we report that human and mouse islets can be efficiently transduced by a cationic peptide transduction domain (PTD-5) without impairment of islet function. PTD mediated delivery of a peptide inhibitor of the IL-1␤-induced I B kinase (IKK), derived from IKK␤ (NBD; Nemo-binding domain), and completely blocked the detrimental effects of IL-1␤ on islet function and NF-B activity, in a similar manner to Ad-I B. We also demonstrate that mouse islets can be transduced in situ by infusion of the transduction peptide through the bile duct prior to isolation, resulting in 40% peptide transduction of the ␤-cells. Delivery of the IKK inhibitor transduction fusion peptide (PTD-5-NBD) in situ to mouse islets resulted in improved islet function and viability after isolation. These results demonstrate the feasibility of using PTD-mediated delivery to transiently modify islets in situ to improve their viability and function during isolation, prior to transplantation.

Bone morphogenetic proteins (BMPs) play important roles in regulating lung development and function although the endogenous regulatory effects of BMP signaling are still controversial. We found that BMP type I receptor Alk3 is expressed predominantly in airway epithelial cells during development. The function of Alk3 in lung development was determined using an inducible knockout mouse model by crossing epithelial cell-specific Cre transgenic mice SPC-rtTA/TetO-Cre and floxed-Alk3 mice. Abrogation of Alk3 in mouse lung epithelia from either early lung organogenesis or late gestation resulted in similar neonatal respiratory distress phenotypes accompanied by collapsed lungs. Early-induction of Alk3 knockout in lung epithelial cells caused retardation of early lung branching morphogenesis, reduced cell proliferation, and differentiation. However, late gestation induction of the knockout caused changes in cell proliferation and survival, as shown by altered cell biology, reduced expression of peripheral epithelial markers (Clara cell-specific protein, surfactant protein C, and aquaporin-5), and lack of surfactant secretion. Furthermore, canonical Wnt signaling was perturbed, possibly through reduced Wnt inhibitory factor-1 expression in Alk3-knockout lungs. Therefore, our data suggest that deficiency of appropriate BMP signaling in lung epithelial cells results in prenatal lung malformation, neonatal atelectasis, and respiratory failure.

Synovial hyperplasia, resulting in erosion of cartilage and bone, represents one of the major pathologies associated with rheumatoid arthritis. To develop an approach for efficient delivery of proteins or agents to synovium to induce targeted apoptosis of hyperplastic synovial tissue, we have screened an M13 peptide phage display library for synovial-specific transduction peptides. We identified a novel synovial-targeted transduction peptide, HAP-1, which is able to facilitate specific internalization of protein complexes into human and rabbit synovial cells in culture and rabbit synovial lining in vivo. HAP-1 and a non-tissue-specific cationic protein transduction domain, PTD-5, were fused to an antimicrobial peptide, (KLAK)(2), to generate two proapoptotic peptides termed DP2 and DP1, respectively. Administration of these peptides was able to induce apoptosis of rabbit and human synovial cells in culture, with DP2 inducing synovial cell-specific apoptosis. Intra-articular injection of DP1 and DP2 into arthritic rabbit joints with synovial hyperplasia induced extensive apoptosis of the hyperplastic synovium, while reducing the leukocytic infiltration and synovitis. These results suggest that proapoptotic peptides and, in particular, DP2 can be clinically useful for treatment of synovial hyperplasia, as well as inflammation. Moreover, the results demonstrate the feasibility of identifying tissue-specific transduction peptides capable of mediating efficient transduction in vivo.

In the first 4-5 days after anthesis, the central cell of the wheat embryo sac undergoes transformation from a multinucleate syncytium to the cellular endosperm. This is accomplished initially by the centripetal growth of wall projections from the central cell wall and the formation of cylindrical, highly vacuolate alveoli. Growth is mediated through the production and planar aggregation of vesicles at the distal tip of the developing projections. The innermost ends of the alveoli are closed by a thin layer of cytoplasm which is bounded on the inner side by the vacuolar membrane of the central cell. Cell wall material is not found in this thin layer of cytoplasm and the alveoli therefore are not complete cells. Following the division of the alveolar nucleus a cross-wall is laid down between the daughter nuclei by a process which is similar to normal cytokinesis and a layer of endosperm cells is formed from the peripheral portions of the alveoli. This pattern of centripetal growth of alveoli and the formation of complete cells from the proximal portions continues until cellularization is completed by the confluence of alveoli originating from opposite sides of the central cell. Further growth of the cellular endosperm is accomplished by the meristematic activity of the peripheral layer of cells. The ultrastructure of the early stages of partitioning of the central cell is discussed in relation to current views on the ontogeny of wheat endosperm.