bluuebunny/crossref_metadata_2025 huggingface dataset:
Motility of a biflagellate sperm: Waveform analysis and cyclic nucleotide activation
Authors: Howard David R. , Trantow Colleen M. , Thaler Catherine D.
Published: August 2004

The sperm of the freshwater clam Corbicula fluminea are unusual in that they have two flagella, both of which are capable of beating. When Corbicula sperm are removed from the gonad and placed into freshwater, most remain immotile. Video microscopy was used to assess signaling molecules capable of activating Corbicula sperm motility. Experiments using the cAMP analogs dbcAMP or 8‐Br‐cAMP show that elevating cAMP activates flagellar motility. Treatments with 8‐Br‐cGMP activated motility in similar numbers of sperm. Treatments with the selective cAMP‐dependent protein kinase (PKA) inhibitor H‐89 block activation by 8‐Br‐cAMP but not by 8‐Br‐cGMP. Similar treatments with the cGMP‐dependent protein kinase (PKG) inhibitor Rp‐8‐pCPT‐cGMPS block activation by 8‐Br‐cGMP but not by 8‐Br‐cAMP. These results suggest that cAMP and cGMP each work through their specific kinase to activate flagellar motility. Analysis of spontaneously activated freely swimming sperm shows that the two flagella beat with different parameters. The A flagellum beats with a shorter wavelength and a higher frequency than the B flagellum. The observed differences in flagellar waveform indicate that the flagella are differentially controlled. Cell Motil. Cytoskeleton 59:120–130, 2004. © 2004 Wiley‐Liss, Inc.
Independent maturation of the GABAB receptor subunits GABAB1 and GABAB2 during postnatal development in rodent brain
Authors: Fritschy Jean‐Marc , Sidler Corinne , Parpan Franziska , Gassmann Martin , Kaupmann Klemens , Bettler Bernhard , Benke Dietmar
Published: August 2004

GABAB receptors mediate slow inhibitory GABAergic neurotransmission. They are encoded by two distinct subunits, GABAB1 (GBR1) and GABAB2 (GBR2), with two major isoforms of GBR1, GBR1a and GBR1b, arising from differential promoter usage. Heterodimerization of GBR1 and GBR2 is essential for GABAB receptor function, as shown in recombinant expression systems and in GBR1−/− mice. GABAB receptors are highly expressed during ontogeny, prior to synaptogenesis, but their developmental function remains elusive. Here we investigated the postnatal development of GABAB receptors in rodent brain, focusing on potential differences in the spatial and temporal expression pattern of GBR1 and GBR2. Immunohistochemistry with subunit‐specific antibodies revealed a widespread staining for GBR1a and GBR2 in neonatal rodent brain. During the first 2 weeks, these two subunits exhibited largely overlapping regional distribution, but with profound distinctions in cellular and subcellular localization. The adult‐like pattern was established during the third week, with a prominent up‐regulation of GBR1b, extensively codistributed with GBR2. Several unexpected features were noted at early stages, notably, a selective GBR2 staining of axonal tracts, such as the corticothalamic projection, and a prominent GBR1 expression in astrocytes. The specificity of the antibody labeling was verified in GBR1‐ and GBR2‐knockout mice. In addition, the analysis of these mutants revealed a partial preservation of GBR2 staining in GBR1−/− mice and vice versa. Altogether, the results suggest a functional role for GBR1 and GBR2 proteins in immature brain in addition to their contribution to dimeric GABAB receptor complexes. J. Comp. Neurol. 477:235–252, 2004. © 2004 Wiley‐Liss, Inc.
Differential distribution of synaptic endings containing glutamate, glycine, and GABA in the rat dorsal cochlear nucleus
Authors: Rubio Maria E.
Published: August 2004

The dorsal cochlear nucleus (DCN) integrates the synaptic information depending on the organization of the excitatory and inhibitory connections. This study provides, qualitatively and quantitatively, analyses of the organization and distribution of excitatory and inhibitory input on projection neurons (fusiform cells), and inhibitory interneurons (vertical and cartwheel cells) in the DCN, using a combination of high‐resolution ultrastructural techniques together with postembedding immunogold labeling. The combination of ultrastructural morphometry together with immunogold labeling enables the identification and quantification of four major synaptic inputs according to their neurotransmitter content. Only one category of synaptic ending was immunoreactive for glutamate and three for glycine and/or γ‐aminobutyric‐acid (GABA). Among those, nine subtypes of synaptic endings were identified. These differed in their ultrastructural characteristics and distribution in the nucleus and on three cell types analyzed. Four of the subtypes were immunoreactive for glutamate and contained round synaptic vesicles, whereas five were immunoreactive for glycine and/or GABA and contained flattened or pleomorphic synaptic vesicles. The analysis of the distribution of the nine synaptic endings on the cell types revealed that eight distributed on fusiform cells, six on vertical cells and five on cartwheel cells. In addition, postembedding immunogold labeling of the glycine receptor α1 subunit showed that it was present at postsynaptic membranes in apposition to synaptic endings containing flattened or pleomorphic synaptic vesicles and immunoreactive for glycine and/or GABA on the three cells analyzed. This information is valuable to our understanding of the response properties of DCN neurons. J. Comp. Neurol. 477:253–272, 2004. © 2004 Wiley‐Liss, Inc.
Patterns of cell–cell coupling in embryonic spinal cord studied via ballistic delivery of gap‐junction‐permeable dyes
Authors: Bittman Kevin S. , Panzer Jessica A. , Balice‐Gordon Rita J.
Published: August 2004

Intercellular communication mediated by gap junctions is developmentally regulated in many tissues, including the nervous system. In rodent lumbar spinal cord, extensive gap junctional coupling among motor neurons innervating the same muscle is present at birth but is no longer present 1 week later. Little is known about how this motor‐pool‐specific coupling arises during embryonic development. To address this question, we developed a novel method of visualizing patterns of cell–cell coupling that can be applied to a wide range of tissues. Gap‐junction‐permeable dyes adsorbed to metal beads were delivered into individual cells in embryonic cerebral cortex or spinal cord using pressure. Dye diffused off of the bead surface into the cytoplasm, crossed gap junctions, and labeled clusters of coupled cells. For embryonic cerebral cortex, this method revealed patterns of cell–cell coupling similar to those reported with other techniques. In embryonic lumbar spinal cord, cell–cell coupling is widespread in the ventricular zone at E11, and the extent of coupling decreases until birth. In the ventral horn, motor neurons are coupled into clusters at E14, with little change in the extent of coupling at E16, and a similar extent of coupling is present at birth. The cell types within clusters, identified by using antibodies against homeodomain transcription factors, were surprisingly heterogeneous in both the ventricular zone and the motor columns. Taken together, these data suggest that the spatial and temporal patterns of cell–cell coupling are dynamic and that cell‐type‐specific gap junctional coupling arises gradually during spinal cord development. J. Comp. Neurol. 480:273–285, 2004. © 2004 Wiley‐Liss, Inc.
Expression and distribution of octopus gonadotropin‐releasing hormone in the central nervous system and peripheral organs of the octopus (Octopus vulgaris) by in situ hybridization and immunohistochemistry
Authors: Iwakoshi‐Ukena Eiko , Ukena Kazuyoshi , Takuwa‐Kuroda Kyoko , Kanda Atshuhiro , Tsutsui Kazuyoshi , Minakata Hiroyuki
Published: August 2004

We recently purified a peptide with structural features similar to vertebrate gonadotropin‐releasing hormone (GnRH) from the brain of Octopus vulgaris, cloned a cDNA encoding the precursor protein, and named it oct‐GnRH. In the current study, we investigated the expression and distribution of oct‐GnRH throughout the central nervous system (CNS) and peripheral organs of Octopus by in situ hybridization on the basis of the cDNA sequence and by immunohistochemistry using a specific antiserum against oct‐GnRH. Oct‐GnRH mRNA‐expressing cell bodies were located in 10 of 19 lobes in the supraesophageal and subesophageal parts of the CNS. Several oct‐GnRH–like immunoreactive fibers were seen in all the neuropils of the CNS lobes. The sites of oct‐GnRH mRNA expression and the mature peptide distribution were consistent with each other as judged by in situ hybridization and immunohistochemistry. In addition, many immunoreactive fibers were distributed in peripheral organs such as the heart, the oviduct, and the oviducal gland. Modulatory effects of oct‐GnRH on the contractions of the heart and the oviduct were demonstrated. The results suggested that, in the context of reproduction, oct‐GnRH is a key peptide in the subpedunculate lobe and/or posterior olfactory lobe–optic gland–gonadal axis, an octopus analogue of the hypothalamo–hypophysial–gonadal axis. It may also act as a modulatory factor in controlling higher brain functions such as feeding, memory, movement, maturation, and autonomic functions. J. Comp. Neurol. 477:310–323, 2004. © 2004 Wiley‐Liss, Inc.
Postnatal development of the cerebellum and the CNS adrenergic system is independent of norepinephrine and epinephrine
Authors: Jin Sung‐Ha , Kim Hyung J.T. , Harris D. Christopher , Thomas Steven A.
Published: August 2004

A fundamental question in the formation of the nervous system is the extent to which a neurotransmitter contributes to the development of the neurons that synthesize and release it. A complementary question is whether neurotransmitter signaling contributes to the development of postsynaptic targets. Prior studies have suggested that adrenergic signaling may promote adrenergic neuronal proliferation or survival and may be critical for the postnatal development of the cerebellum. To test these possibilities genetically, we studied mice that are unable to synthesize norepinephrine and epinephrine (NE/E), the endogenous adrenergic receptor ligands, due to a disruption the gene for dopamine β‐hydroxylase. These mice develop postnatally in the absence of NE/E. Here we report that the adrenergic neurons of these mutant mice are present in normal numbers and locations and exhibit typical innervation patterns throughout the central nervous system (CNS), as assessed by immunostaining for tyrosine hydroxylase and the NE transporter. Furthermore, cerebellar cortical development (size, foliation, layering, cell number, and position), which proceeds to a large degree postnatally, is unaltered in the mutants. These results indicate that the fate and innervation pattern of the adrenergic neurons, as well as the development of the cerebellum, do not depend on postnatal signaling by NE/E. The results also suggest that when restoration of adrenergic signaling is performed in this mutant mouse model (by administering a synthetic precursor of NE), reversal of phenotypes is due to the synthesis and release of NE/E from adrenergic terminals that are distributed normally within the CNS. J. Comp. Neurol. 477:300–309, 2004. © 2004 Wiley‐Liss, Inc.
Mechanical Properties of Single Motor Molecules Studied by Three‐Dimensional Thermal Force Probing in Optical Tweezers
Authors: Jeney Sylvia , Stelzer Ernst H. K. , Grubmüller Helmut , Florin Ernst‐Ludwig
Published: August 2004

A new method combining three‐dimensional (3D) force measurements in an optical trap with the analysis of thermally induced (Brownian) position fluctuations of a trapped probe was used to investigate the mechanical properties of a single molecule, the molecular motor kinesin. One kinesin molecule attached to the probe was bound in a rigorlike state to one microtubule. The optical trap was kept weak to measure the thermal forces acting on the probe, which were mainly counterbalanced by the kinesin tether. The stiffness of kinesin during stretching and compression with respect to its backbone axis were measured. Our results indicate that a section of kinesin close to the motor domain is the dominating element in the flexibility of the motor structure. The experiments demonstrate the power of 3D thermal fluctuation analysis to characterize mechanical properties of individual motor proteins and indicate its usefulness to study single molecule in general.
Electrochemical Deposition of Platinum Nanoparticles on Carbon: A Study by Standard and Anomalous X‐ray Diffraction
Authors: Adora Sébastien , Simon Jean Paul , Soldo‐Olivier Yvonne , Faure René , Chaînet Eric , Durand Robert
Published: August 2004

This paper is devoted to an alternative method to characterize platinum nanoparticles: X‐ray powder diffraction with synchrotron radiation in classical and anomalous dispersion modes. We could straightforwardly determine the mean diameter and the surface concentration of carbon‐supported platinum nanoparticles, even down to diameters of 2–3 nm and catalyst amounts of 0.03 mg cm−2. We could study early stages of the formation of electrochemically prepared platinum nanoparticles from [PtCl4]2− species preadsorbed on carbon inside a carbon–Nafion layer, to obtain a fuel‐cell electrode. Our X‐ray diffraction (XRD) results demonstrate that, provided the superficial concentration is not too high, new and smaller particles appear for each current pulse, since there is not any strong nucleation limitation for the high overvoltages obtained. Hydrogen evolution becomes the main electrochemical phenomenon on particles of sufficient size and it explains the noteworthy size limitation. Better yields of Pt metal are obtained for smaller current densities and longer times: the rate‐determining step is then not electrochemical, but chemical or related to superficial diffusion.
Ethanol Decomposition: CC Cleavage Selectivity on Rh(111)
Authors: Vesselli Erik , Baraldi Alessandro , Comelli Giovanni , Lizzit Silvano , Rosei Renzo
Published: August 2004

Ethanol adsorption, desorption and decomposition on Rh(111) have been studied by X‐ray photoelectron spectroscopy and temperature‐programmed desorption experiments. The evolution of the C 1s and O 1s core level spectra was monitored as a function of ethanol exposure and surface temperature. Ethanol adsorption at 90 K results in two nonequivalent ethanol‐adsorbed species at low surface coverage, while a third species—related to multilayer formation—appears after longer exposures. Upon surface annealing, ethanol undergoes both desorption and dissociation, thus creating intermediate surface species which further decompose to hydrogen, carbon monoxide and atomic carbon. Our results clearly show that CC bond cleavage is the preferential dissociation channel, while CO bond scission is not observed. Calculations performed within the framework of the unity bond index—quadratic exponent potential model, have been used to test and compare different competing dissociation channels, providing an estimate of adsorption energies and dissociation barriers.
Adsorption of Amyloid Beta (1‐40) Peptide to Phosphatidylethanolamine Monolayers
Authors: Maltseva Elena , Brezesinski Gerald
Published: August 2004

The aggregation of soluble, nontoxic amyloid β (Aβ) peptide to β‐sheet containing fibrils is assumed to be a major step in the development of Alzheimer's disease. Interactions of Aβ with neuronal membranes could play a key role in the pathogenesis of the disease. Herein, we study the adsorption of synthetic Aβ peptide to DPPE and DMPE monolayers (dipalmitoyl‐ and dimyristoylphosphatidylethanolamine). Both lipids exhibit a condensed monolayer state at 20 °C and form a similar lattice. However, at low packing densities (at large area per molecule), the length of the acyl chains determines the phase behavior, therefore DPPE is fully condensed whereas DMPE exhibits a liquid‐expanded state with a phase transition at approximately 5–6 mN m−1. Adsorption of Aβ to DPPE and DMPE monolayers at low surface pressure leads to an increase of the surface pressure to approximately 17 mN m−1. The same was observed during adsorption of the peptide to a pure air–water interface. Grazing incidence X‐ray diffraction (GIXD) experiments show no influence of Aβ on the lipid structure. The adsorption kinetics of Aβ to a DMPE monolayer followed by IRRAS (infrared reflection absorption spectroscopy) reveals the phase transition of DMPE molecules from liquid‐expanded to condensed states at the same surface pressure as for DMPE on pure water. These facts indicate no specific interactions of the peptide with either lipid. In addition, no adsorption or penetration of the peptide into the lipid monolayers was observed at surface pressures above 30 mN m−1. IRRAS allows the measurement of the conformation and orientation of the peptide adsorbed to the air–water interface and to a lipid monolayer. In both cases, with lipids at surface pressures below 20 mN m−1 and at the air–water interface, adsorbed Aβ has a β‐sheet conformation and these β‐sheets are oriented parallel to the interface.
The State of the Iron Promoter in Tungstated Zirconia Catalysts
Authors: Carrier Xavier , Lukinskas Povilas , Kuba Stefan , Stievano Lorenzo , Wagner Friedrich E. , Che Michel , Knözinger Helmut
Published: August 2004

The activity and selectivity of tungstated zirconia (WZ) for the conversion of n‐ into isopentane are dramatically enhanced when the catalyst is modified with Pt and Fe. The state of iron in these catalysts was hitherto only poorly characterized. Therefore, in the present work we investigated the structural and electronic properties of iron in WZ catalysts containing 1 wt % Pt and 1 wt % Fe2O3, by a combination of spectroscopic techniques, namely X‐ray absorption spectroscopy (XAS), in situ electron paramagnetic resonance (EPR), and Mössbauer spectroscopy.

In the oxidized catalyst, iron is present as FeIII and predominantly forms a surface solid solution in which the isolated FeIII ions are located in a distorted octahedral environment. A small amount of the total iron (around 10 %) is present in the form of small iron oxide particles. Both iron species can be reduced in H2 and then easily reoxidized on exposure to air at room temperature. We infer that the promoter action of iron in these catalysts is intimately related to its redox properties and specifically affects the dehydrogenation activity of the materials.
The Excited‐State Dynamics of Phycocyanobilin in Dependence on the Excitation Wavelength
Authors: Dietzek Benjamin , Maksimenka Raman , Hermann Gudrun , Kiefer Wolfgang , Popp Jürgen , Schmitt Michael
Published: August 2004

The primary light‐induced processes of phycocyanobilin were studied by means of transient‐grating spectroscopy, whereby the excitation wavelength was varied over the spectral region of the ground‐state absorption. On the basis of the results obtained, both the rate of the photoreaction in phycocyanobilin and the ratio of the decay of different excited‐state species via two decay channels depend on the excitation wavelength. Furthermore, the formation of the photoreaction product is also dependent on the pump color. These data support a recently established model for the primary photoprocesses in phycocyanobilin. In addition, phycocyanobilin protonated at the basic pyrrolenine‐type nitrogen atom was included in the transient absorption study. The decay behavior was found to be almost unchanged when compared with the unprotonated form, and this suggests that protonation of the tetrapyrrole ring structure has no effect on the overall photochemistry.
Noniterative Biexponential Fluorescence Lifetime Imaging in the Investigation of Cellular Metabolism by Means of NAD(P)H Autofluorescence
Authors: Niesner Raluca , Peker Bülent , Schlüsche Peter , Gericke Karl‐Heinz
Published: August 2004

The cofactors NADH and NADPH, hereafter NAD(P)H [NAD(P)=nicotinamide adenine dinucleotide (phosphate)], belong to the principal endogenous indicators of energetic cellular metabolism. Since the metabolic activity of cells is given by the ratio between the concentrations of free and protein‐bound NAD(P)H, the development of autofluorescence techniques which accurately measure the modifications to this ratio is particularly significant. Hitherto the methods applied in the monitoring of cellular metabolism have provided either imprecise results, due to interference of the NAD(P)H signal by perturbing factors, or they have required a complicated internal calibration. We employ biexponential fluorescence lifetime imaging (FLIM) in order to discriminate between the free and protein‐bound NAD(P)H without any previous calibration. Thus, we have obtained directly, and for the first time, a high‐resolution map of cellular metabolism, that is, an image of the contribution of the protein‐bound NAD(P)H to the cumulative NAD(P)H fluorescence signal. Moreover, we demonstrate that protein–NAD(P)H complexes characterised by different fluorescence lifetimes are not uniformly distributed all over the cell, as assumed until now, but are concentrated in certain cellular regions. The different fluorescence lifetimes indicate either different protein–NAD(P)H complexes or different bond strengths between NAD(P)H and the protein in these complexes.

Since an important aspect in biological applications is to monitor the dynamics of the relevant processes (such as cellular metabolism), rapid dynamical techniques, for example, rapid biexponential fluorescence lifetime imaging, are needed. Furthermore, it is necessary to reduce the evaluation effort as much as possible. Most of the evaluation techniques in multiexponential FLIM are time‐expensive iterative methods. The few exceptions are connected with a loss of information, for example, global analysis; or a loss in accuracy, for example, the rapid evaluation technique (RLD). We implement for the first time in FLIM a noniterative, nonrestrictive method originally developed by Prony for approximations of multiexponential decays. The accuracy of this method is verified in biexponential FLIM experiments in time‐domain on mixtures of two chromophores both in homogenous and in heterogeneous media. The resulting fluorescence lifetimes agree (within error margins) with the lifetimes of the pure substances determined in monoexponential FLIM experiments. The rapidity of our evaluation method as compared to iterative pixel‐by‐pixel methods is evidenced by a reduction of the evaluation time by more than one order of magnitude. Furthermore, the applicability of this method for the biosciences is demonstrated in the investigation of cellular metabolism by means of NAD(P)H endogenous fluorescence.
Self‐Assembly and Characterization of Hydrogen‐Bond‐Induced Nanostructure Aggregation
Authors: Liu Yang , Zhuang Junpeng , Liu Huibiao , Li Yuliang , Lu Fushen , Gan Haiyang , Jiu Tonggang , Wang Ning , He Xiaorong , Zhu Daoben
Published: August 2004

A supramolecular system of a perylene derivative containing bis(2,6‐diacylaminopyridine) units and a perylene bisimide bound through three hydrogen‐bonds was synthesized and characterized. 1H NMR spectra confirmed the existence of hydrogen‐bonding interactions between the perylene derivative (3) and the perylene bisimide (7). The photocurrent generation of the self‐assembled 3⋅7 film was measured, and a cathodic photocurrent response was obtained. SEM images indicated that well‐defined long fibers could be fabricated by self‐assembly, by exploiting the hydrogen bonding interactions and π‐π stacking interactions of perylene rings.
Conservation of structure and functional divergence of duplicated Wnt8s in pufferfish
Authors: Ramel Marie‐Christine , Buckles Gerri R. , Lekven Arne C.
Published: August 2004

The zebrafish wnt8 locus differs from its tetrapod counterparts in that it produces two functionally overlapping but distinct Wnt8 proteins. Studies of zebrafish wnt8 have suggested that the two major Wnt8 proteins produced are functionally similar yet may behave differently depending on the assay context. To determine whether the bicistronic wnt8 and its accompanying unique protein activities found in zebrafish are more widespread (and perhaps universal) among teleosts, we have extended our studies to the pufferfish Takifugu rubripes. We have found that Takifugu wnt8 is also bicistronic, indicating that the wnt8 duplication occurred before the divergence of these teleosts approximately 150 million years ago. Furthermore, overexpression assays in zebrafish embryos show that functional differences between the zebrafish Wnt8.1 and Wnt8.2 proteins are conserved in their Takifugu orthologs. Thus, despite the fact that Wnt8.1 and Wnt8.2 proteins are as similar to each other as each is to Xenopus Xwnt‐8, Wnt8 family members can behave quite differently in the context of zebrafish embryos. This finding suggests that zebrafish (and possibly teleost in general) Wnt8 receptors are able to discriminate between highly related ligands. Developmental Dynamics 231:441–448, 2004. © 2004 Wiley‐Liss, Inc.
Expression patterns of claudin family of tight junction membrane proteins in developing mouse submandibular gland
Authors: Hashizume Atsushi , Ueno Tomoo , Furuse Mikio , Tsukita Shoichiro , Nakanishi Yasuo , Hieda Yohki
Published: August 2004

Here, we investigated the expression of the claudin family of tight junction transmembrane proteins in the developing mouse submandibular gland. Data obtained by reverse transcriptase‐polymerase chain reaction, Western blot, and immunofluorescence microscopy showed the expression and localization of claudin‐3 to ‐8, ‐10, and ‐11 at epithelial tight junctions. Examination of the glands taken from embryonic day (E) 14, E16, and newborn mice revealed differential expression patterns of these claudins in the developing epithelium. Claudin‐3, ‐5, and ‐7 were expressed in all of the luminal epithelial cells of the ducts at all of the developmental stages examined and in those of terminal tubules at E16 and later. Claudin‐4 was expressed mainly in the ducts at all the developmental stages. The expression of claudin‐6 and ‐8 was also restricted to the ducts at E14 and E16; but after birth, the former was undetectable, whereas the latter was expressed in both the ducts and terminal tubules. Claudin‐10 and ‐11 were detectable mainly in the terminal tubules at E16 and later. In addition to being found in the epithelium, claudin‐5 was also expressed in certain mesenchymal cells, probably endothelial cells. These results will provide a valuable resource for further investigation of tubulogenesis and physiological regulation of claudin‐based tight junctions. Developmental Dynamics 231:425–431, 2004. © 2004 Wiley‐Liss, Inc.
PP1β9C interacts with trithorax in Drosophila wing development
Authors: Rudenko Andrey , Bennett Daimark , Alphey Luke
Published: August 2004

Type 1 Ser/Thr protein phosphatase (PP1) has many roles in Drosophila: regulating diverse processes from chromatin condensation to transforming growth factor‐β signaling. The presence of four PP1 genes, PP1α87B, PP1β9C, PP1α96A, and PP1α13C, encoding very similar proteins complicates analysis of their particular functions. Here, we report that the minor PP1 isoform PP1β9C binds in vitro and in vivo and genetically interacts with Trithorax (TRX), the archetypal member of the Trx‐G family of epigenetic regulators in Drosophila. Direct binding was demonstrated by GST pull‐down experiments and PP1β9C/TRX interaction in vivo was confirmed by coimmune precipitation from Drosophila embryonic extracts. PP1β9C was found to be present at all TRX sites on the polytene chromosomes. Flies homo‐ and hemizygous for loss‐of‐function alleles of PP1β9C exhibited specific wing defects when combined with various trx mutants, which indicates that PP1β9C and TRX cooperate in Drosophila wing development. Developmental Dynamics 231:336–341, 2004. © 2004 Wiley‐Liss, Inc.
Differential proliferative response of NKT cell subpopulations to in vitro stimulation in presence of different cytokines
Authors: Lin Henry , Nieda Mie , Nicol Andrew J.
Published: August 2004

Human Vα24+Vβ11+ NKT (NKT) cells have immune regulatory activities associated with rejection of tumors, infections and control of autoimmune diseases. They can be stimulated to proliferate using α‐galactosylceramide (KRN7000) and have the potential for therapeutic manipulation. Subpopulations of NKT cells (CD4+CD8–, CD4–CD8+ and CD4–CD8–) have functionally distinctive Th1/Th2 cytokine profiles and their relative numbers following stimulation may influence the Th1/Th2 balance, which may result in or prevent disease. We aimed to determine the effect of different cytokines in culture during stimulation of NKT cells on the relative proportions of NKT cell subpopulations. Our results show that all NKT cell subpopulations expanded following stimulation with KRN7000 and IL‐2, IL‐7, IL‐12 or IL‐15. Expansion capacity differed between subpopulations, resulting in different relative proportions of CD4+ and CD4– NKT cell subpopulations, and this was influenced by the cytokine used for stimulation. A Th1‐biased environment was observed after stimulation of NKT cells. NKT cells expanded under all conditions evaluated demonstrated significant cytotoxicity against U937 tumor cells. In view of the potential for NKT cell subsets to alter the balance of Th1 and Th2 environment, these data provide insights into the effects of NKT cell manipulation for possible therapeutic applications in different disease settings.
Association of hepatitis C virus envelope proteins with exosomes
Authors: Masciopinto Francesca , Giovani Cinzia , Campagnoli Susanna , Galli‐Stampino Luisa , Colombatto Piero , Brunetto Maurizia , Yen T. S. Benedict , Houghton Michael , Pileri Piero , Abrignani Sergio
Published: August 2004

As the human tetraspanin CD81 binds hepatitis C virus (HCV) envelope glycoprotein E2, we addressed the role CD81 may play in cellular trafficking of HCV envelope proteins. Studies on HCV life cycle are complicated by the lack of a robust cell culture system; we therefore transfected mammalian cells with HCV E1‐E2 cDNA, with or without human CD81 (huCD81) cDNA. In the absence of huCD81, HCV envelope proteins are almost completely retained in the endoplasmic reticulum. Instead, when huCD81 is present, a fraction of HCV envelope proteins passes through the Golgi apparatus, matures acquiring complex sugars and is found extracellularly associated with exosomes. These are 60–100‐nm membrane vesicles enriched in tetraspanins, released into the extracellular milieu by many cell types and having fusogenic activity. We also report that human plasma contains exosomes and that in HCV patients, viral RNA is associated with these circulating vesicles. We propose that the HCV‐CD81 complex leaves cells in the form of exosomes, circulates in this form and exploits the fusogenic capabilities of these vesicles to infect cells even in the presence of neutralizing antibodies.
CCL3/MIP‐1α is pro‐inflammatory in murine T cell‐mediated hepatitis by recruiting CCR1‐expressing CD4+ T cells to the liver
Authors: Ajuebor Maureen N. , Hogaboam Cory M. , Le Tai , Proudfoot Amanda E. I. , Swain Mark G.
Published: August 2004

T cell‐mediated hepatitis is associated with significant morbidity and mortality worldwide. Levels of C‐C chemokine ligand 3/macrophage inflammatory protein‐1α (CCL3/MIP‐1α) are elevated in the serum of patients with T cell‐mediated liver diseases, but its role is not fully understood. Con A‐induced hepatitis is a murine liver‐specific inflammation mediated by activated T cells and is driven by an up‐regulation of the hepatic expression of IFN‐γ. In this study, we have used CCL3/MIP‐1α gene‐deficient mice to examine the role of CCL3/MIP‐1α in the pathogenesis of Con A‐induced hepatitis. We demonstrate a novel pro‐inflammatory role for CCL3/MIP‐1α since CCL3/MIP‐1α deficiency significantly attenuated hepatic injury, both biochemically and histologically. Moreover, the recruitment of CCR1‐expressing CD4+ T cells to the liver after Con A treatment was strikingly attenuated by CCL3/MIP‐1α deficiency. Correspondingly, hepatic IFN‐γ produced by the recruited CD4+ T cells was significantly reduced by CCL3/MIP‐1α deficiency during Con A‐induced hepatitis. Furthermore, treatment of mice with a dual CCR1/CCR5 peptide antagonist, methionylated RANTES, also markedly reduced hepatic injury and decreased the numbers of CD4+ T cells within the liver producing IFN‐γ during Con A‐induced hepatitis. These findings demonstrate that blockade of the CCL3/MIP‐1α‐CCR1 pathway may represent a novel therapeutic target for treating T cell‐mediated liver diseases.
Anti‐apoptotic mechanism and reduced expression of phospholipase D in spontaneous and Fas‐stimulated apoptosis of human neutrophils
Authors: Lee Sun‐Young , Oh Ji‐Young , Lee Min‐Jung , Jang Min‐Jung , Park Hae‐Young , Kim Ja‐Woong , Min Do Sik , Park Yeong‐Min , Chang Young‐Chae , Bae Yoe‐Sik , Kwak Jong‐Young
Published: August 2004

Neutrophil apoptosis is a constitutive process that can be enhanced or delayed by signals triggered by various stimuli. In this work, we investigated the action mechanism of phospholipase D (PLD) and its expression in the inhibition of spontaneous and Fas‐mediated apoptosis. Anti‐Fas antibody‐stimulated apoptosis of neutrophils was significantly blocked by the exogenous addition of bacterial PLD from Streptomyces chromofuscus (scPLD), and neutrophils cultured for 24 h in the presence of anti‐Fas antibody showed lower agonist‐stimulated PLD activity compared to untreated cells. The amount of PLD1a protein reduced time‐dependently in cultured neutrophils, but was recovered by treating with LPS or GM‐CSF. The reduction in PLD1a protein level was blocked by caspase inhibitors. The exogenous addition of scPLD blocked the up‐regulation of Fas‐associated death domain expression, mitochondrial permeability, and the cleavages of procaspase‐8, procaspase‐3, and protein kinase C‐δ. We also found that the protein level of apoptosis‐inducing factor was increased in cultured neutrophils but its expression was reduced by scPLD. However, sulfasalazine‐induced apoptosis and change of protein expression were not blocked by scPLD. Taken together, the activity and protein levels of PLD play a role as an anti‐apoptotic factor by acting at multiple levels of the apoptotic cascade in neutrophils.
Cross‐presentation of a human tumor antigen delivered to dendritic cells by HSV VP22‐mediated protein translocation
Authors: Chhabra Arvind , Mehrotra Shikhar , Chakraborty Nitya G. , Mukherji Bijay , Dorsky David I.
Published: August 2004

Dendritic cells (DC) capture antigens from apoptotic and/or necrotic tumor cells and cross‐present them to T cells, and various ways of delivering tumor antigens to DC in vitro and in vivo are being pursued. Since fusions of antigenic proteins with the HSV integument protein VP22 are capable of intercellular trafficking, this approach has been exploited for delivery of antigens to antigen‐presenting cells. Adenoviral vectors were used to express the tumor‐associated‐but‐self‐antigen MART‐1 fused to HSV VP22 in MART‐1‐negative A375 melanoma cells and in DC. When expressed in A375 cells and allowed to spread to DC across a transwell barrier, the VP22‐MART‐1 fusion protein localized to both early and late endosomal structures of the DC. The DC loaded with the VP22‐MART‐1 fusion by intercellular trafficking efficiently presented the MART‐127–35 epitope to MART‐127–35‐specific CTL. Furthermore, transloaded DC were capable of expanding the population of MART‐127–35‐specific CTL. Thus, a tumor antigen acquired by intercellular trafficking can be cross‐presented by DC. This experimental approach should therefore be useful not only for studying the mechanism of cross‐presentation but also for vaccine development.
Heteroatom‐Substituted Group 4 Bis(indenyl)metallocenes
Authors: Leino Reko , Lehmus Petri , Lehtonen Ari
Published: August 2004

The synthesis, characterization and applications of heteroatom‐substituted group 4 bis(indenyl)metallocene complexes is reviewed. In recent years, an increasing number of group 4 metallocenes with electronically modified cyclopentadienyl ligands have been reported with, in some cases, considerably altered catalytic properties. This microreview focuses on bis(indenyl) complexes of titanium, zirconium and hafnium that contain nitrogen, oxygen and sulfur atoms directly bonded to the five‐ or six‐membered rings of the indenyl ligand framework. The catalytic performance is briefly reviewed, with emphasis on catalytic olefin polymerization. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
A Novel Route to Multinuclear d8 Metal−Chalcogen Compounds with Nuclearity Control
Authors: Mas‐Ballesté Rubén , Clegg William , Lledós Agustí , González‐Duarte Pilar
Published: August 2004

We have designed a new synthetic procedure to obtain multinuclear aggregates containing a {Pt2S2}n core. The synthetic strategy involves the reaction of [(dppp)Pt(μ‐S)2Pt(dppp)] with [PtCl2(PhCN)2] and thus the expansion of the {Pt2S2} ring to a [{Pt2S2}PtCl2] fragment. The lability of the chloride anions allows their replacement by bridging sulfide ligands, thus affording a new [{Pt2(μ3‐S)2}{Pt2(μ‐S)2}{Pt2(μ3‐S)2}] core. Its subsequent evolution to [{Pt2S2}3PtCl2] lays the foundation for a new cycle. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
First Tetranuclear CoII Cluster with Planar Triangular Pattern: Crystal Structure and Ferromagnetic Behavior
Authors: Du Miao , Guo Ya‐Mei , Bu Xian‐He , Ribas Joan
Published: August 2004

The preparation, crystal structure and magnetic properties of the first tetrameric CoII cluster with a unique planar triangular motif, [Co4L3(H2O)2](ClO4)2·2H2O (1), based on a carboxylic‐functionalized 1,5‐diazacyclooctane (DACO) bridging ligand [1,5‐bis(2‐carboxyethyl)‐DACO], are reported. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Generation of a conditional allele of the mouse prostaglandin EP4 receptor
Authors: Schneider André , Guan YouFei , Zhang Yahua , Magnuson Mark A. , Pettepher Cathy , Loftin Charles D. , Langenbach Robert , Breyer Richard M. , Breyer Matthew D.
Published: August 2004

Genetic disruption of the mouse EP4 receptor results in perinatal lethality associated with persistent patent ductus areteriosus (PDA). To circumvent this, an EP4 allele amenable to conditional deletion using the Cre/loxP system was generated. The targeting construct was comprised of a floxed exon2 in tandem with the neomycin‐resistance gene in intron 2, flanked by third 3′ LoxP site. Mice homozygous for the targeted allele (EP4lox+neo/lox+neo), or following its Cre‐mediated deletion (EP4del/del), also die within hours of birth with PDA. In contrast, mice homozygous for a partially recombined allele, retaining exon2 but lacking neo (EP4flox/flox), are viable and show no overt phenotype. Postnatal deletion of the floxed EP4 gene is efficiently achieved in the liver and kidney in a transgenic mouse expressing the inducible Mx1Cre recombinase. The EP mouse should provide a useful reagent with which to examine the physiologic roles of the EP4 receptor. genesis 40:7–14, 2004. © 2004 Wiley‐Liss, Inc.
Frizzled‐9 promoter drives expression of transgenes in the medial wall of the cortex and its chief derivative the hippocampus
Authors: Zhao Chunjie , Pleasure Samuel J.
Published: August 2004

The hippocampus develops from the medial wall of the forming cerebral cortex during embryonic life. Morphogenic signals from the Wnt pathway regulate several events during hippocampal development (Galceran et al.: Development 127:469–482, 2000; Lee et al.: Development 127:457–467, 2000; Zhou et al.: J Neurosci 24:121–126, 2004) and we have previously shown that Wnt receptors from the Frizzled (Fzd) family are expressed in discreet cortical domains during development (Kim et al.: Mech Dev 103:167–172, 2001). We generated transgenic mice using the putative control elements of the Fzd9 gene, normally selectively expressed in the developing and adult hippocampus, driving expression of a marker gene. These mice express LacZ in the brain in the same developmental distribution as endogenous Fzd protein. Postnatally, expression remains strong in the dendritic fields of hippocampal principal cells as well as hippocampal efferent axons. These mice provide a genetic and anatomic tool for analyzing development and reorganization in the hippocampus. genesis 40:32–39, 2004. © 2004 Wiley‐Liss, Inc.
Comparing alternative models: log vs Cox proportional hazard?
Authors: Basu Anirban , Manning Willard G. , Mullahy John
Published: August 2004

Health economists often use log models (based on OLS or generalized linear models) to deal with skewed outcomes such as those found in health expenditures and inpatient length of stay. Some recent studies have employed Cox proportional hazard regression as a less parametric alternative to OLS and GLM models, even when there was no need to correct for censoring. This study examines how well the alternative estimators behave econometrically in terms of bias when the data are skewed to the right. Specifically we provide evidence on the performance of the Cox model under a variety of data generating mechanisms and compare it to the estimators studied recently in Manning and Mullahy (2001). No single alternative is best under all of the conditions examined here. However, the gamma regression model with a log link seems to be more robust to alternative data generating mechanisms than either OLS on ln(y) or Cox proportional hazards regression. We find that the proportional hazard assumption is an essential requirement to obtain consistent estimate of the E(y∣x) using the Cox model. Copyright © 2004 John Wiley & Sons, Ltd.
The productivity of health care and health production functions
Authors: Koç Çağatay
Published: August 2004

This paper derives a necessary and sufficient condition under which increased health care productivity must lead to decreased (increased) demand for health care as long as the demand for health care is inelastic (elastic). It is shown that this condition identifies a class of health production functions, which may provide useful guidance to empirical studies that depend wholly or partly on the correct specification of a health production function. As an illustration, it is demonstrated that this class of production functions may be useful for empirical studies that test the hypothesis that schooling, increasing the efficiency of health production, leads to a larger health output from a given set of health inputs. The paper also offers broader classes of production functions that would enable one to test this relationship between the demand elasticity and the effect of health care productivity on health care demand. Copyright © 2004 John Wiley & Sons, Ltd.
Assessing quality of life in the elderly: a direct comparison of the EQ‐5D and AQoL
Authors: Holland Richard , Smith Richard D , Harvey Ian , Swift Louise , Lenaghan Elizabeth
Published: August 2004

As more research is undertaken on the elderly, accurately assessing changes in their quality of life becomes increasingly important. Generic instruments are the most popular method to assess quality of life, and one of the most widely used is the EQ‐5D. However, the range of dimensions, sensitivity of scales and completion rates have been raised as concerns when using this measure with the elderly. The AQoL is a newer instrument which offers greater richness in dimensions of health covered, and potentially offers greater sensitivity to changes in quality of life.

This paper presents the results of a ‘head‐to‐head’ comparison of the EQ‐5D and AQoL in terms of practicality, construct validity, agreement (of absolute scores and their change over time) and sensitivity to change, as part of a randomised controlled trial in the elderly. Poor agreement was found between both the absolute scores from each instrument and change in scores over time. Although the AQoL appeared to have more favourable construct validity, the EQ‐5D was easier to administer, had a higher completion rate, and appeared more sensitive to change. We conclude that the AQoL is probably less well suited to measuring health status in a very elderly population than the EQ‐5D. Copyright © 2004 John Wiley & Sons, Ltd.
The influence of subjective expectations about length and quality of life on time trade‐off answers
Authors: Nooten Floortje van , Brouwer Werner
Published: August 2004

When answering TTO questions respondents sometimes have to imagine being in a certain health state during their remaining lifespan, often based on objective life tables. Respondents however may have subjective expectations about length and quality of life that differ from the objective ones. If respondents do not fully abstract from own expectations, TTO scores may be biased. In this note, we indicate how subjective expectations could influence TTO scores and present some empirical findings suggesting that they do. Our results indicate that subjective expectations may serve as unobserved reference points and as such influence TTO responses. Copyright © 2004 John Wiley & Sons, Ltd.
Rapid Formation of Cholesterol Crystals in Gallbladder Bile Is Associated With Stone Recurrence After Laparoscopic Cholecystotomy
Authors: Jungst D , del Pozo R , Dolu M H , Schneeweiss S G , Frimberger E
Published: August 2004

Laparoscopic cholecystotomy (LCT) with subsequent extraction of gallstones and primary closure of the gallbladder has been introduced as an alternative therapy for patients with cholecystolithiasis and preserved gallbladder function. However, stone recurrence has to be considered as a major drawback that might be related to lithogenic factors of gallbladder bile or the composition of gallbladder stones. Therefore, these were studied in relation to stone recurrence within an observation period of 1 to 5 years (median, 3.6 years) in 50 patients after LCT. The concentrations of total and individual bile acids, phospholipids, cholesterol, total lipids, mucin, protein, and the cholesterol saturation indices in gallbladder bile were not significantly different between 10 patients with and 40 patients without stone recurrence. However, the crystal observation time was significantly (P < .02) shorter (range, 1–2 days; median, 1.5) in the bile of patients with stone recurrence compared to those without (range, 1–21 days, median 3.5). Moreover, all 10 stone recurrences were observed in the 28 patients with a crystal observation time in the bile of less than or equal to 2 days (approximate annual risk: 12%–15%), and no recurrences were observed in the 22 patients with a crystal observation time greater than 2 days (P < .0001) or in patients with pigment stones. The rapid formation of cholesterol monohydrate crystals in bile seems to be the major risk factor for recurrent stones after LCT. These are most likely cholesterol stones and, therefore, are amenable to oral bile–acid prevention or treatment.