Restriction fragment length polymorphisms associated with the gene for the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Cloned cDNAs coding for eye-lens fibre cell-membrane proteins, MIP and MP70, were used to detect restriction fragment length polymorphisms (RFLPs) in genomic DNA from inbred mice with autosomally inherited cataracts. Whereas distinct RFLPs associated with the MIP gene were identified in the Cba Cat and Nct mutants, no such genetic variation was associated with the MP70 gene. RFLPs associated with the mouse MIP gene may provide informative DNA markers in gene linkage studies of murine hereditary cataracts.

Immunochemical comparison of the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Expression of the major intrinsic protein (MIP) of eye-lens fibre cell membranes was compared in normal (DBA), cataractous (CAT, LOP, NCT) and chimaeric (CBA-LOP) mice at different stages of development using immunofluorescence microscopy and immunoblotting techniques. MIP of apparent molecular mass 26 kDa was detected in extracts of adult DBA, LOP and CBA-LOP lenses, but only low molecular mass (less than 26 kDa) immunoreactive proteins were detected in similar extracts from adult CAT and NCT lenses. The corresponding MIP distribution patterns confirmed the highly organised fibre-cell histology in embryonic DBA and adult CBA-LOP lenses and also highlighted the severe fibre-cell degeneration in the LOP lens. In contrast, however, no immunoreactive MIP was detected in situ in embryonic CAT and NCT lenses. These results suggest that a structural alteration of MIP occurs during embryonic lens development in the cataractous CAT (dominant) and NCT (recessive) mutant mice.

In vitro characterization of response to stimulus (wounding) with regard to ageing in human skin fibroblasts.

Confluent cultured normal human skin fibroblasts from neonatal, adult and aged donors have been stimulated to respond to wounding of the cell sheet. The latent period prior to initial migration of cells from the leading edge of the monolayer is correlated with in vitro population doubling level and in vivo donor age. Time-lapse photography of areas along the edge of the cell sheet reveals a specific pattern of migration by which the cells reestablish a confluent monolayer.

Age associated decrease of N-acetyl-beta-glucosaminidase activity in the lens epithelial cells from C57Bl/6 mice.

Lens epithelial cells were isolated from samples of C57BL/6 and CD-1 mice at different ages between 1 and 5 months, and assayed for the level of N-acetyl-beta-glucosaminidase and their ability to replicate in-vitro. The lens epithelial cells from C57BL/6 mice had a reduced replicative potential compared to other normal strains. A statistically significant decrease in active enzyme levels was demonstrated to be associated with increased animal age of C57BL/6 mice.

Age-dependent metabolic changes in cultured human fibroblasts.

The effects of metabolic poisons on the ATP content of cultured human skin fibroblasts at selected in vitro and in vivo ages were studied. Potassium cyanide, iodacetamide, and Arsenate were used to inhibit ATP restoration by glycolysis and oxidative phosphorylation. Cells treated with these metabolic poisons showed an age-dependent change in their ATP content. The decrease in cellular ATP content after exposure to these drugs was taken as an estimate of ATP turnover. It was found that there was a decrease in the ATP turnover with increasing population doubling level (i.e. in vitro age), and cells cultured from a 68-yr-old donor had a lower ATP turnover than those cultured from a neonatal donor. This decreased ATP turnover correlates with a previous finding of a decreased ability of "older" cells to be stimulated to migrate in culture and suggests that there is a metabolic component to this age-related functional deficiency.

Statin expression associated with terminally differentiating and postreplicative lens epithelial cells.

The expression of a nuclear (57 kDa) protein statin has been previously characterized as a specific marker of quiescent or senescent aging human fibroblasts in vitro. In these studies we have shown that the expression of statin is associated specifically with the postreplicative and terminally differentiating lens epithelial cell. By monitoring the synthesis of specific lens crystallin proteins, and the morphological and cellular changes associated with this differentiated system, we have demonstrated a close correlation between statin expression and cell commitment to the G0 nonreplicative cell cycle state.

The shiverer mouse mutation shi/shi: rescue and preimplantation detection.

The shiverer mouse mutation has been used as a model in this series of experiments. Germ-line therapy of this disorder has been demonstrated by producing mice transgenic for the wild-type gene for myelin basic protein (MBP). The mutation has also been diagnosed in preimplantation mouse blastocysts.

Factors modulating mouse lens epithelial cell morphology with differentiation and development of a lentoid structure in vitro.

The morphological and cellular changes that occur with differentiation and development of a lentoid structure from cultured mouse lens epithelial cells have been found to be dependent on the presence of lens capsule in association with the cells. The development of the 'lentoid body' is a multiphase process involving cell replication, synthesis of mucosubstances and a basement collagen membrane, cell aggregation and differentiation. Stage-specific synthesis of lens proteins confirms that the genes regulating normal differentiation in vivo are operating in the in vitro system. The hydrated collagen gel studies described in this report demonstrate that the cuboidal morphology and apical-basal polarity of the lens epithelial cells are dependent on their relationship with the lens capsule. Following a replicative phase the cells assume a mesenchyme-like morphology and migrate into the gel. Trypsinized cells freed from the lens capsule replicate but form colonies on the surface of the gel. The implications of these results are discussed with respect to previous observations made on normal lens development and the abnormalities associated with the congenital cataractous embryonic lens.

An in vivo and in vitro study of the embryonic and adult lop mutant congenital cataractous lens.

A detailed histological study of the embryonic and adult congenital cataract Lop mouse lens has been made. A comparison with the congenital cataract (CatFr) mouse lens has shown that the full range of lens anomalies noted in the congenital cataractous mice are very similar. However, the Lop mutant demonstrates these defects to a greater degree in both the embryonic and adult stages. The lens epithelial cells of the Lop adult lens have been cultured to ascertain their in vitro phenotype. These studies have shown that the proliferation pattern and population doubling level are similar to that of the CatFr lens epithelial cells. A comparison with non-cataractous mouse lens epithelial cells demonstrates that the congenital cataractous mice lens epithelial cells have a very limited life span in vitro. An increase in the nuclear diameter with population doubling level was observed in the cultured mouse lens epithelial cells.

In-vitro studies on 'spare' human preimplantation embryos in culture.

Methods previously used for the biopsy of preimplantation mouse embryos have been applied to individual 'spare' human embryos. Early cleavage-stage human embryos have been cultured and individual blastomeres removed following zonae thinning or drilling. Embryos have also been cultured to the blastocyst stage for the biopsy of three to five trophectoderm cells. Both the biopsied embryo and the biopsied cells have been allowed to develop and/or grow in vitro.

The nature and distribution of serologically detectable alloantigens on the preimplantation mouse embryo.

The nature and distribution of surface alloantigens on preimplantation mouse embryos has been examined by immunofluorescence. Non-H-2 alloantigens were detected at all stages examined, from the 2-cell to the 4 1/2-day blastocyst. Cleaving blastomeres, inner cell mass cells and cells of the primary trophectoderm were all positive. In F1 embryos maternal non-H-2 alloantigens were detectable at all stages, whereas paternal antigens first became evident at the 6- to 8-cell stage. No convincing evidence of the presence of alloantigens associated with the H-2 haplotype was found at any stage or on any cell type, suggesting that if these antigens are present they are in low quantity or are masked.

Replicative potentials of various fusion products between WI-38 and SV40 transformed WI-38 cells and their components.

Hybrid cells derived from whole-cell fusions of replicating phase-II normal fibroblast cells (WI-38s) with SV40 transformed WI-38 fibroblast cells (CL-1s) demonstrated that the majority of the hybrid experimental cells still maintained a finite life-span. Approximately 2% demonstrated sustained and possibly indefinite replication. Experimental binucleate cells and subsequent hybrid synkaryons were also formed by fusing CL-1 karyoplasts into phase-II WI-38 replicating normal fibroblasts. In addition, viable cells were constructed from WI-38 fibroblast cytoplasts with CL-1 karyoplasts. Sustained replication was not observed in these crosses.

Cycling cytoplasmic factors that promote mitosis in the cultured 2-cell mouse embryo.

A cytoplasmic component(s), previously shown to rescue the 'blocked' 2-cell mouse embryo in vitro, has been demonstrated to peak in activity during the transition between G2 and M phase and decline thereafter. The possible significance of this component(s) in the regulation of cleavage of the cultured mouse embryo is discussed.

Rescue of developmental lens abnormalities in chimaeras of noncataractous and congenital cataractous mice.

In the study of the lens of a congenital cataractous mouse mutant (CAT), it has been shown that a loss of growth regulation at the cellular level causes gross lens abnormalities. The phenotypic characteristics of the cataractous mouse lens are similar to those seen in human congenital cataract and thus serves as a model system for medical research. In this present investigation, we have demonstrated that the abnormalities of the congenital cataractous lens can be rescued by forming chimaeras between DBA/2 (a noncataractous strain of mouse) and the CAT mutant. This report describes the histological, cellular and biochemical analysis of the resultant chimaeric eyes, and discusses possible mechanisms by which these results were achieved.