Therapeutic potential of breakers of advanced glycation end product-protein crosslinks

Long-lived structural proteins, collagen and elastin, undergo continual non-enzymatic crosslinking during aging and in diabetic individuals. This abnormal protein crosslinking is mediated by advanced glycation end products (AGEs) generated by non-enzymatic glycosylation of proteins by glucose. The AGE-derived protein crosslinking of structural proteins contributes to the complications of long-term diabetes such as nephropathy, retinopathy, and neuropathy. AGE-crosslinks have also been implicated in age-related cardiovascular diseases. Potential treatment strategies for these AGE-derived complications include prevention of AGE-formation and breaking of the existing AGE-crosslinks. The therapeutic potential of the AGE-inhibitor, pimagedine (aminoguanidine), has been extensively investigated in animal models and in Phase 3 clinical trials. This review presents the pre-clinical and clinical studies using ALT-711, a highly potent AGE-crosslink breaker that has the ability to reverse already-formed AGE-crosslinks. Oral administration of ALT-711 has resulted in a rapid improvement in the elasticity of stiffened myocardium in experimental animals. Topical administration of ALT-711 was effective in improving the skin hydration of aged rats. The therapeutic potential of crosslink breakers for cardiovascular complications and dermatological alterations associated with aging and diabetes is discussed.     

Protein targets of 1,4-benzoquinone and 1,4-naphthoquinone in human bronchial epithelial cells

Many aspects of the toxicity of xenobiotic compounds have been attributed to the consequences of covalent modification of specific proteins, but the nature and specificity of protein targets for classes of electrophilic toxins remain largely uncharacterized. For inhaled toxicants, the point of exposure or absorption lies with epithelial cells lining the pulmonary tree. In this study, abundant proteins in human bronchial epithelial cells that are arylated in vitro by two quinonoid compounds, 1,4-benzoquinone (BQ) and 1,4-naphthoquinone (NQ) have been detected using (14)C-labeled quinones and two-dimensional gel electrophoresis. These proteins were identified using matrix assisted laser desorption/ionization mass spectrometry for tryptic mass mapping followed by sequence database searching. Corroborative identification of protein targets was obtained from the apparent isoelectric points, molecular weights, and the use of antibody probes. There were subtle differences in the protein targets of BQ and NQ, but both associated with the following abundant proteins, nucleophosmin, galectin-1, probable protein disulfide isomerase, protein disulfide isomerase, 60 kDa heat shock protein, mitochondrial stress-70 protein, epithelial cell marker protein, and S100-type calcium binding protein A14. We further delineate the properties of these proteins that make them preferred targets and the evidence these adducts present for delivery of these quinones to subcellular compartments.     

Anoxia tolerance and anaerobic catabolism of aged beetroot storage tissues5

The relationship between anoxia tolerance and rates of anaerobiccatabolism was studied using aged storage tissues of red beetroot.This tissue has substantial experimental advantages over mostother tissues for studies on response to anoxia; even the aeratedtissues do not grow, thus allowing assesment of any possiblePasteur effect. Furthermore, loss of the endogeneous dye, betacyanin,serves as a marker for death of individual cells. A high tolerance to anoxia was achieved by adding glucose andby applying a hypoxic pretreatment for 48 h (02 at 0.003–0.015mol m^– Such tissues survived anoxia for at least 150 h. Alcoholic fermentation was the principal catabolic pathway inanoxic beetroot tissue; ethanol synthesized over 88.5 h of anoxiaaccounted for about 86% of the decrease In endogenous sugarcontent in hypoxically pretreated tissues. During the first24 h of anoxia, rates of alcoholic fermentation were stimulatedby high concentrations of endogenous glucose, supplying exogenousglucose and also by hypoxic pretreatment. In aerobically pretreatedtissues, alcoholic fermentation increased with time over thefirst 24 h of anoxia and these increases were correlated withincreases in activity of pyruvate decarboxylase (PDC). The mostrapid rates of glycolysis under anoxia were observed in thepresence of glucose, c. 50% above the rate in aerated tissues. When anoxia lasted longer than 24 h, the rate of alcoholic fermentationdeclined with time, in all treatments. Furthermore, decreasesin content of endogen ous substrates (sugars + starch), between0 and 88.5 h anoxia, indicated that, in hypoxically pretreatedtissues, glycolysis was 30% lower under anoxia than in air.These decreases in rate of alcoholic fermentation were not dueto injury, or decreases in activity of PDC. Reduced availabilityof endogenous sugar can not be excluded as a cause for the decreasein rate of glycolysis. However, we favour fine control, whichwould regulate glycolysis once requirements for ATP are reduced,after adaptation to anoxia has been completed. We also estimatethat maintenance requirement for ATP is 10–25 times lowerIn anoxic than in aerated tissues. Key words: Anoxia tolerance, alcoholic fermentation, maintenance requirement, storage tissues     

Characterization of Radioiodinated TISCH: A High-Affinity and Selective Ligand for Mapping CNS D1 Dopamine Receptor

In developing CNS D1 dopamine receptor-imaging agents with improved specificity and longer brain retention, an iodinated D1 ligand was synthesized. In vitro and in vivo radiolabeling studies of a new iodinated benzazepine, TISCH [7-chloro-8-hydroxy-1-(3'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1H-3- benzazepine], an analog of SCH 23390 (7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e), were investigated. After an intravenous injection, the R(+) isomer of TISCH showed high brain uptake in rats (2.20 and 0.57% dose per whole brain at 2 and 60 min, respectively). The striatum/cerebellum ratio increased progressively with time (12 at 60 min). Ex vivo autoradiography of rat brain sections, after intravenous injection of R(+)-[125I]TISCH, displayed the highest uptake in striatum and substantia nigra, regions known to have a high concentration of D1 receptors, whereas the S(-) isomer displayed no specific uptake. Furthermore, the specific uptake can be blocked by pretreatment with SCH 23390. In vitro binding studies using the rat striatum tissue preparation showed high specific and low nonspecific bindings (KD = 0.21 +/- 0.03 nM). The rank order of potency exhibiting high specificity to the D1 receptor was SCH 23390 greater than (+/-)-TISCH greater than (+)-butaclamol = (+/-)-FISCH [7-chloro-8-hydroxy-1-(4'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1 H-3-benzazepine] much greater than WB4101 = spiperone greater than dopamine, serotonin, (+/-)-propranolol, and naloxone. Imaging studies in a monkey with the resolved isomer, R(+)-[123I]TISCH, demonstrated a high uptake in the basal ganglia and prolonged retention. The preliminary data suggest that R(+)-TISCH is selective for the CNS D1 receptor and is potentially useful for in vivo and in vitro pharmacological studies. When labeled with iodine-123, it may be suitable for noninvasive imaging in humans.     

Identification and functional characterization of a novel human misshapen/Nck interacting kinase-related kinase, hMINK beta

Misshapen/NIKs-related kinase (MINK) is a member of the germinal center family of kinases that are homologous to the yeast sterile 20 (Ste20) kinases and regulate a wide variety of cellular processes, including cell morphology, cytoskeletal rearrangement, and survival. Here, we present the cloning and functional characterization of a novel human Misshapen/NIKs-related kinase beta (hMINK beta) that encodes a polypeptide of 1312 amino acids. hMINK beta is ubiquitously expressed in most tissues with at least five alternatively spliced isoforms. Similar to Nck interacting kinase (NIK) and Traf2 and Nck-interacting kinase (TNIK), hMINK beta moderately activates c-Jun N-terminal kinase (JNK) and associates with Nck via the intermediate domain in the yeast two-hybrid system and in a glutathione S-transferase (GST) pull-down assay. Interestingly, overexpression of the kinase domain deleted and kinase-inactive mutants of hMINK beta in human fibrosarcoma HT1080 cells enhanced cell spreading, actin stress fiber formation, and adhesion to extracellular matrix, as well as decreased cell motility and cell invasion. Furthermore, these mutants also promoted cell-cell adhesion in human breast carcinoma MCF7 cells, evidenced with cell growth in clusters and increased membrane localization of beta-catenin, a multifunctional protein involved in E-cadherin-mediated cell adhesion. Finally, hMINK beta protein was found to colocalize with the Golgi apparatus, implicating that hMINK beta might exert its functions, at least in part, through the modulation of intracellular protein transport. Taken together, these results suggest that hMINK beta plays an important role in cytoskeleton reorganization, cell adhesion, and cell motility.     

NXS2 murine neuroblastomas express increased levels of MHC class I antigens upon recurrence following NK-dependent immunotherapy

We evaluated recurrent NXS2 neuroblastoma tumors that developed following NK- or T-cell–mediated immunotherapy in tumor-bearing mice. Recurrent tumors developed following an NK-dependent antitumor response using a suboptimal dose of hu14.18-IL2, a humanized IL-2 immunocytokine targeted to the GD₂-ganglioside. This treatment initially induced complete resolution of measurable tumor in the majority of mice, followed, however, by delayed tumor recurrence in some mice. These recurrent NXS2 tumors revealed markedly enhanced (> fivefold) MHC class I antigen expression when compared with NXS2 tumors growing in PBS-treated control mice. A similar level of enhanced MHC class I antigen-expression could be induced on NXS2 cells in vitro by culturing with interferon , and was associated with reduced susceptibility to both NK-cell–mediated tumor cell lysis and antibody-dependent cellular cytotoxicity in vitro. In contrast, Flt3-ligand treatment of NXS2-bearing mice induced a protective T-cell–dependent antitumor memory response. Recurrent NXS2 tumors that developed following Flt3-L therapy revealed a decreased expression of MHC class I antigens. While NXS2 tumors are susceptible to in vivo destruction following either hu14.18-IL2 or Flt3-ligand immunotherapies, these results suggest that some tumor cells may be selected to survive and progress by expressing either higher or lower levels of MHC class I antigen in order to resist either NK- or T-cell–mediated antitumor responses, respectively.Abbreviations ADCC antibody-dependent cellular cytotoxicity - Flt3-L Flt3-ligand - GD₂ GD₂-disialoganglioside - IC immunocytokine - mAb monoclonal antibody - NB neuroblastoma - NXS2 transplantable murine neuroblastoma - s.c. subcutaneous     

Comparison of the power between microsatellite and single-nucleotide polymorphism markers for linkage and linkage disequilibrium mapping of an electrophysiological phenotype

We performed linkage and linkage disequilibrium (LD) mapping analyses to compare the power between microsatellite and single nucleotide polymorphism (SNP) markers. Chromosome-wide analyses were performed for a quantitative electrophysiological phenotype, ttth1, on chromosome 7. Multipoint analysis of microsatellite markers using the variance component (VC) method showed the highest LOD score of 4.20 at 162 cM, near D7S509 (163.7 cM). Two-point analysis of SNPs using the VC method yielded the highest LOD score of 3.98 in the Illumina SNP data and 3.45 in the Affymetrix SNP data around 152-153 cM. In family-based single SNP and SNP haplotype LD analysis, we identified seven SNPs associated with ttth1. We searched for any potential candidate genes in the location of the seven SNPs. The SNPs rs1476640 and rs768055 are located in the FLJ40852 gene (a hypothetical protein), and SNP rs1859646 is located in the TAS2R5 gene (a taste receptor). The other four SNPs are not located in any known or annotated genes. We found the high density SNP scan to be superior to microsatellites because it is effective in downstream fine mapping due to a better defined linkage region. Our study proves the utility of high density SNP in genome-wide mapping studies.     

Changes in Sr/Ca, Ba/Ca and 87Sr/86Sr ratios between trophic levels in two forest ecosystems in the northeastern U.S.A.

The variability and biologicalfractionation of Sr/Ca, Ba/Ca and ⁸⁷Sr/⁸⁶Srratios were studied in a soil–plant–invertebrate–bird food chain in two forested ecosystems withcontrasting calcium availability in the northeasternU.S.A. Chemical measurements were made of the soilexchange pool, leaves, caterpillars, snails, and boththe femurs and eggshells of breeding insectivorousmigratory songbirds. ⁸⁷Sr/⁸⁶Sr values weretransferred up the food chain from the soil exchangepool to leaves, caterpillars, snails and eggshellswithout modification. Adult birds were the oneexception; their ⁸⁷Sr/⁸⁶Sr values generallyreflected those of lower trophic levels at each site,but were lower and more variable, probably becausetheir strontium was derived in part from foods intropical winter habitats where lower⁸⁷Sr/⁸⁶Sr ratios are likely to predominate. Sr/Ca and Ba/Ca ratios decreased at each successive trophiclevel, supporting previous suggestions that Sr/Ca andBa/Ca ratios can be used to identify the trophic levelat which an organism is primarily feeding. The changesin Sr/Ca and Ba/Ca ratios we measured for vegetationand insects were comparable to similar measurementsmade previously (but based on single samples of eachorganism) in an alpine ecosystem. Changes in Sr/Ca andBa/Ca ratios between birds and their food have notpreviously been measured, but the values we obtainedwere similar to those for herbivorous and carnivorousmammals at similar trophic levels. Our results provideevidence that supports the use of Sr/Ca ratios in thedetermination of human paleodiets and suggests thatSr/Ca ratios may also provide a useful tool in studiesof modern food webs. Furthermore, our findings suggestthat ⁹⁰Sr from nuclear fallout will notbioaccumulate in forests and that changes in Sr/Caratios between trophic levels will need to beconsidered in some cases when using⁸⁷Sr/⁸⁶Sr as a tracer of calciumbiogeochemistry.