Modification of adenovirus capsid with a designed protein ligand yields a gene vector targeted to a major molecular marker of cancer

The future of genetic interventions in humans critically depends on the selectivity and efficiency of gene transfer to target tissues. The viral gene vectors explored to date cannot selectively transduce the desired targets. While substantial progress has been made in developing targeting strategies for adenovirus (Ad) vectors, future advances in this direction are severely limited by the shortage of naturally existing molecules available for use as targeting ligands. This shortage is due to fundamental and irresolvable differences at the level of both posttranslational modifications and intracellular trafficking between the Ad structural proteins and those natural proteins that are involved in interactions with the cell surface and could otherwise be considered as potential targeting ligands. We hypothesized that this problem could be resolved by altering the natural tropism of Ad vector through incorporation into its capsid of a rationally designed protein ligand, an affibody, whose structural, functional, and biosynthetic properties make it compatible with the Ad assembly process. We tested this hypothesis by redesigning the receptor-binding Ad protein, the fiber, using affibodies specific for human epidermal growth factor receptor type 2 (Her2), a major molecular marker of human tumors. The biosynthesis and folding of these fiber chimeras were fully compatible with Ad virion formation, and the resultant viral vectors were capable of selective delivery of a dual-function transgene to Her2-expressing cancer cells. By establishing the feasibility of this affibody-based approach to Ad vector targeting, the present study lays the foundation for further development of Ad vector technology toward its clinical use.     

Molecular and genetic analysis of condensin function in vertebrate cells

We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.     

Giardia lamblia EB1 is a functional homolog of yeast Bim1p that binds to microtubules

Giardia lamblia, with two nuclei and a distinct polarized morphology, is an interesting organism for investigating how distribution of its microtubule (MT) is controlled during its cell cycle. In this study, we identified the end-binding protein 1 (EB1) of G. lamblia, a well-known microtubule-associated protein that organizes MTs in eukaryotes. Immunofluorescence assays using recombinant EB1 (rEB1)-specific antibodies demonstrated EB1 localization in nuclear membrane as well as in some cytoskeletal structures such as axomenes and median bodies of trophozoites of G. lamblia. Complementation experiments using the BIM1 knock-out mutant of yeast, the yeast homolog of mammalian EB1, showed that giardial EB1 was able to carry out a homologous function in controlling MT dynamics. In addition, rEB1 of G. lamblia co-precipitated with MTs by an in vitro binding assay, thereby demonstrating that G. lamblia EB1 is a MT-associated protein. These results, taken together, suggest that G. lamblia EB1 is a functional homolog of eukaryotic EB1 and is likely to be a determinant for MT distribution.     

Phosphorylation of Serine 148 in Giardia lamblia End‐binding 1 Protein is Important for Cell Division

Giardia lamblia is a unicellular organism, showing a polarity with two nuclei and cytoskeletal structures. Accurate positioning of these organelles is essential for division of G. lamblia, which is poorly understood. Giardia lamblia end‐binding 1 (GlEB1) protein and G. lamblia aurora kinase (GlAK) have been shown to modulate microtubule (MT) distribution during cytokinesis. A direct association between GlEB1 and GlAK was demonstrated. Like GlEB1, GlAK was also found at nuclear envelopes and median bodies of G. lamblia. In vitro kinase assays using Giardia lysates immunoprecipitated with anti‐GlAK antibodies or recombinant GlAK suggested that GlEB1 is a substrate of GlAK. Site‐directed mutagenesis indicated that threonine‐205 in GlAK was auto‐phosphorylated and that GlAK phosphorylated serine (Ser)‐148 in GlEB1. Ectopic expression of a mutant GlEB1 (with conversion of Ser‐148 into alanine of GlEB1) resulted in an increased number of Giardia cells with division defects. Treatment of G. lamblia with an AK inhibitor triggered cytokinesis defects, and ectopic expression of a phospho‐mimetic mutant GlEB1 (with conversion of Ser‐148 into aspartate) rescued the defects in Giardia cell division caused by the AK inhibitor. These results suggested that phosphorylation of GlEB1 played a role in cytokinesis in G. lamblia.     

A novel WD repeat protein component of the methylosome binds Sm proteins.

We have recently described a large (20 S) protein arginine methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 (PRMT5) and the pICln protein. The methylosome functions to modify specific arginines to dimethylarginines in the arginine- and glycine-rich domains of several spliceosomal Sm proteins, and this modification targets these proteins to the survival of motor neurons (SMN) complex for assembly into small nuclear ribonucleoprotein (snRNP) core particles. Here, we describe a novel component of the methylosome, a 50-kilodalton WD repeat protein termed methylosome protein 50 (MEP50). We show that MEP50 is important for methylosome activity and binds to JBP1 and to a subset of Sm proteins. Because WD repeat proteins provide a platform for multiple protein interactions, MEP50 may function to mediate the interaction of multiple substrates with the methylosome. Interestingly, all of the known components of the methylosome bind Sm proteins, suggesting that in addition to producing properly methylated substrates for the SMN complex, the methylosome may be involved in Sm protein rearrangements or pre-assembly required for snRNP biogenesis.     

The ability of the epithelium of diencephalic origin to differentiate into cells of the ocular lens.

After the discovery that in adult salamanders following lentectomy a new, functional lens develops by transdifferentiation (cell-type conversion) of previously depigmented epithelial cells of the iris (Wolffian lens regeneration), this phenomenon has been intensively studied by various experimental approaches. During the last two decades it was shown that pleiomorphic aggregates of atypical lens cells (lentoids) differentiated in reaggregates of dissociated cells of the chick neural retina and in spread cell cultures of the pigmented epithelium of the iris and retina, of the neural retina and the pineal gland of the chick embryo. The neural retina of human fetuses and adults also displayed this capacity. We showed that lentoids developed at a low incidence in renal isografts of rat embryonic shields or isolated embryonic ectoderm and of lentectomized eyes of rat fetuses, as well as in organ cultures of rat embryonic shields in chemically defined media. The addition of transferrin significantly increased the incidence of differentiation of lentoids in explants. In both renal isografts and explants in vitro a continuous transformation of retinal epithelial cells into atypical lens cells was observed. In renal isografts lentoids were also observed to originate from the ependyma of the brain ventricle. All tissues having the capacity to convert into lens cells belong to the diencephalon in a broad sense. Evolutionary aspects of this feature are discussed.     

Ear cartilage grafts to the nose.

We present our experience in the treatment of secondary nasal deformities caused by excessive resection of the nasal dorsum. We have used ear cartilage autografts, with good results and (on the whole) little or no resorption (some have been followed for 10 years). We consider it essential to correctly shape the transplant and to hold it firmly in the proper position for a period of not less than one week.     

Unrip is a component of SMN complexes active in snRNP assembly

A macromolecular complex containing survival of motor neurons (SMN), the spinal muscular atrophy protein, and Gemin2-7 interacts with Sm proteins and snRNAs to carry out the assembly of these components into spliceosomal small nuclear ribonucleoproteins (snRNPs). Here we report the characterization of unr-interacting protein (unrip), a GH-WD protein of unknown function, as a component of the SMN complex that interacts directly with Gemin6 and Gemin7. Unrip also binds a subset of Sm proteins, and unrip-containing SMN complexes are necessary and sufficient to mediate the assembly of spliceosomal snRNPs. These results demonstrate that unrip functions in the pathway of snRNP biogenesis and is a marker of cellular SMN complexes active in snRNP assembly.