Geroscience-based therapeutics have the opportunity to transform the field of geriatric medicine, yet few training programs afford scholars with the necessary skills, knowledge, and experiences ...needed to successfully design and implement geroscience trials. We have developed a 2 year curriculum with two different training tracks for aging science scholars. The training tracks capitalize on the strengths and skillsets of eligible candidates. Both pathways afford scholars the opportunity to learn the fundamentals of aging research and the opportunity to apply this knowledge via a mentored translational research project. The two training pathways capitalize on existing clinical and research training infrastructures and include required and elective coursework, longitudinal clinical experiences, small group discussions, laboratory experience, and mentored translational research. This first of its kind geroscience training program is a potential feasible, scalable solution to the existing training gap. We believe that the Kogod Scholars Program at the Mayo Clinic can serve as a prototype for other academic aging centers.
To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were ...incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochondria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.
INTRODUCTIONThe severe pain that commonly accompanies appendicular flare-ups of fibrodysplasia ossificans progressiva (FOP) is often ascribed to compartment syndrome, but no documentation exists. ...CASE REPORTWe revisited the case of an adult with classic FOP who underwent measurement of compartment pressure of the thigh during an acute, severely painful flare-up of the thigh. The intracompartmental pressure of the thigh was measured at 95--110 mm of mercury (normal compartment pressure is 0--8 mmHg). A fasciotomy of the thigh was performed. Despite immediate post-operative relief of pain, progressive heterotopic ossification and loss of function of the hip and knee occurred. CONCLUSIONThis unique case documents and confirms the suspected presence of compartment syndrome during an acute flare-up of FOP and has vital implications for understanding the pathophysiology and care of patients with acute appendicular flare-ups of FOP and for the design of emerging clinical trials.
We have examined differential protein expression in serum-stimulated young and senescent WI-38 human fetal lung-derived cells in culture using high-resolution two-dimensional gel electrophoresis. ...Overexpression of a protein with an approximate M(r) of 29,000 and pI of 5.8 was observed in senescent cells during the G0 and throughout the G1 stage of the cell cycle. Automated amino-terminal sequencing of the peptide from polyvinylidene difluoride electroblots showed 100% sequence identity to cathepsin B or pre-procathepsin B in a 12-amino acid overlap, beginning at residue 48 or 129, respectively. The 29-kDa peptide corresponds to the heavy chain of the two-chain enzyme form. Cathepsin B activity was found to be decreased in cells aged in vitro in comparison to that in young controls. Changes in the steady-state levels of both the 4.0- and the 2.2-kb cathepsin B transcripts between young and senescent cells cannot account for the overexpression of the two-chain form of the enzyme. These results suggest that increased proteolysis of a conformationally more labile single-chain form and/or decreased turnover and accumulation of a less active form of this lysosomal protease occur in senescent fibroblasts and may account for the observed decreased cathepsin B activity in senescent cells in culture.
Molecular Changes with in Vitro Cellular Senescence CRISTOFALO, VINCENT J.; PIGNOLO, ROBERT J.; ROTENBERG, MITCH O.
Annals of the New York Academy of Sciences,
November 1992, Letnik:
663, Številka:
1
Journal Article
We have compared the in vitro replicative life span and characteristics of immortalization of skin fibroblast cultures derived from ad libitum-fed and caloric-restricted Fischer 344 rats of 6, 24, ...and 29 months of age. Cells from all 6-, 24-, and 29-month-old animals showed a gradual decline in proliferative potential as evidenced by decreases in harvest density, in the fraction of cells initiating DNA synthesis, and in the number of population doublings per passage. These declines were accompanied by morphological changes including cell enlargement. The replicative life span prior to immortalization decreased significantly with donor age (P less than 0.0001), while caloric restriction had no effect on the cumulative population doubling level. Prior to immortalization mitotic cells from all cultures showed a normal rat karyotype. Postcrisis cultures tended to have more polyploid cells but there were no characteristic or specific chromosomal changes found in the cells with an immortalized phenotype. Interestingly, fibroblasts derived from caloric-restricted animals had a significantly slower growth rate through the tenth week after immortalization (P less than 0.005). When these cultures were seeded at one-quarter the normal seeding density, to favor the outgrowth of the fastest growing cells, a population with a more "transformed" phenotype emerged.
Normal human WI-38 fibroblast-like cells in culture undergo a process of senescence, one feature of which is a gradual decline in proliferative capacity. As these cells reach the end of their ...replicative life span they exhibit decreases in the fraction of cells able to synthesize DNA, in the number of doublings per passage (constant seeding density), and in the cell harvest and saturation densities. They also display increased average cell cycle times, largely at the expense of longer G1intervals. These alterations are accompanied by morphologic changes, including cell enlargement. Before the end of the replicative life span or phase-out, there is a highly reproducible (55/58 sublines) cell loss of approximately 50%; however, a stable population survives that can exist in a viable yet nonproliferative state for many months. This stable population maintains an extremely low saturation density, representing <5% of that achieved by early passage cultures. Further, we show that maximum harvest densities achieved by senescent cells are lower, irrespective of seeding densities, i.e. when placed at cell densities higher than those normally achieved by senescent cultures they display a net decline in cell number. This decline continues until the cell density approximates the density that would have been achieved had the cultures been seeded at standard density ($1 \times 10^4 cells/cm^2$). By measuring the accumulation of an mRNA species, EPC-1, that is expressed when early passage cultures reach a growth-arrested state via density-dependent contact inhibition, we also show that senescent cells are unable to produce this transcript at either their normal confluent density or at high cell density obtained by overseeding. The above results suggest that there are significant alterations in cell-to-cell contact sensitivity and arrest state of senescent cultures. These changes result in both a cytotoxic response to crowding and failure to express at least one molecular marker which is induced as young cells approach growth arrest by contact inhibition.
Nonhereditary heterotopic ossification (NHHO) usually arises in the setting of trauma, certain arthropathies, or following injury, often in the setting of common age-related conditions. In this ...article, we discuss the pathophysioiogy, diagnosis, and clinical findings in periarticular NHHO and the relevance to age-related pathology, as well as the prevention and treatment of NHHO. Except for the precipitating events or clinical conditions in which NHHO occurs, primary etiological mechanisms remain unknown. Many forms of NHHO develop in the context of injury and inflammation, suggesting that the formation of extraskeletal bone may share similar initiating events with ectopic ossification in fibrodysplasia ossificans progressiva.
