BASP1 in the lens.

BASP1 was detected in the embryonic and adult chicken lens, using immunological methods and by peptide sequence analysis. The protein was predominantly expressed in fiber cells and only faintly detected in annular pad cells. Localization of the protein was along the plasma membrane of fiber cells often in discrete areas. The role of BASP1 in the lens requires further study.

Effect of thermal stress on early and late passaged mouse lens epithelial cells.

Cataract is an age related disease of protein aggregation. It has been suggested that aging affects the cells ability to protect protein integrity. The protein integrity, which is essential for cellular homeostasis, is maintained by a complex system of refolding or degradation of damaged proteins. The heat shock proteins (hsps) are the major contributors in the maintenance of protein integrity. The heat shock transcription factor (HSF-1) is the master regulator of all hsp synthesis in response to stress. This investigation examined the role of HSF-1 in the regulation of hsp synthesis in early and late passaged alphaTN-4 cells. Data collected in this study revealed that the nucleotide sequence of HSF-1 mRNA obtained from early and late passaged alphaTN-4 cells were identical. When early and late passaged cell were exposed to thermal stress, their hsp expression were also similar. HSP-40 expression was detected after 2 h of heat stress, whereas HSP-70 and low molecular weight heat shock protein alphabeta crystallin showed significantly increased synthesis 18 h post heat stress. The late passaged alphaTN-4 cells ability to upregulate hsps in response to heat stress could be due to its high replicative activities. The data presented here suggests a relationship between the presence of functional HSF-1 and sustained proliferative activities of the late passaged alphaTN-4 cell.

Lamins of ocular lens epithelial cells.

Experiments were performed to characterize a prominent nuclear matrix (NM) protein isolated from tissue cultured mouse lens epithelial cells. This NM protein was separated by SDS-PAGE and the stained gel band was analyzed by mass spectroscopy. Blast analysis of the amino acid sequence derived by mass spectroscopy revealed the presence of Lamin C in the NM of the mouse lens epithelial cells. We also examined nuclear proteins of adult and fetal human lenses. Data collected from these experiments showed the presence of Lamin C in both adult and fetal lens cells. However fetal lens cells only show Lamin C dimers, whereas adult human lens contained dimers, monomers and degraded Lamin C. Early and late passaged tissue cultured mouse lens epithelial cells also contained Lamin C in the nucleus with a preponderance of the dimer in the early passaged cells. The biological significance of the presence of dimers in human fetal lens cells and early passaged mouse lens cells is not known. However, it could suggest an enhanced docking capability of Lamin C dimers for other physiologically important nuclear proteins.

ERM proteins of the lens.

Ezrin and radixin and protein 4.1 were detected in the lens of the eye. These proteins were mainly present in the young elongating cortical fiber cells and localized to the plasma membranes. Moesin was not detected. Ezrin, radixin, and protein 4.1 provide another means whereby actin is linked to the plasma membrane in addition to the known adherens junctions in the lens.

Paralemnin of the lens.

Paralemmin was identified in the chicken lens as a protein with mol. wt 65 kDa and a splice variant of 60 kDa, both soluble in Triton X-100. Paralemmin is localized to the plasma membrane of fiber cells, and was not detected in the annular pad cells. Thus in the chick lens it is another feature of fiber cell differentiation. Its localization to the short side of the fiber cell and the sites of fiber cell interlocking suggests that paralemmin may play a role in the development of such interdigitating processes.

Associated proteins of lens adherens junction.

Cytoplasmic proteins associated with adherens junctions were identified in the chicken ocular lens. The catenins, alpha, beta, and gamma, were present in epithelial and fiber cells, although their pattern of distribution changed with fiber cell differentiation. The sharp decline in alpha-catenin with fiber cell formation and the increasing Triton-insolubility of N-cadherin suggests that another subtype of alpha-catenin exists in the lens.

Effect of exogenous stress on the tissue-cultured mouse lens epithelial cells.

The effects of heat, oxidative and osmotic stress on heat shock proteins (HSP-70(I), HSC-70, and HSP-40 of tissue cultured mouse lens epithelial cells (alphaTN-4) were investigated. The effect of stress on the heat shock transcription factor (HSF-1), and a nuclear matrix protein (NM-60) of alphaTN-4 cells was also examined. Cells were exposed to heat (45 degrees C), oxidative stress (50 mM H(2)0(2)) and osmotic (600 mM medium) shock for 30 min, and then allowed to recover for 18 h in physiological medium. Control cells were maintained at 37 degrees C in an isosmolar medium. Cellular proteins were isolated and fractionated by SDS-PAGE. Western blot was used to determine the levels of HSP and nuclear proteins. Heat stressed cells were also examined, by immunofluorescence, for the specific localization of NM-60 and HSF-1. The results revealed that both NM-60 and HSF-I were present in specific locations in normal and heat-stressed cell nuclei. Nuclei isolated immediately after stress showed localization of fluorescence near the nuclear membrane. When heat stressed cells were allowed to recuperate at 37 degrees C, most of the fluorescence were relocated in discrete areas of the nucleus. These experiments showed that alphaTN-4 cells responded to stress by overexpression of HSP-70(I) and HSP-40. This increase was not present immediately after the end of the stress period, but clearly evident at 18 h of recovery in physiological medium. Immunofluorescent data suggest that heat stress induced the localization of NM-60 and HSF-1 near the nuclear membrane.

Heat shock proteins of adult and embryonic human ocular lenses.

We investigated the presence and distribution of heat shock proteins, HSP-70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449-10453], HSP-40, HSc-70, HSP-27, and alphabeta-crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS-PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP-27 and alphabeta-crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP-27. The presence of HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age-related cataract.

Heat shock proteins of chicken lens.

The presence of heat shock proteins HSP-40, HSP-70, and HSc-70 in adult and embryonic chicken lenses were determined. The epithelium, cortex, and nucleus of adult chicken lens were separated and tested for the presence of heat shock proteins (hsps) by western blot, using specific antibodies for HSP-40, HSP-70, and HSc-70. Water soluble (WSF) and water insoluble fractions (WIF) of embryonic chicken lenses were isolated and tested for the presence of HSP-40, HSP-70, and HSc-70 by immunoblot. Embryonic chicken lens sections were also analyzed for the presence of heat shock proteins by immunofluorescence technique. Data obtained from these experiments revealed that HSP-40, HSP-70, and HSc-70 are present in all areas of both adult and embryonic chicken lens. Presence of hsps protein in the deep cortex and nucleus is intriguing as no detectable metabolic activities are reported in this area. However it can be proposed that hsps HSP-40, HSP-70, and HSc-70 can interact with protein of these areas and protect them from stress induced denaturation.

A heat shock transcription factor like protein in the nuclear matrix compartment of the tissue cultured mammalian lens epithelial cell.

This investigation characterizes a prominent nuclear matrix protein isolated from tissue cultured mouse lens epithelial cells. The nuclear matrix protein was isolated using a modified Penman technique. Total nuclear matrix proteins were further separated by SDS-polyacrylamide gel electrophoresis. The SDS-PAGE profile of the nuclear matrix proteins displayed a prominent doublet band at 60 kDa region. Nonequilibrium 2D gel electrophoresis revealed that this protein is a basic nuclear protein. This 60 kDa protein was further characterized by comparing its internal peptide amino acid sequence with known protein sequence using the BLAST technique, and this study demonstrated that 60 kDa nuclear matrix protein displays significant sequence similarity with Xenopus Laevis heat shock transcription factor. We also raised antibodies against 60 kDa nuclear matrix protein. Immunofluorescence, studies showed that this 60 kDa nuclear matrix protein preferably decorates nucleus, and puncted pattern of fluorescence suggest presence of this protein in the discrete areas of the nucleus. Heat shock transcription factors upregulate synthesis of heat shock proteins and many of these protein act as molecular chaperones. Thus, presence of a nuclear matrix protein with significant sequence similarity with heat shock transcription factor suggests sustained heat shock protein synthesis in the mouse lens cells.

Up-regulation of novel intermediate filament proteins in primary fiber cells: an indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed "beaded filaments." CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up-regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49(INS), was also detected early in embryonic development and into adulthood. Peptide-specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49(INS) in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up-regulated expression of CP49 and CP95 could serve as pan-specific markers for all vertebrate lens fiber development.

Magnitudes and orientations of interaction tensors determined from rotational resonance MAS NMR lineshapes of a four-(13)C-spin system

Possibilities and limitations of iterative lineshape fitting approaches for the complete determination of magnitudes and orientations of NMR interaction tensors in a four-(13)C-spin system from MAS NMR experiments are investigated. The availability of fast and numerically accurate computational methods is an important prerequisite. The model compound chosen for this investigation is the monoammonium salt of maleic acid. Various selectively and fully (13)C-labeled versions of this compound permit a stepwise reduction of the number of unknown parameters, necessary to fully describe the four-(13)C-spin system in the uniformly (13)C-labeled maleate moiety. This stepwise procedure allows one to monitor reliability and accuracy of multiparameter fits of the four-(13)C-spin system itself, as well as to characterize limitations and requirements for such fitting procedures. Satisfactory (1)H-decoupling performance is an essential experimental requirement; TPPM decoupling yields n = 1, 2 rotational resonance (13)C MAS NMR lineshapes suitable for analysis by iterative lineshape fitting methods. It is demonstrated that assumptions about "typical" chemical shielding tensor orientations, even if not deviating much from the real orientations, lead to severe errors in internuclear distance determinations. Copyright 1999 Academic Press.

Quantification and regulation of mRNAs encoding beaded filament proteins in the chick lens.

PURPOSE: To quantify the expression of beaded filament protein mRNA levels in regions of the chick lens and to examine the in vitro regulation of message and protein levels using cell culture techniques. METHODS: RNase protection assays and Northern blotting were used to quantify beaded filament protein mRNA levels in dissected lenses. Cultured cells were assayed for mRNA with RNase protection and for protein with Western blotting and ELISA techniques after treatment with cAMP analogs. RESULTS: Beaded filament protein message levels were greatly up-regulated in cortical fiber cells compared to annular pad cells. Full length messages were also detected in nuclear fiber cells. The presence of an unusual form of the CP49 message with a lamin-like insert, CP49INS, was also established. Both message and protein levels were subject to regulation in response to elevated intracellular cAMP levels. CONCLUSIONS: The accumulation of beaded filament protein levels during fiber cell development may be due to the increased cAMP-mediated transcription of message. The presence of CP49INS may lend new insight into mechanisms of intermediate filament assembly.

Nonchromatin nuclear proteins of mammalian lens epithelial cells.

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determined possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [35S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development.

Electron microscopic detection of glycoconjugates in the chicken lens.

The cytochemical localization of glycoconjugates in the 14-day old embryonic chick lens was analysed by lectin-gold labelling. Con A/HRP gold particles, specific for D-mannose labelled the interior of the rough endoplasmic reticulum, membranes of the Golgi complex, secretory vesicles and the plasma membranes of the lens epithelial cell. The lens capsule was heavily labelled. Lens fiber cell membranes were also labelled. In contrast LFA, specific for neuraminic acid, did not bind to the endoplasmic reticulum or nuclear membrane. Labelling of the Golgi complex, secretory vesicles and capsule was observed. The plasma membranes of epithelial and fiber cells were extensively labelled, and probably reflects the presence of glycolipids such as gangliosides.

Developmental changes in glycoconjugate composition during chick lens morphogenesis.

The following lectins: Con A, WGA, sWGA, PNA, RCA and UEA were used to study developmental changes in the expression of glycoconjugates during chicken lens morphogenesis. Con A, WGA, sWGA binding epitopes were observed in the lens placode and vesicle. Once the fiber mass was formed, the glycoconjugates were mainly found at the epithelial-fiber-cell junction, on epithelial cell membranes but only weakly on fiber-cell membranes. The PNA reaction was restricted to the apical surface of cells of the lens placode and vesicle and to the epithelial-fiber junction throughout the rest of lens development. The RCA reaction was mainly localized to the apical plasma membrane and moderately at the lateral plasma membrane of cells of the lens placode and vesicle and maintained this staining pattern in the lens epithelial cell during the progressive development of lens. UEA binding was initially localized along the posterior elongating cells of the lens vesicle, and then expressed in both epithelial and fiber cells. Subsequently UEA binding was restricted to the epithelium and central fiber mass. The extensive distribution of glycoconjugates on the surface of the invaginating placode cells suggests a role during invagination and subsequent detachment of the placode from the surface ectoderm. The capsule was labelled by Con A, WGA, sWGA, RCA and PNA but not by UEA. The posterior capsule was more intensely reactive with Con A, RCA and PNA than the anterior capsule.

Identification of a ubiquitin-like protein in the mammalian vitreous humor.

An 8 kDa ubiquitin-like peptide (ULP) was isolated by high performance liquid chromatography from the rabbit vitreous humor, and the N-terminal amino acid sequence of this peptide showed complete homology with ubiquitin. Western blot revealed the presence of free ULP in both the iris-ciliary (IC) complex and the aqueous humor extracts. In the IC complex, fluorescence and immunoelectron microscopy detected high concentrations of ULP in the posterior epithelial cells, suggesting this tissue as a possible source of ULP in the ocular fluids. Significantly, this is the first time that the presence of free ULP has been reported in mammalian extracellular fluids. Furthermore, we recently demonstrated that the 8 kDa fraction of vitreous humor containing ULP is a potent inhibitor of protein synthesis [Banerjee et al. (1992): J Cell Biochem 49:66-73]. These findings taken together suggest a novel biological role for ULP in the control of lens cell growth.

Chicken CP49: significant or paltry? An evolutionary perspective.

The lens-specific intermediate filament (IF) proteins CP49 and filensin have been identified in a large number of evolutionary divergent species. In the chick lens both CP49 and filensin have been identified. Recently a unique variant of CP49, CP49ins has been identified. CP49ins contains an insertion of 49 amino acids in helix 1b giving it characteristics similar to the lamin-like cytoplasmic IF of invertebrates. The presence of this variant form in the chick lens poses some important questions about the co-assembly of the two CP49 forms and also the evolutionary relationship with invertebrate IF proteins.

The predicted structure of chick lens CP49 and a variant thereof, CP49ins, the first vertebrate cytoplasmic intermediate filament protein with a lamin-like insertion in helix 1B.

The full length cDNA sequence for the lens-specific intermediate filament protein, CP49, from chicken is presented. The sequence contains features typical of the other intermediate filament proteins, including two major alpha-helical regions, helix I and II and appropriate linker regions. CP49 lacks a C-terminal non-alpha-helical domain and is only the second intermediate filament protein to be described missing this feature. Comparison to the bovine CP49 shows significant homology in all domains except the N-terminal non-alpha-helical domain. Besides bovine CP49, the other protein most homologous to chicken CP49 in the database was keratin 18, a type I keratin. A variant of CP49 is also described, called CP49ins. Of the 61 positive clones identified in the library, two encoded CP49ins, one of these being a full-length clone. The sequence differed to CP49 by the insertion of 49 amino acids in helix IB. This is the first chordate cytoplasmic intermediate filament protein sequence to be identified with an archetypal lamin-like insertion in this helical subdomain and represents a key discovery in tracing the evolutionary pathway of intermediate filament protein family.