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A Sandilands

Publications and source records attributed to A Sandilands.

9 recordsLinked to original sources
PubMed2000

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.

Animals↗
PubMed1999

The eye lens cytoskeleton.

During lens cell differentiation there are a number of very characteristic morphological changes that occur. These include a 50- to 100-fold increase in cell length as the equatorial lens epithelial cells differentiate into fibre cells and the loss of the cellular organelles such as mitochondria, nuclei, Golgi apparatus and endoplasmic reticulum. Coincident with these changes are dramatic alterations in the organisation of the lens fibre cell cytoskeleton and in particular the lens-specific intermediate filament network comprising CP49 and filensin. Cell shape and cell polarisation as well as tissue integrity are all processes that depend upon the cytoskeleton and are therefore important to the lens. The unique aspects of the lenticular cytoskeleton are the subject of this review.

Animals↗
PubMed1998

Three murine cataract mutants (Cat2) are defective in different gamma-crystallin genes.

A number of murine cataract mutations have been localized to chromosome 1 close to the gamma-crystallin gene cluster (Cryg) (Everett et al., 1994, Genomics 20: 429-434; Löster et al., 1994, Genomics 23: 240-242). Based on the size of the mapping or allelism tests they have not been shown to be genetically distinct and have been assigned to locus symbol Cat2. Here we assign three mutations to the respective gamma-crystallin gene. Using a systematic candidate gene approach to analyze the entire Cryg cluster, an A-->G transition was found in exon 2 of Cryga for the ENU-436 mutation and is designated Cryga1Neu. The mutant allele Crygbnop (formerly Cat2(nop)) is caused by a replacement of 11 bp by 4 bp in the third exon of Crygb, while a C-->G transversion in exon 3 of Cryge has been found for the Cryget (formerly Cat2(t)) mutation. For the mutation Cryga1Neu, an Asp-->Gly exchange is deduced, whereas the mutations Crygbnop and Cryget lead to the formation of in-frame stop codons and give rise to truncated proteins of 144 and 143 amino acids, respectively. The effects of the mutations upon gamma-crystallin structure are likely to be quite different. The Cryga1Neu mutation is expected to affect the link between Greek-key motifs 2 and 3, whereas both Crygbnop and Cryget mutations are supposed to truncate the fourth Greek-key motif. All three mutations are predicted to alter protein folding of the gamma-crystallins and result in lens cataract, but the phenotype for each is quite distinctive.

Amino Acid Sequence↗
PubMed1996

The beaded filament of the eye lens: an unexpected key to intermediate filament structure and function.

In 1959, an unusual filamentous polymer, now called the beaded filament, was described in the lens of the eye. The constituent proteins, assembly properties and functions of the beaded filament have been elusive. The recent publication of the sequences for two major lens filament proteins (CP49 and filensin) and the reconstitution in vitro of structures closely resembling beaded filaments, suggests that the beaded filament is related structurally to intermediate filaments (IFs). The association of the lenticular chaperones, the alpha-crystallins, with the filament contributes to the characteristic beaded morphology, as well as giving important clues to the function of this unusual filament in the lens. These recent results have several implications for IF function and assembly.

Journal Article↗
PubMed1996

The intermediate filament cytoskeleton of the lens: an ever changing network through development and differentiation. A minireview.

In the eye lens, intermediate filament proteins form two morphologically distinct polymers, 10-nm intermediate-sized filaments and beaded filaments. Coincidently, the beaded filament polymer and the proteins filensin and CP49 are specific to lens fibre cells and are therefore excellent markers for fibre cell differentiation. In the adult lens, filensin and CP49 are maintained throughout all stages of lens fibre cell differentiation whilst vimentin is apparently lost at a specific stage from the deeper cortical fibres. The expression of CP49 and filensin is coincident with the presence of beaded filaments suggesting these proteins are filament components. In association with alpha-crystallin, CP49 and filensin form beaded filaments in vitro. During fibre cell differentiation, filensin and CP49 are post-translationally modified. In the case of filensin, proteolysis results in two functionally distinct fragment sets, one derived from the alpha-helical rod domain and the other from the C-terminal tail domain of filensin. It is proposed that both filensin and CP49 are critically involved in organising the cytoplasmic and plasma membrane domains of the fibre cell and therefore essential to the optical properties of the lens.

Animals↗
PubMed1995

Vimentin and CP49/filensin form distinct networks in the lens which are independently modulated during lens fibre cell differentiation.

The cells of the eye lens contain the type III intermediate filament protein vimentin, as well as two other intermediate filament proteins, CP49 and filensin. These two proteins appear to be unique to the differentiated lens fibre cell. Immunoblotting and confocal microscopy were used to describe changes which occur in these three intermediate filament proteins and the networks they form during fibre cell differentiation and maturation. The vimentin network was present in both epithelial cells and some fibre cells. Fibre cells were vimentin positive up to a specific point 2-3 mm in from the lens capsule where the vimentin signal was drastically reduced. The CP49/filensin network was not present in the undifferentiated epithelial cells but emerged in the differentiating fibre cells. This latter network exhibited a principally plasma membrane localization in younger fibre cells but became more cytoplasmic in older fibre cells. This change also occurred at a distinct point in fibre cell differentiation, much earlier than the observed loss of the vimentin network. The subcellular changes in the distributions of these cytoskeletal networks were correlated to the loss of the fibre cell nucleus, another feature of fibre cell differentiation. No correlation was found to changes in the vimentin network but nuclear loss did coincide with changes in the CP49/filensin network. Concomitant with nuclear pyknosis, there were also changes in the nuclear lamina as well as infringement of the nuclear compartment by CP49, as shown by confocal microscopy. This study demonstrates vimentin and the CP49/filensin network to be independent in the lens but both networks undergo dramatic changes in subcellular distribution during the differentiation/maturation of the fibre cell. Only changes in the CP49/filensin network can be correlated to nuclear loss. Thus in the lens, unlike mammalian erythropoiesis which is also characterized by nuclear loss, the vimentin network does not appear linked to nuclear retention.

Animals↗
PubMed1995

Filensin is proteolytically processed during lens fiber cell differentiation by multiple independent pathways.

Filensin is a lens-specific intermediate filament protein, expressed in the lens fiber cells but not the lens epithelium. Using antibodies to filensin and the other lens intermediate filament proteins, vimentin and CP49, the codistribution of filensin with CP49 and independence of this network from the vimentin network was confirmed. Monoclonal and polyclonal antibodies to peptides and specific subdomains of filensin were used to follow changes in the subcellular distribution of filensin during bovine lens fiber cell differentiation. Filensin is shown to be extensively processed during lens fiber cell differentiation to give protein fragments derived from distinct protein domains, one corresponding to the N-terminal non-alpha-helical/and rod domain and the other to the C-terminal non-alpha-helical tail domain. Immunoblotting analysis using anti-filensin peptide polyclonal antibodies suggested that the two fragment sets arose separately. Residues 331 to 430 in filensin have been identified as an important region in the processing pathway(s). Our results clarify previous confusion in the literature regarding the processing of filensin which arose because of the similar relative electrophoretic mobilities by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the different fragment sets. The predicted secondary structure characteristics of the different domains of filensin suggests different functions for the two fragment sets to give filensin a dual role in the lens. This suggestion is supported by the subtly different subcellular distributions in the peripheral and mature fiber cells of the two filensin fragment sets.

Animals↗
PubMed1993

Effect of chlormethiazole, dizocilpine and pentobarbital on harmaline-induced increase of cerebellar cyclic GMP and tremor.

Administration to mice of harmaline (100 mg/kg SC) resulted in a greater than two-fold increase in cyclic GMP in the cerebellum 15 min later. This response was inhibited by pretreatment 5 min before the harmaline with pentobarbital (ED50 6.5 mg/kg), chlormethiazole (ED50 10.4 mg/kg) and dizocilpine (ED50 0.5 mg/kg). Harmaline-induced tremor was inhibited by pentobarbital (ED50 30 mg/kg) and chlormethiazole (ED50 50 mg/kg) but not dizocilpine. The data demonstrate that the harmaline-induced tremor and cerebellar cyclic GMP rise are probably not associated. They also demonstrate that chlormethiazole is able to inhibit a biochemical response (the increase in cerebellar cyclic GMP) which results from increased glutamate function.

Animals↗