Accumulation of mitochondrial DNA deletions in human retina during aging.

PURPOSE: The authors investigated the presence of mitochondrial DNA (mtDNA) mutations in aging human retina. METHODS: A quantitative polymerase chain reaction technique for studying the common mtDNA 4977-deletion (delta mtDNA4977) in retinal pigment epithelium (RPE) and neural retinal (NR) was developed. RESULTS: Although no deletion was detected in the fetus, every adult RPE and NR had this common deletion. The ratio of the deleted delta mtDNA4977 to the total mtDNA increased significantly in elderly persons 60 to 110 years of age and was greater in peripheral than in central RPE. CONCLUSIONS: These results suggest that at least one type of mutation accumulates in the mtDNA in the retina during aging, reflecting a general phenomenon of genomic instability that could influence its function.

FRAR course on laboratory approaches to aging. Cellular aging, in vitro and in vivo.

Human cells grown in culture exhibit exponential growth provided they are regularly provided with fresh medium. This exponential growth is limited, and although the cells eventually cease to divide, they remain viable for long periods of time. Such a culture is deemed to be "senescent", but it is not clear whether this reflects the growth pattern of cells in vivo. Considerable evidence has been obtained to correlate cell senescence in vitro with aging in vivo, but there is no convincing evidence that any organ or tissue ages as a result of senescence of its individual cells. Recently gained insights into the control of cell proliferation through studies of cell senescence, and the relevance of cell senescence as a strategy to prevent carcinogenesis are of particular interest.

Differential localization by in situ hybridization of specific crystallin transcripts during mouse lens development.

The embryonic development of the mammalian lens is well known at the biochemical and histological level. However few data are available at the molecular level concerning gene expression during the continuous differentiation of the lens. In the present study, we have investigated by in situ hybridization the changes in the distribution of mouse crystallin mRNA as a marker of differentiated lens cells, during development of the lens primordium, when tissue interactions are known to be essential. The transcripts of alpha and beta crystallins are first detected at the early elongation stage of primary fibres; gamma-crystallin-transcripts do not appear until the late elongation phase. All areas of the lenses exhibited crystallin mRNA until the beginning of secondary fiber formation at 18 days of development. Hybridization for alpha and beta crystallin is confined at that time to the equatorial part of the lens. The gamma crystallin transcripts are no longer found in the equatorial region after 1 post-natal day, but remain in the lens core, decreasing gradually. A possible mechanism is discussed.

Biomarkers of aging.

This paper summarises a conceptual discussion of some aspects of biomarkers of aging and the logical strategy for developing them. A biomarker of aging is a biological parameter of an organism that either alone or in some multivariate composite will, in the absence of disease, better predict functional capability at some late age than with chronological age. The criteria for putative biomarker measurement and assessment of putative biomarkers are discussed.

Variation in the relative abundance of gamma-crystallin gene transcripts during development and ageing.

The transcripts of gamma-crystallin mRNA were examined during adulthood. The mRNA transcripts were detected by Northern blot technique. Although the total RNA per lens measured remains constant during adulthood, the mRNA transcript size was observed to decline with ageing, specifically for the gamma-crystallin and not for alpha- and beta-crystallins. We could show that the relative amount of mRNA transcripts detected with the four probes decreased with ageing, with gamma 1 and gamma 2 transcripts being found at a higher level than the gamma 3 and gamma 4 transcripts.

Gamma-crystallin family of the mouse lens: structural and evolutionary relationships.

The heterogeneity inherent among gamma-crystallins of the mouse lens was investigated by sequence analysis of three gamma-crystallin-specific cDNAs. Comparison of the nucleotide sequence of these cDNAs and one previously reported by us revealed that the four gamma-cDNAs share 80-90% homology in nucleotide sequence. The entire 3' half of the coding region shows more variability than the 5' half, whereas the greatest variability is observed in the 3' untranslated region where numerous base substitutions, deletions, and insertions seem to have occurred. Alignment of the amino acid sequences of the four mouse gamma-crystallins according to the known four structural motifs of the major calf gamma-crystallin, gamma-II, suggests that all four mouse polypeptides are structurally very similar to calf gamma-II. However, most of the mouse polypeptides differ from gamma-II by the absence of one amino acid residue, resulting in a shorter connecting peptide between the two globular domains of the protein. Primary sequence alignment also revealed that the four mouse gamma-crystallins are most divergent in the third structural motif of the polypeptide. The significance of these differences in terms of the structure and function of the gamma-crystallins in the mouse lens is discussed.

Evidence against gamma-crystallin DNA or RNA sequences in the chicken.

A cloned cDNA (pM gamma 1 Crl) encoding about two-thirds of a gamma-crystallin polypeptide from the murine lens was used as a hybridization probe to search for the presence of gamma-crystallin-like RNA or DNA sequences in the chicken. The 15-day-old chicken lens did not contain any RNA sequences homologous to the murine gamma-crystallin cDNA, as judged by Northern blot hybridization. An approximate 2.3 Kbp (kilobase pair) Bam HI fragment from the chicken genome hybridized to the murine gamma-crystallin cDNA in Southern blots. Cloning and sequencing of this genomic fragment, however, did not reveal any homology with gamma- or beta-crystallin sequences. A stretch of 22 dG: dC nucleotides was present in the cloned DNA fragment and possibly this hybridized to the cloned gamma-crystallin cDNA via its dG:dC nucleotide tails introduced during the cloning procedure. These data support those of McDevitt and Croft (1977) indicating that the chicken lens lacks gamma-crystallin and provide evidence against gamma-crystallin mRNA or a gamma-crystallin gene or pseudogene in the chicken. The absence of gamma-crystallin mRNA in the embryonic chicken lens cells makes them potentially useful recipients for investigating the expression of cloned gamma-crystallin genes.

Analysis of the rat lens epithelial cell nucleus during ageing by scanning cytophotometry.

Nuclei of rat lens epithelial cells were stained with eosin and hematoxylin in order to investigate by cytophotometric analysis the effects of cellular ageing on their morphologic parameters and chromatin compaction. Our results demonstrate a decrease in the size of the nucleus without modification in their shape as a function of age. Older nuclei also have more condensed and heterogeneously distributed chromatin than young nuclei. The homogeneous population of nuclei in the young epithelium gave rise to a highly heterogeneous population in the old animal for both morphometric and densitometric parameters. In very old rats a small percentage of the cell nuclei retained young characteristics. These data support an earlier observation that growth of the lens epithelial cells declines with age.

Evolution of the distribution, proliferation and ultraviolet repair capacity of rat lens epithelial cells as a function of maturation and aging.

The symmetrical organization of lens epithelium was a determining factor in this study whose purpose was to investigate the growth of this tissue during development, maturation and aging. By direct observation it was possible to score all the nuclei, to determine four different zones of cellular density and to monitor them during the whole lifespan. The central zone is mainly quiescent and the proliferation at the periphery accounts for the low steady growth rate of the tissue. With aging there is a continuous decrease in the mitotic index and a cell size enlargement. These cells were able to perform unscheduled DNA synthesis in vitro after ultraviolet irradiation. There is an age-related decrease in DNA repair, but most of it occurs during development (until 59 weeks of age) and then remains constant (59-173 weeks). These results mean that in a differentiated pure cell population aging is not directly related to decline in unscheduled DNA synthesis.

Action of ultrasonic irradiation on the DNA of cultivated bovine epithelial lens cell.

Cultivated epithelial lens cells have been submitted to a 20 kHz continuous ultrasonic irradiation. At relativity high intensity, destruction of the cells was observed. At lower intensities, where no cell destruction appeared, the molecular weight of the single strand DNA of these cells was monitored to determine whether breakage of DNA molecules was induced by the ultrasound. No breakage of the single strand DNA was observed for intensities below 0.1 and 0.4 W cm-2 with irradiation times of less than 20 minutes and 15 seconds, respectively. These intensities and their corresponding irradiation time are higher than those used in current diagnostic practice.