Proteins exposed to glucose over long periods are known to undergo physicochemical changes including crosslinking and formation of brown fluorescent pigments of poorly characterized structure. Acid hydrolysis of both browned poly(L-lysine) and browned bovine serum albumin is found to release a major fluorescent chromophore, which after alkalinization is extractable into organic solvents and which can be purified by. silica gel chromatography. The fluorescence properties of this compound very closely resemble those of the bulk browned polypeptides. By NMR, mass spectroscopy, and chemical derivatization, this compound is assigned the structure 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI). Confirmation was obtained by independent chemical synthesis from furylglyoxal and ammonia. properties.In recent years it has become evident that the Maillard reaction also occurs in vivo. This was first substantiated by the finding that glucose was attached via an Amadori rearrangement to the amino-terminal of the a-chain of hemoglobin (3, 4). Subsequently, glucose-protein adducts have been found in vivo for a number of proteins (5). Brown pigments with spectral and fluorescent properties similar to those of late-stage Maillard products have also been observed in vivo in association with several long-lived proteins-e.g., lens proteins and collagen-from aged individuals (6, 7). In fact, an age-related linear increase in pigment was observed in human dura collagen between the ages of 20 to 90 years (7).Besides imparting a color to these proteins, the pigments, by their crosslinking nature, could modify these long-lived proteins and account for the changes in proteins noted to occur during aging (8). The aging of collagen, for example, can be mimicked in vitro by the crosslinking induced by glucose (9). In addition, the trapping of other proteins to collagen may occur by a crosslinking reaction and account for the accumulation of albumin and antibodies in kidney basement membrane (9).The lack of detailed information on the chemistry of the late-stage Maillard process has made it difficult to form a clear understanding of the possible roles of this reaction in structural and functional changes in tissues. For some time we have sought to identify specific fluorescent chromophores derived from the reaction of glucose with amino groups of polypeptides. In the present paper, we report the isolation and characterization of a highly fluorescent yellowbrown product from the acid hydrolysates of bovine serum albumin and polylysine that have undergone nonenzymatic browning in vitro with glucose. By chemical and spectroscopic analysis and independent chemical synthesis we have identified this compound as 2-(2-furoyl)-4(5)-(2-furanyl)-lHimidazole (FFI). The similarity of the fluorescence properties of FFI to the fluorescence of nonenzymatically glycosylated polypeptides from which it is derived strongly suggests that FFI is representative of a chromophore present in the intact polypeptides. The possible pathways for the formation of...

Abstract. Irreversibly sickled cells (ISCs) remainsickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37°C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in /3-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control/3-actin is the inaccessibility of two cysteine residues in ISC ~/-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine TM and cysteine 373 in ISC/~-actin, or alternatively another modification of cysteine TM and cysteine 373 which is reversible with DTT and adds less than 100 D to the molecular weight of ~-actin.

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Propylene glycol accumulated significantly in pediatric intensive care patients receiving continuous lorazepam infusion, and propylene glycol concentration correlated with the cumulative lorazepam dose the patient received. However, significant laboratory abnormalities due to propylene glycol accumulation were not observed.

We have previously demonstrated that the membrane skeletons of irreversibly sickled cells (ISCs) dissociate more slowly at 37 degrees C, in high ionic strength Triton X-100 buffer, than do the membrane skeletons of reversibly sickled cells or control erythrocytes [Shartava et al. (1995) J. Cell. Biol. 128, 805-818]. Furthermore, we demonstrated that the major cause of this slow dissociation was a single posttranslational modification in ISC beta-actin. Two sulfhydryl groups (Cys284 and Cys373) became inaccessible to thiol reagents because of this modification. We suggested the possibility that the modification was a disulfide bridge between Cys284 and Cys373 since the reducing agent dithiothreitol restored the sulfhydryl groups. In this article, we directly demonstrate the existence of the disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin. We synthesized the associated ISC beta-actin tryptic cystine-peptide (KCF-CDVDIR), characterized it by HPLC, MS. and MSMS, and identified it in the tryptic digest of the ISC beta-actin. These results support our earlier suggestion that the oxidative change in ISC beta-actin is a major cause of the irreversible sickling phenomenon.

We have previously demonstrated that an oxidative change, the formation of a disulfide bridge between two cysteine residues, in the membrane protein beta-actin is primarily responsible for locking the irreversibly sickled red blood cells (ISCs) of sickle cell anemic patients into the sickle shape. To support studies on biological and chemical characterization of the oxidized beta-actin and pharmacological research toward the reversal of the oxidation, we attempted to prepare oxidized beta-actin from normal red blood cell (RBC) beta-actin by a chemical reaction, expecting a product equivalent to that found in ISCs. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB, or Ellman's reagent) was used for the oxidation. We proved the absence of accessible sulfhydryl groups in the oxidized product using liquid chromatography (LC) with both UV and fluorescence detection. Polymerization assays indicated that the chemically produced ISC actin demonstrated the same kinetics as ISC actin obtained from patients with sickle cell disease. The effect of the oxidation could be reversed by the use of the reducing agent tris(carboxyethyl)phosphine (TCEP).

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Electron ionization (EI), chemical ionization (CI) and fast-atom bombardment (FAB) mass spectra of the marine toxin okadaic acid and its synthetic methyl, pentafluorobenzyl, and trimethylsilyl ester and ether derivatives were generated. Several ionization conditions and ion-processing methods were used to obtain positive-and negative-ion conventional spectra and tandem (MSIMS) spectra. The EI and the positive-ion CI spectra provided fragment ions characteristic of the structure, and the negative-ion CI and FAB spectra provided molecular ions. The addition of alkali salts to the FAB matrix resulted in reduced fragmentation and the formation of intense alkali-metal-cationized molecules. Pentafluorobenzyl ester derivatives provided intense carboxylate ions under electron-capture ionization. Analytically useful MSIMS spectra were obtained by low-energy collision-induced decomposition of the carboxylate anion produced from the tetrasilylated pentafluorobenzylokadaate.In recent years, increased efforts to isolate and to identify marine toxins chemically have led to the discovery of several classes of toxins with distinctive chemical structures' and unique physiological activities.' Long-chain pol yethers containing several hydroxyl and occasionally a carboxyl, sulfhydryl, aldehyde or alkylamide group constitute one class with notable members such as the brevet ox in^,^ the ciguatoxin~,~ okadaic acid Author to whom correspondence should be addressed. and analogues,' and palytoxin .' The hydroxylated polyether structures of these toxins are closely related to the ionophoric antibiotics (monensin, salinomycin, nigericin, etc) described from terrestrial streptomycete bacteria.' The marine polyethers are part of a rapidly emerging group of toxins produced by dinoflagellate species of circumglobal distribution.Okadaic acid (OA) is a tetrahydroxylated pentamethyl monomethylene derivative of a C-38 heptaether carboxylic acid (Figure 1