bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the α‐Homo‐DNA:RNA Duplex and the Function of Twist and Slide To Catalogue Nucleic Acid Duplexes
Authors: Nauwelaerts Koen , Lescrinier Eveline , Herdewijn Piet
Published: September 2006

High‐resolution NMR studies of an α‐homo‐DNA:RNA duplex reveal the formation of a right‐handed parallel‐oriented helix. It differs significantly from a standard A‐ or B‐type helix by a small twist value (26.2°), which leads to a helical pitch of 13.7 base pairs per helical turn, a negative inclination (−1.78 Å) and a large x displacement (5.90 Å). The rise (3.4 Å) is similar to that found in B‐DNA. The solution of this new helix structure has stimulated us to develop a mathematical and geometrical model based on slide and twist parameters to describe nucleic acid duplexes. All existing duplexes can be positioned within this landscape, which can be used to understand the helicalization process.
Iridium(III) Silyl Alkyl and Iridacyclic Compounds Supported by 4,4′‐Di‐tert‐butyl‐2,2′‐bipyridyl
Authors: Sau Yiu‐Keung , Lee Hung‐Kay , Williams Ian D. , Leung Wa‐Hung
Published: September 2006

Treatment of IrCl3⋅x H2O with one equivalent of 4,4′‐di‐tert‐butyl‐2,2′‐bipyridyl (dtbpy) in N,N‐dimethylformamide (dmf) afforded [IrCl3(dmf)(dtbpy)] (1). Alkylation of 1 with Me3SiCH2MgCl resulted in CSi cleavage of the Me3SiCH2 group and formation of the IrIII silyl dialkyl compound [Ir(CH2SiMe3)(dtbpy)(Me)(SiMe3)] (2), which reacted with tBuNC to afford [Ir(tBuNC)(CH2SiMe3)(dtbpy)(Me)(SiMe3)] ([2(tBuNC)]). Reaction of 2 with phenylacetylene afforded dimeric [{Ir(CCPh)(dtbpy)(SiMe3)}2(μ‐CCPh)2] (3), in which the bridging PhCC− ligands are bound to Ir in a μ‐σ:π fashion. Alkylation of 1 with PhMe2CCH2MgCl afforded the cyclometalated compound [Ir(dtbpy)(CH2CMe2C6H4)(2‐C6H4CMe3)] (4), which features an agostic interaction between the Ir center and the 2‐tert‐butylphenyl ligand. The cyclic voltammogram of 4 in CH2Cl2 shows a reversible IrIV–IrIII couple at about 0.02 V versus ferrocenium/ferrocene. Oxidation of 4 in CH2Cl2 with silver triflate afforded an IrIV species that exhibits an anisotropic electron paramagnetic resonance (EPR) signal in CH2Cl2 glass at 4 K with g∥=2.430 and g⊥=2.110. Protonation of 4 with HCl and p‐toluenesulfonic acid (HOTs) afforded [{Ir(dtbpy)(CH2CMe2Ph)Cl}2(μ‐Cl)2] (5) and [Ir(dtbpy)(CH2CMe2Ph)(OTs)2] (6), respectively. Reaction of 5 with Li[BEt3H] gave the cyclometalated complex [{Ir(dtbpy)(CH2CMe2C6H4)}2(μ‐Cl)2] (7). Reaction of 4 with tetracyanoethylene in refluxing toluene resulted in electrophilic substitution of the iridacycle by C2(CN)3 with formation of [Ir(dtbpy)(CH2CMe2C6H3{4‐C2(CN)3})(2‐C6H4CMe3)] (8). Reaction of 4 with diethyl maleate in refluxing toluene gave the iridafuran compound [Ir(dtbpy)(CH2CMe2C6H4){κ2(C,O)‐C(CO2Et)CH(CO2Et)}] (9). Treatment of 9 with 2,6‐dimethylphenyl isocyanide (xylNC) led to cleavage of the iridafuran ring and formation of [Ir(dtbpy)(CH2CMe2C6H4){C(CO2Et)CH(CO2Et)}(xylNC)] (10). Protonation of 9 with HBF4 afforded the dinuclear neophyl complex [(Ir(dtbpy)(CH2CMe2Ph){κ2(C,O)‐C(CO2Et)CH(CO2Et)})2][BF4]2 (11). The solid‐state structures of complexes 2–5 and 8–11 have been determined.
N‐Fusion Reaction Sequence of Heptaphyrin(1.1.1.1.1.1.1): Singly, Doubly, and Quadruply N‐Fused Heptaphyrins
Authors: Saito Shohei , Osuka Atsuhiro
Published: September 2006

meso‐Heptakis(pentafluorophenyl) heptaphyrin(1.1.1.1.1.1.1) (1) was prepared by a stepwise route in 39 % yield and its unique N‐fusion reaction (NFR) sequence has been revealed; this reaction leads to singly‐, doubly‐, and quadruply N‐fused heptaphyrins (4, 5, and 6) in good yields. These transformations are facilitated by the inherent conformational distortion of 1 as well as the distorted, folded conformations of N‐fused heptaphyrins 4 and 5. The proximate arrangement of the three pyrrole units in 6 allowed for the formation of the tripyrrolylboron(III) complexes 7, 8, and 9 with unique coordination features. Molecules 1, 5, and 9 were structurally characterized by X‐ray crystallography. In addition, the boron complexes 7, 8, and 9 displayed weak but distinct fluorescence in the near infrared region.
Luminescent PtII(bipyridyl)(diacetylide) Chromophores with Pendant Binding Sites as Energy Donors for Sensitised Near‐Infrared Emission from Lanthanides: Structures and Photophysics of PtII/LnIII Assemblies
Authors: Ronson Tanya K. , Lazarides Theodore , Adams Harry , Pope Simon J. A. , Sykes Daniel , Faulkner Stephen , Coles Simon J. , Hursthouse Michael B. , Clegg William , Harrington Ross W. , Ward Michael D.
Published: September 2006

The complexes [Pt(bipy){CC‐(4‐pyridyl)}2] (1) and [Pt(tBu2bipy){CC‐(4‐pyridyl)}2] (2) and [Pt(tBu2‐bipy)(CC‐phen)2] (3) all contain a Pt(bipy)(diacetylide) core with pendant 4‐pyridyl (1 and 2) or phenanthroline (3) units which can be coordinated to {Ln(diketonate)3} fragments (Ln = a lanthanide) to make covalently‐linked PtII/LnIII polynuclear assemblies in which the PtII chromophore, absorbing in the visible region, can be used to sensitise near‐infrared luminescence from the LnIII centres. For 1 and 2 one‐dimensional coordination polymers [1⋅Ln(tta)3]∞ and [2⋅Ln(hfac)3]∞ are formed, whereas 3 forms trinuclear adducts [3⋅{Ln(hfac)3}2] (tta=anion of thenoyl‐trifluoroacetone; hfac=anion of hexafluoroacetylacetone). Complexes 1–3 show typical PtII‐based 3MLCT luminescence in solution at ≈510 nm, but in the coordination polymers [1⋅Ln(tta)3]∞ and [2⋅Ln(hfac)3]∞ the presence of stacked pairs of PtII units with short Pt⋅⋅⋅Pt distances means that the chromophores have 3MMLCT character and emit at lower energy (≈630 nm). Photophysical studies in solution and in the solid state show that the 3MMLCT luminescence in [1⋅Ln(tta)3]∞ and [2⋅Ln(hfac)3]∞ in the solid state, and the 3MLCT emission of [3⋅{Ln(hfac)3}2] in solution and the solid state, is quenched by Pt→Ln energy transfer when the lanthanide has low‐energy f–f excited states which can act as energy acceptors (Ln=Yb, Nd, Er, Pr). This results in sensitised near‐infrared luminescence from the LnIII units. The extent of quenching of the PtII‐based emission, and the Pt→Ln energy‐transfer rates, can vary over a wide range according to how effective each LnIII ion is at acting as an energy acceptor, with YbIII usually providing the least quenching (slowest Pt→Ln energy transfer) and either NdIII or ErIII providing the most (fastest Pt→Ln energy transfer) according to which one has the best overlap of its f–f absorption manifold with the PtII‐based luminescence.
In Silico ADMET Traffic Lights as a Tool for the Prioritization of HTS Hits
Authors: Lobell Mario , Hendrix Martin , Hinzen Berthold , Keldenich Jörg , Meier Heinrich , Schmeck Carsten , Schohe‐Loop Rudolf , Wunberg Tobias , Hillisch Alexander
Published: September 2006

The need for in silico characterization of HTS hit structures as part of a data‐driven hit‐selection process is demonstrated. A solution is described in the form of an in silico ADMET traffic light and PhysChem scoring system. This has been extensively validated with in‐house data at Bayer, published data, and a collection of launched small‐molecule oral drugs.
A new type of susceptibility‐artefact‐based magnetic resonance angiography: intra‐arterial injection of superparamagnetic iron oxide particles (SPIO) A Resovist® in combination with TrueFisp imaging: a feasibility study
Authors: Maes Robbert M. , Lewin Jonathan S. , Duerk Jeffrey L. , Misselwitz Bernd , Kiewiet Cunera J. M. , Wacker Frank K.
Published: September 2006

The goal of this study was to evaluate the use of super paramagnetic particles of iron oxide (SPIO) as a dark blood contrast agent, in combination with a bright blood steady‐state free precession sequence for magnetic resonance angiography (MRA), in an animal model. The original concentration of the SPIO of 500 mmol Fe/l and dilutions to 250, 125, 60, 30, 10 and 5 mmol Fe/l were intra‐arterially injected into the aorta of a pig. Then the dilution of 10 mmol Fe/l was chosen for repeated intra‐arterial injections into two pigs. During these intra‐arterial SPIO injections MR images were acquired with a 1.5 T scanner. Signal intensity measurements were performed in the aorta. The signal‐to‐noise ratio during SPIO bolus passage was significantly less than during baseline conditions (Fisher's F‐ratio 159.8, p < 0.005) or the recovery signal‐to‐noise ratio (Fisher's F‐ratio 144.6, p < 0.005). Also, confirmation of flow distal to the catheter‐tip position was possible. The use of SPIO as a dark blood agent in combination with a bright blood MR imaging sequence is feasible. Temporary loss of intraluminal signal occurs due to local decrease of the signal because of induction of local inhomogeneities after mixture the present blood and SPIO solution. It provides immediate information about blood flow distal to the catheter and is a potentially useful to guide intravascular MR‐interventional procedures. Copyright © 2006 John Wiley & Sons Ltd.
An HPLC/mass spectrometry platform for the development of multimodality contrast agents and targeted therapeutics: prostate‐specific membrane antigen small molecule derivatives
Authors: Humblet Valerie , Misra Preeti , Frangioni John V.
Published: September 2006

The production of disease‐targeted agents requires the covalent conjugation of a targeting molecule with a contrast agent or therapeutic, followed by purification of the product to homogeneity. Typical targeting molecules, such as small molecules and peptides, often have high charge‐to‐mass ratios and/or hydrophobicity. Contrast agents and therapeutics themselves are also diverse, and include lanthanide chelates for MRI, 99mTc chelates for SPECT, 90Y chelates for radiotherapy, 18F derivatives for PET, and heptamethine indocyanines for near‐infrared fluorescent optical imaging. We have constructed a general‐purpose HPLC/mass spectrometry platform capable of purifying virtually any targeted agent for any modality. The analytical sub‐system is composed of a single dual‐head pump that directs mobile phase to either a hot cell for the purification of radioactive agents or to an ES‐TOF MS for the purification of nonradioactive agents. Nonradioactive agents are also monitored during purification by ELSD, absorbance and fluorescence. The preparative sub‐system is composed of columns and procedures that permit rapid scaling from the analytical system. To demonstrate the platform's utility, we describe the preparation of five small molecule derivatives specific for prostate‐specific membrane antigen (PSMA): a gadolinium derivative for MRI, indium, rhenium and technetium derivatives for SPECT, and an yttrium derivative for radiotherapy. All five compounds are derived from a highly anionic targeting ligand engineered to have a single nucleophile for N‐hydroxysuccinimide‐based conjugation. We also describe optimized column/mobile phase combinations and mass spectrometry settings for each class of agent, and discuss strategies for purifying molecules with extreme charge and/or hydrophobicity. Taken together, our study should expedite the development of disease‐targeted, multimodality diagnostic and therapeutic agents. Copyright © 2006 John Wiley & Sons, Ltd.
Comparison of a brush‐sampling fecal immunochemical test for hemoglobin with a sensitive guaiac‐based fecal occult blood test in detection of colorectal neoplasia
Authors: Smith Alicia , Young Graeme P. , Cole Stephen R , Bampton Peter
Published: September 2006

BACKGROUND.

Fecal immunochemical tests (FIT) are an advanced fecal occult blood test (FOBT) technology that reduces barriers to population screening by simplifying the logistics of stool‐sampling. The current study was conducted to undertake a paired comparison of a sensitive guaiac FOBT (GFOBT; Hemoccult II Sensa, Beckman Coulter, Fullerton, CA) with a brush‐sampling FIT (InSure; Enterix, North Ryde, NSW, Australia), to determine whether this FIT improves detection of significant neoplasia.

METHODS.

Individuals sampled consecutive stools, at home, with both FIT and GFOBT sampling devices while following dietary restrictions appropriate for GFOBT. Study populations included a screening cohort (n = 2351) and a symptomatic diagnostic group (n = 161). Paired comparison of positivity rates was undertaken in those found to have cancer and/or significant adenoma (high‐grade dysplasia, villous change, ≥10 mm, serrated histology or ≥3 polyps), benign pathology, or no pathology.

RESULTS.

Combined results for both cohorts showed that the FIT returned a true‐positive result significantly more often in cancer (n = 24; 87.5% vs. 54.2%) and in significant adenomas (n = 61; 42.6% vs. 23.0%). Of all UICC Stage I cancers, the FIT was positive in 12 of 13 compared with 4 of 13 with the GFOBT (P = .002). In analyses of just the screening cohort, the FIT remained significantly better at detecting cancers and significant adenomas; the false‐positive rate for any neoplasia was marginally higher with the FIT than the GFOBT (3.4% vs. 2.5%; 95% CI of difference, 0–1.8%), whereas positive predictive values were 41.9% and 40.4%, respectively.

CONCLUSIONS.

This brush‐sampling FIT is more sensitive for cancers and significant adenomas than a sensitive GFOBT. As such, it should deliver greater reductions in colorectal cancer mortality and incidence than the GFOBT. Cancer 2006; © 2006 American Cancer Society.
JAK2 (V617F) as an acquired somatic mutation and a secondary genetic event associated with disease progression in familial myeloproliferative disorders
Authors: Rumi Elisa , Passamonti Francesco , Pietra Daniela , Porta Matteo G. Della , Arcaini Luca , Boggi Sabrina , Elena Chiara , Boveri Emanuela , Pascutto Cristiana , Lazzarino Mario , Cazzola Mario
Published: September 2006

BACKGROUND.

A somatic gain‐of‐function mutation of the Janus kinase 2 (JAK2) gene has been identified in chronic myeloproliferative disorders, which appear to have a sporadic occurrence in most individuals. The authors studied the biologic significance of the JAK2 (V617F) mutation in familial myeloproliferative disorders.

METHODS.

Twenty pedigrees with familial chronic myeloproliferative disorders were identified through an investigation of family history in 264 patients with sporadic myeloproliferative disorders. A quantitative real‐time polymerase chain reaction (qRT‐PCR)‐based allelic discrimination assay was employed for the detection of the V617F mutation in circulating granulocytes and T lymphocytes. An analysis of X‐chromosome inactivation pattern was performed in female patients.

RESULTS.

Fourteen families had homogeneous phenotypes, and 6 families had mixed phenotypes. By using a qRT‐PCR‐based allelic discrimination assay, the JAK2 (V617F) mutation was detected in circulating granulocytes from 20 of 31 patients, but the mutation was not detected in T lymphocytes. Granulocyte mutant alleles ranged from 2.1% to 91.5% and, on average, increased with time. Discordant distribution of the JAK2 (V617F) mutation was observed in siblings with polycythemia vera. The proportion of granulocytes that carried the JAK2 (V617F) mutation was lower than the proportion of clonal granulocytes, as determined in an analysis of X‐chromosome inactivation patterns in female patients.

CONCLUSIONS.

The current findings indicated that the JAK2 (V617F) mutation represents an acquired somatic mutation in patients with familial chronic myeloproliferative disorders and probably occurs as a secondary genetic event in the background of preexisting clonal hematopoiesis. Thus, a genetic predisposition to acquisition of JAK2 (V617F) is inherited in families with myeloproliferative disorders. Cancer 2006. © 2006 American Cancer Society.
Plasticity of synaptopodin and the spine apparatus organelle in the rat fascia dentata following entorhinal cortex lesion
Authors: Deller Thomas , Bas Orth Carlos , Vlachos Andreas , Merten Tobias , Del Turco Domenico , Dehn Doris , Mundel Peter , Frotscher Michael
Published: September 2006

Synaptopodin is an actin‐associated molecule essential for the formation of a spine apparatus in telencephalic spines. To study whether synaptopodin and the spine apparatus organelle are regulated under conditions of lesion‐induced plasticity, synaptopodin and the spine apparatus were analyzed in granule cells of the rat fascia dentata following entorhinal denervation. Confocal microscopy was employed to quantify layer‐specific changes in synaptopodin‐immunoreactive puncta densities. Electron microscopy was used to quantify layer‐specific changes in spine apparatus organelles. Within the denervated middle and outer molecular layers, the layers of deafferentation‐induced spine loss, synaptogenesis, and spinogenesis, the density of synaptopodin puncta and the number of spine apparatuses decreased by 4 days postlesion and slowly recovered in parallel with spinogenesis by 180 days postlesion. Within the nondenervated inner molecular layer, the zone without deafferentation‐induced spine loss, a rapid loss of synaptopodin puncta and spine apparatuses was also observed. In this layer, spine apparatus densities recovered by 14 days postlesion, in parallel with plastic remodeling at the synaptic level and the postlesional recovery of granule cell activity. These data demonstrate layer‐specific changes in the distribution of synaptopodin and the spine apparatus organelle following partial denervation of granule cells: in the layer of spine loss, spine apparatus densities follow spine densities; in the layer of spine maintenance, however, spine apparatus densities appear to be regulated by other signals. J. Comp. Neurol. 499:471–484, 2006. © 2006 Wiley‐Liss, Inc.
Structural isoforms of the circadian neuropeptide PDF expressed in the optic lobes of the cricket Gryllus bimaculatus: Immunocytochemical evidence from specific monoclonal antibodies
Authors: Honda Takeshi , Matsushima Ayami , Sumida Kazunori , Chuman Yoshiro , Sakaguchi Kazuyasu , Onoue Hitoshi , Meinertzhagen Ian A. , Shimohigashi Yasuyuki , Shimohigashi Miki
Published: September 2006

Pigment‐dispersing factor (PDF) is an 18‐mer peptide that acts as a principal neurotransmitter of the insect circadian clock. Our previous study, utilizing anti‐Uca β‐PDH polyclonal antibody (pAb) to immunolabel the optic lobe of the cricket Gryllus bimaculatus, suggested the existence of an alternative PDF‐like peptide in the outer cells of the first neuropile, or lamina (La), which were much less immunoreactive than the inner cells of the second neuropile, the medulla (Me). To obtain structural information about such a PDF‐like peptide, we prepared 10 anti‐Gryllus PDF monoclonal (mAb) and pAb antibodies and analyzed their detailed epitope specificities. The PDFMe and PDFLa inner cells and their axonal projections were clearly immunoreactive to all these antibodies, revealing the widespread immunocytochemical organization of the PDF system in the optic lobe, as seen previously with anti‐Uca β‐PDH pAb and anti‐Gryllus PDF mAb, the epitope structures of which were also clarified in this study. The lamina outer cells, which we found lacked a target pdf mRNA, displayed specific immunoreactivities, indicating that the cells contain a distinct PDF‐like peptide possessing both N‐ and C‐terminal structures. These cells were not immunolabeled by some other monoclonal antibodies, however, implying that the PDFLa outer cells have a PDF isoform peptide devoid of Asn at positions 6 and 16. This isoform was also identified in a varicose arborization in the lamina. These results suggest not only the structure of the peptide, but also the possibility of additional functions of this novel PDF isoform. J. Comp. Neurol. 499:404–421, 2006. © 2006 Wiley‐Liss, Inc.
Distinct and gradient distributions of connexin26 and connexin30 in the cochlear sensory epithelium of guinea pigs
Authors: Zhao Hong‐Bo , Yu Ning
Published: September 2006

Connexin26 (Cx26) and Cx30 are predominant isoforms of gap junction channels in the cochlea and play a critical role in hearing. In this study, the cellular distributions of Cx26 and Cx30 in the cochlear sensory epithelium of guinea pigs were examined by immunofluorescent staining and confocal microscopy in whole mounts of the cochlear sensory epithelium and dissociated cell preparations. The expression of Cx26 and Cx30 demonstrated a longitudinal gradient distribution in the epithelium and was reduced threefold from the cochlear apex to base. The reduction was more pronounced in the Deiters cells and pillar cells than in the Hensen cells. Cx26 was expressed in all types of supporting cells, but little Cx30 labeling was seen in the Hensen cells. Cx26 expression in the Hensen cells was concentrated mainly in the second and third rows, forming a distinct band along the sensory epithelium at its outer region. In the dissociated Deiters cells and pillar cells, Cx30 showed dense labeling at the cell bodies and processes in the reticular lamina. Cx26 labeling largely overlapped that of Cx30 in these regions. Cx26 and Cx30 were also coexpressed in the gap junctional plaques between Claudius cells. Neither Cx26 nor Cx30 labeling was seen in the hair cells and spiral ganglion neurons. These observations demonstrate that Cx26 and Cx30 have a longitudinal gradient distribution and distinct cellular expression in the auditory sensory epithelium. This further supports our previous reports that Cx26 and Cx30 can solely and concertedly perform different functions in the cochlea. J. Comp. Neurol. 499:506–518, 2006. © 2006 Wiley‐Liss, Inc.
Immunocytochemical analysis of GABA‐positive and calretinin‐positive horizontal cells in the tiger salamander retina
Authors: Zhang Jian , Zhang Ai‐Jun , Wu Samuel M.
Published: September 2006

By using immunocytochemical techniques, we demonstrate that there are two distinct, nonoverlapping populations of horizontal cells (HCs) in the tiger salamander retina: GABA‐positive cells account for about 72% and GABA‐negative (calretinin‐positive) cells account for 28% of the total HC somas. The calretinin‐positive HCs have relatively sparse and thick dendrites: soma diameter of 19.72 ± 0.29 μm, and soma density of 140 ± 13 cells/mm2, morphological features very much like the A‐type HCs described in the accompanying article. The GABA‐positive HCs have thinner dendritic and coarse axon‐terminal‐like processes of higher density: soma diameter of 18 ± 0.18 μm, and soma density of 364 ± 18 cells/mm2, features that very much resemble the B‐type HCs and B‐type HC axon terminals in the accompanying article. By using double and triple immunostaining techniques we found that only 18% of the non‐GABAergic HC dendritic clusters contact rods, whereas the remaining 82% of the dendritic clusters contact cones. This is consistent with the physiological finding in the accompanying article that the A‐type HCs are cone‐dominated. On the other hand, 32% of GABAergic HC dendrites contact rod pedicles and 68% contact cone pedicles, consistent with the physiological finding that B‐type HCs and B‐type HC axon terminals receive mixed rod/cone inputs. Detailed confocal microscope analysis shows that 4% rods, 6% principal double cones/single cones, and 100% accessory double cones contact calretinin‐positive HCs, and 79% rods, 100% principal double cones, 14% accessory double cones, and 82% single cones contact GABAergic HCs. These results suggest that GABAergic and non‐GABAergic HC input/output synapses differ and they may mediate different functional pathways in the outer retina. J. Comp. Neurol. 499:432–441, 2006. © 2006 Wiley‐Liss, Inc.
Flow cytometry for assessment of the efficacy of siRNA
Authors: Ho Hung‐Yao , Cheng Mei‐Ling , Wang Yi‐Hui , Chiu Daniel Tsun‐Yee
Published: September 2006

Background:

Small interfering RNA (siRNA) has emerged as a powerful tool to study the loss‐of‐function phenotype by specifically silencing a target gene. The success of gene silencing depends on the choice of appropriate target sequence, and requires a rapid and sensitive assay for quantifying siRNA efficiency. Conventional assays include Western blotting and reverse transcription coupled with polymerase chain reaction. However, these methods are somewhat inaccurate owing to the variation in transfection efficiency. To avoid this, we have developed a flow cytometric method to provide a quantitative analysis of siRNA efficacy.

Methods:

We constructed a novel vector pHyper1G, which can express enhanced green fluorescent protein (EGFP). It contains a H1 promoter, which can drive expression of short hairpin RNA (shRNA) directed against a target gene. The target gene was cloned into pDsRed1‐N1. The resulting construct can express a fusion protein between target protein and DsRed. These vectors were co‐transfected into 293T cells. The transfected cells were analyzed by flow cytometry. The percentage of EGFP+, DsRed+cells and the change in mean fluorescence intensity (MFI) of DsRed channel indicate changes in expression of target gene in a cell population, and hence the efficacy of the corresponding shRNA. In addition, the cells transfected with pHyper1G derivative were sorted, and analyzed for the activity of target gene.

Results:

We designed an oligonucleotide duplex encoding shRNA against glucose‐6‐phosphate dehydrogenase (G6PD) gene, and cloned this into pHyper1G. The resulting vector pHyper1G101 effectively knocked down the expression of G6PD‐DsRed from pDsRed301, as shown by significant reduction in both the percentage of EGFP+, DsRed+cells, and MFI. Changes in these parameters were consistent with decreases in protein level and activity of G6PD. Moreover, albeit at low suboptimal transfection efficiency, cells transfected with pHyper1G101 alone were successfully sorted for those expressing shRNA, and their G6PD activity was found to be suppressed.

Conclusion:

Flow cytometric analysis provides a reliable assessment of the efficiency of siRNA in a cell population. This method can be easily automated and used for screening of appropriate shRNAs against certain genes. Moreover, using the present system, we can deliberately sort for and analyze those cells expressing shRNA. This can be applied to the hard‐to‐transfect cell types, and greatly facilitates the analyses of gene silencing in these cells. © 2006 International Society for Analytical Cytology
High‐throughput fluorescent screening of transgenic animals: Phenotyping and haplotyping
Authors: Hellrung Daniel J. , Rossi Gabriela , Link Charles J.
Published: September 2006

Background:

Methods for genotyping transgenic animals currently consist of extracting genomic DNA from blood or tissue followed by PCR or Southern blot analysis. These methods when used to screen large numbers of animals can be time consuming and expensive. Therefore, we developed a novel method that allows high‐throughput screening of phenotypic changes on leukocytes, resulting from the transgenic genotype. This technique allows investigators to quickly screen a large number of animals without the need to extract DNA from each one. Moreover, since blood is collected for the initial screening, putative homozygotes can be confirmed by conventional methods using the same blood samples.

Methods:

We collected blood from wild‐type αgal positive and αgal knockout mice and probed for the presence of Galα(1→3)Gal (αgal) epitopes. Also, alloantigen specific antibodies were used to determine the haplotype of our outbred mouse colony in order to develop an inbred line.

Results:

αgal epitopes were detected in wild‐type but not αgal knock‐out samples. To validate these results, PCR was used to demonstrate the native αgal gene in wild‐type and the pGKneo construct in αgal knock‐out mice. Furthermore, haplotypes were determined and mice divided for backcrosses.

Conclusions:

This screening method is useful for both preliminary screening of transgenic mice and the development of an inbred mouse colony by rapid determination of MHC I haplotype. Here, we demonstrate the use of this technique and show how it can be a valuable tool, saving time and resources in both investigator effort and animal husbandry. © 2006 International Society for Analytical Cytology
Flow cytometric method for the assessment of the minimal inhibitory concentrations of antibacterial agents to Mycoplasma agalactiae
Authors: Assunção Patrícia , Antunes Nuno T. , Rosales Ruben S. , de la Fe Christian , Poveda Carlos , Poveda José B. , Davey Hazel M.
Published: September 2006

In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MIC) of seven antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, oxytetracycline, and tylosin) on Mycoplasma (M.) agalactiae. Flow cytometry was able to detect M. agalactiae inhibition from 6 h postincubation, although it seems that definitive MIC values determined by flow cytometry were only possible at 12‐h postincubation. However, the results obtained by the traditional method were only obtained at 24 h, when a visible change in the medium had occurred. At 24 h, both methods gave the same result for six antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, and oxytetracycline); whereas flow cytometry gave slightly higher MIC for tylosin. This was attributed to the fact that the M. agalactiae growth that had occurred in the tubes containing tylosin was not enough to visibly change the color of the medium. Futhermore, flow cytometry detected that inhibitory concentrations of oxytetracycline, chloramphenicol, and tylosin as judged at 24 h were not able to inhibit the M. agalactiae growth after 48 h. MIC values of enrofloxacin and ciprofloxacin were sufficient only to maintain the total counts per milliliter throughout the time matched samples, whereas higher concentrations of theses antibacterial agents reduced the total counts per milliliter over the course of the experiment. The main advantage of the flow cytometric method is that MIC results for M. agalactiae can be obtained in a shorter time than is possible with the traditional method. The method presented makes identification of resistant populations of M. agalactiae possible and, unlike the traditional method, allows the effect of each antibacterial agent to be determined in real‐time at the single‐cell level. © 2006 International Society for Analytical Cytology
Single and three‐color flow cytometry assay for intracellular zinc ion availability in human lymphocytes with Zinpyr‐1 and double immunofluorescence: Relationship with metallothioneins
Authors: Malavolta Marco , Costarelli Laura , Giacconi Robertina , Muti Elisa , Bernardini Gianni , Tesei Silvia , Cipriano Catia , Mocchegiani Eugenio
Published: September 2006

Background:

The amount of available intracellular zinc is pivotal to regulate many cellular processes, including oxidative stress response and apoptotic mechanisms. Therefore it is not surprising that zinc homeostasis and dyshomeostasis is involved in many physiological and pathological states, respectively. Cell permeable zinc probes allow intracellular applications with microscopy technology, but flow cytometry (FC) applications have been scarcely explored, albeit they can be suited to study zinc homeostasis in different cell types, including rare cells.

Methods:

We describe a FC method able to estimate intracellular zinc ion availability and the intracellular capability to activate a zinc signal after treatment with an NO‐donor (AcOM‐DEA/NO) in human PBMCs, using the fluorescent zinc‐specific probe, Zinpyr‐1 (ZP1), alone or in association with CD4‐PE and CD8‐Cychrome mAb.

Results:

This method was able to detect an increase/decrease of intracellular zinc available in human fresh cultured PBMC and in immune subsets using AcOM‐DEA/NO or TPEN, respectively. ZP1 mean fluorescence on gated histograms was sensitive to the amount of zinc added in the culture medium and significantly correlated to metallothioneins and total intracellular zinc.

Conclusions:

FC applications using ZP1 may be a fast and useful tool to study zinc homeostasis in immune cells. © 2006 International Society for Analytical Cytology
Dynamic quantification of the tyrosine phosphorylation of the sperm surface proteins during capacitation
Authors: Piehler Erik , Petrunkina Anna M. , Ekhlasi‐Hundrieser Mahnaz , Töpfer‐Petersen Edda
Published: September 2006

Background:

Spermatozoa acquire active fertilizing competence only after deposition in the female tract and subsequent capacitation. Recent studies on the cellular location of major sperm phosphoproteins suggest that capacitation is associated with tyrosine phosphorylation of proteins exposed on the sperm surface. However, these changes have not yet been quantified objectively. A calcium influx seems to be required for the completion of tyrosine phosphorylation in some species; however, the exact temporal coordination between these processes is still poorly understood.

Methods:

Flow cytometry was used to quantify the degree of phosphorylation of the sperm surface proteins by probing with fluorescein isothiocyanate‐conjugated anti‐phosphotyrosine (pY) antibody raised in mouse. Dynamic changes in other sperm parameters (calcium influx, membrane integrity, and spontaneous acrosome reaction) were assessed to analyze their temporal coordination.

Results:

The changes in specific phosphotyrosine (pY) fluorescence signal detected in live, nonpermeabilized boar cell suspensions were biphasic during incubation under capacitating conditions. After 120 min of incubation, the degree of pY fluorescence increased threefold, indicating the changes in proteins exposed on sperm surface. At the same time there was a gradual increase in cytosolic calcium ion levels with the maximal rate at 60 min of incubation. This rate slowed immediately before the onset of the massive rise in tyrosine phosphorylation and decreased by 90% after its completion. The integrity of plasma and acrosome membranes decreased only slowly, illustrating that the changes observed were not due to the process of spontaneous acrosome reaction.

Conclusions:

These data provide quantitative evidence for the appearance of tyrosine‐phosphorylated proteins on the surface of live boar spermatozoa during capacitation. An exact temporal coordination exists between cytosolic calcium ion content and protein tyrosine phosphorylation under these conditions. This novel approach has the advantage of making possible a precise quantification and kinetic comparison of molecular processes in different cell subpopulations. © 2006 International Society for Analytical Cytology
Ad hocnetwork routing and security: A review
Authors: Zhang Congzhe , Zhou MengChu , Yu Ming
Published: September 2006

Ad hocnetwork is a collection of wireless mobile nodes dynamically forming a local area network without the use of any existing network infrastructure or centralized administration. One problem in the design of such network is its wireless vulnerabilities and limited physical security. This paper considers the security attributes and goals ofad hocnetwork, reviews the latest developments of current routing and security mechanissm including encryption, protocols, user authentication, and physical security. It also explores the new approaches trying to solve current problems and points out the future research directions. These methods will be helpful in the military and other security‐sensitive fields. Copyright © 2006 John Wiley & Sons, Ltd.
New Copper(II) Complexes as Efficient Catalysts for Olefin Aziridination: The Effect of Ligand Steric Hindrance on Reactivity
Authors: Dhanalakshmi Thirumanasekaran , Suresh Eringathodi , Stoeckli‐Evans Helen , Palaniandavar Mallayan
Published: September 2006

Three new copper(II) complexes derived from the tridentate N3 ligands 4‐methyl‐1‐(pyrid‐2‐ylmethyl)‐1,4‐diazacycloheptane (L1), 1‐(quinol‐2‐ylmethyl)‐1,4‐diazacycloheptane (L2), and 4‐methyl‐1‐(quinol‐2‐ylmethyl)‐1,4‐diazacycloheptane (L3) have been prepared and examined as copper catalysts for olefin aziridination. In the X‐ray crystal structures of the complexes [Cu(L2)Cl2] (2) and [Cu(L3)Cl2] (3) copper(II) adopts a trigonal‐bipyramidal distorted square‐based pyramidal geometry (TBDSBP) as seen from the values of the trigonality index τ (0.08 for 2 and 0.48 for 3). The enhanced trigonal distortion in 3 is due to the presence of an N‐Me group, the lone pair orbital of which is not oriented exactly along the d orbital of copper(II). While [Cu(L1)(H2O)](ClO4)2 (1) assumes a tetragonal geometry in solution, complexes 2 and 3 adopt a distorted tetragonal geometry, as revealed by UV/Vis and EPR spectral studies. The complexes undergo quasi‐reversible CuII/CuI redox behavior in methanol solution. The ability of the complexes to mediate nitrene transfer from PhINTs to olefins to form N‐tosylaziridines has been studied. The complexes are found to be efficient catalysts (in 5 mol‐% amounts) for the aziridination of the reactive olefin styrene, with yields varying from 80 to 90 % (with respect to PhINTs). They exhibit significant catalytic nitrene transfer reactivity (yields of 30 to 60 %) also towards the less reactive olefins cyclohexene and cyclooctene. A remarkable observation is the significantly accelerated rate of aziridination by 3, which is ascribed to the steric crowding around copper(II) imposed by the bulky quinolyl and N‐Me groups of the tridentate ligand L3.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)
A High‐Temperature Multinuclear NMR Study of Na3AlF6–FeO andNa3AlF6–Fe2O3 Melts
Authors: Šimko František , Rakhmatullin Aydar , Boča Miroslav , Daněk Vladimír , Bessada Catherine
Published: September 2006

In the Hall–Héroult process for the industrial production of aluminum, iron oxide impurities are known to lower the current efficiency as well as the metal quality. Iron can be present in the di‐ or trivalent form. The nature of dissolved FeIII and FeII species and the reactions taking place in cryolitic melts have been investigated by high‐temperature NMR spectroscopy. The evolution of 27Al, 23Na, and 19F NMR chemical shifts are reported in the Na3AlF6–FeO and Na3AlF6–Fe2O3 systems for different iron oxide contents. They express the formation of oxofluoroaluminate species. For the Na3AlF6–FeO system, the 19F signal appears only after 14 minutes at 1020 °C. The line position is shifted with heating time. This evolution is associated with a narrowing of the 27Al signal due to a decrease in the content of paramagnetic compounds in the sample. This effect is probably caused by the evaporation of an FeII compound, as already mentioned in the literature. For the Na3AlF6–Fe2O3 system we have never observed the 19F NMR spectra over the full heating time (ca. 30 min). This could be due to the influence of paramagnetic FeIII nuclei, probably in FeF63– species present in the melt. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)
SnCl4‐Mediated Reactions of Cyclopropyl Alkyl Ketones with α‐Keto Esters
Authors: Yang Yong‐Hua , Shi Min
Published: September 2006

This paper reports the full details of Lewis acid SnCl4‐mediated reactions of cyclopropyl alkyl ketones with α‐keto esters under mild conditions. In general, it was found that four products were formed in the SnCl4‐mediated reactions of cyclopropyl alkyl ketones. However, by controlling the aldol reaction process, the corresponding spiro‐γ‐lactone products can be exclusively obtained in moderate to good yields with good stereoselectivities via the sequential nucleophilic ring‐opening reaction of cyclopropane by H2O, aldol type reaction and subsequent cyclic transesterification mediated by the Lewis acid. The mechanism was further confirmed by introducing a substituent at the other α‐position of the cyclopropyl alkyl ketone to produce two or three other products without the formation of spiro‐γ‐lactone products under the same reaction conditions. In summary, SnCl4‐mediated reactions of cyclopropyl alkyl ketones with α‐keto esters afforded a new general method for the synthesis of spiro‐γ‐lactones in moderate to good yields with good stereoselectivities. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)
Simultaneous separation of nitrofuran antibiotics and their metabolites by using micellar electrokinetic capillary chromatography
Authors: Wickramanayake Priyanga U. , Tran Tin C. , Hughes Jeff G. , Macka Mirek , Simpson Nigel , Marriott Philip J.
Published: September 2006

Mixtures comprising nitrofuran antibiotics (NFA) and nitrofuran metabolites (NFM) were resolved for the first time by using MEKC. Sodium deoxycholate (SDC) was chosen as the micelle‐forming surfactant. Optimization of separation conditions was achieved by using a central composite experimental design (CCD) approach. Experimental parameters such as concentration ratio of borate to phosphate in the buffer, pH of the running electrolyte and voltage were investigated. The effect of concentration of the surfactant on resolution was significant. Under optimal conditions of 80 mM SDC, pH 9.0, (20 mM borate + 20 mM phosphate) and 16 kV, the resolution between eight consecutive peak pairs ranged from 1.9 to 11.8. Due to the absence of a UV‐active chromophore in the metabolites, they were derivatized with 2‐nitrobenzaldehyde (2‐NBA). In order to mimic a proposed extraction procedure for the analysis of both NFA and/or derivatized NFM in a sample, aqueous samples (prederivatized with 2‐NBA) were extracted by using C18 SPE cartridges. After washing with H2O, the cartridges were eluted with a small portion of organic solvent with weak elution characteristics to remove excess 2‐NBA (hexane was chosen). Target analytes were then recovered with ACN. Excellent reproducibility of migration time (tmig) was achieved for all analytes using the developed MECC approach, with absolute tmig <1% RSD and tmig ratio <0.2% RSD, and peak area ratio was 4% RSD. The LOD for each compound, calculated by extrapolating to an S/N of 3, were found to be 0.19–2.0 μg/mL.
Application of capillary zone electrophoresis with large‐volume sample stacking to the sensitive determination of sulfonamides in meat and ground water
Authors: Soto‐Chinchilla Jorge J. , García‐Campaña Ana M. , Gámiz‐Gracia Laura , Cruces‐Blanco Carmen
Published: September 2006

A CZE method with UV‐Vis detection has been established and validated for the determination of nine sulfonamides: sulfapyridine, sulfamethazine, sulfamerazine, sulfamether, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfachlorpyridazine, and sulfamethizole. Optimum separation was obtained on a 64.5 cm×75 μm bubble cell capillary using a buffer containing 45 mM sodium phosphate and 10% methanol at pH 7.3, with temperature and voltage of 27°C and 25 kV, respectively. p‐Aminobenzoic acid was used as an internal standard . Taking into account the lack of sensitivity of the UV‐Vis detection, the application of an on‐line preconcentration methodology, such as large‐volume sample stacking with polarity switching has been proposed. This procedure combined with a solvent extraction/SPE method applied for off‐line preconcentration and cleanup provides a significant improvement in the LODs, ranging from 2.59 to 22.95 μg/L for the studied compounds; the quantification of these residues being possible below the levels established by EU legislation in animal food products, such as meat. Satisfactory recoveries were also obtained in the analysis of these compounds in ground water.
Discontinuous native protein gel electrophoresis
Authors: Niepmann Michael , Zheng Junfeng
Published: September 2006

Analysis of the oligomeric state of a native protein usually requires analytical ultracentrifugation or repeated gel filtration to calculate the protein's size. We have developed a discontinuous native protein gel electrophoresis system that allows the separation of even basic proteins according to their size, oligomeric state, and shape. This gel system combines the addition of negative charges to the proteins by Serva Blue G with a discontinuous buffer system and gradient gels. As in SDS‐PAGE, chloride constitutes the high mobility anion in the gel and anode buffer. However, for sample focusing this system employs histidine instead of glycine as the slow dipolar ion following from the cathode buffer to improve migration of basic proteins. In addition, proteins run into gel pores corresponding to their size and shape in the gradient gel. Using this gel system, we show that the polypyrimidine tract‐binding protein (PTB) is a monomer.
Kinetic study of paracetamol on prolidase activity in erythrocytes by capillary electrophoresis with Ru(bpy)32+ electrochemiluminescence detection
Authors: Yuan Jipei , Wei Hui , Jin Wenrui , Yang Xiurong , Wang Erkang
Published: September 2006

We explored the CE with (bpy) 2+3 electrochemiluminescence detection for the kinetic study of drug–enzyme interaction. Effects of four nonsteroidal anti‐inflammatory drugs including aspirin, paracetamol, sodium salicylate and phenacetin on prolidase (PLD) activity in erythrocytes were investigated. Aspirin enhanced PLD activity whereas the other three had inhibiting effects. This may reveal their different effects on the collagen biosynthesis and catabolism that influence tumor invasiveness. Kinetic study of paracetamol on PLD showed that the value of Michaelis constant Km for PLD was 1.23 mM. The mechanism of PLD inhibition by paracetamol is noncompetitive inhibition, and the inhibitor constant Ki value obtained in our research was 9.73×103 μg/L.
A Retrospective lyrical analysis of songs written by adolescents with anorexia nervosa
Authors: McFerran Katrina , Baker Felicity , Patton George C. , Sawyer Susan M.
Published: September 2006

Song‐writing plays a central role in music therapy with adolescents. A retrospective lyrical analysis of songs written by adolescents with anorexia nervosa was undertaken in order to identify common themes solicited through song‐writing interventions. Fifteen participants contributed 17 songs, from which 368 lyrical units were identified. A modified content analysis approach was used with each lyrical unit being categorised to one of six themes determined from the literature and clinical experience. The theme of ‘identity’ was used most frequently (28%), with the sub‐theme of ‘exploring new behaviours, positive self‐talk’ being addressed most often (12.5% of total). The dominance of the developmentally important theme of ‘identity’ may reflect the relationship between adolescents, music and identity or may be related to the way in which anorexia nervosa has been theorised to assist the adolescent in managing challenges to the emerging sense of self. Copyright © 2006 John Wiley & Sons, Ltd and Eating Disorders Association.
Persistent alluvial fanhead trenching resulting from large, infrequent sediment inputs
Authors: Davies Timothy R. H. , Korup Oliver
Published: September 2006

We present laboratory and field evidence that in mountainous catchment‐fan systems persistent alluvial fanhead aggradation and trenching may result from infrequent, large sediment inputs. We suggest that the river‐fan systems along the fault‐bounded range front of the western Southern Alps, New Zealand, are likely to be in a dynamic equilibrium on ≥103‐yr timescales, superimposed on which their fanheads undergo long‐term cumulative episodic aggradation. These fanheads are active only in rare events, do not take part in the usual behaviour of the catchment‐fan system and require much longer to exhibit dynamic equilibrium than the rest of the fan. These findings (1) increase our knowledge of the effects of extreme events on alluvial fan morphodynamics in humid climates, (2) question the general applicability of inferring past climatic or tectonic regimes from alluvial‐fan morphology and stratigraphy and (3) provide a conceptual basis for hazard zonation on alluvial fans. Copyright © 2006 John Wiley & Sons, Ltd.