bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary Graft Nonfunction and Kupffer Cell Activation After Liver Transplantation From Non-Heart-Beating Donors in Pigs
Authors: Monbaliu Diethard , van Pelt Jos , De Vos Rita , Greenwood Joanne , Parkkinen Jaakko , Crabbé Tina , Zeegers Marcel , Vekemans Katrien , Pincemail Joël , Defraigne Jean-Olivier , Fevery Johan , Pirenne Jacques
Published: January 2007

More extensive use of non-heart-beating donors (NHBD) could reduce mortality on liver transplantation waiting lists, but this is associated with more primary nonfunction (PNF). We assessed which parameters are involved in the development of PNF in livers from NHBD in a previously validated pig liver transplantation model, in which livers were transplanted after exposure to incremental periods of warm ischemia. The risk of PNF was unacceptably high (>50%) when livers were exposed to >30 minutes' warm ischemia before a short cold ischemic period. This study examined how PNF is affected by Kupffer cell activation (β-galactosidase), the generation of cytokines tumor necrosis factor alpha and interleukin 6, antioxidant mechanisms (ascorbic acid, α-tocopherol, reduced glutathione), circulating redox-active iron, and sinusoidal endothelial cell function (hyaluronic acid clearance). Kupffer cells were more activated in PNF recipients, as suggested by higher β-galactosidase levels (15 minutes after reperfusion), and secondarily, by higher production of tumor necrosis factor alpha and interleukin 6 (180 minutes after reperfusion). In addition, α-tocopherol and reduced glutathione were lower, and ascorbic acid and redox-active iron higher in PNF recipients. Finally, PNF grafts displayed progressively decreasing hyaluronic acid clearance (suggesting sinusoidal endothelial cell dysfunction) and parenchymal edema. Consequently, a reduced-flow phenomenon was documented. In grafts from NHBD that are destined to fail, β-galactosidase activity (a surrogate of Kupffer cell activation) is higher, proinflammatory cytokines are overproduced, some antioxidant mechanisms fail, and circulating redox-active iron is more rapidly released. A no-flow phenomenon is eventually observed in these failing grafts. Liver Transpl 13:239–247, 2007. © 2007 AASLD.
Cytotoxic T-Cell-Mediated Defense Against Infections in Human Liver Transplant Recipients
Authors: Tanaka Koichi , Uemoto Shinji , Egawa Hiroto , Takada Yasutsugu , Ozawa Kazue , Teramukai Satoshi , Kasahara Mureo , Ogawa Kohei , Ono Masako , Sato Hiroshi , Takai Kenji , Fukushima Masanori , Inaba Kayo
Published: January 2007

Previous studies have shown that postoperative infection is highest in transplant recipients with preexisting high levels of cytotoxic T lymphocytes (CTLs). To study this phenomenon, 106 adult liver transplant recipients were divided into 3 groups, based on hierarchical clustering of the CD3+CD8+CD45 isoform fractions prior to living donor liver transplantation (LDLT). Group I had the highest naive T-cell levels (subset CD45RO−CCR7+), Group II had the highest effector/memory (EM) T-cell levels (subset CD45RO+CCR7−), and Group III had the highest effector T-cell levels (subset CD45RO−CCR7−). In Group I, CTLs upregulated in response to invading pathogens much earlier and more rapidly than the other groups; this response was associated with CD4+ T-cell help, downregulation of CD27+CD28+ subsets, and upregulation of interferon-gamma and perforin expression. In contrast, in Groups II and III, CTLs upregulated slowly following persistent viral infection and did not respond efficiently to acute infection. In addition, Group II's cytolytic responses were due mainly to upregulation of the CD8+ EM T-cell fraction, whereas Group III's cytolytic responses were attributable to upregulation of effector T cells. The prevalence of EM or effector T cells was dependent on differentiation of the CD8+ phenotype before LDLT. In conclusion, in most infected transplant recipients who died, generation of CD8+ CTLs had been suppressed without associated CD4+ T-cell help. Liver Transpl 13:287–293, 2007. © 2007 AASLD.
Incisional Hernia Repair After Liver Transplantation
Authors: Perkins James D.
Published: January 2007

Abstract

Background:

Incisional hernia is a potential complication of orthotopic liver transplantation (OLT), with various options for repair.

Study Design:

We conducted a retrospective review of a series of adult patients with incisional hernias after OLT to identify risk factors and to compare methods of repair.

Results:

Incisional hernia repair was performed in 44 of 959 patients (4.6%) who underwent OLT from 1999 to 2005. Mean age at time of OLT was 53 years, and 73% were men. One or more complications of OLT occurred in 33 patients (75%) and included reoperation for bile leak or hemoperitoneum (34%), pulmonary problems (27%), early acute rejection (7%), and severe ascites and retransplantation (5% each). Incisional hernia was diagnosed at 419 days (range 62 to 1,524 days) and repaired at 471 days (range 109 to 1,581 days) after OLT. Presentation included pain or discomfort (78%) and incarceration or strangulation (5%); 17% were asymptomatic. Herniorrhaphy techniques included fascial repair with onlay polypropylene mesh reinforcement (n = 25, 57%); fascial repair only (n = 15, 34%); or inlay mesh sewn to fascial edges (n = 4, 9%). Complications of repair included recurrence in seven patients (16%) and wound infection and seroma in one patient each. Recurrence occurred in five patients with primary repair and two with mesh techniques (33% versus 6%, p = 0.04).

Conclusions:

Incisional hernia is a late complication of OLT for which male gender and early post-OLT complications are risk factors. Repair is safe when undertaken after acute problems have resolved and is best accomplished using mesh reinforcement of autologous tissue. (J Am Coll Surg 2006;203:421-425.)
Protein–protein recognition and interaction hot spots in an antigen–antibody complex: Free energy decomposition identifies “efficient amino acids”
Authors: Lafont Virginie , Schaefer Michael , Stote Roland H. , Altschuh Danièle , Dejaegere Annick
Published: January 2007

The molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) method was applied to the study of the protein–protein complex between a camelid single chain variable domain (cAb‐Lys3) and hen egg white lysozyme (HEL), and between cAb‐Lys3 and turkey egg white lysozyme (TEL). The electrostatic energy was estimated by solving the linear Poisson–Boltzmann equation. A free energy decomposition scheme was developed to determine binding energy hot spots of each complex. The calculations identified amino acids of the antibody that make important contributions to the interaction with lysozyme. They further showed the influence of small structural variations on the energetics of binding and they showed that the antibody amino acids that make up the hot spots are organized in such a way as to mimic the lysozyme substrate. Through further analysis of the results, we define the concept of “efficient amino acids,” which can provide an assessment of the binding potential of a particular hot spot interaction. This information, in turn, can be useful in the rational design of small molecules that mimic the antibody. The implications of using free energy decomposition to identify regions of a protein–protein complex that could be targeted by small molecules inhibitors are discussed. Proteins 2007. © 2007 Wiley‐Liss, Inc.
Solution structure of ApaG from Xanthomonas axonopodis pv. citri reveals a fibronectin‐3 fold
Authors: Cicero Daniel O. , Contessa Gian M. , Pertinhez Thelma A. , Gallo Mariana , Katsuyama Angela M. , Paci Maurizio , Farah Chuck S. , Spisni Alberto
Published: January 2007

ApaG proteins are found in a wide variety of bacterial genomes but their function is as yet unknown. Some eukaryotic proteins involved in protein‐protein interactions, such as the human polymerase δ‐interacting protein (PDIP38) and the F Box A (FBA) proteins, contain ApaG homology domains. We have used NMR to determine the solution structure of ApaG protein from the plant pathogen Xanthomonas axonopodis pv. citri (ApaGXac) with the aim to shed some light on its molecular function. ApaGXac is characterized by seven antiparallel β strands forming two β sheets, one containing three strands (ABE) and the other four strands (GFCC′). Relaxation measurements indicate that the protein has a quite rigid structure. In spite of the presence of a putative GXGXXG pyrophosphate binding motif ApaGXac does not bind ATP or GTP, in vitro. On the other hand, ApaGXac adopts a fibronectin type III (Fn3) fold, which is consistent with the hypothesis that it is involved in mediating protein‐protein interactions. The fact that the proteins of ApaG family do not display significant sequence similarity with the Fn3 domains found in other eukaryotic or bacterial proteins suggests that Fn3 domain may have arisen earlier in evolution than previously estimated. Proteins 2007 © 2007 Wiley‐Liss, Inc.
Identification of FAKTS as a novel 14‐3‐3‐associated nuclear protein
Authors: Luhn Patricia , Wang Haining , Marcus Adam I. , Fu Haian
Published: January 2007

Through bioinformatics and experimental approaches, we have assigned the first biochemical property to a predicted protein product in the human genome as a new 14‐3‐3 binding protein. 14‐3‐3 client proteins represent a diverse group of regulatory molecules that often function as signaling integrators in response to various environmental cues and include proteins such as Bad and Foxo. Using 14‐3‐3 as a probe in a yeast two‐hybrid screen, we identified a novel 14‐3‐3 binding protein with unknown function, initially designated as clone 546. Confocal microscopy revealed that clone 546 localized to the nucleus of mammalian cells. Additional studies show that the gene encoding clone 546 is expressed in many human tissues, including the thymus, as well as a number of cancer cell lines. The interaction of clone 546 with 14‐3‐3 was confirmed in mammalian cells. Interestingly, this interaction was markedly enhanced by the expression of activated Akt/PKB, suggesting a phosphorylation dependent event. Mutational analysis was carried out to identify Ser479 as the predominant residue that mediates the clone 546/14‐3‐3 association. Phosphorylation of Ser479 by AKT/PKB further supports a critical role for Akt/PKB in regulation of the clone 546/14‐3‐3 interaction. On the basis of these findings, we named this undefined protein FAKTS: Fourteen‐three‐three associated AKT Substrate. Proteins 2007. © 2007 Wiley‐Liss, Inc.
Probing nascent structures in peptides using natural abundance 13C NMR relaxation and reduced spectral density mapping
Authors: Slupsky Carolyn M. , Spyracopoulos Leo , Booth Valerie K. , Sykes Brian D. , Crump Matthew P.
Published: January 2007

The main chain motional properties for a series of peptides that appear to have preferred conformations in solution have been systematically studied using solution‐state nuclear magnetic resonance spectroscopy. The series of peptides were derived from the N‐termini of pro‐inflammatory chemokine proteins and HoxB1, a transcriptional regulator. As an unstructured control, a ten residue peptide was designed, synthesized, and found to be minimally structured from solution NMR data. The dynamic properties of the main chain for the peptides were assessed through longitudinal and transverse main chain 13Cα relaxation rates and the heteronuclear nuclear Overhauser effect. Motional parameters were interpreted using reduced spectral density mapping and compared with those derived from an extended Lipari–Szabo model in which the rotational correlation time was calculated for each main chain site of the peptide. Comparison of spectral density and Lipari–Szabo analyses for the peptides to those of the unstructured control peptide reveals significant differences in the dynamic behavior of the peptides. The amplitude of picosecond to nanosecond timescale motions for the main chain is observed to decrease for all of the chemokine peptides and HoxB1 over the regions that show partial structure at low temperatures. Comparatively, changes in picosecond to nanosecond timescale motions for the unstructured control peptide show no correlation with sequence position. These results indicate that there are distinguishable low temperature motional differences between an intrinsically unstructured peptide and peptides that have an inherent propensity to structure. Proteins 2007; © 2006 Wiley‐Liss, Inc.
Gender ratios in high school science departments: The effect of percent female faculty on multiple dimensions of students' science identities
Authors: Gilmartin Shannon , Denson Nida , Li Erika , Bryant Alyssa , Aschbacher Pamela
Published: January 2007

To examine how school characteristics are tied to science and engineering views and aspirations of students who are underrepresented in science and engineering fields, this mixed‐methods study explores relationships between aspects of students' science identities, and the representation of women among high school science teachers. Quantitative analyses tested the hypothesis that percent female faculty would have a positive effect on girls' science interests, and perceptions in particular, given the potentially greater availability of women role models. Findings indicate that percent female science faculty does not have an effect on a range of science measures for both male and female students, including the ways in which they understand scientific practice, their science self‐concept, and their interest in science‐related college majors. As qualitative data demonstrate, this could reflect practical constraints at schools where female faculty are concentrated and narrow perceptions of science teachers and “real” science. © 2007 Wiley Periodicals, Inc. J Res Sci Teach 44: 980–1009, 2007
Human insulin receptor juxtamembrane domain independent insulin signaling
Authors: Sattar Akm A. , Berhanu Chali , Gebreselassie Surafel , Berhanu Paulos
Published: January 2007

The exon 16‐encoded juxtamembrane (JM) domain of human insulin receptor (hIR) harbors the NPEY motif which couples the insulin‐activated hIR kinase to downstream signal transduction molecules. We sought to determine if signal transduction requires the entire exon 16‐encoded 22‐amino acid JM domain. Transfected CHO cells were generated stably expressing either the wild‐type hIR (hIR‐WT) or two mutant hIRs (hIRΔEx16 in which the JM domain was deleted, and hIRrosJM in which the deleted segment was replaced by the corresponding domain of v‐ros protein). The mutant hIRΔEx16 and hIRrosJM exhibited similar insulin‐binding as the hIRWT. Insulin internalization and insulin dose‐response experiments toward activation of downstream signal transduction molecules demonstrated that: i) the presence of intact hIR‐JM domain which harbors the NPEY motif is essential for Shc phosphorylation but not for IRS‐1 phosphorylation; ii) insulin signal transduction can occur independent of the JM domain of hIR and without participation of the NPEY motif; iii) engagement of this putative alternative downstream signal transduction is Shc independent and is dependent on insulin concentration; and iv) insulin internalization does not necessarily require the hIR specific aa sequence of the JM domain which can be partially substituted by the JM domain of the v‐ros tyrosine kinase.
Medical undergraduate students' beliefs and attitudes toward pain – How do they mature?
Authors: Niemi‐Murola Leila , Nieminen Juha T. , Kalso Eija , Pöyhiä Reino
Published: January 2007

At the University of Helsinki, pain‐related topics are taught throughout medical studies but without a formal pain curriculum. The purpose of this study was to assess medical students’ attitudes towards pain.

A questionnaire using a 6‐point Likert scale was constructed to measure pain‐related attitudes and beliefs described in previous studies. After a pilot study, the questionnaire was developed to assess attitudes towards elderly patients’ pain, prescription of opioids, assessment of pain and anxiety concerning chronic pain and its treatment. An electronic questionnaire with seven demographic and 28 pain‐related items was sent via e‐mail to 680 medical students at the University of Helsinki, Finland. Three reminders were sent with a new letter of encouragement. The students had 3 weeks to answer the questionnaire.

The questionnaire was returned by 63.4% of the students (N = 430). There were statistically significant differences between the students in different study years showing increasing empathy towards elderly patients’ pain (p < 0.001), willingness to prescribe opioids (p < 0.001) and anxiety about meeting patients suffering from chronic pain (p < 0.01). Final year students felt significantly more often anxious about seeing a chronic pain patient (p < 0.05) compared with the first year students. Female students were more anxious about seeing a patient suffering from chronic pain (p < 0.05) and they were less confident of their ability to treat chronic pain patients in primary care (p < 0.001) than the male students.

The general attitudes of students mature as hoped for during medical studies. Attitudes towards treating the pain of cancer patients and elderly patients are positive. Attention should be paid to helping students to cope with their emotions and to reducing their anxiety about meeting patients with chronic pain.
Active Gαq subunits and M3 acetylcholine receptors promote distinct modes of association of RGS2 with the plasma membrane
Authors: Clark Michael A. , Sethi Pooja R. , Lambert Nevin A.
Published: January 2007

RGS proteins accelerate the GTPase activity of heterotrimeric G proteins at the plasma membrane. Association of RGS proteins with the plasma membrane can be mediated by interactions with other membrane proteins and by direct interactions with the lipid bilayer. Here we use fluorescence recovery after photobleaching (FRAP) to characterize interactions between RGS2 and M3 acetylcholine receptors (M3Rs), Gα subunits and the lipid bilayer. Active Gαq and M3Rs both recruited RGS2‐EGFP to the plasma membrane. RGS2‐EGFP remained bound to the plasma membrane between interactions with active Gαq, but rapidly exchanged between membrane‐associated and cytosolic pools when recruited by M3Rs.
New Culture Approaches for Yessotoxin Production from the Dinoflagellate Protoceratium reticulatum
Authors: José Gallardo Rodríguez Juan , del Carmen Cerón García María , Camacho Francisco García , Sánchez Mirón Asterio , Belarbi El Hassan , Grima Emilio Molina
Published: January 2007

Fed‐batch and perfusion cultures were carried out in a traditional glass 2‐L bioreactor with the toxic dinoflagellate Protoceratium reticulatum. The maximum cell concentration obtained was 2.3 × 105 cell·mL−1, which is almost 1 order of magnitude higher than the maximum previously referenced for this species. L1 medium was shown to be clearly deficient in nitrate and phosphate for this strain, and addition of highly concentrated aliquots of these nutrients allowed higher cell concentrations to be obtained. This species consumed high amounts of nitrate and phosphate, 2.1 × 10‐3 and 2.3 × 10‐4 μmol·h−1·cell−1, respectively. However, this consumption produced a very low number of cells compared to other classes of microalgae, indicating that this species is, like other dinoflagellates, a poor competitor in terms of utilization of inorganic nutrients. Higher production of toxins and pigments was strongly associated with cell number in the culture, with maximum values of 700 ng·mL−1 and 1321 μg·mL−1, respectively. Most yessotoxins remained within the cells and not in the cell‐free culture medium, and their production was not related to either the age of the culture or the cell growth phase.
Transient Gene Expression Levels from Multigene Expression Vectors
Authors: Underhill Michèle F. , Smales C. Mark , Naylor Louise H. , Birch John R. , James David C.
Published: January 2007

Multigene expression vectors are commonly utilized whereby the (over)expression of two or more genes is desired simultaneously and often at supposedly equivalent levels. We have characterized dual‐gene and pEE14.4 RlucMFluc expression plasmids in which the second hCMV‐MIE promoter is replaced with a c‐myc IRES to enable one‐step coordinated expression of multiple genes in eukaryotic cells. The efficacy of these plasmids has been tested in Chinese hamster ovary (CHO) cells using renilla and firefly luciferase reporter genes, with the reporter gene in either position 1 or 2 from the 5′ to 3′ direction, respectively. The dual‐gene constructs gave enhanced second position gene expression levels compared to the first gene during transient transfection experiments (4‐ to 50‐fold increase 24 h post‐transfection). The pEE14.4 RlucMFluc plasmid gave enhanced first cistron expression compared to the second cistron (∼19‐fold increase 24 h post‐transfection). More equivalent transient expression levels were obtained by undertaking co‐transfection of the appropriate single gene plasmids. Reporter gene mRNA levels in the transfected cells were also evaluated by qRT‐PCR and compared to the observed protein expression levels. Analysis of the resulting data showed that transcriptional limitation of the first cistron in the dual‐gene expression system and not translational limitation was the major limiting factor. Taken together these data suggest that relative protein expression levels expected from heterologous gene products in a multigene vector cannot be predicted on copy number alone and it is important to characterize multigene or oligocistronic systems prior to use.
Effect of the Engineered Indole Pathway on Accumulation of Phenolic Compounds in Catharanthus roseus Hairy Roots
Authors: Chung Ill‐Min , Hong Seung‐Beom , Peebles Christie A. M. , Kim Jin‐Ae , San Ka Yiu
Published: January 2007

Catharanthus roseus has been well‐known to contain indole alkaloids effective for treatment of diverse cancers. We examined the intracellular accumulation profiles of phenolic compounds in response to ectopic overexpression of tryptophan feedback‐resistant anthranilate synthase holoenzyme (ASαβ) in C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic compounds were detected in late exponential phase ASαβ hairy roots. Uninduced and induced ASαβ hairy roots accumulated up to 1.2 and 4.5 mg/g DW over a 72‐h period, respectively. Upon induction, in parallel with a rapid increase in tryptophan in the first 48 h, accumulation of phenolic compounds tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which phenolic levels decreased back to the uninduced level by 72 h. Naringin was a predominant form that comprised about 72% and 36% of the total content of phenolic compounds in the uninduced and induced lines, respectively. Upon induction, accumulation of catechin drastically increased with the highest level (3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic acid showed no statistical difference. Catechin is a final product of the flavonoid pathway, and thus metabolic flux into this pathway is transiently increased by overexpression of AS. Like catechin, salicylic acid is very sensitive to induction as it began to increase to 5‐fold within 4 h of induction, but unlike catechin, no significant accumulation of salicylic acid was noted after 4 h of induction. The results suggest differential regulation of this particular biosynthesis branch within the phenolic pathway.
Production of IgM Specific Recombinant Dengue Multiepitope Protein for Early Diagnosis of Dengue Infection
Authors: Tripathi Nagesh K. , Shrivastva Ambuj , Pattnaik Priyabrata , Parida Manmohan , Dash Paban K. , Gupta Nimesh , Jana Asha M. , Rao P. V. Lakshmana
Published: January 2007

Dengue virus infections have recently undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Early detection of dengue infection based on the identification of antibodies has emerged as a practical and reliable means of diagnosis of dengue fever. The recombinant dengue multiepitope (rDME‐M) protein specific to IgM in E. coli was produced in a 5‐L fermentor for use in diagnostic purpose. After fermentation, dry cell weight was approximately 11.8 g/L of the culture. The rDME‐M protein was purified under denaturing conditions using single‐step nickel nitrilotriacetate (Ni‐NTA) affinity chromatography. The final yield of purified rDME‐M protein from this method was approximately 68.5 mg/L of the culture. The purity of rDME‐M protein was checked by SDS‐PAGE analysis, and the reactivity of this protein was further checked by Western blotting and enzyme‐linked immunosorbent assay (ELISA). The purified protein was used as an antigen in the development of an in‐house dipstick ELISA and evaluated with a panel of 80 patient sera, characterized using commercially available tests for detection of dengue antibody. The results were in excellent agreement with those of IgM capture ELISA (Pan‐Bio) and rapid immunochromatography (IC) test (Pan‐Bio). These results show that the in‐house dipstick ELISA using rDME‐M protein can be used as a promising kit because of its comparable sensitivity, specificity, field applicability, and low cost.
Effects of levonorgestrel on ovulation and oestrous behaviour in the female tammar wallaby
Authors: Hynes Emily F. , Nave Chris D. , Shaw Geoff , Renfree Marilyn B.
Published: January 2007

Subcutaneous hormone implants are a useful method for managing overabundant marsupials in restricted enclosures in Australia. Levonorgestrel induces long-term infertility in the kangaroo, tammar wallaby and koala, although the contraceptive mechanism of levonorgestrel is unknown for any marsupial. In the present study, it was investigated if insertion of a single levonorgestrel or control implant at the time of reactivation of the diapausing blastocyst affected the subsequent post-partum oestrus or the preceding follicular development. Twenty levonorgestrel-treated and 16 control animals were autopsied the day before birth and the accompanying post-partum oestrus (Day 25), and 10 levonorgestrel-treated and five of the nine control animals were autopsied 3–4 days (Days 29–30) after the expected birth and oestrus. Peripartum behaviour was observed and birth and mating times were recorded. Levonorgestrel treatment did not prevent follicular growth because there was no significant difference between treatment and control animals in the size of the dominant follicle at Day 25. None of the levonorgestrel-treated females autopsied at Days 29–30 had ovulated (n = 10), in contrast to controls, where four of the five that were autopsied had ovulated. Mating occurred in eight of nine control animals but in only three of 10 levonorgestrel-treated females. Males showed a more sustained period of interest in the three that were mated than in the controls, and mating took place significantly later after birth (36 v. 10 h; P = 0.038). Follicular growth and development was not blocked in any female but only one-third of the animals mated and none ovulated after levonorgestrel treatment. These results suggest that levonorgestrel inhibits the preovulatory surge of luteinising hormone.
mGluR5 stimulates gliotransmission in the nucleus accumbens
Authors: D'Ascenzo Marcello , Fellin Tommaso , Terunuma Miho , Revilla-Sanchez Raquel , Meaney David F. , Auberson Yves P. , Moss Stephen J. , Haydon Philip G.
Published: January 2007

Although metabotropic glutamate receptor 5 (mGluR5) is essential for cocaine self-administration and drug-seeking behavior, there is limited knowledge of the cellular actions of this receptor in the nucleus accumbens (NAc). Although mGluR5 has the potential to regulate neurons directly, recent studies have shown the importance of mGluR5 in regulating Ca
2+
signaling in astrocytes and, as a consequence, the Ca
2+
-dependent release of excitatory transmitters from these glia. In this study, we demonstrate that activation of mGluR5 induces Ca
2+
oscillations in NAc astrocytes with the correlated appearance of NMDA receptor-dependent slow inward currents detected in medium spiny neurons (MSNs). Photolysis of caged Ca
2+
loaded specifically into astrocytes evoked slow inward currents demonstrating that Ca
2+
elevations in astrocytes are responsible for these excitatory events. Pharmacological evaluation of these glial-evoked NMDA currents shows that they are mediated by NR2B-containing NMDA receptors, whereas synaptic NMDA receptors rely on NR2A-containing receptors. Stimulation of glutamatergic afferents activates mGluR5-dependent astrocytic Ca
2+
oscillations and gliotransmission that is sustained for minutes beyond the initial stimulus. Because gliotransmission is mediated by NMDA receptors, depolarized membrane potentials exhibited during up-states augment excitation provided by gliotransmission, which drives bursts of MSN action potentials. Because the predominant mGluR5-dependent action of glutamatergic afferents is to cause the sustained activation of astrocytes, which in turn excite MSNs through extrasynaptic NMDA receptors, our results raise the potential for gliotransmission being involved in prolonged mGluR5-dependent adaptation in the NAc.
Hepatitis B virus X protein enhances androgen receptor-responsive gene expression depending on androgen level
Authors: Chiu Chi-Ming , Yeh Shiou-Hwei , Chen Pei-Jer , Kuo Ti-Jung , Chang Ching-Ju , Chen Po-Jen , Yang Wan-Jen , Chen Ding-Shinn
Published: January 2007

Persistent hepatitis B virus (HBV) infection is a major risk of hepatocellular carcinoma (HCC). One intriguing feature of HBV-related HCC is the male predominance, with a male to female ratio of 5–7:1. This dominance has been attributed to the elevated androgen level and the enhanced androgen receptor (AR)-mediated activity in the host. How HBV infection and AR signaling modulate HCC is unknown. We investigated whether the HBV nonstructural protein, X protein (HBx) could cooperate with the AR signaling pathway to enhance carcinogenesis. We found that HBx increased the anchorage-independent colony-formation potency of AR in a nontransformed mouse hepatocyte cell line. We also found that HBx functioned as a positive transcriptional coregulator to increase AR-mediated transcriptional activity. This transcription enhancement was increased in the presence of androgen in a concentration-responsive manner, thus explaining a more prominent effect in males. HBx did not physically associate with ligand-bound AR in the nucleus, and it likely augmented AR activity by increasing the phosphorylation of AR through HBx-mediated activation of the c-Src kinase signaling pathway. Our study documents HBx as a previously undescribed class of noncellular positive coregulators for AR. The results reveal a mechanism for the vulnerability of males to microbial infections and the subsequent development of cancer.
Oviduct-specific expression of two therapeutic proteins in transgenic hens
Authors: Lillico S. G. , Sherman A. , McGrew M. J. , Robertson C. D. , Smith J. , Haslam C. , Barnard P. , Radcliffe P. A. , Mitrophanous K. A. , Elliot E. A. , Sang H. M.
Published: January 2007

Recent advances in avian transgenesis have led to the possibility of utilizing the laying hen as a production platform for the large-scale synthesis of pharmaceutical proteins. Ovalbumin constitutes more than half of the protein in the white of a laid egg, and expression of the ovalbumin gene is restricted to the tubular gland cells of the oviduct. Here we describe the use of lentiviral vectors to deliver transgene constructs comprising regulatory sequences from the ovalbumin gene designed to direct synthesis of associated therapeutic proteins to the oviduct. We report the generation of transgenic hens that synthesize functional recombinant pharmaceutical protein in a tightly regulated tissue-specific manner, without any evidence of transgene silencing after germ-line transmission.
Cholesteric mesophases
Authors: P Sixou  , J M Gilli  , A Ten Bosch  , F Fried  , P Maïssa  , L Varichon  , M H Godinho
Published: January 2007

In the past few years, there have been important developments concerning the role and the use of chirality in liquid crystals. An example which is now relatively well known is the chiral smecticC* low molecular weight liquid crystal (LMWLC) which lowers the response time of displays by a factor between one hundred and one thousand [1-3]. In chiral liquid crystal polymers (cholesteric or twisted nematic and smecticC*), it is necessary to understand the relation between the chemical structure and the properties of mesophases in liquid and solid polymer. Furthermore, chirality is also found in abundance in biopolymers and may be important for life processes. Applications in industry (high modulus fibers, sensors, non linear optics...) have already been realised or are being pursued.

The aim of this paper is to emphasize the polymer character of these liquid crystals. What is the influence of the molecular weight, the degree of substitution, the viscoelasticity and the existence of a glass transition? How to use the polymer character to obtain new informations on the liquid crystal state or to make new devices?

This rapid overview roughly covers the following parts:

(1) Formation and general properties of cholesteric mesophases: we give a rapid discussion of thermotropics and lyotropics, of the influence of degree of polymerisation, degree of substitution, concentration, temperature..., on the cholesteric-isotropic transition and on the order observed by textures or as described more quantitatively by the order parameter.

(2) Optical properties: we give a brief presentation of a theoretical work concerning the pitch prediction taking into account the asymmetric term in the interaction potential and the comparison with experimental results.

(3) Textures and defects which are mainly observed in these mesophases.

(4) Flow properties and rheological behaviour which are fundamental for processing.

(5) Blue phases near the isotropic-cholesteric transition which are discussed with emphasis on the specificity of the polymer state.

It is usual to distinguish main chain and side chain liquid crystal polymers [4, 5], which may be modelled as indicated in Fig. 1.

Cellulosic derivatives are good examples of main chain cholesteric polymers [6]. Cellulose is a very abundant polymer: each year, biosynthesis produces about 1011tons of cellulose. It readily accepts grafting of substituents, thus producing derivatives like cellulose acetate of hydroxypropylcellulose.

Examples of side chain liquid crystal polymers are those obtained with homo- or copolymers of polyacrilate, polymethacrylate or polysiloxane.
Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS
Authors: Thompson Andrew , Prescott Mark , Chelebi Noorhan , Smith John , Brown Tom , Schmidt Günter
Published: January 2007

The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.
OsCSLD1, a Cellulose Synthase-Like D1 Gene, Is Required for Root Hair Morphogenesis in Rice
Authors: Kim Chul Min , Park Sung Han , Je Byoung Il , Park Su Hyun , Park Soon Ju , Piao Hai Long , Eun Moo Young , Dolan Liam , Han Chang-deok
Published: January 2007

Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice.
An Arabidopsis Homolog of YeastATG6/VPS30Is Essential for Pollen Germination
Authors: Fujiki Yuki , Yoshimoto Kohki , Ohsumi Yoshinori
Published: January 2007

Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination.
empty pericarp4Encodes a Mitochondrion-Targeted Pentatricopeptide Repeat Protein Necessary for Seed Development and Plant Growth in Maize
Authors: Gutiérrez-Marcos José F. , Dal Prà Mauro , Giulini Anna , Costa Liliana M. , Gavazzi Giuseppe , Cordelier Sylvain , Sellam Olivier , Tatout Christophe , Paul Wyatt , Perez Pascual , Dickinson Hugh G. , Consonni Gabriella
Published: January 2007

The pentatricopeptide repeat (PPR) family represents one of the largest gene families in plants, with >440 members annotated in Arabidopsis thaliana. PPR proteins are thought to have a major role in the regulation of posttranscriptional processes in organelles. Recent studies have shown that Arabidopsis PPR proteins play an essential, nonredundant role during embryogenesis. Here, we demonstrate that mutations in empty pericarp4 (emp4), a maize (Zea mays) PPR-encoding gene, confer a seed-lethal phenotype. Mutant endosperms are severely impaired, with highly irregular differentiation of transfer cells in the nutrient-importing basal endosperm. Analysis of homozygous mutant plants generated from embryo-rescue experiments indicated that emp4 also affects general plant growth. The emp4-1 mutation was identified in an active Mutator (Mu) population, and cosegregation analysis revealed that it arose from a Mu3 element insertion. Evidence of emp4 molecular cloning was provided by the isolation of four additional emp4 alleles obtained by a reverse genetics approach. emp4 encodes a novel type of PPR protein of 614 amino acids. EMP4 contains nine 35–amino acid PPR motifs and an N-terminal mitochondrion-targeted sequence peptide, which was confirmed by a translational EMP4–green fluorescent protein fusion that localized to mitochondria. Molecular analyses further suggest that EMP4 is necessary to regulate the correct expression of a small subset of mitochondrial transcripts in the endosperm.
Arabidopsis PLDζ2Regulates Vesicle Trafficking and Is Required for Auxin Response
Authors: Li Gang , Xue Hong-Wei
Published: January 2007

Phospholipase D (PLD) and its product, phosphatidic acid (PA), play key roles in cellular processes, including stress and hormonal responses, vesicle trafficking, and cytoskeletal rearrangements. We isolated and functionally characterized Arabidopsis thaliana PLDζ2, which is expressed in various tissues and enhanced by auxin. A PLDζ2-defective mutant, pldζ2, and transgenic plants deficient in PLDζ2 were less sensitive to auxin, had reduced root gravitropism, and suppressed auxin-dependent hypocotyl elongation at 29°C, whereas transgenic seedlings overexpressing PLDζ2 showed opposite phenotypes, suggesting that PLDζ2 positively mediates auxin responses. Studies on the expression of auxin-responsive genes and observation of the β-glucuronidase (GUS) expression in crosses between pldζ2 and lines containing DR5-GUS indicated that PLDζ2, or PA, stimulated auxin responses. Observations of the membrane-selective dye FM4-64 showed suppressed vesicle trafficking under PLDζ2 deficiency or by treatment with 1-butanol, a PLD-specific inhibitor. By contrast, vesicle trafficking was enhanced by PA or PLDζ2 overexpression. Analyses of crosses between pldζ2 and lines containing PIN-FORMED2 (PIN2)–enhanced green fluorescent protein showed that PLDζ2 deficiency had no effect on the localization of PIN2 but blocked the inhibition of brefeldin A on PIN2 cycling. These results suggest that PLDζ2 and PA are required for the normal cycling of PIN2-containing vesicles as well as for function in auxin transport and distribution, and hence auxin responses.
Arabidopsis KAM2/GRV2 Is Required for Proper Endosome Formation and Functions in Vacuolar Sorting and Determination of the Embryo Growth Axis
Authors: Tamura Kentaro , Takahashi Hideyuki , Kunieda Tadashi , Fuji Kentaro , Shimada Tomoo , Hara-Nishimura Ikuko
Published: January 2007

We isolated an Arabidopsis thaliana mutant, katamari2 (kam2), that has a defect in the organization of endomembranes. This mutant had deformed endosomes and formed abnormally large aggregates with various organelles. Map-based cloning revealed that kam2 is allelic to gravitropism defective 2 (grv2). The KAM2/GRV2 gene encodes a homolog of a DnaJ domain–containing RECEPTOR-MEDIATED ENDOCYTOSIS-8, which is considered to play a vital role in the endocytotic pathway from the plasma membrane to lysosomes in animal cells. Immunofluorescent staining showed that KAM2/GRV2 protein localizes on punctate structures, which did not merge with any markers for Golgi, trans-Golgi network, endosomes, or prevacuolar compartments. KAM2/GRV2, which does not have a predicted transmembrane domain, was peripherally associated with the membrane surface of uncharacterized compartments. KAM2/GRV2 was expressed at the early to middle stages of seed maturation. We found kam2 mis-sorted seed storage proteins by secreting them from cells, indicating that KAM2/GRV2 is involved in the transport of the proteins into protein storage vacuoles. kam2 had another defect in embryogenesis. Half of the developing kam2-1 cotyledons grew into the opposite space of the seeds before the walking stick–shaped embryo stage. Our findings suggest that KAM2/GRV2 is required for proper formation of the endosomes involving protein trafficking to the vacuoles and determination of growth axis of the embryo.
ARF7 and ARF19 Regulate Lateral Root Formation via Direct Activation ofLBD/ASLGenes inArabidopsis
Authors: Okushima Yoko , Fukaki Hidehiro , Onoda Makoto , Theologis Athanasios , Tasaka Masao
Published: January 2007

Lateral root formation in Arabidopsis thaliana is regulated by two related AUXIN RESPONSE FACTORs, ARF7 and ARF19, which are transcriptional activators of early auxin response genes. The arf7 arf19 double knockout mutant is severely impaired in lateral root formation. Target-gene analysis in arf7 arf19 transgenic plants harboring inducible forms of ARF7 and ARF19 revealed that ARF7 and ARF19 directly regulate the auxin-mediated transcription of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18 (LBD16/ASL18) and/or LBD29/ASL16 in roots. Overexpression of LBD16/ASL18 and LBD29/ASL16 induces lateral root formation in the absence of ARF7 and ARF19. These LBD/ASL proteins are localized in the nucleus, and dominant repression of LBD16/ASL18 activity inhibits lateral root formation and auxin-mediated gene expression, strongly suggesting that these LBD/ASLs function downstream of ARF7- and ARF19-dependent auxin signaling in lateral root formation. Our results reveal that ARFs regulate lateral root formation via direct activation of LBD/ASLs in Arabidopsis.
The Acyltransferase GPAT5 Is Required for the Synthesis of Suberin in Seed Coat and Root of Arabidopsis
Authors: Beisson Fred , Li Yonghua , Bonaventure Gustavo , Pollard Mike , Ohlrogge John B.
Published: January 2007

Suberin and cutin are fatty acid– and glycerol-based plant polymers that act as pathogen barriers and function in the control of water and solute transport. However, despite important physiological roles, their biosynthetic pathways, including the acyl transfer reactions, remain hypothetical. We report the characterization of two suberin mutants (gpat5-1 and gpat5-2) of Arabidopsis thaliana GPAT5, encoding a protein with acyl-CoA:glycerol-3-phosphate acyltransferase activity. RT-PCR and β-glucuronidase–promoter fusion analyses demonstrated GPAT5 expression in seed coat, root, hypocotyl, and anther. The gpat5 plants showed a 50% decrease in aliphatic suberin in young roots and produced seed coats with a severalfold reduction in very long chain dicarboxylic acid and ω-hydroxy fatty acids typical of suberin but no change in the composition or content of membrane or storage glycerolipids or surface waxes. Consistent with their altered suberin, seed coats of gpat5 mutants had a steep increase in permeability to tetrazolium salts compared with wild-type seed coats. Furthermore, the germination rate of gpat5 seeds under high salt was reduced, and gpat5 seedlings had lower tolerance to salt stress. These results provide evidence for a critical role of GPAT5 in polyester biogenesis in seed coats and roots and for the importance of lipid polymer structures in the normal function of these organs.