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Biomedical subjects

A Spector

Publications and source records attributed to A Spector.

At least 109 records · Page 6Linked to original sources
PubMed1984

Oxidation and cataract.

Previous work has established that oxidation of the constituents of the human lens is an early event in the development of cataract. In old pre-cataractous lenses, oxidation of the fibre membrane polypeptides is observed. Non-disulphide-linked aggregates possibly generated by photo-oxidation are also found. With the development of cataract the oxidation becomes more extensive, affecting many of the proteins of the tissue. High molecular weight, disulphide-linked aggregates are formed, involving cytosol and membrane components. Membrane rupture accompanies the process. Hydrogen peroxide has been found in elevated levels in the aqueous fluid of some cataract patients. H2O2 will cause cataract and has been shown to affect Na+,K+-ATPase. Analyses of available data suggest that (1) oxidation of membrane components may be an initiating event in cataract, and (2) the oxidizing agent may come from the exterior environment of the tissue. The problems involved in proving this hypothesis are discussed and an approach to testing the hypothesis is suggested.

Aging↗
PubMed1984

A simple method to characterize gamma-crystallin synthesized in vitro.

Investigation of the migration of in vitro synthesized gamma-crystallin which has not been heat denatured on polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate indicates that this class of proteins behaves in an anomalous manner. While other in vitro synthesized lens proteins under these conditions migrate as theoretically expected, gamma-crystallin is retarded, having a mobility comparable to a 92 000 dalton component. Only upon heat denaturation does this protein fraction migrate as a 20 000 dalton species. Since other in vitro synthesized lens proteins have molecular weights of approximately 20 000 daltons, it is only with this methodology that gamma-crystallin synthesis can be unequivocally followed in a simple, rapid, one-dimensional fractionation system.

Animals↗
PubMed1984

Phosphorylation of H2O2-treated lens Na+,K+-ATPase.

It has been previously shown that H2O2 inhibits lens 86Rb influx and results in modification of Na+,K+-ATPase with respect to ATP hydrolysis. The effect of H2O2 on ATP hydrolysis was further investigated using [gamma-32P]-ATP to examine the Na+,K+-ATPase phosphorylated intermediate. A Na+-dependent phosphorylated polypeptide with an apparent molecular weight of approximately 100 000 was detected in all lens preparations, irrespective of H2O2 treatment. Similar results were observed for partially purified Na+,K+-ATPase from bovine kidney, porcine brain and canine kidney.

Animals↗
PubMed1984

The phosphorylation of bovine and human lens polypeptides.

The phosphorylation of bovine and human lens polypeptides was examined using an in vitro labeling assay with [gamma-32P]-ATP, followed by SDS-PAGE and radioautography. The major protein phosphorylation in all lens preparations was cAMP-dependent. The effect of cAMP could be depressed by monovalent ions. Phosphorylated polypeptides were detected in capsule-epithelial and outer cortical but not inner cortical-nuclear preparations. Differences in phosphorylated polypeptide distributions and molecular weight patterns were observed between bovine capsule-epithelium and outer cortex fiber cells and bovine and human preparations. Human cataractous lens preparations had the same pattern of phosphorylation as human normal lens preparations but with much less 32P incorporation. Major phosphorylated polypeptides in the 100 000 dalton, 60 000 dalton and 43 000 dalton range were detected in both bovine and human preparations. A 32 000 dalton phosphorylated polypeptide appeared exclusively in the bovine capsule-epithelium and a phosphorylated 23 000 dalton polypeptide was detected only in outer cortical preparations.

Animals↗
PubMed1984

The endopeptidase-resistant protein fraction from human cataractous lenses.

With aging and cataract formation, modifications in absorption and fluorescence of the human lens proteins are observed. These changes have been investigated by the examination of the endopeptidase-resistant fraction isolated from human cataractous lenses. This fraction is highly enriched in atypical fluorescence and absorption (i.e. not attributable to tryptophan, tyrosine or phenylalanine). It has a molecular weight of approximately 3000, is enriched in acidic amino acids and has only a 280 nm shoulder in its u.v. spectrum. The material does not contain detectable levels of malondialdehyde or N-formylkynurenine. Upon acid hydrolysis the fluorescence and u.v. spectra remain unchanged with only a minor degree of cleavage observed. Structural studies on some of the cleavage products indicated the presence of oxindolyl alanine and kynurenine. These compounds could result from photo-oxidation of tryptophan.

Amino Acids↗
PubMed1984

Nondisulfide polymerization of gamma- and beta-crystallins in the human lens.

The water-soluble 43,000-dalton fraction (WS43) of the human lens has been shown to be heterogeneous. It appears to contain, in addition to actin, components related to the crystallins. Immunoblot reactions indicate that this polypeptide fraction is composed of dimers containing beta- and gamma-crystallin components. It has been estimated that 10-30% of this fraction arises by dimerization of gamma-crystallin. A possible route for the formation of the 43,000-dalton fraction is suggested by the observation that photolysis of gamma-crystallin with light greater than 295 nm leads to polymer formation, including the 43,000-dalton fraction. The polymerization products react with anti-WS43. The results suggest that photochemical reactions may lead to the accumulation of polymers of some of the crystallins with aging of the human lens. Similar covalently linked polypeptides have previously been shown to be present in the high molecular weight aggregates associated with cataract formation.

Aging↗
PubMed1984

Complete nucleotide sequence of a cDNA derived from calf lens gamma-crystallin mRNA: presence of Alu I-like DNA sequences.

The nucleotide sequence of a cloned cDNA derived from gamma-crystallin mRNA of calf lens was determined. The cloned cDNA contains the entire coding region 522 bp long, 30 nucleotides of the 5' noncoding region, and 67 residues in the 3' noncoding region followed by a poly(A) tail of 25 nucleotides. The deduced amino acid sequence directly demonstrates for the first time that the calf gamma-crystallin contains 174 residues. The nucleotide sequence contains a number of interesting features including a 32-bp sequence in the 3' region with 70% complementarity to the 3' end of the first monomer unit of the consensus Alu I DNA. Within this region, a 32-bp sequence shows about 80% homology with a segment of hamster 4.5S RNA. The possible evolutionary and regulatory significance of these sequences is discussed.

Amino Acid Sequence↗
PubMed1984

Determination of the solvent accessibility of specific aromatic residues in gamma-crystallin by photo-CIDNP NMR measurements.

The surface or solvent accessibility of certain individual aromatic residues of calf-gamma II crystallin in solution (1 mM) were measured by the dramatic intensity enhancements of NMR lines generated by the interactions of cyclic radical pair formation of the 3-N-carboxymethyl lumiflavin (flavin I) dye excited (488nm) by an argon laser (5 watts) with the protein. This effect is called photo-chemically induced dynamic nuclear polarization: photo-CIDNP. The "light" and "dark" NMR spectra were taken in alternating scans in the pulsed Fourier transform mode on a Bruker 360 MHz instrument. Subtraction results in the photo-CIDNP difference spectrum containing lines of the polarized residues. With flavin dyes only tyrosine, histidine, and tryptophan can be polarized. The respective theoretical static accessibility of these residues based upon van der Waal's contact radii have been calculated from the atomic coordinates and provide a basis for evaluating the dynamic NMR photo-CIDNP results and for assigning the resonances. These results suggest that while the four tryptophan residues are completely buried, His-113 and His-14 of the five histidines; and Tyr-165 and Tyr-62 of the fifteen tyrosines are sufficiently exposed to elicit a photo-CIDNP effect. These results confirm and extend the observations previously obtained with theoretical electrostatic programs and FT-NMR measurements.

Animals↗
PubMed1984

HPLC analysis of lens GSH and GSSG.

A sensitive method for the analysis of reduced and oxidized glutathione in bovine lens fractions is described. Samples which have been derivatized with iodoacetic acid and dinitrofluorobenzene have been analyzed by high pressure liquid chromatography. Quantitation of reduced and oxidized glutathione in the sample at nanomole levels has been accomplished using a commercially available Bondapak-amine column. Values for reduced glutathione of approximately 30 mumoles/gm wet weight have been found for epithelium preparations while only 4 mumoles/gm wet weight were found in the nucleus. Only small amounts of oxidized glutathione were observed, increasing in the inner regions of the lens.

Animals↗
PubMed1984

Identification of two of the major phosphorylated polypeptides of the bovine lens utilizing a lens cAMP-dependent protein kinase system.

Two of the major in vitro phosphorylated polypeptides of the bovine lens have been identified. Analysis by means of two-dimensional gel electrophoresis (IEF) has demonstrated that the lens phosphorylated 57,000 and 43,000 dalton polypeptides correspond in mobility to purified phosphorylated bovine lens vimentin and chicken gizzard actin, respectively. Purified actin and vimentin were phosphorylated by a partially purified cAMP-dependent protein kinase isolated from the outer cortex water soluble fraction. All detectable bovine lens vimentin isoelectric variants were phosphorylated. In both the lens fiber cell and chicken gizzard actin preparations, the phosphorylated actin isoelectric variants did not correspond in mobility to the major actin isoelectric variant, but were more acidic. Phosphorylation in all preparations occurred at serine residues.

Actins↗
PubMed1983

Lens calcium activated proteinase: degradation of vimentin.

The lens has been shown to contain a Ca+2 activated proteinase specific for vimentin. The proteinase is present in the soluble fraction of the cortex but not in the epithelium. It is suggested that this proteinase is expressed during terminal differentiation of the epithelial cells and may be responsible for degradation of the intermediate filaments in the fiber cells. The proteinase is inhibited by EGTA but not by several proteinase inhibitors.

Animals↗
PubMed1983

Polyamines in normal and cataractous human lenses: evidence for post-translational modification.

The polyamine composition of normal and cataractous human lenses has been studied. Polyamines (putrescine, spermidine and spermine) have been shown to occur in the unbound form in the acid-soluble fraction and in the bound form in the acid-insoluble fraction of the lens. It has also been shown that only putrescine occurs in both conjugated and non-conjugated states in the lens, while putrescine as well as spermidine occur in both forms in the aqueous fluid. Estimation of the polyamine content in cataractous lenses indicates elevated levels of both bound and unbound polyamines in these lenses in comparison to the normal lenses. Evidence is presented which indicates an increase in the level of polyamines covalently bound to protein, in a gamma-glutamyl linkage, in the cataractous lens.

Aged↗
PubMed1983

The state of neutral lipids in normal and cataractous human lenses.

A simple and rapid high pressure liquid chromatographic method (HPLC) was developed to analyse the neutral lipid fraction from normal and cataractous human lenses. No differences were found between old normal and cataractous lenses suggesting that membrane deterioration in cataracts is not due to oxidation of these components. Several new lens components were isolated and identified including cholesterol metabolites, and vitamin E (alpha-tocopherol).

Adult↗
PubMed1983

H2O2-induced uncoupling of bovine lens Na+,K+-ATPase.

A 1-hr exposure of bovine lenses in organ culture to H2O2 concentrations in the range found in the aqueous fluid of patients with cataracts inhibits 86Rb+ influx. At 1 mM H2O2, complete inhibition was observed and further investigated. Membrane permeability is slightly decreased. Although lactate concentrations increase 2-fold, lens ATP concentrations decrease approximately equal to 10%, suggesting that glycolysis may be stimulated but ATP production is not able to keep up with the demand for energy. Examination of epithelial cell Mg2+-stimulated Na+,K+-ATPase isolated from the cultured lenses indicates H2O2-induced modification. At 5 mM MgATP, ATP hydrolysis is accelerated 30%; at 3 mM MgATP, hydrolysis is normal; and at 0.75 mM MgATP, it is inhibited 75%. p-Nitrophenyl phosphate hydrolysis and eosin maleimide binding indicate that K+ control of the enzyme is modified. Thus, a very early effect of H2O2 upon the lens, well before the formation of opacity, appears to be the uncoupling of Na+ and K+ transport from ATP hydrolysis.

Adenine Nucleotides↗