Search PubMed⌕ Search

Biomedical subjects

J Kistler

Publications and source records attributed to J Kistler.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

A distinct membrane current in rat lens fiber cells isolated under calcium-free conditions.

PURPOSE: To investigate whether lens fiber cells isolated and maintained under calcium-free conditions exhibit distinct membrane currents. METHODS: Fiber cells were isolated from the cortical portion of neonate rat lenses using a trypsin digestion protocol and were maintained in EDTA-buffered Ringer's solutions. Membrane currents were recorded from fiber bundles using the whole-cell patch-clamp technique. RESULTS: Cortical fiber cells of up to 600-microm length were viable and amenable to whole-cell patch-clamp recording. The major current recorded under these conditions was a slowly activating, voltage-dependent current that was markedly increased on membrane depolarization. This current appeared to be fiber cell specific and had similar properties to currents elicited by gap junction hemichannels previously recorded by others in Xenopus oocytes. CONCLUSIONS: This is the first report of whole-cell patch-clamp recordings from intact elongated fiber cells. Fiber cells kept in calcium-free bath medium appear to be electrically "leaky" and exhibit a distinct membrane current that has not been described previously for lens cells. This current is unlikely to be active in the normal lens but may play a role in the depolarized cataractogenic lens.

Animals↗
PubMed1998

Expression profiles of P2-receptor isoforms P2Y1 and P2Y2 in the rat lens.

PURPOSE: To verify at the molecular level that P2 receptors are expressed in the lens and to determine their expression profiles. METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) was used to screen rat lens epithelial and fiber cells for the expression of the ionotropic P2X2 receptor and the G-protein-linked receptor isoforms P2Y1 and P2Y2. Northern blot analysis was used to confirm the level of expression of P2Y1 and P2Y2. The profile of P2-receptor isoform expression in the lens was identified using an indirect RT-PCR in situ hybridization procedure on paraffin sections of whole rat eyes. RESULTS: P2X2-receptor transcripts could not be detected in lens epithelial or fiber cells. P2Y1- and P2Y2-receptor transcripts were detected in lens fiber cells but not in the epithelial cells. Their expression profiles were maximal in the lens cortex and mostly overlapped each other, except that transcripts for the P2Y2-receptor isoform appeared earlier in the not yet fully elongated fiber cells in the lens bow region. CONCLUSIONS: The molecular data support physiological evidence previously reported by others that P2Y receptors are expressed in the lens and identifies cortical fiber cells as the principal site of expression.

Animals↗
PubMed1997

Polymorphic fibrillar assembly of human amylin.

Human amylin forms fibrillar amyloid between pancreatic islet cells in patients with non-insulin-dependent (type 2) diabetes mellitus. Fibrillar assemblies also form in vitro in aqueous solutions of synthetic human amylin. We now report on the structural polymorphism of these fibrils. The thinnest fibril, referred to as the protofibril, has an apparent width of 5 nm but is only rarely observed by itself. These protofibrils spontaneously assemble into higher order fibrillar structures with distinct morphologies. Prominent among these is an 8-nm fibril with a distinct 25-nm axial crossover repeat which is formed by left-handed coiling of two 5-nm protofibrils. Coiling of more than two 5-nm protofibrils results in cable-like structures of variable width depending on the number of protofibrils involved. Lateral (side-by-side) assembly of 5-nm protofibrils is also observed and produces ribbons which may contain two, three, four, or more protofibrils and occasionally large single-layered sheets. The mass-per-length (MPL) of the 5-nm protofibril is 10 kDa/nm. This has been established in two ways: first, the 8-nm fibril, which is formed by coiling two 5-nm protofibrils around each other, has an MPL of 20 kDa/nm. Second, higher order fibrils differ by increments of 10 kDa/nm. Hence, about 2.6 human amylin molecules (3904 Da) are packed in 1 nm of protofibril length. Similarities exist between amylin fibrils and those formed from other amyloid proteins, suggesting that the in vitro assembly of synthetic protein may serve as a useful model system in advancing our understanding of amyloid formation in disease.

Amyloid↗
PubMed1997

Gap junction channels: new roles in disease.

The importance of intercellular communication to complex cellular processes such as development, differentiation, growth, propagation of electrical impulses and diffusional feeding has long been appreciated. The realization that intercellular communication is mediated by gap junction channels, which are in turn comprised of a diverse family of proteins called the connexins, has provided new tools and avenues for studying the role of intercellular communication in these important cellular processes. The identification of different connexin isoforms has not only enabled the development of specific reagents to study connexin expression patterns, but has also allowed the functional properties of the different connexin isoforms and how they interact with each other, to be explored. Increasingly, the knowledge gained from studying connexin diversity is being used to investigate the role played by gap junction channels in a number of diseases. In this article we highlight selected cases where gap junction channels have been shown or are believed to be directly involved in the disease process.

Animals↗
PubMed1997

Processing of the gap junction protein connexin50 in the ocular lens is accomplished by calpain.

Gap junction channel forming connexins share a common membrane topology which has four transmembrane spanning segments with the amino- and carboxy termini both located on the cytoplasmic side. Both, mutation and truncation of the carboxyl tail of some connexins have been shown previously to have profound effects on channel function. Truncation of the carboxyl tail of connexin50 (Cx50) and connexin46 (Cx46) occurs naturally during the maturation of fiber cells in the mammalian lens. This system therefore offers the unique opportunity to study not only the cleavage process but also the functional role played by the cleaved domain, in a physiologically relevant context. As a first step, we now report on the cleavage of the 70 kDa ovine isoform of Cx50. The calcium-activated neutral protease calpain (EC 3.4.22.17) was identified as the enzyme which removed a 32 kDa carboxyl portion from the Cx50 molecule in mature lens fiber cells. The amino-terminal 38 kDa portion remained embedded in the plasma membrane and was isolated and visualized as channel structures. The amino-terminal sequence of the cleaved 32 kDa portion matched an interior portion of the published amino acid sequence of the ovine Cx50 isoform. Thus, two closely spaced calpain cleavage sites were identified in the Cx50 molecule which were located carboxy-terminal from the predicted exit of the fourth transmembrane spanning segment by 62 or 72 amino acid residues, respectively. These data provide the basic information required for the future construction of Cx50 mutants to explore the functional consequences of this cleavage.

Amino Acid Sequence↗
PubMed1996

Liquefaction of cortical tissue in diabetic and galactosemic rat lenses defined by confocal laser scanning microscopy.

PURPOSE: To investigate whether a histologic link exists between osmotic fiber cell swelling and cortical tissue liquefaction in experimentally induced diabetic and galactosemic cataractogenesis of the rat lens. METHODS: Confocal laser scanning microscopy, in conjunction with specific membrane labels and correlative transmission electron microscopy, was used to image large cortical areas with precise definition of the individual cells. RESULTS: In both cataract models, tissue liquefaction--defined as the disintegration of tissue and the appearance of large fluid-filled spaces--typically was limited to a discrete zone in the lens cortex. The borders of the liquefaction zone were characterized by transitions between normal-appearing cells and swollen cells, which gained in size as plasma membranes ruptured and cytoplasmic contents fused and ultimately burst, thereby contributing to the formation of large fluid-filled spaces. During cataractogenesis, before tissue liquefaction became evident, selected fiber cells appeared swollen and accumulated specifically in the zone destined for tissue liquefaction. With increasing duration of diabetes or galactosemia, swollen fiber cells in this zone became more frequent and enlarged, resulting first in tissue disorder and then in tissue disintegration and the formation of large fluid-filled spaces. CONCLUSIONS: New imaging protocols strongly support a direct involvement of lens fiber cell swelling in the liquefaction of cortical tissue. The appearance of swollen fiber cells in the lens cortex, therefore, can be used as an early indicator of the histopathology of sugar cataractogenesis.

Animals↗
PubMed1995

Ocular lens gap junctions: protein expression, assembly, and structure-function analysis.

Recent advances in understanding lens fiber gap junction formation are reviewed. These include studies of junctional protein expression in the embryonic lens, and of age related changes affecting gap junction structure and composition in the adult lens. An in vitro assembly system based on detergent solubilized pore complexes and endogenous lipids has been developed to provide information on the molecular interactions involved in gap junction formation and to provide material for structure analysis. Important information on the electrical properties of lens gap junction channels is obtained using electrophysiological techniques including planar lipid bilayer analysis and patch clamping.

Animals↗
PubMed1995

Changes in lens connexin expression lead to increased gap junctional voltage dependence and conductance.

The differentiation of mouse lens epithelial cells into fiber cells is a useful model for studying the changes of the electrical properties of gap junction (cell-to-cell) channels that are induced by an alteration in connexin expression patterns. In this model, cuboidal lens epithelial cells differentiate into elongated fiber cells, and the expression of connexin43 (Cx43) in the epithelial cells is replaced with the production of high levels of Cx50 and Cx46 in the fiber cells. We now report a new procedure to isolate mouse lens fiber cell pairs suitable for double whole cell patch-clamp analysis. Analysis was also performed for fiberlike cell pairs differentiated from epithelial cells in culture. Voltage dependence and unitary conductance of fiber cell gap junction channels were determined and compared with the corresponding values previously measured for the channels joining lens epithelial cells and for lens connexin channels formed in Xenopus oocyte pairs. Our results support a differentiation-induced shift toward stronger gap junctional voltage dependence and larger unitary conductances in the fiber cells. Our data further reflect a balanced functional contribution of Cx50 and Cx46 in the fiber cell-to-cell channels rather than a predominance of a single connexin.

Animals↗
PubMed1994

Reconstitution of native-type noncrystalline lens fiber gap junctions from isolated hemichannels.

Gap junctions contain numerous channels that are clustered in apposed membrane patches of adjacent cells. These cell-to-cell channels are formed by pairing of two hemichannels or connexons, and are also referred to as connexon pairs. We have investigated various detergents for their ability to separately solubilize hemichannels or connexon pairs from isolated ovine lens fiber membranes. The solubilized preparations were reconstituted with lipids with the aim to reassemble native-type gap junctions and to provide a model system for the characterization of the molecular interactions involved in this process. While small gap junction structures were obtained under a variety of conditions, large native-type gap junctions were assembled using a novel two-step procedure: in the first step, hemichannels that had been solubilized with octylpolyoxyethylene formed connexon pairs by dialysis against n-decyl-beta-D-maltopyranoside. In the second step, connexon pairs were reconstituted with phosphatidylcholines by dialysis against buffer containing Mg2+. This way, double-layered gap junctions with diameter < or = 300 nm were obtained. Up to several hundred channels were packed in a noncrystalline arrangement, giving these reconstituted gap junctions an appearance that was indistinguishable from that of the gap junctions in the lens fiber membranes.

Animals↗
PubMed1994

Differential expression of two gap junction proteins in corneal epithelium.

Two distinct gap junction proteins (connexins) are expressed in rat corneal epithelium in a way which parallels cellular differentiation processes in this tissue. Connexin43 is restricted predominantly to the basal cells of the corneal epithelium and is present in significantly reduced amounts compared to the situation in the adjacent conjunctival epithelium. In contrast, a gap junction protein recognized by antibodies against MP70 which is the ovine homolog of mouse connexin50, is strongly expressed in the corneal epithelium and is present in the basal cells, wing cells and surface cells. While the functional significance of this differential expression of corneal epithelial connexins has yet to be established, the corneal epithelium is the third avascular tissue besides lens and heart valves which expresses a gap junction protein recognized by anti-MP70 antibodies.

Animals↗
PubMed1994

Electrical properties of mammalian lens epithelial gap junction channels.

PURPOSE: To establish an "electrical fingerprint" for the gap junction channels between mammalian lens epithelial cells. METHODS: The double whole cell patch clamp technique was applied to isolated cell pairs obtained from mouse lens epithelium and a continuous cell line of lens epithelial cells derived from the sheep lens (SLE 2.1). RESULTS: The junctional conductance in mouse lens epithelial cells and in cultured SLE 2.1 cells was found to be moderately voltage dependent. SLE 2.1 cells were analyzed in more detail. The voltage dependence could be described by a Boltzmann distribution with Vo = +/- 63.1 mV and Gmin = 0.34. In cell pairs that exhibited spontaneously low junctional conductance, single channel events could be distinguished. Single gap junction channel currents had a linear current-voltage relationship. A frequency histogram of single channel conductances from eight cell pairs had three major peaks of 35, 60 and 97 pS. CONCLUSION: The electrical properties of gap junction channels between mammalian lens epithelial cells were virtually identical to those previously reported for transfected cell lines expressing connexin43. The authors' physiological data are therefore in agreement with molecular studies that have identified connexin43 as the major connexin of lens epithelial cells.

Animals↗
PubMed1993

Gap junction formation during development of the mouse lens.

We have identified a 60 kDa membrane protein (MP60) as a component of the mouse cortical lens fiber gap junction and a monoclonal antibody recognizing this protein has been used to establish the temporal and spatial patterns of gap junction formation during development of the mouse lens. The initial expression of MP60 during embryonic development of the mouse lens correlates with primary fiber elongation and is first seen on the luminal aspect of the extending cells. About 2 days after birth, the relatively large, antibody-positive macular structures characteristic of late embryonic fiber cells begin to disperse into progressively smaller structures within a centrally located region of the lens. This change in the staining pattern with antibody directed against MP60 is consistent with the dispersion of gap junction plaques as confirmed by freeze fracture analysis. Around 5 days after birth, the 60 kDa gap junctional protein in this central region of the lens undergoes a modification resulting in the alteration to the epitope for the monoclonal antibody and a consequent loss of immunorecognition. Our results suggest that gap junctions in the central region of the developing mouse lens undergo sequential changes in immunoreactivity which may reflect potentially distinct functional phases of intercellular communication.

Age Factors↗
PubMed1992

Reconstitution of channels from preparations enriched in lens gap junction protein MP70.

Detergent-solubilized ovine lens membrane proteins, enriched in the 70-kDa gap junction component (MP70), were reconstituted into planar lipid bilayers and analyzed for channel activities. Three distinct activities were found. Those showing conductance steps of 290 pS (symmetrical 150-mM KCl solutions) had properties similar to those reported earlier for MIP26 (Ehring, G.R., Zampighi, G., Horwitz, J., Bok, D., Hall, J.E. 1990. J. Gen. Physiol. 96:631-664.) of which minor amounts were normally present in the detergent-solubilized preparations. Two novel channel activities had unitary conductances of 90 and 45 pS, were halothane sensitive and did not discriminate between sodium and potassium ions. The 90-pS channel was asymmetrically voltage dependent, and its properties would be consistent with the expected properties of junctional hemichannels.

Animals↗
PubMed1991

In vitro assembly of gap junctions.

Gap junction structures were assembled in vitro from octyl-beta-D-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = b = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.

Animals↗
PubMed1990

MP38 contains the membrane-embedded domain of the lens fiber gap junction protein MP70.

A 70-kDa lens membrane polypeptide (MP70) is a specific component of the fiber gap junctions. The C-terminal portion of MP70 is removed by age-related proteolytic processing, leaving an N-terminal 38-kDa polypeptide (MP38) in the membrane. Membrane association and topology of MP70 and of its processed form MP38 have been studied by hydrophobic labeling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and phenyl isothio[14C]cyanate. Membrane-embedded segments have been identified. They are localized in the N-terminal 30-kDa portion of MP70 and MP38. The C-terminal 40-kDa portion of MP70 appears to be exposed entirely at the cytoplasmic side of the junctional membranes. Hence, potentially poreforming peptide segments in the MP70 molecule are conserved upon age-related processing to MP38.

Amino Acid Sequence↗
PubMed1990

Molecular portrait of lens gap junction protein MP70.

A 70-kDa membrane protein (MP70) is a component of the lens fiber gap junctions. Its membrane topology and its N-terminal sequence are similar to those of the connexin family of proteins. Some features of MP70 containing fiber gap junctions are, however, distinct from gap junctions in other mammalian tissues: (i) Lens connexons form crystalline arrays only after cleavage of junctional proteins in vitro. These hexagonal arrays have a periodicity of 13.6 nm which is significantly larger than the 8- 9-nm spacing of liver and heart gap junctions. (ii) Lens fiber gap junctions dissociate in low concentrations of nonionic detergent and this provides an avenue to purify MP70 directly from a membrane mixture. Isolated MP70 in the form of 17 S structures has an appearance consistent with connexon pairs. (iii) The C-terminal half of MP70 is cleaved in situ by a lens endogenous calcium-dependent protease. The processed from MP38 remains in the membrane and is abundant in the central region of the lens. A testable hypothesis for MP70 function is presented.

Connexins↗