Fibril formation by primate, rodent, and Dutch-hemorrhagic analogues of Alzheimer amyloid beta-protein.

Deposition of extraneuronal fibrils that assemble from the 39-43 residue beta/A4 amyloid protein is one of the earliest histopathological features of Alzheimer's disease. We have used negative-stain electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and fiber X-ray diffraction to examine the structure and properties of synthetic peptides corresponding to residues 1-40 of the beta/A4 protein of primate [Pm(1-40); human and monkey], rodent [Ro(1-40); with Arg5-->Gly, Tyr10-->Phe, and His13-->Arg], and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) [Du(1-40); with Glu22-->Gln]. As controls, we examined a reverse primate sequence [Pm*(40-1)] and an extensively substituted primate peptide [C(1-40); with Glu3-->Arg, Arg5-->Glu, Asp7-->Val, His13-->Lys, Lys16-->His, Val18-->Asp, Phe19-->Ser, Phe20-->Tyr, Ser26-->Pro, Ala30-->Val, Ile31-->Ala, Met35-->norLeu, Gly38-->Ile, Val39-->Ala, and Val40-->Gly]. The assembly of these peptides was studied to understand the relationship between species-dependent amyloid formation and beta/A4 sequence and the effect of a naturally occurring point mutation of fibrillogenesis. The three N-terminal amino acid differences between Pm(1-40) and Ro(1-40) had virtually no effect on the morphology or organization of the fibrils formed by these peptides, indicating that the lack of amyloid deposits in rodent brain is not due directly to specific changes in its beta/A4 sequence. beta-Sheet and fibril formation, judged by FT-IR, was maximal within the pH range 5-8 for Pm(1-40), pH 5-10.5 for Du(1-40), and pH 2.5-8 for Ro(1-40).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome C is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis

The generation of reactive oxygen species has been suggested to occur at increased rates during apoptosis, but the validity and significance of this remain contentious. In several key studies levels of reactive oxygen species have been monitored using the intracellular probe dichlorofluorescin (DCFH(2)), which undergoes oxidation to the fluorescent dichlorofluorescein (DCF). We report here that cytochrome c, which is released from mitochondria during cell death, is a potent catalyst of DCF formation. In a model system using xanthine oxidase to generate superoxide radicals, the rate of DCF formation was insensitive to changes in the rate of superoxide production over a 17-fold range, but extremely sensitive to nanomolar concentrations of cytochrome c. Thus we conclude that the DCF fluorescence observed in dying cells is a reflection of increased cytosolic cytochrome c. Moreover, we suggest that the suppression of DCF formation by the anti-apoptotic oncoprotein Bcl-2, which has been suggested to have antioxidant properties, can be explained on the basis of its prevention of mitochondrial cytochrome c release.     

In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis

Intracellular production of active oxygen in the green alga Haematococcus pluvialis was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate to the fluorescent dye dichlorofluorescein in different algal cell types (i.e., vegetative, immature cyst and mature cyst cells). The increase in formation of dichlorofluorescein by methyl viologen (superoxide anion radical generator) was linear for 2 h in immature cyst cells (low astaxanthin) in a methyl viologen-concentration-dependent manner, while no production was detected in mature (high astaxanthin) cysts. Compared to cyst cells, formation of dichlorofluorescein in vegetative cells (no astaxanthin) was markedly increased by methyl viologen. The formation of dichlorofluorescein in cyst cells was decreased with higher astaxanthin content under excessive oxidative stress. All of the active oxygen species tested (singlet oxygen, superoxide anion radical, hydrogen peroxide and peroxy radical) at 10⁻³ M increased the intracellular dichlorofluorescein formation in immature cysts, but did not increase the dichlorofluorescein content of mature cysts. Therefore, astaxanthin in cyst cells appeared to function as an antioxidant agent against oxidative stress. Received: 26 January 2000 / Received revision: 5 April 2000 / Accepted: 1 May 2000     

植物细胞中蓝光诱导ROS生成的识别和亚细胞定位检测

以2’,7’-二氯二氢荧光素二乙酯(dichlorofluorescein diacetate,H2DCF-DA)为荧光探针孵育拟南芥叶表皮条,利用荧光光谱和激光共聚焦扫描显微技术,对高辐照蓝光诱导下叶肉细胞活性氧(reactive oxygen spe-cies,ROS)的生成,进行了分子识别和亚细胞定位检测。结果表明:植物细胞在蓝光诱导下,可以产生大量的ROS。过氧化氢酶清除实验表明:高辐照蓝光诱导产生的ROS,主要成分是H2O2,并且主要定位在叶绿体和细胞膜上。     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

The role of Tyr605 and Ala607 of thimet oligopeptidase and Tyr606 and Gly608 of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.     

Copper catalyzed oxidation of ascorbate (vitamin C). Inhibitory effect of catalase, superoxide dismutase, serum proteins (ceruloplasmin, albumin, apotransferrin) and amino acids

The inhibitory effect of catalase and superoxide dismutase on copper catalyzed oxidation of ascorbate is probably due to a binding of copper ions. Scavengers of hydroxyl ions and singlet oxygen had no effect on the ascorbate oxidation rate. Copper binding serum proteins reduced the oxidation rate; the order of effectiveness being: Ceruloplasmin greater than human albumin = bovine albumin greater than apotransferrin. The excellent protection obtained with catalase and ceruloplasmin is possibly due to a strong affinity for cuprous ions generated during the reaction. Cupric ion binding amino acids (His, Thr, Glu, Gln, Tyr) had considerably weaker protective effect than the proteins studied. Apparently they do not compete favorably with ascorbate for cupric ions.     

Production of nerve growth factor enhanced in cultured mouse astrocytes by glycerophospholipids, sphingolipids, and their related compounds

The presence of cysteine and methionine groups together with an ability to bind long-chain fatty acid (LCFA) oxidation products makes liver fatty acid binding protein (L-FABP) an attractive candidate against hepatocellular oxidative stress. In this report, we show that pharmacological treatment directed at modulating L-FABP level affected hepatocellular oxidant status. L-FABP expressing 1548-hepatoma cells, treated with dexamethasone or clofibrate, decreased and increased intracellular L-FABP levels, respectively. Oxidative stress was induced by H₂O₂ incubation or hypoxia–reoxygenation. The fluorescent marker, dichlorofluorescein (DCF), was employed to measure intracellular reactive oxygen species (ROS). Hepatocellular damage was assessed by lactate dehydrogenase (LDH) level. Dexamethasone treatment resulted in a significant increase in DCF fluorescence with higher LDH release compared to control cells. Clofibrate treatment, however, resulted in a significant decrease in both parameters (p < 0.05). Drug treatments did not affect cytosolic activites of glutathione peroxidase (GPx), superoxide dismutase (SOD), or catalase suggesting that the differences between treated and control cells may likely be associated with varying L-FABP levels. We conclude that L-FABP may act as an effective endogenous cytoprotectant against hepatocellular oxidative stress.     

Amino acid substitutions of His296 alter the catalytic properties of Zymomonas mobilis 10232 levansucrase

His296 of Zymomonas mobilis levansucrase (EC 2.4.1.10) is crucial for the catalysis of the transfructosylation reaction. The three-dimensional structures of levansucrases revealed the His296 is involved in the substrate recognition and binding. In this study, nine mutants were created by site-directed mutagenesis, in which His296 was substituted with amino acids of different polarity, charge and length. The substitutions of His296 with Arg or Trp retained partial hydrolysis and transfructosylation activities. The positively charged Lys substitution resulted in a 2.5-fold increase of sucrose hydrolysis. Substitutions with short (Cys or Ser), negatively charged (Glu) or polar (Tyr) amino acids virtually abolished both the activities. Analysis of transfructosylation products indicated that the mutants synthesized different oligosaccharides, suggesting that amino acid substitutions of His296 strongly affected both the enzyme activity and transfructosylation products.     

Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements.

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.     

Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides

The photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides has been modified such that the bacteriochlorophyll dimer, when it becomes oxidized after light excitation, is capable of oxidizing tyrosine residues. One factor in this ability is a high oxidation-reduction midpoint potential for the dimer, although the location and protein environment of the tyrosine residue appear to be critical as well. These factors were tested in a series of mutants, each of which contains changes, at residues L131, M160, M197, and M210, that give rise to a bacteriochlorophyll dimer with a midpoint potential of at least 800 mV. The protein environment was altered near tyrosine residues that are either present in the wild type or introduced by mutagenesis, focusing on residues that could act as acceptors for the phenolic proton of the tyrosine upon oxidation. These mutations include Ser M190 to His, which is near Tyr L162, the combination of His M193 to Tyr and Arg M164 to His, which adds a Tyr-His pair, and the combinations of Arg L135 to Tyr with Tyr L164 to His, Arg L135 to Tyr with Tyr L144 to Glu, and Arg L135 to Tyr with Tyr L164 to Phe. Radicals were produced in the mutants by using light to initiate electron transfer. The radicals were trapped by freezing the samples, and the relative populations of the oxidized dimer and tyrosyl radicals were determined by analysis of low-temperature electron paramagnetic resonance spectra. The mutants all showed evidence of tyrosyl radical formation at high pH, and the extent of radical formation at Tyr L135 with pH differed depending on the identity of L144 and L164. The results show that tyrosine residues within approximately 10 A of the dimer can become oxidized when provided with a suitable protein environment.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Photosensitized oxidation of 2',7'-dichlorofluorescin: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2',7'-dichlorofluorescein

2',7'-Dichlorofluorescin (DCFH) is often employed to assess oxidative stress in cells by monitoring the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. We have investigated the photosensitized oxidation of DCFH in solution and elucidated the role played by singlet molecular oxygen (1O(2)) in this reaction. We used rose bengal (RB), protoporphyrin, and DCF as photosensitizers. Irradiation (550 nm) of RB (20 microM) in 50 mM phosphate (pH 7.4) in the presence of DCFH (50 microM) resulted in the rapid formation of DCF, measured as an increase in its characteristic absorbance and fluorescence. The oxidation rate was faster in deoxygenated solution, did not increase in D(2)O, and even increased in the presence of sodium azide. The presence of antioxidants that react with 1O(2), thus removing oxygen, accelerated DCF formation. Such results eliminate any potential direct involvement of 1O(2) in DCF formation, even though DCFH is an efficient (physical) quencher of 1O(2) (k(q) = 1.4 x 10(8) M(-1)s(-1) in methanol). DCF is also a moderate photosensitizer of 1O(2) with a quantum yield of circa phi = 0.06 in D(2)O and phi = 0.08 in propylene carbonate, which unequivocally indicates that DCF can exist in a triplet state upon excitation with UV and visible light. This triplet can initiate photo-oxidization of DCFH via redox-and-radical mechanism(s) similar to those involving RB (vide supra). Our results show that, upon illumination, DCF can function as a moderate photosensitizer initiating DCFH oxidation, which may prime and accelerate the formation of DCF. We have also shown that, while 1O(2) does not contribute directly to DCF production, it can do so indirectly via reaction with cellular substrates yielding peroxy products and peroxyl radicals, which are able to oxidize DCFH in subsequent dark reactions. These findings suggest that DCFH should not be regarded as a probe sensitive to singlet molecular oxygen, and that care must be taken when using DCFH to measure oxidative stress in cells as a result of both visible and UV light exposure.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase

The peroxidative oxidation of extracted rat liver microsomal lipid, assayed as malondialdehyde production, can be promoted by milk xanthine oxidase in the presence of 0.2 mM FeCl₃ and 0.1 mM EDTA. The reaction is inhibited by the superoxide dismutase activity of erythrocuprein. The reaction is also inhibited by 1,3-diphenylisobenzofuran, which reacts with singlet oxygen to yield dibenzoylbenzene. During inhibition of the lipid peroxidation reaction by 1,3-diphenylisobenzofuran, o-dibenzoylbenzene was produced. The rate of superoxide production by xanthine oxidase was not affected by 1,3-diphenylisobenzofuran. Lipid peroxidation promoted by ascorbic acid is not inhibited by either erythrocuprein or 1,3-diphenylisobenzofuran. Therefore it is suggested that the peroxidative oxidation of unsaturated lipid promoted by xanthine oxidase involves the formation of singlet oxygen from superoxide, and the singlet oxygen reacts with the lipid to form fatty acid hydroperoxides.     

Direct oxidation of 2',7'-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production

Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2',7'-dichlorodihydrofluorescein (DCFH2), in cells loaded with the latter compound is often used to detect ROS formation. We previously found that exposure of DCFH2-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production. However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH2 to DCF without an ROS intermediate. Incubation of DCFH2 with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the [pyocyanin] and was not inhibited by SOD or catalase. Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF. Mitoxantrone and ametantrone also produced DCF. However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not. Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2',7'-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate. Under aerobic conditions, the interaction of DCFH2 with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping. Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.     

Chaperone function of mutant versions of alpha A- and alpha B-crystallin prepared to pinpoint chaperone binding sites.

A major stress protein, alpha-crystallin, functions as a chaperone. Site-directed mutagenesis has been used to identify regions of the protein necessary for chaperone function. In this work we have taken some of the previously described mutants produced and assessed their chaperone function by both a traditional heat-induced aggregation method at elevated temperature and using enzyme methods at 37 degrees C. In general the different assays gave parallel results indicating that the same property is being measured. Discrepancies were explicable by the heat lability of some mutants. Most mutants had full chaperone function showing the robust nature of alpha-crystallin. A mutant corresponding to a minor component of rodent alpha A-crystallin, alpha Ains-crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139--> Arg, Thr144-->Arg, Ser59-->Ala mutants of alpha B-crystallin and double mutants Ser45-->Ala/Ser59-->Ala, Lys103--> Leu/His104-->Ile, and Glu110-->His/His111-->Glu. A mutant Phe27-->Arg that was the subject of previous controversy was shown to be fully active at physiological temperatures.     

Histidine-13 is a crucial residue in the zinc ion-induced aggregation of the A beta peptide of Alzheimer's disease.

Metal ions such as Zn(2+) and Cu(2+) have been implicated in both the aggregation and neurotoxicity of the beta-amyloid (Abeta) peptide that is present in the brains of Alzheimer's sufferers. Zinc ions in particular have been shown to induce rapid aggregation of Abeta. Rat Abeta binds zinc ions much less avidly than human Abeta, and rats do not form cerebral Abeta amyloid. Rat Abeta differs from human Abeta by the substitution of Gly for Arg, Phe for Tyr, and Arg for His at positions 5, 10, and 13, respectively. Through the use of synthetic peptides corresponding to the first 28 residues of human Abeta, rat Abeta, and single-residue variations, we use circular dichroism spectroscopy, sedimentation assays, and immobilized metal ion affinity chromatography to show that the substitution of Arg for His-13 is responsible for the different Zn(2+)-induced aggregation behavior of rat and human Abeta. The coordination of Zn(2+) to histidine-13 is critical to the zinc ion induced aggregation of Abeta.     

Oxidation pathways underlying the pro-oxidant effects of apigenin

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (H₂DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H₂DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H₂DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H₂DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.     

Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization

The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).