Jose Alcala

NCAM in the differentiation of embryonic lens tissue

The role of the neural cell adhesion molecule (NCAM)2 in ocular lens differentiation was investigated in chicken embryos. Changes in expression of NCAM were documented by immunohistology of frozen sections. This analysis revealed that NCAM diminished during lens fiber differentiation, in contrast to the gap junction-associated protein MP26 which became more abundant. The form of NCAM expressed was determined by Western blot analysis of proteins extracted from the different regions of the Embryonic Day 6 lenses. All regions expressed NCAM with an apparent molecular weight of 140 kDa and relatively low levels of polysialylation. The function of NCAM in lens differentiation was investigated using antibodies that inhibit NCAM-mediated adhesion. Two parameters that change during maturation of the lens epithelial cells were monitored: the thickness of the tissue, indicating the length of lens cells, and the particle arrangement of gap junctions, reflecting the state of junctional differentiation. When epithelial cell explants of Embryonic Day 6 lenses were cultured for 5 days, the cells elongated and displayed an increase in the loose, random intramembranous particle arrangements characteristic of maturing lens fiber gap junctions. When the explants were cultured in the presence of anti-NCAM Fabs, the epithelia were thinner than in matched controls and had particle arrangements characteristic of a less mature state. The expression of NCAM during lens differentiation and the effects of attenuating NCAM function suggest that adhesion mediated by NCAM is an essential event in lens cell differentiation.

Lipid composition of chick lens fiber cell gap junctions

Chick lens fiber cell gap junctions were isolated to homogeneity by the urea-deoxycholate method, characterized ultrastructurally and biochemically, and their lipid composition determined by quantitative thin layer chromatography (TLC). The junctions were estimated to comprise about 52% of the lens fiber plasma membrane. Unlike the junctions of other organs, the lens gap junctions were found to contain sphingomyelin. The cholesterol/phospholipid molar ratio was 2.1 for total fiber membranes but 3.1 for the fiber gap junctions. The levels of major phospholipids in decreasing order were SPH, PC, PE, PI for fiber junctions and PE, SPH, PC, PI for total fiber membranes. The gap junctions were found to contain about 57% of the total fiber cholesterol and 53% of the total fiber sphingomyelin. The high cholesterol and sphingomyelin content suggests that lens fiber gap junctions constitute highly rigid membrane regions conferring significant constraints to the movement of their intramembrane particles (connexons) in the plane of the membrane. The findings help to explain the resistance to the crystallization of their connexons, observed so far only in lens gap junctions.

Limited proteolysis of MP26 in lens fiber plasma membranes of the U18666A-induced cataract in rats.

Most of the animals treated with U18666A every other day beginning at one-day of age developed permanent nuclear cataracts by 3-4 weeks of age. Lens fiber plasma membranes were isolated from cortical and nuclear areas of untreated controls, treated but clear, and treated cataractous lenses, and analyzed by SDS-PAGE. MP26 was the major intrinsic polypeptide in the plasma membranes of both cortical and nuclear fibers of control lenses. MP26 was largely replaced by MP23-24 in the plasma membranes of nuclear fibers of treated but clear lenses, and in the membranes of both cortical and nuclear fibers of cataractous lenses.

Human beta crystallins: regional and age related changes

The composition of human beta-crystallins displayed specific changes with age and region of the lens. 27 kD and 29 kD human beta-crystallin subunits were singled out for study. The 29 kD beta-crystallin subunit constituted approximately 10% of the total lens crystallins at 8 months of fetal life. Its accumulation decreased steadily to 3.3% during postnatal year 1, to 0.5% by year 5 and to 0.3% thereafter. At all postnatal ages, however, it persisted mainly in the superficial fibers. Thus in a 17-years old lens it made up 1.3% of the superficial fiber soluble protein but was already absent from deep cortical and nuclear fibers. The 27 kD subunit increased steadily from 3.5% at 8 months fetal to 7% at year 5; it then decreased steadily to 1.2% in the 86-year old lens. It persisted in all regions of the lens but decreased markedly in the deep cortical and nuclear fibers with increasing age beginning at 5-17 years of age. Studies on the oligomeric structure of human beta-crystallin must take into account age-related changing quantitative patterns in the subunit polypeptide composition of this lens protein.

Evidence for sequential replacement of gap junctions in chick lens development

Abstract Freeze fracture analysis of differentiating (elongating) primary and secondary chick lens fiber cells revealed two morphologically distinct populations of gap junctions. Primary and secondary fiber cells in initial stages of elongation were joined by infrequent, small gap junctions which were characterized by the crystalline order of their intramembrane particles (connexons). Stages of rapid elongation of primary and secondary fiber cells were characterized by the development of non-crystalline type junctions and the progressive loss of the crystalline type junctions. Occasionally, crystalline, non-crystalline, developing non-crystalline and mixed crystalline/non-crystalline gap junctions were observed to occur on the same fiber cell at these stages. Fully differentiated (elongated) primary and secondary fiber cells displayed only the non-crystalline type junctions identical to those previously observed in adult, mature secondary lens fiber cells. The results suggest that the crystalline gap junctions may be related to the differentiation of fiber cells, while the non-crystalline gap junctions may be the expression of the differentiated state. allowing intercellular communication between the progressively metabolically incompetent lens fiber cells.

Reversal of the limited proteolysis of MP26 during the reversal and prevention of the galactose cataract in rat lenses

The reversal and prevention of the galactose-induced cataract in rats were employed to study their effects on the acceleration of the limited proteolysis of MP26 into MP23-24 previously observed in cataractous lenses of galactose-fed animals. Lenses of rats on a cataract reversal-diet demonstrated the reversal of MP23-24 and MP26 levels to control levels in the clearing cortical areas but not in remaining cataractous nuclear areas. Acceleration of the limited proteolysis of MP26 was observed in the nucleus but not the cortex in the clear lenses of animals on a cataract prevention-diet. The results demonstrated that the limited proteolysis of MP26 may form part of a gradual aging process that although not directly (causally) related to cataractogenesis may at least be accelerated by cataractogenic agents or conditions.

Limited proteolysis of MP26 in lens fiber plasma membranes of the galactose-induced cataract in the rat

Lenses of rats maintained on a 50% galactose diet displayed the development of a progressive cataract which was cortical at 3-11 days, and progressively internalized (nuclear as well) and mature at 16-20 days of feeding. Lens fiber plasma membranes were isolated from female rats subjected to the galactose diet and from controls at 11, 19, and 31 days of feeding, and analyzed by SDS-PAGE. Examination of the fiber plasma membranes from whole lenses of galactose-fed rats demonstrated the limited proteolysis of MP26 into MP23-24, in both the cortical and mature stages of the resultant cataracts. The limited proteolysis of MP26 was first evident in the lens cortex at 11 days of galactose feeding, and was evident as well, and more severe in proportion, in the lens nucleus at 19 days of feeding. The greatest proportion in MP26 limited proteolysis was observed in whole lenses at 31 days of galactose feeding. The regional progression of MP26 limited proteolysis closely paralleled the morphological progression of the galactose-induced cataract in the rat. The proportion of lens MP26 which underwent limited proteolysis into MP23-24 increased the longer the animals were kept on the galactose diet.

Limited proteolysis of gap junction protein is intrinsic in mammlian lens fiber-cell plasma membranes

We demonstrate that the limited proteolysis of the lens fiber-cell gap junction protein, MP26, is intrinsic in mammalian lens fiber plasma membranes. Incubations of isolated intact bovine lens fiber plasma membranes in buffer alone did not elicit proteolysis of MP26. Incubations in the buffer with detergent, however, resulted in the limited proteolysis of MP26 which was totally inhibited by calcium chelators, thiol-alkylating agents, and protease inhibitors. As the limited proteolysis required the presence of detergent, it must depend on an enzymatic activity intrinsic in the lens fiber plasma membranes or in MP26 itself.

δ-Crystallin is a chick lens fiber cell membrane extrinsic protein

Abstract In an attempt to resolve the present controversy concerning the reported presence of δ-crystallin in preparations of chick lens plasma membranes, a study was conducted on the extractability of chick lens fiber cell plasma membranes in the presence or absence of calcium ions. One lens from each animal was homogenized in calcium-containing buffer and the other in (calcium) chelating buffer. Each resulting water insoluble crude cell membrane fraction was evenly divided and membranes isolated from each portion by treatment with urea in either calcium-containing buffer or chelating buffer. The isolated membranes were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Gels of membranes isolated in the presence of calcium showed very prominent δ-crystallin bands while gels of membranes isolated under chelating conditions showed a marked depletion of δ-crystallin. The treatment sequence employed ruled out the possibility that the observed binding of δ-crystallin to the membranes was a result of a spurious association caused by calcium because excess soluble δ-crystallin was removed in discarded chelating buffer washes prior to the isolation of the membranes with urea in chelating buffer. The results of the study showed that δ-crystallin is an extrinsic protein of the chick lens fiber cell plasma membranes, and that its linkage to the membranes is calcium-dependent.

The Main Intrinsic Membrane Polypeptide of Vertebrate Lens Fiber Cells

A polypeptide of molecular weight 24,000 daltons is the principal component of the cortical fiber cell plasma membranes of the Northern frog lens. The corresponding principal polypeptide of the fish (pickerel) lens has a molecular weight of 26,000 daltons. These polypeptides differ in electrophoretic mobility from the main polypeptide of the chick and mammalian lens cortical fiber cell membranes.