bluuebunny/crossref_metadata_2025 huggingface dataset:
Phytosphingosine in combination with ionizing radiation enhances apoptotic cell death in radiation-resistant cancer cells through ROS-dependent and -independent AIF release
Authors: Park Moon-Taek , Kim Min-Jung , Kang Young-Hee , Choi Soon-Young , Lee Jae-Hoon , Choi Jung-A , Kang Chang-Mo , Cho Chul-Koo , Kang Seongman , Bae Sangwoo , Lee Yun-Sil , Chung Hee Yong , Lee Su-Jae
Published: October 2004

The use of chemical modifiers as radiosensitizers in combination with low-dose irradiation may increase the therapeutic effect on cancer by overcoming a high apoptotic threshold. Here, we showed that phytosphingosine treatment in combination with γ-radiation enhanced apoptotic cell death of radiation-resistant human T-cell lymphoma in a caspase-independent manner. Combination treatment induced an increase in intracellular reactive oxygen species (ROS) level, mitochondrial relocalization of B-cell lymphoma-2(Bcl-2)-associated X protein (Bax), poly-adenosine diphosphate (ADP)-ribose polymerase 1 (PARP-1) activation, and nuclear translocation of apoptosis-inducing factor (AIF). siRNA targeting of AIF effectively protected cells from the combination treatment-induced cell death. An antioxidant, N-acetyl-L-cysteine (NAC), inhibited Bax relocalization and AIF translocation but not PARP-1 activation. Moreover, transfection of Bax-siRNA significantly inhibited AIF translocation. Pretreatment of PARP-1 inhibitor, DPQ (3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone), or PARP-1-siRNA also partially attenuated AIF translocation, whereas the same treatment did not affect intracellular ROS level and Bax redistribution. Taken together, these results demonstrate that enhancement of cell death of radiation-resistant cancer cells by phytosphingosine treatment in combination with γ-radiation is mediated by nuclear translocation of AIF, which is in turn mediated both by ROS-dependent Bax relocalization and ROS-independent PARP-1 activation. The molecular signaling pathways that we elucidated in this study may provide potential drug targets for radiation sensitization of cancers refractive to radiation therapy. (Blood. 2005;105:1724-1733)
Hepcidin is decreased in TFR2 hemochromatosis
Authors: Nemeth Elizabeta , Roetto Antonella , Garozzo Giovanni , Ganz Tomas , Camaschella Clara
Published: October 2004

The hepatic peptide hepcidin is the key regulator of iron metabolism in mammals. Recent evidence indicates that certain forms of hereditary hemochromatosis are caused by hepcidin deficiency. Juvenile hemochromatosis is associated with hepcidin or hemojuvelin mutations, and these patients have low or absent urinary hepcidin. Patients with C282Y HFE hemochromatosis also have inappropriately low hepcidin levels for the degree of iron loading. The relationship between the hemochromatosis due to transferrin receptor 2 (TFR2) mutations and hepcidin was unknown. We measured urinary hepcidin levels in 10 patients homozygous for TFR2 mutations, all with increased transferrin saturation. Urinary hepcidin was low or undetectable in 8 of 10 cases irrespective of the previous phlebotomy treatments. The only 2 cases with normal hepcidin values had concomitant inflammatory conditions. Our data indicate that TFR2 is a modulator of hepcidin production in response to iron. (Blood. 2005;105:1803-1806)
Binding of β2–glycoprotein I to anionic phospholipids facilitates processing and presentation of a cryptic epitope that activates pathogenic autoreactive T cells
Authors: Kuwana Masataka , Matsuura Eiji , Kobayashi Kazuko , Okazaki Yuka , Kaburaki Junichi , Ikeda Yasuo , Kawakami Yutaka
Published: October 2004

Antiphospholipid syndrome (APS) is an autoimmune prothrombotic disorder in association with autoantibodies to phospholipid (PL)–binding plasma proteins, such as β2-glycoprotein I (β2GPI). We have recently found that CD4+ T cells autoreactive to β2GPI in patients with APS preferentially recognize a cryptic peptide encompassing amino acid residues 276-290 (p276-290), which contains the major PL-binding site, in the context of DR53. However, it is not clear how previously cryptic p276-290 becomes visible to the immune system and elicits a pathogenics autoimmune response to β2GPI. Here we show that presentation of a disease-relevant cryptic T-cell determinant in β2GPI is induced as a direct consequence of antigen processing from β2GPI bound to anionic PL. Dendritic cells or macrophages pulsed with PL-bound β2GPI induced a response of p276-290–specific CD4+ T-cell lines generated from the patients in an HLA-DR–restricted and antigen-processing–dependent manner but those with β2GPI or PL alone did not. In addition, the p276-290–reactive T-cell response was primed by stimulating peripheral blood T cells from DR53-carrying healthy individuals with dendritic cells bearing PL-bound β2GPI in vitro. Our finding is the first demonstration of an in vitro mechanism eliciting pathogenic autoreactive T-cell responses to β2GPI and should be useful in clarifying the pathogenesis of APS.
Notch ligands Delta1 and Jagged1 transmit distinct signals to T-cell precursors
Authors: Lehar Sophie M. , Dooley James , Farr Andrew G. , Bevan Michael J.
Published: October 2004

Signaling through the Notch pathway plays an essential role in inducing T-lineage commitment and promoting the maturation of immature thymocytes. Using an in vitro culture system, we show that 2 different classes of Notch ligands, Jagged1 or Delta1, transmit distinct signals to T-cell progenitors. OP9 stromal cells expressing either Jagged1 or Delta1 inhibit the differentiation of DN1 thymocytes into the B-cell lineage, but only the Delta1-expressing stromal cells promote the proliferation and maturation of T-cell progenitors through the early double-negative (DN) stages of thymocyte development. Whereas the majority of bone marrow–derived stem cells do not respond to Jagged1 signals, T-cell progenitors respond to Jagged1 signals during a brief window of their development between the DN1 and DN3 stages of thymic development. During these stages, Jagged1 signals can influence the differentiation of immature thymocytes along the natural killer (NK) and γδ T-cell lineages.
Marburg I polymorphism of factor VII–activating protease is associated with idiopathic venous thromboembolism
Authors: Hoppe Berthold , Tolou Farzaneh , Radtke Hartmut , Kiesewetter Holger , Dörner Thomas , Salama Abdulgabar
Published: October 2004

The factor VII–activating protease (FSAP) variant Marburg I is known to attenuate the profibrinolytic system in vitro and was recently shown to be a significant predictor for the evolution and progression of carotid stenosis. The objective of this case-control study was to assess FSAP Marburg I's role in the occurrence of venous thromboembolism (VTE). The frequency of FSAP Marburg I was significantly increased in patients with a history of VTE (17 of 213 patients, 8.0%, P = .014) or idiopathic VTE (12 of 103 patients, 11.7%, P = .002) compared to healthy controls (5 of 213 controls, 2.3%). Logistic regression analysis confirmed FSAP Marburg I to be an independent risk factor for VTE (odds ratio, 3.5; 95% confidence interval [CI], 1.2-10.0) and idiopathic VTE (odds ratio, 6.2; 95% CI, 2.0-18.9).
Integrating linkage and radiation hybrid mapping data for bovine chromosome 15
Authors: Snelling Warren M , Gautier Mathieu , Keele John W , Smith Timothy PL , Stone Roger T , Harhay Gregory P , Bennett Gary L , Ihara Naoya , Takasuga Akiko , Takeda Haruko , Sugimoto Yoshikazu , Eggen André
Published: October 2004

Background

Bovine chromosome (BTA) 15 contains a quantitative trait loci (QTL) for meat tenderness, as well as several breaks in synteny with human chromosome (HSA) 11. Both linkage and radiation hybrid (RH) maps of BTA 15 are available, but the linkage map lacks gene-specific markers needed to identify genes underlying the QTL, and the gene-rich RH map lacks associations with marker genotypes needed to define the QTL. Integrating the maps will provide information to further explore the QTL as well as refine the comparative map between BTA 15 and HSA 11. A recently developed approach to integrating linkage and RH maps uses both linkage and RH data to resolve a consensus marker order, rather than aligning independently constructed maps. Automated map construction procedures employing this maximum-likelihood approach were developed to integrate BTA RH and linkage data, and establish comparative positions of BTA 15 markers with HSA 11 homologs.

Results

The integrated BTA 15 map represents 145 markers; 42 shared by both data sets, 36 unique to the linkage data and 67 unique to RH data. Sequence alignment yielded comparative positions for 77 bovine markers with homologs on HSA 11. The map covers approximately 32% of HSA 11 sequence in five segments of conserved synteny, another 15% of HSA 11 is shared with BTA 29. Bovine and human order are consistent in portions of the syntenic segments, but some rearrangement is apparent. Comparative positions of gene markers near the meat tenderness QTL indicate the region includes separate segments of HSA 11. The two microsatellite markers flanking the QTL peak are between defined syntenic segments.

Conclusions

Combining data to construct an integrated map not only consolidates information from different sources onto a single map, but information contributed from each data set increases the accuracy of the map. Comparison of bovine maps with well annotated human sequence can provide useful information about genes near mapped bovine markers, but bovine gene order may be different than human. Procedures to connect genetic and physical mapping data, build integrated maps for livestock species, and connect those maps to more fully annotated sequence can be automated, facilitating the maintenance of up-to-date maps, and providing a valuable tool to further explore genetic variation in livestock.
i-Genome: A database to summarize oligonucleotide data in genomes
Authors: Lin Feng-Mao , Huang Hsien-Da , Chang Yu-Chung , Horng Jorng-Tzong
Published: October 2004

Background

Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming.

Results

The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes.

Conclusions

This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes.
Erytrocyte membrane anionic charge in type 2 diabetic patients with retinopathy
Authors: Budak Yasemin , Demirci Hakan , Akdogan Muberra , Yavuz Dilek
Published: October 2004

Background

The Steno hypothesis states that changes in basement membrane anionic charge leads to diabetic microvascular complications. In diabetic nephropathy, loss of basement membrane glycosaminoglycans and the association between glomerular basement membrane heparan sulphate and proteinuria has been documented. A correlation between erythrocyte surface and the glomerular capillary wall charges has also been observed.

The aim of this study is to evaluate the relationship between retinopathy and erythrocyte anionic charge and urinary glycosaminoglycan excretion in type 2 diabetic patients.

Methods

49 subjects (58 ± 7 yrs, M/F 27/22) with type 2 diabetes with proliferative retinopathy (n = 13), nonproliferative retinopathy (n = 13) and without retinopathy (n = 23) were included in the study. 38 healthy subjects were selected as control group (57 ± 5 yrs, M/F 19/19). Erythrocyte anionic charge (EAC) was determined by the binding of the cationic dye, alcian blue. Urinary glycosaminoglycan and microalbumin excretion were measured.

Results

EAC was significantly decreased in diabetic patients with retinopathy (255 ± 30 ng alcian blue/106 RBC, 312 ± 30 ng alcian blue/106 RBC for diabetic and control groups respectively, p < 0.001). We did not observe an association between urinary GAG and microalbumin excretion and diabetic retinopathy. EAC is found to be negatively corralated with microalbuminuria in all groups.

Conclusions

We conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients.
Long-term patterns in European brown hare population dynamics in Denmark: effects of agriculture, predation and climate
Authors: Schmidt Niels M , Asferg Tommy , Forchhammer Mads C
Published: October 2004

Background

In Denmark and many other European countries, harvest records suggest a marked decline in European brown hare numbers, a decline often attributed to the agricultural practice. In the present study, we analyse the association between agricultural land-use, predator abundance and winter severity on the number of European brown hares harvested in Denmark in the years 1955 through 2000.

Results

Winter cereals had a significant negative association with European brown hare numbers. In contrast to this, root crop area was positively related to their numbers. Remaining crop categories were not significantly associated with the European brown hare numbers, though grass out of rotation tended to be positively related. The areas of root crop production and of grass out of rotation have been reduced by approximately 80% and 50%, respectively, while the area of winter cereals has increased markedly (>70%). However, European brown hare numbers were primarily negatively associated with the number of red fox. Finally, we also found a positive association between mild winters and European brown hare numbers.

Conclusion

The decline of Danish European brown hare populations can mainly be attributed to predation by red fox, but the development in agricultural land-use during the last 45 years have also affected the European brown hare numbers negatively. Additionally, though mild winters were beneficial to European brown hares, the increasing frequency of mild winters during the study period was insufficient to reverse the negative population trend.
Feasibility of rapid and automated importation of 3D echocardiographic left ventricular (LV) geometry into a finite element (FEM) analysis model
Authors: Verhey Janko F , Nathan Nadia S
Published: October 2004

Background

Finite element method (FEM) analysis for intraoperative modeling of the left ventricle (LV) is presently not possible. Since 3D structural data of the LV is now obtainable using standard transesophageal echocardiography (TEE) devices intraoperatively, the present study describes a method to transfer this data into a commercially available FEM analysis system: ABAQUS©.

Methods

In this prospective study TomTec LV Analysis TEE© Software was used for semi-automatic endocardial border detection, reconstruction, and volume-rendering of the clinical 3D echocardiographic data. A newly developed software program MVCP FemCoGen©, written in Delphi, reformats the TomTec file structures in five patients for use in ABAQUS and allows visualization of regional deformation of the LV.

Results

This study demonstrates that a fully automated importation of 3D TEE data into FEM modeling is feasible and can be efficiently accomplished in the operating room.

Conclusion

For complete intraoperative 3D LV finite element analysis, three input elements are necessary: 1. time-gaited, reality-based structural information, 2. continuous LV pressure and 3. instantaneous tissue elastance. The first of these elements is now available using the methods presented herein.
Association of mitral annulus calcification, aortic valve calcification with carotid intima media thickness
Authors: Sgorbini Luca , Scuteri Angelo , Leggio Massimo , Leggio Francesco
Published: October 2004

Background

Mitral annular calcification (MAC) and aortic annular calcification (AVC) may represent a manifestation of generalized atherosclerosis in the elederly. Alterations in vascular structure, as indexed by the intima media thickness (IMT), are also recognized as independent predictors of adverse cardiovascular outcomes.

Aim

To examine the relationship between the degree of calcification at mitral and/or aortic valve annulus and large artery structure (thickness).

Methods

We evaluated 102 consecutive patients who underwent transthoracic echocardiography and carotid artery echoDoppler for various indications; variables measured were: systemic blood pressure (BP), pulse pressure (PP=SBP-DBP), body mass index (BMI), fasting glucose, total, HDL, LDL chlolesterol, triglycerides, cIMT. The patients were divided according to a grading of valvular/annular lesions independent scores based on acoustic densitometry: 1 = annular/valvular sclerosis/calcification absence; 2 = annular/valvular sclerosis; 3 = annular calcification; 4 = annular-valvular calcification; 5 = valvular calcification with no recognition of the leaflets.

Results

Patient score was the highest observed for either valvular/annulus. Mean cIMT increased linearly with increasing valvular calcification score, ranging from 3.9 ± 0.48 mm in controls to 12.9 ± 1.8 mm in those subjects scored 5 (p < 0.0001). In the first to fourth quartile of cIMT values the respective maximal percentual of score were: score 1: 76.1%, score 2: 70.1%, score 4: 54.3% and score 5: 69.5% (p > 0.0001).

Conclusion

MAC and AVC score can identify subgroups of patients with different cIMT values which indicate different incidence and prevalence of systemic artery diseases. This data may confirm MAC-AVC as a useful important diagnostic parameter of systemic atherosclerotic disease.
Ventilator associated pneumonia: comparison between quantitative and qualitative cultures of tracheal aspirates
Authors: Camargo Luis FernandoAranha , De Marco Fernando Vinícius , Barbas Carmen SílviaValente , Hoelz Cristiane , Bueno Marco AurélioScarpinella , Rodrigues Jr Milton , Amado Verônica Moreira , Caserta Raquel , Martino Marinês DallaValle , Pasternak Jacyr , Knobel Elias
Published: October 2004

Introduction

Deferred or inappropriate antibiotic treatment in ventilator-associated pneumonia (VAP) is associated with increased mortality, and clinical and radiological criteria are frequently employed to establish an early diagnosis. Culture results are used to confirm the clinical diagnosis and to adjust or sometimes withdraw antibiotic treatment. Tracheal aspirates have been shown to be useful for these purposes. Nonetheless, little is known about the usefulness of quantitative findings in tracheal secretions for diagnosing VAP.

Methods

To determine the value of quantification of bacterial colonies in tracheal aspirates for diagnosing VAP, we conducted a prospective follow-up study of 106 intensive care unit patients who were under ventilatory support. In total, the findings from 219 sequential weekly evaluations for VAP were examined. Clinical and radiological parameters were recorded and evaluated by three independent experts; a diagnosis of VAP required the agreement of at least two of the three experts. At the same time, cultures of tracheal aspirates were analyzed qualitatively and quantitatively (105 colony-forming units [cfu]/ml and 106 cfu/ml)

Results

Quantitative cultures of tracheal aspirates (105 cfu/ml and 106 cfu/ml) exhibited increased specificity (48% and 78%, respectively) over qualitative cultures (23%), but decreased sensitivity (26% and 65%, respectively) as compared with the qualitative findings (81%). Quantification did not improve the ability to predict a diagnosis of VAP.

Conclusion

Quantitative cultures of tracheal aspirates in selected critically ill patients have decreased sensitivity when compared with qualitative results, and they should not replace the latter to confirm a clinical diagnosis of VAP or to adjust antimicrobial therapy.
11-Deoxycorticosterone Is a Potent Agonist of the Rainbow Trout (Oncorhynchus mykiss) Mineralocorticoid Receptor
Authors: Sturm A. , Bury N. , Dengreville L. , Fagart J. , Flouriot G. , Rafestin-Oblin M. E. , Prunet P.
Published: October 2004

The teleost fish are thought to lack the mineralocorticoid hormone aldosterone but possess mineralocorticoid receptor (MR) homologs. Here we describe the characterization of two rainbow trout (Oncorhynchus mykiss) MRs, called rtMRa and rtMRb. The open reading frame of rtMRa cDNA encoded a protein of 1041 amino acids. The rtMRb predicted protein sequence is similar, differing in only 10 amino acids in the nonconserved A/B domain and lacking a three-amino acid insertion between the two zinc fingers of the C domain. Expression of rtMR mRNA (sum of both forms), measured in juvenile trout by real-time RT-PCR, shows that the transcripts are ubiquitous. Expression was significantly higher in brain than the other tissues studied (eye, trunk kidney, head kidney, gut, gills, liver, spleen, ovary, heart, white muscle, skin). Hormonal stimulation of receptor transactivation activity was studied in COS-7 cells transiently cotransfected with receptor cDNA and a mouse mammary tumor virus-luciferase reporter. The mineralocorticoids 11-deoxycorticosterone and aldosterone were more potent enhancers of rtMRa transcriptional activity (EC50 = 1.6 ± 0.5 × 10−10 and 1.1 ± 0.4 × 10−10m, respectively) than the glucocorticoids cortisol and 11-deoxycortisol (EC50 = 1.1 ± 0.3 × 10−9 and 3.7 ± 1.9 × 10−9m, respectively). A similar response was observed in transactivation assays with rtMRb. These results are discussed in the view of reported circulating levels of corticosteroids in trout.
Cell-Specific Expression of Glucose-Dependent-Insulinotropic Polypeptide Is Regulated by the Transcription Factor PDX-1
Authors: Jepeal Lisa I. , Fujitani Yoshio , Boylan Michael O. , Wilson Cherrell N. , Wright Christopher V. , Wolfe M. Michael
Published: October 2004

Glucose-dependent insulinotropic polypeptide (GIP) is a potent stimulator of insulin secretion and comprises an important component of the enteroinsular axis. GIP is synthesized in enteroendocrine K-cells located principally in the upper small intestine. The homeobox-containing gene PDX-1 is also expressed in the small intestine and plays a critical role in pancreatic development and in the expression of pancreatic-specific genes. Previous studies determined that the transcription factors GATA-4 and ISL-1 are important for GIP expression. In this study, we demonstrate that PDX-1 is also involved in regulating GIP expression in K-cells. Using immunohistochemistry, we verified the expression of PDX-1 protein in the nucleus of GIP-expressing mouse K-cells and evaluated the expression of PDX-1, serotonin, and GIP in wild-type and PDX-1−/− mice at 18.5 d after conception. Although we demonstrated a 97.8% reduction in the number of GIP-expressing cells in PDX-1−/− mice; there was no statistical difference in the number of serotonin-positive cells. Additionally, PDX-1 transcripts and protein were detected in a GIP-expressing neuroendocrine cell line, STC-1. Electromobility shift assays using STC-1 nuclear extracts demonstrated the specific binding of PDX-1 protein to a specific regulatory region in the GIP promoter. Using chromatin immunoprecipitation analysis, we demonstrated binding of PDX-1 to this same region of the GIP promoter in intact cells. Lastly, overexpression of PDX-1 in transient transfection assays led to a specific increase in the activity of GIP/Luc reporter constructs. The results of these studies indicate that the transcription factor PDX-1 plays a critical role in the cell-specific expression of the GIP gene.
Stimulation of Luteinizing Hormone-Releasing Hormone (LHRH) Gene Expression in GT1–7 Cells by Its Metabolite, LHRH-(1–5)
Authors: Wu T. J. , Mani Shaila K. , Glucksman Marc J. , Roberts James L.
Published: October 2004

Given the central role of the decapeptide LHRH in reproduction and reproductive behavior, it is important to focus on delineating the possible effects of this gene and its products in the regulation of hormone-dependent reproductive processes. In the female, ovulation is preceded by a marked increase in LHRH release; the increase in LHRH release culminates in a preovulatory LH surge, which coincides with a period of sexual receptivity. In contrast to the belief that the proteolytic metabolism of LHRH serves only as a degradative process that removes excess LHRH and attenuates signal transduction through the LHRH receptor, we hypothesized that a metabolite of the decapeptide, LHRH-(1–5), can directly regulate LHRH neuronal function. This study demonstrates the ability of LHRH-(1–5) peptide to regulate LHRH gene expression in the LHRH neuronal cell line, the GT1–7 cell. The results show that LHRH-(1–5) stimulated LHRH gene expression at the posttranscriptional level. In contrast to the LHRH suppression of its own gene expression, the coadministration of LHRH with the metalloendopeptidase, EC 3.4.24.15, an endopeptidase known to cleave LHRH to form LHRH1–5, shows a reversal of effect, a stimulation of LHRH gene expression. Finally, the effect of LHRH-(1–5) on LHRH gene expression appears to be mediated by the calcium/calmodulin-dependent protein kinase. The present study supports the hypothesis that the physiological metabolite of LHRH, LHRH-(1–5), is functionally capable of regulating the reproductive neuroendocrine system.
Hypophysiotropic Thyrotropin-Releasing Hormone and Corticotropin-Releasing Hormone Neurons of the Rat Contain Vesicular Glutamate Transporter-2
Authors: Hrabovszky Erik , Wittmann Gábor , Turi Gergely F. , Liposits Zsolt , Fekete Csaba
Published: October 2004

TRH and CRH are secreted into the hypophysial portal circulation by hypophysiotropic neurons located in parvicellular subdivisions of the hypothalamic paraventricular nucleus (PVH). Recently these anatomical compartments of the PVH have been shown to contain large numbers of glutamatergic neurons expressing type 2 vesicular glutamate transporter (VGLUT2). In this report we presented dual-label in situ hybridization evidence that the majority (>90%) of TRH and CRH neurons in the PVH of the adult male rat express the mRNA encoding VGLUT2. Dual-label immunofluorescent studies followed by confocal laser microscopic analysis of the median eminence also demonstrated the occurrence of VGLUT2 immunoreactivity within TRH and CRH axon varicosities, suggesting terminal glutamate release from these neuroendocrine systems. These data together indicate that the hypophysiotropic TRH and CRH neurons possess glutamatergic characteristics. Future studies will need to address the physiological significance of the endogenous glutamate content in these neurosecretory systems in the neuroendocrine regulation of thyroid and adrenal functions.
New insights into plant development in New England
Authors: Dolan Liam , Langdale Jane A.
Published: October 2004

This year, the biannually organized FASEB meeting `Mechanisms in Plant Development' took place in August in Vermont, USA, organized by Martin Hulskamp (University of Köln, Köln, Germany) and John Schiefelbein(University of Michigan, Ann Arbor, MI, USA). The meeting covered numerous topics, ranging from patterning and differentiation to the evolution of developmental mechanisms. Despite apparent distinctions between the sessions,many of the talks were broad ranging and most highlighted unifying developmental concepts.
Quantitative, Highly Sensitive Liquid Chromatography–Tandem Mass Spectrometry Method for Detection of Synthetic Corticosteroids
Authors: Taylor Robert L , Grebe Stefan K , Singh Ravinder J
Published: October 2004

Background: Measurements of serum or urine concentrations of synthetic glucocorticoids are useful for assessing suspected iatrogenic hypothalamic-pituitary-adrenal axis suppression and Cushing syndrome. We have developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantitative analysis of beclomethasone dipropionate, betamethasone, budesonide, dexamethasone, fludrocortisone, flunisolide, fluorometholone, fluticasone propionate, megestrol acetate, methylprednisolone, prednisolone, prednisone, triamcinolone, and triamcinolone acetonide.

Methods: Stable isotopes of cortisol-9,11,12,12-d4 and triamcinolone-d1 acetonide-d6 were added as internal standards to calibrators, controls, and unknown samples. After acetonitrile precipitation, these samples were extracted with methylene chloride, and the extracts were washed and dried. Reconstituted extract (15 μL) was injected on a reversed-phase column and analyzed by LC-MS/MS in positive-ion mode. Assay precision, accuracy, linearity, and sample stability were determined by use of enriched samples. Clinical validation included analysis of 8 serum and 20 urine samples from patients with undetectable cortisol concentrations and analysis of different types of tablets.

Results: Functional assay sensitivity was as low as 0.6–1.6 nmol/L for all compounds except for triamcinolone (7.6 nmol/L). Interassay CVs were 3.0–20% for concentrations of 0.6–364 nmol/L for all analytes. Recoveries of all analytes (except triamcinolone in serum) were 82–138% at 19.2–693 nmol/L. All but one of the serum and urine samples from patients who were tested because of suppressed cortisol concentrations contained at least one synthetic steroid. Tablet analysis recovered 75% of the synthetic steroids in suspected drugs.

Conclusions: LC-MS/MS allows simultaneous quantitative detection of various synthetic steroids in serum, plasma, urine, and tablets. This provides a valuable tool for evaluating the clinical effects of topical and systemic synthetic corticosteroids.
Immunoassay to Measure Ataxia-Telangiectasia Mutated Protein in Cellular Lysates
Authors: Butch Anthony W , Chun Helen H , Nahas Shareef A , Gatti Richard A
Published: October 2004

Background: Ataxia-telangiectasia (A-T) is a neurologic disorder caused by mutations in the ataxia-telangiectasia mutated (ATM) gene. A clinical diagnosis of A-T is confirmed by radiosensitivity testing and immunoblotting for ATM protein. Because both of these tests have long turnaround times (≥3 months), we developed a rapid immunoassay to measure ATM protein and determined its sensitivity and specificity for diagnosing A-T.

Methods: Recombinant ATM protein was used for standardization. Lysates of lymphoblastoid cell lines (LCLs) and peripheral blood mononuclear cells (PBMCs) from A-T patients, controls, and A-T heterozygotes were tested for ATM protein by immunoassay.

Results: Between-run imprecision (CV) was ≤13%. Nuclear lysates from control LCLs and PBMCs had ATM protein concentrations of 49–610 μg/L and 48–943 μg/L, respectively. ATM protein was not detectable in LCL nuclear lysates from 18 of 21 A-T patients. The three remaining A-T patients had trace amounts of ATM protein, which was confirmed on immuoblots. ATM protein was also detectable in whole-cell lysates from 4 × 106 cells at concentrations of 64–463 μg/L and 42–444 μg/L for control LCLs and PBMCs, respectively. A-T heterozygotes had ATM protein concentrations of 52–98 μg/L. ATM protein was stable in PBMCs stored for 1 month at −70 °C, but rapidly decreased after 1 day in unprocessed blood.

Conclusions: This ATM protein immunoassay can be used to confirm a diagnosis of A-T in 2 days on small numbers of PBMCs and can potentially identify A-T carriers and individuals at increased risk for cancer.
Mapping molecular landscapes inside cells
Authors: Baumeister Wolfgang
Published: October 2004

Cryoelectron tomography opens a window into the inner space of cells. It combines the potential of three-dimensional imaging with a close-to-life preservation of biological samples. Tomograms with molecular resolution are essentially images of the cellular proteome and, in conjunction with advanced pattern recognition techniques, they can be used to map the molecular landscape inside organelles and cells.
Imprinted small RNA genes
Authors: Seitz Hervé , Royo Hélène , Lin Shau-Ping , Youngson Neil , Ferguson-Smith Anne C. , Cavaillé Jérôme
Published: October 2004

Genomic imprinting is an epigenetic phenomenon that results in differential expression of both alleles, depending on their parent of origin. We have recently identified many imprinted small non-coding RNA genes belonging to the C/D RNA and microRNA gene families, both of which are usually known to play key roles in post-transcriptional metabolism of specific genes (e.g. C/D RNAs guide ribose methylation of target RNAs while microRNAs elicit either translational repression or RNA interference). Although the functional and evolutionary significance of this association between C/D RNA genes, microRNA genes and genomic imprinting is still highly elusive, these observations provide a framework for further analysis of the potential role of small non-coding RNAs in epigenetic control.
How do ABC transporters drive transport?
Authors: Van Der Does Chris , Tampé Robert
Published: October 2004

Members of the ATP-binding cassette (ABC) superfamily are integral membrane proteins that hydrolyze ATP to drive transport. In the last two decades these proteins have been extensively characterized on a genetic and biochemical level, and in recent years high-resolution crystal structures of several nucleotide-binding domains and full-length transporters have extended our knowledge. Here we discuss the possible mechanisms of transport that have been derived from these crystal structures and the extensive available biochemical data.
Automation of Urine Sediment Examination: a Comparison of the Sysmex UF-100 Automated Flow Cytometer with Routine Manual Diagnosis (Microscopy, Test Strips, and Bacterial Culture)
Authors: Hannemann-Pohl Kurt , Kampf Sieghard Carsten
Published: October 2004

Urine specimens from 438 patients were examined with the UF-100 flow cytometer (Sysmex TOA Medical Electronics (Europe) GmbH, Hamburg, Germany) and by manual microscopy and test strips. One hundred and forty-two of these were also examined bacteriologically. The measurements with the UF-100 were performed on native urine without prior centrifugation. Intraassay imprecision, CV of 1.3 % (547/μl) to 8.5 % (24/μl) for erythrocytes and CV of 2.4 % ( 218/μl) to 5.6 % (10/μl) for leukocytes, are similar to those usual in clinical chemistry, and are very much better than those seen in manual microscopy of sediment. In routine use, overloading the flow cytometer by an excessive concentration of particles was observed in 9% of specimens. Such specimens should be checked visually. The UF-100 is distinctly more sensitive than manual microscopy for determining leukocytes, erythrocytes, epithelial cells and bacteria. Reference ranges were estimated from the results obtained. These enabled the UF-100 to replace routine manual diagnostic methods. Although the sensitivity is improved over manual microscopy of sediment, it is always necessary to perform parallel test strip examination when determining erythrocytes in order to detect haemolysis.

In our opinion the Sysmex UF-100 is a suitable replacement for manual microscopy of urine sediment. In addition it offers an opportunity to improve standardization of basic urinalysis.
Iron Saturation of Ferritin in the Course of Phlebotomy Treatment in Patients with Haemochromatosis
Authors: ten Kate Joop , Marell Kirstin , Huizinga Reint , Kreeftenberg Herman , van Deursen Cees
Published: October 2004

This paper describes the iron saturation of ferritin haemochromatosis patients during phlebotomy therapy. The iron saturation of ferritin does not change during therapy and cannot be used as a parameter follow therapy. Furthermore, the iron saturation seems to be a constant characteristic of a given person. It does not vary with the body iron stores in patients with haemochromatosis.
Recommendations of the German Working Group on Medical Laboratory Testing (AML) on the Introduction and Quality Assurance of Procedures for Point-of-Care Testing (POCT) in Hospitals
Authors: Briedigkeit L. , Müller-Plathe O. , Schlebusch H. , Ziems J.
Published: October 2004

The following recommendations were drawn up by the Working Group “Point-of-Care Testing” of the DGKC and DGLM which was set up in 1997. This first document from the Working Group sets out the principles which should be observed when introducing point-of-care testing.

These general recommendations are to be followed by further specific recommendations on individual procedures or groups of procedures, e.g. blood glucose, electrolytes in whole blood, blood gases, coagulation, toxicology, quality control and others, which will be drawn up by experts at the suggestion of the Working Group.
Multiplex In-cell Reverse Transcription-Polymerase Chain Reaction for the Simultaneous Detection of p210 and p190 BCR-ABL mRNAs in Chronic Myeloid Leukemia and Philadelphia-Positive Acute Lymphoblastic Leukemia Cell Lines
Authors: Suh Soon Pal , Kee Seung Jung , Lim Woo Hyun , Song Jeong Won , Lee Sang Khoo , Kim Jong Phil , Shin Jong Hee , Ryang Dong Wook
Published: October 2004

We designed a novel multiplex in-cell reverse transcription-
polymerase chain reaction method for the simultaneous
detection and differentiation of p190 and
p210 BCR-ABL mRNAs within single cells from the human
chronic myeloid leukemia and Philadelphia positive
acute lymphoblastic leukemia. Human K562
chronic myeloid leukemia and SUP B-15 Ph+ acute
lymphoblastic leukemia cell lines were used as positive
controls for p210 and p190 BCR-ABL mRNAs, respectively.
HL60 cell line was used as a negative control.
After the leukemia cells were fixed and
permeabilized, without extracting nucleic acids, the
mRNAs were reverse transcribed to cDNAs, and the
cDNAs were amplified by multiplex polymerase chain
reaction with fluorescent primers specific for p190 and
p210 BCR-ABL mRNAs. After transfer onto glass slides
by cytospin, the amplified cells were detected by fluorescence
microscopy. Fluorescence microscopy after
propidium iodide or 4′,6-diamidino-2-phenylindone
counterstaining showed that the positive K562 cells
exhibited a yellow-green fluorescent cytoplasm
around a red nucleus, and that the positive SUP B-15
cells exhibited an orange cytoplasm around a blue nucleus.
Only the red or blue nucleus was visible in respective
negative HL60 cells. The specificity of amplification
was confirmed by the absence of a signal when
control experiments were performed either with
RNase digestion of mRNA or without reverse transcriptase/Taq polymerase. We conclude that the multiplex
in-cell reverse transcription-polymerase chain
reaction method is capable of simultaneously detecting
and differentiating the p210 and p190 BCR-ABL
mRNAs of chronic myeloid leukemia and Philadelphia-positive
acute lymphoblastic leukemia cells, and that it
may be useful in quantitatively monitoring the minimal
residual disease during therapy.
Genetic Polymorphisms and Activity of Cholesterol Ester Transfer Protein (CETP): Should We Be Measuring Them?
Authors: Ordovas Jose M.
Published: October 2004

Cholesteryl ester transfer protein (CETP) is a plasma
glycoprotein that mediates the transfer of cholesteryl
ester from high density lipoproteins (HDL) to triglyceride-rich lipoproteins in exchange for triglycerides.
Several approaches are currently being used in research
laboratories to measure its activity and/or
mass. However, these assays are not standardized and
it is not possible to compare data from different laboratories.
Also, we lack enough information to assess
the value of this variable as a coronary heart disease
(CHD) predictor. Several genetic variants at CETP locus
have been identified and they have been generally
associated with increased HDL-cholesterol concentrations.
However, there is no consensus about the association
of this CETP-related increase in HDL-cholesterol
and protection against CHD. Nevertheless, the
most recent evidence from the common CETP-TaqI-B
polymorphism shows that the lower CETP activity associated
with the presence of this polymorphism decreases
CHD risk in men. Based on this and previous
evidence, there has been an interest in the development
of CETP inhibitors as a tool to increase HDL-cholesterol,
thus reducing CHD risk. However, it should be
noted that the evidence about the cardioprotective
role of these drugs is not yet available.
Detection of Haemoglobins with Abnormal Oxygen Affinity by Single Blood Gas Analysis and 2,3-Diphosphoglycerate Measurement
Authors: Guerrini Giovanni , Morabito Alberto , Samaja Michele
Published: October 2004

The aim is to determine if a single measurement of
blood 2,3-diphosphoglycerate combined with gas
analysis (pH, PCO2, PO2 and saturation) can identify
the cause of an altered blood-oxygen affinity: the presence
of an abnormal haemoglobin or a red cell disorder.
The population (n=94) was divided into healthy
controls (A, n=14), carriers of red cell disorders (B,
n=72) and carriers of high oxygen affinity haemoglobins
(C, n=8). Those variables were measured both in
samples equilibrated at selected PCO2 and PO2 and in
venous blood. In the univariable approach applied to
equilibrated samples, we correctly identified C subjects
in 93.6% or 96.8% of the cases depending on the
selected variable, the standard P50 (PO2 at which 50%
of haemoglobin is oxygenated) or a composite variable
calculated from the above measurements. After
introducing the haemoglobin concentration as a further
discriminating variable, the A and B subjects were
correctly identified in 91.9% or 94.2% of the cases, respectively.
These figures become 93.0% or 86.1%, and
93.7% or 94.9% of the cases when using direct readings
from venous blood, thereby avoiding the blood
equilibration step. This test is feasible also in blood
samples stored at 4°C for 48 h, or at room temperature
for 8 h.
Effect of Acute Phase Response on Cumulative Troponin T Release
Authors: Diris Jart H.C. , Kragten Johannes A. , Kleine Appie H. , Hermens Wim Th. , Dieijen-Visser Marja P. van
Published: October 2004

We studied a possible effect of the extent of the acute
phase response after acute myocardial infarction on
the cumulative release of troponin T. The height of the
acute phase response might influence the cumulative
release of troponin T, bound to the myofibrillar structures
of the heart, in a different way compared to the
free cytoplasmic cardiac marker hydroxybutyrate dehydrogenase
(EC 1.1.1.27). To investigate this, the cumulative
amount of C-reactive protein in plasma, i.e.
the quantified acute phase response, was related to
the cumulative plasma release of hydroxybutyrate dehydrogenase
(an established method for infarct sizing)
on the one hand and to that of troponin T on the other
hand.

The study was performed in patients receiving
(n=16) and in patients not receiving (n=6) thrombolytic
therapy. Cumulative protein release was calculated using
a two-compartment model for circulating proteins.

Conclusions: The cumulative amount of plasma C-reactive
protein is significantly higher in the patients
not receiving thrombolytic therapy, as is in accordance
with earlier studies. The cumulative amount of troponin
T released is significantly related to the cumulated
concentration of C-reactive protein, especially in
patients not receiving thrombolytic therapy. The intensity
of the acute phase response, estimated from cumulative
plasma C-reactive protein response, has no
effect on the relative proportions of troponin T and hydroxybutyrate
dehydrogenase released into plasma.
Personal Experience with the Application of Carbohydrate-Deficient Transferrin (CDT) Assays to the Detection of Congenital Disorders of Glycosylation
Authors: Colomé Catrina , Ferrer Imma , Artuch Rafael , Vilaseca M. Antònia , Pineda Mercè , Briones Paz
Published: October 2004

Congenital disorders of glycosylation (CDG) are genetic
multisystemic diseases due to various defects in
the biosynthesis or processing of glycoproteins. Our
aim is to present our experience in the selective
screening of CDG syndrome in a paediatric population
(421 patients) with clinical suspicion of the disease,
analysing serum carbohydrate-deficient transferrin
(CDT) by radioimmunoassay and/or immunoturbidimetry.
We established the normal values for our
paediatric population. The abnormal results were confirmed
and classified by isoelectric focusing of serum
sialotransferrins, and by enzymatic and molecular
studies. We found 14 patients (3.3%) with abnormal
serum CDT; 11 of them were classified as CDG type Ia
(CDG-Ia) and the other three showed altered isoelectrofocusing
patterns but remain untyped and are under
investigation. In conclusion, both CDT assays
proved to be useful tools for CDG screening. Isoelectric
focusing is a simple procedure but it requires specific
instruments that are not always available. Since the
immunoturbidimetric procedure is commonly used to
monitor for recent excessive alcohol consumption in
clinical laboratories and does not require special
equipment, it may also be reliably used to screen for
CDG in children under clinical suspicion.
Screening for Proteinuria in Japanese Schoolchildren: a New Approach
Authors: Pugia Michael J. , Murakami Mutsumi , Lott John A. , Ohta Yoshihide , Kitagawa Teruo , Yamauchi Kuniaki , Suhara Yutaka , Kasjima Junko
Published: October 2004

By governmental mandate, Japanese school children are screened annually for proteinuria, hematuria, and glucosuria to identify children with possible renal disorders. We added urine dipstick tests for albumin and creatinine to the Japanese screening protocol, and used their dipstick results for blood, glucose and protein. The sulfosalicylic acid precipitation test was used to confirm “trace” positive protein dipsticks. The Japanese and our screening protocol have in common the same data for glucosuria and proteinuria. Their scheme has an algorithm for repeat testing of children with abnormal results, and further testing and medical evaluation for those showing persistently abnormal values. Out of the 23,121 students, we found seven with likely nephritis, one with confirmed nephritis, one with nephrotic syndrome, 170 with persistent unexplained hematuria, 19 with persistent unexplained proteinuria, 14 cases of urinary tract infection, and 20 cases of likely diabetes mellitus. We conclude that dipstick testing for albumin, protein, creatinine, glucose and occult blood has significant value in a multilevel testing scheme for identifying children with urinary tract abnormalities or diabetes. The assay of albumin increases the sensitivity of the screening, and dividing the albumin by the creatinine concentration reduces the potential errors arising from concentrated or dilute urines.