Laura R. Miesbauer

Glutathione adducts, not carbamylated lysines, are the major modification of lens α-crystallins from renal failure patients

Abstractα-Crystallins from the water-soluble and the water-insoluble, guanidine-soluble portions of lenses from four renal failure patients and two normal donors of similar age were isolated and enzymatically digested into peptides. Molecular weights of the peptides, determined by fast atom bombardment mass spectrometry, indicated modifications specifically associated with renal failure. The only modifications observed in theα-crystallins from renal failure patients, but not in the normal old lenses, were glutathione adducts to Cys 131 and Cys 142. These adducts were present in the lenses of all four renal failure patients, but not in the two normal old lenses. The four lenses from the renal failure patients were searched for evidence of carbamylation at lysyl or cysteinyl residues: carbamylation was not detected. Because the same mass spectrometric methods had previously demonstrated sufficient sensitivity and specificity to detect as little as 5% modification in the examination ofin vitro carbamylated bovine lenses, these results indicated that carbamylation is not a major modification of the lensα-crystallins of renal failure patients.

New cytotoxic annonaceous acetogenins: Bullatanocin and cis- and trans-Bullatanocinone, from Annona bullata (Annonaceae)

Abstract Further bioactivity-directed fractionation of the ethanol extract of the bark of Annona bullata Rich. (Annonaceae) has led to the isolation of the new nonadjacent bis-tetrahydrofuran acetogenins, bullatanocin ( 1 ), cis- bullatanocinone ( 2 ) and trans-bullatanocinone ( 3 ). A known adjacent bis-tetrahydrofuran acetogenin, desacetyluvaricin ( 4 ), which is new to this species, was also isolated. Brine shrimp lethality test (BST) data and cytotoxicities against human solid tumor cell lines of 1–4 were compared with those of the diastereoisomers, bullatalicin ( 5 ), cis- and trans-bullatalicinone ( 6 and 7 ) and 4-deoxyasimicin ( 8 ). 1 shows cytotoxic potencies 10,000 times those of adriamycin in the lung and colon cancer cell lines.

Biologically active acetogenins from stem bark of Asimina triloba.

In continuing our research with cytotoxic and pesticidal components from the stem bark of the North American paw paw, Asimina triloba, the novel cytotoxic monotetrahydrofuran Annonaceous acetogenins, cis- and trans-annonacin-A-one, cis- and trans-gigantetrocinone and cis-isoannonacin, in addition to the known compounds, trans-isoannonacin and squamolone, have been identified. Brine shrimp lethality testing was used to direct the fractionation. The structures were elucidated by spectral analysis and/or chemical synthesis. These acetogenins have potent cytotoxicities against the human tumour cell lines of A-549 (lung carcinoma), MCF-7 (breast carcinoma) and HT-29 (colon adenocarcinoma).

A new type of cytotoxic annonaceous acetogenin: Giganin from goniothalamus giganteus

A new cytotoxic Annonaceous acetogenin, giganin (1), was isolated from the bark of Goniothalamus giganteus (Annonaceae). This compound represents a new type of Annonaceous acetogenin lacking tetrahydrofuran or epoxide rings along the aliphatic chain, and its structure was elucidated by 1H and 13C NMR, MS, COSY, and single-relayed COSY.

Mass spectrometric analysis of lens β-crystallins

Abstract A combination of electrospray ionization mass spectrometry (ESIMS), fast atom bombardment mass spectrometry (FABMS), and sodium dodecylsulfate-polyacrylamide get electrophoresis (SDS-PAGE) was used to determine the homegeneity of chromatographic fractions of β-crystallins, separated by reversed-phase HPLC. The molecular weight of the major component of β-crystallins, βB2, was found by ESIMS to be 23 215, in agreement with the accepted value of 23 209. The practical utility of improved resolution and accuracy of ESIMS relative to SDS-PAGE of identifying proteins is discussed. The component βB2 was found in more than one HPLC fraction, suggesting that it migrates through the HPLC column aggregated with itself or other β-crystallins. Several proteins with different molecular weights were found to be unique to each of the two classes of β-crystallins. Tryptic digest of the major HPLC fractions were analyzed by FABMS, on-line HPLC continuous-flow FABMS—MS to give detailed information about the primary structure of βB2. Peptides related to all but one portion of the proposed amino acid sequence of βB2 were found in the tryptic digest, suggesting that the proposed sequence is correct. On-line HPLC continuous-flow FABMS analysis of the proteolytic digest was particularly attractive because it gave a nearly complete peptide molecular weight map in 20 min.

Post translational modification of crystallins isolated from human lenses

Crystallins, the structural proteins of the lens, have been isolated from human lenses using a combination of gel filtration and reversed phase high performance liquid chromatography (HPLC). The molecular weights of the isolated crystallins have been determined by electrospray ionization mass spectrometry. The isolated crystallins have also been proteolytically digested into peptides, the peptides fractionated by HPLC, and the masses of the peptides determined by fast atom bombardment mass spectrometry. With these techniques, it is possible to confirm and/or correct known protein sequences and identify and locate post translational modifications. I NTRODU CTl ON Cataract, which can be defined as an opacity of the eye lens, is the leading cause of blindness worldwide. In countries where surgery to remove a cataractous lens and replace it with a synthetic lens is readily available, cataract may not be a serious impairment; however, in much of the world this surgery is not available, and the development of cataract leads to blindness. The two types of cataract, cortical and nuclear, both involve changes in the lens proteins, called the crystallins. For the lens to be transparent, the crystallins must be tightly and uniformly packed producing a lens with a uniform refractive index. In cortical cataract, the density of the crystallins is altered, causing a non-uniform density with variations in the lens refractive index and consequent light scattering (ref. 1). Nuclear cataract is associated with the formation of insoluble protein aggregates. In both cases, there are modifications to the lens crystallins that either prevent their normal close packing in the cortex or cause their aggregation in the nucleus. The focus of our research is to determine which proteins are modified, where the modifications are located, and the identity of the modifications. Cataract is most commonly associated with old age. Most people over age 80 have at least early indications of cataract. Cataract occurs earlier in people with diabetes, renal failure, chronic diarrhea or who have experienced prolonged exposure to W radiation. The mechanisms that have been proposed for the development of cataract vary, depending on the disease with which it is associated. Cataract associated with old age, diabetes, renal failure and chronic diarrhea are all proposed to occur because of modifications to the lysyl residues of the crystallins. In old age, the concentration of glutathione in the lens is lower. This permits an increase in the concentration of dehydroascorbic acid, which is normally reduced by glutathione in younger lenses. Dehydroascorbic acid can react with the amino groups of the lysyl residues, forming a Schiff base which can then rearrange or possibly form cross-links (ref. 2). In diabetes, the elevated concentrations of glucose may lead to formation of a similar Schiff base between the lysyl residues and glucose (ref. 3). Both renal failure and chronic diarrhea are associated with elevated urea, which forms an equilibrium with isocyanate. Isocyanate can also react with lysyl residues forming a carbamylated protein (refs. 4, 5). A different mechanism, oxidation of the tryptophan residues to form kynurenine, has been proposed