Immunohistochemical detection of 4-hydroxy-2-nonenal adducts in Alzheimer's disease is associated with inheritance of APOE4

Cumulative oxidative damage, including lipid peroxidation, is a central component of cellular aging and is thought to play a role in the pathogenesis of late-onset Alzheimer's disease (AD). Lipid peroxidation produces several cytotoxic aldehydes, one of the most potent being 4-hydroxy-2-nonenal (HNE). We have shown previously that HNE is a potent neurotoxin that covalently modifies and cross-links neuronal cytoskeletal protein in neuroglial cultures, suggesting that HNE may contribute to the pathogenesis of AD. In addition to aging, inheritance of the epsilon 4 allele of APOE is the other major risk factor for development of late-onset AD; however, the mechanisms through which aging and apolipoprotein E isoforms may collaborate in the onset or progression of AD are not known. We tested the hypothesis that HNE may yield a particular type of protein modification, pyrrole adduction, and that this may contribute to the pathogenesis of AD. Our data demonstrated that HNE formed pyrrole adducts with protein. Polyclonal antiserum was raised that specifically recognized HNE pyrrole adducts, and immunohistochemical analysis was performed on hippocampus and temporal cortex of 10 patients with histologically verified AD. Pyramidal neuron cytoplasm was immunoreactive in 4 of 4 APOE4 homozygotes, 2 of 3 APOE3/4 heterozygotes, and none of 3 APOE3 homozygotes (P < 0.05). The pattern of staining was highly suggestive of neurofibrillary tangles as the primary immunoreactive structure. These data suggest that differences in neuronal protein modification by HNE may account in part for the APOE-associated stratification of risk for late-onset AD.     

Epoxidation of trans-4-hydroxy-2-nonenal by fatty acid hydroperoxides and hydrogen peroxide

In this study, we reported that fatty acid hydroperoxides and hydrogen peroxide are capable of epoxidizing 4-hydroxy-2-nonenal, a lipid peroxidation product, to the mutagenic epoxide. The evidence of its formation is provided (i) by trapping with [8-3H]deoxyadenosine for the formation of 7-(1',2'-dihydroxyheptyl)-1,N6-ethenodeoxyadenosine as a pair of diastereomers, (ii) by derivatization with (2,4-dinitrophenyl)hydrazine in acidic methanol, and (iii) by comparing its 1H-nuclear magnetic resonance and mass spectra to those of the authentic standard. After incubating 4-hydroxy-2-nonenal with 9- or 13-linoleic acid hydroperoxide at 37 degrees C for 24 h, the epoxide was produced in 13.4% or 12.5% yield, and with hydrogen peroxide, the yield was 21.5%. In the presence of fatty acid (linoleic acid, gamma-linolenic acid, or arachidonic acid) and lipoxygenase, the epoxide of 4-hydroxy-2-nonenal was formed in 15.3%, 7.2%, or 6.2% yield, respectively. The xanthine/xanthine oxidase/superoxide dismutase system generated the epoxide in 1.2% yield. These yields are estimated on the basis of a standard curve obtained from reactions of deoxyadenosine and epoxide. These results show that 4-hydroxy-2-nonenal is epoxidized by biological oxidants, suggesting a plausible endogenous pathway for the in vivo formation of etheno adducts.     

Simultaneous determination of acrolein, malonaldehyde and 4-hydroxy-2-nonenal produced from lipids oxidized with Fenton's reagent

Ethyl linoleate, ethyl linolenate, ethyl arachidonate and cod liver oil were oxidized with Fenton's reagent. Acrolein, malonaldehyde and 4-hydroxynonenal formed were derivatized to N-methylpyrazoline, N-methylpyrazole and 5-(1'-hydroxyhexyl)-1-methyl-2-pyrazoline with N-methylhydrazine, respectively. The derivatives were simultaneously analysed by gas chromatograph equipped with a fused silica capillary column and a nitrogen-phosphorus detector. The maximum amounts of acrolein (9.7 +/- 2.11 nmol/ml) and malonaldehyde (61.18 +/- 6.51 nmol/ml) were formed from cod liver oil. The highest amount of 4-hydroxynonenal (6.83 +/- 0.53 nmol/ml) was produced from ethyl arathidonate.     

Malondialdehyde and 4-hydroxy-2-nonenal in plant tissue cultures: LC-MS determination of 2,4-dinitrophenylhydrazone derivatives

The cytologically active secondary lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) have been detected as their 2,4-dinitrophenylhydrazone (DNP) derivatives in plant tissue cultures using LC-MS. This paper reports, for the first time, the use of LC-MS methodology to definitively identify 4-hydroxy-2-nonenal in plants. Limits of detection for the two derivatives are approximately 5 pmol (1.2 x 10(-9) g; 1 microM) and 0.1 pmol (3 x 10(-11) g; 20 nM) respectively. Mass spectrometer response was linear in the range from 2-200 microM DNP-MDA and 0.02-10 microM DNP-HNE. This methodology has been used to assess the formation of aldehydic secondary lipid peroxidation products in dedifferentiated callus cultures of Daucus carota. The finding that profiles of MDA and HNE can be correlated with embryogenic competence is of considerable interest as oxidative status has already been implicated as a regulatory factor in animal development.     

Identification of novel urinary metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in rats

Following iv administration of 4-hydroxy-2-nonenal (HNE) and [4-3H]HNE to rats, 15 polar urinary metabolites accounting for about 50% of the urinary radioactivity were separated by HPLC. Among them, eight major compounds and tritiated water were quantified. The metabolites were unequivocally characterized using GC/MS and ESI/MS/MS/MS. Most of "HNE polar metabolites" originate from omega-oxidation of 4-hydroxy-2-nonenoic acid (HNA): 9-hydroxy-HNA, its mercapturic acid conjugate, and two diastereoisomers of the corresponding lactone. The oxidation of 9-hydroxy-HNA by alcohol and aldehyde dehydrogenases leads to the excretion of 9-carboxy-HNA and of the corresponding lactone mercapturic acid conjugate. 1, 4-Dihydroxy-2-nonene (DHN) originating from the reduction of HNE by alcohol dehydrogenase was to a lesser extent omega-hydroxylated, leading to 9-hydroxy-DHN which was excreted as a mercapturic acid conjugate (two diastereoisomers).     

Selective inactivation of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase: reaction of lipoic acid with 4-hydroxy-2-nonenal

Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting alpha-ketoglutarate dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate dehydrogenase (PDH), while other NADH-linked dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in the presence of substrates that reduce the sulfur atoms of lipoic acid covalently bound to the E2 subunits of KGDH and PDH. In addition, loss of enzyme activity induced by HNE correlated closely with a decrease in the availability of lipoic acid sulfhydryl groups. Use of anti-lipoic acid antibodies indicated that HNE modified lipoic acid in both purified enzyme preparations and mitochondria and that this modification was dependent upon the presence of substrates. These results therefore identify a potential mechanism whereby free radical production and subsequent lipid peroxidation lead to specific modification of KGDH and PDH and inhibition of NADH-linked mitochondrial respiration.     

Characterisation of a novel AGE-compound derived from lysine and 3-deoxyglucosone

Dicarbonyl compounds are supposed to be reactive intermediates in the non-enzymatic glycation of proteins. A process that eventually could lead to the development of late diabetic complications. Glyoxal lysine dimer (GOLD) and methylglyoxal lysine dimer (MOLD) have previously been described as such reactive dicarbonyls. Here a new compound 3-deoxyglucosone lysine dimer (DOLD), a cross-link resulting from the reaction between hippuryl-lysine and 3-deoxyglucosone, has been isolated by HPLC and the structure determined by mass spectrometry and NMR.     

Relationships between 4-hydroxy-2-nonenal, 2-thiobarbituric acid reactive substances and n-6 polyunsaturated fatty acids in refrigerated and frozen pork

The relationships between 4-hydroxy-2-nonenal (HNE), 2-thiobarbituric acid reactive substances (TBARS) and n-6 polyunsaturated fatty acids (n-6 PUFAs) were investigated for pork stored at 0, -20 and -80 degrees C. A significant linear correlation was apparent between HNE and TBARS for the pork stored at each temperature. However, the n-6 PUFA content remained unchanged during storage.     

Synthesis and 32P-postlabeling/high-performance liquid chromatography separation of diastereomeric 1,N2-(1,3-propano)-2'-deoxyguanosine 3'-phosphate adducts formed from 4-hydroxy-2-nonenal

4-Hydroxy-2-nonenal (HNE), a major electrophilic byproduct of lipid peroxidation, is mutagenic and cytotoxic. The two pairs of HNE-derived diastereomeric 1,N2-propanodeoxyguanosine 3'-monophosphate adducts were synthesized from reaction of HNE with 2'-deoxyguanosine 3'-monophosphate. After HPLC separation, these adducts were characterized by UV-visible absorption and negative ion electrospray ionization MS/MS analysis. To further characterize the structures, these adducts were dephosphorylated to the corresponding HNE-modified deoxyguanosine adducts and their HPLC retention times and UV spectra were compared with those of the synthetic standards prepared from reaction of HNE with 2'-deoxyguanosine. Separation of these adducts by 32P-postlabeling/HPLC was developed. Reaction of HNE with calf thymus DNA resulted in only one pair of diastereomeric adducts, with one adduct predominantly formed with a modification level of 1.2 +/- 0.5 adducts/10(7) nucleotides.     

Structural and functional characterization of proteolytic fragments derived from the C-terminal regions of bovine fibrinogen

A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been characterized by sequence analysis, differential scanning calorimetry and circular dichroism. Determination of the extremities of the polypeptide chains forming individual fragments allowed the scheme of proteolysis and the borders between domains in the D region of fibrinogen to be established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The alpha-helical content of the newly prepared 21-kDa TSD2 and 16-kDa TSD3 fragments were 82% and 75%, respectively, strongly supporting a coiled-coil structure for this region of the fibrinogen molecule. The DX and DZ fragments, prepared from a chymotryptic digest of the DLA fragment, were found to be similar to the DL and DY fragments, respectively, except for an internal cleavage at K393-T394 in their beta chains. This cleavage leads to destabilization of all thermolabile domains, indicating interaction between them. The DL and DY fragments, containing only one polymerization site in their beta chains, were able to inhibit fibrin polymerization at high concentration. However, these same fragments failed to bind to fibrin-Sepharose under conditions where their structural analogues, DX and DZ, were tightly bound, indicating that cleavage after K393 substantially increases the affinity of this site.     

Preliminary studies of the toxic effects of non-ionic surfactants derived from lysine

The toxic effects of new synthetic monodisperse non-ionic long-chain N alpha, N epsilon-diacyl lysine polyoxyethylene glycol amide compounds with a structural resemblance to natural lecithin phospholipids were studied by the haemolytic method and the test of the chorioallantoic membrane of the hen's egg (HET-CAM). The following compounds were tested: symmetrical N alpha,N epsilon-diacyl lysine homologues (N alpha,N epsilon-dihexanoyl, N alpha,N epsilon-dioctanoyl and N alpha,N epsilon-didecanoyl lysine) with one methyl ether polyoxyethylene glycol chain of different oxyethylene units (dioxyethylene glycol, tetraoxyethylene glycol and hexaoxyethylene glycol) as headgroup; symmetrical N alpha,N epsilon-diacyl lysine homologues with two methyl ether dioxyethylene glycol chains and the asymmetrical N alpha-butanoyl, N epsilon-dodecyl lysine with two hydrophilic methyl ether dioxyethylene glycol chains as headgroup. A commercial (polydisperse) oleoyl polyoxyethylene glycol diethanolamide with an average of eight units of ethylene oxide was used as control. All the synthesized tested compounds appeared to be less haemolytic and less irritant than the control. The synthesized products were studied with regard to their hydrophobic and hydrophilic chains in order to evaluate the influence of their structure on their haemolytic and irritative action. The results of this study show that the acyl chain distribution of these compounds greatly influence toxic effects: the asymmetrical compound N alpha-butanoyl,N epsilon-dodecyl lysine-bis[methyl ether diethylene glycol]amide was found to be the most haemolytic and irritating compound. Among the symmetrical homologues, the shortest-chain compounds N alpha,N epsilon-dihexanoyl lysine methyl ether polyoxyethylene glycol amides present the least haemolytic and irritating activity, independently of the number and length of the hydrophilic methyl ether polyoxyethylene glycol chains. Taking into account their surface activity properties and their less haemolytic and irritant action, the compound N alpha,N epsilon-dioctanoyl lysine-bis[methyl ether diethylene glycol]amide would be the most suitable for practical purposes.     

2-羟基-5-特辛基苯乙酮肟的合成及表征

以四氯乙烯为溶剂,对特辛基苯酚、乙酰氯、无水AlCl3和盐酸羟胺为主要原料,采用分步合成法合成了2-羟基-5-特辛基苯乙酮肟.考察了无水AlCl3用量、乙酰氯的用量、Fries重排温度、肟化反应盐酸羟胺用量以及缚酸剂无水碳酸钠用量对产物产率的影响,实验结果表明,当n(AlCl3)∶n(乙酰氯)∶n(对特辛基苯酚)=1.5∶2∶1,Fries重排温度为120℃,n(Na2CO3)∶n(盐酸羟胺)∶n(对特辛基苯酚)=0.5∶1∶1.产物收率64.9%.对合成产物利用核磁共振进行了结构表征.     

Biological and immunochemical characterization of recombinant human thyrotrophin

Recombinant human thyroid-stimulating hormone (recTSH) has recently been engineered to detect metastatic lesions in patients operated on for thyroid cancer. In this report, we have compared the microheterogeneity, carbohydrate (CHO) content, mitogenic potency and immunoreactivity of the biotechnology product to those of human TSH of pituitary origin (pitTSH). Compositional analysis revealed that recombinant (rec) TSH produced in Chinese hamster ovary cells was overglycosylated compared with the native hormone (21 and 14%, respectively) with a higher amount of sialic acid and lack of N-acetylgalactosamine. Electrofocusing followed by immunoblotting resolved recTSH into six glycoforms with pIs ranging from 6.0 to 8.6, which were converted to a major species of pI 8.9 by sialidase treatment. pitTSH contained five main isoforms of pI 6.5-8.2 distinct from those of recTSH and partially resistant to sialidase. Binding activity of both human TSHs to porcine thyroid membrane receptors was found to be similar, but recTSH appeared to be 20% active compared to pitTSH in eliciting cAMP production and cell growth in rat FRTL-5 cells. Immunoreactivity of the recombinant hormone was investigated using polyclonal and monoclonal antibodies raised against the native hormone or synthetic peptide sequences of its subunits. While rec- and pitTSH were recognized to a similar extent by anti-protein antibodies, they exhibited a different binding pattern to antipeptide antibodies. Serial dilution of anti-alpha 1-25, anti-alpha 26-51, anti-beta 96-112 antisera bound recTSH to a greater extent than pitTSH, while anti-beta 31-51 and anti-beta 53-76 displayed similar recognition toward both preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a neuropathogenic simian immunodeficiency virus derived from a sooty mangabey

Transfusion of blood from a simian immunodeficiency virus (SIV)- and simian T-cell lymphotropic virus-infected sooty mangabey (designated FGb) to rhesus and pig-tailed macaques resulted in the development of neurologic disease in addition to AIDS. To investigate the role of SIV in neurologic disease, virus was isolated from a lymph node of a pig-tailed macaque (designated PGm) and the cerebrospinal fluid of a rhesus macaque (designated ROn2) and passaged to additional macaques. SIV-related neuropathogenic effects were observed in 100% of the pig-tailed macaques inoculated with either virus. Lesions in these animals included extensive formation of SIV RNA-positive giant cells in the brain parenchyma and meninges. Based upon morphology, the majority of infected cells in both lymphoid and brain tissue appeared to be of macrophage lineage. The virus isolates replicated very well in pig-tailed and rhesus macaque peripheral blood mononuclear cells (PBMC) with rapid kinetics. Differential replicative abilities were observed in both PBMC and macrophage populations, with viruses growing to higher titers in pig-tailed macaque cells than in rhesus macaque cells. An infectious molecular clone of virus derived from the isolate from macaque PGm (PGm5.3) was generated and was shown to have in vitro replication characteristics similar to those of the uncloned virus stock. While molecular analyses of this virus revealed its similarity to SIV isolates from sooty mangabeys, significant amino acid differences in Env and Nef were observed. This virus should provide an excellent system for investigating the mechanism of lentivirus-induced neurologic disease.     

Substrate selectivity and biochemical properties of 4-hydroxy-2-keto-pentanoic acid aldolase from Escherichia coli

4-Hydroxy-2-keto-pentanoic acid aldolase from Escherichia coli was identified as a class I aldolase. The enzyme was found to be highly selective for the acetaldehyde acceptor but would accept alpha-ketobutyric acid or phenylpyruvic acid in place of the pyruvic acid carbonyl donor.     

Establishment and characterization of a canine T-lymphoblastoid cell line derived from malignant lymphoma

A canine lymphoma cell line (CL-1) was established in culture from tumor cells found in the pleural fluid of a 7-year old female Japanese terrier with thymic form lymphoma. The CL-1 cells were positive for CD45 and MHC class II and negative for CD4, CD5, CD8, Thy-1 and B-cell specific antigen and surface immunoglobulin. The CL-1 cells had a rearranged T-cell receptor beta-chain gene and a germ-line form immunoglobulin gene, indicating that the CL-1 cells represented a monoclonally expanded population of canine alpha beta T-cell lineage.     

Biochemical and immunochemical characterization of Brassica juncea glyoxalase I

A homogenous preparation of glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1.5) was obtained from Brassica juncea seedlings. The enzyme is a heterodimer with 27,000 and 29,000 M(r) subunits and native M(r) of 56,000. The circular dichroic spectra of the protein showed characteristics of a distinctly helical protein, and magnesium affected the secondary structure. It is a zinc metalloenzyme. Amino acid modification studies suggested the involvement of histidine residues in catalysis. Apo-glyoxalase I was reactivated by divalent cations Mn2+ (0.5 Mm) > Mg2+ (5 Mm) > Zn2+ (0.05 Mm) and Ca2+ (0.01 Mm). Monospecific, polyclonal anti-glyoxalase I antibodies were raised, which showed its presence in seeds, roots, hypocotyl, cotyledon and different flower parts. They showed varied degree of cross reactivity with the extracts from various plants, yeast, bacteria and animal system.     

Structural identification of a major mitogenic lipid derived from Bacillus subtilis as a glycerophosphoglycolipid

Lipids extracted from Bacillus subtilis using a 2:1 mixture of chloroform and methanol have been found to be very mitogenic. These lipids were fractionated on a silica column and eluted with chloroform, acetone, and 60% methanol in chloroform, and the mitogenic activity was recovered in the last fraction. Further purification of the mitogenic components was achieved by HPLC on an amino-isopropyl bonded-phase column using a linear gradient of 5-20 mM ammonium acetate in a mobile phase consisting of hexane, 2-propanol, methanol, and water (5.5:8:1.5:1). Two major and several minor mitogenic peaks were observed. One major mitogenic lipid was isolated in pure form and structurally characterized by chemical degradation analysis, NMR spectroscopy, and mass spectrometry. Mild acid hydrolysis of the lipid released glycerol phosphate and a neutral glycolipid. Saponification of the lipid released a water-soluble head group and C14-C17 branched fatty acids. Total acid hydrolysis of the head group revealed the presence of glycerol and glucose in a ratio of 1:1. Mild acid hydrolysis of the head group to remove the glycerol phosphate produced a neutral partial head group. The partial head group was methylated and then analyzed by GLC-CIMS and by the reductive-cleavage method, which revealed that it was composed of nonreducing terminal glucopyranosyl, 6-linked glucopyranosyl, and 3-linked glycerol residues in equimolar proportions. Finally, the molecular weight of the permethylated head group, obtained by fast atom bombardment mass spectrometry, was 724.3340, which is consistent with the composition of two glucose residues, one glycerol residue, and one glycerol phosphate residue. On the basis of all these results, the intact mitogenic lipid was identified as 1,2-di-O-acyl-3-O-[6-(sn-glycerol-phospho)-beta-D-glucopyranosyl-(1-->6) - beta-D-glucopyranosyl]glycerol. The purified glycolipid possessed very potent mitogenic activity in a murine splenocyte proliferation assay at a concentration of 0.01-0.1 microgram/mL.     

Deoxyribonuclease I from rat urine: affinity purification, characterization, and immunochemical studies

Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000-fold to apparent homogeneity with a 14% yield by affinity chromatography utilizing polyguanylic acid-agarose and DNA-cellulose. The purified enzyme preparation was found to contain no other detectable nucleases. Isoelectric focusing electrophoresis revealed that all six isoelectric forms of the enzyme had been purified, and the resulting bands all contained DNase I activity. Quantitative amino acid analysis and N-terminal amino acid sequencing were performed on the purified DNase I. The N-terminal sequence up to the 15th residue of the enzyme was identical to that of rat parotid DNase I. The enzyme was found to be a glycoprotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine, and at least 3 sialic acid residues per molecule. The isoelectric multiplicity of the enzyme was partly due to differences in the sialic acid content of the isoforms. Gel filtration on Superose 12 and electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicated an approximate molecular mass for DNase I of 32 kDa. The enzyme had an optimum pH of 6.5 and required divalent cations such as Ca2+ for its activity. Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin. An antibody against the purified enzyme was found to be monospecific against rat urine and the pure antigen, and completely blocked the activity of the purified enzyme.