BBA - Molecular Basis of Disease (v.1537, #1)

In the present study the effect of thyroid hormone (T3) on oxidative stress parameters of mitochondria of rat liver is reported. Hypothyroidism is induced in male adult rats by giving 0.05% propylthiouracil (PTU) in drinking water for 30 days and in order to know the effect of thyroid hormone, PTU-treated rats were injected with 20 μg T3/100 g body weight/day for 3 days. The results of the present study indicate that administration of T3 to hypothyroid (PTU-treated) rats resulted in significant augmentation of oxidative stress parameters such as thiobarbituric acid reactive substances and protein carbonyl content of mitochondria in comparison to its control and euthyroid rats. The hydrogen peroxide content of the mitochondria of liver increased in hypothyroid rats and was brought to a normal level by T3 treatment. Induction of hypothyroidism by PTU treatment to rats also resulted in the augmentation of total and CN-sensitive superoxide dismutase (SOD) activities of the mitochondria, which was reduced when hypothyroid rats were challenged with T3. Although CN-resistant SOD activity of the mitochondria remained unaltered in response to hypothyroidism induced by PTU treatment, its activity decreased when hypothyroid rats were injected with T3. The catalase activity of the mitochondria decreased significantly by PTU treatment and was restored to normal when PTU-treated rats were given T3. Total, Se-independent and Se-dependent glutathione peroxidase activities of the mitochondria were increased following PTU treatment and reduced when T3 was administered to PTU-treated rats. The reduced and oxidised glutathione contents of the mitochondria of liver increased significantly in hypothyroid rats and their level was restored to normal when hypothyroid rats were injected with T3. The results of the present study suggest that the mitochondrial antioxidant defence system is considerably influenced by the thyroid states of the body.
Keywords: Liver; Mitochondria; 3,3′,5-Triiodothyronine; Oxidative stress; Antioxidant enzyme; Rat;

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A 330 units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar R f values for the long wavelength-absorbing fluorophores. Glycation with [U-14C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A 330-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A 330-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A 330-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses represent advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo.
Keywords: Human lens; Brunescent cataract; Ascorbic acid; Advanced glycation endproduct; Maillard reaction; Yellow chromophore; Fluorescence; Aging;

Cholera toxin-induced PGE2 activity is reduced by chemical reaction with l-histidine by Johnny W. Peterson; David King; Edward L. Ezell; Mark Rogers; Deborah Gessell; Jennifer Hoffpauer; Luis Reuss; Ashok K. Chopra; David Gorenstein (27-41).
Mediators of cholera toxin (CT)-induced fluid secretion include 3′,5′-adenosine monophosphate (cAMP), prostaglandin E2 (PGE2), and 5-hydroxytryptamine (5-HT). Administration of l-histidine significantly reduced the net secretory response of the small intestine of mice challenged with CT and reduced the capacity of PGE2 to stimulate Na+ transport in Ussing chambers. We demonstrated that l-histidine chemically modified the structure of PGE2 but had no direct effect on cAMP or 5-HT. l-Histidine and imidazole reacted with PGE2 in vitro in cell-free mixtures incubated at 37°C and pH 7.0 under an atmosphere of N2 with the formation of PGE2–imidazole and PGE2–histidine covalent adducts. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analysis of the purified adduct showed that imidazole catalyzed the dehydration of PGE2. A Michael adduct then was formed between C11 of 11-deoxy-Δ10 PGE2 (PGA2) and the tau nitrogen in the imidazole ring of l-histidine. Importantly, the isolated PGE2–imidazole and PGE2–histidine adducts inhibited CT-induced fluid loss and cAMP accumulation in mouse intestinal loops. The protection provided by PGE2–imidazole, PGE2–histidine, and l-histidine against intestinal fluid loss could provide a basis for future therapy against cholera.
Keywords: Prostaglandin; Imidazole; l-Histidine; Structure; Cholera toxin;

Homogeneous assay based on 52 primer sets to scan for mutations of the ABCA1 gene and its application in genetic analysis of a new patient with familial high-density lipoprotein deficiency syndrome by Katarzyna Lapicka-Bodzioch; Marek Bodzioch; Matthias Krüll; Danuta Kielar; Mario Probst; Beata Kiec; Hajnalka Andrikovics; Alfred Böttcher; Jaroslav Hubacek; Charalampos Aslanidis; Norbert Suttorp; Gerd Schmitz (42-48).
Familial high-density lipoprotein (HDL)-deficiency syndromes are caused by mutations of the ABCA1 gene, coding for the ATP-binding cassette transporter 1. We have developed a homogeneous assay based on 52 primer sets to amplify all 50 ABCA1 exons and approximately 1 kb of its promoter. The assay allows for convenient amplification of the gene from genomic DNA and easy mutational analysis through automatic sequencing. It obviates the need to use mRNA preparations, which were difficult to handle and posed a risk to miss splice junction or promoter mutations. The application of the test to the molecular analysis of a new patient with familial HDL-deficiency (Tangier disease) led to a discovery of two novel ABCA1 mutations: C2665del and C4457T.
Keywords: ABCA1; Familial high-density lipoprotein deficiency; Tangier disease; Genetic testing;

The role of Kupffer cell α2-adrenoceptors in norepinephrine-induced TNF-α production by Mian Zhou; Shaolong Yang; Douglas J. Koo; David A. Ornan; Irshad H. Chaudry; Ping Wang (49-57).
Although previous studies have demonstrated that plasma levels of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) increase during early sepsis, the precise mechanism responsible for its upregulation remains to be elucidated. Since recent studies have shown that the gut is an important source of norepinephrine (NE) release during early sepsis and enterectomy prior to the onset of sepsis attenuates TNF-α production, we hypothesized that gut-derived NE plays a major role in upregulating TNF-α via the activation of α2-adrenoceptors on Kupffer cells. To confirm that NE increases TNF-α synthesis and release, Kupffer cells were isolated from normal rats and incubated with NE (20 or 50 nM) or another α2-adrenergic agonist clonidine (50 nM) without addition of Escherichia coli endotoxin. Supernatant levels of TNF-α were then measured. In additional animals, intraportal infusion of NE (20 μM) with or without the specific α2-adrenergic antagonist yohimbine (1 mM) at a rate of 13 μl/min was carried out for 2 h. Plasma and Kupffer cell levels of TNF-α were assayed thereafter. Moreover, the effects of NE and yohimbine on TNF-α production was further examined using an isolated perfused liver preparation. The results indicate that both NE and clonidine increased TNF-α release by approximately 4–7-fold in the isolated cultured Kupffer cells. Similarly, intraportal infusion of NE in vivo or in isolated livers increased TNF-α synthesis and release which was inhibited by co-infusion of yohimbine. Furthermore, the increased cellular levels of TNF-α in Kupffer cells after in vivo administration of NE was also blocked by yohimbine. These results, taken together, suggest that gut-derived NE upregulates TNF-α production in Kupffer cells through an α2-adrenergic pathway, which appears to be responsible at least in part for the increased levels of circulating TNF-α observed during early sepsis as well as other pathophysiologic conditions such as trauma, hemorrhagic shock, or gut ischemia/reperfusion.
Keywords: Isolated perfused liver preparation; Catecholamine; Clonidine; Yohimbine; Proinflammatory cytokine; Endotoxin; Sepsis;

Expression of BRI–amyloid β peptide fusion proteins: a novel method for specific high-level expression of amyloid β peptides by P.A. Lewis; S. Piper; M. Baker; L. Onstead; M.P. Murphy; J. Hardy; R. Wang; E. McGowan; T.E. Golde (58-62).
In order to develop transgenic animal models that selectively overexpress various Aβ peptides, we have developed a novel expression system that selectively expresses Aβ40 or Aβ42 in the secretory pathway. This system utilizes fusion constructs in which the sequence encoding the 23-amino-acid ABri peptide at the carboxyl terminus of the 266-amino-acid type 2 transmembrane protein BRI is replaced with a sequence encoding either Aβ40 or Aβ42. Constitutive processing of the resultant BRI-Aβ fusion proteins in transfected cells results in high-level expression and secretion of the encoded Aβ peptide. Significantly, expression of Aβ42 from the BRI–Aβ42 construct resulted in no increase in secreted Aβ40, suggesting that the majority of Aβ42 is not trimmed by carboxypeptidase to Aβ40 in the secretory pathway.
Keywords: Amyloid β protein; British familial dementia; Alzheimer disease; Fusion proteins;

Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase by Christian C Lee; Francisco J. Medrano; Sydney P Craig; Ann E Eakin (63-70).
Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the enzyme from humans can result in gouty arthritis or Lesch–Nyhan syndrome, and in parasites these enzymes are potential targets for antiparasite chemotherapy. In HPRTs, a long flexible loop (active site loop II) closes over the active site during the enzyme catalyzed reaction. Functional roles for this loop have been proposed but have yet to be substantiated. For the present study, seven amino acids were deleted from loop II of the HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant enzyme (Δloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the enzyme. Loop II deletion resulted in moderate (0.6–2.7-fold) changes in the Michaelis constants (K ms) for substrates other than pyrophosphate (PPi), for which there was a 5.8-fold increase. In contrast, k cat values were severely affected by loop deletion, with rates that were 240–840-fold below those for the wild-type enzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PPi as a product of the salvage reaction.
Keywords: Phosphoribosyltransferase; Hypoxanthine; Mutagenesis; Transition state; Loop deletion;

Novel mutations in ABCA1 gene in Japanese patients with Tangier disease and familial high density lipoprotein deficiency with coronary heart disease by Wei Huang; Kengo Moriyama; Takafumi Koga; Han Hua; Masato Ageta; Seiro Kawabata; Koji Mawatari; Takuro Imamura; Tanenao Eto; Mitsunobu Kawamura; Tamio Teramoto; Jun Sasaki (71-78).
Mutations in the ATP-binding cassette transporter 1 (ABCA1) gene have been recently identified as the molecular defect in Tangier disease (TD) and familial high density lipoprotein deficiency (FHA). We here report novel mutations in the ABCA1 gene in two sisters from a Japanese family with TD who have been described previously (S. Ohtaki, H. Nakagawa, N. Kida, H. Nakamura, K. Tsuda, S. Yokoyama, T. Yamamura, S. Tajima, A. Yamamoto, Atherosclerosis 49 (1983)) and a family with FHA. Both probands of TD and FHA developed coronary heart disease. Sequence analysis of the ABCA1 gene from the patients with TD revealed a homozygous G to A transition at nucleotide 3805 of the cDNA resulting in the substitution of Asp 1229 with Asn in exon 27, and a C to T at nucleotide 6181 resulting in the substitution of Arg 2021 with Trp in exon 47. Sequence analysis of the ABCA1 gene from the FHA patient revealed a homozygous 4 bp CGCC deletion from nucleotide 3787 to 3790 resulting in premature termination by frameshift at codon 1224. These mutations were confirmed by restriction digestion analysis, and were not found in 141 control subjects. Our findings indicate that mutations in the ABCA1 gene are associated with TD as well as FHA.
Keywords: Tangier disease; ABCA1; High density lipoprotein; Coronary heart disease; ATP-binding cassette;

Cisplatin induced apoptosis in regenerating liver after partial hepatectomy (PH). Apoptosis was determined by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was obvious at 4 h and peaked at 8 h after PH. The activity of Jun N-terminal kinase (JNK) transiently increased at 1 h after PH. However, in cisplatin-injected rats, the JNK activity increased at 30 min and the increased level was maintained up to 4 h after PH. The in vivo activation of JNK was confirmed by the increased level of the phosphorylated c-Jun protein. Western blot analysis showed that the phosphorylated c-Jun level increased at 1 h and reached more than 30-fold the control level at 2 h after PH with cisplatin. The c-jun mRNA levels also markedly increased at 1 h after PH with cisplatin. The protein level of p53 increased after 1 h on cisplatin injection, but no significant change in the mRNA level was observed. The rise in the p53 protein level was followed by the upregulation of p21 WAF1/CIP1 mRNA and protein levels. These results suggested that the enhanced and sustained JNK activation and the upregulation of p53 and p21 WAF1/CIP1 were involved in hepatocyte apoptosis induced by PH with cisplatin.
Keywords: Cisplatin; Apoptosis; Jun N-terminal kinase; p53; p21; Liver regeneration;