Arginine-rich peptide conjugation to morpholino oligomers: effects on antisense activity and specificity

Noncharged antisense compounds, such as phosphorodiamidate morpholino oligomers (PMOs), do not readily enter mammalian cells in culture. A simple and effective means for cellular delivery of PMOs is through their conjugation to arginine-rich peptides. Understanding the effect of peptide conjugation on the efficacy, toxicity, and specificity of PMOs is important to the successful application of this antisense delivery method. We investigated the effects of conjugation of arginine-rich peptides to PMO on the thermal stability, efficacy and specificity for targeted RNA of the resulting compound. In vitro translation assays showed that (1) R9F2-PMO generated antisense activity 3-25-fold higher than corresponding nonconjugated PMO, (2) the level of antisense activity enhancement by R9F2-PMO over a corresponding nonconjugated PMO is related to the GC content of the PMO sequence, (3) R9F2 conjugation reduced the minimum length of a PMO required to inactivate a target RNA from 20 bases to 14 bases, and (4) nonspecific effects of R9F2-PMO occur at lower concentrations than corresponding PMO alone. Thermal stability of heteroduplexes of PMO and complementary RNA were increased by conjugation of PMO to R9F2 peptide, likely accounting for the increased specific antisense activity of conjugated over nonconjugated PMO. A cell-culture based assay demonstrated that while conjugation to unnatural peptides increased PMO efficacy without causing nonspecificity at concentrations < or = 10 microM, only L-peptide conjugation retained high specificity at higher concentrations. This study demonstrates that conjugation of PMO to an arginine-rich peptide generally increases the binding affinity of the PMO to complementary RNA and increases its antisense potency. Additionally, it is shown that the enzymatic stability of an L- or unnatural peptide used for PMO conjugation affects the antisense properties of the resulting compound.     

Spatial analysis of within-population microsatellite variability reveals restricted gene flow in the Pacific golden chanterelle (Cantharellus formosus)

We examined the within-population genetic structure of the Pacific golden chanterelle (Cantharellus formosus) in a 50 y old forest stand dominated by Douglas-fir (Pseudotsuga menziesii) and western hemlock (Tsuga heterophylla) with spatial autocorrelation analysis. We tested the null hypothesis that multilocus genotypes possessed by chanterelle genets were randomly distributed within the study area. Fruit bodies from 203 C. formosus genets were collected from a 50 ha study plot. One hundred six unique multilocus genotypes were identified after scoring these collections at five microsatellite loci. Statistically significant positive spatial autocorrelation was detected indicating the presence of fine-scale genetic structure within the area. Repeated autocorrelation analyses with varied minimum distance classes (50-500 m) detected positive spatial genetic structure up to 400 m. Therefore nonrandom evolutionary processes (e.g., isolation by distance) can cause fine-scale genetic structure in C. formosus. The implications of this research for future broad-scale population studies of this species are that population samples should be separated by at least 400 m to be considered statistically independent. Sampling designs that account for fine-scale genetic structure will better characterize heterogeneity distributed across the landscape by avoiding the effects of pseudo replication.     

Targeting the retinoblastoma protein by MC007L, gene product of the molluscum contagiosum virus: detection of a novel virus-cell interaction by a member of the poxviruses

The human pathogenic poxvirus molluscum contagiosum virus (MCV) is the causative agent of benign neoplasm, with worldwide incidence, characterized by intraepidermal hyperplasia and hypertrophy of cells. Here, we present evidence that the MC007L protein of MCV targets retinoblastoma protein (pRb) via a conserved LxCxE motif, which is present in many viral oncoproteins. The deregulation of the pRb pathway plays a central role in tumor pathogenesis. The oncoproteins of small DNA viruses contain amino acid sequences that bind to and inactivate pRb. Isolated expression of these oncoproteins induces apoptosis, cell proliferation, and cellular transformation. The MC007L gene displays no homology to other genes within the poxvirus family. The protein anchors into the outer mitochondrial membrane via an N-terminal mitochondrial targeting sequence. Through the LxCxE motifs, MC007L induces a cytosolic sequestration of pRb at mitochondrial membranes, leading to the inactivation of the protein by mislocalization. MC007L precipitates the endogenous pRb/E2F-1 complex. Moreover, MC007L is able to cooperate to transform primary rat kidney cells. The interaction between MC007L and pRb provides a novel mechanism by which a virus can perturb the cell cycle.     

A human RNA polymerase II subunit is encoded by a recently generated multigene family

Background  

Some origins in eukaryotic chromosomes fire more frequently than others. In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion. The important sequence motifs within this enhancer were not previously localized.     

Kindlin-2 is required for myocyte elongation and is essential for myogenesis

Background

Retrodifferentiation and regained proliferative capacity of growth-arrested human leukemic cells after monocyte-like differentiation requires proteolytic activities together with distinct regulatory factors. The AAA ATPase valosin-containing protein (VCP/p97) contributes to protein degradation and cell cycle regulation, respectively, and it was of interest to study a possible role of VCP/p97 during this myelomonocytic differentiation and retrodifferentiation.

Results

Separation of autonomously proliferating human U937 myeloid leukemia cells by centrifugal elutriation demonstrated unaltered VCP/p97 expression levels throughout distinct phases of the cell cycle. However, phorbol ester-induced G₀/G₁ cell cycle arrest in differentiating human U937 leukemia cells was associated with a significantly increased protein and mRNA amount of this AAA ATPase. These elevated VCP/p97 levels progressively decreased again when growth-arrested U937 cells entered a retrodifferentiation program and returned to the tumorigenic phenotype. Whereas VCP/p97 was observed predominantly in the cytosol of U937 tumor and retrodifferentiated cells, a significant nuclear accumulation appeared during differentiation and G₀/G₁ growth arrest. Analysis of subcellular compartments by immunoprecipitations and 2D Western blots substantiated these findings and revealed furthermore a tyrosine-specific phosphorylation of VCP/p97 in the cytosolic but not in the nuclear fractions. These altered tyrosine phosphorylation levels, according to distinct subcellular distributions, indicated a possible functional involvement of VCP/p97 in the leukemic differentiation process. Indeed, a down-modulation of VCP/p97 protein by siRNA revealed a reduced expression of differentiation-associated genes in subsequent DNA microarray analysis. Moreover, DNA-binding and proliferation-associated genes, which are down-regulated during differentiation of the leukemic cells, demonstrated elevated levels in the VCP/p97 siRNA transfectants.

Conclusion

The findings demonstrated that monocytic differentiation and G₀/G₁ growth arrest in human U937 leukemia cells was accompanied by an increase in VCP/p97 expression and a distinct subcellular distribution to be reverted during retrodifferentiation. Together with a down-modulation of VCP/p97 by siRNA, these results suggested an association of this AAA ATPase in the differentiation/retrodifferentiation program.     

Subretinal delivery of adeno‐associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice

Background

Adeno‐associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long‐term therapy is the development of immune responses against the vector or transgene product.

Methods

We evaluated cellular and humoural responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression.

Results

Following subretinal administration of vector, splenocytes and T‐cells from draining lymph nodes showed minimal activation following stimulation by co‐culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low‐dose AAV.hRPE65.hRPE65 to both eyes of RPE65^?/? mice resulted in transgene expression and functional rescue, but re‐administration of high‐dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye.

Conclusions

These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose‐dependent. Low‐dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration. Copyright © 2009 John Wiley & Sons, Ltd.