Small molecule modulation of splicing factor expression is associated with rescue from cellular senescence

Background

Altered expression of mRNA splicing factors occurs with ageing in vivo and is thought to be an ageing mechanism. The accumulation of senescent cells also occurs in vivo with advancing age and causes much degenerative age-related pathology. However, the relationship between these two processes is opaque. Accordingly we developed a novel panel of small molecules based on resveratrol, previously suggested to alter mRNA splicing, to determine whether altered splicing factor expression had potential to influence features of replicative senescence.

Results

Treatment with resveralogues was associated with altered splicing factor expression and rescue of multiple features of senescence. This rescue was independent of cell cycle traverse and also independent of SIRT1, SASP modulation or senolysis. Under growth permissive conditions, cells demonstrating restored splicing factor expression also demonstrated increased telomere length, re-entered cell cycle and resumed proliferation. These phenomena were also influenced by ERK antagonists and agonists.

Conclusions

This is the first demonstration that moderation of splicing factor levels is associated with reversal of cellular senescence in human primary fibroblasts. Small molecule modulators of such targets may therefore represent promising novel anti-degenerative therapies.

     

Serum Amyloid P Therapeutically Attenuates Murine Bleomycin-Induced Pulmonary Fibrosis via Its Effects on Macrophages

Macrophages promote tissue remodeling but few mechanisms exist to modulate their activity during tissue fibrosis. Serum amyloid P (SAP), a member of the pentraxin family of proteins, signals through Fcγ receptors which are known to affect macrophage activation. We determined that IPF/UIP patients have increased protein levels of several alternatively activated pro-fibrotic (M2) macrophage-associated proteins in the lung and monocytes from these patients show skewing towards an M2 macrophage phenotype. SAP therapeutically inhibits established bleomycin-induced pulmonary fibrosis, when administered systemically or locally to the lungs. The reduction in aberrant collagen deposition was associated with a reduction in M2 macrophages in the lung and increased IP10/CXCL10. These data highlight the role of macrophages in fibrotic lung disease, and demonstrate a therapeutic action of SAP on macrophages which may extend to many fibrotic indications caused by over-exuberant pro-fibrotic macrophage responses.     

Complete genome sequence of Thermanaerovibrio acidaminovorans type strain (Su883)

Thermanaerovibrio acidaminovorans (Guangsheng et al. 1997) Baena et al. 1999 is the type species of the genus Thermanaerovibrio and is of phylogenetic interest because of the very isolated location of the novel phylum Synergistetes. T. acidaminovorans Su883(T) is a Gram-negative, motile, non-spore-forming bacterium isolated from an anaerobic reactor of a sugar refinery in The Netherlands. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from a member of the phylum Synergistetes. The 1,848,474 bp long single replicon genome with its 1765 protein-coding and 60 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Nitratifractor salsuginis type strain (E9I37-1)

Nitratifractor salsuginis Nakagawa et al. 2005 is the type species of the genus Nitratifractor, a member of the family Nautiliaceae. The species is of interest because of its high capacity for nitrate reduction via conversion to N(2) through respiration, which is a key compound in plant nutrition. The strain is also of interest because it represents the first mesophilic and facultatively anaerobic member of the Epsilonproteobacteria reported to grow on molecular hydrogen. This is the first completed genome sequence of a member of the genus Nitratifractor and the second sequence from the family Nautiliaceae. The 2,101,285 bp long genome with its 2,121 protein-coding and 54 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Dura mater secretes soluble heparin-binding factors required for cranial suture morphogenesis

Summary Cranial sutures play a critical role in calvarial morphogenesis, serving as bone growth centers during skull enlargement. Defective suture morphogenesis, resulting in premature osseous obliteration of sutures and their failure to function appropriately, causes severe craniofacial anomalies. Previously published data demonstrated osseous obliteration of coronal suturesin vitro in the absence of dura mater and the rescue of sutures from osseous obliteration in rudiments cocultured with dura mater on the opposite sides of 0.45-μm polycarbonate filters. With thisin vitro culture system, experiments were designed to examine the nature of the soluble signal secreted by dura mater, required for maintaining intact sutures. The signal remained active in conditioned medium produced from dura mater, which was capable of rescuing coronal sutures from osseous obliteration in calvaria cultured without dura mater. When conditioned medium was segregated into heparin-binding and non-heparin-binding fractions, the signal capable of maintaining intact coronal sutures cosegregated with the heparin-binding component and remained functional in the absence of the non-heparin-binding component of conditioned medium. Evidence indicates that soluble, heparin-binding factors secreted by the dura mater act as osteoinhibitory signals at the suture site.     

The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol

Several protein toxins, including the A chain of ricin (RTA), enter mammalian cells by endocytosis and subsequently reach their cytosolic substrates by translocation across the endoplasmic reticulum (ER) membrane. To achieve this export, such toxins exploit the ER-associated protein degradation (ERAD) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates. Toxins that translocate from the ER have an unusually low lysine content. Since lysyl residues are potential ubiquitination sites, it has been proposed that this paucity of lysines reduces the chance of ubiquitination and subsequent ubiquitin-mediated proteasomal degradation [Hazes, B., and Read, R. J. (1997) Biochemistry 36, 11051-11054]. Here we provide experimental support for this hypothesis. The two lysyl residues within RTA were changed to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect, suggesting that the endogenous lysyl residues are not the usual sites for ubiquitin attachment. However, when four additional lysines were introduced into RTA in a way that did not compromise the activity, structure, or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasome inhibitor clasto-lactacystin beta-lactone increased the cytotoxicity of the lysine-rich RTA to a level approaching that of wild-type ricin. The introduction of four additional lysyl residues into a second ribosome-inactivating protein, abrin A chain, also dramatically decreased the cytotoxicity of the holotoxin compared to wild-type abrin. This effect could also be reversed by proteasomal inhibition. Our data support the hypothesis that the evolution of a low lysine content is a degradation-avoidance strategy for toxins that retrotranslocate from the ER.     

Embryonic surface antigens: A “quasi-endodermal” teratoma antigen

Hyperimmune antisera against a teratoma-derived endodermal carcinoma of the mouse were used in cytotoxicity tests and by absorption analysis to define a surface antigen. This antigen was found to be present on all tumor cells tested that were of endodermal origin as well as on embryonic liver, and was therefore designated “Endo.” Further testing, however, revealed that Endo was represented on several inappropriate cell types such as young embryos before the appearance of endoderm, sperm, and a small proportion of non-endodermal tumors. Endo must therefore be considered a “quasi-endodermal” antigen.     

Maintenance of highly contractile tissue-cultured avian skeletal myotubes in collagen gel

Summary Highly contractile skeletal myotubes differentiated in tissue culture are normally difficult to maintain on collagen-coated tissue culture dishes for extended periods because of their propensity to detach as a sheet of cells from their substratum. This detachment results in the release of mechanical tension in the growing cell “sheet” and, consequently, loss of cellular protein. We developed a simple method of culturing high density contractile primary avian myotubes embedded in a collagen gel matrix (collagel) attached to either a stainless steel mesh or nylon support structure. With this system the cells are maintained in a highly contractile state for extended periods in vitro under tension. Structural integrity of the myotubes can be maintained for up to 10 d in basal medium without serum or embryo extract. Total cellular protein and myosin heavy chain accumulation in the cells can be maintained for weeks at levels which are two to three times those found in timematched controls that are under little tension. Morphologically, the myotubes are well differentiated with structural characteristics of neonatal myofibers. This new collagel culture system should prove useful in the analysis of in vitro gene expression during myotube to myofiber differentiation and its regulation by various environmental factors such as medium growth factors, innervation, and mechanical activity. This work was supported by grant AM 36266 from the National Institutes of Health, Bethesda, MD, and grant NAG2-414 from the National Aeronautics and Space Administration, Washington, D.C. Parts of this work have appeared in abstract form, In Vitro 23:24a; 1987.     

Body Size Perceptions and Eating Attitudes in Elderly Men

The majority of studies on eating attitudes, dieting and body size perceptions have focused on young adults and women. This study examined these attitudes in 334 black and white men, ages 55 to 98 years, who were members of the Charleston Heart Study cohort. Associations of the eating attitude variable with race, education and weight status were examined. Eighty-two percent of the overweight white men studied had dieted to lose weight, whereas only 49% of slimmer white men had dieted. In contrast, overweight black men did not diet more than slimmer black men. Overall black men dieted less than white men (37% reported dieting). Black men who were high school graduates were 1.3 times more likely to have dieted than were less educated black men. Overweight white men were over twice as likely as slimmer white men to feel guilty after overeating. This difference was not found in overweight versus slimmer black men. Education was not associated with measured body mass index (BMI) or perceived or ideal body size. However, there were some racial differences in these variables. White men preferred to be slightly thinner than black men (ideal BMI 25.6 vs. 26.1 kg/m²), and the difference between actual and desired BMI was 0.6 kg/m² greater in white than in black men. These results indicate that effects of race and weight status on eating restraint and body size perceptions seen in younger subjects and in women are also present, at least to some degree, in elderly men.