BBA - Molecular Basis of Disease (v.1688, #2)

Oxidatively damaged proteins of heart mitochondrial electron transport complexes by K.B. Choksi; W.H. Boylston; J.P. Rabek; W.R. Widger; J. Papaconstantinou (95-101).
Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I–V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.
Keywords: Oxidative stress; Mitochondrial dysfunction; Carbonylation; Nitration; 4-hydroxynonenal; Lipid peroxidation adduct;

Tear lipocalin: potential for selective delivery of rifampin by Oktay K Gasymov; Adil R Abduragimov; Elshad O Gasimov; Taleh N Yusifov; Alek N Dooley; Ben J Glasgow (102-111).
The potential of ligand binding proteins as drug carriers and delivery systems has recently sparked great interest. We investigated the potential of tear lipocalin (TL) to bind the antibiotic, rifampin, and the environmental conditions for controlled release. To determine if TL binds rifampin, gel filtration was used to isolate protein fractions of tears. Rifampin was detected by absorbance spectroscopy in the elution fractions containing TL. The bound complex of rifampin–TL generates optical activity at about 360 nm, indicating a unique conformation at the binding site. Rifampin has a higher affinity for TL (K d=128 μM) than albumin. Rifampin is released from the TL calyx in acidic conditions and is displaced by palmitic acid. Autooxidation of free rifampin begins in minutes but is delayed by at least 3 h in the presence of TL. These properties are conducive to stabilization and delivery of rifampin to tubercles that are acidic and rich in fatty acids. These studies show the potential of TL as a carrier for rifampin with controlled release to a targeted environment.
Keywords: Tear lipocalin; von Ebner's gland protein; Rifampin; Tuberculosis; Drug delivery;

The effect of glutamine on A549 cells exposed to moderate hyperoxia by Folasade Ogunlesi; Cecilia Cho; Sharon A. McGrath-Morrow (112-120).
The use of high oxygen concentrations is frequently necessary in the treatment of acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD). High oxygen concentrations, however, are detrimental to cell growth and cell survival. Glutamine (Gln) may be protective to cells during periods of stress and recently has been shown to increase survival in A549 cells exposed to lethal concentrations of oxygen (95% O2). We found that supplemental Gln enhances cell growth in A549 cells exposed to moderate concentrations of oxygen (60% O2). We therefore evaluated the effect of moderate hyperoxia on the cell cycle distribution of A549 cells. At 48 h there was no significant difference in the cell cycle distribution between 2 mM Gln cells in 60% O2 and 2 mM cells in room air. Furthermore, 2 mM Gln cells in 60% O2 had stable protein levels of cyclin B1 consistent with ongoing cell proliferation. In contrast, at 48 h, cells not supplemented with glutamine (Gln) in 60% O2 had evidence of growth arrest by both flow cytometry (increased percentage of G1 cells) and by decreased protein levels of cyclin B1. G1 growth arrest in the Gln cells exposed to 60% O2 was not, however, associated with induction of p21 protein. At 72 and 96 h, Gln cells in 60% O2, began to demonstrate a partial loss of G1 checkpoint regulation and an increase in apoptosis, indicating an increased sensitivity to oxygen toxicity. Glutathione (GSH) concentrations were then measured. 2 mM Gln cells in 60% O2 were found to have higher concentrations of GSH compared to Gln cells in 60% O2, suggesting that Gln confers protection to the cell during exposure to hyperoxia through up-regulation of GSH. When cells in 60% O2 were given higher concentrations of Gln (5 and 10 mM), cell growth at 96 h was increased compared to cells grown in 2 mM Gln (P<0.04). Clonal survival was also increased in cells exposed 60% O2 and supplemented with higher concentrations of Gln compared to Gln cells in 60% O2. These studies suggest that supplemental glutamine may improve cell growth and cell viability and therefore may be beneficial to the lung during exposure to moderate concentrations of supplemental oxygen.
Keywords: Glutamine; A549 cell; Hyperoxia;

Modulation of rat erythrocyte antioxidant defense system by buthionine sulfoximine and its reversal by glutathione monoester therapy by Namakkal Surappan Rajasekaran; Niranjali S. Devaraj; Halagowder Devaraj (121-129).
The protective effects of glutathione monoester (GME) on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion and its sequel were evaluated in rat erythrocyte/erythrocyte membrane. Animals were divided into three groups (n=6 in each): control, BSO and BSO+GME group. Administration of BSO, at a concentration of 4 mmol/kg bw, to the albino rats resulted in depletion of blood GSH level to about 59%. GSH was elevated several folds in the GME group as compared to the control (P<0.05) and BSO (P<0.001) groups. Decreased concentration of vitamin E was found in the erythrocyte membrane isolated from BSO-administered animals. Antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were also found to be altered due to BSO-induced GSH depletion in blood erythrocytes. The SOD and CAT activities in BSO group were significantly lower (P<0.001) than the other groups. Lipid peroxidation index and malondialdehyde (MDA) levels in erythrocytes and their membranes were increased to about 45% and 40%, respectively. The activities of Ca2+ATPase, Mg2+ATPase and Na+K+ATPase were lower than those of control group (P<0.05), whereas the activities of these enzymes were found to be restored to normal followed by GME therapy (P<0.05). Cholesterol, phospholipid and C/P ratio and some of the phospholipid classes like phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin were significantly (P<0.05) altered in the erythrocyte membranes of BSO-administered rats compared with those of control group. These parameters were restored to control group levels in GME-treated group. Oxidative stress may play a major role in the BSO-mediated gamma glutamyl cysteine synthetase (γ-GCS) inhibition and hence the depletion of GSH. In conclusion, our findings have shown that antioxidant status decreased and lipid peroxidation increased in BSO-treated rats. GME potentiates the RBC and blood antioxidant defense mechanisms and decreases lipid peroxidation.
Keywords: Buthionine sulfoximine; Antioxidant; Glutathione monoester;

Mössbauer spectroscopy and ELISA studies reveal differences between Parkinson's disease and control substantia nigra by Jolanta Galazka-Friedman; Erika R. Bauminger; Dariusz Koziorowski; Andrzej Friedman (130-136).
The possible role of iron in the degeneration of nervous cells in Parkinson's disease (PD) was studied with the use of Mössbauer spectroscopy (MS) and enzyme-linked immunoabsorbent assay (ELISA). Mössbauer data were obtained at 90 and 4.1 K from 21 samples of control and 9 samples of parkinsonian substantia nigra (SN). Mössbauer spectra were very similar to those observed in ferritin. Small differences were detected between the spectra obtained from PD and from control SN, and could be due to a slight difference in the composition of the ferritin-like iron cores or due to the presence of about 8% of non-ferritin-like iron in parkinsonian SN. ELISA studies from 11 controls and 6 parkinsonian SN showed a decrease in the concentration of L-chains in wet tissues of PD-SN compared to control SN. The decrease in the amount of L subunits may correspond to a decreased ability of this ferritin to keep iron in a safe form. Iron released from ferritin or neuromelanin (NM) may be the source of such iron, which may cause the difference in the Mössbauer spectra and may trigger oxidative stress leading to cell death.
Keywords: Parkinson's disease; Nigral iron; Mössbauer spectroscopy; ELISA; Ferritin; Oxidative stress;

The absence of a functional relationship between ATM and BLM, the components of BASC, in DT40 cells by Wensheng Wang; Masayuki Seki; Makoto Otsuki; Shusuke Tada; Noriaki Takao; Ken-ichi Yamamoto; Makoto Hayashi; Masamitsu Honma; Takemi Enomoto (137-144).
Bloom syndrome (BS) and ataxia-telangiectasia (A-T) are rare autosomal recessive diseases associated with chromosomal instability. The genes responsible for BS and A-T have been identified as BLM and ATM, respectively, whose products were recently found to be components of BRCA1-associated genome surveillance complex (BASC), a supercomplex possibly involved in the recognition and repair of aberrant DNA structures. Based on experiments using BLM −/− DT40 cells and BLM −/−/RAD54 −/− DT40 cells, we previously suggested that BLM functions to reduce the formation of double-strand breaks (DSBs) during DNA replication. To examine whether ATM is involved in the recognition and/or repair of DSBs generated in BLM −/− DT40 cells and to address the functional relationship between the two BASC components, we generated BLM −/−/ATM −/− DT40 cells and characterized their properties as well as those of ATM −/− and BLM −/− DT40 cells. BLM −/−/ATM −/− cells proliferated slightly more slowly than either BLM −/− or ATM −/− cells. The sensitivity of BLM −/−/ATM −/− cells to γ-irradiation was similar to that of ATM −/− cells, while BLM −/− cells were slightly resistant to γ-irradiation compared with wild-type cells. BLM −/− cells showed sensitivity to methyl methanesulfonate (MMS) and UV irradiation while ATM −/− cells did not show sensitivity to either agent. The sensitivity of BLM −/−/ATM −/− cells to MMS and UV was similar to that of BLM −/− cells. Disrupting the function of ATM reduced the targeted integration frequency in BLM −/− DT40 cells. However, a defect in ATM only slightly reduced the increased sister chromatid exchanges (SCEs) in BLM −/− DT40 cells.
Keywords: BLM; ATM; BASC; Targeted integration; SCE; DSB;

Age-adjusted antitumoral therapy based on the demonstration of increased apoptosis as a mechanism underlying the reduced malignancy of tumors in the aged by Orit Itzhaki; Tatiana Kaptzan; Ehud Skutelsky; Judith Sinai; Moshe Michowitz; Annette Siegal; Ginnette Schibi; Monica Huszar; Lya Ben-Dor; Judith Leibovici (145-159).
In view of the constant increase in the aged population, age-adjusted cancer therapy becomes an urgent target. Although cancer incidence rises with age, paradoxically, growth rate and metastasis often proceed at a slower rate in the aged. Determining the mechanism(s) underlying this reduced tumor progression in the old might have implications for a rational design of age-adjusted therapy. Thus far, decreased cell proliferation or immune response modifications were suggested as possible mechanisms. We show here that an increased tendency to apoptotic tumor cell death in the aged could constitute an additional mechanism. Based on this mechanism, we compared the therapeutic efficacy of two apoptosis inducers, hydrocortisone and adriamycin, on AKR lymphoma and B16 melanoma growth in young and old mice. Treatment with hydrocortisone acetate inhibited tumor growth practically only in old mice in the two tumor systems. Similar effects were obtained with adriamycin treatment of AKR lymphoma but opposite results were seen with B16 melanoma. We thus demonstrated, in three of the four tumor-therapeutic modality systems examined, an age-related antitumoral efficacy of two apoptosis-inducing agents, with tendency for a remarkably more pronounced effect in aged mice.
Keywords: Cancer treatment; Aging; Apoptosis induction; Tumor progression; Age-dependent therapy design;

Analysis of common IL-6 promoter SNP variants and the AnTn tract in humans and primates and effects on plasma IL-6 levels following coronary artery bypass graft surgery by Daniel Kelberman; Mark Fife; Matthew V. Rockman; David J. Brull; Patricia Woo; Steve E. Humphries (160-167).
Interleukin-6 (IL-6) is a pro-inflammatory cytokine and major mediator of the acute phase response. Single nucleotide polymorphisms within the 5′ flanking region (−597G>A, −572G>C and −174G>C) have previously been associated with increased risk of coronary heart disease and influencing transcription of IL-6 both in vitro and in vivo. In addition to these, a polymorphic AnTn tract is also present in the promoter of IL-6. Analysis in five different primate species demonstrated a G allele at −597, −572 and −174 in all species. By contrast, the AnTn tract was polymorphic in at least three species, and was roughly conserved in overall length despite an increase in the relative proportion of A versus T in the evolution of the human sequence from that in the ancestor of the great apes. The effect of the AnTn polymorphism on IL-6 levels was examined following coronary artery bypass graft surgery (CABG), a known inflammatory stimulus for IL-6 production. One hundred and thirty-two patients undergoing CABG were genotyped for the AnTn tract by automated sequencing. Four alleles were identified: A8T12 (allele frequency 0.35, 95% CI 0.29–0.40); A9T11 (0.26, 0.21–0.31); A10T11 (0.21, 0.16–0.26); and A10T10 (0.18, 0.14–0.23). Isolation of the effect of different alleles of the AnTn tract on an identical haplotypic background for the other polymorphisms in the promoter showed that individuals homozygous for A9T11 had significantly higher post-operative IL-6 levels than A10T11 homozygotes (275±46 pg/ml versus 152±29; P=0.04). The effect of the A8T12 allele could not be determined separately due to strong allelic association with −174C. The conserved length of the AnTn tract and the association in vivo with IL-6 levels strongly suggest the functionality of the tract on IL-6 expression, independent of contributions from other polymorphic sites within the promoter.
Keywords: Interleukin-6; Promoter polymorphism; Haplotype analysis; Coronary artery bypass graft surgery; Coronary heart disease;

Hereditary sensory neuropathy type I (HSN1) is a common degenerative disorder of peripheral sensory neurons. HSN1 is caused by mutations in the gene, encoding the long chain base 1 of serine palmitoyltransferase (SPT) [Nat. Genet. 27 (2001) 309]. Here, we show a 44% reduction of SPT activity in transformed lymphocytes from HSN1 patients with mutation T399G in the SPTLC1 gene. However, the decrease in SPT activity had no effect on de novo sphingolipid biosynthesis, cellular sphingolipid content, cell proliferation and death (apoptosis and necrosis). The removal of extracellular sphingolipids did not affect viability of HSN1 cells. We also found no significant difference in whole blood counts, viability, and permeability to Triton X-100 of primary lymphocytes from HSN1 patients. These results suggest that, despite the inhibition of mutant allele, the activity of nonmutant allele of STP may be sufficient for adequate sphingolipid biosynthesis and cell viability. Therefore, the neurodegeneration in HSN1 is likely to be caused by subtler and rather long-term effect(s) of these mutations such as loss of a cell-type selective facet of sphingolipid metabolism and/or function, or perhaps accumulation of toxic species, including abnormal protein(s) as in other neurodegenerations.
Keywords: Hereditary sensory neuropathy; Serine palmitoyltransferase; Transformed lymphocyte; Sphingolipid; Cell proliferation; Cell death;

Imaging of colorectal adenocarcinoma using FT-IR microspectroscopy and cluster analysis by Peter Lasch; Wolfgang Haensch; Dieter Naumann; Max Diem (176-186).
In this paper, three different clustering algorithms were applied to assemble infrared (IR) spectral maps from IR microspectra of tissues. Using spectra from a colorectal adenocarcinoma section, we show how IR images can be assembled by agglomerative hierarchical (AH) clustering (Ward's technique), fuzzy C-means (FCM) clustering, and k-means (KM) clustering. We discuss practical problems of IR imaging on tissues such as the influence of spectral quality and data pretreatment on image quality. Furthermore, the applicability of cluster algorithms to the spatially resolved microspectroscopic data and the degree of correlation between distinct cluster images and histopathology are compared.The use of any of the clustering algorithms dramatically increased the information content of the IR images, as compared to univariate methods of IR imaging (functional group mapping). Among the cluster imaging methods, AH clustering (Ward's algorithm) proved to be the best method in terms of tissue structure differentiation.
Keywords: Cluster analysis; IR imaging; Biomedical spectroscopy; FT-IR microspectroscopy; Colorectal adenocarcinoma; Pattern recognition; Tissue classification;