HYDRO is a program for the calculation of sedimentation and diffusion coefficients, rotational relaxation times, and intrinsic viscosities of rigid macromolecules of arbitrary shape that are represented by bead models. Actually, HYDRO contains various FORTRAN callable subroutines that can be linked to the user's own programs to account for variability of shape or flexibility. Some hints are given for the use of HYDRO in various situations.

Werner's syndrome (WS) is an inherited disease characterized by genomic instability and premature aging. The WS gene encodes a protein (WRN) with helicase and exonuclease activities. We have previously reported that WRN interacts with Ku70/80 and this interaction strongly stimulates WRN exonuclease activity. To gain further insight on the function of WRN and its relationship with the Ku heterodimer, we established a cell line expressing tagged WRN H , a WRN point mutant lacking helicase activity, and used affinity purification, immunoblot analysis and mass spectroscopy to identify WRNassociated proteins. To this end, we identified three proteins that are stably associated with WRN in nuclear extracts. Two of these proteins, Ku70 and Ku80, were identified by immunoblot analysis. The third polypeptide, which was identified by mass spectrometry analysis, is identical to poly(ADP-ribose) polymerase-1(PARP-1), a 113-kDa enzyme that functions as a sensor of DNA damage. Biochemical fractionation studies and immunoprecipitation assays and studies confirmed that endogenous WRN is associated with subpopulations of PARP-1 and Ku70/80 in the cell. Protein interaction assays with purified proteins further indicated that PARP-1 binds directly to WRN and assembles in a complex with WRN and Ku70/80. In the presence of DNA and NAD ؉ , PARP-1 poly(ADP-ribosyl)ates itself and Ku70/80 but not WRN, and gel-shift assays showed that poly-(ADP-ribosyl)ation of Ku70/80 decreases the DNA-binding affinity of this factor. Significantly, (ADP-ribosyl)ation of Ku70/80 reduces the ability of this factor to stimulate WRN exonuclease, suggesting that covalent modification of Ku70/80 by PARP-1 may play a role in the regulation of the exonucleolytic activity of WRN.Werner's syndrome (WS) 1 is a human genetic disease with many features of premature aging (1, 2). The first signs of this disorder appear soon after puberty, with the symptoms becoming fully evident in individuals between 20 and 30 years old. Individuals with WS display a high incidence of diseases associated with normal aging, including atherosclerosis, osteoporosis, type II diabetes mellitus, and cancer. Myocardial infarction and cancer are the most common causes of death among WS patients. The median age of death is ϳ47 years (1, 3). Cells isolated from WS patients show genomic instability and a shorter replicative life span (4). The genomic instability is characterized by an elevated rate of chromosomal translocations and extensive genomic deletions (5). These findings suggest that genomic instability underlies the development of the diseases associated with WS. Cultured cells from WS patients are also hypersensitive to some DNA damaging agents (4), suggestive of a defect in the repair of specific DNA lesions.Werner's syndrome is caused by mutations within a single gene, which is located on chromosome 8 (6). The cDNA encodes a protein (Werner's syndrome protein, WRN) with strong homology to a class of enzymes called RecQ helicases (7). In addition, the amino-terminal region of WR...

Natural lung aging is marked by molecular changes that occur during development, maturation, and late-life decline. At the cellular and whole organ level, degenerative changes that are a hallmark of natural aging (shorter telomeres, increased expression of cellular senescence markers, increased DNA damage, oxidative stress, and apoptosis, accompanied by diminished elasticity) reach pathological levels in aging humans in the form of chronic respiratory disease. Aging strongly correlates with the development and incidence of chronic respiratory diseases, including cancer and idiopathic pulmonary fibrosis, but is most strongly linked with development of chronic obstructive pulmonary disease. Lung failure due to aging can be traced to loss of lung stem cell regenerative capacity within the distinctive stem cell niches found within each compartment of the lung. Current knowledge about the identity and function of these stem cell compartments has been largely drawn from a variety of transgenic and spontaneously mutated mouse models that are characterized by rapid rates of aging or have been used to examine regeneration from injury in the context of natural or accelerated aging. While much work has focused on the failure of epithelial cell populations as a key component of the aging process, additional studies have shown that aging, as a global phenomenon in the lung, also impacts resident endothelial, mesenchymal, and immune cell populations. In this review, we examine aging as a process dependent on specific changes in molecular pathways within multiple lung cell populations.

SummaryMutations in the lamin A/C gene cause the rare genetic disorder Hutchinson-Gilford progeria syndrome (HGPS). The prevalent mutation results in the production of a mutant lamin A protein with an internal 50 amino acid deletion which causes a cellular aging phenotype characterized by growth defects, limited replicative lifespan, and nuclear membrane abnormalities. However, the relevance of these findings to normal human aging is unclear. In this study, we demonstrate that increased levels of wild-type lamin A in normal human cells result in decreased replicative lifespan and nuclear membrane abnormalities that lead to apoptotic cell death and senescence in a manner that is strongly reminiscent of the phenotype shown by HGPS cells. In contrast to the accelerated aging defects observed in HGPS cells, the progeroid phenotype resulting from increased expression of wild-type lamin A can be rescued by overexpression of ZMPSTE24, the metalloproteinase responsible for the removal of the farnesylated carboxyl terminal region of lamin A. Furthermore, farnesyltransferase inhibitors also serve to reverse the progeroid phenotype resulting from increased lamin A expression. Significantly, cells expressing elevated levels of lamin A display abnormal lamin A localization and similar alterations in the nuclear distribution of lamin A are also observed in cells from old-age individuals. These data demonstrate that the metabolism of wild-type lamin A is delicately poised and even in the absence of diseaselinked mutations small perturbations in this system are sufficient to cause prominent nuclear defects and result in a progeroid phenotype.

Type 2 alveolar epithelial cells (AEC2) are regarded as the progenitor population of the alveolus responsible for injury repair and homeostatic maintenance. Depletion of this population is hypothesized to underlie various lung pathologies. Current models of lung injury rely on either uncontrolled, nonspecific destruction of alveolar epithelia or on targeted, nontitratable levels of fixed AEC2 ablation. We hypothesized that discrete levels of AEC2 ablation would trigger stereotypical and informative patterns of repair. To this end, we created a transgenic mouse model in which the surfactant protein-C promoter drives expression of a mutant SR39TK herpes simplex virus-1 thymidine kinase specifically in AEC2. Because of the sensitivity of SR39TK, low doses of ganciclovir can be administered to these animals to induce dose-dependent AEC2 depletion ranging from mild (50%) to lethal (82%) levels. We demonstrate that specific levels of AEC2 depletion cause altered expression patterns of apoptosis and repair proteins in surviving AEC2 as well as distinct changes in distal lung morphology, pulmonary function, collagen deposition, and expression of remodeling proteins in whole lung that persist for up to 60 days. We believe SPCTK mice demonstrate the utility of cell-specific expression of the SR39TK transgene for exerting fine control of target cell depletion. Our data demonstrate, for the first time, that specific levels of type 2 alveolar epithelial cell depletion produce characteristic injury repair outcomes. Most importantly, use of these mice will contribute to a better understanding of the role of AEC2 in the initiation of, and response to, lung injury.

High levels of rRNA synthesis by RNA polymerase I are important for cell growth and proliferation. In vitro studies have indicated that the formation of a stable complex between the HMG box factor [Upstream binding factor (UBF)] and SL1 at the rRNA gene promoter is necessary to direct multiple rounds of Pol I transcription initiation. The recruitment of SL1 to the promoter occurs through protein interactions with UBF and is regulated by phosphorylation of UBF. Here we show that the protein kinase CK2 co-immunoprecipitates with the Pol I complex and is associated with the rRNA gene promoter. Inhibition of CK2 kinase activity reduces Pol I transcription in cultured cells and in vitro. Significantly, CK2 regulates the interaction between UBF and SL1 by counteracting the inhibitory effect of HMG boxes five and six through the phosphorylation of specific serines located at the C-terminus of UBF. Transcription reactions with immobilized templates indicate that phosphorylation of CK2 phosphoacceptor sites in the C-terminal domain of UBF is important for promoting multiple rounds of Pol I transcription. These data demonstrate that CK2 is recruited to the rRNA gene promoter and directly regulates Pol I transcription re-initiation by stabilizing the association between UBF and SL1.

Religion is an important determinant in Hispanic Americans (HA) becoming organ donors as HA often believe religion forbids donation. We investigated the effect of an educational program targeting HA organ donation in places of worship. A prospective observational study was conducted at four Catholic churches with a high percentage of HA. A 45 minute ‘culturally sensitive’ educational program, conducted in Spanish, was implemented. Organ donation awareness, knowledge, perception and beliefs, as well as the intent to become an organ donor, were measured before and after the intervention. Differences between before and after the intervention were analyzed. A total of 182 surveys were collected before and 159 surveys were collected after the educational program. A significant increase was observed in organ donation knowledge (54% vs. 70%, p<0.0001), perception (43% vs. 58%, p<0.0001) and beliefs (50% vs. 60%, p=0.0001). However, no significant difference was found in the willingness to discuss donation with family, intent-to-donate, or registering to donate after the intervention. This study demonstrates that a focused educational program in places of worship can significantly improve HA knowledge, perceptions, and beliefs regarding organ donation. Further work is needed to understand why intent-to-donate does not increase despite the increase in organ donation awareness.

A media campaign emphasizing culturally sensitive educational material can significantly influence organ donation awareness and beliefs in HA.