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J Kistler

Publications and source records attributed to J Kistler.

At least 19 recordsLinked to original sources
PubMed2001

MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3.

BACKGROUND: Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator. RESULTS: MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction. CONCLUSIONS: MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.

Animals↗
PubMed2001

Ultrastructural evidence that apoptosis is the mechanism by which human amylin evokes death in RINm5F pancreatic islet beta-cells.

A view is emerging that human amylin (HA) kills pancreatic islet beta-cells by apoptosis. This study strengthens this view by documenting time-dependent morphological and ultrastructural changes in 10 microm HA-treated cultured RINm5F islet beta-cells. Membrane blebbing and microvilli loss were the earliest detectable apoptosis-related phenomena, already evident 1 h after HA exposure. Following 6-12 h of HA-treatment, chromatin margination became evident, consistent with detecting DNA laddering about the same time. Nuclear shrinkage, nuclear membrane convolution and prominent cytoplasmic vacuolization were clearly recognized at 22 h post-treatment. Together, these cellular changes constitute a strong case for HA-induced apoptosis, and further demonstrates that electron microscopy is a more sensitive tool for early apoptosis detection in cultured cells than classical biochemical assays like visualizing DNA laddering. The ultrastructural changes reported here contribute further evidence to be included in the ongoing dissection of molecular mechanisms underlying HA-induced apoptosis, as may occur in type-2 diabetes mellitus.

Amyloid↗
PubMed2001

Utility values associated with blindness in an adult population.

AIM: To ascertain utility values associated with varying degrees of legal blindness. METHODS: A cross sectional study on three group of patients. There were: (1) 15 patients with complete absence of vision (no light perception) in at least one eye who were asked to assume a scenario of no light perception in the second eye as well, (2) 17 patients with light perception to counting fingers in the better seeing eye, and (3) 33 patients with 20/200-20/400 vision in the better seeing eye. Utility values were measured using the time trade-off and standard gamble methods in each of the three groups. RESULTS: The mean time trade-off utility value for the no light perception group with the theoretical scenario of bilateral absence of light perception was 0.26 (95% CI, 0.19-0.33). The mean utility value for the light perception to counting fingers group was 0.47 (95% CI, 0.33-0.61), and the mean utility value for the 20/200-20/400 group was 0.65 (95% CI, 0.58-0.72). Thus, patients with no light perception in one eye, who were presented with the same scenario in the second eye as well, were willing to trade almost 3 out of every 4 years of remaining life in return for perfect vision in each eye. Those with light perception to counting fingers would trade approximately 1 of 2 remaining years and those with 20/200-20/400 would trade approximately 1 of 3 remaining years. CONCLUSIONS: There is a wide range of utility values associated with legal blindness. The utility value decreases dramatically with perceived total loss of vision (absence of light perception in each eye), compared with counting fingers to light perception vision, indicating that the preservation of even small amounts of vision in patients with legal blindness is critically important to their wellbeing and functioning in life.

Adult↗
PubMed2001

Molecular solutions to mammalian lens transparency.

The mammalian lens generates an internal microcirculation that maintains transparency in the avascular lens. Significant progress has been made in characterizing the membrane transport proteins associated with this circulation. By combining physiological and molecular evidence, a more comprehensive understanding of normal lens function and cataractogenesis is emerging.

Animals↗
PubMed2000

Surface tongue-and-groove contours on lens MIP facilitate cell-to-cell adherence.

The lens major intrinsic protein (MIP, AQP0) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells, indicating that it has adhesive properties in addition to its channel function. Using atomic force and cryo-electron microscopy we document that crystalline sheets reconstituted from purified ovine lens MIP mostly consisted of two layers. MIP lattices in the apposing membranes were in precise register, and determination of the membrane sidedness demonstrated that MIP molecules bound to each other via their extracellular surfaces. The surface structure of the latter was resolved to 0.61 nm and revealed two protruding domains providing a tight "tongue-and-groove" fit between apposing MIP molecules. Cryo-electron crystallography produced a projection map at 0.69 nm resolution with a mirror symmetry axis at 45 degrees to the lattice which was consistent with the double-layered nature of the reconstituted sheets. These data strongly suggest an adhesive function of MIP, and strengthen the view that MIP serves dual roles in the lens.

Aluminum Silicates↗
PubMed2000

Utility values and age-related macular degeneration.

OBJECTIVE: To ascertain the utility values associated with age-related macular degeneration and varying degrees of visual loss. DESIGN: A cross-sectional study. PARTICIPANTS: Eighty white patients with unilateral or bilateral age-related macular degeneration in 1 or both eyes, and visual loss to a minimum of the 20/40 level in at least 1 eye. MAIN OUTCOME MEASURES: Utility values were measured in 5 groups according to the visual acuity in the better-seeing eye, 1 (20/20 to 20/25), 2 (20/30 to 20/ 50), 3 (20/60 to 20/100), 4 (20/200 to 20/400), and 5 (counting fingers to light perception), using the time trade-off and the standard gamble methods. Conventionally assigned anchor utility values were 1.0 for perfect health and 0.0 for death. RESULTS: The mean utility value for the total group with age-related macular degeneration was 0.72 (95% confidence interval [CI], 0.66-0.78) using the time trade-off method and 0.81 (95% CI, 0.76-0.86) using the standard gamble method. Using the time trade-off method correlated with the visual acuity in the better-seeing eye, the results were as follow: group 1, 0.89 (95% CI, 0.82-0.96), group 2, 0.81 (95% CI, 0.73-0.89), group 3, 0.57 (95% CI, 0.47-0.67), group 4, 0.52 (95% CI, 0.38-0.66), and group 5, 0.40 (95% CI, 0.29-0.50). Thus, those patients in group 1 were willing to trade 11% of their remaining lifetime in return for perfect vision in each eye, whereas those in group 5 were willing to trade 60% of their remaining lifetime in return for perfect vision in each eye. CONCLUSION: Age-related macular degeneration causes a substantial decrease in patient utility values and is highly dependent on the degree of visual loss in the better-seeing eye.

Aged↗
PubMed2000

Amyloid fibril formation from full-length and fragments of amylin.

Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1-37) versus fragments of this peptide-hA(8-37) and hA(20-29)-is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to beta-sheet/alpha-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20-29) formed flat ribbons consisting of numerous 3. 6-nm-wide protofibrils. In contrast, hA(1-37) and hA(8-37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1-37), the predominant fibril type contained three protofibrils and for hA(8-37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1-37) and hA(8-37) fibrils. hA(20-29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20-29 contribute to the fibrils' beta-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.

Amyloid↗
PubMed2000

Galectin-3 is associated with the plasma membrane of lens fiber cells.

PURPOSE: To discover proteins that have the potential to contribute to the tight packing of fiber cells in the lens. METHODS: Crude fiber cell membranes were isolated from ovine lens cortex. Proteins were separated by two-dimensional gel electrophoresis, and selected protein spots identified by micro-sequencing. The identification of galectin-3 was confirmed by immunoblotting with a specific antibody. The association of galectin-3 with the fiber cell plasma membrane was investigated using immunofluorescence microscopy, solubilization trials with selected reagents, and immunoprecipitation to identify candidate ligands. RESULTS: A cluster of three protein spots with an apparent molecular weight of 31,000 and isoelectric points ranging between 7 and 8.5 were resolved and identified as galectin-3. This protein was associated peripherally with the fiber cell plasma membrane and interacted with MP20, an abundant intrinsic membrane protein that had been identified previously as a component of membrane junctions between fiber cells. CONCLUSIONS: The detection of galectin-3 in the lens is a novel result and adds to the growing list of lens proteins with adhesive properties. Its location at the fiber cell membrane and its association with the junction-forming MP20 is consistent with a potential role in the development or maintenance of the tightly packed lens tissue architecture.

Amino Acid Sequence↗
PubMed2000

Blocking chloride channels in the rat lens: localized changes in tissue hydration support the existence of a circulating chloride flux.

PURPOSE: To investigate the effects of inhibitors of chloride channels on lens volume and tissue architecture under isotonic conditions. METHODS: Rat lenses were maintained in organ culture under isotonic conditions in the presence of various putative chloride channel inhibitors. The effect of an inhibitor on lens wet mass and tissue morphology was determined by weighing and histologic examination, respectively. RESULTS: Exposure to 100 microM of either 5-nitro-2- (3-phenylpropylamino) benzoic acid (NPPB) or 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) caused an increase in wet mass and severe tissue disruption in the lens equatorial region. Two distinctly different zones of tissue damage were evident: a peripheral zone of fiber cell swelling and an inner zone of extensive tissue breakdown. Extracellular space dilations caused the extensive tissue damage in the inner zone and preceded the peripheral fiber cell swellings. That the observed effects were a consequence of the inhibition of chloride channels was supported by (1) the effectiveness of NPPB at the lower dose of 10 microM, (2) the absence of any NPPB effect in chloride-free medium, and (3) an identical effect after exposure to tamoxifen, an inhibitor of the chloride channel regulator p-glycoprotein. CONCLUSIONS: Study results indicate that chloride channels are active in the lens under isotonic conditions. The spatial and temporal pattern of morphologic changes that was observed is consistent with a steady state efflux of chloride ions and water from peripheral fiber cells and a corresponding influx into fiber cells deeper in the lens. These observations may therefore represent the first visualization of the chloride flux postulated by others to be a component of the lens internal circulation system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Quantitative determination of gap junctional permeability in the lens cortex.

We have developed a simple dye transfer method, which allows the gap junction permeability of lens fiber cells to be quantified. Two fixable fluorescent dyes (Lucifer yellow and rhodamine-dextran) were introduced into peripheral lens fiber cells via mechanical damage induced by removing the lens capsule. After a defined incubation period, lenses were fixed, sectioned, and the distribution of the dye recorded using confocal microscopy. Rhodamine-dextran and Lucifer yellow both labeled the extracellular space between fiber cells and the cytoplasm of fiber cells that had been damaged by capsule removal. For the gap junctional permeable dye Lucifer yellow, however, labeling was not confined to the damaged cells and exhibited intercellular diffusion away from the damaged cells. The extent of dye diffusion was quantified by collecting radial dye intensity profiles from the confocal images. Effective diffusion coefficients (D eff) for Lucifer yellow were then calculated by fitting the profiles to a series of model equations, which describe radial diffusion in a sphere. D eff is the combination of dye diffusion through the cytoplasm and through gap junction channels. To calculate the gap junctional permeability (Pj) an estimate of the cytoplasmic diffusion coefficient (Dcyt = 0.7 x 10(-6) cm2/sec) was obtained by observing the time course of dye diffusion in isolated elongated fiber cells loaded with Lucifer yellow via a patch pipette. Using this approach, we have obtained a value for Pj of 31 x 10(-5) cm/sec for fiber-fiber gap junctions. This value is significantly larger than the value of Pj of 4.4 x 10(-6) cm/sec reported by Rae and coworkers for epithelial-fiber junctions (Rae et al., 1996. J. Membrane Biol. 150:89-103), and most likely reflects the high abundance of gap junctions between lens fiber cells.

Animals↗
PubMed1999

Watching amyloid fibrils grow by time-lapse atomic force microscopy.

Late-onset diabetes is typically associated with amyloid deposits of fibrillar amylin in the pancreatic islets. Aqueous synthetic human amylin spontaneously forms polymorphic fibrils in vitro, and this system was used to examine the dynamics of fibril assembly. By time-lapse atomic force microscopy (AFM), the growth of individual amylin fibrils on a mica surface was observed over several hours. Prominent was the assembly of a protofibril with an elongation rate in these experiments of 1.1(+/-0.5) nm/minute. The assembly of higher order polymorphic fibrils was also observed. Growth of the protofibrils was bidirectional, i.e. it occurred by elongation at both ends. This ability of AFM to continuously monitor growth, directionality, and changes in morphology for individual fibrils, provides a significant advantage over spectroscopy-based bulk methods which average the growth of many fibrils and typically require 100 to 1000-fold more protein. The time-lapse AFM procedure used for human amylin here is thus likely to be applicable to fibril formation from other amyloid proteins and peptides.

Aluminum Silicates↗
PubMed1999

Connexins in the lens: are they to blame in diabetic cataractogenesis?

The pathohistology of the diabetic lens is an enigma. Under normal conditions the lens behaves as a functional syncitium, whereas the diabetic lens exhibits a localized zone of fibre cell swelling and rupture that is confined to the lens outer cortex. Because the lens fibre cells are extensively coupled by gap junction channels, it is believed that the abnormal closure of these channels is responsible for this phenomenon. New evidence concerning regional differences in gap junction gating supports this contention, and it is used to propose a new hypothesis that may explain the cellular changes observed in the diabetic lens.

Animals↗
PubMed1999

Localised fibre cell swelling characteristic of diabetic cataract can be induced in normal rat lens using the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid.

Chloride channels are known to be involved in the regulated volume decrease that occurs when the rat lens is exposed to hypotonic challenge. We now report that chloride channel blockage makes the rat lens gain water under isotonic conditions, suggesting that chloride and water fluxes may also play an important role under resting conditions. Histological comparison of hypotonically and isotonically swollen rat lenses revealed a significant difference: in the former, fibre cells were swollen from the periphery inwards, while in the latter, swollen fibre cells were confined to a discrete cortical zone which was located 150-200 micron from the lens surface with cells on either side of this zone appearing unaffected. This localised fibre cell swelling is remarkable because of its similarity to the situation in the diabetic rat lens.

Animals↗
PubMed1999

Differential expression of facilitative glucose transporters GLUT1 and GLUT3 in the lens.

PURPOSE: To determine the expression patterns for members of the facilitative glucose transporter family (GLUT1-4) in the rat lens. METHODS: An initial molecular profiling of GLUT expression in lens fiber cells was achieved using reverse transcription-polymerase chain reaction (RT-PCR). The presence of isoform-specific transcript detected by RT-PCR was then confirmed using northern blot analysis and in situ hybridization. The presence of transporter protein was verified by western blot analysis and immunocytochemistry. RESULTS: Transcripts for GLUT1 and GLUT3, but not for GLUT2 and GLUT4, were detected by RT-PCR of fiber cell mRNA. Transcript for GLUT3, but not for GLUT1, was detected by northern blot analysis of fiber cell total RNA, indicating that GLUT3 was the predominant isoform in the fiber cells. In situ hybridization and immunolocalization in rat lens sections confirmed this result at the transcript and protein levels, respectively. In contrast, GLUT1 was predominantly expressed in the lens epithelium and only to a limited extent in the equatorial fiber cells. CONCLUSIONS: GLUT1 and GLUT3 are differentially expressed in the rat lens. The presence of the high-affinity transporter GLUT3 in fiber cells indicates that these cells have the capacity to take up glucose independently from the epithelium.

Animals↗
PubMed1998

Purified lens major intrinsic protein (MIP) forms highly ordered tetragonal two-dimensional arrays by reconstitution.

Lens major intrinsic protein (MIP) is the founding member of the MIP family of membrane channel proteins. Its isolation from ovine lens fibre cell membranes and its two-dimensional crystallization are described. Membranes were solubilized with N-octyl-beta-D-glucoside and proteins fractionated by sucrose gradient centrifugation containing decyl-beta-D-maltoside. MIP was purified by cation exchange chromatography, and homogeneity was assessed by mass analysis in the scanning transmission electron microscope. Purified MIP reconstituted into a lipid bilayer at a low lipid-to-protein ratio formed highly ordered tetragonal two-dimensional crystals. The square unit cell had a side length of 6.4 nm, and exhibited in negative stain four stain-excluding elongated domains surrounding a central stain-filled depression. Projection maps of freeze-dried crystals exhibited a resolution of 9 A, and revealed a monomer structure of MIP consisting of distinct densities. Despite significant differences in the packing of tetramers in the crystals, the projection map of the MIP monomer was similar to that of aquaporin-1 (AQP1), the first member of the MIP family which had its structure resolved to 6 A. Our protocols for the purification and reconstitution of MIP establish the feasibility for future work to visualize structure elements which determine the diverse functional properties of the MIP family members.

Animals↗
PubMed1998

2D crystallization of membrane proteins: rationales and examples.

The difficulty in crystallizing channel proteins in three dimensions limits the use of X-ray crystallography in solving their structures. In contrast, the amphiphilic character of integral membrane proteins promotes their integration into artificial lipid bilayers. Protein-protein interactions may lead to ordering of the proteins within the lipid bilayer into two-dimensional crystals that are amenable to structural studies by electron crystallography and atomic force microscopy. While reconstitution of membrane proteins with lipids is readily achieved, the mechanisms for crystal formation during or after reconstitution are not well understood. The nature of the detergent and lipid as well as pH and counter-ions is known to influence the crystal type and quality. Protein-protein interactions may also promote crystal stacking and aggregation of the sheet-like crystals, posing problems in data collection. Although highly promising, the number of well-studied examples is still too small to draw conclusions that would be applicable to any membrane protein of interest. Here we discuss parameters influencing the outcome of two-dimensional crystallization trials using prominent examples of channel protein crystals and highlight areas where further improvements to crystallization protocols can be made.

Bacterial Proteins↗
PubMed1998

Spatial differences in gap junction gating in the lens are a consequence of connexin cleavage.

Gap junctions in the vertebrate lens exhibit spatial differences in pH gating: those in the cortical fibre cells close upon tissue acidification while those in the core region do not. It has been speculated that this difference in channel gating is a consequence of the cleavage of the connexins (Cx) that form the gap junction channels. We report the construction of a truncation mutant of ovine Cx50 which mimicks the cleavage in the intact lens. The construct when expressed in Xenopus oocytes results in the formation of functional channels. Comparison with full-length Cx50 revealed a significant reduction in the pH-sensitivity of the truncated form. This is the first evidence linking the non-uniform gating of gap junction channels in the lens with connexin cleavage. It also reveals how fibre cells in the core region remain connected despite the acidic environment caused by elevated lactate levels.

Animals↗