bluuebunny/crossref_metadata_2025 huggingface dataset:
1,25-Dihydroxyvitamin D3decreases podocyte loss and podocyte hypertrophy in the subtotally nephrectomized rat
Authors: Kuhlmann Alexander , Haas Christian S. , Gross Marie-Luise , Reulbach Udo , Holzinger Marc , Schwarz Ute , Ritz Eberhard , Amann Kerstin
Published: February 2004

1,25(OH)2D3has antiproliferative effects and promotes cell differentiation. This consideration has provided the rationale for studies in subtotally nephrectomized rats showing that 1,25(OH)2D3interferred with glomerulosclerosis. The cellular mechanisms involved have remained obscure, however. It was the purpose of the present study to assess glomerular structure and cellular composition in subtotally nephrectomized (SNX) rats treated with nonpharmacological doses of 1,25(OH)2D3. Male Sprague-Dawley rats were sham operated (sham) or underwent SNX under general anesthesia and received either solvent or 1,25(OH)2D3(3 ng·100 g body wt-1·day-1sc). Blood pressure (BP) and albuminuria were measured. After 16 wk, the remnant renal tissue was perfusion fixed and morphometric and stereological measurements were carried out. The expression of proliferating cellular antigen (PCNA), cyclin-dependent kinase inhibitor p27, Wilms tumor gene (WT1), and desmin, a marker of early podocyte damage, was investigated by immunohistology. BP, serum creatinine, and urinary albumin excretion were significantly higher in SNX than in sham rats. Albuminuria was significantly lower in SNX+1,25(OH)2D3compared with SNX+solvent rats. Mean glomerular tuft volume was significantly higher in SNX+solvent (2.69 ± 0.21 g× 106μm3) than in sham rats (1.44 ± 0.17 and 1.28 ± 0.14 × 106μm3); it was significantly ( P < 0.05) lower in SNX+1,25(OH)2D3rats (1.81 ± 0.16 × 106μm3). The main finding was a significantly higher number of podocytes in SNX+1,25(OH)2D3(88 ± 9) and sham (98 ± 17) compared with SNX+solvent rats (81 ± 8.7). In parallel, the increase in podocyte volume in SNX+solvent rats was abrogated by treatment with 1,25(OH)2D3, and immunohistochemistry revealed less expression of desmin, PCNA, and p27, suggesting less podocyte injury and activation of the cyclin cascade. This study identifies the podocyte as an important target cell for the renoprotective action of 1,25(OH)2D3. This notion is suggested by less evidence of podocyte injury, decreased podocytes loss, and abrogation of podocyte hypertrophy, findings that may also explain less pronounced albuminuria and glomerulosclerosis.
Microarray experimental design: power and sample size considerations
Authors: Yang M. C. K. , Yang J. J. , McIndoe R. A. , She J. X.
Published: February 2004

Gene expression analysis using high-throughput microarray technology has become a powerful approach to study systems biology. The exponential growth in microarray experiments has spawned a number of investigations into the reliability and reproducibility of this type of data. However, the sample size requirements necessary to obtain statistically significant results has not had as much attention. We report here statistical methods for the determination of the sufficient number of subjects necessary to minimize the false discovery rate while maintaining high power to detect differentially expressed genes. Two experimental designs were considered: 1) a comparison between two groups at a single time point, and 2) a comparison of two experimental groups with sequential time points. Computer programs are available for the methods discussed in this paper and are adaptable to more complicated situations.
Transcriptome profiling of aSaccharomyces cerevisiaemutant with a constitutively activated Ras/cAMP pathway
Authors: Jones D. L. , Petty J. , Hoyle D. C. , Hayes A. , Ragni E. , Popolo L. , Oliver S. G. , Stateva L. I.
Published: February 2004

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2Δ mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of ∼11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.
Pancreatic triacylglycerol lipase in a hibernating mammal. I. Novel genomic organization
Authors: Squire Teresa L. , Andrews Matthew T.
Published: February 2004

Pancreatic triacylglycerol lipase ( PTL) is expressed in novel locations during hibernation in the thirteen-lined ground squirrel ( Spermophilus tridecemlineatus). PTL cDNAs isolated from two of these locations, heart and white adipose tissue (WAT), contain divergent 5′-untranslated regions (5′-UTRs) suggesting alternative promoter usage or the possibility of multiple PTL genes in the ground squirrel genome. In addition, cDNAs isolated from WAT contain tracts of retroviral sequence in their 5′-UTRs. Our examination of PTL genomic clones isolated from a thirteen-lined ground squirrel genomic DNA library, coupled with genomic Southern blot analysis, enabled us to conclude that PTL mRNAs expressed in heart and WAT are the products of the same single-copy gene. The 5′ portion of this gene spans 9.2 kb, is composed of 6 exons, and contains a full-length endogenous retroviral genome with conserved long terminal repeats (LTRs). Alignment of the ground squirrel PTL gene with the mouse, rat, and human PTL genes indicates that this retrovirus inserted into the ground squirrel genome ∼200 bases upstream of the original PTL transcriptional start site. The insertion is a relatively recent event based on largely intact open-reading frames containing minimal frame-shift and nonsense mutations. The high-percentage identity (99.2%) shared between the 5′- and 3′-LTRs of this endogenous retrovirus suggests that the insertion occurred as recently as 300,000 years ago.
Pancreatic triacylglycerol lipase in a hibernating mammal. II. Cold-adapted function and differential expression
Authors: Squire Teresa L. , Lowe Mark E. , Bauer Vernon W. , Andrews Matthew T.
Published: February 2004

Thirteen-lined ground squirrels ( Spermophilus tridecemlineatus) exploit the low-temperature activity of pancreatic triacylglycerol lipase (PTL) during hibernation. Lipolytic activity at body temperatures associated with hibernation was examined using recombinant ground squirrel and human PTLs expressed in yeast. Both the human and ground squirrel enzymes displayed high activity at temperatures as low as 0°C and showed Q10values of 1.2–1.5 over a range of 37–7°C. These studies indicate that low-temperature lipolysis is a general property of PTL and does not require protein modifications unique to mammalian cells and/or the hibernating state. Western blots show elevated levels of PTL protein during hibernation in both heart and white adipose tissue (WAT). Significant increases in PTL gene expression are seen in heart, WAT, and testes; but not in pancreas, where PTL mRNA levels are highest. Upregulation of PTL in testes is also accompanied by expression of the PTL-specific cofactor, colipase. The multi-tissue expression of PTL during hibernation supports its role as a key enzyme that shows high activity at low temperatures.
Identification of genomic islands in the genome ofBacillus cereusby comparative analysis withBacillus anthracis
Authors: Zhang Ren , Zhang Chun-Ting
Published: February 2004

Horizontal gene transfer has been recognized as a universal event throughout bacterial evolution. The availability of both complete genome sequences of Bacillus cereus and B. anthracis provides the possibility to perform comparative analysis based on their genomes. By using a windowless method to display the distribution of the genomic GC content of B. cereus and B. anthracis, we have found three genomic islands in the genome of B. cereus, i.e., BCGI-1, BCGI-2, and BCGI-3, respectively, which are absent in the genome of B. anthracis. All the genomic islands have abrupt changes in GC content compared with that of surrounding regions. BCGI-1 has many conserved features of genomic islands, e.g., a Val-tRNA gene is utilized as the integration site, and a site-specific recombinase gene is located at the 3′ end. BCGI-2 has a large percentage of phage protein, suggesting a phage-related recombination is involved. BCGI-3 contains a ferric anguibactin transport system, which is likely to be involved in the iron transport that enables the bacterium to overcome the iron limitation in the host. In addition, BCGI-3 also contains a cluster of genes related to lantibiotics, which may play a role during the evolution of the genome. Furthermore, the integrations of the genomic islands, BCGI-1 and BCGI-3, result in deletions of DNA sequence fragments; therefore, such integrations lead to both gene gain and gene loss simultaneously.
Resolvent estimates of elliptic differential and finite‐element operators in pairs of function spaces
Authors: Bakaev Nikolai Yu.
Published: February 2004

We present some resolvent estimates of elliptic differential and
finite‐element operators in pairs of function spaces, for which
the first space in a pair is endowed with stronger norm. In this
work we deal with estimates in (Lebesgue, Lebesgue), (Hölder,
Lebesgue), and (Hölder, Hölder) pairs of norms. In
particular, our results are useful for the stability and error
analysis of semidiscrete and fully discrete approximations to parabolic partial differential problems with rough and distribution‐valued data.
Absence of Dystrophin in Mice Reduces NO-Dependent Vascular Function and Vascular Density: Total Recovery After a Treatment with the Aminoglycoside Gentamicin
Authors: Loufrani Laurent , Dubroca Caroline , You Dong , Li Z. , Levy Bernard , Paulin Denise , Henrion Daniel
Published: February 2004

Objectives—

Mutations in the dystrophin gene causing Duchenne’s muscular dystrophy (DMD) lead to premature stop codons. In mice lacking dystrophin (mdx mice), a model for DMD, these mutations can be suppressed by aminoglycosides such as gentamicin. Dystrophin plays a role in flow (shear stress)-mediated endothelium-dependent dilation (FMD) in arteries. We investigated the effect of gentamicin on vascular contractile and dilatory functions, vascular structure, and density in mdx mice.

Methods and Results—

Isolated mice carotid and mesenteric resistance arteries were mounted in arteriographs allowing continuous diameter measurements. Mdx mice showed lower nitric oxide (NO)-dependent FMD and endothelial NO synthase (eNOS) expression as well as decreased vascular density in gracilis and cardiac muscles compared with control mice. Treatment with gentamycin restored these parameters. In contrast, smooth muscle–dependent contractions as well as endothelium-dependent or -independent dilation were not affected by dystrophin deficiency or by gentamicin treatment.

Conclusion—

Dystrophin deficiency induces a selective defect in flow-dependent mechanotransduction, thus attenuating FMD and eNOS expression, and may contribute to low arteriolar density. These findings open important perspectives regarding the mechanism involved in the pathophysiology of genetic diseases related to premature stop codons such as DMD.
Angiotensin-Converting Enzyme D/I Gene Polymorphism and Age-Related Changes in Pulse Pressure in Subjects with Hypertension
Authors: Safar Michel E. , Lajemi Malika , Rudnichi Annie , Asmar Roland , Benetos Anathase
Published: February 2004

Background—

Few studies have examined the possible influence of gene polymorphisms on the increase of systolic blood pressure (SBP) and pulse pressure (PP) with age, although in older populations, SBP>160 mm Hg or PP>60 mm Hg are strong mechanical factors predicting cardiovascular mortality.

Methods and Results—

This cross-sectional study involved 315 men and 154 women with either systolic–diastolic or isolated systolic hypertension. Using polymerase chain reaction, the angiotensin-converting enzyme (ACE) D/I gene polymorphism was investigated separately in men and women, enabling us to determine the relationships between age and PP, SBP, and diastolic blood pressure (DBP) for each genotype in each population. In men, most of which were under 50 years of age, the slope of the age–PP and age–SBP (but not age–DBP) relationships differed significantly between genotypes (
P
=0.0096 and 0.0175). The interslope difference was unmodified after adjustments involving all of the following factors together: plasma glucose, cholesterol, creatinine, potassium, body weight, tobacco consumption, mean blood pressure, and previous antihypertensive therapy. Adjustment of the two latter parameters alone significantly attenuated the interslope difference. Based on logistic regressions, the DD genotype was shown to independently predict a PP>60 mm Hg but not a SBP>160 mm Hg.

Conclusions—

In men, the ACE D/I gene polymorphism independently modulates age-related increase of PP, and potentially modulates the resulting cardiovascular risk. This finding requires the development of long-term follow-up.
The “CholesteROR” Protective Pathway in the Vascular System
Authors: Boukhtouche Fatiha , Mariani Jean , Tedgui Alain
Published: February 2004

Retinoic acid receptor-related Orphan Receptor α (RORα) is a member of the nuclear hormone receptor superfamily. RORα has long been considered as a constitutive activator of transcription in the absence of exogenous ligand; however, cholesterol has recently been identified as a natural ligand of RORα. The spontaneousstaggerer(sg/sg) mutation is a deletion in the Rora gene that prevents the translation of the ligand-binding domain (LBD), leading to the loss of RORα activity. The homozygous Rorasg/sgmutant mouse, of which the most obvious phenotype is ataxia associated with cerebellar degeneration, also displays a variety of other phenotypes, including several vascular ones; in particular, dysfunction of smooth muscle cells and enhanced susceptibility to atherosclerosis. Moreover, RORα appears to participate in the regulation of plasma cholesterol levels, and has been shown to positively regulate apolipoprotein (apo)A-I and apoC-III gene expression. Yet its activity is regulated by cholesterol itself, making RORα an intracellular cholesterol target.
Subclasses of Low-Density Lipoprotein and Very Low-Density Lipoprotein in Familial Combined Hyperlipidemia: Relationship to Multiple Lipoprotein Phenotype
Authors: Georgieva A.M. , van Greevenbroek M.M.J. , Krauss R.M. , Brouwers M.C.G.J. , Vermeulen V.M.M.-J. , Robertus-Teunissen M.G. , van der Kallen C.J.H. , de Bruin T.W.A.
Published: February 2004

Objective—

The present study addresses the presence of distinct metabolic phenotypes in familial combined hyperlipidemia (FCHL) in relation to small dense low-density lipoprotein (sd LDL) and very low-density lipoprotein (VLDL) subclasses.

Methods and Results—

Hyperlipidemic FCHL relatives (n=72) were analyzed for LDL size by gradient gel electrophoresis. Pattern B LDL (sd LDL, particle size <258 Å) and pattern A LDL (buoyant LDL, particle size ≥258 Å) were defined. Analyses showed bimodal distribution of LDL size associated with distinct phenotypes. Subjects with predominantly large, buoyant LDL showed a hypercholesterolemic phenotype and the highest apo B levels. Subjects with predominantly sd LDL showed a hypertriglyceridemic, low high-density lipoprotein (HDL) cholesterol phenotype, with moderately elevated apoB, total cholesterol level, and LDL cholesterol level. Subjects with both buoyant LDL and sd LDL (pattern AB, n=7) showed an intermediate phenotype, with high normal plasma triglycerides. VLDL subfraction analysis showed that the sd LDL phenotype was associated with a 10-times higher number of VLDL1 particles of relatively lower apo AI and apo E content, as well as smaller VLDL2 particles, in combination with increased plasma insulin concentration in comparison to pattern A.

Conclusions—

The present observations underscore the importance of the VLDL triglyceride metabolic pathway in FCHL as an important determinant of the phenotypic heterogeneity of the disorder.
Implications of Upstream Glycoprotein IIb/IIIa Inhibition and Coronary Artery Stenting in the Invasive Management of Unstable Angina/Non–ST-Elevation Myocardial Infarction
Authors: Sabatine Marc S. , Morrow David A. , Giugliano Robert P. , Murphy Sabina A. , Demopoulos Laura A. , DiBattiste Peter M. , Weintraub William S. , McCabe Carolyn H. , Antman Elliott M. , Cannon Christopher P. , Braunwald Eugene
Published: February 2004

Background—

TIMI IIIB and TACTICS-TIMI 18 were 2 trials of an early invasive strategy in unstable angina (UA)/non–ST-elevation myocardial infarction (NSTEMI) that were conducted nearly a decade apart but with virtually identical enrollment criteria and designs, except that upstream glycoprotein IIb/IIIa inhibition was mandated and coronary artery stenting was routinely used in TACTICS-TIMI 18. We sought to examine the effect of these advances on clinical outcomes and the benefits of an early invasive strategy in UA/NSTEMI.

Methods and Results—

Patients were stratified on the basis of their TIMI risk score into low-, intermediate-, and high-risk categories. Within each risk category, the rates of clinical outcomes and the benefit of an early invasive strategy were compared. Compared with patients in TIMI IIIB and adjusting for baseline risk, patients in TACTICS-TIMI 18 had lower rates of death, MI, or rehospitalization for acute coronary syndromes (OR, 0.62;
P
<0.0001). Across both trials, the benefit of an early invasive strategy was significantly greater with increasing baseline risk: OR, 1.39 in low-risk, 0.80 in intermediate-risk, and 0.57 in high-risk patients (
P
≤0.004 for interactions). After adjustment for baseline risk, an early invasive strategy tended toward a more favorable result in TACTICS-TIMI 18 than in TIMI IIIB (OR, 0.79; 95% CI, 0.56 to 1.11).

Conclusions—

Advances in the care of patients with UA/NSTEMI, including glycoprotein IIb/IIIa inhibition and stenting, were associated with lower rates of death, MI, and rehospitalization for acute coronary syndromes and a trend toward a greater benefit of an early invasive strategy.
Coronary Bypass Surgery Performed off Pump Does Not Result in Lower In-Hospital Morbidity Than Coronary Artery Bypass Grafting Performed on Pump
Authors: Légaré Jean-Francois , Buth Karen J. , King Sharon , Wood Jeremy , Sullivan John A. , Friesen Camille Hancock , Lee John , Stewart Kier , Hirsch Gregory M.
Published: February 2004

Background—

There is increasing evidence that cardiopulmonary bypass (CPB) may be responsible for the morbidity associated with coronary artery bypass grafting (CABG) surgery. Recent developments in cardiac stabilization devices have made CABG without CPB feasible. However, there is conflicting evidence to date from published trials comparing outcomes between CABG performed with and without CPB, with some trials indicating an advantage to the avoidance of CPB and others showing little benefit.

Methods and Results—

In a single-center randomized trial, 300 patients requiring CABG surgery at a single institution were prospectively randomized to have the procedure performed with CPB (n=150) or on the beating heart (n=150). Exclusion criteria for the trial included emergency procedure, concomitant major cardiac procedures, ejection fraction <30%, and reoperation. In-hospital outcomes were analyzed on an intention-to-treat basis. A mean of 3.0±0.9 grafts were performed in the CPB group compared with 2.8±0.9 grafts in the beating-heart group (
P
=0.06). There were no significant differences between the CPB group and the beating-heart group in mortality (0.7% versus 1.3%;
P
=1.0), transfusion (8.7% versus 9.3%), perioperative myocardial infarction (0.7% versus 2.7%;
P
=0.37), permanent stroke (0% versus 1.3%;
P
=0.50), new atrial fibrillation (32% versus 25%;
P
=0.20), and deep sternal wound infection (0.7% versus 0%;
P
=1.0). The mean time to extubation was 4 hours, the mean stay in the intensive care unit was 22 hours, and the median length of hospitalization was 5 days in both groups (
P
=NS).

Conclusions—

In contrast to published trials, we were unable to demonstrate any advantage with CABG performed without CPB in terms of patient morbidity. Excellent results can be obtained with either surgical approach.
Aberrant quantity and localization of Aurora-B/AIM-1 and survivin during megakaryocyte polyploidization and the consequences of Aurora-B/AIM-1–deregulated expression
Authors: Zhang Ying , Nagata Yuka , Yu Guangyao , Nguyen Hao G. , Jones Matthew R. , Toselli Paul , Jackson Carl W. , Tatsuka Masaaki , Todokoro Kazuo , Ravid Katya
Published: February 2004

Megakaryocytes skip late anaphase and cytokinesis during endomitosis. We found normal expression and localization of a fundamental regulator of mitosis, Aurora-B/AIM-1, during prophase in polyploidizing mouse bone marrow megakaryocytes. At late anaphase, however, Aurora-B/AIM-1 is absent or mislocalized. Megakaryocytes treated with a proteasome inhibitor display Aurora-B/AIM-1 properly expressed and localized to the midzone, suggesting that protein degradation contributes to this atypical appearance. In contrast, survivin, an Aurora-B/AIM-1 coregulator of mitosis, is not detected at any stage of the endomitotic cell cycle, and in most megakaryocytes proteasome inhibition does not rescue this phenotype. To further explore the importance of reduced Aurora-B/AIM-1 for polyploidization, it was overexpressed in megakaryocytes of transgenic mice. The phenotype includes increased transgenic mRNA, but not protein, in polyploidy megakaryocytes, further suggesting that Aurora-B/AIM-1 is regulated at the protein level. Aurora-B/AIM-1 protein is, however, elevated in diploid transgenic megakaryocytes. Transgenic mice also exhibit enhanced numbers of megakaryocytes with increased proliferative potential, and some mice exhibit mild decreases in ploidy level. Hence, the molecular programming involved in endomitosis is characterized by the mislocalization or absence of at least 2 critical mitotic regulators, Aurora-B/AIM-1 and survivin. Future studies will examine the impact of survivin restoration on mouse megakaryocyte polyploidization.
Smallpox: Clinical Features, Prevention, and Management
Authors: Guharoy Roy , Panzik Robert , Noviasky John A , Krenzelok Edward P , Blair Donald C
Published: February 2004

OBJECTIVE

To describe a general overview of smallpox, clinical presentation, diagnosis, adverse events, and management of both pre- and postexposure vaccination.

DATA SOURCES

Literature was identified by search of MEDLINE (1966–June 2003) and International Pharmaceutical Abstracts (1966–May 2003) databases using the key terms smallpox, bioterrorism, biological warfare, and smallpox vaccine.

STUDY SELECTION AND DATA EXTRACTION

Articles identified from data sources were evaluated, and relevant information was included in this review.

DATA SYNTHESIS

Smallpox is spread by human-to-human contact with an infected host and therefore can be contagious. The mortality rate for smallpox is approximately 30%. While the disease was completely eradicated by 1980 with successful use of smallpox vaccine, concern has been raised that smallpox may emerge as a tool of bioterrorism. This concern, combined with the reality of current smallpox vaccination programs in the military and selected civilian populations, mandates a clear understanding of vaccination-related adverse events and contraindications by all healthcare professionals. The vaccine may cause moderate to severe adverse events such as eczema vaccinatum, progressive vaccinia, and generalized vaccinia.

CONCLUSION

The balance between the risks and benefits of mass vaccination in prevention of an epidemic is not clear. The Centers for Disease Control and Prevention has established a guideline for appropriate use of smallpox vaccine in the civilian population.
Performance of Bilirubin Determinations in US Laboratories—Revisited
Authors: Lo Stanley F , Doumas Basil T , Ashwood Edward R
Published: February 2004

Background: The diagnosis and management of hyperbilirubinemia in the newborn requires accurate and precise bilirubin determinations. We evaluated the current status of bilirubin measurements in US laboratories by examining data submitted by laboratories participating in the College of American Pathologists (CAP) Neonatal Bilirubin (NB) and Chemistry (C) Surveys.

Methods: We analyzed specimens from the CAP NB and C Surveys by the reference method for total bilirubin in three laboratories. The reference method bilirubin values were compared with bilirubin values reported by Survey participants.

Results: The imprecision (CV) for all instruments combined (CAP-All Instruments) ranged from 4.7% to 5.6% at the bilirubin concentrations tested. The CVs of the four most commonly used instruments were smaller, ranging from 1.9% to 4.5%. Differences between bilirubin values by the reference method and mean values from the four most common instruments ranged from −21.6% to 10.9%. When the same specimens from the NB Surveys were used in the C Surveys, the Vitros values were strikingly different from those of the NB Surveys. The use of different methods in the NB and C Surveys coupled with the presence of a nonhuman protein base and ditaurobilirubin (DTB) in the Survey specimens accounted for the discrepant results.

Conclusions: The evaluation of accuracy is impossible from the CAP Surveys because the specimens consist of bovine serum containing a mixture of unconjugated bilirubin and DTB. For the evaluation of accuracy, we recommend that Survey specimens consist of human serum enriched with unconjugated bilirubin.
N-Acetylated Metabolites in Urine: Proton Nuclear Magnetic Resonance Spectroscopic Study on Patients with Inborn Errors of Metabolism
Authors: Engelke Udo F H , Liebrand-van Sambeek Maria L F , de Jong Jan G N , Leroy Jules G , Morava Éva , Smeitink Jan A M , Wevers Ron A
Published: February 2004

Background: There is no comprehensive analytical technique to analyze N-acetylated metabolites in urine. Many of these compounds are involved in inborn errors of metabolism. In the present study, we examined the potential of proton nuclear magnetic resonance (1H-NMR) spectroscopy as a tool to identify and quantify N-acetylated metabolites in urine of patients with various inborn errors of metabolism.

Methods: We performed 1H-NMR spectroscopy on a 500 MHz spectrometer. Using a combination of one- and two-dimensional correlation spectroscopy (COSY) 1H-NMR spectra, we were able to assign and quantify resonances of characteristic N-acetylated compounds products in urine of patients with 13 inborn errors of metabolism.

Results: The disease-specific N-acetylated metabolites were excreted at concentrations >100 μmol/mmol of creatinine in the patients’ urine. In control urine samples, the concentration of individual N-acetyl-containing compounds was <40 μmol/mmol of creatinine. The combination of one- and two-dimensional COSY NMR spectroscopy led to the correct diagnosis of nine different inborn errors of metabolism. No abnormalities were observed in the spectra of urine from patients with GM1- or GM2-gangliosidosis. We also determined the 1H-NMR characteristics of N-acetylated metabolites that may be relevant to human metabolism.

Conclusion: 1H-NMR spectroscopy may be used to identify and quantify N-acetylated metabolites of diagnostic importance for the field of inborn errors of metabolism.
Improved Accuracy of Detection of Nasopharyngeal Carcinoma by Combined Application of Circulating Epstein–Barr Virus DNA and Anti-Epstein–Barr Viral Capsid Antigen IgA Antibody
Authors: Leung Sing-fai , Tam John S , Chan Anthony T C , Zee Benny , Chan Lisa Y S , Huang Dolly P , Van Hasselt Andrew , Johnson Philip J , Lo Y M Dennis
Published: February 2004

Background: Circulating Epstein–Barr viral (EBV) DNA and anti-EBV capsid antigen IgA (IgA VCA) represent two of the most sensitive peripheral blood markers of nasopharyngeal carcinoma (NPC), but direct comparative studies of these two markers are lacking.

Methods: The sensitivities and specificities of IgA-VCA and EBV DNA for diagnosis of NPC were determined in 139 new cases of NPC and 178 healthy individuals, respectively. EBV DNA was also assessed in 36 healthy family members identified as having false-positive IgA-VCA results at a screening clinic. EBV DNA was measured by a real-time quantitative PCR assay with a detection limit of 60 copies/mL. IgA-VCA was measured by semiquantitative indirect immunofluorescent method; a titer ≥1/10 was taken as positive.

Results: The sensitivities of EBV DNA and IgA-VCA for diagnosis of NPC were 95% (95% confidence interval, 91–98%) and 81% (73–87%), respectively. The combined marker panel had an overall sensitivity (positive result by either marker) of 99%. The concentrations of both markers showed dependence on cancer stage. The specificities of EBV DNA and IgA-VCA were 98% (96–99%) and 96% (91–98%), respectively. Among 36 healthy family members with false-positive IgA-VCA results, three-fourths had undetectable EBV DNA, whereas the others had increased EBV DNA concentrations that were significantly lower than in NPC patients.

Conclusions: For diagnosis of NPC, EBV DNA identifies almost all false-negative IgA-VCA cases and gives a 99% diagnostic sensitivity when combined with IgA-VCA. In the screening setting, EBV DNA identifies three-fourths of false-positive IgA-VCA cases. The selective application of EBV DNA in an IgA-VCA-based screening protocol could improve screening accuracy with only moderate increases in cost.
The Interleukin-6 G(−174)C Promoter Polymorphism Does Not Determine Plasma Interleukin-6 Concentrations in Experimental Endotoxemia in Humans
Authors: Endler Georg , Marsik Claudia , Joukhadar Christian , Marculescu Rodrig , Mayr Florian , Mannhalter Christine , Wagner Oswald F , Jilma Bernd
Published: February 2004

Background: Interleukin 6 (IL-6) is a pleiotropic cytokine that plays an essential role in the pathogenesis of acute and chronic infections. As the role of the IL-6 G(−174)C polymorphism in determining serum concentrations of IL-6 is controversial, we studied the genotype-specific IL-6 response in a well-standardized model of systemic inflammation.

Methods: A total of 76 healthy young males (age range, 19–35 years) received a single bolus of 2 ng/kg endotoxin [lipopolysaccharide (LPS)] intravenously. Plasma IL-6 was measured by enzyme immunoassay at 0, 2, 6, and 24 h after LPS infusion, and the IL-6 promoter genotype was analyzed by a mutagenic separated PCR assay.

Results: IL-6 increased 300-fold 2 h after LPS challenge and returned almost to normal within 24 h. Neither basal IL-6 nor the IL-6 response to LPS was significantly affected by the IL-6 promoter genotype.

Conclusions: The IL-6 G(−174)C promoter polymorphism does not significantly influence basal concentrations of IL-6 or peak IL-6 in human endotoxemia.
β-Thalassemia Microelectronic Chip: A Fast and Accurate Method for Mutation Detection
Authors: Foglieni Barbara , Cremonesi Laura , Travi Maurizio , Ravani Anna , Giambona Antonino , Rosatelli Maria Cristina , Perra Chiara , Fortina Paolo , Ferrari Maurizio
Published: February 2004

Background: β-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the β-thalassemia alleles in the Mediterranean area.

Methods: We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, −87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes.

Results: Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip.

Conclusions: The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing β-thalassemia in other regions of the world.
Validation of a Rapid and Sensitive Liquid Chromatography–Tandem Mass Spectrometry Method for Free and Total Mycophenolic Acid
Authors: Streit Frank , Shipkova Maria , Armstrong Victor William , Oellerich Michael
Published: February 2004

Background: Because mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography–tandem mass spectrometry is ideally suited for such a method.

Methods: Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C18 column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column.

Results: A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 μg/L, and the method was linear to 1000 μg/L. Interassay imprecision (CV) was <10% for free MPA (0.5–333 μg/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing–Bablok regression line was: y = 0.95x + 0.27 μg/L for free MPA and y = 0.98x + 0.03 mg/L for total MPA.

Conclusions: The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug.
Quantification of Serum and Urinary S-Adenosylmethionine and S-Adenosylhomocysteine by Stable-Isotope-Dilution Liquid Chromatography-Mass Spectrometry
Authors: Stabler Sally P , Allen Robert H
Published: February 2004

Background: We have developed an assay that uses stable-isotope-dilution liquid chromatography–mass spectrometry to assess S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in body fluids to investigate the relationship of these metabolites to hyperhomocysteinemia.

Methods: Commercially obtained SAM (D3 methyl) and 13C5-SAH uniformly labeled in the adenosyl moiety, which was synthesized using S-adenosylhomocysteine hydrolase, were added to samples followed by perchloric acid protein precipitation, C18 chromatography, and analysis by liquid chromatography–mass spectrometry with quantification by comparison of the areas of internal standard and endogenous peaks.

Results: Estimates of intraassay imprecision (CV) were 5% and 17% for SAM and SAH, respectively (n = 10). SAM decreased and SAH increased in serum and plasma samples at both 4 °C and room temperature over 80 h. SAM and SAH were unstable in samples stored longer than 2 years at −20 °C. In 48 volunteers, the estimated reference intervals [from mean (2 SD) of log-transformed data] for serum SAM and SAH were 71–168 and 8–26 nmol/L, respectively. Fractional excretion of SAM was higher than that of SAH, and the urinary SAM:SAH ratio was much higher than the serum or erythrocyte SAM:SAH ratios.

Conclusions: Stable-isotope-dilution liquid chromatography–mass spectrometry can be used to quantify SAM and SAH in biological fluids and tissues. Sample handling and storage must be stringently controlled for any epidemiologic or clinical use of such assays.
Detection of Hemoglobin-Based Oxygen Carriers in Human Serum for Doping Analysis: Screening by Electrophoresis
Authors: Lasne Françoise , Crepin Nathalie , Ashenden Michael , Audran Michel , de Ceaurriz Jacques
Published: February 2004

Background: Hemoglobin-based oxygen carriers (HBOCs) have recently been included in the International Olympic Committee and World Anti-Doping Agency lists of substances and methods prohibited in sports. To enforce this rule and deter abuse of HBOCs in elite sports, it is necessary to develop HBOC-specific screening and confirmation tests that are the usual steps in antidoping control analysis.

Methods: We developed a screening method based on electrophoresis of serum samples cleared of haptoglobin (Hp). Four successive steps (immunoprecipitation of Hp, electrophoresis of the cleared serum, Western blotting of the separated proteins, and detection of hemoglobin-related molecules based on the peroxidase properties of the heme moiety), provided electropherograms that could be easily interpreted in terms of the presence of HBOCs. This method was tested with serum samples enriched with various types of HBOCs: polymerized, conjugated, and cross-linked hemoglobins. It was also applied to blood samples collected from 12 healthy volunteers who had been infused with either 30 or 45 g of Hemopure, a glutaraldehyde-polymerized bovine hemoglobin.

Results: The method clearly detected the presence in serum of the various types of HBOCs tested and demonstrated no possible confusion with endogenous hemoglobin that may be present in cases of hemolysis. The test was able to detect Hemopure for 4–5 days after administration of 45 g to healthy individuals.

Conclusions: The electrophoretic method is a simple, fast, and sensitive procedure that appears to fulfill the criteria of a screening test for the presence of HBOCs in antidoping control samples.
Rates of Positive Cardiac Troponin I and Creatine Kinase MB Mass among Patients Hospitalized for Suspected Acute Coronary Syndromes
Authors: Lin Joseph C , Apple Fred S , Murakami MaryAnn M , Luepker Russell V
Published: February 2004

Background: Cardiac troponin I (cTnI) is a more specific and sensitive biomarker than creatine kinase MB (CKMB) for detection of myocardial damage. We report the prevalence of positive cTnI and CKMB mass among patients hospitalized with suspected acute coronary syndrome (ACS) and the potential impact of use of different reference cutoffs, particularly those proposed by European Society of Cardiology/American College of Cardiology (ESC/ACC) consensus guidelines, on rates of diagnosis of acute myocardial infarction (AMI).

Methods: We analyzed 1719 consecutive patients with suspected ACS admitted to an urban acute care hospital over a 6-month period. Patients (≥18 years of age) had at least two separate sets of plasma biomarkers (cTnI and CKMB) measured more than 12–24 h after admission to determine the potential rates of AMI based on different biomarker cutoff concentrations.

Results: The prevalence of cTnI-positive cases ranged from 10.6%, based on a cutoff of twice the ROC curve (cTnI ≤1.2 μg/L), to 25.0%, using the ESC/ACC-recommended 99th percentile cutoff (cTnI <0.1 μg/L). The prevalence of CKMB-positive cases ranged from 10.4%, with the cutoff of twice the ROC curve (CKMB ≤10.0 μg/L) to 21.7%, with the 99th percentile cutoff (CKMB <3.9 μg/L). Use of the 10% CV cutoff (cTnI ≤0.3 μg/L and CKMB <3.9 μg/L) instead of the ROC cutoff produced a 26% increase in all cTnI-positive cases. Use of the 99th percentile reference cutoff instead of the ROC curve-derived cutoff produced an 85% increase in all cTnI-positive cases. A substantial proportion of the increase in total cTnI-positive cases was derived from cTnI-positive/CKMB-negative cases: 71 (4.1%), 73 (4.2%), 98 (5.7%), and 209 (12.2%) of cTnI-positive cases were CKMB-negative, as determined by the twice the ROC, ROC, 10% CV, and 99th percentile reference cutoffs, respectively. At the 99th percentile cutoffs, 8.8% of cases were CKMB-positive/cTnI-negative.

Conclusions: Use of lower reference cutoffs for plasma biomarkers, as recommended by ESC/ACC guidelines, markedly increases the rates of cTnI-positive cases overall. A substantial proportion of the increase in total cTnI-positive cases was derived from the creation of additional cTnI-positive/CKMB-negative cases. CKMB-positive/cTnI-negative cases are likely false positive for myocardial injury.
An organizing framework for the implementation of environmental voluntary approaches
Authors: Cunningham James A. , Clinch J. Peter
Published: February 2004

There is a rich terminology associated with environmental voluntary approaches. The richness in terminology is matched by the pervasive nature of the implementation of voluntary approaches into the fabric of European environmental policy and regulation. The lack of a universal definition of what an environmental voluntary approach is has made the implementation task more difficult. The richness in terminology dealing with environmental voluntary approaches is categorized and the characteristics, context and implementation of environmental voluntary approaches are discussed. This paper proposes an organizing framework for the implementation of environmental voluntary approaches, which includes context assessment; instrument rationale; negotiation process; content; implementation; and post‐implementation, enforcement and monitoring. Copyright © 2004 John Wiley & Sons, Ltd and ERP Environment.
A holistic approach for construction with respect to environmental law
Authors: Siddiqi Khalid M. , Oberle Rita , Thomas‐Mobley Linda , Kangari Roozbeh
Published: February 2004

The impetus for widespread change within the private‐sector building industry is about no single owner retaining a constructed project during its entire life cycle. This impetus will be difficult to establish as statutory relief because of changes in private‐sector ownership. This is not true in most cases for federal agencies. However, with the advent of sustainability in construction and bioremediation in environmental construction, federal agencies also need to address the problem in an integrated fashion by improving their project management, planning, and implementation strategies. Thus, federal agencies have a responsibility and duty to take the lead in developing processes to ensure environmentally friendly construction. Currently the motivations are only voluntary, not regulatory. The divided nature of the construction industry complicates the problem further. Construction will become substantially more environmentally friendly if private‐sector owners are motivated through rewards and federal agencies through more integrated life‐cycle planning and programming, built within the socioeconomic and regulatory framework. In this article, the building industry, which currently stands fragmented, is analyzed in the context of the incentives and structural changes it needs to implement environmental laws fully. This article presents the motivation for adopting a holistic approach toward environmental compliance by modeling the strategic interventions required. These interventions, within and outside the construction industry, are also used to compare economic parity issues between existing building stock and future environmentally compliant buildings. Fundamental internal and external changes have been identified in the manner business is presently conducted to reward and benefit those who comply with the environmental laws and regulations. The authors further encourage federal agencies to take the lead role, through both voluntary and required processes, in changing the construction industry to be environmentally compliant in an integrated approach. © 2004 Wiley Periodicals, Inc.
Dual role of tumor necrosis factor-α in hepatic ischemia-reperfusion injury: Studies in tumor necrosis factor-α gene knockout mice
Authors: Teoh Narci , Field Jacqueline , Sutton Jaim , Farrell Geoffrey
Published: February 2004

Although hepatic ischemia-reperfusion (IR) injury is partially mediated by tumor necrosis factor-α (TNF), we recently found that low-dose TNF before IR is hepatoprotective. We examined the seemingly conflicting roles of TNF in mediating liver injury in a partial hepatic IR model using TNF gene knockout (TNF ko) mice to allow TNF replacement at specified times. Compared with wild-type mice, TNF ko mice exhibit minimal alanine aminotransferase release and few hepatonecrotic lesions during the early (time, 2 hours) and late (time, 24 hours) phases of IR. TNF ko mice differed from wild-type mice in that TNF ko mice exhibited no activation or induction of nuclear factor-κ B, p38, cyclin D1, or proliferating cell nuclear antigen after IR. A single low-dose TNF injection 1 minute before the onset of hepatic ischemia restored hepatic IR injury in TNF ko mice. To clarify the importance of TNF for hepatoprotection, preconditioning (10 minutes of ischemia and 10 minutes of reperfusion) was performed before the onset of IR for TNF ko mice whose capacity to undergo IR injury had been restored by TNF replacement. Ischemic preconditioning failed to protect these mice from TNF-augmented IR injury; however, following the administration of intravenous TNF (1 μg per kg body weight, which mimics the early increase in hepatic and plasma TNF levels that is mobilized by ischemic preconditioning), significant hepatoprotection against both the early and late phases of TNF-augmented IR injury was observed. In conclusion, TNF appears to mediate both the early and late phases of liver injury in hepatic IR, but it also is an essential mediator of hepatoprotective effects brought about by ischemic preconditioning. (Hepatology 2004;39:412-421.)