bluuebunny/crossref_metadata_2025 huggingface dataset:
A randomized trial of recruitment methods for older African American men in the Prostate, Lung, Colorectal and Ovarian (PLCO) cancer screening trial
Authors: Ford Marvella E , Havstad Suzanne L , Davis Shawna D
Published: August 2004

Background Incidence rates for many types of cancer are higher among African American men than in the general population, yet African American men are less likely to participate in cancer screening trials. This paper describes the outcomes of a randomized trial (the AAMEN Project) designed to recruit African American men aged 55–74 years to a prostate, lung and colorectal cancer screening trial.

Methods The recruitment interventions address four types of barriers to clinical trial participation: sociocultural barriers, economic barriers, individual barriers and barriers inherent in study design. Subjects were randomized to a control group or one of three increasingly intensive intervention arms, which used different combinations of mail, phone and in person church-based recruitment.

Results Of the 39 432 African American men residing in the geographically defined study population (southeastern Michigan and northern Ohio), 17 770 men (45%) could be contacted, and 12 400 (31% of 39 432) were found to be eligible to participate. No statistically significant differences in age, education or income level were found among participants in the four study arms. A significantly greater enrollment yield (3.9%) was seen in the most intensive, church-based intervention arm, compared to the enrollment yields in the other two intervention arms (2.5 and 2.8%) or the control group (2.9%) (P, 0.01).

Conclusions The intervention that involved the highest rate of face-to-face contact with the study participants produced the highest enrollment yield, but several strategies that were thought could improve yield had no effect. These findings, which are consistent with current literature on population-based recruitment, should facilitate the development of future recruitment efforts involving older African American men.
Nonparametric prediction of event times in randomized clinical trials
Authors: Ying Gui-shuang , Heitjan Daniel F , Chen Tai-Tsang
Published: August 2004

In clinical trials with planned interim analysis, it can be valuable for logistical reasons to predict the times of landmark events such as the 50th and 100th event. Bagiella and Heitjan (Stat Med 2001; 20: 2055–63) proposed a parametric prediction model for failure-time outcomes assuming exponential survival and Poisson enrollment. When little is known about the distributions of interest, there is concern that parametric prediction methods may be biased and inefficient if their underlying distributional assumptions are invalid. We propose nonparametric approaches to make point and interval predictions for landmark dates during the course of the trial. We obtain point predictions using the Kaplan–Meier estimator to extrapolate the survival probability into the future, selecting the time when the expected number of events is equal to the landmark number. To construct prediction intervals, we use a simulation strategy based on the Bayesian bootstrap. Monte Carlo results demonstrate the superiority of the nonparametric method when the assumptions underlying the parametric model are incorrect. We demonstrate the methods using data from a trial of immunotherapy of chronic granulomatous disease.
Are missing outcome data adequately handled? A review of published randomized controlled trials in major medical journals
Authors: Wood Angela M , White Ian R , Thompson Simon G
Published: August 2004

Background Randomized controlled trials almost always have some individuals with missing outcomes. Inadequate handling of these missing data in the analysis can cause substantial bias in the treatment effect estimates. We examine how missing outcome data are handled in randomized controlled trials in order to assess whether adequate steps have been taken to reduce nonresponse bias and to identify ways to improve procedures for missing data.

Methods We reviewed all randomized trials published between July and December 2001 in BMJ, JAMA, Lancet and New England Journal of Medicine, excluding trials in which the primary outcome was described as a time-to-event. We focused on trial designs, how missing outcome data were described and the statistical methods used to deal with the missing outcome data, including sensitivity analyses.

Results We identified 71 trials of which 63 (89%) reported having partly missing outcome data: 13 trials had more than 20% of patients with missing outcomes. In 26 trials that measured the outcome at a single time point, 92% performed a complete case analysis and 8% imputed the missing outcomes using baseline values or the worst case value. In 37 trials with repeated measures of the outcome, 46% performed complete case analyses, potentially excluding individuals with some follow-up data, while 14% performed a repeated measures analysis, 19% used the last observation carried forward, 11% imputed with the worst case value and 2% imputed using regression predictions. Thirteen (21%) of trials with missing data reported a sensitivity analysis.

Conclusions Our review shows that missing outcome data are a common problem in randomized controlled trials, and are often inadequately handled in the statistical analysis in the top tier medical journals. Authors should explicitly state the assumptions underlying the handling of the missing outcomes and justify them through data descriptions and sensitivity analyses.
Optimizing clinical trial supply requirements: simulation of computer-controlled supply chain management
Authors: Peterson Magnus , Byrom Bill , Dowlman Nikki , McEntegart Damian
Published: August 2004

Background Computer-controlled systems are commonly used in clinical trials to control dispensing and manage site inventories of trial supplies. Typically such systems are used with an interactive telephone or web system that provide an interface with the study site. Realizing the maximum savings in medication associated with this approach has, in the past, been problematic as it has been difficult to fully estimate medication requirements due to the complexities of these algorithms and the inherent variation in the clinical trial recruitment process.

Methods We describe the traditional and automated methods of supplying sites. We detail a simulation approach that models the automated system. We design a number of simulation experiments using this model to investigate the supply strategy properties that influence medication overage and other strategy performance metrics.

Results The computer-controlled medication system gave superior performance to the traditional method. In one example, a 75% overage of wasted medication in the traditional system was associated with higher supply failure than an automated system strategy with an overage of 47%. In a further example, we demonstrate that the impact of using a country stratified as opposed to site stratified scheme affects the number of deliveries and probability of supply failures more than the amount of drug wasted with respective increases of 20, 2300 and 4%. Medication savings with automated systems are particularly significant in repeat dispensing designs. We show that the number of packs required can fall by as much as 50% if one uses a predictive medication algorithm.

Conclusions We conclude that a computer-controlled supply chain enables medication savings to be realized and that it is possible to quantify the distribution of these savings using a simulation model. The simulation model can be used to optimize the prestudy medication supply strategy and for midstudy monitoring using real-time data contained in the study database.
Up-Regulation of Basal Transcriptional Activity of the Cytochrome P450 Cholesterol Side-Chain Cleavage (CYP11A) Gene by Isoform-Specific Calcium-Calmodulin-Dependent Protein Kinase in Primary Cultures of Ovarian Granulosa Cells
Authors: Seals Richard C. , Urban Randall J. , Sekar Natesampillai , Veldhuis Johannes D.
Published: August 2004

Intracellular calcium ions (Ca2+) regulate steroidogenesis in the placenta, adrenal gland, testis, and ovary. Earlier data indicate that Ca2+/calmodulin-dependent protein kinase (CamK) may mediate Ca2+-dependent up-regulation of CYP11A (cholesterol side-chain cleavage). To examine this notion further, we assessed the expression and actions of isotype-specific CamK on in vitro transcription of the swine CYP11A gene promoter in primary cultures of ovarian granulosa-luteal cells. RT-PCR and oligodeoxynucleotide sequencing identified gene transcripts encoding CamKII and IV in granulosa and theca cells and corpora lutea. DNA sequence homology with the cognate human and rat genes was 97 and 94% (CamKII) and 96 and 88% (CamKIV), respectively. SDS-PAGE and isoform-specific immunoblotting corroborated expression of CamKII (∼52 kDa) and CamKIV (∼60 kDa) proteins. To monitor transcriptional control, granulosa-luteal cells were transfected transiently with a putative 5′-upstream regulatory region of the homologous CYP11A gene −2320 to +23 bp from the transcriptional start site driving luciferase (CYP11A/luc). Coexpression of constitutively active CamKIV elevated basal transcription by 3.5 ± 0.2-fold (P < 0.001), whereas inactive mutant CamKIV and native CamKII had no effect. Forskolin, an activator of adenylyl cyclase, stimulated expression of CYP11A/luciferase by 4.5 ± 0.9-fold (P < 0.001) and did not enhance transcriptional drive by exogenous CamKIV. Preliminary promoter-deletional analyses showed that a proximal 5′-fragment −100 to +23 bp, but not −50/+23 bp, retained full responsiveness to CamKIV (4.5 ± 0.4-fold; P < 0.001). Threefold cotransfection of −100/+23 bp CYP11A/luciferase, active CamKIV, and a dominant-negative mutant of the cAMP-responsive element binding protein (10, 100, and 250 ng) inhibited CamKIV-stimulated transcriptional activity by 17, 47, and 48% (pooled sem± 2%) [P < 0.01]. The dominant-negative mutant of the cAMP-responsive element binding protein also repressed forskolin’s stimulation of −100/+23 CYP11A/luciferase by 12, 38, and 52% (P < 0.01). Based on these ensemble outcomes, we postulate that endogenous CamKIV may serve as a Ca2+-dependent effector mechanism to maintain basal CYP11A gene expression in ovarian granulosa-luteal cells.
Nuclear Factor of Activated T Cells (NFAT) Is Involved in the Depolarization-Induced Activation of Growth Hormone-Releasing Hormone Gene Transcription in Vitro
Authors: Asai Masato , Iwasaki Yasumasa , Yoshida Masanori , Mutsuga-Nakayama Noriko , Arima Hiroshi , Ito Masafumi , Takano Koji , Oiso Yutaka
Published: August 2004

GHRH plays a pivotal role in the regulation of both synthesis and secretion of GH in the anterior pituitary. In this study, we examined the molecular mechanism of depolarization-induced GHRH gene transcription using the hypothalamus cell line, Gsh+/+, revealing the involvement of the transcription factor called nuclear factor of activated T cells (NFAT). GHRH, NFAT1, NFAT4, and related genes were endogenously expressed in Gsh+/+ cells and the rat arcuate nucleus, where NFAT1 and GHRH were colocalized. Cellular excitation with high potassium potently stimulated endogenous GHRH gene 5′-promoter activity as well as the NFAT-mediated gene transcription, the former being further enhanced by coexpression of NFAT. On the other hand, cyclosporin A (a calcineurin-NFAT inhibitor) or EGTA (a calcium chelator) significantly blocked the depolarization-induced GHRH gene transcription. EMSA and site-directed mutagenesis experiments showed the direct binding of NFAT at five sites of the GHRH promoter, among which the relative importance of three distal sites (−417/−403, −402/−387, −317/−301) was suggested. Finally, elimination of all five sites completely abolished the NFAT-induced GHRH gene up-regulation. Altogether, our results suggest that the transcription factor NFAT is involved in the depolarization-induced transcriptional activation of GHRH gene in the neuronal cells.
Endothelin B receptor Ca2+ signaling in shark vascular smooth muscle: participation of inositol trisphosphate and ryanodine receptors
Authors: Fellner Susan K. , Parker Laurel A.
Published: August 2004

In mammals, endothelin receptors are sub-classified into ETAreceptors (ETAR), which are purely constrictive in vascular smooth muscle (VSM), and ETBR, which may produce constriction in VSM or dilatation by stimulating the production of nitric oxide (NO) from endothelial cells. In contrast, previous studies suggested that shark VSM is stimulated exclusively by ETBR. The Ca2+ signaling pathways utilized by shark VSM in response to stimulation by endothelin-1 (ET-1) have not previously been investigated. We measured cytosolic Ca2+concentration ([Ca2+]i) in fura-2-loaded VSM of anterior mesenteric artery of Squalus acanthias and show that the ETBR agonists IRL 1620 and sarafotoxin S6c (SRX) increase[Ca2+]i in VSM to the same extent as ET-1 and ETBR appears to be the only ETR subtype in sharks. To investigate the participation of the inositol trisphosphate (IP3) receptors(IP3R), we utilized two inhibitors of the mammalian IP3R, TMB-8 and 2-APB. In Ca2+-free Ringer, these agents inhibit the response to ETBR agonist stimulation by 71%. The ryanodine-sensitive receptor (RyR) may be activated by low concentrations of ryanodine, by abrupt local increases of [Ca2+]i,(calcium-induced calcium release) or by cyclic adeninediphosphate ribose(cADPR). We employed three inhibitors of activation of the RyR, Ruthenium Red,8-Br cADPR and high concentrations of ryanodine; these agents blocked the[Ca2+]i response to ETBR agonist stimulation by a mean of 39%. These data show for the first time that in VSM of the shark,ETBR activation stimulates both IP3R and RyR, and that cADPR is involved in RyR activation.
Evidence that thyroid hormone induces olfactory cellular proliferation in salmon during a sensitive period for imprinting
Authors: Lema Sean C. , Nevitt Gabrielle A.
Published: August 2004

Salmon have long been known to imprint and home to natal stream odors, yet the mechanisms driving olfactory imprinting remain obscure. The timing of imprinting is associated with elevations in plasma thyroid hormone levels,with possible effects on growth and proliferation of the peripheral olfactory system. Here, we begin to test this idea by determining whether experimentally elevated plasma levels of 3,5,3′-triiodothyronine (T3)influence cell proliferation as detected by the 5-bromo-2′-deoxyuridine(BrdU) cell birth-dating technique in the olfactory epithelium of juvenile coho salmon (Oncorhynchus kisutch). We also explore how natural fluctuations in thyroxine (T4) relate to proliferation in the epithelium during the parr-smolt transformation. In both studies, we found that BrdU labeled both single and clusters of mitotic cells. The total number of BrdU-labeled cells in the olfactory epithelium was significantly greater in fish with artificially elevated T3 compared with placebo controls. This difference in proliferation was restricted to the basal region of the olfactory epithelium, where multipotent progenitor cells differentiate into olfactory receptor neurons. The distributions of mitotic cluster sizes differed significantly from a Poisson distribution for both T3 and placebo treatments, suggesting that proliferation tends to be non-random. Over the course of the parr-smolt transformation, changes in the density of BrdU cells showed a positive relationship with natural fluctuations in plasma T4. This relationship suggests that even small changes in thyroid activity can stimulate the proliferation of neural progenitor cells in the salmon epithelium. Taken together, our results establish a link between the thyroid hormone axis and measurable anatomical changes in the peripheral olfactory system.
Phenotypic deconstruction reveals involvement of manganese transportermalvolioin honey bee division of labor
Authors: Ben-Shahar Yehuda , Dudek Nichole L. , Robinson Gene E.
Published: August 2004

Molecular analysis of a complex behavioral phenotype is facilitated by dissecting it into simpler behavioral components. Using this approach, we present evidence implicating increased manganese transport by the malvolio (mvl) gene into brain cells as one factor that influences age-related division of labor in honey bee colonies. We studied mvl because manganese affects sucrose responsiveness in Drosophila melanogaster, and sucrose responsiveness is related to division of labor in honey bee colonies. Honey bee foragers are more responsive to sucrose in the laboratory than are younger nurse bees, and pollen foragers are more responsive to sucrose than nectar foragers. Levels of mvl mRNA in the brain and manganese in the head were higher in pollen foragers compared with nurses, with nectar foragers intermediate. Manganese treatment increased honey bee sucrose responsiveness and caused precocious foraging. Manganese levels showed a similar pattern to mvl mRNA but manganese treatment did not increase pollen foraging. These results suggest that, while there are molecular pathways common to sucrose responsiveness and division of labor, linkages between a complex behavior and some of its simpler behavioral components are not obligatory. Together with previous findings,these results support the idea that some feeding-related genes in Drosophila have been used in social evolution to regulate division of labor.
Colour constancy in diurnal and nocturnal hawkmoths
Authors: Balkenius Anna , Kelber Almut
Published: August 2004

Diurnal and nocturnal hawkmoths have been shown to use colour vision for flower discrimination. Here, we present evidence that the nocturnal hawkmoth Deilephila elpenor and the diurnal hawkmoth Macroglossum stellatarum also have colour constancy. Colour constancy was shown in D. elpenor in two multiple-choice experiments with five different bluish colour patches under white and blue illumination and with five yellowish colour patches under white, blue and yellow illumination. The mechanism underlying colour constancy in both species was investigated in two dual-choice experiments. The choice behaviour is consistent with the use of the von Kries coefficient law. Although the moths have colour constancy, they react to the colour of the illumination. They make fewer choices when tested under the changed illumination, where they never receive a reward, compared with the training illumination. Even if colour constancy can be explained by a von Kries adaptation mechanism, the fact that the animals discriminate between different illuminations indicates that some additional process must be involved.
Navigation in wood ants Formica japonica: context dependent use of landmarks
Authors: Fukushi Tsukasa , Wehner Rüdiger
Published: August 2004

Wood ants Formica japonica can steer their outbound (foraging) and inbound (homing) courses without using celestial compass information, by relying exclusively on landmark cues. This is shown by training ants to run back and forth between the nest and an artificial feeder, and later displacing the trained ants either from the nest (when starting their foraging runs:outbound full-vector ants) or from the feeder (when starting their home runs:inbound full-vector ants) to various nearby release sites. In addition, ants that have already completed their foraging and homing runs are displaced after arrival either at the feeder (outbound zero-vector ants) or at the nest(inbound zero-vector ants), respectively, to the very same release sites. Upon release, the full-vector ants steer their straight courses by referring to panoramic landmark cues, while the zero-vector ants presented with the very same visual scenery immediately search for local landmark cues defining their final goal. Hence, it depends on the context, in this case on the state of the forager's round-trip cycle, what visual cues are picked out from a given set of landmarks and used for navigation.
Novel Palladium Catalyst Supported on GaAs(001) Passivated by Ammonium Sulfide
Authors: Takamiya Ikuko , Tsukamoto Shiro , Shimoda Masahiko , Miyashita Naoki , Arisawa Mitsuhiro , Arakawa Yasuhiko , Nishida Atsushi
Published: August 2004

A more reactive palladium catalyst than homogeneous Pd(PPh3)4 catalyst for the Heck reaction supported on a sulfur-terminated GaAs(001) plate was developed. Sulfur termination using (NH4)2Sx at 60 °C and Pd absorption in acetonitrile at 100 °C is essential for the preparation of an active and stable catalyst. The catalyst could be reused in this reaction up to ten times.
High Stability in Organic Solvent of Heme Proteins Immobilized in the Interlayers of Magadiite Nanoparticles
Authors: Peng Shuge , Gao Qiuming , Shi Jianlin
Published: August 2004

Enzymatic activities of heme proteins, myoglobin (Mb) and hemoglobin (Hb), immobilized in the interlayers of magadiite nanoparticles under mild condition, toward the oxidation of o-phenylenediamine (OPD) in an organic solvent were firstly reported. The immobilized heme proteins showed higher stability than that of free Mb and Hb in organic toluene solution.
The First Enantiomerically Pure Synthesis of (S)- and (R)-Naftopidil Utilizing Hydrolytic Kinetic Resolution of (±)-(α-Naphthyl) Glycidyl Ether
Authors: Kothakonda Kiran Kumar , Bose D Subhas
Published: August 2004

Hydrolytic Kinetic Resolution (HKR) of (±)-(α-Naphthyl) glycidyl ether with (R,R)-salen Co(III) OAc complex provided enatiomerically pure (S)-naphthyl glycidyl ether and (R)-1-naphthyl glycerol; opening of the corresponding pure terminal epoxide with 1-(2-methoxyphenyl)piperizine gave the enantiomerically pure (S)- and (R)-Naftopidil.
Synthesis of Novel Organic–Inorganic Hybrid Cages via Cobalt-catalyzed Cyclotrimerization of Dimethylethynylsilyl Groups on a Silsesquioxane
Authors: Wada Kenji , Yamasaki Takuro , Kondo Teruyuki , Mitsudo Take-aki
Published: August 2004

Novel organic–inorganic hybrid cages composed of a half-caged silsesquioxane skeleton and a benzene ring have been synthesized by cobalt-catalyzed intramolecular cyclotrimerization of a silsesquioxane bearing three dimethylethynylsilyl groups.
Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet
Authors: Johnson Scott C , Marshall David J , Harms Gerda , Miller Christie M , Sherrill Christopher B , Beaty Edward L , Lederer Scott A , Roesch Eric B , Madsen Gary , Hoffman Gary L , Laessig Ronald H , Kopish Greg J , Baker Mei Wang , Benner Steven A , Farrell Philip M , Prudent James R
Published: August 2004

Background: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening.

Methods: MultiCode® PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in ∼3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns.

Results: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls.

Conclusions: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting.
Screening for Serum Total Homocysteine in Newborn Children
Authors: Refsum Helga , Grindflek Anne W , Ueland Per M , Fredriksen Åse , Meyer Klaus , Ulvik Arve , Guttormsen Anne B , Iversen Ole E , Schneede Jørn , Kase Bengt F
Published: August 2004

Background: Newborn screening for total homocysteine (tHcy) in blood may identify babies with vitamin B12 (B12) deficiency or homocystinuria, but data on the causes of increased tHcy in screening samples are sparse.

Methods: Serum concentrations of tHcy, cystathionine, methionine, folate, and B12 and the methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism were determined in 4992 capillary blood samples collected as part of the routine screening program in newborn children. Methylmalonic acid (MMA), gender (SRY genotyping), and the frequency of six cystathionine β-synthase (CBS) mutations were determined in 20–27% of the samples, including all samples with tHcy >15 μmol/L (n = 127), B12 <100 pmol/L (n = 159), or methionine >40 μmol/L (n = 154).

Results: The median (5th–95th percentile) tHcy concentration was 6.8 (4.2–12.8) μmol/L. B12 status, as determined by serum concentrations of B12, tHcy, and MMA, was moderately better in boys than in girls. tHcy concentrations between 10 and 20 μmol/L were often associated with low B12, whereas tHcy >20 μmol/L (n = 43) was nearly always explained by increased methionine. tHcy did not differ according to folate concentrations or MTHFR 677C>T genotypes. None of the babies had definite CBS deficiencies, but heterozygosity led to low cystathionine, increased methionine, but normal tHcy concentrations.

Conclusion: Increased tHcy is a common but not specific finding in newborns. The metabolite and vitamin profiles will point to the cause of hyperhomocysteinemia. Screening for tHcy and related factors should be further evaluated in regions with high prevalence of homocystinuria and in babies at high risk of B12 deficiency.
Miniaturized Multiplex Label-Free Electronic Chip for Rapid Nucleic Acid Analysis Based on Carbon Nanotube Nanoelectrode Arrays
Authors: Koehne Jessica E , Chen Hua , Cassell Alan M , Ye Qi , Han Jie , Meyyappan Meyya , Li Jun
Published: August 2004

Background: Reducing cost and time is the major concern in clinical diagnostics, particularly in molecular diagnostics. Miniaturization technologies have been recognized as promising solutions to provide low-cost microchips for diagnostics. With the recent advancement in nanotechnologies, it is possible to further improve detection sensitivity and simplify sample preparation by incorporating nanoscale elements in diagnostics devices. A fusion of micro- and nanotechnologies with biology has great potential for the development of low-cost disposable chips for rapid molecular analysis that can be carried out with simple handheld devices.

Approach: Vertically aligned multiwalled carbon nanotubes (MWNTs) are fabricated on predeposited microelectrode pads and encapsulated in SiO2 dielectrics with only the very end exposed at the surface to form an inlaid nanoelectrode array (NEA). The NEA is used to collect the electrochemical signal associated with the target molecules binding to the probe molecules, which are covalently attached to the end of the MWNTs.

Content: A 3 × 3 microelectrode array is presented to demonstrate the miniaturization and multiplexing capability. A randomly distributed MWNT NEA is fabricated on each microelectrode pad. Selective functionalization of the MWNT end with a specific oligonucleotide probe and passivation of the SiO2 surface with ethylene glycol moieties are discussed. Ru(bpy)2+-mediator-amplified guanine oxidation is used to directly measure the electrochemical signal associated with target molecules.

Summary: The discussed MWNT NEAs have ultrahigh sensitivity in direct electrochemical detection of guanine bases in the nucleic acid target. Fewer than ∼1000 target nucleic acid molecules can be measured with a single microelectrode pad of ∼20 × 20 μm2, which approaches the detection limit of laser scanners in fluorescence-based DNA microarray techniques. MWNT NEAs can be easily integrated with microelectronic circuitry and microfluidics for development of a fully automated system for rapid molecular analysis with minimum cost.
Maternal Serum Invasive Trophoblast Antigen (Hyperglycosylated hCG) as a Screening Marker for Down Syndrome during the Second Trimester
Authors: Palomaki Glenn E , Neveux Louis M , Knight George J , Haddow James E , Pandian Raj
Published: August 2004

Background: Approximately two million pregnancies in the United States are screened for Down syndrome annually by use of second-trimester maternal serum markers. At present, a combination of four markers can identify 75% of affected pregnancies when 5% of screened women are classified as candidates for amniocentesis. Although not currently included in screening panels, invasive trophoblast antigen (ITA) is a promising screening marker in serum or urine in both the second and first trimesters. This study aims at better defining the screening performance of serum ITA in the second trimester.

Methods: In an earlier study, serum samples from an unbiased sampling of 45 Down syndrome (cases) and 238 unaffected (control) pregnancies between 14 and 20 weeks of gestation were collected from various centers in the United States. Samples were aliquoted and stored at −20 °C for 8 years. We measured ITA in these samples and determined the screening performance both univariately and in combination with other screening markers.

Results: The median ITA in Down syndrome pregnancies was >3.00 multiples of the median, higher than that found for human chorionic gonadotropin (hCG) or free β-hCG. At a 5% false-positive rate, ITA univariately detected 38% and 40% of Down syndrome pregnancies, respectively, when assigned by date of last menstrual period or ultrasound date. Modeling yielded rates of 45% and 48%. ITA correlated strongly with hCG and free β-hCG. When substituted for either of these in a multiple marker panel, ITA performed comparably.

Conclusions: This study indicates that serum ITA is an effective marker for Down syndrome. It is highly correlated with both hCG and free β-hCG and could replace either of them in a multiple marker panel.
Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster
Authors: Przibilla S. , Hitchcock W. W. , Szécsi M. , Grebe M. , Beatty J. , Henrich V. C. , Spindler-Barth M.
Published: August 2004

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acidbinding residue within the ligand-binding pocket, I323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.
Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy
Authors: Toivola J. , Michel P. O. , Gilbert L. , Lahtinen T. , Marjomäki V. , Hedman K. , Vuento M. , Oker-Blom C.
Published: August 2004

Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and pastimmunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7x10exp-7 cm(2)s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5x10exp-8 cm(2)s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a pastimmunity serum sample gave an average diffusion coefficient of 3.5x10exp-8 cm(2)s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7x10exp-7 to 1.6x10exp-7 cm(2)s(-1), which is much smaller than that observed with acute phase or pastimmunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and pastimmunity sera. FCS measurement of VLPimmune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.
The Btk inhibitor LFM-A13 is a potent inhibitor of Jak2 kinase activity
Authors: Akker E. van den , Dijk T.B. van , Schmidt U. , Felida L. , Beug H. , Löwenberg B. , Lindern M. von
Published: August 2004

LFMA13, or α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide, was shown to inhibit Brutons tyrosine kinase (Btk). Here we show that LFM-A13 efficiently inhibits erythropoietin (Epo)-induced phosphorylation of the erythropoietin receptor, Janus kinase 2 (Jak2) and downstream signalling molecules. However, the tyrosine kinase activity of immunoprecipitated or in vitro translated Btk and Jak2 was equally inhibited by LFM-A13 in in vitro kinase assays. Finally, Epo-induced signal transduction was also inhibited in cells lacking Btk. Taken together, we conclude that LFM-A13 is a potent inhibitor of Jak2 and cannot be used as a specific tyrosine kinase inhibitor to study the role of Btk in Jak2-dependent cytokine signalling.
Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages
Authors: Lecaille F. , Muno D. , Kominami E. , Ishidoh K.
Published: August 2004

The mammalian legumain is a recently identified lysosomal cysteine proteinase belonging to the clan CD and homologous to plant legumain. This enzyme has the characteristic of specifically hydrolyzing peptide bonds after asparagine residues. As in the case of papain-type cysteine proteinases, legumain is synthesized as an inactive zymogen, and processed into a mature form localized in lysosomes. However, the mechanism of its activation remains unclear. In this study, we analyze which types of proteinases may participate in the processing of legumain in rat primary cultured macrophages using various proteinase inhibitors after 24 h treatment with Bafilomycin A1, a vacuolar ATPase inhibitor. The processing of legumain in macrophages was accomplished by papain-type cysteine proteinases other than cathepsin B.
Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family
Authors: Dubin G. , Stec-Niemczyk J. , Dylag T. , Silberring J. , Dubin A. , Potempa J.
Published: August 2004

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties ofStaphylococcus epidermidisstaphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinantS. epidermidisstaphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases fromS. aureuscleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue,S. aureusstaphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by nontarget cysteine proteases. Conversely,S. aureusstaphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.
Metalloproteases with EGF, CUB, and thrombospondin-1 domains function in molting of Caenorhabditis elegans
Authors: Suzuki M. , Sagoh N. , Iwasaki H. , Inoue H. , Takahashi K.
Published: August 2004

Functional analysis using RNAi was performed on eleven genes for metalloproteases of the M12A family inCaenorhabditis elegansand the interference of the C17G1.6 gene (nas-37) was found to cause incomplete molting. The RNAi of the C26C6.3 gene (nas-36) also caused a similar molting defect but not so severely as that of the nas-37 gene. Both the genes encode an astacinlike metalloprotease with an epidermal growth factor (EGF) like domain, a CUB domain, and a thrombospondin-1 domain, in this order. The promoterdriven green fluorescent protein (GFP) expression analysis suggested that they are expressed in hypodermal cells throughout the larval stages and in the vulva of adult animals. In the genetic background of rde-1(ne219), where RNAi does not work, the molting defect caused by the nas-37 interference was observed when the transgenic wildtype rde-1 gene was expressed under the control of the dpy-7 promoter, known to be active in the hypodermal cells, but not under the control of the myo-3 promoter, active in the muscular cells. Therefore these proteases are thought to be secreted by the hypodermal cells and to participate in shedding of old cuticles.
Regulation of gene expression by α-tocopherol
Authors: Azzi A. , Gysin R. , Kempná P. , Munteanu A. , Villacorta L. , Visarius T. , Zingg J.-M.
Published: August 2004

Several genes are regulated by tocopherols which can be categorized, based on their function, into five groups: genes that are involved in the uptake and degradation of tocopherols (Group 1) include α-tocopherol transfer protein (α-TTP) and cytochrome P450 (CYP3A); genes that are associated with lipid uptake and atherosclerosis (Group 2) include CD36, SRBI and SRAI/II. Genes that modulate the expression of extracellular proteins (Group 3) include tropomyosin, collagenα1, MMP-1, MMP-19 and connective tissue growth factor (CTGF). Genes that are related to inflammation, cell adhesion and platelet aggregation (Group 4) include Eselectin, ICAM-1, integrins, glycoprotein IIb, Il-2, IL-4 and IL-β. Group 5 comprises genes coding for proteins involved in cell signaling and cell cycle regulation and consists of PPARγ, cyclin D1, cyclin E, Bcl2-L1, p27 and CD95 (Apo-1/Fas ligand). The expression of P27, Bcl2, α-TTP, CYP3A, tropomyosin, Il-2, PPAR-γ, and CTGF appears to be up-regulated by one or more tocopherols whereas all other listed genes are down-regulated. Several mechanisms may underlie tocopherol-dependent gene regulation. In some cases protein kinase C has been implicated due to its deactivation by α-tocopherol and its participation in the regulation of a number of transcription factors (NFκB, AP-1). In other cases a direct involvement of PXR/ RXR has been documented. The antioxidant responsive element (ARE) appears in some cases to be involved as well as the transforming growth factor β responsive element (TGF-β-RE). This heterogeneity of mediators of tocopherol action suggests the need of a common element that could be a receptor or a co-receptor, able to interact with tocopherol and with transcription factors directed toward specific regions of promoter sequences of sensitive genes. Here we review recent results of the search for molecular mechanisms underpinning the central signaling mechanism.
Cell type-dependent release of nitric oxide and/or reactive nitrogenoxide species from intracellular SIN-1: effects on cellular NAD(P)H
Authors: Swintek A.U. , Christoph S. , Petrat F. , Groot H. de , Kirsch M.
Published: August 2004

SIN-1 is frequently used in cell culture studies as an extracellularly operating generator of peroxynitrite. However, little is known about the nature of the reactive species produced intracellulary from SIN-1. SIN-1 can easily penetrate cells as exemplified for both L-929 mouse fibroblasts and bovine aortic endothelial cells (BAECs) by utilizing capillary zone electrophoresis. In L-929 cells, SIN-1 produced nitric oxide (NO) as monitored by the fluorescent NO scavenger FNOCT-1 and by means of a NO electrode, as well as reactive nitrogenoxide species (RNOS, e.g. peroxynitrite, nitrogen dioxide, dinitrogen trioxide), as detected with the fluorescent indicator DAF-2. Laser scanning microscopy revealed that in L-929 cells SIN-1-derived species initially oxidized the major fraction of the NAD(P)H within the cytosol and the nuclei, whereas the mitochondrial NAD(P)H level was somewhat increased. In marked contrast to this, in BAECs no evidence for NO formation was found although the intracellular amount of SIN-1 was fourfold higher than in L-929 cells. In BAECs, the level of NAD(P)H was slightly decreased within the first 10 min after administration of SIN-1 in both the cytosol/nuclei and mitochondria. These observations reflect the capability of SIN-1 to generate intracellularly either almost exclusively RNOS as in BAECs, or RNOS and freely diffusing NO as in L-929 cells. Nitric oxide as well as RNOS may decisively affect cellular metabolism as indicated by the alterations in the NAD(P)H level. Hence, care should be taken when applying SIN-1 as an exclusively peroxynitrite-generating compound in cell culture systems.
Cathepsin L is involved in cathepsin D processing and regulation of apoptosis in A549 human lung epithelial cells
Authors: Wille A. , Gerber A. , Heimburg A. , Reisenauer A. , Peters C. , Saftig P. , Reinheckel T. , Welte T. , Bühling F.
Published: August 2004

Cathepsins are implicated in a multitude of physiological and pathophysiological processes. The aim of the present study was to investigate the function of cathepsin L (catL) in the proteolytic network of human lung epithelial cells and its role in the regulation of apoptosis. We found that catL-deficient A549 cells as well as lung tissue extracts of catL[-/-] mice express increased amounts of single-chain cathepsin D (catD). Degradation experiments indicate that catL specifically degrades the singlechain isoform of catD. Furthermore, we found that catLdeficient cells showed increased sensitivity to apoptosis. Finally, we demonstrate that the inhibition of catD activity by pepstatin A decreased the number of apoptotic cells in catLdeficient A549 cells after anti-Fas treatment. In conclusion, catL is involved in catD processing and the accumulation of catD isoforms in catL-deficient cells is associated with increased rates of spontaneous and anti-Fas-induced apoptosis.
Current concepts in standardization of cardiac marker immunoassays
Authors: Panteghini Mauro
Published: August 2004

Cardiac markers are measured by a number of different immunoassays using specific antibodies directed to the respective antigens. Lacking assay standardization, different results from different assays measuring the same marker may be obtained and this problem may cloud interpretations of reported data. Presently, there are no reference procedures for cardiac markers; certified reference materials should still be established and, at least for cardiac troponins, the analyte in the patients' blood is significantly different from newly synthesized protein. It is therefore clear that the problems of cardiac marker standardization will not be quickly solved. A number of projects are, however, underway under the auspices of the IFCC and other organizations. The aim of this opinion is to reflect on some concepts related to the implementation of a metrologically correct measurement system, giving practical examples on how these concepts can be applied to immunoassays measuring cardiac markers.
High prevalence of non-HFE gene-associated haemochromatosis in patients from southern Italy
Authors: Marco F. De , Liguori R. , Giardina M. G. , D'Armiento M. , Angelucci E. , Lucariello A. , Morante R. , Cimino L. , Galeota-Lanza A. , Tarantino G. , Ascione A. , Budillon G. , Vecchione R. , Martinelli R. , Matarazzo M. , Simone V. De
Published: August 2004

Hereditary haemochromatosis is an autosomal recessive disorder of iron regulation that results in abnormal intestinal iron absorption with progressive iron overloading of parenchymal cells. Two specific, single point mutations of the
Blood alcohol testing: comparison of the performance obtained with the different methods used in the Belgian external quality assessment schemes
Authors: Devleeschouwer Nicole , Libeer Jean Claude , Martens Frank K. , Neels Hugo , Damme Marc Van , Verstraete Alain , Deveaux Marc , Wallemacq Pierre E.
Published: August 2004

This study describes the performance of the different methods used for blood alcohol analysis by the Belgian laboratories participating in the external quality assessment scheme. The best performance was achieved by the gas chromatography (GC) and the enzymatic methods. The enzymatic methods are easier to use but more apparatus-dependent, whereas GC methods display higher precision, especially at concentrations near the critical legal limit of 0.5 g/l. The study highlights the need to introduce a methodology other than the chemical oxidation-based method of Casier, the sole legal method for forensic analyses in drinking-driving cases in Belgium. This method with poor intra-laboratory, inter-laboratory and linearity performances has definitely become obsolete. Even though GC clearly appears as the method of choice, mainly due to its higher specificity, the present study also shows the need for practical guidelines to improve the quality of GC alcohol analyses used in Belgium.
Determination of lipase catalytic activity in two reference materials: BCR 693 and BCR 694 by titrimetry at constant pH
Authors: Lessinger Jean-Marc , Parashou Stella , Arzoglou Panteleimon , Ramos Paul , Chapus Catherine , Dufaux Jeanine , Junge Wolfgang , Profilis Christos , Férard Georges
Published: August 2004

Because routine assays for pancreatic lipase catalytic activity are not yet standardized, between-method comparability is very poor. This is mainly due to the lack of reference materials (RMs). The aim of this study was to assign values of catalytic concentration to two human pancreatic lipase RMs, one prepared from human pancreatic juice (BCR 693), the other obtained by recombinant technology (BCR 694). Lipase catalytic activity was assayed in five experienced laboratories, using aliquots from the same lot of triolein emulsion and a standardized titrimetric procedure, optimized with regard to substrate, cofactors and pH. The accepted sets of data (n=4) gave a mean ± the corresponding uncertainty expressed as the 0.95 confidence interval of 1732±72 U/l and 1043±60 U/l for BCR 693 and 694, respectively. Transferability of the whole operating procedure proved to be quite satisfactory. The authors conclude that both RMs can be used to verify the correct implementation of the standardized measurement procedure and to assign values to secondary lipase materials (commercial calibrators, control products) which, in turn, ensures traceability to the standardized procedure in this study, and contributes to the harmonization of laboratory results according to the Directive for in vitro Diagnostic Medical Devices.