Biomedical subjects

R Kopito

Publications and source records attributed to R Kopito.

7 recordsLinked to original sources

PubMed2003

[Correlation between refraction and ocular biometry].

PURPOSE: To study the correlation between subjective refraction and biometry obtained by Orbscan and echography in normal eyes. To compare biometric parameters with the subjective spherical equivalent. MATERIAL AND METHODS: Subjective refraction, biometric parameters using Orbscan, and echography were recorded in 190 normal eyes (including eyes with ametropia) of 95 patients. Biometric parameters (i.e., corneal diameter, anterior chamber depth, central pachymetry, iridocorneal angle, corneal and lens radii of curvature, and axial length of the eye) were compared in different refractive groups and were correlated with the subjective refraction. RESULTS: Corneal biometric parameters did not correlate with the subjective spherical equivalent and showed no differences between the refractive groups except for the central pachymetry. In the high myopic group (<-6D), the central cornea was significantly thinner (531 micro m versus 549 micro m, p=0.016). The correlation between corneal radius and axial length was strong in emmetropic eyes (r(s)=0.63, p<0.001) and poor but significant in ametropic eyes (r(s)=0.28, p=0.002). Axial length, anterior chamber depth, and iridocorneal angle showed significant differences between the refractive groups (p<0.001) and correlated with the subjective spherical equivalent (r(s[[/INF=0.44, p<0.001). Subjective spherical equivalent showed the strongest correlation with the axial length (rs)=0.82, p<0.001). Subjective spherical equivalent, central corneal thickness, axial length, anterior chamber depth, and anterior corneal radius showed a strong correlation between both eyes (rs[[/INF=0.94, p<0.001). CONCLUSION: Biometric characteristics of the eye (excluding cornea characteristics) vary with subjective spherical equivalent. Axial length presents the strongest correlation with the subjective spherical equivalent and correlates with the other ocular biometric parameters. Axial length plays a major role in the ocular biometry and refraction.

Adult↗

PubMed1998

Topology of the region surrounding Glu681 of human AE1 protein, the erythrocyte anion exchanger.

AE1 protein transports Cl- and HCO3- across the erythrocyte membrane by an electroneutral exchange mechanism. Glu681 of human AE1 may form part of the anion translocation apparatus and the permeability barrier. We have therefore studied the structure of the sequence surrounding Glu681, using scanning cysteine mutagenesis. Residues of the Ser643 (adjacent to the glycosylation site) to Ser690 region of cysteineless mutant (AE1C-) were replaced individually with cysteine. The ability of mutants to mediate Cl-/HCO3- exchange in transfected HEK293 cells revealed that extracellular mutants, W648C, I650C, P652C, L655C, and F659C have an important role in transport. By contrast, only transmembrane mutation E681C fully blocked anion exchange activity. The topology of the region was investigated by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent 3-(N-maleimidylpropionyl)biocytin, with or without prior labeling with membrane-impermeant lucifer yellow iodoacetamide (LYIA). Two regions readily label with 3-(N-maleimidylpropionyl)biocytin (Ser643-Met663 and Ile684-Ser690). We propose that poorly labeled Met664-Gln683 corresponds to transmembrane segment 8 of AE1. Regions Ser643-Met663 and Ile684-Ser690 localize, respectively, to extracellular and intracellular sites on the basis of accessibility to LYIA. On the basis of LYIA accessibility, we propose that the Arg656-Met663 region forms a "vestibule" that leads anions to the transport channel. Glu681 is located 3 amino acids from the C terminus of transmembrane segment 8, which places the membrane permeability barrier within 5 A of the intracellular surface of the membrane.

Amino Acid Sequence↗

PubMed1997

Immuno and functional characterization of CFTR in submandibular and pancreatic acinar and duct cells.

Cystic fibrosis results from defective Cl- channel activity mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) gene product. In the gastrointestinal tract this is manifested in abnormal salivary secretion and pancreatic insufficiency. This is generally attributed to defective Cl- transport by the ductal system of the glands. We provide the first immunocytochemical and functional evidence for expression of CFTR protein and Cl- current in rat and mouse submandibular gland (SMG) and pancreatic acinar cells, a site proximal to the ductal system of these secretory glands. Monoclonal and polyclonal antibodies recognizing COOH-terminal epitopes of CFTR show that duct and acinar cells from the two glands express CFTR in the luminal membrane. Specificity of the polyclonal antibody was verified by absence of staining in duct and acinar cells of the SMG of cf-/cf- and delta F/delta F mice. Identification of CFTR in acinar cells was aided by demonstrating coexpression of CFTR and type 3 inositol 1,4,5-trisphosphate receptors in the luminal pole of acini and absence of type 3 inositol 1,4,5-trisphosphate receptors in ducts. Electrophysiological characterization in single SMG duct and acinar cells shows the presence of a protein kinase A-activated, voltage- and time-independent, ohmic Cl- current and absence of repolarization-dependent tail currents, all of which are kinetic properties of the CFTR-dependent Cl- channel. In addition, the channel was activated by the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate and the benzimidazalone NS-004. Channels activated by all activators were inhibited by glibenclamide and a known inhibitory antiserum [anti-CFTR-(505-511)]. Combined immunologic, functional, and pharmacological evidence allows us to conclude that acinar cells of the SMG and pancreas express functional CFTR-dependent Cl- channels. Because this site is proximal to the duct, modification of activity of this channel in acinar cells is likely to contribute to abnormal salivary secretion and pancreatic insufficiency typical of cystic fibrosis.

Animals↗

PubMed1996

Mapping of ankyrin binding determinants on the erythroid anion exchanger, AE1.

The association of ankyrin with the AE1 anion exchanger contributes an essential function to the mechanical and viscoelastic properties of the erythrocyte and constitutes the best understood link between the plasma membrane and the underlying membrane skeleton. The AE1 binding domain of ankyrin consists of 24 tandem repeats of a 33-amino acid motif that is present on a wide variety of otherwise unrelated proteins. The experiments described in this paper are aimed at identifying the specific amino acid sequences in AE1 that comprise the ankyrin binding site. We have exploited a cell-free binding assay to quantify the binding affinity of anion exchangers and a recombinant fragment of ANK1, R13-H. Our previous study (Ding, Y., Casey, J. R. and Kopito, R. R. (1995) J. Biol. Chem. 269, 32201-32208) identified an essential role of the amino-terminal 79 AE1 residues in ankyrin binding. The present study extends these findings to show that these 79 amino acids, although necessary, are not sufficient for ankyrin binding. Using chimeras between AE1 and the closely related anion exchanger AE2, which does not bind ankyrin, we have defined a 40-residue region of AE1 between positions 155 and 195 that is also essential for ankyrin binding.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1995

High level expression, partial purification, and functional reconstitution of the human AE1 anion exchanger in Saccharomyces cerevisiae.

Human erythroid anion exchanger AE1 (Band 3) was expressed in the yeast Saccharomyces cerevisiae under the control of the constitutive promoter and transcriptional terminator of the yeast phosphoglycerate kinase gene. AE1 expression in stable yeast transformants was estimated to be approximately 0.7 mg AE1 per liter. Density gradient sedimentation analysis indicated that the AE1 protein was associated with a membrane fraction distinct from plasma membrane, most likely the endoplasmic reticulum. AE1 protein was solubilized from yeast membranes with lysophosphatidyl choline, and the protein, tagged with six histidines at its amino terminus, was purified to 35% homogeneity by metal chelation affinity chromatography. Size-exclusion chromatography in the presence of octaethylene glycol monododecyl ether indicated that the solubilized yeast-expressed AE1, like endogenous erythroid AE1, eluted at a stokes radius of 77 A, consistent with a dimeric oligomeric state. Binding of partially purified yeast-expressed AE1 to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate resin was competitive with the transportable substrate chloride but not the nontransported anion citrate, suggesting that the structure of the anion binding site is preserved. The specific activity of sulfate transport by partially purified yeast AE1 was determined in proteoliposomes to be similar to that of authentic AE1 purified from erythrocyte membranes. These data show that this expression system has the capacity to produce functional mammalian plasma membrane anion exchangers at levels sufficient for biochemical and biophysical analysis.

Amino Acid Sequence↗

PubMed1989

A PstI polymorphism for the human erythrocyte surface protein band 3 (EPB3) demonstrates close linkage of EPB3 to the nerve growth factor receptor.

Erythrocyte surface protein band 3 (EPB3) plays an important role in CO2 transport in the blood. We have isolated a recombinant lambda bacteriophage that contains coding sequence for the human gene. Sequence analysis demonstrated that the human insert contains a portion of exon 13. A 1.1-kb BamHI fragment revealed a two-allele polymorphism with PstI. Alleles of 1.4 and 0.9/0.5 kb were present in Caucasoids at frequencies of 0.74 and 0.26, respectively. EPB3 has previously been mapped to 17q21-qter by in situ hybridization, and linkage analysis showed that EPB3 is tightly linked to the gene for the nerve growth factor receptor (NGFR). The maximum likelihood estimate of recombination (theta) is 0.00, with a lod score of 11.40 and confidence interval of 0.00 to 0.04.

Alleles↗

PubMed1988

A 115-kD polypeptide immunologically related to erythrocyte band 3 is present in Golgi membranes.

Band 3 multigene family consists of several distinct but structurally related polypeptides which are probably involved in the transport of anions across the plasma membrane of both erythrocytes and nonerythroid cells. A novel member of this family of polypeptides that resides in the Golgi complex was identified with antibodies to Band 3. The Golgi antigen had a larger molecular size and was antigenically distinct from Band 3 in the amino-terminal domain. It was expressed most prominently in cells that secrete large amounts of sulfated proteins and proteoglycans. This polypeptide may participate in sulfate transport across Golgi membranes.

Animals↗