bluuebunny/crossref_metadata_2025 huggingface dataset:
GObar: A Gene Ontology based analysis and visualization tool for gene sets
Authors: Lee Jason SM , Katari Gurpreet , Sachidanandam Ravi
Published: July 2005

Background

Microarray experiments, as well as other genomic analyses, often result in large gene sets containing up to several hundred genes. The biological significance of such sets of genes is, usually, not readily apparent.

Identification of the functions of the genes in the set can help highlight features of interest. The Gene Ontology Consortium [1] has annotated genes in several model organisms using a controlled vocabulary of terms and placed the terms on a Gene Ontology (GO), which comprises three disjoint hierarchies for Molecular functions, Biological processes and Cellular locations. The annotations can be used to identify functions that are enriched in the set, but this analysis can be misleading since the underlying distribution of genes among various functions is not uniform. For example, a large number of genes in a set might be kinases just because the genome contains many kinases.

Results

We use the Gene Ontology hierarchy and the annotations to pick significant functions and pathways by comparing the distribution of functions in a given gene list against the distribution of all the genes in the genome, using the hypergeometric distribution to assign probabilities. GObar is a web-based visualizer that implements this algorithm.

The public website for GObar [2] can analyse gene lists from the yeast (S. cervisiae), fly (D. Melanogaster), mouse (M. musculus) and human (H. sapiens) genomes. It also allows visualization of the GO tree, as well as placement of a single gene on the GO hierarchy. We analyse a gene list from a genomic study of pre-mRNA splicing to demonstrate the utility of GObar.

Conclusion

GObar is freely available as a web-based tool at http://katahdin.cshl.org:9331/GO[2] and can help analyze and visualize gene lists from genomic analyses.
Dynamin-dependent NMDAR endocytosis during LTD and its dependence on synaptic state
Authors: Montgomery Johanna M , Selcher Joel C , Hanson Jesse E , Madison Daniel V
Published: July 2005

Background

The N-methyl-D-aspartate (NMDA)-type glutamate receptor expressed at excitatory glutamatergic synapses is required for learning and memory and is critical for normal brain function. At a cellular level, this receptor plays a pivotal role in triggering and controlling synaptic plasticity. While it has been long recognized that this receptor plays a regulatory role, it was considered by many to be itself immune to synaptic activity-induced plasticity. More recently, we and others have shown that NMDA receptor-mediated synaptic responses can be subject to activity-dependent depression.

Results

Here we show that depression of synaptic transmission mediated by NMDA receptors displays a state-dependence in its plasticity; NMDA receptors are resistant to activity-induced changes at silent and recently-silent synapses. Once synapses transition to the active state however, NMDA receptors become fully 'plastic'. This state-dependence is identical to that shown by the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor. Furthermore, the down-regulation of NMDAR-mediated responses during synaptic depression is prevented by disruption of dynamin-dependent endocytosis.

Conclusion

NMDA receptor-mediated synaptic responses are plastic in a state-dependent manner. Depending on the plasticity state in which a synapse currently resides, NMDA receptors will either be available or unavailable for down-regulation. The mechanism underlying the down-regulation of NMDA receptor-mediated synaptic responses is endocytosis of the NMDA receptor. Other potential mechanisms, such as receptor diffusion along the plane of the membrane, or changes in the activity of the channel are not supported. The mechanisms of AMPA receptor and NMDA receptor endocytosis appear to be tightly coupled, as both are either available or unavailable for endocytosis in the same synaptic states. Endocytosis of NMDA receptors would serve as a potent mechanism for metaplasticity. Such state-dependent regulation of NMDAR endocytosis will provide fundamental control over downstream NMDA receptor-dependent plasticity of neuronal circuitry.
Oblique decision trees for spatial pattern detection: optimal algorithm and application to malaria risk
Authors: Gaudart Jean , Poudiougou Belco , Ranque Stéphane , Doumbo Ogobara
Published: July 2005

Background

In order to detect potential disease clusters where a putative source cannot be specified, classical procedures scan the geographical area with circular windows through a specified grid imposed to the map. However, the choice of the windows' shapes, sizes and centers is critical and different choices may not provide exactly the same results.

The aim of our work was to use an Oblique Decision Tree model (ODT) which provides potential clusters without pre-specifying shapes, sizes or centers. For this purpose, we have developed an ODT-algorithm to find an oblique partition of the space defined by the geographic coordinates.

Methods

ODT is based on the classification and regression tree (CART). As CART finds out rectangular partitions of the covariate space, ODT provides oblique partitions maximizing the interclass variance of the independent variable. Since it is a NP-Hard problem in RN, classical ODT-algorithms use evolutionary procedures or heuristics. We have developed an optimal ODT-algorithm in R2, based on the directions defined by each couple of point locations. This partition provided potential clusters which can be tested with Monte-Carlo inference.

We applied the ODT-model to a dataset in order to identify potential high risk clusters of malaria in a village in Western Africa during the dry season. The ODT results were compared with those of the Kulldorff' s SaTScan™.

Results

The ODT procedure provided four classes of risk of infection. In the first high risk class 60%, 95% confidence interval (CI95%) [52.22–67.55], of the children was infected. Monte-Carlo inference showed that the spatial pattern issued from the ODT-model was significant (p < 0.0001).

Satscan results yielded one significant cluster where the risk of disease was high with an infectious rate of 54.21%, CI95% [47.51–60.75]. Obviously, his center was located within the first high risk ODT class. Both procedures provided similar results identifying a high risk cluster in the western part of the village where a mosquito breeding point was located.

Conclusion

ODT-models improve the classical scanning procedures by detecting potential disease clusters independently of any specification of the shapes, sizes or centers of the clusters.
Application of affymetrix array and massively parallel signature sequencing for identification of genes involved in prostate cancer progression
Authors: Oudes Asa J , Roach Jared C , Walashek Laura S , Eichner Lillian J , True Lawrence D , Vessella Robert L , Liu Alvin Y
Published: July 2005

Background

Affymetrix GeneChip Array and Massively Parallel Signature Sequencing (MPSS) are two high throughput methodologies used to profile transcriptomes. Each method has certain strengths and weaknesses; however, no comparison has been made between the data derived from Affymetrix arrays and MPSS. In this study, two lineage-related prostate cancer cell lines, LNCaP and C4-2, were used for transcriptome analysis with the aim of identifying genes associated with prostate cancer progression.

Methods

Affymetrix GeneChip array and MPSS analyses were performed. Data was analyzed with GeneSpring 6.2 and in-house perl scripts. Expression array results were verified with RT-PCR.

Results

Comparison of the data revealed that both technologies detected genes the other did not. In LNCaP, 3,180 genes were only detected by Affymetrix and 1,169 genes were only detected by MPSS. Similarly, in C4-2, 4,121 genes were only detected by Affymetrix and 1,014 genes were only detected by MPSS. Analysis of the combined transcriptomes identified 66 genes unique to LNCaP cells and 33 genes unique to C4-2 cells. Expression analysis of these genes in prostate cancer specimens showed CA1 to be highly expressed in bone metastasis but not expressed in primary tumor and EPHA7 to be expressed in normal prostate and primary tumor but not bone metastasis.

Conclusion

Our data indicates that transcriptome profiling with a single methodology will not fully assess the expression of all genes in a cell line. A combination of transcription profiling technologies such as DNA array and MPSS provides a more robust means to assess the expression profile of an RNA sample. Finally, genes that were differentially expressed in cell lines were also differentially expressed in primary prostate cancer and its metastases.
Phase I dose-escalating study of docetaxel in combination with 5-day continuous infusion of 5-fluorouracil in patients with advanced gastric cancer
Authors: Park Se Hoon , Bang Soo-Mee , Cho Eun Kyung , Shin Dong Bok , Lee Jae Hoon , Lee Woon KI , Chung Min
Published: July 2005

Background

Published data suggests that docetaxel combined with 5-fluorouracil (5-FU) may have synergistic activity in treating advanced gastric cancer. We performed a phase I study of docetaxel and 5-FU to determine the maximum tolerated dose (MTD), the recommended dose for phase II studies, and the safety of this combination.

Methods

Eligible patients had recurrent and/or metastatic advanced gastric cancer with normal cardiac, renal and hepatic function. Traditional phase I methodology was employed in assessing dose-limiting toxicity (DLT) and MTD. On day 1 every 3 weeks, docetaxel 75 mg/m2 (fixed dose) was infused over 1-h, followed immediately by 5-FU as a 5-day continuous infusion.

Results

Dose escalation schema was as follows: dose level (DL) 1 (5-FU 250 mg/m2/day), 2 (500), 3 (750), and 4 (1000). Three patients were enrolled on DL1, without DLT. On DL2, 1 DLT (grade 3 stomatitis) was developed in first 3 patients, and this cohort was expanded to 6 patients. Three patients had been enrolled on DL3. Because two out of 3 patients had DLTs, the MTD was reached at DL3.

Conclusion

The recommended phase II dose of this combination is 75 mg/m2 docetaxel on day 1 immediately followed by a 5-day continuous infusion of 5-FU 500 mg/m2/day.
Creating an "enabling environment" for taking insecticide treated nets to national scale: the Tanzanian experience
Authors: Magesa Stephen M , Lengeler Christian , deSavigny Don , Miller Jane E , Njau Ritha JA , Kramer Karen , Kitua Andrew , Mwita Alex
Published: July 2005

Introduction

Malaria is the largest cause of health services attendance, hospital admissions and child deaths in Tanzania. At the Abuja Summit in April 2000 Tanzania committed itself to protect 60% of its population at high risk of malaria by 2005. The country is, therefore, determined to ensure that sustainable malaria control using insecticide-treated nets is carried out on a national scale.

Case description

Tanzania has been involved for two decades in the research process for developing insecticide-treated nets as a malaria control tool, from testing insecticides and net types, to assessing their efficacy and effectiveness, and exploring new ways of distribution. Since 2000, the emphasis has changed from a project approach to that of a concerted multi-stakeholder action for taking insecticide-treated nets to national scale (NATNETS). This means creating conditions that make insecticide-treated nets accessible and affordable to all those at risk of malaria in the country. This paper describes Tanzania's experience in (1) creating an enabling environment for insecticide-treated nets scale-up, (2) promoting the development of a commercial sector for insecticide-treated nets, and (3) targeting pregnant women with highly subsidized insecticide-treated nets through a national voucher scheme. As a result, nearly 2 million insecticide-treated nets and 2.2 million re-treatment kits were distributed in 2004.

Conclusion

National upscaling of insecticide-treated nets is possible when the programme is well designed, coordinated and supported by committed stakeholders; the Abuja target of protecting 60% of those at high risk is feasible, even for large endemic countries.
Cost-effectiveness of models for prevention of vertical HIV transmission – voluntary counseling and testing and choices of drug regimen
Authors: Teerawattananon Yot , Vos Theo , Tangcharoensathien Viroj , Mugford Miranda
Published: July 2005

Objectives

From a health care provider prospective, to assess the cost-effectiveness of four Antiretroviral therapy (ART) regimens given in addition to voluntary counselling and testing (VCT) for preventing mother-to-child transmission of HIV: a) Zidovudine (AZT); b) Nevirapine (NVP); c) a combination of AZT for early antenatal attenders and NVP for late arrivals; and d) combined administration of AZT and NVP and to assess the incremental cost-effectiveness of adding a second VCT session in late pregnancy.

Design & Setting

We examine a hypothetical cohort of 100,000 pregnancies as a decision model. Cost and outcome parameters are estimated as they would apply under Thai routine health service conditions. Effectiveness probabilities are based on best available evidence, from systematic reviews where possible. The main outcome is the number of cases of paediatric HIV averted.

Results

The combining administration of AZT and NVP is the most cost-effective drug option. One VCT session with AZT+NVP averts 337 cases of infection at 556 USD per case averted, while two VCT with the same drug regimen averts 16 additional cases at cost of 1,266 USD per infection averted. The incremental cost-effectiveness ratio of moving from 1VCT, AZT+NVP to 2VCT, AZT+NVP is 16,000 USD per additional averted case, which is much lower than the recommended threshold value for HIV infection averted in Thailand. Multivariate uncertainty analysis supports the findings, showing that at a threshold of 35,000 USD, 2VCT, AZT+NVP is preferable to other VCT and drug strategies.

Conclusion

Interventions for preventing mother-to-child transmission of HIV are cost-effective. Further costs and negative effects of drug resistance, are unlikely to outweigh the social benefits of reduce transmission of HIV. This model suggests that the new drug regimen is a cost-effective option in the Thai health system at currently accepted thresholds for adopting health technologies.
Psychometric properties of the CDC Symptom Inventory for assessment of Chronic Fatigue Syndrome
Authors: Wagner Dieter , Nisenbaum Rosane , Heim Christine , Jones James F , Unger Elizabeth R , Reeves William C
Published: July 2005

Objectives

Validated or standardized self-report questionnaires used in research studies and clinical evaluation of chronic fatigue syndrome (CFS) generally focus on the assessment of fatigue. There are relatively few published questionnaires that evaluate case defining and other accompanying symptoms in CFS. This paper introduces the self-report CDC CFS Symptom Inventory and analyzes its psychometric properties.

Methods

One hundred sixty-four subjects (with CFS, other fatiguing illnesses and non fatigued controls) identified from the general population of Wichita, Kansas were enrolled. Evaluation included a physical examination, a standardized psychiatric interview, three previously validated self-report questionnaires measuring fatigue and illness impact (Medical Outcomes Survey Short-Form-36 [MOS SF-36], Multidimensional Fatigue Inventory [MFI], Chalder Fatigue Scale), and the CDC CFS Symptom Inventory. Based on theoretical assumptions and statistical analyses, we developed several different Symptom Inventory scores and evaluated them on their ability to differentiate between participants with CFS and non-fatigued controls.

Results

The Symptom Inventory had good internal consistency and excellent convergent validity. A Total score (all symptoms), Case Definition score (CFS case defining symptoms) and Short Form score (6 symptoms with minimal correlation) differentiated CFS cases from controls. Furthermore, both the Case Definition and Short Form scores distinguished people with CFS from fatigued subjects who did not meet criteria for CFS.

Conclusion

The Symptom Inventory appears to be a reliable and valid instrument to assess symptoms that accompany CFS. It is a positive addition to existing instruments measuring fatigue because it allows other dimensions of the illness to be assessed. Further research is needed to confirm and replicate the current findings in a normative population.
Shared expression of phenotypic markers in systemic sclerosis indicates a convergence of pericytes and fibroblasts to a myofibroblast lineage in fibrosis
Authors: Rajkumar Vineeth S , Howell Kevin , Csiszar Katalin , Denton Christopher P , Black Carol M , Abraham David J
Published: July 2005

The mechanisms by which microvascular damage leads to dermal fibrosis in diffuse cutaneous systemic sclerosis (dcSSc) are unclear. We hypothesized that microvascular pericytes constitute a cellular link between microvascular damage and fibrosis by transdifferentiating into myofibroblasts. We used a combination of immunohistochemistry and double immunofluorescence labelling of frozen skin biopsies taken from normal and dcSSc patients to determine whether a phenotypic link between pericytes and myofibroblasts exists in dcSSc. Using α-smooth muscle actin, the ED-A splice variant of fibronectin (ED-A FN) and Thy-1 to identify myofibroblasts, we demonstrated the presence of myofibroblasts in fibrotic dcSSc skin. Myofibroblasts were totally absent from control skin, atrophic stage dcSSc skin and non-lesional skin. Using double immunofluorescence labelling, both myofibroblasts and pericytes were shown to express ED-A FN and Thy-1 in dcSSc skin but not in control skin. Proliferating cell nuclear antigen was also expressed by myofibroblasts and pericytes in dcSSc skin while being absent in control skin. These observations suggest that the presence of myofibroblasts may represent a transitional phase during the fibrotic stages of dcSSc and that Thy-1+ve pericytes participate in the fibrogenic development of dcSSc by synthesizing ED-A FN, which may be associated with a proliferation and transition of pericytes and fibroblasts to myofibroblasts, thus linking microvascular damage and fibrosis.
Mannan-binding lectin and procalcitonin measurement for prediction of postoperative infection
Authors: Siassi Michael , Riese Jutta , Steffensen Rudi , Meisner Michael , Thiel Steffen , Hohenberger Werner , Schmidt Joachim
Published: July 2005

Introduction

Postoperative infection is a major cause of morbidity and mortality. We investigated two serum markers for their ability to identify patients at risk for postoperative infection. Mannan-binding lectin (MBL) is a central molecule of the innate immune system and MBL deficiency is known to predispose to infection. Procalcitonin (PCT) is a sensitive marker for bacterial infection.

Methods

We investigated 162 patients undergoing elective surgery for cancer of the gastrointestinal tract. Patients were classified as having no complications (group A), having infection for unknown reason (group B) or having sepsis after events like aspiration or anastomotic leakage (group C). Analysis was done pre- and postoperatively for serum levels of MBL, PCT and C-reactive-protein. DNA was preoperatively sampled and stored and later analysed for genetic polymorphisms of MBL.

Results

The preoperative serum levels of MBL were significantly lower in group B patients than in group A patients (1332 ± 466 ng/ml versus 2523 ± 181 ng/ml). PCT measured on day one post-surgery was significantly higher in group B patients than in group A (3.33 ± 1.08 ng/ml versus 1.38 ± 0.17 ng/ml). Patients with an aberrant MBL genotype had a significantly higher risk of postoperative infections than wild-type carriers (p < 0.05).

Conclusion

Preoperative MBL and early postoperative PCT measurement may help identify patients at risk for postoperative infection.
Physical activity and modulation of systemic low-level inflammation
Authors: Bruunsgaard Helle
Published: July 2005

It has been recognized for some time that cardiovascular disease and type 2 diabetes are, to a major extent, inflammatory disorders associated with an environment characterized by a sedentary lifestyle together with abundant intakes of calories. Systemic low-level inflammation is suggested to be a cause as well as consequence of pathological processes with local tumor necrosis factor α production as an important biological driver. It is hypothesized that physical inactivity contributes to an enhanced proinflammatory burden independently of obesity, as regular muscle contractions mediate signals with myokines/cytokines as important messengers, which suppress proinflammatory activity at distant sites as well as within skeletal muscle. Muscle-derived interleukin (IL)-6 is considered to possess a central role in anti-inflammatory activities and health beneficial effects in relation to physical exercise. It is discussed how this fits the consistent observation that enhanced plasma levels of IL-6 represent a strong risk marker in chronic disorders associated with systemic low-level inflammation and all-cause mortality.
Activation, cytokine production, and intracellular survival of bacteria inSalmonella-infected human monocyte-derived macrophages and dendritic cells
Authors: Pietilä Taija E , Veckman Ville , Kyllönen Päivi , Lähteenmäki Kaarina , Korhonen Timo K , Julkunen Ilkka
Published: July 2005

Salmonella enterica serovar typhimurium (S. typhimurium) is an intracellular pathogen causing localized gastroenteritis in humans. Macrophages (Mφs) and dendritic cells (DCs) play an important role in innate immunity against Salmonella. In this report, we have compared the consequences of infection of human Mφs and DCs with wild-type S. typhimurium and an isogenic PgtE-defective strain. PgtE is an outer membrane protein hypothesized to have a role in intracellular survival of Salmonella. We observed that DCs undergo full maturation in response to Salmonella infection, as indicated by up-regulation of cell-surface marker proteins CD80, CD83, CD86, and human leukocyte antigen class II. CC chemokine ligand 5 (CCL5), CXC chemokine ligand 10, tumor necrosis factor α, interleukin (IL)-12, and IL-18 gene expression and protein production were readily induced by Salmonella-infected Mφs and DCs. CCL20 was preferentially produced by Mφs, whereas DCs secreted higher levels of CCL19 as compared with Mφs. DCs and Mφs infected with S. typhimurium also produced high levels of interferon-γ (IFN-γ). Cytokine neutralization and stimulation experiments suggest that the production was partly regulated by Salmonella-induced type I IFNs, IL-12, and IL-18. DC cytokine production induced by Salmonella was much higher as compared with the responses induced by Salmonella lipopolysaccharide or flagellin. Mφs and DCs were capable of internalizing and harboring Salmonella for several days. S. enterica PgtE provided no survival advantage for the bacteria in human Mφs or DCs. Our results demonstrate that although Mφs and DCs share similar functions, they may have different roles during Salmonella infection as a result of differential production of certain chemokines and cytokines.
Relation of postmortem blood alcohol and drug concentrations in fatal poisonings involving amitriptyline, propoxyphene and promazine
Authors: Koski A , Vuori E , Ojanperä I
Published: July 2005

Drugs and alcohol often occur together in fatal poisonings, complicating the process of determining the cause of death. Especially when found in concentrations generally regarded as toxic but not lethal, the question arises whether the combination of sublethal amounts was the likely cause of death.

In this study, we examined poisoning deaths involving amitriptyline, propoxyphene and promazine, which are, after benzodiazepines, the most frequently occurring drugs in Finnish alcohol-related poisonings. From the forensic toxicology database, covering the years 1995- 2002, we extracted 332 fatal poisonings, calculated median blood alcohol and drug concentrations, constructed concentration-concentration and concentration-response curves and evaluated the significance of the presence of therapeutic amounts of benzodiazepines.

Median amitriptyline and propoxyphene concentrations were lower in alcohol-related cases than in clean drug poisonings. Correspondingly, the median blood alcohol concentrations in all drug-related poisonings were 1.5-2.2 mg/g lower than that found in clean alcohol poisonings. Alcohol concentration proved to be a more sensitive indicator of alcohol-drug interaction than drug concentration. This result suggests that when alcohol is present, relatively small overdoses of the studied drugs may result in fatal poisoning. In this context, fatal drug and alcohol concentrations and the issue of determining the most important agent in fatal drug-alcohol intoxications are discussed.
Pathological effects of formalin (37% formaldehyde) feeding in female Japanese quails (Coturnix coturnix japonica)
Authors: Khan A , Bachaya H A , Khan M Z , Mahmood F
Published: July 2005

The aim of this study was to determine the pathological effects of formalin on female quails. Seventy-five 1-day-old female Japanese quails (Coturnix coturnix japonina) were divided into five equal groups (A–the eighth week. No clinical signs were observed in quails fed 2.5 mL formalin/kg feed. Depression, dullness and anorexia were prominent in quails fed 20 mL formalin/kg feed. Feed intake, body weight, egg production and egg weight together with absolute and relative weight of organs, erythrocyte and leukocyte counts, hemoglobin concentration and hematocrit were decreased at higher doses, i.e., 10 and 20 mL formalin/kg feed. Total serum proteins and globulin significantly increased (P B / 0.05) in all formalin fed quails compared to control birds. No gross lesions were observed in the 2.5 mL formalin/kg fed group. At higher doses (10 and 20 mL formalin/kg feed) hemorrhages on the thigh muscles, decreased weight and reduction in area and folds of different segments of oviduct were recorded. Reduced size of liver, heart and kidneys was recorded in quails fed 20 mL formalin/kg feed compared to controls and other birds. The histopathological changes in oviduct consisted of degeneration of mucosal glands characterized by vacuolation of nuclei of cells. It was concluded from the study that formalin feeding to female quails at 2.5 mL/kg feed is without harmful effects, however, higher doses are not without health risks.
Current Status of Acrylamide Research in Food: Measurement, Safety Assessment, and Formation
Authors: BLANK IMRE
Published: July 2005

Abstract: Relatively high amounts of acrylamide have recently been reported in carbohydrate‐rich foods under low moisture conditions. This sparked intensive investigations into acrylamide, encompassing its occurrence, chemistry, and toxicology/potential health risk in the human diet. Robust and efficient analytical methods have been developed to obtain reliable quantitative data. Recent epidemiological studies failed to evidence an association of cancer incidence and dietary acrylamide exposure. The link of acrylamide in foods to Maillard‐type reactions and, in particular, to asparagine has been a major step in elucidating feasible chemical formation routes. Decarboxylation of the Schiff base derived from asparagine and a carbonyl reactant plays a key role in acrylamide formation leading to azomethine ylide intermediates, which offer an attractive mechanistic explanation of the acrylamide amounts found experimentally, including the fact that acrylamide is preferentially formed in the presence of fructose, as compared to glucose or α‐dicarbonyls. However, the physical state of the reaction system may also affect acrylamide formation by influencing molecular mobility, particularly under low moisture conditions. Current research deals mainly with mitigation studies to reduce acrylamide during food processing and the role of water.
Dicarbonyl Intermediates in the Maillard Reaction
Authors: THORNALLEY PAUL J.
Published: July 2005

Abstract: The complexity of the Maillard reaction arises partly from multiple fragmentation reactions of the sugar moiety, constituting branch points in the reaction progress and establishing many parallel reaction pathways. Reactive intermediates produced by these processes are often α‐oxoaldehydes. The formation of α‐oxoaldehydes enhances and redirects glycating activity in the Maillard reaction since α‐oxoaldehydes are up to 20,000‐fold more reactive than glucose in glycation processes and are predominantly arginine‐directed glycating agents. α‐Oxoaldehydes bypass a requirement for a fructosamine precursor in the formation of advanced glycation end products (AGEs) since α‐oxoaldehydes react with proteins (also nucleotides and basic phospholipids) to form AGEs directly. The major AGE formed from α‐oxoaldehydes is generally a hydroimidazolone with other products—although for glyoxal, Nω‐carboxymethylarginine is a major product. α‐Oxoaldehyde formation also occurs in the absence of an amine substrate, particularly during heat processing of sugar solutions and lipid peroxidation processes—in the latter case, the glycation adducts are advanced lipoxidation products (ALEs). Hydroimidazolones are quantitatively important AGEs in cellular and extracellular proteins in physiological systems. Hydroimidazolone free adducts are liberated by cellular proteolysis and digestion. They are released into blood plasma for urinary excretion. Modification of arginine residues by α‐oxoaldehydes may be particularly damaging since arginine residues have high‐frequency occurrence in ligand and substrate recognition sites in receptor and enzyme active sites. Along with fructosamine formation, α‐oxoaldehyde intermediates of the Maillard reaction represent a major source of damage to the proteome and genome.
Methylglyoxal Can Modify GAPDH Activity and Structure
Authors: LEE HYON JAE , HOWELL SCOTT K. , SANFORD REBECCA J. , BEISSWENGER PAUL J.
Published: July 2005

Abstract:The activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) can play an important role in regulating multiple upstream pathways relating to the development of diabetic complications. GAPDH can be modified by a number of metabolic factors, including oxidative and glycation products. To study the effect of glycation on GAPDH we have measured GAPDH structure and activity after exposure of the enzyme to the potent alpha dicarbonyl sugar methylglyoxal (MG). Rabbit GAPDH was incubated with 10‐1000 μM MG for 96 hours, and enzyme activity was measured at intervals by a spectrophotometric assay. Isoelectric focusing of purified and cellular GAPDH was performed with a PROTEAN IEF system and the bands visualized by Western blotting. The mass of glycated and native GAPDH was determined by MALDI with a Applied Biosystems Voyager System 6235. GAPDH activity (at 96 h) was decreased by 20% with 1.0 micromolar MG and showed progressively greater suppression of activity with increasing concentrations up to 1 mM, where activity was decreased by 97%. Reduction in GAPDH activity was rapidly decreasing by 69.2% by two hours with 1 mM MG. IEF showed an isoelectric point (IEP) of 8.5 for native GAPDH, while measurable changes were seen with modification by MG levels of 1 mM (IEP 7.5) and 50 μM (IEP 8.0). With MALDI, GAPDH mass increased from 36.012 kDa to 37.071 after exposure to 50 μM MG and to 40.625 following 1 mM MG. This indicates addition of 12.75 and 55.6 MG residues, respectively, to GAPDH. GAPDH can be modified by methylglyoxal intracellular concentrations close to those previously observedin vivo, with measurable changes in isoelectric point and mass. These modifications can lead to decreased enzyme activity, suggesting that conditions associated with elevated intracellular MG could modify GAPDH activityin vivo.
Assessment of Protein Function following Cross‐Linking by α‐Dicarbonyls
Authors: MILLER ANTONIA G. , GERRARD JULIET A.
Published: July 2005

Abstract: Protein cross‐linking via the Maillard reaction with α‐dicarbonyl compounds has been the subject of intense scrutiny in the literature. We report here a study of the impact of this cross‐linking on enzyme function. Protein function following glycation was examined by treating ribonuclease A with methylglyoxal, glyoxal, and diacetyl, which cross‐linked the enzyme and impaired its activity. The effects of two reported Maillard reaction inhibitors, aminoguanidine and 3,5‐dimethylpyrazole‐1‐carboxamidine, on the cross‐linking reaction were assessed, with a parallel measurement of the effect on enzyme activity. The results demonstrate that preventing protein cross‐linking does not necessarily preserve enzyme activity. These results cast doubt on the likely efficacy of some purported antiaging compounds in vivo.
Advanced Glycation End Products/Peptides: An in Vivo Investigation
Authors: LAPOLLA A , FEDELE D , REITANO R , BONFANTE L , PASTORI G , SERAGLIA R , TUBARO M , TRALDI P.
Published: July 2005

Abstract: Advanced glycation end products/peptides (AGE/peptides) originate by in vivo enzymatic digestion of nonenzymatically glycated proteins, which are produced by reaction of glucose with primary amino groups present in the protein chain following the Maillard pattern. AGE/peptides are highly reactive species and can interact with tissue and circulating proteins, leading to tissue modification and impaired protein functionality. Serum levels of AGE/peptides are reported to be particularly high in diabetes (in terms of higher production) or in end‐stage renal disease (in terms of accumulation). For these reasons, their structural identification is of high interest, giving information on their relationship with the pathological state and allowing the design of possible therapeutic interventions. We report here some preliminary results obtained by liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) and matrix‐assisted laser desorption ionization MS (MALDI‐MS) investigations carried out on the low‐molecular‐weight serum peptide fraction from 10 healthy subjects, 10 patients with poorly controlled diabetes, and 10 patients with end‐stage nephropathy.
Tandem Mass Spectrometry Analysis of Amadori‐Glycated Phosphatidylethanolamine in Human Plasma
Authors: MIYAZAWA TERUO , OAK JEONG‐HO , NAKAGAWA KIYOTAKA
Published: July 2005

Abstract: Amadori‐glycated phosphatidylethanolamine (Amadori‐PE), a nonenzymatically glycated lipid formed under hyperglycemic conditions, is known as a reliable indicator of lipid glycation in vivo. We have quantified the Amadori‐PE concentration in human plasma samples using a reverse‐phase liquid chromatography‐tandem mass spectrometry with neutral loss scan or multiple reaction monitoring. Amounts of Amadori‐PE in plasma of diabetic patients (0.15 mol% of PE), diabetic patients with chronic hemodialysis (0.29 mol% of PE), and nondiabetic patients with chronic hemodialysis (0.13 mol% of PE) are higher than that of the control group (0.08 mol% of PE). In addition, the concentration of Amadori‐PE was proportional to that of phosphatidylcholine hydroperoxide, a reliable indicator of membrane lipid peroxidation, in human plasma (P < 0.05). These results indicate that plasma Amadori‐PE‐glycated lipid product formed under hyperglycemic conditions is an inducer of membrane lipid peroxidation, and therefore lipid glycation plays an active part in the development of human disease.
Accumulation of Advanced Glycation End Products, Measured as Skin Autofluorescence, in Renal Disease
Authors: HARTOG JASPER W. L. , DE VRIES AIKO P. J. , LUTGERS HELEN L. , MEERWALDT ROBBERT , HUISMAN ROEL M. , VAN SON WILLEM J. , DE JONG PAUL E. , SMIT ANDRIES J.
Published: July 2005

Abstract: Advanced glycation end products (AGEs) accumulate during renal failure and dialysis. Kidney transplantation is thought to reverse this accumulation by restoring renal function. Using a noninvasive and validated autofluorescence reader, we evaluated AGE levels in 285 transplant recipients (mean age, 52 years; range, 41 to 60 years), 32 dialysis patients (mean age, 56 years; range, 43 to 65 years), and 231 normal control subjects (mean age, 51 years; range, 40 to 65 years). Measurements in transplant recipients were performed for a mean of 73 months (range, 32 to 143 months) after transplantation. Dialysis patients were on dialysis therapy for a mean of 42 months (range, 17 to 107 months). Fluorescence was significantly increased in dialysis patients compared with normal control subjects (2.8 vs. 2.0 arbitrary units [a.u.], P < .0001). Although fluorescence levels were significantly decreased in transplant recipients compared with dialysis patients (2.5 vs. 2.8 a.u., P < .0001), fluorescence in transplant recipients was higher than in controls (2.5 vs. 2.0 a.u., P < .0001). In transplant recipients, fluorescence correlated positively with the duration of dialysis prior to transplantation (R= 0.21, P < .0001), and negatively with creatinine clearance (R=−0.34, P < .0001). No correlation was found between time after transplantation and fluorescence in transplant recipients (R=−0.10, P= .10). Fluorescence in dialysis patients was positively correlated with duration of dialysis (R= 0.36, P= .042). Our results, like those of others, suggest that kidney transplantation does not fully correct increased AGE levels found in dialysis patients. The increased AGE levels in kidney transplant recipients cannot be explained by the differences in renal function alone. The availability of a simple, noninvasive method (AGE‐Reader) to measure AGE accumulation may be used to monitor AGE accumulation in a clinical setting as well as in a study setting.
Isolevuglandins, Oxidatively Truncated Phospholipids, and Atherosclerosis
Authors: SALOMON ROBERT G.
Published: July 2005

Abstract: Isolevuglandins (isoLGs) and oxidatively truncated phospholipids are products of lipid peroxidation. Some of these, especially isoLGs and γ‐hydroxyalkenal analogues (e.g., the 5‐hydroxy‐8‐oxo‐6‐octenoic acid and 9‐hydroxy‐12‐oxo‐10‐dodecenoic acid esters of 2‐lysophosphatidylcholine, HOOA‐PC or HODA‐PC, respectively) of 4‐hydroxy‐2(E)‐nonenal (HNE), damage proteins by covalent adduction, thereby interfering with their normal functions. These lipid‐derived protein modifications may serve as dosimeters of oxidative injury. Elevated plasma levels of isoLG‐protein epitopes are associated with atherosclerosis but are independent of total cholesterol, a classical risk factor. Both protein adducts and oxidatively truncated phospholipids (oxPL) can also elicit receptor‐mediated cellular responses that include endocytosis of oxidized low‐density lipoprotein (LDL) and expression of chemokines, which may foster infiltration of monocyte macrophages into the subendothelial space, where they become foam cells through unregulated endocytosis of oxidatively damaged LDL.
Role of Glucoxidation and Lipid Oxidation in the Development of Atherosclerosis
Authors: SCHLEICHER ERWIN , WEIGERT CORA , ROHRBACH HELMUT , NERLICH ANDREAS , BACHMEIER BEATRICE , FRIESS ULRICH
Published: July 2005

Abstract: Previous data have indicated that modification of proteins/lipids by glucoxidation and/or lipid oxidation may initiate/propagate the formation of atherosclerotic plaques. Although the biomarker carboxymethyllysine (CML) has been detected in these lesions, the origin of the reactive oxygen species (ROS) leading to its formation and the source of its carbon backbone are unknown. As presented here, the stimulation of cultured monocytes by phorbol‐12‐myristate‐13‐acetate (TPA), an activator of protein kinase C that can mimic the effects of high glucose, angiotensin II, and other physiological stimuli, leads to cellular ROS generation and concomitant formation of intracellular CML. Inhibitors of ROS‐generating cellular systems such as NO synthase, xanthine oxidase, or cytochrome P450 oxidase had no effect on CML formation. Likewise, in cells with inactive NAD(P)H oxidase no reduced CML formation was found. In cells exhibiting a high glycolysis rate, CML formation was unaffected. Because we found rapid CML formation in the presence of unsaturated fatty acids, it appears that lipid oxidation is quantitatively more important. In vivo studies revealed strong intracellular CML staining in areas of histiocytic/monocytic infiltration or proliferation, mostly associated with atheroma formation. Corresponding CML staining patterns were found in healing wounds of different ages, indicating that formation of atherosclerosis is a chronic wound repair associated with a low‐grade inflammatory reaction. In summary, CML is formed concomitantly with oxidative stress in activated monocytes and can be regarded as a biomarker for a low‐grade inflammatory tissue reaction in the atherosclerotic plaque. Its formation via lipid oxidation may be involved in the development of atherosclerosis.
Is Atherosclerosis a Multifactorial Disease or Is It Induced by a Sequence of Lipid Peroxidation Reactions?
Authors: SPITELLER GERHARD
Published: July 2005

Abstract: The delivery of not only free cholesterol but also cholesterol esters to cells by low‐density lipoprotein (LDL) has hitherto been unstudied. Minor compounds present in mammalian‐derived food include cholesterol linoleate and arachidonate. Evidence is presented that these esters are directly incorporated into VLDL and are responsible for the deleterious effects of atherosclerosis. Cholesterol esterified with these polyunsaturated fatty acids (PUFAs) is readily oxidized at the PUFA residue during storage and heating. Apparently, the liver is unable to distinguish between nonoxidized and oxidized cholesterol PUFA esters and also incorporates the latter into VLDL, which is transformed to LDL. When this LDL is transferred to endothelial cells, the toxic products are liberated and induce cell damage. Cell damage is combined with structural changes that influence neighboring cells and cause an influx of Ca2+ ions and activation of phospholipases and lipoxygenases, resulting in production of lipid hydroperoxides (LOOHs). When the level of free PUFAs generated by phospholipases exceeds a certain limit, lipoxygenases commit suicide, causing liberation of iron ions. The latter react with LOOHs and thus induce a switch from enzymatic to nonenzymatic generation of lipid peroxidation (LPO) products. Although the LOO· radicals produced in enzymatic reactions are deactivated within the enzyme complex, LOO· radicals generated in nonenzymatic reactions are able to attack any biological compound, inducing severe damage. Apparently, iron ions and LOOH molecules at the surface of injured cells transfer the nonenzymatic LPO reactions to the phospholipid layer of bypassing lipoproteins, thus explaining why inflammatory diseases, such as diabetes, are combined with atherogenesis.
Glycation Does Not Alter LDL‐Induced Secretion of Tissue Plasminogen Activator and Plasminogen Activator Inhibitor‐1 from Human Aortic Endothelial Cells
Authors: KLEIN RICHARD L. , SEMLER ANDREA J. , BAYNES JOHN W. , THORPE SUZANNE R. , LYONS TIMOTHY J. , JENKINS ALICIA J.
Published: July 2005

Abstract: Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low‐density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor‐1 (PAI‐1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI‐1 production were determined after incubation with LDL (50 to 500 μg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N‐LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N‐LDL (83 ± 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G‐LDL (73 ± 7), MO‐LDL (74 ± 13), or MOG‐LDL (66 ± 15) and was not influenced by LDL concentration. The rate of PAI‐1 release was similar in HAECs incubated with N‐LDL (5.7 ± 0.6 μg/mg cell protein/24 h), G‐LDL (5.7 ± 0.7), MO‐LDL (5.5 ± 0.8), or MOG‐LDL (5.7 ± 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 μg/mL) modified extensively by oxidation, and averaged 45.2 ± 5.0 and 43.7 ± 9.9 ng/mg/24 h for HO‐LDL and HOG‐LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI‐1 release was not significantly decreased relative to N‐LDL in cells incubated with low concentrations (5 to 50 μg/mL) of HO‐LDL and HOG‐LDL, but was decreased to 3.2 ± 0.5 and 3.1 ± 0.7 μg/mg/24 h for HO‐LDL and HOG‐LDL at 200 μg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI‐1 release did not differ between subfractions. Release of tPA averaged 5.11 ± 0.6 and 5.12 ± 0.7 ng/mg/24 h, whereas release of PAI‐1 averaged 666 ± 27 ng/mg/24 h and 705 ± 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI‐1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.
Covalent Binding of Acetone to Aminophospholipids in Vitro and in Vivo
Authors: KUKSIS ARNIS , RAVANDI AMIR , SCHNEIDER MICHAEL
Published: July 2005

Abstract: We have determined the ions characteristic of acetone adducts of reference aminophospholipids and have used them as markers for identification of acetone adducts of aminophospholipids in commercial lecithin, acetone extracts of tissue lipids, and in plasma and red blood cells of diabetic subjects. The acetonation products were determined by normal‐phase high‐performance liquid chromatography (HPLC) with on‐line electrospray‐mass spectrometry, and electrospray/collision‐induced dissociation in the negative ion mode. The major acetone complexes of PtdEtn and PtdSer were identified as the diacetone derivatives [PtdEtn+116‐H2O]− and [PtdSer+116‐H2O]−, respectively, although ions corresponding to monoacetone [PtdEtn+58‐H2O]− and doubly dehydrated diacetone adducts [PtdSer+116‐2x18]− were also observed. Upon increase of the capillary exit voltage (CapEx) from −160 to −300 V, new ions appeared with the original retention time but with 58 masses (one acetone molecule) lower than the mass of the parent compounds, along with fragment ions corresponding to lysoGPE+40 and free fatty acids. Scanning of chloroform/methanol extracts of red blood cell lipids of two of five diabetic subjects examined yielded elevated levels (in relation to nondiabetic subjects) for ions corresponding to the diacetone adducts [M+98]− of the major molecular species of PtdEtn and PtdSer. Because of possible overlap with major molecular species of PtdIns, the identification of the acetonated PtdSer in diabetic blood requires further confirmation.