Albumin-like proteins are critical regulators of vascular redox signaling.

This laboratory previously identified an albumin-like protein (denoted as p70) as a component of the macromolecular complex assembled within the 5'-regulatory region of redox-sensitive genes in vascular smooth muscle cells (vSMCs). Here we show that p70 is present in the cytosolic and nuclear compartments of vSMCs and dynamically responsive to redox status. Intense cytoplasmic and perinuclear staining, coupled with enhanced nuclear localization, was observed in vSMCs, but not HepG2 cells, treated with benzo(a)pyrene (BaP), H(2)O(2), or N-acetylcysteine, agents known to modulate redox status. 3' RACE indicated that p70 is not generated as a product of endogenous gene expression, but rather taken up from the extracellular compartment. While p70 was undetectable in cells grown for 24 hours under serum-free conditions, cell-associated, acid-resistant albumin was detected 30 min after the addition of exogenous albumin. vSMCs incubated at 4°C with 100 µ g/mL unlabeled BSA and 10 µ g/mL FITC-BSA for 60 minutes and switched to 37°C to examine temperature-sensitive label uptake showed punctate structures throughout the cell consistent with albumin internalization at the higher temperature. Albumin was found to influence redox-signaling, as evidenced by modulation of cyp1a1 gsta1 and Ha-ras gene inducibility. Together, these results implicate albumin and albumin-like proteins as critical regulators of vascular redox signaling.

Molecular imaging reveals a progressive pulmonary inflammation in lower airways in ferrets infected with 2009 H1N1 pandemic influenza virus.

Molecular imaging has gained attention as a possible approach for the study of the progression of inflammation and disease dynamics. Herein we used [(18)F]-2-deoxy-2-fluoro-D-glucose ([(18)F]-FDG) as a radiotracer for PET imaging coupled with CT (FDG-PET/CT) to gain insight into the spatiotemporal progression of the inflammatory response of ferrets infected with a clinical isolate of a pandemic influenza virus, H1N1 (H1N1pdm). The thoracic regions of mock- and H1N1pdm-infected ferrets were imaged prior to infection and at 1, 2, 3 and 6 days post-infection (DPI). On 1 DPI, FDG-PET/CT imaging revealed areas of consolidation in the right caudal lobe which corresponded with elevated [(18)F]-FDG uptake (maximum standardized uptake values (SUVMax), 4.7-7.0). By days 2 and 3, consolidation (CT) and inflammation ([(18)F]-FDG) appeared in the left caudal lobe. By 6 DPI, CT images showed extensive areas of patchy ground-glass opacities (GGO) and consolidations with the largest lesions having high SUVMax (6.0-7.6). Viral shedding and replication were detected in most nasal, throat and rectal swabs and nasal turbinates and lungs on 1, 2 and 3 DPI, but not on day 7, respectively. In conclusion, molecular imaging of infected ferrets revealed a progressive consolidation on CT with corresponding [(18)F]-FDG uptake. Strong positive correlations were measured between SUVMax and bronchiolitis-related pathologic scoring (Spearman's ρ = 0.75). Importantly, the extensive areas of patchy GGO and consolidation seen on CT in the ferret model at 6 DPI are similar to that reported for human H1N1pdm infections. In summary, these first molecular imaging studies of lower respiratory infection with H1N1pdm show that FDG-PET can give insight into the spatiotemporal progression of the inflammation in real-time.

Osteopontin regulates α-smooth muscle actin and calponin in vascular smooth muscle cells.

vSMCs (vascular smooth muscle cells) lose differentiation markers and gain uncontrolled proliferative activity during the early stages of atherosclerosis. Previous studies have shown that OPN (osteopontin) mRNA and protein levels increase significantly on induction of proliferative activity by allylamine (an atherogenic amine) and that this response can be inhibited by OPN antibodies. We have investigated the role of OPN in vSMC differentiation. Primary cultures of aortic mouse vSMCs were transfected with an OPN expression plasmid and several vSMC differentiation markers including α-SM actin (α-smooth muscle actin), SM22-α, tropomyosin and calponin were monitored in this cellular model. α-SM actin and calponin protein levels were significantly decreased by OPN overexpression. Down-regulation of α-SM actin and calponin was also observed on extracellular treatment of mouse vSMCs with recombinant OPN. In addition, calponin mRNA was significantly decreased under serum-restricted conditions when OPN mRNA was dramatically increased, while α-SM actin mRNA remained unchanged. These data indicate that OPN down-regulates α-SM actin and calponin expression through an extracellular signalling pathway. Functional connectivity between OPN and vSMC differentiation markers has been established. Since vSMCs lose differentiation features during early atherosclerosis, a mechanistic basis for OPN functions as a critical regulator of proliferative cardiovascular disease has been presented.

Epigenetic control of mammalian LINE-1 retrotransposon by retinoblastoma proteins.

Long interspersed nuclear elements (LINEs or L1 elements) are targeted for epigenetic silencing during early embryonic development and remain inactive in most cells and tissues. Here we show that E2F-Rb family complexes participate in L1 elements epigenetic regulation via nucleosomal histone modifications and recruitment of histone deacetylases (HDACs) HDAC1 and HDAC2. Our experiments demonstrated that (i) Rb and E2F interact with human and mouse L1 elements, (ii) L1 elements are deficient in both heterochromatin-associated histone marks H3 tri methyl K9 and H4 tri methyl K20 in Rb family triple knock out (Rb, p107, and p130) fibroblasts (TKO), (iii) L1 promoter exhibits increased histone H3 acetylation in the absence of HDAC1 and HDAC2 recruitment, (iv) L1 expression in TKO fibroblasts is upregulated compared to wild type counterparts, (v) L1 expression increases in the presence of the HDAC inhibitor TSA. On the basis of these findings we propose a model in which L1 sequences throughout the genome serve as centers for heterochromatin formation in an Rb family-dependent manner. As such, Rb proteins and L1 elements may play key roles in heterochromatin formation beyond pericentromeric chromosomal regions. These findings describe a novel mechanism of L1 reactivation in mammalian cells mediated by failure of corepressor protein recruitment by Rb, loss of histone epigenetic marks, heterochromatin formation, and increased histone H3 acetylation.

From genomics to mechanistic insight: a global perspective on molecular deficits induced by environmental agents.

As the postgenomic era continues to unfold, a new wave of scientific investigation is upon us focusing on the application of genomic technologies to study the meanings encrypted on the DNA code and the responses of living organisms to changes in their environment. Recent functional genomics studies in this laboratory have focused on the role of the aryl hydrocarbon receptor, a ubiquitous transcription factor, in genetic programming during renal development. Also of interest is the application of genomics investigations to the study of chronic medical conditions associated with early life exposures to environmental contaminants. Molecular evidence is discussed in this review within the framework of human molecular medicine.

Rpm2p, a component of yeast mitochondrial RNase P, acts as a transcriptional activator in the nucleus.

Rpm2p, a protein subunit of yeast mitochondrial RNase P, has another function that is essential in cells lacking the wild-type mitochondrial genome. This function does not require the mitochondrial leader sequence and appears to affect transcription of nuclear genes. Rpm2p expressed as a fusion protein with green fluorescent protein localizes to the nucleus and activates transcription from promoters containing lexA-binding sites when fused to a heterologous DNA binding domain, lexA. The transcriptional activation region of Rpm2p contains two leucine zippers that are required for transcriptional activation and are conserved in the distantly related yeast Candida glabrata. The presence of a mitochondrial leader sequence does not prevent a portion of Rpm2p from locating to the nucleus, and several observations suggest that the nuclear location and transcriptional activation ability of Rpm2p are physiologically significant. The ability of RPM2 alleles to suppress tom40-3, a temperature-sensitive mutant of a component of the mitochondrial import apparatus, correlates with their ability to transactivate the reporter genes with lexA-binding sites. In cells lacking mitochondrial DNA, Rpm2p influences the levels of TOM40, TOM6, TOM20, TOM22, and TOM37 mRNAs, which encode components of the mitochondrial import apparatus, but not that of TOM70 mRNA. It also affects HSP60 and HSP10 mRNAs that encode essential mitochondrial chaperones. Rpm2p also increases the level of Tom40p, as well as Hsp60p, but not Atp2p, suggesting that some, but not all, nucleus-encoded mitochondrial components are affected.