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At least 217 records · Page 12Linked to original sources
PubMed2002

The U1 snRNP protein U1C recognizes the 5' splice site in the absence of base pairing.

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals, which engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 snRNP proteins also contribute to U1 snRNP activity. Here we show that U1 snRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. We also show that recombinant yeast U1C protein, a U1 snRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. We propose that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway.

Animals↗
PubMed2001

Selective enhancement of gene transfer by steroid-mediated gene delivery.

The incorporation of transgenes into the host cells' nuclei is problematic using conventional nonviral gene delivery technologies. Here we describe a strategy called steroid-mediated gene delivery (SMGD), which uses steroid receptors as shuttles to facilitate the uptake of transfected DNA into the nucleus. We use glucocorticoid receptors (GRs) as a model system with which to test the principle of SMGD. To this end, we synthesized and tested several bifunctional steroid derivatives, finally focusing on a compound named DR9NP, consisting of a dexamethasone backbone linked to a psoralen moiety using a nine-atom chemical spacer. DR9NP binds to the GR in either its free or DNA-crosslinked form, inducing the translocation of the GR to the nucleus. The expression of transfected DR9NP-decorated reporter plasmids is enhanced in dividing cells: expression of steroid-decorated reporter plasmids depends on the presence of the GR, is independent of the transactivation potential of the GR, and correlates with enhanced nuclear accumulation of the transgene in GR-positive cells. The SMGD effect is also observed in cells naturally expressing GRs and is significantly increased in nondividing cell cultures. We propose that SMGD could be used as a platform for selective targeting of transgenes in nonviral somatic gene transfer.

Active Transport, Cell Nucleus↗
PubMed2005

Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity.

We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

Animals↗
PubMed2003

Epigenetic silencing of RNA polymerase I transcription.

The genes that encode ribosomal RNA exist in two distinct types of chromatin--an 'open' conformation that is permissive to transcription and a 'closed' conformation that is transcriptionally refractive. Recent studies have provided insights into the molecular mechanisms that silence either entire nucleolus organizer regions (NORs) in genetic hybrids or individual rRNA genes within a NOR. An emerging theme from these studies is that epigenetic mechanisms operating at the level of DNA methylation and histone modifications alter the chromatin structure and control the ratio of active and inactive rRNA genes.

Animals↗
PubMed2006

The role of BRCA2 in replication-coupled DNA interstrand cross-link repair in vitro.

Using a defined substrate DNA with a single psoralen interstrand cross-link (ICL), we studied the molecular mechanism of human ICL repair. In vitro ICL repair by human extracts is dependent on replication and is a largely error-free process. Extracts from a human BRCA2-defective mutant cell line, CAPAN-1, are severely compromised in ICL repair. Specifically, 'unhooked' but not fully repaired products accumulate in the reaction with CAPAN-1, and transient expression of BRCA2 in CAPAN-1 restores repair activity. Together, these results reveal that BRCA2 participates in repair of replication-mediated double-strand breaks generated when replication forks encounter ICLs. We also show that nucleotide excision repair is essential for the removal of the lesion left behind on one strand after unhooking. This study provides new mechanistic insights into the repair of ICLs in human cells.

BRCA2 Protein↗
PubMed2003

Treating refractory chronic graft-versus-host disease with extracorporeal photochemotherapy.

Extracorporeal photochemotherapy (ECP; photopheresis), an immunomodulatory therapy, has previously demonstrated promising results in treating chronic graft-versus-host disease (cGvHD). We treated six patients (ages 33-54 years) with long-standing refractory extensive-stage cGvHD. ECP was performed thrice weekly initially in all patients. Concomitant therapies included prednisone (n=6), tacrolimus (n=5), cyclosporin A (n=2), hydroxychloroquine (n=2), mycophenolate mofetil (n=1), and psoralen plus ultraviolet A radiation (n=1). After an average of 7.2 months (range, 2-13 months) of ECP, all patients experienced either improvement or stabilization in sclerodermatous skin changes, as well as partial improvements in liver enzyme levels. Skin softening occurred in four patients and was noted as early as 3-8 weeks into treatment. Two patients were able to taper steroid therapy, and two patients were able to taper ECP to twice weekly. ECP was well tolerated. Our results support those of previous studies, suggesting that ECP may be beneficial in patients with refractory cGvHD.

Adult↗
PubMed2000

Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant.

Tumor-directed gene therapy, such as "suicide gene" therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (10(6) MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 microg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.

Animals↗
PubMed2005

Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells.

DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. ICLs are induced in DNA by intercalating compounds such as the widely used therapeutic agent psoralen. In bacteria, both nucleotide excision repair (NER) and homologous recombination are required for the repair of ICLs. The processing of ICLs in mammalian cells is not clearly understood. However, it is known that processing can occur by NER, which for psoralen ICLs can be an error-generating process conducive to mutagenesis. We show here that another repair pathway, mismatch repair (MMR), is also involved in eliminating psoralen ICLs in human cells. MMR deficiency renders cells hypersensitive to psoralen ICLs without diminishing their mutagenic potential, suggesting that MMR does not contribute to error-generating repair, and that MMR may represent a relatively error-free mechanism for processing these lesions in human cells. Thus, enhancement of MMR relative to NER may reduce the mutagenesis caused by DNA ICLs in humans.

Base Pair Mismatch↗
PubMed2003

Induction of a caffeine-sensitive S-phase cell cycle checkpoint by psoralen plus ultraviolet A radiation.

Induction of interstrand crosslinks (ICLs) in chromosomal DNA is considered a major reason for the antiproliferative effect of psoralen plus ultraviolet A (PUVA). It is unclear as to whether PUVA-induced cell cycle arrest is caused by ICLs mechanically stalling replication forks or by triggering cell cycle checkpoints. Cell cycle checkpoints serve to maintain genomic stability by halting cell cycle progression to prevent replication of damaged DNA templates or segregation of broken chromosomes. Here, we show that HaCaT keratinocytes treated with PUVA arrest with S-phase DNA content. Cells that had completed DNA replication were not perturbed by PUVA and passed through mitosis. Cells treated with PUVA during G1-phase continued traversing G1 until arresting in early S-phase. PUVA induced rapid phosphorylation of the Chk1 checkpoint kinase at Ser345 and a concomitant decrease in Cdc25A levels. Chk1 phosphorylation, decrease of Cdc25 A levels and S-phase arrest were abolished by caffeine, demonstrating that active checkpoint signaling rather than passive mechanical blockage by ICLs causes the PUVA-induced replication arrest. Overexpression of Cdc25A only partially overrode the S-phase arrest, suggesting that additional signaling events implement PUVA-induced S-phase arrest.

Animals↗
PubMed2006

Vibronic absorption, fluorescence, and phosphorescence spectra of psoralen: a quantum chemical investigation.

Excited state potential energy hypersurfaces of 7H-furo[3,2-g][1]benzopyran-7-one (psoralen) have been explored employing (time-dependent) Kohn-Sham density functional theory. At selected points, we have determined electronic excitation energies and electric dipole (transition) moments utilizing a combined density functional/multireference configuration interaction method. Spin-orbit coupling has been taken into account employing an efficient, non-empirical spin-orbit mean-field Hamiltonian. Franck-Condon factors have been computed for vibrational modes with large displacements in the respective Dushinsky transformations. The simulated band spectra closely resemble experimental band shapes and thus validate the theoretically determined nuclear structures at the S(0), S(1), and T(1) minima. In the S(1) (pi(HOMO)-->pi*(LUMO)) state, the lactone bond of the pyrone ring is significantly elongated. From excited vibrational levels of the S(1) state a conical intersection between a (pi-->sigma*) excited state and the electronic ground state may be energetically accessible. Fast non-radiative decay via this relaxation pathway could explain the low fluorescence quantum yield of psoralen. The T(1) (pi(HOMO-1)-->pi*(LUMO)) exhibits a diradicaloid electronic structure with a broken C(5)-C(6) double bond in the pyrone ring. A variational multireference spin-orbit configuration interaction procedure yields a phosphorescence lifetime of 3 s, in excellent agreement with experimental estimates.

Absorption↗