bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal and quantitative changes in sexual reproductive cycling of the cladoceran Daphnia magna by a juvenile hormone analog
Authors: Olmstead Allen W. , LeBlanc Gerald A.
Published: August 2002

Cyclic parthenogens, such as the cladoceran, Daphnia magna, utilize both asexual (parthenogenetic) and sexual reproduction in order to maximize population fitness in variable environments. Parthenogenetic reproduction is the default strategy among D. magna, while various environmental cues trigger cycles of sexual reproduction. Experiments were conducted with the juvenile hormone analog methoprene to test the hypothesis that members of the insect juvenile hormone/vertebrate retinoic acid family of transcription factors are involved in the regulation of sexual reproduction in daphnids. Neither methoprene, food reduction, or crowding independently stimulated entry into the sexual reproductive phase of the daphnids. However, the combination of food deprivation and crowding stimulated entry into the sexual reproductive phase characterized by an initial high production of males and the subsequent intermittent production of haploid egg‐containing ephippia. Exposure to 160 nM methoprene along with food deprivation and crowding caused a significant reduction in the percentage of males produced during the early phase of the sexual cycle and significantly increased the percentage of males produced during the later stages of the cycle. Methoprene concentrations as low as 6.4 nM significantly reduced the number of resting eggs produced and proportionately increased the production of parthenogenetically‐produced neonates. These experiments demonstrate that methoprene uncouples the coordinate production of males and resting eggs during the sexual reproductive period of D. magna. Methoprene stimulates male offspring production and defers their production to latter stages of the sexual reproductive period, while inhibiting the production of resting eggs and promoting the continuance of parthenogenetic reproduction. J. Exp. Zool. 290:148–155, 2001. © 2001 Wiley‐Liss, Inc.
Monotreme IGF2 expression and ancestral origin of genomic imprinting
Authors: Killian J. Keith , Nolan Catherine M. , Stewart Niall , Munday Barry L. , Andersen Niels A. , Nicol Stewart , Jirtle Randy L.
Published: August 2002

IGF2 (insulin‐like growth factor 2) and M6P/IGF2R (mannose 6‐phosphate/insulin‐like growth factor 2 receptor) are imprinted in marsupials and eutherians but not in birds. These results along with the absence of M6P/IGF2R imprinting in the egg‐laying monotremes indicate that the parental imprinting of fetal growth‐regulatory genes may be unique to viviparous mammals. In this investigation, we have cloned IGF2 from two monotreme mammals, the platypus and echidna, to further investigate the origin of imprinting. We report herein that like M6P/IGF2R, IGF2 is not imprinted in monotremes. Thus, although IGF2 encodes for a highly conserved growth factor in chordates, it is only imprinted in therian mammals. These findings support a concurrent origin of IGF2 and M6P/IGF2R imprinting in the late Jurassic/early Cretaceous period. The absence of imprinting in monotremes, despite apparent interparental conflicts over maternal–offspring exchange, argues that a fortuitous congruency of genetic and epigenetic events may have limited the phylogenetic breadth of genomic imprinting to therian mammals. J. Exp. Zool. (Mol. Dev. Evol.) 291:205–212, 2001. © 2001 Wiley‐Liss, Inc.
Effect of melatonin on hemolymph glucose and lactate levels in the fiddler crab Uca pugilator
Authors: Tilden Andrea , McGann Liz , Schwartz Janine , Bowe Adina , Salazar Christina
Published: August 2002

Melatonin was injected into intact and eyestalk‐ablated fiddler crabs (Uca pugilator), and its effects on hemolymph glucose and lactate levels were studied. In intact crabs, glucose and lactate levels cycled simultaneously, with peaks occurring during early and late photophase. Melatonin caused a shift in the glucose and lactate cycles, with only one peak occurring closer to mid‐photophase. In eyestalk‐ablated animals, the glucose rhythmicity was lost; lactate cycled, but levels were significantly lower than in intact animals. Melatonin caused a delayed hyperglycemia in eyestalk‐ablated animals, with concurrent but much lower increases in lactate. Overall, melatonin demonstrated delayed hyperglycemic effects that do not appear to be mediated solely via eyestalk factors such as crustacean hyperglycemic hormone (CHH), though involvement of the eyestalks cannot be ruled out. An influence on extra‐eyestalk CHH secretion is a potential mechanism of melatonin activity. J. Exp. Zool. 290:379–383, 2001. © 2001 Wiley‐Liss, Inc.
Searching for missing pieces of the sex‐determination puzzle
Authors: Bullejos Monica , Bowles Josephine , Koopman Peter
Published: August 2002

Little is known of the mechanisms whereby the mammalian indifferent gonad develops into a testis or ovary. In XY individuals, Sry, the mammalian testis‐determining gene, is expressed in the pre‐Sertoli cells, which then differentiate into Sertoli cells. Other cell types, which include the germ cells, the steroidogenic cells and the connective tissue cells, must then be instructed to develop in a male‐specific manner. Although some genes involved in sex‐determination and differentiation processes have been identified, we know little of how they interact and cooperate to orchestrate the development of a testis or ovary. We have initiated an expression‐screening program designed to identify additional genes, known or novel, which play a role in these processes. This approach is based on our belief that many of the genes we seek will be expressed in a sex‐specific manner during the period of sex‐determination and differentiation. Most of the genes identified previously are transcription factors and so we aim, in particular, to find genes involved in cell‐to‐cell communication, signal transduction, and transcriptional regulation, downstream of the differentiation of Sertoli cells. We have used a suppression subtractive‐hybridization method to generate male‐ and female‐enriched probes and libraries. Clones are validated as being sex‐specific in their expression patterns by array screening and in situ hybridization. Here we report on our progress to date and the general applicability of the approach for studies in other systems. J. Exp. Zool. 290:517–522, 2001. © 2001 Wiley‐Liss, Inc.
N‐acetylcysteine augments adenovirus‐mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry
Authors: Jornot L. , Morris M. A. , Petersen H. , Moix I. , Rochat T.
Published: August 2002

Background

It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N‐acetylcysteine (NAC) in adenovirus‐mediated gene transfer has been investigated.

Methods

HUVECs were pretreated or not with NAC, and infected with E1E3‐deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV‐LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of β‐Gal activity, and western blot), mRNA level (real‐time RT‐PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real‐time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis.

Results

Pretreatment of cells with NAC prior to Ad infection enhanced β‐Gal activity by two‐fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post‐infection period, a five‐fold increase in β‐Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post‐infection period, β‐Gal activity was further enhanced, by 15‐fold. Augmentation of β‐Gal activity was paralleled by an increase in β‐Gal protein and mRNA levels. NAC did not affect the half‐life of LacZ mRNA.

Conclusion

Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post‐infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases. Copyright © 2001 John Wiley & Sons, Ltd.
Therapeutic levels of human Factor VIII in mice implanted with encapsulated cells: potential for gene therapy of haemophilia A
Authors: García‐Martín Carmen , Chuah Marinee K. L. , Van Damme An , Robinson Kelly E. , Vanzieleghem Beatrijs , Saint‐Remy Jean‐Marie , Gallardo Dominique , Ofosu Frederick A. , Vandendriessche Thierry , Hortelano Gonzalo
Published: August 2002

Background

A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein has been evaluated. The microcapsules are implanted intraperitoneally. In order to prevent cell immune rejection, cells are enclosed in non‐antigenic biocompatible alginate microcapsules prior to their implantation into mice. It has been shown that encapsulated myoblasts can deliver therapeutic levels of Factor IX (FIX) in mice. The delivery of human Factor VIII (hFVIII) in mice using microcapsules was evaluated in this study.

Methods

Mouse C2C12 myoblasts and canine MDCK epithelial kidney cells were transduced with MFG‐FVIII (B‐domain deleted) vector. Selected recombinant clones were enclosed in alginate microcapsules. Encapsulated recombinant clones were subsequently implanted intraperitoneally into C57BL/6 and immunodeficient SCID mice.

Results

Plasma of mice receiving C2C12 and encapsulated MDCK cells had transient therapeutic levels of FVIII in immunocompetent C57BL/6 mice (up to 20% and 7% of physiological levels, respectively). In addition, FVIII delivery in SCID mice was also transient, suggesting that a non‐immune mechanism must have contributed to the decline of hFVIII in plasma. Quantitative RT‐PCR analysis confirmed directly that the decline of hFVIII is due to a reduction in steady‐state hFVIII mRNA, consistent with transcriptional repression. Furthermore, encapsulated cells retrieved from implanted mice were viable, but secreted FVIII ex vivo at three‐fold lower levels than the pre‐implantation levels. In addition, antibodies to hFVIII were detected in immunocompetent C57BL/6 mice.

Conclusions

Implantable microcapsules can deliver therapeutic levels of FVIII in mice, suggesting the potential of this gene therapy approach for haemophilia A. The findings suggest vector down‐regulation in vivo. Copyright © 2002 John Wiley & Sons, Ltd.
Transitive tournaments and self‐complementary graphs
Authors: Gyárfás András
Published: August 2002

A simple proof is given for a result of Sali and Simonyi on self‐complementary graphs. © 2001 John Wiley & Sons, Inc. J Graph Theory 38: 111–112, 2001
The electronic dissemination of policy information; experiences from the livestock–environment toolbox
Authors: Morton John
Published: August 2002

Among the many implications for development of the global revolution in Information and Communication Technologies (ICTs) is the possibility of using ICTs to assist policy makers. This paper reflects on the author's experience drafting the policy modules of the ‘Livestock‐Environment Toolbox.’ Using an electronic web‐based format for the Toolbox allowed it to be highly compact and non‐linear, while remaining accessible through Web and CD‐ROM versions. Some policy issues for the application of ICTs are raised: no field of policy is self‐contained. There are trade‐offs between global outreach and regional specificity, and between participation and authority. The electronic dissemination of policy information must be supported through training and capacity‐building. Copyright © 2001 John Wiley & Sons, Ltd.
How institutional quality and economic factors impact technological deepening in developing countries
Authors: Clarke George R. G.
Published: August 2002

This paper assesses the effect of institutional and economic factors on research and development (R&D) expenditures in developing countries. The paper finds that several measures of institutional quality are correlated with R&D expenditures. Since the institutional variables increase as institutional quality improves, this suggests that stronger institutions and better protection of property rights encourage greater R&D expenditures. In addition, foreign direct investment is negatively correlated within R&D expenditures, while defense expenditures are positively correlated with R&D expenditures in some model specifications. Although R&D is not the primary way that developing countries gain access to technology, these results are of interest since R&D might encourage technological deepening better than other methods that developing countries use to gain access to technology (e.g., through FDI or capital goods imports). Copyright © 2001 John Wiley & Sons, Ltd.
Scaling‐up information services for development: a framework of increasing returns for telecentres
Authors: Tschang Ted , Chuladul Mathurot , Le Thuy Thu
Published: August 2002

Telecentres are an important type of information and communication technology (ICT) system particularly assisting rural and urban communities in developing countries, but the focus is often put on social development, sometimes on static economic returns, and rarely on dynamic economic aspects. This paper develops a basis for understanding the factors behind financial success in a dynamic setting based on the concept of increasing returns. This is applied to telecentres according to the stage of maturity of their information services. The framework is illustrated with literature‐ and interview‐based cases, and will help an understanding of ways in which telecentres can scale‐up their operation and impact. Copyright © 2002 John Wiley & Sons, Ltd.
Synthesis of 3,4,3 LI 1,2 HOPO labelled with 14C
Authors: Burgada R. , Bailly T. , Noël J. P. , Gomis J. M. , Valleix A. , Ansoborlo E. , Hengé‐Napoli M. H. , Paquet F. , Gourmelon P.
Published: August 2002

3,4,3 LI 1,2 HOPO 1 or LIHOPO (1,5,10,14‐tetra (1‐hydroxy‐2‐pyridone‐6‐oyl)‐1,5,10,14‐tetraazatetradecane.) is a decorporation agent used against actinides. It was synthesised labelled with carbon 14 with a view to metabolism studies. [6,9‐14C]‐LIHOPO 2 was obtained in two radioactive steps through a very convenient synthesis in 50% radioactive yield from commercially available [1,4‐14C]‐spermine, hydrochloride and 1‐benzyloxy‐6‐oxo‐1,6‐dihydropyridine‐2‐carbonyl chloride 7. The synthesis of 7 is also described. Copyright © 2001 John Wiley & Sons, Ltd.
Radioiodinated α1‐adrenergic receptor ligands
Authors: Hamill Terence G. , Burns H. Donald , Gibson Raymond E.
Published: August 2002

The synthesis and preliminary characterization of three radioiodinated α1‐adrenergic receptor ligands [125I]5a–c are described. These tracers are analogs of L‐760,478, 1b, itself an analog of prazosin, 1a. The highest affinity tracer, [125I]5a, has six fold higher affinity for the α1 receptor subtypes than prazosin. These ligands could be useful for autoradiographic studies of the α1 receptor subtypes. Copyright © 2001 John Wiley & Sons, Ltd.
Synthesis and characterization of [phenyl‐3H] clonidine hydrochloride at high specific activity
Authors: Filer Crist N. , Ahern David G.
Published: August 2002

p‐Aminoclonidine hydrochloride 1 was converted to m‐dibromo‐p‐iodoclonidine 3 and the latter was used as a precursor to prepare [phenyl‐3H] clonidine hydrochloride 4 at high specific activity via a selective catalytic dehalogenation with tritium. Copyright © 2001 John Wiley & Sons, Ltd.
Syntheses of [14C]‐detergents: octaethylene‐glycol‐[1‐14C]‐dodecylether, [1‐14C]‐dodecyl‐β‐D‐maltoside and dibromo‐analogues
Authors: Georgin Dominique , le Maire Marc , Noël Jean Pierre
Published: August 2002

Octaethylene‐glycol‐dodecylether and dodecyl‐β‐D‐maltoside are two widely used detergents in membrane protein studies. We describe here the synthesis of the 14C‐labelled brominated analogues, and of the 14C‐labelled forms. [1‐14C]‐5,6‐Dibromo‐dodecylether was prepared by coupling [1‐14C]‐(Z)‐1‐bromo‐dodec‐5‐ene with octaethylene glycol followed by bromination. [1‐14C]‐5,6‐Dibromo‐dodecyl‐β‐D‐maltoside was synthesised from [1‐14C]‐(Z)‐dodec‐5‐en‐l‐ol via a coupling with α‐bromohepta‐O‐acetyl‐maltose followed by a deprotection step and bromination. Following similar methods, octaethylene‐glycol‐[1‐14C]‐dodecylether and [1‐14C]‐dodecyl‐β‐D‐maltoside were also obtained. Copyright © 2001 John Wiley & Sons, Ltd.
Fast [18F]FDG synthesis by alkaline hydrolysis on a low polarity solid phase support
Authors: Lemaire C. , Damhaut Ph. , Lauricella B. , Mosdzianowski C. , Morelle J.‐L , Monclus M. , Van Naemen J. , Mulleneers E. , Aerts J. , Plenevaux A. , Brihaye C. , Luxen A.
Published: August 2002

The synthesis of 2‐deoxy‐2‐[18F]fluoro‐D‐glucose ([18F]FDG) has been simplified by the use of a tC18 Sep Pak cartridge to effect purification and hydrolysis of the tetraacetylated [18F]fluoro‐glucose compound ([18F]TAG). After radiolabelling, this derivative was trapped on a solid phase extraction (SPE) cartridge and the residual reaction solvent (CH3CN), reagents (K222, K2CO3,…) and by‐products removed by washing the support with water. After this cleaning step, the acetyl groups were cleaved on the same tC18 column using 2N sodium hydroxide. This fast reaction proceeded near quantitatively (>98%) at room temperature in less than 2 min. The [18F]FDG was then recovered with a small amount of water, neutralized with a slight excess of 2N hydrochloric acid, buffered for pH with a citrate solution and finally purified on a neutral alumina oxide and a second tC18 column. After filtration, the radiochemical yield of this [18F]FDG isotonic solution after more than 100 production runs was found to be very reliable and reproducible (70±6% decay corrected). The synthesis time was about 22 min. Quality controls showed that the radiochemical purity was higher than 98% and in any case no [18F]FDM was detected. Only traces of 2‐chloro‐2‐deoxy‐glucose (ClDG) were found in the final sample (64±9 μg/ batch of 16 ml). [18F]FDG specific activity averaged between 1 and 20 Ci/µmol (EOS). No evaporation and use of ion retardation resin (AG11A8) are required. Moreover, this new approach is suitable for complete remote operation using available single use medical components. Copyright © 2002 John Wiley & Sons, Ltd.
Molecularly imprinted polymer using­p‐hydroxybenzoic acid, p‐hydroxyphenylacetic acid and p‐hydroxyphenylpropionic acid as templates
Authors: Sun Bao Wei , Li Yuan Zong , Chang Wen Bao
Published: August 2002

Molecularly imprinted polymers (MIPs) using p‐hydroxybenzoic acid (p‐HB), p‐hydroxyphenylacetic acid (p‐HPA) and p‐hydroxyphenylpropionic acid (p‐HPPA) as templates were synthesized. The performance of the templates and their analogues on polymer‐based high performance liquid chromatography (HPLC) columns was studied. The imprinting effect of the MIP using p‐HB as template is more obvious than that of MIP using either p‐HPA or p‐HPPA as template, and the mixture of p‐HB and p‐HPA can be well separated on the MIP using p‐HB as template, but not on the blank. Interestingly, the recognition of MIP (p‐HB as the template) to p‐HB showed a synergistic effect. The retention factor of p‐HB is not the sum of those of phenol and benzoic acid. We also found that the imprinting effect decreased when increasing the concentration of acetic acid in mobile phase. The possible reason is that acetic acid molecules occupied the binding sites of the polymer, thereby decreasing the concentration of binding sites. Furthermore, polymers, which showed specificity to 3,4‐dihydroxybenzoic acid, can be prepared with p‐HB as template. It is thus possible to synthesize a specific polymer for a compound that is either expensive or unstable by using a structurally similar compound as template. Copyright © 2001 John Wiley & Sons, Ltd.
Rapid quantitative lung 1H T1 mapping
Authors: Jakob Peter M. , Hillenbrand Claudia M. , Wang Tungte , Schultz Georg , Hahn Dietbert , Haase Axel
Published: August 2002

In this contribution, a rapid and robust technique for quantitative T1 mapping of the human lung is presented. Based on a series of Snapshot FLASH tomograms acquired after a single inversion pulse, high quality and quantitative T1 parameter maps acquired in under five seconds were obtained from six healthy volunteers. The measured T1 values are in good agreement with previously reported literature values. T1 maps were also acquired with the volunteers breathing room air or 100% O2. The T1 difference between breathing room air and 100% O2 is statistically significant at P < 0.0001. J. Magn. Reson. Imaging 2001;14:795–799. © 2001 Wiley‐Liss, Inc.
Ovarian veins: Magnetic resonance imaging findings in an asymptomatic population
Authors: Nascimento Alessandra B. , Mitchell Donald G. , Holland George
Published: August 2002

Purpose

To evaluate the magnetic resonance (MR) venographic appearance of the ovarian veins in a healthy population, correlated with surgical findings. Our data can be used as a basis for comparison to patients with suspected pelvic congestion syndrome (PCS).

Materials and Methods

We retrospectively reviewed exams of 22 women who had MR angiography for potential renal donation evaluation (age range, 19.3–60.5 years; mean, 38 years). We evaluated the diameter of the ovarian veins on dynamic multiphasic postcontrast coronal images, the phase of contrast appearance in these veins, the apparent direction of flow, and the presence of ovarian or pelvic varices. Surgical correlation was obtained in 12 patients. Clinical charts were reviewed and patients were asked about symptoms of chronic pelvic pain and the number of their children.

Results

Of the 22 exams, 21 left gonadal veins were identified. Passive reflux from the left renal vein into the left gonadal vein was suggested in eight exams (38%). These veins had a mean diameter of 6.4 ± 1.6 mm, significantly larger than 4.5 ± 1.3 mm for the other 13 left gonadal veins without reflux (P < 0.005). The right gonadal vein was identified in 8 of 22 women, with a mean diameter of 4.4 ± 0.5 mm. No patient reported symptoms of chronic pelvic pain.

Conclusion

Passive reflux from the left renal vein to the left gonadal vein can occur in asymptomatic women. Diagnosis of PCS depends heavily on appropriate clinical history. J. Magn. Reson. Imaging 2002;15:551–556. © 2002 Wiley‐Liss, Inc.
Automated registration of dynamic MR images for the quantification of myocardial perfusion
Authors: Bidaut Luc M. , Vallée Jean‐Paul
Published: August 2002

Cardiac dynamic magnetic resonance imaging (MRI) after contrast media injection suffers from motion induced by free breathing during acquisition. This work presents an automated approach for motion correction of the heart. The registration is based on the multipass/multiresolution iterative minimizing of intrinsic differences between each image and a reference image coupled to a two‐dimensional/3 parameters rigid body correction. The efficiency of this correction method was evaluated with anatomical landmarks, various cost functions, and for a compartment model fit of the data with 2 parameters: K1, the blood to myocardium transfer coefficient; and Vd, the distribution volume of the contrast media. The variability of K1 and Vd, derived from the fit of the registered images (using the manual correction as a gold standard), was significantly reduced by comparison with the variability obtained from the uncorrected images (P < 0.04). This motion correction method also clearly improves the analysis of dynamic cardiac MRI after contrast media injection in comparison to manual correction. J. Magn. Reson. Imaging 2001;13:648–655. © 2001 Wiley‐Liss, Inc.
Diffusion tensor MRI of the human kidney
Authors: Ries Mario , Jones Richard A. , Basseau Fabrice , Moonen Chrit T.W. , Grenier Nicolas
Published: August 2002

This study characterizes the diffusion anisotropy of the human kidney using a diffusion‐weighted, single‐shot echo planar imaging (EPI) sequence in order to access the full apparent diffusion tensor (ADT) within one breathhold. The fractional anisotropy (FA) of the cortex and the medulla were found to be 0.22 ± 0.12 and 0.39 ± 0.11, respectively (N = 10), which emphasizes the need for rotationally invariant diffusion measurements for clinical applications. Additional limitations for clinical diffusion imaging on the kidney are the strong susceptibility variations within the abdomen that restrict the use of imaging techniques employing long echo trains, and the severe motion sensitivity that limits the available imaging time to one breath‐hold. To overcome these problems an isotropic, diffusion‐weighted, segmented EPI protocol that facilitates the acquisition of high‐resolution diffusion‐weighted images within a single breath‐hold was implemented. Using this method, the apparent diffusion coefficient (ADC) of the cortex and medulla were found to be 2.89 ± 0.28 · 10−9 m2/s and 2.18 ± 0.36 · 10−9 m2/s (N = 10). J. Magn. Reson. Imaging 2001;14:42–49. © 2001 Wiley‐Liss, Inc.
Spatial variation in cartilage T2 of the knee
Authors: Smith Harvey E. , Mosher Timothy J. , Dardzinski Bernard J. , Collins Belinda G. , Collins Christopher M. , Yang Qing X. , Schmithorst Vincent J. , Smith Michael B.
Published: August 2002

Technical limitations imposed by resolution and B1 homogeneity have thus far limited quantitative in vivo T2 mapping of cartilage to the patella. The purpose of this study is to develop T2 mapping of the femoral/tibial joint and assess regional variability of cartilage T2 in the knee. Quantitative in vivo T2 mapping of the knee was performed on 15 asymptomatic adults (age, 22–44) using a 3T MR scanner. There is a consistent pattern of spatial variation in cartilage T2 with longer values near the articular surface. The greatest variation occurs in the patella, where T2 increases from 45.3 ± 2.5 msec at a normalized distance of 0.33–67 ± 5.5 msec at a distance of 1.0. These results demonstrate feasibility of performing in vivo T2 mapping of femoral tibial cartilage. Except for the superficial 15% where T2 values are lower, the spatial variation in T2 of femoral and tibial cartilage is similar to patellar cartilage. J. Magn. Reson. Imaging 2001;14:50–55. © 2001 Wiley‐Liss, Inc.
Multi‐slice dynamic imaging: Complete functional cardiac MR examination within 15 seconds
Authors: Bornstedt Axel , Nagel Eike , Schalla Simon , Schnackenburg Bernhard , Klein Christoph , Fleck Eckart
Published: August 2002

A new magnetic resonance (MR) sequence was developed to acquire real‐time images in a multi‐slice dynamic imaging mode to cover the complete heart in 15 seconds without the need for electrocardiogram (ECG) triggering and multiple breath holds. In 34 patients, left ventricular function was assessed with the new technique and a standard technique. The new technique proved to be feasible and accurate for functional cardiac examinations. J. Magn. Reson. Imaging 2001;14:300–305. © 2001 Wiley‐Liss, Inc.
Mass spectrometry of steroid glucuronide conjugates. I. Electron impact fragmentation of 5α‐/5β‐androstan‐3α‐ol‐17‐one glucuronides, 5α‐estran‐3α‐ol‐17‐one glucuronide and deuterium‐labelled analogues
Authors: Thevis Mario , Opfermann Georg , Schmickler Hans , Schänzer Wilhelm
Published: August 2002

Owing to the developments of analytical instruments and interfaces (e.g. coupling high‐performance liquid chromatography to mass spectrometry), there has been increased interest in new reference materials, for example in doping analysis with steroid glucuronide conjugates. The synthesized reference material has to pass several characterization steps including the use of gas chromatography/mass spectrometry (GC/MS) for its structure confirmation. In the present study, the fragmentation and mass spectrometric behaviour of several steroid glucuronide conjugates of endogenous and anabolic steroids after derivatization to pertrimethylsilylated products and to methyl ester pertrimethylsilylated products were investigated using GC/MS ion trap and GC/MS quadrupole instruments. The mass spectra of the derivatives of androsterone glucuronide, d5‐androsterone glucuronide, epiandrosterone glucuronide, etiocholanolone glucuronide, 11β‐hydroxy etiocholanolone glucuronide, 19‐norandrosterone glucuronide, d4‐19‐norandrosterone glucuronide and 1α‐methyl‐5α‐androstan‐3α‐ol‐17‐one glucuronide are presented and the origin of typical fragment ions of the glycosidic and steroidal moieties is proposed, based on different derivatization techniques including derivatization with d18‐bistrimethylsilylacetamide, methyl ester and trimethylsilyl ester derivatization and selected reaction monitoring. Typical fragmentation patterns which are related to the steroid structure are discussed. Copyright © 2001 John Wiley & Sons, Ltd.
Gabapentin quantification in human plasma by high‐performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study
Authors: Ifa Demian R. , Falci Márcio , Moraes Maria E. , Bezerra Fernando A. F. , Moraes Manoel O. , Nucci Gilberto de
Published: August 2002

A rapid, sensitive and specific analytical method was developed and validated to quantify gabapentin in human plasma using acetaminophen as an internal standard. The method employs a single plasma protein precipitation. The analytes are chromatographed on a C4 reversed‐phase chromatographic column and analyzed by mass spectrometry in the multiple reaction monitoring (MRM) mode. The method has a chromatographic run time of 4 min and a linear calibration curve over the range 50–10 000 ng ml−1 (r > 0.999). The between‐run precision, based on the relative standard deviation for replicate quality controls, was ≤4.8 % (200 ng ml−1), 6.0% (1000 ng ml−1) and 4.4% (5000 ng ml−1). The between‐run accuracy was ±2.6, 4.4 and 0.5% for the above‐mentioned concentrations, respectively. This method was employed in a bioequivalence study of two gabentin capsule formulations (Progresse from Biosintética, Brazil, as a test formulation, and Neurotin from Parke‐Davis, as a reference formulation) in 24 healthy volunteers of both sexes who received a single 300 mg dose of each formulation. The study was conducted using an open, randomized, two‐period crossover design with a 7‐day washout interval. The 90% confidence interval (CI) of the individual ratio geometric mean for Progresse/Neurotin was 87.9–115.6% for AUC(0–36 h) and 88.6–111.7% for Cmax. Since both 90% CI for AUC(0–36 h) and Cmax were included in the 80–125% interval proposed by the US Food and Drug Administration, Progresse was considered bioequivalent to Neurotin according to both the rate and extent of absorption. Copyright © 2001 John Wiley & Sons, Ltd.
Electrospray ionization mass spectrometric studies on the amphoteric surfactant cocamidopropylbetaine
Authors: Eichhorn Peter , Knepper Thomas P.
Published: August 2002

After liquid chromatographic (LC) separation, electrospray ionization mass spectrometry (ESI‐MS) was investigated for the determination of the amphoteric surfactant cocamidopropylbetaine (CAPB). In the positive ion mode the molecule formed the adduct ions [M + H]+, [M + Na]+and [M + K]+. Adducts of these cations were also detected with decreasing abundance as dimer and trimer clusters. Additionally, doubly charged molecular ions with different combinations of cations were identified. It was noticed that the relative abundances of individual cation adducts were not reproducible, apparently owing to varying contents of alkali metal ions originating from the solvent and the sample. Under negative ionization, the major molecular ion was [M − H]−. Higher clusters formed by two and three surfactant molecules, i.e. [2M − H]−and [3M − H]−were likewise registered. The tendency to form clusters in both positive and negative ion modes, even at 0.1 mg l−1levels, was attributed to strong electrostatic interactions between the zwitterionic head groups. Further evidence for this assumption was provided by the detection of a fragment formed from [2M − H]−which contained the two charged head groups. Studies were undertaken in the negative ion mode on the concentration‐ and orifice voltage‐dependent monomer, dimer and trimer formation of C12‐CAPB in order to evaluate potential issues in using the ion [M − H]−mode for quantitative analysis. Finally, the established (−)‐LC/ESI‐MS method was applied to follow up the primary degradation of CAPB in a laboratory‐scale fixed‐bed bioreactor (FBBR) spiked with a test concentration of 10 mg l−1. Direct analysis without sample pretreatment revealed that higher alkyl homologues were more prone to adsorption. Primary biodegradation of all alkyl homologues was completed after a period of 4 days. Selected lyophilized FBBR samples were examined for the presence of transient or stable degradation intermediates, but no metabolite could be identified. Copyright © 2001 John Wiley & Sons, Ltd.
Quantification of nimesulide in human plasma by high‐performance liquid chromatography/tandem mass spectrometry. Application to bioequivalence studies
Authors: Barrientos‐Astigarraga Rafael Eliseo , Vannuchi Yuri Borges , Sucupira Mauro , Moreno Ronilson Agnaldo , Muscará Marcelo Nicolás , Nucci Gilberto De
Published: August 2002

A method based on liquid chromatography with negative ion electrospray ionization and tandem mass spectrometry is described for the determination of nimesulide in human plasma. Liquid–liquid extraction using a mixture of diethyl ether and dichloromethane was employed and celecoxib was used as an internal standard. The chromatographic run time was 4.5 min and the weighted (1/x) calibration curve was linear in the range 10.0–2000 ng ml−1. The limit of quantification was 10 ng ml−1, the intra‐batch precision was 6.3, 2.1 and 2.1% and the intra‐batch accuracy was 3.2, 0.3 and 0.1% for 30, 300 and 1200 ng ml−1 respectively. The inter‐batch precision was 2.3, 2.8 and 2.7% and the accuracy was 3.3, 0.3 and 0.1% for 30, 300 and 1200 ng ml−1 respectively. This method was employed in a bioequivalence study of one nimesulide drop formulation (nimesulide 50 mg ml−1 drop, Medley S/A Indústria Farmacêutica, Brazil) against one standard nimesulide drop formulation (Nisulid, 50 mg ml−1 drop, Astra Médica, Brazil). Twenty‐four healthy volunteers (both sexes) took part in the study and received a single oral dose of nimesulide (100 mg, equivalent to 2 ml of either formulation) in an open, randomized, two‐period crossover way, with a 2‐week washout interval between periods. The 90% confidence interval (CI) for geometric mean ratios between nimesulide and Nisulid were 93.1–109.6% for Cmax, 87.7–99.8% for AUClast and 88.1–99.7% for AUC0–∞. Since the 90% CI for the above‐mentioned parameters were included in the 80–125% interval proposed by the US Food and Drug Administration, the two formulations were considered bioequivalent in terms of both rate and extent of absorption. Copyright © 2001 John Wiley & Sons, Ltd.
Real‐time PCR for detection and quantitation of hepatitis B virus DNA
Authors: Chen Ren Wei , Piiparinen Heli , Seppänen Mikko , Koskela Pentti , Sarna Seppo , Lappalainen Maija
Published: August 2002

A sensitive and reproducible real‐time PCR assay based on TaqMan technology was developed for the detection and quantitation of hepatitis B virus (HBV) DNA in serum, and compared with an “in‐house” qualitative PCR assay. HBV DNA was measured in 125 serum samples from 76 hepatitis B patients, consisting of 22 patients with an acute infection, 20 patients with a previous history of hepatitis B infection, and 34 patients with a chronic hepatitis B. Four patients with a chronic infection were treated with either an IFN‐alpha monotherapy or a combination of IFN‐alpha and lamivudine. Twenty‐nine sera from healthy individuals and non‐hepatitis B patients served as negative controls. The assay was validated by using a 10‐fold dilution series of the World Virological Quality Control (VQC) sample containing 3.73 × 107 genome equivalents per ml. The detection limit for the real‐time PCR was 3.73 × 102 genome equivalents per ml (geq/ml), while it was 3.73 × 103 geq/ml for the in‐house PCR. The real‐time PCR assay had an 8‐logarithm dynamic range spanning from 102 to 1010 geq/ml. In clinical serum samples, the real‐time PCR and the in‐house PCR detected HBV DNA in 81% (101/125) and 66% (83/125) of samples, respectively. HBV DNA was not detected among the negative controls by either of these assays. In conclusion, real‐time PCR is a sensitive, specific, and a reproducible approach for the detection and quantitation of HBV DNA in clinical serum samples, useful also for monitoring the efficacy of antiviral treatment. J. Med. Virol. 65:250–256, 2001. © 2001 Wiley‐Liss, Inc.
Molecular epidemiology of calicivirus infections in Norway
Authors: Vainio Kirsti , Stene‐Johansen Kathrine , Jonassen Tom Øystein , Bruu Anne‐Lise , Grinde Bjørn
Published: August 2002

The national reference laboratory for calicivirus diagnostics monitors the epidemiology of calicivirus infections in Norway. During winter 1998‐1999, 406 fecal samples were received from patients with suspected calicivirus infection. Of these, 76 (19%) were calicivirus positive by a nested reverse transcription‐polymerase chain reaction. A number of alternative PCR designs were employed to disclose false negatives, but none were found. One half of the PCR positive samples were sequenced in order to investigate whether various cases represented the same outbreak, and to what extent a single or multiple subtypes were responsible for the morbidity during this season. The sequence data revealed that the majority of cases represented a genotype related to the Lordsdale strain, whereas the remaining cases seemed more sporadic. Most often, samples from particular outbreaks were highly homogenous. However, in a few cases, samples connected with the same outbreak proved to contain epidemiologically independent strains. J. Med. Virol. 65:309–314, 2001. © 2001 Wiley‐Liss, Inc.
HTLV‐II infection associated with a chronic neurodegenerative disease: Clinical and molecular analysis
Authors: Silva Edimilson A. , Otsuki Koko , Leite Ana Claudia B. , Alamy Alexandre H. , Sá‐Carvalho Daniel , Vicente Ana Carolina P.
Published: August 2002

HTLV II is a retrovirus endemic in some Amerindian tribes and spread worldwide with a high prevalence among intravenous drug abusers. It has three different genetic subtypes a, b, and d, defined mainly by the long terminal repeat (LTR) region. HTLV II has been associated with a neurodegenerative disease in few cases. We describe the first well‐documented case in Brazil where the virus is endemic in isolated ethnic groups. The patient is a 55‐year‐old woman with a chronic and painful syndrome characterized by spastic paraparesis, hyperactive reflexes and spastic bladder. Somatosensory evoked potential indicates a thoracic spinal cord lesion. Computer tomography showed periventricular demyelination. Enzyme‐linked immunosorbent assay was positive for HTLV I/II whereas the discriminatory Western blot was indeterminate. Molecular analysis of the Tax region revealed a HTLV II pattern that was also confirmed through sequencing the LTR region. Phylogenetic analysis of the LTR sequence shows an HTLV IIa subtype that clustered with the virus isolated from Kayapo Indians and Brazilian urban intravenous drug users. Indeterminate Western blots are frequently found using commercial kits, therefore we recommend that all cases in which a myelopathy is associated with an indeterminate serological result should be evaluated by PCR to determine the actual number of HTLV II associated myelopathy cases. J. Med. Virol. 66:253–257, 2002. © 2002 Wiley‐Liss, Inc.
Risk factors and mechanism of transplacental transmission of hepatitis B virus: A case‐control study
Authors: Xu De‐Zhong , Yan Yong‐Ping , Choi Bernard C.K. , Xu Jian‐Qiu , Men Ke , Zhang Jing‐Xia , Liu Zhi‐Hua , Wang Fu‐Sheng
Published: August 2002

Intrauterine hepatitis B virus (HBV) infection has been suggested to be caused by transplacental transmission that cannot be blocked by hepatitis B vaccine. This would decrease the effectiveness of hepatitis B vaccine. This study examined the risk factors and mechanism of transplacental HBV transmission. A case‐control study included 402 newborn infants from 402 HBsAg‐positive pregnant women. Among these, 15 newborn infants infected with HBV by intrauterine transmission were selected as cases, and the rest as controls. A pathology study included 101 full‐term placentas from the HBsAg‐positive pregnant women above and 14 from HBsAg‐negative pregnant women. Immunohistochemistry staining and HBV DNA in situ hybridization were used to estimate the association of intrauterine HBV infection and HBV infection in the placentas. HBeAg positivity in mothers' sera (OR = 17.07, 95%CI 3.39–86.01) and threatened preterm labor (OR = 5.44, 95%CI 1.15–25.67) were found to be associated with transplacental HBV transmission. The intrauterine infection rate increased linearly and significantly with maternal serum HBsAg titers (trend test P = 0.0117) and HBV DNA concentration (trend test P < 0.01). Results of the pathology study showed that HBV infection rates decreased gradually from the maternal side to the fetal side (trend test P = 0.0009) in the placental cell layers. There was a significant association between intrauterine HBV transmission and HBV infection in villous capillary endothelial cells (VCEC) in the placenta (OR = 18.46, P = 0.0002). The main risk factors for intrauterine HBV infection are maternal serum HBeAg positivity, history of threatened preterm labor, and HBV in the placenta especially the villous capillary endothelial cells. Previous reports of transplacental leakage of maternal blood causing intrauterine infection are confirmed. In addition, there appears to be a “cellular transfer” of HBV from cell to cell in the placenta causing intrauterine infection. This latter hypothesis needs to be confirmed. J. Med. Virol. 67:20–26, 2002. © 2002 Wiley‐Liss, Inc.
Human cytomegalovirus as a direct pathogen: Correlation of multiorgan involvement and cell distribution with clinical and pathological findings in a case of congenital inclusion disease
Authors: Bissinger A.L. , Sinzger C. , Kaiserling E. , Jahn G.
Published: August 2002

The human cytomegalovirus (HCMV), a member of the Herpesviridae, is the most frequent cause of congenital virus infections and a major cause of morbidity and mortality in immunocompromised patients. Due to the lack of an appropriate animal model, insight into the pathogenesis of HCMV infections originates primarily from in situ examination of HCMV‐infected tissues. Although in immunocompromised adults such tests are complicated frequently by the presence of additional misleading pathogens, the absence of additional pathogens renders congenital inclusion disease the most suitable access for investigation of pathogenetic aspects of HCMV infections. Immunohistochemical examination of tissue sections from a boy with fatal congenital inclusion disease was undertaken to detect the extent of multiorgan and cell involvement. Adrenal gland, bone marrow, diencephalon, heart, kidney, liver, lung, pancreas, placenta, small bowel and spleen were included in this study. Detection of virus antigens from different phases of viral replication revealed that all investigated organs were infected by HCMV. Simultaneous detection of cell type specific marker molecules showed that a variety of cell types stained positive for HCMV antigens including endothelial cells, epithelial cells, smooth muscle cells, mesenchymal cells, hepatocytes, monocytes/macrophages and granulocytes. The lung, the pancreas, the kidneys and the liver were the major target organs with a high number of HCMV infected cells. This correlated with multiorgan failure as the cause of death and strongly indicates direct pathogenetic effects of HCMV. J. Med. Virol. 67:200–206, 2002. © 2002 Wiley‐Liss, Inc.
Reduced phospholipase C‐β activity and isoform expression in the cerebellum of TS65DN mouse: A model of down syndrome
Authors: Ruiz de Azúa Iñigo , Lumbreras María Ángeles , Zalduegui Amaia , Baamonde Carmela , Dierssen Mara , Flórez Jesús , Sallés Joan
Published: August 2002

Agonist‐ and guanine‐nucleotide‐stimulated phospholipase C‐β (PLC) activity was characterized in crude plasma membrane preparations from cerebral cortex, hippocampus and cerebellum of Ts65Dn mice, a model for Down syndrome, and their control littermates. The levels of expression of PLC‐β(1–4) isoforms and G‐protein αq/11 subunits were also quantified by Western blot analysis to establish their contribution to the patterns of PLC functioning. PLC activity regulated by G‐proteins and muscarinic and 5‐HT2 receptors presented a regional distribution in both control and Ts65Dn mice. In both groups of mice, the intensity of PLC responses to maximal activation by calcium followed the sequence cerebellum > cortex > hippocampus. Both basal and maximal PLC activities, however, were significantly lower in cerebellar membranes of Ts65Dn than in control mice. This difference was mostly revealed in crude plasma membranes prepared from cerebellum at the level of G‐protein‐dependent‐PLC activity because the concentration‐response curve to GTPγS showed a reduction of the maximal effect in Ts65Dn mice, with no change in sensitivity (EC50). Western blot analysis showed a heterogeneous distribution of PLC‐β(1–4) isoforms in both groups of mice. The levels of PLC‐β4 isoform, however, were significantly lower in the cerebellum of Ts65Dn than in control mice. We conclude that the cerebellum of Ts65Dn mice has severe deficiencies in PLC activity stimulated by guanine nucleotides, which are specifically related to a lower level of expression of the PLC‐β4 isoform, a fact that may account for the neurological phenotype observed in this murine model of Down syndrome. J. Neurosci. Res. 66:540–550, 2001. © 2001 Wiley‐Liss, Inc.
Glutamine transport in C6 glioma cells: Substrate specificity and modulation in a glutamine deprived culture medium
Authors: Dolińska Monika , Dybel Anna , Hilgier Wojciech , Zielińska Magdalena , Zabłocka Barbara , Bużańska Leonora , Albrecht Jan
Published: August 2002

A previous study has shown that glutamine (Gln) uptake in C6 cells grown in a standard medium containing 2 mM Gln, is predominantly mediated by a sodium‐dependent system that is inhibited by ASC system substrates alanine (Ala), serine (Ser), cysteine (Cys) and threonine (Thr), shows pH sensitivity and partial tolerance to substitution of Na+by Li+, features compatible with system ASCT2 that is strongly expressed in cultured astrocytes. The uptake was not inhibited by the model system A substrate α‐(methylamino)isobutyric acid (MeAiB), and glycine (Gly) or proline (Pro), indicating that the substrate‐regulated system A as defined by routine criteria is relatively inactive in these cells (Dolińska et al.,2000). In this study we compared the uptake of radiolabeled Gln and a model ASC substrate ‐Thr in cells grown to the same density in Gln‐containing and Gln‐deprived media. Cells grown in the absence of Gln showed a reduced activity of system ASC‐mediated Gln uptake, and the system lost tolerance for Li+and became somewhat more resistant to lowering pH of the medium. In contrast to cultured astrocytes deprived of Gln, the overall Gln uptake activity in C6 cells adapted to grow in a medium without Gln was lower than in cells grown in a Gln containing medium, and the uptake by system A remained inactive. C6 cells cultured both in the presence and absence of Gln expressed ASCT2 mRNA, indicating that system ASCT2‐mediated Gln uptake is modulated at a posttranscriptional level. In contrast to Gln uptake, Thr uptake was more active in cells cultured in the absence of Gln and showed neither pH dependence nor lithium tolerance in either medium, which is typical of an uptake mediated by the widespread ASCT1 isoform of system ASC. In C6 cells grown in the presence or absence of Gln alike, ≈20% of the sodium‐dependent Gln uptake was resistant to MeAiB+Thr, indicating contribution of system N. The N system‐mediated uptake in C6 cells grown in the absence, but not in the presence of Gln was not inhibited by glutamate (Glu) that conforms to the characteristics of the glial N system variant, SN1. © 2001 Wiley‐Liss, Inc.