bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen-specific human T-cell responses and T cell–dependent production of human antibodies in a humanized mouse model
Authors: Tonomura Noriko , Habiro Katsuyoshi , Shimizu Akira , Sykes Megan , Yang Yong-Guang
Published: February 2008

Humanized mice with a functional human immune system would be very useful for in vivo studies of human immunobiology. We have previously shown that cotransplantation of human fetal thymus/liver tissues and CD34+ fetal liver cells into immunodeficient nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice leads to the development of multiple lineages of human lymphohematopoietic cells and formation of secondary lymphoid organs with normal architecture. Here, we evaluated the ability of these humanized mice to develop antigen-specific, T cell–dependent antibody responses after in vivo immunization with T-dependent antigen, 2,4-dinitrophenyl hapten-keyhole limpet hemocyanin (DNP23-KLH). Human T cells from DNP23-KLH–immunized mice showed strong proliferation in response to KLH in vitro. Furthermore, T cell–dependent production of DNP-specific human antibodies (mainly IgG1 and IgG2) was detected in all immunized mice. These results confirm that a functional human immune system can be established in immunodeficient mice through cotransplantation of human fetal thymus/liver tissues and CD34+ hematopoietic stem/progenitor cells.
Modeling Escherichia coli signal peptidase complex with bound substrate: determinants in the mature peptide influencing signal peptide cleavage
Authors: Choo Khar Heng , Tong Joo Chuan , Ranganathan Shoba
Published: February 2008

Background

Type I signal peptidases (SPases) are essential membrane-bound serine proteases responsible for the cleavage of signal peptides from proteins that are translocated across biological membranes. The crystal structure of SPase in complex with signal peptide has not been solved and their substrate-binding site and binding specificities remain poorly understood. We report here a structure-based model for Escherichia coli DsbA 13–25 in complex with its endogenous type I SPase.

Results

The bound structure of DsbA 13–25 in complex with its endogenous type I SPase reported here reveals the existence of an extended conformation of the precursor protein with a pronounced backbone twist between positions P3 and P1'. Residues 13–25 of DsbA occupy, and thereby define 13 subsites, S7 to S6', within the SPase substrate-binding site. The newly defined subsites, S1' to S6' play critical roles in the substrate specificities of E. coli SPase. Our results are in accord with available experimental data.

Conclusion

Collectively, the results of this study provide interesting new insights into the binding conformation of signal peptides and the substrate-binding site of E. coli SPase. This is the first report on the modeling of a precursor protein into the entire SPase binding site. Together with the conserved precursor protein binding conformation, the existing and newly identified substrate binding sites readily explain SPase cleavage fidelity, consistent with existing biochemical results and solution structures of inhibitors in complex with E. coli SPase. Our data suggests that both signal and mature moiety sequences play important roles and should be considered in the development of predictive tools.
Stability of the core domain of p53: insights from computer simulations
Authors: Madhumalar Arumugam , Smith Derek John , Verma Chandra
Published: February 2008

Background

The tumour suppressor protein p53 protein has a core domain that binds DNA and is the site for most oncogenic mutations. This domain is quite unstable compared to its homologs p63 and p73. Two key residues in the core domain of p53 (Tyr236, Thr253), have been mutated in-silico, to their equivalent residues in p63 (Phe238 and Ile255) and p73 (Phe238 and Ile255), with subsequent increase in stability of p53. Computational studies have been performed to examine the basis of instability in p53.

Results

Molecular dynamics simulations suggest that mutations in p53 lead to increased conformational sampling of the phase space which stabilizes the system entropically. In contrast, reverse mutations, where p63 and p73 were mutated by replacing the Phe238 and Ile255 by Tyr and Thr respectively (as in p53), showed reduced conformational sampling although the change for p63 was much smaller than that for p73. Barriers to the rotation of sidechains containing aromatic rings at the core of the proteins were reduced several-fold when p53 was mutated; in contrast they increased when p73 was mutated and decreased by a small amount in p63. The rate of ring flipping of a Tyrosine residue at the boundary of two domains can be correlated with the change in stability, with implications for possible pathways of entry of agents that induce unfolding.

Conclusion

A double mutation at the core of the DNA binding domain of p53 leads to enhanced stability by increasing the softness of the protein. A change from a highly directional polar interaction of the core residues Tyr236 and Thr253 to a non-directional apolar interaction between Phe and Ile respectively may enable the system to adapt more easily and thus increase its robustness to structural perturbations, giving it increased stability. This leads to enhanced conformational sampling which in turn is associated with an increased "softness" of the protein core. However the system seems to become more rigid at the periphery. The success of this methodology in reproducing the experimental trends in the stability of p53 suggests that it has the potential to complement structural studies for rapidly estimating changes in stability upon mutations and could be an additional tool in the design of specific classes of proteins.
SynechoNET: integrated protein-protein interaction database of a model cyanobacterium Synechocystis sp. PCC 6803
Authors: Kim Woo-Yeon , Kang Sungsoo , Kim Byoung-Chul , Oh Jeehyun , Cho Seongwoong , Bhak Jong , Choi Jong-Soon
Published: February 2008

Background

Cyanobacteria are model organisms for studying photosynthesis, carbon and nitrogen assimilation, evolution of plant plastids, and adaptability to environmental stresses. Despite many studies on cyanobacteria, there is no web-based database of their regulatory and signaling protein-protein interaction networks to date.

Description

We report a database and website SynechoNET that provides predicted protein-protein interactions. SynechoNET shows cyanobacterial domain-domain interactions as well as their protein-level interactions using the model cyanobacterium, Synechocystis sp. PCC 6803. It predicts the protein-protein interactions using public interaction databases that contain mutually complementary and redundant data. Furthermore, SynechoNET provides information on transmembrane topology, signal peptide, and domain structure in order to support the analysis of regulatory membrane proteins. Such biological information can be queried and visualized in user-friendly web interfaces that include the interactive network viewer and search pages by keyword and functional category.

Conclusion

SynechoNET is an integrated protein-protein interaction database designed to analyze regulatory membrane proteins in cyanobacteria. It provides a platform for biologists to extend the genomic data of cyanobacteria by predicting interaction partners, membrane association, and membrane topology of Synechocystis proteins. SynechoNET is freely available at http://synechocystis.org/ or directly at http://bioportal.kobic.kr/SynechoNET/.
Comparing estimates of influenza-associated hospitalization and death among adults with congestive heart failure based on how influenza season is defined
Authors: Sandoval Carolyn , Walter Stephen D , Krueger Paul , Loeb Mark B
Published: February 2008

Background

There is little consensus about how the influenza season should be defined in studies that assess influenza-attributable risk. The objective of this study was to compare estimates of influenza-associated risk in a defined clinical population using four different methods of defining the influenza season.

Methods

Using the Studies of Left Ventricular Dysfunction (SOLVD) clinical database and national influenza surveillance data from 1986–87 to 1990–91, four definitions were used to assess influenza-associated risk: (a) three-week moving average of positive influenza isolates is at least 5%, (b) three-week moving average of positive influenza isolates is at least 10%, (c) first and last positive influenza isolate are identified, and (d) 5% of total number of positive isolates for the season are obtained. The clinical data were from adults aged 21 to 80 with physician-diagnosed congestive heart failure. All-cause hospitalization and all-cause mortality during the influenza seasons and non-influenza seasons were compared using four definitions of the influenza season. Incidence analyses and Cox regression were used to assess the effect of exposure to influenza season on all-cause hospitalization and death using all four definitions.

Results

There was a higher risk of hospitalization associated with the influenza season, regardless of how the start and stop of the influenza season was defined. The adjusted risk of hospitalization was 8 to 10 percent higher during the influenza season compared to the non-influenza season when the different definitions were used. However, exposure to influenza was not consistently associated with higher risk of death when all definitions were used. When the 5% moving average and first/last positive isolate definitions were used, exposure to influenza was associated with a higher risk of death compared to non-exposure in this clinical population (adjusted hazard ratios [HR], 1.16; 95% confidence interval [CI], 1.04 to 1.29 and adjusted HR, 1.19; 95% CI, 1.06 to 1.33, respectively).

Conclusion

Estimates of influenza-attributable risk may vary depending on how influenza season is defined and the outcome being assessed.
Identification of a novel resistance (E40F) and compensatory (K43E) substitution in HIV-1 reverse transcriptase
Authors: Huigen Marleen CDG , van Ham Petronella M , de Graaf Loek , Kagan Ron M , Boucher Charles AB , Nijhuis Monique
Published: February 2008

Background

HIV-1 nucleoside reverse transcriptase inhibitors (NRTIs) have been used in the clinic for over twenty years. Interestingly, the complete resistance pattern to this class has not been fully elucidated. Novel mutations in RT appearing during treatment failure are still being identified. To unravel the role of two of these newly identified changes, E40F and K43E, we investigated their effect on viral drug susceptibility and replicative capacity.

Results

A large database (Quest Diagnostics database) was analysed to determine the associations of the E40F and K43E changes with known resistance mutations. Both amino acid changes are strongly associated with the well known NRTI-resistance mutations M41L, L210W and T215Y. In addition, a strong positive association between these changes themselves was observed. A panel of recombinant viruses was generated by site-directed mutagenesis and phenotypically analysed. To determine the effect on replication capacity, competition andin vitroevolution experiments were performed. Introduction of E40F results in an increase in Zidovudine resistance ranging from nine to fourteen fold depending on the RT background and at the same time confers a decrease in viral replication capacity. The K43E change does not decrease the susceptibility to Zidovudine but increases viral replication capacity, when combined with E40F, demonstrating a compensatory role for this codon change.

Conclusion

In conclusion, we have identified a novel resistance (E40F) and compensatory (K43E) change in HIV-1 RT. Further research is indicated to analyse the clinical importance of these changes.
Identification of CD4int progenitors in mouse fetal spleen, a source of resident lymphoid cells
Authors: Desanti Guillaume E , Cumano Ana , Golub Rachel
Published: February 2008

Hematopoiesis occurs in different tissues during adult and fetal life. Splenic hematopoiesis arises in the fetal period until the first weeks of life. We have analyzed the hematopoietic progenitor content of the fetal spleen (FS) at the embryonic days 14.5–15.5. We first demonstrate that the hematopoietic content of the FS differs largely from its fetal liver (FL) counterpart. The difference mainly concerns the distribution of the different pool of progenitors, as most of the splenic progenitors are comprised in the lineage−Sca1−cKitlo contrary to the FL. We have divided the fetal hematopoietic pool into smaller fractions to enable characterization of the earliest lymphoid progenitors. Among the lymphoid progenitors that already represent a rare population, we were able to separate a population, respectively, enriched in B or T/NK progenitors. Lineage restriction of the different developmental intermediates was tested by clonal assays. We propose a model for fetal splenic hematopoietic progenitors and their distribution.
MicroRNA Expression Profiling of Thyroid Tumors: Biological Significance and Diagnostic Utility
Authors: Nikiforova Marina N. , Tseng George C. , Steward David , Diorio Donna , Nikiforov Yuri E.
Published: February 2008

Objective: MicroRNA (miRNA) expression is deregulated in many types of human cancers. We sought to investigate the expression patterns of miRNA in all major types of thyroid tumors, including tumors carrying distinct oncogenic mutations, and to explore the utility of miRNA profiling for the preoperative diagnosis of thyroid nodules.

Design: miRNA expression levels were detected in 60 surgically removed thyroid neoplastic and nonneoplastic samples and in 62 fine-needle aspiration (FNA) samples by RT-PCR using TaqMan MicroRNA Panel or individual miRNA sequence-specific primers. miRNA expression levels were calculated relative to normal thyroid tissue. All tumors were genotyped for most common mutations.

Results: Various histopathological types of thyroid tumors, including those deriving from the same cell type, showed significantly different profiles of miRNA expression. Oncocytic tumors, conventional follicular tumors, papillary carcinomas, and medullary carcinomas formed distinct clusters on the unsupervised hierarchical clustering analysis. Significant correlation between miRNA expression patterns and somatic mutations was observed in papillary carcinomas. A set of seven miRNAs (miR-187, miR-221, miR-222, miR-146b, miR-155, miR-224, and miR-197) that were most differentially overexpressed in thyroid tumors vs. hyperplastic nodules in the surgical samples was validated in the FNA samples, showing high accuracy of thyroid cancer detection.

Conclusions: In this study, we demonstrate that various histopathological types of thyroid tumors have distinct miRNA profiles, which further differ within the same tumor type, reflecting specific oncogenic mutations. A limited set of miRNAs can be used diagnostically with high accuracy to detect thyroid cancer in the surgical and preoperative FNA samples.
Preparation of polylactide-co-glycolide and chitosan hybrid microcapsules of amifostine using coaxial ultrasonic atomizer with solvent evaporation
Authors: Pamujula Sarala , Graves Richard A , Moiseyev Raisa , Bostanian Levon A , Kishore Vimal , Mandal Tarun K
Published: February 2008

The objective of this study was to evaluate the effect of various processing and formulation factors on the characteristics of amifostine hybrid microcapsules. Amifostine-loaded hybrid microcapsules were prepared using PLGA and chitosan. In short, amifostine powder was dissolved in de-aerated water with or without chitosan. The amifostine solution was later emulsified into PLGA solution in dichloromethane containing phosphatidylcholine. The resultant emulsion was fed through the inner capillary of a coaxial ultrasonic atomizer. The liquid fed through the coaxial outer capillary was either water or chitosan solution. The atomized droplets were collected into PVA solution and the droplets formed microcapsules immediately. The hybrid microcapsules prepared with chitosan solution only as an outer layer liquid showed the maximum efficiency of encapsulation (30%). The median sizes of all three formulations were 33–44 μm. These formulations with chitosan showed positive zeta-potential and sustained drug release with 13–45% amifostine released in 24 h. When chitosan was incorporated into inner as well as outer liquid layers, the drug release increased significantly, 45% (compared with other formulations) released in 24 h and almost 100% released in 11 days. Hybrid microcapsules of amifostine showed moderately high efficiency of encapsulation. The cationic charge (due to the presence of chitosan) of these particles is expected to favour oral absorption and thus overall bioavailability of orally administered amifostine.
Cell culture models of the human cornea — a comparative evaluation of their usefulness to determine ocular drug absorption in-vitro
Authors: Reichl Stephan
Published: February 2008

Cell culture models of the cornea are continually developed to replace the isolated animal cornea for transcorneal drug absorption studies. The aim of this study was to determine and compare epithelial tightness and permeability of currently available corneal cell culture models to avoid interlaboratory variability and to assess their usefulness for in-vitro permeation studies. Pure epithelial cell culture models (CEPI, SIRC and HCE-T cell lines), primary cultures of human corneal epithelium (HCEpiC) and the two commercially available models (RHC and Epiocular), as well as organotypic human cornea constructs (HCC, HCC-HCE-T), were investigated and data were compared with those obtained from the excised bovine cornea. Barrier properties were assessed by measurements of transepithelial electrical resistance (TEER) and permeability of three passively absorbed substances (mannitol, testosterone and timolol maleate) with different physico-chemical properties. TEER experiments revealed weak barrier functions for all of the investigated epithelial models (≤100–200 Ω cm2), except the HCE-T cell line. Transport studies confirmed TEER results insofar that models showing low TEER values also had higher permeation rates in comparison with the excised bovine cornea. However, models based on HCE-T cells demonstrated similar barrier properties to isolated corneal tissue. The corneal models investigated in our laboratory show clear differences in epithelial barrier function. In-vitro systems comprising the HCE-T cell line seem to be most appropriate to replace excised animal cornea for assessing corneal permeability.
Antibodies to S100 proteins have anxiolytic-like activity at ultra-low doses in the adult rat
Authors: Castagne Vincent , Lemaire Martine , Kheyfets Irina , Dugina Julia L , Sergeeva Svetlana A , Epstein Oleg I
Published: February 2008

S100 proteins are small calcium-binding proteins interacting with numerous intra- and extra cellular targets involved in diverse physiological functions. In particular, S100 proteins may be involved in the regulation of anxiety-related behaviour. In the present study, the effects of affinity-purified antibodies to S100 proteins administered orally at ultra-low doses were evaluated in pre-clinical tests for anxiolytic-like activity in the adult rat. In the Vogel conflict test in the rat, antibodies to S100 proteins increased punished drinking (anti-conflict effect) at 5 and 7.5 mL kg−1, but not at 2.5 or 10 mL kg−1. Antibodies to S100 proteins increased the percentage of entries into the open arms of an elevated plus-maze at 10 mL kg−1, but not at lower doses. Taken together, these results indicate the presence of anxiolytic-like activity for antibodies to S100 proteins over the dose range 5–10 mL kg−1 in the adult rat.
Ethanol consumption increases blood pressure and alters the responsiveness of the mesenteric vasculature in rats
Authors: Tirapelli Carlos R , Leone Andreia F C , Yogi Alvaro , Tostes Rita C , Lanchote Vera L , Uyemura Sergio A , Resstel Leonardo B M , Corrêa Fernando M A , Padovan Cláudia M , de Oliveira Ana M , Coelho Eduardo B
Published: February 2008

Chronic ethanol consumption and hypertension are related. In the current study we investigated whether changes in reactivity of the mesenteric arterial bed could account for the increased blood pressure associated with chronic ethanol intake. Changes in reactivity to phenylephrine and acetylcholine were investigated in the perfused mesenteric bed from rats treated with ethanol for 2 or 6 weeks and their age-matched controls. Mild hypertension was observed in chronically ethanol-treated rats. Treatment of rats for 6 weeks induced an increase in the contractile response of endothelium-intact mesenteric bed to phenylephrine, but not denuded rat mesenteric bed. The phenylephrine-induced increase in perfusion pressure was not altered after 2 weeks' treatment with ethanol. Moreover, acetylcholine-induced endothelium-dependent relaxation was reduced by ethanol treatment for 6 weeks, but not 2 weeks. Pre-treatment with indometacin, a cyclooxygenase inhibitor, reduced the maximum effect induced by phenylephrine (Emax) in endothelium-intact mesenteric bed from both control and ethanol-treated rats. No differences in the Emax values for phenylephrine were observed between groups in the presence of indometacin. l-NNA, a nitric oxide (NO) synthase (NOS) inhibitor, increased the Emax for phenylephrine in endothelium-intact mesenteric bed from control rats but not from ethanol-treated rats. Levels of endothelial NOS (eNOS) mRNA were not altered by chronic ethanol consumption. However, chronic ethanol intake strongly reduced eNOS protein levels in the mesenteric bed. This study shows that chronic ethanol consumption increases blood pressure and alters the reactivity of the mesenteric bed. Moreover, the increased vascular response to phenylephrine observed in the mesenteric bed is maintained by two mechanisms: an increased release of endothelial-derived vasoconstrictor prostanoids and a reduced modulatory action of endothelial NO, which seems to be associated with reduced post-transcriptional expression of eNOS.
Neuroprotective effects of breviscapine against apoptosis induced by transient focal cerebral ischaemia in rats
Authors: Yiming Liu , Wei He , Aihua Lin , Fandian Zeng
Published: February 2008

Breviscapine, a flavonoid isolated from the traditional Chinese medicinal herb Erigerin breviscapus, has been proved to be effective in protecting the brain against ischaemic damage, but the mechanisms remain unknown. In this study, we have demonstrated the effects of breviscapine on neuronal apoptosis in a rat model of transient focal cerebral ischaemia. Rats were administered with breviscapine (50 or 100 mg kg−1/day) intragastrically for seven successive days, then subjected to 2-h brain ischaemia induced by middle cerebral artery occlusion, followed by 24-h reperfusion. After reperfusion, the rats were killed and the brain samples were collected. Haematoxylin-eosin staining was used to evaluate the histopathological changes. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) and flow cytometry (FCM) analysis were used to detect the level of apoptosis. The expressions of bcl-2 and caspase-3 in the cortex were determined by Western blot. Significant increases in the number of haematoxylin-eosin- and TUNEL-positive staining cells and DNA fragmentation were observed at 24h after reperfusion, and the increases were inhibited by breviscapine (50 and 100 mg kg−1). Breviscapine at both doses markedly inhibited the expression and activation of caspase-3 and up-regulated the expression of bcl-2. These findings suggested that breviscapine attenuated neuroapoptosis and regulated the protein expression related to apoptosis after transient focal cerebral ischaemia, which may have contributed, in part, to the protective effects of breviscapine on cerebral ischaemic damage.
Quercetin does not alter lipopolysaccharide-induced fever in rats
Authors: Kanashiro Alexandre , Machado Renes R , Malvar David do C , Aguiar Fernando A , Souza Glória E P
Published: February 2008

Fever is considered an important component of the acute phase response of the body in defence against invading organisms such as bacteria. Quercetin, an important representative of the flavonoid class, has been extensively studied as an anti-inflammatory agent. In the present study, we investigated the effect of quercetin, administered orally (5, 25 and 50 mg kg−1) or intraperitoneally (50 mg kg−1), on the febrile response induced by either intraperitoneally (50 μg kg−1) or intravenously (5 μg kg−1) injected lipopolysaccharide (LPS from Escherichia coli) in rats. In contrast with the well known anti-inflammatory activity of quercetin, the results demonstrate that quercetin, at the doses used, did not alter the fever induced by LPS, regardless of the route of administration.
Transport ofSchisandra chinensisextract and its biologically-active constituents across Caco-2 cell monolayers — an in-vitro model of intestinal transport
Authors: Madgula Vamsi L M , Avula Bharathi , Choi Young W , Pullela Srinivas V , Khan Ikhlas A , Walker Larry A , Khan Shabana I
Published: February 2008

We have determined the intestinal transport of Schisandra chinensis extract and its lignans (gomisin A, gomisin N and schisandrin C) in the Caco-2 cell monolayer model. The transport across monolayers was examined for 2 h in absorptive and secretory directions. Quantitation of lignans was performed by HPLC. Out of the three lignans, gomisin A exhibited bi-directional transport, with Papp values in the range of 25–29 × 10−6 cm s−1, indicating a passive diffusion. Gomisin N, mixture and Schisandra extract displayed a higher transport in the secretory direction with efflux ratios in the range of 2.2–5.2. The efflux was decreased in the presence of inhibitors of multidrug resistance protein (MRP) transporter (MK-571) and P-glycoprotein (verapamil) indicating a possible involvement of an efflux pump and MRP in the transport of Schisandra lignans. Poor transport of schisandrin C was observed which could not be quantitated. The permeability of gomisin A in the isolated form was significantly different compared with the mixture or extract.
Thiolated chitosans: useful excipients for oral drug delivery
Authors: Werle Martin , Bernkop-Schnürch Andreas
Published: February 2008

To improve the bioavailability of orally administered drugs, formulations based on polymers are of great interest for pharmaceutical technologists. Thiolated chitosans are multifunctional polymers that exhibit improved mucoadhesive, cohesive and permeation-enhancing as well as efflux-pump-inhibitory properties. They can be synthesized by derivatization of the primary amino groups of chitosan with coupling reagents bearing thiol functions. Various data gained in-vitro as well as in-vivo studies clearly demonstrate the potential of thiolated chitosans for oral drug delivery. Within the current review, the synthesis and characterization of thiolated chitosans so far developed is summarized. Features of thiolated chitosans important for oral drug delivery are discussed as well. Moreover, different formulation approaches, such as matrix tablets and micro-/nanoparticles, as well as the applicability of thiolated chitosans for the oral delivery of various substance classes including peptides and efflux pump substrates, are highlighted.
A novel HSP90 chaperone complex regulates intracellular vesicle transport
Authors: Lotz Gregor P. , Brychzy Alexander , Heinz Stefan , Obermann Wolfgang M. J.
Published: February 2008

Heat shock protein 90 (HSP90) is considered a specialized molecular chaperone that controls the folding of cell-regulatory proteins such as steroid receptors and kinases. However, its high abundance is suggestive of a more general function in other fundamental processes. Here, we show that HSP90 is required for vesicular protein transport in the cell. We have identified a novel chaperone complex comprising HSP90 and TPR1 that is recruited to the membrane protein VAP-33. Depletion of the TPR1 protein in mammalian cells inhibits transport of vesicular stomatitis virus glycoprotein (VSVG) and leads to accumulation of this cargo protein in the Golgi apparatus. Furthermore, trafficking of VSVG between Golgi stacks is dependent on the ATPase function of HSP90 and can be inhibited by drugs specific for HSP90. Our results identify a new role for HSP90 in protein sorting, pointing to a central role for this molecular chaperone in the cell.
NEP-A and NEP-B both contribute to nuclear pore formation in Xenopus eggs and oocytes
Authors: Salpingidou Georgia , Rzepecki Ryszard , Kiseleva Elena , Lyon Carol , Lane Birgit , Fusiek Kasia , Golebiewska Anja , Drummond Shoena , Allen Terry , Ellis Juliet A. , Smythe Carl , Goldberg Martin W. , Hutchison Christopher J.
Published: February 2008

In vertebrates, the nuclear envelope (NE) assembles and disassembles during mitosis. As the NE is a complex structure consisting of inner and outer membranes, nuclear pore complexes (NPCs) and the nuclear lamina, NE assembly must be a controlled and systematic process. In Xenopus egg extracts, NE assembly is mediated by two distinct membrane vesicle populations, termed NEP-A and NEP-B. Here, we re-investigate how these two membrane populations contribute to NPC assembly. In growing stage III Xenopus oocytes, NPC assembly intermediates are frequently observed. High concentrations of NPC assembly intermediates always correlate with fusion of vesicles into preformed membranes. In Xenopus egg extracts, two integral membrane proteins essential for NPC assembly, POM121 and NDC1, are exclusively associated with NEP-B membranes. By contrast, a third integral membrane protein associated with the NPCs, gp210, associates only with NEP-A membranes. During NE assembly, fusion between NEP-A and NEP-B led to the formation of fusion junctions at which >65% of assembling NPCs were located. To investigate how each membrane type contributes to NPC assembly, we preferentially limited NEP-A in NE assembly assays. We found that, by limiting the NEP-A contribution to the NE, partially formed NPCs were assembled in which protein components of the nucleoplasmic face were depleted or absent. Our data suggest that fusion between NEP-A and NEP-B membranes is essential for NPC assembly and that, in contrast to previous reports, both membranes contribute to NPC assembly.
Distribution and lateral mobility of DC-SIGN on immature dendritic cells–implications for pathogen uptake
Authors: Neumann Aaron K. , Thompson Nancy L. , Jacobson Ken
Published: February 2008

The receptor C-type lectin DC-SIGN (CD209) is expressed by immature dendritic cells, functioning as an antigen capture receptor and cell adhesion molecule. Various microbes, including HIV-1, can exploit binding to DC-SIGN to gain entry to dendritic cells. DC-SIGN forms discrete nanoscale clusters on immature dendritic cells that are thought to be important for viral binding. We confirmed that these DC-SIGN clusters also exist both in live dendritic cells and in cell lines that ectopically express DC-SIGN. Moreover, DC-SIGN has an unusual polarized lateral distribution in the plasma membrane of dendritic cells and other cells: the receptor is preferentially localized to the leading edge of the dendritic cell lamellipod and largely excluded from the ventral plasma membrane. Colocalization of DC-SIGN clusters with endocytic activity demonstrated that surface DC-SIGN clusters are enriched near the leading edge, whereas endocytosis of these clusters occurred preferentially at lamellar sites posterior to the leading edge. Therefore, we predicted that DC-SIGN clusters move from the leading edge to zones of internalization. Two modes of lateral mobility were evident from the trajectories of DC-SIGN clusters at the leading edge, directed and non-directed mobility. Clusters with directed mobility moved in a highly linear fashion from the leading edge to rearward locations in the lamella at remarkably high velocity (1420±260 nm/second). Based on these data, we propose that DC-SIGN clusters move from the leading edge–where the dendritic cell is likely to encounter pathogens in tissue–to a medial lamellar site where clusters enter the cell via endocytosis. Immature dendritic cells may acquire and internalize HIV and other pathogens by this process.
Indoor Measurements of Noise at Low Frequencies — Problems and Solutions
Authors: Pedersen Steffen , Møller Henrik , Waye Kerstin Persson
Published: February 2008

Due to standing waves, the sound pressure level within a room may vary as much as 20–30 dB at low frequencies. Principal properties of low-frequency sound in rooms are illustrated by simulations, and sound pressure distributions as well as the performance of Swedish and Danish measurement methods are studied by measurements in three rooms. For assessment of annoyance, mainly areas of the room with high sound pressure levels are of interest, since persons present in such areas are not helped by the existence of lower levels in other areas. The level that is exceeded in 10% of the room (L10) is proposed as a rational and objective target for a measurement method. The Swedish method showed results close to the target, but, due to a doubtful use of C-weighting in the scanning, it may give results below the target in case of complex sounds. The Danish method was found to have a high risk of giving results substantially below the target, unless complainants can precisely appoint measurement positions, where the sound is loudest/most annoying. An alternative method using measurements in four three-dimensional corners of the room is proposed. This easy and straightforward method seems to give reliable results close to the proposed target.
Design Optimization of Long-Span King-Post Trusses
Authors: Gong Yanglin
Published: February 2008

With a unique arch and tie feature, king-post trusses can be built economically and efficiently and quickly for long-span roof structures. It has gained much popularity since its advent in early 1990s. The optimal design of the king-post truss system involves not only weight of the material but also factors such as fabrication, transportation and erection. This paper presents a design optimization method for long-span king-post roof truss systems. Through two example trusses built in North America, the main parameters affecting the cost of the truss system are studied thoroughly. Design guidelines, which aim to obtain a cost-effective design, are given for the determination of the depths of the overall truss and arched top truss. The result of the study is of great assistance to practicing engineers.
Development of Strut-And-Tie Models in Deep Beams with Web Openings
Authors: Guan Hong , Doh Jeung-Hwan
Published: February 2008

The strut-and-tie models of a total of fourteen (14) concrete deep beams with varying size and location of web openings are developed herein using a topology optimisation approach. By systematically eliminating inefficient materials from an over-designed discretized domain, the load transfer mechanism in deep beams is progressively characterised by the residual part of the structure. Both von Mises stress and displacement sensitivity number are used as deletion criteria with the aim of maximising the material efficiency and overall stiffness of the structural beam. The performance indices in terms of von Mises stress and nodal displacement are evaluated to monitor the optimisation process and to determine the optimal topology which is transformed to the strut-and-tie model. The relationship between the strut-and-tie models and the ultimate load-carrying capacity of the beams are discussed in some detail. A series of comparisons offer insight into the varying characteristics of the strut-and-tie models influenced by the size and location of web openings. To demonstrate the effectiveness of the resulting strut-and-tie model in aiding the design of deep beams, a worked design example of a representative beam is also presented where the practical layout of main steel reinforcement is determined.
Evolutionary Structural Topology Optimization for Continuum Structures with Structural Size and Topology Variables
Authors: Rong Jian Hua , Jiang Jie Sheng , Xie Yi Min
Published: February 2008

In this paper, a topology optimization method mixed structures with continuum and discrete elements is proposed. First, based on the stress and displacement formulations of the finite element analysis, a set of stress sensitivity numbers for beam and plane-stress structures are derived. Then, relative difference quotients for topology and size variables of mixed continuum structures with several different types of components are formulated. The optimization criteria, which incorporate the element stress level and relative different quotients, are developed for the topology optimization of continuum domains and the sizing optimization of discrete elements. A new optimization procedure based on the bi-directional evolutionary structural optimization method is proposed. Two examples are provided to demonstrate the validity and effectiveness of the proposed method.
Smaller Dendritic Spines, Weaker Synaptic Transmission, but Enhanced Spatial Learning in Mice Lacking Shank1
Authors: Hung Albert Y. , Futai Kensuke , Sala Carlo , Valtschanoff Juli G. , Ryu Jubin , Woodworth Mollie A. , Kidd Fleur L. , Sung Clifford C. , Miyakawa Tsuyoshi , Bear Mark F. , Weinberg Richard J. , Sheng Morgan
Published: February 2008

Experience-dependent changes in the structure of dendritic spines may contribute to learning and memory. Encoded by three genes, the Shank family of postsynaptic scaffold proteins are abundant and enriched in the postsynaptic density (PSD) of central excitatory synapses. When expressed in cultured hippocampal neurons, Shank promotes the maturation and enlargement of dendritic spines. Recently, Shank3 has been genetically implicated in human autism, suggesting an important role for Shank proteins in normal cognitive development. Here, we report the phenotype of Shank1 knock-out mice. Shank1 mutants showed altered PSD protein composition; reduced size of dendritic spines; smaller, thinner PSDs; and weaker basal synaptic transmission. Standard measures of synaptic plasticity were normal. Behaviorally, they had increased anxiety-related behavior and impaired contextual fear memory. Remarkably, Shank1-deficient mice displayed enhanced performance in a spatial learning task; however, their long-term memory retention in this task was impaired. These results affirm the importance of Shank1 for synapse structure and functionin vivo, and they highlight a differential role for Shank1 in specific cognitive processes, a feature that may be relevant to human autism spectrum disorders.
The Activation Gate and Gating Mechanism of the NMDA Receptor
Authors: Chang Huai-Ren , Kuo Chung-Chin
Published: February 2008

The NMDA receptor opens in response to binding of NMDA and glycine. However, it remains unclear where and how gating of the NMDA receptor pore is accomplished. We show that different point mutations between S645 and I655 (thus including the highly conserved SYTANLAAF motif) of M3c in NR2B lead to constitutively open channels. The current through these constitutively open channels are readily blocked by external Mg2+and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]. Also, the open-channel blocker MK-801 can no longer be trapped in these channels when NMDA and glycine are washed off. Moreover, M3c residues at or below A651(NR2B, A7 in SYTANLAAF) react with external methanethiosulfonate (MTS) reagents ∼500 to 1000-fold faster in the presence than in the absence of agonists NMDA and glycine. In fact, the MTS modification rate shows exactly the same NMDA concentration dependence as channel activation. In contrast, those residues external to A651 are always modified with similar kinetics whether NMDA and glycine are present or not. Interestingly, MTS modification of A651C(NR2B) holds the channel constitutively open. Mutations of A651(NR2B) into arginine, tryptophan, or phenylalanine, and similar mutations of the corresponding A652 in NR1 also lead to constitutively open channels. Double-mutant cycle analysis further shows that the effects of A652(NR1) and A651(NR2B) mutations are evidently non-additive (i.e., cooperative) if mutated into residues with large side chains or with compensatory charges [e.g., A652E(NR1)+A651R(NR2B)]. The side chain of A7 thus plays a determinant role in the intersubunit distance at this level, which is directly responsible for the activation gate and activation–deactivation gating of the NMDA receptor.
Persistent Na+and K+-Dominated Leak Currents Contribute to Respiratory Rhythm Generation in the Pre-Bötzinger ComplexIn Vitro
Authors: Koizumi Hidehiko , Smith Jeffrey C.
Published: February 2008

A central problem in analyzing neural circuit function is establishing how intrinsic neuronal conductances contribute to the generation of network activity. We used real-time calcium activity imaging combined with whole-cell patch-clamp recording to analyze contributions of subthreshold conductances in the excitatory rhythm-generating network in the respiratory pre-Bötzinger complex (pre-BötC) of neonatal ratin vitrobrainstem slice preparations. Voltage-clamp ramp recordings from imaged pre-BötC neurons revealed that persistent sodium (NaP) and K+-dominated leak currents primarily contribute to subthresholdI–Vrelations. We quantified NaP and leak conductance densities (g/Cm) in intrinsic oscillatory bursters and intrinsically nonbursters, the two main electrophysiological phenotypes of inspiratory neurons within the pre-BötC. Densities ofgNaPwere significantly higher for intrinsic bursters, whereas leak conductance densities were not significantly different between intrinsic bursters and nonbursters. By pharmacologically manipulatinggNaPand/orgLeakdirectly within the pre-BötC, we could modulate network oscillation frequency over a wide dynamic range and cause transitions between oscillatory and quiescent states. These results were consistent with models of the pre-BötC excitatory network consisting of heterogeneous mixtures of intrinsic bursters and nonintrinsic bursters incorporatinggNaPandgLeakwith parameter values found experimentally. We propose a paradigm whereby NaP and Leak represent a functional set of subthreshold conductances that endow the pre-BötC with rhythmogenic properties and represent targets for modulatory control of inspiratory rhythm generation.
Presynaptic Opioid and Nicotinic Receptor Modulation of Dopamine Overflow in the Nucleus Accumbens
Authors: Britt Jonathan P. , McGehee Daniel S.
Published: February 2008

Behaviorally relevant stimuli prompt midbrain dopamine (DA) neurons to switch from tonic to burst firing patterns. Similar shifts to burst activity are thought to contribute to the addictive effects of opiates and nicotine. The nucleus accumbens DA overflow produced by these drugs is a key element in their pathological effects. Using electrochemical techniques in brain slices, we explored the effects of opioids on single-spike and burst stimuli-evoked DA overflow in the dorsal and ventral striatum. In specific subregions of the nucleus accumbens, μ-opioids inhibit DA overflow elicited with single-spike stimuli while leaving that produced by burst stimuli unaffected. This is similar to published effects of nicotinic receptor blockade or desensitization, and is mediated by opioid receptor-induced inhibition of cholinergic interneurons. Whereas δ-opioids have similar effects, κ-opioids inhibit evoked DA overflow throughout the striatum in a manner that is not overcome with high-frequency stimuli. These observations reveal remarkable mechanistic overlap between the effects of nicotine and opiates within the dopamine reward pathway.
Necdin Plays a Role in the Serotonergic Modulation of the Mouse Respiratory Network: Implication for Prader-Willi Syndrome
Authors: Zanella Sébastien , Watrin Françoise , Mebarek Saïda , Marly Fabienne , Roussel Michel , Gire Catherine , Diene Gwenaëlle , Tauber Maïté , Muscatelli Françoise , Hilaire Gérard
Published: February 2008

Prader-Willi syndrome is a neurogenetic disease resulting from the absence of paternal expression of several imprinted genes, includingNECDIN. Prader-Willi children and adults have severe breathing defects with irregular rhythm, frequent sleep apneas, and blunted respiratory regulations. For the first time, we show that Prader-Willi infants have sleep apneas already present at birth. In parallel, in wild-type and Necdin-deficient mice, we studied the respiratory system within vivoplethysmography,in vitroelectrophysiology, and pharmacology. Because serotonin is known to contribute to CNS development and to affect maturation and function of the brainstem respiratory network, we also investigated the serotonergic system with HPLC, immunohistochemistry, Rabies virus tracing approaches, and primary culture experiments. We report first that Necdin-deficiency in mice induces central respiratory deficits reminiscent of Prader-Willi syndrome (irregular rhythm, frequent apneas, and blunted respiratory regulations), second that Necdin is expressed by medullary serotonergic neurons, and third that Necdin deficiency alters the serotonergic metabolism, the morphology of serotonin vesicles in medullary serotonergic neurons but not the number of these cells. We also show that Necdin deficiency in neonatal mice alters the serotonergic modulation of the respiratory rhythm generator. Thus, we propose that the lack of Necdin expression induces perinatal serotonergic alterations that affect the maturation and function of the respiratory network, inducing breathing deficits in mice and probably in Prader-Willi patients.
Coordinate Transformation is First Completed Downstream of Primary Motor Cortex
Authors: Yanai Yuval , Adamit Nofya , Israel Zvi , Harel Ran , Prut Yifat
Published: February 2008

It was suggested previously that the transformation of action to muscle-based coding is completed in the primary motor cortex (M1). This is consistent with a predominant direct pathway leading from M1 to motoneurons. Accordingly, spinal segmental interneurons that are located downstream to M1 are expected to show muscle-like coding properties. We addressed this hypothesis using simultaneous recording of cortical and spinal activity in primates performing an isometric wrist task with multiple targets and two hand postures. Here we show that while the motor cortex follows an intermediate coordinate frame, spinal interneurons already follow a muscle-like coordinate frame. We thus suggest that the final steps in coordinate transformation of motor commands take place downstream of M1 via corticospinal interactions.